Eye, Retina, and Visual System of the Mouse

MD DALIM #964302 5/11/08 RED GREEN BLUE BLK

EYE, RETINA, AND VISUAL SYSTEM OF THE MOUSE

EYE, RETINA, AND VISUAL SYSTEM OF THE MOUSE Edited by Leo M. Chalupa and Robert W. Williams THE MIT PRESS CAMBRIDGE, MASSACHUSETTS LONDON, ENGLAND

© 2008 Massachusetts Institute of Technology All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means (including photocopying, recording, or information storage and retrieval) without permission in writing from the publisher. For information about special quantity discounts, please email [email protected] This book was set in Baskerville by SNP Best-set Typesetter Ltd., Hong Kong. Printed and bound in the United States of America. Library of Congress Cataloging-in-Publication Data Eye, retina, and visual system of the mouse / Leo M. Chalupa and Robert W. Williams, editors. p. cm. Includes bibliographical references and index. ISBN 978-0-262-03381-7 (hardcover : alk. paper) 1. Mice—Sense organs. 2. Eye. 3. Visual pathways. I. Chalupa, Leo M. II. Williams, Robert W., 1952– QL737.R6E94 2008 573.8′819353—dc22 2007047445 10 9 8 7 6 5 4 3 2 1

CONTENTS

Preface

xi

I

INTRODUCTION TO THE MOUSE AS A SPECIES AND RESEARCH MODEL 1

1

Evolutionary History of the Genus Mus PRISCILLA K. TUCKER

2

Visual and Other Sensory Abilities of Mice and Their Influence on Behavioral Measures of Cognitive Function 13 Aimée A. Wong and RICHARD E. BROWN

3

Comparative Survey of the Mammalian Visual System with Reference to the Mouse 35 Catherine A. Leamey, Dario A. Protti, and BOGDAN DREHER

4

Survey of the Research Opportunities Afforded by Genetic Variation in the Mouse Visual System 61 LAWRENCE H. PINTO and John B. Troy

3

II OPTICS, PSYCHOPHYSICS, AND VISUAL BEHAVIORS OF MICE 71 5

The Mouse as a Model for Myopia, and Optics of Its Eye FRANK SCHAEFFEL

6

Characteristics and Applications of Mouse Eye Movements JOHN S. STAHL

73

87

v

7

Measuring Vision in the Awake Behaving Mouse GLEN T. PRUSKY and Robert M. Douglas

107

8

Electroretinographic Correlates of Normal and Abnormal Retinal Ganglion Cell Activity 119 VITTORIO PORCIATTI

III ORGANIZATION OF THE ADULT MOUSE EYE AND CENTRAL VISUAL SYSTEM 127 9

Aqueous Humor Dynamics and Trabecular Meshwork ERNST R. TAMM and Antonia Kellenberger

10

Recent Advances in the Investigation of Mouse Cone Photoreceptors ARKADY LYUBARSKY, SERGEI S. NIKONOV, LAUREN L. DANIELE, and EDWARD N. PUGH, JR.

11

Mosaic Architecture of the Mouse Retina BENJAMIN E. REESE

12

Synaptic Organization of the Mouse Retina ENRICA STRETTOI

13

Distribution and Functional Roles of Neuronal Gap Junctions in the Mouse Retina 165 STEWART A. BLOOMFIELD and Béla Völgyi

14

Neurotransmission in the Mouse Retina 175 MAUREEN A. MCCALL, Neal S. Peachey, and Ronald G. Gregg

15

Morphological, Functional, and Developmental Properties of Mouse Retinal Ganglion Cells 189 Julie L. Coombs and LEO M. CHALUPA

16

The Lamina Cribrosa Region and Optic Nerve of the Mouse CHRISTIAN-ALBRECHT MAY

17

Photoentrainment of the Circadian Oscillator SATCHIDANANDA PANDA

18

Physiology of the Mouse Dorsal Lateral Geniculate Nucleus MATTHEW S. GRUBB

19

Superior Colliculus and Saccade Generation in Mice Tomoya Sakatani and TADASHI ISA

20

Interconnections of Visual Cortical Areas in the Mouse ANDREAS BURKHALTER and Quanxin Wang

vi

contents

129

135

147

157

201

207

219

233

245

IV DEVELOPMENT OF THE MOUSE EYE

255

21

Chronology of Development of the Mouse Visual System: Comparisons with Human Development 257 BARBARA L. FINLAY and Barbara Clancy

22

Developmental Studies of the Mouse Lens: Past, Present, and Future MICHAEL L. ROBINSON

23

Development of the Retinal Vasculature and the Effects of High Oxygen (Retinopathy of Prematurity) 285 MICHAEL R. POWERS

24

Specification, Histogenesis, and Photoreceptor Development in the Mouse Retina 299 DEEPAK Lamba, Branden Nelson, Mike O. Karl, and THOMAS A. REH

25

Rb and the Control of Retinal Development MICHAEL A. DYER

26

Gene Regulatory Networks and Retinal Ganglion Cell Development XIUQIAN MU and WILLIAM H. KLEIN

27

Cell Death in the Mouse Retina 333 LUCIA GALLI-RESTA and Maria Cristina Cenni

28

The Function of the Retina prior to Vision: The Phenomenon of Retinal Waves and Retinotopic Refinement 343 MARLA B. FELLER and Aaron G. Blankenship

29

ON and OFF Pathways in the Mouse Retina and the Role of Stimulation NING TIAN

30

Retinoic Acid Function in Central Visual Pathways 363 URSULA C. DRÄGER, Tuanlian Luo, and Elisabeth Wagner

267

311

321

353

V DEVELOPMENT AND PLASTICITY OF RETINAL PROJECTIONS AND VISUOTOPIC MAPS 377 31

Intraretinal Axon Guidance 379 LYNDA ERSKINE and Hannah Thompson

32

Early Development of the Optic Stalk, Chiasm, and Astrocytes TIMOTHY J. PETROS and CAROL A. MASON

33

Axon Growth and Regeneration of Retinal Ganglion Cells JEFFREY L. GOLDBERG

389

401

contents

vii

34

Development of the Retinogeniculate Pathway WILLIAM GUIDO

415

35

Developmental Synaptic Remodeling: Insights from the Mouse Retinogeniculate Synapse 429 CHINFEI CHEN

36

Ocular Dominance Plasticity 439 MARK HÜBENER, SONJA B. HOFER, and THOMAS D. MRSIC-FLOGEL

37

Environmental Enrichment and Visual System Plasticity 449 Alessandro Sale, Nicoletta Berardi, and LAMBERTO MAFFEI

38

Bidirectional Experience-Dependent Plasticity in Primary Visual Cortex Mikhail Y. Frenkel and MARK F. BEAR

VI MOUSE MODELS OF HUMAN EYE DISEASE

465

477

39

Mouse Models: A Key System in Revolutionizing the Understanding of Glaucoma 479 Gareth R. Howell, Jeffrey K. Marchant, and SIMON W. M. JOHN

40

Cataract Genetics JOCHEN GRAW

41

Mouse Models of Infectious Eye Diseases LINDA D. HAZLETT

42

Mouse Models of Autoimmune and Immune-Mediated Uveitis RACHEL R. CASPI

43

Mouse Models of Norrie Disease WOLFGANG BERGER

44

The Lipofuscin of Retinal Pigment Epithelial Cells: Learning from Mouse Models of Retinal Disease 539 JANET R. SPARROW, So R. Kim, Young P. Jang, and Jilin Zhou

45

Studies of Diabetic Retinopathy Using Mice 547 Rose A. Gubitosi-Klug and TIMOTHY S. KERN

46

Roles of Oxygen in the Stability of Photoreceptors: Evidence from Mouse and Other Models of Human Disease 559 JONATHAN STONE and Krisztina Valter

47

Complex Genetics of Photoreceptor Light Damage MICHAEL DANCIGER

48

Age-Related Eye Diseases BO CHANG

viii

contents

493

581

505

513

527

573

VII ADVANCED GENOMIC TECHNOLOGIES: APPLICATIONS TO THE MOUSE VISUAL SYSTEM 591 49

New Genetic Technologies for Studying the Morphology, Physiology, and Development of Mouse Retinal Neurons 593 Tudor C. Badea and JEREMY Nathans

50

Adenoassociated Virus Gene Therapy in Mouse Models of Retinal Degeneration 605 Shannon E. Boye, Sanford L. Boye, and WILLIAM W. HAUSWIRTH

51

Delivery of Plasmids into the Visual System Using Electroporation Takahiko Matsuda and CONSTANCE L. CEPKO

52

Genetic Knockouts in Ophthalmic Research and Drug Discovery DENNIS S. RICE

53

Beyond Positional Cloning of Single Gene Mutations: Use of Mouse Models to Examine Allelic Variance and to Identify Genetic Modifiers 649 PATSY M. NISHINA and Juergen K. Naggert

54

The Mouse Eye Transcriptome: Cellular Signatures, Molecular Networks, and Candidate Genes for Human Disease 659 ELDON E. GEISERT and Robert W. Williams

55

Mouse Models, Microarrays, and Genetic Networks in Retinal Development and Degenerative Disease 675 Sunil K. Parapuram and Anand SWAROOP

56

Retinal Vascular and Retinal Pigment Epithelium Gene Expression Michael I. Dorrell and MARTIN FRIEDLANDER

57

Gene Expression in Cornea and Lens 697 Shivalingappa K. Swamynathan and JORAM PIATIGORSKY

58

Proteomics of the Mouse Lens 711 WOLFGANG HOEHENWARTER and PETER R. JUNGBLUT

59

Genetic and Proteomic Analyses of the Mouse Visual Cycle John C. Saari and JOHN W. CRABB

Contributors Index

621

635

685

721

733

737

contents

ix

PREFACE The publication of this volume on the mouse eye and visual system seems overdue, given the burst of studies that have appeared on this topic in recent years. As the chapters in this volume illustrate, mouse models have already contributed to a wide range of scientific breakthroughs for a significant number of ocular and neurological diseases. They have also allowed us to address fundamental issues that could not be pursued using other experimental models. As is often the case, technical rather than conceptual advances have induced or driven the move to the mouse. The major factor has been the comparatively recent ability to produce mice with precisely defined changes in gene sequence—so-called transgenic and knockout mice. As a result of this powerful technology, genes can now be turned on or off in specific cell types at specific stages of development. Of equal importance, mouse genes can now be swapped for their human equivalents. This advance should ultimately permit us to humanize cells and tissues as complex as the retina, which will allow us to test new therapies in a controlled experimental context. Although the power of mouse models is now taken for granted, it is worth noting that until fairly recently, study of the mouse visual system was a backwater of vision research. Size matters, and the large eyes of rabbits, cats, and cows were much more tractable for biochemical, histological, and functional studies. However, a small and dedicated cadre of radiation biologists and geneticists began to exploit mice for vision research, collecting and analyzing the stream of spontaneous and induced mutations at the Jackson Laboratory, at Oak Ridge National Laboratory, at the MRC Harwell, at the German Research Center for Environmental Health (GSF, Munich), and at universities and mouse breeding and research centers. Richard Sidman, Jin Kinoshita, Matthew LaVail, Ursula Dräger, and others helped to build the mouse resource infrastructure that we now take for granted, mapping and characterizing some of the first retinal degeneration, cataract, and pigmentation mutations that disturbed visual system function. The main activity in vision research following the catalytic studies of David Hubel and Torsten Wiesel in the 1960s and 1970s centered on fundamental problems in visual neurophysiology, development, plasticity, and pathology. Cats and macaques were the dominant species used in this program of discovery. These models are still making major contributions to our understanding of mechanisms of vision and visual system development. Because of its small size, experimental fragility, and low acuity, the mouse was generally regarded as a compromised model for this kind of systems neuroscience. Those scientists interested in development typically followed the lead of Roger Sperry and colleagues and turned to cold-blooded vertebrates, particularly fishes and frogs. The sudden introduction of transgenic and knockout technology by Rudolph Jaenisch, Mario Capecchi, Martin Evans, and Oliver Smithies radically restructured experimental possibilities. What once seemed risky or even foolish—for example, functional studies of mouse retinal ganglion cells and visual cortex—now seemed well warranted if only the experimental methods could be miniaturized to deal with an eye with an axial length of 2 mm and a brain with a total mass of 450 mg. It soon became obvious just how informative

xi

it would be to exploit this species to modify gene sequences and to test effects on development, function, plasticity, and disease progression. This book can be considered a compendium of results from the first wave of studies. We hope that it will answer the question of just how useful mouse models can be as part of the exchange between experimental and clinical research. Study of these mouse models has already demonstrated real translational prowess. However, it is just a beginning, since large gaps in our knowledge remain to be filled, some of which, no doubt, are still blissfully outside our awareness. We owe thanks to many colleagues. First, we are grateful to Drs. Patsy Nishina and Maureen McCall, as well as the National Eye Institute of the National Institutes of Health and the Jackson Laboratory, for having organized and supported meetings and workshops in 2004 and 2006 on the laboratory mouse in vision research. These meetings helped motivate the assembly of this book. We also thank the staff at MIT Press. For several decades, MIT Press has helped promote vision research by publishing wide-ranging texts on vision from engineering, computational, biological, psychological, and theoretical points of view. In particular, we are grateful to Barbara Murphy and Meagan Stacey for their expert assistance in bringing this project to fruition. Finally, we thank the authors whose work is included herein, for their contributions to this volume and for the sacrifices they made to meet the deadlines necessary to bring this project to a successful conclusion.

xii

preface

I INTRODUCTION TO THE MOUSE AS A SPECIES AND RESEARCH MODEL

1

Evolutionary History of the Genus Mus PRISCILLA K. TUCKER

Knowing the evolutionary history of the house mouse is valuable not just for evolutionary and systematic biologists, who study the diversity of life, but also for biomedical researchers, who use the mouse as a research model with the assumption that certain discoveries can be generalized to humans. Understanding the biological significance of a new finding ultimately requires comparisons among populations and species in the context of their evolutionary history. For example, a newly discovered feature of the mouse may be unique to a population or to the species, it may be conserved only among species closely related to the mouse, or it may be conserved across more distantly related species such as mouse and human. Comparisons among taxa in the context of their evolutionary history also provide the researcher the ability to determine how genes, genetic or developmental pathways, physiological features, or complex traits have changed over time, and whether observed similarities between the mouse and other species are due to common ancestry or to convergence (independent evolution). This chapter introduces the reader to the evolutionary history of species in the genus Mus, to which house mice and laboratory inbred strains belong. It includes an overview of species diversity in the genus and a more comprehensive account of the evolutionary history of the house mouse, the Mus musculus complex, from which laboratory strains are derived. It concludes with a summary of our current understanding of the evolutionary relationships among members of the genus as depicted in a phylogeny. The house mouse is a member of the Order Rodentia, one of 29 currently recognized orders of mammals (Wilson and Reeder, 2005). The common laboratory rat (Rattus norvegicus) is also a member of this order. Rodentia and the Order Lagomorpha (rabbits, hares, and pikas) are considered sister taxa, each other’s closest relatives, and together are thought to be closely related to a group comprising the Orders Primates, Dermoptera (flying lemurs), and Scandentia (tree shrews) (Murphy et al., 2001; Carleton and Musser, 2005; Kriegs et al., 2006). In contrast, the cat, a taxon that is also used in vision research, is a member of the Order Carnivora, a taxonomic group proposed to be more closely related to the Orders Perissodactyla (horses), Artiodactyla (even-toed ungulates), and Cetacea (whales, dolphins, and

porpoises) than to either Rodentia or Primates (Murphy et al., 2001; Kriegs et al., 2006). The genus Mus Linnaeus, 1758, includes a monophyletic group of 30 or more species belonging to the subfamily Murinae in the speciose rodent family Muridae (Musser and Carleton, 2005; Cucchi et al., 2006; Shimada et al., 2007). Mus species are found throughout the Old World from southern Africa to eastern Asia. They occupy ecologically diverse habitats such as tropical montane forests, grasslands, and deserts (Suzuki et al., 2004; Musser and Carleton, 2005; Veyrunes et al., 2005). Like many other murine rodents, most are thought to be active at night. The genus can be distinguished from other murine genera using a combination of morphological features (Marshall, 1977a, 1997b, 1978, 1981) and molecular characters (reviewed in Suzuki et al., 2004, and Veyrunes et al., 2005). Utilizing a molecular clock, a combination of mitochondrial and nuclear genes, and calibration points based on fossil dates, researchers have estimated the divergence of the genus Mus from other murine lineages as occurring between 11 and 8 million years ago (mya) (Chevret et al., 2005; Veyrunes et al., 2005). Fossil evidence (Flynn and Jacobs, 1983; Jacobs and Downs, 1994) and phylogeographic considerations (Suzuki et al., 2004; Veyrunes et al., 2005) place the origin of the genus in Asia.

Diversity in the genus Mus Based on morphological characters and diploid chromosome numbers, Marshall (1977a, 1977b, 1978, 1981, 1998) recognized four subgenera: Coelomys, Nannomys, Pyromys, and Mus. Biochemical and molecular studies (Bonhomme et al., 1984; She et al., 1990; Lundrigan and Tucker, 1994; Sourrouille et al., 1995; Lundrigen et al., 2002) based on limited taxonomic sampling verified that the subgenera represent discrete evolutionary lineages (figure 1.1). The monophyly of each of these subgenera, with one exception, described subsequently, was also confirmed with morphological and molecular analyses of multiple species from each subgenus (Chevret et al., 2003, 2005), and a suite of key morphological characters to distinguish among the three Eurasian subgenera, Pyromys, Coelomys, and Mus, was

3

Species Group

Subgenera

musculus 0.5**

castaneus domesticus

(2.3 - 1.5)

spretus 0.5***

musculus

spicilegus cypriacus macedonicus lepidoides

lepidoides

(4.8 - 4.3)

Mus

famulus booduga

(3.3 - 1.5)

nitidulus

booduga

terricolor fragilicauda caroli cookii cervicolor

(4 - 1.6)

cervicolor

platythrix phillipsi

(6.7 - 3.4)

saxicola

Pyromys

shortridgei fernandoni setulosus

(5.2 - 3.0)

haussa

2.6*

mattheyi

3.2*

(8 - 6.5 mya)

indutus

2.5* 1.6*

Nannomys

minutoides musculoides crociduroides

(7 - 3.4)

mayori pahari

Coelomys

vulcani

Figure 1.1 A synthetic phylogeny depicting evolutionary relationships among subgenera and species in the genus Mus. Species belonging to the four subgenera are bracketed. The phylogeny is based on recent studies using different combinations of molecular characters (Lundrigan et al., 2002; Auffray et al., 2003; Chevret et al., 2003, 2005; Suzuki et al., 2004; Tucker et al., 2005; Veyrunes et al., 2005; Cucchi et al., 2006; Shimada et al., 2007) and chromosomal characters (Veyrunes et al., 2006). Polytomies (unresolved nodes) indicate uncertainty of relationship. Estimates for divergence times (in parentheses) are ranges based on the variation in estimates calculated using DNA-DNA hybridization (She et al.,

1990) or different combinations of genes, taxa, and fossil calibration dates (Suzuki et al., 2004; Chevret et al., 2005; Veyrunes et al., 2005; Cucchi et al., 2006). Where ranges are not given, divergence time estimates are from *Veyrunes et al., 2005, **Suzuki et al., 2004, and ***Cucchi et al., 2006. The recent addition of molecular data from Mus lepidoides to the total data (K. Aplin, pers. comm.) suggests a topology for the subgenus Mus in which the M. cervicolor group is basal and the M. booduga group is sister to a lineage giving rise to M. lepidoides and M. musculus species groups. Dashed lines indicate taxa not represented in any of the phylogenetic analyses already cited.

proposed (Chevret et al., 2003). These characters include qualitative features of the skull and dentition and the presence or absence of spinous guard hairs. Some authors (Bonhomme et al., 1984; She et al., 1990) have argued that the four subgenera should be elevated to the rank of genera based on the degree of difference in biochemical and molecular characters. Others contend that the level of genetic divergence among subgenera does not merit a change in rank (Chevret et al., 2005). Utilizing a molecular clock, different combinations of mitochondrial and nuclear genes, and different calibration points based on fossil dates,

researchers have estimated the divergence of the four subgenera from 8 to 6.5 mya (Suzuki et al., 2004; Chevret et al., 2005; Veyrunes et al., 2005). For comparison, human and chimpanzee are thought to have diverged from a common ancestor approximately 6–7 mya (Brunet et al., 2002).

4

the mouse as a research model

The Subgenus COELOMYS Thomas 1915: Shrew Mice Mice of this subgenus are large with a shrewlike appearance characterized by small eyes and long noses. There are four recognized species, all of which inhabit mountain forests.

They are Mus mayori, from Sri Lanka; Mus pahari, from northern India (Sikkim) to Vietnam; Mus crociduroides, from Sumatra; and Mus vulcani from Java (Marshall, 1977b). Estimates for diversification within the subgenus range from 7 to 3.4 mya (She et al., 1990; Chevret et al., 2005; Veyrunes et al., 2005). The Subgenus NANNOMYS Peters 1876: African Pygmy Mice These are small-sized ( 16 mo. of age BALB/cJ Inbred 000651 Normal Albino C3H/HeJ Inbred 000659 Normal Pde66 rd1 C57BL/6J Inbred 000664 Normal Deaf > 16 mo. of age CAST/EiJ Wild-derived 000928 Normal Unknown DBA/2J Inbred 000671 Deaf < 3 mo. of age Glaucoma > 9 mo. of age FVB/NJ Inbred 001800 Normal Pde66 rd1 MOLF/EiJ Wild-derived 000550 Normal Pde66 rd1 SJL/J Inbred 000686 Normal Pde66 rd1 SM/J Inbred 000687 Normal Unknown SPRET/EiJ Wild-derived 001146 Normal Unknown Note: The Jackson Laboratory ( JAX) catalogue number is given. Pde66 rd1 is the gene for retinal degeneration, so these mice are blind by weaning age.

without the retinal degeneration gene to create a congenic strain that carries the mutation on a desired genetic background and does not have retinal degeneration. For example, FVB/N mice are an albino strain that is commonly used for the generation of transgenic mice because of their large, strong pronuclei and high breeding performance, but they have retinal degeneration, which impairs their performance on behavioral tasks. A pigmented, sighted FVB/N strain (named FVBS/Ant) was created by repeated backcrossing of an FVB/N × 129P2/OldHsd F1 hybrid with FVB/N mice while selecting against albinism and homozygosity of the retinal degeneration mutation (Pde6b). The FVBNS/Ant mouse is suitable for behavioral analysis as it has normal eye histology, an positive visual evoked potential response, and improved performance on visuospatial learning tasks (Errijgers et al., 2006).

Behavioral tasks used to measure sensory functions in mice

Table 2.2 Sensory demands of behavioral tasks used to measure higher order cognitive functions Task Vision Hearing Olfaction Taste Morris water Yes No No No maze Cued and context Yes Yes Yes No conditioning Passive and active Yes No No No avoidance Conditioned taste No No No Yes aversion Barnes maze Yes Yes No No Olfactory tubing No Yes Yes No maze Open field test Yes No No No Light-dark Yes No No No transition test Elevated plus Yes No No No maze

For its behavioral phenotype to be determined, a mouse must be tested in a series of behavioral tasks (Bailey et al., 2006). Performance on behavioral tasks is dependent on the sensory, motor, and cognitive abilities of the mouse. Every behavioral task depends on at least one of the sensory abilities for the mouse to correctly execute the task, but surprisingly little information is available on the sensory abilities of mice. The goal of detecting sensory deficits is to eliminate artifacts that could confound the results of tasks used to test higher cognitive function. Many commonly used strains of mice have vision or hearing deficits (table 2.1), and these

sensory impairments may affect performance on behavioral tasks, as most behavioral experiments measure motor responses to sensory information. Table 2.2 shows some of the behavioral tasks most commonly used in mice and their corresponding sensory demands. This chapter summarizes some of the behavioral tasks that are most commonly used to evaluate the sensory capabilities of mice (with an emphasis on vision), highlights strain

wong and brown: sensory abilities and their influence on cognitive function

Pain No Yes Yes No No No No No No

15

differences in sensory function, and discusses how strain differences in sensory ability may confound the results of learning and memory and anxiety-related behavioral tasks. The need for proper controls and for developing tasks that exploit the natural abilities of the mouse is also discussed.

visual function (Crawley, 2000; Pinto and Enroth-Cugell, 2000). The visual cliff test (Fox, 1965) measures the ability of a mouse to detect the appearance of a drop-off at the edge of a horizontal plane and was designed to detect depth perception, and thus provides only a crude measure of visual acuity (Crawley, 2000).

Vision Mice are not usually considered visual animals because they are nocturnal and rely principally on olfactory, auditory, and tactile information to sense predators, food, and conspecifics in their environment. Although the laboratory setting provides an unnatural environment for the mouse (food and water are provided ad libitum, constant temperature is maintained, and physical activity is greatly reduced), the eye of the laboratory mouse remains anatomically adapted for natural conditions. For example, Shupe et al. (2006) found no significant differences in outer nuclear layer thickness, linear density of the ganglion cell layer, and cone opsin expression in the eyes of wild M. musculus, three inbred strains (C57BL/6J, NZB/BINJ, and DBA/1J), and wild and outbred laboratory-domesticated stock of the deer mouse, Peromyscus maniculatus. Therefore, the laboratory mouse’s visual system remains adapted to natural conditions, and use of the mouse as a model system to investigate properties of the visual system and visual disorders is justified. The visual system of the mouse serves many functions, ranging from adjusting pupillary diameter in response to changes in light levels to tracking moving targets and discerning spatial details of objects in the environment (Pinto and Enroth-Cugell, 2000). For this reason, no one behavioral task provides a comprehensive test of visual function in mice, and different techniques have been used to assess gross visual ability, visual detection, visual pattern discrimination, and visual acuity. Many mouse strains, especially albino mice such as A/J, AKR/J, BALB/cByJ, and BALB/cJ, suffer from lightinduced retinal degeneration, while other strains, such as C57BL/6J, do not (LaVail et al., 1987a, 1987b). This means that when mice are housed under bright lights or under 24-hour light conditions, some strains of mice experience retinal degeneration while others will not, and as they age, the retinal degeneration becomes more severe (Danciger et al., 2000, 2003, 2007). Thus, the lighting conditions under which mice are housed constitute an environmental factor that interacts with genetic differences between strains to alter visual ability, and it is important that the lighting conditions under which mice are housed be reported. Gross Measures of Visual Ability Reflex responses to visual stimuli such as the eye blink reflex and pupillary constriction in response to light can be used to measure gross

16

the mouse as a research model

Brightness/Visual Detection The ability of mice to detect visual stimuli as ascertained by behavioral testing was pioneered by Yerkes (1907), who developed a special discrimination box in which mice were trained to discriminate between black and white stimulus cards (figure 2.1A). The mice were trained to choose the box with the white card and received a mild electrical shock when they entered the box with the black card (Yerkes and Dodson, 1908). Mice achieved a perfect score (100% correct choices) after only 50 trials (Yerkes, 1907). Many adaptations of the Yerkes apparatus have been used to test black versus white discrimination in mice. One of these was a modified water T-maze with rounded arms, one of which was painted black and the other white (Wimer and Weller, 1965). Mice were trained to escape from the black or the white arm using an escape ladder, and the correct number of responses in the 10 trials on the fourth day served as the learning measure. On this procedure, AKR/J, C57BL/6J, DBA/2J, and RF/J strains exhibited significant learning, but A/HeJ mice did not. The performance of mice with retinal degeneration (C3H/HeJ, SJL/J, SWR/J) was significantly inferior to that of all other stains tested except A/HeJ mice. Since A/J mice are the strain most sensitive to light-induced retinal degeneration (Danciger et al., 2007), and since A/J and A/HeJ mice are from the same stock (Fox and Witham, 1997), their performance may be related to light-induced retinal degeneration. An adaptation of the water T-maze, the six-choice water maze (Balkema et al., 1983), was used to investigate brightness discrimination. The maze was painted white, and mice were trained to detect a black stimulus placed at the end of one of the six arms, which contained an escape ramp. Under normal lighting conditions, mice were trained for 10 trials per day to swim to the black stimulus. Once they reached a criterion of 90% correct, the room luminance was lowered until the success criterion was no longer achievable. C57BL/6J pe/pe mice have decreased dark-adaptive sensitivity but no photoreceptor degeneration (Balkema et al., 1983), and when tested on the six-choice water maze, these mice performed as well as the wild-type mice in the lightadapted state (>10−3 cd/m2), but their performance was significantly impaired in the dark-adapted state. This suggests that C57BL/6J pe/pe mice may provide a good model for some forms of human stationary night blindness. The visual water box, developed by Prusky, West, and Douglas (2000a), assesses visual detection (Wong and Brown,

A

e

e

w

B

140 cm

w

Computer monitor A (S+) 75 cm

b

l

w

r

40 cm b

o

Hidden platform Divider (41 cm) Water Release 90 cm Chute

Computer monitor B (S–)

o

80 cm a k

C

D 5.5 cm d = 48.7 cm Choice Arm

Choice Arm

23.1 cm Start Arm

A

B

C

D

E

F

12.8 cm

E

Figure 2.1 Examples of apparatus used to test for visual ability in mice. A, The discrimination box developed by Yerkes (1907) to test for black (b)-white (w) discriminations in mice. This box was 94 cm long, 30 cm wide, and 11.5 cm deep and was divided into a nest box (a), an entrance chamber (b), and two boxes (l, r) with wires (w) on the floor connected to a battery (c) and a switch (k), which could deliver foot shocks if mice entered the wrong compartment. Mice exited (e) from the boxes into an alley (o), which led back to the nest box. B, The visual water box designed to test vision in

F

Virtual Cylinder Projected Image

rodents by Prusky et al. (2000a) as used by Wong and Brown (2006, 2007). C, The radial maze used by Hyde and Denenberg (1999) to test pattern vision in mice and the six patterns used as stimuli. D, The modified water T-maze used for horizontalvertical discriminations by Balogh et al. (1999). E, The twoalternative-choice apparatus used by Gianfranceschi et al. (1999) to measure visual acuity in mice. (From Wiesenfeld and Branchek, 1976.) F, The optomotor test apparatus used by Prusky et al. (2004) to test visual acuity in mice.

wong and brown: sensory abilities and their influence on cognitive function

17

2006) on a task requiring mice to swim to a hidden platform, the location of which is predicted by a visual cue displayed on a computer screen (see figure 2.1B). Mice were trained for eight trials per day to discriminate between a low spatial frequency (0.17 c/deg) sinusoidal vertical grating (S+), and a gray screen (S−). The percentage of correct choices on each day was used as a measure of visual ability, with 70% correct set as the learning criterion. Using this procedure, Wong and Brown (2006) tested 14 strains of mice with different visual abilities as predicted by the physiology of their visual system (figure 2.2). Mice with reportedly normal vision at 3–4 months of age (129S1/SvImJ, C57BL/6J, DBA/2J) performed very well on this task, and mice with poor visual abilities due to albinism (AKR/J, A/J, BALB/cByJ, BALB/ cJ) performed worse than mice with normal vision except AKR/J, which performed as well as mice with normal vision. Mice with retinal degeneration (C3H/HeJ, FVB/NJ, MOLF/EiJ, SJL/J) performed at chance levels, as did mice with uncharacterized visual abilities (CAST/EiJ, SM/J, SPRET/EiJ). Pattern Discrimination Behavioral tasks have also been used to study pattern discrimination in mice. Using a modified version of the eight-arm radial maze, AmmassariTeule and de Marsanich (1996) trained mice to collect food from four baited arms displaying distinctive visual patterns. CD-1 mice adopted a 45-degree angle turn strategy to locate the food instead of associating the visual patterns with the presence of food; however, C57BL/6NCrlBR and DBA/ 2NCrlBR mice successfully learned this pattern discrimination task, and there was no difference in performance between these strains (Passino and Ammassauri-Teule, 1999). The results of these experiments indicate there are strain differences in visual pattern discrimination ability, but discrimination ability could have been masked by the complexity of the task. Using escape from an eight-arm water maze modified into a T-maze, Hyde and Denenberg (1999) found that BXSB mice could learn to discriminate between different pairs of visual patterns and could reach a criterion of 80% correct after 5 days of training with 10 trials per day with each of the nine pattern discrimination tasks (see figure 2.1C). On a probe trial in which all six patterns were available in the eight-arm maze, the mice showed a preference for the S+ over the S− patterns, indicating they were capable of complex pattern discrimination. The modified radial arm maze pro-

Figure 2.2 Mean (± SEM) percent correct for each of the 14 mouse strains on each of the eight days of testing in the visual detection task (left column) and the pattern discrimination task (middle column) and calculation of visual acuity threshold from mean (± SEM) percent correct for each strain on each of the eight spatial

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the mouse as a research model

tocol employed by Hyde and Denenberg (1999) exploited the natural inclination for the mouse to escape from water, and the results on this task suggest that escape from water is a more successful motivation than food reward as used by Passino and Ammassauri-Teule (1999) for visual discrimination tasks in mice. Balogh et al. (1998, 1999) found that C57BL/6J, ectopic BXSB mice, which have neocortical malformations and are used as a model of developmental learning difficulties, nonectopic BXSB mice, and 129/SvEvTac mice, which have the pink-eyed dilution allele, could be trained to discriminate between horizontal and vertical patterns in a water-escape T maze (see figure 2.1D). The 129/SvEvTac mice, however, did not perform as well as the BXSB and C57BL/6J strains, presumably because of deficits in visual acuity. Using the visual water box, Wong and Brown (2006) trained mice of 14 strains to discriminate between vertical and horizontal gratings. Mice with reportedly normal vision (129S1/SvImJ, C57BL/6J, DBA/2J) were capable of pattern discrimination as they reached the criterion of 70% correct within the 8 days of testing. Albino mice (AKR/J, A/J, BALB/cByJ, and BALB/cJ) performed worse than mice with normal vision, taking longer to reach criterion, while mice with retinal degeneration (C3H/HeJ, FVB/NJ, MOLF/EiJ and SJL/J) and mice with uncharacterized visual abilities (CAST/EiJ, SM/J and SPRET/EiJ) performed at chance levels, a finding indicating they could not differentiate between the vertical and horizontal grating patterns (see figure 2.2). Visual Acuity Visual acuity is the ability to detect small distances separating two points, and the threshold for visual acuity is reached when the observer can no longer determine a separation between the two points. Spatial frequency is defined by how rapidly a stimulus changes across space and is measured in term of cycles per degree (c/deg). For a grating stimulus, spatial frequency refers to the frequency with which the grating repeats itself per degree of visual angle (Goldstein, 1999). One procedure for determining the visual acuity of mice is a two-alternative forced choice apparatus (see figure 2.1E) consisting of a start box that leads to a choice area with two parallel alleys, each with a food cup and a stimulus card at the end (Wiesenfeld and Branchek, 1976). Food-deprived mice were trained for 80 trials per day to discriminate

frequencies that were tested in the visual acuity task (right column). For clarity, mice were grouped into those with normal vision (A), albinos (B), those with retinal degeneration (C), and those of unknown visual abilities (D). (From Wong and Brown, 2006, p. 394.)

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between pairs of vertical and horizontal stripes of the same spatial frequency (0.15 c/deg) using a food reward. During testing, the spatial frequencies were increased or decreased by 0.1 c/deg until the mouse reached a criterion of 90% correct choices. Using this protocol, Gianfranceschi et al. (1999) determined that the visual acuity of C57BL/6J mice was between 0.5 and 0.6 c/deg. Using their visual water box, Prusky, West, and Douglas (2000a) trained mice as young as 40 days of age to swim to an escape platform signaled by a sinusoidal wave grating (S+) versus a homogeneous gray screen (S−). During testing, the spatial frequency of the grating was increased by one cycle at a time until the mouse made an incorrect response. A visual acuity threshold was obtained when the mouse performed below 70% correct at a particular spatial frequency. With this protocol, it was determined that C57BL/6J mice had a visual acuity of 0.51 c/deg. Different test procedures, however, result in different visual acuity thresholds in the same strains of mice. Wong and Brown (2006) modified the procedure of Prusky et al. (2000a) by employing a standard set of eight spatial frequencies that were tested daily for 8 days. Using this procedure, mice with no known visual defects (129S1/SvImJ, C57BL/6J, DBA/2J) and two albino strains (AKR/J and BALB/cByJ) had defined visual acuity thresholds of 0.375, 0.245, 0.375, 0.375, and 0.320 c/deg, respectively. The other albino strains (A/J, BALB/cByJ, BALB/cJ) did not reach the criterion of 70% correct and so had thresholds below 0.17 c/ deg (the largest stimulus used), whereas mice with retinal degeneration (C3H/HeJ, FVB/NJ, MOLF/EiJ, SJL/J) and mice with uncharacterized visual abilities (CAST/EiJ, SM/J, SPRET/EiJ) performed at chance levels. Using the same apparatus and procedure, however, Wong and Brown (2007) determined the visual acuity of 6-month-old C57BL/6J mice as 0.48 c/deg and that of DBA/2J mice as 0.54 c/deg. There are also age-related differences in visual acuity within strains. As DBA/2J mice age, they develop glaucomalike visual defects, and after 12 months of age they perform at chance, and no visual acuity threshold can be measured. C57BL/6J mice, on the other hand, retain their visual ability to at least 24 months of age, when it was measured at 0.38 c/ deg (Wong and Brown, 2007) (figure 2.3). Because BALB/ cByJ albino mice also show age-related retinal degeneration (Danciger et al., 2000, 2003), measurements of visual acuity obtained in young mice (3–6 months) may change as these mice age (see discussion in Wong and Brown, 2007). Using the virtual optokinetic system, which records head movements in response to the “movement” of computergenerated vertical gratings in an optical cylinder (see figure 2.1F), Prusky et al. (2004) determined that C57BL/6J mice had a visual acuity threshold of 0.39 c/deg. The virtual optokinetic system is much less time-consuming than the visual

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water box, as it can provide reliable, consistent visual acuity thresholds in a single 25- to 40-minute test. Because it measures a reflex response and does not rely on swimming ability or involve the stress of immersion in water, as occurs with the visual water box, it seems to be a better way of measuring visual acuity, but this method is less sensitive than the visual water box in determining visual acuity thresholds in mice. Using an optokinetic drum, Abdeljalil et al. (2005) determined the visual acuity of C57BL/6J mice as 0.26 c/deg under scotopic conditions and 0.52 c/deg under photopic conditions, while 129/SvPas mice had a visual acuity threshold of 0.52 c/deg under both conditions. These measures are only crude estimates, because the steps were 0.26, 0.52, and 1.25 c/deg. CD-1 albino mice and C3HeB/FeJ mice, which have retinal degeneration, had no observable optomotor responses; thus their visual acuity was not measurable. In general, behavioral tests indicate that mice without visual defects (C57BL/6J) have a visual acuity between 0.5 and 0.6 c/deg and albino mice have reduced visual acuity thresholds of 0.17–0.38 c/deg (Gianfranceschi et al., 1999; Prusky et al., 2000a; Sinex et al., 1979; Wong and Brown, 2006). This is relatively low compared to the visual acuity of 0.92 c/deg for the rat (Prusky et al., 2000a), 6–7 c/deg for cats (Blake et al., 1974; Smith, 1936), and 60 c/deg for humans (Oyster, 1999).

Hearing The most sensitive and objective measure of hearing ability in mice is the measurement of the auditory evoked brainstem response (ABR), which is an evoked potential measurement of activity in the auditory nerve, fiber tracts, and nuclei within the auditory brainstem pathways. The ABR is a useful measure for determining hearing sensitivity in mice (Zhou et al., 2006), because analysis of ABR threshold, amplitude, and latency provides important information regarding the peripheral hearing status and the integrity of the brainstem pathways. The ABR is elicited using clicks and tones of varying frequency and intensity that are channeled through the ear canals of the mouse. Measurement of the ABR requires mice to be deeply anesthetized and subcutaneously implanted with a recording electrode below the pinna, superficial to the auditory nerve. Various clicks and tones ranging from 8 to 32 kHz are presented through earphones on both ears and the loudness of each tone is varied in 5-dB steps. Auditory nerve responses are filtered through a preamplifier and amplified 25,000–100,000 fold. The ABR is presented as a waveform for each stimulus, and an auditory threshold is defined as the lowest stimulus intensity that will evoke a normal ABR wave. In a survey of 80 inbred mouse strains, Zheng et al. (1999) found that 61 strains had normal ABR thresholds and 19

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Figure 2.3 Mean (± SEM) percent correct for each strain on each of the eight days of testing in the visual detection task (A, D, G, and J) and the pattern discrimination task (B, E, H, and K) when mice were 6, 12, 18, and 24 months old. Visual acuity curves (C, F, I,

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and L) from mean (± SEM) percent correct for each strain on each of the eight spatial frequencies that were tested in the visual acuity task. (From Wong and Brown, 2007.)

wong and brown: sensory abilities and their influence on cognitive function

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strains had elevated ABR thresholds before 13 weeks of age. The 129/J, 129/ReJ, 129SvJ, C57BR/cdJ, C57L/J, DBA/ 2HaSmn, I/LnJ, and MA/MyJ strains had a mild hearing impairment (defined as 20–40 dB sound pressure level [SPL] above normal threshold); the A/J, DBA/2J, and SKH2/J strains had an intermediate impairment (41–60 dB SPL above normal threshold), and the BUB/BnJ, NOR/LtJ, and NOD/LtJ strains had a severe hearing impairment (>60 dB SPL above normal threshold). They also found that 16 strains with normal hearing when measured before 13 weeks of age exhibited age-related hearing loss. ABR thresholds for all hearing impaired strains increased with age, and for some strains, ABR thresholds varied significantly, depending on stimulus frequency (Zheng et al., 1999). Tone detection thresholds have been evaluated behaviorally in mice using a testing cage divided into two compartments: a listening area, which has a speaker above it, and a response area, containing a water dipper. A water-deprived mouse is placed in the listening area, and when it hears a sequence of 250 ms tone bursts presented at a rate of two bursts per second, it must move into the response area within 3 s to receive a water reward. Tone detection thresholds are determined by gradually decreasing the tone levels in intervals of 5 dB. A tone detection threshold is calculated in terms of d ′ values, where d′ = (z score for the percentage of hits for particular tone level) − (z score for the percentage of false alarms). An absolute threshold is defined as the sound pressure level that elicits a d ′ value of 1. Using this procedure, age-related hearing loss for high-frequency tones (16 kHz) in C57BL/6J mice was shown to develop between 21 and 33 weeks of age, while hearing loss for low-frequency tones (8 kHz) developed between 27 and 50 weeks of age. CBA/ CaJ mice did not develop age-related hearing loss (Prosen et al., 2003). Another widely used behavioral test for measuring auditory ability in the mouse is the acoustic startle test, which measures the startle response to a sudden loud noise. The startle reflex is measured by the amplitude of the whole-body flinch, and the acoustic startle amplitude is defined as the minimum decibel level that elicits a flinch (Crawley, 2000). Mice are placed in a small restraining tube in a chamber with white noise of 75–120 dB present as background noise. Brief tones of 70–120 dB are randomly delivered through a speaker on the wall of the chamber. Mice with normal hearing will show a startle response to tones of 100 dB or more, mice with impaired hearing will respond only to louder tones (100–120 dB), and deaf mice will respond only to acoustic stimuli of 120 dB or louder owing to their vibrational effects (Crawley, 2000). A more sensitive measure of hearing is the prepulse inhibition of the acoustic startle response, which is also used as a behavioral measure of sensorimotor gating. First, a weak tone of 70–90 dB of varying frequencies (4, 12, or 20 kHz) is

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the mouse as a research model

delivered, followed by a loud 100–120 dB tone 100 ms later. If mice can hear the weaker tone, they will inhibit their response to the louder tone, thus providing a measure of auditory ability. Willott et al. (2003) found significant differences among 40 inbred strains of mice tested for both acoustic startle and prepulse inhibition of acoustic startle. For example, DBA/2J mice, which have a hearing impairment, and C3H/HeJ mice had the smallest acoustic startle responses, BALB/c and FVB/NJ mice, which have normal hearing, had relatively large acoustic startle responses, and the responses of AKR/J mice fell in the middle. Willott et al. (2003), however, demonstrated that there is only a weak relationship between hearing sensitivity as measured by the ABR and acoustic startle and prepulse inhibition of acoustic startle responses. Some strains of mice that demonstrate hearing loss at an early age (BUB/BnJ, A/J) show large acoustic startle amplitudes and relatively good prepulse inhibition, and conversely, some strains of mice that hear well (MOLF/Ei, C3H/HeJ, SJL/J) exhibit very small acoustic startle responses and weak prepulse inhibition. This could be due to the fact that measures of acoustic startle and prepulse inhibition of acoustic startle are not pure measures of hearing but have emotional and motor components that may confound the interpretation of the results. Also, a large body of literature suggests that dopamine is involved in sensory gating and prepulse inhibition (for a review, see Geyer, 2006). Strain differences in dopaminergic function could thus be responsible for this contradictory relationship between different measures of hearing ability.

Olfaction The simplest behavioral test of olfactory ability in mice is the buried food test, in which the time taken for a fooddeprived mouse to locate a piece of food buried in bedding material is recorded (Crawley, 2000). Using this test, Luo et al. (2002) showed that mice deficient in the G protein (Go) were unable to locate a piece of food buried in 3 cm of bedding, while CD-1 hybrid and 129Sv-ter/C57BL/6 hybrid mice were able to do so. The buried food test is a measure of olfactory ability and cannot be used to measure strain differences in olfactory acuity or discrimination. Odor discrimination ability can be tested using habituation-dishabituation or the habituation-discrimination tests, which are based on the observation that mice decrease their investigatory behavior when presented with a familiar odor and increase their investigatory behavior when presented with a novel odor (Slotnick et al., 2005). The habituationdishabituation test involves the presentation of odor A for multiple trials (the habituation phase) and then a novel odor B is introduced (the dishabituation phase). In the habituation-discrimination test, odors A and B are both presented

following habituation to odor A. Using the habituationdishabituation test, Luo et al. (2002) found that mice deficient in Go were unable to discriminate between geraniol (a flower odor) and citralua (lemon odor), whereas CD-1 hybrid and 129Sv-ter/C57BL/6 hybrid mice were able to discriminate between the two odors. Likewise, Macknin et al. (2004) found that transgenic mice overexpressing human tau protein (Tα1-3RT τTG mice) were unable to discriminate between vanilla and peach odors or between peppermint and vanilla orange spice odors in a habituationdishabituation test, while their wild-type littermates were able to make this discrimination. Staggerer mutant (sg/sg) mice and their nonmutant littermates on a C57BL/6 background were both able to discriminate vanillin from amyl acetate odors in the habituation-dishabituation test (Deiss and Baudoin, 1999). Using the habituation-dishabituation test to determine olfactory acuity, Lee et al. (2003) showed that BALB/c mice could discriminate between conspecific urine odors at lower concentrations than C57BL/6J or 129/ S1 mice. A criticism of these tests is that they depend on the intrinsic motivation of the mouse to investigate odors, and failure to show discrimination or dishabituation does not lead to the conclusion that the mice are unable to discriminate between odor A and odor B. They may be able to discriminate but may not show a motor investigatory response (Slotnick et al., 2005). A more powerful method for studying the ability of mice to detect and discriminate between odors is to use an operant olfactometer, which delivers odors with controlled concentration, flow, and temporal parameters and measures operant responses (Slotnick et al., 2005). Water-deprived mice are trained to nose-poke into a glass odor port to sample the odor stimuli and receive a water reward when they detect the correct odor by licking the water spout (Bodyak and Slotnick, 1999). After the initial odor–no odor training, mice are trained in a go/no-go discrete trials procedure to discriminate between an odor that results in a water reward (CS+) and an odor that is not associated with reward (CS−). The operant olfactometer can also be used in mice to test odor detection thresholds, odor sampling behavior, discrimination between odor mixtures, odor masking, and odor memory. Using this protocol, it was shown that CF-1 mice readily acquired four separate two-odor discrimination tasks, and when all eight odors were randomly presented in the same session, the mice continued to perform well. Furthermore, memory for the eight odors after a 32-day rest period was at least 90% correct (Bodyak and Slotnick, 1999). Mice with retinal degeneration, albino mice, and mice with age-related blindness are all able to learn to discriminate between odors in a conditioned odor preference task and remember these odors for up to 6 months (Brown and Schellinck, 2007; Brown and Wong, 2007; Schellinck et al., 2001; Wong and Brown, 2007).

Taste The ability to discriminate different tastes is usually detected using a two-bottle choice test, in which two different taste solutions are presented in identical bottles in the home cage (Crawley, 2000; Ritchter 1939). To control for place preference, the position of the two bottles is switched over testing days and the volume of each taste solution consumed is measured over a fixed period of days. A difference between the volumes of each taste solution consumed is an index of taste discrimination ability, because mice will drink more of the preferred taste solution. Usually, a sweet (saccharin) or a bitter (quinine) solution is paired with normal tap water to investigate flavor discrimination ability; however, a twobottle test of two different concentrations of the same flavor can be used to evaluate taste acuity. In a survey of 28 mouse strains that were tested in a twobottle preference test with different concentrations of NaCl, KCl, CaCl2, or NH4Cl versus water, Bachmanov et al. (2002) found significant strain differences in solution intakes and preferences. For example, CBA/J, C3H/HeJ, and AKR/J mice showed the strongest avoidance of NaCl solutions, while CAST/Ei, BUB/BnJ, and NZB/BINJ mice demonstrated the highest preference for NaCl solutions. AKR/J mice had the highest avoidance scores for KC1 solutions, and C57BL/6J and SM/J mice had the highest preference scores for these solutions. All strains avoided the highest concentration of CaCl2 solutions, with 129P3/J mice displaying the highest intake of CaCl2. All strains also avoided the strongest concentration of NH4Cl solution, with NZB/ B1NJ and C57L/J mice having the highest intakes of NH4Cl (Bachmanov et al., 2002). There have been modifications of the two-bottle test that use three or six bottles (Owings et al., 1967). Tordoff and Bachmanov (2003) compared C57BL/6J and 129X1/SvJ mice on the standard two-bottle test and two forms of the three-bottle test, one in which the middle bottle contained flavored solution and the bottles on either side contained water and one in which two bottles of flavored solution were placed on either side of a bottle in the middle containing water. The results indicated that taste solution preferences were highest on the three-bottle test containing two flavored solutions and lowest on the standard two-bottle test. Similar results were found when the two-bottle test was compared with a six-bottle test, when more than half of the bottles on the six-bottle test contained a flavored solution. Thus, the relative availability of taste solution seems to determine taste preference scores, and Tordoff and Bachmanov (2003) suggest that the three-bottle test is more sensitive than the two-bottle test. The two-bottle test is also subject to other confounds such as position preference, postingestional influences, and strain differences in thirst demand and water deprivation (Kotlus

wong and brown: sensory abilities and their influence on cognitive function

23

and Blizard, 1998). To avoid these confounds, one can use a short-term test that measures fluid intake in a 6-hour period from a single graduated cylinder given to non-waterdeprived mice. Mice can be given a variety of flavored solutions over consecutive days, and water intake is measured three times before flavored solutions are introduced and twice after the last flavored solution is introduced. Using this protocol, Kotlus and Bizard (1998) found that among 10 inbred strains of mice (AKR/J, A/J, C3H/HeJ, C57BL/6J, C57/L, DBA/2J, NZB/BINJ, SJL/J, SM/J, SWR/J), there were significant strain differences in the intake of all flavored solutions (sucrose, saccharin, quinine, HCl, NaCl, and monosodium glutamate). Interestingly, unlike all other strains tested, DBA/2J and A/J mice did not increase their consumption of the sucrose- or saccharin-flavored solution, and DBA/2J mice were the only strain that did not decrease their intake of the quinine solution. DBA/2J mice (quinine insensitive) and C57BL/6J mice (quinine sensitive) have been used to study the genetic basis of bitter taste (Nelson et al., 2005). Glendinning et al. (2002) developed a brief-access taste test that involves repeatedly presenting various flavor stimuli for 5–30 s to food- and water-deprived mice. This paradigm requires the use of a gustometer, an apparatus equipped with a taste stimulus delivery system that records the licking behavior of the mouse. When they tested C57BL/6J mice on three different flavored solutions (quinine hydrochloride, NaCl, and sucrose), Glendinning et al. (2002) demonstrated that a concentration–response curve for NaCl or sucrose could be generated in a single 30-minute session, and for quinine hydrochloride in three 30-minute sessions. This method is high throughput and can be used as a screening protocol to identify mice with either enhanced or diminished gustatory responsiveness. Furthermore, the data can be used to compare the gustatory responses of mutant mice maintained on a C57BL/6J background with wild-type C57BL/ 6J mice.

Tactile and vibrissae sensitivity Tactile sensitivity can be measured by placing mice on a wire mesh platform and pushing von Frey monofilaments (e.g., bending forces of 0.3, 0.7, 1.6. 4.0, 9.8, 22.0, and 53.9 nM) through the mesh onto their hind feet from below using a single, steady application of more than 1 s. The median foot withdrawal threshold is a measure of tactile sensitivity (Crawley, 2000). In the von Frey filament test of mechanical sensitivity, C57BL/6J, CBA/J, and SM/J mice showed a below-average foot withdrawal threshold, 129/J, C3H/HeJ, C58/J, DBA/2J, and RIIIS/J mice showed an average response, and A/J, AKR/J, and BALB/cByJ mice showed an elevated foot withdrawal threshold (Mogil et al., 1999a). The tactile version of the startle reflex can also be

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the mouse as a research model

used to measure the flinch response to a puff of air (Plappert et al., 2006). To test tactile discrimination, two different types of textures can be placed on the floor in the arms of a Y-maze, and differences in the amount of time a mouse spends in each arm are recorded as an indication of tactile discrimination preference (Lipp and van der Loos, 1991). Tactile discrimination learning can also be tested using a Y-shaped modified Lashley jumping stand. A start platform is located approximately 6 cm away from the two choice platforms, which are lined with different textures (e.g., diamond vs. square wire mesh). Using only their vibrissae to detect these textures, mice are trained to jump to one of the platforms to receive a food reward (Cybulska-Klosowicz and Kossut, 2001; Mazarakis et al., 2005). Using this test, it was demonstrated that presymptomatic transgenic Huntington disease mice (R6/1) had a severe impairment of tactile discrimination learning compared with wild-type CBA × C57BL/6 mice (Mazarakis et al., 2005). Visual-Somatosensory Interaction The topographic maps of visual input and somatosensory input from the vibrissae of mice in the superior colliculus (SC, optic tectum) are highly correlated (Drager and Hubel, 1975). In fact, the vibrissae projections to the SC follow “the roles dictated by the visual projections” (Drager and Hubel, 1976, p. 99), and in blind mice, the use of vibrissae and the somatosensory representation of the whiskers in the SC are both increased (Benedetti, 1992; Raushecker et al., 1992). This suggests that mice may be able to develop a tactile as well as a visual “cognitive map” of their environment, and that this vibrissae “map” may allow blind mice to perform many behavioral tasks.

Pain sensitivity Pain sensitivity in mice can be measured in response to thermal, chemical, or neuropathic pain-inducing procedures. Thermal Nociception The latency to withdraw from a hot or cold stimulus can be used as a measure of pain sensitivity. In the tail flick test, a mouse is placed on a platform with its tail positioned under a hot light beam and the latency to flick the tail out of the light beam is used as a measure of pain sensitivity. Adaptations of the tail flick test, such as the Hargreaves test (Hargreaves et al., 1988), involve directing the beam of light onto the skin of the hind paw instead of the tail. The hot water tail flick and cold water tail flick tests measure the latency to remove the tail from a beaker of hot (49.0–52.5°C) or ice water, respectively (Janssen et al., 1963). The hot-plate test involves placing the mouse on a metal plate that has been heated to 52–55°C.

The latency to lick or shake the paw or to jump in response to the heat is used as a measure of pain sensitivity (Crawley, 2000). Chemical Nociception The formalin test consists of injecting formalin subcutaneously into the dorsal surface of the hind paw and scoring the responses to the affected paw (lifting, shaking, licking) as measures of pain sensitivity (Crawley, 2000; Tjolsen et al., 1992). Hind paw sensitivity to subcutaneous carrageenan injections is another method used to measure chemical pain sensitivity in mice. Carrageenan is a mucopolysaccharide that produces inflammation, hypersensitivity, and spontaneous pain (Tonussi and Ferreira, 1992). Four hours after injection, mice are observed for changes in sensitivity of the injected paw, using the Hargreaves test as previously described (Mogil et al., 1999a). Neuropathic Pain Pain resulting from hind limb denervation is used as an animal model of anesthesia dolorosa (numbness accompanied by constant pain) or phantom limb (Mogil et al., 1999a; Wall et al., 1979). The Chung peripheral nerve injury model (Kim and Chung, 1992) is also used to produce neuropathic pain in mice. One of the three spinal nerves serving the hind limb is ligated, leaving partial innervation of the hind limb. Mice can be evaluated for hypersensitivity of the operate paw using the Hargreaves test of thermal nociception to compare paw withdrawal latencies in the operate paw and nonoperate paw (Mogil et al., 1999a). There are strain differences in responses to all of these different pain-inducing stimuli (Kazdoba et al., 2007; Mogil et al., 1999a, 1999b), but strains have different rankings in pain sensitivity on each test. Thus, the comparisons of inbred mouse strains in terms of their sensitivity to pain depends on the type of pain stimulus used. For example, BALB/cJ, FVB/NJ, and DBA/2J mice are more sensitive to foot shock pain than C57BL/6J, 129X1/SvJ and C3H/HeJ mice (Kazdoba et al., 2007) and C57BL/6J and 129/J mice are more sensitive than C3H/He and DBA/2 mice to thermal pain on the hot-plate test (Mogil et al., 1999a). C57BL/6J mice are more sensitive than 129/J, BALB/c, C3H/HeJ, and DBA/2 mice on the tail withdrawal test of thermal sensitivity, but 129/J, BALB/cJ, C57BL/6J and DBA/2J mice are less sensitive than C3H/HeJ mice on the acute response to formalin injection test (Mogil et al., 1999a). There is considerable evidence that pain responses are influenced by laboratory environmental variables such as experimenter effects, housing, time of day, seasonal effects, and test order (Chesler et al., 2002), as well as by differences in stimuli used to elicit pain, but there is no evidence from these studies that pain responses are influenced by visual ability in mice. However, if pain such as shock is used as

punishment on a vision test, as in the Yerkes discrimination box (see figure 2.1A), strain differences in pain responsiveness may confound measures of strain differences in visual ability.

The importance of vision in tasks evaluating higher order cognitive functions Behavioral tasks used to study higher order cognitive functions, such as learning and memory and anxiety-related behaviors, are crucial in the search for the genetic, neural, and molecular bases of these complex behaviors. These tasks, however, are dependent on the sensory abilities of the mouse, and if it is to be demonstrated that strain differences in complex behavioral responses are due to differences in cognitive function, it must be shown that these differences are not confounded by strain differences in sensory functions (Bailey et al., 2006; Brown and Wong, 2007; Crawley, 1999). The following section reviews of some of the tasks commonly used to study complex behavior, with examples of how performance on these tasks can be confounded by visual impairments.

Learning and memory tasks Some learning and memory tasks depend on visual ability, others do not (see table 2.2). The following descriptions are divided into tasks that depend on visual ability and tasks that involve other sensory abilities to execute the task correctly. Visually Dependent Tasks The Morris water maze task. The Morris water maze (Morris, 1984) has become the most widely used task to evaluate spatial learning and memory in mice. It consists of a circular pool filled with opaque water, with a submerged platform in a hidden location. Over multiple trials, mice are trained to locate the hidden platform using spatial information from distal visual cues around the testing room, and the latency to find the platform is recorded. There are many different protocols for using the Morris water maze (Crawley, 2000). To evaluate reversal learning, the position of the platform can be moved to the opposite quadrant after 3 days of training, and to evaluate cued learning, a visible platform can be used (Wong and Brown, 2007). To evaluate memory, mice are tested in a probe trial in which the platform is removed from the pool and the time spent swimming in the quadrant where the platform was previously located is used as an index of spatial memory. The Morris water maze relies on visual ability because the mouse must locate the hidden platform using visual cues. Thus, the experimenter must be sure that the mice being tested do not have any vision problems that would result in

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The radial arm maze. The radial arm maze consists of a central start box with 8 or 12 radiating arms. Food-restricted mice are trained to find a food reward in one or more of the arms of the maze (Crusio et al., 1995; Olton and Samuelson, 1976). Different training protocols can be used, depending on the type of memory being evaluated (MacDonald and

Figure 2.4 Regression plots showing the correlation of strain means between percent correct on day 8 of the visual detection task (VD 8) and mean reversal latency (s) (A), mean reversal swim distance (cm) (B), number of annulus crossings (C), and mean visible platform latency (s) (D) on the Morris water maze task. Visual ability was significantly correlated with all four measures of performance in the water maze. (From Brown and Wong, 2007, p. 141.)

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A MWM Reversal Latency (s)

60 55 50 45 40 35 30 25 20 15

r = –0.74, P < 0.01 SPRET/EiJ MOLF/EiJ FVB/NJ

SJL/J A/J C3H/HeJ CAST/EiJ

SM/J C57BL/6J DBA/2J

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1400 FVB/NJ SPRET/EiJ

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MWM Annulus Crossings (acq. + rev.)

MWM Reversal Distance (cm)

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r = 0.60, P < 0.02

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CAST/EiJ SJL/J MOLF/EiJ C3H/HeJ SPRET/EiJ A/J

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D MWM Visible Platform Latency (s)

poor performance in the Morris water maze. Although researchers often use a visual platform test to control for visual deficits, the visual platform may not be adequate for detecting small differences in visual ability that may be responsible for poor performance (Prusky et al., 2000b; Robinson et al., 2001). For example, visual ability, as measured in the visual water box, was highly correlated with learning (swim latency and distance to find the platform) and memory (number of annulus crossings in the probe trial), indicating that mice with poor visual ability also performed poorly in the Morris water maze (Brown and Wong, 2007) (figure 2.4). Furthermore, when the performance of C3H/ HeJ and CBA/J mice, which have the rd1 mutation of the Pde6b gene causing retinal degeneration, on the Morris water maze task was compared with the performance of two Pde6b wild-type strains that do not have retinal degeneration (C3A. BLiA-Pde6b+/J and CBA/CaJ), only the wild-type strains showed spatial learning (Clapcote et al., 2005). Transgenic anophthalmic mice displayed persistent thigmotaxic behavior and severe spatial learning impairments on the Morris water maze task when compared to their wild-type counterparts (Buhot et al., 2001). Likewise, Garcia et al. (2004) demonstrated that the presence of the retinal degeneration gene in three strains of transgenic Alzheimer’s disease mice (Appsw, Tau, and Tau + APPsw) and their wild-type controls resulted in profound impairment in learning the Morris water maze. The Morris water maze also relies on adequate motor ability, because mice may have to swim for up to 60 s to find the platform. Thus, researchers who use the Morris water maze to evaluate learning and memory in the mouse must be sure that their mice are healthy enough to perform the task and have no obvious abnormalities in motor ability that would affect task performance. For example, many transgenic mice are smaller than their wild-type littermates, and smaller body weight could result in hypothermia in the Morris water maze (Iivonen et al., 2003).

60

SPRET/EiJ

r = –0.72, P < 0.01

MOLF/EiJ

50

SJL/J FVB/NJ C3H/HeJ

40

A/J 129S1/SvImJ

30 CAST/EiJ

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AKR/J DBA/2J C57BL/6J

10 0 40

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White, 1993). For example, to assess working memory, every arm is baited with food, and errors are scored when the mouse visits arms more than once (win-shift). To assess reference memory, only particular arms contain food, with the same arms being baited over successive trials (win-stay). The mouse is required to learn the location of the baited arms, and errors are scored when mice enter nonbaited arms. To assess conditioned-cue preference, only two arms of the radial arm maze are used. Each arm has a cue (such as a light versus no light) and mice are trained to receive food reward in either the “light” or the “dark” arm. The stimulusreward association memory is assessed by the time spent in each arm in a preference test in which neither arm contains food. Mice that remember the stimulus–reward association should spend more time in the arm that was previously paired with food. The radial arm maze relies on visual ability because researchers often place visual cues, such as lights, patterns, or small objects, between arms to orient the mouse. Mice with poor vision (CBA, C3H/He, BALB/c) exhibited poor performance in a radial arm maze using distal visual cues to signal food reward, whereas mice with normal vision (C57BL/6 and DBA/2) were able to learn the task (Roullet and Lassalle, 1995). Mice with retinal degeneration were impaired in learning to find the platform in a six-arm radial swim maze (Garcia et al., 2004). The Barnes maze. The Barnes maze was developed to evaluate spatial learning and memory in rats (Barnes, 1979) and has been modified for use with mice (Bach et al., 1995; Holmes et al., 2002). It consists of a circular, white, brightly lit platform with 12 or more holes around the periphery, one of which leads to a small enclosed escape box. The mouse must learn to escape the bright light by finding the location of the escape hole using distal visual cues placed around the testing room. Thus the Barnes maze relies on visual ability as well as auditory ability, as many researchers use a buzzer to make the task more aversive. Many mice suffer from noise-induced hearing loss, so it is important to test mice in the Barnes maze after all other behavioral tasks that rely on hearing ability are completed. Although the Barnes maze relies on adequate motor ability to execute the task, it is less stressful than the Morris water maze, because swimming is not necessary. Furthermore, it offers an advantage over the radial arm maze task in that mice are not required to undergo food deprivation. CBA/J mice (a strain of mice with retinal degeneration) and DBA/2J mice (a strain that is deaf by 3 months of age) were significantly worse at locating the escape hole in the Barnes maze than C57BL/6J or 129/SvEms mice (Nguyen et al., 2000). Garcia et al. (2004) found that performance in the Barnes maze was not affected by homozygosity of the retinal degeneration gene in three strains of transgenic Alzheimer’s disease mice (Appsw, Tau, and Tau + APPsw) and

their wild-type controls, although there were clear trends for an rd effect in each genotype. However, this study used an adaptation of the original Barnes maze design, in which a black wall was placed around the periphery of the maze (Pompl et al., 1999), and the addition of this wall causes mice to adopt a nonvisual search strategy, as mice use tactile information rather than visual cues to navigate around the wall (O’Leary and Brown, 2005). Cued and contextual fear conditioning. Cued and contextual fear conditioning measures the ability of the mouse to learn and remember environmental cues associated with an aversive experience (Fanselow, 1980; LeDoux, 1995). The major advantage of using the cued and context conditioning paradigm is that it provides a measure of learning and memory in only two test days. Each type of conditioning (cued versus contextual) places different sensory demands on the mouse, allowing the researcher to determine whether sensory deficits are the underlying cause for poor performance. The standard cued and contextual conditioning procedure entails placing the mouse in a visually distinct conditioning chamber and exposing it to an auditory stimulus followed by a mild foot shock. Approximately 24 hours later, the mouse is returned to the same conditioning chamber and observed for bouts of freezing. The number of seconds that the mouse spends freezing is a measure of memory for the context in which fear was conditioned. This task relies on visual, tactile, and olfactory ability, because the mouse must be able to recognize that it has been placed in the same chamber that had previously delivered the foot shock. To test for memory of the auditory cue, mice are placed in a novel chamber with different visual, tactile, and/or odor cues from the conditioning chamber, and the auditory cue used in the conditioning phase is presented. The number of seconds spent freezing is an indication of memory for the auditory cue to which fear was conditioned. This task depends on auditory ability, because the mouse must be able to hear the auditory cue, and deaf mice will fail to freeze to the tone. Although visual ability plays some role in cued conditioning, changes in odor and texture of the chamber may be robust enough to elicit fear behavior in blind mice. Strain differences have been reported in both cued and context conditioning, and three strains of mice with retinal degeneration (C3H/HeIbg, CBA/J, and SJL/J) had impaired memory for the context, while DBA/2J mice (which have age-related hearing loss) displayed memory impairment on the cued version of the task (Balogh and Wehner, 2003; Owen et al., 1997). However, Bolivar et al. (2001) found no influence of vision on cued or contextual fear conditioning. Active and passive avoidance learning. Active and passive avoidance tasks require the mouse to avoid the location where it

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received a mild foot shock, either by exiting the chamber where it received the foot shock (active avoidance) or by refraining from entering the chamber in which the foot shock was delivered (passive avoidance) (Crawley, 2000; Mathis et al., 1994; McGaugh, 1966). Testing active avoidance entails placing the mouse in the dark compartment of a two-compartment chamber and giving foot shock until the mouse enters the light compartment to escape the foot shock. Twenty-four hours later, the mouse is reintroduced to the dark compartment and latency to enter the light compartment is used as a measure of memory. In the passive avoidance task, the mouse is placed in the light compartment, and when the door between the two compartments is lifted the mouse enters the dark chamber. Once the mouse has entered the dark compartment, the door is closed and a single foot shock is administered. Twenty-four hours later, the mouse is reintroduced to the light compartment and latency to enter the dark compartment is used as a measure of memory (Crawley, 2000). Adaptations of the avoidance paradigms include step-down passive avoidance, in which the mouse must learn that stepping down from a small platform will elicit a foot shock, and Y-maze or T-maze avoidance learning, in which mice must learn to avoid or escape the arm of the maze that delivers a foot shock. Avoidance learning requires mice to have normal pain perception and motor abilities to execute the task. Although visual ability is required for mice to differentiate between the two compartments, mice with retinal degeneration are capable of brightness detection (Nagy and Misinin, 1970). Blind mice are not impaired in learning an active avoidance response in a T-maze (Farr et al., 2002), but mice with retinal degeneration (C3H and CBA strains) were impaired in learning a one-way active avoidance task (Wahlsten, 1973). FVB/NJ mice (which have retinal degeneration), 129S6/SvEvTac mice (which have visual impairment), and C3H mice (which also have retinal degeneration) were the poorest learners in a lever press escape/avoidance task (Brennan, 2004). Visually Independent Tasks Conditioned odor preference. Schellinck et al. (2001) developed an olfactory discrimination task that entails training mice to associate an odor with a sugar reward. Food-deprived mice are presented with a rose odor paired with a sugar reward and a lemon odor paired with no reward (or vice versa) for four training trials per day over 4 days. They are then tested for a conditioned odor preference by presenting both odors with no reward, and the time spent digging in each odor pot is measured. Mice able to discriminate between a rose odor and a lemon odor will dig in the odor pot that was associated

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with the sugar reward (CS+) during training. Mice of three strains (CD-1, C57BL/6NCrlBR, and DBA/2NCrlBR) have been shown to learn this task (Schellinck, 2001), and both C57BL/6J and DBA/2J mice, a strain of mice that exhibits age-related blindness, can perform this task at 12–24 months of age, indicating that blind mice can perform this task as well as sighted mice (Wong and Brown, 2007). Indeed, blind mice appear to have a better memory for odors than sighted mice (Brown and Wong, 2007). The olfactory tubing maze. The olfactory tubing maze is an ethologically relevant way to study learning and memory in mice, independent of their visual ability (Roman et al., 2002). The olfactory tubing maze consists of four chambers joined together by plastic elbow tubes. The testing chambers are composed of two plastic tubes connected in a plus shape and having an odor port, water well, and buzzer at one end and a fan above the center to exhaust the odor stimuli from the maze. After habituation to the apparatus, water-deprived mice are placed in the maze and a CS+ odor stimulus (associated with a drop of water in the water well) and a CS− odor stimulus (associated with a 3-second buzzer) are presented. The mouse is required to enter the tube with the CS+ odor in order to get a water reward. The mouse must then move to the second testing chamber to start the next trial. During the intertrial interval, clean air is flushed through the apparatus. Mice are tested for 20 trials per day for 7 days, and the percentage of correct responses and the latency to respond to an odor stimulus are analyzed to evaluate performance. Using this protocol, Restivo et al. (2006) tested five strains of mice (BALB/c, CD-1, 129/SvPasCrl, C57BL/6, and DBA/2J) and found that all strains except DBA/2J mice were able to successfully acquire the odor–reward associations by the seventh day of testing. Because it has been previously demonstrated that DBA/2J mice are able to acquire simple odor discriminations (Schellinck et al., 2001; Wong and Brown, 2007), the poor performance of DBA/2J mice in the olfactory tubing maze may reflect an impairment of the odor–reward associations. However, DBA/2J mice suffer from hearing loss at an early age (see table 2.1), and therefore poor performance in the olfactory tubing maze could result from inability to hear the buzzer associated with an incorrect choice. Conditioned taste aversion. Conditioned taste aversion is a classic conditioning paradigm that relies on the ability of the mouse to remember and avoid the taste of foods that had been associated with sickness (Palmerino et al., 1980). In this test, a novel taste, such as saccharin or sucrose, is added to the drinking water and paired with an intraperitoneal injection of a malaise-inducing agent such as lithium chloride.

The suppression of drinking liquid containing the taste paired with illness versus water in a two-bottle choice test is used as a measure of associative learning and memory. The advantages of using the conditioned taste aversion paradigm is that it has a short training period of one or two trials, associative learning is maintained after long delays between consumption of the novel taste and administration of the malaise-inducing drug, and performance relies mainly on normal taste perception. Mice readily learn the conditioned taste aversion to a variety of malaise-inducing agents, including lithium chloride (Ingram, 1982), nicotine (Risinger and Brown, 1996), and ethanol (Broadbent et al., 2002). There is no evidence that visual impairment influences conditioned taste aversion ability, as mice with retinal degeneration (C3H, SJL) and albino mice with poor vision (BALB/cJ and A/J) perform as well as sighted mice (Broadbent et al., 2002).

Anxiety-related behavior tasks Three different behavioral tasks, the elevated plus or zero maze, the open field test, and the light-dark transition test, are commonly used to assess anxiety-related behavior. All involve some form of visual cues. The Elevated Plus and Elevated Zero Mazes The elevated plus maze is based on the principle that mice that are more anxious should avoid open, brightly lit areas and prefer dark, enclosed compartments. The elevated plus maze consists of two enclosed arms and two open arms, joined at the middle in the shape of a plus. All four arms of the maze are divided into equal areas, in order to determine activity levels. The maze is elevated approximately 1 m from the floor to emphasize the aversiveness of the open arms. Mice are placed in the middle compartment and observed for locomotor activity (line crosses, rears), risk assessment (head dips, stretch-attend postures), and anxiety-related behaviors (closed-arm duration, urinations, defecations) (Brown et al., 1999; Podhorna and Brown, 2002). The elevated plus maze relies on visual ability because the aversive stimuli used to provoke anxiety-related behaviors entail visual cues such as height and light. Nontransgenic mice with retinal degeneration showed less anxiety on the elevated plus maze than those with normal vision (Garcia et al., 2004), as did transgenic anophthalmic mice (Buhot et al., 2001). C3H/HeJ, a strain with retinal degeneration, showed the least anxiety (spent the most time in the open arms of an elevated plus maze) compared to 129S2/Sv, BALB/c, C57BL/6J, CBA/Ca, and DBA/2 mice (Brooks et al., 2005). Mice with retinal degeneration also spent more time in the open areas of an elevated zero maze (an adaptation of the elevated plus maze) than mice with normal vision (Cook et al., 2001).

The Open Field Test The open field test consists of a square or circular arena, brightly lit from above, with the floor divided into a grid of equal squares with lines or photocells. Mice are placed in the apparatus for 5 minutes per day for 2 days, and the frequency of spontaneous behaviors such as line crosses, rearing, grooming, stretchattend postures, time spent in the center square, urinations, and defecations are measured (Brown et al., 1999; Podhorna and Brown, 2002). From these behaviors, measures of locomotion (number of line crosses and rearing), risk assessment (frequency of stretch-attend postures), and anxiety (frequency of entries, duration in the center square, and number of defecations) can be obtained. Anxious mice have fewer entries into and duration in the center square. The open field test involves visual ability, because the apparatus must be seen by the mouse to provoke anxious behaviors. For example, among 13 strains of mice, Wong and Brown (2004) found a significant negative correlation between visual ability (as measured by performance on the visual water box) and frequency of center square entries, indicating that mice with poor visual ability (e.g., strains of mice with retinal degeneration: C3H/HeJ, FVB/NJ, MOLF/EiJ, SJL/J) entered the center square in the open field more often than mice that performed well in the visual water box. Thus, blind mice may venture into the center square because they are unaware that it is aversive. Albino mice behave differently from pigmented mice in the open field owing to differences in their visual system, and these strain differences depend on the light levels used during testing (DeFries et al., 1966). The Light-Dark Transition Task The light-dark transition task exploits the natural inclination of the mouse to avoid brightly lit areas. The apparatus consists of a box with a smaller, dark compartment and a larger, brightly lit compartment, and an opening between the two compartments. Mice are placed in the center of the brightly lit compartment facing the opening and allowed to explore the apparatus for 5 minutes. A mouse that is more anxious will spend less time in the illuminated compartment and make fewer transitions between the two compartments (Bourin and Hascoet, 2003). The benefit of using the light-dark transition task to test anxiety in mice is that even mice with retinal degeneration are capable of brightness detection (Nagy and Misanin, 1970) and modify their activity levels in response to pulses of bright light (Mrosovsky et al., 2000). This may be due to the presence of melanopsin in photosensitive retinal ganglion cells (Panda et al., 2003). Wong and Brown (2004) found there was no significant correlation between visual ability (as measured by performance in the visual water box) and time spent in the light zone of the light-dark transition test among 13 strains of mice, some of which had retinal degeneration (C3H/HeJ, FVB/NJ, MOLF/EiJ, SJL/J).

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Future directions for using behavioral tasks in mice with normal visual ability and with deficits in visual ability: Exploiting the natural abilities of mice It is important to develop behavioral tasks that exploit the natural abilities of the mouse, which can be used to test cognitive functions independently of visual ability. Although olfactory-based tasks seem to be the best suited for the mouse, tasks that rely on olfactory ability present problems in the generation, control, and measurement of the odor stimuli that are not encountered with stimuli for other sensory modalities (Slotnick et al., 2005). A behavioral test battery in which many tasks are used to measure the same underlying cognitive function is probably the best approach. In this way, scores from tasks that measure the same higher order functions but rely on different sensory abilities can be compared. For example, discrimination learning and memory ability can be measured on tasks that use visual, auditory, olfactory, gustatory, or tactile stimuli. Mice with visual impairments but normal cognitive ability will fail on the visual discrimination task but learn auditory, olfactory, gustatory, and tactile discriminations. Bailey et al. (2006) recommend a multitiered approach to phenotyping transgenic mice that begins with evaluating basic measures of general health, followed by examining neurological, sensory, and motor functions. The last stages of the phenotyping strategy involve testing mice on behavioral tasks that are aimed to address the hypothesized function of the gene of interest, such as tests for anxiety-related behaviors, drug abuse, motor function, social interactions, learning and memory, and nociception. By determining sensory and motor abilities first, one can select complex tests of cognitive function that are not confounded by sensory or motor impairments (Bailey et al., 2006). Similarly, the SHIRPA test battery (Rodgers et al., 1997) is a systematic and hierarchical protocol used to detect mouse mutants. It consists of a primary screen of approximately 30 rapid tests for neurological and neuropsychological deficits, such as deficits in muscle and lower motor neuron function, spinocerebellar function, sensory function, neuropsychiatric function, and autonomic function. The secondary screen consists of a comprehensive behavioral screening battery and pathological analysis. Finally, the tertiary screen involves more sophisticated tests to assess behavior, such as the Morris water maze, electroencephalography, and nerve conduction. However, many mouse test batteries do not have systematic tests for sensory function (e.g., Takao et al., 2007). Behavioral paradigms that are used to phenotype mice should be standardized and validated. Standardization is necessary to increase replicability within a given laboratory and is also required for comparison of results between laboratories (Wahlsten et al., 2003, 2006). Tasks for a specific behavioral domain should also be assessed for construct

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validity (how well a measure of a particular trait, process, or state reflects theoretical assumptions), predictive validity (how well a manipulation predicts performance in the condition that is to be modeled), and face validity (the degree of similarities between the model and the actual disorder) (van der Staay and Steckler, 2001, 2002). Furthermore, behavioral tasks that were originally developed for rats and adapted for mice require extensive validation (Arndt and Surjo, 2001).

Conclusions Many of the behavioral tasks that are used to measure cognitive functions in mice depend on normal visual function. Thus, it is critical to first evaluate the sensory functioning of the mouse, and then compare the data with results obtained on behavioral tasks to determine higher order cognitive function. This is especially true for studies in transgenic mice that aim to determine the genetic contribution of human neurological diseases. Transgenic and knockout mice have the phenotypic traits of the background strain, and differences between inbred strains, which are independent of the gene of interest, may significantly affect the behavioral phenotype of a mouse. For this reason, it is important to examine phenotypic differences between inbred strains of mice used as background strains for transgenic mice (Bothe et al., 2005; Brooks et al., 2005; Gerlai 1996; Nguyen and Gerlai, 2002; Tarantino et al., 2000). In addition, the background strain used to maintain the genetic disruption must be carefully selected, so that noncognitive factors such as sensory deficits do not affect task performance and thus decrease the usefulness of a particular animal model.

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Comparative Survey of the Mammalian Visual System with Reference to the Mouse CATHERINE A. LEAMEY, DARIO A. PROTTI, AND BOGDAN DREHER

Until very recently, the house mouse (Mus musculus) was not a preferred model for study of the mammalian visual system. However, the power of transgenic and knockout mice as models for the study of the central nervous system (CNS) has led to important questions concerning the overall properties of the visual system of the house mouse compared with those of other mammalian species. Indeed, the power of knockout mice models was formally recognized by the award of the Nobel Prize in Physiology or Medicine for 2007 to Mario R. Capecchi, Martin J. Evans, and Oliver Smithies, the principal developers of the knockout mouse model. This chapter addresses similarities and differences between the visual system of the mouse and that of other mammalian (especially eutherian) species. Available information indicates great similarity to other species, especially the closely related laboratory rat (Rattus norvegicus; see Arnason et al., 2002). However, substantial differences do exist between the visual systems of nocturnal rodents and those of more highly visual mammals, such as primates (including humans) and carnivores. Thus, although transgenic and knockout mice can provide useful and elegant models with which to assess visual function, there is sound justification to maintain a comparative approach.

The eye, the retina, and visual acuity The adult house mouse is a very small nocturnal mammal with a relatively small eye having an axial length from anterior cornea to anterior choroid of about 3.4 mm (Remtulla and Hallett, 1985). As is typical for nocturnal mammals (see Walls, 1942), the mouse eye is characterized by a relatively large cornea and lens, the latter accounting for 60% of the axial length (Remtulla and Hallett, 1985). Apart from limitations imposed by the optical properties of the eye, the limits of visual acuity depend on the arrays of neurons in the retinal network. Several factors determine the limits of the retinal acuity. These factors include the retinal sampling frequency, which is determined by the size

and density of photoreceptors and their degree of convergence onto retinal ganglion cells (RGCs), the output cells of the retina. As is characteristic of nocturnal mammals, the mouse retina is strongly rod-dominated, with rods making up 97% of all photoreceptors. Cones constitute a much smaller proportion of photoreceptors (Carter-Dawson and LaVail, 1979; Jeon et al., 1998) in the house mouse than in diurnal rodents such as the ground squirrel (Spermophilus beecheyi; Kryger et al., 1998), diurnal primates such as macaque monkeys or humans, or nocturno-diurnal carnivores such as the domestic cat (Felis catus). Nevertheless, the cones appear to play an important role in mouse vision. A high degree of convergence combined with the small size of the eye results in rather small numbers of RGCs (48,000– 65,000: Dräger and Olsen, 1980; ca. 45,000 RGCs out of 110,000 neurons in the ganglion cell layer: Jeon et al., 1998). Despite their relatively small numbers, the peak RGC density is about 8,000 cells/mm2 (Dräger and Olsen, 1981). This figure is not only higher than the peak RGC density in the rabbit (Oryctolagus cuniculus, a crepuscular lagomorph with only partially vascularized retina; Robinson et al., 1990); it is also higher than that in other nocturnal rodents with strongly vascularized retinas, such as the rat (McCall et al., 1987) or the golden hamster (Mesocricetus auratus; Tiao and Blakemore, 1976b; Métin et al., 1995). Indeed, the peak RGC density in the mouse appears rather similar to that of the domestic cat (Stone, 1978; Hughes, 1981). The structure of the mouse retina has been thoroughly studied anatomically using Golgi staining (Ramón y Cajal, 1972), Nissl staining, electron microscopy (Carter-Dawson and LaVail, 1979; Dräger and Olsen, 1981; Jeon et al., 1998; Tsukamoto et al., 2001), and differential interference contrast microscopy (Jeon et al., 1998). Apart from the photoreceptors and retinal neurons, three types of glial cells are found in the mouse retina (as in other mammals with vascularized retinas): astrocytes are found in the optic nerve fiber layer, microglial cell bodies form a regular array in the inner nuclear layer (INL), and Müller cells span the entire retina vertically (Haverkamp and Wässle, 2000). The Müller cells described in mice are morphologically similar

35

to those in other mammals (Dreher, Robinson, et al., 1992). As in all mammals other than primates (see Walls, 1942; Rowe and Dreher, 1982; Rowe, 1991; Provis et al., 1998), the mouse retina does not have a fovea centralis (a pitted region in the center of the retina with the highest density of cones, but no rods or other retinal neurons). In nonfoveate mammals, including mice, the density of rods and cones, as well as the density of the RGCs, peaks in the area centralis (Dräger and Olsen, 1980) and decreases more peripherally. In mice, the peak rod density is about 100,000/mm2 and the peak cone density is 16,000/mm2 (Jeon et al., 1998), similar to the average densities of rods and cones in the peripheral retina of macaque monkey (Packer et al., 1989) and domestic cat (Steinberg et al., 1973). The centroperipheral RGC density ratio in the mouse, at around 3.5 : 1 (Dräger and Olsen, 1980), is somewhat smaller than that in other murid rodents such as laboratory rat (McCall et al., 1987) or a critecid rodent, the golden hamster, in which it is approximately 5 : 1 (Tiao and Blakemore, 1976b; Métin et al., 1995). Photoreceptors The spectral sensitivity of mouse rod photoreceptors (estimated using three different methods: rhodopsin absorption, the b-wave of electroretinograms [ERGs], and recordings of ganglion cell activity), peaks at 497–500 nm (Soucy et al., 1998; Toda et al., 1999; Fan et al., 2005). There are two types of cone photoreceptors, differing in their photopigments and their absorption spectra. One is maximally responsive to ultraviolet (UV) light (peak sensitivity at 360 nm); the other is to medium (M)-wavelength light (peak sensitivity at 508 nm; Nikonov et al., 2006). Most cones express both UV and M photopigments but are maximally sensitive at 360 nm (Nikonov et al., 2006). In the dorsal retina, however, cones are maximally sensitive at 508 nm, while the highest density of UV-sensitive cones occurs in the ventral retina (Szél et al., 1992, 1993). Retinal Network The basic functional organization of the mouse retina is largely similar to that of other mammalian species (figure 3.1A). Scotopic signals are transmitted via the classical rod pathway, in which rod photoreceptors synapse onto rod bipolar cells. These bipolar cells do not transmit light signals directly to RGCs but rather piggyback them via AII amacrine cells onto the evolutionary preexisting cone pathways by making sign-inverting synapses with OFF cone bipolar cells and sign-conserving synapses with ON cone bipolar cells. Cone bipolar cells in turn synapse onto RGCs. In addition, both anatomical and physiological evidence indicates the existence of alternative pathways for the transmission of scotopic signals. A second pathway by which rod signals could “leak” into the cone pathway via gap junctions

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the mouse as a research model

Figure 3.1A Signaling pathways in the mouse retina. Cone circuit: cone photoreceptors contact both ON and OFF bipolar cells. ON bipolar cells synapse onto ON ganglion cells (an α-like ganglion cell with a large dendritic tree is represented here). OFF bipolar cells synapse onto OFF ganglion cells. In the rod circuit, light signals can follow three different pathways: in the classical rod pathway, signals initiated in rod photoreceptors (Rod PRs) are transferred to a single type of bipolar cell, the rod bipolar cell (RB). RB cells transmit their signal to AII amacrine cells (AII), which split the signal into the ON and OFF channels by making gap junctions with the axons of ON cone bipolar cells (ON CB) in sublamina b (sub b) of the inner plexiform layer (IPL) and inhibitory synapses (glycinergic) in sublamina a (sub a) with the axons of OFF cone bipolar cells (OFF CB), respectively. ON CBs and OFF CBs synapse with ON and OFF ganglion cells, respectively. Alternatively, rod signals may flow via gap junctions into cone photoreceptors (Cone PRs), from which they are fed into ON CB and OFF CB cells and subsequently onto their respective ganglion cells. A third pathway has been described in which some rod PRs (shaded Rod PR) synapse directly onto the dendrites of a particular type of OFF CB cell, which transfers this signal to OFF ganglion cells. These pathways are likely to operate under different lighting conditions. (Modified from Protti et al., 2005, with permission.) B, Schematic drawing of three morphologically and physiologically identified ganglion cell types in the mouse retina. Left, Bistratified ON-OFF direction-selective ganglion cell with one dendritic plexus branching in the ON sublamina and the other in the OFF sublamina. (Based on Weng et al., 2005.) Center, ON α–ganglion cell with a large soma and several robust, radiating primary dendrites that expand to form a relatively wide dendritic tree covering between 6° and 12°. (Based on Pang et al., 2003.) Right, Melanopsin-containing ganglion cell with dendritic tree covering a large area of retina (>15°) and branching mainly in the outermost part of the inner plexiform layer. Scale bar in B corresponds only to part B. (B, Based on Berson et al., 2002.)

between the two classes of photoreceptors has been described anatomically (Tsukamoto et al., 2001). This pathway has been shown to exist both anatomically and functionally in the cat (Kolb, 1977; Nelson, 1977) and macaque monkey (Raviola and Gilula, 1973; Schneeweis and Schnapf, 1995).

A third pathway involving direct synaptic contacts between rods and one type of OFF cone bipolar cell has also been described for the flow of scotopic signals (Soucy et al., 1998; Hack et al., 1999; Tsukamoto et al., 2001; Protti et al., 2005), although the relative contribution of this mechanism remains to be established. The anatomical basis for such a pathway has also been demonstrated in the cat (Fyk-Kolodziej et al., 2003), although thus far there are no reports of a similar circuit in primates. Tsukamoto and colleagues (2001) proposed that in nocturnal rodents, the transmission of scotopic light OFF rather than light ON signals might be favored because when these animals are active in twilight conditions, objects would appear dark on a brighter background and consequently would be most efficiently detected by the OFF pathway. The structure of the photopic pathways in the mouse is also typical of that in other mammals. Cone photoreceptors connect to cone bipolar cells, which pass light signals directly to RGCs. Cone bipolar cells are subdivided into ON and OFF cells, according to their level of stratification in the inner plexiform layer (figure 3.1B). The generation of the ON and OFF channels of the visual system, optimized to respond to light increments and light decrements, respectively, relies on different types of glutamate receptors expressed in the dendritic tips of bipolar cells. ON bipolar cells express the metabotropic glutamate receptor mGluR6 (group 3) in the outer plexiform layer postsynaptic to photoreceptors (Masu et al., 1995), while OFF bipolar cells express AMPA/KA ionotropic glutamate receptors (Hack et al., 2001; Sun and Kalloniatis, 2006). So far, nine different types of cone bipolar cells, comprising four OFF and five ON cone bipolar cells, have been morphologically identified (Ghosh et al., 2004). Light-evoked responses also indicate the existence of two functionally distinct cone bipolar cell types, one responding to light by depolarizing, the other displaying hyperpolarizing light responses (Berntson and Taylor, 2000; Pang et al., 2004), consistent with their morphological identity. Recently, a bipolar cell type with morphological characteristics similar to the blue ON bipolar cells of the primate retina (Mariani, 1984; Kouyama and Marshak, 1992; Ghosh et al., 1997) has been identified in the mouse (Haverkamp et al., 2005). As postulated for the primate (Boycott and Wässle, 1999; Wässle, 2004), bipolar cell diversity may constitute the basis for segregation of parallel pathways of information. Although currently there is no precise information regarding the nature of these pathways in the mouse visual system, the wiring of the mouse retina could, therefore, provide higher visual centers with information concerning different aspects of the visual world carried by parallel channels via different classes of RGCs. Retinal Ganglion Cells A number of different morphological types of RGCs have been distinguished in the

mouse retina. This diversity seems to reflect a similar pattern to that observed in other mammals, including primates and domestic cats. Whereas earlier studies (Dräger and Olsen, 1981; Doi et al., 1995) distinguished only three to four principal morphological classes of RGCs, more recent surveys (Jeon et al., 1998; Sun et al., 2002; Kong, 2005; Coombs et al., 2006) indicate greater morphological diversity, with up to 11–14 morphological types identified. Similar trends in the classification of RGCs are present in the literature for the domestic cat and macaque monkey (Boycott and Wässle, 1974; Wässle and Boycott, 1991; Rowe, 1991; Rodieck and Watanabe, 1993; Pu, 1999; O’Brien et al., 2002; Dacey and Packer, 2003). In the cat (Boycott and Wässle, 1974; Peichl and Wässle, 1979; Rowe and Dreher, 1982; Rowe, 1991) and macaque monkey (Leventhal et al., 1981; Rowe, 1991; Wässle and Boycott, 1991), the sizes of the somata and dendritic trees of RGCs increase with eccentricity concomitantly with a reduction in RGC density. This trend is present, although substantially weaker, in the ferret (Vitek et al., 1985) and rat (Perry and Linden, 1982; Dreher et al., 1985). In contrast, Sun and colleagues (2002) reported that in the mouse, neither somal sizes nor dendritic tree sizes of RGCs increase significantly with a reduction in RGC density. Consistent with the aforementioned cone spectral sensitivity, in photopic conditions mouse RGCs are driven by signals originating from UV- and M-wavelength-sensitive cones. The majority of RGCs receive mixed cone input, with a preponderance of RGCs in ventral retina receiving UV cone input, while 18% of RGCs receive input from only UV and 3% from only M cones (Ekesten et al., 2000; Ekesten and Gouras, 2005). Interestingly, a small number of cells (ca. 2%) receive mixed input from UV and M cones of opposite polarity (ON vs. OFF), suggesting the existence of a pathway capable of carrying chromatic information (Ekesten and Gouras, 2005). The functional properties of some mouse RGCs have been characterized both in vivo and in vitro (Stone and Pinto, 1993; Sagdullaev and McCall, 2005). Overall, mouse RGCs have relatively large receptive fields, 2–10° in diameter, with antagonistic center-surround organization (Stone and Pinto, 1992). Stone and Pinto (1993) identified mouse RGCs as X-like when they displayed linear spatial summation (compare X cells in cat retina; Enroth-Cugell and Robson, 1966; Hochstein and Shapley, 1976) or as Y-like when they displayed nonlinear spatial summation dynamics (compare Y cells in cat retina; Enroth-Cugell and Robson, 1966; Hochstein and Shapley, 1976). The high cutoff spatial frequency of both X-like and Y-like cells’ RGCs in the central retina was found to be rather low, about 0.2 c/deg (Stone and Pinto, 1993). A more recent study found no clear separation between linear and nonlinear modes of operation in

leamey, protti, and dreher: mammalian visual system with reference to the mouse

37

mouse RGCs but rather a continuum characterized by different degrees of nonlinearity (Carcieri et al., 2003). Since a correlation with their morphological identity has not yet been done, it is unknown whether Y-like cells in mice correspond to a type similar to α–like ganglion cells of other mammals (see Peichl et al., 1987), but RGCs with morphological characteristics similar to those of α–cells have been described in the mouse (Sun et al., 2002; Pang et al., 2003). Both ON and ON-OFF direction-selective (DS) ganglion cells with functional and morphological characteristics similar to the rabbit DS ganglion cell have been described in the mouse (Yoshida et al., 2001; Weng et al., 2005; Sun et al., 2006). Furthermore, the mechanisms involved in the generation of DS responses seem to be similar to those in the well-characterized rabbit DS RGCs (Weng et al., 2005). The recently described non-image-forming retinofugal pathway constituted by melanopsin-containing RGCs is also present in the mouse. A total of about 700 intrinsically photosensitive melanopsin-containing ganglion cells (ipRGCs), which are involved in the regulation of circadian rhythms and pupillary reflexes, has been reported in the mouse (Hattar et al., 2002). These cells project to the suprachiasmatic nucleus (SCN) of the hypothalamus, the ventral lateral geniculate nucleus (vLGN), and the intergeniculate leaflet (IGL) of the ventral thalamus (i.e., structures involved in the regulation of circadian rhythms). Other target regions of ipRGCs include the preoptic region, the hypothalamic subparaventricular zone, the superior colliculus (SC) in the midbrain, and the nucleus of the optic tract (NOT) in the pretectum (Gooley et al., 2003; Hattar et al., 2003; Hannibal and Fahrenkrug, 2004). Recently, projections to the perisupraoptic nucleus of the hypothalamus, medial amygdala, margin of the lateral habenula, posterior limitans nucleus, and periaqueductal gray have also been described (Hattar et al., 2006). Moreover, projections to the margins of the dorsal lateral geniculate nucleus (dLGN) were also observed, a finding consistent with the reported projection of ipRGCs to the dLGN in primates (Dacey et al., 2005). In knockout mice in which the melanopsin gene has been deleted, RGCs that normally express melanopsin are no longer intrinsically photosensitive, and both the pupillary light reflex (Lucas et al., 2003) and the circadian clock (Ruby et al., 2002; Panda et al., 2003) are severely impaired. Interestingly, despite great differences in the total number of RGCs among different mammalian species (see Dreher et al., 1985; Robinson and Dreher, 1990b), the total number of melanopsin-expressing RGCs seems to be highly conserved (ca. 2,300–2,600 ipRGCs per retina in the rat, corresponding to ca. 2.3%– 2.6% of RGCs: Hattar et al., 2002; ca. 1,700 ipRGCs per retina in the cat, corresponding to ca. 1% of RGCs: Semo et al., 2005; ca. 3,000 ipRGCs per retina in humans, corresponding to 0.2%–0.3% of RGCs: Dacey et al., 2005).

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the mouse as a research model

Spatial Resolution Using the focal length of the eye, the retinal magnification factor (RMF), and assumptions of the sampling theorem, it is possible to derive the limits of animals’ spatial acuity from the peak density of the RGC mosaic (e.g., Hughes, 1986; compare Pettigrew et al., 1988). Applying these calculations gives theoretical upper limits of visual acuity (in cycles/degree) of 1.3 in the mouse (Remtulla and Hallett, 1985; Hallett, 1987), and 9.1–9.8 in cats (Vakkur et al., 1963; Stone, 1978; Hughes, 1981). Since there are no RGCs in the fovea, estimates of visual acuity in primates are based on peak cone density, which give values of 42 in macaques (Perry and Cowey, 1985) and 56.5 in humans (Østerberg, 1935; Duke-Elder, 1953). In carnivores and primates, the estimates based on the peak ganglion cell or cone densities and the RMF are close to those obtained behaviorally (7–9 c/deg in cat: Blake et al., 1974; Jacobson et al., 1976; Mitchell et al., 1977; 46 c/deg in macaque: De Valois et al., 1974; 50–64 c/deg in humans: Reymond and Cook, 1984). Estimates obtained behaviorally in mice, using either optokinetic reflexes (Sinex et al., 1979; Abdeljalil et al., 2005; Schmucker et al., 2005) or visual discrimination tasks (Gianfranceschi et al., 1999; Prusky et al., 2000) at 0.4.–0.5 c/deg (with peak sensitivities at 0.125– 0.26 c/deg), are, however, substantially lower than the theoretical limits estimated on the basis of peak RGC density and RMF. It is possible that the current estimates of RGC density in the mouse are overestimates due to the relatively large amount of shrinkage of the small retina during histological processing. The discrepancy between the theoretical estimates of acuity based on the peak RGC density and RMFs and behavioral data may also be related to the fact that in mice (Hofbauer and Dräger, 1985) as in other nocturnal rodents (rat: Linden and Perry, 1983; Dreher et al., 1985; hamster: Chalupa and Thompson, 1980) and rabbit (Vaney et al., 1981), virtually all RGCs project to the SC, whereas only a subpopulation projects to the visual cortex via relays in the dLGN; this is different from what is seen in carnivores and primates, where the majority of RGCs project to the geniculocortical system. Indeed, estimates of visual acuity based on peak RCG density and RMF for a number of nocturnal rodents are substantially higher than estimates derived from behavioral testing (see Pettigrew et al., 1988). In contrast, there is a good correlation between measurements of visual acuity based on behavioral studies and those extrapolated from the visually evoked potentials (VEPs) recorded from area 17 in mouse (Porciatti et al., 1999), rat (Boyes and Dyer, 1983), and rabbit (Kulikowski, 1978). It is likely, therefore, that estimates of visual acuity based on the peak density of the RGCs contributing to the geniculocortical system may be a better predictor of behavioral acuity than peak RGC density per se. Interestingly, the behaviorally determined acuity in the mouse also correlates well with visual acuity in the mouse estimated on the basis of RMF

and the spatial extent of the smallest dendritic trees of RGCs (ca. 30 μm, equivalent to ca. 1°; Sun et al., 2002; Coombs et al., 2006). Spatial resolution in the mouse may be limited at an early stage of the visual system by the low spatial resolution of the array of retinal bipolar cells (0.3 c/deg; Berntson and Taylor, 2000).

Ipsilateral and contralateral retinofugal projections and the visual field In all mammals studied so far, the optic nerves meet at the optic chiasm. In all mammalian species, the majority of RGC axons carrying information from a given retina cross at the chiasm and project to the contralateral retinorecipient nuclei. The proportion of RGC axons that do not cross and thus project to the ipsilateral retinorecipient nuclei, however, varies considerably among species and is closely related to the position of the eyes in the skull. The visual field in virtually all mammals contains at least some region of binocular overlap. Table 3.1 lists the proportion of ipsilaterally projecting RGCs in a variety of mammalian species for comparison. In species with laterally positioned eyes and therefore a relatively small binocular field, the proportion of ipsilaterally projecting RGCs tends to be small. In particular, in the rabbit, with its very laterally positioned eyes, the binocular

portion of the visual field is correspondingly small (ca. 25° out of the large 360° panoramic visual field; Hughes, 1971; Hughes and Vaney, 1982). In this species, only about 0.6% of RGCs project ipsilaterally (Robinson and Dreher, 1990a). In the mouse as in other nocturnal rodents (for a review, see Rhoades et al., 1991; see also table 3.1), the eyes are more frontally positioned, the binocular field is larger (30–40°; Dräger, 1978; Dräger and Olsen, 1980; Wagor et al., 1980), and the proportion of RGCs projecting ipsilaterally is slightly higher (2%–3%; Dräger and Olsen, 1980). The correlation between the extent of binocular visual field and the proportion of RGCs that project ipsilaterally is also apparent in carnivores. The eyes of the ferret are positioned more laterally than those of the cat. In ferrets, only about 7% of RGCs project ipsilaterally (Morgan et al., 1987), while in domestic cats, with their frontally positioned eyes and large binocular visual field (ca. 90° out of a ca. 180° visual field; Vakkur et al., 1963), the proportion of RGCs projecting ipsilaterally is much higher (ca. 25%–30%; Stone and Fukuda, 1974; Wässle and Illing, 1980; Illing and Wässle, 1981; Mastronarde, 1984; FitzGibbon and Burke, 1989; Tassinari et al., 1997). The proportion of ipsilaterally projecting RGCs is very high (ca. 40%) in diurnal primates with frontally positioned eyes, such as the Old World macaque monkey (Perry and Cowey, 1984; Perry et al., 1984).

Table 3.1 Proportions of ipsilaterally and contralaterally projecting RGCs in different mammalian species relative to proportions of binocular cells, proportions of cells receiving exclusive or dominant input from the contralateral eye and the presence or absence of ocular dominance columns % Binocular % Cells in Binocular Cells in Region of V1 Driven % Ipsilaterally Binocular Region Exclusively or Dominated Ocular Dominance Species Projecting RGCs of V1 by Contralateral Inputs Columns Present? References* Mouse 2–3 66 65 No 1–3 Rat 3 85 68 No 4–6 Syrian hamster 1–2.5 89 56 No 7–9 Rabbit 0.6 80 65 No 10–12 Opossum 85 37 ? 13 Brushtail possum

65

46

?

14

Sheep

6.5

54

Maybe

15 (but see 16)

72 57

53 64

Yes Yes

17–19 20, 21

Ferret Mink

7

Cat 25–30 63 43 Yes 22–27 Cat (area 18) 25–30 64 57 Yes 28 Tree shrew 71 73 No 29, 30 Macaque 40 72 33 Yes 31–35 * 1, Dräger, 1974, 2, Gordon and Stryker, 1996; 3, Antonini et al., 1999; 4, Jeffery, 1984; 5, Dreher et al., 1985; 6, Fagiolini et al., 1994; 7, Tiao and Blakemore, 1976a; 8, Hsiao et al., 1984; 9, Métin et al., 1995; 10, Robinson et al., 1990; 11, Van Sluyters and Stewart, 1974; 12, Holländer and Hälbig, 1980; 13, Rocha-Miranda et al., 1976; 14, Crewther et al., 1984; 15, Clarke et al., 1976; 16, Pettigrew et al., 1984; 17, Morgan et al., 1987; 18, Law et al., 1988; 19, White et al., 1999; 20, McConnell and LeVay, 1986; 21, LeVay et al., 1987; 22, Stone and Fukuda, 1974; 23, Illing and Wässle, 1981; 24, Mastronarde, 1984; 25, FitzGibbon and Burke, 1989; 26, Burke et al., 1992; 27, Tassinari et al., 1997; 28, Dreher, Michalski, et al., 1992; 29, Hubel, 1975; 30, Humphrey et al., 1977; 31, Hubel and Wiesel, 1968; 32, Stone et al., 1973; 33, Leventhal et al., 1988; 34, Fukuda et al., 1989; 35, Chalupa and Lia, 1991.

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In all mammalian species studied so far, almost all ipsilaterally projecting RGCs are located within the temporal retina. In the mouse, virtually all ipsilaterally projecting RGCs are located in the peripheral ventrotemporal region known as the ventrotemporal crescent (VTC). Only a minority (ca. 15%) of RGCs located within the VTC project ipsilaterally, however, with the great majority projecting contralaterally (see figure 3.2; Dräger and Olsen, 1980). Because of the position of the eyes in the mouse skull, this means that the binocular field is approximately the central 30% of visual space contained within the receptive fields of RGCs in the VTC of both eyes (Dräger, 1978; Dräger and Olsen, 1980; figures 3.2 and 3.3). Since the ipsilateral projection arises from the VTC region, in mice the binocular field widens dorsally (see figure 3.2). This is not the case in carnivores with frontally positioned eyes, such as the domestic cat (Illing and Wässle, 1981), where ipsilateral projections arise from all dorsoventral levels of the temporal retina. Overall, in carnivores, such as the domestic cat or ferret, unlike in nocturnal rodents, contralaterally projecting cells constitute a significant minority of RGCs located in the temporal retina (Cooper and Pettigrew, 1979; Leventhal, 1982; Vitek et al., 1985; Tassinari et al., 1997). By contrast,

in macaques (Chalupa and Lia, 1991) and humans, virtually all RGCs in the temporal retina project ipsilaterally. Thus, in primates, and to a lesser extent in carnivores, the region of binocular overlap is viewed by the nasal retina of one eye and the temporal retina of the other eye. In primates and carnivores there is also a more clearly defined line of decussation that passes through the region of highest acuity, the fovea centralis (Stone et al., 1973; Leventhal et al., 1988; Fukuda et al., 1989; Chalupa and Lia, 1991) or area centralis (Illing and Wässle, 1981), respectively. This is different from the situation in mice, in which RGCs from all regions of the retina project contralaterally and the ipsilateral projection arises exclusively from a subregion of peripheral retina (Dräger and Olsen, 1980).

Retinorecipient nuclei In the mouse, there are direct retinal projections to the SCN of the hypothalamus, the dLGN of the dorsal thalamus, and the vLGN and IGL of the ventral thalamus (Dräger, 1974; Provencio et al., 1998). There is also a direct retinal projection to several nuclei in the pretectal complex of the epithalamus (Provencio et al., 1998). The most massive direct retinal

A

B

Figure 3.2A The visual field of the mouse. Projection of the mouse visual field onto a sphere, assuming that the mouse is sitting inside the sphere and facing intersection of zero vertical and zero horizontal meridian. Star indicates location of the optic disc. Note that the binocular field widens dorsally. (From Dräger, 1978, with

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the mouse as a research model

permission.) B, Approximate positions of binocularly corresponding regions in mouse retinas. The small ipsilateral retinofugal projection originates from these regions. (From Dräger and Olsen, 1980, with permission.)

18b

17 (M) 17 (B) 18a

dLGN

vLGN

IGL

1 mm

V/T

V/T

D/N

D/N

Monocular visual field

Monocular visual field

Binocular field

Figure 3.3 Schematic diagram showing the organization of the ipsilateral and contralateral visual pathways in mice. As shown in color plate 1, blue and red indicate fibers and regions representing the left and right eyes, respectively. Purple indicates binocular regions. Ipsilateral projections arising from the ventrotemporal retina terminate in dorsomedial dLGN. Contralateral retinal projections fill the rest of the dLGN. The locations of other retinorecipient nuclei in the dorsal thalamus, the intergeniculate leaflet (IGL), and ventral LGN (vLGN) are also shown. The dLGN projects topographically to primary visual cortex (area 17). The medial two-thirds of area 17 receives monocular input from the contralateral eye (17M). The lateral one-third receives binocular inputs (17B). Adjacent to area 17 laterally is area 18a; area 18b is medial. See color plate 1.

projection, however, is to the SC in the midbrain (Dräger, 1974; Hofbauer and Dräger, 1985; Provencio et al., 1998). Overall, the pattern of retinal projections in mouse is remarkably similar to that in the laboratory rat (Arnason et al., 2002; for a review, see Sefton et al., 2004). A schematic diagram illustrating the retinal projection to the thalamus is given in figure 3.3. Suprachiasmatic Nucleus, Intergeniculate Leaflet, and Ventrolateral Geniculate Nucleus In nocturnal rodents, the SCN, IGL, and vLGN appear to be associated with circadian responses to light (Cassone et al., 1988; Sefton

et al., 2004). Furthermore, the IGL and vLGN jointly with olivary pretectal nuclei (OPT) are also involved in the pupillary light reflex (for a review, see Sefton et al., 2004). Unlike the retinal input to the SC, the proportions of contralateral and ipsilateral RGCs that innervate the SCN are very similar (ca. 50/50; Abrahamson and Moore, 2001; Youngstrom and Nunez, 1986; Balkema and Drager, 1990; see also Dräger, 1974). The SCN, IGL, and vLGN receive direct retinal input from small but distinct subpopulations of RGCs (Provencio et al., 1998). Most of the direct retinal input to these nuclei is provided by melanopsin-expressing RGCs, which are thought to function as luminosity detectors (Berson, 2003; Dacey et al., 2005). By contrast, RGCs that express the Brn3a transcription factor do not project to these nuclei and innervate the SC and dLGN (Quina et al., 2005). Pretectum and Accessory Optic Tract Nuclei There is a direct retinal input to the anterior and posterior OPT that originates mainly but not exclusively from the contralateral retina (Pak et al., 1987). In the anterior OPT the terminals from the contralateral and ipsilateral retinas occupy distinct parts of the nucleus. The anterior OPT is larger than the posterior OPT (Pak et al., 1987). In rat (Trejo and Cicerone, 1984), carnivores (Distler and Hoffmann, 1989), and primates (Gamlin et al., 1995), the OPT is the principal nucleus involved in the pupillary light reflex and receives its principal retinal input most from the melanopsinexpressing RGCs. Two other pretectal nuclei, the posterior pretectal nucleus (PP) and the nucleus of optic tract (NOT), also receive direct retinal input, but only from the contralateral retina (Pak et al., 1987). In the mouse, all the accessory optic tract terminal nuclei—that is, the dorsal, lateral, and medial terminal nuclei (DTN, LTN, and MTN, respectively)—receive retinal input from the contralateral but not the ipsilateral eye (Pak et al., 1987). The DTN is located caudolateral to the NOT of pretectum between the stratum griseum superficiale of the SC and the medial geniculate nucleus (Pak et al., 1987). The LTN is a very small group of cells located just at the border of the medial geniculate nucleus and cerebral peduncles, while the MTN, the largest of the accessory optic tract nuclei, is located in the ventral midbrain tegmentum medial to the cerebral peduncle and the substantia nigra (Pak et al., 1987). The overall plan of organization of mouse accessory optic tract nuclei conforms well to that of other mammals (Cooper and Magnin, 1986). The accessory optic tract nuclei are strongly interconnected with each other as well as with the NOT. In all mammals studied so far, the DTN and NOT appear to play crucial roles in driving horizontal optokinetic nystagmus, while the LTN and MTN are associated with vertical optokinetic nystagmus (reviewed in Grasse and Cynader, 1991; Sefton et al., 2004; Ibbotson and Dreher, 2005).

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Superior Colliculus The SC constitutes a part of the tectum of the mesencephalon (midbrain) and is the mammalian homologue of the optic tectum of other vertebrates. In all mammals, including the mouse, seven distinct alternating cellular and fibrous layers are apparent in Nissl-stained and myelin-stained coronal or sagittal sections through the SC (Paxinos and Franklin, 2004). In the mouse (Dräger and Hubel, 1975b) as in other nocturnal rodents (Sefton et al., 2004) and in virtually all other mammals studied so far (for reviews, see Rhoades et al., 1991; Stein and Meredith, 1991), the retinal input terminates almost exclusively in the three superficial layers of the SC, that is, the most superficial, thin, and relatively cell-poor stratum zonale (zonal layer), the adjacent thicker and cell-rich stratum griseum superficiale (superficial gray layer), and the white fibrous layer, the stratum opticum (optic layer). As mentioned earlier, the ipsilateral projection in mice arises from a minority of RGCs in the VTC; these terminate in distinct clumps in the lower stratum griseum superficiale and stratum opticum in the rostral and medial parts of the SC (Dräger and Hubel, 1975b; Godement et al., 1984). In the mouse (Hofbauer and Dräger, 1985) as in the laboratory rat (Linden and Perry, 1983; Dreher et al., 1985), hamster (Chalupa and Thompson, 1980), rabbit (Vaney et al., 1981), and polyprotodont marsupials such as North American opossum (Rapaport et al., 2004), virtually all RGCs (and thus presumably all morphological and functional classes of RGCs) project to the SC. On the other hand, in carnivores such as the domestic cat only about 50% of RGCs project to the SC, while 80% project to the dLGN (Illing and Wässle, 1981). Of the RGCs that project to the SC, about half (25% of all RGCs) also send a collateral to the dLGN (Wässle and Illing, 1980; Illing and Wässle, 1981). In primates such as macaque monkeys, while most, if not all, RGCs project to the dLGN, only a small minority ( Cryget) as the decrease in the relative content of water-soluble lens protein, which might be used as a rough indicator of the severity of cataractogenesis (Graw and Liebstein, 1993). Although Cryg mutation-mediated mechanisms of cataract formation are not fully understood, it is known that the mutations interfere with the breakdown of the lens fiber cell nuclei during terminal differentiation. The alteration of this process was demonstrated for three mouse Cryg mutants (Crygbnop, Crygeelo, Cryget); in these mutants, it was shown that the mutant γ-crystallins contributed to the formation of amyloid-like fibers in the lens fiber cell nuclei (Sandilands et al., 2002). Some of the inherited cataracts in humans are also related to mutations in the γ-crystallin-encoding genes. In humans, two of the six CRYG genes on chromosome 2 are pseudo-

genes, but mutations associated with a clinical phenotype have been found up to now only in CRYGC and CRYGD. Surprisingly, the CRYGD-P23T mutation was observed in five independent families from different continents and reported to be causative for phenotypically diverse cataractous features. In all cases, the wild-type sequence CCAC CCCAA changes to CCACACCAA. Moreover, one of the dominant human CRYGD mutations (W156X) is identical to the dominant mouse Lop12 mutation; the common single base-pair exchange is G→A, which cannot be explained by a slippage mechanism during DNA replication. Consequently, in mouse and human, 18 amino acids are missing at the C-terminus, leading to a dominant phenotype (in contrast to the 16 amino acids that are missing in the recessive mouse Crygs mutation).

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Cytoskeletal Proteins There are three major lens cytoskeletal proteins, filensin (CP94 or beaded filament structural protein 1; gene symbol Bfsp1), phakinin (also referred to as CP49 or beaded filament structural protein 2; gene symbol Bfsp2), and vimentin. Since vimentin knockout mutants do not show an ocular phenotype, the two Bfsp genes are important for lens transparency. CP49 and filensin, together with α-crystallins, have been localized at unique cytoskeletal structures within the lens fiber cells known as

beaded filaments. They are considered to be important in facilitating the chaperone activity of α-crystallin assemblies. Mutations in these two genes lead to cataract formation. Disruption of the Bfsp1 gene reduced levels of filensin’s assembly partner CP49 and prevented the assembly of beaded filaments. These knockouts began to show evidence of light scattering by 2 months and worsened with age. Heterozygous animals exhibited an intermediate phenotype, showing a moderate light scattering at 5 months (Alizadeh et al., 2003). However, the knockout of the Bfsp2 gene does not lead to cataracts, even if the absence of CP49 causes a subtle loss of optical clarity in the lens (Alizadeh et al., 2002; Sandilands et al., 2003). Moreover, a deletion of the splice-acceptor site in exon 2 of the mouse Bfsp2 results in a splicing of exon 1 to exon 3 and causes a frameshift in the reading frame as well as the introduction of a stop codon at position 2 of exon 3 in the Bfsp2 transcript. The phenotype of this mutation is also subtle, as described for the knockout of the entire gene. Since this mutation is present in several mouse strains (129, 101, and CBA), it might interfere with other mutations or targeted deletions, and therefore it might have important implications for lens studies using these strains (Sandilands et al., 2004). Mutations in CP49-encoding gene BFSP2 were shown to be responsible for dominant cataracts in humans (Conley et al., 2000; Jakobs et al., 2000); their phenotypes, however, seem to be variable ranging from congenital nuclear, sutural, or stellate cataracts to juvenile-onset cataracts. Another protein, one associated with the lens cytoskeleton, is the Nhs1 protein, which is affected in the human Nance-Horan syndrome (NHS). Recently, a large insertion between exon 1 and exon 2 of the mouse Nhs1 gene was shown to underlie the X-linked dominant cataract Xcat, which was recovered after parental irradiation. Histological analysis during embryonic development revealed that in the affected embryos, the primary fiber cells are irregularly arranged and show small foci of cellular disintegration; the fibers progressively degenerate. Molecularly, the insertion inhibits the expression of the Nhs1 isoform containing exon 1 and results in exclusive expression of the alternative isoform containing exon 1A. The presence of Nhs1 exon 1 is critical for localization of the protein to the cytoplasm, whereas proteins lacking Nhs1 exon 1 are predominantly nuclear. These results indicate that the first exon of Nhs1 contains crucial information required for the proper expression and localization of Nhs1 protein (Huang et al., 2006).

Mouse models for metabolic cataracts Sugar-Induced Cataracts in the Mouse Inborn errors in the galactose pathway and diabetes are known risk factors for the development of cataracts in humans. Sugar is

converted to the corresponding sugar alcohol, which accumulates in the lens and creates osmotic problems, eventually leading to cataract. For a long time, appropriate mouse models were missing. One hypothesis was that the enzyme aldose reductase (responsible for the conversion of sugar to its alcohol) has a very low activity in the mouse compared to human. Therefore, it was not surprising that the galactokinase (Glk1) knockout in the mouse does not suffer from cataracts at all. However, the introduction of a human aldose reductase transgene into a Glk1-deficient background resulted in cataract formation within the first postnatal day (Ai et al., 2000). This result highlights the importance of aldose reductase in sugar-dependent cataract formation. Another candidate for cataract formation under diabetic conditions came from investigation of the bifunctional protein DCoH (dimerizing cofactor for HNF1). It acts as an enzyme in intermediary metabolism (gene symbol Pcbd1: pterin 4α-carbinolamine dehydratase) and as a binding partner of the HNF1 family of transcriptional activators. Knockout mutants of Pcbd1 are viable and fertile but display hyperphenylalaninemia and a predisposition to cataracts. Lens opacities were visually detectable in about 20% of the Pcbd1 null mice, if maintained on the outbred CD1 genetic background. The age at onset varied widely, with the earliest detection at 12 days; most of the affected animals had developed cataracts by the age of 24 weeks. The incidence of cataract formation was reduced in the C57BL/6J inbred genetic background (Bayle et al., 2002). Protein-Bound Carbohydrates and Cataract Besides the free sugars of the intermediate metabolic pathways, sugar residues are present in a variety of glycoproteins. One of the corresponding enzymes is the α(1,3)-galactosyltransferase, which catalyzes the addition of galactose in an α(1,3) configuration to particular glycoproteins. Therefore, it is also referred to as glycoprotein galactosyltransferase α1,3 (gene symbol Ggta1); the corresponding gene is mapped to chromosome 2. The transfer of galactose to particular glycoproteins creates a highly immunogenic epitope that is present in all mammals except humans, apes, and Old World monkeys. Ggta1 knockout mice have impaired glucose tolerance and decreased insulin sensitivity, and develop cataracts. A white pinhead opacity was observed in one eye at an average age of 36–37 days and in the second eye 1–2 days later. Rapid progression to full opacities occurred on average within 7–8 days. Early nuclear and posterior cortical changes, as well as fiber folds and swollen sutures, have been observed (see Dahl et al., 2006, and references therein). Another interesting mouse model for congenital cataracts was characterized by targeting the gene coding for perlecan (Hspg2, mapped to chromosome 4). Perlecan is a large,

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multidomain, heparan sulfate proteoglycan found in all basement membranes; besides type IV collagen and laminin, it is a core protein of the lens capsule. Therefore it is not surprising that the homozygous deletion of the exon 3 by gene targeting leads to leakage of cellular material through the lens capsule and degeneration of the lens within 3 weeks after birth. In detail, loss of exon 3 removes the attachment sites for three heparan sulfate side chains composed of linear polysaccharides. It is speculated that this deletion might also change the affinity of perlecan for basic fibroblast growth factor. The cataractogenic potential of deletion within the Hspg2 gene is dramatically enhanced in double knockouts, including both Hspg2 and Col18a1; the Col18a1 knockouts do not have any eye phenotype (Rossi et al., 2003). Cholesterol Metabolism and Cataract One of the “old” syndromic, dominant mouse cataract mutants is the X-linked bare patches (Bpa). Whereas hemizygous males die before birth, heterozygous females have patches of bare skin. Lens cortical “frost figure” opacities are present. Molecular analysis showed that mutations in the gene Nsdhl (encoding an NAD(P)H steroid dehydrogenase-like protein) are responsible for the phenotype in two independent Bpa and three independent striated (Str) alleles (Liu et al., 1999). At the time it was published, it was the first mammalian locus associated with an X-linked dominant, male-lethal phenotype. In total, 10 alleles are reported (three spontaneous, two chemically induced, and five irradiation induced). It is also the first cataract phenotype shown to be related to the cholesterol pathway.

are characterized by an earlier onset and more rapid advancement of senescence resulting from a significantly shorter life span. Cataracts have been found in the SAM-P/1 and SAM-P/9 strains. The earliest change was the appearance of a ripple mark body at about 3 months of age. The number of rippled rings increased with age. These changes later induced refractive distortion of retinal vessels. Wholemount flat preparations of the epithelium showed that the number of cells was markedly decreased in advanced stages of cataract. In the late stages of life, the lens cortex became liquefied and developed into a mature cataract (Nishimoto et al., 1993). The mode of inheritance and the linkage to a particular chromosome still remain to be investigated. Recently, the phenotypic characterization of a conditional knockout of the murine Nbn gene (encoding nibrin) was reported. The Nbn gene is the mouse homologue for the human Nijmegen breakage syndrome gene. All Nbn-deficient lenses develop cataracts at an early age due to altered lens fiber cell differentiation, including disruption of normal lens epithelial and fiber cell architecture and incomplete denucleation of fiber cells. In addition, Nbn-deficient lenses show dysregulated transcription of various crystalline genes. These features implicate a function of Nbn in terminal differentiation of the lens fiber cells and cataractogenesis (Yang et al., 2006). The encoded protein nibrin has a role in DNA double-strand-break sensing in response to DNA damage and repair. Since defects in DNA damage repair are frequently associated with premature aging processes, the mutation in the Nbn gene might be a first hint at the participation of the corresponding repair and cell cycle control proteins in the formation of age-related cataracts.

Mouse models for senile cataracts The Emory mouse is a well-characterized genetic model for age-onset cataract. Emory mice develop cataracts at 5–6 months (early cataract strain) or 6–8 months (late cataract strain). Emory mouse cataracts increase in severity with age and first develop in the anterior superficial cortex region of the lens. They eventually progress into the anterior deep cortex region and ultimately result in complete lens opacification. Emory mouse cataracts are also associated with changes in numerous biochemical parameters and gene expression levels in the lens (see Sheets et al., 2002, and references therein). The first unpublished results from our laboratory concerning the mapping of the underlying mutation strongly suggest that the late Emory cataract (gene symbol Em) is a complex genetic disease that results from mutations in several genes, which must interact to produce this particular type of cataract. Another genetic mouse model for senile cataracts is the senescence-accelerated mouse (SAM), which was identified at Kyoto University in 1970 on an AKR/J background strain. There are eight senescence-prone (SAM-P) strains, which

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Open questions and conclusions Database searches indicate additional mouse mutants exhibiting diverse forms of cataract that have not yet been characterized with respect to their mutations. One of them is Tim (translocation-induced circling mutation), which is associated with a reciprocal chromosomal translocation between chromosomes 4 and 17. Affected mice develop an anterior subcapsular cataract that appears after birth and is progressive and accompanied by abnormal head tossing and circling behavior. The most likely explanation for the phenotype is that the translocation breakpoint disrupted a gene or its regulation; this breakpoint remains to be determined (Smith et al., 1999). Other mutants include the following (details and references are available on the Web site of the Jackson Laboratory [www.informatics.jax.org]): • The mutation vacuolated lens (vl) is mapped to mouse chromosome 1 and leads to opaque white lenses. Additionally, the mutants are characterized by a white belly spot and spina bifida. Small lens vacuoles are present at birth.

• The mutant blind-sterile (bs) is characterized by bilateral nuclear cataracts, microphthalmia, and glossy coats. The cataracts are detectable at E16. Females are fertile, but males are sterile. The mutation was mapped to mouse chromosome 2. • The Tcm mutation (total cataract with microphthalmia), a cataract with iris dysplasia and coloboma, and the Ccw mutation, cataract and curly whiskers, are localized to mouse chromosome 4. • The nuclear posterior polar opacity (Npp) maps to chromosome 5. • Cat5 (previously To2), a total opacity, is located close to the centromer on chromosome 10. • Two alleles of Cat3 (Cat3vl, vacuolated lens; Cat3vao, cataract with anterior opacity) arose independently in the F1 generation after paternal γ-irradiation and map to the central region of chromosome 10. • The so-called rupture of lens cataract (rlc) was mapped to chromosome 14; a similar form, lr2 (lens rupture 2), was mapped to a close position. The opacity in the rlc/rlc mice becomes apparent at 35–60 days of age; there are no developmental changes reported. • Finally, a form of cataract that forms postnatally without observed developmental alterations is the Nakano cataract (nct). The mutation was mapped to chromosome 16. A more detailed analysis of these mutants should allow a more precise description of the mechanisms leading to cataracts. The list of already characterized mutants and the list of not yet characterized mutants (which is still increasing, owing to ongoing mutagenesis screens and improved phenotyping strategies) underline the power of this particular genetic system. REFERENCES Ai, Y., Zheng, Z., O’Brien-Jenkins, A., Bernard, D. J., WynshawBoris, T., Ning, C., Reynolds, R., Segal, S., Huang, K., et al. (2000). A mouse model of galactose-induced cataracts. Hum. Mol. Genet. 12:1821–1827. Alizadeh, A., Clark, J., Seeberger, T., Hess, J., Blankenship, T., and FitzGerald, P. G. (2003). Targeted deletion of the lens fiber cell-specific intermediate filament protein filensin. Invest. Ophthalmol. Vis. Sci. 44:5252–5258. Alizadeh, A., Clark, J. I., Seeberger, T., Hess, J., Blankenship, T., Spicer, A., and FitzGerald, P. G. (2002). Targeted genomic deletion of the lens-specific intermediate filament protein CP49. Invest. Ophthalmol. Vis. Sci. 43:3722–3727. Aso, S., Ishikawa, A., Wakana, S., Baba, R., Fujita, M., and Namikawa, T. (2001). The eye lens aplasia (elap) maps to mouse chromosome 2. Exp. Anim. 50:97–98. Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L., Vivian, N., and Lovell-Badge, R. (2003). Multipotent cell lineages in early mouse development depend on SOX2 function. Genes. Dev. 17:126–140. Bayle, J. H., Randazzo, F., Johnen, G., Kaufman, S., Nagy, A., Rossant, J., and Crabtree, G. R. (2002). Hyperphenylalanin-

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and homeodomain in brain development. Development 131: 6131–6140. Hill, R. E., Favor, J., Hogan, B. L. M., Ton, C. C. T., Saunders, G. F., Hanson, I. M., Prosser, J., Jordan, T., Hastie, N. D., et al. (1991). Mouse Small eye results from mutations in a paired-like homeobox-containing gene. Nature 354:522–525. Hong, H. K., Lass, J. H., and Chakravarti, A. (1999). Pleiotropic skeletal and ocular phenotypes of the mouse mutation congenital hydrocephalus (ch/Mf1) arise from a winged helix/forkhead transcription factor gene. Hum. Mol. Genet. 8:625–637. Huang, K. M., Wu, J., Duncan, M. K., Moy, C., Dutra, A., Favor, J., Da, T., and Stambolian, D. (2006). Xcat, a novel mouse model for Nance-Horan syndrome inhibits expression of the cytoplasmic-targeted Nhs1 isoform. Hum. Mol. Genet. 15: 319–327. Hwang, D. Y., Ardayfio, P., Kang, U. J., Semina, E. V., and Kim, K. S. (2003). Selective loss of dopaminergic neurons in the substantia nigra of Pitx3-deficient aphakia mice. Mol. Brain. Res. 114:123–131. Jakobs, P. M., Hess, J. F., FitzGerald, P. G., Kramer, P., Weleber, R. G., and Litt, M. (2000). Autosomal-dominant congenital cataract associated with a deletion mutation in the human beaded filament protein BFSP2. Am. J. Hum. Genet. 66:1432–1436. Jiang, H., Jin, Y., Bu, L., Zhang, W., Liu, J., Cui, B., Kong, X., and Hu, L. (2003). A novel mutation in GJA3 (connexin46) for autosomal dominant congenital nuclear pulverulent cataract. Mol. Vis. 9:579–583. Johnson, G. J., and Foster, A. (2004). Prevalence, incidence and distribution of visual impairment. In G. J. Johnson, D. C. Minassian, R. A. Weale, and S. K. West (Eds.), The epidemiology of eye disease (pp. 3–28). London: Arnold. Kamachi, Y., Uchikawa, M., and Kondoh, H. (2000). Pairing SOX off with partners in the regulation of embryonic development. Trends Genet. 16:182–187. Kiyozumi, D., Sugimoto, N., and Sekiguchi, K. (2006). Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects. Proc. Natl. Acad. Sci. U.S.A. 103:11981– 11986. Klopp, N., Favor, J., Löster, J., Lutz, R. B., Neuhäuser-Klaus, A., Prescott, A., Pretsch, W., Quinlan, R. A., Sandilands, A., et al. (1998). Three murine cataract mutants (Cat2) are defective in different γ-crystallin genes. Genomics 52:152–158. Kratochvilova, J., and Ehling, U. H. (1979). Dominant cataract mutations induced by γ-irradiation of male mice. Mutat. Res. 63:221–223. Litt, M., Kramer, P., LaMorticella, D. M., Murphey, W., Lovrien, E. W., and Weleber, R. G. (1998). Autosomaldominant congenital cataract associated with a missense mutation in the human alpha-crystallin gene CRYAA. Hum. Mol. Genet. 7:471–474. Liu, H., Du, X., Wang, M., Huang, Q., Ding, L., McDonald, H. W., Yates, J. R., III, Beutler, B., Horwitz, J., et al. (2005). Crystallin γB-I4F mutant protein binds to α-crystallin and affects lens transparency. J. Biol. Chem. 280:25071–25078. Liu, X. Y., Dangel, A. W., Kelley, R. I., Zhao, W., Denny, P., Botcherby, M., Cattanach, B., Peters, J., Hunsicker, P. R., et al. (1999). The gene mutated in bare patches and sdtriated mice encodes a novel 3β-hydroxysteroid dehydrogenase. Nat. Genet. 22:182–187. Lyon, M. F., Jamieson, R. V., Perveen, R., Glenister, P. H., Griffiths, R., Boyd, Y., Glimcher, L. H., Favor, J., Munier,

F. L., et al. (2003). A dominant mutation within the DNAbinding domain of the bZIP transcription factor Maf causes murine cataract and results in selective alteration in DNA binding. Hum. Mol. Genet. 12:585–594. Mackay, D. S., Andley, U. P., and Shiels, A. (2003). Cell death triggered by a novel mutation in the αA-crystallin gene underlies autosomal dominant cataract linked to chromosome 21q. Eur. J. Hum. Genet. 11:784–793. Mackay, D. S., Boskovska, O. B., Knopf, H. L. S., Lampi, K. J., and Shiels, A. (2002). A nonsense mutation in CRYBB1 associated with autosomal dominant cataract linked to human chromosome 22q. Am. J. Hum. Genet. 71:1216–1221. Mackay, D., Ionides, A., Kibar, Z., Rouleau, G., Berry, V., Moore, A., Shiels, A., and Bhattacharya, S. (1999). Connexin46 mutations in autosomal dominant congenital cataract. Am. J. Hum. Genet. 64:1357–1364. Min, J. N., Zhang, Y., Mosykophidis, D., and Mivechi, N. F. (2004). Unique contribution of heat shock transcription factor 4 in ocular lens development and fiber cell differentiation. Genesis 40:205–217. Mörner, C. T. (1893). Untersuchungen der Proteinsubstanzen in den lichtbrechenden Medien des Auges. Z. Physiol. Chem. 18: 61–106. Nishiguchi, S., Wood, H., Kondoh, H., Lovell-Badge, R., and Episkopou, V. (1998). Sox1 directly regulates the γ-crystallin gene and is essential for lens development in mice. Genes Dev. 12: 776–781. Nishimoto, H., Uga, S., Miyata, M., Ishikawa, S., and Yamashita, K. (1993). Morphological study of the cataractous lens of the senescence accelerated mouse. Graefes Arch. Clin. Exp. Ophthalmol. 231:722–728 Perng, M. D., and Quinlan, R. A. (2005). Seeing is believing! The optical properties of the eye lens are dependent upon functional intermediate filament cytoskeleton. Exp. Cell. Res. 305: 1–9. Peterson, C. A., and Piatigorsky, J. (1986). Preferential conservation of the globular domains of the βA3/A1-crystallin polypeptide of the chicken eye lens. Gene 45:139–147. Piatigorsky, J. (2003). Crystallin genes: Specialization by changes in gene regulation may precede gene duplication. J. Struct. Funct. Genomics 3:131–137. Pras, E., Frydman, M., Levy-Nissenbaum, E., Bakhan, T., Raz, J., Assia, E. I., Goldman, B., and Pras, E. (2000). A nonsense mutation (W9X) in CRYAA causes autosomal recessive cataract in an inbred Jewish Persian family. Invest. Ophthalmol. Vis. Sci. 41:3511–3515. Reddy, M. A., Bateman, O. A., Chakarova, C., Ferris, J., Berry, V., Lomas, E., Sarra, R., Smith, M. A., Moore, A. T., et al. (2004). Characterization of the G91del CRYBA1/3-crystallin protein: A cause of human inherited cataract. Hum. Mol. Genet. 13:945–953. Rees, M. I., Watts, P., Fenton, I., Clarke, A., Snell, R. G., Owen, M. J., and Gray, J. (2000). Further evidence of autosomal dominant congenital zonular pulverulent cataracts linked to 13q11 (CZP3) and a novel mutation in connexin 46 (GJA3). Hum. Genet. 106:206–209. Rieger, D. K., Reichenberger, E., McLean, W., Sidow, A., and Olsen, B. R. (2001). A double-deletion mutation in the Pitx3 gene causes arrested lens development in aphakia mice. Genomics 72:61–72. Ring, B. Z., Cordes, S. P., Overbeek, P. A., and Barsh, G. S. (2000). Regulation of mouse lens fiber cell development and differentiation by the Maf gene. Development 127:307–317.

Rossi, M., Morita, H., Sormunen, R., Airenne, S., Kreivi, M., Wang, L., Fukai, N., Olsen, B. R., Tryggvason, K., et al. (2003). Heparan sulfate chains of perlecan are indispensable in the lens capsule but not in the kidney. EMBO J. 22:236– 245. Ruiz-Ederra, J., and Verkman, A. S. (2006). Accelerated cataract formation and reduced lens epithelial water permeability in aquaporin-1-deficient mice. Invest. Ophthalmol. Vis. Sci. 47: 3960–3967. Sandilands, A., Hutcheson, A. M., Long, H. A., Prescott, A. R., Vrensen, G., Löster, J., Klopp, N., Lutz, R. B., Graw, J., et al. (2002). Altered aggregation properties of mutant γ-crystallins cause inherited cataract. EMBO J. 21:6005–6014. Sandilands, A., Prescott, A. R., Wegener, A., Zoltoski, R. K., Hutcheson, A. M., Masaki, S., Kuszak, J. R., and Quinlan, R. A. (2003). Knockout of the intermediate filament protein CP49 destabilises the lens fibre cytoskeleton and decreases lens optical quality, but does not induce cataract. Exp. Eye Res. 76:385– 391. Sandilands, A., Wang, X., Hutcheson, A. M., James, J., Prescott, A. R., Wegener, A., Pekny, M., Gong, X., and Quinlan, R. A. (2004). Bfsp2 mutation found in mouse 129 strains causes the loss of CP49′ and induces vimentin-dependent changes in the lens fibre cell cytoskeleton. Exp. Eye Res. 78:875–889. Santhiya, S. T., Abd-Alla, S. M., Löster, J., and Graw, J. (1995). Reduced levels of γ-crystallin transcripts during embryonic development of murine Cat2nop mutant lenses. Graefes Arch. Clin. Exp. Ophthalmol. 233:795–800. Santhiya, S. T., Manisastry, S. M., Rawlley, D., Malathi, R., Anishetty, S., Gopinath, P. M., Vijayalakshmi, P., Namperumalsamy, P., Adamski, J., et al. (2004). Mutation analysis of congenital cataracts in Indian families: Identification of SNPs and a new causative allele in CRYBB2 gene. Invest. Ophthalmol. Vis. Sci. 45:3599–3607. Semina, E., Murray, J. C., Reiter, R., Hrstka, R. F., and Graw, J. (2000). Deletion in the promoter region and altered expression of Pitx3 homeobox gene in aphakia mice. Hum. Mol. Genet. 9: 1575–1585. Sheets, N. L., Chauhan, B. K., Wawrousek, E., Hejtmancik, J. F., Cvekl, A., and Kantorow, M. (2002). Cataract- and lens-specific upregulation of ARK receptor tyrosine kinase in Emory mouse cataract. Invest. Ophthalmol. Vis. Sci. 43:1870– 1875. Shiels, A., and Bassnett, S. (1996). Mutations in the founder of the MIP gene family underlie cataract development in the mouse. Nat. Genet. 12:212–215. Sinha, D., Wyatt, M. K., Sarra, R., Jaworski, C., Slingsby, C., Thaung, C., Pannell, L., Robison, W. G., Favor, J., et al. (2001). A temperature-sensitive mutation of Crygs in the murine Opj cataract. J. Biol. Chem. 276:9308–9315. Smidt, M. P., Smits, S. M., Bouwmeester, H., Hamers, F. P., van der Linden, A. J., Hellemons, A. J., Graw, J., and Burbach, J. P. (2004). Early developmental failure of substantia nigra dopamine neurons in mice lacking the homeodomain gene Pitx3. Development 131:1145–1155. Smith, R. S., Johnson, K. R., Hawes, N. L., Harris, B. S., Sundberg, J. P., and Davisson, M. T. (1999). Lens epithelial proliferation cataract in segmental trisomy involving mouse chromosomes 4 and 7. Mamm. Genome 10:102–106. Steele, E. C., Jr., Kerscher, S., Lyon, M. F., Glenister, P. H., Favor, J., Wang, J., and Church, R. L. (1997). Identification of a mutation in the MP19 gene, Lim2, in the cataractous mouse mutant To3. Mol. Vis. 3:5.

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Steele, E. C., Jr., Lyon, M. F., Favor, J., Guillot, P. V., Boyd, Y., and Church, R. L. (1998). A mutation in the connexin 50 (Cx50) gene is a candidate for the No2 mouse cataract. Curr. Eye Res. 17:883–889. Vanita, Sarhad, V., Reis, A., Jung, M., Singh, D., Sperling, K., Singh, J. R., and Burger, J. (2001). A unique form of autosomal dominant cataract explained by gene conversion between β-crystallin B2 and its pseudogene. J. Med. Genet. 38:392– 396. Verkman, A. S. (2003). Role of aquaporin water channels in eye function. Exp. Eye Res. 76:137–143. Vicart, P., Caron, A., Guicheney, P., Li, Z., Prévost, M. C., Faure, A., Chateau, D., Chapon, F., Tomé, F., et al. (1998). A missense mutation in the αB-crystallin chaperone gene causes a desmin-related myopathy. Nat. Genet. 20:92–95. White, T. W., Goodenough, D. A., and Paul, D. L. (1998). Targeted ablation of connexin50 in mice results in microphthalmia and zonular pulverulent cataracts. J. Cell. Biol. 143:815– 825.

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Xia, C., Liu, H., Chang, B., Cheng, C., Cheung, D., Wang, M., Huang, Q., Horwitz, J., and Gong, X. (2006a). Arginine 54 and tyrosine 118 residues of αA-crystallin are crucial for lens formation and transparency. Invest. Ophthalmol. Vis. Sci. 47:3004– 3010. Xia, C., Liu, H., Cheung, D., Cheng, C., Wang, M., Du, X., Beutler, B., Lo, W. K., and Gong, X. (2006b). Diverse gap junctions modulate distinct mechanism for fiber cell formation during lens development and cataractogenesis. Development 133:2033– 2040. Yang, Y. G., Frappart, P. O., Frappart, L., Wang, Z. Q., and Tong, W. M. (2006). A novel function of DNA repair molecule Nbs1 in terminal differentiation of the lens fibre cells and cataractogenesis. DNA Repair 5:885–893. Zaki, P. A., Collinson, J. M., Toraiwa, J., Simpson, T. I., Price, D. J., and Quinn, J. C. (2006). Penetrance of eye defects in mice heterozygous for mutation of Gli3 is enhanced by heterozygous mutation of Pax6. BMC Dev. Biol. 6:46; doi:10.1186/1471-213X6-46.

41

Mouse Models of Infectious Eye Diseases LINDA D. HAZLETT

The pathogenesis of ocular infectious disease is determined by the virulence of the microorganism, the host immune response, and the anatomical features of the site of infection. In the eye, the cornea is unique anatomically, as it must remain optically clear and avascular, yet able to respond rapidly to microbial insult, protecting itself and the interior structures of the eye, such as the retina, from sight-threatening microbially initiated damage. Understanding innate and acquired immune response mechanisms during viral, bacterial, fungal, and parasitic eye infections remains fundamental to the rational design of therapeutic strategies to eliminate the microorganisms, control inflammatory responses, and minimize the action of their virulence factors, thus preventing permanent structural damage to the cornea and interior ocular structures, which would render them optically dysfunctional. Numerous animal models have provided much information about immune system responses to infection. Among them, the mouse is often regarded as a model of choice. Mice are susceptible to a similar range of microbial infections as humans, and marked differences between inbred strains can be exploited to analyze the genetic basis of infections. In addition, the genetic tools that are available for use in the laboratory mouse, and new techniques to monitor the expression of genes (e.g., bacterial) in vivo, make the mouse the principal experimental animal model for studying mechanisms of infection and immunity (Buer and Balling, 2003).

Herpetic infections of the cornea Herpetic viral infection of the cornea caused by herpes simplex virus (HSV) is a leading nontraumatic cause of blindness, despite the availability of both immunosuppressive and antiviral drugs (O’Brien and Hazlett, 1996). Acute infection of the corneal surface is a manifestation of virusinduced cytolysis. The corneal stroma may also be involved as a result of recurring infections associated with reactivation from latency. Stromal inflammation reflects an immunopathological process that often leads to corneal scarring, neovascularization, permanent endothelial dysfunction, and vision impairment. HSV also may be causative in dermatitis, blepharitis, conjunctivitis, iridiocyclitis, and retinitis.

The mouse model has greatly furthered our understanding of the role of the immune response in tissue damage. Mice are experimentally infected with certain strains of HSV and develop keratitis. Wounding of the cornea is usually required, and not all mouse strains are susceptible. Herpes stromal keratitis (HSK) in the mouse is a model of disciform nonnecrotizing keratitis in which the inflammation may be dominated eventually by lymphocytes and other mononuclear cells (O’Brien and Hazlett, 1996). HSK is T cell mediated, and in BALB/c mice infected with HSV-1, tissuedestructive inflammation in the cornea (HSK) and other periocular lesions develop about 7 days after viral infection. CD4+ T cells and T helper 1 (Th1)-type cytokines contribute both to the immunopathology in the cornea and to the eradication of viral replication in the skin. Studies in CD4+ T cell–deficient mice showed that these cells preferentially mediate HSK, but in their absence, a high infectious dose of HSV-1 can induce histologically similar but transient HSK that is mediated by CD8+ T cells (Lepisto et al., 2006). Disruption of CD40/154 signaling does not affect the initial expansion of CD4+ T cells in the draining lymph nodes but dramatically reduces the persistence and Th1 polarization of these cells. It was concluded that CD154 signaling is required during the inductive but not the effector phase of the Th1 immune response in the infected cornea, and therefore local disruption of this signaling pathway would not likely be a useful therapy for HSK (Xu et al., 2004). In contrast, others have used mice to show that CD86, a member of the costimulatory family of molecules, is important in the development of cytotoxic T cells and in reducing viral replication in the eyes of HSV-1-infected mice (Osorio et al., 2005). In mice, low delayed-type hypersensitivity (DTH) responses were found to be associated with less severe disease, while high DTH responsive mice exhibited worse disease. IL-10 knockout mice also had worsened disease, while mice given recombinant (r) IL-10 protein by ocular and intraperitoneal routes had less severe lesions (Keadle and Stuart, 2005). Recurrent HSV-1 usually results from reactivation of latent virus in sensory neurons, followed by transmission to peripheral sites (Liu et al., 2000). The mechanism of this has been extensively studied in mouse models. It was shown that CD8+ T cells that are present in the trigeminal ganglion at

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the time of its excision can maintain HSV-1 in a latent state in sensory neurons in ex vivo trigeminal ganglion cultures (Liu et al., 2000). The use of T cell transgenic mice with severe combined immunodeficiency syndrome (SCID) or mice on a recombinase-activating gene–deficient background lacking both T and B lymphocytes also suggests that CD8+ T cell control is expressed in the trigeminal ganglion, serving to curtail the source of the virus to the cornea (Banerjee et al., 2004). The pathogenesis of corneal scarring and vascularization in HSK is uncertain but appears to reflect a complex interaction of various cytokines, chemokines, and growth factors brought in by inflammatory cells or produced locally in the cornea in response to HSV-1 infection. Evidence suggests that HSV-1 infection disrupts the normal equilibrium between angiogenic and antiangiogenic stimuli, leading to vascularization of the normally avascular cornea. Thrombospondin 1 and 2, matrix proteins involved in wound healing, are potent antiangiogenic factors and may be among the critical players involved. It has been suggested that elucidating their role in corneal scarring and vascularization may lead to improved therapies for HSK (Kaye and Choudhary, 2006). In this regard, matrix metalloproteinase-9 (MMP-9), a type IV collagenase, has been shown to contribute to corneal neovascularization in the mouse corneal stroma infected with HSV. Neutrophils (PMNs), which invade the cornea soon after infection, were considered a likely source of the matrix-degrading enzyme, since using a PMN-specific monoclonal antibody (mAb) diminished MMP-9 expression, as well as the extent of angiogenesis. MMP-9 knockout mice also had diminished disease, while use of tissue inhibitor of metalloproteinase-1 (TIMP-1), a specific MMP-9 inhibitor, reduced disease in wild-type mice. These data suggest that targeting MMP-9 for inhibition may improve therapy for HSK (Lee et al., 2002). Rapid improvement of HSV-1induced keratitis also was noted after amniotic membrane transplantation onto the cornea. It was postulated that this was caused by reduced expression of MMP-8 and -9 and increased expression of TIMP-1 (Heiligenhaus et al., 2005). Others have shown that in the virus-infected cornea, interleukin-6 (IL-6) promotes corneal infection by acting in an autocrine-paracrine fashion to induce resident corneal cells to make macrophage inhibitory protein-2 (MIP-2) and macrophage inflammatory protein-1α (MIP-1α/CCL3), which in turn recruit PMNs to the virus infection site in the murine cornea (Fenton et al., 2002). Use of a transgenic mouse that overexpresses the IL-1 receptor antagonist (IL-1ra) protein revealed that these mice were markedly resistant to HSK, compared with IL-1ra knockout and C57BL/6 WT control mice. Resistance was the consequence of reduced expression of molecules such as IL-6 and MIP-2, as well as vascular endothelial growth factor (VEGF) production, normally

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upregulated directly or indirectly by IL-1. These mice also had a marked reduction in angiogenesis, an essential step in HSK pathogenesis (Biswas et al., 2004a). Mice receiving IL-1ra protein also had diminished disease severity (Biswas et al., 2004b). A better understanding of the role of VEGF in induction of angiogenesis and in the pathogenesis of HSK was derived from experiments in which mice were injected with the VEGF inhibitor mFlt(1-3)-immune globulin G. These mice had diminished angiogenesis, and the severity of lesions after HSV infection was less (Zheng et al., 2001). Administration of a cyclooxygenase 2 (COX-2) selective inhibitor also was tested and was found to decrease PMN infiltration into the HSV-infected cornea and diminished corneal VEGF levels, likely accounting for the reduced angiogenic response noted in these mice (Biswas et al., 2005). In another study, application of plasmid DNA encoding IL18 to the cornea of mice before HSV-1 ocular infection also reduced angiogenesis and diminished HSK lesions (Kim et al., 2005). Systemic injection of VEGF pathway-specific silencing (si) RNAs before infection was another confirmation of the role of VEGF in disease, as knocking down the gene for VEGF inhibited ocular angiogenesis induced by HSV (Kim et al., 2004). The absence of the chemokine receptor CCR5, a shared receptor for the beta chemokines CCL3 and CCL5, appeared to play a role in regulating leukocyte trafficking and control of virus burden, but was not critical to prevent mortality after corneal HSV-1 infection (Carr et al., 2006). Findings from mouse studies have also suggested that HSK may be the result of either molecular mimicry or bystander activation phenomenon (Wickham and Carr, 2004). Molecular mimicry has been difficult to prove and is based on the tenet that causative viruses express epitopes that cross-react with a host protein and that the initial immune response to viruses carries over to include antihost reactivity. Bystander activation represents a complex that could include the release of normally sequestered antigens from damaged cells that have become immunogenic, alteration of host protein structure, and the subversion of host cells, causing pro-inflammatory mediator production or the synthesis of abnormal products such as autoantibodies (Wickham and Carr, 2004).

Bacterial infections and keratitis Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that rapidly induces bacterial keratitis, a disease often associated with extended-wear contact lens use (O’Brien and Hazlett, 1996). The host innate immune response to this pathogen includes local PMN recruitment, which is essential to control bacterial replication, bacterial spread, and host survival. Nonetheless, PMN persistence in the cornea is also associated with destructive pathology, including stromal

scarring and perforation, potentially requiring corneal transplantation (Steuhl et al., 1987; Hazlett, 2004). PMN infiltration into inflamed tissue is largely controlled by local production of inflammatory mediators. In the mouse, two members of the C-X-C family of chemokines, MIP-2 (functional homologue of human IL-8) and KC, are potent chemoattractants and activators of PMN. In corneal infections, MIP-2 was shown to be the major chemokine that attracts PMNs into the P. aeruginosa-infected cornea, and the persistence of PMNs in the cornea of susceptible (cornea perforates) C57BL/6 versus resistant BALB/c (no corneal perforation) mice was found to correlate with higher MIP-2 chemokine levels (both mRNA and protein) (Kernacki et al., 2000). IL-1, produced by macrophages, monocytes, and resident corneal cells (Niederkorn et al., 1989; Dinarello, 1996), also influences PMN infiltration into tissues (Dinarello and Wolff, 1993). When tested, levels of IL-1α and IL-1β (mRNA and protein) were elevated in the infected cornea of C57BL/6 (susceptible) over BALB/c (resistant) mice. Neutralization of IL-1β in infected C57BL/6 mice (Rudner et al., 2000) reduced disease severity, as evidenced by a reduction in PMNs in the cornea (MPO assay), a decreased bacterial load, and decreased levels of MIP-2 at both the mRNA and protein levels. The use of caspase-1 inhibitor treatment in C57BL/6 mice confirmed these data, even when inhibitor treatment was initiated after disease onset. In addition, improvement was augmented when the caspase1 inhibitor was given after infection together with the antibiotic ciprofloxacin (Thakur et al., 2004). A live attenuated P. aeruginosa vaccine has also been tested and been found to elicit outer membrane protein-specific active and passive protection against corneal infection (Zaidi et al., 2006). The role of T cells in P. aeruginosa corneal infection was first studied in inbred C57BL/6 WT and CD8+ T-deficient, β2-microglobulin knockout mice (on the C57BL/6 background) (Kwon and Hazlett, 1997). Corneas of both groups of mice perforated by 7 days post infection and the histopathology was similar, with infiltration of PMNs within 24 hours post infection. In contrast, corneas of wild-type mice antibody depleted of CD4+ T cells and infected with P. aeruginosa did not perforate at 7 days post infection, versus mice depleted of CD8+ T cells or treated with an irrelevant antibody. Antibody neutralization of IFN-γ before infecting C57BL/6 mice also prevented corneal perforation and was associated with a lower DTH response when compared with C57BL/6 mice similarly treated with an irrelevant antibody. These data support that a CD4+ T cell (Th1)–dominant response following P. aeruginosa infection is associated with genetic susceptibility and corneal perforation in C57BL/6 mice (Kwon and Hazlett, 1997) and provided the first evidence that CD4+ T cells are important in the development of keratitis. In addition, the use of gene array studies confirmed a Th1 versus Th2 bias of C57BL/6 versus BALB/c

mice to infection with Pseudomonas (Huang and Hazlett, 2003). Other studies investigated whether IL-12 (IL-12 p40) was associated with IFN-γ production and the susceptibility response of C57BL/6 mice after P. aeruginosa challenge. IL12 p40 knockout mice (C57BL/6 background) versus wildtype mice were tested to examine disease progression in the endogenous absence of the cytokine. When tested, both groups of mice were susceptible to corneal challenge with P. aeruginosa, with corneal perforation observed 5–7 days post infection. Semiquantitative RT-PCR and ELISA analyses confirmed that IL-12 p40 message and protein levels were elevated after infection in the wild-type mice over the expected absence of IL-12 p40 in the knockout mouse cornea. Immunostaining for IL-12 in wild-type C57BL/6 mice revealed that stromal PMNs were at least one source of the cytokine (Hazlett et al., 2002). The role of IL-18 and IFN-γ production in the resistance response of the predominantly Th2 responding BALB/c mouse was also tested. Semiquantitative RT-PCR detected IFN-γ mRNA expression levels in the cornea of infected mice at 1–7 days post infection. Cytokine levels were significantly upregulated when compared with control, uninfected normal mouse corneas (Huang et al., 2002). With RT-PCR, IL-18 mRNA expression was detected constitutively in the normal, uninfected cornea, but levels were significantly elevated 1–7 days post infection. To test whether IL-18 regulated IFN-γ production, BALB/c mice were injected with an anti-IL-18 mAb. Treatment decreased corneal IFN-γ mRNA (Huang et al., 2002) levels, and both bacterial load and disease severity increased when compared with IgG-injected control mice. These data provide evidence that IL-18 is critical to the resistance response of BALB/c mice by induction of IFN-γ and that IFN-γ is required for bacterial killing or stasis in the cornea (Hazlett, 2002; Hazlett et al., 2005). Another study showed that the killing effect of IFN-γ was indirect, through regulation of nitric oxide levels (McClellan et al., 2006). Further study of the resistance response in BALB/c mice examined the role of the pro-inflammatory neuropeptide, substance P (SP), in IFN-γ production. Natural killer cells were required to produce IFN-γ; the cells expressed the neurokinin-1 receptor (a major SP receptor) and directly regulated IFN-γ production through interaction with this receptor (Lighvani et al., 2005), suggesting a unique link between the nervous system and the development of innate immunity in the cornea. Staphylococcus aureus is a gram-positive organism that is seen in patients with epithelial defect, especially those on prolonged corticosteroid treatment (O’Brien and Hazlett, 1996). The disease progresses gradually and induces stromal opacity. A mouse model of the disease has been developed (Girgis et al., 2003) that should be usable in a large range of studies that were not feasible in a rabbit keratitis model,

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particularly those requiring a genetically altered host or specific immunologic reagents. It was reported that different strains of mice had disparate cytokine responses correlated with disease severity (Hume et al., 2005). Aged mice also were more susceptible to disease than young mice (Girgis et al., 2004), and bacterial toxins, especially α-toxin, could mediate corneal disease in mice (Girgis et al., 2005). Differences in severity of the keratitis in aged versus young mice correlated with their susceptibility to α-toxin. In the mouse model of S. aureus-induced endophthalmitis (a serious and potentially blinding ocular infection usually associated with recent intraocular surgery), it was shown that Fas ligand, but not complement, is critical for control of the disease (Engelbert and Gilmore, 2005). The mouse has also been used as a model to study conjunctival infection. Immune responses in mice after conjunctival exposure to Chlamydia trachomatis have been examined (Barsoum et al., 1988). Chlamydia was consistently isolated from the conjunctiva and draining lymph node at 1 and 7 days after conjunctival exposure, but by about 5 weeks, blastogenic responsiveness was very low, perhaps reflecting a state of immunosuppression (Barsoum et al., 1988). The introduction of Listeria monocytogenes into the anterior chamber of mice was used to test whether immune privilege (anterior chamber–associated immune deviation, ACAID) of the anterior chamber, resulting in reduced DTH, extended to this bacterial pathogen (Li and Niederkorn, 1997). The results showed that immune privilege is not extended to all foreign antigens that enter the anterior chamber, and as a result, some intraocular antigens can provoke a strong systemic DTH response. Mouse eye infection models have also been used to study the role of Toll receptors in disease. The Toll family of receptors (TLR), conserved throughout evolution from flies to humans, is central in initiating innate immune responses. This family of receptors, composed of transmembrane molecules, links the extracellular compartment where contact and recognition of microbial pathogens occur, and the intracellular compartment, where signaling cascades leading to cellular responses are initiated. Gene array data showed that the expression of TLRs and related molecules, including CD14, soluble IL-1ra, TLR6, and IL-18R-accessory protein, were significantly elevated in susceptible (C57BL/6) versus resistant (BALB/c) mice following challenge with P. aeruginosa (Huang and Hazlett, 2003). In a sterile keratitis model (Khatri et al., 2002), when C3H/HeJ (TLR4 point mutation) and control mice were treated with lipopolysaccharide from P. aeruginosa, a significant increase in stromal thickness and haze was seen in the cornea of control mice but not in TLR4 mutant mice, and the severity of the disease coincided with PMN stromal infiltration. Another study showed that the corneal epithelium has functional TLR2 and TLR9, and that TLR2, TLR4, and TLR9 signal

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through myeloid differentiation factor 88 (MyD88) (Johnson et al., 2005). Recent evidence showed that a single Ig IL-1R related molecule (SIGIRR) is differentially expressed in BALB/c (resistant) compared to B6 (susceptible) mice and that this Toll receptor is critical in resistance to P. aeruginosa infection in BALB/c animals, functioning to downregulate type 1 immunity and negatively regulating IL-1 and TLR4 signaling (Huang et al., 2006). siRNA treatment to knockdown TLR9 was also found to influence bacterial keratitis, leading to reduced inflammation but also decreased bacterial killing (Huang et al., 2005). Modulation of bacterial factors has also been shown to reduce ocular virulence (Zolfaghar et al., 2006; Parks and Hobden, 2005).

Mouse models of fungal infection Compared with bacterial infections, there are relatively few studies on the pathogenesis of oculomycoses, and a complete understanding of appropriate therapy is lacking. A murine model of keratomycosis has been established, despite mice being innately resistant to the fungal pathogen, Candida albicans. Moderate to severe keratomycosis was established using immunocompromised mice and a route of corneal scarification (Wu et al., 2003). A mouse model of corneal fusariosis by infection with the fungal pathogen Fusarium solani has also been established and permits evaluation of fungal infection and pathogenesis. Immunosuppression and surface scarification were also needed for this model to establish infection (Wu et al., 2004).

Mouse models of parasitic infection Parasitic infections of the eye are a leading cause of blindness in many parts of the world. Inflammation of the corneal stroma is a serious complication of infection with the nematode parasite, Onchocerca volvulus (Pearlmann et al., 1996), a cause of river blindness in humans. An essential role for PMNs and eosinophils, as well antibody, has been shown for their recruitment (Pearlman et al., 1998; Hall et al., 1999). Further studies showed that disease is exacerbated in BALB/c IL-4 knockout mice and that both IL-4 and Th2 cells were required for disease development (Pearlman et al., 1995). Use of this mouse model of river blindness in which soluble extracts of filarial nematodes were injected into the corneal stroma also made it possible to demonstrate that the predominant inflammatory response in the cornea was due to a species of endosymbiotic Wolbachia bacteria and that the inflammatory response induced was dependent on Tolllike 4 receptors on host cells (Saint Andre et al., 2002), as well as signaling through MyD88 (Gillette-Ferguson et al., 2006). Critical to infection with another parasite genus, Acanthamoeba, small, free-living protozoa that are widely distrib-

uted in nature, is the ability of the parasite to bind to the corneal surface. Epidemiological studies have shown that contact lens wear is the most significant risk factor, while trauma is the second most significant factor. Poor lens hygiene is frequently encountered in association with tap water rinsing of the lens (O’Brien and Hazlett, 1996). Eleven host species of Acanthamoeba were tested, and parasites failed to bind to or damage intact epithelium in the mouse cornea. Results indicate that A. castellani, the species tested, exercises rigid host specificity at the host cell surface (Niederkorn et al., 1992). Intracorneal inoculation of nude, athymic (lacking T cells) mice with A. hatchetti, however, was capable of inducing keratitis (Paniagua-Crespo et al., 1989).

Mouse models of retinal infection Herpes simplex may be a cause of viral retinitis or encephalitis in immunologically competent and incompetent adults (O’Brien and Hazlett, 1996). The full-thickness necrotizing retinitis presents as diffuse retinal opacification and edema with perivascular inflammation. In AIDS patients, selected rampant cases of cytomegalovirus retinopathy and encephalitis have also been documented to be coinfected with HSV (O’Brien and Hazlett, 1996). Herpetic retinitis in humans is characterized by a high frequency of bilateral localization. The mouse has been used to examine HSV-1 retinitis after anterior chamber inoculation. Modulation of disease by CD4+ T cells was detected in the uninoculated eye (Azumi et al., 1994); natural killer cells prevented direct anteriorposterior spread of virus (Tanigawa et al., 2000), and spread of HSV-2 to the suprachiasmatic nuclei and retina was detected in T cell–depleted mice (Matsubara and Atherton, 1997). The observation that in the inoculated eye, T cells arrive in the sensory retina at the onset of retinal necrosis and not during acute retinitis and the peak of viral replication, suggests that T cells play a role in the development of retinal necrosis; they may also have a role in resolution of the disease, as they were observed as late as 63 days post infection in the uninoculated eye (Azumi and Atherton, 1998). Injection of HSV-1 into the vitreous of BALB/c mice was also used to examine the occurrence of contralateral virus spread, and the data suggested that spread was mediated by local (nonsynaptic) transfer in the optic chiasm from infected to uninjected axons in the optic nerves (Labetoulle et al., 2000). Nectin-1, a member of the immune globulin superfamily, has been shown to be a receptor for HSV, and this molecule was found widely expressed in murine ocular tissues (Valyi-Nagy et al., 2004). Murine cytomegalovirus infections (MCMV) (AIDSrelated in human retinitis) were examined in the mouse, and the virus was found to spread to and replicate in the retina after LPS-induced disruption of the blood-retinal barrier in immunosuppressed mice (Zhang et al., 2005a). Apoptosis in

the retina was detected in uninfected retinal cells in this model (Bigger et al., 2000). The mouse has served to allow detection of ocular reactivation of MCMV after immunosuppression of latently infected mice (Zhang et al., 2005b). Therapy for MCMV used a SCID mouse model, and the efficacy of therapy with cidofovir was successfully monitored with electroretinography (Garneau et al., 2003). MCMV retinitis in the mouse was examined in retrovirus-induced immunodeficiency (MAIDS) in mice, and loss of the perforin cytotoxic pathway appeared to predispose mice to retinitis (Dix et al., 2003); susceptibility also correlated with intraocular levels of TNF-α and IFN-γ (Dix and Cousins, 2004). Murine coronavirus, mouse hepatitis virus (MHV), JHM strain, induces disease in mice, and a genetic predisposition to disease induction of the retina has been shown (Wang et al., 1996). Intraocular coronavirus injection results in a biphasic retinal disease in susceptible BALB/c mice characterized by acute inflammation and retinal degeneration associated with autoimmune reactivity. Resistant CD-1 mice develop only the early phase of the disease (Vinores et al., 2001). Retinal apoptosis may be one of the host mechanisms contributing to limiting this retinal infection (Wang et al., 2000). Toxoplasma gondii, a protozoan parasite, can cause severe, life-threatening disease, especially in newborns and immunosuppressed (e.g., HIV-infected) patients. The active phase of retinal disease is self-limited, but it can recur, and the destructive nature of the infection places patients at risk for blindness if tissues critical for vision, such as the optic nerve or macula, are involved (O’Brien and Hazlett, 1996). Toxoplasmosis, caused by an obligate intracellular parasite, is established in mice by either an intravitreal or an instillation route of infection using T. gondii Me-49 strain. The route of inoculation influences the inflammatory pattern and the instillation route was preferred, as it avoids extensive needle damage in a small animal (Tedesco et al., 2005). The effects of sulfamethoxazole treatment have been assessed using other toxoplasmosis strains in IFN-γ knockout mice. This model allowed the quantitative assessment of treatment effects (Norose et al., 2006).

Summary The mouse provides a valuable animal model for infectious disease studies in the eye. The ability to manipulate genetically based experimental systems because of the availability of numerous inbred strains, the wealth of gene knockout and transgenic animals, and the easy availability of immunological and molecular reagents are only a few of the reasons that this is the case. acknowledgments

Work was supported by NIH grant nos. R01EY02986, R01EY016058, and P30EY04068 and by a grant from Ciba-Vision.

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Mouse Models of Autoimmune and Immune-Mediated Uveitis RACHEL R. CASPI

Uveitis, or inflammation of the uvea, is a generic term that denotes ocular inflammation. The condition may be of infectious or noninfectious origin. Infectious uveitis is discussed in chapter 41. Noninfectious uveitis, the subject of this chapter, was initially referred to as idiopathic but is now known to be of autoimmune or immune system–mediated etiology in many cases. Several human diseases typically characterized by detectable responses to protein antigens found in the retina and uvea fall in this category (Body et al., 2001; Nussenblatt and Whitcup, 2004). Some of these diseases are part of a generalized systemic syndrome in which the eye is only one of the organs involved. Examples of such diseases are anterior uveitis associated with juvenile rheumatoid arthritis or with ankylosing spondylitis (HLA-B27 related), and uveitides involving the posterior pole, such as Behçet’s disease, VogtKoyanagi-Harada disease, and ocular sarcoidosis. Other types of uveitis target primarily the eye, with no signs of disease observed in other organs. Examples are sympathetic ophthalmia and birdshot retinochoroidopathy. Traditionally, the rat (particularly the Lewis strain) served as a model for these diseases, but in the past two decades, mouse models of anterior and posterior uveitis have been developed and have given a tremendous push to basic studies of disease mechanisms. Models of uveitis can be divided into autoimmune, where a particular self antigen is known to be the target of an immunological attack, as opposed to immunemediated, where inflammation is not driven by a response to a particular antigen. Autoimmune models can be further subdivided into induced models, with disease being precipitated by immunization with a retinal or uveal protein, and “spontaneous,” which occur in genetically or surgically altered animals that have not, however, been actively immunized. A graphical representation of mechanistic relationships in rodent models of uveitis is shown in figure 42.1. The various uveitis models are reviewed by Caspi (2006a). This chapter concentrates on mouse models of experimental autoimmune uveitis (uveoretinitis) (EAU), induced and spontaneous, and also mentions endotoxin-induced uveitis (EIU), an important nonautoimmune model of anterior segment inflammation. Although there are differences in the structure and composition of the retina between human and mouse, in the case

of immune-mediated uveitis, anatomical interspecies differences are thought to be secondary. Of primary importance is the relationship between the eye and the immune system, which is believed to be similar in both species. This permits extrapolation from mouse models to the human in terms of immunological mechanisms involved in the pathogenesis and resolution of ocular autoimmune and inflammatory disease. The following discussion addresses the rapidly expanding uveitis research exploiting mouse models, its contribution to the understanding of fundamental mechanisms driving the disease, and the translational potential of using mouse models in terms of approaches to therapy.

Endotoxin-induced uveitis: Nonautoimmune anterior uveitis Endotoxin-induced uveitis (EIU) is a model of anterior uveitis induced by peripheral injection of bacterial endotoxin (lipopolysaccharide, LPS), with primary involvement of the innate immune response elements, such as resident ocular macrophages that secrete cytokines and chemokines (Smith et al., 1998). EIU, initially developed in rats (Rosenbaum et al., 1980), was first reported in endotoxin-sensitive C3H/HeN mice by Kogiso et al. in 1992, and subsequently C3H/SW and FVB/N were also found to be susceptible (Li et al., 1995). EIU typically develops and peaks within 24 hours of a peripheral injection of endotoxin. A second peak of disease has been described 5 days after the first (Kozhich et al., 2000). Although the EIU model is very robust, its limitation is that it may be “a model in search of a disease.” Namely, there is some debate over which human disease it should represent. There may be parallels to anterior uveitis without systemic disease. It has also been used to represent anterior uveitis associated with rheumatic disease; however, LPS uveitis is of short duration and self-limiting, unlike anterior uveitis associated with rheumatic syndromes. A human equivalent to EIU that is not usually considered may be what is known as toxic anterior chamber syndrome, a sterile inflammation seen occasionally after intraocular surgery (e.g., cataract extraction) that is usually transient and is

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Figure 42.1 Inflammatory and autoimmune animal models of uveitis. Except as noted, all models are induced (endotoxin or immunization with the specified antigen). , disease inducible in Lewis rats; , disease inducible in both mice and rats. BSA, bovine serum albumin; EAAU, experimental autoimmune anterior

uveitis; EAPU, experimental autoimmune posterior uveitis; EIU, endotoxin-induced uveitis; EMIU, experimental melanin-protein induced uveitis; RAU, recurrent anterior uveitis; VKH, VogtKoyanagi-Harada syndrome.

Figure 42.2 Typical histology of EIU and EAU in the mouse. Healthy compared to diseased eye tissue in EIU (A and B) and in EAU (C and D). A, Healthy anterior chamber. Shown is the angle where the iris and ciliary body connect to the sclera. C, cornea; CB, ciliary body; I, iris; S, sclera. B, EIU in a C3H mouse induced by subcutaneous injection of LPS. Note infiltration of inflammatory cells in the angle and around the iris and ciliary processes. Original magnification ×1200. C, Healthy mouse retina.

C, choroid; G, ganglion cell layer; P, photoreceptor cell layer; R, retinal pigment epithelium; S, sclera; V, vitreous. D, EAU in the B10.RIII mouse induced by immunization with IRBP. Note structural disorganization and loss of nuclei in the ganglion and photoreceptor cell layers, retinal folds, subretinal exudate, vasculitis, damage to the retinal pigment epithelium, and choroid inflammation. Original magnification ×1000. See color plate 39. (Photomicrographs courtesy of Dr. Chi-Chao Chan, NEI, NIH.)

attributed to improper composition of surgical solutions (nonphysiologic or containing an irritant) or inadequate sterilization (endotoxin contamination) of solutions and instruments (Mamalis et al., 2006; Holland et al., 2007). It seems that a variety of noxious stimuli, not just LPS, can precipitate this syndrome. The release of pro-inflammatory cytokines could conceivably be triggered by a variety of noxious stimuli that cause tissue damage and constitute endogenous “danger”

signals. In this scenario, endotoxin would be a model of a spectrum of causes having in common the triggering of local cytokine and chemokine release from innate immune and resident ocular cells. Irrespective of its exact human parallel, EIU continues to provide a useful model for the study of mechanisms of inflammation and inflammatory cell recruitment into the eye. Typical histological appearance of EIU in a C3H mouse can be seen in figure 42.2B.

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A limitation to using the mouse as a model of anterior uveitis is that no autoimmune model of anterior uveitis in mice has yet been developed. Experimental autoimmune anterior uveitis (EAAU) or experimental melanin-induced uveitis (EMIU), two closely related models of anterior uveitis induced by melanin-associated antigens, so far are inducible only in rats. EAAU/EMIU is felt to be a more relevant model of human anterior uveitis than EIU because of its autoimmune etiology and recurrent nature (Smith et al., 1998). A robust model of mouse anterior uveitis on an autoimmune basis is needed.

Experimental autoimmune uveoretinitis and related models EAU is autoimmune in nature and is thought to be a reasonably accurate representation of those (mainly posterior) uveitic diseases in which patients exhibit responses to retinal or uveal antigens. Although no single animal model can reproduce the complexity of human uveitis, there are now a number of EAU variants that may be useful for studying distinct aspects of uveitis or that provide unique insights into basic mechanisms of pathogenesis. The target antigen in EAU is usually a retinal antigen; therefore, it manifests as a uveoretinitis. However, the terms uveitis and uveoretinitis are used here interchangeably. Experimental Autoimmune Uveoretinitis Induced by Active Immunization with Retinal Antigen Most patients who respond to retinal antigen, respond to a limited number of fragments (epitopes) of the retinal soluble antigen (S-Ag, retinal arrestin), although in advanced disease, additional specificities may be recognized (de Smet et al., 2001). This may suggest that S-Ag has a primary involvement and that responses to other antigens are secondary, resulting from autoimmunization to tissue breakdown products. EAU can be elicited in rats by immunization with S-Ag injected in emulsion with complete Freund’s adjuvant (CFA) and has served for many years as a model of human uveitis. Unfortunately, and not for lack of trying by many laboratories, immunization with S-Ag has failed to reliably induce EAU in the commonly available strains of laboratory mice. A compounding difficulty was that many strains of laboratory mice carry the rd gene and have no photoreceptor cells by weaning time, eliminating them from evaluation. Among these are strains such as SJL/J and PL/J, which are susceptible to related autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE). In 1987 it was found that another retinal antigen, interphotoreceptor retinoid-binding protein (IRBP), is uveitogenic in rats (Gery et al., 1986), and subsequently we found that it also induces uveitis in several strains of mice (Caspi et al., 1988, 1990). As in rats, EAU in mice is induced by active immunization with IRBP in CFA, and a coinjection of

pertussis toxin (PT) as additional adjuvant is usually needed. An exception is the B10.RIII strain (Jax strain B10.RIII-H2r H2-T18b/(71NS)SnJ; stock no. 000457), the most susceptible mouse strain currently known, which develops severe EAU after immunization with IRBP in CFA without the need for PT (Silver et al., 1999). Antibodies are produced as a result of immunization and have an amplifying role (Pennesi et al., 2003). Typical pathology of EAU in a B10.RIII mouse can be seen in figure 42.2D. Because it is induced by immunization with a soluble protein, the EAU model as described earlier is MHC class II restricted; that is, the antigenic fragments of IRBP are presented by class II (mouse I-A and/or I-E) molecules and recognized by CD4+ T lymphocytes. The uveitogenic fragments of IRBP that induce disease in mice of the H-2r, H-2b, and H-2a haplotypes have been identified, and disease can be induced by immunization with the synthetic peptides or by transfer of cells from donors immunized with the peptides, obviating the need to purify IRBP from natural sources or to produce it recombinantly. Table 42.1 summarizes the uveitogenic fragments of IRBP defined to date. Experimental Autoimmune Uveoretinitis Induced by Adoptive Transfer of Antigen-Specific T Cells EAU is a cell-mediated response with central involvement of T lymphocytes with specificity for the immunizing retinal antigen. The full-blown disease can be induced by transferring T cells from donors immunized for induction of uveitis to genetically compatible recipients (Tarrant et al., 1998). Antigen-specific T cells can be propagated in vitro as longterm antigen-specific T cell lines (Silver et al., 1995). The adoptively transferred model is useful to represent the efferent (effector) phase of disease without any confounding effects of a continuing induction (priming) phase or effects of CFA. Experimental Autoimmune Uveoretinitis in Mice Expressing a Transgenic Target Antigen in the Retina A variation on the theme of traditional EAU is the use of transgenic mice that are genetically engineered to express a foreign protein under control of a retina-specific promoter. Mice have been created that express β-galactosidase (β-gal) under the arrestin promoter, or that express hen egg lysozyme (HEL) under the rhodopsin promoter (Gregerson et al., 1999; Ham et al., 2004). This neo-self antigen can serve as an antigenic target for uveitis induced by active immunization or by adoptive transfer of the specific T cells, similarly to the native target IRBP. The advantages of such neo-antigendriven EAU models are that they can permit more sophisticated approaches and questions that cannot be addressed in the more traditional EAU models, for example, by combining use of such mice with mice expressing a transgenic T cell receptor (TCR) specific for the retinal

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Strain B10.RIII

Class II Haplotype r

C57BL/6

b

B10.A, B10.BR

k

Table 42.1 Uveitogenic peptides of IRBP Peptide Position and Amino Acid Sequence 51–70: QTLASVLTAGVQSSLNDPRL 161–180: SGIPYIISYLHPGNTILHVD 541–560: SLGWATLVGEITAGNLLHTR 1–20: GPTHLFQPSLVLDMAKVLLD 461–480: LRHNPGGPSSAVPLLLSYFQ 651–670: LAQGAYRTAVDLESLASQLT 681–700: RLLVFHSPGELWEEAPPPP 201–216: ADKDVVVLTSSRTGGV

neo-self antigen. A disadvantage of the use of neo-self antigen transgenic models is that expression of transgenic antigens is subject to integration effects, potentially resulting in unexpected expression patterns and artifacts. Transgenic mice expressing IRBP-specific TCRs are under development (Caspi et al., 2008). Experimental Autoimmune Uveoretinitis Induced by Injection of Antigen-Pulsed Dendritic Cells An alternative model of IRBP-EAU that has recently been developed is elicited by adoptive transfer of in vitro matured dendritic cells (DCs) pulsed with the uveitogenic 161–180 peptide of IRBP (Tang et al., 2007). DCs have been identified as the most important antigen-presenting cells and are believed to be the only antigen-presenting cells that prime naive T cells that respond to their antigen for the first time. Understanding the requirements to prime a T cell for effector function versus regulatory function is of paramount importance. It is of interest that the disease induced by matured and antigenpulsed DCs has a different clinical course and appearance and is also associated with a different type of effector response. The limitation of this model is the requirement for large numbers of DCs that have to be administered twice to elicit good disease scores (Tang et al., 2007). Its special advantage is that it provides an alternative model of EAU with unique characteristics and permits direct manipulation of DCs to study requirements for uveitogenic antigen presentation and possibly for tolerogenic presentation to already primed cells. “Humanized” Experimental Autoimmune Uveoretinitis Model in Human Lymphocyte Antigen Transgenic Mice Although animal models can teach us about pathogenesis of disease, they cannot serve as templates for dissecting the antigenic specificity of the response, for the simple reason that they recognize different antigens, and different regions in the same antigens. This necessarily follows from the fact that their MHC molecules, which bind and present antigenic fragments to T lymphocytes, are different from those of humans. To address this issue, a number of laboratories have generated genetically engineered

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Reference Cortes et al. (in press) Silver et al., 1995 Cortes et al. (in press) Avichezer et al., 2000 Cortes et al. (in press) Cortes et al. (in press) Cortes et al. (in press) Namba et al., 1998

mice that transgenically express human MHC molecules (human lymphocyte antigen, HLA) in lieu of mouse MHC molecules (known as H-2). We examined susceptibility to EAU of single-transgenic mice expressing HLA-DR3, HLADR2, HLA-DQ6, or HLA-DQ8 (Pennesi et al., 2003). The HLA-Tg mice, similarly to wild-type mice, developed EAU after immunization with IRBP. Interestingly, HLA-DR3 Tg mice, and to a lesser extent other HLA transgenic mice, developed EAU with S-Ag, to which the parental wild-type strains are resistant. As mentioned earlier, S-Ag is the retinal antigen to which human patients most often exhibit lymphocyte responses. Interestingly, double-transgenic mice carrying a DR and a DQ molecule, which more closely mimics the situation in humans, exhibited changes in epitope recognition and enhanced susceptibility compared to the single transgenic counterparts (Mattapallil and Caspi, 2008). Thus, the “humanized” EAU model may help to identify the antigenic molecules and their critical regions that might be involved in driving human uveitis, and can help dissect the epistatic effects of HLA antigens on disease susceptibility and epitope recognition. Spontaneous Experimental Autoimmune Uveoretinitis– Like Models of Uveitis Several models of spontaneous uveitis have been described. They differ from the induced models by not requiring active immunization or transfer of immune cells to trigger the disease, although they develop only in mice that have been genetically or surgically manipulated. In humans, a type of uveitis known as birdshot retinochoroidopathy is highly associated with HLA-A29. Relative risks between 50 and 249 have been reported in different ethnic groups in various studies (Nussenblatt and Whitcup, 2004). Szpak et al. (2001) made an HLA-A29 Tg mouse and found that it spontaneously develops a posterior uveitis that closely resembles birdshot retinochoroidopathy. The target antigen has not been identified, but, owing to association with HLA-A29, an MHC class I molecule, it is expected to be an epitope(s) recognized by CD8+ effector T cells. Two other spontaneous models, although developing in a very different way, probably share an underlying etiology,

namely, spontaneous development of responsiveness to IRBP. (1) Ichikawa and collaborators (1991) reported that nude mice implanted with a neonatal rat thymus, which are known to develop a variety of autoimmune diseases, also develop spontaneous uveitis accompanied by humoral and cellular responses to IRBP. (2) A similar situation exists in mice deficient in the gene encoding the AIRE protein (AutoImmune REgulator). AIRE is a molecule that controls ectopic expression of tissue antigens in the thymus, including several retinal antigens. As they age, AIRE knockout mice develop spontaneous uveitis also associated with responses to IRBP (Anderson et al., 2002; Devoss et al., 2006). The insights into basic mechanisms of uveitic disease gained from these models are discussed later in the chapter. Uveitis Associated with Antitumor Response to Melanoma Antigen Gp100 (Si) is an antigen associated with melanin pigment that serves as a vaccination target for immunotherapy of melanoma. The antitumor effector cells induced by the vaccination regimen are CD8+ cytotoxic lymphocytes specific to gp100. Successful eradication of experimental melanoma in mouse models as well as in ongoing clinical trials of human melanoma immunotherapy is typically associated with autoimmune vitiligo (Kawakami and Rosenberg, 1997; Overwijk et al., 2003). It has recently been appreciated that these mice, in addition to generalized vitiligo, also develop a uveitis that can be quite severe (D. Palmer and N. P. Restifo, pers. comm., 2007). Of note, some melanoma patients given cellular immunotherapy targeting

Figure 42.3 Critical checkpoints in EAU pathogenesis. Schematic representation of critical events and checkpoints in the pathogenesis of EAU as revealed by studies in mouse models. See

gp100 also were observed to develop uveitis (Robinson et al., 2004). This condition is reminiscent of Vogt-KoyanagiHarada disease, in which patients develop vitiligo and uveitis and exhibit responses to melanin-related antigens (Boyd et al., 2001).

Basic mechanisms involved in uveitic disease as revealed by mouse models Over the years, the various EAU models have provided invaluable insights into the fundamental mechanisms involved in acquisition of self tolerance to ocular antigens, its breakdown, the subsequent events that result in pathology, and the processes involved in resolution (figure 42.3). Based on combined evidence from many studies, the following discussion presents a putative scenario of the pathogenesis of uveitis. In a nutshell, retina-specific T cells that escape negative selection in the thymus cannot be efficiently tolerized in the periphery, owing to relative inaccessibility of retinal antigens, thus leaving a circulating pool of potentially uveitogenic T cells. “Natural” thymic-derived T-regulatory cells keep them under control, but upon encountering antigen-specific signals in the context of innate immune stimuli (“danger” signals), they can differentiate to an autoaggressive Th1 or Th17 effector phenotype. When they reach the eye and recognize their specific antigen there, they initiate a cascade of recruitment and amplification that culminates in characteristic EAU. Ocular antigens released from inflamed tissue subsequently induce antigen-specific

color plate 40. (This figure was previously published in Caspi, 2006. Copyright does not apply.)

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T-regulatory cells in a process requiring the spleen, which helps to reestablish homeostasis and limits recurrence of disease (reviewed in Caspi 2006b). Susceptibility to Experimental Autoimmune Uveoretinitis Starts from Defects in T Cell Repertoire Selection Self tolerance to tissue antigens, including uveitogenic retinal antigens such as S-Ag and IRBP, is first established through thymic selection (Kyewski and Klein, 2006). Lymphocytes passing through the thymus during their natural maturation process are selected for survival or death according to their ability to recognize self molecules. Many if not most tissue-specific antigens, including the retinal antigens S-Ag and IRBP, are expressed by specialized thymic epithelial cells. Immature lymphocytes that fail to recognize any self antigens undergo death “by neglect”; lymphocytes that recognize self with low affinity are selected for survival (positive selection), and lymphocytes possessing highaffinity antigen receptors for a self antigen are eliminated or tolerized (negative selection). Egwuagu et al. (1997) demonstrated that mice express relatively high levels of S-Ag in the thymus but low levels of IRBP, which correlates with their relative resistance to uveitis induced by S-Ag and susceptibility to IRBP-induced EAU. The lowest expression of IRBP was seen in B10.RIII mice, the mouse strain most susceptible to IRBP-induced EAU. These findings support the notion that inefficient thymic elimination of uveitogenic cells due to low expression of the selecting antigen predisposes to uveitis. (However, this does not explain the susceptibility to S-Ag-EAU of HLA-DR3 transgenic mice, unless their thymic expression of S-Ag is in some way altered by expression of human, instead of mouse, class II MHC molecules!) Direct evidence that insufficient thymic elimination of retinal antigen-specific cells predisposes to autoimmune uveitis comes from the observation that T cells originating from IRBP-deficient mice are much more highly uveitogenic than T cells from wild-type mice, suggesting that a repertoire that has not been properly depleted of IRBP-reactive T cells indeed predisposes to autoimmunity (Avichezer et al., 2003). The two models of spontaneous uveitis described in the previous section lend further support to this notion (it should be noted that these models develop also other tissue-specific autoimmune manifestations, which are disregarded for the purposes of this discussion). Antiretina antibodies and cellular responses develop in mice deficient in the AIRE molecule, a transcription factor controlling ectopic expression of tissue antigens in the thymus. These mice fail to express retinal antigens in the thymus, and therefore, similarly to IRBP knockouts, cannot cull their lymphocyte repertoire of high-affinity uveitogenic T cells (Anderson et al., 2002; Devoss et al., 2006). This is reminiscent of the spontaneous uveitis that develops in athymic (nude) mice implanted

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with neonatal rat thymus. Although there is proper expression of retinal antigens in the thymus, they apparently cannot be presented to the mouse T cells because of an incompatibility between the participating thymic elements of the donor and the immune system of the recipient (Ichikawa et al., 1991). Of note, in both these spontaneous models the antiretina response that develops targets IRBP, even though negative selection fails to other retinal antigens as well. Even in the absence of IRBP (double knockouts for AIRE and for IRBP), no other retinal antigens are recognized as surrogate targets for autoimmunity. This supports the concept of a speciesspecific primary autoantigen, which may help to explain why S-Ag is by far the most frequent antigen recognized by uveitis patients. The thymus also selects “natural” regulatory T cells, which characteristically coexpress the molecules CD4 and CD25 (IL-2Rα chain, gene symbol Il2ra), which have a role in raising the threshold of susceptibility to EAU by keeping the potentially uveitogenic T cells in check (Avichezer et al., 2003; Grajewski et al., 2006). Interestingly, although IRBP expression in the thymus is required to select natural Tregulatory cells specific to IRBP, “polyclonal” T-regulatory cells specific to other antigens can also be harnessed to inhibit induction of uveitogenic effector T cells by innate stimuli (Grajewski et al., 2006), which might overlap with the innate stimuli necessary to trigger the induction of effector T cells. Deficient Peripheral Tolerance as a Factor in Susceptibility to Experimental Autoimmune Uveoretinitis Negative selection in the thymus is never fully efficient and, as discussed previously, is affected by the quantitative aspects of self-antigen expression in the thymus. Potentially autoreactive T cells that exit the thymus into the periphery normally have a “second chance” at tolerance as they recirculate through the tissues and encounter self antigens under noninflammatory conditions. However, unlike other organs, the eye becomes separated from the immune system early in ontogeny by an efficient blood-organ barrier that restricts free entry of lymphocytes into the eye, and also limits free exit of antigenic molecules from the eye, especially from the posterior segment. This separation from the immune system results in a circulating pool of uveitogenic lymphocytes that are not truly tolerant but merely “ignorant” of retinal antigens, and that can potentially be triggered into action by an (in)appropriate stimulus. The notion that inadequate peripheral tolerance contributes to susceptibility is borne out by the finding that if these circulating, potentially uveitogenic lymphocytes are tolerized by expressing the uveitogenic retinal antigen outside of the eye, thus making it accessible to peripheral tolerance mechanisms, such mice become profoundly resistant to

EAU. Mice transgenic for β-gal on the S-Ag (arrestin) promoter express β-gal as a neo-self antigen in the retina, where it can serve as a target for EAU. Mice that also express β-gal in the periphery (ROSA26, JAX strain ID 004847) are tolerant and resist EAU induction by immunization with β-gal (Gregerson et al., 1999). In adult animals, infusion of genetically compatible B cells retrovirally transduced to express a uveitogenic fragment of IRBP also results in resistance to EAU (Agarwal et al., 2000). Peripheral tolerance thus constitutes a “weak link” in homeostasis of tolerance to retinal antigens, which might be successfully targeted therapeutically once the inciting retinal antigens in humans are positively identified. Antigen Presentation to Uveitogenic Lymphocytes: What, Where, and by Whom? It is believed that the triggering event for uveitis is exposure of the circulating “ignorant” retina-specific T cells to their cognate antigen. A prototypic autoimmune disease of the eye that is believed to be triggered in this way is sympathetic ophthalmia, where a penetrating wound to one eye is followed after weeks or months by a destructive inflammation in the contralateral, “sympathizing” eye, apparently as a result of autoimmunization to retinal or uveal proteins (Boyd et al., 2001). However, for most types of autoimmune uveitis an exposure to antigen originating from the eye cannot be demonstrated, and it is believed that cross-reactive microbial antigens may provide such a stimulus through antigenic mimicry. A number of antigenic substances have been identified that cross-react with retinal S-Ag and induce EAU in rats, supporting the antigenic mimicry hypothesis (Wildner and Diedrichs-Mohring, 2004), but there are no reports as yet of molecular mimics that induce EAU in mice. Where does the exposure to retinal antigens occur? The retina resides behind an efficient blood-organ barrier that restricts passage of molecules and of cells. Those lymphocytes that do enter the eye encounter a hostile environment composed of a variety of cell-bound and soluble inhibitory factors, as well as a paucity of professional antigen-presenting cells (APCs). Therefore, it is unlikely that priming of errant lymphocytes in the eye could occur and be a cause of uveitis. Rather, it is believed that lymphocyte priming takes place in the periphery, that is, in the lymph nodes, likely in conjunction with an adjuvant effect provided by a concomitant infection. Although the inside of the healthy eye lacks lymphatic drainage, antigen from an injured eye escapes into the subconjunctival, scleral, and periocular space, which is drained by the submandibular and cervical lymph nodes. In support of this notion, Camelo et al. (2006) demonstrated that injected antigen from the anterior chamber of the eye initially travels in a soluble form to secondary lymphoid organs via lymphatic and vascular routes. A microbial mimic antigen encountered through a wound or infection else-

where in the body would drain to the relevant local lymph node, and would in addition provide its own adjuvant effect. Who are the antigen-presenting cells? In order to present antigen for acquisition of effector function, the APC must express appropriate costimulatory molecules, which are induced by microbial or endogenous “danger” signals. Hence the need for adjuvant, whose role is discussed in more detail in the next section. In the draining lymph nodes, resident and migrating DCs serve as APCs to naive T cells, and the ability to induce EAU with antigen-pulsed DCs (Tang et al., 2007) is in line with this being a mechanism of priming in EAU. However, experimental evidence indicates that although the uveitogenic cells have been primed in the periphery, they must recognize their antigen within the eye for EAU to be induced (Prendergast et al., 1998; Chen et al., 2004). Although in the healthy eye there are few or no functional APCs in the retina, it is conceivable that those initial effector T cells that infiltrate the eye might induce a microenvironment through local cytokine secretion that would result in “arming” of resident cells such as microglia to acquire APC function. However, resident APCs may not be essential in the case of peripherally primed T cells, as APCs recruited from the circulation are sufficient to support EAU induction with activated retina-specific T cells (Gregerson and Kawashima, 2004). Interestingly, and arguably of functional relevance to induction of uveitis, retinal antigens may facilitate their own presentation to uveitogenic T and B cells. We recently reported that both S-Ag and IRBP induce migration of immature DCs, as well as T and B lymphocytes, by binding to the chemotactic receptors CXCR3 and CXCR5 (Howard et al., 2005). This would accomplish a dual role, on one hand attracting immunocompetent cells to the site where antigen is present, and on the other hand promoting antigen presentation, as association of antigens with cells’ surface receptors strongly enhances their uptake by APCs and their immunogenicity (Biragyn et al., 2004; Bonifaz et al., 2004). Innate Immune Signals Trigger Autoimmunity: Toll Receptors and Other Danger Signals EAU is elicited by immunization with the retinal antigen in emulsion with CFA. The role of the adjuvant, through its mycobacterial component, is to provide innate immune danger signals to DCs, which then become activated and direct the ensuing adaptive T cell response toward a pro-inflammatory, tissuedestructive, effector phenotype. In the past decade much attention was focused on Toll-like receptors (TLRs) that recognize conserved pathogen-associated molecular patterns (PAMPs) and activate cells of the innate immune system, including macrophages, DCs, and granulocytes, although TLRs are by no means the only receptors that activate

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innate immune responses. The antigen-pulsed DCs used to elicit EAU in the model described earlier must be activated (“matured”) in vitro using LPS (a TLR4 agonist) and at the same time crosslinking the CD40 molecules (costimulatory tnfsf5) by a monoclonal antibody, to acquire the ability to prime uveitogenic T cells for effector function (Tang et al., 2007). Antigen-pulsed DCs that have not been matured prime retinal antigen-specific T cells into a nonpathogenic, or even overtly regulatory, phenotype, and protect mice from a uveitogenic challenge with IRBP in CFA, presumably through induction of regulatory T cells (Jiang et al., 2003; Siepmann et al., 2007). It is currently not clear whether such immature DCs could convert to regulatory function T cells that had already acquired effector function, or whether the T-regulatory cells they induce are capable of inhibiting already primed effector cells, which would be important from the clinical point of view. CFA provides its innate stimulatory effect not only through TLRs. The extent of dependence of EAU induction on TLR stimulation currently appears somewhat contradictory. In a transgenic uveitis model using mice that express HEL in their retina and receive an infusion of TCR transgenic T cells that express a HEL-specific TCR, coinjection of HEL with any one of several TLR ligands is sufficient to induce EAU (Fujimoto et al., 2006). Interestingly, PT, which in addition to being a TLR4 ligand also has other biological activities, was superior to “pure” TLR ligands in supporting induction of disease. In some situations, TLR signaling may be redundant with other innate signals. For example, in EAU induced with IRBP in CFA, single knockout mice deficient in TLR4, TLR9 or TLR2 (as well as double knockout mice lacking any two of these receptors) remain fully susceptible (Su et al., 2005). However, abrogation of IL-1 signaling (which, like the TLRs, also uses the MyD88 signaling pathway) prevented induction of EAU, indicating that whereas IL-1 signaling is necessary and nonredundant for effector T cell generation in EAU, signaling through TLRs may be replaced by other innate stimuli. Recent data indicate that innate signaling is also important in activating natural T-regulatory cells (nT-regs) that protect from EAU. nT-regs can be activated by mycobacterial components present in CFA (Grajewski et al., 2006) and limit induction of uveitogenic effector T cells, though it is obviously the latter that “win out” when EAU is induced. Preliminary data implicate TLR signaling in nT-reg activation (Grajewski, 2006), but it is currently not clear whether the effect is direct or indirect. Pathogenic Effectors: Th1 or Th17? Retinal antigenspecific T cells that have encountered a uveitogenic stimulus acquire a pro-inflammatory, tissue-destructive effector phenotype. This depends strictly on the presence of the innate danger signals discussed earlier, many of which are

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still probably unidentified, that induce appropriate cytokine production and costimulatory molecule expression on APCs. It has long been known that Th1-like cells, which are induced in the presence of IL-12 and whose hallmark is the production of IFN-γ, are a pathogenic effector phenotype (Xu et al., 1997). EAU can be transferred with a few million primary IRBP-specific IFN-γ-producing effector T cells, or less than a million cells from a polarized IRBP-specific Th1 cell line (Silver et al., 1999). IRBP-specific T cells that do not produce IFN-γ and are not pathogenic can be converted in the presence of IL-12 into a pathogenic, IFN-γ-producing phenotype (Tarrant et al., 1998). Recently a new effector phenotype has been described and shown to be centrally involved in several autoimmune and inflammatory diseases. Its induction and maintenance depend, respectively, on TGF-β + IL-6 and on IL-23, and its hallmark cytokine is IL-17 (Bowman et al., 2006). Some studies have gone so far as to suggest that it is the IL-17producing “Th17” effectors that are the main players in tissue destructive autoimmunity, and that Th1 cells play at best only a minor role. However, recent data in the EAU model support a distinct role for the Th1 effector phenotype. In murine EAU, both Th1 and Th17 antigen-specific effectors are induced in mice by uveitogenic immunization with IRBP. Studies aimed at elucidating their relative roles suggest that both participate in pathogenesis, and conceivably complement each other. On one hand, a small number of cells from an IRBP-specific Th1 cell line, which is unable to produce IL-17, elicits severe EAU. This speaks strongly in favor of an important role for Th1 effector T cells. On the other hand, polarized Th17 cell lines unable to produce IFN-γ elicit EAU, and neutralization of IL-17 in IRBP/ CFA-immunized hosts aborts EAU even when started 7 days after immunization, implicating IL-17 as an effector cytokine (Luger et al., in press). The question of whether IFN-γ is an effector cytokine or just a hallmark of Th1 cells is more complex to answer, as neutralization of IFN-γ in immunized mice exacerbates EAU, and IFN-γ knockout mice develop severe disease. This appears to be due at least in part to inhibition of Th1 effector cell generation by a feedback effect of IFN-γ in a process involving induction of nitric oxide and apoptosis (Tarrant et al., 1999). However, when EAU is induced by adoptive transfer of polarized IFN-γ-producing Th1 effector lymphocytes, treatment with anti-IFN-γ inhibits disease, suggesting that IFN-γ does have an effector function(s) (Luger et al., in press). Additionally, antigenpulsed mature DCs from wild-type mice fail to induce EAU in IFN-γ knockout recipients, even though these mice produce large amounts of IL-17, suggesting that an IFN-γproducing antigen-specific effector is important (Tang et al., 2007). IFN-γ is thus a double-edged sword, and its effects on disease depend on where and when it is produced.

Effector Cell Migration, Infiltration, and Inflammatory Cell Recruitment Activated antigen-specific T cells that had been primed in peripheral lymphoid tissues must migrate and find their way to the eye if uveitis is to occur. Although often we refer to this as “homing” to the eye, the term is probably not correct, as it is unlikely that immune cells are able to detect retinal antigens on the other side of the blood-retinal barrier in a healthy, unmanipulated eye (Gregerson et al., 1999). It is likely that extravasation of antigen-specific T cells primed elsewhere into the eye occurs at random (Prendergast et al., 1998; Caspi, 2006b). Because of the small size of the retina it is possible to retrieve essentially all the cells that have entered the target tissue. By a simple calculation of how many cells were injected versus how many can be retrieved from the retina it appears that only a few such cells (fewer than 15) are needed to trigger the sequence of events leading to EAU (Caspi, 2006b). Release of inflammatory mediators by these initial cells “activates” the vascular endothelium of the retina and uvea, promoting adhesion of passing cells and facilitating recruitment of all types of leukocytes from the circulation. Since the great majority of the infiltrating cells found in a uveitic eye are the recruited leukocytes (Pennesi et al., 2003; Chen et al., 2006), it is clear that this recruitment provides a massive amplification mechanism. When antigen-specific donor T cells are transferred into a naive host with a healthy eye, there is a period of several days after the initial antigen-specific donor T cells enter the retina when nothing much seems to happen in the eye. However, changes undetectable by current methods must be taking place in the local microenvironment, including changes in the blood-retinal barrier and the production of chemotactic substances in the target tissue, that will facilitate subsequent massive recruitment of leukocytes from the periphery. This is evident from the finding that only transfer of retinal antigen-specific T cells, but not of activated T cells that do not recognize retinal antigens, is followed by a massive entry of additional donor T cells and of recruited host cells into the eye on the fourth day after transfer (Prendergast et al., 1998; Chen et al., 2004, 2006). Studies in a transgenic EAU model showed that during this seemingly quiescent period, the bulk of the uveitogenic T cells accumulate in the recipient’s spleen, where they proliferate and activate nonspecific host CD4+ T cells. They also undergo (and induce) changes in chemokine and adhesion molecule expression, which culminates on day 4 in a mass emigration of donor and host cells from the spleen to the eye and EAU induction (Chen et al., 2004, 2006). Owing to the transparency of ocular media, the eye offers special advantages as a model for analyzing cellular interactions, leukocyte flow, adhesion, and extravasation of cells by intravital microscopy. Unlike in other tissues, intravital microscopy in the eye does not require invasive procedures,

which by themselves may modify cellular interactions and behavior. Thus, it is possible to easily visualize sticking, rolling, and extravasation of leukocytes, and to track the T cell infiltration into the eye and the cells’ interactions with putative APCs there (Rosenbaum et al., 2002). Similar techniques are being applied to the human eye and should yield important information about the pathogenic processes in human uveitis. Natural Resolution Mechanisms In humans, uveitis can remit spontaneously even without treatment, and the disease in animal models is self-limiting without necessarily proceeding to destruction of the entire retina and elimination of the source of inciting antigen. The mechanisms that bring about remission are not completely understood. Unraveling natural resolution mechanisms of disease can give us clues to the types of processes that we would wish to promote via immunotherapy. An important mechanism that is being actively studied in mouse models is regulatory T cells. Several types of T-regulatory cells related to uveitis have been described (Stein-Streilein and Taylor, 2007), but their origin and the relationships between them remain unclear. As mentioned earlier, CD4+CD25+ thymic-derived nT-regs protect from induction of EAU (Grajewski et al., 2006), and although in disease their threshold of protection has obviously been overcome, they may proliferate and constitute a homeostatic mechanism that would help to bring about remission. Tregulatory cells can also be induced or converted from CD25− populations, which also give rise to effector T lymphocytes, and under some conditions functional effectors may also convert to acquire T-regulatory properties. As an example, IRBP-specific T cells may be converted from effectors to regulators by aqueous humor, a phenomenon that has been attributed to the effects of TGF-β and inhibitory neuropeptides (Stein-Streilein and Taylor, 2007). Natural T-regulatory cells as well as some types of induced Tregulatory cells express the transcription factor FoxP3, but although expression of this molecule is generally recognized to be indicative of T-regulatory cell function, it does not follow that the regulatory cells that express it are necessarily descended from CD4+CD25+ nT-regs (Ziegler, 2006). The uniquely eye-related phenomenon of anterior chamber–associated immune deviation (ACAID) has been well studied. It is induced by injection of antigen into the anterior chamber of the eye and results in development of regulatory CD4+ and CD8+ T cells that inhibit, respectively, the induction and the expression of antigen-specific immune responses (Stein-Streilein and Streilein, 2002). Although elicitation of ACAID to IRBP protects mice from EAU (Hara et al., 1992), it is difficult to conclude that ACAID represents a natural mechanism of protection from autoimmunity used by the intact eye. This is because ACAID

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results from injection of an antigen into the anterior chamber of the eye, necessarily perturbing its integrity. It is possible, in fact, that such mechanisms come into play as a result of damage to the eye. IRBP-specific T-regulatory cells, though apparently distinct from ACAID-induced T-regulatory cells, are induced as a result of EAU. Such cells are found in mice that have recovered from EAU and limit reinduction of disease (Kitaichi et al., 2005). Interestingly, their induction is dependent on the presence of the eye, indicating that something originating in the eye is crucial to their development. The eye could be necessary as a source of antigen, or of inhibitory factors such as α-MSH, which has been shown to convert primed IRBP-specific T cells to a regulatory phenotype (Stein-Streilein and Taylor, 2007). Other mechanisms of suppression could conceivably include production of inhibitory cytokines, such as IL-10 and TGF-β, or inhibition by contact-driven mechanisms. IL-10 is detected in the retina of mice recovering from EAU, and can inhibit activation and function of already immune effector T cells, which are impervious to the inhibitory effects of TGF-β (Xu et al., 1997; Rizzo et al., 1998; Xu et al., 2003). They could also include contact with ocular resident cells, such as retinal glial Müller cells, which proliferate during uveitis as part of the process of healing and retinal scar formation, and whose proliferation in vitro is induced by activated lymphocyte/monocyte products (Caspi et al., 1987). Müller cells are able to inhibit activation and function of T cells, even if these T cells have previously been activated, and similar inhibitory effects are exerted by retinal pigment epithelial cells and iris/ciliary body epithelial cells (Caspi et al., 1987; Yoshida et al., 2000). In some cases interaction with ocular resident cells additionally induces conversion of T cells to a regulatory phenotype (Ishida et al., 2003; Sugita et al., 2006). Only some of the molecular interactions involved in these phenomena have been characterized.

Translational potential Many of critical checkpoints in pathogenesis of uveitis, as defined in mouse models and depicted in figure 42.3, can serve as targets for immunotherapeutic intervention. To mimic the clinical situation in which the patient already has acquired immunity to retinal autoantigens, ideally the experimental approaches should be able not only to prevent but also to reverse disease. However, approaches that target only acquisition of immunity may also be of value, since chronic autoimmunity involves constant priming of new lymphocytes and their entry into the effector pool. This section provides some examples of possible immunotherapeutic approaches, drawing on what we have learned about disease pathogenesis, but it is by no means an exhaustive discussion of the translation of experimental approaches to the clinic.

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Deficits in peripheral tolerance could be corrected in the adult animal by expressing the retinal antigen outside the eye in a tolerance-inducing form. Agarwal et al. showed that infusion of B cells, made to express a uveitogenic fragment of IRBP into susceptible mice, resulted in a profound and long-term induction of resistance to EAU, and moreover was able not only to prevent but also to reverse the disease process (Agarwal et al., 2000). Adapting this regimen to autologous human B cells using S-Ag, which appears to be the major retinal antigen recognized by humans, could represent a viable approach to therapy of uveitis, a notion that is supported by the ability of S-Ag-transduced B cells to inhibit EAU in HLA-DR3 transgenic mice (Liang et al., 2006). Another antigen-based approach to prevent EAU that has been successful in animal models is induction of oral tolerance by feeding retinal antigens. In uveitis patients, oral administration of S-Ag showed promise in a double-blind, placebo-controlled Phase I/II clinical trial; however, larger trials are required to establish efficacy (Nussenblatt, 2002). A limitation of antigen-based therapy is that it requires knowledge of the inciting retinal antigen. Although uveitisassociated antigens are increasingly being identified, there are still many unknowns. Furthermore, the phenomenon of epitope and antigen spreading, which can accompany chronic autoimmune disease, could make identification of the antigen(s) driving pathology at a given time a moving target. Finally, there are concerns about introducing the putative disease-inciting antigen into an already immune individual. Immunotherapeutic approaches that target common pathways of lymphocyte activation do not require knowledge of the inciting antigen, but are not as selective, and therefore have the potential to also inhibit immunity to pathogens. Several such approaches have been examined in mouse models of uveitis, and some appear to have clinical potential. For example, costimulatory molecule blockade targeting the B7 or CD40 pathways inhibits induction of EAU pathology in mice (Silver et al., 2000; Bagenstose et al., 2005). However, it does not induce long-term tolerance, contrary to reports in transplantation models. Thus, in theory, continuing treatment would be needed to maintain protection. A related approach is blockade of the IL-2 receptors, which are present on all activated T cells. This approach was shown to inhibit EAU in animals, and IL-2 receptor blockade with humanized anti-CD25 antibodies (Daclizumab or Zenapax) showed efficacy in clinical trials (Nussenblatt et al., 1999). Surprisingly, the mechanism of action of this treatment in humans appears to involve CD56high regulatory natural killer (NK) cells rather than elimination of the CD25+ effector T cells, as had initially been assumed (Li et al., 2005). Migration and infiltration of recruited cells into the eye, which constitutes a necessary mechanism for disease

induction and progression, could be targeted by blockade of adhesion molecules or of chemokines and chemokine receptors. Blockade of the adhesion molecules ICAM-1 and LFA-1 in mice inhibited EAU, presumably by blocking inflammatory cell recruitment (Whitcup et al., 1993), as does blockade of the chemokine receptor CXCR3, which is important for migration of uveitogenic and recruited cells into the eye (Chen et al., 2004). Finally, the effector cytokines produced by pathogenic T cells and the inflammatory leukocytes recruited by them may serve as targets for inhibition or neutralization. Neutralization of IL-17 by monoclonal antibodies in mice aborts disease even when instituted after the uveitogenic effectors have already been generated (Luger et al., in press). Similarly, TNF-α neutralization, which had been shown in animal models to be protective, is showing efficacy in clinical trials for some types of uveitis (Greiner et al., 2004). These examples underscore the contribution of research in animal models to understanding the fundamental mechanisms driving ocular autoimmune and inflammatory disease, and their importance in devising novel therapies to combat sight-threatening uveitic diseases.

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Cytotoxic T lymphocyte–associated antigen 4 blockade in patients with metastatic melanoma: A new cause of uveitis. J. Immunother. 27:478–479. Rosenbaum, J. T., McDevitt, H. O., Guss, R. B., and Egbert, P. R. (1980). Endotoxin-induced uveitis in rats as a model for human disease. Nature 286:611–613. Rosenbaum, J. T., Planck, S. R., Martin, T. M., Crane, I., Xu, H., and Forrester, J. V. (2002). Imaging ocular immune responses by intravital microscopy. Int. Rev. Immunol. 21:255–272. Siepmann, K., Biester, S., Plskova, J., Muckersie, E., Duncan, L., and Forrester, J. V. (2007). CD4(+)CD25(+) T regulatory cells induced by LPS-activated bone marrow dendritic cells suppress experimental autoimmune uveoretinitis in vivo. Graefes. Arch. Clin. Exp. Ophthalmol. 245:221–229. Silver, P. B., Chan, C. C., Wiggert, B., and Caspi, R. R. (1999). The requirement for pertussis to induce EAU is straindependent: B10.RIII, but not B10.A mice, develop EAU and Th1 responses to IRBP without pertussis treatment. Invest. Ophthalmol. Vis. Sci. 40:2898–2905. Silver, P. B., Hathcock, K. S., Chan, C. C., Wiggert, B., and Caspi, R. R. (2000). Blockade of costimulation through B7/ CD28 inhibits experimental autoimmune uveoretinitis, but does not induce long-term tolerance. J. Immunol. 165:5041–5047. Silver, P. B., Rizzo, L. V., Chan, C. C., Donoso, L. A., Wiggert, B., and Caspi, R. R. (1995). Identification of a major pathogenic epitope in the human IRBP molecule recognized by mice of the H-2r haplotype. Invest. Ophthalmol. Vis. Sci. 36:946–954. Smith, J. R., Hart, P. H., and Williams, K. A. (1998). Basic pathogenic mechanisms operating in experimental models of acute anterior uveitis. Immunol. Cell Biol. 76:497–512. Stein-Streilein, J., and Streilein, J. W. (2002). Anterior chamber associated immune deviation (ACAID): Regulation, biological relevance, and implications for therapy. Int. Rev. Immunol. 21: 123–152. Stein-Streilein, J., and Taylor, A. W. (2007). An eye’s view of T regulatory cells. J. Leukoc. Biol. 81:593–598. Su, S. B., Silver, P. B., Grajewski, R. S., Agarwal, R. K., Tang, J., Chan, C. C., and Caspi, R. R. (2005). Essential role of the MyD88 pathway, but nonessential roles of TLRs 2, 4, and 9, in the adjuvant effect promoting Th1-mediated autoimmunity. J. Immunol. 175:6303–6310. Sugita, S., Ng, T. F., Lucas, P. J., Gress, R. E., and Streilein, J. W. (2006). B7+ iris pigment epithelium induce CD8+ T regulatory cells; both suppress CTLA-4+ T cells. J. Immunol. 176: 118–127.

Szpak, Y., Vieville, J. C., Tabary, T., Naud, M. C., Chopin, M., Edelson, C., Cohen, J. H., Dausset, J., de Kozak, Y., et al. (2001). Spontaneous retinopathy in HLA-A29 transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 98:2572–2576. Tang, J., Zhu, W., Silver, P. B., Su, S.-B., Chan, C.-C., and Caspi, R. R. (2007). Autoimmune uveitis elicited with antigenpulsed dendritic cells has a distinct clinical signature and is driven by unique effector mechanisms: Initial encounter with autoantigen defines disease phenotype. J. Immunol. 178:7072– 7080. Tarrant, T. K., Silver, P. B., Chan, C. C., Wiggert, B., and Caspi, R. R. (1998). Endogenous IL-12 is required for induction and expression of experimental autoimmune uveitis. J. Immunol. 161:122–127. Tarrant, T. K., Silver, P. B., Wahlsten, J. L., Rizzo, L. V., Chan, C. C., Wiggert, B., and Caspi, R. R. (1999). Interleukin 12 protects from a T helper type 1–mediated autoimmune disease, experimental autoimmune uveitis, through a mechanism involving interferon gamma, nitric oxide, and apoptosis. J. Exp. Med. 189:219–230. Whitcup, S. M., DeBarge, L. R., Caspi, R. R., Harning, R., Nussenblatt, R. B., and Chan, C. C. (1993). Monoclonal antibodies against ICAM-1 (CD54) and LFA-1 (CD11a/CD18) inhibit experimental autoimmune uveitis. Clin. Immunol. Immunopathol. 67:143–150. Wildner, G., and Diedrichs-Mohring, M. (2004). Autoimmune uveitis and antigenic mimicry of environmental antigens. Autoimmun. Rev. 3:383–387. Xu, H., Rizzo, L. V., Silver, P. B., and Caspi, R. R. (1997). Uveitogenicity is associated with a Th1-like lymphokine profile: Cytokine-dependent modulation of early and committed effector T cells in experimental autoimmune uveitis. Cell. Immunol. 178:69–78. Xu, H., Silver, P. B., Tarrant, T. K., Chan, C. C., and Caspi, R. R. (2003). Tgf-beta inhibits activation and uveitogenicity of primary but not of fully polarized retinal antigen-specific memory-effector T cells. Invest. Ophthalmol. Vis. Sci. 44:4805– 4812. Yoshida, M., Kezuka, T., and Streilein, J. W. (2000). Participation of pigment epithelium of iris and ciliary body in ocular immune privilege. 2. Generation of TGF-betaproducing regulatory T cells. Invest. Ophthalmol. Vis. Sci. 41:3862– 3870. Ziegler, S. F. (2006). FOXP3: Of mice and men. Annu. Rev. Immunol. 24:209–226.

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43

Mouse Models of Norrie Disease WOLFGANG BERGER

Norrie disease is a severe X-linked recessive trait with the hallmark features of congenital blindness, deafness, and mental retardation. The disorder is caused by mutations in a gene encoding norrin, a small extracellular protein. To characterize the function of norrin and to study the pathophysiology of Norrie disease, a knockout mouse model was generated and examined. The results of these studies showed that abnormal retinal angiogenesis during development is one of the most prominent observations in mice lacking norrin. This causes severe retinal hypoxia, which leads to profound tissue damage. The disease phenotype was rescued by breeding knockout mice with transgenic animals with ectopic norrin expression in the lens. In addition, transgenic lenses induced proliferation of microvascular endothelial cells in coculture. These and other findings identified norrin as a key regulator of angiogenic processes in the retina. Most of the ocular symptoms in human patients may also be attributed to oxygen deficiency during retinal development, and the mouse lines significantly contributed to a better understanding of the primary events of this severe neurological disorder.

Clinical background Norrie disease was named after Gordon Norrie, a consultant ophthalmologist at the Eye Clinic of the Institute for the Blind in Copenhagen. Norrie was the first to describe this trait as a nosological entity, in the late 1920s (Norrie, 1927). The medical condition was given its name by Mette Warburg, another Danish ophthalmologist, who reported the clinical picture and X-linked inheritance in a comprehensive monograph (Warburg, 1966). She decided to use this eponym in part to avoid a descriptive name that might reflect only secondary clinical features. Norrie realized that the disease was familial and that only boys were affected. Warburg then provided evidence that the disease is inherited in a sex chromosome–linked fashion (Andersen and Warburg, 1961; Warburg et al., 1965). In an X-linked recessive mode of inheritance, only males are affected, but a few exceptions exist (Chen et al., 1993b; Woodruff, et al., 1993; Kellner et al., 1996; Sims et al., 1997; Yamada et al., 2001). The most prominent clinical finding in affected patients is early-onset bilateral blindness. Soon after birth, leukokoria is observed, a white pupillary reflex due to vascularized retrolental membranes and masses. This is a cardinal symptom

but not pathognomonic, as it is present in a number of other eye diseases (Francois, 1978). Additional ocular manifestations consist of vitreoretinal hemorrhages, retinal folding and detachment, bilateral and congenital pseudotumor of the retina (pseudoglioma), and atrophy of the bulbus (phthisis bulbi) late in the disease. The appearance of the vitreal cavity alludes to persistent primary vitreous and hyaloid vessels. The lens becomes cataractous during the first months or years of life. Also, the cornea, iris, ciliary body, and retinal pigment epithelium may be affected by the disease. The extraocular features of Norrie disease include deafness and mental retardation. The latter also occurs early in childhood in one-third to one-half of patients. The onset of progressive hearing loss was observed in the second or third decade of life. Initially, deafness was reported in about onethird of patients (Warburg, 1966), but recent data suggest an almost 100% occurrence of hearing loss in patients (Halpin et al., 2005). Some histological studies of affected tissues (eye, ear, brain) available in the literature describe advanced stages of the disease. Hypotheses regarding the pathophysiology of Norrie disease involve an early arrest in neuroectodermal development, as well as abnormal vasoproliferative processes (Warburg, 1966; Parsons et al., 1992).

The molecular basis of Norrie disease With the advent of molecular genetic mapping in humans, the responsible locus has been localized to the short arm of the human X chromosome (Xp11.4). Linkage mapping was consistent with deletions of the same chromosomal segment in affected male patients (L. M. Bleeker-Wagemakers et al., 1985; Gal et al., 1985, 1986; E. M. Bleeker-Wagemakers et al., 1988; Diergaarde et al., 1989). Detailed analysis of the corresponding genetic interval led to the identification of the mutation-carrying gene by positional cloning (Berger, Meindl, et al., 1992; Chen et al., 1992). The official gene symbol is NDP (Norrie disease pseudoglioma), and the protein was designated norrin. The human gene spans a genomic region of approximately 25 kb (24,729 bps) and consists of three exons. The length of the transcript is 1,833 nucleotides. It codes for a protein of 133 residues, including a signal peptide of 24 amino acids. The small size of the gene allows rapid diagnostic testing. Around 100 mutations have been described so far. Point mutations in NDP represent missense and nonsense mutations, as well as frameshifting

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and splice site mutations (Berger, van de Pol, et al., 1992; Meindl et al., 1992, 1995). Approximately 20% of patients have complete or partial gene deletions (see Berger, 1998, and references therein). NDP mutations were also found in the DNA of patients with X-linked recessive familial exudative vitreoretinopathy (XLEVR), or Coats’ disease, and were associated with advanced stages of retinopathy of prematurity (Black et al., 1999; Chen et al., 1993a; Shastry et al., 1997). Thus, the clinical expressivity of mutations in the NDP gene is highly variable, even within a family, and both genetic and environmental modifiers are likely to influence the disease course (Zaremba et al., 1998). However, all these traits have the common feature of retinal blood vessel malformations. Therefore, a role of norrin in vascular development and homeostasis seems reasonable. It should be noted, however, that only classic Norrie disease is associated with deafness and mental retardation; the allelic traits do not show this pleiotropic effect.

Evolutionary conservation of the NDP gene and its expression pattern

nuclear layer (INL) as well as the ganglion cell layer (GCL) (Chen et al., 1995; Berger et al., 1996; Hartzer et al., 1999), and might also be expressed in the outer nuclear layer (ONL) and the choroid (Hartzer et al., 1999). In mouse and rabbit brain, transcripts were detected by RISH in the olfactory bulbus and epithelium, cerebellum, cortex, and hippocampus (Chen et al., 1995; Berger et al., 1996; Hartzer et al., 1999). Transcriptional activity in the ear has been reported in neurons of the spiral ganglion cells and marginal cells of the stria vascularis of the cochlea (Chen et al., 1995; Hartzer et al., 1999). Furthermore, norrin has also been shown to be expressed in the endometrium of pregnant female mice (Luhmann et al., 2005b). In addition to these RISH studies, the more sensitive RT-PCR has also revealed norrin mRNA in other tissues, including several cell lines, as well as mouse endometrium and human placenta (Luhmann et al., 2005b), muscle, liver, and lung (Chen et al., 1995). The expression of norrin in mouse endometrium and human placenta is particularly interesting, because homozygous knockout mice show a characteristic infertility phenotype, as discussed later in the chapter.

Putative norrin function Norrin shows high conservation in different species, as revealed by multiple protein sequence alignment (figure 43.1). The human sequence showed 95% identity on the amino acid level with rat, mouse, and dog, and 94% with cow. Lower levels of sequence identity of 87%, 60%, 57%, and 54% were observed with chicken, zebrafish, tetraodon, and fugu, respectively. This high degree of conservation implies an important function of norrin in vertebrates. In particular, the number and positions/spacing of 11 cysteine residues are highly conserved in all the species just mentioned. They form a cystine knot motif, which is a characteristic feature of a structural superfamily of growth factors that includes nerve growth factor (NGF), platelet-derived growth factor-β (PDGF-β), vascular endothelial growth factor (VEGF)-A and -B, placental growth factor (PlGF), and transforming growth factor-β (TGF-β) (McDonald and Hendrickson, 1993). The first cysteine residue occurs at position 39 in the human peptide sequence of norrin. The degree of conservation from this position toward the C-terminus of the protein is conspicuously higher in comparison to the N-terminal part, which contains the signal peptide and shows less conservation (see figure 43.1). To date, tissue- or cell type–specific expression of the NDP gene has been studied only at the transcriptional level. Protein expression by immunohistochemistry has not yet been reported. RNA in situ hybridizations (RISH) detected transcripts of the NDP gene in different tissues, including eye, ear, and brain. Thus, the gene is expressed in all tissues or organs that are affected in Norrie disease. In mouse, rabbit, and human retina, norrin mRNA is expressed in the inner

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Computational modeling of the gene product, norrin, revealed a three-dimensional structure very similar to TGF-β (Meitinger et al., 1993). The amino acid sequence contains a cystine knot motif that is present in several other proteins, including several growth factors, as well as in mucins, the Drosophila slit protein, von Willebrandt factor, and others. The common feature of the cystine knot in all these proteins is responsible for protein interaction and, most likely, dimerization of the respective molecules. NDP mutations lead to exudative vitreoretinopathy, a disease characterized by an avascular retinal periphery, leakiness of retinal blood vessels, and vitreoretinal hemorrhages. Similar symptoms are observed in patients with a mutation in the Wnt receptor Frizzled-4 (FZD4) and its coreceptor LRP5 (low-density lipoprotein receptor–related protein 5) (Robitaille et al., 2002; Toomes et al., 2004). This observation and the fact that Frizzled-4 knockout mice show a similar phenotype prompted Xu, Wang, and co-workers to analyze norrin as a ligand for FZD4/LRP5 (Xu et al., 2004). The results of these experiments revealed that norrin is able to activate the canonical Wnt β-catenin pathway and to drive reporter gene transcription in a cell line (HEK293) coexpressing norrin, FZD4/LRP5, and a luciferase reporter gene under the control of transcription factor–binding sites (LEF/TCF). They concluded that norrin is a high-affinity ligand for FZD4/LRP5 and transcriptionally activates downstream target genes. Ligand binding depends on coexpression of both receptor molecules Lrp5 and Fzd4. No significant or very little activation of the Wnt pathway was

A

B Figure 43.1 Multiple norrin protein sequence alignment from different species using the Clustal algorithm (Clustal W [1.83], www.ebi.ac.uk/clustalw). A, Alignment of the peptide sequences clearly shows a much higher conservation after the first of 11 cysteine residues (indicated by arrows above the mouse sequence of norrin). Asterisks in the bottom line indicate identical amino acids; dots designate similar properties of the residues. The putative signal

peptide is highlighted in gray in the human sequence. B, The phylogenetic tree shows the evolutionary relationship of norrin protein sequences in different species. Sequence identity between human, dog, mouse, and rat is 95%. Identity with cow, chicken, zebrafish, tetraodon, and fugu is 94%, 87%, 60%, 57%, and 54%, respectively.

observed when cell lines were transfected with either of the two receptors. Mutations in LRP5 are also associated with autosomal recessive osteoporosis-pseudoglioma (OPPG) syndrome, which is characterized by congenital or early childhood onset visual loss and bone fragility (Gong et al., 2001; Ai et al., 2005).

Generation of a knockout mouse model for Norrie disease Given the high conservation between human and mouse and the tissue-specific transcription of the mouse norrin gene (gene symbol: Ndph, Norrie disease pseudoglioma homo-

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logue) in eye, brain, and ear, the mouse may provide an adequate model for Norrie disease and allelic disorders. Reverse genetics is a powerful tool to establish models for human gene defects and use them to study the pathology at the phenotypic and molecular levels. The gene-targeting approach has been used to generate a knockout mouse model for Norrie disease (Berger et al., 1996). The targeting construct replaced the coding part of exon 2 with a neomycin cassette in opposite transcriptional orientation to Ndph. As a consequence of this replacement, the translation start codon, the splice donor site of exon 2 and 55 additional N-terminal amino acids, including the signal peptide for extracellular transport, are removed from the gene. After homologous recombination of this construct in embryonic stem cells, blastocysts were injected to produce chimeric embryos. Chimeric male offspring were crossed to wild-type females to obtain female carriers for the knockout allele. Approximately half of the male offspring were hemizygous for the knockout allele, which is consistent with Mendelian transmission of a viable mutation. In an initial survey to characterize the corresponding phenotype, the retinal morphology of hemizygous knockout mice (Ndph−/y) was compared with that in wild-type (Ndph+/y) littermates (Berger et al., 1996). One of the most prominent findings in knockout mice was the presence of fibrous masses containing blood vessels in the vitreous of all nine mice analyzed. This is probably the anatomical correlate to the precipitate-like structures that had been observed on slit lamp biomicroscopy. Likewise, an abnormal organization of nuclei in the GCL had been reported in all Ndph−/y mice examined. Occasionally, outer retinal layers, including the ONL and outer segments of photoreceptor cells, also showed a characteristic pattern of disorganization. Heterozygous female carriers (Ndph+/−) were inconspicuous. These initial data revealed a clear retinal phenotype in Ndph knockout mice. Histopathological data from human patients in whom the clinical diagnosis was confirmed by molecular testing are rare. There is one report in the literature in which retinal and vitreal tissues were examined after vitreoretinal surgery in a 6-month-old boy with a frameshift mutation in the NDP gene (Schroeder et al., 1997). In this case, a reduction in the number of ganglion cells was observed, along with a largely disarranged INL but otherwise normally differentiated retina. Massive fibrovascular proliferation was present in the vitreous.

Retinal physiology of knockout mice To examine the consequences of the knockout mutation and the morphological changes on retinal physiology, electroretinography (ERG) was performed. This analysis allows direct measurement of the activity of photoreceptor cells and second-order neurons in the retina as a response to light stimuli. The two major ERG components are the a- and

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b-waves, which reflect photoreceptor and second-order neuron (bipolar, amacrine, and horizontal cells) activities, respectively. Also, the two photoreceptor systems, rods and cones, can be discriminated by using dark adaptation and excitation with different light intensities. When the retina is dark adapted and the photoreceptors receive light stimuli of low to moderate intensity, the corresponding a-wave mainly represents the response of rod photoreceptors. In the lightadapted state, when bright flashes are utilized to excite photoreceptors, the corresponding a-wave primarily reflects cone photoreceptor activity. Therefore, the ERG is a suitable tool to characterize retinal function. ERG measurements were performed in hemizygous knockout mice from 7 months of age up (Ruether et al., 1997). Data from hemizygous knockouts were compared with data from a group of heterozygous females and wildtype male litters. Both a- and b-waves were attenuated in hemizygous knockout mice but not in wild-type mice or female carriers. An interesting finding was that the a-waves of the three different genotypes (wild type, hemizygous knockout, heterozygous) were not much different at low intensities but became prominent at higher intensities of light stimuli. Conversely, the b-wave amplitudes were much lower in the group of hemizygous knockout mice at all intensities. Oscillatory potentials, which reflect the response of part of the second-order neurons in the retina, were severely reduced in male mice with the knockout allele. In summary, the ERG recordings revealed a more severe functional effect of the knockout mutation on the inner retina compared to photoreceptor cells. This is in agreement with molecular studies in which a late involvement of photoreceptor cells was found (Lenzner et al., 2002). In this study, the expression of photoreceptor-specific gene transcripts was normal until the age of 12 months in knockout mice and significantly reduced only after 2 years, indicating a rather slow progression of photoreceptor cell degeneration.

Abnormal retinal vascular development in norrin knockout mice In normal mouse development, three vascular networks, superficial, deep, and intermediate, appear in the retina. The superficial retinal vasculature, located within the GCL/ nerve fiber layer, spreads from the entry point of the large artery and vein from the central retina (at the optic nerve head) toward the periphery between days 3 and 17 post partum (P3–P17). Around P7 or P8, the deep retinal vasculature starts forming by angiogenic sprouting from the superficial network. After completion, the deep vascular system will be present in the outer plexiform layer (OPL). From P12, an intermediate vascular system develops between the superficial and deep networks, close to the boundary between inner nuclear and plexiform layers.

Abnormalities and changes in the retinal vasculature in norrin knockout mice have been reported by several authors (Richter et al., 1998; Rehm et al., 2002; Luhmann et al., 2005a). Vascular development between P5 and P21 in knockout mice was studied in detail by Luhmann and co-workers and was correlated with molecular analyses of angiogenic signaling pathways. The outgrowth of the superficial vascular system was temporally retarded and never completed. In wild-type mice, the retina was entirely covered by superficial vessels at P21. The periphery of the retina in hemizygous knockout mice remained avascular, and only three-quarters of the retina was supplied with vessels. The development of the deep capillary network, which normally starts at P7, was completely abolished in norrin-deficient mice (figures 43.2 and 43.3). Although the vascular tubes of the superficial network branched out and tried to initiate angiogenic sprouting, this process was never completed but was entirely blocked at this point, leading to microaneurysmlike capillary lesions. Of note, the astrocytic network, which is important for vessel guidance, seemed to be normal in knockout mice in early disease phases. Rather, a primary effect on endothelial cells has been suggested by Luhmann and co-workers. This view is supported by some molecular findings. The transcript (mRNA) levels of Tie1, Tie2, and Pdgfb, which are involved in angiogenesis and vessel maturation and represent endothelial markers, were significantly lower in norrin-deficient mice at P5 and P10 than in wildtype controls. The defect in vascular development in norrin knockout mice can be summarized as delayed and incomplete out-

growth of the superficial retinal blood vessels, leading to an avascular retinal periphery. Additionally, the deep and intermediate vessel networks fail to develop, owing to an arrest in sprouting angiogenesis.

Failure in sprouting angiogenesis leads to retinal hypoxia and leakiness of blood vessels, but not retinal neovascularization As noted, the retinal vasculature in norrin-deficient mice does not develop properly. As a consequence, oxygen deficiency occurs in the inner retina. This hypoxic condition leads to an activation of angiogenic cascades, as shown by the transcriptional upregulation of Vegfa from P10 onward (Luhmann et al., 2005a). At P15, several other factors in the retina are transcriptionally activated: angiopoietin-2 (Agpt2), ephrin-B4 (EphB4), frizzled-4 (Fzd4), integrins α-V and βIII (Itga5, Itgb3), pdgfb and its receptor (pdgfrb), as well as Tie1. All of them also show higher transcript levels at P21 in addition to the Vegf receptors 1 and 2 (Vegfr1/Flt1 and Vegfr2/Flk1). This transcriptional activation of angiogenic factors indicates a response of retinal cells to hypoxia and provides an explanation for microaneurysms and the leakiness of blood vessels, which leads to retinal and vitreal hemorrhages and exudates. Another excellent indicator of hypoxia is the presence of hypoxia-inducible factor 1α (Hif1α), which is known to be stabilized at the protein level under hypoxic conditions. Indeed, the Hif1-α protein level was increased in norrin knockout mice compared to wild-type litters at P15 and P21, but not at P5 and P10 (Luhmann

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Figure 43.2 Sections of the central retina from wild-type and knockout mice at P21 after staining with a collagen IV antibody (red), which detects the extracellular matrix of endothelial cells, and diamidinophenylindol/DAPI (blue), which labels cell nuclei. Blood vessels (red) in wild-type mice are present in three layers, indicating completed development of the superficial (ganglion cell layer [GCL]), deep (outer plexiform layer [OPL]), and intermediate (border between the inner plexiform layer [IPL] and inner nuclear

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layer [INL]) vessel networks. In contrast, age-matched knockout mice show only enlarged superficial vessels, while the deeper and intermediate networks failed to develop. In addition, the presence of nuclei in the IPL (white arrowheads) may reflect the characteristic disorganization in the GCL. See color plate 41. (Images acquired and provided courtesy of Nikolaus Schäfer, Division of Medical Molecular Genetics, Institute of Medical Genetics, University of Zurich, Switzerland.)

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Figure 43.3 Three-dimensional microscopy of retinal flat mounts stained with anticollagen IV from wild-type and knockout mice at P7. Top panels show an area of approximately 300 × 400 μm from wild-type and knockout mice. Vessels in the knockout are dilated, and the capillary network is less dense than in the wild-type control. Bottom panels (z-axis) show how blood vessels enter the deeper retinal

layers in wild-type mice by angiogenic sprouting. In contrast, this process is abolished in knockout mice, and the vessels do not invade the deeper retinal layers. See color plate 42. (Images acquired using the Zeiss ApoTome technology and provided courtesy of Nikolaus Schäfer, Division of Medical Molecular Genetics, Institute of Medical Genetics, University of Zurich, Switzerland.)

et al., 2005a). Particularly Hif1-α and Vegfa clearly indicate retinal hypoxia, and many aforementioned angiogenic factors are known to be regulated by them, including Pdgfb. Strikingly, reduced Pdgfb, as shown in norrin knockout mice at P10, results in the formation of microaneurysms, as reported previously (Enge et al., 2002). In many other cases of retinal hypoxia, neovascularization occurs as a result of increased activity of pro-angiogenic signaling cascades. Obviously, this does not apply to norrindeficient mice. Retinal neovascularization has never been reported, not even in late stages of the disease. Thus, high VEGF-α alone is not sufficient to induce the formation of new blood vessels in the retina if norrin is lacking.

were found to grow into the peripheral retina of knockout mice (Ohlmann et al., 2004; Luhmann et al., 2005a). There are two alternative hypotheses regarding the primary defect in norrin-deficient mice: (1) Norrin causes persistence of the hyaloid vessels that supply oxygen to the retina, and thus retinal vascular development is dispensable (Ohlmann et al., 2004). (2) Norrin deficiency in the retina results in an arrest of vascular development of the deep capillary networks, which leads to hypoxia and subsequent upregulation of VEGF. This excess of VEGF provides an antiapoptotic signal to the endothelial cells of the hyaloid vasculature and its persistence or extremely delayed regression (Luhmann et al., 2005a). The second alternative is supported by the fact that hyaloid vessels are obliterated and most likely nonfunctional. Moreover, it has been shown in mice that, by blocking PGF, functional hyaloid vessels can persist without significantly affecting retinal vascular development (Feeney et al., 2003).

Regression of hyaloid vessels versus angiogenesis of retinal blood vessels In addition to the characteristic abnormal pattern of retinal vasculature, norrin is required for normal regression of the hyaloid vessels, which is completed by P21 in control mice but heavily delayed in norrin knockout mice (figure 43.4) (Ohlmann et al., 2004; Luhmann et al., 2005a). Nevertheless, these hyaloid vessels are obliterated and most likely nonfunctional in knockout mice, as shown by angiography (Luhmann et al., 2005a). Between P14 and P21, the number and diameter of hyaloid vessels were reduced. This obliteration continued until the age of 6–8 weeks, when most of them became nonfunctional. Occasionally, hyaloid vessels

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The two phases of Norrie disease in the eye Examination of blood vessel development in norrin knockout versus wild-type mice has revealed a delayed outgrowth of the superficial retinal vasculature, an arrest in sprouting angiogenesis that forms the deep and intermediate retinal capillary networks, and a delay in hyaloid regression. According to Luhmann et al. (2005a), there are two phases of

P5

P10

P15

P21

L

wt

R H/V O

ko

Figure 43.4 Hematoxylin-eosin-stained sections of whole eyes from wild-type (wt) and norrin knockout (ko) mice at P5, P10, P15, and P21. The hyaloid vessel system (H) in the vitreous body (V) is clearly present in wild-type mice at P5 and P10, and remnants are evident at P15. In knockout mice, the hyaloid vasculature persists

through P21 and beyond, and regression of this vascular network is profoundly delayed. L, lens; O, optic nerve; R, retina. See color plate 43. (Images acquired and provided courtesy of Dr. Ulrich Luhmann, Division of Medical Molecular Genetics, Institute of Medical Genetics, University of Zurich, Switzerland.)

pathology in knockout mice. The first phase is characterized by the abnormal vascular development and may last until P7 or P8. After this, hypoxia-driven activation of angiogenic signaling cascades lead to severe lesions in the vitreous and retina and the characteristic disease symptoms, which include vitreoretinal hemorrhages and exudates caused by leaky blood vessels in the vitreous and retina, an avascular retinal periphery, and retinal folding and detachment. Very similar symptoms were described in human patients. Thus, the Ndph knockout mouse may represent a suitable model not only for the classic form of Norrie disease but also for allelic traits such as exudative vitreoretinopathy or Coats’ disease, as well as more common vasoproliferative retinal diseases (retinopathy of prematurity, diabetic retinopathy, age-related macular dystrophy).

cochlear structures, including the stria vascularis, spiral ganglion, outer and inner hair cells, and cochlear vasculature. These analyses revealed a progressive hearing loss in hemizygous knockout mice across all frequencies (5–45 kHz). At younger ages (3–4 months), hearing impairment was more severe in the higher frequencies, but it progressed to a profound loss at all frequencies by the age of 15 months. This functional decline in the auditory system in knockout mice was found to be consistent with morphological changes in the cochlea, which was completely normal at P12 (Rehm et al., 2002). Thus, cochlear structures progressively degenerate over time, while normal development is not affected by the knockout mutation. The most affected structure was the stria vascularis in all three age groups. Vessels were significantly enlarged, particularly in the apical cochlear turn. The loss of outer hair cells progressed consistently, with an increase in auditory threshold. With progression of hearing loss, neurons in the spiral ganglion degenerated. Obviously, the outer hair cells of knockout mice are more susceptible to premature cell death than the inner hair cells, which show significant cell death at 15 months but not in the two groups of animals aged 3–4 months and 6.5 months. In addition, characteristic changes were found in the cochlear vasculature. The average vessel size was larger in knockout mice than in wild-type mice, and the number of vessels decreased significantly over time. Differences in vessel quantity between control animal and knockout mice were marginal at 3–4 months. However, enlarged vessels in the stria vascularis and a sparse capillary network in the spiral ganglion were clearly visible at this age. At 15 months, two-thirds of vessels had disappeared. To summarize these findings, the stria vascularis might be the primarily affected cochlear structure, followed by the spiral ganglion. Hearing loss is progressive in knockout mice,

Hearing loss and abnormal cochlear vasculature in Ndph knockout mice Because one of the clinical hallmarks of Norrie disease is progressive deafness in human patients, knockout mice were examined with respect to auditory performance. The auditory brainstem response (ABR) test is a useful diagnostic tool for measuring hearing performance. ABR records activities in the auditory centers of the brain in response to sound stimuli of different frequencies (e.g., 5–45 kHz). The test is considered reliable and objective, because it does not depend on subjective feedback as other, more conventional hearing tests do. ABR test results were compared for hemizygous knockout mice at three ages (3–4, 6.5, and 15 months) and wild-type littermates (Rehm et al., 2002). ABR recordings were correlated with cochlear pathology that had been scored by histological approaches, namely, conventional histology of

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with only mild symptoms or almost normal hearing present at younger ages, although the expressivity of hearing impairment shows a high degree of interindividual variability even in mice (Rehm et al., 2002). These observations suggest normal development of the auditory system but a defect in its homeostasis, probably resulting from degenerative processes in the cochlear vasculature. This is in contrast to the retina, where part of the vasculature fails to develop properly because of a defect in angiogenic sprouting. Thus, norrin might have different effects in eye and ear. Still, a major role in angiogenesis is reconcilable as the mechanism of angiogenic sprouting is necessary in both processes, development and homeostasis.

Generation and analysis of a transgenic mouse line with norrin overexpression in the lens The role of norrin in vascular development has also been demonstrated in transgenic mice overexpressing norrin in the lens. The transgenic lines have been established by pronucleus injection of a transgene construct containing the chicken βB1-crystallin promoter in front of the coding region of the mouse norrin cDNA (Ohlmann et al., 2005). In addition, a polyadenylation signal was present downstream of the translation termination codon of the norrin cDNA. Four founder lines were obtained containing 3, 9, 10, and 11 copies of the transgene, respectively. At the transcript and protein level, norrin expression from the transgene construct approximately correlated with the copy number. The line with 3 copies of the transgene showed less norrin mRNA and protein than the transgenic lines with 9–11 copies but more than wild-type littermates. Expression studies also showed that norrin expression in transgenic animals was restricted to the lens; other ocular structures did not express the transgene. At the morphological level, the lenses and eye bulbs of transgenic mice carrying between 9 and 11 copies were smaller than those of wild-type mice or the line with 3 transgene copies (approximately 30%). The tunica vasculosa lentis, an extensive blood vessel network of capillaries spreading over the posterior and lateral lens surface, in transgenic mice contained significantly more capillaries than in wildtype litters at P0–P2. Ultrastructurally, the capillaries were very similar in wild-type and transgenic mice. They were unfenestrated and surrounded by pericytes. Quantitatively, the number of capillaries was 1.5- to 2-fold higher in transgenic animals with 9–11 gene copies and increased by 20% in the transgenic line with 3 copies of the transgene. Lenses with overexpression of norrin were also examined with respect to stimulation of growth and proliferation of endothelial cells. Indeed, there was a positive effect of lenses expressing norrin on human dermal microvascular endothelial cells (HDMECs). The presence of norrin resulted in a

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66% or 40% increase in proliferation of HDMECs when the culture medium was preconditioned with lenses from transgenic mice at P1 or P7, respectively, which might also suggest a direct effect of norrin on endothelial cells.

Rescue of the knockout phenotype by ectopic norrin expression in the lens Norrin-deficient mice (Berger et al., 1996) were crossed with transgenic mice carrying 11 copies of the transgene, showing norrin expression at the transcript and protein levels (Ohlmann et al., 2005). Offspring were examined morphologically and electrophysiologically (ERG). These tests showed a rescue of the vascular and ERG phenotype of norrin knockout mice. The deep and intermediate vascular systems present in the OPL and IPL, respectively, were restored in knockout mice with ectopic norrin expression in the lens at P21 (Ohlmann et al., 2005). Also, disorganization of the GCL, which is seen in knockout animals, disappeared. The ERG pattern of knockout mice, characterized by a reduced bwave and almost absent oscillatory potentials, was rescued completely, although the amplitudes of the responses were lower than in wild-type litters, probably due to the reduced size of the bulbus. These findings were surprising, since diffusion of norrin from the lens to the retina, a prerequisite for the observed rescue effects, might be limited by the high affinity of norrin for the extracellular matrix (ECM). However, this retarded spatial spreading had been reported for norrin expressed and secreted from COS-7 (African green monkey kidney fibroblasts) cells (Perez-Vilar and Hill, 1997). The differences in ECM composition between cultured cells and in the vitreous body might explain this observation. Over and above the rescue effects of ectopic norrin, it might have a sort of neurotrophic or neuroprotective effect. Ohlmann and co-workers (2005) observed an increase in thickness of the ONL and INL, as well as an increase in the number of nuclei in the GCL. It is not yet clear whether these observations are mediated by Vegfa, which was shown to be more abundant in the retinas of transgenic mice. Compensation for a smaller eye size in transgenic mice may be another reason for this finding.

Fetal loss in homozygous knockout female mice Almost complete infertility of female mice homozygous for the knockout allele (Ndph−/−) has been reported (Luhmann et al., 2005b). Breeding of Ndph−/− females revealed a mean litter size of 0.3, while the control litters had seven offspring. The number of implantation sites was not different in homozygous knockout females, indicating normal implantation. However, massive bleeding at the implantation sites was reported at all embryonic stages from E9 onward, and

occasionally at E7. Embryonic tissues were highly disorganized and surrounded by blood. A normal chorioallantoic placenta was never observed, but yolk sac and trophoblast derivatives were differentiated. However, the spatial pattern of the trophoblast was severely disrupted, and the number of spongiotrophoblast cells was reduced. The labyrinthine area failed to develop and was filled with blood. Implantation of blastocysts and decidual reaction still occurred in homozygous females, but decidualization was compromised, which led to severely reduced fertility. The observed bleeding, which occurred before normal development of the chorioallantois placenta, might be a result of defects in decidual angiogenesis and blood vessel remodeling due to impaired angiogenic processes similar to those observed in the retina. Homozygous Frizzled-4 knockout female mice were also reported to be infertile, but as a result of failure of corpora lutea formation and function, which causes a defect in implantation (Hsieh et al., 2005). The expression of norrin has also been shown in human placenta at the RNA level (Luhmann et al., 2005b), implicating a role for norrin in human female reproduction. However, because of the low frequency of NDP gene mutations, homozygous females have never been described, and the function of this gene in human female reproductive tissues remains elusive. It might be noteworthy in this context that genotypic wild-type (for NDP) male and female offspring of NDP gene mutation carriers showed abnormal vascular patterns in the peripheral retina reminiscent of mild retinopathy of prematurity (Mintz-Hittner et al., 1996). This might be caused by the expression of norrin in the placenta of female mutation carriers from the mutant allele. The mildness of the phenotype can be explained by the presence of the wild-type allele in the embryos.

Summary Taken together, the data that have emerged from these knockout and transgenic mouse models indicate that Norrie disease is caused by a primary defect in the inner retina and vitreous. In the retina, the failure of proper blood vessel development leads to oxygen deficiency (hypoxia), which activates angiogenic signaling cascades. Activation of these pathways causes subsequent leakiness of blood vessels, hemorrhages, and fibrovascular membranes. The vitreal phenotype may be caused primarily by a delayed regression of the hyaloid vasculature and leaky blood vessels. The data obtained in mice can explain the leading ocular symptoms in patients with Norrie disease, exudative vitreoretinopathy, Coats’ disease, and retinopathy of prematurity, and indicate a role for norrin in vascular development in the eye. In contrast, its function in the auditory system might be vascular maintenance in the stria vascularis and spiral ganglion rather than vascular development in these structures, as

shown in the knockout mouse. This is also consistent with the human condition, where auditory function is normal in the beginning and the onset of sensorineural hearing loss or impairment occurs in adolescence. acknowledgments

I thank Dr. Ulrich Luhmann and Nikolaus Schäfer for critical reading of the manuscript and helpful comments and suggestions, as well as for providing the images in figures 43.2, 43.3, and 43.4. REFERENCES

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44

The Lipofuscin of Retinal Pigment Epithelial Cells: Learning from Mouse Models of Retinal Disease JANET R. SPARROW, SO R. KIM, YOUNG P. JANG, AND JILIN ZHOU

Within the visual cycle, 11-cis-retinal, the chromophore of visual pigment in photoreceptor cells, absorbs a photon of light, is isomerized to all-trans-retinal, and is then regenerated through a series of biochemical reactions within the retinal pigment epithelium (RPE). Not all of the highly reactive all-trans-retinal generated by photoisomerization of 11-cis-retinal within photoreceptor cells remains within the visual cycle, however; a minor portion inadvertently reacts to form retinoid-derived fluorophores that reside in photoreceptor outer segments until the latter are phagocytosed by RPE cells, whereupon the compounds accumulate as the lipofuscin of RPE (Sparrow and Boulton, 2005). Accordingly, all of the RPE lipofuscin fluorophores that have been isolated to date, including A2E, the A2E photoisomer isoA2E, minor cis-isomers of A2E, and the all-trans-retinal dimerconjugates, all-trans-retinal dimer-phosphatidylethanolamine (atRAL-dimer-PE), and all-trans-retinal dimer-ethanolamine (atRAL-dimer-E) (figure 44.1), are generated by reactions of all-trans-retinal. Because of their unusual structures, including that of a pyridinium bisretinoid in the case of A2E, these pigments cannot be enzymatically degraded, and thus they accumulate. The excessive accumulation of the retinoid-derived nondegradable lipofuscin in RPE cells is considered the primary cause of RPE atrophy in autosomal recessive Stargardt disease (arSTGD), a form of macular degeneration having onset in the early decades of life; RPE lipofuscin may also be a factor contributing to age-related macular degeneration (Sparrow and Boulton, 2005).

Rpe65 −/− mouse: Absence of all-trans-retinal-derived fluorophores Important insight into the origin of RPE lipofuscin as a by-product of the visual cycle was provided by studies of mice carrying a null mutation in Rpe65 (Rpe65−/−) (Redmond et al., 1998), an essential protein of the visual

cycle that is reported to be the isomerohydrolase responsible for generating 11-cis-retinol from all-trans-retinyl esters (Jin et al., 2005; Moiseyev et al., 2006). Without Rpe65, the 11-cis-retinal chromophore of rhodopsin is not generated, photoisomerization does not occur, and all-trans-retinal is not produced. We compared the constituents present in chloroform-methanol extracts of eyecups obtained from 3month-old wild-type C57BL/6J mice to that from agematched Rpe65 −/− mice (figure 44.2), the latter lacking the 11-cisRAL and atRAL chromophores (Redmond et al., 1998). When eluent was monitored for absorbance at 440 nm using a C4 column (figure 44.2A and B), Rpe65 −/− samples generated a chromatographic profile of reduced complexity as compared to C57BL/6J mice samples. Representative chromatograms are presented in figure 44.2. By simple visual subtraction analysis it was apparent that there were several peaks in the C57BL/6J profile for which there was no match in chromatograms generated from Rpe65 −/− mice samples. In particular, peaks in the C57BL/6J profile were attributed to the bisretinoid lipofuscin fluorophores A2E (λmax 335, 437 nm), atRAL dimer-PE (λmax 271, 518 nm), and the A2E precursors, NRPE (λmax 440 nm) and A2PE (λmax 332, 450 nm; 333, 448 nm), all of which were identified by coelution with synthetic standard and all of which we have previously identified. None of these pigments was detectable in Rpe65 −/− eyes. Further analysis of the eyecup extracts with a reverse phase C18 column (figure 44.2C and D) revealed peaks corresponding to all-transretinal (λmax 378 nm), A2E (λmax 335, 439 nm), iso-A2E (λmax 330, 445 nm), and a conjugate of all-trans-retinal with ethanolamine (NRE; λmax 440 nm), all of which were absent in Rpe65 −/− mice. These results are consistent with the observation that lipofuscin-specific autofluorescence in the RPE is reduced by more than 90% in Rpe65 −/− mice (Katz and Redmond, 2001), and they indicate that chloroform/methanol-soluble RPE lipofuscin fluorophores are derived primarily from retinoid precursors (Sparrow, Fishkin, et al.,

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Figure 44.1 RPE lipofuscin pigments, precursors, and photooxidation products. Shown are the structures of A2E and its photoisomer, iso-A2E, and the precursor of the latter pigments, A2PE, all-trans-retinal dimer-phosphatidylethanolamine (atRAL dimer-

PE), all-trans-retinal dimer-ethanolamine (atRAL dimer-E), and the precursor all-trans-retinal dimer (atRAL dimer). Monofurano-A2E and peroxy-A2E are photo-oxidation products of A2E.

Figure 44.2 HPLC detection of RPE lipofuscin pigments in C57BL/6J mouse eyecups and the absence of these compounds in Rpe65 null mutant mice. Shown are chromatograms generated from hydrophobic extracts of eyecups (6 eyecups per sample) from 3-month-old C57BL/6J (A and C ) and Rpe65 −/− (B and D) mice using reverse phase C4 (A and B) and C18 (C and D) columns with monitoring at 440 nm. Compounds were identified by UV/visible

absorbance and by coinjection of authentic standards. The two peaks attributable to A2PE (A) have retention times and UV/visible absorbance that correspond to the synthesized standard dipalmitoyl-A2PE and likely reflect A2PE of varying fatty acid composition. (Rpe65−/− mice were a gift from Dr. Michael Redmond, National Eye Institute, Bethesda, MD.)

2003). Indeed, a number of observations over the years have also shown that the deposition of lipofuscin fluorophores in RPE is dependent on vitamin A availability (Katz et al., 1986, 1987). Significantly, in patients with early-onset retinal dystrophy associated with mutations in RPE65, RPE lipofuscin is also lacking (Lorenz et al., 2004).

Stargardt macular degeneration and the Abca4/Abcr null mutant mice

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The relationship between Stargardt macular degeneration and RPE lipofuscin was elucidated by studies in Abca4/Abcr null mutant mice (Weng et al., 1999; Mata et al., 2000, 2001, 2002; Kim et al., 2004) (figure 44.3). ABCA4/ABCR (rim

Figure 44.3 Autofluorescence of RPE lipofusin in Abca4/Abcr−/− mouse retina. Cross section of mouse retina viewed by epifluorescence microscopy (excitation 425 ± 45 nm, emission 510 nm long pass). Nuclei are stained with DAPI. The yellow-gold autofluorescence in RPE cells is associated with lipofuscin in RPE cells. The less pronounced autofluorescence in photoreceptor outer segments (POS) is attributable to lipofuscin precursors that form in this location. The albino mouse was 8 months old and homozygous for leucine at amino acid 450 of Rpe65. GC, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium. See color plate 44.

Figure 44.4 RPE lipofuscin pigments in a mouse model of Stargardt disease. A, A2E/iso-A2E (left) increases with age in Abca4/ Abcr−/− mice; A2PE, the precursor of A2E in photoreceptor outer segments, does not accrue with age. Compared with A2E/iso-A2E, atRAL dimer-PE and atRAL dimer-E (right) exhibit increase with

protein) is an ATP-binding-cassette transporter and the protein product of the Stargardt disease gene, ABCA4/ABCR (Allikmets et al., 1997). Studies of purified and reconstituted ABCA4/ABCR protein (Sun and Nathans, 1997, 2001a, 2001b; Sun et al., 1999; Ahn et al., 2000; Beharry et al., 2004), together with work in Abca4/Abcr−/− mice (Weng et al., 1999), have shown that this photoreceptor-specific protein aids in the movement of all-trans-retinal to the cytosolic side of the disc membrane so that it can be reduced to all-trans-retinol by retinol dehydrogenase. The ligand recognized by ABCA4/ABCR is probably N-retinylidenephosphatidylethanolamine (NRPE), the conjugate formed by Schiff base reaction between a single all-trans-retinal molecule and phosphatidylethanolamine. The reaction of NRPE with a second molecule of all-trans-retinal leads to a series of random/nonenzymatic reactions and the formation of an unstable dihydropyridinium (dihydro-A2PE) (Kim et al., 2007a) (see figure 44.1) compound. Simulation by timedependent density functional theory reveals that dihydroA2PE exhibits a UV-visible spectrum with two absorbance maxima at approximately 494 and 344 nm. Dihydro-A2PE is an unstable intermediate that automatically undergoes oxidative aromatization to yield A2PE (see figure 44.1), the precursor of A2E detectable in photoreceptor outer segments (Parish et al., 1998; Liu et al., 2000). A2PE is internalized by RPE cells concomitant with outer segment phagocytosis; within RPE cell lysosomes, A2PE subsequently undergoes phosphate cleavage to generate A2E. Due to its unusual pyridinium bisretinoid structure (Sakai et al., 1996), A2E cannot be enzymatically degraded and thus is amassed with age (figure 44.4A). Conversely, A2PE does not accumulate with age; this would be expected of a precursor that is continually being cleaved to generate A2E (see figure 44.4A).

age. B, A2E increases with age and is more abundant in Abca4/ Abcr−/− mice than in wild-type mice (Abca4/Abcr+/+). Compounds were quantified from HPLC chromatograms and external standards to calibrate analyte peak areas.

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Chromatographic studies of extracts of eyecups harvested from Abca4/Abcr null mutant mice have consistently demonstrated that the availability of all-trans-retinal resulting from a loss of Abca4/Abcr activity leads to levels of A2E that are increased manyfold relative to age-matched wild-type eyes (Parish et al., 1998; Mata et al., 2000; Kim et al., 2004, 2006; Fishkin et al., 2005) (figure 44.4B). As a model of Stargardt disease, these increases in A2E in Abca4/Abcr−/− mice parallel similar increases in individuals diagnosed with Stargardt disease (Delori et al., 1995). A2E has an amphiphilic structure that is conferred by hydrophobic side-arms extending from a positively charged aromatic head group. A number of different approaches have shown that A2E can perturb cell membrane integrity when present in sufficient concentration (Sparrow et al., 1999, 2006; De and Sakmar, 2002; Sparrow, Fishkin, et al., 2003). This detergent-like behavior may be one means by which lipofuscin constitutents exert adverse effects on RPE cells. A2E also has an absorbance maximum in the blue range of the visible spectrum (ca. 440 nm). Photosensitization of A2E leads to the generation of singlet oxygen and perhaps other reactive forms of oxygen. In the process, A2E is photo-oxidized along the retinoid-derived side-arms of the molecule, with the result that reactive photolytic products are generated (Sparrow et al., 2000, 2002; Schutt et al., 2000; Ben-Shabat et al., 2002; Sparrow, Vollmer-Snarr, et al., 2003; Dillon et al., 2004; Jang et al., 2006). Studies in Abca4/Abcr−/− mice have enabled detection of photooxidized forms of A2E. Specifically, analysis of eyecups of Abca4/Abcr−/− mice ( Jang et al., 2006) by liquid chromatography-mass spectrometry permitted the detection of both monofuran-A2E (Dillon et al., 2004), a product generated by the addition of one atom of oxygen, and monoperoxyA2E, resulting from the addition of molecular oxygen (O2) at two carbon-carbon double bonds (see figure 44.1). In vitro studies indicate that the photo-oxidation of A2E can involve the incorporation of as many as nine oxygens into the retinoid-derived side-arms of A2E; thus we anticipate that a complex mixture of oxidized products may form in vivo, with multiple oxygen-containing moieties situated within an A2E molecule. Moreover, mass spectral studies also demonstrate that irradiation-induced oxidation can lead to A2E fragmentation. Evidence indicates that the production of reactive oxidized forms of A2E may account at least in part for the cellular damage ensuing from photochemical mechanisms initiated by blue light excitation of A2E (Sparrow, Vollmer-Snarr, et al., 2003). Indeed, the amount of A2E that undergoes photo-oxidation and cleavage in a lifetime may be significant: high performance liquid chromatography (HPLC) quantitation of A2E and its precursor, A2PE, in posterior eyecups of young mice (9 weeks) revealed that the amounts of A2PE that form are

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greater than is reflected in the levels of A2E that accumulate, an observation that indicates that some portion of A2E may undergo degradation (Kim et al., 2006), perhaps subsequent to oxidation. Work in the Abca4/Abcr−/− mouse has further served in the detection and characterization of diretinal RPE lipofuscin constituents other than A2E. In particular, we found that condensation reactions between two all-trans-retinal leads to the generation of an aldehyde-bearing dimer (all-trans-retinal dimer, atRAL dimer), which then forms conjugates with primary amines such as phosphatidylethanolamine via Schiff base linkages (Fishkin et al., 2004, 2005; Kim et al., 2007b) (see figure 44.1). The pigment all-trans-retinal dimer-PE that forms through this unique biosynthetic pathway has a structure that is distinct from A2E/isoA2E and an absorbance maximum in the visible spectrum that is red-shifted relative to A2E (A2E/iso-A2E, λmax ca. 435 nm; atRAL dimer-PE, λmax ca. 510 nm). Another ca. 510 nm absorbing dimerconjugate, atRAL dimer-E, forms by phosphate cleavage of atRAL dimer-PE. Both atRAL dimer-PE and atRAL dimer-E have been identified in the lipofuscin-filled RPE of Abca4/Abcr−/− mice and are present at higher levels in the mutant mice than in wild-type mice (see figure 44.4A).

The Leu450Met variant in murine Rpe65 is associated with reduced levels of retinal pigment epithelial lipofuscin As part of our effort to understand factors that modulate RPE lipofuscin formation, we have studied the relationship between the kinetics of the visual cycle and A2E formation. The Rpe65 polymorphism present in inbred strains of mice is such that the amino acid variant at residue 450 is either leucine or methionine (Danciger et al., 2000). From biochemical and electroretinographic studies it was apparent that in C57BL/6J mice that express methionine at codon 450, regeneration of rhodopsin is slowed and photon catch is reduced as compared to BALB/cByJ mice, which have leucine at that position (Wenzel et al., 2001, 2003; Nusinowitz et al., 2003). To determine whether the reduced availability of all-trans-retinal is accompanied by a decrease in RPE lipofuscin formation, we relied on quantitative HPLC to measure A2E and iso-A2E levels in mice homozygous for either the Leu-450 or Met-450 allele (Kim et al., 2004) (figure 44.5). Accordingly, whether the comparison was made between C57BL/6J-c2J (Met-450) and BALB/cByJ (Leu-450) mice or between Leu-450 and Met-450 in Abca4/ Abcr−/− mice, the levels of A2E in the presence of the methionine variant were consistently 25%–30% of that occurring with the leucine variant. The difference in the quantity of A2E associated with the Leu-450 variant versus the Met-450 variant occurred in pigmented and albino mice and in mice of varying ages. It is likely that the reduced flux of all-

trans-retinal that accompanies slowing of the visual cycle is responsible for the decreased formation of A2E in the presence of the methionine variant in C57BL/6J-c2J mice. The slowing of the visual cycle at the stage involving RPE65, a protein that has a rate-determining role in the visual cycle, can clearly provide protection against RPE lipofuscin formation.

Targeting the visual cycle to inhibit the formation of retinal pigment epithelial lipofuscin fluorophores

Figure 44.5 The levels of A2E and iso-A2E in mouse eyecups varies in relation to the Rpe65 Leu450Met variant. A2E and isoA2E were measured in wild-type (BALB/cByJ and C57BL/6J-c2J) mice (A) and in Abca4/Abcr−/− mice expressing either the Leu-450 or Met-450 variant of Rpe65 (B). Levels (mean ± SEM) were determined from HPLC chromatograms as integrated peak areas normalized to an external standard of A2E.

Given the adverse behavior of the diretinoid pigments that form RPE lipofuscin (Sparrow et al., 1999, 2000; Schutt et al., 2000; De and Sakmar, 2002; Sparrow et al., 2002; Sparrow, Fishkin, et al., 2003; Sparrow, Vollmer-Snarr, et al., 2003; Sparrow et al., 2006), there is considerable interest in retarding A2E formation as a means to prevent vision loss in Stargardt disease and perhaps in age-related macular degeneration. Accordingly, isotretinoin (13-cis-retinoic acid), the acne medication that induces night blindness and protects against light damage by retarding 11-cis-retinal regeneration (Sieving et al., 2001), can reduce A2E deposition in RPE of Abca4/ Abcr−/− mice (Radu et al., 2003). Isotretinoin is suggested to act, at least partially, by inhibiting 11-cis-retinol dehydrogenase (Sieving et al., 2001). Nonetheless, 13-cis-retinoic acid has severe side effects, including teratogenicity (Sparrow, 2003), and thus is not appropriate for long-term therapy. The evidence garnered from studies of the Rpe65 Leu450Met variant suggested that RPE65 would be an excellent target for therapeutic interventions aimed at reducing A2E formation in arSTGD and other retinal disorders characterized by aberrant RPE lipofuscin accumulation. Thus, in a collaborative effort, we tested nonretinoid isoprenoid compounds for their ability to compete with retinyl esters for binding to RPE65, thereby interfering with visual cycle kinetics (Maiti et al., 2006). Following a single intraperitoneal injection (50 mg/kg) of the compounds TDT (figure 44.6) and TDH (not shown) in mice, recovery of darkadapted ERG b-wave amplitudes to prebleach levels was delayed and 11-cis-retinal regeneration was slowed. Moreover, twice-weekly treatment of Abca4/Abcr−/− mice for 1 or 2 months resulted in substantial decreases in A2E accumulation (see figure 44.6). Thus, these nontoxic isoprenoid RPE65

Figure 44.6 Nonretinoid RPE65 antagonists suppress A2E/isoA2E formation in a mouse model of recessive Stargardt disease. Shown is the quantitation of A2E and iso-A2E in eyecups of Abca4/ Abcr−/− mice. Abca4/Abcr−/− mice were treated with TDT (50 mg/kg twice weekly, intraperitoneally) for 1 or 2 months beginning at age 10 weeks, and A2E and iso-A2E levels at the end of treatment (age

14 weeks or 18 weeks) were compared to levels in 10-week-old vehicle-treated (control) mice and 14- or 18-week-old vehicle-treated (control) mice. A2E/iso-A2E levels were determined from HPLC chromatograms by integrating peak areas and normalizing to external standards. Values are expressed as picomoles per eye and are based on single samples obtained by pooling data from four eyes.

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antagonists are candidates for the treatment of forms of macular degeneration in which lipofuscin accumulation is an important risk factor. In an alternative approach, the excessive accumulation of A2E in Abca4/Abcr−/− mice has been reduced by daily administration of the retinoic acid analogue N-(4-hydroxyphenyl)retinamide (HPR) for 1 month (Radu et al., 2005). As shown by the investigators, HPR acts by competing for binding sites on retinol-binding protein, thus reducing serum retinol levels. As a result, retinol uptake by the eye is reduced and visual cycle retinoids are decreased. The accompanying decrease in atRAL leads to retarded A2E formation.

Future directions Mouse models of retinal disease have elucidated the relationship between the visual cycle and RPE lipofuscin formation and will continue to be central to the development of gene- and drug-based therapies that can combat vision loss in disorders in which aberrant lipofuscin accumulation threatens RPE cell function and survival. Studies have shown that RPE lipofuscin in ABCR-associated disorders is similar in composition to age-related lipofuscin, although in ABCR-associated disorders the formation and accumulation occur at a faster rate. Work in mice should also clarify mechanisms involved in the higher levels of RPE lipofuscin reported in autosomal dominant Stargardt-like (STGD3) macular degeneration (Karan et al., 2005), an early-onset disorder due to mutations in ELOVL4 (Zhang et al., 2001; Bernstein et al., 2001; Maugeri et al., 2004), and in Best vitelliform macular dystrophy (BMD), an autosomal dominant form of macular degeneration caused by mutations in VMD2 (Petrukhin et al., 1998), the gene encoding bestrophin-1 (hBest1), a protein in RPE (Marmorstein et al., 2000). acknowledgments

Work was supported by National Institutes of Health grant no. EY 12951, Foundation Fighting Blindness, the American Health Assistance Foundation, the Steinbach Fund, and Research to Prevent Blindness. JRS is a recipient of an Alcon Research Institute Award.

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Parish, C. A., Hashimoto, M., Nakanishi, K., Dillon, J., and Sparrow, J. R. (1998). Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium. Proc. Natl. Acad. Sci. U.S.A. 95:14609–14613. Petrukhin, K., Koisti, M. J., Bakall, B., Li, W., Xie, G., Marknell, T., Sandgren, O., Forsman, K., Holmgren, G., et al. (1998). Identification of the gene responsible for Best macular dystrophy. Nat. Genet. 19:241–247. Radu, R. A., Han, Y., Bui, T. V., Nusinowitz, S., Bok, D., Lichter, J., Widder, K., Travis, G. H., and Mata, N. L. (2005). Reductions in serum vitamin A arrest accumulation of toxic retinal fluorophores: A potential therapy for treatment of lipofuscin-based retinal diseases. Invest. Ophthalmol. Vis. Sci. 46:4393– 4401. Radu, R. A., Mata, N. L., Nusinowitz, S., Liu, X., Sieving, P. A., and Travis, G. H. (2003). Treatment with isotretinoin inhibits lipofuscin and A2E accumulation in a mouse model of recessive Stargardt’s macular degeneration. Proc. Natl. Acad. Sci. U.S.A. 100:4742–4747. Redmond, T. M., Yu, S., Lee, E., Bok, D., Hamasaki, D., Chen, N., Goletz, P., Ma, J.-X., Crouch, R. K., et al. (1998). Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat. Genet. 20:344–351. Sakai, N., Decatur, J., Nakanishi, K., and Eldred, G. E. (1996). Ocular age pigment “A2E”: An unprecedented pyridinium bisretinoid. J. Am. Chem. Soc. 118:1559–1560. Schutt, F., Davies, S., Kopitz, J., Holz, F. G., and Boulton, M. E. (2000). Photodamage to human RPE cells by A2-E, a retinoid component of lipofuscin. Invest. Ophthalmol. Vis. Sci. 41:2303– 2308. Sieving, P. A., Chaudhry, P., Kondo, M., Provenzano, M., Wu, D., Carlson, T. J., Bush, R. A., and Thompson, D. A. (2001). Inhibition of the visual cycle in vivo by 13-cis retinoic acid protects from light damage and provides a mechanism for night blindness in isotretinoin therapy. Proc. Natl. Acad. Sci. U.S.A. 98:1835–1840. Sparrow, J. R. (2003). Therapy for macular degeneration: Insights from acne. Proc. Natl. Acad. Sci. U.S.A. 100:4353–4354. Sparrow, J. R., and Boulton, M. (2005). RPE lipofuscin and its role in retinal photobiology. Exp. Eye Res. 80:595–606. Sparrow, J. R., Cai, B., Jang, Y. P., Zhou, J., and Nakanishi, K. (2006). A2E, a fluorophore of RPE lipofuscin, can destabilize membrane. Adv. Exp. Med. Biol. 572:63–68. Sparrow, J. R., Fishkin, N., Zhou, J., Cai, B., Jang, Y. P., Krane, S., Itagaki, Y., and Nakanishi, K. (2003). A2E, a byproduct of the visual cycle. Vision Res. 43:2983–2990. Sparrow, J. R., Nakanishi, K., and Parish, C. A. (2000). The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells. Invest. Ophthalmol. Vis. Sci. 41:1981–1989. Sparrow, J. R., Parish, C. A., Hashimoto, M., and Nakanishi, K. (1999). A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture. Invest. Ophthalmol. Vis. Sci. 40:2988–2995. Sparrow, J. R., Vollmer-Snarr, H. R., Zhou, J., Jang, Y. P., Jockusch, S., Itagaki, Y., and Nakanishi, K. (2003). A2Eepoxides damage DNA in retinal pigment epithelial cells. Vitamin E and other antioxidants inhibit A2E-epoxide formation. J. Biol. Chem. 278:18207–18213. Sparrow, J. R., Zhou, J., Ben-Shabat, S., Vollmer, H., Itagaki, Y., and Nakanishi, K. (2002). Involvement of oxidative mechanisms in blue light induced damage to A2E-laden RPE. Invest. Ophthalmol. Vis. Sci. 43:1222–1227.

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Weng, J., Mata, N. L., Azarian, S. M., Tzekov, R. T., Birch, D. G., and Travis, G. H. (1999). Insights into the function of Rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in Abcr knockout mice. Cell 98:13–23. Wenzel, A., Grimm, C., Samardzija, M., and Reme, C. E. (2003). The genetic modified Rpe65Leu450: Effect on light damage susceptibility in c-Fos-deficient mice. Invest. Ophthalmol. Vis. Sci. 44:2798–2802. Wenzel, A., Reme, C. E., Williams, T. P., Hafezi, F., and Grimm, C. (2001). The Rpe65 Leu450Met variation increases retinal resistance against light-induced degeneration by slowing rhodopsin regeneration. J. Neurosci. 21:53–58. Zhang, K., Kniazeva, M., Han, M., Li, W., Yu, Z., Yang, Z., Li, Y., Metzker, M. L., Allikmets, R., et al. (2001). A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy. Nat. Genet. 27:89–93.

45

Studies of Diabetic Retinopathy Using Mice ROSE A. GUBITOSI-KLUG AND TIMOTHY S. KERN

Diabetic retinopathy is a progressive retinal disease that threatens patients with blindness. The retinopathy develops slowly, over decades in humans, making investigation of its pathogenesis challenging. Many animal models have been developed to study the evolution of the retinopathy (Kern and Mohr, 2008). The rate of development of the early stages of the retinopathy seems to vary among species, with at least the early stages of the retinal disease apparently progressing faster in smaller species such as the mouse. The relatively short interval until pathology presents, coupled with the ability to genetically engineer the mouse, has prompted an increased interest in mouse models. This chapter reviews mouse models of diabetic retinopathy, compares pathological changes in the retina of the diabetic mouse with those in humans, and highlights advances made using mice to understand the pathogenesis of the retinopathy.

Diabetic retinopathy in humans Diabetic retinopathy has classically referred to alterations in the retinal microvasculature visualized on ophthalmoscopic examination (Davis et al., 1997). Over the course of disease progression, two stages of retinal pathology have been defined: 1. Retinal lesions observed clinically in early retinopathy, or the nonproliferative stage of retinopathy (previously referred to as background retinopathy), consist of nonperfused capillaries, microaneurysms, retinal hemorrhages, cotton wool spots, exudates, and edema. Each of these lesions is believed to represent local alteration in the microvascular circulation of the retina, which in some cases results in damage to the surrounding retinal cells. Histological studies using techniques to isolate the retinal vasculature have demonstrated that the vascular histopathology includes capillary microaneurysms, pericytedeficient capillaries, basement membrane thickening, and degenerate (acellular) capillaries (Aguilar et al., 2003; Cogan and Kuwabara, 1967; Engerman, 1989; Kohner and Henkind, 1970). In addition, histological analysis of the human retina has demonstrated changes in the nonvascular retina as well, including ganglion cell loss (Barber et al., 1998; Bloodworth and Molitor, 1965; Bresnick and Palta, 1987a,

1987b; Lieth et al., 2000). Currently, several different therapies, including intensive insulin therapy and blood pressure medications, are available to inhibit the development of the nonproliferative stage of diabetic retinopathy (The Diabetes Control and Complications Trial Research Group, 1993; Jandeleit-Dahm and Cooper, 2006; Zhang et al., 2001). 2. Subsequently, a proliferative retinopathy develops in about 10%–15% of diabetic patients, with marked growth of new blood vessels growing out of the existing retinal vasculature onto the surface of the retina and into the vitreous. These newly formed blood vessels (as well as other blood vessels in the retina) leak, occasionally resulting in vitreous hemorrhages, which can obstruct vision (Cunha-Vaz et al., 1975). In addition, with the development of these new vessels a preretinal fibrovascular membrane can form that also can obstruct vision. The neovascularization, preretinal hemorrhage, and membranes currently can be corrected using photocoagulation or vitrectomy, respectively. In addition to vitreous hemorrhages, other late retinal changes that lead to visual impairment in diabetic retinopathy include retinal detachments (caused by traction of the fibrovascular membrane associated with new vessel formation) and retinal edema secondary to breakdown of the blood-retinal barrier and leakage of plasma from the microvasculature. The evolution of these stages of diabetic retinopathy in humans occurs over years, with nonproliferative changes first detected at 5–10 years of diabetes and proliferative changes developing later.

Diabetic retinopathy in mice The desire to investigate the pathogenesis of diabetic retinopathy has driven the development and evaluation of many animal models of diabetic retinopathy. Of course, the better the human disease is replicated, the better the animal model. To date, the diabetic dog is the animal model that best reproduces the nonproliferative stages of diabetic retinopathy, reproducibly developing lesions seen rarely or not at all in rodent models (Engerman, 1989; Engerman and Bloodworth, 1965; Kern and Mohr, 2007). Because retinopathy in the dog requires 3–5 years to develop, however, the associated time and cost make using this model cumbersome.

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Moreover, the availability of antibodies and reagents to conduct molecular studies is very limited for dogs. Mice develop degenerate, acellular capillaries in the retina, a critical step in the progression of diabetic retinopathy, as well as some other lesions characteristic of the early stages of retinopathy (Feit-Leichman et al., 2005). The mouse model has a number of advantages over larger models, including lesser space requirements and cost. The mouse also allows genetic manipulation and relatively easy administration of limited pharmacological therapies. Anatomical differences between the mouse and human retina do exist. The mouse retina has multiple arteries emanating from the optic nerve that feed the different regions of the retina (figure 45.1), whereas the human retina receives its blood supply from one main artery, the ophthalmic artery, which divides only after entering the retina. In addition, retinas from mice (as well as all other nonprimate species) differ from the human retina in that only humans and primates possess a macula (see figure 45.1). Initial investigations using the mouse as a model of diabetic retinopathy yielded controversial results, but, more recent results have shown the mouse model to reliably develop capillary degeneration, pericyte loss, and capillary basement membrane thickening (Feit-Leichman et al., 2005; Hammes et al., 2002; Joussen et al., 2004; Midena et al., 1989). Diabetes can be induced experimentally using drugs such as streptozotocin and alloxan to disrupt pancreatic beta cells, thus reproducing an insulinopenia like that of type I diabetes. Several strains of mice (including the C57BL/6JIns2Akita [Akita], db/db, KK.Cg-Ay/J [KKAY], and NOD strains) develop diabetes spontaneously and have microvascular changes of the retina consistent with nonproliferative retinopathy (capillary degeneration, pericyte loss, thickening of capillary basement membrane) over a period of 6–9 months (Barber et al., 2005; Cheung et al., 2005; FeitLeichman et al., 2005; Naeser and Agren, 1978; Ning et al.,

2004; Whetzel et al., 2006). The Akita and NOD strains are models of type I diabetes, and db/db and KKAY strains are models of type II diabetes. Retinal function, as demonstrated by the electroretinogram (ERG), becomes impaired in diabetic mice and other models (Barile et al., 2005; Zheng et al., 2007). Whether or not diabetes causes degeneration of retinal neurons in mice is an area of controversy. Some investigators have reported extensive degeneration of retinal ganglion cells (RGCs) in C57BL/6J mice after diabetes duration as little as 14 weeks (Martin et al., 2004), whereas others have not detected retinal neurodegeneration in that strain even after durations of up to 1 year (Feit-Leichman et al., 2005). Retinal neurodegeneration has been detected in a different diabetic mouse strain, the Akita. Whether or not neuronal cell loss plays an important role in diabetic retinopathy and eventual visual impairment remains an open question. To date, diabetic mice have not been found to develop preretinal neovascularization secondary to diabetes. (Likewise, neither experimentally diabetic dogs, cats, rats, nor other species have been found to develop the proliferative retinopathy; Kern and Mohr, 2007.) The relatively short duration of diabetes in most studies and the resulting modest extent of capillary degeneration in studies of mice are believed to be the reason that neovascularization has not been observed in these models. Advanced proliferative retinopathy similar to that seen in diabetic patients does develop in nondiabetic mice, however, under specific stresses or gene alterations (Lai et al., 2005; Ruberte et al., 2004; Smith et al., 1999). This is discussed further in a later section. The mouse models used to study the nonproliferative vascular lesions, neurodegenerative changes, and proliferative stages of diabetic retinopathy are detailed in the next section. Each model is evaluated based on the histopathological changes observed and the pathogenic mechanisms tested using these models.

Mouse models of vascular lesions of nonproliferative retinopathy

Figure 45.1 The retinal vasculature of the mouse, following isolation by proteolytic digestion of the formalin-fixed retina. See color plate 45.

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Chemically Induced Diabetes The C57BL/6J mouse is the strain of mouse most used in studies of diabetic retinopathy. Typically, diabetes is induced as a result of a 3- to 5-day course of streptozotocin (50–75 mg/kg of body weight), although other single-dose regimens exist. Several days after chemical induction, randomly sampled blood glucose levels of 250 mg/dL or higher denote diabetes. Long-term monitoring of glycohemoglobin (HbA1c or GHb) ensures comparable degrees of hyperglycemia among experimental groups. Vascular changes consistent with diabetic retinopathy, including acellular capillaries, pericyte loss, and capillary cell apoptosis, become apparent beginning about 6 months after the onset of diabetes (figure 45.2)

Figure 45.2 Degenerate (acellular) capillaries (arrows) in the diabetic mouse retina. The degenerate vessels lack vascular cell nuclei and have an irregular or shrunken diameter compared to surrounding healthy capillaries. See color plate 46.

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Figure 45.3 Frequency of acellular capillaries (A) and pericyte loss (B) increase with duration of diabetes in the C57BL/6J mouse.

(Feit-Leichman et al., 2005). The number of acellular capillaries and pericyte ghosts increases with diabetes duration (figure 45.3). Maintaining diabetes for 12 months or more is difficult, owing to increasing mortality, but administration of insulin in doses sufficient to prevent body weight loss but not reduce hyperglycemia (0.1–0.2 U; 2–3 times per week) seems to reduce mortality. Several characteristic lesions of early diabetic retinopathy in humans (including microaneurysms and retinal hemorrhages) have not been reproducibly detected in diabetic mice over the 6–12 months that the animals commonly are studied.

Spontaneous Diabetes Akita mouse. The Akita mouse develops type I diabetes spontaneously due to a dominant point mutation in the insulin 2 gene (Barber et al., 2005). Heterozygote male mice show hyperglycemia and hypoinsulinemia by 4 weeks of age. By 12 weeks of age, the diabetic mice develop increased vascular permeability compared to sibling wild-type mice. Acellular capillary formation and pericyte loss become statistically greater than in nondiabetic animals at about 6 months of diabetes, and increase with longer diabetes duration (Barber et al., 2005).

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db/db mouse. The db/db mouse is a genetic model of type II diabetes. A mutation in the leptin receptor gene leads to obesity and subsequent obesity-induced type II diabetes mellitus. These mice have been reported to develop an increased endothelial cell:pericyte ratio (Midena et al., 1989). This was interpreted as a reduction in the number of capillary pericytes, but this ratio could also increase as a result of endothelial cell proliferation (Cuthbertson and Mandel, 1986). Diabetes-induced vessel leakage, degeneration of capillaries, thickening of retinal capillary basement membrane, and increased expression of vascular endothelial growth factor (VEGF, the protein encoded by gene Vegfa) and platelet/ endothelial cell adhesion molecule-1 (PECAM-1, the protein encoded by gene Pecam1) have been detected at 15 months of age (Cheung et al., 2005). db/db mice were crossed with apolipoprotein E (Apoe)deficient mice to generate insulin-deficient and hyperlipidemic animals (Barile et al., 2005). The superimposition of hyperlipidemia on diabetes resulted in accelerated development of acellular capillaries and pericyte loss at 6 months of diabetes (Barile et al., 2005). This same hyperglycemic, hyperlipidemic model was used to study the role of advanced glycation end products (AGEs) in the development of diabetic retinopathy. Diabetes-induced capillary degeneration and pericyte loss could be inhibited by the administration of soluble receptors for AGEs (sRAGE) daily from 8 weeks to 6 months of diabetes (Barile et al., 2005), thus suggesting that AGEs play a role in the development of retinal histopathology in this model. Consistent with the conclusions of that study, the diabetes-induced thickening of retinal capillary basement membranes in the outer plexiform layer (OPL) of the retina from diabetic db/db mice was not observed after intraperitoneal injection with antibodies against glycated albumin (Clements et al., 1998). Whether or not this therapeutic approach also had effects on other aspects of the microvascular disease was not determined. Aldose reductase deficiency in db/db mice, achieved by crossing db/db mice and aldose reductase null mutation mice, resulted in less of the diabetes-induced increase in vascular permeability, VEGF expression, and PECAM-1 expression than that seen in control db/db mice (Cheung et al., 2005). The effect of aldose reductase deficiency on the development of retinal histopathology has not been reported to date in diabetic mice, but results in other species have been mixed (Dagher et al., 2004; Engerman and Kern, 1993). KKAY mouse. The KKAY mouse develops diabetes mellitus when fed a high fat diet, thus, it is considered a genetic model of type II diabetes mellitus (Siracusa, 1994). Control animals are fed a standard diet. After 3 months of diabetes, KKAY mice demonstrated microvascular changes that included variable basement membrane thickening, mitochondrial swelling, capillary peripheral cell edema and

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degeneration, and endothelial cell hyperplasia, as detected by transmission electron microscopy (Ning et al., 2004). These animals have not been studied with respect to susceptibility to developing capillary degeneration, microaneurysms, or other characteristic vascular histopathology. NOD mouse. The nonobese diabetic mouse (NOD) develops diabetes through an autoimmune-mediated destruction of pancreatic beta cells (Leiter et al., 1987). The resulting insulitis leads to spontaneous hyperglycemia, again modeling type I diabetes in humans. Retinas from the NOD mouse have not yet been examined for the development of the characteristic microvascular lesions of diabetic retinopathy. ob/ob mouse. ob/ob mice are leptin deficient, and phenotypically are obese by 4 weeks of age. Regression of pancreatic islets results in severe diabetes and early death when on the C57BLKS background. In control animals (C57BL/6J background), the mice are only transiently hyperglycemic, blood glucose returning to normal by 14–16 weeks. Retinas from these obese, hyperglycemic mice were morphologically the same as in littermate controls, with a similar endothelial: pericyte ratio and absence of microaneuryms (Naeser and Agren, 1978). Nondiabetic Mouse Models That Develop Diabetic-Like Retinopathy Galactose feeding. Feeding nondiabetic mice a high-galactose diet results in systemic elevation of blood galactose levels, but glucose levels remain normal. This model confirms the causative role of elevated hexose in the pathogenesis of diabetic retinopathy, since the other metabolic alterations characteristic of insulin deficiency, including altered lipid metabolism, are not observed (Engerman and Kern, 1984). In mice fed 30% galactose, the retinopathy can be followed for up to 24 months because the animals generally are quite healthy. Mice (C57BL/6J or BALB/cJ) fed 30% galactose develop a diabetic-like retinopathy with acellular capillaries, pericyte ghosts, basement membrane thickening, and occasional saccular microaneurysms ( Joussen et al., 2004; Kern and Engerman, 1996). The advantages of this model are that experimental galactosemia is easy to establish and the animals require less care than diabetic animals. A possible deficiency of the model is that galactosemic retinopathy might differ in several ways from that in diabetes (Frank et al., 1997; Kern et al., 2000; Mohr et al., 2002). Moreover, the cost of the galactose diet can be expensive for large experimental groups. Plasminogen activator inhibitor-1(PAI-1). PAI-1 regulates serine proteases known as plasminogen activators (PAs). PAs convert plasminogen to plasmin, which then degrades fibrin, as well as basement membrane components such as laminin and fibronectin. A transgenic mouse model was developed

with the human PAI-1 gene sequence (SERPINE1) under regulation of the metallothionein promoter (Grant et al., 2000). Administration of ZnSO4, which allows for continuous expression of the transgene, resulted in increased PAI-1 immunoreactivity in the retinal capillaries. Electron microscopy demonstrated increased basement membrane thickening in the PAI-1-overexpressing mice compared to control animals. Whole-mount retinal analysis revealed an increased endothelial cell:pericyte ratio (even in the absence of diabetes), suggesting that PAI-1 might contribute to alterations in vascular structure. Degeneration of capillaries in diabetes has not been determined in this model. Platelet-derived growth factor(PDGF-B). PDGF-B (encoded by gene Pdgfb) is an important factor in pericyte recruitment, and studies support a role for pericytes in the formation and stabilization of vessels. Mice that are heterozygous for Pdgfb (i.e., with one functional allele) have fewer pericytes and a significant increase in the number of acellular capillaries in their retinas compared to wild-type littermates (Hammes et al., 2002). Under diabetic conditions, the retinopathy of the Pdgfb heterozygous mice was worsened, with increased formation of acellular capillaries and lesions regarded as microaneurysms. In addition, studies of nondiabetic mice deficient in endothelium-specific PDGF-B demonstrated pericyte deficiency associated with capillaries of variable diameter, density, and ring structure, as well as microaneurysms (Enge et al., 2002). This suggests an important role for PDGF-B in the development of diabetic retinopathy.

Use of mouse models to study diabetes-induced retinal neurodegeneration Of the many experimental models of developing retinal microvascular lesions described in the previous section, only a few have been examined for neurodegenerative changes to date. The mouse models evaluated thus far are reviewed in this section. Techniques to quantify neurodegenerative changes in the retina include counting the number of cells in the ganglion cell layer (GCL) per length of retina, quantifying the number of retinal neurons that are immunopositive for activated caspase-3 (encoded by gene Casp3) or TUNEL stain, or measuring the thickness of the retina or neuroretinal layers. C57BL/6J mouse. The C57BL/6J mouse strain has been evaluated for diabetes-induced neurodegeneration in the retina. Some investigators reported a 20%–25% reduction in cells of the GCL of the retina as early as 14 weeks of diabetes (Martin et al., 2004). In contrast, others did not detect any GC loss at diabetes durations of up to 1 year (FeitLeichman et al., 2005). No explanation for these different conclusions has yet been put forth. Retinal glia cells also have

been reported to show signs that suggest cell death in diabetes (Kusner et al., 2004). Translocation of glyceraldehyde-3phosphate dehydrogenase (GAPDH) to the nucleus, which is strongly associated with cell death in cerebral injury (Tatton et al., 2000), has also been demonstrated in retinal Müller cells of diabetic mice and humans (Kusner et al., 2004). Akita mouse. In the Akita mouse at 22 weeks of hyperglycemia, neurodegenerative changes detected included a reduction in (1) the thickness of the inner plexiform layer in the central and peripheral portions of the retina, (2) the thickness of the inner nuclear layer, and (3) the number of nuclei in the RGC layer (Barber et al., 2005). Although morphological alterations in the astrocytes and microglia were observed, no changes in glial fibrillary acidic protein (GFAP) immunoreactivity were noted. After 4 weeks of diabetes, caspase-3 immunoreactivity increased, suggesting increased apoptosis of some cells of the neuroglial retina. db/db mouse. At 15 months of age, db/db mice have been reported to demonstrate accelerated ganglion cell apoptosis and glial cell activation as detected by GFAP staining (Cheung et al., 2005). At 6 months of diabetes, db/db mice crossed with Apoedeficient mice had abnormal function of retinal neurons as characterized by abnormal electroretinograms (ERGs) (Barile et al., 2005). Aldose reductase deficiency in the db/db mouse resulted in a reduction in the number of caspase-3 immunoreactive cells and GFAP-positive cells compared to the wild-type db/db diabetics (Cheung et al., 2005). KKAY mouse. In this model, neurodegeneration was quantified by the use of TUNEL staining of ultrathin sections of fixed retinas at 1 and 3 months of diabetes (Ning et al., 2004). An increased rate of neuronal cell loss in the ganglion cell in diabetic compared to control animals has been reported. The number of apoptotic cells increased with the duration of diabetes in both the RGC layer and the inner nuclear layer.

Biochemical changes in the mouse retina that contribute to the development of diabetic retinopathy In both clinical trials and experimental studies of diabetic models, hyperglycemia has been found to be closely associated with the development of the spectrum of lesions regarded as diabetic retinopathy (Engerman and Kern, 1995; Zhang et al., 2001). Diabetes-induced death of retinal capillary and neuroglial cells likely is due to (1) metabolic abnormalities within those cells or (2) extracellular events, such as capillary obstruction by leukocytes, platelets, or erythrocytes, or receptor-mediated processes initiated by binding of AGE or

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insulin-like growth factor I (IGF-I) (figure 45.4). Oxidative stress, protein kinase C, the sequelae of nonenzymatic glycation, altered metabolism of sugar through glycolysis and the polyol pathways, and inflammatory processes have been postulated to be intracellular abnormalities contributing to the pathogenesis of the retinopathy ( Joussen et al., 2004; Kowluru, 2002; Moore et al., 2003; Ruberte et al., 2004; Vlassara, 2001). The respective contributions of these sequelae of hyperglycemia to the pathogenesis of diabetic retinopathy remain under investigation. A comparison of metabolic abnormalities in the retinas of diabetic mice and rats has demonstrated both similarities and differences. At 2 months’ duration of diabetes, diabetesinduced reductions in glutathione and increases in nitric oxide, lipid peroxide, and protein kinase C activity in the mouse retina paralleled results in the rat model (Kowluru, 2002). Another study compared the mouse and rat models using a different panel of biochemical changes at 6 weeks of diabetes (Obrosova et al., 2006). Whereas the rat model showed enhanced VEGF expression, increased poly(ADPribosyl)ation, and decreased antioxidant enzyme activities, the mouse model reflected none of these changes. However, documentation of long-term hyperglycemia was not presented in the latter publication, and differences in the severity of hyperglycemia between the studies might have contributed to the differences between the rat and mouse models. Overall, it seems that the biochemical alterations in the mouse model are less severe than in the rat model, although in both models animals develop a similar severity of capillary degeneration at approximately the same duration of diabetes (6–8 months). The administration of pharmacological therapies that can inhibit a particular biochemical defect or the induction of diabetes in mice having selective gene deficiencies has pro-

Figure 45.4 Leukocyte adherence, or leukostasis (arrows), in the retinal microvasculature of the diabetic mouse. See color plate 47.

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vided novel insights into the pathogenesis of the early stages of diabetic retinopathy. Several of these areas of investigation are summarized here. Advanced glycation end products. AGEs form and accumulate in the retinal microvascular cells of diabetic animals and have been linked to generation of oxidative stress and activation of NFkB, a transcription factor that regulates expression of a variety of pro-inflammatory proteins, including inducible nitric oxide synthase (iNOS, which is encoded by gene Nos2) and intracellular adhesion molecule-1 (ICAM-1, the protein encoded by gene Icam1) (Goldin et al., 2006; Vlassara, 2001). To assess in vivo the role of AGEs in retinal microvascular disease, nondiabetic C57BL/6J mice received 7 daily intraperitoneal injections of AGE-albumin or albumin alone as control (Moore et al., 2003). The mice receiving AGE-albumin demonstrated a threefold increase in NFkB expression compared to control animals, as well as a significant increase in Icam1 mRNA, leukocyte adherence, and blood-retinal barrier breakdown. Moreover, hyperlipidemic diabetic mice were protected from capillary degeneration, pericyte loss, and neural dysfunction by administration of sRAGE (a soluble form of the receptor for AGE, which is a therapy that reduces binding of AGEs to cellular receptors) (Barile et al., 2005). Aldose reductase. Aldose reductase (AR) is the first enzyme in the polyol pathway. During hyperglycemia, excess glucose is converted by AR to sorbitol and then potentially to fructose by sorbitol dehydrogenase. AR activity is increased in diabetes, although more modestly so in the mouse retina than in the rat retina (Kowluru, 2002; Obrosova et al., 2006). Transgenic mice overexpressing human AR (AKR1B1) fed a galactose-rich diet for 7 days demonstrated occlusion of the retinochoroidal vessels. AR (encoded by gene Akr1b3)deficient mice crossed with db/db mice were protected from diabetes-associated microvascular and neurodegenerative changes (Cheung et al., 2005). Angiopoietin. Pericyte loss is heralded as an early morphological change in diabetic retinopathy, in part because pericytes are believed to regulate endothelial proliferation. Evidence indicates that angiopoietin-1 and -2 regulate vessel sprouting and regression through pericyte recruitment. Both angiopoietin-1 and -2 have been evaluated in mouse models for roles in diabetic retinopathy pathogenesis. At 16 weeks of diabetes, mice injected via tail vein with an adenovirus coding for human angiopoietin-1 (gene ANGPT1) demonstrated less leukocyte adherence, reduced endothelial cell damage or death (as detected by propidium iodide staining), and inhibition of blood-retinal barrier breakdown ( Joussen et al., 2002). Recently, heterozygous deficiency of angiopoietin-2 (mouse gene Angpt2) in Ang-2 lacZ knock-in mice

prevented loss of pericytes and acellular capillary formation at 26 weeks of diabetes (Hammes et al., 2004). This suggests that angiopoietin-1 and -2 are involved in various proposed pathogenic mechanisms leading to diabetic retinopathy. Eicosanoids. Eicosanoids, metabolites of arachidonic acid, are known inflammatory mediators. 5-Lipoxygenase (Alox5) products, including leukotriene B4 and leukotriene C4/ D4/E4, function in leukocyte recruitment and vascular permeability, respectively. Over a period of 9 months of diabetes, Alox5 deficiency inhibited the degeneration of retinal capillaries (Gubitosi-Klug and Kern, 2006). In addition, diabetes-induced leukostasis (Gubitosi-Klug and Kern, 2006) and superoxide generation (Gubitosi-Klug and Kern, 2008) in the retina were significantly inhibited in the Alox5-deficient mice compared to diabetic wild-type controls. Intracellular adhesion molecule-1 and CD18. ICAM-1 (encoded by gene Icam1) and its ligand CD18 (encoded by gene Itgb2) are involved in the adherence of leukocytes to the vascular endothelium, especially in inflammatory conditions. The role of these molecules and leukostasis in diabetic retinopathy was explored using ICAM-1- and CD18-deficient mice ( Joussen et al., 2004). In both the streptozotocin-induced diabetic and galactosemic models, permeability of the retinal vasculature and the number of leukocytes adhering to the retinal vasculature were less in the diabetic ICAM-1- or CD18-deficient mice than in diabetic wild-type controls. Of note, capillary degeneration and pericyte loss also were inhibited in these genetically deficient animals compared to wild-type diabetic controls ( Joussen et al., 2004). Inducible nitric oxide synthase. Nitric oxide (NO) is an important signaling molecule in many physiological processes such as inflammation and cell survival. It is synthesized by two types of nitric oxide synthase, one that is expressed constitutively and another that is regulated or induced (iNOS) by cellular stimuli such as cytokines. After 9 months of diabetes, mice deficient in iNOS (encoded by gene Nos2) had developed significantly less degeneration of retinal capillaries than wild-type diabetic control animals (Zheng et al., 2007). Diabetic iNOS-deficient mice had less leukostasis and less production of nitric oxide, PGE2, and superoxide. Interleukin-1b. IL-1β (encoded by gene IL1b) is a proinflammatory cytokine that acts through IL-1β receptors on cells. The role of IL-1β in the development of diabetic retinopathy has been investigated in two mouse models. Inhibition of caspase-1 activity (the enzyme that activates IL-1β) by administration of minocycline inhibited retinal levels of IL-1β and inhibited the degeneration of retinal capillaries in C57BL/6J mice having chemically induced diabetes or

galactosemia (Vincent and Mohr, 2007). As a further confirmation of the role of IL-1β in degeneration of retinal capillaries, mice lacking the IL-1β receptor likewise were protected from degeneration of retinal capillaries in diabetes (Vincent and Mohr, 2007). Superoxide dismutase. It has been demonstrated in rats (Du et al., 2003) and mice (unpublished results) that diabetes induces an increase in superoxide production. Retinal mitochondrial superoxide dismutase activity has been found to be subnormal in diabetic rats (Du et al., 2003), likely contributing to this increase in superoxide. A recent report examined the effect of mitochondrial superoxide dismutase (encoded by gene Sod2) overexpression on retinal oxidative and nitrative stress parameters at 7 weeks of diabetes duration (Kowluru et al., 2006). In contrast to wild-type diabetic mice, diabetic mice overexpressing Sod2 did not show subnormal glutathione levels or elevated nitrotyrosine levels. Possible effects on the development of retinal lesions in diabetes have not been reported to date.

Mouse models of proliferative retinopathy To date, diabetic mice have not been found to develop proliferative (preretinal neovascularization) retinopathy without the superimposition of some other stress. Thus, studies of retinal neovascularization have relied on other pathological processes that result in new vessel growth in the retina. The nondiabetic models of retinal neovascularization are being used as models of the neovascular process in diabetic proliferative retinopathy. Available evidence suggests that VEGFmediated neovascularization in diabetes is similar to that underlying other conditions of retinal neovascularization, such as retinopathy of prematurity. Non-VEGF-mediated mechanisms of neovascularization have been identified recently, and the ability of the nondiabetic and acute models to reproduce the complexity of all the stimuli of neovascularization induced by diabetes remains to be learned. Oxygen-induced retinopathy. This model differs from that of diabetic retinopathy in that the retina in oxygen-induced retinopathy is immature and poorly differentiated. Newborn mice, typically C57BL/6J, are exposed from P7 to P12 to hyperoxic conditions, during which interval vascular development is arrested. The mice then are returned to room air, the retinas thus becoming relatively “hypoxic” as a result of the inadequate vascularization of the retina. Preretinal neovascularization and upregulation of VEGF develop during the subsequent days in room air (Smith et al., 1994). Using this mouse model, multiple in vivo studies have been done to investigate candidate antiangiogenic drugs. For example, inhibition of neovascularization has been achieved by intravitreal injection of soluble VEGF receptor/IgG

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fusion proteins, Vegfa antisense oligonucleotides, neutralizing VEGF antibodies, soluble erythropoietin receptor, and intravitreal injection of MAE 87, a receptor tyrosine kinase inhibitor (Agostini et al., 2005; Aiello et al., 1995; Robinson et al., 1996; Sone et al., 1999; Watanabe et al., 2005). In addition, insulin receptor and IGF-I receptor gene deficient mice are protected from hypoxia-induced retinal neovascularization (Kondo et al., 2003). Lentivirus-mediated expression of angiostatin, a derivative of plasminogen, also inhibited this hypoxia-induced retinal neovascularization (Igarashi et al., 2003). An important role of nitric oxide in the neovascular response and retinal development has been demonstrated in mice lacking iNOS. Oxygen-induced retinopathy induced less apoptosis and thinning in the inner nuclear layer and retina, respectively, of newborn iNOS knockout mice than in wild-type controls (Sennlaub et al., 2002). Fewer TUNELpositive cells were found in the avascular retina and ONL and GCLs of the retina of these animals. Decreased 3nitrotyrosine production also was detected in retinas from the iNOS-deficient mice compared to wild-type controls. The iNOS inhibitor 1400W injected intravitreally gave similar results with less apoptosis in the INL and less intravitreal neovascularization. Increased cyclooxygenase-2 (COX-2) activity also has been implicated in angiogenesis in tumors, and neovascularization in cornea and retina. In the mouse model of oxygen-induced retinopathy, preretinal (intravitreal) neovascularization was inhibited using selective COX-2 inhibitors (Sennlaub et al., 2003). Surprisingly, COX-2 (encoded by gene Ptgs2)-deficient mice showed an increased area of retinal nonperfusion, suggesting that COX-2 also might have a vasoprotective effect in the ischemic retina (Cryan et al., 2006). This was not confirmed using the COX-2 inhibitor (Sennlaub et al., 2003). Endothelial cell interactions with extracellular matrix proteins, such as fibronectin, are a critical step in angiogenesis. This integrin-mediated adhesive interaction can be inhibited by small peptides, such as the α-defensins, which are activated during inflammatory processes. Administration of αdefensins systemically or locally blocked neovascularization in the oxygen-induced retinopathy mouse model (Economopoulou et al., 2005). ob/ob mice develop less neovascularization than controls in the oxygen-induced retinopathy model. Opposite of these results in leptin-deficient mice, overexpression of leptin in this mouse model worsened neovascularization (Suganami et al., 2004). The role of leptin in the development of diabetes-induced retinal neovascularization remains to be clarified. VEGF overexpression. VEGF has been implicated in diabetic retinopathy and is a known potent angiogenic factor in

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normal retinal development (Saint-Geniez and D’Amore, 2004). Intravitreal injection of human VEGF (VEGFA) causes severe neovascularization in several animal models (Ozaki et al., 1997; Tolentino et al., 1996; Wong et al., 2001). Transgenic mice in which VEGFA expression was under the control of the mouse rhodopsin promoter developed intraretinal and subretinal neovascularization ranging from mild to severe (Lai et al., 2005; Vinores et al., 2000). VEGFA expression directed by a lens-specific promoter resulted in some abnormal growth of vessels on the retinal surface but lacked extension of the vessels into the vitreous, which is characteristic of the preretinal neovascularization in diabetes. The more severe phenotype included more extensive subretinal neovascularization with hemorrhages and retinal detachment (Lai et al., 2005). Insulin-like growth factor-I. IGF-I (encoded by gene IgfI ) stimulates retinal endothelial cell growth, and IGF-I signaling through the IGF-I receptor leads to increased VEGF levels (Smith et al., 1999). Retinas from nondiabetic mice that overexpress an IgfI transgene reportedly demonstrate lesions characteristic of nonproliferative diabetic retinopathy, including pericyte loss, thickened capillary basement membrane, and intraretinal microvascular abnormalities. In addition, these models have been reported to progress to proliferative retinopathy and retinal detachment (Ruberte et al., 2004).

Summary The use of mice to model diabetic retinopathy has allowed investigators to creatively test the contributions of various mechanisms in the pathogenesis of early, nonproliferative diabetic retinopathy (e.g., the roles of various pro-inflammatory mediators) and more advanced proliferative retinopathy (e.g., VEGF). Studies using pharmacological therapies to inhibit retinopathy have generated considerable excitement, but the lack of absolute specificity of most available pharmacological therapies complicates the interpretation of how they might be acting. Genetically modified mice will continue to be an important tool to further dissect the pathogenesis of retinopathy. Additional studies in the mouse hold the potential of offering new insight into the pathogenesis of diabetic retinopathy, and thus a means to identify new therapeutic approaches to inhibit the retinopathy. REFERENCES Agostini, H., Boden, K., Unsold, A., Martin, G., Hansen, L., Fiedler, U., Esser, N., and Marme, D. (2005). A single local injection of recombinant VEGF receptor 2 but not of Tie2 inhibits retinal neovascularization in the mouse. Curr. Eye Res. 30(4):249–257.

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Du, Y., Miller, C. M., and Kern, T. S. (2003). Hyperglycemia increases mitochondrial superoxide in retina and retinal cells. Free Radic. Biol. Med. 235(11):1491–1499. Economopoulou, M., Bdeir, K., Cines, D. B., Fogt, F., Bdeir, Y., Lubkowski, J., Lu, W., Preissner, K. T., Hammes, H. P., et al. (2005). Inhibition of pathologic retinal neovascularization by alpha-defensins. Blood 106(12):3831–3838. Enge, M., Bjarnegard, M., Gerhardt, H., Gustafsson, E., Kalen, M., Asker, N., Hammes, H. P., Shani, M., Fassler, R., et al. (2002). Endothelium-specific platelet-derived growth factor-B ablation mimics diabetic retinopathy. EMBO J. 21(16): 4307–4316. Engerman, R. L. (1989). Pathogenesis of diabetic retinopathy. Diabetes 38(10):1203–1206. Engerman, R. L., and Bloodworth, J. M., Jr. (1965). Experimental diabetic retinopathy in dogs. Arch. Ophthalmol. 73:205– 210. Engerman, R. L., and Kern, T. S. (1984). Experimental galactosemia produces diabetic-like retinopathy. Diabetes 33(1):97–100. Engerman, R. L., and Kern, T. S. (1993). Aldose reductase inhibition fails to prevent retinopathy in diabetic and galactosemic dogs. Diabetes 42(6):820–825. Engerman, R. L., and Kern, T. S. (1995). Retinopathy in galactosemic dogs continues to progress after cessation of galactosemia. Arch. Ophthalmol. 113(3):355–358. Feit-Leichman, R. A., Kinouchi, R., Takeda, R. M., Fan, Z., Mohr, S., Kern, T. S., and Chen, D. F. (2005). Vascular damage in a mouse model of diabetic retinopathy: Relation to neuronal and glial changes. Invest. Ophthalmol. Vis. Sci. 46(11):4281– 4287. Frank, R. N., et al. (1997). An aldose reductase inhibitor and aminoguanidine prevent vascular endothelial growth factor expression in rats with long-term galactosemia. Arch. Ophthalmol. 115(8):1036–1047. Goldin, A., Amin, R., Kennedy, A., and Hohman, T. C. (2006). Advanced glycation end products: Sparking the development of diabetic vascular injury. Circulation 114(6):597–605. Grant, M. B., Spoerri, P. E., Player, D. W. D., Bush, M., Ellis, E. A., Caballero, S., and Robison, W. G. (2000). Plasminogen activator inhibitor (PAI)-1 overexpression in retinal microvessels of PAI-1 transgenic mice. Invest. Ophthalmol. Vis. Sci. 41(8):2296– 2302. Gubitosi-Klug, R., and Kern, T. (2006). A role for 5-Lipoxygenase in diabetic retinopathy. Presented at the annual meeting of the ADA, Washington, DC. Gubitosi-Klug, R., and Kern, T. (2008). [5-Lipoxygenase, but

not 12/5-lipoxygenase, contributes to degeneration of retinal capillaries in a mouse model of diabetic retinopathy.] Unpublished results. Hammes, H. P., Lin, J., Renner, O., Shani, M., Lundqvist, A., Betsholtz, C., Brownlee, M., and Deutsch, U. (2002). Pericytes and the pathogenesis of diabetic retinopathy. Diabetes 51(10):3107–3112. Hammes, H. P., Lin, J., Wagner, P., Feng, Y., Vom Hagen, F., Krzizok, T., Renner, O., Breier, G., Brownlee, M., et al. (2004). Angiopoietin-2 causes pericyte dropout in the normal retina: Evidence for involvement in diabetic retinopathy. Diabetes 53(4):1104–1110. Igarashi, T., Miyake, K., Kato, K., Watanabe, A., Ishizaki, M., Ohara, K., and Shimada, T. (2003). Lentivirus-mediated expression of angiostatin efficiently inhibits neovascularization in a murine proliferative retinopathy model. Gene Ther. 10(3):219– 226.

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Whetzel, A. M., Bolick, D. T., Srinivasan, S., Macdonald, T. M., Morris, M. A., Ley, K., and Hedrick, C. C. (2006). Sphingosine-1 phosphate prevents monocyte/endothelial interactions in type 1 diabetic NOD mice through activation of the S1P1 receptor. Circ. Res. 99(7):731–739. Wong, C. G., Rich, K. A., Liaw, L. H., Hsu, H. T., and Berns, M. W. (2001). Intravitreal VEGF and bFGF produce florid retinal neovascularization and hemorrhage in the rabbit. Curr. Eye Res. 22(2):140–147. Yamaoka, T., Nishimura, C., Yamashita, K., Itakura, M., Yamada, T., Fujimoto, J., and Kokai, Y. (1995). Acute onset of diabetic pathological changes in transgenic mice with human aldose reductase cDNA. Diabetologia 38(3):255–261. Zhang, L., Krzentowski, G., Albert, A., and Lefebvre, P. J. (2001). Risk of developing retinopathy in Diabetes Control and Complications Trial type 1 diabetic patients with good or poor metabolic control. Diabetes Care 24(7):1275–1279. Zheng, L., Du, Y., Miller, C., Gubitosi-Klug, R. A., Kern, T. S., Ball, S., and Berkowitz, R. A. (2007). Critical role of inducible nitric oxide synthase in degeneration of retinal capillaries in mice with streptozotocin-induced diabetes. Diabetologia 50(9):1987–1996.

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Roles of Oxygen in the Stability of Photoreceptors: Evidence from Mouse and Other Models of Human Disease JONATHAN STONE AND KRISZTINA VALTER

Photoreceptors are the most fragile of retinal neurons in the face of genetic and environmental stress, and oxygen and oxidative stress are emerging as major environmental factors affecting their stability and contributing to retinal degeneration. This chapter addresses the question of why photoreceptors are vulnerable to a wider range of mutations than any other neuron class of the retina or brain. Increasing evidence suggests that this fragility results in great part from environmental factors such as tissue oxygen levels. Although studies of the mouse retina have been prominent in the growing literature on the subject, this chapter also considers relevant findings from other species, including humans. Photoreceptors are selectively vulnerable not only to genetic mutations but also to a range of environmental stresses, metabolic toxins (Eells et al., 2003; Graymore and Tansley, 1959), hypoxia and hyperoxia (Wellard et al., 2005), trauma such as retinal detachment (Fisher et al., 2001; Mervin et al., 1999), and the cumulative stresses of aging (Gao and Hollyfield, 1992; Jackson et al., 2002). Genetic mutations act on a background of stress to and degeneration of photoreceptors. The phenotypes that emerge—the many forms of retinitis pigmentosa, the normal aging of the retina, the edge-specific degeneration that begins in childhood, the collapse of the macula with age— occur on a normal background. Appropriate therapy requires knowledge of both the normal and mutation-driven degeneration of photoreceptors and of the interaction between environmental and genetically induced stress. In this chapter, we focus on the delivery of oxygen to the retina, the regulation and dysregulation of that delivery, and the consumption of oxygen by photoreceptors. Clinically, there are two reasons for this focus. First, tissue oxygen levels affect the stability of the normal retina, from neonatal life to advanced age. Second, the role of environmental stress in genetically driven degeneration has long gone unrecognized and provides an unexplored avenue for therapy of the degenerating retina.

The delivery of oxygen to the retina The mouse retina has a retinal vasculature, like the human and many other mammalian retinas. Oxygen is delivered from two sources, the retinal and choroidal circulations. These circulations have distinct properties, and the interaction between them is critical in determining oxygen levels across the retina, and therefore the stability of photoreceptors. The Retinal Circulation The retinal circulation is a conventional system of arteries, capillaries, and veins. It forms developmentally from the hyaloid artery, which forms to supply the lens vesicle well before the retina requires its own circulation. At birth in the rat (Cairns, 1959), the retina is almost avascular, but the hyaloid artery passes from the optic disc to the developing lens. Over the first 10 days of postnatal life, the hyaloid circulation regresses and the retinal circulation forms by growth from the hyaloid vessels, following a template of astrocytes that spread from the optic disc across the retinal surface (Chan-Ling et al., 1990; Stone et al., 1995), expressing potent angiogenic factors (reviewed in Stone and Maslim, 1997). In the mouse, the retinal circulation begins to form before birth (Connolly et al., 1988) and also forms along a preexisting astrocyte template (Dorrell et al., 2002). Once the branches of the hyaloid vessels that supply the lens have regressed, the stem vessels are termed retinal vessels, and supply the inner layers of the retina. In the adult retina, arteries radiate from the optic disc across the inner surface of the retina and branch to form to arterioles and capillaries, which spread through the inner half of the retina. The capillaries space themselves through the inner layers of the retina and are described as concentrating in three layers, in the ganglion cell layer (GCL) and at the inner and outer surfaces of the inner nuclear layer (INL). Capillaries drain to venules, and these in turn drain to veins, which converge on the optic disc, to leave the eye.

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Physiologically, arterial and venous oxygen levels have been measured in the human (Alm, 1992) and cat (Alm and Bill, 1970, 1972a, 1972b). The consistent outcome is that oxygen extraction from the retinal circulation is 30%–40%, a level of oxygen extraction similar to that in the cerebral circulation. Rates of blood flow through retinal tissue have been estimated for a range of animals (reviewed in Paques et al., 2003), including rodents and primates. The rate of capillary flow in the mouse retinal circulation was estimated by Paques and colleagues at 1.26 mm/s, within the range reported for cat, rat, and monkey but higher than in other rodent tissues, including the rat brain (0.7 mm/s). Paques et al. note that the relatively high flow rate would be advantageous for oxygen delivery. Retinal vessels show blood-brain barrier properties, induced by their contact with neuroglial cells (Chan-Ling and Stone, 1992; Janzer and Raff, 1987; Tout et al., 1993; reviewed in Stone and Maslim, 1997). Further, the capillaries of the retinal circulation show the conventional property of autoregulation (Alm and Bill, 1970, 1972a, 1972b; Eperon et al., 1975); that is, they constrict, reducing flow, when oxygen levels in the tissue they supply rise, and dilate when oxygen levels fall. The mechanism of constriction is believed to lie in muscular control of arteriolar diameter, or in the control of capillary flow by pericytes (Peppiatt et al., 2006). Paques and colleagues (2003) note that the architecture of the mouse retinal circulation (which may be representative of mammalian retinal circulations) has features that favor control of capillary flow by local tissue conditions. Some superficial capillaries, for example, connect directly to large veins, and could provide a shunt past the deeper capillary beds. The one distinctive feature of the retinal circulation is that its capillaries reach only halfway across the thickness of the retina. From arteries at the inner surface of the retina, capillaries extend radially as far as the outer plexiform layer (OPL), and never (normally) into the outer nuclear layer (ONL). Their growth into that layer is robustly inhibited by a mechanism that has been explored in the mouse. The development of the retinal circulation is severely disturbed by antibodies to R-cadherin (Dorrell et al., 2002; reviewed in Stone et al., 2006), which presumably prevent the normal adherence of vessels to cells that express cadherin. Cadherin is strongly expressed in the OPL of the mouse, and Dorrell and colleagues suggest that vessels growing radially outward in the retina are diverted to follow this line of expression, along the OPL. Vessels grow past the OPL into the ONL only if this barrier is disturbed (by masking cadherin with antibodies) or overcome by an abnormal expression of angiogenic factors by cells of the ONL (Tobe et al., 1998). As a consequence, the ONL and the layers of inner and outer segments—effectively, the photoreceptors—are normally avascular, the only avascular parts of the CNS. Oxygen

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diffuses to the photoreceptors from the deepest capillaries of the retinal circulation, but the major oxygen supply to photoreceptors reaches them from the external surface of the retina, by diffusion (Yu and Cringle, 2001). This dual supply of oxygen to photoreceptors is distinctive and an important factor in their relative fragility. The Choroidal Circulation In all vertebrates, a rich vascular bed forms just external to the neural retina. Known as the choroidal circulation, this vascular bed is highly distinctive structurally and functionally. Anatomically, the choroidal circulation is a rich vascular bed that forms the tunica vasculosa of the eyeball, between the retina (tunica nervosa) and sclera (tunica fibrosa). The capillaries of the choroidal circulation (the choriocapillaris) are coarse and profuse and form a lake of blood at the outer surface of the retina. The choriocapillaris abuts the outer surface of the basement membrane of the retinal pigmented epithelium (RPE) (Bruch’s membrane), and oxygen diffuses across Bruch’s membrane, across the RPE and subretinal space to reach the mitochondria of the inner segments. The capillaries of the choriocapillaris do not have barrier properties but are highly fenestrated and permeable (Bill et al., 1980). Further, the rates of blood flow through the choroidal circulation are very high. When expressed in milliliters per minute per gram of tissue supplied, the choroidal blood flow is several times higher than in other high-rate tissues, such as the cortex of the kidney, cardiac muscle, the inner retina, and cerebral cortex (see Alm, 1992, fig. 6.15 therein). Veins converge into distinctively convoluted vortex veins, which emerge from the eyeball near its equator and join orbital veins. As a result of the very high rates of blood flow through the choroidal circulation, the partial pressure of oxygen in vortex vein blood is only 3%–4% below arterial levels (Alm, 1992; Bill et al., 1983); that is, the level of oxygen extraction is much lower than in retinal and cerebral circulations. As a consequence, the capillary bed of the choroidal circulation (the choriocapillaris) forms a lake of near-arterial blood around the outer surface of the retina, which is the main source of oxygen to photoreceptors. An important feature of the anatomy of the choroidal circulation is that its capillaries do not lie in the tissue they supply and do not respond to conditions in that tissue. Choroidal blood flow shows some evidence of regulation, in response, for example, to experimental increases in arterial pressure (Riva et al., 1997), but is not responsive to oxygen or metabolite levels in the outer retina, the tissue it supplies. As a consequence, oxygen levels in the outer retina can fluctuate more widely than in any other retinal layer, and this fluctuation underlies the pathogenesis of a range of retinal diseases, among them retinopathy of prematurity, retinopathy of detachment, and retinitis pigmentosa.

Oxygen Consumption in the Adult The consumption of oxygen by the retina has been scrutinized with a range of technologies. For the mouse, the most direct and recent data are oxygen tension measurements obtained in vivo with oxygen-sensitive electrodes. These data (figure 46.1A) demonstrate that in the mouse (Yu and Cringle, 2006), as in the rat, cat, and monkey (reviewed by Yu and Cringle, 2001), oxygen flows into the retina from two sources, one in inner retina and corresponding to the retinal circulation and the other at the outer surface of the retina and corresponding to the choriocapillaris. Oxygen is consumed in the retina at locations corresponding to the sites of mitochondria. Most of the oxygen flowing from the choriocapillaris is consumed by the dense concentration of mitochondria in the inner segment. In the darkadapted retina, the oxygen tension in the ONL is near zero;

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oxygen from the choriocapillaris does not reach beyond the inner segments. The oxygen sinks of the inner retina are more distributed, in ganglion cells, in the IPL and OPL. As already noted, the flow of blood through the choriocapillaris and the level of oxygen in choroidal blood do not vary with the level of oxygen in the tissue being supplied. As a consequence, oxygen flows from the choroid to the outer

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Finally, the choroid develops early, well before the neural differentiation of the retina. Choroidal development has been described in most detail for the human (Allende et al., 2006). It shows a centroperipheral pattern of formation and forms well before the photoreceptors (which in the normal adult retina consume all the oxygen delivered by the choroid) have begun to function. The development of the retinal circulation occurs much later, in the mouse in the first 2 weeks of postnatal life (Connolly et al., 1988), well after vessels invade the cerebral cortex (in midgestation; Stone and Maslim, 1997), and is strongly influenced by oxygen reaching the retina from the choroid.

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Figure 46.1 Oxygen levels within the retina, measured with oxygen-sensitive electrodes stepped through the retina, and the effect of depletion. In all three graphs the inner surface of the retina is at left, and the outer (choroidal) surface is at right. A, In the mouse, oxygen levels are maximal at the outer surface, close to the choriocapillaris. A second area of increased oxygen level is apparent at the inner surface, representing oxygen delivered by the retinal circulation. Asterisk marks a minimum in the region of the ONL; the sharp fall in tissue oxygen tension between the choroidal maximum and this minimum results from intense oxygen consumption by photoreceptor inner segments. B, Corresponding measurements in the naturally degenerative RCS rat. This is a developmental series. The minimum produced by photoreceptor oxygen consumption is evident in the youngest age studied (P20) but disappears as the animals age and the photoreceptor population is exhausted. C, Comparison of oxygen tension profiles obtained from a nondegenerative Sprague-Dawley (SD) rat and the degenerative P23H-3 transgenic strain. The minimum produced by photoreceptor consumption of oxygen is absent from the P23H-3 strain. (A, From Yu and Cringle, 2006. B, From Yu et al., 2000. C, From Yu et al., 2004.)

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retina (i.e., to photoreceptors) following physical gradients; there is no mechanism to regulate that flow in response to the needs of photoreceptors. The Developmental Onset of Oxygen Consumption The onset of oxygen consumption by photoreceptors has been well documented in rodents. Graymore (1959, 1960, 1963) used as a measure of metabolism the retina’s production of lactic acid, an end product of glycolysis. He showed that the retina’s production of lactate is low at birth and increases dramatically, in the rat between P15 and P20. This period corresponds to the onset of several measures of photoreceptor function, including the development of the electroretinogram (Fulton et al., 1995), the growth of the inner and outer segments of photoreceptors (Weidman and Kuwabara, 1968), the onset of oxygen consumption by the photoreceptors (Cringle et al., 2006), and the onset of a period of physiological hypoxia (Chan-Ling et al., 1995a), which induces the expression of angiogenic factors in glial templates on which the retinal blood vessels form (Dorrell et al., 2002; Stone et al., 1995). The development of the retinal vasculature is thus driven by the development of photoreceptor function (reviewed in Stone and Maslim, 1997). The mechanism that links photoreceptor function to vascular development is hypoxia; the onset of photoreceptor function and oxygen consumption reduces the flow of choroidal oxygen to the inner retina, and the resulting hypoxia serves as a signal for vasogenesis. Neuroglobin: An Oxygen-Binding Globin Prominent in the Retina Neuroglobin is a member of the globins, heme proteins found in plants, bacteria, fungi, and animals, with the property of reversibly binding oxygen. Neuroglobin was first described in the mouse and human (Burmester and Hankeln, 2004) and was named for its specific location in brain tissue (Burmester and Hankeln, 2004; Sun et al., 2001); it is a candidate protein for providing an oxygen store in brain tissue, as hemoglobin does in blood. Neuroglobin expression is upregulated in hypoxia (Sun et al., 2001) and is expressed roughly 100 times more strongly in the retina than in brain tissue (Schmidt et al., 2003), arguably reflecting the high oxygen utilization of the retina. The distribution of neuroglobin in the retina, in the IPL and OPL, and in the inner segments of photoreceptors correlates with the distribution of mitochondria, the organelles in which oxygen is recruited in the energy-producing mechanisms of oxidative phosphorylation (Schmidt et al., 2003). Correspondingly, neuroglobin levels are low in the inner retina of the guinea pig (Bentmann et al., 2005), in which species a retinal vasculature is lacking and oxygen metabolism cannot be detected (Yu and Cringle, 2001). The cellular and laminar distribution of neuroglobin in humans (Rajendram and Rao, 2007) and dogs (Ostojic et al., 2006) is similar to

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that observed in mice, and its amino acid sequence is highly conserved within mammals (Zhang et al., 2002).

Consequences of lack of regulation of oxygen supply to the outer retina As we have argued previously (Chan-Ling and Stone, 1993; Lewis et al., 1999; Stone and Maslim, 1997; Stone et al., 1999), the inability of the choroidal circulation to regulate its delivery of oxygen to the retina underlies the pathogenesis of several retinal diseases, including retinopathy of prematurity, retinopathy of detachment, and the photoreceptor degenerations. The following sections trace the underlying mechanisms. Inhaled Oxygen Reaches the Retina Selectively The lack of regulation of the choroidal circulation is evident in the effect that raising oxygen levels in inhaled air has on body tissues. The two body tissues most directly affected by hyperoxia are the epithelium of the lung (where hyperoxia induces a dysplasia; Barazzone et al., 1998; O’Brodovich and Mellins, 1985) and the retina, where effects include a narrowing of retinal vessels and the death of photoreceptors. Other tissues appear to be protected from hyperoxia by autoregulatory mechanisms induced by the hyperoxia. The contrast between autoregulation and the lack of it is evident in studies of the effect of hyperoxia on tissue oxygen levels across the retina, where there is a retinal circulation (Yu and Cringle, 2001; figure 46.2). As the level of oxygen in inhaled air is increased, oxygen levels rise throughout the retina, but much more so in the outer retina. The flow of higher than normal levels of oxygen from the choroid to the retina has two spectacular effects on the structure of the retina. It causes the death of photoreceptors, and it causes the constriction and in some circumstances the closure of vessels of the retinal circulation. Inhaled oxygen kills photoreceptors. Because inhaled oxygen reaches the outer retina from the choroid, simply breathing oxygen-enriched air can kill photoreceptors. The first evidence of this toxicity came from Noell’s brief report in 1955 that inhalation of 100% oxygen causes the total degeneration of photoreceptors in the rabbit retina within 3 days. The inner retinal layers appeared unaffected. The selective vulnerability of photoreceptors was subsequently confirmed in the mouse (Walsh, Bravo-Nuevo, et al., 2004; Yamada et al., 2001) and rat (Geller et al., 2006; Wellard et al., 2005) (figure 46.3). The effect takes longer (2 weeks) in rodents than in the rabbit, but it is also highly specific to photoreceptors and is accompanied by an upregulation of stress-inducible proteins (fibroblast growth factor-2 [FGF-2], glial fibrillary acidic protein [GFAP]). Wellard et al. (2005) reported that the vulnerability of photoreceptors to hyperoxia is part

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Figure 46.2 Effects of inhaled oxygen and light on intraretinal oxygen, measured in the eye of the rat (left) and cat (right). Left, The lower-most curve is equivalent to the SD data in figure 46.1C, the oxygen tension profile across the retina, in normoxia. As the proportion of oxygen in the air inhaled increases, oxygen levels rise all across the retina. The rise is much greater near the choriocapillaris (at right) than in the inner retina; this greater rise results from the lack of regulation of the choroidal circulation (From Cringle et al., 2002). Right, The lower pair of curves shows the oxygen tension profiles across the cat retina, in normoxic conditions, light- and dark-adapted. The dark-adapted curve shows the same general

features as reported for other mammals (see figure 46.1); in this graph the choroidal side of the retina is to the right. Light adaptation causes a rise (shown by the bracket) in tissue oxygen tension in the outer retina, the result of a light-induced reduction in the consumption of oxygen by photoreceptors. The upper pair of curves shows the oxygen tension profiles obtained when the animal breathed oxygen-enriched air. Oxygen tension is raised all across the retina, but again more markedly so at the choroidal side, and light adaptation increases tissue oxygen tension further (bracketed area). That is, the effects of hyperoxia and light on oxygen tension are additive. (From Linsenmeier and Yancey, 1989.)

of a wider pattern in which photoreceptors are maximally stable at 21% oxygen (room air) and are destabilized (i.e., the rate of their death is increased) by both hypoxia and hyperoxia. Finally on this point, it has been reported that in mice, the vulnerability of photoreceptors to oxygen is strain dependent (Walsh et al., 2004a), being less in the Balb/c strain than in the C57BL/6 strain. These strain differences are currently being used in our laboratory to identify genes that regulate the oxygen vulnerability of photoreceptors.

normal development of retinal vessels and causes the thinning or obliteration of vessels already formed. When the baby or pup or kitten is returned to room air, the inner retina, lacking its normal complement of vessels, becomes abnormally hypoxic, triggering the death of astrocytes (the cells that normally express angiogenic factors such as vascular endothelial growth factor, VEGF) and upregulation of the same factors by neurons (Stone et al., 1996). The resulting rapid growth of vessels in the absence of astrocytes, which both induce barrier properties in new vessels and form part of the inner limiting membrane, results in the formation of leaky vessels, which grow into the vitreous humor. The use of supplemental oxygen to allow the late, nearly normal growth of vasculature has been explored (Chan-Ling et al., 1995b). In recent decades, the incidence of retinopathy of prematurity has been greatly reduced with the careful monitoring of arterial oxygen levels. Vessels in the developing retina are particularly vulnerable to oxygen. In the rat, molecular events associated with the spread of pericytes at the end of the first postnatal month make adult vessels resistant to obliteration (Benjamin et al., 1998).

Inhaled oxygen blocks vasogenesis and thins or obliterates formed vessels. The impact of choroidal oxygen on the retinal vasculature was first detected in the analysis of the causes of retinopathy of prematurity, a neovascularizing disease of the infant retina associated with the use of oxygen to relieve respiratory difficulties in prematurely born infants (reviewed in Chan-Ling and Stone, 1993). Work in mouse (Browning et al., 1997; Smith et al., 1994), rat (Penn et al., 1994), and cat (Chan-Ling and Stone, 1992, 1993; Chan-Ling et al., 1992; Stone et al., 1996) models established that oxygen flowing from the choroid in the neonatal retina inhibits the

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Figure 46.3 Oxygen is selectively toxic to photoreceptors. A–D, Sections across the retina of C57BL/6 mice exposed to high oxygen levels in inhaled air. Before exposure, dying (red, TUNEL-labeled) cells were rare. Exposure to hyperoxia for 14–35 days (B–D) increased the frequency of dying cells, selectively in the ONL (o). By 35 days, the ONL had thinned and TUNEL+ debris appeared

in the inner nuclear layer (i), probably ingested by Müller cells. The blue dye is a DNA label (bisbenzamide). E and F, When the sheaths of cones were labeled with PNA lectin, the nuclei of some were TUNEL labeled (red), indicating that cones as well as rods were oxygen sensitive. See color plate 48. (From Geller et al., 2006.)

Retinal Detachment Causes Intense Hypoxia of the Outer Retina: The Use of Oxygen Relieves the Retinopathy of Detachment The viability of the retina is threatened when it becomes detached from the outer walls of the eyeball. This detachment can be spontaneous or trauma induced; typically the neural retina detaches from the RPE, separating photoreceptors from their major source of oxygen. The results of detachment have been well studied (Fisher et al., 2001) and include photoreceptor death and proliferation and hypertrophy of neuroglia, which in humans can result in the total loss of retinal function. The effects of detachment are greatly reduced by giving supplemental oxygen (i.e., enriching the inhaled air with

oxygen). Photoreceptor death is reduced (Mervin et al., 1999), glial proliferation and hypertrophy are limited (Lewis et al., 1999), and the neurochemistry of the retina is less disturbed. This indicates that hypoxia plays a major role in inducing the pathological changes, and that hypoxic damage occurs because the choroid has no mechanism to detect or respond to abnormal hypoxia in the outer layers of the detached (or attached) retina.

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A Reduction in Photoreceptor Metabolism Raises Oxygen Levels in the Outer Retina Once it is accepted that the flow of oxygen from the choroid to the outer retina is unregulated, it follows that any reduction in the oxidative

metabolism of photoreceptors will, because the choroidal circulation cannot autoregulate, lead to an increase in the oxygen reaching the retina (Stone et al., 1999). This prediction was formalized in models of oxygen supply and consumption in the retina developed from the laminar analysis of oxygen distribution in the retina (Haugh et al., 1990; Yu and Cringle, 2001). These models include a major oxygen sink at the inner segments of photoreceptors. If that sink weakens in intensity, then, the models predict, oxygen from the choroid will flow into the cellular layers of the retina. Further, the thinning of the outer retina caused when photoreceptors degenerate will reduce the required diffusion path and add to this effect.

Clinically, photoreceptor degeneration protects against retinal neovascularization. Confirmation of the idea that, when photoreceptors degenerate, oxygen from the choroidal circulation reaches the inner layers of retina comes from reports that retinal degeneration is protective against hypoxic neovascularizing diseases of the retina, in particular diabetic retinopathy (Arden, 2001; Sternberg et al., 1984). The protective effect has been confirmed (Lahdenranta et al., 2001) in a mouse model of photoreceptor degeneration; these authors noted that the normal hypoxia-induced expression of the angiogenic factor VEGF during development, which is critical for vessel formation, does not occur in the degenerative mouse retina.

Experimentally, light raises oxygen levels in the outer retina. Photoreceptors generate an electrical response to light (beginning the coding of vision) by reducing their dark current. Molecules generated when rhodopsin absorbs light sequester cyclic guanosine monophosphate (cGMP) from the cytoplasm, closing cGMP-gated Na+ channels, and reducing the need for Na+ pumps in the inner segment to expel Na+ from the cell. These pumps utilize adenosine triphosphate (ATP), so that light reduces the photoreceptors’ requirement of ATP, and therefore for oxygen, which is used in oxidative phosphorylation to produce ATP. As a consequence, a maintained increase in light absorbed by photoreceptors causes a maintained increase in oxygen tension in the outer retina (reviewed in Wangsa-Wirawan and Linsenmeier, 2003; see figure 46.2). The rise is reversible; decreasing light falling on the retina decreases retinal oxygen tension. The dynamics of the oxygen change are relatively rapid; oxygen levels rise and fall with increases and decreases in light, with a time constant of seconds with dim light, and of minutes with bright (photopic) levels of light (see Linsenmeier, 1986, fig. 46.4 therein).

Clinically, laser burns limit retinal neovascularization. The still current use of laser burns to peripheral retina to protect central retina from neovascularization, particularly in diabetic retinopathy, provides further evidence that photoreceptor depletion results in increased oxygen levels in the retina. Laser treatment was developed initially without a theory of its mechanism (reviewed in Benson et al., 1988). Several authors (Stefansson et al., 1981, 1986; Stone and Maslim, 1997) have argued that the well-established protective effects of laser treatment arise from depletion of the photoreceptor layer at the site of the burn and a consequent flow of oxygen from the choroid to the inner retina. This oxygen counteracts the hypoxia caused in diabetic retinopathy by capillary closure, reducing or delaying the damaging neovascularization. The recent study of Yu and colleagues (2005) addressed this question, demonstrating that partial laser burns at the level of the RPE and outer segments in rabbit retina caused increased retinal oxygen levels internal to the lesions.

Experimentally, photoreceptor depletion raises oxygen levels in the outer retina. A sustained rise in oxygen tension in the outer (photoreceptor) layers of retina has now been demonstrated in three animal models of photoreceptor degeneration, the RCS rat (Yu et al., 2000) (see figure 46.1B), P23H-3 rat (Yu et al., 2004) (see figure 46.1C ), and Abyssinian cat (Padnick-Silver et al., 2006). These degenerations are all genetic in origin, but the mutations are diverse. In the RCS rat, a mutation in the receptor tyrosine kinase Mertk is associated with failure of the RPE to phagocytose the discarded membrane of photoreceptors (D’Cruz et al., 2000); in the P23H-3 rat, a transgene generates rhodopsin with a single amino acid substitution (Machida et al., 2000). The mutation in the Abyssinian cat (Ehinger et al., 1991) remains unidentified but appears not to involve rhodopsin (Gould and Sargan, 2002). All these studies relate the rise in oxygen levels in the outer retina to a decrease in oxygen consumption by photoreceptors.

Depletion-Induced Hyperoxia Is Inhibitory to Vessels and May Be Toxic to Rods and Cones If the increases in retinal oxygen levels known to be induced by photoreceptor depletion are sufficient to influence the cell biology of the retina, then effects similar to those induced by inhaling oxygen (the death of photoreceptors and the thinning of vessels) should be apparent. Retinal vessels are thinned. Evidence of depletion-induced thinning of retinal vessels is substantial. Clinicians had long noted that vessels in the degenerating human retina are abnormally thin (Heckenlively, 1988), and thinning of the retinal vessels has been documented in several animal models of photoreceptor degeneration, including the rd mouse (Blanks and Johnson, 1986), the P23H-3 mouse (Penn et al., 2000), and the Abyssinian cat (Nilsson et al., 2001). Penn and colleagues noted that the effect was reduced by hypoxia, confirming that the thinning is caused by hyperoxia. Nilsson and colleagues noted further that as the degeneration of

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photoreceptors progresses in the Abyssinian cat, the volume of blood passing through the retinal circulation decreases markedly, while the choroidal circulation continues without reduction. Cones are damaged by rod dysfunction. Evidence that the depletion of rods can damage surviving photoreceptors is clearest in the rod-cone dystrophies, in which photoreceptor death occurs first among rods, perhaps owing to mutations in a protein expressed only in rods (e.g., rhodopsin), and later involves cones. Arguably, the tendency of macular degeneration to follow relatively severe loss of rod function in normal human retina (Curcio et al., 2000; Jackson et al., 2002) represents a form of rod-cone dystrophy. Two explanations, one specific and one general, have been developed to explain the impact of rod loss on cone viability. The specific explanation is that rods produce a cone survival factor (Hicks and Sahel, 1999; Mohand-Said et al., 1998), for want of which cones die when rods are depleted. The more general explanation is the oxygen toxicity hypothesis (Stone et al., 1999), namely, that the depletion-induced hyperoxia will damage all surviving photoreceptors, rods, and cones. These two mechanisms are not exclusive. Shen and colleagues (2005) reported evidence, from a rod-degenerative pig model, that surviving cones are subject to oxidative stress, which they related to the depletion of rods, giving support to the idea that hyperoxia may be the factor linking rod death to cone damage. There is no reason, moreover, to expect surviving rods to be immune to the oxidative stress caused by rod depletion, and evidence is available to demonstrate that rods surviving in a rod-specific mutant degeneration, such as the P23H-3 rat, are damaged. Comparing nondegenerative (Sprague-Dawley) and P23H-3 retinas raised in identical conditions, Walsh and colleagues (2004b) showed that the P23H-3 photoreceptors have shorter, more disorganized outer segments, with a higher rate of cell death and a smaller a-wave. Evidence that this damage to P23H-3 photoreceptors is at least partly due to an environmental factor came from the demonstration that, when the ambient light to which the retina is exposed was raised, damage to surviving rods increased, and the damage could be reversed by reducing ambient illumination ( Jozwick et al., 2006). Light Accelerates Many Forms of Photoreceptor Degeneration The effect of light experience on the progress of retinal degenerations varies with the underlying mutations. Light exposure has been known for many years to accelerate retinal degenerations in the RCS rat (Dowling and Sidman, 1962; Kaitz and Auerbach, 1978, 1979), in the P23H-3 mouse (Naash et al., 1996), and in the P23H-3 rat (Walsh et al., 2004b). Evidence in the human is limited but suggests that light restriction may slow degenerations in

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some cases (reviewed in Stone et al., 1999). The variability in the human response to light exposure has yet to be related to the underlying genetic defect. In animal rodent models as in humans, light either exacerbates the degeneration or has no effect (Paskowitz et al., 2006); there appear to be no reports of light being protective to photoreceptors. As Paskowitz and colleagues note, the effect of light is now well understood in a number of animal models, down to the molecular level. This evidence (which is beyond our present scope) makes clear how photoreceptor signaling is disturbed by mutations in proteins related to phototransduction, but the link between the disturbed signaling and cell death remains elusive. Recent analyses of the P23H-3 rat model, which is light sensitive (Jozwick et al., 2006; Walsh et al., 2004b), showed that the damaging effect of light can occur quickly (within a week) and in response to very modest increases in just the daylight part of the day-night cycle. Further, the effects were reversible; within 2–5 weeks of a reduction in ambient light, the P23H-3 photoreceptors rebuilt their outer segments and partially (30%–40%) regained their sensitivity to light. We have recently demonstrated (Chrysostomou et al., 2008) that the same modest rise in ambient illumination that caused severe rod damage in the P23H-3 retina also damaged cones, and the damage to cones was also reversible. Since the transgene is in rhodopsin and cones do not express rhodopsin, the toxic factors that damaged cones must all have been environmental. One possibility, as we argued when proposing the idea of oxygen toxicity in 1999 (Stone et al., 1999), is that the toxic effect of increased ambient light on both rods and cones is mediated by a rise in oxygen tension in the outer retina induced by light. The Edge of the Normal Retina Degenerates The normal (wild-type) retina degenerates at its edge. The degeneration has been shown in rodent models to begin as soon as photoreceptors begin to function (Mervin and Stone, 2002b; reviewed in Stone et al., 2005) (figure 46.4). In humans, a cystoid degeneration of the edge of the retina becomes evident in childhood and progresses with age until it involves the peripheral several millimeters of retina (Bell and Stenstrom, 1983). There is considerable evidence that this “normal” degeneration of the edge of the retina is driven by hyperoxia. The structural factors that control retinal oxygen levels in the retina are perturbed at the edge of the retina by the sharp discontinuity of the retina itself. Specifically, the choroidal circulation extends past the edge, and as a result, oxygen can be predicted to reach the edge of the retina by diffusing around the edge. Working with the C57BL/6 mouse, Mervin (Mervin and Stone, 2002a, 2002b) demonstrated that the adult mouse retina shows an edge-specific degeneration. Retinal layers are thinned at the edge; photoreceptor mor-

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Figure 46.4 Photoreceptors at the most peripheral margin of the retina are subject to stress from early postnatal life. A–C, The edge of the retina of C57BL/6 mouse retina, with dying cells (bright red, TUNEL labeled) clustering at the edge of P14, P16, and P18. All are in the ONL. D and E, This cluster of dying cells colocalizes with the upregulation of two stress-induced proteins. GFAP is normally expressed (red) only at the inner surface of the retina, by astrocytes (E and G). At the edge, GFAP is upregulated also in Müller cells. FGF-2 is strongly upregulated (green) in photorecep-

tors, in a gradient that is maximal at the end. H, Quantification of the frequency of dying cells in the peripheral-most 100 μm of retina. I, Oxygen influences this distribution of dying cells at the edge of the early postnatal retina. Hypoxia increases photoreceptor death throughout most of the retina; at the edge, however, hyperoxia reduces death, suggesting that the edge-related death is driven by oxygen stress (see the text). Hyperoxia causes a small increase in the frequency of dying cells at the edge of the retina. See color plate 49. (From Mervin and Stone, 2002a, 2002b.)

phology is disturbed, and stress-sensitive proteins (FGF-2, GFAP) are sharply upregulated (figures 46.4D–G). Second, a focus of accelerated photoreceptor death was detected at the edge, occurring in early postnatal life, between P12 and P20, as photoreceptors start to function. Third, the acceleration was slowed by hypoxia, indicating that it is caused by

hyperoxia. Throughout the rest of the retina, by contrast, hypoxia increased the rate of photoreceptor death. The oxygen status of the most peripheral 100 μm of the retina is distinctive, however; the region is degenerative and the degeneration is progressive, and related to high levels of oxygen.

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Edge-related degeneration is evident in all species in which it has been studied (rats, mice, and humans; reviewed in Stone et al., 2005). In humans, the edge of the retina is eroded throughout life by a process called cystoid degeneration (Bell and Stenstrom, 1983; Vrabec, 1967). The degeneration is most severe at the edge of the retina, where the layered structure of the retina breaks down; cysts form within the retina, and eventually, pigmented cells invade from the RPE. This invasion mimics the pigmentation of the prematurely degenerating retina, which led to the term retinitis pigmentosa. In the area of degeneration, vessels are absent, a symptom of raised oxygen levels. Central to the cystoid degeneration, extending some millimeters toward the center of the retina, the layered structure of the retina is intact, but vessels are absent or reduced, and the morphology of photoreceptors is degraded (Stone et al., 2005). More centrally, the morphology of photoreceptors and their content, the metabolic enzyme cytochrome oxidase, and the retinal circulation are normal. This grading of the degeneration from the edge of the retina suggests that the source of stress is at the edge. Further, the extent of the degeneration increases with age (Stone et al., 2005), indicating, as we originally suggested, that the normal retina is unstable under hyperoxic stress and the degeneration is progressive.

Oxygen stress during development: The concept of a critical period in photoreceptor development The relationship between photoreceptor stability and retinal oxygen levels described in the previous section—that photoreceptors are maximally stable in normoxia and are destabilized by both hypoxia and hyperoxia—is true for the adult. Different relationships hold during development, however. In early postnatal life in mouse (Mervin and Stone, 2002a) and rat (Maslim et al., 1997), a period of accelerated photoreceptor death has been described. This “critical period” of development (P15–P20 in the mouse, P15–P25 in the rat) coincides with a period of physiological hypoxia caused by a rapid increase in photoreceptor metabolism (Graymore, 1960). This period of physiological hypoxia (Chan-Ling et al., 1995a) is critical in inducing angiogenesis of the retinal circulation. Testing whether the death of photoreceptors in the developing retina is driven by hypoxia, Maslim and colleagues (1997) demonstrated that the rate is sharply increased by hypoxia and decreased by hyperoxia. Mervin and Stone (2002) extended these observations to the C57BL/6 mouse. After about P30 in both species, the effects of oxygen levels on photoreceptor death shift subtly but significantly, in two ways. First, photoreceptors become relatively resistant to variations in oxygen levels; some still unidentified stabilizing factor ends the developmental period of high naturally

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occurring death rates. Second, the sign of the effect of hyperoxia changes. During the critical period, hyperoxia is protective; in the adult, hyperoxia is toxic. Finally, it has become clear that many forms of mutationinduced retinal degeneration accelerate during the critical period, suggesting an interaction between environmental or cell biological factors (the critical period) and the gene mutation (RCS rat: Valter et al., 1998; P23H-3 rat: Walsh et al., 2004b; the rd mouse: Hafezi et al., 1998). In our original (1999) review, we speculated on the teleological significance of a period of development during which photoreceptors are hypoxia sensitive. We suggested that, like most classes of retinal cells, photoreceptors are overproduced during development and then culled to adult populations by a process of cell death. In photoreceptors but not other retinal cells, the culling is driven by the supply of oxygen to the retina, with the effect (we suggested) of matching the photoreceptor population to the supply of oxygen available.

Implications for the retinal degenerations The analysis just developed draws attention to environmental factors, such as tissue oxygen levels, that influence the course of retinal degenerations. How powerful is this influence? Can purely environmental factors precipitate degenerations? Can they provide effective therapy? Evidence that environmental factors can precipitate degenerations has come from two sources. Earlier we referred to evidence that age-related macular degeneration may be preceded by significant loss of rods. Curcio and colleagues (2000) suggest that the rod loss follows age-related changes in the recycling of retinoids, that rod loss contributes somehow to the degeneration of the cone-rich macula, and that improving rod survival could be the key to the stability of the macula. Considering the possibility of significant environmentally induced photoreceptor degeneration early in life, we (Stone et al., 2001) examined a cohort of cases of retinal degeneration, separating them into groups in which a familial pattern could and could not be detected. Our working hypothesis was that perinatal stress, which is associated with a wide range of adult-onset diseases, might also contribute to retinal degenerations, perhaps by precipitating degeneration in the developing photoreceptor population. A history of perinatal stress was significantly more common in the nonfamilial group, suggesting that in a proportion of nonfamilial cases, perinatal stress (which includes hypoxic stress) contributes to the cause of photoreceptor degenerations. We have mentioned the ability of light restriction not only to slow photoreceptor loss in a degenerative model, the P23H-3 rat, but also to restore photoreceptor structure and function. This recovery of function stems from a capacity of

individual cells to repair their outer segments and restore their functional organization. Obviously, this self-repair capacity has therapeutic potential; but is it special to this model? In normal (wild-type) retina, photoreceptors also respond to sustained (over weeks) increases in ambient light by decreasing the length of their outer segments. The result is a drop in rhodopsin levels in the retina and a reduction in the dark current and a-wave. The effects are reversible, and the effect and its reversal have been summarized in the principle of photostasis (reviewed in Williams, 1998), which states that the retina regulates the amount of active rhodopsin deployed to keep the daily quantum catch constant. The effect of light on wild-type outer segments goes beyond shortening and lengthening, however. Reports of the effect of ambient light on normal photoreceptors (Kuwabara, 1970; Penn and Anderson, 1991) describe significant disorganization of the outer segment membrane, very like that seen in the P23H-3 model. This damage is also reversed, as the cell replaces its outer segment membrane. It thus seems likely that the ability of photoreceptors to self-repair is general, and likely to be available in any retina. These examples of environmentally induced damage, degeneration, and recovery of photoreceptors are unlikely to be all oxygen mediated. The level of oxygen in the outer retina is only one measure of the impact of light, photoreceptor depletion, and age-related factors such as the recycling of retinoids on the stability of photoreceptors. Nevertheless, oxidative stress is prominent in many recent ideas of the mechanisms that precipitate photoreceptor death. Photoreceptors produce reactive oxygen species when exposed to light (especially blue light; Yang et al., 2003), and their production follows the activation of rhodopsin, before the downstream events of visual transduction (Demontis et al., 2002). Thus, oxidative stress may be intrinsically linked to the absorption of visible light by rhodopsin, and photoreceptors are rich in antioxidant molecules, such as docosahexaenoic acid (Rotstein et al., 2003). As a consequence, measures such as light restriction, filtering out just blue light (Margrain et al., 2004), dietary supplementation with antioxidants (Militante and Lombardini, 2004), and infrared radiation to repair oxidatively damaged mitochondria (Eells et al., 2003) are being debated and studied. As emphasized in this chapter, environmental factors can add significantly to the stresses that cause photoreceptor degeneration, and consequently, management of these factors can be valuable therapeutically.

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47

Complex Genetics of Photoreceptor Light Damage MICHAEL DANCIGER

The understanding of music is in the understanding of a single note. —Unknown

Retinal degeneration (RD) can be caused by a number of different factors: gene mutations, such as those responsible for retinitis pigmentosa (RP) or other forms of RD; environmental exposures, such as toxic levels of light; and a combination of environmental factors and genetic tendencies, such as those producing age-related macular degeneration (AMD). Regardless of cause, the end stage is loss of photoreceptors due to apoptosis. Therefore, RD in experimental animals (in this case, mice) caused by toxic levels of light is a good model in which to study photoreceptor death because it is easy to manage in the laboratory. In addition, patients with some retinal diseases who are exposed to high levels of light for extended periods, such as fishermen or skiers, may have their disease course worsened (Taylor et al., 1990; Cruickshanks et al., 1993; Simons, 1993; Cideciyan et al., 1998; Mata et al., 2000). Supporting this is the fact that in some animal models of inherited RD, the disease process is accelerated by light exposures (Sanyal and Hawkins, 1986; Wang et al., 1997; LaVail et al., 1999; Organisciak et al., 1999). Therefore, it is not surprising that there has been a tremendous amount of work on light damage to the retina in many different fields. In this chapter, we cover work on the complex genetics of light-induced RD in the mouse model. In the late 1980s, LaVail and colleagues published two studies on photoreceptor (PR) light damage of albino mice (LaVail et al., 1987a, 1987c). Several albino strains were exposed to constant light at about 115–130 foot-candles (ft-c) (1,265–1,400 lux) for 2 weeks. After exposure, the PR outer nuclear layer (ONL) was measured and used as an indicator of PR loss. Compared to unexposed control mouse retinal ONLs, the B6(Cg)-Tyrc-2J/J albino (B6alb) still had 80% of the PR ONL, while the other strains (NZWLacJ, A/J, BALB/c, AKR, Ma/My, RF/J, RIIIs/J) had only 40% or less (LaVail et al., 1987a, 1987c). The B6alb strain is coisogenic with the standard pigmented C57BL/6J strain; the only genetic difference is the homozygous mutation in the tyrosinase gene carried by B6alb, causing it to be albino. (Co-isogenic or congenic refers to nearly identical strains of an organism that vary at only a single locus.) Why were B6alb

PRs so much more resistant to light insult than those of the other strains? Insofar as the mice were all raised and maintained under the same circumstances, the answer had to be genetics. Of course, this is what the LaVail group thought and demonstrated in a second study in which they performed the same type of light damage protocol on B6alb, BALB/c (C), and F1 mice (in this case, F1 = progeny of a B6alb × C cross). The results confirmed the substantial and significant difference in loss of PRs to light insult between the B6alb and C strains, with an intermediate response from the F1s (skewed a bit toward the C phenotype), demonstrating the genetic influence on the trait (LaVail et al., 1987b). We became interested in this work and set up a collaboration with Dr. LaVail (Beckman Research Center, University of California–San Francisco) to try to identify the gene (or genes) and alleles responsible for this remarkable difference in light-induced PR loss between the C and B6alb strains.

Identification and verification of a gene modifier that has a strong influence on photoreceptor light damage We started with the simplest explanation: a single gene is responsible for the difference in phenotype between the strains. The B6alb allele protects the PRs from light insult, and the C allele makes them more sensitive. We performed a backcross with the B6alb and C strains and attempted to score the ONL of the progeny after the same constant light exposure as before. The term backcross refers to a cross of the F1 of two strains (for example, strains A and B) with one of the original strains. Thus, F1(A × B) × B or F1(A × B) × A, or, in our case, F1(B6alb × C) × B6alb. If one gene was responsible for the difference, there should have been only two phenotypes, resistant (B6alb/B6alb) and sensitive (B6alb/C). Linkage analysis with genetic markers evenly spanning the genome at regular intervals of ≤30 centimorgans (cM) within chromosomes and ≤15 cM from the telomere or centromere (mouse chromosomes are acrocentric—they have the centromere at one end) should have revealed the chromosomal location of the gene. It did not work. First, it was difficult in many individual cases

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to score the light damage phenotype as resistant or sensitive, and second, no marker cosegregated with the phenotype; that is, no marker was B6alb/B6alb for all mice with the resistant phenotype and B6alb/C for all mice with the sensitive phenotype (data not published). Therefore, we hypothesized that PR damage in response to constant light exposure is a complex trait. The definition of a complex trait in the circumstance in which the environment is controlled—that is, it is not a differentiating factor—is a trait or phenotype that is governed by two or more genes. Since the number of genes that govern the trait is unknown at the outset, the number of phenotypes is unknown as well. For example, one gene governing a trait with alleles that are additive would produce three phenotypes. Additive means that the alleles are not dominant or recessive but instead, when both are present, the phenotype is intermediate. Two additive genes would produce nine phenotypes, three genes 27 phenotypes, and so on. Therefore, the phenotype is measured or quantified. In this case, the phenotype was the average of 54 measurements of the ONL thickness along a central retinal section running far superior through the optic nerve head to far inferior. To do the experiment, we used the same backcross protocol and light exposure as before, but instead of linkage analysis, quantitative genetics analysis was performed, using the Map Manager QTX program (Manly et al., 2001). This software identifies quantitative trait loci (QTL) by regression analysis. Thus, it computes the degree of significance for the association of a marker with the phenotype by evaluating the genotype of all markers comprising a genome-wide scan in all progeny animals and comparing that with the phenotypes of all progeny animals. Significance is identified when a high proportion of progeny with a particular genotype for a marker (for example, B6alb/B6alb) tend to have a particular phenotype, and at the same time when the genotype is different for that marker (B6alb/C, in this case) the phenotype tends to be opposite. In this way, utilizing the Map Manager program, we identified several QTL that influenced PR light damage in this cross. The most significant of these was on distal chromosome 3, which accounted for nearly 50% of the genetic effect and had a probability of 10−19 of being a random, chance phenomenon (LOD score was ca. 19) (Danciger et al., 2000). When we examined that locus on the gene map for candidates, we found the Rpe65 gene, a gene present in the retinal pigmented epithelium (RPE) but known to be involved in the response of the PR cell to light. When we sequenced the Rpe65 cDNA, we found an A for the first nucleotide of codon 450 in B6alb and a C in the BALB/c strain, predicting a methionine for B6alb and a leucine for the BALB/c. Since both methionine and leucine are nonpolar amino acids, the change from one to the other is conservative and would not predict the normal function of RPE65 to be

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affected. This was supported by studies showing normal responses of B6alb mice in water mazes and by studies showing normal electrophysiological responses of B6alb retinas (Balkema and Dräger, 1991; Hayes and Balkema, 1993; Nusisnovitz et al., 2003). Our hypothesis was that under the oxidative conditions of constant light, however, the methionine would be susceptible to oxidation to methionine sulfoxide, rendering the RPE65 protein more susceptible to degradation, while the leucine 450 would not be. Oxidation of key methionines in other proteins has been shown to disrupt function (Vogt, 1995). Lower levels of RPE65 would decrease the cycles of rhodopsin regeneration and thereby protect the PRs by slowing down the vision transduction process and its concomitant hyperpolarization and depolarization cycling. The PR response to light involves the stimulation of rhodopsin involving the conversion of 11-cis-retinal to all-trans-retinal and separation from the opsin protein. The activated protein interacts with the G protein transducin as the first step in a biochemical pathway resulting in the hyperpolarization of the PR cell with a concomitant alteration in neurotransmission to the next level of neurons in the retina. To stop the process, both rhodopsin kinase and arrestin interact with rhodopsin, stopping its interaction with transducin and preparing it for ligation with 11-cis-retinal. 11-cis-retinal is recycled by a pathway that involves RPE65 in the RPE cells and appropriate transport from and to the PRs (for reviews, see Chabre and Deterre, 1989; Chen, 2005). Support for our hypothesis came from two publications demonstrating the same PR sensitivity to perturbations in the visual transduction process under different circumstances. In one case, PRs were protected from intense light exposures by the absence of RPE65 protein in the corresponding knockout mice (Grimm et al., 2000), and in the other, PRs degenerated even in the presence of dim light, owing to the absence of rhodopsin kinase (Grk1) in the corresponding knockout mice (Chen et al., 1999a). In the first case, the cycling of rhodopsin and the hyperpolarization and depolarization cycles of PRs were stopped, and in the second case the PRs were overstimulated because activated rhodopsin could not be shut off in a timely way. Verification of the hypothesis and a more precise explanation of the protective effect of the RPE65 met450 variant were demonstrated in studies by Wenzel et al. (2001b, 2003). The sum of the two studies (done on wild-type and c-FOS −/− backgrounds) was severalfold. Mice homozygous for RPE65 leu450, for met450, or heterozygous were exposed to intense light after dilation of their pupils producing a bleach of a high number of PRs (a high percentage of the retinal rhodopsin) and then held in the dark. At various time points the amount of rhodopsin regenerated was measured spectrophotometrically and compared to the baseline before the bleach application. The presence of the met450 variant significantly slowed

the regeneration of rhodopsin. The speed of regeneration was directly correlated to the amount of RPE65 protein present in the RPE measured by Western blot and to the PR damage to the retina measured in stained histological sections. Thus, mice homozygous for RPE65 met450 had lower levels of RPE65 protein in their RPE, slower regeneration of rhodopsin, and less PR loss after intense light exposure (Wenzel et al., 2001b, 2003). The amount of Rpe65 mRNA, however, was the same in eyes with RPE65 met450 and RPE65 leu450 (Danciger et al., 2000; Wenzel et al., 2001b, 2003). An additional piece of information was the fact that the protective effect of the met/met genotype was not great enough to account for the resistance of B6 mice to damage following toxic light exposure (Wenzel et al., 2001b). This substantiated the original quantitative genetics study demonstrating the presence of several other B6alb gene alleles that also protect against light-induced damage (Danciger et al., 2000). In addition, several other QTL were identified in a light damage study of an F1 intercross of the BALB/c and 129S1/SvImJ strains (Danciger et al., 2004). These QTL were on chromosomes 1, 4, 6, and 2, representing a completely different set of QTL than in the original light damage study of BALB/c and B6alb. However, in the earlier study the mice were exposed to 2 weeks of constant light at an intensity of approximately 115–130 ft-c (1,265–1,400 lux) and sacrificed immediately after exposure. In the 129 × BALB/c study the mice were exposed (with dilated pupils) to 1 hour of 15,000 lux, allowed to stay in the dark for 16 hours, and then kept in dim cyclic light for about 12 days before evaluation of retinal damage by measurement of the amount of rhodopsin remaining (Danciger et al., 2004). To evaluate the argument that the visual transduction response was slowed in the PRs of mice carrying the RPE65 met450 variant at the physiological level, an electroretinogram study was carried out as follows: BALB/c and B6alb mice were exposed to a bleaching light and then placed in the dark. At various time points, the mice were tested for the rod response to light and compared to normal. For both the a-wave and b-wave, the BALB/c retinas recovered significantly sooner than those of B6alb (Nusinowitz et al., 2003). The slower physiological recovery of the response to light in B6alb mice compared to BALB/c mice corresponded perfectly to results of the previous biochemical studies showing a slower regeneration of rhodopsin (Wenzel et al., 2001b). A further substantiation of the RPE65 met/leu450 variant as a modifier of PR light damage was demonstrated when RPE65 protein activity and concentration were measured in the eyes of various breeding combinations of BALB/c, B6 and RPE65−/− (knockout) mice (the knockout mice were on a mixed background of 129S1/SvImJ and B6). The order of concentration of RPE65 in the eye from highest to lowest was BALB/c (leu/leu) > (BALB/c × B6)F1 (leu/met) > BALB ×

KO (leu/−) > B6alb (met/met) > B6alb × KO (met/−). The rate of rhodopsin regeneration matched that order, but the rate of rhodopsin regeneration per unit RPE65 protein was not influenced by the leu/met variant; it was the different amount of RPE65 protein present (Lyubarsky et al., 2005). This same conclusion can be extracted from two very recent cell expression studies. In the first case, it took 5–10 × met450 cDNA to express the same amount of RPE65 protein as leu450 cDNA in 293T-LC cells, but both RPE65 proteins had the same activity (Jin et al., 2006). In the second case, amounts of RPE65 protein expressed by cell transfection of cDNA were directly related to the presence of the met450 or leu450 variant, whether it was in cDNA from mouse or from dog. Based on predicted protein structure, the authors of this work hypothesized that the RPE65 met450 variant made the protein much less stable than the leu450 variant (Redmond et al., 2007). The evidence cited so far demonstrated that the met450 variant of RPE65 reduced the amount of RPE65 protein present in the eye relative to the leu450 variant. This reduction protected the PRs from light-induced damage by slowing the recycling of rhodopsin, and therefore slowing the recovery of the capability to respond to light physiologically. There was, however, a fly in the ointment (something inconsistent). The met450 variant of RPE65 is rare (figure 47.1). However, the albino NZW/LacJ mouse (NZW) carries the met variant yet is almost as sensitive to light-induced retinal damage as BALB/c mice, and far more sensitive than B6alb mice (LaVail et al., 1987a, 1987c; Danciger et al., 2005). We could think of three explanations for this: (1) The met450 variant of RPE65 is not responsible for the retinal light damage resistance found in B6alb. It is instead a nonfunctional sequence variant that segregates with the actual nearby gene responsible for the effect. The protective allele of this gene is present in B6alb and the sensitive allele in BALB/c and NZW. (2) There is a second change in the NZW Rpe65 gene or a nearby sequence that suppresses the protective effect of RPE65 met450. (3) There are other NZW susceptibility gene alleles that overcome the protective effect of the met450 variant. To evaluate these possibilities, we repeated the constant light–induced retinal damage protocol on a small test cross between B6alb and NZW. If the first or second explanation was true, we would expect a strong and highly significant QTL at distal chromosome 3 with a B6alb protective allele because the NZW allele of the gene in this QTL would not be present either because met450 was suppressed or because the allele of the true gene responsible for the QTL was not present. We found no such distal chromosome 3 QTL and did find other QTL supporting the third explanation (Danciger et al., 2005). In addition, we bred the met450 variant from NZW to BALB/c to the N8 generation and tested it under the same conditions of light exposure and by

danciger: complex genetics of photoreceptor light damage

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Mouse Strains - From the MGI mouse SNP database (http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=snpQF)

Inbred C57BL/6J

(AA residue)

#450

GLNHFVPDKLCKMNVKTKE

NZW/LacJ

450

GLNHFVPDKLCKMNVKTKE

129S1/SvImJ

450

GLNHFVPDKLCKLNVKTKE

A/J

450

GLNHFVPDKLCKLNVKTKE

AKR/J

450

GLNHFVPDKLCKLNVKTKE

BALB/cByJ

450

GLNHFVPDKLCKLNVKTKE

BTBR tf

450

GLNHFVPDKLCKLNVKTKE

C3H/HeJ

450

GLNHFVPDKLCKLNVKTKE

DBA/2J

450

GLNHFVPDKLCKLNVKTKE

FVB/NJ

450

GLNHFVPDKLCKLNVKTKE

KK/HIJ

450

GLNHFVPDKLCKLNVKTKE

NOD/LtJ

450

GLNHFVPDKLCKLNVKTKE

MOLF/EiJ

450

GLNHFVPDKLCKMNVKTKE

CAST/EiJ

450

GLNHFVPDKLCKLNVKTKE

WSB/EiJ

450

GLNHFVPDKLCKLNVKTKE

Wild

Animal species – From NCBI (http://www.ncbi.nlm.nih.gov/)

C57-mouse

450

GLNHFVPDKLCKMNVKTKE

Rat

450

GLNHFVPDKLCKLNVKTKE

Dog

450

GLNHFVPDRLCKLNVKTKE

Human

450

GLNHFVPDRLCKLNVKTKE

Rhesus Monkey

450

GLNHFVPDRLCKLNVKTKE

Vervet monkey

450

GLNHFVPDRLCKLNVKTKE

Cow

450

GLNHFVPDRLCKLNVKTKE

Gray Opossum

791

GLNHFVPDRLCKLNVKTKE

Chicken

450

GLNHFVPDRLCKLNVKTKE

Japanese Firebelly Newt

450

GLNHFVPDRLSKLNVKTKE

Chimpanzee

467

GLNHFVPDRLCKLNVKTKE

Tiger Salamander

450

GLNHFVPDRLSKLNVKTKE

Aquatic frog

450

GLNHFVPDRLTKLNVKTKE

Zebrafish

450

GLNHFVPDRICKLNVKSKE

Pufferfish

451

GLNHFIPDRICKLNVKTKE

Figure 47.1 The RPE65 met/leu450 variant in wild and inbred strains of mice and from other animal species.

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mouse models of human eye disease

the same ONL thickness measurements described earlier in the text. Figure 47.2 shows that the RPE65 met450 coming from NZW and operating in the BALB/c background now protected the PRs from light-induced damage. To make the N8 generation, BALB/c mice were crossed with NZW. The offspring (F1) were crossed into BALB/c mice to produce N2 mice. Tail DNAs from these mice were tested for the presence of the met450 variant by PCR, and those that were positive (RPE65 heterozygous met/leu450) were bred to BALB/c again to produce N3 offspring. The same process was repeated until N8. N refers to the number of generations of one strain (in this case BALB/c) bred into the F1 such that the higher the N, the greater the percentage of the mouse that is BALB/c while still carrying the selected allele—in this case, around 99.6% BALB/c background and heterozygous for met/leu450. The N8, BALB/c, RPE65 met/leu450 mice were intercrossed to produce mice homozygous for leu450, homozygous for met450, and heterozygous. Glazier et al. (2002) recently published a set of criteria that must be demonstrated to establish that a gene is responsible for a QTL (quantitative trait gene, or QTG). These include: (1) the demonstration of a linkage or association between the locus of the gene and the trait; (2) a significant sequence variant in the QTG between two strains with different phenotypes; (3) functional tests of the candidate QTG variants; and (4) an explanation of any complicating or confounding factors. A QTL on distal mouse chromosome 3, the locus of the Rpe65 gene, was demonstrated to be associated with differences in a retinal light damage phenotype between the BALB/c and B6alb albino mouse strains (point 1). The BALB/c sequence of the Rpe65 gene has the CTG code for leucine at residue 450, while the B6alb sequence has ATG for methionine (point 2). There is less RPE65 protein when met450 is present (demonstrated by Western blot), resulting

in a slower recovery of the visual transduction response of the PR demonstrated physiologically, a slower regeneration of rhodopsin after bleach demonstrated biochemically, and a decreased level of PR damage after light insult demonstrated histologically (point 3). The presence of the “protective” met450 variant of RPE65 in the NZW strain that is relatively sensitive to light insult was explained by the presence of other QTL genes that negate the protective effect (point 4). The types and quality of data cited in this review match or exceed the criteria for establishing a variant gene to be one QTG or one modifier of several comprising the genetic portion of the complex trait of PR light damage.

Some genes identified in microarray expression studies and studies of single genes are candidates for light damage quantitative trait loci Differential or comparative studies of mRNA expression and studies of individual genes bring much information about the light damage process in their own right, and also provide candidates for QTL based on chromosomal map locations. In the first approach, the upregulation (or downregulation) of retinal mRNAs in response to damaging levels of light is determined by comparing expression before and after light exposure, generally using microarray chips. The second approach involves measuring the effect on light damage of an increase or decrease or elimination of the expression of a gene or genes. The point of these studies is to identify the genes that govern the response to light insult, the protective defensive response, and the pathophysiological process of PR death. Three quantitative genetics studies designed to identify the chromosomal loci of genes that are involved in the retinal light damage process have been carried out, and, along with the chromosome 3 QTL (shown to be the Rpe65

Average ONL thickness of PRs after light exposure from N8, BALB/c mice with variations in the RPE65 met/leu450 allele.

ONL in microns

40 35 30 25 20 15 10 5 0 leu/leu

Figure 47.2 The number of mice measured for ONL was 15 for leu/leu, 18 for met/leu, and 5 for met/met. Probabilities derived from the student’s unpaired t-test were 0.03 between leu/leu and

met/leu

met/met

met/met, 0.31 between leu/leu and met/leu, and 0.01 between met/leu and met/met.

danciger: complex genetics of photoreceptor light damage

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gene), 11 other QTL have been described (Danciger et al., 2000, 2004, 2005). Are any of the genes implicated in light damage and discovered by microarray or single-gene studies located in the loci of these 11 QTL? Three parallel studies asked what changes occur in levels of mRNA after a toxic light exposure (Chen et al., 2004; Roca et al., 2004; Rattner and Nathans, 2005). The BALB/c albino was the mouse strain, the retina was the source of the mRNA, and a comprehensive Affymetrix (Santa Clara, Calif.) genome chip was used for mRNA probe hybridization in all three studies. The conditions before and after light exposure and the intensity and duration of light differed. Dark adaptation ranged from 24 hours (Chen et al., 2004) to 1–2 weeks (Rattner and Nathans, 2005) to rearing in the dark (Roca et al., 2004). Light exposures ranged from 1,500– 2,000 lux for 3 hours to 6,000 lux for 6 hours to 10,000 lux for 7 hours. Mice were sacrificed immediately (Chen et al., 2004; Roca et al., 2004) or kept in the dark for 24 hours first (Rattner and Nathans, 2005). Despite differences in the light exposure protocol, many genes were upregulated in common, particularly in the category of transcription factors. Many of the genes most highly upregulated appeared to be induced in Müller cells, even though light damage occurs primarily in PRs (Rattner and Nathans, 2005). At least part of the function of retinal Müller cells is thought to be to help maintain PRs and protect them from stress. The total number of upregulated genes in the three studies was greater than 300. Searching among 300 genes for those that are located in any of 11 chromosomal QTL loci would certainly provide candidate genes just by chance. In one of the studies, only 15 genes were upregulated (Roca et al., 2004); in another study, well over 200 were (Rattner and Nathans, 2005), and in the third study, about 70 genes were upregulated (Chen et al.,

2004). The first study had more stringent criteria for considering a gene upregulated. Therefore, we considered the 15 genes most highly upregulated (the only 15 genes, in one case) from each study. Of the 45 genes, 6 were upregulated in more than one study and 12 were located within the loci of retinal light damage QTL. One gene (Cebpd) was shown to be upregulated in all three microarray reports. This gene was located in a QTL from one retinal light damage study. Two more upregulated genes were each located in QTL present in two light damage studies (table 47.1). Examples of single-gene studies show that many genes influence retinal light damage. Bax and Bak1 double knockout mice were protected from intense light exposure (Hahn et al., 2004). Bax and Bak1 are pro-apoptotic genes of the bcl-2 family. Inhibition of the apoptotic factor AP-1 also protected PR cells from light-induced damage (Wenzel et al., 2001a). AP-1 is either a heterodimer of any of several Fos and Jun proteins or a homodimer of any of several Jun proteins (Curran and Franza, 1988). The absence of the antioxidant glutathione peroxidase-1 (Gpx1) in knockout mice left the retinas more sensitive to light-induced damage (Gosbell et al., 2006). The absence of the apoptotic regulator p53 (Trp53) or the absence of the Müller cell neurotrophin receptor p75 (Ngfr) in knockout mice had no effect on retinal light damage (Marti et al., 1998; Rohrer et al., 2003). Overexpression of erythropoietin (Epo) by hypoxic preconditioning protected the retina (Grimm et al., 2004), while overexpression of the EAT/mcl-1 gene (Mcl1), another member of the bcl-2 family, worsened light damage to the retina (Shinoda et al., 2001). These examples are apoptotic or other genes that are widely expressed and have a function that is not specific to the retina. On the other hand, the mouse orthologue of human RDH12 (Rdh12), a gene involved

Table 47.1 Upregulated genes after damaging light exposure that map to retinal light damage QTL Retinal Light Damage Study Gene Symbol Gene Name Klf10 B6alb × BALB/c Kruppel-like factor 10—TGF-β-inducible early growth response Ifi204 B6alb × NZW Interferon-γ-inducible gene 204 Activating transcription factor 3 Atf 3 Ctg f 129 × BALB/c

B6alb × BALB/c and B6alb × NZW 129 × BALB/c and B6alb × NZW

578

QTL 12 proximal 1 distal 1 distal

Fibroblast-inducible secreted protein

10 proximal

Cebpd

CCAAT/enhancer-binding protein δ

16 proximal

Edn2 Egr1

Endothelin 2 Early growth response 1

4 distal 4 distal

Pdpn

Podoplanin; glycoprotein 38

4 distal

Lcn2

Lipocalin 2

2 proximal

Mthfd2

Methylenetetrahydrofolate dehydrogenase (NAD+ dependent)

6 middle

Ednrb Chi3l1

Endothelin receptor type B Chitinase 3-like 1

14 distal 1 middle

mouse models of human eye disease

in the specific retinal function of visual transduction, particularly in the vitamin A recycling system (Thomson et al., 2005), protected the mouse retina from light-induced damage (Maeda et al., 2006). Specifically, the absence of mouse Rdh12 did not cause RD in the mouse, but it did make the mouse retina more sensitive to intense light. The absence of Rdh12 caused 11-cis-retinal to be regenerated more rapidly under excessive light (Maeda et al., 2006), increasing the cycles of rhodopsin regeneration and increasing the levels of all-trans-retinaldehyde in the inner segments. The increased levels of all-trans-retinal made PRs more sensitive to light. On the other hand, an opposite mechanism was involved when RPE65 protein was removed (Grimm et al., 2000) or decreased due to the met450 variant (Danciger et al., 2000; Wenzel et al., 2001b, 2003) with opposite effect. As mentioned earlier, mice homozygous for RPE65 met450 or with no RPE65 had lower or no levels of RPE65 protein in their RPE, slower regeneration of rhodopsin, and less PR loss after intense light exposure (Danciger et al., 2000; Grimm et al., 2000; Wenzel et al., 2001b, 2003). In summary, quantitative genetics studies have demonstrated the presence of many genes that affect light damage by the discovery of QTL. Microarray studies show the upregulation of many genes after damaging light exposure. Some of these genes are located in QTL and may be considered light damage effector gene candidates. Not surprisingly, single-gene knockout or enhanced gene expression studies have implicated apoptotic genes in PR light damage. Such genes are involved in cell death initiated in many other tissues as well. So far, only one specific eye gene expressed in the RPE, Rpe65, has been shown to be a naturally occurring complex trait gene or gene modifier. The influence of the met450 variant of RPE65 is to protect PRs from light damage by slowing down the visual cycle (Wenzel et al., 2001b, 2003). However, this same mechanism is in play in several genetically modified mice. The absence of Rdh12 speeds up regeneration of rhodopsin during intense light exposure, exacerbating PR damage (Thompson et al., 2005). Absence of the α -subunit of transducin (Gnat1) protects PRs from light damage under some circumstances (Hao et al., 2002). α-Transducin is an integral part of the visual cycle, and without it there is no hyperpolarization-depolarization cycle. The absence of either rhodopsin kinase (Grk1) (Chen et al., 1999a) or arrestin (Sag) (Chen et al., 1999b), both needed to shut off rhodopsin, brings about RD even in dim cyclic light because the visual cycle is kept on. Therefore, one component of the complex trait of light damage to PRs is perturbation of the very biochemical-physiological cycle that mediates vision under normal circumstances. Thus, in the retina of a mouse expressing the naturally occurring but rare RPE65 met450 modifier, damage is modulated under the stressed conditions of toxic light, while under nonstressed conditions the retina is normal. This is a very nice example

of an allele or gene variant that perturbs a system only under abnormal conditions and thereby modifies the effect of the abnormal conditions. REFERENCES Balkema, G. W., and Dräger, U. C. (1991). Impaired visual thresholds in hypopigmented animals. Vis. Neurosci. 6:577–585. Chabre, M., and Deterre, P. (1989). Molecular mechanism of visual transduction. Eur. J. Biochem. 179:255–266. Chen, C. K. (2005). The vertebrate phototransduction cascade: Amplification and termination mechanisms. Rev. Physiol. Biochem. Pharmacol. 154:101–121. Chen, C. K., Burns, M. E., Spencer, M., Niemi, G. A., Chen, J., Hurley, J. B., Baylor, D. A., and Simon, M. I. (1999a). Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase. Proc. Natl. Acad. Sci. U.S.A. 96:3718– 3722. Chen, J., Simon, M. I., Matthes, M. T., Yasumura, D., and LaVail, M. M. (1999b). Increased susceptibility to light damage in an arrestin knockout mouse model of Oguchi disease (stationary night blindness). Invest. Ophthalmol. Vis. Sci. 40:2978– 2982. Chen, L., Wu, W., Dentchev, T., Zeng, Y., Wang, J., Tsui, I., Tobias, J. W., Bennett, J., Baldwin, D., et al. (2004). Light damage induced changes in mouse retinal gene expression. Exp. Eye Res. 79:239–247. Cideciyan, A. V., Hood, D. C., Huang, Y., Banin, E., Li, Z. Y., Stone, E. M., Milam, A. H., and Jacobson, S. G. (1998). Disease sequence from mutant rhodopsin allele to rod and cone photoreceptor degeneration in man. Proc. Natl. Acad. Sci. U.S.A. 95: 7103–7108. Cruickshanks, K. J., Klein, R., and Klein, B. E. (1993). Sunlight and age-related macular degeneration: The Beaver Dam Eye Study. Arch. Ophthalmol. 111:514–518. Curran, T., and Franza, B. R., Jr. (1988). Fos and Jun: The AP-1 connection. Cell 55:395–397. Danciger, M., Lyon, J., Worrill, D., Hoffman, S., Lem, J., Reme, C. E., Wenzel, A., and Grimm, C. (2004). New retinal light damage QTL in mice with the light-sensitive RPE65 LEU variant. Mamm. Genome 15:277–283. Danciger, M., Matthes, M. T., Yasamura, D., Akhmedov, N. B., Rickabaugh, T., Gentleman, S., Redmond, T. M., LaVail, M. M., and Farber, D. B. (2000). A QTL on distal chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors. Mamm. Genome 11:422–427. Danciger, M., Yang, H., Handschumacher, L., and LaVail, M. M. (2005). Constant light-induced retinal damage and the RPE65-MET450 variant: Assessment of the NZW/LacJ mouse. Mol. Vis. 11:374–379. Glazier, A. M., Nadeau, J. H., and Aitman, T. J. (2002). Finding genes that underlie complex traits. Science 298:2345–2349. Gosbell, A. D., Stefanovic, N., Scurr, L. L., Pete, J., Kola, I., Favilla, I., and de Haan, J. B. (2006). Retinal light damage: Structural and functional effects of the antioxidant glutathione peroxidase-1. Invest. Ophthalmol. Vis. Sci. 47:2613–2622. Grimm, C., Wenzel, A., Hafezi, F., Yu, S., Redmond, T. M., and Reme, C. E. (2000). Protection of Rpe65-deficient mice identifies rhodopsin as a mediator of light-induced retinal degeneration. Nat. Genet. 25:63–66. Grimm, C., Wenzel, A., Stanescu, D., Samardzija, M., Hotop, S., Groszer, M., Naash, M., Gassmann, M., and Reme, C.

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(2004). Constitutive overexpression of human erythropoietin protects the mouse retina against induced but not inherited retinal degeneration. J. Neurosci. 24:5651–5658. Hahn, P., Lindsten, T., Lyubarsky, A., Ying, G. S., Pugh, E. N., Jr., Thompson, C. B., and Dunaief, J. L. (2004). Deficiency of Bax and Bak protects photoreceptors from light damage in vivo. Cell Death Differ. 11:1192–1197. Hao, W., Wenzel, A., Obin, M. S., Chen, C. K., Brill, E., Krasnoperova, N. V., Eversole-Cire, P., Kleyner, Y., Taylor, A., et al. (2002). Evidence for two apoptotic pathways in light-induced retinal degeneration. Nat. Genet. 32:254–260. Hayes, J. M., and Balkema, G. W. (1993). Visual thresholds in mice: Comparison of retinal light damage and hypopigmentation. Vis. Neurosci. 10:931–938. Jin, M., Li, S., Moghrabi, W. N., Philp, A. R., and Travis, G. H. (2006). Mutational analysis to determine key residues essential for activity and membrane association of Rpe65 isomerohydrolase. ARVO Abstract. LaVail, M. M., Gorrin, G. M., and Repaci, M. A. (1987a). Strain differences in sensitivity to light-induced photoreceptor degeneration in albino mice. Curr. Eye Res. 6:825–834. LaVail, M. M., Gorrin, G. M., Repaci, M. A., Thomas, L. A., and Ginsberg, H. M. (1987b). Genetic regulation of light damage to photoreceptors. Invest. Ophthalmol. Vis. Sci. 28:1043– 1048. LaVail, M. M., Gorrin, G. M., Repaci, M. A., and Yasumura, D. (1987c). Light-induced retinal degeneration in albino mice and rats: Strain and species differences. Prog. Clin. Biol. Res. 247: 439–454. LaVail, M. M., Gorrin, G. M., Yasumura, D., and Matthes, M. T. (1999). Increased susceptibility to constant light in nr and pcd mice with inherited retinal degenerations. Invest. Ophthalmol. Vis. Sci. 40:1020–1024. Lyubarsky, A. L., Savchenko, A. B., Morocco, S. B., Daniele, L. L., Redmond, T. M., and Pugh, E. N., Jr. (2005). Mole quantity of RPE65 and its productivity in the generation of 11cis-retinal from retinyl esters in the living mouse eye. Biochemistry 44:9880–9888. Maeda, A., Maeda, T., Imanishi, Y., Sun, W., Jastrzebska, B., Hatala, D. A., Winkens, H. J., Hofmann, K. P., Janssen, J. J., et al. (2006). Retinol dehydrogenase (RDH12) protects photoreceptors from light-induced degeneration in mice. J. Biol. Chem. 281:37697–37704. Manly, K. F., Cudmore, R. H., Jr., and Meer, J. M. (2001). Map Manager QTX, cross-platform software for genetic mapping. Mamm. Genome 12:930–932. Marti, A., Hafezi, F., Lansel, N., Hegi, M. E., Wenzel, A., Grimm, C., Niemeyer, G., and Reme, C. E. (1998). Light-induced cell death of retinal photoreceptors in the absence of p53. Invest. Ophthalmol. Vis. Sci. 39:846–849. Mata, N. L., Weng, J., and Travis, G. H. (2000). Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCRmediated retinal and macular degeneration. Proc. Natl. Acad. Sci. U.S.A. 97:7154–7159. Nusinowitz, S., Nguyen, L., Radu, R., Kashani, Z., Farber, D., and Danciger, M. (2003). Electroretinographic evidence for altered phototransduction gain and slowed recovery from photobleaches in albino mice with a MET450 variant in RPE65. Exp. Eye Res. 77:627–638.

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Organisciak, D. T., Li, M., Darrow, R. M., and Farber, D. B. (1999). Photoreceptor cell damage by light in young Royal College of Surgeons rats. Curr. Eye Res. 19:188–196. Rattner, A., and Nathans, J. (2005). The genomic response to retinal disease and injury: Evidence for endothelin signaling from photoreceptors to glia. J. Neurosci. 25:4540–4549. Redmond, T. M., Weber, C. H., Poliakov, E., Yu, S. S., and Gentleman, S. (2007). Effect of leu/met variation at residue 450 on isomerase activity and protein expression of RPE65 and its modulation by variation at other residues. Mol. Vis. 13:1813– 1821. Roca, A., Shin, K. J., Liu, X., Simon, M. I., and Chen, J. (2004). Comparative analysis of transcriptional profiles between two apoptotic pathways of light-induced retinal degeneration. Neuroscience 129:779–790. Rohrer, B., Matthes, M. T., LaVail, M. M., and Reichardt, L. F. (2003). Lack of p75 receptor does not protect photoreceptors from light-induced cell death. Exp. Eye Res. 76:125–129. Sanyal, S., and Hawkins, R. K. (1986). Development and degeneration of retina in rds mutant mice: Effects of light on the rate of degeneration in albino and pigmented homozygous and heterozygous mutant and normal mice. Vision Res. 26:1177– 1185. Shinoda, K., Nakamura, Y., Matsushita, K., Shimoda, K., Okita, H., Fukuma, M., Yamada, T., Ohde, H., Oguchi, Y., et al. (2001). Light induced apoptosis is accelerated in transgenic retina overexpressing human EAT/mcl-1, an anti-apoptotic bcl2 related gene. Br. J. Ophthalmol. 85:1237–1243. Simons, K. (1993). Artificial light and early-life exposure in agerelated macular degeneration and in cataractogenic phototoxicity. Arch. Ophthalmol. 111:297–298. Taylor, H. R., Munoz, B., West, S., Bressler, N. M., Bressler, S. B., and Rosenthal, F. S. (1990). Visible light and risk of agerelated macular degeneration. Trans. Am. Ophthalmol. Soc. 88:163– 173 [discussion 173–178]. Thompson, D. A., Janecke, A. R., Lange, J., Feathers, K. L., Hubner, C. A., McHenry, C. L., Stockton, D. W., Rammesmayer, G., Lupski, J. R., et al. (2005). Retinal degeneration associated with RDH12 mutations results from decreased 11-cis retinal synthesis due to disruption of the visual cycle. Hum. Mol. Genet. 14:3865–3875. Vogt, W. (1995). Oxidation of methionyl residues in proteins: Tools, targets, and reversal. Free Radic. Biol. Med. 18:93–105. Wang, M., Lam, T. T., Tso, M. O., and Naash, M. I. (1997). Expression of a mutant opsin gene increases the susceptibility of the retina to light damage. Vis. Neurosci. 14:55–62. Wenzel, A., Grimm, C., Samardzija, M., and Reme, C. E. (2003). The genetic modifier Rpe65Leu(450): Effect on light damage susceptibility in c-Fos-deficient mice. Invest. Ophthalmol. Vis. Sci. 44:2798–2802. Wenzel, A., Grimm, C., Seeliger, M. W., Jaissle, G., Hafezi, F., Kretschmer, R., Zrenner, E., and Reme, C. E. (2001a). Prevention of photoreceptor apoptosis by activation of the glucocorticoid receptor. Invest. Ophthalmol. Vis. Sci. 42:1653– 1659. Wenzel, A., Reme, C. E., Williams, T. P., Hafezi, F., and Grimm, C. (2001b). The Rpe65 Leu450Met variation increases retinal resistance against light-induced degeneration by slowing rhodopsin regeneration. J. Neurosci. 21:53–58.

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Age-Related Eye Diseases BO CHANG

Age-related eye diseases are the leading causes of vision impairment and blindness throughout the world. With the world’s population of senior citizens growing rapidly, the issue of vision loss in the older population is of paramount importance. Age-related eye diseases are costly to treat, threaten the ability of older adults to live independently, and increase the risk for accidents and falls. Of the age-related eye diseases, age-related macular degeneration (AMD) increases dramatically with age in men and women and is the most important cause of irreversible visual loss in the elderly. Cataracts are the leading cause of blindness in the world, affecting nearly 20.5 million Americans age 40 and older (Congdon et al., 2004; Blindness and visual impairment [editorial], 2004). From the available data, it appears that age-related eye diseases are caused by environmental factors triggering disease in genetically susceptible subjects. Identifying the genetic factors would contribute to understanding the pathogenesis. If those at risk could be identified, it might be possible to modify lifestyle or develop novel therapies in the presymptomatic stage to prevent disease or decrease severity. However, direct research on human ocular conditions is impeded by the poor availability of tissues and the impossibility of performing genetic manipulation in humans. Human eye tissue (including biopsy material) in most ocular diseases is seldom available, because it is difficult to obtain eye tissue samples without the risk of damaging the patient’s vision. Since genetic and biochemical experiments in human patients are not possible, animal models serve an important and unique role. Historically, mouse models have been especially useful in determining biochemical mechanisms in human ocular diseases. In this chapter, I focus on mouse age-related retinal degeneration disorders as possible models for human AMD and mouse age-related cataract mutants as models for human cataracts.

Mice as models for human age-related eye diseases The mouse lens and retina are remarkably similar in structure to the human lens and retina, and both species experience similar ocular disorders (Chang et al., 2005). Not only are developmental and invasive studies possible in mice, but the mouse’s accelerated life span and generation time (one mouse year equals about 30 human years, based on a ratio of average life span for each species) make it possible to

follow the natural progression of eye diseases in a relatively brief period. Studying aging diseases in mice has many advantages: typically mice live 2 years and thus age quickly, and they are relatively inexpensive and easy to maintain. The availability of inbred strains provides a population that is genetically the same from one mouse to the rest; each inbred strain is like an infinite set of monozygotic or identical twins. All mice of the same inbred strain generally can be expected to have the same phenotype. By contrast, differing environmental factors combined with many genetic variations between affected and unaffected individuals make it difficult to identify specific genes responsible for age-related eye diseases in humans. Environmental factors can play an especially strong confounding role in the etiology of lateonset eye diseases in humans because of the long period of life with exposure to different variables before any agerelated eye diseases are detected. In the mouse, environmental factors can be controlled to a high degree by raising mice in standard conditions of diet, light-dark cycle, cagechanging schedules, experienced animal handlers, and constant and clean air exchange in the mouse rooms, and with a high standard of disease prevention and animal health care. With environmental variance controlled, researchers can focus on the underlying genetic causes. Genetically homogeneous inbred mice, accessible at any age, offer an opportunity to study the histopathological and biochemical changes that occur during age-related eye disease formation and permit controlled study of single gene variations in a constant genetic background. The high level of homology between mouse and human genomes (>95% of genes are conserved) means that similar disease manifestations are often identified in mice and humans. Preservation of gene function is the reason that mice are often useful in studies of disease mechanisms in humans (Davisson et al., 1991; Nadeau et al., 1992; Quiring et al., 1994). Thus, the high degree of conservation between human and mouse chromosomes makes the mouse a powerful research tool with which to identify human genes, which can then be used in presymptomatic testing and for developing preventive treatment.

The search for age-related eye diseases in mice The Jackson Laboratory (TJL), having the world’s largest collection of mouse mutant stocks and genetically diverse inbred strains—more than 3,000 strains in 2006—is an ideal

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place to discover genetically determined eye variations and disorders. While screening mouse strains and stocks at TJL for genetic mouse models of human ocular disorders, we have identified numerous spontaneous or naturally occurring mutations (table 48.1). However, the search for lateonset eye disease has never been done, and the best place to do this is at TJL. TJL has the best collection of genetically unrelated inbred strains available anywhere. For clinical characterization, a primary search for gross eye abnormalities has been done by examining the eyelids, globe, cornea, and iris, first with visual inspection and then using a Nikon biomicroscope (slit lamp). The cornea is checked for clarity, size (bupthalmos vs. microcornea), surface texture, and vascularization. The iris is examined for pupil size, constriction, reflected luminescence, and synechiae. The eye is then dilated with 1% atropine and the lens is examined for cataracts. Finally, an indirect ophthalmoscope is used to examine the fundus for signs of retinal degeneration. In mice, the typical changes are retinal vessel constriction or retinal pigment epithelial disturbance, drusen, or other retinal deposits. Mice with a suspected abnormality are followed up with a secondary examination that includes electroretinography (ERG) and histological investigation. For genetic characterization, an initial genetic analysis to determine the mode of inheritance is carried out by making outcrosses and backcrosses or intercrosses. Once an eye disorder has been shown to be due to a new mutation, a linkage cross is set up to determine the mutant gene’s chromosomal location by doing a genome scan of DNA markers using PCR analysis. Mice with age-related eye diseases often are past the breeding age. Because all mice in an inbred strain are genetically identical, this can be overcome by doing the genetic crosses with young mice from the same strain, because they will acquire the same eye disease. Dominant and semidominant mutations are mapped by backcrosses or outcrosses to wildtype (+/+) mice. Recessive mutations are mapped using intercrosses, which are more efficient, because each F2 mouse obtains two potentially recombinant chromosomes, one from each parent. Modifying genes often can be detected in the initial cross by finding more than one chromosomal region associated with the disease phenotype. Polygenic traits are recognized by phenotype loss in outcrosses. If a disease turns out to be polygenic—that is, influenced by more genes than can be identified in standard genetic crosses—mice of the strain are still histologically and clinically characterized to provide a model for clinically similar human ocular disorders.

Age-related retinal degenerations in human and mouse eyes For many people, retirement means more time to read, watch television, sew, play cards, or drive to places they have

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always longed to visit. Yet by the time they reach age 65, many retirees find they no longer have those options because they have lost much of their vision to age-related eye disorders, such as age-related macular degeneration (AMD) and many forms of late-onset retinal degeneration, the most common uncorrectable causes of vision loss in the elderly. Although mice do not have a macula, mice do have homologues of genes that are associated with human AMD, and mutations in such genes in mice likely cause age-related retinal degeneration (ARRD). For example, partial loss of the ABCA4 or rim protein is sufficient to cause a phenotype in mice similar to recessive Stargardt disease and AMD, a common cause of visual loss in the human elderly (Mata et al., 2001). In the Abca4tm1Ght homozygous mouse model, the presence of A2E and lipofuscin granules is seen, along with shortening of the photoreceptor outer segments (Weng et al., 1999; Mata et al., 2000; Radu et al., 2004). Another example is that the ELOVL4 gene is associated with two related forms of human autosomal dominant macular dystrophy, and in situ hybridization on mouse retinal sections shows a strong uniform signal in the photoreceptor layer, particularly in the region corresponding to the photoreceptor inner segments (Zhang et al., 2001). Mutant mice carrying an Elovl4 transgene demonstrate an accumulation of A2E and lipofuscinlike material, but they also show photoreceptor outer segment disc disorganization and geographic atrophy (Karan et al., 2005). Mice with a 5 bp deletion knock-in of Elovl4 develop progressive photoreceptor degeneration (Vasireddy et al., 2006). Recently, a known polymorphism in human complement factor H (CFH), T → C substitution in exon 9, which resulted in the substitution of an uncharged tyrosine with a positively charged histidine (Y402H), was shown to be associated with increased risk for AMD (Edwards et al., 2005; Hageman et al., 2005; Haines et al., 2005; Klein et al., 2005; Zareparsi et al., 2005). The human and mouse CFH proteins contain 20 repetitive units of 60 amino acids, referred to as the short consensus repeat or the complement control protein module, arranged in a continuous fashion like a string of 20 beads (Rodriguez de Cordoba et al., 2004). In human and mouse, the CFH expression pattern was found to be similar, with the highest level of expression in the liver. In ocular tissue, CFH was detected in the distalmost optic nerve (3 mm) cut from the scleral surface of the eyeball, sclera, RPE-choroid, retina, lens, and ciliary body. In mouse, Cfh expression was observed from early embryonic stages, and in the eye, its expression increased with age (Mandal and Ayyagari, 2006). Historically, mouse models have been especially useful in determining biochemical mechanisms in retinal diseases. For example, invaluable information about the molecular and pathological basis of some of these diseases has been provided by the discoveries of gene mutations in several mouse retinal degeneration models. Discovery of the mouse

Gene (or Strain) Retinal Models rd3 Cln8mnd Rd4 Tubtub Mfrprd6 Nr2e3rd7 Cln6nclf Crb1rd8 Rd9 Pde6brd10 rd11 Rpe65rd12 rd13 rd14 rd15 rd16 rd17 B6-Trp53tm B6-Nr2e1frc Col2a1sed nob2 lvi nob3 cpfl1 Cpfl2 cpfl3 Cpfl4 cpfl5 cpfl6 cpfl7 Vldlrtm1Her Nm2621* Nm2641 nm3344 Rico Cataract Models Gja8Lop10 lop11 CrygdLop12 lop13 lop14 Nm1853 lop16 Cryaalop18 Dstncorn1 Dstncorn1-2J

Table 48.1 Mouse models of ocular disease for which chromosomal locations have been established Location Phenotype Description Chr 1 Chr 8 Chr 4 Chr 7 Chr 9 Chr 9 Chr 9 Chr 1 Chr X Chr 5 Chr 13 Chr 3 Chr 15 Chr 18 Chr 7 Chr 10 Chr 9 Chr 11 Chr 10 Chr 15 Chr X Chr 18 Chr 11 Chr 19 Chr 3 Chr 3 Chr 17 Chr 1 Chr 13 Chr 19 Chr 19 Chr 6 Chr 19 Chr 13 Chr 13

Retinal degeneration 3 Retinal degeneration discovered in mnd mice Retinal degeneration 4 Retinal degeneration 5 discovered in tub mice Retinal degeneration 6 Retinal degeneration 7 Retinal degeneration discovered in nclf mice Retinal degeneration 8 Retinal degeneration 9 Retinal degeneration 10 Retinal degeneration 11 Retinal degeneration 12 Retinal degeneration discovered in nmf5 mice Retinal degeneration 14 Retinal degeneration 15 Retinal degeneration 16 No ERG a-wave and retinal degeneration 17 Abnormal retinal layers, extra tissue and vessels in posterior chamber No retinal vessels and bad ERG response Retinoschisis Missing retinal outer plexiform layer (OPL) and no ERG b-wave 2 Light-induced visual impairment and missing retinal OPL No ERG b-wave 3 Cone photoreceptor function loss 1 White retinal spots with cone photoreceptor function loss 2 Cone photoreceptor function loss 3 White retinal spots with cone photoreceptor function loss 4 Cone photoreceptor function loss 5 Cone photoreceptor function loss 6 Cone photoreceptor function loss 7 Develop retinal spots and subretinal neovascularization Severe retinal pigment loss, it is allelic to Mitf mi Dominant optic nerve coloboma Recessive retinal degeneration Retina and iris coloboma

Chr 3 Chr 8 Chr 1 Chr 15 Chr 14 Chr 1 Chr 10 Chr 17 Chr 2 Chr 2

Homozygous mice are microphthalmic with dense white cataracts Recessive, vacuolated cataract Dominant, nuclear lumpy irregular cataract Early white cataract (former name nuc) Extruded lens cataract Dominant cataract Muscle paresis and cataract, mutants die at 1 month of age Recessive cataract Corneal epithelial dystrophy and white cataract Mild corneal epithelial dystrophy and white cataract Continued

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Gene (or Strain) bs2 lop20 Lop21 Nm2249 Nm2520 Nm2541 Nm2620 nm2897 Nm3062 nm3347 nm3364 nm3365 Glaucoma Models

Table 48.1 Mouse models of ocular disease for which chromosomal locations have been established (continued) Location Phenotype Description Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr Chr

2 1 16 3 5 5 10 2 1 5 2 17

Gpnmbipd Chr 6 Chr 4 Tryp1isa Nm2702 Chr 11 Other Eye Disease Models

Blind-sterile-2 with microphthalmia and cataract Cataract with variable expression Transgenic cataract Fetal cataract with microphthalmia Cataract and coloboma Dominant cataract Dominant cataract Recessive cataract Dominant cataract Recessive cataract starts at 1 month of age Recessive cataract Recessive cataract Iris pigment dispersion Iris stromal atrophy Swollen eyes, inner retina loss, and optic nerve cupping

Low-set ear, vascular hazy cornea; homozygotes have cataracts Wavy coat, eyelids fail to develop, enlarged heart and esophagus Anophthalmos in 80% of offspring; mice with intact globes have microophthalmia and frequently congenital corneal perforations, with collapse of the anterior chamber A/J Chr X Crystal deposits in iris nm1863 Chr 12 Cataract, abnormal iris and cornea nm2619 Chr 9 Mouse model for persistant hyperplastic primary vitreous Nm2557 Chr X X-linked model for aniridia: heterozygous females have a small iris, homozygous females and males have a cataract Nm3408 Chr 2 Eyeless, lens-cornea synechiae JR4326 Chr 2 Eyeless, lens-cornea synechiae * New mutation (NM, Nm or nm) numbers are assigned in TJL’s program as temporary unique identifiers until sufficient information is available to name the mutant allele or the mutated gene is identified. Lse wa3 eyeless

Chr 7 Chr 12 Chr 3

retinal degeneration 1 (rd1) mutation in the gene for the β-subunit of cGMP-phosphodiesterase (Pde6b) (Bowes et al., 1990) led to the identification of mutations in the human homologue (PDE6B) in similar human disorders (McLaughlin et al., 1993), and identification of the tubby (tub) mouse gene family (Noben-Trauth et al., 1996) led to the finding of tubby-like protein 1 (TULP1) alterations in individuals affected with autosomal recessive RP (Hagstrom et al., 1998; Banerjee et al., 1998). Similarly, discovery of the mutations Rds (retinal degeneration slow) in the peripherin 2 (Prph2) (Travis et al., 1989) and shaker 1 (sh1) in the myosin 7A (Myo7a) (Gibson et al., 1995) mouse genes paved the way for the identification of defects in the RDS-peripherin gene in patients with autosomal dominant RP, and various cone, cone-rod, and macular dystrophies (Keen and

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Inglehearn, 1996) and in MYO7A in patients with Usher’s syndrome type 1b (Weil et al., 1995). Discovery of 15 Strains of Mice with Age-Related Retinal Degeneration At TJL we have a productive screening and evaluation program for identifying new mouse models of ocular diseases. A component of this program, the study of age-related genetic eye diseases in mice, was funded by the Foundation Fighting Blindness. We aged 10 mice from each of 35 different inbred strains and screened them from 6 months to 2 years of age and up for late-onset retinal disorders. The 35 strains listed in table 48.2 have known abnormalities (low ERG, iris atrophy, cataracts) as strain characteristcs or are wild-derived strains that have not been characterized for age-related eye diseases.

Table 48.2 Eye disorders in aged 35 inbred strains Laboratory Strains C57BL/6J A/J RIII/DmMobJ 129 P3/J DBA/1J BALB/cByJ BALB/cJ LP/J NZW/LacJ SJL/J YBR/EiJ CE/J RF/J SWR/J CBA/CaJ C57BR/cdJ

Cataract* and normal retina at 30 months old ARRD at 26 months old and iris calcium deposits at 6 months old, normal lens† at 26 months old Subcapsular cataract‡ and ARRD at 24 months old ARRD and normal lens† at 23 months old Pigment in angle, thin iris, cataract§ and normal retina at 25 months old ARRD and normal lens† at 25 months old ARRD and normal lens† at 25 months old ARRD and cataract§ at 25 months old ARRD and normal lens† at 25 months old Pdebrd1 and normal lens† at 24 months old Cornea opacity (dystrophy), normal lens† and ARRD at about 24 months old Subcapsular cataract§ and normal retina at 24 months old Normal lens† and retina at about 18 months old Pdebrd1, normal lens† at 24 months old Nuclear cataract‡ and normal retina at 26 months old Iris transillumination, cataract,* and normal retina at 26 months old

Wild-Derived Strains CASA/RkJ (M.m. castaneus) CAST/EiJ (M.m. castaneus) CZECHII/EiJ (Mus musculus) AU/SsJ LEWES/EiJ (Mus domesticus) MOLC/RkJ (M.m. molossinus) MOLD/RkJ (M.m. molossinus) MOLF/EiJ (M.m. molossinus) Mus caroli/EiJ PANCEVO/EiJ (Mus hortulanus) PERA/EiJ (Peru-Atteck) PERC/EiJ (Peru-Coppock) SF/CamEiJ (San Francisco) SKIVE/EiJ(Mus musculus/domesticus) SPRET/EiJ (Mus spretus, Spain) TIRANO/EiJ (Mus domesticus) WSB/EiJ (Mus domesticus) WMP/PasDnJ (Mus domesticus) ZALENDE/EiJ (Mus domesticus) * Cataract starting at 14 months of age. † Clear lenses throughout their lives. ‡ Lens opacity starting at 8 months of age. § Mild lens opacity starting at 22 months of age. ARRD, age-related retinal degeneration.

Subcapsular cataract‡ and normal retina at 23 months old Subcapsular cataract‡ and normal retina at 25 months old Cataract* and ARRD at 23 months old Pupils don’t fully dilate, cataract* and normal retina at 24 months old Nuclear cataract‡ and ARRD at 30 months old Cataract* and normal retina at 24 months old Iris holes, Pdebrd1 and subcapsular cataract* at 24 months old Pdebrd1 and normal lens† at 24 months old Areas of disrupted ONL and RPE, normal lens† and ARRD at 28 months old Posterior cataract§ and normal retina at 26 months old Cortical cataract‡ and ARRD at 24 months old Cataract* and ARRD at 24 months old Pdebrd1, normal lens† at 30 months old Cataract* and normal retina at 24 months old Cortical cataract§ and normal retina at 24 months old Cortical cataract‡ and normal retina at 26 months old Subcapsular cataract§ and ARRD at 30 months old Cornea spots, normal lens† and ARRD at 30 months old Retinal cupping and detachment, normal lens† at about 20 months old

We have discovered 15 strains in which all mice screened had ARRD (late-onset retinal degeneration). Seven of these were wild-derived strains (PERA/EiJ, PERC/EiJ, CZECHII/EiJ, LEWES/EiJ, Mus Caroli/EiJ, WSB/EiJ, WMP/PasDnJ) and eight were laboratory strains (A/J,

129P3/J, BALB/cJ, BALB/cByJ, RIII/DmMobJ, LP/J, YBR/EiJ, NZW/LacJ) (see table 48.2). Wild-derived strains have been inbred from mice captured in natural populations and are expected to harbor different mutations from those found in long-inbred laboratory strains. Mice from three of

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the 15 strains in which ARRD was found were blind at 2 years of age as confirmed by ERG (no response) and histology (no outer nuclear layer, ONL), and mice of the other 12 strains experienced slow retinal degeneration with very poor vision, as confirmed by low ERG response and reduced ONL (2–6 layers compared to the normal 10 layers of ONL) at 2 years of age. The data presented here document the

disease phenotype in seven wild-derived ARRD strains with retinal degeneration (figure 48.1) and eight laboratory ARRD strains of mice with ONL cell loss (figure 48.2). In six of these laboratory strains the retinal degeneration may be caused by light damage during aging, because these six strains of mice are albino and lack a pigmented iris to protect the retina from light during the daily 14 hours of light in the

Figure 48.1 Retinal histological sections in seven wild-derived ARRD strains showing relatively normal retinal structure at relatively younger ages and retinal degeneration at older ages. The strain names are labeled at the top and the ages are labeled within each retinal section. GCL, ganglion cell layer; INL, inner nu-

clear layer; IPL, inner plexiform layer; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer segments; RPE, retinal pigment epithelial cells.

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light-dark cycle of the mouse room. Retinal degeneration can be induced by exposure to excessive doses of light and is used as a model to study photoreceptor apoptosis, the common final pathway of cell loss in human AMD and some forms of inherited retinal degenerations (Rome et al., 1998).

Genetic factors reducing the light damage susceptibility (LDS) of retinal photoreceptors in C57BL/6J mice were postulated in 1987 (LaVail et al., 1987); however, the underlying molecular mechanisms have not yet been identified. Recently, by comparing progeny from a cross between

Figure 48.2 Retinal histological sections in eight laboratory ARRD strains showing relatively normal retinal structure at relatively younger ages and retinal degeneration at older ages. The strain names are labeled at the top and the ages are labeled within each retinal section. GCL, ganglion cell layer; INL, inner nucle-

ar layer; IPL, inner plexiform layer; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer segments; RPE, retinal pigment epithelial cells.

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BALB/cJ and C57BL/6J mice, a sequence variation in the Rpe65 gene cosegregated with the low LDS of C57BL/6J mice (Danciger et al., 2000). We have typed the six albino strains of mice with ARRD identified in our screening study for sequence variation in the Rpe65 gene and found that two of them (RIII/DmMobJ and NZW/LacJ) have the same Rpe65 genotype as C57BL/6J. The other four carry the susceptibility allele.

Cataracts in human and mouse eyes Early in life the lens is transparent, and incoming light encounters no difficulty in its passage through the eye. With aging, the lens becomes less clear, incoming light is scattered to an increasing degree, and, if loss of lens clarity is severe enough, vision is affected. When clouding of the lens impairs vision, a clinically significant cataract is present. More than half of Americans over age 65 have a cataract ( Jacques et al., 1997). Cataracts account for 42% of all vision loss, making the disorder the leading cause of blindness worldwide (National Eye Institute report, Vision Research: A National Plan: 1999–2003). Based on our preliminary studies, agerelated cataracts are also very common in mice. At TJL, when we screened aged mice from 35 strains for models of human age-related ocular diseases, we identified 21 strains with a high incidence of late-onset cataracts. Onset ranged from 8 to 22 months, and 14 strains of mice had clear lenses throughout their lives (see table 48.2). There is increasing epidemiological evidence that agerelated cataracts are a multifactorial disease, with environmental and genetic components interacting (West and Valmadrid, 1995). Epidemiological studies also suggest a major inherited genetic component in the etiology of both anterior cortical and nuclear cataracts (Heiba et al., 1993, 1995), and twin studies suggest that 50% of age-related cataracts can be accounted for by inheritance (Hammond et al., 2000). Inheritance is already known to be the major contributor to congenital cataract occurring in childhood, and several mutated genes causing human congenital cataracts have already been identified (Francis et al., 2000). Because cataracts are easily detected by external clinical examination, the number of described mouse cataract models has grown rapidly. A 1982 review listed 18 spontaneous and induced mutations that cause cataracts as part of the resulting phenotype (Foster et al., 1982). By 1997 the number had grown to 46 (Smith et al., 1997). Research by our group at TJL has identified the spontaneous mutation lop18 in the mouse Cryaa (α-crystallin) gene (Chang et al., 1999), the spontaneous mutation Lop12 in the Crygd (γDcrystallin) gene (Smith et al., 2000), and the spontaneous mutation Lop10 in the Gja8 (connexin 50) gene (Chang et al., 2002). Currently, human mutations have been identified in the genes encoding some lens-specific crystallins,

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connexins, aquaporin, and beaded filament protein, BFSP2 (Sheils et al., 1998; Heon et al., 1999; Mackay et al., 1999; Berry et al., 2000; Jakobs et al., 2000; Rees et al., 2000). In most cases the genetic mutations causing cataracts have been identified using a candidate gene approach once the chromosomal location of the mutation has been determined. It has been estimated that there might be as many as 40 genes contributing to congenital cataracts in the mouse, and it would be reasonable to assume a similar number in humans (Hejtmancik and Kantorow, 2004). Thus, the concept of genetic mechanisms causing earlyonset cataracts is well established, but to date, very little is known concerning the identity of genes that, either alone or together with environmental factors, confer susceptibility to age-related cataracts. Discovery of Age-Related Cataracts in Mouse In our search for late-onset ocular diseases in mice, we have found that the majority of eye diseases occur in aged mice, just as they do in humans. In the study described earlier in which we evaluated aged mice of 35 selected, genetically diverse inbred strains at greater than 2 years of age for age-related eye disorders, we also screened for lens opacity. As part of this program, we have found seven strains of mice with lens opacity starting at eight months of age (RIII/DmMobJ, CBA/CaJ, CASA/RkJ, CAST/EiJ, LEWES/EiJ, PERA/ EiJ, TIRANO/EiJ), eight strains of mice with cataract starting at 14 months of age (C57BL/6J, C57BR/cdJ, AU/ SsJ, CZECHII/EiJ, MOLC/RkJ, MOLD/RkJ, PERC/EiJ, SKIVE/EiJ), and six strains of mice with a mild lens opacity starting at 22 months of age (DBA/1J, LP/J, CE/J, PANCEVO/EiJ, SPRET/EiJ, WSB/EiJ). Mice of another 14 strains have clear lenses throughout their lives (see table 48.2).

Summary Because the 35 strains of mice in our age-related vision loss study were all inbred, the individuals in a strain are genetically identical (like monozygotic twins), and all mice were maintained in the same standard environment, our findings demonstrate that genetic mechanisms contribute to the etiology and pathogenesis of ARRD and cataracts. No matter what the mode of inheritance or number of genes involved, the strains themselves provide reproducible models for research on similar human eye disorders. acknowledgments

Work was supported by the Foundation Fighting Blindness. I am grateful to Norm Hawes, Ron Hurd, Jieping Wang, Muriel Davisson, Tom Roderick, and John Heckenlively for their excellent help on the mouse aging project, and Melissa Berry for her critical reading of the manuscript.

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Hagstrom, S. A., North, M. A., Nishina, P. L., Berson, E. L., and Dryja, T. P. (1998). Recessive mutations in the gene encoding the tubby-like protein TULP1 in patients with retinitis pigmentosa. Nat. Genet. 18:174–176. Haines, J. L., Hauser, M. A., Schmidt, S., Scott, W. K., Olson, L. M., Gallins, P., Spencer, K. L., Kwan, S. Y., Noureddine, M., et al. (2005). Complement factor H variant increases the risk of age-related macular degeneration. Science 308:419– 421. Hammond, G. J., Sneider, H., Spector, T. D., and Gilbert, G. E. (2000). Genetic and environmental factors in age-related nuclear cataracts in monozygotic and dizygotic twins. N. Engl. J. Med. 342:1786–1790. Heiba, I. M., Elston, R. C., Klein, B. E., and Klein, R. (1993). Genetic etiology of nuclear cataract: Evidence for a major gene. Am. J. Med. Genet. 47:1208–1214. Heiba, I. M., Elston, R. C., Klein, B. E., and Klein, R. (1995). Evidence for a major gene for cortical cataract. Invest. Ophthalmol. Vis. Sci. 36:227–235. Hejtmancik, J. F., and Kantorow, M. (2004). Molecular genetics of age-related cataract. Exp. Eye Res. 79(1):3–9. Heon, E., Priston, M., Schorederet, D., Billingsley, G., Girard, P., Lubsen, N., and Munier, F. L. (1999). The gamma crystallins and human cataracts: A puzzle made clearer. Am. J. Hum. Genet. 65:1261–1267. Jacques, P. F., Taylor, A., Hankinson, S. E., Willett, W. C., Mahnken, B., Lee, Y., Vaid, K., and Lahav, M. (1997). Longterm vitamin C supplement use and prevalence of early agerelated lens opacities. Am. J. Clin. Nutr. 66:911–916. Jakobs, P. M., Hess, J. F., Fitzgerald, P. G., Kramer, P., Weleber, R. G., and Litt, M. (2000). Autosomal dominant congenital cataract associated with a deletion mutation in the human beaded filament protein gene BFSP2. Am. J. Hum. Genet. 66: 1432–1436. Karan, G., Lillo, C., Yang, Z., Cameron, D. J., Locke, K. G., Zhao, Y., Thirumalaichary, S., Li, C., Birch, D. G., et al. (2005). Lipofuscin accumulation, abnormal electrophysiology and photoreceptor degeneration in mutant ELOVL4 transgenic mice: A model for macular degeneration. Proc. Natl. Acad. Sci. U.S.A. 102:4164–4169. Keen, T. J., and Inglehearn, C. F. (1996). Mutations and polymorphisms in the human peripherin-RDS gene and their involvement in inherited retinal degeneration. Hum. Mutat. 8: 297–303. Klein, R. J., Zeiss, C., Chew, E. Y., Tsai, J. Y., Sackler, R. S., Haynes, C., Henning, A. K., San Giovanni, J. P., Mane, S. M., et al. (2005). Complement factor H polymorphism in age-related macular degeneration. Science 308:385–389. LaVail, M. M., Gorrin, G. M., and Repaci, M. A. (1987). Genetic regulation of light damage to photoreceptors. Invest. Ophthalmol. Vis. Sci. 28:1043–1048. Mackay, D., Ionides, A., Kibar, Z., Rouleau, G., Berry, V., Moore, A., Shiels, A., and Bhattacharya, S. (1999). Connexin46 mutations in autosomal dominant congenital cataract. Am. J. Hum. Genet. 64:1357–1364. Mandal, M. N., and Ayyagari, R. (2006). Complement factor H: Spatial and temporal expression and localization in the eye. Invest. Ophthalmol. Vis. Sci. 47:4091–4097. Mata, N. L., Tzekov, R. T., Liu, X., Weng, J., Birch, D. G., and Travis, G. H. (2001). Delayed dark-adaptation and lipofuscin accumulation in abcr +/− mice: Implications for involvement of ABCR in age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 42:1685–1690.

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Mata, N. L., Weng, J., and Travis, G. H. (2000). Biosynthesis of a major lipofuscin fluorophore in mice and humans with abcrmediated retinal and macular degeneration. Proc. Natl. Acad. Sci. U.S.A. 97:7154–7159. McLaughlin, M. E., Sandberg, M. A., Berson, E. L., and Dryja, T. P. (1993). Recessive mutations in the gene encoding the beta-subunit of rod phosphodiesterase in patients with retinitis pigmentosa. Nat. Genet. 4:130–134. Nadeau, J. H., Davisson, M. T., Doolittle, D. P., Grant, P., Hillyard, A. L., Kosowsky, M. R., and Roderick, T. H. (1992). Comparative map for mice and human. Mamm. Genome 3:480–536. Noben-Trauth, K., Naggert, J. K., North, M. A., and Nishina, P. M. (1996). A candidate gene for the mouse mutation tubby. Nature 380:534–538. Quiring, R., Walldor, U., Kloter, U., and Gehring, W. J. (1994). Homology of the eyeless gene of Drosophila to the small eye gene in mice and aniridia in human. Science 265:765–769. Radu, R. A., Mata, N. L., Bagla, A., and Travis, G. H. (2004). Light exposure stimulates formation of A2E oxiranes in a mouse model of Stargardt’s macular degeneration. Proc. Natl. Acad. Sci. U.S.A. 101:5928–5933. Rees, M. I., Watts, P., Fenton, I., Clarke, A., Snell, R. G., Owen, M. J., and Gray, J. (2000). Further evidence of autosomal dominant congenital zonular pulverulent cataracts linked to 13q11 (CZP3) and a novel mutation in connexin 46 (GJA-3). Hum. Genet. 106:206–209. Rodriguez de Cordoba, S., Esparza-Gordillo, J., Goicoechea de Jorge, E., Lopez-Trascasa, M., and Sanchez-Corral, P. (2004). The human complement factor H: Functional roles, genetic variations and disease associations. Mol. Immunol. 41: 355–367. Rome, C. E., Grimm, C., Hafezi, F., Marti, A., and Wenzel, A. (1998). Apoptotic cell death in retinal degenerations. Prog. Retin Eye Res. 17:443–464. Sheils, A., Mackay, D., Ionides, A., Berry, V., Moore, A., and Bhattacharya, S. (1998). A missense mutation in the human connexin 50 gene underlies autosomal dominant “zonular

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pulverulent” cataract, on chromosome 1q. Am. J. Hum. Genet. 62:526–532. Smith, R. S., Hawes, N. L., Chang, B., Roderick, T. H., Akeson, E. C., Heckenlively, J. R., Gong, X., Wang, X., and Davisson, M. T. (2000). Lop12, a new mouse mutation causing lens opacity similar to human Coppock cataract. Genomics 63:314–320. Smith, R. S., Sundberg, J. P., and Linder, C. C. (1997). Mouse mutations as models for studying cataracts. Pathobiology 65: 146–154. Travis, G. H., Brennan, M. B., Danielson, P. E., Kozak, C. A., and Sutcliffe, J. G. (1989). Identification of a photoreceptorspecific mRNA encoded by the gene responsible for retinal degeneration slow (rds). Nature 338:70–73. Vasireddy, V., Jablonski, M. M., Mandal, M. N., Raz-Prag, D., Wang, X. F., Nizol, L., Iannaccone, A., Musch, D. C., et al. (2006). Elovl4 5-bp-deletion knock-in mice develop progressive photoreceptor degeneration. Invest. Ophthalmol. Vis. Sci. 47:4558–4568. Weil, D., Blanchard, S., Kaplan, J., Guilford, P., Gibson, F., Walsh, J., Mburu, P., Varela, A., Levilliers, J., et al. (1995). Defective myosin VIIA gene responsible for Usher syndrome type 1B. Nature 374:60–61. Weng, J., Mata, N. L., Azarian, S. M., Tzekov, R. T., Birch, D. G., and Travis, G. H. (1999). Insights into the function of rimprotein in photoreceptors and etilogy of Stargardt’s disease from the phenotype in abcr knockout mice. Cell 98:13–23. West, S. K., and Valmadrid, C. T. (1995). Epidemiology of risk factors for age-related cataract. Surv. Ophthalmol. 39:323– 334. Zareparsi, S., Branham, K. E., Li, M., Shah, S., Klein, R. J., Ott, J., Hoh, J., Abecasis, G. R., and Swaroop, A. (2005). Strong association of the Y402H variant in complement factor H at 1q32 with susceptibility to age-related macular degeneration. Am. J. Hum. Genet. 77:149–153. Zhang, K., Kniazeva, M., Han, M., Li, W., Yu, Z., Yang, Z., Li, Y., Metzker, M. L., Allikmets, R., et al. (2001). A 5-bp deletion in ELOVL4 is associated with two related forms of autosomal dominant macular dystrophy. Nat. Genet. 27:89–93.

VII ADVANCED GENOMIC TECHNOLOGIES: APPLICATIONS TO THE MOUSE VISUAL SYSTEM

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New Genetic Technologies for Studying the Morphology, Physiology, and Development of Mouse Retinal Neurons TUDOR C. BADEA AND JEREMY NATHANS

Despite its comparatively reduced visual abilities, the mouse is becoming one of the most important organisms for investigating the physiology, organization, and development of the mammalian visual system. This trend reflects the ease with which genetic manipulations can be performed in the whole animal, including manipulation of defined subsets of neurons. Mouse studies have made significant contributions to the three main and interrelated areas of vision research: the molecular mechanisms of neuronal function and development, circuits and information coding, and pathological mechanisms of disease. In each of these areas, the modern mouse geneticist’s approach consists largely in introducing or removing various genetic elements and asking how neuronal or glial structure, function, or development has been modified. This chapter surveys current genetic approaches used to visualize subpopulations of cells or to alter their function or development. The emphasis is on retinal neurons, although the experimental approaches we discuss apply equally to glia and to visual pathways beyond the retina. Rather than providing an exhaustive summary, we highlight examples that illustrate general principles.

Gene transfer and gene targeting methods In some situations, it is feasible to directly deliver DNA or RNA to target cells in the living animal, for example by injecting them into the eye as recombinant viruses or by using electroporation (Matsuda and Cepko, 2004; Kachi et al., 2005; Bi et al., 2006). With these approaches the researcher can achieve a decrement in gene expression by RNA interference (RNAi), or the ectopic production of reporters, light-activated channels, or other proteins. This is especially attractive for animals in which germ-line manipulation is difficult or impossible or in which the eye is particularly accessible to injection. In addition, ex vivo experiments,

in which isolated retinas are maintained in culture, allow gene delivery by the aforementioned approaches, as well as by particle-mediated gene transfer (the “gene gun”) (Wellmann et al., 1999; Gan et al., 2000; O’Brien and Lummis, 2004). Individual neurons can also be labeled using particlemediated transfer of lipophilic dyes (Sun et al., 2002; Pignatelli and Strettoi, 2004). Applications of somatic cell gene transfer technologies include visualizing cell lineages and neuronal morphology by expressing histochemical reporters (Price et al., 1987; Turner et al., 1990), and modifying or monitoring function, for example, by RNAi or expression of a fluorescent calcium indicator. In some cases of somatic cell gene transfer, gene expression can be limited to a defined subset of neurons with a cell type–specific promoter. More commonly, the inefficient gene delivery process produces a sparse population of transduced cells, permitting the identities of individual cells to be determined later based on morphology and position, a strategy that was used in the retroviral marking of neuronal lineages (Turner et al., 1990). Because somatic cell gene transfer approaches are not specific to the mouse, they are not the focus of this review. However, they represent valuable alternatives to germ-line manipulation and can also be used in conjunction with the germ-line approaches discussed in this chapter. Among techniques for altering the germ line, the insertion of transgenes (figure 49.1) is conceptually the simplest. Classically, this has involved the random insertion of relatively small constructs in which promoters with more or less restricted expression patterns control reporter genes (such as E. coli β-galactosidase; human placental alkaline phosphatase [PLAP], or fluorescent proteins such as green fluorescent protein [GFP]), genetic modulators (such as Cre recombinase or the ligand-controlled reverse tetracycline transactivator [rtTA]), or, more recently, modulators of physiological function (such as drug- or light-activated ion channels). Reporter genes are usually enzymes with

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Cell specific promoter

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Diphtheria Toxin

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Figure 49.1 Transgenic approaches. Transgenic constructs are stably inherited genetic elements that integrate at random chromosomal locations and with a variable number of copies at the site of integration. A, Typical transgenes are composed of promoter elements that can direct transcription in more or less restricted cell populations and various cDNAs encoding reporter genes (such as PLAP or GFP), genetic regulators (represented here by the Cre recombinase), or other functional effectors (represented here by the α subunit of diphtheria toxin, which ablates the cell in which it is expressed). B, Bacterial artificial chromosome (BAC) transgenes are also randomly integrated in the genome; however, they carry larger segments of DNA and often reproduce more faithfully the expression pattern of the endogenous gene. The desired genetic elements can be placed under the control of the transgene by means of an internal ribosomal entry site (IRES) as a second translation unit (top), or they can replace the transgene coding region (bottom).

well-established histochemical stains, such as β-galactosidase or PLAP, or fluorescent proteins such as GFP. Transgene constructs are injected into the pronuclei of fertilized eggs and the resulting founders are screened by polymerase chain reaction (PCR) or Southern blotting for the presence of the transgene (Hogan et al., 1986). These founders will transmit the stably integrated transgene through the germ line to their offspring, which can then be analyzed to determine the pattern of transgene expression. In general, only a fraction of the lines express the injected transgene, and the expression pattern may vary substantially from line to line. In addition, transgene expression patterns are often variegated (i.e., expressed in only a subset of the expected cells), an effect that appears to reflect the chromatin structure at the site of integration. Finally, many transgenes are expressed in subsets of cells distinct from the ones in which the promoter segment used to drive expression is normally active (Xiang

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et al., 1996). In some situations, variegation or misexpression has proved useful, as described later in the chapter. The utility of the classic transgenic approach depends on the availability of promoter segments that can drive expression in defined subsets of neurons. So far, this has been the case in only a few instances, most notably in primary sensory neurons that express receptor proteins in a cell type–specific manner. In the mouse retina, rhodopsin is produced in rod photoreceptors, short-wavelength-sensitive (S) photopigment is produced in S cones, middle-wavelength-sensitive (M) photopigment is produced in M cones, and melanopsin is produced in a special class of light-responsive retinal ganglion cells (RGCs). Even in this relatively simple system, however, there is an imperfect correlation between cell type and molecular marker: in mice and other rodents, some cones coexpress both S and M photopigment genes (Rohlich et al., 1994; Applebury et al., 2000). A second route to cell type–specific promoters for transgene expression in the retina is through the use of genes that are expressed by many neuronal populations elsewhere in the CNS but are restricted in the retina to one or a few cell types. Two examples are the genes encoding choline acetyltransferase and tyrosine hydroxylase, which are expressed in starburst and dopaminergic amacrine cells, respectively (Tauchi and Masland, 1984; Gustincich et al., 1997). Many other promoters are available for broad classes of neurons; for example, the Crx promoter is expressed in all photoreceptors (Furukawa et al., 2002). Bacterial artificial chromosome (BAC) transgenesis (figure 49.1B) represents a variant strategy in which a large chromosomal fragment containing the gene of interest is typically engineered to direct the expression of a foreign protein (Heintz, 2001). The rationale for this strategy is that a large (100–200 kb) fragment, often containing the entire gene of interest, will more faithfully reproduce the endogenous expression pattern than a small promoter fragment, and will therefore direct reporter expression more precisely to that cell population. In some instances in which a transgene is normally expressed in multiple cell types or at multiple stages of development, it is desirable to activate the transgene in a subset of these cells or only during a limited temporal window. This can be achieved by using conditional expression systems, which are discussed at the end of the chapter. The second major class of germ-line genetic manipulation involves targeted alterations using embryonic stem (ES) cells. This typically involves reporter insertion (knock-in), target gene deletion (knockout), or a combination of the two (figure 49.2). Briefly, in this approach, the construct of interest is introduced into ES cells, where it recombines with the target locus by homologous recombination. The potentially difficult steps are (1) construction of the targeting plasmid, (2) screening for the correct homologous recombination event among many ES clones, and (3) the subsequent generation

loxP

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excision by Cre recombination

loxP

Figure 49.2 Knock-in and knockout strategies. In the example shown here, the endogenous exons of the target gene are flanked by loxP sites, and the endogenous transcription unit is followed by a reporter gene that is transcriptionally silent prior to Cre-mediated recombination. Recombination induced by Cre deletes the endogenous gene’s exons and thereby allows the expression of the reporter gene under the same 5′ transcriptional regulatory elements as the

endogenous gene. We note here that any regulatory elements located within introns would be deleted. This technique can be used to guide functional and morphological analyses of the cells that normally express the target gene. These cells can be studied in the heterozygous or homozygous mutant state, depending on whether the other allele of the gene is wild type or mutant (assuming that the gene is autosomal).

of the mutant mouse by injection of ES lines into early embryos. However, the advantages are substantial. Knock-in lines generally exhibit less variegation and more faithful patterns of reporter gene expression than transgenic lines, the dosage of the modified gene is well defined, and the line-toline variability in expression that is seen in transgenic lines (reflecting the different locations and copy numbers of the transgene) is eliminated. As a result of this last characteristic, the number of lines to be screened is substantially reduced. Targeted gene manipulations are increasingly being used in combination with site-specific DNA rearrangement catalyzed by sequence-specific Cre or Flp recombinases (Branda and Dymecki, 2004), which can be expressed (from a second locus) in defined subsets of cells and, in the case of the CreER(T) fusion protein, can also be controlled pharmacologically. This approach has been used to circumvent problems of lethality or other severe phenotypes associated with the ubiquitous loss of gene function that in many cases is observed with the standard knockout allele. With the availability of new mouse lines that express the recombinases under cell type–specific control, this approach is increasingly being used to produce changes in gene function in defined subsets of cells and to map the cell types and time windows in which a particular gene acts. In the following sections we describe specific examples of these approaches as they have been applied to different retinal cell types. At the end of the chapter we discuss techniques for temporal control of gene expression.

galactosidase or PLAP reporters in transgenic mice. These experiments showed that photopigment promoter transgenes from various mammals generally recapitulate the expression patterns of the corresponding endogenous genes. However, the expression of these transgenes exhibits substantial variegation, and, in the case of the human S cone photopigment promoter, misexpression was also observed in a subset of cone bipolar cells (Chen et al., 1994; Chiu and Nathans, 1994a). Interestingly, the human long-wavelength (L) pigment promoter and 5′ enhancer drives transgene expression in both M and S cones in the mouse (Wang et al., 1992). With larger photopigment promoter segments, expression is generally observed in a larger fraction of the expected target cells (Zack et al., 1991). For example, transgenic constructs carrying 11 kb of the mouse rhodopsin locus, including 4.5 kb of 5′ flanking sequences and all of the introns and exons, efficiently express rhodopsin in rod photoreceptors, which facilitated the study of a rhodopsin mutant responsible for retinitis pigmentosa (Sung et al., 1994). One of the most informative genetic approaches is the targeted ablation of a specific cell type to assess its role in the circuitry of the system. Soucy et al. (1998) used this strategy to assess the role of cones in the scotopic (dim light) visual pathway. Prior studies had shown that rods transmit their signals via metabotropic glutamate receptors on rod bipolar cells, all of which are of the ON type; these bipolar cells then synapse onto AII amacrine cells in the innermost division of the inner plexiform layer (IPL). The AII amacrine cells in turn feed the rod signals into the cone pathway by means of gap junctions with ON cone bipolar cells and by sign-inverting glycinergic synapses with OFF cone bipolar cells. From this circuitry, it would be predicted that the glutamate analogue L-(+)-2-amino-4-phosphonobutyric acid (APB), which selectively activates metabotropic glutamate receptors, and therefore blocks both rod-to-rod bipolar and

Photoreceptors Promoter sequences of bovine (Zack et al., 1991), human (Wang et al., 1992; Chen et al., 1994; Chiu and Nathans, 1994a), and murine (Chiu and Nathans, 1994b) photopigment genes have been used to drive expression of β-

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cone-to-ON-cone bipolar synaptic signals, should inhibit both ON and OFF responses of RGCs under scotopic conditions. However, Soucy and colleagues observed that applying APB to the retina ex vivo effectively abolished only ON responses under scotopic conditions; OFF responses remained largely intact. Soucy and colleagues also observed that strychnine, which blocks glycinergic signaling from AII amacrine cells to OFF cone bipolar cells, failed to inhibit the scotopic OFF RGC response. A second, APB-resistant pathway involving electrical coupling between rods and cones had previously been identified in the rabbit retina and could, in theory, have accounted for these data (DeVries and Baylor, 1995). To address this possibility, a human red pigment gene promoter was used to drive the expression of the α subunit of diphtheria toxin in cones, but not in rods or other cell types (Soucy et al., 1998). This transgene selectively eliminates nearly all cones, leaving only a few survivors in the inferior retina. Not surprisingly, retinas prepared from these animals failed to respond to light stimuli in the photopic (high light level) range. Under scotopic conditions, the RGC responses were nearly normal, as assayed by multielectrode array recordings. Interestingly, the scotopic APBresistant OFF pathway responses were comparable to those in wild-type mice, indicating that, under these experimental conditions, cones are not required for the transmission of rod signals to OFF RGCs. These data implicated a third and previously unknown pathway leading from rod photoreceptors to OFF RGCs, which has since been characterized as a direct synaptic contact between rods and OFF cone bipolar cells (Hack et al., 1999; Tsukamoto et al., 2001; FykKolodziej et al., 2003; Li et al., 2004).

Bipolar cells The role of gap junctions in the bipolar-AII amacrine cell circuit has been investigated with the use of transgenic and knockout/knock-in mice. Connexins are the building blocks of gap junctions, providing a direct electrical connection between neurons. In the mouse retina, connexin 36 and connexin 45 are expressed in distinct subsets of neurons. For example, an ON cone bipolar cell type specifically labeled by GFP in the BPGus-GFP transgenic mouse line expresses connexin 36 but not connexin 45 (Han and Massey, 2005; Lin and Masland, 2005). Replacing the open reading frame of the connexin 36 gene with a bicistronic gene encoding both PLAP and β-galactosidase revealed connexin 36 gene expression in photoreceptors, several classes of bipolar cells, and AII amacrine cells (Deans et al., 2001, 2002). In retinas from a connexin 36 knockout mouse, scotopic responses for both ON- and OFF-center RGCs were abolished, and there was a defect in gap junctional coupling between AII amacrine cells and cone bipolar cells. Although the RGC defect might well be a direct consequence of the AII amacrine-

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bipolar cell defect, connexin 36 is also expressed at other points within this circuit, such as the rod-to-cone gap junction, which might contribute to the phenotype (Deans et al., 2002; Völgyi et al., 2004). Conditionally knocking out connexin 36 in defined subsets of neurons should resolve this issue. More recently, the role of connexin 45 has been investigated using a conditional knock-in strategy. Since a knockout of connexin 45 is embryonic lethal, a knock-in allele was constructed in which exon 3 (encompassing the entire open reading frame) was flanked by loxP sites and followed by the coding region for GFP (see figure 49.2 for an analogous knock-in strategy). After crossing in a Nestin-Cre transgene, which leads to Cre-mediated recombination principally in the CNS and is compatible with viability, several types of OFF and ON cone bipolar cells, but not rod bipolar cells, were seen to express this connexin gene, as judged by the localization of GFP. In addition, in the conditional mutant mouse, the electroretinogram (ERG) b-wave, which is thought to largely represent bipolar cell depolarization, was greatly reduced under scotopic but not photopic conditions, suggesting an involvement of connexin 45 in the pathway leading from rods to cone bipolar cells (Maxeiner et al., 2005; Schubert et al., 2005). In the retina, expression of the metabotropic glutamate receptor mGluR6 is restricted to rod and ON cone bipolar cells. In mGluR6 knockout mice, the ERG b-wave is abolished, and visually evoked potentials in the superior colliculus are severely reduced. Nevertheless, the general architecture of the retina is preserved, and the mice can be trained to respond to light stimuli (Masu et al., 1995; Tagawa et al., 1999). Signaling through mGluR6 appears to require the Goα subunit of heterotrimeric G proteins, since the Goα gene knockout phenocopies the mGluR6 phenotype in the retina (Dhingra et al., 2000, 2002). A 9.5 kb upstream region encompassing the promoter of the mGluR6 gene is sufficient to drive β-galactosidase expression in the correct cell types (Ueda et al., 1997). This information was used to construct the corresponding GFP line, and by imaging the GFPexpressing retinas ex vivo, Morgan et al. (2006) were able to observe the development of axonal and dendritic processes of bipolar cells and to show that they derive from basal and apical processes of the postmitotic precursor. This example nicely illustrates the utility of ex vivo imaging with a fluorescent reporter that is limited to one or a few cell types. Several other useful genetic tools have been generated for the study of bipolar cells. A BAC transgenic line has been constructed in which the Pcp-2 gene (Purkinje cell protein-2, a protein/gene of unknown function, also known as L7) was modified to carry an internal ribosome entry site (IRES)-Cre coding region in the 3′ untranslated region, resulting in Cre expression specifically in rod bipolar cells (Zhang et al.,

2005). To trace synaptic connections and projection patterns, wheat germ agglutinin (WGA), a plant lectin known to be transported anterogradely along axons (Yoshihara et al., 1999), has been introduced as a transgene under the control of the L7 promoter, which drives expression in rod bipolar cells (Hanno et al., 2003). The level of WGA production is high enough to observe retrograde transsynaptic transport to photoreceptors, as well as anterograde transsynaptic transport of the protein to RGCs, revealing their axonal projections into the brain. The intracellular concentrations of ions associated with signaling and membrane voltage changes can be visualized with subcellular spatial resolution using genetically encoded fluorescent reporters. Kuner and Augustine in 2000 reported that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) is dependent on chloride concentration. If a CFP-YFP fusion protein (Clomeleon) is excited with a wavelength suitable for CFP, the energy can be efficiently transferred to YFP, but the efficiency of energy transfer decreases with increasing chloride concentration. By expressing this construct in neurons one can monitor the opening of chloride channels such as GABA receptors. In one application of this technique, the Thy-1 promoter, which is discussed in detail in the next section, was used to drive the expression of transgenic Clomeleon in a diverse population of retinal neurons to study the ionic basis of ON versus OFF bipolar cell responses (Duebel et al., 2006). At the synapse between photoreceptors and bipolar cells, ON bipolar cells invert the polarity of the membrane potential (by using mGluRs) and OFF bipolar cells conserve the polarity (by using ionotropic glutamate receptors). It was previously inferred that ON and OFF bipolar cells have different dendrite:soma internal chloride (iCl) ratios. The YFP : CFP emission ratio of the transgenic Clomeleon showed that ON bipolar neurons have a high iCl in their dendrites, and therefore the opening of chloride channels (following GABA release from horizontal cells) leads to an outward chloride flux and membrane depolarization. By contrast, OFF bipolar cells have a low dendritic iCl concentration and respond to chloride channel opening with chloride influx and membrane hyperpolarization. By this simple mechanism, GABA release from horizontal cells produces receptive field antagonism in both the ON and OFF channels. In cone bipolar cells, the transgenic Clomeleon was used to measure iCl in various cell compartments at resting membrane potential. Even though the iCl at the resting potential varies among different cells, the dendritic iCl was consistently higher and the axonal (i.e., output) arbor iCl was consistently lower than that of the soma. Moreover, two morphologically distinct bipolar cell types, type 7 and type 9, had distinct dendrite:soma iCl ratios, with type 9 having significantly higher dendritic iCl. Application of GABA to

these two cell types had different effects, inducing chloride influx in type 7 and efflux in type 9 cells. Interestingly, one of the Thy-1–Clomeleon lines expresses the transgene in a bipolar cell subpopulation in which the dendritic arbors exclusively contact S cones (Haverkamp et al., 2005).

Amacrine cells As noted earlier in this chapter, transgene expression patterns can vary considerably among different mouse lines carrying the same construct, depending on the location of transgene integration. Although this is not always a desirable characteristic, sometimes the “wrong” expression pattern turns out to be useful. As an example, expression of a βgalactosidase transgene driven by the vasoactive intestinal peptide (VIP) promoter was observed in a subpopulation of amacrine cells that do not normally express VIP (Nirenberg and Meister, 1997). Since these cells appeared quite uniform with respect to their distribution across the retina and dendritic arborization, they were considered to be members of one specific amacrine cell class, V-amacrine cells. The Vamacrine cells were photoablated with approximately 95% efficiency by incubating with the cell-permeant β-galactosidase substrate fluorescein-d-galactopyranoside, followed by intense illumination in the presence of aminoethyl carbazole (Nirenberg and Cepko, 1993). Multielectrode array recordings revealed a significant increase in response latency and response duration in ON-transient RGCs in the photoablated retinas (Nirenberg and Meister, 1997). Immunohistochemical characterization indicated that V-amacrine cells are GABA-ergic, consistent with the observation that picrotoxin, a GABA receptor antagonist, has effects on response latency and duration that resemble the effects of photoablation. These data suggest that V-amacrine cells shape RGC responses via GABA release, although their exact place in the inner retinal circuitry remains to be elucidated. A second subpopulation of amacrine cells that have been labeled genetically consists of dopaminergic amacrine cells. Gustincich et al. (1997) isolated a transgenic mouse line that expresses PLAP under the control of the tyrosine hydroxylase (TH) promoter in dopaminergic amacrine cells. Interestingly, PLAP expression was also observed in a second class of wide-field amacrine cells, with dendritic arbors stratifying in the middle of the IPL; the dendritic arbors of dopaminergic amacrine cells stratify only in the outermost strata of the IPL. After enzymatic dissociation of the transgenic retina, live dopaminergic amacrine cells were identified by staining with fluorescent anti-PLAP antibody (PLAP is a glyosyl phosphatidyl inositol [GPI]-anchored protein that accumulates on the extracellular face of the plasma membrane) and studied electrophysiologically. With this approach, Gustincich et al. showed that these cells fire spontaneous action potentials that are repressed by GABA. Moreover, by

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immunopurifying the PLAP-expressing neurons, it was possible to analyze their mRNAs and identify additional genes that may be involved in dopaminergic amacrine cell function (Gustincich et al., 2004). A similar transgenic approach has recently been used to generate a mouse line expressing Cre recombinase in dopaminergic neurons in the retina and brain (Gelman et al., 2003). Ever since the original descriptions of cholinergic amacrine cells (also called starburst amacrine cells), there has been debate as to the physiological function of these cells (Hayden et al., 1980; Masland et al., 1984). Starburst amacrine cells have radially symmetrical dendritic arbors that stratify in either of two narrow zones within the IPL, they release GABA as well as acetylcholine, and they have a retinal coverage factor estimated to be between 10 and 70, depending on the species. Because of the unusual distribution of inputs and outputs and their costratification with the two arbors of direction-selective (DS) RGCs, starburst amacrine cells have long been thought to be involved in DS responses to moving stimuli. A variety of approaches have been taken to test this hypothesis. In one approach, calcium imaging of DS responses in the dendritic arbors of starburst amacrine cells showed that these arbors have highly asymmetrical responses to stimuli moving across the retina in different directions (Euler et al., 2002). A second approach used a genetic tagging technique to ablate starburst amacrine cells from the mouse retina and then determined the effect of this ablation on DS RGC responses (Yoshida et al., 2001). A human interleukin-2 receptor α subunit (hIL2Rα)–GFP fusion protein was expressed as a transgene under the control of the mGluR2 promoter. One of the transgenic lines expressed hIL-2Rα–GFP only in starburst amacrine cells, a subset of the cells that express mGluR2. A monoclonal anti-hIL-2Rα antibody conjugated to a bacterial toxin was injected intraocularly in this transgenic line, resulting in an almost complete ablation of starburst amacrine cells. Concomitant with this ablation, there was an almost complete loss of DS RGC responses and an increase in the spiking rate of presumptive DS RGCs regardless of the direction of the stimulus. In addition, the optokinetic reflex was abolished, while pupillary constriction was preserved. This study demonstrated an essential role for starburst amacrine cells in the DS circuitry, and of DS RGC responses in the optokinetic reflex. Conditional gene deletion has been used to define the role of cholinergic signaling by starburst amacrine cells in the spontaneous waves of activity that characterize the developing retina. Cre-mediated recombination can be induced throughout much of the retina by using a transgene expressing Cre under the control of an intronic enhancer from the Pax6 gene (Pax6αCre) (Marquardt et al., 2001). This transgene is active in proliferating retinal precursors beginning at embryonic day E9.5, prior to the differentiation of all retinal

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cell types. Interestingly, the expression pattern of this transgene excludes a wedge-shaped domain in the center of the retina. Using the Pax6αCre line, it is possible to ablate any loxP-flanked gene in the retina while largely avoiding the deleterious effects of gene deletion in the rest of the embryo. To eliminate cholinergic signaling in the retina, the gene encoding choline acetyltransferase (ChAT), an enzyme required for the synthesis of acetylcholine, was deleted using the Pax6αCre line (Stacy et al., 2005). ChAT is expressed in all cholinergic neurons in the body, but in the retina it is expressed only by starburst amacrine cells. This elimination of retinal cholinergic signaling had little or no effect on retinal morphology but delayed by several days the appearance of the spontaneous waves of activity. It would be interesting to investigate the long-term effects of this manipulation on DS RGCs. In the context of new genetic tools for visualizing neurons, special mention should be made of mouse lines carrying fluorescent proteins under the control of the Thy-1 promoter (Feng et al., 2000). Thy-1 is a GPI-anchored cell-surface protein of uncertain function that was originally identified on thymocytes (T cells) in both mice and humans. It was subsequently found in the retina, where it decorates the surface of all or nearly all RGCs. Feng and colleagues made the extremely useful observation that transgenic lines in which any of a variety of fluorescent reporters—GFP, CFP, YFP, RFP—are driven by a Thy-1 promoter typically show reporter expression in relatively small numbers of neurons, with different lines expressing the reporter in different neuronal subtypes. Each of these lines generally carries multiple transgene copies integrated at a single site, and, perhaps because of this high copy number, several of the lines produce readily detectable fluorescence in both large and small arbors in those retinal neurons that express the transgene. The sparse expression that is a characteristic of the Thy-1 transgenic lines is also important for tracing the full extent and ramification of individual arbors. In one application of this system, the GFP-M line has been used to visualize a variety of wide-field amacrine cells that would have been difficult to completely fill by tracer injection (Lin and Masland, 2006).

Retinal ganglion cells RGCs are the projection neurons of the vertebrate retina. They have been a focus of intense interest for over a century not only because they encode and transmit all of the information captured by the eye but also because they offer an excellent system for studying axonal pathfinding and target selection by projection neurons (Hartline, 1938; Kuffler, 1953; Attardi and Sperry, 1963; Schnitzer and Meister, 2003). At the time of this writing, a literature search with the key words “retinal ganglion cell” and “mouse” yielded 178 papers, of which 140 were published in the past 10 years.

Although a majority of these studies focus on RGC development, the ability to discriminate individual RGCs using genetic techniques will likely lure an increasing number of physiologists and systems neuroscientists to ask their favorite questions in this system. Several classification systems for RGC cell types derived by genetic and nongenetic approaches have been published (Sun et al., 2002; Badea and Nathans, 2004; Kong et al., 2005; Coombs et al., 2006). Here we describe several genetic approaches that have proved useful in the study of RGCs. It has been known for some time that ipsilateral RGC projections are diminished in albino mice (Guillery et al., 1973). To more precisely assess the role of melanin (or lack of melanin) in RGC axonal pathfinding, Cronin et al. (2001, 2003) adapted the components of the lac repressor-lac operator system, one of the best-studied prokaryotic gene regulatory systems, to generate an inducible gene expression system in the mouse. In E. coli, the lac repressor protein (lacI) binds to and inhibits transcription from promoter elements containing the lac operator. This transcriptional repression can be abolished by addition of isopropyl-thiogalactoside (IPTG), a synthetic lactose analogue that binds the repressor protein and decreases its affinity for the operator. Cronin and colleagues synthesized a DNA segment encoding the lacI gene (the natural E. coli sequence is extensively methylated and transcriptionally silenced when introduced in mammalian cells) and then created transgenic mice in which the synthetic lacI DNA (synlacI) is under the control of the chicken β-actin promoter, leading to its expression in many tissues and cell types. These transgenic mice were crossed to a line carrying a tyrosinase transgene with three lac operators inserted near the promoter. In the absence of the synlacI transgene, the tyrosinase transgene produces tyrosinase, and therefore induces coat and retinal pigmented epithelium melanin production (i.e., pigmentation) in an albino genetic background. In the presence of the synlacI transgene, coat color and eye pigmentation revert to the albino phenotype. However, if IPTG is administered in the food, the tyrosinase transgene is derepressed, resulting in pigmentation. Using this system, the authors were able to demonstrate that tyrosinase activity is required during an early time window when RGC axons are growing (Cronin et al., 2003). The Thy-1-GFP-H line, similar to the M line mentioned earlier in the context of amacrine cell analysis, has also proved useful in the study of RGC morphology. In one study, RGC dendritic morphologies were quantified during the period surrounding eyelid opening, and the effect of dark rearing on the stratification of RGC dendritic arbors within the IPL was measured (Tian and Copenhagen, 2003). It was found that in P10 retinas (just before eye opening), about 50% of labeled RGCs were bistratified, and that under normal light exposure conditions this number decreased to 30% by P30. However, when animals were reared in the

dark, the fraction of labeled RGCs that were bistratified was still around 50% at P30, indicating that light exposure plays a role in RGC stratification or differentiation. These data were further supported by multielectrode array recordings which showed a decrease in the number of ON-OFF RGCs between P10 and P30 under normal rearing conditions and the persistence of ON-OFF RGCs in dark-reared animals. By crossing the Thy-1-GFP alleles with other transgenes expressing fluorescent proteins, one can begin to ask questions about the wiring of various cell types, such as bipolar cells and RGCs (Lin and Masland, 2005). Moreover, crossing these transgenes into various gene knockout backgrounds can provide insights into the development of the labeled cell types (Lin et al., 2004). We note that one potential challenge with a strategy of this type is that the promoter that drives the transgenic reporter may be under the control of the gene that is knocked out, in which case a change in transgene expression may reflect this regulatory relationship rather than a change in the fate of the labeled cell. An especially useful reporter for the study of projection neurons is the τ-β-galactosidase fusion protein (tau-lacZ). Tau is a microtubule-associated protein, and as a result, taulacZ nicely labels axonal microtubules. The tau-lacZ reporter has been knocked into the genes encoding several transcription factors expressed in RGCs (Pak et al., 2004; Pratt et al., 2004; Quina et al., 2005), as well as the gene encoding melanopsin, a photopigment expressed in a subset of RGCs (Hattar et al., 2002). In melanopsin tau-lacZ knock-in mice, β-galactosidase accumulation has been used to catalogue the central targets for these RGCs (Hattar et al., 2006). Genetic cell-labeling techniques have also made contributions to our understanding of RGC physiology. For example, reporters of intracellular calcium have been expressed in RGCs using the tetracycline-inducible system (Hasan et al., 2004; described in the next section), allowing real-time measurements of intracellular calcium in conjunction with the electrical activity of the cells. These genetically encoded fluorescent reporters should facilitate optical measurements across large cell populations.

Genetic systems for site-specific recombination and drug-controlled gene expression The Cre-lox recombinase system has been noted at several points in the preceding sections. An application of the Crelox technology that was not previously discussed involves using site-specific recombination as an indelible marker of lineage. In one version of this lineage analysis, the Cre coding region is inserted into a gene that is expressed in the progenitors of a particular lineage, for example a gene encoding one of the basic helix-loop-helix (bHLH) transcription factors that are transiently expressed during neural development. By crossing in a reporter that is expressed only

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following Cre-mediated deletion of a loxP-flanked transcription termination signal, one can visualize all the progeny of the cells in which the bHLH gene was previously active (Zirlinger et al., 2002; Yang et al., 2003; Ma and Wang, 2006). A somewhat more flexible version of this approach uses a Cre-recombinase that is fused to the ligand-binding domain of a mutated estrogen receptor (ER(T)) that recognizes the synthetic ligand 4-hydroxytamoxifen (4-HT) instead of the endogenous ligand estrogen (Feil et al., 1996; Brocard et al., 1997). The Cre-ER(T) fusion protein is sequestered in the cytosol in the absence of 4-HT; systemic or local administra-

tion of 4-HT releases the fusion protein, which then migrates to the nucleus and catalyzes site-specific recombination (figure 49.3B). By using CreER(T), the timing of the sitespecific recombination event can be controlled to within 1 day at any time in the life of the mouse simply by administering 4-HT; for prenatal exposure, 4-HT is administered to the mother. The efficiency of this process depends on several factors: (1) the abundance of the CreER(T) fusion protein, (2) the availability of the target locus for recombination, which appears to be determined by local chromatin environment, (3) the amount of 4-HT delivered, and (4) the differentiation state of the target cell.

Cell specific promoter rtTA

A

rtTA inactive doxycycline rtTA active

rtTA regulated promoter

Reporter Gene

B Promoter 1

Promoter 2 loxP

loxP

CreER(T)

ORF 1

ORF 2 STOP excision by Cre recombination

inactive

4-HT

active loxP ORF 2

Figure 49.3 Pharmacological control of gene expression. Pharmacological approaches can be used to express genetic elements with improved spatial and temporal control or in a more restricted cell population. A, A transgene driven by a cell-specific promoter expresses rtTA, a doxycycline-sensitive transcriptional regulator. Unliganded rtTA is inactive but can be activated by the administration of doxycycline, causing it to bind to a specific DNA recognition site (tetO), and induce the transcription of a second genetic element. Thus, by generating one cell-specific transgene and crossing it to various rtTA-sensitive reporters, one can activate a reporter or a functional modulator of the target cells in a timed fashion, depending on doxycycline administration. B, A target locus (controlled by promoter 2) can be modified by the activity of a drug-sensitive Cre

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recombinase, CreER(T), expressed under the control of promoter 1. CreER(T) is produced in an inactive form and is activated by the administration of 4-HT (4-hydroxytamoxifen). The active recombinase then deletes open reading frame (ORF) 1, allowing ORF 2 to be transcribed under the control of promoter 2. This strategy can be used for conditional gene ablation, if the second locus is arranged as shown in figure 49.2, or for the conditional overexpression of reporter genes or other elements. Since the final pattern of ORF 2 expression is controlled by the spatial intersection of the expression patterns of promoters 1 and 2, as well as by 4-HT, this combinatorial strategy exhibits great flexibility for labeling and manipulating neuronal targets.

In an initial application of this method for lineage tracing, a CreER(T) line with ubiquitous expression was generated by knocking CreER(T) coding sequences into the ROSA26 locus (Zambrowicz et al., 1997; Badea et al., 2003). ROSA26 is a genetic locus identified by Soriano and colleagues during an insertional mutagenesis screen. The ROSA26 locus becomes transcriptionally active early in development, and transcription persists in all or nearly all cells in the body from that time on. Crossing the ROSA26-CreER(T) line to one that carries a ubiquitously expressed and Cre-activated PLAP (the Z/AP line constructed by Lobe et al., 1999) permits histochemical identification of those cells that have inherited a Cre-mediated recombination event (Badea et al., 2003). As expected, administering 4-HT at earlier or later developmental times generates, respectively, larger or smaller clones. Also as expected, the ROSA26-CreER(T);Z/AP combination generates labeled clones throughout the body at a density that depends on the 4-HT dose. Lineage tracing with ROSA26-CreER(T);Z/AP revealed the previously defined patterns of radial clones of retinal neurons (Price et al., 1987; Turner et al., 1990), and it also showed that retinal capillaries are composed of the intermingled progeny of a relatively small number of endothelial progenitors (Badea et al., 2003). A second application of the CreER(T) technology is in the production of tissues with very low densities of genetically labeled neurons for morphological analysis. This approach has been used to generate a catalogue of all the major neuronal cell types in the retina by using ROSA26-CreER(T);Z/ AP mice with both systemic and intraocular 4-HT injections (Badea and Nathans, 2004). The resulting set of labeled neurons has provided the raw material for a quantitative analysis of the patterns of stratification and dendritic arborization for all the major cell types. For example, in analyzing the arbors of polyaxonal amacrine cells, the data show that the lengths of individual dendrites can be comparable to the diameter of the retina, a finding also obtained with Thy-1 transgenic mice (Lin and Masland, 2006). The ROSA26-CreER(T);Z/AP labeling method has more recently been applied to the study of neuronal morphologies in various mutant retinas (Badea and Nathans, unpublished results). Finally, we note that reversible drug control of gene expression, as described for synlacI/IPTG control of melanin production, is still a largely untapped technology for visual system studies. The rtTA is currently the system most widely used in both mammalian cell cultures and mice (Urlinger et al., 2000; Hasan et al., 2004; figure 49.3A). In this system, the tetracycline repressor (TetR), which in E. coli maintains the gene encoding the tetracycline efflux pump in a repressed state in the absence of tetracycline, has been engineered to bind rather than release its DNA target in the presence of tetracycline or related drugs, such as doxycycline (hence the

adjective “reverse” in rtTA). The rtTA protein also carries a eukaryotic transcriptional activation domain, and therefore, when it binds to its DNA target (tetO), previously engineered into the gene of interest, transcription ensues. Coding sequences for rtTA, as well as for Cre, have recently been introduced into transgenic mice under the control of the Thy-1 promoter (Campsall et al., 2002; Kerrison et al., 2005).

Conclusion We hope that the examples presented here make a convincing argument for using the mouse as a genetic model organism for the study of visual system function and development. However, the examples also illustrate the need to develop additional tools to target more refined subpopulations of neurons. We anticipate that combinations of gene expression cassettes with partially overlapping expression patterns—for example, one cell type–specific promoter controlling CreER(T) and a second cell type–specific promoter controlling a Cre-dependent reporter—will be developed in the near future to provide improved specificity by the intersection of their expression patterns. These and other advances should ultimately provide investigators with the ability to precisely control both cell type and temporal patterns of neuronal gene expression.

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Kachi, S., Oshima, Y., Esumi, N., Kachi, M., Rogers, B., Zack, D. J., and Campochiaro, P. A. (2005). Nonviral ocular gene transfer. Gene Ther. 12:843–851. Kerrison, J. B., Duh, E. J., Yu, Y., Otteson, D. C., and Zack, D. J. (2005). A system for inducible gene expression in retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 46:2932–2939. Kong, J. H., Fish, D. R., Rockhill, R. L., and Masland, R. H. (2005). Diversity of ganglion cells in the mouse retina: Unsupervised morphological classification and its limits. J. Comp. Neurol. 489:293–310. Kuffler, S. W. (1953). Discharge patterns and functional organization of mammalian retina. J. Neurophysiol. 16:37–68. Kuner, T., and Augustine, G. J. (2000). A genetically encoded ratiometric indicator for chloride: Capturing chloride transients in cultured hippocampal neurons. Neuron 27:447–459. Li, W., Keung, J. W., and Massey, S. C. (2004). Direct synaptic connections between rods and OFF cone bipolar cells in the rabbit retina. J. Comp. Neurol. 474:1–12. Lin, B., and Masland, R. H. (2005). Synaptic contacts between an identified type of ON cone bipolar cell and ganglion cells in the mouse retina. Eur. J. Neurosci. 21:1257–1270. Lin, B., and Masland, R. H. (2006). Populations of wide-field amacrine cells in the mouse retina. J. Comp. Neurol. 499:797– 809. Lin, B., Wang, S. W., and Masland, R. H. (2004). Retinal ganglion cell type, size, and spacing can be specified independent of homotypic dendritic contacts. Neuron 43:475–485. Lobe, C. G., Koop, K. E., Kreppner, W., Lomeli, H., Gertsenstein, M., and Nagy, A. (1999). Z/AP, a double reporter for Cre-mediated recombination. Dev. Biol. 208:281–292. Ma, W., and Wang, S. Z. (2006). The final fates of neurogenin2expressing cells include all major neuron types in the mouse retina. Mol. Cell Neurosci. 31:463–469. Marquardt, T., Ashery-Padan, R., Andrejewski, N., Scardigli, R., Guillemot, F., and Gruss, P. (2001). Pax6 is required for the multipotent state of retinal progenitor cells. Cell 105:43– 55. Masland, R. H., Mills, J. W., and Cassidy, C. (1984). The functions of acetylcholine in the rabbit retina. Proc. R. Soc. Lond. B. Biol. Sci. 223:121–139. Masu, M., Iwakabe, H., Tagawa, Y., Miyoshi, T., Yamashita, M., Fukuda, Y., Sasaki, H., Hiroi, K., Nakamura, Y., et al. (1995). Specific deficit of the ON response in visual transmission by targeted disruption of the mGluR6 gene. Cell 80:757– 765. Matsuda, T., and Cepko, C. L. (2004). Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc. Natl. Acad. Sci. U.S.A. 101:16–22. Maxeiner, S., Dedek, K., Janssen-Bienhold, U., Ammermuller, J., Brune, H., Kirsch, T., Pieper, M., Degen, J., Kruger, O., et al. (2005). Deletion of connexin 45 in mouse retinal neurons disrupts the rod/cone signaling pathway between AII amacrine and ON cone bipolar cells and leads to impaired visual transmission. J. Neurosci. 25:566–576. Morgan, J. L., Dhingra, A., Vardi, N., and Wong, R. O. (2006). Axons and dendrites originate from neuroepithelial-like processes of retinal bipolar cells. Nat. Neurosci. 9:85–92. Nirenberg, S., and Cepko, C. (1993). Targeted ablation of diverse cell classes in the nervous system in vivo. J. Neurosci. 13: 3238–3251. Nirenberg, S., and Meister, M. (1997). The light response of retinal ganglion cells is truncated by a displaced amacrine circuit. Neuron 18:637–650.

O’Brien, J., and Lummis, S. C. (2004). Biolistic and diolistic transfection: Using the gene gun to deliver DNA and lipophilic dyes into mammalian cells. Methods 33:121–125. Pak, W., Hindges, R., Lim, Y. S., Pfaff, S. L., and O’Leary, D. D. (2004). Magnitude of binocular vision controlled by islet-2 repression of a genetic program that specifies laterality of retinal axon pathfinding. Cell 119:567–578. Pignatelli, V., and Strettoi, E. (2004). Bipolar cells of the mouse retina: A gene gun, morphological study. J. Comp. Neurol. 476: 254–266. Pratt, T., Tian, N. M., Simpson, T. I., Mason, J. O., and Price, D. J. (2004). The winged helix transcription factor Foxg1 facilitates retinal ganglion cell axon crossing of the ventral midline in the mouse. Development 131:3773–3784. Price, J., Turner, D., and Cepko, C. (1987). Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 84:156–160. Quina, L. A., Pak, W., Lanier, J., Banwait, P., Gratwick, K., Liu, Y., Velasquez, T., O’Leary, D. D., Goulding, M., and Turner, E. E. (2005). Brn3a-expressing retinal ganglion cells project specifically to thalamocortical and collicular visual pathways. J. Neurosci. 25:11595–11604. Rohlich, P., van Veen, T., and Szel, A. (1994). Two different visual pigments in one retinal cone cell. Neuron 13:1159–1166. Schnitzer, M. J., and Meister, M. (2003). Multineuronal firing patterns in the signal from eye to brain. Neuron 37:499–511. Schubert, T., Maxeiner, S., Kruger, O., Willecke, K., and Weiler, R. (2005). Connexin45 mediates gap junctional coupling of bistratified ganglion cells in the mouse retina. J. Comp. Neurol. 490:29–39. Soucy, E., Wang, Y., Nirenberg, S., Nathans, J., and Meister, M. (1998). A novel signaling pathway from rod photoreceptors to ganglion cells in mammalian retina. Neuron 21:481–493. Stacy, R. C., Demas, J., Burgess, R. W., Sanes, J. R., and Wong, R. O. (2005). Disruption and recovery of patterned retinal activity in the absence of acetylcholine. J. Neurosci. 25:9347–9357. Sun, W., Li, N., and He, S. (2002). Large-scale morphological survey of mouse retinal ganglion cells. J. Comp. Neurol. 451: 115–126. Sung, C. H., Makino, C., Baylor, D., and Nathans, J. (1994). A rhodopsin gene mutation responsible for autosomal dominant retinitis pigmentosa results in a protein that is defective in localization to the photoreceptor outer segment. J. Neurosci. 14: 5818–5833. Tagawa, Y., Sawai, H., Ueda, Y., Tauchi, M., and Nakanishi, S. (1999). Immunohistological studies of metabotropic glutamate receptor subtype 6-deficient mice show no abnormality of retinal cell organization and ganglion cell maturation. J. Neurosci. 19:2568–2579. Tauchi, M., and Masland, R. H. (1984). The shape and arrangement of the cholinergic neurons in the rabbit retina. Proc. R. Soc. Lond. B. Biol. Sci. 223:101–119. Tian, N., and Copenhagen, D. R. (2003). Visual stimulation is required for refinement of ON and OFF pathways in postnatal retina. Neuron 39:85–96. Tsukamoto, Y., Morigiwa, K., Ueda, M., and Sterling, P. (2001). Microcircuits for night vision in mouse retina. J. Neurosci. 21:8616–8623. Turner, D. L., Snyder, E. Y., and Cepko, C. L. (1990). Lineageindependent determination of cell type in the embryonic mouse retina. Neuron 4:833–845. Ueda, Y., Iwakabe, H., Masu, M., Suzuki, M., and Nakanishi, S. (1997). The mGluR6 5′ upstream transgene sequence directs a

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cell-specific and developmentally regulated expression in retinal rod and ON-type cone bipolar cells. J. Neurosci. 17:3014–3023. Urlinger, S., Baron, U., Thellmann, M., Hasan, M. T., Bujard, H., and Hillen, W. (2000). Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. U.S.A. 97:7963–7968. Völgyi, B., Deans, M. R., Paul, D. L., and Bloomfield, S. A. (2004). Convergence and segregation of the multiple rod pathways in mammalian retina. J. Neurosci. 24:11182–11192. Wang, Y., Macke, J. P., Merbs, S. L., Zack, D. J., Klaunberg, B., Bennett, J., Gearhart, J., and Nathans, J. (1992). A locus control region adjacent to the human red and green visual pigment genes. Neuron 9:429–440. Wellmann, H., Kaltschmidt, B., and Kaltschmidt, C. (1999). Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun. J. Neurosci. Methods 92:55–64. Xiang, M., Zhou, L., and Nathans, J. (1996). Similarities and differences among inner retinal neurons revealed by the expression of reporter transgenes controlled by Brn-3a, Brn-3b, and Brn-3c promotor sequences. Vis. Neurosci. 13:955–962. Yang, Z., Ding, K., Pan, L., Deng, M., and Gan, L. (2003). Math5 determines the competence state of retinal ganglion cell progenitors. Dev. Biol. 264:240–254. Yoshida, K., Watanabe, D., Ishikane, H., Tachibana, M., Pastan, I., and Nakanishi, S. (2001). A key role of starburst amacrine

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cells in originating retinal directional selectivity and optokinetic eye movement. Neuron 30:771–780. Yoshihara, Y., Mizuno, T., Nakahira, M., Kawasaki, M., Watanabe, Y., Kagamiyama, H., Jishage, K., Ueda, O., Suzuki, H., et al. (1999). A genetic approach to visualization of multisynaptic neural pathways using plant lectin transgene. Neuron 22:33–41. Zack, D. J., Bennett, J., Wang, Y., Davenport, C., Klaunberg, B., Gearhart, J., and Nathans, J. (1991). Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas. Neuron 6:187–199. Zambrowicz, B. P., Imamoto, A., Fiering, S., Herzenberg, L. A., Kerr, W. G., and Soriano, P. (1997). Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc. Natl. Acad. Sci. U.S.A. 94:3789– 3794. Zhang, X. M., Chen, B. Y., Ng, A. H., Tanner, J. A., Tay, D., So, K. F., Rachel, R. A., Copeland, N. G., Jenkins, N. A., et al. (2005). Transgenic mice expressing Cre-recombinase specifically in retinal rod bipolar neurons. Invest. Ophthalmol. Vis. Sci. 46:3515–3520. Zirlinger, M., Lo, L., McMahon, J., McMahon, A. P., and Anderson, D. J. (2002). Transient expression of the bHLH factor neurogenin-2 marks a subpopulation of neural crest cells biased for a sensory but not a neuronal fate. Proc. Natl. Acad. Sci. U.S.A. 99:8084–8089.

50

Adenoassociated Virus Gene Therapy in Mouse Models of Retinal Degeneration SHANNON E. BOYE, SANFORD L. BOYE, AND WILLIAM W. HAUSWIRTH

A variety of therapeutic strategies aimed at combating inherited retinal diseases have recently been developed. In general, these strategies can be divided into three categories: gene therapy, pharmacological neuroprotection, and stem or precursor cell therapy. Of these approaches, only gene therapy is capable of curing a disease state. The others seek either to halt or slow the progression of an existing disorder or to replace key retinal cells once they are lost or become dysfunctional. Of course, if tissue replacement utilizes a sufficient number of normal cells, a cure may also be effected. This chapter focuses on gene-based therapeutic approaches for treating retinal degeneration (RD) in mouse models.

Gene therapy vectors The history of gene therapy is best illustrated by the attempts to develop a lasting treatment for cystic fibrosis (CF). The earliest CF gene therapy trials utilized recombinant adenoassociated virus (AAV), which was found to be ineffective, owing to problems with delivery to the target cell and insufficient expression levels (Griesenbach et al., 2006; Flotte et al., 2007). In addition to AAV, two other candidate gene therapy vectors, adenovirus and lentivirus, were investigated for their therapeutic potential for treating CF. However, the occurrence of two high-profile adverse events in humans involving these vectors affected their utility in CF and other clinical studies. The systemic administration of an adenovirus vector for ornithine transcarbamylase (OCT) deficiency in which adenovirus vector was delivered to the liver resulted in a fatality (Raper et al., 2003). It was concluded that the high dose of vector (required because of poor expression from vector) given to this patient was immunogenic. Additionally, administration of a lentiviral vector to patients with X-linked severe combined immunodeficiency syndrome resulted in site-specific integration of the vector near a specific pro-oncogene, which resulted in three cases of leukemia, one of which was ultimately fatal (Williams, 2006). AAV’s relatively nonimmunogenic properties and apparent

lack of chromosomal integration indicated that it was inherently safer and more effective than the aforementioned viral vectors. Newer AAV vectors have been designed to address earlier shortcomings and are now in the process of being evaluated for the treatment of CF (Flotte et al., 2005). These same advances in AAV vector technology are being applied to the gene therapy of various other disorders, including disorders of the eye (Warrington and Herzog, 2006).

Adenoassociated virus vectors Although there are a number of strategies for delivering genes to cells, the nature of the target tissue and the therapeutic requirements for the disease normally dictate which choice is optimal. Viruses of the Parvoviridae family have shown the most promise in gene therapy, particularly for retinal disease. A member of this family, human AAV has been widely exploited as a vector for gene delivery because of its advantages over other viral gene therapy vectors, namely, safety, long-term expression, the ability to transduce terminally differentiated cells, and selective (as well as broad) tropism through the use of the numerous AAV serotypes currently available. AAV is a nonpathogenic, replicationdeficient dependovirus that contains a single-stranded genome of 4.7 kb flanked by inverted terminal repeats (ITRs), each 145 bases in length (Srivastava et al., 1983; Flotte and Berns, 2005). Specific sites within these ITRs control the conversion of single-stranded AAV genomes to their double-stranded DNA state necessary for subsequent transcription and translation (Qing et al., 1997, 1998; Mah et al., 1998). Wild-type AAV is not associated with any pathological condition in humans. Recombinant AAV (rAAV) is produced by removing all native AAV coding sequences, leaving only the short ITRs flanking the promoter and cDNA of interest. This eliminates the virus’s ability to integrate site specifically into the genome; the integration events that do occur are very infrequent and require chromosomal breakage (Miller et al., 2004). Thus, the overall risk of an AAV

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integration event activating an oncogene is considered low. AAV vectors do, however, promote long-term transgene expression by remaining in the transduced cell in a circular, double-stranded episomal form (Song et al., 2001, 2004; Duan et al., 2003). In other words, AAV-delivered DNA remains in the host cell nucleus as an independent genetic unit and not as part of the host cell’s genome. In vivo expression has been documented to persist for more than 6 years in a dog model of Leber congenital amaurosis (Acland et al., 2005; G. Acland and G. Aguirre, pers. comm., 2007). Additionally, AAV vectors have been shown to efficiently transduce terminally differentiated, nondividing cells. This is clearly vital for treating retinal diseases, most of which involve malfunction of photoreceptors or other terminally differentiated retinal cells. There are more than 100 different variants of AAV, categorized into serotypes and genomovars (Gao et al., 2004). Among these genomic variants is a broad diversity of AAV serotypes that utilize a range of different receptors (Flotte and Berns, 2005). The nine AAV serotypes currently in wide use, AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, and 9, differ from each other to varying degrees in their capsid protein sequence critical for serotype determination. Capsid variations confer distinct tissue and cell affinities and, as a result, define the speed of expression onset and the overall intensity of transgene expression by AAV vectors. AAV serotype 2 (AAV2) was the first used for gene transfer to the rodent retina via subretinal injection and was subsequently shown to promote broad transduction of photoreceptors and retinal pigmented epithelium (RPE) (Bennett et al., 1997; Flannery et al., 1997; Ali et al., 1998). Subsequently other serotypes have shown potential for targeting transgene expression to specific subsets of cells in the mouse retina. Typically, non-serotype 2 AAV vectors are made by packaging the vector DNA flanked by serotype 2 ITRs into the desired capsid serotype, a process termed pseudotyping. Almost all non-serotype 2 vectors to date have been made by this technique. AAV5, like AAV2, targets photoreceptors and RPE after subretinal injection, but does so with greater efficiency than AAV2 (Yang et al., 2002). AAV1 and AAV6, two closely related serotypes, have both been shown to transduce primarily RPE (Xiao et al., 1999). AAV4 is the only serotype to be expressed solely in RPE, based on results in rat, dog, and nonhuman primate, and would therefore be expected to do the same in mouse (Weber et al., 2003). AAV3 does not appear to transduce retinal cells at all after subretinal injection (Yang et al., 2002). Although AAV7 and AAV8 have not been as comprehensively evaluated in the mouse retina as other serotypes, there is some evidence that both transduce RPE and photoreceptors, with AAV8 being more efficient (Lauramore, 2004). The transduction characteristics of AAV9 in mouse retina have yet to be reported in the literature. The use of ITRs from serotypes other than AAV2 has been

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reported for an AAV5 capsid containing serotype 5 ITRs (Yang et al., 2002). This vector had a similar transduction pattern to that for AAV5 pseudotyped with serotype 2 ITRs, in that it was more efficient at targeting photoreceptors and RPE than standard AAV2. When injected into the vitreous of mouse and rat, AAV2 has proven to be the most efficient serotype for targeting expression to the inner retina, primarily retinal ganglion cells (RGCs; Ali et al., 1998; Auricchio et al., 2001; Liang et al., 2001a; Martin et al., 2003). In addition to targeting expression to the inner retina, limited spread of expressed transgene to the optic nerve and brain has also been observed (Dudus et al., 1999). The transduction patterns of various AAV serotypes following subretinal or intravitreal delivery are summarized in table 50.1. In addition to serotype selection, promoter choice aids significantly in defining the retinal cell specificity of transgene expression. For broad transgene expression in the retina, the cytomegalovirus (CMV) immediate early promoter has historically been used (Ali et al., 1998). More recently, the chimeric CMV-chicken β-actin promoter/ CMV enhancer (CBA) has been used in applications where high-level, long-term expression of protein in a broad variety of cells is required (Raisler et al., 2002; Pang et al., 2006). In cases where a single cell type is the target of therapy, nonviral cell-specific promoters enhance the safety of AAV vectors by reducing possible toxicity associated with the transduction of nontarget cell types. AAV-mediated expression targeted to photoreceptors was first achieved in rat using 472 bps (−386/+85) of the proximal mouse rhodopsin promoter (Rho, also commonly referred to as mOP)

Table 50.1 Transduction characteristics of various AAV serotypes following subretinal or intravitreal administration to the mouse retina AAV Serotype* Injection Route Transduction Pattern AAV2/2 Subretinal Photoreceptors, RPE AAV2/2 Intravitreal Ganglion cells AAV2/5 Subretinal Photoreceptors, RPE AAV2/5 Intravitreal None AAV2/1 Subretinal Primarily RPE AAV2/1 Intravitreal None AAV2/6 Subretinal Primarily RPE AAV2/4 Subretinal Solely RPE AAV2/3 Subretinal None AAV2/7 Subretinal Photoreceptors, RPE AAV2/8 Subretinal Photoreceptors, RPE AAV5/5 Subretinal Photoreceptors, RPE * The first number indicates the serotype of the ITRs flanking vector DNA, and the second number represents the serotype of capsid protein.

in conjunction with AAV2 (Flannery et al., 1997). It has subsequently been shown to be the case for mouse as well (Min et al., 2005; Pawlyk et al., 2005). Some controversy exists as to whether mOP targets both rods and cones, but several recent studies have provided convincing evidence that cones are indeed transduced when using this promoter, particularly in conjunction with AAV5 (Glushakova et al., 2006a; Haire et al., 2006). A human blue cone opsin promoter originally shown to preferentially target cone photoreceptors in rat has also been shown to effectively target cones in mouse retina when used with AAV5 (Glushakova et al., 2006b). In addition, a human red cone opsin promoter fragment used in conjunction with AAV5 has also been shown to preferentially target cones in mouse retina (Alexander et al., 2005). Promoter analysis indicates that RPE-specific expression can be achieved using portions of the vitelliform macular dystrophy 2 (VMD2) promoter of the bestrophin gene (the gene responsible for Best disease) or the RPE65 promoter (Boulanger et al., 2000, 2002; Esumi et al., 2004, 2007). This has been confirmed in mouse when these promoters have been used in conjunction with AAV1 (Glushakova and Hauswirth, 2004). Regulatable systems of gene expression are desirable in cases where continuous, high levels of expression of a therapeutic agent may be harmful to retina. Two have been used in conjunction with AAV vectors targeted in the retina: the tetracycline-inducible and the rapamycin-inducible transcriptional regulatory systems. Although both have been studied in rat (McGee-Sanftner et al., 2001; Auricchio, Rivera, et al., 2002; Smith et al., 2005), it is reasonable to expect that the results are equally applicable to mouse. This chapter focuses on the application of AAV-mediated gene therapy in several different mouse models of inherited RD. In all studies mentioned, AAV-mediated somatic gene transfer resulted in significant functional improvement, as assessed by electroretinography (ERG) or behavior, or by regeneration or stabilization of retinal structure. In some cases, knowledge gained from initial work in mice has set the stage for the development of human clinical trials. A comprehensive list of these studies and the type of vector, promoter, and transgene used is given in table 50.2.

Antiangiogenic gene therapy Although many clinical RDs are the result of mutations in genes expressed in the retina, some are secondary to systemic disease. The mammalian retina is one of the most metabolically active tissues in the body and therefore demands very high levels of oxygen via both the retinal and choroidal blood vessels. Systemic disease such as diabetes or retinopathy of prematurity (ROP) can result in retinal hypoxia that subsequently leads to retinal neovascularization. Age-related macular degeneration (AMD), the leading

cause of blindness in developed countries, in its “wet form” is characterized by neovascularization originating in the choroidal vasculature. Normal retinal and choroidal vessel beds are maintained by a balance of several endogenous proteins, including positive growth factors such as vascular endothelial growth factor (VEGF) and antiangiogenic proteins such as pigmented epithelium–derived factor (SERPINF1, more commonly referred to as PEDF). Traditional treatments (laser, photodynamic therapy) seek to delay the progression of the disease by destroying new, pathogenic blood vessels. They do not, however, address the underlying cause, that being the recurring, inappropriate proliferation of the retinal or choroidal vasculature. The delivery of proteins with antiangiogenic properties has proved somewhat successful in patients with various neovascular or neovascular-associated diseases (O’Reilly et al., 1994, 1997; Stellmach et al., 2001). Two such therapies currently available to patients with neovascular AMD are Pfizer’s Macugen (pegaptanib), a pegylated aptamer of VEGF, and Genentech’s Lucentis (ranibizumab), an anti-VEGF antibody. However, both require repeated intraocular injections for extended periods of time, thus posing risk and compliance issues for patients. In animal models, AAV-mediated gene transfer of antiangiogenic proteins to various cells of the retina has been employed to combat both retinal and choroidal neovascularization, thus suggesting AAV-vectored genes may be an attractive, long-term alternative for the treatment of local neovascular disease.

Therapy for retinal neovascularization The oxygen-induced retinopathy (OIR) mouse model, more commonly referred to as the ROP mouse, has been developed for the study of retinal neovascular disease (Smith et al., 1994). Newborn mice are placed in a hyperoxic (ca. 75% oxygen) chamber for 5 days. This high-oxygen environment induces retinal capillary ablation, which, when mice are returned to normoxic conditions, results in relative hypoxia and retinal ischemia. This in turn leads to VEGF-mediated retinal neovascularization like that seen in humans with neovascular retinal disease. A variety of antiangiogenic treatments have been tested in the ROP mouse. In one study, AAV2 was used to deliver PEDF, under the control of the CBA promoter, to the intravitreal or subretinal space of a newborn mouse eye (Raisler et al., 2002). The contralateral control eye received an equivalent injection of PBS. Retinal neovascularization was quantified in both treated and untreated (contralateral) eyes by counting the number of vascular endothelial cell nuclei above the inner limiting membrane in P17 eyes. The number of neovascular nuclei observed in eyes treated with AAV2PEDF was significantly reduced relative to that in control eyes. Similar results were obtained when AAV serotype 1

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Mouse Model ROP

Description of Model Oxygen-induced retinopathy

ROP

Oxygen-induced retinopathy

ROP

Oxygen-induced retinopathy

ROP

Oxygen-induced retinopathy

ROP

Oxygen-induced retinopathy

ROP

Oxygen-induced retinopathy

ROP

Oxygen-induced retinopathy

CNV laser

Laser-induced choroidal neovascularization Knockout of peripherin2

Prph2Rd2/Rd2*

Table 50.2 Summary of mouse models treated with AAV gene therapy Related Disease or Disorder in Human AAV Vector Serotype Retinopathy of 2 CBA-PEDF prematurity and other vasculopathologies Retinopathy of 1 CMV-PEDF prematurity and other vasculopathologies Retinopathy of 2 CBA-VEGF prematurity (exon 6–7) and other vasculopathologies Retinopathy of 2 CMV-sFlt1 prematurity and other vasculopathologies Retinopathy of 1 CMV-Timp3 prematurity and other vasculopathologies Retinopathy of 1 CMV-endostatin prematurity and other vasculopathologies Retinopathy of 2 CBA-K1K3 prematurity (angiostatin) and other vasculopathologies 2 Age-related macular CBA-PEDF degeneration

Auricchio, Behling, et al., 2002

Angiogenic regulator

Deng et al., 2005

Inhibitor of VEGF, antiangiogenic

Bainbridge et al., 2002

Regulates structure of basement membrane

Auricchio, Behling, et al., 2002

Antimitogenic factor

Auricchio, Behling, et al., 2002

Angiogenic regulator

Raisler et al., 2002

Angiogenic regulator

Mori et al., 2002 Ali et al., 2000, and Sarra et al., 2001 Zeng et al., 2004

Rho-prph2

2

Stabilization of discs in outer segments

CMV-Rs1

2

Structural and synaptic integrity of retinal neurons Structural and synaptic integrity of retinal neurons cGMP production, phototransduction

Knockout of retinoschisin

Rs1−/−

Knockout of retinoschisin

X-linked retinoschisis

mOP-RS1

5

GC1−/−

LCA 1

mOP-bGC1/ CBA-bGC1

5

Rpe65 −/−

Knockout of guanylate cyclase-1 Knockout of RPE65

LCA 2

CMV-RPE65

1

rd12

Mutation in RPE65

LCA 2

CBA-RPE65

5

Rpgrip−/−

Knockout of RP GTPase Regulator (RPGR)– interacting protein

Leber congenital amaurosis (LCA)

mOP-Rpgrip

2

advanced genomic technologies

Reference Raisler et al., 2002

Angiogenic regulator

Autosomal dominant RP, macular dystrophies X-linked retinoschisis

Rs1−/−

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Function of Transgene Delivered Angiogenic regulator

Retinal isomerohydrolase Retinal isomerohydrolase Protein trafficking through connecting cilia of rods and cones

Min et al., 2005

Haire et al., 2006 Dejneka et al., 2003 Pang et al., 2006 Pawlyk et al., 2005

Mouse Model

Table 50.2 Summary of mouse models treated with AAV gene therapy (continued) Related Disease or Function of Description of Model Disorder in Human AAV Vector Serotype Transgene Delivered

MPS VII

Mutation in βglucuronidase (GUSB)

Mucopolysaccharidosis type VII

CBA-GUSB

2

Ppt1−/−

Knockout of palmitoyl protein thioesterase1 Mutation in α subunit of cone transducin Knockout of rhodopsin Mutation (insertional gene disruption) in peripherin2

Infantile neuronal ceroid lipofuscinosis (INCL) Achromatopsia

CMV-PPT1

2

PR2.1-Gnat2

CMV-CNTFIRES-GFP CBA-GDNF and Rho-Prph2

Gnat2Cpfl3

Rho−/− Prph2Rd2/Rd2

Retinitis pigmentosa Autosomal dominant RP, macular dystrophies

Reference

Degradation of chondroitin, heparan, and dermatan sulfate proteoglycans Lysosomal hydrolase

Hennig et al., 2004

5

G protein involved in phototransduction

2

Neurotrophic factor

2

Neurotrophic factor and stabilization of discs in outer segments

Hauswirth, unpublished data Liang, Dejneka, et al., 2001 Buch et al., 2006

Griffey et al., 2005

* Also referred to as rds. bOp, bovine rhodopsin promoter; CBA, chimeric CMV-chicken beta actin promoter; CMV, cytomegalovirus immediate early promoter; mOP, murine opsin promoter; PR2.1, human red cone opsin promoter.

was used to deliver PEDF under the control of the CMV promoter (Auricchio, Behling, et al., 2002). These were the first studies to show that intravitreal AAV conferred stable and effective expression of an antiangiogenic transgene. VEGF has been demonstrated to be a key mediator of retinal neovascularization. VEGF isoforms bind two tyrosine kinase receptors expressed on the surface of endothelial cells, VEGF receptor 1 (FLT1) and VEGF receptor 2 (KDR) (De Vries et al., 1992; Gitay-Goren et al., 1992; Jakeman et al., 1992). When activated by VEGF, these receptors stimulate endothelial cell proliferation and migration, vasopermeability, and vasculature organization and modeling. VEGF has been shown to be upregulated by hypoxia in various types of neovascular retinopathies (Adamis et al., 1994; Aiello et al., 1994), as well as in animal models of retinal ischemia (Miller et al., 1994; Pierce et al., 1995; Shima et al., 1996). Various anti-VEGF strategies have been developed that target either the expression of the protein or its interaction with its receptors. Antisense oligonucleotides, soluble VEGF receptors, receptor mimics, and small interfering RNAs targeting VEGF have all been shown to inhibit neovascularization in mouse models (Aiello et al., 1995; Robinson et al., 1996; Reich et al., 2003). However, the transient nature of all of these reagents may well hinder their therapeutic application. In vitro studies have shown that VEGF peptides derived from exons 6 and 7 inhibit VEGF-mediated angiogenesis

(Soker et al., 1997; Jia et al., 2001). These peptides were found to block the interaction of VEGF with the KDR receptor and coreceptor, neuropilin-1 (NPN1), thereby inhibiting VEGF-induced mitogenesis and migration of endothelial cells ( Jia et al., 2001). Taking advantage of the antiangiogenic properties of these peptides by delivering both to the ROP mouse retina via an AAV serotype 2 vector under the control of a CBA promoter effectively inhibited retinal neovascularization (Deng et al., 2005). In this study, vector was administered to newborn pups prior to exposing them to the hyperoxic environment. A related study sought to prevent ocular neovascularization by expressing a soluble version of the VEGF receptor 1, SFLT1, in the ROP mouse retina. AAV2 was used to deliver SFlt1 under the control of the CMV promoter to the intravitreal space of ROP mouse eyes (Bainbridge et al., 2002). In this case, vector was delivered after the mouse was exposed to the hyperoxic environment. Utilizing a secretable gene product expressed by retinal cells not normally responsible for production of SFLT1 allowed vector injection into the vitreous rather than the subretinal space, a much less challenging technique. Treatment resulted in a 50% reduction of retinal neovascularization, with efficacy quantified as previously described. Another vascular retinopathy characterized by early-onset ocular neovascularization is Sorsby’s fundus dystrophy. Mutations in the gene for tissue inhibitor of matrix metalloproteinase-3 (TIMP3) have been identified in Sorsby’s

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patients (Weber et al., 1994). The antiangiogenic properties of TIMP3 in vitro (Anand-Apte et al., 1997) prompted AAV1-mediated delivery of this transgene, under the control of the CMV promoter, to the subretinal space of newborn pups prior to placing them in the hyperoxic environment. The number of neovascular retinal endothelial cell nuclei in eyes treated with AAV1-Timp3 was significantly less than in those injected with control vector (AAV1-GFP or AAV1lacZ), proving that TIMP3 exerts angiostatic effects in vivo (Auricchio, Behling, et al., 2002). The same study also addressed whether endostatin exhibited anti-angiogenic properties in the ROP mouse. Endostatin, encoded by a natural fragment of collagen XVIII (Col18a1), has been shown to inhibit ocular angiogenesis when secreted intravascularly from hepatocytes transduced by viral vectors (O’Reilly et al., 1997; Mori et al., 2001). Using the same protocol as in their TIMP3 experiment, it was shown that AAV1-mediated endostatin expression, driven by the CMV promoter, was also able to reduce angiogenesis in the ROP mouse (Auricchio, Behling, et al., 2002). Another potent inhibitor of angiogenesis is angiostatin, a proteolytic fragment of plasminogen. Kringle domains 1 through 3 of angiostatin (K1K3), has been shown to have angiostatic effects in tumor bearing mice (O’Reilly et al., 1994, 1996; Wu et al., 1997). AAV2 was used to deliver K1K3 under the control of a CBA promoter to either the subretinal or intravitreal space of newborn mouse pups with contralateral eyes receiving only PBS (Raisler et al., 2002). In ROP mice, the number of inner retinal vascular endothelial cells treated intravitreally or subretinally with AAV2-CBA-K1K3 was again significantly less than in control eyes.

Therapy for choroidal neovascularization The laser-induced choroidal neovascularization protocol was developed first in nonhuman primates (Ryan, 1982). Subsequently used in other mammalian species, including mouse (Miller et al., 1990), this model employs the use of a laser to rupture Bruch’s membrane, which leads to recruitment of inflammatory and neovascular mediators promoting the proliferation of choroidal vessels into the retina proper. This pathology mimics that seen in the late stages of wet AMD. The extent of CNV is measured in the mouse 2–4 weeks post laser treatment in choroidal flat mounts prepared from animals that have been perfused with a fluorescent dye (Raisler et al., 2002). In a study using the laser CNV mouse model to test the effects of the antiangiogenic protein PEDF, mice were given intravitreal or subretinal injections of AAV2 containing a CBA promoter driving expression of either PEDF or GFP as a control (Mori et al., 2002). At 4–6 weeks post injection, the Bruch’s membranes of these mice were

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ruptured by laser photocoagulation at three sites per eye. After 14 days, the area of CNV at the laser hole was measured by imaging choroidal flat mounts of mice that had been perfused with fluorescein-labeled dextran. Mice treated with AAV-CBA-PEDF before laser treatment, either subretinally or intravitreally, showed significantly smaller mean areas of CNV than mice injected with control vector. Thus, the principle of AAV-vectored antiangiogenic gene therapy utilizing soluble factors has been well established in mouse models for both retinal and choroidal neovascularization.

Gene replacement therapy Gene replacement strategies are primarily designed for autosomal recessive diseases in which a defective gene is complemented, in trans, with a wild-type counterpart that will produce a normal functional protein, thereby preventing the pathology. It is usually important to express the desired transgene in the cell type in which it is normally expressed, and autosomal recessive retinal diseases are no exception. A number of successful retinal gene replacement studies have been performed in mice using genes that all play significant roles in the structural integrity or function of the retina. Several of these proof-of-principle mouse studies hold great promise for human application. Therapy for Structural Defects in Retinal Disease The oldest and most studied mouse model for structural disorders of the retina is the retinal degeneration slow (rds) or peripherin-2 (Prph2Rd2/Rd2) mouse. The Prph2Rd2/Rd2 mouse is homozygous for a null mutation (large DNA insertion) in the Prph2 gene, encoding a photoreceptor-specific membrane glycoprotein (Travis et al., 1989) necessary for the stabilization of disc rims in photoreceptor outer segments. As a consequence of its absence, the Prph2Rd2/Rd2 mouse is incapable of developing normal photoreceptor discs and outer segments (Sanyal and Jansen, 1981; Molday 1994). In addition to loss of photoreceptor function, rhodopsin is downregulated, and photoreceptors eventually suffer apoptotic cell death (Reuter and Sanyal, 1984; Nir et al., 1990; Chang et al., 1993). In humans, mutations in PRPH2 cause autosomal dominant retinitis pigmentosa and macular dystrophy. As in the Prph2Rd2/Rd2 mouse, these human diseases are characterized by progressive photoreceptor cell death and loss of visual function (Gregory-Evans and Bhattacharya, 1998). AAV2 carrying a normal mouse Prph2 under the control of a bovine rhodopsin promoter was subretinally injected at P10 into Prph2Rd2/Rd2 mice and resulted in preservation of vestigial rhodopsin-containing outer segments and improved photoreceptor function (Ali et al., 2000). However, subsequent experiments noted that ultrastructural improvements in photoreceptors depended on the age at which animals were

treated (Sarra et al., 2001). Also, in all cases, there was no significant slowing of the rate of photoreceptor cell loss. Attempts at promoting photoreceptor cell survival in the Prph2Rd2/Rd2 mouse via AAV delivered neurotrophic factors are discussed later. X-linked juvenile retinoschisis (XLRS) is caused by mutations in the gene for retinioschisin-1 (RS1) (Sauer et al., 1997) that encodes a protein secreted from photoreceptor and bipolar cells as a multimeric complex of identical subunits linked by disulfide bonds (Molday et al., 2001; Reid et al., 2003; Wu and Molday, 2003). Each subunit contains a discoidin domain that has been implicated in cell adhesion and cell-cell interactions (Baumgartner et al., 1998). Patients with XLRS have intraretinal macular voids that form in a spoke-wheel-like pattern, peripheral retinal schisis, and functional changes classically illustrated by a depressed rod ERG b-wave and a fairly normal a-wave. The net effect is a single electronegative ERG waveform. The murine orthologue of the human retinoschisis gene, when knocked out in the mouse (Rs1−/−), mimics some of the pathology seen in human patients, including disorganization of multiple retinal layers, duplication and mislocalization of ganglion cells, shortening of photoreceptor inner and outer segments, and mislocalization of photoreceptor nuclei (Weber et al., 2002). In one study, AAV2 was used to deliver RS1 under the control of a CMV promoter to the intravitreal space of 13-week-old Rs1−/− mice (Zeng et al., 2004). Contralateral eyes received an injection of PBS. Treatment resulted in retinoschisin protein distributed throughout the retina, as well as a reversal of the electronegative waveform through restoration of the normal, positive b-wave. However, treatment failed to improve retinal structure (Zeng et al., 2004). In another study, in order to target RS1 properly to photoreceptors, AAV5 containing human RS1 under the control of the mOP promoter was delivered to the subretinal space of P15 Rs1−/− mice. Treatment led to a normal distribution of retinoschisin and progressive and significant improvement in both retinal function (ERG) and retinal morphology (Min et al., 2005). Photoreceptors were preserved and ERG signals improved for at least 5 months post treatment (Min et al., 2005). Thus, it appears that replacement of either the mouse or human form of RS1 conferred varying levels of synaptic integrity, depending on what AAV serotype, promoter, and delivery route was used. Taken together, these results provide hope that this type of therapy may be useful for XLRS patients. Therapy for Functional Defects in the Retina Leber congenital amaurosis. Leber congenital amaurosis (LCA) is an early-onset form of congenital blindness. Thus far, 10 genes associated with LCA have been identified or mapped.

The first to be discovered, and therefore assigned to the LCA1 locus, was the retinal-specific guanylate cyclase gene (GUCY2D) (Perrault et al., 1996). Since its discovery nine others, including RPE65 and RP1GRIP1, have been identified (Marlhens et al., 1998; Perrault et al., 1999; Dryja et al., 2001; Gerber et al., 2001). All LCA patient pedigrees are consistent with an autosomal recessive inheritance pattern, yet each gene has proved to be involved in strikingly different physiological pathways. A mouse model of LCA1 was created by knocking out guanylate cyclase-1 (GC1) (Yang et al., 1999). Rods in GC1−/− mice do not degenerate, whereas cone loss progresses from central to peripheral retina and peaks between 9 and 16 weeks of age, with very few cones remaining at 6 months of age (Coleman et al., 2004). Cone-mediated ERGs are barely detectable at 1 month of age, while rods exhibit ERG amplitudes 30%–50% of those in congenic control mice but decrease until they plateau at 5 months of age (Yang et al., 1999). Additionally, cone arrestin and cone α-transducin fail to properly translocate between photoreceptor outer and inner segments in response to light. Recent studies have shown that subretinal AAV5-mediated delivery of bovine GC1 under control of either mOP or CBA promoters to P21 GC1−/− mice resulted in restoration of correct lightactivated cone arrestin translocation (Haire et al., 2006). These results suggest that early therapeutic intervention may be required for the optimal treatment of LCA1. A mouse model of LCA2, the Rpe65 knockout mouse, was generated in 1998 (Redmond et al., 1998). RPE65 is a 61-kd protein that is primarily expressed in the microsomal membrane fraction of the retinal pigmented epithelium (RPE) and is the isomerohydrolase responsible for converting alltrans-retinyl ester to 11-cis-retinol in the visual cycle (Mata et al, 2004; Moiseyev et al., 2005). Rod outer segment discs in the Rpe65−/− mouse are disorganized and rod ERGs abolished, while an apparent cone signal remains. However, this ERG signal is not a true cone response but rather a highly desensitized rod response that is being detected under standard cone assay conditions (Seeliger et al., 2001). Rpe65−/− mice lack rhodopsin but maintain opsin apoprotein, and, owing to their inability to isomerize all-trans-retinal ester to its cis-form, all-trans-retinyl esters accumulate in the RPE (Redmond et al., 1998). AAV1 was used for in utero delivery of human RPE65, driven by a CMV promoter, to Rpe65−/− mice (Dejneka et al., 2004). Gene transfer into E14 fetuses resulted in efficient transduction of the RPE, restoration of ERG signals, and measurable rhodopsin after P6. Some animals responded with nearly normal ERG amplitudes and levels of rhodopsin. A naturally occurring model of LCA2, the rd12 mouse, contains a nonsense mutation in the Rpe65 gene rendering the enzyme dysfunctional (Pang et al., 2005). As was noted in Rpe65−/− mice, rd12 mice have profoundly

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diminished rod ERGs, an absence of 11-cis-retinaldehyde and rhodopsin, massive accumulation of retinyl esters in the RPE, and slow photoreceptor degeneration (Pang et al., 2005). Subretinal treatment of P14 mice with a serotype 5 vector containing human RPE65 cDNA, driven by the CBA promoter, resulted in RPE65 expression only in RPE cells, restoration of 11-cis-retinaldehyde, the presence of substantial levels of rhodopsin, and near normal rod and cone ERG amplitudes (Pang et al., 2006). Retinyl ester levels were also reduced to normal, and related to this, funduscopy and retinal morphology remained normal. All parameters of restored retinal health remained stable for at least 7 months. Perhaps most important, visually cued behavioral tests revealed that mice treated in only one eye performed as well as normal, wild-type mice. These results confirm that gene therapy can restore normal vision-dependent behavior in a congenitally blind animal. The promising results obtained in both LCA2 mouse models and the earlier Briard dog results (Acland et al., 2001), coupled with the preclinical safety studies done in rat, dog, and nonhuman primate, have laid the groundwork for upcoming clinical trials for LCA2, likely the first AAV human gene therapy trial in the eye ( Jacobson et al., 2006a, 2006b). A third mouse model of LCA is the retinitis pigmentosa GTPase regulator interacting protein (Rpgrip) knockout mouse (Hong et al., 2000). RPGRIP is a protein located in the ciliary axoneme of rod and cone photoreceptors (Hong et al., 2000, 2003) that serves to tether retinitis pigmentosa GTPase regulator (RPGR) to this structure (Zhao et al., 2003). In turn, RPGR is proposed to play a role in regulating protein transport through the connecting cilium of photoreceptors to outer segments (Hong et al., 2000). Rpgrip−/− mice display photoreceptor degeneration beginning at P15, as well as early-onset loss of ERG signals. AAV2-mediated delivery of RPGRIP driven by a 236 bp (−218/+17) mOP promoter and delivered to the subretinal space of P18–P20 Rpgrip−/− mice resulted in RPGRIP localized normally to the connecting cilium, photoreceptor preservation, and ERG recovery (Pawlyk et al., 2005). Lysosomal storage disease. Lysosomes are responsible for the breakdown of intracellular macromolecules and are characterized by the nature of the material stored within. Defects in the enzymes that degrade lysosomal contents can lead to inability to turn over lysosomal material, resulting in a broad spectrum of more than 40 clinical disorders, two of which are mucopolysaccharidosis type VII (MPS VII) and infantile neuronal ceroid lipofuscinosis (INCL). MPS VII is a member of a group of diseases caused by deficiencies in enzymes responsible for the breakdown of glycosaminoglycans (GAGs). MPS VII is caused by a deficiency in the enzyme β-glucuronidase (Sly et al., 1973) and leads to progressive RD and vision impairment. MPS VII mice lack

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β-glucuronidase activity due to a single base pair deletion in Gusb (Birkenmeier et al., 1989; Sands and Birkenmeier, 1993) and display progressive shortening of photoreceptor outer segments, apoptotic photoreceptor death, accumulation of lysosomal storage bodies in the RPE, and diminished ERG amplitudes (Birkenmeier et al., 1989; Lazarus et al., 1993; Ohlemiller et al., 2000; Birkenmeier et al., 1991; Stramm et al., 1990). AAV2 was used to deliver GUSB, driven by the CBA promoter, to the intravitreal space of 4-weekold MPS VII mice. At 16 weeks of age, vector-treated eyes had near normal levels of β-glucuronidase, preservation of cells in the outer nuclear layer, decreased RPE lysosomal storage, and significantly improved rod ERGs (Hennig et al., 2004). A similar vector administered systemically to newborn MPS VII mice prior to the initiation of disease resulted in almost complete ERG protection (Daly et al., 1999a, 1999b, 2001). A second lysosomal storage disease having an ocular pathology is INCL, a neurodegenerative disorder described as the earliest onset form of Batten disease. INCL is caused by mutations in the palmitoyl protein thioesterase-1 gene (PPT1), a lysosomal hydrolase that is trafficked to the lysosome via the mannose-6-phosphate receptor (Vesa et al., 1995; Sleat et al., 1996). A Ppt1 knockout mouse model of INCL displays significant rod and cone ERG deficits as early as 2 months of age, as well as progressive RD relative to congenic controls (Gupta et al., 2001; Griffey et al., 2005). Intravitreal delivery of AAV2 carrying human PPT1 driven by the CMV promoter into P18–P21 PPT −/− mice resulted in increased PPT1 expression, significant improvements in rod and cone ERGs, and a slower rate of degeneration relative to that in untreated controls (Griffey et al., 2005). It is likely that use of an AAV5 vector or subretinal injection may increase the amount of PPT1 expression in photoreceptors and lead to improved histopathological protection. Achromatopsia. Achromatopsia, also known as rod monochromatism, is an early-onset form of severe color blindness that results from defects in cone photoreceptors. A mouse model of recessive achromatopsia was found by ERG screening in the mouse strain collection at the Jackson Laboratory. The mutation, termed cpfl3, for cone photoreceptor function loss-3, was mapped to mouse chromosome 3, the same location as human GNAT2 (Chang et al., 2006). GNAT2 encodes the α subunit of cone transducin, the G protein involved in phototransduction, and, when mutated in the Cpfl3 mouse, renders the G protein nonfunctional. As a result, cone photoreceptors are unable to sustain the phototransduction cascade in response to light, leading to a very weak photopic cone response at 3 weeks of age that is lost by 9 weeks (Chang et al., 2006). Rod ERG responses are initially normal but also diminish somewhat with age. Cone transducin α immunolabeling was minimally detectable and

was accompanied by some photoreceptor outer segment disorganization. Subretinal injection of AAV5 containing Gnat2 cDNA driven by a cone-targeting PR2.1 promoter partially preserved retinal structure, completely restored photopic cone responses, and restored visual acuity to near normal (Alexander et al., 2007). These studies, which targeted transgene expression specifically to cones, hold promise for analogous cone therapy in other models of achromatopsia, as well as for other cone-targeted retinal diseases. Neurotrophic factor gene therapy. Neurotrophic cell survival factors have shown promise in preserving photoreceptor cells from apoptosis, the common fate of photoreceptors in many RT diseases. In surveys of neurotrophic factors injected directly into the vitreous of various rodent models of photoreceptor degeneration, a number have been shown to promote photoreceptor survival (Faktorovich et al., 1990; LaVail et al., 1992, 1998). AAV-mediated delivery of cDNAs for such factors is an attractive option that would not require multiple injections and could supplement gene replacement therapies. AAV2-mediated delivery of ciliary neurotrophic factor (CNTF) has been evaluated in several mouse models of RD, including the rhodopsin knockout mouse (Rho−/−), the peripherin knockout (PrphRd2/Rd2) mouse, and a heterozygous Prph2 mouse with a transgenic copy of the P216L mutant Prph2 allele (rds+/−P216L) (Liang et al., 2001a, 2001b; Bok et al., 2002; Schlichenbrede et al., 2003; Buch et al., 2006). All studies showed that vectored CNTF expression promotes photoreceptor survival. Although not evaluated in the Rho−/− mouse (Liang et al., 2001a), in both Prph2Rd2/Rd2 and rds+/−P216L mice, photoreceptor function, as evaluated by ERG, was either not improved or reduced in vector injected eyes relative to untreated controls (Bok et al., 2002; Schlichenbrede et al., 2003; Buch et al., 2006). This treatment-related loss in photoreceptor function also appeared to be dose dependent and occurred in normal eyes as well (Bok et al., 2002; Buch et al., 2006). Additionally, in the Prph2Rd2/Rd2 mouse, when AAV-mediated CNTF expression was supplemented with either AAV2-Prph2 or AAV2-rhodopsin, there was still no improvement relative to untreated control eyes. Taken together, these results suggest that CNTF dosing may be crucial to avoid any negative effects of the neurotrophin (Schlichenbrede et al., 2003). Indeed, in a recent study, AAV-mediated CNTF expression was found to be deleterious to photoreceptor function in wild-type, C57BL6, mice (Rhee et al., 2007). What remains to be determined is the dosage of CNTF delivered in vivo by AAV vectors relative to that expected in a clinical trial, and to what extent toxicity seen in one species, the mouse, can be carried over into the human. In contrast, AAV-mediated glial cell line–derived neurotrophic factor (GDNF) expression driven by the CBA promoter in the Prph2Rd2/Rd2 mouse slowed photoreceptor loss

and, more importantly, did not have the adverse effects on photoreceptor function that were seen with CNTF. In addition, when used in combination with the appropriate gene replacement therapy, enhanced photoreceptor function was observed (Buch et al., 2006).

Conclusions and future directions for adenoassociated virus gene therapy in the mouse retina A large body of evidence supports the use of AAV as an effective vector for gene delivery to the mouse retina. Optimization of gene therapy strategies, as well as the development of new mouse models of human retinal disease, suggests that appropriately designed AAV-mediated gene therapy may eventually be applied broadly to human subjects. Current research is focusing on mouse models that are more “human-like” and therefore may address the species differences between man and mouse. One such study is the development of the neural retina leucine zipper knockout (Nrl −/−) mouse. As determined by structural and functional characteristics, the Nrl −/− mouse, in contrast to the normal roddominant mouse retina, contains an all cone-like retina (Daniele et al., 2005). This mouse may be a better rodent model of the cone-rich human macula, suggesting that subjecting the Nrl −/− mouse to ROP conditioning or laserinduced CNV, for example, may provide a better model for central field loss in neovascular disorders such as diabetic retinopathy and AMD. Additionally, crossing the Nrl −/− mouse with known cone degeneration models may aid in the development of more effective cone-targeted gene therapies. Although this chapter has summarized gene replacement strategies in mouse models of RD that are predominantly inherited in an autosomal recessive fashion, progress has also been made toward addressing autosomal dominantly inherited RD. To date, most of this work has been carried out in the P23H rhodopsin rat, the primary current model for a common form of autosomal dominant retinitis pigmentosa (Dresner et al., 1998; Lewin et al., 1998; LaVail et al., 2000). A mouse model mimicking autosomal dominant macular dystrophy is the ELOVL4 mouse (Raz Praq et al., 2006; Vasireddy et al., 2006), which contains a heterozygous knock-in carrying the same 5 bp deletion in the ELOVL4 gene associated with dominant Stargardtlike macular degeneration (STGD3) in humans. The ELOVL4 mouse has a retinal phenotype similar to STGD3 patients (Vasireddy et al., 2006), and attempts are currently under way to mediate the mouse disease using AAV vectors. Another recent advance in the field of AAV gene delivery is the development of self-complementary AAV vectors (scAAV), which package double-stranded DNA in the viral capsid, in contrast to single-stranded DNA in standard AAV vectors. This modification overcomes the rate-limiting step

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for AAV-vectored transgene expression, the conversion of vector single-stranded DNA into double-stranded DNA by the host cell (McCarty et al., 2001, 2003). Studies have shown that scAAV mediates quicker and more efficient transgene expression than standard AAV vectors in mouse brain and retina (Fu et al., 2006; Hauswirth et al., 2006). scAAV-mediated gene therapy may therefore be of use in the treatment of disorders that require immediate, high-level intervention, particularly for some very rapidly degenerating mouse models of retinal disease. In summary, based on a broad variety of successfully treated mouse models of retinal RD, the clinical future for AAV-vectored gene therapy seems bright. REFERENCES Acland, G. M., Aguirre, G. D., Bennett, J., Aleman, T. S., Cideciyan, A. V., Bennicelli, J., Dejneka, N. S., PearceKelling, S. E., Maguire, A. M., et al. (2005). Long-term restoration of rod and cone vision by single dose rAAV-mediated gene transfer to the retina in a canine model of childhood blindness. Mol. Ther. 12:1072–1082. Acland, G. M., Aguirre, G. D., Ray, J., Zhang, Q., Aleman, T. S., Cideciyan, A. V., Pearce-Kelling, S. E., Anand, V., Zeng, Y., et al. (2001). Gene therapy restores vision in a canine model of childhood blindness. Nat. Genet. 28:92–95. Adamis, A. P., Miller, J. W., Bernal, M. T., D’Amico, D. J., Folkman, J., Yeo, T. K., and Yeo, K. T. (1994). Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am. J. Ophthalmol. 118:445– 450. Aiello, L. P., Avery, R. L., Arrigg, P. G., Keyt, B. A., Jampel, H. D., Shah, S. T., Pasquale, L. R., Thieme, H., Iwamoto, M. A., et al. (1994). Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med. 331:1480–1487. Aiello, L. P., Pierce, E. A., Foley, E. D., Takagi, H., Chen, H., Riddle, L., Ferrara, N., King, G. L., and Smith, L. E. (1995). Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGFreceptor chimeric proteins. Proc. Natl. Acad. Sci. U.S.A. 92: 10457–10461. Alexander, J. J., Li, Q., Timmers, A., Hawes, N., Chang, B., and Hauswirth, W. W. (2005). Restoration of cone function in a mouse model of achromatopsia by rAAV mediated promoter targeted expression of cone specific α-transducin. Invest. Ophthalmol. Vis. Sci. 46. ARVO E-Abstract 5207. Alexander, J. J., Umino, Y., Everhart, D., Chang, B., Min, S. H., Li, Q., Timmers, A. M., Hawes, N. L., Pang, J. J., et al. (2007). Restoration of cone function in a mouse model of achromatopsia. Nat. Med. May 21 (Epub ahead of print). Ali, R. R., Reichel, M. B., De Alwis, M., Kanuga, N., Kinnon, C., Levinsky, R. J., Hunt, D. M., Bhattacharya, S. S., and Thrasher, A. J. (1998). Adeno-associated virus gene transfer to mouse retina. Hum. Gene Ther. 9:81–86. Ali, R. R., Sarra, G. M., Stephens, C., Alwis, M. D., Bainbridge, J. W., Munro, P. M., Fauser, S., Reichel, M. B., Kinnon, C., et al. (2000). Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy. Nat. Genet. 25:306–310.

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Yang, G. S., Schmidt, M., Yan, Z., Lindbloom, J. D., Harding, T. C., Donahue, B. A., Engelhardt, J. F., Kotin, R., and Davidson, B. L. (2002). Virus-mediated transduction of murine retina with adeno-associated virus: Effects of viral capsid and genome size. J. Virol. 76:7651–7560. Yang, R. B., Robinson, S. W., Xiong, W. H., Yau, K. W., Birch, D. G., and Garbers, D. L. (1999). Disruption of a retinal guanylyl cyclase gene leads to cone-specific dystrophy and paradoxical rod behavior. J. Neurosci. 19:5889–5897. Zeng, Y., Takada, Y., Kjellstrom, S., Hiriyanna, K., Tanikawa, A., Wawrousek, E., Smaoui, N., Caruso, R., Bush, R. A., et al.

(2004). RS-1 gene delivery to an adult Rs1h knockout mouse model restores ERG b-wave with reversal of the electronegative waveform of X-linked retinoschisis. Invest. Ophthalmol. Vis. Sci. 45:3279–3285. Zhao, Y., Hong, D. H., Pawlyk, B., Yue, G., Adamian, M., Grynberg, M., Godzik, A., and Li, T. (2003). The retinitis pigmentosa GTPase regulator (RPGR)- interacting protein: Subserving RPGR function and participating in disk morphogenesis. Proc. Natl. Acad. Sci. U.S.A. 100:3965–3970.

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Delivery of Plasmids into the Visual System Using Electroporation TAKAHIKO MATSUDA AND CONSTANCE L. CEPKO

The ability to alter gene expression in vivo has opened a wide range of investigations into gene structure and function. In addition, it has provided a means to visualize specific cells, track cells with a particular history of gene expression, and alter cellular physiology, and it has allowed the production of models of disease. To facilitate the rapid introduction of gene constructs into the visual system, we used electroporation for delivery to the embryonic and early postnatal retina and brain. Plasmids that allow controlled expression of genes or short hairpin RNAs for RNA interference (RNAi) vectors have been developed. These methods open up the possibility of relatively rapid assessment of gene structure and function.

Comparison of gene transfer methods Electroporation can be compared to other methods of gene delivery in vivo. Several types of viral vectors, including murine oncoretrovirus (Price et al., 1987; Turner and Cepko, 1987), lentivirus (Miyoshi et al., 1997), adenovirus (Bennett et al., 1994; Jomary et al., 1994; Li et al., 1994), and adenoassociated virus (Ali et al., 1996), are in use. There are advantages and disadvantages inherent in the use of viral vectors. The disadvantages are these: (1) It is time-consuming to prepare high-titer virus stocks to achieve efficient gene transfer. (2) Viral vectors have a size limitation for insert DNA. (3) In general, such vectors do not readily allow introduction of more than two genes into the same cells. (4) Biosafety is a concern for some viral vectors with broad host ranges. However, an advantage that cannot be overlooked for some applications (e.g., lineage analysis) is that integration of the oncoretroviral genome gives stable gene expression throughout all progeny of an infected cell. Electroporated DNA does not integrate efficiently. For some species, transgenic animals can be made by injecting DNA into fertilized eggs. However, producing such strains is invariably slow and often very expensive. Electroporation bypasses many of the disadvantages cited earlier (Matsuda and Cepko, 2004). This method is faster

and in some cases safer than viral gene transfer methods. The efficiency of electroporation into at least some areas of the developing visual system is quite good, and transgene expression persists for more than a month. There does not appear to be a limitation in terms of the species that can be targeted. Various types of DNA constructs, including RNAi vectors as well as conventional gene expression vectors, are readily introduced into the retina without DNA size limitation. We have even successfully electroporated bacterial artificial chromosome (BAC) constructs, although with more difficulty and low levels of expression (Cherry and Cepko, 2008). More than two different DNA constructs can be introduced at once. We found that at least five plasmids can be coelectroporated into the same cells without a significant reduction in coelectroporation frequency. We have also generated a series of cell type–specific promoter constructs that direct expression to specific retinal cell types and have achieved temporal control using recombinases. When considering the options for gene transfer, one should carefully evaluate whether transient or stable, clonal or nonclonal gene expression is desirable, as well as the feasibility of applying the various methods to the species under study. In addition, it is important to recognize that electroporation, like most viral transduction methods, does not transduce 100% of cells in a targeted area. Transgenic animals can provide more uniform transduction, and if this is required, then electroporation and viral methods cannot be used.

Method of electroporation Retina in Vivo The basic strategy for in vivo electroporation into the retinas of newborn mouse and rat pups is to inject DNA into the subretinal space between the retina and retinal pigment epithelium (RPE) (figure 51.1A). Electrodes are then placed on the heads of the pups and electric pulses are applied to the eyes (figure 51.2). The DNA constructs are transduced into the scleral side of the retina, where undiffentiated mitotic and newly postmitotic cells

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exist (figure 51.3). Because DNAs are preferentially transduced into undifferentiated progenitor/precursor cells, only late-born cell types (rod, bipolar, Müller glia, and a subset of amacrine cells) are labeled by electroporation at P0 (figures 51.4 and 51.5). It is not clear whether electroporation

Figure 51.1 Strategy for in vivo electroporation A, Electroporation to the scleral (RPE) side of the retina. B, Electroporation to the vitreal side of the retina. See color plate 50.

into progenitor/precursor cells is more effective than into mature neurons because of an inherent difference in cell types or whether this is simply due to the location of progenitor/precursor cells adjacent to the DNA injection site, the subretinal space. In addition to this strategy, it is also theoretically possible to transfect DNAs from the vitreous side of the retina by injecting DNAs into the vitreous chamber, and applying electric pulses in the direction opposite to that used for subretinal injections (see figure 51.1B). Indeed, other groups reported that DNA constructs could be transduced to ganglion cells, which line the surface of the retina facing the vitreous body, by in vivo electroporation using this strategy (Dezawa et al., 2002; Huberman et al., 2005; Kachi et al., 2005). However, our data show that the transfection efficiency of the vitreal side (ganglion cells) of the neonatal retina, as well as of the adult retina, is much lower than of the scleral side (progenitor/precursor cells) of the neonatal retina (see figure 51.8). Again, it is not clear why this injection site results in less successful transduction. It could result from physiological differences between ganglion cells/displaced amacrine cells and progenitor/ precursor cells; for example, it could be the case that DNA plasmids may not be readily transcribed in these neurons. Alternatively, or in addition, it could result from such things as access of the electroporated plasmids to the nuclear

Figure 51.2 Electrodes and procedure for in vivo electroporation. Tweezer-type electrodes (A) are placed to hold the head of newborn (P0) rat or mouse (B). See color plate 51.

Figure 51.3 Whole-mount preparation of rat retina in vivo electroporated at P0 with CAG-GFP (Matsuda and Cepko, 2004), a GFP expression vector driven by the CAG (chicken β-actin pro-

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moter with cytomegalovirus enhancer) promoter and harvested at P21. Images are from the scleral side. Bright-field (A), GFP (B), and merged (C ) images are shown. See color plate 52.

Figure 51.4 In vivo electroporated retina (P0 electroporation, section). Rat retinas were in vivo electroporated with CAG-GFP at P0 and harvested at P2 (top panel) or P20 (bottom panel). At P2, most of the GFP-positive cells have the morphology of progenitor/precursor cells, suggesting that DNAs are preferentially transfected to progenitor/precursor cells. Retinogenesis is completed within the

first 2 weeks after birth. At P20, GFP is observed in four differentiated cell types: rod photoreceptors, bipolar cells, amacrine cells, and Müller glia. Early-born cell types (cone, horizontal, and ganglion cells) are not labeled by P0 electroporation. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment; VZ, ventricular zone. See color plate 53.

compartment of the neurons. For example, electroporated plasmids may enter a cellular compartment, such as axons, Müller glial endfeet, or blood vessels, or even get trapped in basement membrane rather than go directly into the ganglion cell cytoplasm. To deliver DNAs into early-born cell types (cone, horizontal, ganglion, and amacrine cells) whose progenitor/ precursor cells exist primarily in the embryonic retina, one needs to electroporate DNAs into embryonic retinas (see figure 51.5). Two approaches are used for in vivo (in utero) electroporation into embryonic retinas. One utilizes ultrasound to guide delivery of the plasmid DNA to the subretinal space, or early optic vesicle, such as in murine embryos at E9.5. One can also deliver plasmid DNA to pups in utero without ultrasound, from about E13 for mouse or E14 for rat. This approach involves a learning period during which

the practitioner becomes familiar with the landmarks of the embryonic structures as seen through the uterine wall (Turner et al., 1990). Delivery to the embryonic tissue again appears to result in uptake of the DNA primarily by cycling cells or newborn neurons. Because embryonic cells tend to undergo more rounds of cell division than postnatal progenitor cells, the DNA is diluted out more rapidly. Retention of the plasmid in the neurons that are generated soon after electroporation is apparent, because those are the cells that are most strongly marked; an example is the amacrine cells and cone photoreceptors following delivery at E14 in the mouse (figure 51.6). Retina in Vitro In organ cultures of embryonic or neonatal retina, progenitor cells differentiate into neurons and glia and form three layers, mimicking normal development.

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Figure 51.5 Timing of electroporation and labeled cell types. Birth order of retinal cells in the mouse retina is shown. As DNAs are preferentially transfected into undifferentiated progenitor/precursor cells by electroporation, electroporation at P0 labels only late-born cell types (rod, bipolar, Müller glia, and a subset of ama-

crine cells), which are generated from P0 retinal progenitors. On the other hand, electroporation at E14 can label early-born cell types (cone, horizontal, ganglion, and amacrine cells), which are generated from E14 retinal progenitors.

Figure 51.6 In vivo electroporated retina (E14 electroporation, section). Mouse embryonic retinas were electroporated with UBGFP (Matsuda and Cepko, 2004), a GFP expression vector driven by the human ubiquitin promoter, at E14 in utero, and harvested at P20. Early-born cell types (cone, amacrine, horizontal, and ganglion cells) are clearly labeled with GFP, while late-born cell types (bipolar and Müller glial cells), which are generated from E14

RPCs after several rounds of cell division, are poorly labeled. This is probably due to dilution of introduced plasmids. Red arrowheads indicate the labeled cone photoreceptors. Yellow arrowhead indicates the labeled horizontal cell. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment. See color plate 54.

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Taking advantage of this propensity, we also developed a system to electroporate DNAs into isolated retinas (in vitro electroporation; see Matsuda and Cepko, 2004) using a micro-electroporation chamber (figures 51.7 and 51.8). Electroporated retinas are cultured for a few days to weeks. Compared with in vivo electroporation, in vitro electroporation has several advantages. First, in vitro electroporation is easier and less skill dependent than in vivo electroporation. All retinas subjected to electroporation become GFP (RFP) positive when GFP (RFP) expression

vectors are used. Second, it is relatively easy to handle a large number of retinas in a day. Third, in vitro electroporation can be easily applied not only to postnatal retinas, but also to embryonic retinas, to which in vivo electroporation (in utero electroporation) is more difficult to apply. Fourth, realtime monitoring of GFP (RFP)-transduced cells is possible under a fluorescent microscope. However, in vitro electroporation has several disadvantages inherent to organ culture. First, the morphology of cultured retina is frequently poor, and photoreceptor outer segments are poorly formed. Second, it is hard to culture retinas for a long period. In our

Figure 51.7 A, Microchamber for in vitro electroporation. B, Orientation of the retina in the chamber. Maximum transduction efficiency can be obtained when the scleral side is facing the minus electrode. See color plate 55.

Figure 51.8 In vitro electroporated retinal explant (whole mount). Mouse retinas of P0 CD1 (A and B), adult CD1 (C and D), or adult Swiss Webster mice with a retinal degeneration mutation (E and F) were in vitro electroporated with CAG-GFP from the scleral side (A, C, and E) or from the vitreal side (B, D, and F) and cultured for

5 days. Images A, C, and E are from the scleral side and images B, D, and F are from the vitreal side. Note that only the scleral side of developing retina or of degenerated retina is highly transfectable. In E, most of the GFP-positive cells are Müller glial cells. See color plate 56.

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experience, retinas tend to become unhealthy when cultured for more than 2 weeks. Brain in Vivo The delivery of plasmid DNAs to the brain has been used by several groups previously (FukuchiShimogori and Grove, 2001; Saito and Nakatsuji, 2001; Tabata and Nakajima, 2001). The same issues discussed earlier pertain here as well, such as dilution of DNA following early delivery (Shimogori et al., 2004). Marking of visual cortex has been achieved (Akaneya et al., 2005; Huberman et al., 2005). It is likely that most areas adjacent to a ventricle will allow successful plasmid uptake and expression following electroporation. Ventricular zones are adjacent to the ventricular lumina, into which DNA can be readily injected, and they are also the area where progenitor cells reside. As discussed earlier for the retina, it appears that these two criteria offer the ideal circumstances for successful electroporation. Injection into tissue rather than into a lumen does not result in effective electroporation, likely because only the DNA that remains in the needle track is available for uptake. In addition, it is unclear whether the neurons, which are more mature in developing tissue, will successfully take up and express such plasmids.

Controlling expression following electroporation One advantage to using transgenic animals rather than viral vectors or even (often) electroporation is the ability to use regulatable promoters and recombinases to control gene expression. We have created a series of plasmids to realize these advantages for electroporated genes, or RNAi contstructs (Matsuda and Cepko, 2007). Temporal Regulation Conditionally active Cre recombinases are composed of Cre and the mutated ligand binding domains of the human estrogen receptor (ERT2). They are activated by 4-hydroxytamoxifen (4-OHT) and have been used to temporally control gene expression in transgenic mice (Branda and Dymecki, 2004). We tested the 4-OHTresponsible Cre recombinases, CreERT2 (single fusion; Feil et al., 1997) and ERT2CreERT2 (double fusion; Casanova et al., 2002), in vivo in the rat retina. These recombinases were expressed under control of the ubiquitous CAG promoter (Niwa et al., 1991) (figure 51.9A). A Cre-dependent expression vector (Kanegae et al., 1995) containing the CAG promoter, a floxed stop cassette, and a reporter gene (DsRed) was used as a recombination indicator (termed CALNLDsRed; figure 51.9B). When P0 rat retinas were coelectroporated with CAG-CreERT2, CALNL-DsRed (recombination indicator), and CAG-GFP (transfection control) and harvested at P21, very high background recombination (DsRed expression) was detected even without 4-OHT stimulation (figure 51.9D). ERT2Cre (N-terminal ERT2 fusion)

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also had very high background recombination activity. In contrast, ERT2CreERT2 double fusion had no detectable recombination activity without 4-OHT (figure 51.9E). When 4-OHT was injected intraperitoneally into the transfected rats at P20, an induction of DsRed expression was clearly detected 24 hours after 4-OHT administration (figure 51.9F). Similar results were observed when CreERT2 and ERT2CreERT2 were transfected into 293 T cells or E14.5 mouse brain. These results indicate that at least for in vivo electroporation studies, ERT2CreERT2, but not CreERT2 (ERT2Cre), can lead to tight regulation of the onset of transgene expression. It is not clear why the double ERT2 domain construct was required for tight regulation of recombinase activity. The heat shock protein 90 (Hsp90) interacts with the ER domain in the cytosol and thereby prevents the translocation of CreER fusion protein to the nucleus where DNA recombination occurs (Picard, 1994). The double fusion may have a higher affinity for Hsp90 to form a tighter complex. Alternatively, the ERT2CreERT2 fusion may have less activity due to the double fusion, and thus less background activity. It is also possible that degradation of CreERT2 (ERT2Cre) results in generation of “active Cre” lacking the regulatory domain, while ERT2CreERT2 is still inactive even after losing one regulatory domain. Cell Type–Specific Regulation Using Specific Promoters To restrict transgene expression to specified cell types in the retina, several retinal cell type–specific promoters were obtained using the literature to guide construction or were developed in our laboratory. Regulatory sequences for rhodopsin (expressed in rods; Zack et al., 1991), Nrl (expressed in rods; Swaroop et al., 1992), Crx (expressed in photoreceptors and weakly in bipolars; Chen et al., 1997; Furukawa et al., 1997b), calcium-binding protein 5 (Cabp5, expressed in subsets of bipolar cells; Haeseleer et al., 2000), N-myc downstream-regulated gene 4 (Ndrg4, expressed in amacrines; Punzo and Cepko, 2008), cellular retinaldehyde-binding protein (Cralbp, expressed in Müller glia; Kennedy et al., 1998), clusterin (expressed in Müller glia; Blackshaw et al., 2004), Rax (expressed in progenitors and Müller glia; Furukawa et al., 1997a; Mathers et al., 1997), and Hes1 (expressed in progenitors and Müller glia; Tomita et al., 1996) were characterized for this purpose. When these promoters were used to express DsRed, DsRed was detected only in specific cell types in the retina (figure 51.10 and table 51.1). Using fluorescent protein variants (CFP, YFP, and DsRed) as reporters, we could visualize different cell types simultaneously (figure 51.11). The cell type–specific promoters were also used to regulate expression of Cre recombinase (figure 51.12). This type of construct was coelectroporated with CALNL-DsRed, a recombination indicator. Following the action of Cre, DsRed

Figure 51.9 Temporal regulation of gene expression in the retina using inducible Cre recombinases. A, CAG-CreERT2: Fusion protein (CreERT2) between Cre recombinase and the mutated ligand-binding domain (ERT2) of the human estrogen receptor is expressed under the control of the CAG promoter. CAG-ERT2 CreERT2: Fusion protein (ERT2CreERT2) composed of Cre and two ERT2 domains is expressed under the control of the CAG promoter. CreERT2 and ERT2CreERT2 are conditionally activated in response to 4-OHT. B, CALNL-DsRed: Cre/loxP-dependent inducible expression vector. DsRed is expressed only in the presence of Cre. C, A scheme of the experiment. D–I, P0 rat retinas were coelectroporated with three plasmids: CAG-GFP (transfection control),

CALNL-DsRed (recombination indicator), and CAG-CreERT2 (D) or CAG-ERT2CreERT2 (E and F). The retinas were stimulated without 4-OHT (D and E) or with 4-OHT (F) by IP injection at P20 and then harvested at P21. Whole-mount preparations of the harvested retinas are shown. G–I, Sections of the retinas shown in D–F. Cell nuclei were stained with DAPI. When CreERT2 was used, significant background recombination was observed even in the absence of 4-OHT. On the other hand, ERT2CreERT2 had no detectable basal activity in the absence of 4-OHT. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. See color plate 57.

would be expressed from the ubiquitous CAG promoter. In all cases, DsRed expression levels in the retina were higher when DsRed was expressed from the CAG promoter rather than any of the cell type–specific promoters. The rhodopsin promoter-Cre construct specifically induced the expression of DsRed in rods. Similarly, the Nrl promoter-Cre construct led to the expression of DsRed only in rods, indicating that both promoters are restricted to rods and are not even tran-

siently active in other cell types, including multipotent progenitors. Interestingly, when the Cabp5 promoter-Cre construct was used, a subset of rods, as well as bipolars, was labeled with DsRed. The ratio of the number of DsRedpositive rods to that of DsRed-positive bipolars was approximately 1 : 1. This might suggest that the Cabp5 promoter is active in the progenitors that produce rod and bipolar cells. The Ndrg4 promoter-Cre construct induced the expression

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Figure 51.10 Spatial regulation of gene expression in the retina using cell type–specific promoters. Retinal cell type–specific promoters were fused to DsRed cDNA and electroporated into P0 rat retinas. The retinas were harvested at P20 (A–F and H) or P2 (G),

sectioned, and stained with DAPI. Promoters of rhodopsin (A), Nrl (B), Cabp5 (C ), Ndrg4 (D), Cralbp (E), clusterin (F), and Hes1 (G and H) were used to express DsRed. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. See color plate 58.

Table 51.1 Labeled cell types observed following electroporation with retinal cell type–specific promoters Reporter Construct Cell Types (Signal Intensity) Rhodopsin promoter 2.2kb-DsRed Rod (+++++) Nrl promoter 3.2kb-DsRed Rod (+++) Crx promoter 5.0kb-DsRed Rod (+) + Bip (+++) Cabp5 promoter 4.7kb-DsRed Bip (+++) Ndrg4 promoter 6.2kb-DsRed Ama (++++) Cralbp promoter 4.0kb-DsRed MG (+++) Clusterin promoter 6.2kb-DsRed MG (+++) Rax promoter 7.2kb-DsRed (P2) Prog (+) Rax promoter 7.2kb-DsRed (P20) Not detected Hes1 promoter 1.1kb-DsRed (P2) Prog (+++) Hes1 promoter 1.1kb-DsRed (P20) MG (++) Rhodopsin promoter 2.2kb-Cre Rod (++++++) Nrl promoter 3.2kb-Cre Rod (++++) Crx promoter 5.0kb-Cre Rod (++++) + Bip (++++) Cabp5 promoter 4.7kb-Cre Rod (+++) + Bip (++++) Ndrg4 promoter 6.2kb-Cre Rod (+++) + Ama (+++++) Cralbp promoter 4.0kb-Cre Rod (+++) Bip (+++) + MG (++++) Clusterin promoter 6.2kb-Cre Rod (+++) Bip (+++) + MG (++++) Rax promoter 7.2kb-Cre Rod (+++) Bip (+++) + Ama (+++) + MG (+++) Hes1 promoter 1.1kb-Cre Rod (+++) Bip (+++) + Ama (+++) + MG (+++) Note: Results of figure 51.10 (expression patterns of DsRed directly driven by cell type–specific promoters) and figure 51.12 (expression patterns of DsRed induced by Cre recombinase driven by cell type–specific promoters) are summarized in the table. DNA constructs were transfected at P0, and P2 or P20 retinas were analyzed. Cell types were determined based on cell morphology and location. Relative expression levels of DsRed in each experiment are indicated. Ama, amacrine cells; Bip, bipolar cells; MG, Müller glial cells; Prog, progenitor cells.

Figure 51.11 Multicolor labeling of the retina using cell type– specific promoters. P0 rat retina was electroporated with three reporter constructs: rhodopsin promoter 2.2K-CFP (specific for rods), Cabp5 promoter 4.7K-YFP (specific for a subset of bipolar cells), and Cralbp promoter 4.0K-DsRed (specific for Müller glia). The retina was harvested at P20, sectioned, and stained with DAPI. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment. See color plate 59.

of DsRed in amacrines as well as in a subset of rods. The Cralbp promoter-Cre construct and the clusterin promoterCre construct induced the expression of DsRed in Müller glia and a small population of rod and bipolar cells. The Hes1 promoter-Cre construct and the Rax promoter-Cre construct induced the expression of DsRed in rod, bipolar, amacrine, and Müller glial cells. The expression patterns of DsRed were analyzed at P20, with the results summarized in table 51.1. Temporal and Cell Type–Specific Regulation By combining the temporal regulation afforded by ERT2CreERT2 with the cell type–specific regulation provided by the promoters, we could inducibly express a reporter gene specifically in a desired cell type, such as “differentiated” Müller glia. As shown in figure 51.13, the clusterin promoter was cloned upstream of ERT2CreERT2, and the resulting plasmid was coelectroporated with CALNLDsRed (recombination indicator) and CAG-GFP (transfection control) into P0 rat retinas. The retinas were stimulated with 4-OHT at P14, the time point when retinogenesis is complete (Young, 1985; Rapaport et al., 2004), and then analyzed at P16. Without 4-OHT stimulation, DsRed expression was not detected. When 4-OHT was injected into the rats, clear DsRed expression was detected in a subset of GFP-positive cells. The retinal sections showed that all the DsRed-positive cells examined were morphologically Müller glia. These results show that ERT2CreERT2 can be used with retinal cell type–specific promoters to achieve precise temporal and cell type–specific regulation in the retina.

Conditional RNAi To conditionally knock down gene expression in the retina, Cre-dependent inducible RNAi vectors utilizing the micro-RNA30 (mir30)-based shRNA expression system (Zeng et al., 2002; Paddison et al., 2004) were made. The mir30-based shRNA expression system has several advantages over conventional RNAi vectors expressing shRNAs directly from pol III promoters, such as the U6 promoter. First, shRNA can be expressed using pol II promoters (e.g., CAG promoter). Second, it is technically simple to put the regulatory elements (e.g., transcriptional stop cassette) into the expression vectors. Figure 51.14 shows the mir30-based vectors expressing shRNA under the control of the CAG promoter (CAG-mir30 and CALSL-mir30). CALSL-mir30 is a Cre-dependent inducible RNAi vector carrying a floxed transcriptional stop cassette immediately after the CAG promoter. CALSL-mir30 also was used with the rhodopsin promoter-Cre construct in the retina. P0 rat retinas were coelectroporated with four plasmids, CALSLmir30(GFPshRNA) expressing an shRNA against GFP (GFPshRNA), CAG-GFP, CAG-DsRed, and the rhodopsin promoter-Cre, and analyzed at P20. Without the Cre construct, transfected retinal cells were clearly labeled by both GFP and DsRed. With the rhodopsin promoter-Cre construct, GFP expression in the ONL was specifically silenced while that in the INL cells was not affected, demonstrating that CALSL-mir30 can be used to conditionally knock down gene expression in the retina. These experiments showed there is a very high cotransfection efficiency. In order for cells to be both red and green, they must have been successfully electroporated with both CAGGFP and CAG-DsRed. The coexpression rate of these two genes was nearly 100%, both in this case, where four plasmids were coelectroporated, and in many previous cases with two or three coelectroporated plasmids (Matsuda and Cepko, 2004). The Cre/loxP recombination-dependent inducible vectors with the CAG promoter have several useful features. First, it is possible to inducibly express genes in specific cell types at specific time points. Second, after Cre/loxPmediated recombination, strong “output signals” driven by the CAG promoter can be obtained. Expression of fluorescent reporter genes directly from cell type–specific promoters frequently results in such low levels of expression as to render the constructs unusable for some applications, such as the detection of live-labeled cells. We found there was a much greater sensitivity for the detection of expression when cell type–specific promoters were used with Cre plus CALNL-DsRed. Finally, these vectors can be used to trace the fate of progenitor and precursor cells labeled upon Cre/loxP-mediated recombination. Using these vectors, we showed that several cell type–specific promoters are weakly active in progenitors and/or other cell types. Two explanations for this observation, which are not mutually

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Figure 51.12 Lineage tracing experiments in the retina using the Cre/loxP system and cell type–specific promoters. P0 rat retinas were coelectroporated with retinal cell type–specific promoter Cre and CALNL-DsRed (recombination indicator). DsRed expression, induced by Cre/loxP-mediated recombination, was driven by the ubiquitous CAG promoter. The retinas were harvested at P20,

sectioned, and stained with DAPI. Promoters of rhodopsin (A), Nrl (B), Cabp5 (C ), Ndrg4 (D), Cralbp (E), clusterin (F), Hes1 (G), and Rax (H) were used to express Cre. Yellow arrowheads indicate the labeled rods. Blue arrowhead indicates the labeled bipolar cell. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. See color plate 60.

exclusive, can be considered. Lineage analyses using retroviral vectors have shown that clones with bipolar, Müller glia, or amacrine cells almost always also contain rods, even if the clone is of only two cells (Turner and Cepko, 1987). If these promoters are weakly active in progenitors that give rise to multiple cell types, then one might see labeling of rods, along with labeling of the cell type that normally is the only cell type to express a particular promoter. This idea is supported by two previous observations. Most of the known Müller glia–specific genes of the adult retina are also expressed in progenitors (Blackshaw et al., 2004), and two genes thought to be restricted to amacrine and horizontal cells could be observed in progenitors (Alexiades and Cepko, 1997). An additional explanation is that these promoters, transiently introduced

into the retina by electroporation, are slightly “leaky,” and such leakiness was detected by the more sensitive reporter (Cre plus CALNL-DsRed).

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Future applications There are many obvious applications for electroporation in studies of the visual system. Standard gain- and loss-offunction protocols are being employed in which a gene is over- or misexpressed or is targeted with RNAi. However, the flexibility offered by the high efficiency of coelectroporation enables many more types of experiments to be performed. For example, the best control for the specificity of an RNAi event is gene rescue; that is, one supplies a version of the endogenous gene that is targeted by RNAi but that

Figure 51.13 Inducible expression in the differentiated Müller glia (A). Clusterin promoter was fused to ERT2CreERT2 cDNA and coelectroporated into P0 rat retinas with CALNL-DsRed (recombination indicator) and CAG-GFP (transfection control). Retinas were stimulated with or without 4-OHT at P14 by IP injection, then harvested at P16. B, Whole-mount preparation of the trans-

fected retina harvested at P16 without 4-OHT stimulation. No DsRed expression was detected. C, Whole-mount preparation of the transfected retina stimulated with 4-OHT at P14 and harvested at P16. D, The retina shown in C was sectioned and stained with DAPI. Only Müller glial cells are labeled with DsRed. See color plate 61.

does not encode the targeted sequence. This is easily done by coelectroporating the plasmid encoding the RNAi and the resistant version of the targeted gene. One can also perform epistasis experiments. For example, one can target a transcription factor by RNAi and simultaneously supply a putative target of that transcription factor whose expression is controlled by a noncognate promoter to look for rescue. One can use the recombinases to turn on expression of either the RNAi or the rescue cassette to perform rescue or knockdown in specific cells or at specific times. Promoter constructs also allow one to mark subsets of cells and follow their migration, and so on. Their physiology can be controlled by the controlled expression of genes that might silence their neuronal activity. Another application of interest to neuroscientists is to track the synaptic partners of an electroporated cell. Since

the transient expression following electroporation can be relatively high level, one can provide enough of a transsynaptic tracer (e.g., WGA) to find the synaptically connected partners of an electroporated cell, which itself can be readily identified by labeling with an electroporation reporter, for example, GFP. In addition, electroporation of Cre plasmids, RNAi plasmids, and the like into mice in which engineering of the germ line has been carried out is being done. For example, rather than create a mouse strain that expresses Cre under control of a specific promoter, one can simply electroporate a Cre plasmid into a mouse that has a germ line with a floxed allele of a gene of interest. Many additional applications will be discovered through the creative use of these protocols and reagents as more neuroscientists discover the ease and flexibility of this system of gene transfer.

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Figure 51.14 Inducible RNAi in the retina. A, CAG-mir30: The mir30 expression cassette is expressed under the control of the CAG promoter. The mir30 expression cassette has the hairpin stem composed of siRNA sense and antisense strands (22nt each), a loop derived from human mir30 (19nt), and 125nt mir30 flanking sequences on both sides of the hairpin. The mir30 primary transcript is processed to generate the mature shRNA. CALSLmir30: Cre-dependent inducible shRNA expression vector carrying a floxed transcriptional stop cassette (3xpolyA signal sequences).

Only in the presence of Cre, the mir30 expression cassette is expressed under the control of the CAG promoter. B and C, Conditional GFP knockdown in the retina. CAG-GFP and CAGDsRed and CALSL-mir30(GFPshRNA) expressing an shRNA against GFP were coelectroporated without (B) or with (C ) the rhodopsin promoter Cre into P0 rat retinas. The retinas were harvested at P20, sectioned, and stained with DAPI. Rod-specific GFP knockdown was observed in the presence of the rhodopsin promoter Cre. See color plate 62.

REFERENCES

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Miyoshi, H., Takahashi, M., Gage, F. H., and Verma, I. M. (1997). Stable and efficient gene transfer into the retina using an HIVbased lentiviral vector. Proc. Natl. Acad. Sci. U.S.A. 94:10319– 10323. Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199. Paddison, P. J., Cleary, M., Silva, J. M., Chang, K., Sheth, N., Sachidanandam, R., and Hannon, G. J. (2004). Cloning of short hairpin RNAs for gene knockdown in mammalian cells. Nat. Methods 1:163–167. Picard, D. (1994). Regulation of protein function through expression of chimaeric proteins. Curr. Opin. Biotechnol. 5:511–515. Price, J., Turner, D., and Cepko, C. (1987). Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc. Natl. Acad. Sci. U.S.A. 84:156–160. Punzo, C. and Cepko, C. L. (2008). Unpublished results. Rapaport, D. H., Wong, L. L., Wood, E. D., Yasumura, D., and LaVail, M. M. (2004). Timing and topography of cell genesis in the rat retina. J. Comp. Neurol. 21:304–324. Saito, T., and Nakatsuji, N. (2001). Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev. Biol. 240:237–246. Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., and Grove, E. A. (2004). Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex. Development 131:5639–5647. Swaroop, A., Xu, J. Z., Pawar, H., Jackson, A., Skolnick, C., and Agarwal, N. (1992). A conserved retina-specific gene encodes a basic motif/leucine zipper domain. Proc. Natl. Acad. Sci. U.S.A. 89:266–270. Tabata, H., and Nakajima, K. (2001). Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex. Neuroscience 103:865–872. Tomita, K., Ishibashi, M., Nakahara, K., Ang, S. L., Nakanishi, S., Guillemot, F., and Kageyama, R. (1996). Mammalian hairy and Enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis. Neuron 16:723–734. Turner, D. L., and Cepko, C. L. (1987). A common progenitor for neurons and glia persists in rat retina late in development. Nature 28:131–136. Turner, D. L., Snyder, E. Y., and Cepko, C. L. (1990). Lineageindependent determination of cell type in the embryonic mouse retina. Neuron 4:833–845. Young, R. W. (1985). Cell differentiation in the retina of the mouse. Anat. Rec. 212:199–205. Zack, D. J., Bennett, J., Wang, Y., Davenport, C., Klaunberg, B., Gearhart, J., and Nathans, J. (1991). Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas. Neuron 6:187–199. Zeng, Y., Wagner, E. J., and Cullen, B. R. (2002). Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells. Mol. Cell 9: 1327–1333.

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52

Genetic Knockouts in Ophthalmic Research and Drug Discovery DENNIS S. RICE

The study of mouse genetics has had a tremendous impact on medical research over the last few decades. Ophthalmology is no exception and has benefited from the mouse, which exhibits significant homology with the human genome (Waterston et al., 2002). In fact, the first scientific publication of an inheritable defect in the central nervous system (CNS) in mice was rodless (subsequently shown to be a mutation in the β subunit of cGMP phosphodiesterase). Mutations in the human homologue result in retinitis pigmentosa and blindness. More recently, knockout strains have been used to predict drug actions in humans (Zambrowicz et al., 2003). Phenotypic screens are used to examine mouse knockouts for physiological parameters that are beneficial in the clinic. This chapter provides a general introduction to the importance of mouse genetics in understanding ocular development and the physiology of particular cell types and highlights several examples of significant contributions by mouse models to the discovery and development of new therapies in human ophthalmic disease.

Cell types in the retina The neural retina and the retinal pigmented epithelium (RPE) are derived from neuroectodermal precursors in the optic vesicle, which emerges from the ventral diencephalon (Mann, 1964). The vesicle invaginates, forming the optic cup, in which the inner layer becomes the neural retina and the outer layer becomes the RPE. The neural retina in mice is organized into three cellular layers separated by plexiform layers that contain synaptic connections among retinal neurons. The laminar organization of the mouse retina was described in a monograph entitled The Structure of the Retina, first published in 1892 by the legendary anatomist, Santiago Ramón y Cajal (1972). Relying primarily on the Golgi method to label cellular elements in detail, Ramón y Cajal described the major cell types of the mouse retina. This description of retinal cytoarchitecture established the foundation for future studies aimed at understanding anatomical and physiological relationships in the vertebrate retina. In routine histological sections, the mouse retina appears relatively simple and neatly organized into three cellular layers (figure 52.1A). Cells within these layers can be grouped

into neuronal or glial cell types. Within a given general cell type, there are often multiple morphologies and neurochemical signatures that expand the list to 50–60 different cell types (Haverkamp and Wässle, 2000). The ganglion cell layer (GCL) in mice contains one or two rows of cells, depending on the central or peripheral location of the field in view (figure 52.1B). The GCL is populated by ganglion cells, the sole projection neurons of the retina that transmit signals through the optic nerve to the central visual nuclei. The GCL also contains retinal astrocytes, displaced amacrine cells, and cells that make up the superficial retinal vasculature, endothelial cells and mural cells. The inner nuclear layer (INL) is located in the neural retina and is five to seven cell layers thick, depending on the retinal locale in view (see figure 52.1A). The INL is home to a diverse population of retinal cells, such as amacrine cells, Müller glial cells, bipolar neurons, and horizontal cells (figure 52.1B–D). The outer nuclear layer (ONL) contains rod and cone photoreceptors that detect illumination gradients (see figure 52.1A). Approximately 10–15 rows of nuclei populate the ONL, with rods far outnumbering cones in this nocturnal species (Carter-Dawson and LaVail, 1979). With few exceptions, cone cell nuclei are located at the periphery of the ONL (figure 52.2A). Photoreceptors are further specialized into outer and inner segments. These specializations are easily observed in routine sections. Inner segments enable the synthesis and transport of proteins destined for the outer segments. The outer segments contain stacked discs of membranes that capture photons and convert this energy to chemical messengers that travel back toward the INL to ultimately affect synaptic transmission of RGCs. Cones account for approximately 2%–3% of the photoreceptor population in mice (Carter-Dawson and LaVail, 1979; Jeon et al., 1998). Figure 52.2B shows a reliable histological marker for cone photoreceptors, the lectin peanut agglutinin (PNA), which labels the interphotoreceptor matrix associated with cone but not rod photoreceptors ( Johnson et al., 1986). Although mice lack a central specialization observed in other mammals, the density of cones (approximately 3,500 cones/mm2) is comparable to the approximately 3,000 cones/mm2 observed in the parafoveal primate retina (Curcio et al., 1990; Jeon et al., 1998). Rod

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Figure 52.1 Anatomy and cell types in the adult mouse retina. A, The neural retina contains three cellular layers known as the ganglion cell layer (GCL), inner nuclear layer (INL), and outer nuclear layer (ONL). Two synaptic layers connect cells in the nuclear layers, the inner plexiform layer (IPL) and the outer plexiform layer (OPL). The retinal pigmented epithelium (RPE) contains melanin and protects and supports the photoreceptors in the ONL. The choriocapillaris (CC) is also pigmented and provides vascular supply to the RPE. The sclera (Sc) supports the outer eye. B, Higher magnification view of the inner retina. Ganglion cells (gc) are located in the GCL. Amacrine cells (ac) reside in the inner

portion of the INL and stain much less intensely than the neighboring Müller glia (mg), located in the middle region of the INL. Bipolar cells (bc) are located on the outer portion of the INL (asterisks indicate capillaries). C, Many immunohistochemical markers can be used to identify individual cell types in the mouse retina. Anti-PKCα antibodies recognize the rod bipolar cell bodies (arrows) and their synaptic terminals (asterisks) in the IPL, near the GCL. D, Anticalbindin recognizes horizontal cells (arrows) in the outer portion of the INL. For a detailed list of molecular reagents to study individual cell types in the mouse retina, the reader should consult Haverkamp and Wässle (2000).

Figure 52.2 Anatomy of the outer retina. A, Phototransduction occurs in photoreceptors located in the outer nuclear layer (ONL). Cone photoreceptors are easily distinguished from the much more numerous rod photoreceptors by their heterochromatin (arrow) and location within the ONL, which is close to the inner segments (is). Outer segments (os) contain the light-sensitive pigments that capture photons. Their tips are engulfed by apical microvilli (mv)

that extend from the retinal pigmented epithelium (RPE). The section in this image was obtained from an albino mouse that lacked pigmentation in the RPE or choriocapillaris (CC). B, The PNA-lectin conjugated to FITC is an excellent marker to visualize cone outer segments and inner segments in the mouse retina. C, Rods can be visualized with antirhodopsin antibodies.

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photoreceptor density averages 437,000 cells/mm2 and therefore represents approximately 97% of total photoreceptors. Rods can be identified with a variety of antibodies; antirhodopsin is one of the most commonly used (figure 52.2C ). The first synaptic relay in light sensation occurs at the level of the outer plexiform layer (OPL) and involves the transmission between rod photoreceptors and rod bipolar cells during low light or nocturnal conditions, and between cones and cone bipolar cells under photopic or bright-light conditions. Rod bipolar cells are routinely visualized with anti-PKCα antibodies (see figure 52.1C). These synaptic connections mark the interface between the ONL and the INL. Unlike other neuronal cell types in the INL, horizontal cells are defined by a single morphology, the axon-bearing horizontal cell. Calbindin immunoreactivity (see figure 52.1D) is an excellent marker for horizontal cells in the mouse retina (Haverkamp and Wässle, 2000). Rod photoreceptors establish contact at the axon terminal system and cone photoreceptors provide synaptic input to the horizontal cell dendrite. Horizontal cells provide reciprocal communication to photoreceptors and input to bipolar cells. Cone and rod bipolar cells relay visual stimuli from photoreceptors to a synaptic-rich region known as the inner plexiform layer (IPL), which contains connections formed among bipolar, amacrine, and ganglion cells (see figure 52.1A and B).

The retinal pigmented epithelium Photoreceptors are intimately associated with an adjacent layer of cells in the outer retina known as the retinal pigmented epithelium. This epithelium is derived from neuroectoderm that also differentiates into the neural retina (Strauss, 2005). Thus, both neural retina and RPE are lateral extensions of the CNS. RPE cells appear cuboidal in cross section and hexagonal when viewed in whole-mount preparations. The RPE is one of the most intriguing cell types in the eye because of its location and functions within the retina proper. RPE cells are highly polarized, exhibiting a basal side that faces Bruch’s membrane and an apical side that extends microvilli to surround the adjacent photoreceptor outer segments (see figure 52.2A). As the name implies, this epithelium contains numerous melanin granules, which give the cell a dark brown appearance in routine histological preparations (see figure 52.1A). The RPE is the first cell type in the body to express pigmentation, which is visible in the dorsal eye cup at approximately embryonic day 11.5 (E11.5; Strongin and Guillery, 1981). The basal side of the RPE is characterized by numerous infoldings that increase the surface area of the RPE membrane and facilitate transport and secretion (Strauss, 2005). These general characteristics of the RPE cell provide an adequate preamble to the important role this cell type serves in retinal development, physiology, and disease.

The RPE is positioned between the highly vascular choriocapillaris and the avascular photoreceptor layer of the neural retina (see figure 52.2A). Tight junctions that are present between apical surfaces of adjacent RPE cells establish the blood-retinal barrier at this location, while the endothelium is responsible for the barrier within the neural retina. Insofar as the RPE represents a barrier in the retina, one major function of this cell type is to transport water, electrolytes, and metabolic waste from the subretinal space to the choriocapillaris and import nutrients such as glucose and retinol from the systemic circulation (Strauss, 2005). Retinol (vitamin A) is important in visual sensation because it is a major source of 11-cis-retinaldehyde, the chromophore that binds opsins in photoreceptors. Light isomerizes 11-cisretinal to all-trans-retinaldehyde, which initiates the signal transduction cascade and photoreceptor membrane hyperpolarization. The 11-cis-retinaldehyde–opsin receptor complex is restored in a process known as the retinoid visual cycle, and the RPE plays an important role in this process (Dowling, 1960). A number of genes identified in humans and mice have been shown to encode critical components of the retinoid visual cycle, and mutations in these genes result in poor visual sensitivity and photoreceptor degeneration (Besch et al., 2003). Recent studies in mice have suggested that modulation of the retinoid cycle may be a new therapeutic strategy to treat RPE and retinal diseases. For example, the RPE65 gene encodes a protein of 533 amino acids that is required for conversion of all-trans-retinyl esters to 11-cisretinol in the RPE (Redmond et al., 1998). Mutations that affect RPE65 activity result in Leber congenital amaurosis in humans, which is characterized by a severe, early-onset retinal dystrophy (Gu et al., 1997; Marlhens et al., 1997; Redmond et al., 2005). The RPE65 protein is specifically expressed in RPE cells, and knockout of RPE65 in mice results in very low levels of 11-cis-retinal and its esters, while trans-retinyl esters accumulate in the RPE (Bavik et al., 1992; Redmond et al., 1998). Electroretinograms (ERGs) are severely attenuated, despite the presence of photoreceptors and outer segments (Redmond et al., 1998; Pang et al., 2005). The most obvious pathology is the shortening of outer segments in mice lacking RPE65, confirming that proteins that are intrinsic to RPE cells can often affect neighboring photoreceptors. Photoreceptor cell loss occurs in the absence of RPE65, but this event is likely secondary to biochemical changes in the visual cycle (Woodruff et al., 2003). Oral administration of 9-cis-retinal in mice lacking RPE65 led to recovery of light sensitivity and rod function (Van Hooser et al., 2000). Administration of 9-cis-retinoid also restored visual pigment and retinal function in mice lacking the enzyme lecithin:retinol acyltransferase (LRAT), which is critical for the esterification of all-trans-retinol (Batten et al., 2005).

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Mutations in LRAT result in early-onset retinal dystrophy in humans (Thompson et al., 2001). The product of LRAT activity is a substrate for RPE65, which produces 11-cisretinol that is further oxidized to 11-cis-retinal in the RPE (Maeda et al., 2006a). Recently, functional recovery of photoreceptors was observed following 9-cis-retinal treatment in mice lacking the oxidative enzymes required for the final step in the reconversion of the bleached chromophore (Maeda et al., 2006b). These studies suggest that improvement in photoreceptor sensitivity is possible regardless of the primary genetic defect affecting the RPE biochemical synthetic pathway for 11-cis-retinal. A major function of the RPE cells is to phagocytose photoreceptor outer segments, which are shed daily by the photoreceptors (Young and Bok, 1969). As a result, proteins that function in photoreceptors also affect neighboring RPE cells. ABCA4 is a member of the ATP-binding cassette transporter family and is localized in the outer segments of rod and cone photoreceptors (Cideciyan et al., 2004). Mutations in the ABCA4 gene are responsible for a number of photoreceptor degenerations in humans, such as Stargardt disease, atypical retinitis pigmentosa, and cone-rod dystrophy (Allikmets, 2000). Stargardt disease shares pathological features with AMD and is associated with accumulation of lipofuscin pigments in RPE cells and loss of central vision (Cideciyan et al., 2004). Mice lacking ABCA4, also known as rim protein (RmP), exhibit delayed dark adaptation with relatively normal retinal anatomy and photoreceptor sensitivity (Weng et al., 1999; Radu et al., 2003). Most important, these mice exhibit a dramatic accumulation of N-retinylidene-N-retinylethanolamine (A2E), the major fluorophore in lipofuscin that accumulates in RPE cells (Weng et al., 1999). Biophysical studies using recombinant protein and analysis of ABCA4 knockout mice suggest that ABCA4 functions as a flippase for N-retinylidene-phosphatidylethanolamine (Nret-PE), the Schiff-base conjugate of all-trans-retinal and phosphatidylethanolamine. Hydrolysis of N-ret-PE in the photoreceptor cytoplasm releases all-trans-retinaldehyde, which is reduced to all-trans-retinol, which enters the RPE and is subsequently esterified by LRAT (Weng et al., 1999; McBee et al., 2001; Maeda et al., 2006b). In the absence of flippase activity, the N-ret-PE is thought to accumulate in the inner disc compartment. The models predict that following phagocytosis, outer segments containing high levels of the N-ret-PE adduct enter the phagolysosomal pathway in the RPE, which promotes the formation of A2E in lipofuscin upon light exposure. Accumulation of lipofuscin in the ABCA4 knockout is blocked when mice are reared in total darkness, confirming the light cycle dependency of this pathobiology (Mata et al., 2000). Mutations in a different photoreceptor-specific protein known as elongation of very long chain fatty acids (ELOVL4) result in Stargardt disease in humans (Zhang et al., 2001).

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Knockout of ELOVL4 in mice results in a lethal phenotype, while heterozygous mice exhibit very mild morphological defects in the retina (Raz-Prag et al., 2006). Mice genetically engineered to overexpress the human mutation in ELOVL4 exhibit lipofuscin accumulation in the RPE and progressive photoreceptor degeneration (Karan et al., 2005; Vasireddy et al., 2006). Although more work needs to be done to establish the biochemical activity of this gene, this evidence suggests that factors involved in long chain polyunsaturated fatty acid content in photoreceptors could play a significant role in the propensity for lipofuscin accumulation and retinal degeneration. Lipofuscin appears in the RPE during senescence as a by-product of the retinoid visual cycle and phagocytosis of the outer segments (Dorey et al., 1989). Studies in the ABCA4 and ELOVL4 genetic models implicate phospholipid biosynthesis and appropriate processing of retinoid cycle by-products as key cellular events that affect accumulation of lipofuscin in the RPE. High levels of lipofuscin and A2E in RPE cells are toxic to cells and may contribute to degenerations observed in retinal disease patients (Sparrow et al., 2000; Suter et al., 2000). Recently, the photooxidation products of A2E have been shown to activate the complement cascade in vitro (Zhou et al., 2006). Strong genetic associations with AMD have been described recently for complement factor alleles in humans (Edwards et al., 2005; Haines et al., 2005; Klein et al., 2005). The light dependency of lipofuscin accumulation, along with other environmental factors, suggests that modulation of the visual cycle and complement cascade are potential therapeutic strategies for macular degeneration. Recent studies have shown that small molecule antagonists of the visual cycle protect against light-induced degeneration and lipofuscin accumulation in disease models (Maiti et al., 2006). This pharmacological approach recapitulated the phenotype in the RPE65 knockout mice, which exhibited a reduction in lipofuscin during senescence (Katz and Redmond, 2001). Moreover, mice lacking RPE65 are resistant to the light-induced degeneration challenge assay (Grimm et al., 2000). Importantly, inhibiting the visual cycle protects against light-induced degeneration and attenuates lipofuscin accumulation in the ABCR knockout mouse as well (Sieving et al., 2001; Radu et al., 2003, 2005; Maiti et al., 2006). Most knockouts are maintained on a mixed genetic background between 129/SvJ and C57BL/6J. These two inbred strains are homozygous for RPE65 alleles that render mice either susceptible (Leu450) or resistant (Met450) to lightinduced degeneration, most likely as a result of altered rhodopsin regeneration kinetics (Danciger et al., 2000; Kim et al., 2004). Indeed, the B6;129 F2 hybrid background shows different sensitivity to light damage when the RPE65 alleles are compared (figure 52.3). The ability to combine

different alleles of RPE65 in mice also harboring targeted mutations in other genes can reveal molecular modulators in the retinoid visual cycle. Collectively, these studies highlight the important interactions that occur between photoreceptors and RPE cells, and they identify new strategies to modulate human retinal diseases associated with lipofuscin accumulation and deficiencies in the visual cycle. The intricate relationship that exists between the RPE and adjacent photoreceptors is critical to maintain normal visual function. The RPE is also important during retinal development (Strauss, 2005). Detailed studies of retinal development in hypopigmentation mutants have demonstrated that RGCs that normally project to ipsilateral central visual targets instead misroute at the optic chiasm and cross the midline, inappropriately reaching the contralateral central nuclei (LaVail et al., 1978; Dräger and Olsen, 1980; Jeffery, 1997). There is a delay in retinal development and rod number is decreased in albinos lacking functional tyrosinase, the key enzyme in the synthesis of melanin (Ilia and Jeffery, 1999). These studies implied that the enzymatic product of tyrosinase, which could be melanin, controls important aspects of retinal development. Enzyme replacement therapy or product supplementation, as demonstrated in the retinoid cycle, is a viable

Room Light

strategy and mouse models have provided examples of proof of concept–type experiments. For example, during melanin synthesis, tyrosinase oxidizes L-tyrosine to L-dopaquinone, which is further converted to melanin. One product of this enzymatic reaction is L-3,4-dihydroxyphenylalanine (Ldopa). Transgenic mice that express tyrosine hydroxylase in the RPE rescue the retinal abnormalities in albino mice (Lavado et al., 2006). This result was attributed to direct action of L-dopa during retinal development as the transgene was expressed in mice lacking functional tyrosinase and, therefore, could not convert L-dopa to melanin. This clever genetic experiment demonstrates that either L-dopa or a metabolite is critical for normal retinal development and that supplementation could rescue defects that arise due to a deficiency in tyrosinase (Ilia and Jeffery, 1999).

Vascular cell types Cells in the inner retina are nourished by three vascular beds that develop during the first 2 postnatal weeks in mice. Cellular regulation of vascular retinal development in mice remains one of the most intensely studied biological questions, owing to the accessibility of the developing vasculature in the postnatal eye. All aspects of angiogenesis occur in this organ, and the increasing availability of mouse genetic

Light-Induced Degeneration

ONL/INL

1.5

1.0 0.5

0.0 A

B-Leu450

Figure 52.3 Light-induced degeneration in 129;B6 mice that are homozygous for either the Leu450 allele or Met450 allele of the RPE65 gene. Each dot represents the mean thickness of the ONL/ INL from measurements obtained 400 μm on either side of the optic nerve head. A, Mice exposed to room light (40 lux) exhibit an ONL : INL ratio greater than 1.0 (n = 8). B, Mice homozygous

C-Met450

for the Leu450 allele in the RPE65 gene are sensitive to 5 hours of light damage (5,000 lux) as shown in this histological section (n = 6). Sections were generated 7 days post light exposure. C, Mice (n = 7) homozygous for the Met450 allele in the RPE65 gene are resistant to light damage.

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models and the ability to apply agents directly to the postnatal eye have enabled detailed studies of cell types and mechanisms involved in physiological angiogenesis. Pathological angiogenesis contributes to diseases in humans such as cancer and is a serious threat to vision in “wet” AMD and diabetic retinopathy (Folkman, 1995). Therefore, studies of the mouse intraocular vasculature have provided some of the most exciting insights into cellular regulation of blood vessel development, maintenance, and pathobiology. The process of vascularization in the mouse eye is well described, and several excellent reviews are available (Fruttiger, 2002; Dorrell and Friedlander, 2006). Before birth, the developing neural retina is avascular, although the hyloid vasculature nourishes the developing lens and surrounding tissues. As neuronal differentiation proceeds, increasing metabolic activity of the inner retina results in physiological hypoxia that is thought to initiate formation of the retinal vasculature (Zhang et al., 1999, 2003). The first vascular bed arises during postnatal days P1–P10 and is known as the superficial vascular plexus, which supports ganglion cells (Connolly et al., 1988). During the second postnatal week, the superficial vessels branch to descend toward the outer retina, where they elaborate into a second vascular plexus, which nourishes cells located in the distal INL. During the second and third postnatal weeks, a third vascular plexus forms through angiogenic sprouting at the level of the innermost region of the INL (figure 52.4). The orderly appearance of retinal vasculature during the first 3 postnatal weeks is orchestrated by specific cell types and guidance cues. Evidence obtained in a number of species, including mice, indicates that the initial vascular plexus arises through the directed migration of endothelial cells along a preassembled grid of astrocytes (Dorrell and Friedlander, 2006). These astrocytes originate from the optic stalk and migrate radially prior to the arrival of endothelial cells at the optic nerve head. Genetic models have been instrumental in elucidating molecular details of these complex cellular interactions. For example, mice lacking platelet-derived growth factor-A (PDGF-A) exhibit a decrease in the complexity of the astrocytic grid, and consequently the coverage of the superficial vascular plexus is also decreased (Gerhardt et al., 2003). PDGF-A is expressed by RGCs and interacts with its receptor, PDGFR-α, present on astrocytes (Mudhar et al., 1993). Transgenic mice that overexpress PDGF under the control of the neuron-specific enolase promoter exhibit overgrowth of retinal vasculature due to increased density of astrocytes. Conversely, blocking PDGF-A function attenuates retinal vascular development by perturbing the complexity of the astrocyte network (Fruttiger et al., 1996). The cell adhesion molecule Rcadherin also appears to be important for the astrocytemediated guidance of the superficial vascular plexus (Dorrell

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et al., 2002). Mice deficient in the nuclear hormone receptor Tlx exhibit impaired astrocyte development and loss of Rcadherin expression, with concomitant disruption in vascular development, further confirming the intimate association between astrocytes and the developing capillaries (Miyawaki et al., 2004). Astrocytes continue their important role in vascular biology in adult retina by enabling the formation of the blood-retina barrier (Janzer and Raff, 1987). The relative hypoxia of the newborn retina is believed to stimulate expression of various pro-angiogenic factors that drive endothelial cell proliferation and migration (Stone et al., 1995; Gerhardt and Betsholtz, 2005; Dorrell and Friedlander, 2006). VEGF-A is among one of the best characterized of these pro-angiogenic factors, and alternative splicing of Vegf mRNA generates multiple isoforms that have different diffusion capabilities within the extracellular matrix (Ferrara et al., 2003). VEGF is produced by astrocytes prior to the arrival of endothelial cells (Stone et al., 1995; Gariano, 2003; Gerhardt et al., 2003). At the leading edge of the developing capillaries, specialized endothelial cells termed tip cells extend and retract actin-rich filopodia along the glial cell network and the surrounding matrix (Dorrell et al., 2002; Gerhardt et al., 2003). These endothelial tip cell pioneers are followed closely by endothelial cells in the stalk, which are proliferating to form a patent capillary network superimposed on the astrocytic grid (Gariano, 2003; Gerhardt and Betsholtz, 2005). Studies in mice designed to express individual isoforms of VEGF-A demonstrate that tip cell migration and normal vascular patterning in the developing retina and brain are dependent on the establishment of tightly regulated VEGF-A gradients (Ruhrberg et al., 2002; Stalmans et al., 2002; Gerhardt et al., 2003). Moreover, VEGF-A signaling through VEGF receptor 2 (VEGFR2) affects simultaneously tip cell migration and stalk cell proliferation, and the cellular mechanism for this effect remains an area of intense research (Gerhardt et al., 2003; Gerhardt and Betsholtz, 2005). VEGFR2 protein is highly expressed in tip cells, and antagonism of VEGFR2 signaling retracts tip cell filopodia, providing further support that VEGF-A is involved in the guided migration of tip cells. Genetic knockout studies targeting a single allele of VEGF-A in mice result in lethality between E11 and E12. The heterozygous embryos displayed defective vascularization in several organs, confirming the essential role of VEGF in blood vessel formation (Carmeliet et al., 1996; Ferrara et al., 1996). Moreover, knockout of the VEGFR2 results in embryonic lethality around day 9 and is characterized by failed angiogenesis and hematopoiesis, along with disorganized blood vessels (Shalaby et al., 1995). These studies established a clear role for VEGF-A signaling in physiological angiogenesis. The clinical utility of VEGF in pathological angiogenesis is evidenced as VEGF-

Figure 52.4 The retinal vasculature in mice contains three vessel beds. A, Whole-mount retinal preparation from a wild-type mouse stained with isolectin B4 conjugated to fluorescein. The retinal vasculature covers the entire retinal surface. B–D, Images taken at different depths through the retinal vasculature, starting at the most superficial layer, the nerve fiber layer (NFL [B]), which provides capillaries to the inner plexiform layer (IPL [C]), which are continuous with those capillaries in the outer plexiform layer (OPL [D]). E, Retinal whole mount obtained from a P6 mouse and stained

with isolectin B4. The optic nerve head is at the lower left of the image, and the retinal periphery is in the upper right. The vasculature is developing toward the retinal periphery through the movement of the migration front (arrows). F, A higher magnification image of the migration front shows pioneer endothelial cells, termed tip cells, that direct the migration of the vasculature by extending actinrich filipodia. Endothelial cells in the stalk form the nascent capillary bed.

blocking antibodies are now approved for wet AMD and colorectal cancer. Of note, the phenotypes observed in the heterozygous knockout of VEGF predicted the clinical efficacy of VEGF inhibition and provide a benchmark of the embryonic lethal phenotypes in mouse knockouts as a reliable indicator for potentially very potent modulators of vascular development and homeostasis. The endothelial network, initially sculpted by astrocytes, is further remodeled into the mature vasculature that exhibits a stereotypical pattern. This process involves vascular

pruning to establish a mature vessel density concurrent with the establishment of hemodynamic flow and increased oxygen levels in perfused tissue (Alon et al., 1995; Benjamin et al., 1998). Stabilization into a functional vessel requires intimate association with mural cells, such as pericytes and smooth muscle cells (Fischer et al., 2006). The dynamic interactions that occur between pericytes and endothelial cells are little understood at the molecular level, and studies using mouse genetic models are making inroads into this complex interaction among these cell types ( Jain, 2003;

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Armulik et al., 2005). The clinical significance of this question is great, as loss of pericytes and the appearance of microaneurysms are early hallmarks of retinopathy in diabetic patients (Speiser et al., 1968). Continued vessel occlusion results in increased vascular permeability, edema, and the formation of new vessels that proliferate into the vitreous. Pericyte recruitment lags behind vessel formation (Benjamin et al., 1998). Pericyte coverage progresses from arterioles to venules, and the process does not reach completion before 3 weeks from birth. Remodeling of retinal vessels is complete before the establishment of pericyte coverage, indicating that mural cell investment marks the end of vascular remodeling capacity. Several gene knockout studies have shown that mural cells affect blood vessel branching, remodeling, and stabilization. For example, mice with a targeted deletion in either angiopoietin-1 or its receptor Tie2 exhibit defects in angiogenesis and vessel remodeling and maturation (Sato et al., 1995; Suri et al., 1996). Loss of pericytes and smooth muscle cells is evident in these animals. Most of the evidence suggests that these defects arise via an intrinsic block in signaling within endothelial cells, which specifically express Tie2, although alternative models have been suggested (Armulik et al., 2005). Endothelial tip cells secrete platelet-derived growth factor-B (PDGF-B), presumably in response to VEGF signaling (Lindahl et al., 1997; Dorrell and Friedlander, 2006). Mice deficient in PDGF-B are embryonic lethal and exhibit attenuation in pericyte proliferation and decreased vessel coverage. Vascular hemorrhaging, presumably as a result of vessels that are not fully invested by pericytes, is a common pathology observed in these mice. Pericyte loss, microvascular aneurysms, and lethality occur in mice lacking the PDGF-B receptor, which is expressed on mural cells (Benjamin et al., 1998; Hellstrom et al., 2001). In the absence of appropriate pericyte coverage, endothelial cells proliferate and become permeable, leading to impaired perfusion and hypoxia. These highlighted examples, and many others identified through mouse genetic studies, demonstrate a reciprocal cell-cell communication in which endothelial cells promote mural cell proliferation and migration and mural cells help support and stabilize endothelial cells (Hellstrom et al., 1999; Armulik et al., 2005). Retinopathy of prematurity in premature infants is a pathogenesis of retinal blood vessels that arises as a result of high oxygen exposure to compensate for the underdeveloped lungs. Exposure of developing retinal vasculature to high oxygen decreases VEGF expression, leading to dropout of newly formed capillaries (Alon et al., 1995; Benjamin et al., 1998). Upon return to room air (normoxic conditions), VEGF is upregulated and neovascularization occurs. Pericyte investment is thought to be important for the ability of endothelial cells to respond to increasing oxygen tension.

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Fully invested capillaries, as is the case in mature vasculature, are largely refractory to the oxygen-induced vasobliteration (Alon et al., 1995; Benjamin et al., 1998). Novel strategies aimed at modulating cellular interactions between pericytes and endothelial cells are likely to emerge, given their important role in establishing and maintaining vascular integrity. Additional guidance cues have been discovered through knockout mice that act to repel and keep tip cell endothelial pioneers on track toward formation of the complex vascular network. Using a knock-in reporter gene to monitor Unc5B expression, Lu and colleagues demonstrated high levels of Unc5B in endothelial tip cells in the retina and arteriole endothelial cells in more mature vessels (Lu et al., 2004). Unc5 proteins are membrane-bound receptors for axon guidance molecules known as netrins. Depending on the genetic background, homozygous Unc5B deficient mice were lethal by E12.5, exhibiting severe vascular phenotypes. Exuberant branching of vessels was observed in several organs, and endothelial tip cells demonstrated increased filopodial extensions in mice lacking Unc5B. Vascular patterning arises through cellular interactions with both attractive and repulsive cues, which is reminiscent of guidance mechanisms that control the trajectories of neuronal projections during CNS development. Additional molecules involved in axon guidance have been shown to be involved in vascular patterning through gene knockout studies in mice. This list includes plexins, semaphorins, neuropilins, ephrins, and their receptors (Carmeliet and Tessier-Lavigne, 2005; Eichmann et al., 2005). The discovery of tip cells in endothelium that closely resemble growth cones of developing axons suggests that mechanisms acting during axonal guidance may directly apply to those controlling blood vessel patterning. Conversely, manipulations using cellular or molecular strategies in the relatively simple architecture of mouse retinal vasculature may yield insights into mechanisms that could be applied to promote reestablishment of neuronal connections in neurodegenerative conditions affecting the eye, such as glaucoma. The availability of large numbers of genetic knockouts, combined with reliable markers to identify individual cell types, will continue to illustrate the power of mouse models to reveal additional cell types and molecules that affect both physiological and pathological angiogenesis (Jain, 2003). Many gene deletions in mice result in prenatal lethality or reduced viability as a consequence of blood vessel defects. In viable animals, genes affecting blood vessel formation and vascular integrity can be identified using noninvasive imaging techniques, such as angiography. For example, disruption of the endothelial cell–specific protein EGFL7 results in abnormal ocular blood vessel anatomy in some adult mice (figure 52.5A). EGFL7 is a secreted protein

Figure 52.5 EGFL7 affects vascular anatomy. A, Angiography of wild-type mice (control) and mice lacking the secreted protein EGFL7 (−/−). In control eyes, blood vessels project directly toward the retinal periphery, while branches dive down toward the deeper levels of the retina to form the capillary beds. In the EGFL7−/− retina, vessels often meander on their way toward the retinal periphery. B–E, In situ hybridization of Egfl7 in the postnatal mouse eye. B, At P3, Egfl7 expression in endothelial cells is present in the migrating front of the developing capillaries (arrow). Hyloid

vessels and the choriocapillaris surround the eye, and iris vessels also express Egfl7. C, No signal in the P3 eye is observed when an Egfl7 sense probe is used in the hybridization. D, At P6, the developing vasculature approaches the retinal periphery and Eglf7 is present in these developing endothelial cells (arrows). E, Higher magnification of the P6 retina shown in D. Egfl7 expression is present on the nerve fiber layer and the choriocapillaris (arrows), consistent with a developing retinal vasculature in the mouse eye.

that is highly expressed during embryonic and neonatal development (figure 52.5B–E). Expression levels decrease in mature vasculature, but Egfl7 expression is upregulated in vascular injury and in tumor models (Parker et al., 2004; Campagnolo et al., 2005). The abnormal vasculature ob-

served in the eye of adult animals is due to a developmental defect in endothelial cell migration. Therefore, genetic screens in mice using embryonic lethality or noninvasive imaging in viable mice will lead to the identification of new targets for pathological angiogenesis.

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Cell types in the anterior chamber Glaucoma is a disease that affects RGCs, amacrine cells, and the optic nerve. This sight-threatening condition is often associated with increased ocular pressure, a major risk factor for glaucomatous disease. Regulation of intraocular pressure (IOP) is a balance between the production of aqueous humor at the ciliary body and drainage of aqueous humor at the iridocorneal angle (Civan and Macknight, 2004). Aqueous humor is produced by cells in the ciliary body that are nourished by a rich vascular supply. The humor travels through the pupil into the anterior chamber, where it encounters a series of cell types derived from the neural crest and mesoderm (Gould et al., 2004). These cells collectively form the drainage structures known as the conventional and uveoscleral (sometimes referred to as the nonconventional) outflow pathways. Traditionally, the conventional outflow pathway is thought to be dependent on pressure and involves cell types present in the trabecular meshwork, Schlemm’s canal, and episcleral veins (Crowston and Weinreb, 2005). Aqueous humor is also drained through the uveoscleral pathway, which is a route through extracellular spaces in the iris, ciliary muscle, choroid, and sclera, where it ultimately collects in the lymphatics. The facility of the uveoscleral outflow remains an area of intense investigation (Crowston and Weinreb, 2005). Although cell types involved in fluid dynamics have been well described, the molecular mechanisms involved in this process are only now being discovered. This exciting area of research is made possible by the development of several methods to measure IOP, determine the aqueous humor flow and episcleral venous pressure, and calculate total outflow facility in mice (John et al., 1998; Aihara et al., 2003a, 2003b; Crowston et al., 2004a). Mice have IOPs that are consistent with those in normotensive humans (Savinova et al., 2001). The dynamics of humor production and turnover are also comparable to those in humans (Aihara et al., 2003a), and mice respond to pharmacological agents that affect human aqueous humor production and outflow facility (Avila et al., 2001a, 2002; Aihara et al., 2002; Husain et al., 2006). These characteristics are leading to great interest in using mouse models to study processes associated with regulation of IOP and glaucoma. Prostaglandin analogues are among the most widely prescribed drugs to manage ocular hypertension and glaucoma in the clinic. The mechanism by which these analogues lower IOP is still under investigation, but evidence suggests that IOP reductions occur by increasing outflow facility (Weinreb et al., 2002; Crowston and Weinreb, 2005). Prostaglandin analogues have been tested in a number of species, and a single topical application in mice lowers IOP within 6 hours (Aihara et al., 2002; Ota, Murata, et al., 2005; Husain et al., 2006). The intraocular metabolites of these

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analogues bind with high affinity to the FP receptor expressed primarily in cell types associated with aqueous production and flow (Anthony et al., 2001; Weinreb et al., 2002). Mice deficient in several of the prostanoid receptors have been studied to clarify the role of individual receptors involved in IOP effects of these hypotensive agents (Crowston et al., 2004b, 2005; Ota, Aihara, et al., 2005; Ota et al., 2006a, 2006b). A single application of latanoprost, travaprost, bimatoprost, or unoprostone to wild-type mice or mice deficient in the EP1 or EP2 receptors was effective at lowering IOP, indicating that EP receptors are not involved in the pharmacological actions of these analogues. In contrast, when these agents were applied to mice lacking the FP receptor, their ocular hypotensive action was no longer observed. These results clearly demonstrate that the FP receptor is required during the initial IOP-lowering effects of these clinical agents. In the previous example, genetic knockouts provided the ability to determine the molecular target of clinically important compounds. Application of pharmacological agents to mice that lack specific genes also enables the direct test of on- versus off-target side effects (Zambrowicz et al., 2003). However, the phenotype of the knockout is also capable of predicting the potential utility of modulators for clinical applications. For example, several secreted and membrane proteins, such as type 1 collagen, aquaporin 1 and aquaporin 4 (AQP1 and AQP4, respectively), and the adenosine receptor 3 (AR3) have been shown to affect IOP in knockout mice. Mice deficient in type 1 collagen exhibited elevated IOP, while knockout of AQP1, AQP4, or AR3 resulted in lower IOP (Avila et al., 2001b; Zhang et al., 2002; Aihara et al., 2003c). The few studies that have focused on cellular mechanisms that control aqueous humor facility have concluded that mice appear to be excellent models in which to study the dynamics of pressure regulation (Lindsey and Weinreb, 2005). Combined with the increasing number of mouse genetic knockouts and new techniques amenable to the small mouse eye, the future looks very bright for new therapies aimed at specific cell types in the outflow pathway. Ocular cell types are derived from the neural ectoderm, surface ectoderm, and mesenchymal and neural crest. Mouse genetics have played a leading role in elucidating cell migrations and communications that form and maintain the complexity and function of this organ. Therapeutic strategies under investigation or in clinical practice have established proof of concept in genetic models, and the predictive efficacy of the therapeutic was revealed through studies of gene knockouts. Mouse knockouts in challenge assays for pathological stresses, such as ischemia, ocular inflammation, and dry eye, can reveal individual genes with important functions in disease mechanisms (Da and Verkman, 2004; Pflugfelder et al., 2005; Liao et al., 2006). Mouse genetics

will continue to be the core technology to study cell-cell interactions and physiological responses in mammalian biology and contribute to the next generation of therapeutics aimed at treating ocular disease. acknowledgments

The author thanks Kim Paes (Lexicon), Claire Gelfman (Lexicon), Weilan Ye (Genentech), and Maike Schmidt (Genentech) for scientific contributions to this chapter and Alex Turner for critical review of the manuscript.

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Beyond Positional Cloning of Single Gene Mutations: Use of Mouse Models to Examine Allelic Variance and to Identify Genetic Modifiers PATSY M. NISHINA AND JUERGEN K. NAGGERT

Mouse models have proven to be extremely useful in the study of heritable ocular diseases. They have provided candidate genes for similar human diseases and whole animal systems to test potential therapeutic interventions. Environmental factors influencing vision, such as light and diet, can be easily studied in mice. In addition, as a robust renewable resource, they have allowed the systematic exploration of disease etiology, progression, and pathologies, which in turn has led to the generation of hypotheses about the functions of particular molecules and mechanisms underlying disease. Finally, mouse models can be an important tool for examining allelic effects (e.g., variability in phenotypes attributable to different mutations within a single gene) and for identifying genetic background modifiers (e.g., variability in phenotypes attributable to nonallellic interactions). Both are discussed in this chapter. Four key factors make mouse models particularly effective for dissecting allelic effects and identifying modifier loci. First, mice are inbred, that is, all animals from a given strain are genetically identical. Therefore, phenotypic variability observed when comparing different mutations within the same molecule in the same inbred strain background would suggest allelic effects, barring differences in animal husbandry, whereas phenotypic variability observed in two different, genetically defined strains carrying the same mutant allele would suggest the presence of genetic modifier loci. Second, methods have been developed to induce mutations through chemical mutagenesis or by germ-line manipulation through genetic engineering. These approaches, particularly the latter, allow researchers to affect spatial or temporal expression to examine the function or roles of particular domains within genes. Third, the reagents and resources in the form of genomic information are tremendous, and new

data in the form of sequences and single nucleotide polymorphisms over multiple strains are forthcoming with ever increasing frequency (Pletcher et al., 2004; Rudd et al., 2005; Shifman et al., 2006; see also www.jax.org/phenome/ snp.html). These advances in particular will assist in the actual identification of the modifying genes and the pathways through which these genes function. Finally, a large number of noninvasive clinical tools have been adapted to the small eyes of the mouse, allowing for monitoring of phenotypic variability. These tools include slit lamp biomicroscopy, indirect ophthalmoscopy, fluorescein angiography, fundus photography, and electroretinography (ERG). In addition, methods to assess visually evoked potentials (VEPs; Ridder and Nusinowitz, 2006) and behavioral parameters such as visual acuity and contrast sensitivity (Douglas et al., 2005) are available. Finally, new noninvasive methods to access the morphological features of the retina by optical coherence tomography in a living animal are currently being developed by a number of groups. These methods will also extend our ability to carry out repeated measures to study disease progression and treatment modalities in a single mouse over time.

Allelic effects as a cause of phenotypic variation Particular alleles may define clinical outcome or disease progression. They may also reveal the functional importance of a particular domain of a gene or protein and the response to environmental stresses or treatment modalities. The availability of mouse models bearing different alleles of the same gene is likely to increase as more animals are generated through chemical mutagenesis and targeted transgenesis to introduce clinically relevant disease alleles from human

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patients into the mouse genome. These models will be instrumental in elucidating the function of molecules and will provide an in vivo system in which to identify subpopulations of patients who harbor different mutations in the same molecule, and to test genotype-specific therapeutic modalities. Defining Disease Progression, Clinical Outcome, or Important Domains In general, loss-of-function alleles produce a different phenotype than do missense mutations effecting hypomorphic or gain-of-function alleles. It can be argued that insofar as most mutations identified in humans are generally not null mutations, to elucidate the role that molecules may play in vision an array of alleles for each protein should be studied. The best example of an allelic series affecting disease progression was generated by chemical mutagenesis in the gene encoding phosphodiesterase 6b (Hart et al., 2005). Four of the seven mutations identified by the MRC Harwell ENU Mutagenesis program were phenotypically identical to the original Pdeb6brd1 mutation, which is found in many standard inbred strains (e.g., C3H/HeJ, SJL/J, FVB/N) and leads to a rapid panretinal rod photoreceptor degeneration by 3 weeks of age (Bowes et al., 1990). Three of the four predicted loss-of-function alleles were nonsense mutations leading to the introduction of premature stop codons, the fourth was a splice site mutation that would severely disrupt normal splicing. Two of the three remaining alleles that led to a slower progressing phenotype were missense mutations and the third was a splice site mutation that still allowed a small percentage of normal transcript to be formed. Interestingly, mice with the Pdeb6batrd3 allele, which carries an amino acid change from asparagine to serine in the highly conserved residue 606, near a putative catalytic domain, showed earlier impaired vision and a greater loss of rod photoreceptors than did Pdeb6batrd1 mice, which carry a histidine-620glutamine mutation that occurs in a relatively conserved residue within a putative catalytic domain. Further study of these alleles may provide additional insight into the functional significance of the domains in which these missense mutations occur. Although mutations in Pde6b can lead to photoreceptor degeneration, they can also lead to congenital stationary night blindness (CSNB) in humans without apparent morphological changes. Recently, Tsang et al. (2007) introduced a CSNB allele, Pde6bH258N, by transgenic means into the Pdeb6brd1 background and rescued the photoreceptors. ERG abnormalities were observed; however, the actual amplitude of the waveforms appeared to depend on the genetic background. Another recent example of alternative alleles providing insight into the potential function of a gene similarly arose from a chemical mutagenesis screen (http://nmf.jax.org/

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index.html). Through positional candidate cloning, nmf247 was identified as a point mutation in the splice acceptor site in intron 6 of Rpgrip1 that leads to a deletion of exon 7 and a premature stop codon in exon 9 (Won et al., 2007). Interestingly, the Rpgrip1nmf247 allele appeared to be more severe than the previously reported null allele (Zhao et al., 2003) in that outer segments were rarely observed and the rate of photoreceptor degeneration was extremely rapid, with most cell bodies gone by 3 weeks of age. In the rare case in which outer segments developed, they were enlarged and vertically oriented, as described in the null allele (Zhao et al., 2003). The recent report of a shorter murine splice variant of Rpgrip1 (Lu and Ferreira, 2005) and closer inspection of the null allele, which was an insertional mutation into exon 14, indicated that the original null allele potentially affected only the full-length form of Rpgrip1, whereas both splice variants would have been missing in Rpgrip1nmf247 mice. This observation suggests that the short form of RPGRIP1 or an otherwise unidentified splice variant may be important in the initiation of normal outer segment development, a hypothesis that is testable with the availability of the nmf247 mouse model. Defining Responses to Environmental Influences It is perhaps not surprising that different alleles of the same gene produce different responses to environmental influences. Understanding these different responses to nongenetic factors such as light exposure may yield insights into clinical prognosis as well as the function of the molecules studied in relation to the environmental influence tested. Four alleles of Rpe65, a molecule that is abundantly expressed in the retinal pigmented epithelium (RPE), have been described in mice (Hamel et al., 1993; Redmond et al., 1998; Pang et al., 2005). A targeted null mutation of the Rpe65 gene demonstrated that in the absence of functional RPE65 protein, outer segments become disorganized and a slow photoreceptor degeneration ensues (Redmond et al., 1998). The findings that Rpe65−/− mice do not have 11-cisretinyl esters in their RPE and accumulate all-trans-retinyl esters suggested a disruption of the isomerization of all-transretinyl esters to 11-cis-retinal (Redmond et al., 1998). Moiseyev et al. (2005) hypothesized and provided evidence that RPE65 acts as an isomerohydrolase in the retinoid visual cycle. Perhaps it is not surprising, then, that alterations in RPE65 might affect retinal responses to light exposure. Danciger et al. (2000) noted that of nine albino mouse strains, the C57BL/6J-c2J (c2J) strain demonstrated marked resistance to light-induced photoreceptor damage. A genome-wide scan for the resistance/susceptibility alleles in progeny of a (c2J × BALB/c)F(1) × c2J backcross revealed a major locus (accounting for 50% of the protective effect) on chromosome 3 and three other weak but significant contributing regions on chromosomes 9, 12, and 14. The pro-

tective effect observed in the c2J background was determined to result from a single nucleotide polymorphism (SNP) in the Rpe65 gene. The c2J strain has methionine at residue 450, while the other eight susceptible albino strains studied carried leucine at codon 450. BALB/cBy retinas, which were more susceptible to light damage, regenerate rhodopsin at a faster rate than retinas from c2J (Wenzel et al., 2001), indicating that the kinetics of regeneration, and not the absolute levels of rhodopsin, are important in light-induced degeneration. These observations are clinically relevant, as they suggest that naturally occurring variants that do not in themselves cause significant overt disease may influence visual outcome when challenged with environmental stresses. Another example of allelic variants that affect different phenotypes on light exposure are those in the rhodopsin gene. Mutations in the rhodopsin gene, a G protein–coupled receptor, are responsible for 30% of autosomal dominant cases of retinitis pigmentosa (RP) in humans. Rhodopsindeficient mice generated by homologous recombination showed that the presence of rhodopsin is necessary for proper outer segment development and photoreceptor survival (Humphries et al., 1997). Panretinal loss of photoreceptor cell bodies was observed in Rho−/− mice by 3 months of age. Mutational screening of the rhodopsin gene in a dog model of RP found an amino acid change from threonine to arginine at residue 4 (Kijas et al., 2002). The dogs showed a very slow recovery after exposure to bleaching light levels and local areas of degeneration. Of direct clinical relevance is the observation that moderate levels of light, such as those used in clinical examinations, increased the rate of retinal degeneration in these dogs (Cideciyan et al., 2005). We have recently identified two new rhodopsin alleles in our Models for Translational Vision Research (MTVR) program, RhoMtvr1 and RhoMtvr4, which carry missense mutations induced by chemical mutagenesis that are phenotypically similar to the dog model (E. Budzynski, pers. comm., 2007). Degeneration of photoreceptors is focal and occurs only on direct light exposure. The observations from these models have important clinical implications, as individuals with this type of rhodopsin mutation may be more susceptible to lightinduced damage, and early identification of these individuals may delay vision impairment if a reduction in light exposure is included in their treatment plan. Defining Response to Treatment Because particular mutations at similar sites in the rhodopsin gene result in different clinical outcomes, the response to treatment may also be genotype dependent. RHO P23H is one of the most frequently occurring mutations associated with autosomal dominant RP in humans. A transgenic mouse, VPP, made with mutations in three amino acids, including P23H, exhibits a slow degeneration of both rods and cones, as assessed by ERG amplitudes and histology (Naash et al.,

1993). Administration of recombinant human erythropoietin had no effect on the rate of photoreceptor degeneration in VPP mice but was able to protect mice from light-induced damage (Grimm et al., 2004). On the other hand, vitamin A supplementation slowed the rate of retinal degeneration in T17M transgenic mice but not in mice expressing the P347S allele of rhodopsin (Li et al., 1998).

Genetic modifiers as a cause of phenotypic variation The phenotypic outcomes of a particular mutation may also be influenced by specific alleles of other genes in the genome, the so-called genetic modifiers. The phenomenon is also referred to as epistatic or nonlinear interaction between genes, so that the effects of variation in gene A are observed only in the presence of a particular variant of gene B. In mice, the existence of genetic modifiers was originally postulated from the variation in phenotype observed when spontaneous mutations were crossed onto different inbred backgrounds (Hummel et al., 1972). An early example of genetic modification in the visual system (LaVail et al., 1978) was demonstrated in mutants of the c locus in which differences in retinal ganglion cell projections could not be fully explained by allelic effects. Additionally, as large numbers of models with targeted mutations created on a 129Sv/J and C57BL/6 (B6) background have been generated, researchers have reported phenotypic variability, as the genetically engineered alleles were moved from the mixed 129/B6 background onto B6 (Ikeda et al., 1999; Humphries et al., 2001). In these cases, the effects of the modifier are observed only in the context of a primary mutation. The phenomenon of phenotypic modifications is currently recognized as a useful tool for identifying factors that interact with genes involved in known pathways or for providing entry points into the function of novel genes (Nadeau, 2003; Vincent, 2003; Linder, 2006). That is, they may provide additional information about genetic contributions to the phenotype for which treatment may already be available, or they may reveal additional steps in a biological pathway that may be more amenable to treatment. As more genes modifying the progression of ocular diseases caused by specific mutations are discovered, it is envisioned that these modifiers will unlock doors to new treatment modalities and help physicians make better diagnoses and treatment plans, perhaps by defining subgroups within a disease population. Examples of phenotypic variability in different genetic backgrounds where the primary mutation is the same are given in table 53.1. Examples of cases in which genetic modifier loci have been mapped or identified are less frequent; these are given in table 53.2. With the recent increase in available genomic information, the actual identification of genetic modifiers is expected to increase. Potential strategies for the identification of genetic modifiers are discussed later in the chapter.

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Table 53.1 Genetic modification of ocular phenotypes observed in different strain backgrounds Strains Involved in Modification Model Effect of Genetic Modification (Resistant → Susceptible) Variability in onset and progression of RBF/Dn, Meta-In(1)Rk, Retinal degeneration 3 (rd3) photoreceptor degeneration Rb(11.13)4Bnr and In-30 Variable rate of photoreceptor C57BL/6 and 129/SvJ Rhodopsin−/− degeneration Suppression of retinal spotting and B6.Cg-rd7/rd7 and CAST/EiJ Nr2e3rd7 photoreceptor degeneration Attenuation of vitreal opacity, retinal 129/SvJ and C57BL/6 Trp53 folds and retrolental fibroplasia in the 129/SvJ background Anterior segment dysgenesis with C57BL/6J, BliA Bmp4 elevated intraocular pressure CAST/Ei, C3H/HeJ AKR/J, BALB/C 129/SvEvTac Increased cell death AKXD-28/Ty isa, iris stromal atrophy, DBA/2J Milder phenotype AKXD-28/Ty ipd, iris pigment dispersion Thinning of optic layers, differences in B6129F1, 129P3/JEms, and Nr2e1frc ERG responses, and retinal vascular C57BL/6 development Retinal dysplasia and photoreceptor C57BL/6 and C3H/HeJ Crb1rd8 degeneration

Model tubby (tub or rd5) Light-induced photoreceptor damage model isa, iris stromal atrophy, DBA/2J ipd, iris pigment dispersion Chx10orJ VPP and Rpe65Leu450Met

Table 53.2 Genetic modifier loci that have been mapped and/or Strains Involved Effect of Genetic in Modification Modification (Resistant → Susceptible) Delay of photoreceptor AKR/J and C57BL/6 degeneration Resistance to light damage BALB/c2J and albino strains

Humphries et al., 2001 Akhmedov et al., 2000 Ikeda et al., 1999

Chang et al., 2001

Anderson et al., 2001 Anderson et al., 2001 Young et al., 2002

Mehalow et al., 2003

References Ikeda et al., 2002a Danciger et al., 2000; Wenzel et al., 2001

Increased cell death

AKXD-28/Ty

Anderson et al., 2001

Milder phenotype

AKXD-28/Ty

Anderson et al., 2001

Partial recovery of visual function Reduced photoreceptor degeneration

CASA/Rk and 129/SvJ

Examples of Genetic Modification of Retinal Degenerative Disease in Mice Recent figures indicate the existence of at least 181 cloned or mapped genes that, when mutated, lead to retinal disease in humans (Retnet: www. sph.uth.tmc.edu/Retnet/disease.htm). Although modifiers have been reported for a small percentage of these genes, because genes do not act in isolation, one would expect that most mutations are modified to some extent by inter-

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cloned Map Location and/or Modifier Identity Chrs 11 (motr1), 2, and 8 Rpe65 Leu450Met

References Heckenlively, 1993

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Chrs 6 and 14 Rpe65

Leu450Met

Wong, et al., 2006 Naash et al., 1993; Samardzija et al., 2006

action with other genes. In mice, an example of variable expressivity in photoreceptor degeneration as a result of genetic background was reported in 1993 for retinal degeneration 3 (rd3) mutant mice (Heckenlively, 1993). The onset and progression of degeneration differed among strains RBF/Dn, Meta-In(1)Rk, Rb(11.13)4Bnr, and In-30, all carrying the same rd3 allele. Scotopic ERGs were extinguished in the first two strains by 6 weeks of age, and

outer nuclear layer (ONL) degeneration was observed as early as 14 days of age and progressed through 8 weeks. In contrast, mice homozygous for the rd3 mutation on strain In-30 manifested milder retinal dysfunction in which scotopic ERGs were not extinguished until 16 weeks of age, and ONL degeneration began at 3 weeks and progressed through 16 weeks of age (Heckenlively, 1993; Linberg et al., 2005). Since this first report of genetic modification, genetic background modification of photoreceptor degenerations has been reported for a number of models. Mice deficient in rhodopsin (Rho−/− mice) were protected by modifiers from the B6 genetic background when compared with the 129Sv background (Humphries et al., 2001). In the B6 background, Rho−/− mice were reported to have greater cone photoreceptor function and a greater number of photoreceptor nuclei. However, modification is specific for the mutant gene. Unlike the genetic modification observed for Rho−/− mice, in tubby mice (Ikeda et al., 2002a), the B6 background is more susceptible to photoreceptor degeneration and the AKR and CAST strain backgrounds afford some protection. Modifier genes may also completely suppress a mutant phenotype (Ikeda et al., 1999; Akhmedov et al., 2000). In an intercross between strains B6.Cg-Nr2e3rd7/rd7 and CAST/ Ei, some F2 mice homozygous for the Nr2e3rd7 mutation were free of the characteristic retinal spotting phenotype (Akhmedov et al., 2000). Subsequent studies have shown that suppression of retinal spotting and subsequent photoreceptor degeneration correlates with suppression of the excess blue cone production that underlies the retinal whorls and folds (Haider et al., 2006). A similar suppression of the disease phenotype was observed in some F2 Nr2e3rd7 mice from intercrosses with the AKR/J and NON.NODn2b/J strains. Interestingly, none of the modifier regions identified from intercrosses between B6.Cg-Nr2e3rd7/Nr2e3rd7 and any of these strains overlapped (Haider et al., in press). Therefore, not only are genetic modifiers specific to mutations, as described with Rho−/− and tubby mice, they may also be different in different strain combinations. Some modifiers have also demonstrated new phenotypes (Mehalow et al., 2003). The retinal degeneration 8 (rd8) mutation in crumbs1 (Crb1), originally identified in a mixed background between strains C57BL/6 and C3H/HeJ, exhibited large spots in the inferior nasal quadrant of the retina. These spots corresponded to areas of retinal dysplasia as shown by histological analysis. When the Crb1rd8 mutation was moved onto the C57BL/6 background by 10 backcross generations, the dysplastic phenotype disappeared, suggesting that unlinked C3H/HeJ alleles were lost during the process of introgressing the mutation onto the B6 background. Subsequent intercrosses with the B6.C3H-Crb1rd8/ Crb1rd8 mice and C3H/HeJ mice restored the dysplastic phenotype, indicating that the genetic modification is likely conferred by C3H/HeJ (M. M. Edwards, pers. comm., 2007).

In studies with mice where both environmental factors and allelic variability of primary mutations can be controlled, genetic modification may be more evident and tractable. However, genetic modification can still be complex, and for a given disorder, a combination of modifier genes may act together to create a cumulative effect on the expression of a phenotype. For example, in F2 tub/tub progeny of an intercross between B6.Cg-tub/tub and the AKR/J strain, a significant range of photoreceptor cell survival was observed. Several chromosomal regions were identified to cosegregate with the thickness of the ONL (Ikeda et al., 2002a). At 20 weeks of age, B6.Cg-tub/tub mice normally have 5%–10% of residual ONL thickness remaining, whereas ONL thickness in F2 mice from the intercross ranged from 5% to 80%. One modifier on chromosome 11, modifier of tubby retinal degeneration 1 (motr1), was detected with high statistical significance in a genome-wide scan, and two additional loci on chromosomes 2 and 8 showed suggestive linkage. Protective alleles came from both the AKR (motr1 and the chromosome 2 locus) and B6 backgrounds (the chromosome 8 locus). Interestingly, Williams et al. (1998) were able to map the natural variation in ganglion cell number in two recombinant inbred strain sets, BXD and BXH, to the same genomic regions containing the tubby modifiers. Identification of these and other modifier loci should offer insight into the particular pathways through which the primary mutant gene functions and into the pathways involved in degeneration.

Strategies for cloning genetic modifiers As mentioned in the previous section, some genetic modifiers have been identified. The number is small when compared with the number of disease genes known to cause ocular disease, however. Therefore, it seems probable that many modifiers await discovery. Identifying these genes could help improve treatment for individuals affected by ocular diseases by defining pathways through which disease-causing genes function. At this juncture, it seems appropriate to consider methods that are evolving to identify variants that modify phenotypes or that have been used in other systems to identify modifier genes. An integrated approach using a combination of strategies is likely to provide the best opportunity for efficiently identifying genetic modifiers (Cervino et al., 2006). Chromosomal Localization of Modified Traits The first step in identifying a genetic modifier is to establish a robust, reproducible method for phenotyping the modification, either qualitative (e.g., the presence or absence of a particular phenotype) or quantitative (e.g., the onset, rate, or severity of a particular phenotype). Because severity is characterized as a continuous distribution rather

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than in discrete subsets, it is grouped with the quantitative traits. In the next step, the source of modification is chromosomally localized in progeny from backcrosses or intercrosses of mice that carry the primary mutation with another strain whose genetic makeup is able to modify the phenotype. Phenotypic modification that is observed only in mice carrying the primary mutation is mapped by comparing phenotypic variation to sequence variation throughout the genome (e.g., genome-wide scan). Once a region is found in which there is significant skewing of a genotype that correlates with the phenotypic modification, the region can be narrowed by further recombinational linkage analysis and testing for sequence differences among biologically relevant genes contained within the chromosomal area that might explain the modifying effects. If multiple regions are identified, candidate regions can be investigated individually for their ability to modify a given phenotype by construction of congenic strains in which the modifier region and the primary mutation are placed in the same genetic background by successive backcrossing. If the primary mutation is in the C57BL/6 background and modification is observed with strains A/J or PWD/Ph, then the congenic process may be hastened by intercrossing the primary mutation to the consomic or chromosomal substitution strain (Silver, 1995) bearing the chromosome on which the modification is observed. In addition, existing congenic lines that cover the modifier region in question can be used as a resource to verify or to identify the modifier (Ikeda et al., 2002a). Segregating Crosses with Multiple Strains Although quantitative trait loci (QTL) mapping and recombinational linkage analysis are powerful tools, these traditional methods require large numbers of animals. It is important, therefore, that these methods be made more efficient. One way in which the efficiency of QTL mapping can be improved is by performing multiple crosses with different strains in which the primary mutation is segregating. Detecting the same modifier in multiple strains suggests that it may be derived from a common ancestral allele. Although recombinations were previously thought of as random events, hot spots for recombination have been shown in various strain combinations (Kelmenson et al., 2005); the use of multiple crosses narrows the confidence interval significantly by increasing the number of recombinations, both ancestral and new, within the region (Wang et al., 2004). Haplotype Analysis A modifier region identified through multiple cross-mappings can be further narrowed by comparing the genomic sequences of the parental strains (reviewed in DiPetrillo et al., 2005; Flint et al., 2005). Haplotype analysis is one method used for such a comparison.

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This method utilizes the fact that inbred mouse strains are derived from a limited set of ancestors, so that alleles of genes are shared between several inbred strains. These alleles are part of an ancestral haplotype surrounding that gene, and haplotype blocks can be identified by the identity of marker alleles within the haplotype between inbred strains. Since these haplotype blocks are on the order of 40 kb to 1.5 Mb long, the genomic area in which the modifier locus resides can be narrowed by identifying the shared ancestral haplotype block in the strains that modify the phenotype. Conversely, areas where no haplotype sharing is observed can be excluded as candidate regions for the modifier. Practically, a dense SNP map is compiled across the map position from the multiple strain cross and is compared between the strains that modify the phenotype and those that do not. Regions where all the modifying strains share the same SNP alleles and are different from the alleles of the nonmodifying strains can be considered candidate regions. The attraction of this technique is that it can reduce the necessity to generate large numbers of mice to narrow a region considerably, and in many cases the SNP information may already be available through the mouse resequencing project (www.informatics.jax.org/menus/strain_menu. shtml; http://genome.perlegen.com/browser/index.html). This technique has been used in mice to narrow modifier regions in studies investigating cancer (Wang and You, 2005) and cardiomyopathy (Wheeler et al., 2005). The power of haplotype analysis was shown in a study of hypertension, in which a QTL region was narrowed from 18 cM to 2.3 cM using haplotype analysis (DiPetrillo et al., 2004). Not all modifiers, however, are captured by ancestral haplotypes. Genetic variation is continually acquired, and at least some modifier alleles are due to mutations that occurred after the common inbred strains were established and are private to one or a few inbred strains (Ikeda et al., 2002b). Gene Expression Profiling or Microarray Analysis A growing body of literature supports the usefulness of combining the mapping of modifier loci with expression profiling, especially in the identification of complex traits (Wayne and McIntyre, 2002; Tabakoff et al., 2003). In an elegant study, Dyck et al. (2003) combined these two techniques to understand the development of gallstones in C57L/J mice that carry the Lith1 gallstone-susceptibility locus. This study used expression profiling, through microarray analysis, to identify differences in gene expression between the C57L/J mice and gallstone-resistant AKR/J mice. Numerous genes involved in fatty acid metabolism were identified. Through literature searches of common regulatory elements within antioxidant systems, the nuclear transcription factor Nrf2, which maps to the Lith1 locus, was identified. Thus, the combination of modifier mapping and expression profiling in conjunction with pathway analysis is

a powerful tool. Locus mapping identifies chromosomal regions that are associated with a known phenotype but may contain many genes. Expression profiling identifies genes whose expression levels differ between two populations (i.e., modified and unmodified) but whose association with the observed phenotype is unknown. By combining these two techniques, it is possible to use data acquired in the mapping phase to filter data acquired in the expression profiling phase, either by identifying a misregulated gene within the modifier region or by identifying a misregulated pathway, a component of which resides in the modifier region, suggesting it as a candidate gene. Modifier Screens Using Mutagenesis Although mutagenesis screens have been used for years to identify modifiers in lower organisms (Carrera et al., 1998; Therrien et al., 2000; Mutsuddi et al., 2004), it has only recently been used for this purpose in mice. Mice with mutations in a gene that is believed to interact with genetic modifiers can be mutated using N-ethylnitrosurea (ENU), a chemical mutagen, and the resulting offspring screened for an altered phenotype. For example, an ENU modifier screen was used to identify genes that suppress thrombocytopenia (lack of blood platelets) in a mouse model Mpl−/− (Carpinelli et al., 2004). In this study, mutant males were treated with ENU and mated to untreated females. Blood collected from G1 progeny were assessed for platelet levels. Mice that showed improved platelet levels were backcrossed to Mpl −/− mice to verify heritability of suppression. DNA from males with heritable suppression was then used to map candidate modifiers. This study produced two candidate modifier alleles of the c-Myb gene. These alleles were shown to reduce c-MYB activity, subsequently suppressing the thrombocytopenia phenotype (Carpinelli et al., 2004). More recently, a sensitized screen to identify alterations in dopaminergic homeostasis was carried out in mice with a disrupted dopamine transporter (Speca et al., 2006). Seven phenodeviant lines with abnormal locomotor activity were identified, two of which were dependent on the presence of the DAT mutation and two others that affected dopamine neurotransmission. Hence, sensitized mutagenesis-driven modifier screens in mice could also be a powerful adjunct for identification of genetic modifiers for retinal disease genes.

Summary The variability in onset, progression, severity, phenotypic expression, or response to treatment, which is a common observation for many diseases, may be due to interactions of mutant alleles with genetic modifiers, or alternatively to environmental or allelic effects. The heterogeneity within the human population makes it difficult to assess the underlying cause of the phenotypic disease variability. However,

understanding the nature of the variability may be important in effecting optimum treatment modalities. Model organisms, such as the mouse, can play an important and necessary role in elucidating the cause of variation in phenotype and in response to treatment. REFERENCES Akhmedov, N. B., Piriev, N. I., Chang, B., Rapoport, A. L., Hawes, N. L., Nishina, P. M., Nusinowitz, S., Heckenlively, J. R., Roderick, T. H., et al. (2000). A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse. Proc. Natl. Acad. Sci. U.S.A. 97(10): 5551–5556. Bowes, C., Li, T., Danciger, M., Baxter, L. C., Applebury, M. L., and Farber, D. B. (1990). Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMPphosphodiesterase. Nature 347(6294):677–680. Carpinelli, M. R., Hilton, D. J., Metcalf, D., Antonchuk, J. L., Hyland, C. D., Mifsud, S. L., Di Rago, L., Hilton, A. A., Willson, T. A., et al. (2004). Suppressor screen in Mpl−/− mice: c-Myb mutation causes supraphysiological production of platelets in the absence of thrombopoietin signaling. Proc. Natl. Acad. Sci. U.S.A. 101(17):6553–6558. Carrera, P., Abrell, S., Kerber, B., Walldorf, U., Preiss, A., Hoch, M., and Jackle, H. (1998). A modifier screen in the eye reveals control genes for Kruppel activity in the Drosophila embryo. Proc. Natl. Acad. Sci. U.S.A. 95(18):10779–10784. Cervino, A. C., Darvasi, A., Fallahi, M., Mader, C. C., and Tsinoremas, N. F. (2006). An integrated in-silico gene mapping strategy in inbred mice. Genetics, ePub ahead of print. Cideciyan, A. V., Jacobson, S. G., Aleman, T. S., Gu, D., PearceKelling, S. E., Sumaroka, A., Acland, G. M., and Aguirre, G. D. (2005). In vivo dynamics of retinal injury and repair in the rhodopsin mutant dog model of human retinitis pigmentosa. Proc. Natl. Acad. Sci. U.S.A. 102(14):5233–5238. Danciger, M., Matthes, M. T., Yasamura, D., Akhmedov, N. B., Rickabaugh, T., Gentleman, S., Redmond, T. M., LaVail, M. M., and Farber, D. B. (2000). A QTL on distal chromosome 3 that influences the severity of light-induced damage to mouse photoreceptors. Mamm. Genome 11(6):422–427. DiPetrillo, K., Tsaih, S. W., Sheehan, S., Johns, C., Kelmenson, P., Gavras, H., Churchill, G. A., and Paigen, B. (2004). Genetic analysis of blood pressure in C3H/HeJ and SWR/J mice. Physiol. Genomics 17(2):215–220. DiPetrillo, K., Wang, X., Stylianou, I. M., and Paigen, B. (2005). Bioinformatics toolbox for narrowing rodent quantitative trait loci. Trends Genet. 21(12):683–692. Douglas, R. M., Alam, N. M., Silver, B. D., McGill, T. J., Tschetter, W. W., and Prusky, G. T. (2005). Independent visual threshold measurements in the two eyes of freely moving rats and mice using a virtual-reality optokinetic system. Vis. Neurosci. 22(5):677–684. Dyck, P. A., Hoda, F., Osmer, E. S., and Green, R. M. (2003). Microarray analysis of hepatic gene expression in gallstonesusceptible and gallstone-resistant mice. Mamm. Genome 14(9): 601–610. Flint, J., Valdar, W., Shifman, S., and Mott, R. (2005). Strategies for mapping and cloning quantitative trait genes in rodents. Nat. Rev. Genet. 6(4):271–286. Grimm, C., Wenzel, A., Stanescu, D., Samardzija, M., Hotop, S., Groszer, M., Naash, M., Gassmann, M., and Reme, C.

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perturb dopaminergic homeostasis in mice. Genes Brain Behav. 5(1):19–28. Tabakoff, B., Bhave, S. V., and Hoffman, P. L. (2003). Selective breeding, quantitative trait locus analysis, and gene arrays identify candidate genes for complex drug-related behaviors. J. Neurosci. 23(11):4491–4498. Therrien, M., Morrison, D. K., Wong, A. M., and Rubin, G. M. (2000). A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in Drosophila. Genetics 156(3):1231–1242. Tsang, S. H., Woodruff, M., Jun, L., Mahajan, V., Yamashita, C. K., Pedersen, R., Lin, C. S., Goff, S. P., Rosenberg, T., et al. (2007). Transgenic mice carrying the H258N mutation in the gene encoding the beta-subunit of phosphodiesterase-6 (PDE6B) provide a model for human congenital stationary night blindness. Hum. Mutat. 28(3):243–254. Vincent, A. L. (2003). Searching for modifier genes. Clin. Exp. Ophthalmol. 31(5):374–375. Wang, D., and You, M. (2005). Five loci, SLT1 to SLT5, controlling the susceptibility to spontaneously occurring lung cancer in mice. Cancer Res. 65(18):8158–8165. Wang, X., Korstanje, R. Higgins, D., and Paigen, B. (2004). Haplotype analysis in multiple crosses to identify a QTL gene. Genome Res. 14(9):1767–1772.

Wayne, M. L., and McIntyre, L. M. (2002). Combining mapping and arraying: An approach to candidate gene identification. Proc. Natl. Acad. Sci. U.S.A. 99(23):14903–14906. Wenzel, A., Reme, C. E., Williams, T. P., Hafezi, F., and Grimm, C. (2001). The Rpe65 Leu450Met variation increases retinal resistance against light-induced degeneration by slowing rhodopsin regeneration. J. Neurosci. 21(1):53–58. Wheeler, F. C., Fernandez, L., Carlson, K. M., Wolf, M. J., Rockman, H. A., and Marchuk, D. A. (2005). QTL mapping in a mouse model of cardiomyopathy reveals an ancestral modifier allele affecting heart function and survival. Mamm. Genome 16(6):414–423. Williams, R. W., Strom, R. C., and Goldowitz, D. (1998). Natural variation in neuron number in mice is linked to a major quantitative trait locus on Chr 11. J. Neurosci. 18(1):138–146. Won, J., Gifford, E. J., Smith, R. S., Hicks, W. L., Naggert, J. K., and Nishina, P. M. (2007). Allelic variation suggests Rpgrip1 is important for outersegment development. Invest. Ophthalmol. Vis. Sci. 48. E-abstract, 4490. Zhao, Y. D., Hong, H., Pawlyk, B., Yue, G., Adamian, M., Grynberg, M., Godzik, A., and Li, T. (2003). The retinitis pigmentosa GTPase regulator (RPGR)-interacting protein: Subserving RPGR function and participating in disk morphogenesis. Proc. Natl. Acad. Sci. U.S.A. 100(7):3965–3970.

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54

The Mouse Eye Transcriptome: Cellular Signatures, Molecular Networks, and Candidate Genes for Human Disease ELDON E. GEISERT AND ROBERT W. WILLIAMS

The laboratory mouse occupies a unique position at the intersection of basic vision science and clinical medicine. The advantages of this species include its small size and rapid rate of reproduction, its extremely well-characterized genome, and the many well-honed techniques used to engineer gene variants, knockouts, and knock-ins. Temporal and spatial patterns of gene expression can be controlled with impressive precision in eye, retina, brain, and many other tissues of mice (see chapter 39, this volume). Human gene variants, usually those that cause disease, can be inserted into the mouse genome to study mechanisms of normal function, disease progression, and drug efficacy (Pennesi et al., 2003). In a dramatic recent example, Jacobs et al. (2007) inserted the human red opsin gene (OPN1LW) into mice, thereby converting this normally color-blind species into a functional trichromat able to discriminate between red and green. Even more radical engineering is practical. For example, it is now possible to produce so-called humanized mice in which the proteins in an organ such as the liver (Kondo et al., 2002) or the immune system (Shultz et al., 2007) are replaced through genetic engineering by human equivalents. One can only speculate that comparable work humanizing the mouse eye and retina is not far behind. The future holds the potential for having mouse models that will exactly recapitulate human disease affecting vision, allowing us the opportunity to define the fundamental defect in disease states and to develop and test new therapies to prevent the loss of sight. There are also more subtle and perhaps equally important ways in which mice are having an impact on biomedical research and clinical medicine. Populations of diverse strains of mice can be used to model human genetic diversity. Although most biologists use mice to model actions of specific genes—each in isolation on a fixed genetic background (usually C57BL/6J)—a growing number of geneticists are using groups of different strains of mice, particularly off-

spring of the 16 strains that have been sequenced (Frazer et al., 2007), to model the complexity of human populations. Diverse strains of mice and their progeny can be used to test the utility and predictability of personalized medicine. Approximately 10 million common sequence variants, including single nucleotide polymorphisms (SNPs), insertions, deletions, duplications, and even retrotransposons, contribute to much of human biological diversity. Roughly the same types and numbers of genomic sequence variants account for phenotypic differences among common inbred strains of mice, which, unlike humans, can be raised in nearly identical environments. For example, the two oldest strains of mice, the glaucoma-prone DBA/2J and the comparatively normal C57BL/6J, differ at roughly 1.5 million SNPs, as well as an additional large number of microsatellites and copy number variants. It is now practical to use a relatively modest number of strains, from 10 to 100, to model the response diversity and wide spectrum of disease burden of complex human populations rather than just to model that action of a single gene in a single inbred strain. It is finally practical to exploit variation among strains as if it were a “natural” genomic manipulation (Williams et al., 1996). Variation of this type can provide insight into interactions among genes, transcripts, and proteins expressed in the eye, retina, and brain. This more holistic approach tends to scale better to the whole genome than do studies of single alleles on single genetic backgrounds. In the first half of this chapter, we discuss the current state of mouse genomics with special reference to the expression of genes in the mouse eye. How many genes are expressed in different parts of the eye? What online resources are now available to track progress in eye genomics in mouse and human? How well are researchers able to use online resources to computationally annotate or infer gene function in the same way that protein function is often inferred by common structural motifs? In the second half of this chapter, we

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return to the topic of the population genetics of the mouse eye and review a powerful new data set, the Hamilton Eye Institute Mouse Eye Database, which includes many analytical tools to study associations between variation in gene expression and functional differences in the eye, retina, and brain of a large number of highly diverse strains of mice.

Mouse and human genomes Early in 2001, initial draft sequences of the human genome were published nearly simultaneously by the International Human Genome Sequencing Consortium (McPherson et al., 2001) and Celera Genomics (Venter et al., 2001). A few months after release of the human sequence, Celera assembled a commercial draft of the mouse genome that combined public and private sequence data from five strains of mice (A/J, C57BL/6J, DBA/2J, 129X1/SvJ, and 129S1/ SvImJ). The first public assembly of the genome of strain C57BL/6J was released late in 2002 (Waterston et al., 2002). The strong position of C57BL/6J as the most important and widely used inbred strain was reinforced by this public release. This accounts for the now almost obligatory step of transferring (or introgressing, to use the geneticist’s term) all interesting mutations into the C57BL/6 (B6 or “black-six”) genome. The revelation of human and mouse genomes has catalyzed a large and unanticipated revolution in the production and use of online genomic databases. Use was initially confined mainly to molecular biologists and bioinformaticists. At present, however, a significant proportion of the biomedical research community has come to take these resources for granted as a vital research infrastructure on par with PubMed. Over the last few years, dynamic Web editions of human, mouse, rat, chimpanzee, dog, yeast, Drosophila, and C. elegans genomes at NCBI, UCSC, and Ensemble have had an enormous impact on the way biomedical research is conducted. These Web portals or knowledgebases represent dynamic encyclopedias that form a framework for most genetic, genomic, molecular, cellular, systems, and clinical research. Learning how to use and evaluate these resources is becoming as important as learning how to read and critique the literature. Initial comparative analysis of mammalian genomes found that the mouse genome is slightly shorter than the human genome (2.6 billion nucleotides vs. 3.0 billion nucleotides), probably due to “trivial” differences in noncoding sequence and repetitive DNA. As expected, the mouse and human genomes contain very close to the same number of classic protein-coding genes, now estimated at 27,000 ± 3,000 in mammals. A large part of the mouse genome can be partitioned into chromosome strands that are roughly equivalent to homologous strands of human chromosomes, a feature that is referred to as conserved synteny (similar strings). Like

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many interesting research efforts, our improved understanding of genome sequence and the process of transcription has revealed how much we now do not understand. While the genome is often referred to metaphorically as an encyclopedia, or even as a simple string of letters, it is actually a deviously clever and an incomplete patchwork of code. This code cannot be understood except in the context of a whole organism and its environmental history. Having sequence and global transcriptome data in hand has taught molecular biologists and bioinformaticists to be humble in their claims of having seen a path to a brave new postgenomic world. To give one example, we now have a much greater appreciation of the complex role of noncoding RNAs, micro-RNAs, guide RNAs, and antisense RNA sequences in regulating transcription and translation. There is now good evidence that more of the genome (perhaps as much as 30%), including intergenic “junk DNA,” is transcribed, and that only a fraction of transcripts are destined to be translated into mature protein. There is also strong evidence of far more alternative transcript use, with estimates that up to 80%– 90% of genes have two or more variants. The supposedly bland world of DNA and RNA has suddenly become much more of a jungle and may even rival the proteome in its intricacy and complexity (Kampa et al., 2004; Kapranov et al., 2007). Sequence Differences Among Mice: A Great New Resource With genomic sequence data from five different inbred strains, Celera was able to systematically extract close to 3 million SNPs (Waterston et al., 2002; Lindblad-Toh et al., 2005). This resource introduced a new approach to genomics in which missense mutations and premature stop codons can be hunted down directly in databases. For example, among the 1.5 million SNPs that distinguish between the two oldest inbred strains of mice, C57BL/6J and DBA/2J, a subset of 42 is located in the tyrosine-related protein 1 (Tyrp1, also known as the brown locus), including two missense mutations that alter protein sequence (figure 54.1). This first wave of SNP variants for five strains was followed three years later (Frazer et al., 2007) by a massive amount of array-based sequence data generated by Perlegen Sciences for the National Institute for Environment Health Sciences (table 54.1). Perlegen used massive wafers covered with 25-nucleotide DNA strands (68 wafers per genome) to sequence precisely the same gene-rich 1.5 billion base pairs of DNA across a panel of 15 highly diverse strains, including four wild subspecies from which virtually all common strains trace their descent—WSB/EiJ (Mus musculus domesticus), PWD/PhJ (M. m. musculus), MOLF/EiJ (M. m. molossinus), and CAST/EiJ (M. m. castaneus). The result of this massive effort is an open database that provides access to about 10 million high-quality SNPs that can be downloaded and sorted at several sites, including

the SNP browser at GeneNetwork, www.genenetwork.org/ webqtl/snpBrowser (see figure 54.1). Although these new SNPs have not garnered the attention of the original sequencing efforts, they are extremely powerful resources. The millions of SNPs that are now a fixed resource for inbred strains neatly match the 8–10 million common SNPs segregating in human populations (www.hapmap.org). In both mouse and human these SNPs are collectively responsible for much of the phenotypic diversity and differences in health and disease. For example, the first missense mutations in Tyrp1 (figure 54.1, top) change the wild-type TGT cysteine codon at position 86 in exon 2 (note the Gs and As in the top row) present in virtually all mammals to a TAT codon in DBA/2J that codes for a mutant tyrosine residue. This SNP is responsible for the brown locus phenotype. The second missense SNP in exon 5 (rs28091461, the less severe b-“light” allele) results in an arginine (R) to histidine (H) replacement at

amino acid position 326. These mutations in a key melanocyte catalase disturb melanin production and lead to oculocutaneous albinism type 3 (OCA3) in humans. In mice, these mutations also contribute significantly to glaucoma susceptibility (see chapter 39, this volume). To ensure a low error rate, both Celera and Perlegen used stringent criteria to define SNPs. This conservative approach and the focus on gene-rich regions have led to the prediction that there are actually in excess of 40 million common SNPs fixed in the genomes of these 16 common strains (Frazer et al., 2007). There are also many other types of genetic polymorphism variants, including insertions, deletions, polymorphic transposons, duplications, and inversions. In sum, just a small cohort of inbred strains provides direct access to roughly four times the level of common variation segregating in humans. Before the genome projects, this genetic variation remained undiscovered, but after sequencing several

Figure 54.1 The SNPs within the exons of the tyrosine-related protein 1 (Tyrp1) are shown from the SNP browser page of GeneNetwork. This is a list limited to the 11 SNPs found in the exons of Tyrp1 (total of 126 SNPs in the gene) within the 17 strains of mice examined. At the top of the page the critical information can be selected. For this analysis we examined Tyrp1. The chromosomal location is listed. We also selected all strains in the database,

exons only, and all functions of the SNPs. The listing at the bottom of the page is self-explanatory, with SNP ID, SNP RS ID, chromosome (Chr), megabase pair (Mb), domain, gap, gene, function of the SNP (Function), conservation, alleles, source, and the specific SNPs in the strains examined. Notice that four of the SNPs create missense mutations, and the remainder of those found in exons are silent.

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Strain C57BL/6J

DBA/2J

A/J 129S1/SvImJ 129X1/SvJ CAST/EiJ BTBR T+ tf/J MOLF/EiJ KK/HlJ AKR/J PWD/PhJ NZW/LacJ BALB/cByJ WSB/EiJ

Table 54.1 Top strains of mice currently sequenced, along with several pertinent ocular phenotypes Sequencing Status Ocular Trait 8X shotgun Pigmented appearance: black; related genotype: a/a. C57BL/6J was the DNA source for the international collaboration that generated the first high-quality draft sequence of the mouse genome. Dilute brown. Glaucoma model, large eye; interacting loci cause severe iris atrophy and 1.3X + Perlegen glaucoma in DBA/2J mice. Retinal histopathology reveals a loss of RGCs, as well as GABAergic and cholinergic amacrine cells (Moon et al., 2005). Tyr-negative albino 1.3X + Perlegen Tyr-negative albino white-bellied agouti 1.3X + Perlegen Celera Tyr chinchilla pink eye ocular albino Perlegen Agouti Perlegen Black and tan, tufted Perlegen White-bellied agouti; related genotype: Aw/Aw. Homozygous for the retinal degeneration allele Pde6brd1. Perlegen Tyr-negative albino KK/HlJ male mice exhibit diabetic symptoms that include hyperglycemia, hyperinsulinemia, and insulin resistance. Perlegen Tyr-negative albino Perlegen Agouti Perlegen Tyrp1b/Tyrp1b p Tyrc/p Tyrc albino; Pde6brd1 Perlegen Tyr-negative albino White-bellied agouti

C3H/HeJ Perlegen Agouti carries Pde6brd1, resulting in retinal degeneration by weaning age FVB/NJ Perlegen Tyr-negative albino carries Pde6brd1, resulting in retinal degeneration NOD/LtJ Perlegen Tyr-negative albino Note: The level of coverage and quality produced by Perlegen Sciences’ array-based resequencing technology is equivalent to that of 1.5 × whole-genome shotgun sequencing. For mouse strains and phenotypes, see www.informatics.jax.org/external/festing/mouse/ STRAINS.shtml.

different genomes we are finding that the genome contains a surprising number of variations. With the online genomic resources available to investigators, scientists are able to mine these data sources, producing a rapidly evolving understanding of the structure of the genome.

Gene expression in the mouse eye: Results and resources Recent advances in genomic technologies have facilitated the large-scale examination of tissues, defining transcript abundance for tens of thousands of genes simultaneously in single experiments. There are two basic technological approaches. In the first, mRNA is isolated, processed, and directly sequenced (expressed sequence tags [ESTs] or serial analysis of gene expression [SAGE]). In the second approach, mRNA or its cDNA derivative is hybridized to a complementary probe (microarray methods), and rather than counting mRNA molecules, one measures the binding affinity as an indirect measure of mRNA concentration. In both of these applications mRNA is isolated using oligo-dT to hybridize the poly-A tail of the message. This approach selectively isolates RNA that will be translated into protein (approximately 3% of the total RNA in the cell), eliminating

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the highly abundant ribosomal RNA and transfer RNA. The advantage to these methods is the rapid interrogation of all mRNAs. The disadvantage is that untranslated RNAs are not interrogated by these methods and we are not examining the full complexity of transcriptional activity. The EST analysis and the SAGE analysis directly sequence copies of mRNA and can accurately measure the levels of abundant messages. Furthermore, the quality of the database and detection of rare messages can be increased by increasing the number of sequenced clones or cDNA fragments. Microarray has the advantage of examining a large number of transcripts in a single hybridization step. However, microarray methods have two significant disadvantages. For abundant transcripts, the probe can be saturated and will not reflect the true level of message, and for rare transcripts the hybridization may be below the level of detection.

Expressed sequence tag and serial analysis of gene expression analysis Many different genes are expressed in the diverse tissue types that make up the eye. Early estimates based on the short sequences (300–500 base pairs long) derived from mRNAs

(ESTs) indicated that retina alone produced around 15,000 unique transcripts (Williams, Strom, Zhou, et al., 1998). The UniGene EST collection binds together a set of ESTs that trace back to the same genome location, and often to a single well-known gene. This puts the UniGene collection at a halfway point between the modest number of canonical protein-coding genes and the high number of transcript variants. In the mouse there are a total of around 86,000 UniGene EST clusters (build 163), of which 24,000 are derived from mouse eye tissues. This is roughly twice the number found in organs such as liver (12,000) and heart (11,000), but somewhat less than that found in whole brain (30,000). Unfortunately, differences among tissue and organs have as much to do with ascertainment biases and database coverage as they do with inherent differences in mRNA and cellular diversity. This point is driven home by the fact that the number of UniGene clusters in humans is 50% greater than that in mice (124,000), a difference that is almost certainly technical. However, as in the mouse, the number of human UniGene clusters associated with ocular tissues is close to 24,000. The National Eye Institute’s NEIBank is an excellent resource to explore the fine details of EST sets extracted from different ocular tissues in several species (http:// neibank.nei.nih.gov). NEIBank includes EST/UniGene collection libraries from whole eye and several discrete ocular tissue types, including the ciliary body (human only), the choroid/retinal pigmented epithelium (human, mouse, other), cornea (mouse, human, other), iris (human, rat, zebrafish), lacrimal gland (human and mouse), lens (human, mouse, rat, kangaroo, dog, zebrafish, rabbit, guinea pig, cow, rhesus monkey), optic nerve (human only), trabecular meshwork (human and rat), and retina (human, mouse, rat, dog, zebrafish, rabbit, guinea pig). The NEIBank retinal mRNA library for adult mouse (NbLib0027) consists of approximately 35,000 UniGene clusters and ESTs. Only one-third (12,224) are represented by multiple hits or clones. Not surprisingly, at the top of the list is rhodopsin, represented by 1,050 counts. In other words, rhodopsin sequence was identified in 1,050 independently sequenced clones. If we sum the unique transcripts in NEIBank across all mouse eye libraries, the total reaches about 28,000 (table 54.2). This sum does not include those genes, ESTs, and UniGene clusters that are only active during development. What we can conclude is that a significant proportion of the genome, certainly well over half of all coding genes, is expressed in the eye, with an exceedingly high level of expression in the retina. SAGE analysis, like EST analysis, directly sequences short segments of cDNA, offering the ability to improve the quality of the data set by increasing the number of cDNAs sequenced. For the eye, the SAGE database of the Cepko group (http:// itstgp01.med.harvard.edu/retina) examines the expression

Table 54.2 Numbers of clones in different EST libraries in the NEIBank Mouse Eye Human Eye Mouse Retina Number of Clones 28,300 14,342 34,885 1 or more 8,347 5,277 12,224 2 or more 5,240 3,193 8,413 3 or more 3,619 2,058 6,207 4 or more 2,644 1,442 4,757 5 or more 1,997 1,040 3,697 6 or more 1,494 801 2,955 7 or more 1,175 628 2,374 8 or more 956 516 1,920 9 or more 782 433 1,576 10 or more 673 374 1,330 11 or more 570 326 1,110 12 or more 370 241 712 15 or more 221 143 397 20 or more 133 97 230 25 or more 99 68 178 30 or more Note: Shown are the EST copy number in the mouse eye (NbLib0032 353 highest copy number for Cryg), the human (NbLib0079 NEI site 339 highest copy number for Cryg), and the mouse retina (NbLib0027 1050 max, for Rho). Data were extracted from the NEIBank (http://neibank.nei.nih.gov).

patterns in the developing mouse eye. In the case of the Cepko database, SAGE tags are 14 bases in length and are used to identify specific transcripts in the developing C57BL/6 retina (Blackshaw et al., 2004). This high-quality database identifies transcripts expressed at very high levels and low levels. The data are presented in an easy-to-use, highly interactive format presenting SAGE data for different tissues of the eye and at different developmental stages ranging from embryonic day 12.5 to adult. Using this resource one can examine the level of expression of individual transcripts to uncover developmental expression patterns. In addition to the SAGE database, in situ hybridizations of retinal sections are included for many of the mouse transcripts. The data presented at the Cepko site are a starting point for functional analysis of the role of genes in retinal development. For example, Blackshaw et al. (2004) identified genes expressed in the adult retina that formed a photoreceptor-enriched catalogue of transcripts within the inner retina. This group of genes has a correlated developmental expression pattern with rhodopsin and may offer prime candidate genes for human disease.

Microarray analysis The advent of microarray technology in the 1990s led to a revolution in the way data are collected and analyzed. With a single chip, one can interrogate tens of thousands of tran-

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scripts. This created the need for massive databases and the skilled management of bioinformatic resources. These databases contain significant amounts of data for transcript levels within the eye, retina, and other ocular tissues. In the case of the eye and retina, the data are confounded by the fact that the structures are composed of many different tissue types formed by unique cells. From these data one can extract some unique cellular signatures; however, the results may not accurately reflect each of the cell types and may include lack of cell-level specificity. Furthermore, the data do not provide the investigator with any information concerning translational regulation (protein expression) or posttranslational modifications important in protein regulation. Nonetheless, several microarray databases offer unique opportunities for vision researchers to investigate the regulation of the gene expression during eye development and in different strains of the mouse. For the development of the eye there is a wonderful microarray-based Web site maintained by the Friedlander group at Scripps Institute. This site is a source of information for the postnatal developmental expression pattern of transcripts in mouse retina. The Friedlander group used Affymetrix microarrays to identify the relative levels of gene expression during the postnatal development of the mouse eye (Dorrell et al., 2004). (The data are presented at www. scripps.edu/cb/friedlander/gene_expression.) The developmental expression patterns of groups of genes are illustrated in a format that allows the investigator to query the developmental expression patterns of a specific gene or groups of genes. These Web sites can serve as references to define the molecular signatures of different tissues in the eye, along with the developmental regulation of gene expression. They also provide an excellent resource with which to follow the developmental expression patterns of genes expressed in the eye or retina. Their analysis provides the investigator with developmental expression patterns of groups of genes. These clusters of genes are specifically related to the developmental patterns of cell birth and maturation. For example, the last group of genes to be upregulated is related to phototransduction and the maturation of rods. The Friedlander database, like many vision-related mouse databases, is based on information generated from only one strain of mouse, the C57BL/6 mouse. One important goal of genomic research is to combine expression data with variation in phenotype. In the mouse, this can be accomplished by examining strain differences as related to differential susceptibility to disease or naturally occurring differences in phenotype. For the mouse there are several resources that define phenotypic variability. Two examples are the Jackson Laboratory mouse phenotype database (http://phenome.jax.org/pub-cgi/phenome/ mpdcgi?rtn=docs/home) and GeneNetwork Phenotypes (www.genenetwork.org). The mouse strains also serve as a

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rich genomic resource, with expression genetics allowing us to correlate phenotype variation with genomic locus. Quantifying mRNA levels with microarray-based systems has allowed for a rapid interrogation or transcript expression in the mouse. Expression genetics allows for a conceptually unique approach of using this transcript expression data to locate genomic loci controlling disease or phenotype. Furthermore, by using mouse genetic panels such as the BXD strain set, we can provide insights into the genetic networks controlling differences in gene expression and phenotypic variation. The Hamilton Eye Institute Mouse Eye Database We have created the Hamilton Eye Institute Mouse Eye Database (HEIMED, available at www.genenetwork.org) to bring expression genetics to the vision research community. By examining variations at the transcript level we can define correlations in gene expression to genomic loci revealing higher-order transcriptional networks, as well as loci capable of modulating morphological features in the eye, for example, retinal ganglion cell (RGC) number. This approach can lead to dissection of entire networks of genes controlling transcriptional networks within the eye that dictate specific ocular phenotypes. To bring this power of expression genetics and the mouse strain set to the study of the eye, we developed a highly interactive database, HEIMED. This database estimates mRNA expression in whole eyes of young adult mice of many lines generated using the Affymetrix M430 2.0 array, which contains more than 45,000 probe sets representing more than 39,000 transcripts. Within the HEIMED, data from a total of 98 mouse strains is presented, including 67 BXD strains; the two parental strains (C57BL/6J and DBA/2J), along with their reciprocal F1s; and 27 strains from the mouse diversity panel (plus B6 and D2 for a total of 29 common strains) were generated by crossing C57BL/6J with DBA/2J. The BXDs are particularly useful for systems genetics because all of the BXDs are fully mapped and both parental strains are fully sequenced. The BXD Genetic Reference Panel of Mice The BXD strain set is an integral part of HEIMED and our eye databases. This unique strain set was originally developed in the laboratory of Benjamin Taylor and was transferred to the Jackson Laboratory from Dr. Taylor’s research colony at his retirement (Taylor et al., 1999). The two parental mouse strains, C57BL/6J and DBA/2J, were inbred strains developed relatively early. These strains have contrasting characteristics. The C57BL/6J is a very widely used inbred strain that was originally developed to be refractory to tumors; in contrast, the DBA/2J strain was developed to be susceptible to tumors. These inbred genetic differences have produced one strain (C57BL/6J) that is resistant to CNS damage and another (DBA/2J) that is susceptible to injury (Inman et al., 2002). The DBA/2J mice used in the traditional

set of BXD strains carried a mutation in the Tyrp1 gene that contributed to iris stromal atrophy. In 1997, a second mutation in Gpnmb resulted in a significant increase in iris diseases, causing pigment dispersion in the DBA/2J strain (Chang et al., 1999). In the advanced BXD RI strain set, which was developed from DBA/2J mice carrying both mutations (Peirce et al., 2004), 15 strains carry both the Tyrp1 and Gpnmb mutations and should develop iris disease similar to that observed in the DBA/2J strain. Since the BXD RI strain set is fully mapped and since each strain can be sampled an unlimited number of times, these mice are ideal for use as a mapping panel to identify genomic loci controlling specific phenotypes such as ganglion cell number (Williams et al., 1996) or controlling genetic networks (Vazquez-Chona et al., 2005). The BXD RI strain set is at the core of our novel analytical of tool set and QTL mapping algorithms (www. genenetwork.org). This includes an SNP database for sequenced mouse strains, along with unique sequence analysis software developed by the Williams group. The collective purpose of these tools and techniques is to extract and test molecular networks that affect gene expression in the eye and the retina. Examples of the innovative use of this approach are described in articles published in Science (Brem et al., 2002) and Nature (Schadt et al., 2003) that have highlighted the power of treating expression data as a quantitative trait and using QTL mapping methods to systematically identify upstream controllers that are responsible for individual differences in transcript abundance ( Jansen and Nap, 2001). Our group has been using QTL mapping methods of this type for more than 6 years (Williams et al., 1996; Hittalmani et al., 2003; Wang et al., 2003; Chesler et al., 2004; Vazquez-Chona et al., 2005), and we welcome the opportunity to bring these approaches to the vision research community, specifically for study of the retina (Vazquez-Chona et al., 2005) (see www.genenetwork.org and the HEIMED). The precision with which we can map QTLs that modulate retina transcript networks is determined by the quality of genetic maps that we assemble for RI strains. With the sequence data, we can now completely define all breakpoints in the set of 80 BXD RI strains. These strains incorporate approximately 7,000 recombinations. Ideally, we would know the precise location of each of these breakpoints within 100,000 base pairs. This would have been almost unthinkable a few years ago. Now it is simply a matter of finding polymorphic loci that distinguish the parental strains. Using HEIMED, we can extract the molecular signatures of specific tissues or cell types by looking for genes that correlate across the entire genome and are coexpressed with tissue-specific or cell-specific marker genes. If we examine these molecular signatures in the HEIMED database, we can begin to unravel the unique genetic networks that regulate tissue-specific expression patterns. An example of this

type of analysis is genes that covary with rhodopsin (Rho). Unlike other approaches that identify all of the genes expressed in a cell type (in the case of rhodopsin it is rods), HEIMED generates a list of genes that are uniquely expressed in rods, forming a unique signature for this cell type. If we examine the top 100 transcripts that correlate with Rho, we can observe a unique molecular signature for photoreceptors with genes expressed in rods (e.g., Pde6b, Gnat1, Guca1b, Nrl, Pde6g, Pdc, Vtn, Rp1, and Cabp4) with similar expression patterns across the reference panel of mice. The genetic covariance with the rod-specific transcripts not only defines a unique rod signature; it can also be used to define genes associated with human retinal diseases. Pearson and Spearman correlations were used to rank candidates with expression tightly coupled (r > 0.9 for the first 100 probe sets) with rhodopsin across all strains. Thirty of the top 100 covariates with rhodopsin are genes currently associated with retinal disease in humans. For example, Rdh12 (r = 0.98 with rhodopsin) has a well-characterized association with Leber’s congenital amaurosis (LCA3). Examples of other known disease genes that covary with rhodopsin are listed in table 54.3. The genomic locations of other genes that covary with rhodopsin suggest that they are candidate genes for human disease. Their genomic locations in the mouse were converted to human chromosome locations using mouse/ human synteny maps. We examined the location of human diseases with human disease loci with unidentified disease-causing genes in the RetNet database (www.sph.uth.tmc.edu/Retnet/disease. htm). This analysis allowed us to generate strong biological candidates for uncloned human disease loci, using HEIMED in combination with GeneNetwork. The eight candidate genes for human retinal diseases are listed in table 54.4. These results are one example of the power and potential of mouse genomics for understanding retinal disease in the human. An example of a tissue-specific molecular signature is the cornea. If we use Aldh3a1, the gene for aldehyde dehydrogenase family 3, subfamily A1, as a marker for the cornea and run this on the trait correlation function of GeneNetwork, we then retrieve a set of transcripts that appears to be a molecular signature of the cornea. The transcripts in the list include Aldh3a1, keratin 12, members of the KLF transcription factor family, and the corneal crystalline Tkt. If we examine the list of genes from HEIMED, we find they represent a series of genes that are expressed in the cornea, with some genes being unique to the cornea. By comparing these genes with the total gene expression in the cornea as represented in the NEIBank (NbLib0116, adult mouse cornea; http://neibank.nei.nih.gov/index.shtml), we find that 9 of the top 10 genes on the HEIMED corneal list are highly represented in the NEIBank list. These include aldehyde dehydrogenase family 3, subfamily A1 (Aldh3a1, 32 independent

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Gene Crb1

Sag Rho Gnat1 Pde6b Cnga1 Pde6a Guca1b Guca1a Tulp1 Rp1 Kcnv2 Cabp4 Rom1 Nrl Rdh12 Nr2e3 Cngb1 Aipl1 Unc119 Fscn2 Crx Rs1

Table 54.3 Cloned human disease genes found in our correlative analysis of rhodopsin expression in the mouse, with 23 cloned human disease genes and references for the original and cloning of the gene Disease* Human Locus Mapping Reference Cloning Reference Leber congenital amaurosis, AR 1q31.3 den Hollander et al., 1999b den Hollander et al. 1999a Retinitis pigmentosa, AR Other AD retinopathies Retinitis pigmentosa, AR 2q37.1 Ngo et al., 1990 Ngo et al., 1990 CSNB, AR CSNB, AD 3q22.1 Nathans et al., 1986 Nathans and Hogness, 1984 Retinitis pigmentosa, AR, AD CSNB, AD 3p21.31 Sparkes et al., 1987 Lerman and Minna, 2000 CSNB, AD 4p16.3 Bateman et al., 1992 Pittler et al., 1993 Retinitis pigmentosa, AR Retinitis pigmentosa, AR 4p12 Dhallan et al., 1991 Kaupp et al., 1989 Retinitis pigmentosa, AR 5q33.1 Ovchinnikov et al., 1987 Pittler et al., 1990 Macular degeneration, AD 6p21.1 Surguchov et al., 1997 Payne et al., 1999 Cone or cone-rod dystrophy, AD 6p21.1 Subbaraya et al., 1994 Subbaraya et al., 1994 Leber congenital amaurosis, AR 6p21.3 North et al., 1997 North et al., 1997 Retinitis pigmentosa, AR Retinitis pigmentosa, AR, AD 8q12.1 Pierce et al., 1999 Pierce et al., 1999 Cone or cone-rod dystrophy, AR 9q24.2 Ottschytsch et al., 2002 Ottschytsch et al., 2002 CSNB, AR 11q13.1 Haeseleer et al., 2004 Haeseleer et al., 2000 Retinitis pigmentosa, AD 11q12.3 Bascom et al., 1990 Bascom et al., 1989 Retinitis pigmentosa, AD, AR 14q11.2 Yang-Feng and Swaroop, 1992 Swaroop et al., 1991 Leber congenital amaurosis, AR 14q24.1 Haeseleer et al., 2002 Haeseleer et al., 2002 Retinitis pigmentosa, AR 15q23 Kobayashi et al., 1999 Kobayashi et al., 1999 Other AR retinopathy Retinitis pigmentosa, AR 16q13 γ subunit: Ardell et al., 1995 γ subunit: Ardell et al., 1995 β subunit: Ardell et al., 1996 β subunit: Ardell et al., 1996 Cone or cone-rod dystrophy, AD 17p13.2 Sohocki et al., 1999 Sohocki et al., 1999 Cone or cone-rod dystrophy, AD 17q11 Swanson et al., 1998 Higashide et al., 1996 Macular degeneration, AD 17q25.3 Tubb et al., 2000 Saishin et al., 1997 Retinitis pigmentosa, AD 19q13.32 Freund et al., 1997 Freund et al., 1997 Cone or cone-rod dystrophy, AD XL retinoschisis Xp22.13 Dahl et al., 1987 Sauer et al., 1997

AD, autosomal dominant; AR, autosomal recessive; CSNB, congenital stationary night blindness; XL, x-linked.

Gene RP32

Disease Severe AR RP

Table 54.4 Eight candidate genes for mapped human disease Human Locus Mouse Locus 1p34.3–p13.3 4 at 154.132529 Mb

Mapping Reference Zhang et al., 2005

AR ataxia 1q31.1–1q32.3 1 at 134.282002 Mb Higgins et al., 1997 AXPC1 AR RP 4q32.1–4q34.3 8 at 26.0 cM Hameed et al., 2001 RP29 AD macular dystrophy 5pter–5p13.1 15 at 6.994909 Mb Michaelides et al., 2003 MCDR3 ARMD, complex etiology 10q26.13 Chr 7 F3 Jakobsdottir et al., 2005 LOC387715 AR Usher syndrome, French 14q32.11–14qter 4 at 150.799494 Mb Kaplan et al., 1991 USH1A, USH1 AR RP 16p12.3–16p12.1 7 at 60.0 cM Finckh et al., 1998 RP22 AD central areolar choroidal dystrophy 17pter–17p13.1 11 at 68.798082 Lotery et al., 1996 CACD Note: Most of the data came from the website RetNet. The eye signal is the average expression level across all strains of the specific transcript in the eye expressed on a log scale. The clones are expressed at a level of 2 log units less in BXD24, which has acquired a mutation that causes photoreceptor degeneration. AR, autosomal recessive; RP, retinitis pigmentosa.

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nificant variation in phenotypes. These genetic heterogeneities can result in differences in behavior, neural structure, number of neurons, protein expression, and relative abundance of mRNA species. The simplest example is a Mendelian trait with a specific phenotype such as albinism. With the number of inbred strains available, the expression levels of specific transcripts can easily be examined. For example we can look at the levels of tyrosinase (Tyr) across a number of strains (figure 54.2). The lack of tyrosinase function is directly related to a common mouse phenotype, albinism. In figure 54.2, the level of Tyr is illustrated for 21 inbred strains (the data are from HEIMED). The pigmented strains are represented by solid bars and the albino strains by white bars. In general, strains with lower levels of Tyr are animals that are albino and not all albino mice have low levels of Tyr transcript. This points out the simple nature of most mutations. The lack of pigment is related not to the level of the Tyr transcript but to the normal functioning of the tyrosinase protein. This example of Tyr serves as a simple caution always to relate the analysis of data to the biology of the system, especially when examining levels of transcripts. This serves as a word of caution as we begin to examine the differences in the genomes of inbred strains and specific transcriptome profiles within the eye. These genomic or genetic differences may or may not be fully reflected in

clones); keratin complex 1; acidic gene 12 (Krt1-12, 44 independent clones); transketolase (Tkt, five independent clones); and uroplakin 1B (Upk1b, three independent clones). Furthermore, many of the genes in the list are key features of the corneal proteome (see chapter 57, this volume). These include the corneal crystallins, Aldh3A1 and Tkt, which are highly expressed in the cornea (Piatigorsky, 2000; Estey et al., 2007). The list also includes genes of unknown function in the cornea, such as Upk1b. Uroplakin1b is thought of as a bladder protein expressed in the transitional epithelium as part of a complex with the other two known uroplakin proteins (Min et al., 2006; Sun, 2006). The ability to extract this corneal transcriptome signature from the whole eye database illustrates the power of HEIMED. The minor but consistent variations in corneal gene expression across the strains of mice in the data set allow the software to extract a group of genes uniquely expressed in the cornea from a data set consisting of the transcriptome of the whole eye.

Genetic networks in the mouse eye In addition to providing molecular signatures of different cell types and tissues of the eye, HEIMED can reveal genetic networks that underlie phenotypic differences in the mouse eye. The genetic heterogeneity in inbred mice results in sig-

Eye M430v2 09/06 RMA : 1417717_a_at by Case (ranked) 12.0

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*

Value

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Figure 54.2 Expression levels of tyrosinase (Tyr) are shown for many of the inbred strains from the mouse diversity panel (see names along the ordinate). The scale to the left is a log scale of transcript level expressed as a mean and standard deviation as

CAST/EiJ

CAST/Ei

DBA/2J

C3H/HeJ

WSB/EiJ

MOLF/EiJ

NZO/HILtJ

C57BL/6J

KK/HIJ

B6D2F1

LG/J

BALB/cByJ

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129S1/SvImJ

D2B6F1

A/J

FVB/NJ

BXSB/MpJ

NZW/LacJ

PWD/PhJ

9.0

PWK/PhJ

9.5

defined by the Affymetrix chip. Note the low levels of Tyr in most of the albino mice (white bars). The strains that have not been sequenced are indicated by an asterisk above the histogram bar.

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protein expression levels or alterations in functional interactions of the protein. In most cases, the ultimate predictor of phenotype is the protein, not the transcript. Unlike the simple Mendelian trait of albinism, where a single base mutation can cause a complete lack of protein function, most physical traits are regulated by complex genetic interactions that result in modest variation in different transcripts. The genomic loci regulating these complex traits can be difficult to extract from a genetically diverse outbred population. The genetic variability in inbred strains and recombinant inbred strains (such as the BXD strain set) offers unique opportunities to define genomic loci regulating complex traits. The study of these complex traits is facilitated by the availability of many different inbred strains of mice and specific Web-based genomic tools, many of which can be found at GeneNetwork. In this highly interactive database, numerous phenotypes are presented from sets of inbred panels of mice, including CNS phenotypes for cerebellum (Neumann et al., 1993), hippocampus (Lu et al., 2001; Kempermann et al., 2006), striatum (Rosen and Williams, 2001), and amygdala (Mozhui et al., 2007). Of specific interest to the vision community are data sets related to the eye, the retina, and the central visual system. At the University of Tennessee, the Williams group has used these genetically variable populations of mouse strains to define regulatory loci controlling specific phenotypes. These phenotypic characterizations include variability in retinal ganglion cell (RGC) number, eye weight, and cell number in the lateral geniculate nucleus (Williams, Strom, and Goldowitz, 1998; Seecharan et al., 2003). For example, the population of RGCs in the mouse has a bimodal distribution, from a low of approximately 45,000–50,000 in CAST/Ei and BXD27 to a high of 75,000 in BXD5 and BXD32 (Williams et al., 1996; Williams, Strom, and Goldowitz, 1998; Seecharan et al., 2003). A major QTL on chromosome 11 that modulates RGC population size, Nnc1, has been mapped to a 4 Mb interval (peak at 98 Mb, 1.5 LOD confidence interval 97–101 Mb). Within this locus are genomic elements (potential candidate genes) that are involved in regulation of the number of RGCs in the mouse retina (Williams et al., 1996). In addition, Williams, Strom, and Goldowitz (1998) mapped two other contributing loci on chromosome 2 and chromosome 8 by simply quantifying the number of RGCs in 38 recombinant inbred strains and using composite interval-mapping techniques. This type of quantitative method was also used to map genomic loci controlling eye weight (Zhou and Williams, 1999) and cell number in the lateral geniculate nucleus (Seecharan et al., 2003). The traditional QTL mapping strategy defines loci that regulate a physical trait. This approach allows a systematic approach to defining a major genomic loci regulating a complex trait, such as RGC number in the mouse. Furthermore, we can use the QTL mapping methods as the basis

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for defining genetic networks controlling gene expression and phenotypes. The power of inbred strains extends beyond their use as a tool to map genomic loci controlling physical traits. These reference panels of mice can also be used to unravel genetic networks controlling the structure of the eye, its development, and its response to diseases. By treating the changes in mRNA levels as a physical phenotype, differences in transcriptional control can be evaluated using traditional QTL mapping methods to define groups of transcripts with similar patterns of expression. These similar patterns of expression reveal commonality in regulatory networks that are correlated to specific genomic loci. By combining gene expression profiling with genetic linkage analysis, the location of genetic elements that modulate expression levels of specific groups of genes can be identified (Darvasi, 1998; Broman, 2005). This type of expression genetics has been used to identify genes underlying complex traits, diseases, and behavioral phenotypes (Morley et al., 2004; Bystrykh et al., 2005). Examples of this type of integrated gene expression profiling and linkage analysis are studies that have identified genes predisposing individuals to hypertension (Cd36) and atherosclerosis (ABCG5 and ABCG8) (Aitman et al., 1999; Berge et al., 2000). These studies illustrate the power of combining gene expression profiling with linkage analysis to study gene expression.

Quantitative trait locus mapping in the Hamilton Eye Institute Mouse Eye Database The QTL mapping functions in GeneNetwork can be used as a tool to understand the genetic networks controlling changes observed in traditional micoarray studies. As stated earlier, the genetic variability in inbred strains and mouse diversity (BXD) strain sets can be used to define groups of transcripts with similar patterns of expression. These similar patterns reveal commonality in regulatory networks that are correlated with specific genomic loci. By combining gene expression profiling with genetic linkage analysis, the location of genetic elements that modulate expression levels of specific groups of genes can be identified (Darvasi, 1998; Broman, 2005). The high levels of heritable variation in gene expression allow researchers to correlate the expression variability with one or more regions of the genome. Some of these loci modulate genes within the interval where they are located (cis-acting), although most loci modulate genes from a distance, at a different genomic location (trans-acting). HEIMED allows exploration of the genetic variability in the BXD RI strains and selected inbred strains. To examine regulatory networks, the BXD RI strain set plays a preeminent role in identifying QTLs controlling the level of gene expression. This analysis can be used to define cis-acting and trans-acting QTLs.

Three transcripts (Tyrp1, Gpnmb, and Tyr) were selected. The Tyrp1 gene and the Gpnmb gene show strong cis-QTLs (figure 54.3A and C ); Tyr demonstrates a strong trans-QTL. The Tyr gene is quite variable across mouse strains; there is a twofold difference in gene expression across the BXD RI strains and other inbred strains. Running a genome-wide scan by selecting the interval mapping function reveals a linkage of expression variability of Tyr with a locus on chromosome 4 (see figure 54.3A). Note that the peak linkage on chromosome 4 likelihood ratio statistic (LRS) is more than 40 megabases and is at the same locus as the Tyrp1 gene itself. The strong cis-QTL of Tyrp1 at the same genomic locus as the trans-QTL for Tyr makes Tyrp1 a potential candidate gene controlling the gene expression variability of Tyr.

Defining genetic networks in traditional microarray experiments Like many groups, we have used microarray methods to profile global changes following specific experimental manipulations. In this process, changes in gene expression are identified and analyzed to define functional patterns of expression. For most microarray studies, this is the end of the process. However, with the bioinformatics tools offered on GeneNetwork, the analysis of microarray gene clusters can be extended to define genetic networks regulating these changes in gene expression. To illustrate this process, we take an example from our research group. We have used microarray techniques to obtain a picture of the global changes occurring in the retina following injury (Vazquez-Chona et al., 2004). The transcriptome-wide analysis was designed to look at the temporal regulation of transcripts controlling retinal wound healing. This gene expression profiling identified clusters of genes that are regulated in specific temporal patterns (Vazquez-Chona et al., 2004). From all these data, the response of the retina to injury could be subdivided into three temporal phases of gene expression: early acute, delayed subacute, and late chronic. Transcripts in each phase appear to be functionally related and reflect known cellular changes. The global changes occurring after injury are similar across different injury models, including mechanical trauma, ischemia, and increased intraocular pressure. The similarities in these profiles suggest that the changes in gene expression are part of common biochemical and cellular processes involved in the response of the retina to injury. As with many microarray experiments, this description of changes in the transcriptome leaves one with clusters of genes and little understanding of the basic mechanisms involved in regulating gene expression. To take the analysis to the next level, we used the databases and bioinformatics tools on GeneNetwork to examine the common regulatory elements controlling the clusters of genes in the temporal response to injury.

The basic approach to extract the underlying genetic networks involves, in sequence, identifying robust changes in gene expression following injury; defining regulatory loci (trans-QTLs), using genetic analysis of transcript data at GeneNetwork; and predicting candidate regulators, using bioinformatic resources that are available online. We defined a set of acute phase genes that are commonly expressed in the retina, brain, and spinal cord after traumatic injury (Vazquez-Chona et al., 2005). We then used the expression QTL analysis combined transcriptome profiling with linkage analysis to reveal variability in the chromosomal loci modulating gene expression. The analysis was made using the BXD RI strain sets and the bioinformatics tools available at genenetwork.org. Expression of acute phase genes in BXD RI mouse forebrains is modulated by a major QTL on chromosome 12 between 15 and 32 Mb. With the loci identified, the next step was to identify the candidate genes. The identification of candidate genes within specific intervals of the BXD strain set is simplified by the nature of the strain set and the expression data set. For a gene to have a QTL that can be mapped, there must be significant variability in gene expression across the BXD strain set. In HEIMED, 10,000 probe sets have significant variability and have a significant LRS score. The second feature that identifies a potential candidate gene is that it must either be within the locus or have a cis-QTL. Within HEIMED, 5,000 probe sets have significant cis-QTLs. Within the chromosome 12 interval, there are three good candidate genes, Id2, Lpin1, and Sox11. All these transcripts have strong cis-QTLs and are known either to be transcription factors (Yokota and Mori, 2002; Jankowski et al., 2006) or to have the potential to localize to the cell’s nucleus (Peterfy et al., 2005). Thus, these three genes are ideal candidates to be upstream regulators of the injury network. The novel combination of microarray analysis, expression genetics, and bioinformatics provides a new and powerful approach to defining regulatory elements in the genome. Using this approach, we were able to generate specific, testable hypotheses to define the pathways that regulate proliferative and reactive responses in the retina and elsewhere in the CNS. As more diverse gene expression data sets become available, a comparison of gene expression and regulation in different biological contexts should help identify the regulatory elements controlling the reactive response in the retina.

Conclusion The preceding analysis provides a glimpse into the revolution in genomics and genetics that is allowing us to examine and compare gene expression in different tissues. By defining the genetic variability among strains, we can now account

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Figure 54.3 Interval mapping computes linkage maps for the entire genome. The numbers across the top of the three panels indicate mouse chromosomes, from Chr 1 to Chr X. We used the genetic analysis of transcript expression at GeneNetwork to define genomic loci that control transcript abundance variability in mouse eye. The variability in transcript abundance in the segregating population of BXD RI strains makes it possible to map transcript abundance to a specific chromosomal locus. Genetic linkage maps (A–C ) show genes with significant LRS scores (the significance level is indicated by the upper line). A, Individual genome-wide maps for Tyrp1 demonstrating a strong cis-QTL (the location of the gene

is indicated by the black triangle). B, Genome-wide scan for Tyr, a gene with a strong trans-QTL. C, Genome-wide scan for Gpnmb, an additional gene with a strong cis-QTL. Maps were generated by linking transcript variability against 8,222. The linkage between transcript variation and genetic differences at a particular genetic locus is measured in terms of likelihood ratio statistic (LRS, solid line). Dashed horizontal lines mark transcript-specific significance thresholds for genome-wide P < 0.05 (upper band) and genomewide P < 0.63 (lower band). The triangle indicates gene location. Linkage maps were generated using the interval mapping tool at GeneNetwork.

for many phenotypic differences in the mouse eye. The mouse eye is genetically unique in that findings in the mouse can directly apply to understanding gene expression in human conditions leading to the loss of sight. Defining the genetic variability and differences in gene expression across strains of mice that are fully mapped and sequenced allows a direct correlation between eye phenotypes and disease states with genomic loci and candidate genes. Transcriptome-wide analysis of the eye allows us to define genetic variability between strains not only to specific cellular phenotypes, but also to the response of cells to changes in the state of the eye. Using HEIMED allows the vision research community to define molecular signatures within the tissues of the eye, identify candidate genes for human disease, begin to understand genetic networks regulating tissue-specific gene expression, and identify the complex interactions of genomic loci that underlie the complex structures of the eye. As technology progresses, it should be possible to use cell type–specific analysis of mRNA and protein expression across large genetically defined and manipulated panels of mice. The use of these methods in large genetic reference panels of mice and the multiscale integration of the resultant data should allow for the definition of genes responsible for complex genetic diseases. We are currently using this approach to define genomic loci and candidate genes associated with susceptibility and resistance retinal ganglion to cell death in human diseases. acknowledgments

Support for the acquisition of microarray data sets was generously provided by Dr. Barrrett Haik, chair of the Department of Ophthalmology and director of the Hamilton Eye Institute. This work is supported by the National Eye Institute (RO1EY17841), the Integrative Neuroscience Initiative on Alcoholism (U01AA13499, U24AA13513), the Human Brain Project (P20-DA21131), NCI (U01CA105417), and the Biomedical Informatics Research Network, NCRR (U24RR021760). All arrays were processed at the VA Medical Center, Memphis, by Dr. Yan Jiao and Dr. Weikuan Gu. We thank Dr. Lu Lu for his contributions to every aspect of the HEIMED project, Bill Orr for his assistance, and Dr. Mohamed Nassr for his assistance in data analysis.

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Sun, T. T. (2006). Altered phenotype of cultured urothelial and other stratified epithelial cells: Implications for wound healing. Am. J. Physiol. 291(1):F9–F21. Surguchov, A., Bronson, J. D., Banerjee, P., Knowles, J. A., Ruiz, C., Subbaraya, I., Palczewski, K., and Baehr, W. (1997). The human GCAP1 and GCAP2 genes are arranged in a tailto-tail array on the short arm of chromosome 6 (p21.1). Genomics 39(3):312–322. Swanson, D. A., Chang, J. T., Campochiaro, P. A., Zack, D. J., and Valle, D. (1998). Mammalian orthologs of C. elegans unc119 highly expressed in photoreceptors. Invest. Ophthalmol. Vis. Sci. 39(11):2085–2094. Swaroop, A., Xu, J. Z., Agarwal, N., and Weissman, S. M. (1991). A simple and efficient cDNA library subtraction procedure: Isolation of human retina-specific cDNA clones. Nucl. Acids Res. 19(8):1954. Taylor, B. A., Wnek, C., Kotlus, B. S., Roemer, N., MacTaggart, T., and Phillips, S. J. (1999). Genotyping new BXD recombinant inbred mouse strains and comparison of BXD and consensus maps. Mamm. Genome 10(4):335–348. Tubb, B. E., Bardien-Kruger, S., Kashork, C. D., Shaffer, L. G., Ramagli, L. S., Xu, J., Siciliano, M. J., and Bryan, J. (2000). Characterization of human retinal fascin gene (FSCN2) at 17q25: Close physical linkage of fascin and cytoplasmic actin genes. Genomics 65(2):146–156. Vazquez-Chona, F., Khan, A. N., Chan, C. K., Moore, A. N., Dash, P. K., Hernandez, M. R., Lu, L., Chesler, E. J., Manly, K. F., et al. (2005). Genetic networks controlling retinal injury. Mol. Vision 11:958–970. Vazquez-Chona, F., Song, B. K., and Geisert, E. E., Jr. (2004). Temporal changes in gene expression after injury in the rat retina. Invest. Ophthalmol. Vis. Sci. 45(8):2737–2746. Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., Smith, H. O., Yandell, M., Evans, C. A.,

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et al. (2001). The sequence of the human genome. Science 291 (5507):1304–1351. Wang, X., Le Roy, I., Nicodeme, E., Li, R., Wagner, R., Petros, C., Churchill, G. A., Harris, S., Darvasi, A., et al. (2003). Using advanced intercross lines for high-resolution mapping of HDL cholesterol quantitative trait loci. Genome Res. 13(7): 1654–1664. Waterston, R. H., Lindblad-Toh, K., Birney, E., Rogers, J., Abril, J. F., Agarwal, P., Agarwala, R., Ainscough, R., Alexandersson, M., et al. (2002). Initial sequencing and comparative analysis of the mouse genome. Nature 420(6915):520– 562. Williams, R. W., Strom, R. C., and Goldowitz, D. (1998). Natural variation in neuron number in mice is linked to a major quantitative trait locus on Chr 11. J. Neurosci. 18(1):138–146. Williams, R. W., Strom, R. C., Rice, D. S., and Goldowitz, D. (1996). Genetic and environmental control of variation in retinal ganglion cell number in mice. J. Neurosci. 16(22):7193–7205. Williams, R. W., Strom, R. C., Zhou, G., and Yan, Z. (1998). Genetic dissection of retinal development. Semin. Cell Development. Biol. 9(3):249–255. Yang-Feng, T. L., and Swaroop, A. (1992). Neural retina-specific leucine zipper gene NRL (D14S46E) maps to human chromosome 14q11.1–q11.2. Genomics 14(2):491–492. Yokota, Y., and Mori, S. (2002). Role of Id family proteins in growth control. J. Cell. Physiol. 190(1):21–28. Zhang, Q., Zulfiqar, F., Xiao, X., Riazuddin, S. A., Ayyagari, R., Sabar, F., Caruso, R., Sieving, P. A., Riazuddin, S., et al. (2005). Severe autosomal recessive retinitis pigmentosa maps to chromosome 1p13.3–p21.2 between D1S2896 and D1S457 but outside ABCA4. Hum. Genet. 118(3–4):356–365. Zhou, G., and Williams, R. W. (1999). Eye1 and Eye2: Gene loci that modulate eye size, lens weight, and retinal area in the mouse. Invest. Ophthalmol. Vis. Sci. 40(5):817–825.

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Mouse Models, Microarrays, and Genetic Networks in Retinal Development and Degenerative Disease SUNIL K. PARAPURAM AND ANAND SWAROOP

Retinal degenerative diseases (RDs) are a major cause of untreatable blindness in the Western world (Bressler et al., 2003; Hartong et al., 2006). A vast majority of RDs are categorized as orphan diseases, affecting fewer than 200,000 individuals in the population; nevertheless, these diseases collectively represent an important health problem (Weleber, 2005). Of almost 200 mapped RD loci, as many as 130 genes have been identified (www.sph.uth.tmc.edu/Retnet/sumdis.htm). These studies have assisted in better clinical diagnosis and management (Kalloniatis and Fletcher, 2004; Hartong et al., 2006). However, the precise molecular events and cellular pathways of disease pathogenesis are relatively less well understood, and effective treatment paradigms are still not available for most retinopathies. A major challenge has been the heterogeneity of mutations resulting in photoreceptor degeneration, making it economically impractical for pharmaceutical companies to invest in therapies that may alleviate the pathology in small subsets of populations. Mutational heterogeneity is illustrated most noticeably in the case of rhodopsin, where at least 88 mutations can cause the autosomal dominant type of retinitis pigmentosa (RP) yet two alleles lead to RP only in a homozygous state (Gal et al., 1997). Another impediment is that the affected individuals exhibit extensive variations in disease phenotype even though the same gene may be involved. Thus, the design of therapeutic strategies must take into account the complexity of retinal phenotypes and the heterogeneity of gene defects. Gene therapy, a paradigm for individualized treatment, has been used extensively to deliver a functional gene to counter the effect of a nonfunctional gene or to eliminate a mutant allele in animal models of RD (Acland et al., 2001; Hauswirth and Lewin, 2000; Mori et al., 2002; Dinculescu et al., 2005). A number of generalized approaches are also being developed to surmount the complex diversity of RDs; these include the surgical implantation of optoelectronic retinal prostheses (Zrenner, 2002; Humayun et al., 2003;

Rizzo et al., 2004), transplantation of cells (Gouras et al., 1994; Lund et al., 2001; MacLaren et al., 2006), and treatment with growth or survival factors (Faktorovich et al., 1990; Frasson et al., 1999; Delyfer et al., 2004). All of these therapeutic strategies suffer from certain limitations. Gene therapy protocols need to address extensive heterogeneity in gene mutations, apart from safety and efficacy problems associated with viral vectors. For electronic chips, limited field of vision and resolution are among some of the difficulties. Transplantation of cells faces different kinds of hurdles, including functional integration into the retina, biocompatibility, long-term survival of cell transplants, and ethical issues. In the case of treatments involving growth and survival factors, the delivery of optimal amounts and their usefulness or side effects over the long term constitute primary concerns. We therefore need to incorporate additional paradigms for effective design of therapies for RDs. Comprehensive studies have established that dysfunction or death of rod and cone photoreceptors are the primary cause of blindness in the vast majority of RDs (Rattner et al., 1999; Pacione et al., 2003). A better understanding of photoreceptor development, function, and survival would greatly assist in effective design of treatments for blinding retinal diseases. This chapter first discusses gene profiling using microarrays and the construction of regulatory networks underlying photoreceptor differentiation. We then describe a systematic approach to delineating molecular pathways of photoreceptor cell death caused by inherited gene defects, with a goal of developing novel treatment paradigms.

Expression profiling using microarrays Microarrays of oligonucleotides or DNA fragments derived from expressed sequence regions allow simultaneous measurement of expression changes in thousands of genes under a given condition (Duggan et al., 1999; Zareparsi et al.,

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2004). Temporal gene profiles of a specific tissue or cell type can provide valuable information for constructing networks or pathways involved in a complex biological process, such as differentiation or disease pathogenesis. The choice of a specific microarray platform (e.g., cDNA array, short vs. long oligonucleotide arrays) for expression profiling depends on the purpose of the experiments. Because our goal is to compare gene profiles from multiple different experiments (i.e., profiles from the retina or photoreceptors of multiple mutant mice at different stages of disease progression), it is highly desirable to employ stringent quality control and minimize intersample experimental variations. Therefore, we have optimized conditions for microarray analysis using mouse GeneChips MOE430.2.0 (www.affymetrix.com); these microarrays include 45,101 probe sets corresponding to about 39,000 transcripts of 34,000 annotated genes. Discussions of microarrays and associated methods can be found in several recent publications (Duggan et al., 1999; Livesey et al., 2000; Mu et al., 2001; Farjo et al., 2002; Yoshida et al., 2002; Diaz et al. 2003; Hackam et al., 2004; Zareparsi et al., 2004). A major challenge in gene profiling experiments is the extraction of useful information from enormous data sets. The mammalian retina consists of six major types of neurons and one type of glia; however, many distinct subtypes can be identified based on morphology and function (Masland, 2001). Global profiling of the retina would reveal average changes in expression of genes in many different cell types; several of these would reflect manifestations secondary to RD. Notably, a number of photoreceptor-specific genes are mutated in RDs, and even when the mutant gene is widely expressed, photoreceptors are considered the primary site of disease in a majority of patients with RD. In patients with RP and in most rd mouse mutants, rods appear to be the first retinal cell type that is affected. Even in age-related macular degeneration (AMD), increased loss of rods is observed in the central visual field during early stages (Curcio et al., 1996). It is therefore likely that gene profiling of purified rod (and cone) photoreceptors rather than the whole retina will yield more useful data for network or pathway construction to fully comprehend differentiation or degenerative disease.

NRL and gene profiling of purified rod (and cone) photoreceptors The basic motif-leucine zipper transcription factor (TF), NRL (Swaroop et al., 1992), is essential for rod photoreceptor differentiation; loss of NRL in mice (Nrl−/−) results in transformation of postmitotic rod precursors to cones (Mears et al., 2001; Daniele et al., 2005; Akimoto et al., 2006), whereas ectopic expression of NRL converts cones to rods (Oh et al., 2007). NRL is expressed preferentially in rods and

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the pineal gland (Swain et al., 2001; Akimoto et al., 2006). We recently used Nrl promoter to direct the expression of GFP (Nrlp-EGFP transgene) in mice (wt-Gfp) and successfully marked the rod photoreceptors with GFP at the time of birth (Akimoto et al., 2006). GFP tagging allowed us to identify and purify rod photoreceptors at different stages of development using flow cytometry (FACS analysis) (figure 55.1). Evaluation of retinas from the Nrl−/−-Gfp mice (generated by breeding wt-Gfp mice with the Nrl−/− mice) provided a direct demonstration of the transformation of rods to cones in the absence of NRL, and this also led to efficient enrichment of cone photoreceptors by FACS (Akimoto et al., 2006). To produce gene signatures of developing and mature rods or cones, we used total RNA from 1–5 × 105 FACSpurified cells for linear amplification. Biotin-labeled fragmented cDNAs derived from the amplification were then hybridized to mouse GeneChips (Affymetrix). Profiling of rods (from wt-Gfp retina) and cones (from Nrl−/−-Gfp retina) at five distinct stages of development revealed comprehensive sets of differentially expressed genes that distinguish photoreceptors from other cell types (Akimoto et al., 2006). This expression analysis also uncovered the advantages of profiling purified cells, as many changes were missed when profiles of the entire retina were studied. Differential gene expression patterns at specific developmental stages provide basic data sets for higher-order analysis, such as clustering, to identify genes that behave similarly and those unique to a particular cell. However, construction of regulatory networks or pathways is a major challenge, requiring sophisticated statistical tools to bring out features such as key regulators (nodes or hubs), the hierarchies they sustain, and the genes they regulate.

Characteristics of biological networks and relevance to photoreceptors The rationale for generating regulatory networks is to explain the spatial and temporal execution of specific cellular functions (Davidson et al., 2003). Networks consist of nodes representing molecules (such as protein, DNA, RNA and metabolites) and edges that explain the association between nodes. Networks offer a quantifiable description based on factors such as degree or connectivity between hubs, degree distribution, path length, and clustering coefficient between the various elements (Barabasi and Oltvai, 2004; Blais and Dynlacht, 2005). A majority of biological networks are scalefree and, unlike random networks, are nonuniform, having few highly connected hubs and many nodes that have only a few links (Barabasi and Albert, 1999). Many networks also display the “small world” property with high clustering but small characteristic path lengths between two nodes (Watts and Strogatz, 1998). Transcriptional and posttranscription regulatory networks can describe many of the cellular

Figure 55.1 A, A single rod photoreceptor isolated from adult wt-Gfp mice shows the expression of GFP. B, The same cell shows staining for rhodpsin (red) in the outer segment (OS) and inner segment (IS) regions and bis-benzimide staining (blue) for the

nucleus. C, Samples of dissociated cells are viewed under the microscope before (shown) and after flow sorting. D, Flow cytometry allows the GFP-positive population of cells to be gated (R3) and sorted separately. See color plate 63.

activities and are subdivided into physical and functional networks. Although physical networks are represented by interactions such as protein-protein/DNA/RNA or RNARNA, the functional networks represent the outcome of these interactions, such as regulation of gene expression or signaling pathways (Walhout, 2006). Analysis of highthroughput yeast two-hybrid data has revealed about 2,800 interactions among 8,100 human proteins tested, discovering more than 300 new connections to more than 100 disease-associated proteins (Rual et al., 2005). Alterations in transcriptome and proteomic signatures in metastatic and clinically localized prostate cancer, along with the concordance between the protein and transcript levels, were able to predict clinical outcomes in prostate cancer and other solid tumors (Varambally et al., 2005). Though biological network assemblies are still in their infancy, numerous recent studies represent important steps toward understanding complex physiological processes. At this stage, it is not conceivable to decipher the single master network that would describe all the transcribed genes, expressed proteins, and protein-protein interactions in the entire retina (or even in rod and cone photorecep-

tors) at a single specific developmental stage. Hence, our goal is to establish gene regulatory networks in rod photoreceptors under normal and disease conditions. This information can then be integrated into protein expression and interaction networks. These subnetworks are not independent; instead, they link with each other to form more complex patterns. Hence, the biological process can be visualized as the highly regulated and progressive generation of subnetworks that combine together to describe the unique properties of a specific cell type during differentiation or disease (Blais and Dynlacht, 2005; Fraser and Marcotte, 2004).

Hierarchy in regulatory networks underlying photoreceptor development A key question pertaining to photoreceptor development is how NRL expression in postmitotic photoreceptor precursors initiates the cascade of molecular events that suppress the cone-specific differentiation pathway and establish rod lineage and function. In many cases TFs form key hubs of a regulatory network and display a hierarchical organization

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because they regulate the expression of one or more genes. Extensive pyramid-shaped hierarchical structures have been described in the regulatory networks of E. coli and S. cerevisiae, with most of the transcription factors occupying the lower stratum and only a few master transcription factors occupying the top echelon (Yu and Gerstein, 2006). A similar hierarchical structure was identified in C. elegans, where three or more layers of transcriptional control existed (Deplancke et al., 2006). Establishing such hierarchies in the regulatory network of rod development would require integration of large gene expression data sets, obtained from as many stages of differentiation as possible (from birth to maturation). It would also be necessary to perform comprehensive gene profiling of purified rods after altering the function (e.g., by RNAi, transgenics, or knockout) of one or more key NRL-interacting proteins (such as CRX) and of NRL’s downstream targets (such as NR2E3). This can be easily accomplished by breeding the wt-Gfp mice with various mutant strains (figure 55.2). To dissect the role of NRL and other transcriptional regulators (such as NR2E3 and CRX) in photoreceptor differentiation, we have also generated a number of transgenic mouse lines on wild-type (wt) or Nrl−/− background (Yoshida et al., 2004; Cheng et al., 2006; Oh et al., 2007). We are now producing gene profiles of FACS-purified rods with specific genetic mutation or transgenes and have begun constructing cluster networks. We have developed methods using transitive coexpression analysis to tightly cluster functionally related genes by simultaneously including their biological and statistical importance, even when their expression profiles are dissimilar (Zhu et al., 2005). These clusters would form initial components in constructing networks that could

explain the role of transcription factors and signaling molecules in controlling rod differentiation.

Mouse models of retinal degenerative disease and pathways of photoreceptor cell death Mouse models of RDs provide candidate genes for human retinopathies, insights into the mechanisms of disease pathogenesis, and comprehensive evaluation of treatment techniques. Careful ocular phenotyping of naturally occurring inbred strains of mice have already identified at least 35 mutants that exhibit different types of RD (Rattner and Nathans, 1999; Frederick et al., 2000; Bessant et al., 2001; Chang et al., 2002; Pacione et al., 2003). Several of the rd strains (e.g., rd1, rd2 [rds], rd3, rd7, rd16) have led to the discovery of Pde6b, Prph2, Rd3, Nr2e3, and Cep290/Nphp6, respectively, as genes involved in photoreceptor biology (Travis et al., 1989; Bowes et al., 1990; Akhmedov et al., 2000; Chang et al., 2006; Friedman et al., 2006); their corresponding homologues in humans have similar biological functions and are mutated in retinopathies (McLaughlin et al., 1993; Travis and Hepler, 1993; Haider et al., 2000; Wright et al., 2004; den Hollander et al., 2006; Friedman et al., 2006; Sayer et al., 2006). Numerous transgenic and knockout mouse models have also been generated in various laboratories to investigate the physiological function of respective genes and associated disease mechanisms (Humphries et al., 1997; Redmond et al., 1998; Furukawa et al. 1999; Hong et al., 2000; Mears et al., 2001; Zhao et al., 2003; Cheng et al., 2006; Lee et al., 2006). With a goal of developing knowledge-based treatments and therapies, we proposed a comprehensive approach to

Rods with OS

Nrl +/+ , Cr Crx +/+ , Nr2e3 +/+ WT

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Nrl -/- , Cr Crx +/+ , Nr2e3 -/-

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Committed to Rod Lineage? [Post Mitotic Cells]

Committed to Rod Lineage [Rod Precursors]

Crx-ko

Nrl +/+ , Cr Crx +/+ , Nr2e3 -/-

Hybrid / abnormal rods

rd7

Figure 55.2 Role of regulatory genes (NRL, CRX, and NR2E3) in the network(s) leading to rod and/or cone differentiation can be studied by loss of their function in mice. Breeding mutant mice with

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the wt-Gfp mice can provide GFP-tagged rods that can be isolated for gene profiling.

examine commonalities in pathways of photoreceptor cell death caused by mutations in different mutant genes (Yu et al., 2004) (figure 55.3). Though multiple, distinct mutations in more than 100 genes result in RD, it is believed that apoptosis is the final step in photoreceptor cell death (PorteraCailliau et al., 1994; Wong, 1995; Travis, 1998). Thus, it is intuitive to hypothesize that diverse genetic defects eventually converge at a few specific signaling pathways that finally induce apoptosis. The failure to prevent degeneration by inhibition of certain apoptotic pathways (Doonan et al., 2005) indicates that it might either be too late to rescue a cell if apoptotic pathways are set in motion or all apoptotic pathways may need to be simultaneously blocked. We reasoned that delineation of early steps in the cell death pathway induced by distinct genetic mutations would identify common pre-apoptotic signaling (PAS) molecules (see figure 55.3). These molecules can serve as better targets for drug discovery, as photoreceptors may not yet be committed to apoptosis. In addition, PAS-targeted drugs may benefit a larger group of affected RD individuals. Given that photoreceptor degeneration in most rd mutant mice (akin to affected individuals) manifests over a relatively long time frame, an adaptive cellular response should result in altered gene expression profiles as disease progresses. We therefore propose that the temporal profiling of gene expression during disease progression would reveal how a specific genetic mutation causes the death of photoreceptors. Metaanalysis of temporal gene profiles from multiple mouse mutants should then uncover common PAS molecules.

Rod

Mutant Gene Rhodopsin Peripherin Pde6b Rpgr Cep290 Rp1

Crx Nrl Nr2e3 Rd3

Probable Dysfunction

Degeneration network(s) by expression profiling of photoreceptors from rd mice The key question is how inherited mutation(s) in a large number of genes cause rod (or cone) dysfunction, eventually leading to cell death. We can analyze this in the context of networks. A scale-free network has the ability to withstand errors because of the redundancy of the wiring and the robustness of various modules that function in a cell (Hartwell et al., 1999; Albert et al., 2000). However, the capacity for error tolerance also means that the system is prone to attacks (Albert et al., 2000). It is not difficult to imagine the breakdown of cellular functions due to a deleterious mutation in a critical component. Usually, the more connected a gene or protein is in a regulatory network, the more essential it is for the survival and function of the cell (Jeong et al., 2001; Deplancke et al., 2006). Thus, if and when regulatory networks of a cell, such as a rod photoreceptor, are delineated, one might be able to trace the problem and the effects that a mutation has on the module or the hub or the entire network, leading to RD. We need not simulate the mutation leading to the network breakdown, as numerous RD mouse models are already available. Our goals are therefore to produce gene profiles of rod photoreceptors isolated from various mouse models of RD and to compare these with the gene patterns or networks of normal rods. To accomplish this, we are breeding rd mouse strains with wtGfp mice to obtain homozygous rd mutants that have the GFP transgene, permitting FACS purification of mutant rod

Converging Signals

Cell Death

Outer segment biogenesis Ca++/ Metabolic overload

PAS 1

Trafficking

PAS 2

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PAS 3

Apo 1

Apo 2

Apo 3 Unknown

Figure 55.3 Apoptosis is the final step in photoreceptor cell death, even though RDs are caused by defects in different genes. We hypothesize that signaling pathways generated by different gene mutations (as indicated) will eventually converge on a few pre-apoptotic signals (PAS 1, 2, 3, . . .), which then induce apop-

totic (Apo 1, 2, 3, . . .) pathways, resulting in photoreceptor cell death. Genetic mutations that affect similar pathways may induce the same PAS, which can be targeted for drug therapy (Modified from Yu et al., 2004.)

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photoreceptors. In the case of autosomal recessive mutations, a relevant mouse strain that is homozygous for both the Nrlp-EGFP transgene and the rd mutant gene can be obtained by the F3 generation. In fact, a single copy of a GFP transgene is sufficient for rod enrichment by FACS. Gene profiling of rods carrying a specific gene mutation during various stages of disease progression will generate extensive data sets that can be used for constructing the pathways or networks leading to photoreceptor cell death. As indicated in figure 55.3, we compare temporal rod gene expression profiles from various rd mutants, with a goal of elucidating commonalities in cell death pathways or crosstalk in signaling networks. One can speculate that pathways from mutant genes that have similar functions may share more commonalities. Likewise, mice with early-onset RD may share more common signaling molecules than rd mice exhibiting slower photoreceptor disease. Identification of PAS molecules can lead to novel discovery targets, which could be modulated by drugs to prevent network errors and to alter the course of photoreceptor dysfunction or death. In most RDs, it is the rods that degenerate first, and the degeneration of cones is due to a by-stander effect (Ripps, 2002); thus, many laboratories have attempted to preserve cone function (Mohand et al., 2000; Leveillard et al., 2004). PAStargeted drugs, therefore, may contribute to cone survival and vision.

Future considerations In the field of ocular development and pathology, numerous investigators have amassed impressive data on gene expression patterns under normal and disease (mutant) conditions (Blackshaw et al., 2001; Sharon et al., 2002; Yoshida et al., 2002, 2004; Chowers et al., 2003; Farkas et al., 2004; Hackam et al., 2004; Akimoto et al., 2006; Cheng et al., 2006). However, progress in network construction has been relatively slow. A major problem is the lack of coordinated efforts with standard criteria and uniform gene profiling platforms that allow meta-analysis of large data sets obtained by different investigators. Even though we have focused on generating gene networks for rod differentiation and degeneration by exploiting our research on NRL, this approach can be applied to other cell types and tissues and to other disease paradigms. Studies of retinal proteome (Ethen et al., 2006) and protein interactions will undoubtedly complement the gene profiling data sets. It is our hope that collaborative studies will be undertaken by different laboratories using uniform standard profiling platforms to create integrative networks for better understanding of retinal (or photoreceptor) development, function, and disease. acknowledgments

Work was supported by grants from the National Institutes of Health (EY011115, EY007961, and

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EY007003), the Foundation for Fighting Blindness, Research to Prevent Blindness, and the Elmer and Sylvia Sramek Foundation. We thank Andy Goldberg and the members of the Swaroop laboratory, particularly Matthew Brooks, Hong Cheng, Radu Cojocaru, James Friedman, Hemant Khanna, Ritu Khanna, and Edwin Oh, for comments and discussions.

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56

Retinal Vascular and Retinal Pigment Epithelium Gene Expression MICHAEL I. DORRELL AND MARTIN FRIEDLANDER

The highly ordered, compact structure of the mammalian retina is well suited for the unimpeded transmission of light and its subsequent transduction into electrical signals that will be transformed into images in the visual cortex of the brain. The processing and orderly transmission of these signals from the posteriorly located photoreceptors through multiple intraretinal synapses with a variety of other neuronal cell types is critically dependent on maintenance of this highly ordered retinal structure. For reviews of neural retinal structure and synaptic formations, see Lukasiewicz (2005) and Mumm et al. (2005). This orderly development of the retinal structure is dependent on early cell guidance mechanisms to mediate appropriate cell-cell relationships, followed by long-term mechanisms that maintain retinal homeostasis and ensure that the striated retinal plexuses and neuronal synapses are not disrupted. Both the retinal pigment epithelium (RPE) and the retinal vasculature play important roles in maintaining retinal homeostasis and ultimately retinal function. Anatomical or physiological abnormalities of either system can lead to retinal dysfunction and loss of vision. To better understand how structural and functional homeostasis is established and maintained by various cellular components of the retina, numerous studies have focused on the RPE and retinal vasculature. A discussion of all the experiments defining individual gene expression and function in the RPE or retinal vasculature is well beyond the scope of this chapter. Thus, we mainly discuss large-scale gene expression analyses, focusing specifically on challenges faced in obtaining interpretable RPE and retinal vascular gene expression data, strategies to overcome these challenges, and future applications of RPE and retinal vascular gene expression studies. The RPE is a pigmented monolayer of polarized cells located just posterior to the photoreceptors, with many important retinal functions (figure 56.1). The RPE basolateral surface lies on Bruch’s membrane and mediates selective transport of nutrients from the blood vessels of the choriocapillaris to the outer neural retina, as well as removal

of water and waste products from the subretinal space to the blood. Tight junctions between the RPE cells allow the photoreceptor environment to remain separate from the fenestrated choriocapillaris while facilitating selective molecular transport between the two tissue beds, thus maintaining the outer blood-retinal barrier. The apical membranes of RPE cells form numerous long processes that partially envelop photoreceptor outer segments. This close interaction allows RPE cells to play a central supporting role for photoreceptor function by (1) regulating retinal (vitamin A) metabolism and distribution, a process required for the visual cycle and maintenance of photoreceptor excitability, and (2) phagocytosis of the photoreceptor outer segments that are shed daily, a process essential for photoreceptor survival (Futter, 2006). (For good reviews of RPE and visual function, see de Jong, 2006, and Strauss, 2005.) When RPE function is lost, retina function is affected dramatically. For example, mutations in RPE-specific genes are linked to numerous forms of retinal degeneration (Chong and Bird, 1999), and abnormalities in the RPE are associated with age-related macular degeneration (AMD; de Jong, 2006; Zarbin, 2004). Appropriate development and function of the retinal vasculature are also critical for maintaining proper visual function. The very high metabolic activity of the retina demands a substantial blood supply to provide oxygen, nutritional elements, and removal of metabolic waste products. Equally important, it is now believed that paracrine support functions are provided by vascular endothelial cells (ECs) for retinal neuronal elements (Otani and Friedlander, 2005). During development, retinal vessels in the mouse retina form three vascular plexuses in a highly reproducible manner, leading to the formation of distinct vascular and avascular zones within the inner neural retina (see figure 56.1). This process is similar to retinal vascular development in humans during the third trimester in utero. The choriocapillaris forms posterior to the RPE monolayer and Bruch’s membrane and supplies the vast majority of oxygen and nutrients to the retinal photoreceptors in the outer nuclear layer. Mechanisms of vascular guidance in the mouse retina

685

superficial GCL IPL

intermediate

INL deep

OPL ONL

photoreceptor outer segments

Light Absorption OS

Epithelial Transport

Visual Cycle

Glia

k

h·γ

Phagocytosis

Secretion

+

11-cis-retinal

MV

PEDF

photoreceptor outer segments RPE Bruch’s membrane choriocapillaris

Fenestrated Capillary Bed Glucose Vitamin A

CI –

H2O

VEGF

Figure 56.1 Top, The retina is a complex, striated tissue with highly organized neuronal layers. The inner retinal vasculature forms three distinct plexuses: The superficial plexus forms within the ganglion cell layer (GCL), the intermediate plexus forms at the inner edge of the inner nuclear layer (INL), and the deep plexus

forms at the outer edge of the INL. Bottom, Posterior to the retina are the RPE, Bruch’s membrane, and the choriocapillaris. The RPE performs many functions that are critical to retinal function. See color plate 64. (Top, From Dorrell and Friedlander, 2006. Bottom, From Strauss, 2005.)

have been extensively reviewed elsewhere (Dorrell and Friedlander, 2006). If any of the retinal or choroidal vascular layers fail to develop, the retina will lack adequate oxygen and nutrients, leading to cellular death and vision loss. Improper vascular organization or excessive neovascularization can also be quite detrimental to retinal function. For proof of the importance of maintaining a normal, appropriate retinal vasculature, one need only consider the vast majority of diseases that can cause a catastrophic loss of vision. Most of these diseases, which include AMD, diabetic retinopathy, retinopathy of prematurity, and others, have vascular components that contribute directly to pathological manifestation of the disease (Adamis et al., 1999; Eichler et al., 2004). Proper functioning of the RPE and vascular endothelium are critical to maintaining normal retinal function. Thus, analysis of gene expression in these cells should provide insight into how they facilitate normal retinal function and contribute to disease when they malfunction.

Methods of testing gene expression in the retina

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Methods to study gene expression have improved vastly in the past decade, particularly with the advent of and subsequent improvements in microarray and SAGE technologies. Microarray gene chips now allow researchers to study the expression of thousands of different genes simultaneously. Commercial products such as Affymetrix gene arrays now offer comprehensive analysis of genome-wide expression in human and mouse, as well as many other species often used for experimental purposes (for more information on Affymetrix chips, see www.affymetrix.com). These arrays mediate non-hypothesis-driven analysis of the expression levels of multiple, if not all, genes rather than a specific subset of predescribed genes. However, both technical and biological variability need to be considered when analyzing microarray data. Technical variation is caused by differences arising from nonbiological, technical aspects of the experiment, such

as differences in RNA preparation, RNA degradation, array labeling, irregularities in individual arrays, hybridization, washing, and image analysis. Biological variation results from natural variability within the biological system itself. These variables can arise from individual differences, litter effects, or other variables such as pathogen load or diet. Replication is essential to overcome these variables and to increase the likelihood of identifying true positives and reduce the incidence of false positives. In an optimal situation, technical and biological variability are tested separately by (1) testing the same RNA sample or RNA prepared from the same source, and applying to separate arrays (testing technical variability), and (2) testing RNA samples from separate individuals or sources (testing biological variability). Because of the cost of microarray experiments and limited sample availability, particularly when analyzing retinal material, extensive replicates may not be practical. In these instances, biological replications often suffice, since technical variability is innately accounted for within biological replicates; RNA must be prepared separately from each source and ultimately hybridized to separate chips. However, important information regarding the source of variability (i.e., is the variability due to technical problems or natural biological variability?) will be lost. It is also important to validate microarray results using more traditional techniques such as RT-PCR, in situ hybridization, and in vivo analysis of transgenic and knockout mice. In evaluating published reports on gene expression using large-scale array analysis, these major issues should be considered: (1) Were adequate numbers and controls used? (2) Were conclusions validated using other techniques of gene expression analysis? For further details on the use of microarrays for ocular gene expression analysis and important discussions about analyzing and validating results obtained using microarray analysis, see the review by Zareparsi et al. (2004) and chapter 55, this volume. Another form of nonbiased, large-scale gene expression analysis known as serial analysis of gene expression (SAGE) was described in 1995 (Velculescu et al., 1995). SAGE is a sequencing-based method for gene expression profiling that facilitates the global and quantitative characterization of a transcriptome by specifically targeting the 3′ region of expressed gene transcripts. Standard protocols utilize predetermined restriction enzymes (most utilize the NlaIII site CATG) to release 14 base-pair sequences, referred to as SAGE tags, located after the last defined restriction site in the 3′ part of the detected transcript. The 14 bp tags are then ligated together into longer concatemers, which are cloned for DNA sequencing. The sequence of each tag is detected and compared with a preconstructed reference database that predicts SAGE tags for each known gene. In this way, most individual SAGE tags can be assigned to specific genes using available NCBI sequences (SAGEmap). Each tag detected in a sample represents a single parent transcript (expressed

gene), and the frequency of detection of each transcript is directly related to the gene expression level. Thus, SAGE data give both qualitative (identification of expressed genes) and quantitative data (absolute measures of gene expression levels are possible). When a specific SAGE tag does not map to a sequence in the reference database, it suggests that the SAGE tag has detected a novel transcript. For a comprehensive review of SAGE data, see Wang (2006). Unlike microarray analysis, which relies on specific gene sequences immobilized to each chip, SAGE does not rely on previous knowledge of the gene sequence. Thus, SAGE can be useful to detect any expressed gene and historically has been very successful for detecting novel genes that are expressed in the retina (Blackshaw et al., 2004) or various other tissues. However, SAGE is generally more expensive than microarray analysis, and as more and more genomes have been sequenced and as our understanding of individual genes within those genomes has increased, microarrays are becoming all-inclusive for many different species. SAGE can detect alternatively spliced variants and antisense transcripts, which is generally not possible using standard microarray analysis, although specific arrays can be made to detect both of these products for specific genes. The sensitivity of SAGE is better than that of microarrays, but microarrays generally provide better specificity for each gene; SAGE analysis can misidentify a sequence because of sequence redundancy in multiple genes or sequencing errors. Controls for both technical and biological variability, similar to those previously described for microarray analysis, are also required for the effective use of SAGE analysis. SAGE analysis has been used effectively to determine gene expression during early retinal development in the mouse (Blackshaw et al., 2004). Based on the temporal onset of specific gene expression in relation to neuronal cell differentiation, this study was able to define subsets of retinal progenitor cells and provided useful methods for predicting genes whose function might be related to retinal neuronal cell differentiation (Blackshaw et al., 2004). In another study, SAGE was used to specifically profile genes expressed in RPE isolated from human retinas (Sharon et al., 2002). This study demonstrated that the gene expression profile of RPE was significantly different from the rest of the human retina. Interestingly, relatively high percentages (ca. 9.5%) of the identified RPE genes with known functions were involved in phagocytosis and protein degradation. In contrast, the neural retina devotes less than 3% of its transcripts to these processes, demonstrating a strong commitment of the RPE to the critical function of rod outer segment phagocytosis (Sharon et al., 2002). This study also identified 22 previously unknown elements that appear to be exclusively expressed by the RPE. Ongoing studies and new SAGE data will continue to increase our understanding of the role of specific gene expression in RPE and retinal vascular function.

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Isolation of retinal pigment epithelium or vasculature from retinas Recent advances in analytical techniques have greatly enhanced gene expression research, and it is now possible to obtain large amounts of data from single experiments. However, separating the retinal vascular cells or RPE cells from other more prominent neuronal and glial components remains one of the main difficulties in the specific analysis of RPE or vascular gene expression in the retina. This is particularly true when using large-scale, “unbiased” techniques such as array or SAGE analysis. Although RPE and vascular components of the retina are absolutely critical to normal retinal function, the relative percentage of these cell types in the retina is low. The RPE is a monolayer, and the retinal vasculature exists as three small vascular plexuses in the inner neural retina and the single-layer choriocapillaris. Because the RPE and vascular beds constitute only a small percentage of the retinal cells, important information about gene expression in the RPE or retinal vasculature can easily be lost among genes expressed in more abundant retinal neuronal and glial cells when analyzing gene expression using RNA isolated from whole retinas. One such study used microarray analysis to analyze gene expression during various stages of retinal vascular development by isolating mRNA from dissected whole retinas at various stages during postnatal development (Dorrell et al., 2004). Multiple genes involved in neuronal development and the transfer of visual signals also change dramatically during these developmental stages, and these genes are generally expressed at higher abundance. Thus, it is likely that many important vascular-related or RPE-specific genes remained “hidden” within background levels. Deciphering which observed changes in gene expression correlated with important roles during various stages of vascular development rather than roles in other, nonvascular retina developmental processes was difficult. Specific patterns of gene expression changes in relation to developmental progression were eventually linked to various functions such as neuronal differentiation, visual signal transfer, or vascular development (figure 56.2A) (Dorrell, Aguilar, et al., 2004). By correlating the expression profiles with potential function, an important function for R-cadherin in endothelial cell guidance during retinal vascular development emerged from this gene expression analysis study (figure 56.2B–D) (Dorrell et al., 2002). Using the large-scale gene expression analysis, R-cadherin expression was found to be correlated with major events during retinal vascular development. R-cadherin expression is high at birth and throughout the first week, when the superficial vascular plexus is actively forming. R-cadherin expression also peaks just prior to and during formation of the deep and intermediate vascular plexuses. Subsequent immunohistochemical and functional analyses demonstrated

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that R-cadherin expression is localized to sites of vascular development and is required for normal vascular guidance to the characteristic retinal plexuses (see figure 56.2B–D). Many other factors are likely to be involved in retinal vascular guidance during development and maintenance of vascular homeostasis, and future gene expression studies may help further characterize these mechanisms. However, specific isolation of retinal vascular components or RPE cells would greatly enhance the use of large-scale gene expression analysis in these particular fields. Retinal Pigment Epithelium Isolation and Gene Expression Studies Multiple strategies are currently being used to isolate RPE or vascular components from the retina for subsequent gene expression analysis. Techniques for specifically isolating RPE cells free of other contaminating retinal cells have been described. Most of these techniques use various proteolytic enzymes to dissociate the extracellular matrix, allowing the RPE to be mechanically dissected free of the choroid and underlying neural retina (Castillo et al., 1995; Mayerson et al., 1985). Because pure RPE cells can be isolated, these techniques have been valuable for specifically studying RPE gene expression in vivo, particularly gene expression in RPE isolated from diseased human retinas. In 2002, cDNA libraries from native human RPE were used to profile RPE gene expression (Buraczynska et al., 2002). This study identified several RPE-specific genes, along with multiple genes expressed in RPE as well as in other ocular and nonocular tissues. As expected, a majority of the RPE-expressed, classifiable genes included genes involved in the metabolic pathway (21%) or gene regulation and protein expression (23%). A surprisingly large percentage of expressed genes were identified as growth factors and cell signaling molecules (13%), membrane transport molecules (5%), and molecules involved in protein trafficking (7%). This is consistent with a role for RPE in maintaining retinal homeostasis through controlled cytokine expression, transport of nutrients and ions, and maintenance of the interphotoreceptor matrix (Buraczynska et al., 2002). RPE isolation protocols have also facilitated studies that assess changes in gene expression associated with various in vivo mouse models of disease. Using large-scale microarray analysis, RPE gene expression was compared between young (2-month-old) and older (2-year-old) mice to analyze changes in RPE gene expression associated with aging (Ida et al., 2003). A number of genes, mainly involving inflammation, stress response to oxidative stress, DNA damage, and heat shock, were found to be upregulated in isolated RPE cells from older mice. Interestingly, genes involved in stimulating neovascularization were also upregulated, supporting the hypothesis that changes in RPE function related to aging may contribute to the pathogenesis of “wet” AMD (Ida et al., 2003). Few genes were found to be downregulated in

A No correlation with retinal developmental functions

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Figure 56.2 A, Gene expression profile clustering of mRNA from whole mouse retinas at different postnatal developmental stages. Based on expression profile, gene function could be classified as having probable involvement in the onset of vision (mouse retinal development nears completion and vision begins around P14), neuronal development and differentiation (nears completion around P14), or potential involvement in postnatal retinal vascular development (a process that undergoes multiple changes during these

INL IPL GCL Deep vascular plexus formation

times, and thus gene expression would vary throughout). B, The expression profile of R-cadherin was determined to have a potential relationship to retinal vascular development. When R-cadherin function was blocked, the retinal vasculature failed to form normally in the superficial plexus (C), and guidance to the normal deep vascular plexuses was disrupted (D). Vessels migrated through the photoreceptor layer and into the subretinal space. See color plate 65. (Adapted from Dorrell et al., 2002, 2004.)

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treatment of the RPE cells, a process used to mimic the repair response observed in vivo due to proliferative vitreoretinopathy (Fan et al., 2002). Time-dependent changes in gene expression on phagocytosis of rod outer segments (Chowers et al., 2004), the response of RPE to oxidative stress (Alizadeh et al., 2001; Weigel et al., 2002), RPE cell differentiation (Alizadeh et al., 2000), and many other gene expression studies have also been performed using the ARPE-19 spontaneously transformed human RPE cell line. As with any cell line, immortalization of the RPE cells, whether spontaneous or directed, can alter function and gene expression when compared to native RPE. Using microarray analysis, significant differences were found in the gene expression profiles of cultured primary human RPE cells from four separate donors compared to ARPE-19 cells. Hierarchical clustering demonstrated that despite being obtained from a wide range of ages (48–82 years), each of the primary RPE cell groups clustered together, and no significant overlap was observed with the ARPE-19 cell lines (figure 56.3) (Cai and Del Priore, 2006). Thus, despite the definite utility of these cell lines for determining alterations in gene expression due to specific controlled changes, actual gene expression changes to similar effects may differ in vivo, and caution should be used when generalizing results from ARPE-19 cells. It should be noted, however, that no clear differences were observed in the normal (nonstimulated) expression level of genes with functions related to angiogenesis, phagocytosis, or apoptosis (Cai and Del Priore, 2006), the major RPE functions for which ARPE-19 cells are often used. Thus, ARPE-19 cells might be useful for many important studies regarding RPE gene expression, but, as with most in vitro studies, the results ultimately need to be confirmed in vivo. The differentiation state of RPE cells, and ultimately gene expression and cellular function, can be altered by different culture conditions, particularly when grown with different

ahRPE1

this study, although the study was not comprehensive, and important downregulation of certain genes may have been missed by the stringent constraints applied during the data analysis. Although retina dissociation and subsequent isolation of RPE cells has facilitated many important studies on RPE gene expression, one must consider the possible effects of using proteolytic enzymes to dissociate the retina. Gene expression can change rapidly on removal of cells from their native environment, an effect that occurs as extracellular matrix components are broken down or as the cells’ own membrane proteins are cleaved by enzymatic digestion. Thus, care should be taken to dissociate the retina using as gentle a proteolytic process as possible, and isolated RPE cells should be placed into RNA preservatives such as RNALater (Ambion) or Trizol (Invitrogen) quickly to minimize changes in gene expression that occur due to isolation techniques. In addition, it is critical that RPE isolation from all control and test samples is stringently performed in exactly the same manner so as not to increase the levels of technical variability discussed earlier. Although RPE isolation is valuable for studying gene expression changes in vivo, the limited life span of normal, primary RPE cells in culture represents a substantial obstacle for cell and molecular analysis in vitro. However, the analysis of gene expression changes due to specific, controlled variables often requires in vitro studies. Several RPE cell lines have been developed that maintain many of the behavioral and physiological characteristics of primary RPE and express similar markers, such as RPE-65, cellular retinaldehyde–binding protein (CRALBP), and pigmented epithelium–derived factor (PEDF) (Kanuga et al., 2002; Lund et al., 2001). These cell lines, such as the commonly used ARPE-19 cell line, which is currently available from ATCC (catalogue no. CRL-2302), have been used in many in vitro studies to identify important RPE-related genes. Using the ARPE-19 cell line, researchers found correlative changes in gene expression following injury of the cultured monolayer. Not surprisingly, genes involved in wound repair, including DNA synthesis, DNA repair, cellular adhesion, cytokine expression, and signal transduction, were greatly upregulated during repair of the RPE monolayer. Also highly upregulated were interesting inflammatory associated genes such as mitogen-activated kinase, CD44, monocyte chemotactic protein (MCP-1), thymosin β-10, and hepatoma-derived growth factor (Singh et al., 2001). Upregulation of these genes suggests that the RPE may play a significant role during the early inflammatory response that accompanies many ocular diseases, such as proliferative vitreoretinopathy. In another study using ARPE-19 cells, various cellular adhesion molecules, growth factors, and chemokines generally involved in inflammation and repair, including MCP-1, were also greatly upregulated on vitreous

Figure 56.3 Hierarchic clustering analysis of human adult RPE from four different samples (ages ranging from 48 to 82 years) and five ARPE-19 cultures. The gene expression profiles from the four primary human samples and the five ARPE-19 samples cluster into two distinct groups with no discernable overlap, demonstrating significant differences between primary RPE and ARPE-19 cells. Thus, caution should be exercised when making conclusions of RPE gene expression from cultured, immortalized RPE cells. (Adapted from Cai and Del Priore, 2006.)

extracellular matrix components (Turowski et al., 2004). This should also be considered when using cell lines, since different levels of gene expression may be caused simply by differences in culture conditions. Another consideration is the use of specific culture conditions to help maximize similarities to RPE in vivo. For example, culture on porcine lens capsule membranes has been shown to maintain an in vivolike differentiated state in ARPE-19 cells; apical microvilli formation is induced, and rod outer segment phagocytosis properties are maintained (Turowski et al., 2004). These factors should all be considered when using cultured RPE cells for gene expression studies. Isolation of Retinal Vasculature and Subsequent Gene Expression Studies Isolating retinal ECs can be very difficult because much of the retinal vasculature is intricately associated with nonvascular tissue within the neural retinal layers (see figure 56.1B). Numerous EC lines are available, and isolation of primary ECs, such as human umbilical vein ECs (HUVECs), is common. However, analysis of cultured ECs is problematic because these cells, in culture, are significantly different from such cells in vivo, where the tissue microenvironment has a significant impact on the expression and downregulation of various genes. In addition, microvascular ECs from different organs, and even from different blood vessels within these organs (arterial, venous, microvascular capillaries, etc.), behave differently in response to certain stimuli such as cytokines or growth factors (Gumkowski et al., 1987). Thus, for obtaining useful retinal vascular gene expression data, the specific use of retinal microvascular ECs may be important. Several protocols for isolating retinal ECs have been published (Antonetti and Wolpert, 2003), although the isolation of retinal microvascular ECs from mice is quite difficult. In general, isolation protocols utilize proteolytic digestion of the retina, usually with solutions containing collagenase mixed with various other combinations of extracellular matrix digestion enzymes or trypsin, followed by purification of the ECs using immunoprecipitation with vascular-specific markers such as platelet-derived endothelial cellular adhesion molecule (PECAM; CD31). Few studies have been published that specifically address gene expression from retinal ECs in mouse models of disease, including no known large-scale gene expression studies to date, probably owing to the difficulty of obtaining pure primary ECs from mouse retinas. Certain retinal microvascular EC lines have been created, such as rat JG2/1 cells. These maintain many of the characteristics of primary ECs, such as expression of GLUT-1, the transferrin receptor, von Willebrand factor, and the RECA-1 antigen, high-affinity uptake of acetylated LDL, and isolectin Griffonia simplicifolia binding (Greenwood et al., 1996). However, a similar retinal EC line from mice has yet to be developed.

Many nonendothelial cells, such as astrocytes, pericytes, microglia, and perivascular macrophages, also play critical roles in both developmental and pathological neovascularization (Provis et al., 1997). Thus, studies analyzing gene expression related to changes in the retinal vasculature should also consider the role of these cells, since changes in their gene expression profiles may significantly affect retinal neovascularization. One of the main problems with isolating mouse vasculature from retinas is that the strong enzymes required to completely remove retinal ECs from the neural retina often result in EC death. Newer isolation techniques take advantage of gentle retinal dissociation techniques with lower concentrations of dissociation enzymes in an attempt to isolate small fragments of intact vessels ( J. Greenwood, pers. comm.). Although complete purification of ECs or even vessel fragments may not be achieved, the substantial enhancement of vascular-associated cells and thus vascularrelated RNA would represent an important step for retinal vascular gene expression studies. In addition, the coisolation of closely associated astrocytes, pericytes, and microglia, which remain attached to ECs during the gentle dissociation and purification methods, may actually be advantageous. This allows culture and subsequent analysis of vascular fragments rather than ECs alone, and thus is likely to more closely recapitulate the in vivo retinal microvascular environment by including multiple vascular-related cells. Another method of isolating retinal vasculature for subsequent gene expression analysis uses laser capture microdissection technology. Using microdissection, desired cells or tissue can be isolated from frozen sections. Generally, retinal sections are fixed and the vessels are stained using an endothelial marker such as PECAM or von Willebrand factor. Prior to laser capture, the slides are dehydrated in graded ethanol solutions and dried using acetone and xylene. For most laser capture microdissection techniques, the slide must be completely dry for efficient laser-induced capture of the desired cells. Moisture results in inefficient capture of the cells and lower resolution capture, causing nearby, undesired cells to be captured as well. Because gene expression analysis requires intact mRNA, extreme caution should be used throughout dissection, sectioning of the tissue, and laser capture, and all solutions and materials should be RNase free to ensure that the RNA is not degraded. If the RNA has been protected appropriately, RNA can be subsequently extrapolated from the isolated cells using any of a number of commercial micro-RNA isolation kits (e.g., Ambion, Qiagen). Advances in technique have made RNA amplification and subsequent gene array analysis a feasible method of evaluating gene expression from limited tissue (Luzzi et al., 2003), although certain variables are inevitably added when RNA is amplified, such as incomplete amplification of mRNA ends. Laser capture microdissection and subsequent microarray gene expression analysis has already been applied

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to brain microvascular studies (Liu et al., 2006), and similar studies are likely to be valuable in the retina.

Use of transgenics in gene expression analysis The use of transgenic mice and genetic knockouts has also substantially enhanced our knowledge of gene function in the retina. As genes are identified whose expression patterns indicate a role in retinal function, the results of altering gene expression can be very informative. Analysis of mutants with altered gene expression levels, such as those that overexpress or underexpress a specific gene, or that have altered expression localization, can give important insights into a gene’s function. For example, a particular role for VEGF isoforms during retinal vascular development and pathological neovascularization was demonstrated using mice engineered to express specific VEGF isoforms. These studies demonstrated that a combination of diffusible and relatively immobile isoforms of VEGF was required for normal vascular development. Either the combined expression of VEGF120 (readily diffusible) and VEGF188 (largely immobile) (Ishida et al., 2003) or the single-factor expression of VEGF165 (Stalmans et al., 2002), which is partially diffusible through the retina, was required for normal retinal vascular development. Mice that solely expressed either VEGF120 or VEGF188 developed severely abnormal retinal vasculature characterized by abnormal vessel organization and size and inappropriate arterial and venous differentiation (Stalmans et al., 2002). Mice engineered to overexpress VEGF in the photoreceptors (by placing the VEGF gene behind a rhodopsin promoter) also developed abnormal retinal vascularization characterized by the formation of vascular sprouts that grew from the inner retinal vasculature toward the outer photoreceptor segments (Tobe et al., 1998). These studies demonstrate the utility of altered gene expression in determining functional roles for genes in retinal neovascularization. Many genes expressed in the eye, particularly developmental genes, also have fundamental roles elsewhere that can lead to early lethality in mutants. Unfortunately, in many cases this has rendered conventional gene knockouts uninformative for analysis of specific gene functions in later stages of eye development. In addition, it is often difficult to analyze specific vascular- or RPE-related roles of genes in mice with global gene deficiencies. For these reasons, conditional knockouts have become necessary to elucidate the specific role of retinal vascular- or RPE-related genes. The advent of Cre/Lox technology has provided an important tool for manipulating gene expression in mice by allowing cell-specific activation or inactivation of genes. Conditional knockouts have now been created for vascular-associated cells, including ECs (Bjarnegard et al., 2004), astrocytes (Hirrlinger et al., 2006), myeloid cells (Cramer et al., 2003), and smooth muscle actin-expressing cells, such as arterial

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pericytes (Regan et al., 2000). Studies using these mice have already been instrumental in advancing our understanding of the critical roles for different genes and vascularassociated cell types in general neovascularization. As cellspecific Cre mice are combined with more Lox gene knockouts, and as these mice are further exploited for retinal research, we will continue to learn more about the importance of specific gene expression in relation to retinal vascular development and disease. Cre recombinase has also recently been linked to the tyrosinase related protein-1 (Trp-1) promoter, making future RPE specific knockouts possible (Marneros et al., 2005). Although the absolute specificity of this gene promoter for RPE cells remains somewhat controversial, it should suffice for eye-specific studies, and these and future RPE specific knockouts should quickly advance the studies of RPE specific gene expression and function as well.

Correlation of retinal pigment epithelium gene expression and retinal vascularization A strong relationship between the RPE and retinal vasculature, both during retinal development and throughout adulthood, is becoming evident. The RPE clearly induces development of the choroidal vessels. VEGF120 and VEGF165 are both expressed by mouse RPE cells, and when VEGF expression was specifically eliminated in the RPE, the choroid became absent throughout development and adulthood; scleral tissue was observed just posterior to the RPE where the choroidal vascular plexus would normally be found (Marneros et al., 2005). Although multiple retinal abnormalities were observed in these mice, including microphthalmia and an absence of Bruch’s membrane, the RPE seemed to differentiate normally, indicating that the vascular abnormalities were due to a lack of VEGFmediated pro-angiogenic signaling by the RPE. Other growth factors, such as basic fibroblast growth factor (FGF2), are also expressed by the RPE and have been suggested to play a critical role during normal retinal vascular development (Rousseau et al., 2000). RPE abnormalities have also been correlated with the onset of age-related macular degeneration (AMD) (Campochiaro et al., 1999). VEGF overexpression in the RPE has been correlated with AMD progression in excised human diseased retinal tissue (Kliffen et al., 1997) as well as mouse models (Schwesinger et al., 2001; Spilsbury et al., 2000). In addition to the expression of pro-angiogenic factors that may promote choroidal neovascularization when expressed at abnormal levels, the lack of normal RPE expression of certain antiangiogenic factors may also contribute to abnormal retinal neovascularization. The RPE is known to express factors such as pigmented epithelium–derived factor (PEDF), which may help to maintain the normally quiescent retinal vasculature. PEDF expression is upregulated as retinal

vascular development is completed (Behling et al., 2002), and retinas from patients with AMD have demonstrated a lack of normal PEDF expression in the RPE compared with age-matched donors without AMD (Bhutto et al., 2006). In addition to the definite role of the RPE during normal and pathological choroidal neovascularization, new evidence suggests that the RPE may also play an underappreciated role during development of the vascular plexuses in the inner retina. In the postnatal Balb/c mouse, vessels branch from the superficial plexus around P8 and form the deep plexus at the outer edge of the inner nuclear layer (INL). Although the precise factors that initiate this process are not known, hypoxia in the deeper retina, caused by retinal thickening, photoreceptor maturation, and the concomitant increase in neuronal activity, is likely to be a main driving force (Zhang et al., 2003). As a result, the production of growth factors in the neural retina is increased, subsequently initiating the formation of the deep vascular plexuses. As the vessels migrate toward the deep retina, the ECs must be guided to the appropriate plexuses at the outer and inner edges of the INL. Filopodial processes, which can contact and respond to preexisting guidance cues, have been observed at the tips of ECs migrating toward the deep vascular plexus (Dorrell, et al., 2002). R-cadherin expression at the outer and inner edges of the INL correlates with vascularization of the deep and intermediate plexuses, and when R-cadherin mediated adhesion is disrupted, retinal vessels migrate directly past the deep plexus, into the normally avascular photoreceptor layer and subretinal space (Dorrell, et al., 2002; Dorrell, Otani, et al., 2004) (see figure 56.2D). A similar phenotype whereby vessels invade the ONL was also observed in transgenic mice with photoreceptor-induced overexpression of VEGF (Tobe et al., 1998). This indicates that normal guidance cues can be overcome either by overexpression of growth factors beyond the normal deep vascular plexus or by masking specific adhesion molecules critical for EC guidance. Abnormal vascular sprouts invading the outer retina and the subretinal space are also observed in mice lacking the very-low-density lipoprotein receptor (VLDLR) (Heckenlively et al., 2003). Unlike the subretinal vessels that form when R-cadherin mediated adhesion is blocked, which directly bypass the deep layers, these subretinal vessels develop a few days later as sprouts from the normal deep and intermediate retinal plexuses. VLDLR is known to mediate neuronal guidance during development of the neocortex in a mechanism involving R-cadherin (Rice et al., 2001; Trommsdorff et al., 1999) and these results suggest that VLDLR expression in the retina could potentially be functioning in a similar fashion during retinal vascular guidance. In each of these situations, retinal vessels migrate through the photoreceptor layer and into the subretinal space alongside the RPE. Although many of the growth factors that

initiate formation of the deep vascular plexuses definitely come from retinal glial and neuronal cells, these observations suggest that deep vascular sprouting may be promoted by the RPE as well. The pro-angiogenic cytokine gradient that induces development of the choroid may also participate in the development of inner retinal vascular plexuses, particularly the formation of the deep vascular plexuses. According to this hypothesis, ECs may be attracted toward the cytokine gradient formed by growth factor expression in the RPE, but normally they are guided to the deep and intermediate plexuses as they migrate. However, when these guidance cues are lost, such as the effects observed when normal R-cadherin or VLDLR function is lost, the vessels migrate past the normal deep and intermediate plexuses, through the photoreceptors, and into the subretinal space by default. More experimental evidence is certainly required to confirm or reject this hypothesis. The continued study of gene expression in both the RPE and the retinal vasculature should help answer this particular question and will continue to advance our understanding of the normal role of the RPE during normal and pathological retinal neovascularization.

Conclusion and future directions Large-scale genomic analyses are very useful for identifying genes whose expression may be critical to various biological processes. However, gene expression analysis of the RPE and retinal vasculature is only a first step toward understanding the role these genes play in regulating normal and pathological retinal processes. As discussed in this chapter, the results of large-scale gene expression analyses need to be confirmed using other techniques, and protein expression levels must be tested before firm conclusions about the relevance of gene expression to biological function can be made. In addition, the biological effects of a particular gene’s expression, and the effects of differing expression levels in varying circumstances, must be considered. For example, many housekeeping genes, which are generally expressed at high levels, can demonstrate differential gene expression levels. However, even statistically significant differences in the expression of these genes usually have low biological relevance. Because of high expression levels and constitutive functions, even relatively substantial changes in expression may not dramatically affect retinal vascular or RPE functions. By contrast, the function of many genes critical to normal RPE and retinal vascular function, such as cytokines and growth factors, can be significantly altered by marginal differences in expression levels. Small changes in the expression of these genes may have dramatic effects leading to the onset or progression of various diseases. Thus, the biological effects of differential gene expression need to be addressed.

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Finally, many genes are regulated beyond the transcriptional level. For example, HIF1-α is a critical cellular oxygen sensor. It mediates a cell’s response to hypoxia by turning on various stress response pathways and by initiating the expression of VEGF and other pro-angiogenic factors to initiate vessel growth and ultimately alleviate hypoxia. However, HIF1-α is constitutively expressed and is regulated by posttranslational degradation. Under normal oxygen conditions, HIF1-α is rapidly degraded, but in hypoxic conditions it becomes stabilized. Gene expression analysis alone would not detect the important changes in HIF1-α protein levels in cells exposed to different oxygen levels. Thus, whereas gene expression analysis is very useful, it is the amount of functional protein produced by cells that ultimately makes a difference. As techniques for detecting protein expression, such as large-scale proteomic analysis, continue to improve they will become more and more useful for the analysis of RPE and retinal vascular functions. Since the early 1990s, gene expression analysis has seen a massive boom, largely helped by the advent of large-scale analysis techniques. As microarray technologies evolve to provide more sensitive temporal expression information, and as other techniques continue to provide information about spatial expression at the cellular level (i.e., microarray of laser-captured specimens), our understanding of normal and abnormal biological processes in the retina should be greatly enhanced. These large-scale techniques, such as microarray or SAGE, along with other more conventional techniques, such as RT-PCR and in situ hybridization, have already been instrumental in studying retinal gene expression. With the use of isolation techniques such as microdissection, specific RPE and retinal vascular gene expression analyses will continue to be possible. These studies will advance our understanding of the roles that RPE and the retinal vasculature play in normal retinal homeostasis, as well as help determine what effects alterations in normal gene expression levels might have on the progression of various ocular diseases. acknowledgments

Work was supported by the National Eye Institute (grant no. EY11254), the MacTel Foundation, Scripps Fonseca/Mericos Fund, the Robert Mealey Program for the Study of Macular Degenerations, the V. Kann Rasmussen Foundation, and the Horner Family Fund. MID was supported by a California Institute for Regenerative Medicine fellowship. We thank the entire Friedlander laboratory staff for their helpful contributions to the preparation of this chapter.

REFERENCES Adamis, A. P., Aiello, L. P., and D’Amato, R. A. (1999). Angiogenesis and ophthalmic disease. Angiogenesis 3:9–14.

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Alizadeh, M., Gelfman, C. M., Bench, S. R., and Hjelmeland, L. M. (2000). Expression and splicing of FGF receptor mRNAs during APRE-19 cell differentiation in vitro. Invest. Ophthalmol. Vis. Sci. 41:2357–2362. Alizadeh, M., Wada, M., Gelfman, C. M., Handa, J. T., and Hjelmeland, L. M. (2001). Downregulation of differentiation specific gene expression by oxidative stress in ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 42:2706–2713. Antonetti, D. A., and Wolpert, E. B. (2003). Isolation and characterization of retinal endothelial cells. Methods Mol. Med. 89: 365–374. Behling, K. C., Surace, E. M., and Bennett, J. (2002). Pigment epithelium-derived factor expression in the developing mouse eye. Mol. Vis. 8:449–454. Bhutto, I. A., McLeod, D. S., Hasegawa, T., Kim, S. Y., Merges, C., Tong, P., and Lutty, G. A. (2006). Pigment epitheliumderived factor (PEDF) and vascular endothelial growth factor (VEGF) in aged human choroid and eyes with age-related macular degeneration. Exp. Eye Res. 82:99–110. Bjarnegard, M., Enge, M., Norlin, J., Gustafsdottir, S., Fredriksson, S., Abramsson, A., Takemoto, M., Gustafsson, E., Fassler, R., et al. (2004). Endothelium-specific ablation of PDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities. Development 131:1847–1857. Blackshaw, S., Harpavat, S., Trimarchi, J., Cai, L., Huang, H., Kuo, W. P., Weber, G., Lee, K., Fraioli, R. E., et al. (2004). Genomic analysis of mouse retinal development. PLoS Biol. 2: E247. Buraczynska, M., Mears, A. J., Zareparsi, S., Farjo, R., Filippova, E., Yuan, Y., MacNee, S. P., Hughes, B., and Swaroop, A. (2002). Gene expression profile of native human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 43:603–607. Cai, H., and Del Priore, L. V. (2006). Gene expression profile of cultured adult compared to immortalized human RPE. Mol. Vis. 12:1–14. Campochiaro, P. A., Soloway, P., Ryan, S. J., and Miller, J. W. (1999). The pathogenesis of choroidal neovascularization in patients with age-related macular degeneration. Mol. Vis. 5:34. Castillo, B. V., Jr., Little, C. W., del Cerro, C., and del Cerro, M. (1995). An improved method of isolating fetal human retinal pigment epithelium. Curr. Eye Res. 14:677–683. Chong, N. H., and Bird, A. C. (1999). Management of inherited outer retinal dystrophies: Present and future. Br. J. Ophthalmol. 83:120–122. Chowers, I., Kim, Y., Farkas, R. H., Gunatilaka, T. L., Hackam, A. S., Campochiaro, P. A., Finnemann, S. C., and Zack, D. J. (2004). Changes in retinal pigment epithelial gene expression induced by rod outer segment uptake. Invest. Ophthalmol. Vis. Sci. 45:2098–2106. Cramer, T., Yamanishi, Y., Clausen, B. E., Forster, I., Pawlinski, R., Mackman, N., Haase, V. H., Jaenisch, R., et al. (2003). HIF-1alpha is essential for myeloid cell-mediated inflammation. Cell 112:645–657. de Jong, P. T (2006). Age-related macular degeneration. N. Engl. J. Med. 355:1474–1485. Dorrell, M. I., Aguilar, E., and Friedlander, M. (2002). Retinal vascular development is mediated by endothelial filopodia, a preexisting astrocytic template and specific R-cadherin adhesion. Invest. Ophthalmol. Vis. Sci. 43:3500–3510. Dorrell, M. I., Aguilar, E., Weber, C., and Friedlander, M. (2004). Global gene expression analysis of the developing postnatal mouse retina. Invest. Ophthalmol. Vis. Sci. 45:1009–1019.

Dorrell, M. I., and Friedlander, M. (2006). Mechanisms of endothelial cell guidance and vascular patterning in the developing mouse retina. Prog. Retin. Eye Res. 25:277–295. Dorrell, M. I., Otani, A., Aguilar, E., Moreno, S. K., and Friedlander, M. (2004). Adult bone marrow–derived stem cells use R-cadherin to target sites of neovascularization in the developing retina. Blood 103(9):3420–3427. Eichler, W., Yafai, Y., Wiedemann, P., and Reichenbach, A. (2004). Angiogenesis-related factors derived from retinal glial (Müller) cells in hypoxia. Neuroreport 15:1633–1637. Fan, W., Zheng, J. J., Peiper, S. C., and McLaughlin, B. J. (2002). Changes in gene expression of ARPE-19 cells in response to vitreous treatment. Ophthalmic. Res. 34:357–365. Futter, C. E. (2006). The molecular regulation of organelle transport in mammalian retinal pigment epithelial cells. Pigment Cell Res. 19:104–111. Greenwood, J., Pryce, G., Devine, L., Male, D. K., dos Santos, W. L., Calder, V. L., and Adamson, P. (1996). SV40 large T immortalised cell lines of the rat blood-brain and blood-retinal barriers retain their phenotypic and immunological characteristics. J. Neuroimmunol. 71:51–63. Gumkowski, F., Kaminska, G., Kaminski, M., Morrissey, L. W., and Auerbach, R. (1987). Heterogeneity of mouse vascular endothelium: In vitro studies of lymphatic, large blood vessel and microvascular endothelial cells. Blood Vessels 24:11–23. Heckenlively, J. R., Hawes, N. L., Friedlander, M., Nusinowitz, S., Hurd, R., Davisson, M., and Chang, B. (2003). Mouse model of subretinal neovascularization with choroidal anastomosis. Retina 23:518–522. Hirrlinger, P. G., Scheller, A., Braun, C., Hirrlinger, J., and Kirchhoff, F. (2006). Temporal control of gene recombination in astrocytes by transgenic expression of the tamoxifen-inducible DNA recombinase variant CreERT2. Glia 54:11–20. Ida, H., Boylan, S. A., Weigel, A. L., and Hjelmeland, L. M. (2003). Age-related changes in the transcriptional profile of mouse RPE/choroid. Physiol. Genomics 15:258–262. Ishida, S., Usui, T., Yamashiro, K., Kaji, Y., Amano, S., Ogura, Y., Hida, T., Oguchi, Y., Ambati, J., et al. (2003). VEGF164mediated inflammation is required for pathological, but not physiological, ischemia-induced retinal neovascularization. J. Exp. Med. 198:483–489. Kanuga, N., Winton, H. L., Beauchene, L., Koman, A., Zerbib, A., Halford, S., Couraud, P. O., Keegan, D., Coffey, P., et al. (2002). Characterization of genetically modified human retinal pigment epithelial cells developed for in vitro and transplantation studies. Invest. Ophthalmol. Vis. Sci. 43:546–555. Kliffen, M., Sharma, H. S., Mooy, C. M., Kerkvliet, S., and de Jong, P. T. (1997). Increased expression of angiogenic growth factors in age-related maculopathy. Br. J. Ophthalmol. 81:154– 162. Liu, X. S., Zhang, Z. G., Zhang, L., Morris, D. C., Kapke, A., Lu, M., and Chopp, M. (2006). Atorvastatin downregulates tissue plasminogen activator-aggravated genes mediating coagulation and vascular permeability in single cerebral endothelial cells captured by laser microdissection. J. Cereb. Blood Flow Metab. 26:787–796. Lukasiewicz, P. D. (2005). Synaptic mechanisms that shape visual signaling at the inner retina. Prog. Brain Res. 147:205–218. Lund, R. D., Adamson, P., Sauve, Y., Keegan, D. J., Girman, S. V., Wang, S., Winton, H., Kanuga, N., Kwan, A. S., et al. (2001). Subretinal transplantation of genetically modified human cell lines attenuates loss of visual function in dystrophic rats. Proc. Natl. Acad. Sci. U.S.A. 98:9942–9947.

Luzzi, V., Mahadevappa, M., Raja, R., Warrington, J. A., and Watson, M. A. (2003). Accurate and reproducible gene expression profiles from laser capture microdissection, transcript amplification, and high density oligonucleotide microarray analysis. J. Mol. Diagn. 5:9–14. Marneros, A. G., Fan, J., Yokoyama, Y., Gerber, H. P., Ferrara, N., Crouch, R. K., and Olsen, B. R. (2005). Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function. Am. J. Pathol. 167:1451–1459. Mayerson, P. L., Hall, M. O., Clark, V., and Abrams, T. (1985). An improved method for isolation and culture of rat retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 26:1599–1609. Mumm, J. S., Godinho, L., Morgan, J. L., Oakley, D. M., Schroeter, E. H., and Wong, R. O. (2005). Laminar circuit formation in the vertebrate retina. Prog. Brain Res. 147:155–169. Otani, A., and Friedlander, M. (2005). Retinal vascular regeneration. Semin. Ophthalmol. 20:43–50. Provis, J. M., Leech, J., Diaz, C. M., Penfold, P. L., Stone, J., and Keshet, E. (1997). Development of the human retinal vasculature: Cellular relations and VEGF expression. Exp. Eye Res. 65:555–568. Regan, C. P., Manabe, I., and Owens, G. K. (2000). Development of a smooth muscle-targeted Cre recombinase mouse reveals novel insights regarding smooth muscle myosin heavy chain promoter regulation. Circ. Res. 87:363–369. Rice, D. S., Nusinowitz, S., Azimi, A. M., Martinez, A., Soriano, E., and Curran, T. (2001). The reelin pathway modulates the structure and function of retinal synaptic circuitry. Neuron 31: 929–941. Rousseau, B., Dubayle, D., Sennlaub, F., Jeanny, J. C., Costet, P., Bikfalvi, A., and Javerzat, S. (2000). Neural and angiogenic defects in eyes of transgenic mice expressing a dominantnegative FGF receptor in the pigmented cells. Exp. Eye Res. 71: 395–404. Schwesinger, C., Yee, C., Rohan, R. M., Joussen, A. M., Fernandez, A., Meyer, T. N., Poulaki, V., Ma, J. J., Redmond, T. M., et al. (2001). Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium. Am. J. Pathol. 158:1161– 1172. Sharon, D., Blackshaw, S., Cepko, C. L., and Dryja, T. P. (2002). Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE). Proc. Natl. Acad. Sci. U.S.A. 99:315–320. Singh, S., Zheng, J. J., Peiper, S. C., and McLaughlin, B. J. (2001). Gene expression profile of ARPE-19 during repair of the monolayer. Graefes Arch. Clin. Exp. Ophthalmol. 239:946–951. Spilsbury, K., Garrett, K. L., Shen, W. Y., Constable, I. J., and Rakoczy, P. E. (2000). Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization. Am. J. Pathol. 157:135–144. Stalmans, I., Ng, Y. S., Rohan, R., Fruttiger, M., Bouche, A., Yuce, A., Fujisawa, H., Hermans, B., Shani, M., et al. (2002). Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J. Clin. Invest. 109:327–336. Strauss, O. (2005). The retinal pigment epithelium in visual function. Physiol. Rev. 85:845–881. Tobe, T., Okamoto, N., Vinores, M. A., Derevjanik, N. L., Vinores, S. A., Zack, D. J., and Campochiaro, P. A. (1998). Evolution of neovascularization in mice with overexpression of

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vascular endothelial growth factor in photoreceptors. Invest. Ophthalmol. Vis. Sci. 39:180–188. Trommsdorff, M., Gotthardt, M., Hiesberger, T., Shelton, J., Stockinger, W., Nimpf, J., Hammer, R. E., Richardson, J. A., and Herz, J. (1999). Reeler/Disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2. Cell 97:689–701. Turowski, P., Adamson, P., Sathia, J., Zhang, J. J., Moss, S. E., Aylward, G. W., Hayes, M. J., Kanuga, N., and Greenwood, J. (2004). Basement membrane-dependent modification of phenotype and gene expression in human retinal pigment epithelial ARPE-19 cells. Invest. Ophthalmol. Vis. Sci. 45:2786–2794. Velculescu, V. E., Zhang, L., Vogelstein, B., and Kinzler, K. W. (1995). Serial analysis of gene expression. Science 270:484– 487.

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57

Gene Expression in Cornea and Lens SHIVALINGAPPA K. SWAMYNATHAN AND JORAM PIATIGORSKY

Optimal vision depends on the transparence and refractive abilities of both cornea and lens, through which light traverses before reaching the retina. Embryonic induction, development, and postnatal maturation of the mouse lens and cornea involve a series of well-coordinated interactions between the neuroectoderm that forms the retina and the surface ectoderm that forms the lens and cornea (Chow and Lang, 2001; Cvekl and Tamm, 2004). One of the currently emerging unifying themes in developmental ophthalmology is that the transcription factors required for embryonic development of cornea and lens are also used for expression of crystallins, the specialized proteins characteristic of the differentiated states of lens and cornea, in the adult tissues. This chapter summarizes current knowledge and recent developments related to the regulation of gene expression in the mouse lens and cornea during embryonic and adult stages, with appropriate references to the human congenital eye defects associated with mutations in genes regulating eye development.

Development of lens and cornea The lens arises by invagination of the thickened lens placode on the surface ectoderm in response to signals from the underlying optic vesicle (future retina) around embryonic day 11 (E11) in mice (figure 57.1). The anterior cells of the lens vesicle divide and remain cuboidal, while the posterior cells elongate, lose organelles, and differentiate into nondividing primary fiber cells, ultimately losing their nuclei. As development proceeds, proliferation of the anterior epithelial cells becomes progressively confined to the lens equator, where they continue to divide slowly throughout life and give rise to the cortical fiber cells. The lens cells accumulate high proportions of a few water-soluble proteins called crystallins. The transparent primary and secondary cortical fiber cells form the bulk of the lens and are responsible for its optical properties. Three major groups of crystallin gene families, α, β, and γ, each including several members, account for 90% of the water-soluble proteins in the mouse lens (Bloemendal and de Jong, 1991). It is of interest that different species often accumulate different proteins as lens crystallins,

which are known as taxon-specific crystallins (Piatigorsky and Wistow, 1991; Wistow and Piatigorsky, 1988). The cornea has a more complex cellular composition than the lens. The surface ectoderm anterior to the lens vesicle gives rise to the corneal epithelium. Underneath the epithelium, the cornea develops a thick extracellular collagen-filled stroma littered with keratocytes and a posterior monolayer of endothelial cells derived from the neural crest (Hay, 1979; Zieske, 2004). A significant number of corneal basal epithelial cells derived from the embryonic surface ectoderm remain undifferentiated in neonatal mice and serve as corneal epithelial progenitor cells. Following eye opening, around postnatal (PN) day 15, the progenitor cells divide and stratify to form the mature cornea by about 6 weeks of age (Hay, 1979; Zieske, 2004). This proliferation and differentiation continue in the adult mouse, allowing steady replacement of the sloughed off superficial epithelial cells by the slow division of basal cells; basal cells are replenished by stem cells originating from the limbal epithelium at the periphery of the cornea (Collinson et al., 2002; Cotsarelis et al., 1989; Nagasaki and Zhao, 2003). The corneal epithelial cells and keratocytes, like the lens fiber cells, accumulate a high proportion of a few intracellular proteins, called corneal crystallins, and these often differ in different species (Jester et al., 1999, 2005; Piatigorsky, 1998). In the mouse, the corneal crystallins aldehyde dehydrogenase 3A1 (Aldh3a1) and transketolase (Tkt) account for 50% and 10% of watersoluble proteins, respectively. In addition, the corneal epithelial cells accumulate high concentrations of cytokeratins (Krt12 in the case of mice) (Tanifuji-Terai et al., 2006). The abundance of crystallins indicates that they serve specific optical roles (known for lens, hypothesized for cornea), yet they are also expressed in lower amounts in many tissues where they have strictly enzymatic or other cellular functions. In other words, the widely expressed lens and corneal crystallins gain optical functions by virtue of their high tissue-specific expression, a situation that has been called gene sharing (Piatigorsky and Wistow, 1989; Piatigorsky et al., 1988). The gene sharing relationship between tissue-specific expression and function of a gene and its encoded protein adds an intriguing general aspect to gene expression in lens and cornea (Piatigorsky, 2007).

697

Head surface ectoderm

E8

Lens placode

Optic vesicle

E10

Presumptive corneal epithelium

Lens pit

E10.5

Lens vesicle

E11.5

Corneal epithelium

Keratocytes Stroma

Endothelium

{

Mesenchymal cells from neural crest

Presumptive corneal epithelium

E12.5

}

Lens epithelium

Presumptive stroma Embryonic lens nucleus with primary fiber cells

Lens capsule

6 week-old adult

Cortical lens with secondary fiber cells

E15.5 Presumptive corneal endothelium

E14

Presumptive retina

Presumptive optic nerve

Figure 57.1 Schematic overview of the developing mouse eye. Progressive steps in the development of mouse eye at different embryonic days (E8, E10, . . .) and the 6-week-old adult lens and cornea are illustrated. Illustrations are not drawn to scale.

Large-scale analyses of gene expression in the cornea and lens The basal layer of limbal epithelium between the cornea and the conjunctiva is enriched in stem cells that give rise to transient amplifying cells that migrate to the central corneal epithelium and become the basal epithelial cells. Laser capture microdissection followed by microarray analysis identified about 100 differentially expressed genes in limbal compared to corneal epithelial basal cells (Zhou et al., 2006). In another study, comparison of the rat limbal and central corneal transcripts by serial analysis of gene expression (SAGE) identified 759 transcripts specific for the limbus and 844 transcripts specific for the central cornea, with 2,292 transcripts present in both (Adachi et al., 2006). Comparison of PN9 and 6-week-old adult mouse corneas by SAGE demonstrated dynamic changes in gene expression during postnatal corneal maturation (Norman et al., 2004). Roughly one-third of the transcripts expressed in the cornea are present exclusively in the PN9 or mature corneas, and the remaining one-third are expressed at both stages. Abundantly expressed transcripts in the cornea are associated with

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diverse functions such as metabolism, redox activities, and barrier integrity (Norman et al., 2004). In the lens, younger and transcriptionally active differentiating cells envelop a core of mature fiber cells lacking nuclei and cytoplasmic organelles. Microarray comparison of gene expression profiles of young elongating and mature fiber cells captured by laser microdissection identified 65 differentially expressed genes important for lens cell differentiation (Ivanov et al., 2005). In humans, the expression of as many as 1,196 transcripts is elevated and the expression of 1,278 transcripts is decreased by twofold or more in lens epithelial cells compared to cortical fiber cells (Hawse et al., 2005). These gene expression changes correspond to distinct pathways and functions important for the formation of lens fiber cells. Endogenous noncoding microRNAs (miRNAs) inhibit the translation and affect the stability of target mRNAs, and thus have a role in the regulation of development and differentiation (Kloosterman and Plasterk, 2006). Studies of miRNAs in the eye are just beginning. So far, several miRNAs expressed in a distinct tissue- and cell-type-specific manner in different tissues of the eye have been detected using

tion in studies on gene regulation during eye development, in view of their critical contribution. As indicated in the following discussion, in addition to their developmental roles, the homeobox transcription factors are also important regulators of crystallin gene expression.

miRNA microarrays (Ryan et al., 2006). MiRNA (mir)-184, one of the abundant miRNAs in cornea and lens, is expressed in the corneal epithelium and in the lens germinative zone epithelial cells. Mir-205 is widely expressed in the anterior segment epithelia and epidermis (Ryan et al., 2006). Specific functions of these miRNAs are not known yet.

Pax6. Pax6, a paired domain-homeobox transcription factor, is essential for the inductive interactions between the neuroectoderm and the surface ectoderm during early embryonic eye development (see figure 57.2) (Simpson and Price, 2002). Of historical interest, the importance of Pax6 for eye development was first noted in Drosophila, where its homologue is known as eyeless (Gehring, 2004). In humans, mutations in PAX6 result in severe eye defects (Graw, 2003; Hanson, 2003; Hanson et al., 1994). Homozygous Pax6 mutant mice develop only rudiments of the optic vesicle and die in the neonatal stage (Hogan et al., 1988). In heterozygotes, lens placode formation is delayed, resulting in a smaller lens with vacuolated fiber cells, frequently fused to

Transcription factors involved in the development of cornea and lens Studies employing transgenic mice and targeted deletion of genes helped accumulate useful information about the network of transcription factors influencing the development of cornea and lens (figure 57.2). Here we briefly review the contribution of different transcription factors to the development of cornea and lens. Homeobox Transcription Factors Homeobox-containing transcription factors have received preferential atten-

Surface Ectoderm

Development of Lens E7.5

Meis 1 & 2

E8.5

Presumptive Lens Ectoderm

E10

Lens Placode

E11

Development of Cornea Six3

Pax6 + pRb, Maf, Mitf, Sox2, Sox3 or AP2α

Foxe3

Maf + Pax6, or Sox2 Sip1 Foxc1 Sox1 Foxc2 Lens Foxe3

Pitx2 Prox1 Sox2 AP1 Pitx3 Sp1 Sox3 Klf4 Presumptive AP2α Elf3 corneal IκBζ epithelium Klf6

Mesenchymal cell migration between lens vesicle and corneal epithelium

Vesicle

Hsf4

E12.5 Primary fiber cells

Expression of crystallins and other genes

E15

E10

E11

E12.5 Condensation of mesenchymal cells begins Mesenchymal cells E15 form corneal stroma and endothelium

Secondary fiber cells

Maturation PN15

Lens Figure 57.2 Transcription factors regulating the development of mouse lens and cornea. The network of transcription factors regulating the development of lens (red), or cornea (blue), or both (green) is shown, along with the embryonic age and the devel-

Cornea

Proliferation and PN15 stratification of corneal epithelial cells

opmental stages of lens and cornea. Many of these transcription factors remain active in the mature lens and cornea. See color plate 66.

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the cornea, resembling Peter’s anomaly (Collinson et al., 2001). In the mouse, Pax6 expression, first detected at E8 at the optic pit, head surface ectoderm, and neural ectoderm, is restricted to the lens placode, optic vesicle, and stalk at E9.5. After E13.5, Pax6 is expressed in the proliferating anterior epithelial cells of the lens vesicle and the surface ectoderm, which gives rise to cornea, conjunctiva, and eyelids. In the adult mouse, Pax6 is expressed in the lens epithelial cells, cornea, conjunctiva, iris, ciliary body, and retina (Koroma et al., 1997). Homeobox transcription factors Meis1 and Meis2, expressed in a pattern similar to Pax6, directly regulate Pax6 expression in presumptive lens ectoderm by binding the Pax6 lens placode enhancer (see figure 57.2) (Zhang et al., 2002). Pax6 upregulates its own expression and influences eye development both directly, by controlling different genes encoding structural proteins required for eye formation, and indirectly, by controlling a number of genes encoding developmental transcription factors, including Six3, c-Maf, MafA/L-Maf, Prox1, Sox2, and FoxE3 (see figure 57.2) (Aota et al., 2003; Ashery-Padan et al., 2000; Brownell et al., 2000; Chauhan et al., 2002; Cvekl and Tamm, 2004; Cvekl et al., 2004; Duncan et al., 2000; Furuta and Hogan, 1998; Marquardt et al., 2001). Pax6 is autonomously required for formation of corneal epithelium, stroma, and endothelium between E10.5 and E16.5 (Collinson et al., 2003; Davis et al., 2003). The corneal epithelium in the heterozygous Pax6 (Small eye, Sey) mouse is thinner, with a reduced number of cell layers, despite increased cell proliferation. The levels of desmoglein, β-catenin, γ-catenin, and the intermediate filament keratin-12 are reduced in the Sey cornea, suggesting defective intercellular adhesion (Davis et al., 2003), an idea supported by a recent report showing that Pax6 heterozygote corneal epithelial cells have multiple glycoconjugate defects on the cell surface that restrict their ability to initiate migration in response to wound healing (Kucerova et al., 2006). Corneal epithelial cell proliferation is inhibited on overexpression of Pax6 (Ouyang et al., 2006). The distribution of neural crest–derived cells is abnormal in Sey mouse from early developmental stages to the adult, indicating that normal distribution and integration of neural crest–derived cells depend on a proper dosage of Pax6 (Kanakubo et al., 2006). Pax6 is actively involved in the repair and maintenance of the adult corneal surface. During corneal wound healing, the amount of Pax6 increases at the migrating front of the resurfacing corneal epithelium, where it upregulates matrix metalloproteinase gelatinase B (gelB; MMP-9). Two Pax6binding sites exist within the gelB −522/+19 bp promoter fragment, which contains the necessary cis- elements for appropriate expression (Sivak et al., 2000). Pax6 paired domain controls the gelB promoter activity by interacting directly with one of these sites, and indirectly with the other

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site, through cooperative interactions with AP2α. A reduced Pax6 dosage in heterozygous Sey mice results in a loss of gelB expression at the migrating epithelial front, which correlates with an increase in inflammation (Sivak et al., 2004). Pax6 influences target gene expression both independently and in association with other transcription factors such as pRb, MafA, MitF, Sox2, and Sox3 in a cooperative manner (see figure 57.2). Sox2 and Sox3 interact with Pax6, leading to synergistic transcriptional activation (Aota et al., 2003). In the lens, both aA- and aB-crystallin genes are upregulated (Cvekl and Piatigorsky, 1996; Cvekl et al., 1994, 1995; Gopal-Srivastava et al., 1996; Haynes et al., 1996), and bB1-crystallin (Duncan et al., 1998) and g-crystallin (Yang et al., 2004) genes are downregulated by Pax6. Unlike the synergistic activation of aB-crystallin by Pax6 and c-Maf, Pax6 has no effect on c-Maf-mediated αA-crystallin promoter activation (Yang et al., 2004). Compound heterozygous mice with mutations in both Pax6 and Gli3, a zinc finger transcription factor gene expressed in the embryonic eye, develop more extensive abnormalities in the retina, iris, lens, and cornea than do single Gli3+/− or Pax6+/− mutants (Zaki et al., 2006). The requirement for a normal Gli3 gene dosage is greater in the absence of normal Pax6 gene dosage, suggesting that these two transcription factors cooperate during eye morphogenesis. Six3. Six3, a Six domain containing homeobox transcription factor, is expressed in the anterior neural ectoderm at E7, developing retinal field at E8, and head ectoderm that forms the lens placode at E9 in the mouse (Lagutin et al., 2001; Oliver et al., 1995). After E9, Pax6 and Six3, expressed in an overlapping manner, regulate the expression of each other (see figure 57.2) (Goudreau et al., 2002). Conditional deletion of mouse Six3 in the presumptive lens ectoderm disrupts lens induction, resulting in the absence of the lens placode and lens in severe cases. Six3 influences eye development directly, by regulating the expression of structural and metabolic genes required for eye formation, and indirectly, by activating the expression of Pax6 in the lens preplacodal ectoderm (see figure 57.2) (Liu et al., 2006). Prox1. Another well-conserved homeobox transcription factor influencing lens fiber cell elongation in the lens vesicle is Prox1 (see figure 57.2). Prox1 is expressed in the mouse at E9.5 in the lens placode, at E10.5 in the lens vesicle, and at E12.5 onward in the anterior epithelium, lens fiber cells, and surface ectoderm (Tomarev et al., 1998). Homozygous Prox1 null mice, which die around E14.5, show defective fiber cell elongation, abnormal cellular proliferation, downregulated expression of the cell cycle inhibitors Cdkn1b (p27KIP1) and Cdkn1c (p57KIP2), misexpression of Ecadherin, inappropriate apoptosis, and absence of gD- and gB-crystallin expression (Wigle and Oliver, 1999). As the

mutant lens cells fail to polarize and elongate properly, a hollow lens is formed. Prox1 binds and activates the mouse γD-crystallin promoter in vitro (Lengler et al., 2001). POU homeodomain transcription factors. Bicoid-related POU homeodomain transcription factors Pitx2 and Pitx3 also regulate eye development (see figure 57.2). Deletion of Pitx2, normally expressed in the neural crest– and the mesodermderived precursors of the periocular mesenchyme, results in severe disruption of periocular mesenchyme structures and extrinsic defects in early optic nerve development. Studies with neural crest–specific Pitx2 null mice (Pitx2-ncko) indicate that Pitx2 is required in neural crest for specification of the corneal endothelium and stroma and the sclera (Evans and Gage, 2005). Central corneal thickness is reduced in heterozygous Pitx2 mutant mice (Asai-Coakwell et al., 2006). Overexpression of Pitx2a isoform in the mouse corneal mesenchyme and iris lead to corneal opacification, corneal hypertrophy, irido-corneal adhesions, and severely degenerated retina resembling glaucoma and Axenfeld-Rieger syndrome (Homberg et al., 2004). In humans, PITX2 or FOXC1 mutations account for up to 50% of the AxenfeldRieger malformations of the anterior segment (Graw, 2003). Mutations in Pitx3, normally expressed from E10 in the thickening lens placode and later in the lens vesicle, are responsible for the aphakia mutant mice in which lens differentiation is affected after E11 (Semina et al., 1997, 2000). In humans, PITX3 mutations lead to posterior polar cataract and variable anterior segment mesenchymal dysgenesis (Addison et al., 2005; Semina et al., 1998). Sip1. Sip1, a Smad-interacting zinc-finger homeodomain transcription factor, is expressed after lens placode induction in the lens epithelium and immature lens fibers of the bow region (Yoshimoto et al., 2005). Conditional deletion of Sip1 in the lens results in a small hollow lens connected to the surface ectoderm (Yoshimoto et al., 2005). Sip1 is required for lens fiber cell maturation, and γ-crystallin, a marker for mature fiber cells, is absent in the Sip1 null lenses (Yoshimoto et al., 2005). Sip1 activates Foxe3 expression in a Smadbinding domain–dependent manner in the lens (Yoshimoto et al., 2005). Non-homeobox Transcription Factors Regulating the Development of Lens and Cornea High mobility group transcription factors Sox1 and Sox2. Sox transcription factors belonging to the high mobility group (HMG) family of DNA-binding proteins are involved in the regulation of diverse developmental processes. The targeted deletion of Sox1 in mice causes microphthalmia and cataract (Nishiguchi et al., 1998). Mutant lens fiber cells fail to elon-

gate and lack expression of γ-crystallins, as direct interaction of Sox1 protein with γ-crystallin promoters is required for their expression. Both Sox1 and Sox2 bind upstream lens enhancer elements in the mouse aB-crystallin gene (Ijichi et al., 2004). Transcription factors AP2, Pax6, and Prox1 upregulate Sox2 gene expression (see figure 57.2) (Lengler et al., 2001). Hmgn1. The nucleosome-binding HMG protein Hmgn1, capable of altering the structure and activity of chromatin, affects the development of the corneal epithelium in mice (Birger et al., 2006). Hmgn1 null mice develop a thin, poorly stratified corneal epithelium depleted of suprabasal wing cells and a disorganized basement membrane. Epithelial cell–specific markers glutathione-S-transferase (GST)-α4 and -ω1 are reduced in Hmgn1 null corneas, while the components of adherens junctions—E-cadherin and α-, β- and γ-catenin—are upregulated (Birger et al., 2006). Winged helix-forkhead transcription factors. Human congenital primary aphakia, a rare developmental disorder characterized by microphthalmia, anterior segment dysgenesis, and in severe cases the complete absence of lens, is caused by null mutations in FOXE3, a winged helix-forkhead transcription factor (Valleix et al., 2006). In the mouse, mutations in Foxe3 result in the dysgenetic lens (dyl) mutant strain that has several defects in lens development and altered patterns of crystallin expression (Blixt et al., 2007; Brownell et al., 2000; Medina-Martinez et al., 2005). Foxe3 is initially expressed in the developing brain and the lens placode and later restricted to the anterior lens epithelium in a Pax6- and Sip1-dependent manner, and is turned off on fiber cell differentiation (Yoshimoto et al., 2005). Targeted disruption of mouse Foxe3 results in an abnormal eye with a small, vacuolated lens. The anterior lens epithelium and the cornea do not separate, forming an unusual, multilayered tissue. These defects in lens development are accompanied by changes in the expression of DNase II-like acid DNase, Prox1, p57, and PDGF-α receptor (Blixt et al., 2007; Medina-Martinez et al., 2005). Two other forkhead transcription factors, Foxc1 (Mf1) and Foxc2 (Mfh1), have nearly identical DNA binding domains, and largely overlapping expression patterns and functions in the developing eye (Hiemisch et al., 1998; Smith et al., 2000). Foxc1, expressed in the mesenchymal cells in the eye (Kidson et al., 1999; Kume et al., 1998), is restricted to the future trabecular meshwork by E16.5 (Kidson et al., 1999). Foxc1 null mice die at birth with multiple abnormalities, including severe anterior segment developmental defects, such as fused lens and cornea, a thickened corneal epithelium, disorganized stroma, and missing endothelium (Hong et al., 1999; Kidson et al., 1999; Kume et al., 1998). Foxc1 heterozygous mice are viable, with milder anterior

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segment defects (Hong et al., 1999; Smith et al., 2000). Foxc1 and Foxc2 double heterozygous mice have malformations of the ciliary body not seen in either heterozygous mouse alone (Smith et al., 2000). In humans, FOXC1 mutations cause anterior segment dysgenesis and glaucoma (Honkanen et al., 2003; Mears et al., 1998; Mirzayans et al., 2000; Nishimura et al., 1998). Heat shock factor 4. The heat shock factors (HSFs) belonging to the basic domain/leucine zipper family of transcription factors consist of three members in mammals (Hsf1, 2, and 4). HSFs upregulate the stress-inducible promoters by interacting with the heat shock responsive elements. In the embryonic mouse lens, Hsf1 and Hsf2 are expressed at high levels. Their levels decrease in the adult lens, where Hsf4 takes over (Somasundaram and Bhat, 2004). αB-Crystallin promoter is upregulated by Hsf4, but not by Hsf1 or Hsf2 (Somasundaram and Bhat, 2004). Hsf4 null mice show increased proliferation, premature, defective differentiation of the lens epithelial cells, and increased expression of growth factors FGF-1, FGF-4, and FGF-7, and develop early postnatal cataract with abnormal lens fiber cells containing inclusion-like structures (Fujimoto et al., 2004; Min et al., 2004). The human HSF4 locus is associated with autosomal recessive cataract (Smaoui et al., 2004) and autosomal dominant lamellar and Marner cataract (Bu et al., 2002). Maf. Maf transcription factors belong to the basic domain/ leucine zipper family whose consensus target site, T-MARE, is an extended version of an AP1 site. Three members of the large Maf family, c-Maf, MafB, and Nrl, are expressed in the mouse lens (Kawauchi et al., 1999). The expression of c-Maf is earliest and most prominent in lens fiber cells and persists throughout lens development (see figure 57.2) (Kawauchi et al., 1999; Ring et al., 2000). Homozygous Maf mutant embryos and newborns show defective lens fiber cell differentiation, with severely impaired expression of crystallin genes. Posterior lens cells in Maf(lacZ) mutant mice do not elongate, do not express aA- and any of the b-crystallin genes, and display inappropriately high levels of DNA synthesis at E11.5 (Kawauchi et al., 1999; Kim et al., 1999; Ring et al., 2000). Maf interacts with Pax6 and Sox to synergistically upregulate the mouse αB-crystallin and γFcrystallin promoter activities, respectively (see figure 57.2) (Rajaram and Kerppola, 2004; Yang et al., 2004). Synergistic activation of these promoters by Maf and Sox and their subnuclear localization are disrupted by a mutation in Maf that causes cataract (Rajaram and Kerppola, 2004). Sp1/Krüppel-like transcription factors. Many Sp1/Krüppel-like transcription factors, members of the zinc finger family of DNA binding proteins, are expressed in the ocular surface and lens (see figure 57.2) (Chiambaretta et al., 2004; Norman

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et al., 2004). Expression of Sp1 in the ectoderm and lens vesicle at E11 is much lower than that in the cornea from E15.5 to late stages (Nakamura et al., 2005). Sp1 levels in the cornea decline gradually following eyelid opening. Evidence for the involvement of Sp1 in the regulation of corneal gene expression comes from the finding that Sp1 activates involucrin gene expression in the differentiating corneal epithelium (Adhikary et al., 2005b). Expression of Krüppel-like transcription factor Klf4, one of the most highly expressed transcription factors in the mouse cornea (Norman et al., 2004), is detectable in the ocular surface from around E10 and is sustained in the adult cornea (see figure 57.2). Conditional deletion of Klf4 in the surface ectoderm-derived structures of the eye leads to fragile corneal epithelium, swollen, vacuolated basal epithelial and endothelial cells, vacuolated lens, edematous stroma, and loss of conjunctival goblet cells (Swamynathan et al., 2007). Klf4 binds and activates keratin-12 and aquaporin-5 promoters in the corneal epithelium (Swamynathan et al., 2007). Klf4 cooperates with Oct3/4 and Sox2 and acts as a mediating factor that specifically binds to the proximal element to activate the Lefty1 core promoter in embryonic stem cells (Nakatake et al., 2006). Pluripotent stem cells could be derived from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under embryonic stem cell culture conditions (Takahashi and Yamanaka, 2006). As each of these factors is present in the corneal limbus, it is likely that they are involved in the maintenance of limbal stem cells, the source of epithelial cells in the mature cornea. Another Krüppel-like factor, Klf6, is expressed in the lens pit at E10.5, in the ectoderm, mesenchyme, and the lens epithelium at E12.5, and in the lens and corneal epithelium and corneal stroma at E15.5 (Nakamura et al., 2004). Later on, the expression of Klf6 remains high in the cornea but decreases in the lens. In humans, expression of KLF6 is elevated in keratoconus, a progressive disease associated with thinning and scarring of the cornea (Chiambaretta et al., 2006). KLF6 binds and downregulates the a1-proteinase inhibitor (a1-PI) gene in corneal epithelial cells and may thereby be involved in keratoconus (Chiambaretta et al., 2006). Human KLF6 binds and activates keratin-12 promoter in cultured cells (Chiambaretta et al., 2002). I-kB-z. I-κB-ζ, a regulator of the transcription factor NFκB, is expressed in the ocular surface epithelium, a part of the mucosal defense system (see figure 57.2) (Ueta et al., 2005). I-κB-ζ negatively regulates the pathological progression of ocular surface inflammation (Ueta et al., 2005). I-kB-z null mouse ocular surface shows chronic inflammation accompanied by loss of conjunctival goblet cells, indicating that I-κB-ζ is an integral part of the network of transcription factors required for ocular surface development (Ueta et al., 2005).

AP1. AP1, a group of dimeric complexes formed by the various Jun, Fos, Fra, and ATF proteins that regulate cell proliferation in response to various stimuli, is expressed in the lens and cornea (see figure 57.2) (Adhikary et al., 2005a; Ilagan et al., 1999; Norman et al., 2004; Okada et al., 2003; Shirai et al., 2004). AP1 activates mouse aA-crystallin expression through the conserved +25/+32 bp AP1 binding site (Ilagan et al., 1999). AP1 is necessary for expression of involucrin, a structural protein that is selectively expressed in differentiating corneal epithelial cells (Adhikary et al., 2005a, 2005b). In transgenic mice, removal of the AP1 site by truncation or point mutation results in a loss of involucrin expression, confirming the importance of AP1 for involucrin promoter activity during corneal epithelial cell differentiation. AP2. The activating protein-2 (AP2) family of transcription factors, consisting of five different members, AP2α, AP2β, AP2γ, AP2δ, and AP2ε, stimulates proliferation and suppresses terminal differentiation in a cell-type-specific manner during embryonic development (Eckert et al., 2005). AP2α is expressed in the lens and corneal epithelium (see figure 57.2) (Ohtaka-Maruyama et al., 1998; West-Mays et al., 1999, 2003). Ap2a null embryos exhibit a range of phenotypes from a complete lack of eyes to defective lens attached to the overlying surface ectoderm (West-Mays et al., 1999). Conditional deletion of Ap2a in lens placode derivatives, including the corneal epithelium, results in a decrease in the expression of the cell-cell adhesion molecule E-cadherin, misexpression of laminin, entactin, and type IV collagen, and disruption of stromal collagen fibril organization, showing that AP2α is required for proper formation of the mouse cornea (Dwivedi et al., 2005; West-Mays et al., 2003). Pax6 and AP2α interact with each other and coordinate the expression of gelatinase-B (matrix metalloproteinase 9) and corneal epithelial repair (Sivak et al., 2004). Elf3. Ese-1/Elf3, an epithelium-specific transcription factor, is upregulated in differentiating mouse corneal epithelium and in immortalized human corneal epithelial cells (HCEs), and transactivates keratin-12 through Ets binding sites (Yoshida et al., 2000). Suppression of Ese-1/Elf3 by antisense RNA in HCE cells affects their differentiation, suggesting the involvement of Ese-1/Elf3 in differentiation of corneal epithelial cells (see figure 57.2).

Regulation of expression of specific genes in the lens and cornea Crystallin Gene Expression in the Lens Differentiation of the lens is characterized by lens-preferred expression and accumulation of water-soluble crystallins, essential lens structural proteins required for light refraction and

transparency (Piatigorsky, 1998; Piatigorsky and Wistow, 1989). Much effort in the last two decades shows that the lens-preferred expression of crystallins is an outcome of synergistic interactions between developmentally regulated transcription factors such as Pax6, c-Maf, MafA/L-Maf, MafB, Nrl, Sox1, Sox2, Rarβ/Rxrβ, Prox1, Six3, and Hsf4, ubiquitously expressed factors such as AP1, Creb, pRb, and Usf, and chromatin remodeling proteins such as Asc-2 and Cbp/p300 (figure 57.3) (Cvekl and Piatigorsky, 1996; Cvekl and Tamm, 2004; Cvekl et al., 2004). aA-Crystallin. Mouse αA-crystallin, a marker of lens fiber cell differentiation and one of the most abundant mouse lens crystallins, is expressed at high levels in the developing lens, beginning around E10.5, and continuing in the adult lens (Robinson and Overbeek, 1996; Wawrousek et al., 1990). The mouse αA-crystallin promoter contains a Maf responsive element (MARE) at −110/−98 bp position, and overlapping binding sites for αACryBP1/PrdII/Mbp-1 (Brady et al., 1995; Kantorow et al., 1993; Nakamura et al., 1990), Maf (Wawrousek et al., 1990; Yang and Cvekl, 2005), and Pax6 (Cvekl et al., 1995) at −88 to +46 bp position (see figure 57.3). Pax6 and c-Maf activate aA-crystallin moderately and strongly, respectively, in a nonsynergistic manner (Yang and Cvekl, 2005; Yang et al., 2004, 2006). Three conserved distal control regions (DCR1, −7706/ −7492 bp; DCR2, −1900/−1670 bp; DCR3, +3650/ +3856 bp) regulate mouse αA-crystallin promoter activity (see figure 57.3) (Yang et al., 2004). DCR1, bound by Pax6, stimulates αA-crystallin promoter activity in primary lens explants. DCR1/DCR2/αA-crystallin promoter activity resembles the native αA-crystallin promoter activity in lens epithelium and fiber cells. In contrast, the DCR3/αAcrystallin promoter activity is restricted to late lens fibers (Yang et al., 2004, 2006). Pax6, c-Maf, and CREB recruit chromatin-remodeling enzymes such as Brg1, Snf2h, CBP, and/or p300, thereby leading to localized histone acetylation and upregulation of aA-crystallin gene expression (Chen et al., 2002; Yang et al., 2006). aB-Crystallin. Expression of αB-crystallin, a member of the small heat shock protein (sHSP) family, begins in the lens placode at E10 and continues at high levels in the lens epithelium and the lens fibers, and in moderate levels in many tissues (Haynes et al., 1996; Iwaki et al., 1989). A skeletal muscle–preferred, orientation-dependent enhancer containing five distinct cis-acting regulatory elements (αBE-1, αBE-4 αBE-2, αBE-3, and MRF, an E box) is present at −427/−259 bp position (see figure 57.3) (Gopal-Srivastava and Piatigorsky, 1993; Gopal-Srivastava et al., 1995; Swamynathan and Piatigorsky, 2002). The −426/+44 bp, −339/+44 bp, and −164/+44 bp promoter fragments retain activity in the lens as well as corneal epithelium, indicating

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A. Promoters αA-crystallin

αB-crystallin

γ-crystallin β-crystallin

-200

-150

-100

-50

+1

+50

-200

-150

-100

-50

+1

+50

-200

-150

-100

-50

+1

+50

-200

-150

-100

-50

+1

+50

B. Enhancers -7706/-7492 DCR1

+3650/+3856 DCR3

-1900/-1670 DCR2

αA-crystallin −8000

−4000

+1

E1 HspB2-E2

αB-crystallin −3000

Pax6 Maf

HspB2-E1

−2000

+4000

E4

E2

E3

−1000

-2656/-2267

RAR/RXR

Sox

Creb

Six3

AP1

Hsf4

MRF

-427/-259

αB-crystallin

+1

Sp1 Myogenic Factors

Figure 57.3 Organization of different transcription factor binding sites on crystallin promoters (A) and enhancers (B). Note the use of similar transcription factor binding sites on diverse crystallin pro-

moters. DCR, distal control region. DNA fragments are not drawn to scale.

that the αB-crystallin enhancer is not required for basal promoter activity in the lens and cornea (Gopal-Srivastava et al., 2000). Another well-conserved lens-specific enhancer (−2656/−2267 bp) containing four putative Sox1- and Sox2binding sites activates the αB-crystallin promoter, even though the HspB2 gene is located in between this enhancer and the αB-crystallin promoter (see figure 57.3) (Ijichi et al., 2004). Two well-conserved lens-specificity regions, LSR1 (−147/ −118 bp) and LSR2 (−78/−46 bp), are bound by Pax6, Maf, and RARs and are required for the lens activity of the mouse αB-crystallin promoter (see figure 57.3) (Gopal-Srivastava et al., 1996; Yang et al., 2004). The −164/+44 bp and −115/ +44 bp mouse αB-crystallin promoter activities are lens specific in transgenic mice, although the average activity is 30 times lower for the shorter fragment lacking LSR1. Sitespecific mutation of LSR1 eliminates both Pax6 binding and the −164/+44 bp promoter activity in transgenic mice (Gopal-Srivastava et al., 1996). Pax6-mediated activation of the αB-crystallin −162/+45 bp promoter is more robust in association with any of the Mafs and RARβ/RXRβ (Yang et al., 2004).

b- and g-Crystallins. β- and γ-crystallins are expressed in a lens fiber cell–specific manner. Several cis-elements required for lens-specific expression of the βB1-crystallin promoter have been identified by phylogenetic footprinting of the 5′ flanking sequence of the mouse, rat, human, and chicken bB1-crystallin genes (see figure 57.3) (Chen et al., 2001). The mouse bB1-crystallin −1493/+44 CAT transgene is expressed in the lens fiber cells (Chen et al., 2001). Transcription factor c-Maf directly activates many b-crystallin genes (see figure 57.3) (Ring et al., 2000). Mutations in γ-crystallins lead to many dominant inherited cataracts in humans and mice (Garber et al., 1985; Graw, 1999). Sox1 binds a conserved element in the γcrystallin promoter, and targeted deletion of Sox1 causes reduced g-crystallin expression, impaired fiber cell elongation, microphthalmia, and cataract (Nishiguchi et al., 1998). The heat shock factor HSF4 supports gF-crystallin expression in the lens fiber cells (Fujimoto et al., 2004). Pax6 represses the activation of the γF-crystallin promoter by large Mafs, Sox, and RARβ/RXRβ proteins in transiently transfected lens cells (Yang et al., 2004).

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Gene Expression in the Cornea Unlike the lens, where a large body of work has focused on understanding the regulation of a handful of crystallin genes, in the cornea there is a limited amount of work, spread among different genes. Keratocan. Keratocan (Ktcn), a keratan sulfate proteoglycan of the extracellular matrix, is first expressed at E13.5 in the periocular mesenchymal cells and after E14.5 is restricted to the stromal keratocytes (Liu et al., 1998). In adult transgenic mice with b-geo transgene driven by 3.2 kb 5′ flanking sequence, exon 1 and 0.4 kb of intron 1 of Ktcn, β-Gal activity is detected only in cornea. Spatiotemporal expression patterns of the −3.2 kb fragment transgene recapitulate that of endogenous Ktcn, suggesting that the 3.2 kb Ktcn upstream sequence contains the necessary cis-elements to regulate keratocan gene expression (Liu et al., 2000). Keratins. Keratin-12 (Krt12), one of the more than 30 different keratins (intermediate filament components), each with a specific expression pattern in different epithelial cells, is expressed specifically in the stratified corneal epithelium (Tanifuji-Terai et al., 2006). Heterozygous mutations in KRT12 cause Meesmann corneal dystrophy, an autosomal dominant disorder affecting the human corneal epithelium. Krt12 null mice develop fragile corneal epithelium resembling Meesmann corneal dystrophy (Kao et al., 1996). During embryonic development, Krt12 expression is restricted to the suprabasal and/or superficial cells of the corneal epithelium from E15.5 to P10 in mice. After PN30, Krt12 expression is detected sporadically in the basal corneal epithelium, and the number of Krt12-positive basal cells increases as the mice grow older (Tanifuji-Terai et al., 2006). Pax6, KLF4, and KLF6 stimulate Krt12 promoter activity (Liu et al., 1999). Expression of Krt12 is delayed and downregulated in the Pax6+/− corneal epithelium, implying abnormal differentiation (Ramaesh et al., 2005). Corneal crystallins. An underlying assumption in many gene regulation–related studies is that gene expression is controlled mainly at the level of transcription. However, posttranscriptional regulation may play a significant role in the expression of abundant corneal crystallins aldehyde dehydrogenase IIIA1 (Aldh3a1) and transketolase (Tkt), which constitute roughly 50% and 10% of the water-soluble protein, respectively, and only about 1% each of the total mRNA in the adult cornea (Norman et al., 2004). Aldh3a1 expression in the mouse is at least 500-fold higher in the corneal epithelial cells than in other tissues (Kays and Piatigorsky, 1997). In transgenic mice, a 13 kb mouse Aldh3a1 promoter fragment is active selectively in tissues that express the endogenous Aldh3a1 gene; however, unlike the promoter activity of the endogenous wild-type gene, the

promoter activity of the 13 kb fragment in the transgene is higher in the stomach and bladder than in the cornea. By contrast, a 4.4 kb mouse Aldh3a1 promoter fragment drives transgene expression in transgenic mice specifically in the corneal epithelial cells and not in other tissues, indicating that cis-control elements for corneal promoter activity reside within this smaller DNA fragment (Kays and Piatigorsky, 1997). Current experiments indicate that Aldh3a1 promoter activity is controlled by transcription factors that also control lens crystallin genes (Pax6, Oct1) as well as additional transcription factors expressed highly in the cornea (KLF4, KLF5). In addition, a suppressor sequence resides in the first intron of the mouse Aldh3a1 gene, although it is not known at present how it might function in regulating endogenous gene expression (Davis et al., in press). Another mouse corneal crystallin is Tkt, which is expressed at 30–50 times higher levels in the mature mouse cornea than in other tissues (Sax et al., 1996). Tkt mRNA levels increase six-fold in the mouse cornea in vivo within 1–2 days of eye opening. Both exposure to light and oxidative stresses appear to play a role in the up-regulation of Tkt gene expression after eye opening and during corneal maturation (Sax et al., 2000). The Tkt gene contains two transcription initiation sites separated by 630 bp, with a common initiator ATG codon (West-Mays et al., 1999). The distal transcription initiation site is used weakly in liver and the proximal GCrich transcription initiation site (within intron 1) lacking a TATA box is used in the liver and for high corneal expression. There is little knowledge yet of the molecular basis for the high expression of Tkt in the mouse cornea.

Concluding remarks The studies summarized in this chapter indicate that the mouse lens and cornea utilize many similar transcription factors for their development and maintenance. One of the striking similarities in gene expression in these two tissues is that many (not all) of these developmental transcription factors are also employed to express their highly specific multifunctional proteins, the crystallins. In view of the similarities between lens and cornea in their (1) origin from ectodermal cells during embryogenesis, (2) utilization of common transcription factors during embryonic development and postnatal maturation, (3) accumulation of crystallins, and (4) optical function, it has been proposed that the cornea and lens be considered as a single unit called the refracton (Piatigorsky, 2001). The refracton concept is not meant to equate the needs for and extents of refraction served by the lens and/or cornea of different species, both of which may differ considerably among species (especially aquatic versus terrestrial species) or even within the same species at different developmental times (such as an aquatic larva and terrestrial vertebrate, i.e., a toad) or at different

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behavioral times (such as an amphibious species in water or land, i.e., a frog or penguin). Rather, unifying the lens and cornea as the refracton underlines the numerous developmental and functional similarities of these two transparent eye structures, including especially the accumulation of taxon-specific, soluble proteins called collectively the lens or corneal crystallins, respectively. It is our hope that the refracton concept will stimulate cross-fertilization of ideas and knowledge concerning these refractive tissues of the eye by increasing dialogue and information exchange between basic scientists and clinicians presently specializing in the lens or the cornea. We have witnessed substantial progress in our understanding of gene expression and its consequences for the development of the cornea and lens in the recent past. Many studies have utilized germ-line deletions or conditional inactivation of specific genes in the surface ectoderm–derived tissues of the eye. Improvements in our ability to delete or mutate specific genes in a spatiotemporally controlled manner will further aid this effort. We can look forward to the identification of additional roles for the transcription factors currently known to influence eye development, as well as to the discovery of novel transcription factors and new interactions among the transcription factors, in our quest to unravel the molecular basis, biological consequences, and medical implications of gene expression in the lens and cornea. acknowledgments

Work was supported by the intramural research program of the National Eye Institute, NIH. We are grateful to Dr. Janine Davis for a critical reading of the manuscript.

REFERENCES Adachi, W., Ulanovsky, H., Li, Y., Norman, B., Davis, J., and Piatigorsky, J. (2006). Serial analysis of gene expression (SAGE) in the rat limbal and central corneal epithelium. Invest. Ophthalmol. Vis. Sci. 47:3801–3810. Addison, P. K., Berry, V., Ionides, A. C., Francis, P. J., Bhattacharya, S. S., and Moore, A. T. (2005). Posterior polar cataract is the predominant consequence of a recurrent mutation in the PITX3 gene. Br. J. Ophthalmol. 89:138–141. Adhikary, G., Crish, J. F., Bone, F., Gopalakrishnan, R., Lass, J., and Eckert, R. L. (2005a). An involucrin promoter AP1 transcription factor binding site is required for expression of involucrin in the corneal epithelium in vivo. Invest. Ophthalmol. Vis. Sci. 46:1219–1227. Adhikary, G., Crish, J. F., Gopalakrishnan, R., Bone, F., and Eckert, R. L. (2005b). Involucrin expression in the corneal epithelium: An essential role for Sp1 transcription factors. Invest. Ophthalmol. Vis. Sci. 46:3109–3120. Aota, S., Nakajima, N., Sakamoto, R., Watanabe, S., Ibaraki, N., and Okazaki, K. (2003). Pax6 autoregulation mediated by direct interaction of Pax6 protein with the head surface ectodermspecific enhancer of the mouse Pax6 gene. Dev. Biol. 257:1–13.

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with an autosomal recessive congenital cataract. Invest. Ophthalmol. Vis. Sci. 45:2716–2721. Smith, R. S., Zabaleta, A., Kume, T., Savinova, O. V., Kidson, S. H., Martin, J. E., Nishimura, D. Y., Alward, W. L., Hogan, B. L., et al. (2000). Haploinsufficiency of the transcription factors FOXC1 and FOXC2 results in aberrant ocular development. Hum. Mol. Genet. 9:1021–1032. Somasundaram, T., and Bhat, S. P. (2004). Developmentally dictated expression of heat shock factors: Exclusive expression of HSF4 in the postnatal lens and its specific interaction with alphaB-crystallin heat shock promoter. J. Biol. Chem. 279: 44497–44503. Swamynathan, S. K., Katz, J. P., Kaestner, K. H., AsheryPadan, R., Crawford, M. A., and Piatigorsky, J. (2007). Conditional deletion of mouse Klf4 gene results in corneal epithelial fragility, stromal edema and loss of conjunctival goblet cells. Mol. Cell Biol. 27:182–194. Swamynathan, S. K., and Piatigorsky, J. (2002). Orientationdependent influence of an intergenic enhancer on the promoter activity of the divergently transcribed mouse Shsp/alpha Bcrystallin and Mkbp/HspB2 genes. J. Biol. Chem. 277:49700– 49706. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676. Tanifuji-Terai, N., Terai, K., Hayashi, Y., Chikama, T., and Kao, W. W. (2006). Expression of keratin 12 and maturation of corneal epithelium during development and postnatal growth. Invest. Ophthalmol. Vis. Sci. 47:545–551. Tomarev, S. I., Zinovieva, R. D., Chang, B., and Hawes, N. L. (1998). Characterization of the mouse Prox1 gene. Biochem. Biophys. Res. Commun. 248:684–689. Ueta, M., Hamuro, J., Yamamoto, M., Kaseda, K., Akira, S., and Kinoshita, S. (2005). Spontaneous ocular surface inflammation and goblet cell disappearance in I kappa B zeta gene-disrupted mice. Invest. Ophthalmol. Vis. Sci. 46:579–588. Valleix, S., Niel, F., Nedelec, B., Algros, M. P., Schwartz, C., Delbosc, B., Delpech, M., and Kantelip, B. (2006). Homozygous nonsense mutation in the FOXE3 gene as a cause of congenital primary aphakia in humans. Am. J. Hum. Genet. 79: 358–364. Wawrousek, E. F., Chepelinsky, A. B., McDermott, J. B., and Piatigorsky, J. (1990). Regulation of the murine alpha Acrystallin promoter in transgenic mice. Dev. Biol. 137:68–76. West-Mays, J. A., Sivak, J. M., Papagiotas, S. S., Kim, J., Nottoli, T., Williams, T., and Fini, M. E. (2003). Positive influence of

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AP2alpha transcription factor on cadherin gene expression and differentiation of the ocular surface. Differentiation 71:206–216. West-Mays, J. A., Zhang, J., Nottoli, T., Hagopian-Donaldson, S., Libby, D., Strissel, K. J., and Williams, T. (1999). AP2alpha transcription factor is required for early morphogenesis of the lens vesicle. Dev. Biol. 206:46–62. Wigle, J. T., and Oliver, G. (1999). Prox1 function is required for the development of the murine lymphatic system. Cell 98: 769–778. Wistow, G. J., and Piatigorsky, J. (1988). Lens crystallins: The evolution and expression of proteins for a highly specialized tissue. Annu. Rev. Biochem. 57:479–504. Yang, Y., Chauhan, B. K., Cveklova, K., and Cvekl, A. (2004). Transcriptional regulation of mouse alphaB- and gammaFcrystallin genes in lens: Opposite promoter-specific interactions between Pax6 and large Maf transcription factors. J. Mol. Biol. 344:351–368. Yang, Y., and Cvekl, A. (2005). Tissue-specific regulation of the mouse alphaA-crystallin gene in lens via recruitment of Pax6 and c-Maf to its promoter. J. Mol. Biol. 351:453–469. Yang, Y., Stopka, T., Golestaneh, N., Wang, Y., Wu, K., Li, A., Chauhan, B. K., Gao, C. Y., Cveklova, K., et al. (2006). Regulation of alphaA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin. EMBO J. 25:2107– 2118. Yoshida, N., Yoshida, S., Araie, M., Handa, H., and Nabeshima, Y. (2000). Ets family transcription factor ESE-1 is expressed in corneal epithelial cells and is involved in their differentiation. Mech. Dev. 97:27–34. Yoshimoto, A., Saigou, Y., Higashi, Y., and Kondoh, H. (2005). Regulation of ocular lens development by Smad-interacting protein 1 involving Foxe3 activation. Development 132:4437– 4448. Zaki, P. A., Collinson, J. M., Toraiwa, J., Simpson, T. I., Price, D. J., and Quinn, J. C. (2006). Penetrance of eye defects in mice heterozygous for mutation of Gli3 is enhanced by heterozygous mutation of Pax6. BMC Dev. Biol. 6:46. Zhang, X., Friedman, A., Heaney, S., Purcell, P., and Maas, R. L. (2002). Meis homeoproteins directly regulate Pax6 during vertebrate lens morphogenesis. Genes Dev. 16:2097–2107. Zhou, M., Li, X. M., and Lavker, R. M. (2006). Transcriptional profiling of enriched populations of stem cells versus transient amplifying cells: A comparison of limbal and corneal epithelial basal cells. J. Biol. Chem. 281:19600–19609. Zieske, J. D. (2004). Corneal development associated with eyelid opening. Int. J. Dev. Biol. 48:903–911.

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Proteomics of the Mouse Lens WOLFGANG HOEHENWARTER AND PETER R. JUNGBLUT

Proteomics, the science surrounding all of a sample’s protein constituents, and the eye lens have a long mutual history (Hoehenwarter et al., 2006b). The lens is a tissue whose function is to concentrate light and help produce a sharp visual image. It is completely avascular and consists mostly of the proteins of one superfamily, the crystallins (Wistow and Piatigorsky, 1988). Their short-range order and precise molecular arrangement in solution establish the refractive index and ultimately its function (Delaye and Tardieu, 1983). Proteomics has extensively characterized these proteins in many species. A high-definition separation of all the ureasoluble protein constituents of the mouse lens was produced using large-scale carrier ampholyte-based two-dimensional gel electrophoresis (2DE) (Jungblut et al., 1998). This work produced 1,940 distinct dye-stained protein spots, many of which were excised from the gel and analyzed with matrixassisted laser desorption ionization time-of-flight mass spectrometry. It became clear that most of the polypeptides of the eye lens of the mouse are indeed different modified crystallin proteins, or more appropriately crystallin protein species. In-depth studies have been completed in several wildtype and naturally occurring and induced mutant mouse strains and have identified numerous posttranslational modifications in the healthy lens and in the lens affected by cataract, and have elucidated some of the factors implicated in this disease. Future studies should establish the totality of the protein species, resolve proteins specifically involved in the onset and progression of cataract, and give an in-depth understanding of the tissue as a whole.

Major structural proteins of the lens: The crystallins The proteins of the crystallin superfamily make up 80%– 90% of the entire mass of lens proteins. They were originally classified according to their native size, and the first three letters of the Greek alphabet were assigned to them in descending order of their molecular weight (Mörner, 1893). α-Crystallin is the largest native crystallin, with a molecular weight of 300–>1,000 kd; β-crystallin is the next largest, with a molecular weight of between 40 and 200 kd; and γcrystallins are the smallest crystallins, with a molecular weight of around 20 kd. Native α- and β-crystallins are multimers composed of several types of monomeric subunits; native γ-crystallins do not polymerize. More recent size

exclusion chromatography has separated the native watersoluble crystallins of the adult lens more precisely into a large high-molecular-weight (HMW) fraction containing mostly α-crystallin and other proteins on the verge of insolubilization, a fraction containing α-crystallin, a higher and a lower molecular weight fraction of β-crystallin, β high (βH) and β low (βL), and a small fraction containing the γS-crystallin monomer, followed by the larger and lowest molecular weight γ-crystallin fraction. The native crystallin proteins are in various stages of solution and can be thought of as bulk material that fills the otherwise empty lens cells. This material is transparent due to the supramolecular arrangement and short-range order of the proteins. This order is not random but is precisely defined at a molecular level. A disturbance of this homeostasis can lead to unfolding, improper folding, insolubilization, and aggregation of proteins. The insoluble aggregates diffract incident light, resulting in opacification of the lens and visual impairment. This is what is known as cataract (Harding, 1972; Carrell and Lomas, 1997). The peculiarities of the lens, namely, the little protein biosynthesis and reduced metabolism and the exposure to light and its effects, have imparted a high degree of stability to the crystallins. They are also connected to the stress response (de Jong et al., 1989) next to their function as transparent structure. The protein concentration and the stochiometry of the crystallins in the regions of the lens vary, so a refractive index gradient is created. In most species the refractive index declines from center to periphery, which increases the convexity of the lens and can eliminate spherical and chromatic aberration (Fernald and Wright, 1983). This is especially so in the mouse, which has a lens nucleus with a very high protein content and little water. The α-crystallins are clearly distinct from the β- and γcrystallins in molecular evolution. In contrast, the β- and γcrystallin proteins seem to have a common ancestor. This leads to the assumption of an initial α-crystallin and β-/γcrystallin gene in an early archetype lens that differentiated into the multiple genes of the current crystallin protein families. The 16 major proteins of the α-, β-, and γ-crystallin protein families are highly conserved and ubiquitous in the lens of mammals (Lubsen et al., 1988), and are also present in many other species. Two in an evolutionary context, more distant members of the γ-crystallin protein family, are also known in the mouse, γN- and γS-crystallin (van Rens et al.,

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1989; Wistow et al., 2005). In addition, a number of less abundant crystallin proteins with specific functions related to enzymes have been detected in the lens of various species (Wistow, 1993). α-Crystallin Protein Family α-Crystallin is the most abundant protein in the lens. It constitutes between 30% and 50% of the total mass of lens proteins and is evenly distributed throughout the organ in most species. In the mouse, there are three unmodified α-crystallin polypeptides, αA-crystallin, αB-crystallin, and αA-insert crystallin. αA- and αB-crystallin are 57% identical in primary structure. αA-insert crystallin is unique to mammals and common in rodents and is also a product of the αA-crystallin gene. It is produced by alternative splicing of an insert exon in the first intron of the gene (Cohen et al., 1978). α-Crystallin has never been crystallized, so its threedimensional (3D) structure could not yet be determined by X-ray diffraction. The native proteins’ large size also precludes nuclear magnetic resonance (NMR) measurements. However, the secondary and tertiary structure of the individual α-crystallin gene products are known. Circular dichroism and infrared measurements have determined that they are composed primarily of β strands with little α helix structure (Thomson and Augusteyn, 1989; Farnsworth et al., 1997). They are subdivided into a hydrophobic, globular N-terminal domain, a hydrophilic C-terminal domain in β sheet conformation, and a C-terminal extension. The amino acid composition and tertiary structure of the N-terminal domain are relatively varied in the polypeptides and between species but include three structure-function regions in αhelical conformation (Smith et al., 1996; Pasta et al., 2003). A large part of the C-terminal domain is known as the αcrystallin domain (Caspers et al., 1995) and is conserved in the protein family. The C-terminal extension is variable. We have constructed a tertiary structure model of αAcrystallin of the mouse using ab initio protein structure prediction algorithms (figure 58.1; Hoehenwarter et al., 2006a). α-Crystallins undergo extensive posttranslational modification beginning at the earliest stages of lens development. These are mainly deamidation, phosphorylation, and Nand C-terminal truncation. The unmodified and modified α-crystallin monomers are the minimal subunits that assemble the functional native protein. Several models have been proposed that agree that they exchange dynamically (van den Oetelaar et al., 1990; Gesierich and Pfeil, 1996) and form small multimers as the building blocks of higher molecular order (Bova et al., 2000). Posttranslational modification of the monomers affects subunit multi- and oligomerization (Merck et al., 1992; Bova et al., 2000, Pasta et al., 2003; Thampi and Abraham 2003), quaternary structure dynamics (Bova et al., 2000; Pasta et al., 2003), and protein function

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Figure 58.1 Model of αA-crystallin secondary and tertiary structure, colored according to the hydrophobicity of its amino acid residues. The most hydrophobic residues are colored dark blue; the least hydrophobic residues are colored red, as shown in color plate 67. The Rosetta algorithm on the HMMSTR server (www.bioinfo. rpi.edu/~bystrc/hmmstr/server.php) (Bystroff and Shao, 2002) available on the ExPASY (http://au.expasy.org/) home page was used for molecular modeling. Full-length αA-crystallin secondary structure was calculated at 29.5% α helix and 32% β sheet content. The N-terminal globular domain is organized into three helices, displayed as ribbons with hydrophobic side chains buried. Structure-function regions identified earlier (Smith et al., 1996; Pasta et al., 2003) make up the first two of these N-terminal α helices. The highly conserved residues 102–117 of the “α crystallin domain” (Caspers et al., 1995), containing the substantial first part of a DNA-binding motif (Singh et al., 1998), as well as an arginine residue 116 shown to be critical for molecular integrity (Bera et al., 2002), are predicted to have α-helical conformation and are displayed as ribbons. This is consistent with an older 3D model (Farnsworth et al., 1998) and makes the α-helical prediction that is somewhat higher than previous calculations (Farnsworth et al., 1997; Horwitz et al., 1998; Bova et al., 2000) seem plausible. However, it is inconsistent with site-directed spin label studies that demonstrate β sheet conformation for residues 109–120 (Berengian et al., 1997). The model confirms the β sheet secondary structure of residues 67–101, determined to be an alcohol dehydrogenase (ADH) and 1,1′-bi (4-anilino) naphtalene-5,5′-disulfonic acid (bisANS) binding site and to exhibit extensive chaperone activity (Farnsworth and Singh, 2004). See color plate 67.

(Takemoto et al., 1993; Pasta et al., 2003). The small heat shock protein HSP27 can also coassemble with the αcrystallin monomers. Thus, α-crystallin is a polydisperse and highly dynamic protein whose size, structure, and function vary according to the composition of its subunits.

α-Crystallin is a molecular chaperone (Horwitz, 1992; Jakob et al., 1993). The “α-crystallin domain” is implicit in all small heat shock proteins, and it was shown that αBcrystallin is indeed a member of this protein family (Klemenz et al., 1991). The small heat shock proteins interact with denatured proteins, keeping them in solution and in a refoldable conformation independently of ATP. The substrates of α-crystallin in the lens are the β- and γ-crystallins (Wang and Spector, 1994; Bloemendal et al., 2004), some enzymes, and elements of the cytoskeleton, such as the intermediate filament protein vimentin and the beaded filament. It is reasonable to assume that next to its role as transparent structural material, α-crystallin induces proper cytoskeleton architecture and prevents protein insolubilization, and that it is a major factor in upholding the clarity and function of the lens over years. β- and γ-Crystallin Protein Families The β- and γcrystallins are the other abundant major proteins in the lens. The families have very similar protein and gene structure and thus presumably have a common ancestor. In the lens of the mouse and most other mammals, there are seven unmodified β-crystallin polypeptides, four relatively acidic, termed βA1 through A4, and three more basic, termed βB1 through βB3. βA1- and βA3-crystallin are the products of the same gene and are the result of alternate translation initiation starting points. In the mouse, βA1-crystallin lacks the first 17 N-terminal amino acid residues of βA3-crystallin. There are six highly conserved, unmodified γ-crystallin polypeptides, γA–γF, whose genes are linked in one gene cluster, and two more distantly related polypeptides, γN- and γS-crystallin. The primary function of both the β- and γcrystallins is as transparent material. Data on the protein structure for both families are abundant. β- and γ-crystallins have similar secondary and tertiary structure. The monomers are composed mostly of β strands organized into four motifs of four antiparallel β strands each. The motifs are of the Greek key type, with the β strands 1, 2, and 4 forming a β sheet and strand 3 in a proximal position. This motif structure leaves many hydrophobic amino acid residue side chains exposed. The isolated β strand 3 interacts with β strand 4 of the β sheet of the neighboring motif, and the two motifs assemble into a complete globular domain with hydrophobic side chains buried. The monomers are subdivided into two globular domains separated by an unorganized stretch of primary structure, the connecting peptide. Also, primary structure can extend beyond the domains, which are then known as either the N- or Cterminal extensions. The β-crystallin monomers are subunits that assemble the native higher molecular weight β-crystallin protein. The protein can be homo- or heteromeric (Bax et al., 1990; Slingsby and Bateman, 1990; Bateman et al., 2003) and is

mostly a dimer (Slingsby and Bateman, 1990) or tetramer; however, further oligomerization is also known (Bateman et al., 2003). The connecting peptide is extended, which mediates the interaction of two globular domains from individual molecules in pseudo-twofold symmetry. The βB-crystallins have long unstructured N- and C- terminal extensions, the βA-crystallins have only N-terminal extensions. Their function is not clear, but there is evidence that the extensions are involved in promoting higher-order assembly of β-crystallin tetra- and oligomers, and that they may act as “spacers” in the supramolecular arrangement of the proteins (Nalini et al., 1994; Bateman et al., 2003). The native protein is found throughout the fiber cells of the lens; however, the distribution of the individual βcrystallin monomers varies. βB2-crystallin is the major β-crystallin monomer in mammals, and synthesis of the βcrystallins is increased postnatally, so that the native protein is more abundant in the cortex than in the nucleus of the lens. The γ-crystallin proteins are strictly monomers. The connecting peptide is bent, which brings the two globular domains of one molecule into close proximity and promotes intermolecular domain interaction around a pseudo-twofold axis (Blundell et al., 1981). The two motifs that constitute a domain have adopted a slight asymmetry that allows rows of hydrophobic residues to interdigitate much as in a zipper and creates an extra degree of close packing. The γ-crystallins lack N-terminal extensions, and their C-terminal extensions are very short, so the molecules as a whole are very compact, with only limited regions exposed for proteolysis. This guarantees maximum protein stability and allows a very close association, to a high degree excluding water (Mayr et al., 1994). Indeed, the γ-crystallins achieve the highest protein density and are particularly suited for the dehydrated conditions in the nucleus of the lens, where they are most abundant and create the highest refractive index. In this context, the proteins can only conditionally be considered monomeric; it seems more plausible that they have entered a state of macromolecular crowding in an almost completely dehydrated environment (Stevens et al., 1995). The γ-crystallins also have an unusually high content of cysteine residues, which may be involved in molecular bonds with other molecules in this tightly packed arrangement.

Proteomics and eye lens proteomics: An introduction The high abundance and easy accessibility of the proteins of the lens have made the tissue a frequent subject of proteomics studies. Proteomics is a science dedicated to the comprehensive understanding of the totality and the dynamics of all of the protein constituents of a sample. Arbitrarily it began in 1975, when O′Farrell developed the 2DE technique combining isoelectric focusing (IEF) and sodium

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dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) for large-scale separation of the proteins of biological samples (O′Farrell, 1975). With this technique, denatured proteins migrate to positions termed protein spots on the 2DE gel, as dictated by the relation of their chemical parameters to the applied 2DE parameters (Jungblut et al., 1997). High quality 2DE has a remarkable resolution capacity, being able to separate up to 10,000 sample constituents distinguished by a single amino acid or posttranslational modification (Klose and Kobalz, 1995). The separated proteins in the gel are visualized by staining them with a dye or by blotting them onto membranes and then staining them, resulting in a 2DE protein spot pattern. The other premier technique employed for large-scale protein and peptide separation in proteomics is liquid chromatography (LC). LC is one of the oldest and best techniques for molecular separation in biochemical analysis, earning Archer Martin and Richard Synge the Nobel Prize for Chemistry in 1952. It is based on the individual behavior of soluble analytes in a mobile phase passed over and interacting differentially with a solid phase or matrix. Various combinations of liquid and solid phases featuring distinct molecular interaction and separation properties have been developed and tried over the years, leading to several popular LC approaches, among them affinity chromatography, ion exchange chromatography, reverse phase chromatography, and size exclusion chromatography. The combination of two or more of these techniques, in conjunction with relatively long durations (several hours) and low flow rates (nano-LC), has remarkable resolution power. Following separation, it is desirable to identify as many proteins as completely as possible. Originally, purified or separated proteins were mostly analyzed by Edman degradation, a procedure that realizes protein primary structure beginning with a polypeptide’s N-terminus (Edman, 1949). Primary structure information is the most basic and meaningful form of polypeptide characterization and presents the least degree of ambiguities. A major breakthrough was reached in the late 1980s, when mass spectrometry became available for polypeptide analysis. Originally developed late in the nineteenth century, it is a technique that measures an ionized molecule’s mass and charge. Ionized molecules from an ion source are manipulated by electric fields in an analyzer, where their recorded behavior prior to detection by an ion detector allows calculation of their physical parameters. So-called soft ionization techniques convert polypeptides to gas phase ions without damaging the molecules. The principal soft ionization techniques employed in proteomics are matrixassisted laser desorption/ionization (MALDI) (Karas and Hillenkamp, 1988; Tanaka et al., 1988) and electrospray ionization (ESI) (Fenn et al., 1989). It is common to digest proteins either with enzymes or with chemicals and to

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analyze the resulting peptides with mass spectrometry, because mass accuracy, resolution, and charge minimization are all improved in the low mass range, under 3 kd. The data acquired by mass spectrometry must be converted into meaningful information. A polypeptide’s mass and charge allow the calculation of its primary structure; however, in light of the 20 proteinogenic amino acids and modifications, the possibilities for large peptides and proteins are considerable. In theory, the sequenced genomes of organisms contain the primary structures of all of an organism’s primary translation products. This is an enormous asset for protein identification. Polypeptide masses recorded in mass spectra are used to search databases of conceptually translated genomics data according to primary structure segment masses, in many cases resulting in matches of the mass spectrometric data to amino acid sequences, producing peptide sequence suggestions and conclusive protein identification. This is termed peptide mass fingerprinting or peptide mass mapping (Henzel et al., 1993, Jungblut et al., 1997; Thiede et al., 2005). Modifications can be assessed from mass shifts using the identification as a reference. A number of software suites available commercially or free of charge on the Web expedite this process. Today’s generation of mass spectrometers deliver compositional and conformational information by fragmenting or promoting dissociation of the analyzed ions and applying a second round of mass spectrometry to the fragment ions. This is termed tandem mass spectrometry, or MS/MS (Senn et al., 1966; Biemann et al., 1966). Metastable dissociation of polypeptides occurs mainly at the peptide bond. If a peptide ion’s charge is retained at the N-terminus following dissociation, the resulting fragment ion is termed b ion, while if the charge is retained at the C-terminus, the fragment ion is termed y ion. These ions allow the additive calculation of mass differences between fragment ion masses and the correlation of the mass differences with amino acid residues. A combination of mass, charge, and MS/MS data—or exclusively MS/MS data, if sufficiently available—can be used to determine protein primary structure, including modifications and modification sites. Two-dimensional electrophoresis and lens proteomics go together from the outset. Already in 1975 Kibbelaar and Bloemendal applied the water-soluble and water-insoluble proteins of the whole lens, the size exclusion chromatography α, βH, βL, and γ fractions, and the urea-soluble proteins from the calf lens to urea PAGE combined with SDS-PAGE. They produced a first rudimentary protein spot pattern of the major lens proteins, the unmodified crystallins, and suggested α-crystallin interaction with membrane components (Kibbelaar and Bloemendal, 1975). With improvement in 2DE techniques, a definitive protein spot pattern or reference map for the crystallins and other components was achieved in 1982 for the water-soluble proteins of the bovine

lens cortex, with a final definitive nomenclature for β-crystallins added in 1984 (Berbers et al., 1982, 1984). Similar investigations in chicken, mouse, rat, and human yielded comparable crystallin reference patterns and α-crystallin characterization with nomenclature and demonstrated the presence of the cytoskeleton proteins vimentin and actin in the urea-soluble fraction, presumably from the anterior epithelial cell layer and outer cortex (Garadi et al., 1983; Garber et al., 1984; Datiles et al., 1992). Newer investigations have become increasingly comprehensive, often further characterizing crystallin proteins, confirming amino acid sequences predicted from cDNA or detecting discrepancies and annotating sequences and creating higher-quality reference maps, as well as detecting numerous noncrystallin lens proteins (Shih et al., 1998; Lampi et al., 2002; Hoehenwarter et al., 2005). A detailed reference map of the entire adult mouse lens proteome using high-resolution large-scale 2DE separated the urea-soluble proteins into 1,940 spots (Jungblut et al., 1998) and was ultimately followed by an in-depth analysis of the watersoluble and water-insoluble proteins at different ages (Ueda et al., 2002). A 2DE protein spot pattern of the young ureasoluble mouse lens proteome with the characteristic crystallin pattern produced in our laboratory is shown in figure 58.2. An accessible repository for mouse lens and other proteomics data is under preparation in the form of our 2DE database (www.mpiib-berlin.mpg.de/2D-PAGE). Major LC investigations have characterized the infant to adult water-soluble and the adult water-insoluble human lens proteomes. Following size exclusion chromatography, water-soluble α, βH, βL, and γ fractions were applied to reverse phase chromatography on C4 columns for further fractionation. Whole proteins were then analyzed offline with ESI mass spectrometry. The crystallin proteins αA and αB, βB1, βB2, and βB3, A1, A3, and A4, and γC, γD, and γS were detected and changes in their abundance as well as posttranslational modifications with age were characterized (Ma et al., 1998). Another investigation subjected the water-insoluble monomeric α-crystallins to cation exchange chromatography before reverse phase chromatography and offline ESI mass spectrometry. A high degree of separation was achieved, and it was shown that α-crystallin is the major component of the water-insoluble lens proteins in humans, constituting about half of protein abundance. In addition, numerous α-crystallin protein species were identified and distinguished from the water-soluble fraction (Lund et al., 1996). Large-scale LC-based proteomic investigations of the mouse lens proteome have not been published. In summary, the investigations described in this section and others not mentioned have proven invaluable for characterizing the proteins of the healthy lens as well as of morphological sections (Garland et al., 1996), and in elucidating some of the complex factors involved in cataract develop-

ment (Garber et al., 1984; David et al., 1994; Calvin et al., 1996; Li et al., 2002) in the mouse and various other species.

Proteomics beyond genomics The proteins in an organism are not limited to the primary translation products of its genes. Many proteins undergo posttranslational modification. Posttranslational modifications are covalent modifications to a protein’s primary structure that can alter the function of the original unmodified protein. This introduces an additional vast level of functional activity that goes completely beyond the genome and cannot be assayed by genomics- or transciptomics-based research. The taxonomic term protein species was introduced in 1996 (Jungblut et al., 1996) to express this context. A protein species is defined as a polypeptide and possibly one or more other chemical groups that are covalently bonded. As such, it is the most basic term in protein taxonomy. It distinguishes proteins, posttranslationally modified forms of a protein, protein isoforms, different allelic forms of a protein, and any other primary structure variants as mature distinct molecules. The proteome-wide identification of protein species is one of the major challenges facing proteomics today. A protein species is considered identified only when the entirety of its primary structure is known, so that it can be distinguished from another protein species that differs in only one primary structure element, such as one amino acid or chemical group or, ultimately, one atom. Current techniques, particularly 2DE and mass spectrometry, can achieve this on a small scale (Okkels et al., 2004). LC procedures are also able to separate the protein species of a proteome but stop short of making them as readily accessible as 2DE. Their primary advantage is the relative speed at which complex protein mixtures can be assayed, thanks to the development of the electrospray ionization technique. Together with a connecting apparatus, it allows polypeptides to be eluted online or semi-online from chromatography into a mass spectrometer (Yates, 1998). To maximize separation performance, multiple steps of different types of chromatography can be combined. This is known as multidimensional protein identification technology, or MudPIT (Washburn et al., 2001). Strategies of this type that analyze peptides from protein digestion or internal cleavage generate between 10,000 and 100,000 distinct mass spectra, making comprehensive evaluation equally as laborious as the complete analysis of spots from 2DE (Swanson and Washburn, 2005; Hoehenwarter et al., 2006a). Although rapid detection of modifications is possible, the assignment of the identified peptides and modifications to individual molecules and thus the identification of the protein species are not.

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Figure 58.2 Protein spot pattern of the urea-soluble proteins of the 10-day-old mouse lens separated with large-scale 2DE and visualized with silver staining. Some identified proteins are indicated; the crystallin proteins are named as a token only.

The lens proteins of the crystallin superfamily undergo extensive posttranslational modification in all species. This process begins before birth and increases as the organism ages, with early-onset modifications becoming more abundant and additional modification types appearing. Indeed, the water-soluble proteins of the nucleus and cortex of the adult human lens were analyzed separately with 2DE, which revealed a nuclear protein spot pattern that is quite distinct from the previously described cortical or whole-lens pattern (Garland et al., 1996). This is evidence of a high degree of

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posttranslational modification in the very young lens, as the development of the nucleus is essentially prenatal, and so the nuclear protein species must be derived from posttranslational modification of the primary crystallin proteins. As this report and numerous others have made clear, many types of posttranslationally modified crystallins exist and are abundant in the healthy lens. The fact that the protein species are mature molecules with individual primary structures implies that they have defined and possibly distinct functions in the supramolecular

order of the transparent mass of proteins in the lens. This is illustrated for some αA-crystallin protein species separated with 2DE (figure 58.3). The protein species were analyzed with mass spectrometry; it was ascertained that they were products of the αA-crystallin gene, and a nomenclature was introduced (Hoehenwarter et al., 2008). One of the protein species, α-A_B, was analyzed in detail (Hoehenwarter et al., 2006a). The protein species’s N-terminus is serine residue 42 of the full-length αA-crystallin protein and very probably the result of truncation by the calcium-dependent calpastatin protease Lp82. Its position on calibrated 2DE gels is in accordance with its theoretical molecular weight and pI. Nevertheless, the protein species was not identified, as only 50% sequence coverage was achieved with mass spectrometry. A model of its secondary and tertiary structure was produced with ab initio structure prediction algorithms (Bystroff and Shao, 2002). The insights gained from the model allowed the formulation of a hypothesis for the function of the protein species in the healthy lens. It is involved in the regulation of the size and function of the native protein. Together with other evidence, this discovery suggests that the protein species related to the primary translation products of the α-crystallin genes are also α-crystallin subunits, and that many more subunits than previously suspected influence the properties of the native HMW αcrystallin oligomer. In contrast to the number of protein species apparently necessary for the development of the healthy lens, some crystallin protein species are known to induce cataract. Their conformation is incompatible with their environment, so

they become insoluble, leading to opacities in the lens and visual impairment. Tryptophan oxidation to kynurenine was observed in γB- and γC-crystallins of adult mice ( Jungblut et al., 1998), but a clear connection of this modification to cataract is missing. Certain protein species such as truncated βB1- or γ-cystallin (David et al., 1994; Gong et al., 1997; Descamps et al., 2005; Hoehenwarter et al., 2008) are specifically connected to the onset and development of cataract, their appearance in the lens unequivocally leading to the disease. Other protein species may be involved in cataractogenesis; however, they are also found in the healthy tissue. This duality is not fully understood, but it is conceivable that the protein species are deleterious only when their abundance is perpetuated beyond a certain threshold. Also, it has been shown that modifying factors are present in the lens of the mouse, and that the interactions of the crystallin protein species with these protein species affect the onset and development of cataract as well as healthy lens development (Hoehenwarter et al., 2008). The concept of protein species with individual functions introduces a new level of complexity that is not unrealistic. These functions must be elucidated for a true understanding of the biological processes in the lens and other tissues, organs, and organisms. The first big step will be the complete identification of the proteome at the protein species level, meaning an exact description of native components. One of the techniques that could achieve this in the near future is the combination of multidimensional LC for protein species separation and MS/MS mass spectrometry of whole proteins for protein species identification. This approach,

Figure 58.3 Section of a protein spot pattern of the urea-soluble proteins of the 10-day-old mouse lens separated with large-scale 2DE and visualized with silver staining that shows the full length αA-crystallin protein and some other αA-crystallin protein species.

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known as top-down proteomics, has delivered encouraging results (Sze et al., 2002; Ge et al., 2002). Just recently the strategy’s main limitation, that it could not be applied to proteins larger than 50 kd, was alleviated (Han et al., 2006), bringing routine proteome characterization within reach. Other modern mass spectrometry techniques have been used to determine the seminative quaternary structure of αcrystallin and could be applied to other protein species as well (Aquilina et al., 2003). A technique known as imaging mass spectrometry can be used to determine the distribution of proteins and protein species in tissues or organs (Crecelius et al., 2005). The combination of these and other technologies with an established proteome should allow the functional analysis of the totality of the protein species and make a true, in-depth understanding of the native situation conceivable in the eye lens and other tissues.

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Resolution power of two-dimensional electrophoresis and identification of proteins from gels. Electrophoresis 17:839–847. Karas, M., and Hillenkamp, F. (1988). Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 60:2299–2301. Kibbelaar, M., and Bloemendal, H. (1975). The topography of lens proteins based on chromatography and two-dimensional gel electrophoresis. Exp. Eye Res. 21:25–36. Klemenz, R., Frohli, E., Steiger, R. H., Schafer, R., and Aoyama, A. (1991). Alpha B-crystallin is a small heat shock protein. Proc. Natl. Acad. Sci. U.S.A. 88:3652–3655. Klose, J., and Kobalz, U. (1995). Two-dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome. Electrophoresis 16:1034– 1059. Lampi, K., Shih, M., Ueda, Y., Shearer, T. R., and David, L. L. (2002). Lens proteomics: Analysis of rat crystallin sequences and two-dimensional electrophoresis map. Invest. Ophthalmol. Vis. Sci. 43:216–224. Li, W., Calvin, H. I., David, L. L., Wu, K., McCormack, A. L., Zhu, G. P., and Fu, S. C. (2002). Altered patterns of phosphorylation in cultured mouse lenses during development of buthionine sulfoximine cataracts. Exp. Eye Res. 75:335–346. Lubsen, N. H., Aarts, H. J., and Schoenmakers, J. G. (1988). The evolution of lenticular proteins: The beta- and gamma-crystallin super gene family. Prog. Biophys. Mol. Biol. 51:47–76. Lund, A. L., Smith, J. B., and Smith, D. L. (1996). Modifications of the water-insoluble human lens alpha-crystallins. Exp. Eye Res. 63:661–672. Ma, H., Fukiage, C., Azuma, M., and Shearer, T. R. (1998). Cloning and expression of mRNA for calpain Lp82 from rat lens: Splice variant of p94. Invest. Ophthalmol. Vis. Sci. 39:454– 461. Mayr, E. M., Jaenicke, R., and Glockshuber, R. (1994). Domain interaction and connecting peptides in lens crystallins. J. Mol. Biol. 235:84–88. Merck, K. B., De Haard-Hoekman, W. A., Oude Essink, B. B., Bloemendal, H., and De Jong, W. W. (1992). Expression and aggregation of recombinant alpha A-crystallin and its two domains. Biochim. Biophys. Acta 1130:267–276. Mörner, C. T. (1893). Untersuchung der Proteinsubstanzen in den leichtbrechenden Medien des Auges. Z. Physiol. Chem. 18: 61–106. Nalini, V., Bax, B., Driessen, H., Moss, D. S., Lindley, P. F., and Slingsby, C. (1994). Close packing of an oligomeric eye lens beta-crystallin induces loss of symmetry and ordering of sequence extensions. J. Mol. Biol. 236:1250–1258. O’Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007–4021. Okkels, L. M., Muller, E. C., Schmid, M., Rosenkrands, I., Kaufman, S. H., Andersen, P., and Jungblut, P. R. (2004). CFP10 discriminates between nonacetylated and acetylated ESAT-6 of Mycobacterium tuberculosis by differential interaction. Proteomics 4:2954–2960. Pasta, S. Y., Raman, B., Ramakrishna, T., and Rao, C. M. (2003). Role of the conserved SRLFDQFFG region of alpha-crystallin, a small heat shock protein: Effect on oligomeric size, subunit exchange and chaperone-like activity. J. Biol. Chem. 278: 51159–51166. Senn, M., Venkataraghavan, R., and McLafferty, F. W. (1966). Mass spectrometric studies of peptides. 3. Automated determination of amino acid sequences. J. Am. Chem. Soc. 88:5593– 5597.

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Shih, M., Lampi, K. J., Shearer, T. R., and David, L. L. (1998). Cleavage of beta crystallins during maturation of bovine lens. Mol. Vis. 4:4–11. Singh, K., Groth-Vasselli, B., and Farnsworth, P. N. (1998). Interaction of DNA with bovine lens alpha-crystallin: Its functional implications. Int. J. Biol. Macromol. 22:315– 320. Slingsby, C., and Bateman, O. A. (1990). Quaternary interactions in eye lens beta-crystallins: Basic and acidic subunits of betacrystallins favor heterologous association. Biochemistry 29:6592– 6599. Smith, J. B., Liu, Y., and Smith, D. L. (1996). Identification of possible regions of chaperone activity in lens alpha-crystallin. Exp. Eye Res. 63:125–128. Stevens, A., Wang, S. X., Caines, G. H., and Schleich, T. (1995). 13C-NMR off resonance rotating frame spin-lattice relaxation studies of bovine lens gamma-crystallin self association: Effect of “macromolecular crowding.” Biochim. Biophys. Acta 1246:82– 90. Swanson, S. K., and Washburn, M. P. (2005). The continuing evolution of shotgun proteomics. Drug Discov. Today 10:719– 725. Sze, S. K., Ge, Y., Oh, H., and McLafferty, F. W. (2002). Topdown mass spectrometry of a 29-kDa protein for characterization of any posttranslational modification to within one residue. Proc. Natl. Acad. Sci. U.S.A. 99:1774–1779. Takemoto, L., Emmons, T., and Horwitz, J. (1993). The Cterminal region of alpha-crystallin: Involvement in protection against heat-induced denaturation. 294:435–438. Tanaka, K., Waki, H., Ido, Y., Akita, S., Yoshida, Y., and Yoshida, T. (1988). Protein an polymer analysis up to m/z 100,000 by laser ionization time of flight mass spectrometry. Rapid Commun. Mass Spectrom. 2:151–153. Thampi, P., and Abraham, E. C. (2003). Influence of the Cterminal residues on oligomerization of alpha A-crystallin. Biochemistry 42:11857–11863.

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59

Genetic and Proteomic Analyses of the Mouse Visual Cycle JOHN C. SAARI AND JOHN W. CRABB

The vertebrate eye paints upon the retina with bleached photopigments a picture of the luminous world outside. —W. A. H. Rushton

Our retinas have evolved to provide useful detection of light over an extraordinary range of illumination intensities by using two sets of photoreceptors, rods and cones, which complement each other in sensitivity and in the speed and dynamic range of response. With this system, differentiation of the horizon from the moonless night sky is possible even though each rod photoreceptor of the human retina absorbs a photon on average once every 85 minutes. At the other end of the scale, a white ptarmigan can still be seen on a snowy slope in bright daylight, when the average cone photoreceptor absorbs more than 106 photons per second (Rodieck, 1998). Despite enormous differences in physiological responses, the chemistry of light detection involved in both rods and cones of all vertebrate species is the same and involves photoisomerization of 11-cis-retinal, or a closely related derivative, to all-trans-retinal. Enzymatic regeneration of 11cis-retinal takes place in all vertebrates in cells adjacent to the photoreceptor cells in a process originally called the visual cycle and more recently the retinoid cycle. Great advances have been made in the past decade in our understanding of the molecular events responsible for the regeneration of rod visual pigments. Detailed knowledge of a cone visual cycle remains elusive, although the evidence for involvement of Müller cells continues to accumulate. This chapter considers advances in our understanding of the visual cycle that critically depended on the use of mouse genetics, alteration of the mouse genome, and proteomics. The rich history associated with many visual cycle components is mentioned only in passing, with relevant reviews cited. Throughout this chapter, we use standard conventions for denoting genes and gene products (examples: human gene, RPE65; mouse gene, Rpe65; protein, RPE65). A variety of techniques have been employed over the years to study molecular aspects of the rod visual cycle. Early investigators made use of the color changes that accompanied photoisomerization of 11-cis-retinal, bound to opsin, to all-trans-retinal (purple to yellow) and reduction of all-trans-

retinal to all-trans-retinol (yellow to colorless). Later, classic enzymology provided valuable information about the various reactions that make up the cycle, primarily relying on bovine tissue extracts. Molecular cloning of cDNAs and ectopic cDNA expression allowed the assignment of enzymatic activities to molecular entities. The sequencing of complete genomes identified orthologues of key enzymes and opened up comparative sequence analysis for mechanistic studies. Assignment of an in vivo activity to a molecular entity was not a trivial matter and required techniques for generating transgenic and knockout mice. For one enzyme of the cycle (lecithin:retinol acyltransferase, LRAT), the enzymatic activity in vitro clearly pointed to an in vivo function, as proven by the phenotype of mice with targeted disruption of the gene. In other cases, disruption of the genes had either little effect (Rdh5, Rdh11, prRDH, Rdh12, Irbp) or a partial effect (Rlbp1) on the rate of rhodopsin regeneration, suggesting functional redundancy at these steps. Analysis of diseasecausing mutations in humans was not always helpful, presumably because of differences between the human and mouse visual systems. Additional information regarding the visual cycle resulted from the identification and characterization of proteins using mass spectrometry, methodology referred to as proteomics. Indeed, the sensitivity and accuracy of these techniques have revolutionized structural component analysis, and future utilization promises to open up our understanding of the cell biology of retinoid processing.

Genetic analysis of the mouse visual cycle The rod visual cycle is made up of a mixture of wellestablished and novel reactions occurring in two adjacent cell types. Diffusion of retinoids between the two cell types links the reactions into a cycle. Decades of research have established the reaction sequence in rod photoreceptors and retinal pigmented epithelium (RPE), and many of the enzymes that carry out these reactions in vivo have been identified. Nonetheless, details regarding the mechanisms of the reactions and possible reaction controls are lacking, and detailed information about the cell biology of retinoid processing in RPE is almost nonexistent. Furthermore, the

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nature of the diffusion of retinoids between the cell types is poorly understood. The phototransduction cascade is activated by the absorption of a photon by 11-cis-retinal, the chromophore attached to rod opsin (figure 59.1). The photoisomerization product, all-trans-retinal, dissociates from opsin and is reduced to all-trans-retinol by NADPH, catalyzed by one or more retinol dehydrogenases (RDHs) of the rod outer segment. All-trans-retinol diffuses from the rod cell through the interphotoreceptor matrix and enters the RPE, where LRAT catalyzes its esterification with a long chain fatty acid. Isomerohydrolase (RPE65) simultaneously hydrolyzes the ester bond of all-trans-retinyl ester and isomerizes the double bond at position 11–12 to yield 11-cis-retinol and a fatty acid. Oxidation of 11-cis-retinol to 11-cis-retinal by NAD is catalyzed by one or more cis-specific RDHs (cisRDHs) and returned to the rod outer segment for conjugation with opsin and regeneration of the visual pigment. Cellular retinol-binding protein type I (CRBPI) and cellular retinaldehyde-binding protein (CRALBP) are present in RPE and facilitate esterification and isomerization/oxidation of retinoids, respectively. Interphotoreceptor retinoid-

All-TRANS-Retinol Dehydrogenase Following photoisomerization of 11-cis-retinal, all-trans-retinal is released from opsin and reduced to all-trans-retinol by NADPH in the outer segments of photoreceptor cells. This reaction is accompanied by a change in color from yellow (all-transretinal) to colorless (all-trans-retinol) and was the first enzymatic visual cycle reaction to be studied in detail (Wald and Hubbard, 1949). Based on their kinetic properties and substrate specificities, several short chain dehydrogenases/reductases (SDRs) have been proposed to catalyze the reduction of all-transretinal in vivo. However, studies of knockout mice have failed to identify a single enzyme whose absence has a major effect on the rate of visual pigment regeneration in vivo. This example points to the importance of mouse genetics in assigning in vivo functions to in vitro activities.

Figure 59.1 Hypothetical schematic of the rod photoreceptor visual cycle. Transcellular diffusion and intracellular enzymatic processing of visual cycle retinoids are shown. A retinal pigmented epithelial (RPE) cell is depicted with apical process extending toward a rod outer segment, with one disc membrane. Reactions and processes are as discussed in the text. Circular insets depict putative roles of components in RPE apical processes. cis-RDH, 11-cisretinal dehydrogenase; CRALBP, cellular retinaldehyde-binding protein; CRBPI, cellular retinol-binding protein type I; EBP50,

ERM-binding phosphoprotein of 50 kd, also known as NHERF-1 (sodium hydrogen exchanger regulatory factor type 1); IRBP, interphotoreceptor retinoid-binding protein; ISOMERO, isomerohydrolase, recently identified as RPE65; LRAT, lecithin : retinol acyltransferase; PDZ, a scaffold domain protein; RGR, retinal G protein–coupled receptor; RDH, all-trans-retinol dehydrogenase; Rho, rhodopsin; Rho*, activated rhodopsin (metarhodopsin II); RPE65, retinal pigmented epithelial protein of 65 kd. See color plate 68.

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binding protein (IRBP) is present in the interphotoreceptor matrix (IPM) and may protect retinoids during diffusion between cells and reduce their toxic effects. A brief description of our current understanding of the reactions of the rod visual cycle follows.

A cDNA encoding RetSDR1 was cloned during a search of a retinal cDNA library for SDR homologues (Haeseleer et al., 1998). In situ hybridization and immunocytochemistry revealed the gene to be highly expressed in cone outer segments; however, expression was also noted in cells of the inner retina. The expressed enzyme catalyzed the reduction of all-trans- but not 11-cis-retinal and required NADPH. Several hydroxysteroids were also substrates for the enzyme, and transcripts were noted in several other tissues (Cerignoli et al., 2002). At present, the function of this enzyme in cone outer segments is not known. A cDNA-encoding prRDH (RDH8) was cloned during a screen of retina-specific genes (Rattner et al., 2000). Antibodies revealed that the enzyme was expressed in rod and cone photoreceptor outer segments. The expressed enzyme was specific for all-trans-retinal and NADPH, similar to the activity in rod outer segments. prRdh−/− mice developed normally, and their retinal morphology was normal (Maeda et al., 2005). As predicted, the rate of reduction of all-transretinal was reduced in prRdh−/− mouse extracts following a flash; however, the rate of rhodopsin regeneration was normal. This result is an indication that the overall rate of the visual cycle is controlled by a step downstream of RDH, presumably the isomerohydrolase step, and that another dehydrogenase remained to be identified. Mutations in the RDH12 gene in humans resulted in a severe form of retinal degeneration beginning in early childhood (Janecke et al., 2004). However, the enzyme is found in rod inner segments and not in outer segments, and disruption of the Rdh12 gene in mice did not affect the rate of visual pigment regeneration or ERG responses (Kurth et al., 2007). Rdh12−/− mice also exhibited accelerated 11-cis-retinal production and increased susceptibility to light-induced photoreceptor degeneration (Maeda et al., 2006). The cause of the severe form of retinal dystrophy in humans with mutations in this gene remains unexplained but may be related to light damage. ATP-Binding Cassette A4 The disc membrane of the rod photoreceptor cell is equipped with a transporter of the ATP-binding cassette (ABC)-transporter family that retrieves all-trans-retinal from the luminal side of the disc membrane. Topologically, the compartment within the discs is equivalent to the outside of the cell, removed from the cytosolic site of production of NADPH and presumably from the active site of all-trans-RDH. The transporter, originally known as ABCR and now called ABCA4, utilizes ATP to “flip” alltrans-retinal from the luminal side of the disc to the cytosolic side, where it can be reduced to all-trans-retinol. The actual substrate for the translocation may be all-trans-retinal linked to phosphatidylethanolamine as a Schiff base. Characterization of the phenotype of Abca4−/− mice (Weng et al., 1999) revealed that the rate of rhodopsin regeneration

was normal, indicating that most of the all-trans-retinal must be released on the cytosolic side of the disc membrane. However, Abca4−/− mice accumulated all-trans-retinal and A2E, a novel pyridinium bis-retinoid characterized earlier as a component of lipofuscin. Mutations in the human ABCA4 gene cause autosomal recessive Stargardt disease (Allikmets et al., 1997), a form of macular dystrophy with large accumulations of lipofuscin in RPE. Based on these observations, it seems likely that the main physiological role of ABCR is to retrieve the small (in comparison to the total flux of the visual cycle) amount of all-trans-retinal that is released on the lumenal side of the discs and thus to minimize accumulation of its toxic condensation products. Lecithin:Retinol Acyltransferase In RPE, retinyl esters are primarily synthesized by LRAT via transfer of a fatty acyl group from the sn-1 position of phosphatidylcholine to the hydroxyl of retinol (Berry et al., 1989; Saari and Bredberg, 1989). Molecular characterization of LRAT from retina revealed a small hydrophobic protein (25.3 kd) (Ruiz et al., 1999) encoded by a single gene in mouse and humans (Ruiz et al., 2001). Lrat−/− mice were normal in appearance and grossly unremarkable; however, reduced fertility was noted in males. Development of the retina in Lrat−/− mice appeared normal; however, rod outer segments were shorter than normal in 4.5-month-old animals. Rhodopsin and retinoids, except for small amounts of all-trans-retinol, were virtually absent from retinas. ERG responses were grossly diminished, and the scotopic threshold was elevated by 5–6 log units (Batten et al., 2004). The virtual absence of retinyl esters and 11-cis-retinal in retinas of Lrat−/− mice is consistent with the proposed role of LRAT in esterification of all-trans-retinol and of all-transretinyl ester as the substrate for the isomerohydrolase in the visual cycle (Deigner et al., 1989). Thus, in the absence of LRAT, the visual cycle would be blocked at the isomerohydrolase step by the absence of substrate. Other studies ruled out impaired uptake of blood-borne retinoids by RPE because oral gravage of 9-cis-retinyl acetate or succinate esters resulted in formation of isorhodopsin in the retina (Batten et al., 2005). Isomerohydrolase The search for an isomerase is a fascinating story in which investigators followed two different paths for years, a search for a protein associated with the isomerase activity, and a search for a function for isomerohydrolase (RPE65), before realizing the two paths intersected. The isomerase activity, which regenerates the 11-cisretinoid configuration, is the signature enzyme of the visual cycle. Although many investigators assumed that all-transretinal would be isomerized directly to 11-cis-retinal, Rando’s

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laboratory determined that the isomerization took place at the oxidation level of all-trans-retinol and discovered an enzymatic activity that converted all-trans-retinyl esters to 11-cis-retinol and a free fatty acid, a reaction termed an isomerohydrolase (Bernstein and Rando, 1986; Bernstein et al., 1987). Hydrolysis of the ester bond was proposed to provide the energy needed for generation of the hindered 11-cis-retinoid (Deigner et al., 1989). Attempts to purify the enzyme or to isolate a cDNA encoding the enzyme were unsuccessful for nearly two decades following the discovery of its activity. Meanwhile, attempts to identify a retinol-binding protein (RBP) receptor led to the characterization of a major protein of RPE microsomes called p63 (Båvik et al., 1991). Subsequent studies demonstrated that its localization was not consistent with its proposed function. Other investigators, interested in proteins exclusively expressed by RPE cells, characterized a major protein of RPE microsomes, RPE65, which was identical to p63 (Hamel et al., 1993). Rpe65 −/− mice were unable to synthesize 11-cis-retinoids and accumulated large amounts of all-trans-retinyl esters in their RPE (Redmond et al., 1998). Both rod and cone functions were affected, indicating that RPE65 was involved in rod and cone visual cycles (Seeliger et al., 2001). Although this phenotype would be expected if RPE65 were the isomerohydrolase, attempts to provide direct evidence for this contention were unsuccessful until 2005, when three laboratories, using different approaches, demonstrated that RPE65 was responsible for isomerohydrolase activity (Jin et al., 2005; Redmond et al., 2005; Moiseyev et al., 2006). Differences in susceptibility to light damage observed in several strains of mice were traced to a L450M sequence variation in the Rpe65 gene (Danciger et al., 2000). Subsequent studies revealed that this sequence variation also correlated with differences in the rate of rhodopsin regeneration. Strains of mice with L450 regenerated their visual pigment approximately four times faster and were more susceptible to light damage than strains of mice with M450 (Wenzel et al., 2001). The RPE65 content of M450 strains of mice was significantly lower than that of L450 strains, suggesting that the sequence variation affected the stability of RPE65. The rate of the isomerase reaction is the slow step in the mouse visual cycle (see discussion of retinoid flow). Thus, a decrease in the amount of RPE65 would be expected to reduce the flux of retinoid through this reaction, allow retinoid to accumulate as relatively innocuous all-trans-retinyl esters, and reduce the amount of visual pigment available for photoisomerization. (See also the discussion of CRALBP and light damage.) The isomerohydrolase reaction also requires a lipidbinding protein for activity. Apo-cellular retinaldehydebinding protein (CRALBP) performs this function most efficiently in vitro (Winston and Rando, 1998), but other proteins are effective at much higher concentrations. Pre-

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sumably, the retinoid-binding protein relieves inhibition of the enzyme by binding the product of the reaction, 11-cisretinol (Winston and Rando, 1998). This explanation is in keeping with the visual phenotype of Rlbp1−/− (Cralbp−/−) mice, which show a delay in the visual cycle at the isomerohydrolase step (Saari et al., 2001). However, the requirement for CRALBP is not absolute because the visual cycle functions in its absence, although very slowly (see later discussion of CRALBP). Detailed examination of RPE65 structure has provided considerable insight into molecular aspects of the catalytic activity (Moiseyev et al., 2006). RPE65 is a member of the carotenoid-cleavage oxygenase family of enzymes, which includes β-carotene 15,15′-monooxygenase (carotene cleavage enzyme). These enzymes coordinate an active site Fe2+ via four histidines, which are conserved in RPE65 and other members of the family. The crystal structure of an apocarotenoid 15,15′-oxygenase suggests that all-trans-apocarotenoids are transiently isomerized during catalysis of cleavage (Kloer et al., 2005). Mutation of the corresponding histidine residues in RPE65 abolished the isomerohydolase activity of the protein (Redmond et al., 2005). The addition of chelating agents to native RPE65 enzyme reduced the activity of the enzyme and the addition of Fe(II) restored the activity. Finally, native RPE65 bound Fe(II) with a nearly 1 to 1 stoichiometry. These studies suggest, in an evolutionary sense, that an ancestral Fe-binding protein gave rise to a branch with isomerase and carotenoid cleavage activity (e.g., carotene cleavage enzyme) and a branch with just isomerase activity (e.g., RPE65) and demonstrate a role for iron in the regeneration of visual pigments. Retinal G Protein–Coupled Receptor Retinal G protein–coupled receptor (RGR) was first detected during a screen for RPE-specific proteins (Jiang et al., 1993). It was later found to be present in Müller cells also. The importance of RGR for retinal function and health was emphasized by the observation that mutations in the RGR gene in humans resulted in degeneration of the retina (Morimura et al., 1999). Sequence analysis revealed it to be a member of the G protein–coupled receptor family and to bind all-transretinal. Illumination of an RGR·all-trans-retinal complex resulted in photoisomerization of the retinoid to 11-cisretinal, similar to the photoisomerization of all-trans-retinal bound to squid retinochrome. This striking result immediately suggested that a function of the protein might be to provide 11-cis-retinal during illumination via a photoisomerization reaction. Initially, characterization of Rgr−/− mice provided conflicting results regarding the effect of the absence of RGR on the rate of regeneration in the dark. More recently, careful attention to the sequence variation of the Rpe65 gene at position 450 (see discussion of RPE65) in control and experimental groups of animals clearly revealed that the rate

of regeneration of rhodopsin was three times slower in Rgr−/− mice (Wenzel et al., 2005). Surprisingly, the absence of RGR in mice affected regeneration both during light exposure and in the dark, indicating that RGR was not a photoisomerase. Molecular details about the role of RGR remain to be determined. 11-CIS-Retinol Dehydrogenase In RPE, 11-cis-retinol, produced by the isomerohydrolase, is oxidized to 11-cisretinal by an SDR. As with the SDRs in the outer segment, there is functional redundancy at this step. Two SDRs have been identified in extracts of RPE. Surprisingly, neither enzyme alone or in combination accounts completely for 11-cis-retinol oxidizing activity in vivo in the mouse. Shortly after its discovery, RPE65, then known as p63, was noted to interact with another protein in RPE microsomes (Simon et al., 1995). Cloning and studies of the expressed protein revealed it to be a member of the SDR family of proteins (RDH5) and to catalyze the oxidation of 11-cis-retinol by NAD. Humans with mutations in the RDH5 gene have a condition called fundus albipunctatus, which is a slowly progressing form of retinitis pigmentosa characterized by delayed dark adaptation and, in some cases, lateonset cone dystrophy (Yamamoto et al., 1999; Nakamura et al., 2000). Thus, it was a surprise when the rate of rhodopsin regeneration was found to be normal in Rdh5 −/− mice (Driessen et al., 2000). The only abnormality noted in these animals was an accumulation of 13-cis-retinyl esters in RPE. These results suggested that another SDR catalyzed the oxidation of 11-cis-retinol in mice. RDH11 is expressed in prostate and also in RPE cells of the retina (Haeseleer et al., 2002). Rdh11−/− mice showed a phenotype similar to that of Rdh5 −/− mice (Kim et al., 2005). The phenotype of Rdh5 −/−/Rdh11−/− (double knockout) mice was similar to that of Rdh5 −/− mice but also displayed an abnormality in cone function. However, the flow of retinoids in the visual cycle was normal when moderate amounts of visual pigment were bleached (Kim et al., 2005), suggesting the existence of yet additional 11-cis-RDHs. No disease-causing mutations in the RDH11 gene in humans have been reported. RDH10 is expressed in RPE and Müller cells but appears to be specific for all-trans-retinols (B. X. Wu et al., 2004). Several lines of evidence suggest that oxidation of 11-cisretinol by cis-RDHs of RPE microsomes is facilitated by CRALBP. When bound to CRALBP, the aldehyde group of 11-cis-retinal is sequestered from water-soluble carbonyl reagents yet is readily reduced by NADH and RPE microsomes (Saari and Bredberg, 1982). 11-cis-retinol bound to CRALBP is oxidized to 11-cis-retinal by the cis-retinol dehydrogenase(s) of RPE microsomes more rapidly than free retinol (Saari et al., 1994) and has a lower Km (higher affinity) than free retinol for purified recombinant RDH5 (Golovleva et al., 2003).

Retinol-Binding Protein and the RBP Receptor RBP is a small protein (21 kd) secreted into the blood conjugated with all-trans-retinol, where it circulates associated with transthyretin. RBP is synthesized primarily in the liver and in lesser amounts in several tissues, including adipose tissue and RPE. In Rbp−/− mice, which are unable to mobilize hepatic vitamin A but develop normally, dietary vitamin A reaches peripheral tissues as retinyl esters carried by plasma lipoproteins. Retinal function is markedly diminished for the first few months after birth (Quadro et al., 1999), but Rbp−/− mice attain full visual function thereafter if maintained on a vitamin A-sufficient diet. Thus, it appears that for developmental purposes, most tissues are able to obtain vitamin A via circulating dietary retinyl esters, but the eye requires an RBP-dependent mechanism for the amount of vitamin A required for vision (Vogel et al., 2002). Uptake of vitamin A from RBP was suggested to involve a receptor-mediated process in the 1970s (Bok and Heller, 1976), and decades later an RBP-receptor, STRA6, was identified (Kawaguchi et al., 2007). STRA6 was originally discovered as a gene responsive to retinoic acid. Homozygous mutations in STRA6 in humans result in severe developmental malformations and mental retardation (Passuto et al., 2007). Based on the patterns of STRA6 expression in embryo and adult, it appears that STRA6 may play an important role in delivering vitamin A to many tissues in the embryo and a more restricted role in the adult, where it is primarily expressed in tissues making up blood-brain or blood-organ barriers (e.g., RPE, choroid plexus). Cellular Retinol-Binding Protein Type I CRBPI is a small, water-soluble retinoid-binding protein with high affinity for all-trans-retinol and all-trans-retinal (Napoli, 1999). CRBPI is expressed in many tissues, including RPE and Müller cells of the eye (Bok et al., 1984; Saari et al., 1984). Evidence obtained in vitro suggested that the protein could be involved in promoting esterification and oxidation of retinol and hydrolysis of retinyl esters (Herr and Ong, 1992; Napoli, 1999). However, its physiological role in vivo was not clear until analysis of CrbpI −/− mice. Normal development, reproduction, and behavior of these animals ruled out a major role for CRBPI in retinoic acid production because retinoic acid signaling is required for all these processes. A reduced content and six times faster turnover of hepatic retinyl esters was in keeping with a role for the protein in delivery of all-trans-retinol for esterification by LRAT (Ghyselinck et al., 1999). Stores of retinyl esters in RPE were also reduced relative to wild type, and all-trans-retinol accumulated transiently during recovery from a flash (Saari et al., 2002). Overall, the results were in accord with a role for CRBPI in delivery of all-trans-retinol to LRAT for esterification in RPE and liver.

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Interphotoreceptor Retinoid-Binding Protein Interphotoreceptor retinoid-binding protein (IRBP) is a watersoluble protein synthesized by photoreceptor cells and secreted into the interphotoreceptor matrix (IPM) (reviewed in Gonzalez-Fernandez, 2003). Localization of the protein in the extracellular compartment between photoreceptor and RPE cells (Bunt-Milam and Saari, 1983) and its ability to load with all-trans-retinol following bleaches of visual pigment suggested that the protein would be involved in transcellular diffusion of retinoids (reviewed in Pepperberg et al., 1993). The retinas of Irbp−/− mice showed a loss of photoreceptor nuclei and changes in the structural integrity of photoreceptor outer segments from P11 to P30. However, the rate of progression of the condition was quite slow, and was unaffected by raising the animals in the dark (Ripps et al., 2000). Even after 6 months, the number of photoreceptor nuclei was reduced to only about 50% of the number in wild-type mice (Liou et al., 1998; Ripps et al., 2000). Surprisingly, the rate of visual pigment regeneration was unaffected (Palczewski et al., 1999) or was even modestly faster (Ripps et al., 2000) in the absence of IRBP. Attempts to detect other retinoid-binding proteins in IPM that might have substituted for IRBP have been unsuccessful. IRBP appears to be necessary for release of 11-cis-retinal from cultured RPE cells (Carlson and Bok, 1992). One explanation for this finding is that a receptor mechanism is involved. However, the rate of visual pigment regeneration in Irbp−/− mice was nomal, and molecular information regarding this receptor has not yet appeared in the literature. The role of IRBP in visual physiology remains enigmatic. The small enhancement of the rate of rhodopsin regeneration in the absence of IRBP and the slow progression of the associated retinal degeneration are consistent with a role for the protein in buffering the concentration of free (unbound) all-trans-retinal or 11-cis-retinal in the IPM during retinoid diffusion between RPE and photoreceptor cells. However, the absence of protection from retinal degeneration in darkreared animals does not fully accommodate such a function. IRBP binds several hydrophobic substances in addition to retinoids, including docosahexaenoic acid and other fatty acids (Bazan et al., 1985), and may be involved in intercellular diffusion of these substances, including scavenging of oxidatively damaged retinoids and fatty acids. Cellular Retinaldehyde-Binding Protein CRALBP is a water-soluble protein with high affinity for 11-cis-retinol or 11-cis-retinal, found in abundance in RPE and Müller cells (Saari and Crabb, 2005). These features and the protein’s pronounced effects on enzymatic reactions of the visual cycle in vitro (Saari et al., 1994; Golovleva et al., 2003; Winston and Rando, 1998), suggested that it was involved in the regeneration of visual pigments.

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No differences were detected in the gross retinal morphologies of control and Rlbp1−/− mice raised in the dark (Saari et al., 2001). However, the rate of visual pigment regeneration following flash illumination was slower by about 15 times in the Rlbp1−/− animals. PCR analysis of the Rpe65 gene indicated that differences in the rate of regeneration of control and knockout mice could not be accounted for by the L450M sequence variation in the Rpe65 gene (see section on RPE65). Analysis of visual cycle retinoids revealed that retinyl ester accumulated during the delay, suggesting that the isomerohydrolase step in the visual cycle was affected by the absence of CRALBP. Dark adaptation was delayed in both cone and rod visual pathways in Rlbp1 −/− animals as measured by ERG. These results are consistent with studies in humans with mutations in the RLBP1 gene demonstrating dramatic delays in both rod and cone branches of dark adaptation curves (Bursedt et al., 2001). The results of these studies for the rod system are most readily understood if apo-CRALBP accepts 11-cis-retinol from the isomerohydrolase, a role for the protein that is well established in vitro (Winston and Rando, 1998). However, it is important to note that the absence of CRALBP in mice resulted only in a delay in visual pigment regeneration, not a complete block. Some other molecule may be present or induced in the knockout that can fulfill the role of CRALBP, if less efficiently. Albino Rlbp1 −/− mice were protected from light damage (Saari et al., 2001). Other laboratories also observed that delays in the visual cycle resulted in protection from light damage (Sieving et al., 2001; Grimm et al., 2000). Retinoid analysis of Rlbp1−/− mice established that a diminished regeneration rate caused photoreceptor retinoids to accumulate as the relatively innocuous retinyl esters in RPE during constant illumination (Garwin and Saari, 2008). This and the resulting diminished amount of potentially toxic all-transretinaldehyde in flux are likely to account for this protective effect. It is interesting to note that pharmacological inhibition of the visual cycle has been proposed as a potential means of delaying the onset of some inherited retinal diseases (reviewed in Travis et al., 2006). Retinoid Flow in the Visual Cycle The kinetics of appearance and disappearance of visual cycle retinoids during recovery from a flash or from constant illumination has been followed over the years using methods with increasing resolving power and sensitivity. Initial studies reported the flow of retinoids between neural retina and RPE long before sophisticated methods of retinoid separation were available (Dowling, 1960). More recent studies employing high-performance liquid chromatography (HPLC) analysis have identified slow steps based on the accumulation and decay of visual cycle intermediates. Early HPLC studies (Saari et al., 1998) were done in mice of mixed

genetic backgrounds, before the effect of Rpe65 sequence variations on regeneration rates was understood (Wenzel et al., 2001). Nonetheless, these and other studies clearly demonstrated the accumulation and slow decay of all-transretinal during recovery in the dark following a flash (Dowling, 1960; Saari et al., 1998; Palczewski et al., 1999; Qtaishat et al., 1999). Later studies with inbred strains of mice corroborated the slow decay of all-trans-retinal and also noted the accumulation and slow decay of all-trans-retinyl esters during recovery in the dark (Saari et al., 2001). Thus, there appeared to be two slow steps in the flow of retinoids leading to regeneration of rhodopsin following a flash, one related to the reduction of all-trans-retinal and the other related to the isomerohydrolase step. Based on the apparent decay rates for the two intermediates, processing of all-transretinyl ester, presumably by isomerohydrolase, appeared to be the slowest step. A similar conclusion was reached via mathematical analysis of flash-recovery data (Lamb and Pugh, 2004). Other studies had demonstrated that the rate of visual pigment regeneration characteristic of various strains of mice was proportional to the amount of RPE65 (isomerohydrolase) in their retinas (Grimm et al., 2000). Retinoid analysis of mice during steady illumination provided additional information (Garwin and Saari, 2008). All-trans-retinal rose to a maximum and decayed to low levels during the illumination period, whereas all-trans-retinyl esters accumulated to a plateau and remained elevated until the light was extinguished. These results demonstrate the two slow steps but indicate that decay of all-trans-retinyl esters (isomerohydrolase reaction) is the slower of the two. Thus, retinoid analyses identified visual cycle intermediates that accumulated during recovery from either flash or steady illumination. Because both reactions that process these intermediates are complicated, we do not know what limits the rates. For instance, the rate of reduction of all-transretinal by NADPH could be controlled by several processes, including the intrinsic turnover rate and amount of the enzyme, the rate of production of NADPH, the rate of release of all-trans-retinal from opsin, or an active control process. Similarly, the rate of isomerization and cleavage of all-trans-retinyl ester could be limited by processes including the rate of the isomerohydrolase reaction itself, removal of product by apo-CRALBP, substrate delivery of all-transretinyl ester to the enzyme, or active control of the reaction rate.

Cone visual cycle The cone visual cycle has been more difficult to approach experimentally because of the relatively few cones in most retinas used in the laboratory for biochemical studies. Circumstantial evidence has implicated Müller cells in cone

visual pigment regeneration for decades. Recently, considerable progress has been made in characterizing reactions that may be involved in a cone visual cycle. It will be of great interest to learn whether novel reactions occur in Müller cells. Our understanding of this putative pathway lags behind that of the rod regeneration pathway. It is not surprising that many aspects of visual pigment regeneration in rod and cone photoreceptor cells are dramatically different. Rod and cone visual pigments were designed to function in totally different illumination environments. In addition, rods outnumber cones in mammalian retinas by about 30 to one, setting up the possibility of competition for 11-cis-retinal. Bleached salamander cone photoreceptors in vitro will regenerate their visual pigment with exogenous 11-cis-retinol, whereas rods require 11-cis-retinal ( Jones et al., 1989). It is possible that this specificity reduces the competition between rods and cones for 11-cis-retinoids. In salamander retina, isolated cones regenerate when 11-cisretinal is applied to either their inner or outer segments, whereas rods regenerate only when 11-cis-retinal is applied to the outer segments (Jin et al., 1994). This implies that cones could utilize a source of 11-cis-retinoid near their inner segments, possibly Müller cells. Bleached cone photoreceptors in neural retina, separated from RPE, resensitize in the dark, whereas bleached rod photoreceptors require the apposition of RPE (Goldstein and Wolf, 1973), again implying that a source of 11-cis-retinol must exist within cells of the neural retina. Müller cells contain both CRBPI and CRALBP (Saari and Crabb, 2005), and cultured chicken Müller cells synthesize 11-cis-retinol from all-trans-retinol (Das et al., 1992; Muniz et al., 2006). Recently, enzyme activities of a putative cone visual cycle have been identified in retinal extracts of ground squirrels and chickens (Arshavsky, 2002; Mata et al., 2002). Thus, considerable indirect evidence exists for a distinct cone visual cycle in Müller cells. However, much of the evidence comes from studies of amphibian or avian retina, and it is not certain that the same features apply to mammalian retina. The cone visual cycle depicted in figure 59.2 is based on that proposed by Travis and co-workers (Mata et al., 2002).

Proteomic analysis of the visual cycle Proteomics, within the context of this article, refers to the identification of the proteins within a tissue, organ, organelle, or tissue fraction using mass spectrometric analysis. Few studies have directly addressed questions related to the visual cycle with this approach. However, the information obtained from studies with other primary goals has often provided useful insight regarding visual cycle components. Analysis of Interacting Components As discussed elsewhere in this chapter, apo-CRALBP is likely to bind 11-cis-

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Figure 59.2 Hypothetical schematic of the cone photoreceptor visual cycle. Transcellular diffusion and intracellular enzymatic processing of cone visual cycle retinoids are shown. Structure at left depicts a portion of the inner and outer segments of a cone photoreceptor cell. Structure at right depicts the apical end of a Müller

cell. The cells are joined by structures of the external limiting membrane (short wavy line). ARAT, acylCoA : retinol acyltransferase; HYDROLASE, 11-cis-retinyl ester hydrolase; ISOM, isomerase. See also the abbreviation key for figure 59.1. See color plate 69. (After Mata et al., 2002.)

retinol, the product of the isomerohydrolase reaction, and relieve product inhibition of the enzyme. Proteomic analyses have been used to analyze protein components interacting with CRALBP. In one approach, a component with high affinity for CRALBP was detected with a gel overlay assay and identified as EBP50/NHERF-1 (ERM-binding phosphoprotein50/sodium-hydrogen exchanger regulatory factor 1) by mass spectrometric analysis of the corresponding component from a two-dimensional (2D) gel (Nawrot et al., 2004). EBP50/NHERF-1 is a multivalent organizer that can link various proteins with affinity for its two PDZ domains to the cytoskeleton via a C-terminal domain that binds ezrin, radixin, or moesin, proteins with affinity for actin (Bretscher et al., 2002). CRALBP, EBP50/NHERF-1, ezrin, and actin are all found in the apical processes of RPE cells, indi-

cating the potential for interaction in vivo (Nawrot et al., 2004). EBP50/NHERF-1 may organize a retinoid-processing complex in the apical processes of RPE (Nawrot et al., 2006). In a second proteomic approach, a preparation enriched in mouse RPE apical processes was isolated using wheat germ agglutinin-affinity chromatography, and protein components within it were identified using mass spectrometry (Bonilha et al., 2004a). Several visual cycle components were identified, including IRBP, CRALBP, EBP50/NHERF-1, CRBPI, 11-RDH, and ezrin. Furthermore, this approach, in combination with immunohistochemistry, allowed identification of more than 200 proteins from preparations of mouse RPE apical microvilli that may participate in the maintenance, support, and biochemical processes occurring

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at this critical interface with photoreceptors (Bonilha et al., 2004b). These results support other studies providing evidence for a retinoid-processing complex in apical RPE. In a third and ongoing approach, immunoprecipitation (IP) with or without iTRAQ quantitative mass spectrometric methods has been used to probe for visual cycle protein interactions in bovine RPE microsomes. IP experiments with the purified recombinant proteins RDH5 and CRALBP support a structural interaction in a C-terminal region of CRALBP (Wu et al., 2005). As noted earlier in the discussion of 11-cis-retinol dehydrogenase, several lines of evidence support a functional interaction between CRALBP and RDH5 (Saari and Bredberg, 1982; Saari et al., 1994; Golovleva et al., 2003). iTRAQ technology utilizes amine-specific tags and yields diagnostic reporter ions during MS/MS analysis of the labeled peptides, the intensity of which provides relative protein quantification. iTRAQ quantitative analysis of anti-CRALBP IP products from RPE microsomes supports a possible retinoid-processing protein complex composed of CRALBP, RPE65, LRAT, RDH5, and RGR (Gu et al., 2006). Comparative quantification of visual cycle reciprocal IP products is being used to further evaluate the composition of this putative RPE protein complex. In other studies, proteomics has been instrumental in identifying posttranslational modifications and functional domains within visual cycle proteins. Two noteworthy examples include RPE65 and CRALBP. MALDI TOF mass spectrometric analyses of affinity purified RPE65 provided the first compelling evidence that RPE65 was posttranslationally modified and supported the presence in RPE of both a cytosolic, lower-mass isoform and a membrane-associated, higher-mass isoform (Ma et al., 2001). More recently, Rando and co-workers used mass spectrometry to characterize tryptic peptides from RPE65 and reported the presence of three palmitoylated cysteine residues in the membraneassociated isoform. They suggested that palmitoylation may serve as a switching mechanism controlling RPE65 ligand-binding selectivity (Xue et al., 2004). However, others have reported that mutating these three cysteine residues has little effect on isomerase activity, so the role of posttranslational modifications in RPE65 functions remains to be determined (Redmond et al., 2005; Takahashi et al., 2006). Photoaffinity labeling and proteomic high-resolution topological analyses have been particularly useful in understanding the functional domains of CRALBP involved in ligand and protein interactions (Z. Wu et al., 2004). Eight photoaffinity-modified residues in human CRALBP were identified by liquid chromatography tandem mass spectrometry, several of which had been independently identified using other protein chemical methods and site-directed mutagenesis. Topological analysis of apo- and holo-CRALBP

by hydrogen-deuterium exchange and mass spectrometry demonstrated that residues 198–255 incorporated significantly less deuterium when the retinoid-binding pocket was occupied with 11-cis-retinal. This hydrophobic region encompasses all but one of the photolabeled residues. In a structural model of CRALBP based on the crystal structures of three CRAL_TRIO family members, all of the photolabeled residues lined the ligand-binding cavity except one, which appears to reside in a flexible loop at the entrance/ exit of the ligand cavity. The topological analyses also supported the solvent accessibility of CRALBP residues 147– 157 and 262–275 and are consistent with a positively charged groove in the structural model that may be involved in protein interactions as proposed for other CRAL_TRIO family members (Z. Wu et al., 2004).

Conclusion The past decade has produced an astounding amount of information on the pathway for regeneration of rod visual pigments, thanks to advances in mouse genetics, genetic manipulation, and proteomics. The pathway for regeneration of cones has been more difficult to approach experimentally because of the relatively small proportion of cones in mouse and bovine retinas. Despite these great advances, much remains to be learned about rod visual pigment regeneration. For example, the molecular entities responsible for the dehydrogenase activities of the visual cycle remain to be assigned, and the cone pathway remains poorly understood. In addition, the cell biology of retinoid processing and transfer remains to be worked out. We expect many of these problems to be solved as increasingly clever ways of utilizing mouse genomics and proteomics evolve. acknowledgments

Work was supported by NIH grant nos. EY02317, EY06603, EY014239, and EY015638 and by Research to Prevent Blindness, Inc., Foundation Fighting Blindness, the Cleveland Clinic Foundation, and the Milton and Ruth Steinbach fund. The authors thank Gregory G. Garwin for help with the illustrations. REFERENCES

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CONTRIBUTORS Badea, Tudor C. Johns Hopkins University Medical School, Baltimore, Maryland Bear, Mark F. Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, Massachusetts Berardi, Nicoletta Università di Firenze del Consiglio Nazionale delle Ricerche, Pisa, Italy Berger, Wolfgang Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Zurich, Switzerland Blankenship, Aaron G. Neurobiology Section, Division of Biological Sciences, University of California, La Jolla, California Bloomfield, Stewart A. Department of Ophthalmology, New York University School of Medicine, New York, New York Boye, Sanford L. Department of Ophthalmology and Molecular Genetics, University of Florida, Gainesville, Florida Boye, Shannon E. Department of Ophthalmology and Molecular Genetics, University of Florida, Gainesville, Florida Brown, Richard E. Department of Psychology, Dalhousie University, Nova Scotia, Canada Burkhalter, Andreas Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri Caspi, Rachel R. Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland Cenni, Maria Cristina Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, Pisa, Italy Cepko, Constance L. Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts Chalupa, Leo M. Section of Neurobiology, Physiology, and Behavior, University of California, Davis, California Chang, Bo The Jackson Laboratory, Bar Harbor, Maine Chen, Chinfei Division of Neuroscience and Neurobiology Program, Children’s Hospital, Harvard Medical School, Boston, Massachusetts Clancy, Barbara Department of Biology, University of Central Arkansas, Conway, Arkansas Coombs, Julie L. Section of Neurobiology, Physiology, and Behavior, University of California, Davis, California Crabb, John W. Department of Ophthalmic Research, Cole Eye Institute, Cleveland, Ohio Danciger, Michael Department of Biology, Loyola Marymount University, Los Angeles, California Daniele, Lauren L. Department of Ophthalmology, F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Dorrell, Michael I. Department of Cell Biology, Scripps Research Institute, La Jolla, California Douglas, Robert M. Department of Ophthalmology and Vision Sciences, University of British Columbia, Vancouver, Canada

Dräger, Ursula C. Eunice Kennedy Shriver Center for Mental Retardation, University of Massachusetts Medical School, Waltham, Massachusetts Dreher, Bogdan School of Medical Sciences and Bosch Institute, University of Sydney, Sydney, Australia Dyer, Michael A. Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, Memphis, Tennessee Erskine, Lynda School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland Feller, Marla B. Neurobiology Section, Division of Biological Sciences, University of California, Berkeley, California Finlay, Barbara L. Department of Psychology, Cornell University, Ithaca, New York Frenkel, Mikhail Y. Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, Massachusetts Friedlander, Martin Department of Cell Biology, Scripps Research Institute, La Jolla, California Galli-Resta, Lucia Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, Pisa, Italy Geisert, Eldon E. Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee Goldberg, Jeffrey L. Bascom Palmer Eye Institute, University of Miami, Miami, Florida Graw, Jochen Institute of Developmental Genetics, German Research Center for Environmental Health, Helmholtz Center Munich, Neuherberg, Germany Gregg, Ronald G. Departments of Ophthalmology and Visual Sciences and Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky Grubb, Matthew S. Medical Research Council Centre for Developmental Neurobiology, King’s College London, London, England Gubitosi-Klug, Rose A. Departments of Medicine and Ophthalmology, Case Western Reserve University, Cleveland, Ohio Guido, William Department of Anatomy and Neurobiology, Virginia Commonweath University School of Medicine, Richmond, Virginia Hauswirth, William W. Departments of Ophthalmology and Molecular Genetics, University of Florida, Gainesville, Florida Hazlett, Linda D. Department of Anatomy/Cell Biology, Wayne State University School of Medicine, Detroit, Michigan Hoehenwarter, Wolfgang Max Planck Institute for Infection Biology, Berlin, Germany Hofer, Sonja B. Max Planck Institute of Neurobiology, Martinsried, Germany Howell, Gareth R. The Jackson Laboratory, Bar Harbor, Maine Hübener, Mark Max Planck Institute of Neurobiology, Martinsried, Germany Isa, Tadashi Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki, Japan

733

Jang, Young P. Department of Ophthalmology, Columbia University, New York, New York John, Simon W. M. The Jackson Laboratory, Bar Harbor, Maine Jungblut, Peter R. Max Planck Institute for Infection Biology, Berlin, Germany Karl, Mike O. University of Washington, School of Medicine, Seattle, Washington Kellenberger, Antonia Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg, Germany Kern, Timothy S. Medicine and Ophthalmology, Case Western Reserve University, Cleveland, Ohio Kim, So R. Department of Ophthalmology, Columbia University, New York, New York Klein, William H. Department of Biochemistry and Molecular Biology, University of Texas, Houston, Texas Lamba, Deepak University of Washington, School of Medicine, Seattle, Washington Leamey, Catherine A. School of Medical Sciences and Bosch Institute, University of Sydney, Sydney, Australia Luo, Tuanlian Eunice Kennedy Shriver Center for Mental Retardation, University of Massachusetts Medical School, Waltham, Massachusetts Lyubarsky, Arkady Department of Ophthalmology, F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Maffei, Lamberto Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, Pisa, Italy Marchant, Jeffrey K. Department of Anatomy and Cell Biology, Tufts University School of Medicine, Boston, Massachusetts Mason, Carol A. Department of Pathology and Cell Biology, Center for Neurobiology and Behavior, Columbia University, New York, New York Matsuda, Takahiko Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts May, Christian-Albrecht Institut für Anatomie, Technological University of Dresden, Dresden, Germany Department of Psychological McCall, Maureen A. and Brain Sciences, University of Louisville, Louisville, Kentucky Mrsic-Flogel, Thomas D. Max Planck Institute of Neurobiology, Martinsried, Germany Mu, Xiuqian Department of Biochemistry and Molecular Biology, University of Texas, Houston, Texas Naggert, Juergen K. The Jackson Laboratory, Bar Harbor, Maine Nathans, Jeremy Department of Molecular Biology and Genetics, Johns Hopkins University Medical School, Baltimore, Maryland Nelson, Branden University of Washington, School of Medicine, Seattle, Washington Nikonov, Sergei S. Department of Ophthalmology, F. M. Kirby Center for Molecular Ophthalmology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania Nishina, Patsy M. The Jackson Laboratory, Bar Harbor, Maine Panda, Satchidananda Regulatory Biology Laboratory, Salk Institute for Biological Studies, La Jolla, California Parapuram, Sunil K. W. K. Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan

734

contributors

Peachey, Neal S. Cleveland Veterans Affairs Medical Center, Cleveland Clinic Foundation, Cleveland, Ohio Petros, Timothy J. Department of Pathology and Cell Biology, Center for Neurobiology and Behavior, Columbia University, New York, New York Piatigorsky, Joram Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland Pinto, Lawrence H. Department of Neurobiology and Physiology and Center for Functional Genomics, Northwestern University, Evanston, Illinois Porciatti, Vittorio Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida Powers, Michael R. Oregon Health and Science University, Portland, Oregon; Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida Protti, Dario A. School of Medical Sciences and Bosch Institute, University of Sydney, Sydney, Australia Prusky, Glen T. Department of Neuroscience, Canadian Centre for Behavioural Neuroscience, University of Lethbridge, Alberta, Canada Pugh, Edward N., Jr. Department of Ophthalmology, F. M. Kirby Center for Molecular Ophthalmology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Reese, Benjamin E. Neuroscience Research Institute, University of California, Santa Barbara, California Reh, Thomas A. Health Sciences Center, University of Washington, School of Medicine, Seattle, Washington Rice, Dennis S. Lexicon Pharmaceuticals, The Woodlands, Texas Robinson, Michael L. Zoology Department, Miami University, Oxford, Ohio Saari, John C. Department of Ophthalmology, University of Washington, Seattle, Washington Sakatani, Tomoya Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki, Japan Sale, Alessandro Scuola Normale Superiore, Laboratory of Neurobiology, Pisa, Italy Schaeffel, Frank Section of Neurobiology of the Eye, University Eye Hospital Tübingen, Tübingen, Germany Sparrow, Janet R. Department of Ophthalmology, Columbia University, New York, New York Stahl, John S. Department of Neurology, Case Western Reserve University, Cleveland, Ohio Stone, Jonathan Research School of Biological Sciences, Australian National University, Canberra City, Australia Strettoi, Enrica Istituto di Neuroscienze del Consiglio Nazionale delle Ricerche, Laboratorio di Neurofisiologia, Pisa, Italy Swamynathan, Shivalingappa K. Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland Swaroop, Anand Neurobiology Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, Maryland; W. K. Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan Tamm, Ernst R. Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg, Germany Thompson, Hannah Institute of Ophthalmology, University College London, London, England

Tian, Ning Departments of Ophthalmology and Visual Science and Neurobiology, Yale University School of Medicine, New Haven, Connecticut Troy, John B. Department of Biomedical Engineering, Northwestern University, Evanston, Illinois Tucker, Priscilla K. Museum of Zoology, University of Michigan, Ann Arbor, Michigan Valter, Krisztina Research School of Biological Sciences, Australian National University, Canberra City, Australia Völgyi, Béla Department of Ophthalmology, New York University School of Medicine, New York, New York

Wagner, Elisabeth Eunice Kennedy Shriver Center for Mental Retardation, University of Massachusetts Medical School, Waltham, Massachusetts Wang, Quanxin Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri Williams, Robert W. Departments of Anatomy and Neurobiology and Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee Wong, Aimée A. Department of Psychology, Dalhousie University, Nova Scotia, Canada Zhou, Jilin Department of Ophthalmology, Columbia University, New York, New York

contributors

735

INDEX Note: Page numbers followed by f indicate figures; page numbers followed by t indicate tables.

A A and A/J strain auditory evoked brainstem response in, 22 conditioned taste aversion in, 29 hearing and visual abilities in, 15t, 16 mutation and effect in, 62t pattern discrimination in, 18 taste ability in, 24 visual acuity of, 20 ABCA4 mutations, 723 Abca4/Abcr null mutant mice, Stargardt macular degeneration in, 540–542, 541f Ablation, targeted, to assess role of photoreceptors, 595–596 ab-LIM, directed growth of retinal ganglion cell axons toward optic disc and, 384 AC Master, 78, 78f Acanthamoeba infections, 508–509 αA/CAT transgenic mice, 273 Achromatopsia, gene replacement therapy for, 612–613 Acoustic startle test, 22 Adenoassociated virus (AAV) gene therapy, 605–614 antiangiogenic, 607 for choroidal neovascularization, 610 future directions for, 613–614 gene replacement strategies and, 610–613 for functional defects in retina, 611–613 for structural defects in retinal disease, 610–611 for retinal neovascularization, 607, 609–610 vectors for, 605–607, 606t, 608t–609t Advanced glycation end products (AGEs), diabetic retinopathy and, 552 African pygmy mice, 3, 5, 8 Age-related eye diseases, 581–588 cataracts as, in human and mouse eyes, 588 in mice, search for, 581–582, 583t–584t mouse models for, 581 Age-related macular degeneration (AMD) gene therapy for, 607 in human and mouse eyes, 582, 584–588 Age-related retinal degeneration (ARRD), in human and mouse eyes, 582, 584–588, 585f–587f, 585t Akita mice, diabetes in, 549 retinal neurodegeneration induced by, 551 AKR/J strain auditory evoked brainstem response in, 22 hearing and visual abilities in, 15t, 16 pattern discrimination in, 18

taste ability in, 23 visual acuity of, 20 Albinism, retinal ganglion cell projections and, 397 Albino mice, 61 pattern discrimination in, 18 visual acuity of, 20 Aldose reductase, diabetic retinopathy and, 552 Alleles. See also specific alleles defining disease progression, clinical outcome, or important domains, 650 defining response to treatment, 651 defining responses to environmental influences, 650–651 phenotypic variation due to, 649–651 Allen Brain Atlas, search of, for RALDH3 colocalized genes, 370–371, 372f All-trans-retinol dehydrogenase, in visual cycle, 722–723 Amacrine cells cholinergic (starburst) mosaic architecture of, 150–151, 151f study approaches for, 598 dopaminergic genetic labeling of, 597–598 mosaic architecture of, 151–153 gap junctions of, 167 glucagon, in mouse versus chicken eye, 82–83 transgenic mice for study of, 597–598 V-amacrine, 597 AMPA, excitatory lateral geniculate nucleus responses and, 420, 422 AMPA receptors (AMPARs), 223–225 long-term depression and, 470 in reticulogeniculate synapses, 431, 439 Anaphase-promoting complex (APC), retinal ganglion cell axon growth ability and, 408 Anesthesia dolorosa, model of, 25 Angioblasts, 285 Angiogenesis, 285–286, 639–641 hyaloid vessel regression versus, in norrin deficient mice, 532, 533f sprouting, failure in, in norrin deficient mice, 531–532 VEGF-A in, 640–641 Angiopoietin, diabetic retinopathy and, 552–553 Angiopoietin/Tek system, retinal vascularization and, 292 Angiostatin, anti-angiogenic properties of, 610 Angular vestibulo-ocular reflex (aVOR), 88–91, 89f, 90f

gain and response axis of, adaptation of, 96–98, 97t Anophthalmic mice, 269, 271 Anterior chamber, cell types in, 644–645 Anterior chamber–associated immune deviation (ACAID), 521–522 pigmentary glaucoma and, 483 Antiangiogenic gene therapy, 607 Antibodies, against neurofilament H, as retinal ganglion cell marker, 193 Antigen-presenting cells (APCs), experimental autoimmune uveitis and, 519 Anxiety-related behavior tasks, 29 AP1, in cornea and lens development, 703 AP2, in cornea and lens development, 703 AP2α, lens development and, 277 AP2α-Cre mice, 275 Apo-cellular RBP (CRALBP), 724 Apoptosis. See Cell death Aquaporin, cataracts and, 495 Aqueous humor inflow of, 129, 129f outflow of, 129–131 uveoscleral, 132–133 Astrocyte(s) in optic nerve, 201–202, 202f retinal vascularization and, 287 Astrocyte progenitor cells (APCs), 390, 390f Atoh7, retinal ganglion cell development and, 194 ATP-binding cassette A4, in visual cycle, 723 Atropine, myopia development and, 83 Auditory evoked brainstem response (ABR), 20, 22 Autism spectrum disorders, visual cortical functions in, 245 Autoimmune uveitis. See Experimental autoimmune uveitis (EAU) Automated visual responses, measures of, 111–115, 114f, 115f Avoidance conditioning, active and passive, 27–28 a-waves, cone, 138 Axial length, changes in, measurement, 77–79, 78f Axons, of retinal ganglion cells. See Retinal ganglion cells (RGCs)

B Bacterial artificial chromosome (BAC) transgenesis, 594, 594f Bacterial infections, 506–508 Bagg, Halsey J., 13

737

BALB strains, 62t BALB/c anxiety-related behavior tasks and, 29 auditory evoked brainstem response in, 22 conditioned odor preference in, 28 learning and memory in, 27 pain sensitivity in, 25 photoreceptor response damage by light and, 575, 577 BALB/cByJ hearing and visual abilities in, 15t, 16 pattern discrimination in, 18 visual acuity of, 20 BALB/cJ conditioned taste aversion in, 29 hearing and visual abilities in, 15t, 16 pain sensitivity in, 25 pattern discrimination in, 18 visual acuity of, 20 Barnes maze, 27 Basic fibroblast growth factor (bFGF), retinal ganglion cell axon growth and, 401 Batten disease, 612 Bcl-2 Bcl-2-overexpressing retina, 152 cell death and, 337f, 337–338 retinal neovascularization and, 293 Beaded filaments, 499 Behavior, environmental enrichment and, 450 Behavioral studies of mouse vision, 107–116 applied measurement of, 115–116, 116f idiosyncrasies associated with whole animal behavior and, 108–109 measures of, 109–115 of automated visual responses, 111–115, 114f, 115f of visual perception in reinforcementbased tasks, 109–111, 111f–113f rationale for, 107–108 Behavioral tasks, for sensory function measurement, 15t, 15–16 future directions for use of, 30 of hearing, 20, 22 learning and measurement tasks and, 25–29 of olfaction, 22–23 of pain sensitivity, 24–25 of tactile and vibrissae sensitivity, 24 of taste, 23–24 of vision, 16–20 BEN, directed growth of retinal ganglion cell axons toward optic disc and, 382–383 Bfsp1, cataracts and, 499 Bfsp2, cataracts and, 499 bHLH transcription factors, retinal development and, 303 BH3-only protein, cell death and, 338 Binocularity, in primary visual cortex, 49–51, 50f transcallosal cells and, 50–51 Bipolar cells Clomeleon in, 597 depolarizing, 136

738

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synaptic transmission in outer retina and, 176f, 176–179, 177t gap junctions of, 167 hyperpolarizing, 136 synaptic transmission in outer retina and, 176f, 176–179, 177t ON and OFF pathway segregation and, 353–354, 357 in Rb-deficient retinas, 315 role of gap junctions in, transgenic mice to study, 596–597 synaptic output of, control by GABACRmediated inhibition, 182–183 Birdshot retinochoroidopathy, 516 Blind-sterile mutant, 501 Bmp4, lens development and, 276 Bmp7, lens development and, 276 Bone morphogenic protein (BMP) receptor 1b, targeting of retinal ganglion cell axons to optic disc and exit from eye and, 385 Brain electroporation and, 626 environmental enrichment and, 450 retinoic acid actions in, 364–365 Brain Gene Expression Map (BGEM), search of, for RALDH3 colocalized genes, 370 Brain-derived neurotrophic factor (BDNF) acceleration of visual system development and, environmental enrichment compared with, 453, 454 cell death and, 336 environmental enrichment and, increase due to, 457–458 retinal ganglion cell axon growth and, 401 Brief-access taste test, 24 Brightness detection, measurement of, 16, 17f, 18 Brn-3b, chiasm patterning and, 396–397 Brn-3b−/− mice, retinal ganglion cell axon growth ability and, 407–408 Brn-3b−/− Brn-3c−/− mice, retinal ganglion cell axon growth ability and, 407–408 Brn-3c, chiasm patterning and, 397 Brn-3.2, directed growth of retinal ganglion cell axons toward optic disc and, 383–384 Bruch’s membrane, plate 64 BUB strain, mutation and effect in, 62t BUB/BnJ auditory evoked brainstem response in, 22 taste ability in, 23 Buried food test, 22 b-waves, cone-driven, 137, 137f BXD Genetic Reference Panel of Mice, 664–665, 666t, 667 BXSB strain, pattern discrimination in, 18

C C strain, 13 Cacna 1a mutations, 100–101 CaCna1f nob2 mice, retinal ganglion cell responses in, 179–181, 180f

Cadherin4, retinal vascularization and, 287 Calcium retinal ganglion cell axon growth ability and, 408 synaptic transmission in outer retina and, 177–178 Calcium currents, in dorsolateral geniculate nucleus, 220 Calcium imaging of direction-selective retinal ganglion cell responses, 598 for retinal wave measurement, 343 with two-photon microscopy, for ocular dominance plasticity assessment, 441–442, 442f Calmodulin kinases (CaMKs), retinal ganglion cell axon growth ability and, 408 cAMP, retinal ganglion cell axon growth ability and, 408 cAMP response element–binding protein (CREB), retinoic acid and, 366 Candida albicans infections, 508 Capecchi, Mario R., 35 Carbohydrates, protein-bound, cataracts and, 499–500 Carrageenan, subcutaneous injections of, 25 Caspases, cell death and, 338 CAST/Ei strain, taste ability in, 23 CAST/EiJ hearing and visual abilities in, 15t pattern discrimination in, 18 Cat3, 501 Cat5, 501 Cataracts age-related, in human and mouse eyes, 588 gene expression leading to formation of, 273 genetics of, 493–501 mouse models for metabolic cataracts and, 499–500 mouse models for senile cataracts and, 500 mutations affecting lens membranes and, 495–496 mutations affecting structural proteins of lens and, 496–499 mutations at early stages of lens development and, 493–494, 494f pattern electroretinography and, 124 β-Catenin, inhibition of lens induction by, 277–278 Caviness, Vernon, 245 CBA strains, 13 avoidance learning in, 28 CBA/Ca, anxiety-related behavior tasks and, 29 CBA/CaJ, auditory evoked brainstem response in, 22 CBA/J cued and contextual fear conditioning in, 27 learning and memory in, 26, 27 tactile sensitivity in, 24 taste ability in, 23

CBA strains (continued) learning and memory in, 27 mutation and effect in, 62t C57BL/6 and C57BL/6J strain, 271–272 anxiety-related behavior tasks and, 29 auditory evoked brainstem response in, 22 circadian photoentrainment and, 210 conditioned odor preference in, 28 cones of, anatomy of, 135–137 diabetes-induced retinal neurodegeneration in, 551 hearing and visual abilities in, 15t, 16 learning and memory in, 27 mutation and effect in, 62t olfactory ability in, 22, 23 pain sensitivity in, 25 pattern discrimination in, 18 as standard strain, 62 tactile sensitivity in, 24 taste ability in, 23, 24 visual acuity of, 20 C57BL/6NCrlBR strain conditioned odor preference in, 28 pattern discrimination in, 18 C57BL/10 strain, 271–272 C57BR/cdJ strain, auditory evoked brainstem response in, 22 CCCTC binding factor, eye field transcription factor regulation by, 301 CCL2 protein, cell death inhibition by, 122, 123f CD-1 outbred strain conditioned odor preference in, 28 olfactory ability in, 22, 23 pattern discrimination in, 18 CD18, diabetic retinopathy and, 553 Cell adhesion molecules (CAMs), retinal ganglion cell axon guidance through visual pathway and, 393 Cell cycle progression, in retinal progenitor cells, Math5 and, 324 Cell death of developmental cells, retinal ganglion cells and, 122 of endothelial cells, oxygen-induced retinopathy and, 293 of horizontal cells, 149 in mouse retina, 333–339 contribution to shaping of retinofugal and retinal circuits, 334–336, 335f major phases of, 333–334, 334f molecules involved in signaling, regulating, and executing, 336–339, 337f neurotrophic hypothesis of, 336 of retinal ganglion cells, 333, 334 in glaucoma, 485–487, 487f, plate 33, plate 34 matching sizes of interconnected neuronal populations and, 335, 335f molecules involved in signaling, regulating, and executing, 337, 338

Cell type–specific promoters lineage tracing in retina using, plate 60 multicolor labeling of retina using, plate 59 spatial regulation of gene expression using, plate 58 Cell-labeling techniques, for study of retinal ganglion cells, 599 Cellular retinaldehyde-binding protein (CRALBP), in visual cycle, 726 Cellular retinol-binding protein type 1 (CRBP1), in visual cycle, 725 Central nervous system. See also Brain plasticity of, pattern electroretinographic measurement of, 121–122 Central retinal artery (CRA), anatomy of, 203–204, 204f Central retinal vein, anatomy of, 204 Centrifugal model of evolution, 6, 7f cGMP, retinal ganglion cell axon growth ability and, 408 C3H strains, 13 avoidance learning in, 28 conditioned taste aversion in, 29 mutation and effect in, 62t C3HeB/FeJ strain, visual acuity of, 20 C3H/HeIbg strain, cued and contextual fear conditioning in, 27 C3H/He and C3H/HeJ strain anxiety-related behavior tasks and, 29 auditory evoked brainstem response in, 22 hearing and visual abilities in, 15t, 16 learning and memory in, 26, 27 pain sensitivity in, 25 pattern discrimination in, 18 tactile sensitivity in, 24 taste ability in, 23 visual acuity of, 20 Chase, Herman, 269 Chemical nociception, measurement of, 25 CHI strain, 13 Chlamydia trachomatis infections, 508 Cholera toxin β subunit (CTB), development of eye-specific segregation in lateral geniculate nucleus and, 418 Cholesterol metabolism, cataracts and, 500 Cholinergic amacrine cells mosaic architecture of, 150–151, 151f study approaches for, 598 Chondroitin sulfate proteoglycans (CSPGs) control of initial retinal ganglion cell axon outgrowth polarity and, 381 ocular dominance plasticity and, 454–455 Choriocapillaris, plate 64 Choroidal circulation, oxygen delivery to retina and, 560–561 Choroidal neovascularization, gene therapy for, 610 Chromatin, rod development and, 317 Chronology of mouse visual system development, 257–264 comparison with development in rhesus macaque, 261 future research directions for, 264 human developmental timing versus, 261–262, 262f

model for, 257, 258t, 259, 259f, 260f individual variation and observational error and, 260–261 technical and qualitative evaluation of model’s predictions and, 259–261 mouse models for, 262–263 scaling of eye and cortex and, 263–264 variability of mouse strains and, 262 Chung peripheral nerve injury model, 25 Chx10, retinal development and, 303 Chx10-Cre;RbLox/− mice retinal synaptogenesis in, 317–318, 318f rod development in, 313–314 Chx10-Cre;RbLox/+ mice, retinal synaptogenesis in, 317 Chx10-Cre;RbLox/Lox mice, rod development in, 314 Chx10-Cre;RbLox/Lox;Z/AP mice, rod development in, 315 C12I strain, 13 Ciliary body, position of, aqueous humor outflow and, 129 Ciliary epithelium nonpigmented, 129 structure of, 129 Ciliary muscle, 129 Ciliary neurotrophic factor (CNTF), 613 retinal ganglion cell axon growth and, 401 Ciliary processes, 129, 130f Circadian behavior, mouse models for, 215 Circadian photoentrainment, 207–215 circadian behaviors in mice and, 207–209, 208f circadian photoentrainment pathway neuroanatomy and, 209–210 intrinsically sensitive retinal ganglion cells and, 211–212 morphology, ontogeny, and functional diversity of, 211–212, 212f melanopsin and, 213–214 phototransduction mechanism and, 213–214 melanopsin-expressing retinal ganglion cell role in circadian photoentrainment and, 210–211 molecular basis of, 214f, 214–215 mouse as model system for human circadian rhythm and, 207 photosensitivity and, 209 target brain regions innervated by intrinsically photosensitive retinal ganglion cells and, 212f, 212–213 C57L/J strain, auditory evoked brainstem response in, 22 C58/J strain, tactile sensitivity in, 24 Clock gene, 65 Clomeleon, in bipolar cells, 597 Cloning of genetic modifiers, 653–655 chromosomal localization of modified traits and, 653–654 gene expression profiling and, 654–655 haplotype analysis and, 654 microarray analysis and, 654–655 modifier screens using mutagenesis and, 655

index

739

Cloning of genetic modifiers (continued) segregating crosses with multiple strains and, 654 c-Maf, lens fiber differentiation and, 278 Coats’ disease, 528 Cochlear vasculature, abnormalities of, in Ndph knockout mice, 533–534 Cognitive functions, higher order, vision in tasks evaluating, 25 Collagen I, primary open-angle glaucoma and, 482 Color blindness, gene replacement therapy for, 612–613 Commensal mice, 5 Compensatory eye movements, 87, 88 adaptive properties of, 96–99, 97t, 98f, 99f, 100 Complete Freund’s adjuvant (CFA), experimental autoimmune uveitis induced by, 515, 520 Conditioned odor preference, 28 Conditioned taste aversion, 28–29 Cone(s), 135–144. See also Photoreceptors anatomy of, 135–137 numerosity and retinal disposition and, 135–136, 136f, plate 2 retinal connectivity of cones and, 136 S- and M-opsins and, 136–137 ultrastructure and, 136 damaging of, by rod dysfunction, 566 dim-flash responses of, plate 3 electroretinographic studies of, 137–139 a-wave and, 138 b-wave and, 137, 137f functional coexpression of S- and M-opsins and, 138–139 phenotypes of mice with cone phototransduction genes inactivated and, 138, 138f rapid recovery from bleaching stimuli and, 138 functions of, 135 gap junctions of, 165–166, 166f of mouse compared to other mammalian species, 36 Nrl −/−, 139, 141–144 caveats for investigations of, 142 electrophysiological recordings and, 141–142, 143f histochemical and ultrastructural evidence and, 141, 142t prospects for studies using, 143–144 role in scotopic visual pathway, targeted ablation to assess, 595–596 scaling of, 263 single-cell recordings and, 139, 140f–142f spatial vision and, 81–82 Cone pedicles, 136 Cone synapses, 136 Cone visual cycle, 727, 728f, plate 69 Congenital stationary night blindness (CSNB), 650 Connective tissue, in optic nerve, 202, 203f Connexins in lens, cataracts and, 495–496, 496f as retinal ganglion cell markers, 193

740

index

retinal gap junctions and, 165 of amacrine cells, 167 of bipolar cells, 167 of ganglion cells, 169, 171 of horizontal cells, 167 of photoreceptors, 165–166 in proximal rod pathways, 167–169, 168f retinal waves and, 347 Cornea development of, 697, 698f transcription factors involved in, 699f, 699–703, plate 66 gene expression in. See Gene expression, in cornea and lens neovascularization of, matrix metalloproteinase-9 and, 506 refractive power of, 73 Corpus callosum, visual cortex connections with, 46–47, 47f Coulombre, Alfred and Jane, 269 Cre expression, in Le-Cre, MLR10, and MLR39 strains, plate 12 Cre recombinases cell type–specific regulation using, 626–627, 629, 630f conditional gene alterations using, 274–275, 275f conditional RNAi and, 629–630, 632f temporal and cell–type specific regulation using, 629, 631f temporal regulation using, 626, 627f, plate 57 Cre-lox recombinase system for drug-controlled gene expression, 600f, 600–601 for site-specific recombination, 599–600 Cre-lox-P system, 274 Critical periods, experience-dependent plasticity and, 473 Cross-axis adaptation, 98–99, 99f Crx, rod development and, 316 Cry, circadian photoentrainment and, 214, 214f Cry−1− mice, circadian photoentrainment and, 215 Cry−2− mice, circadian photoentrainment and, 215 Cryaa cataracts and, 496, 496f, 588 mutations of, plate 37 Cryab, cataracts and, 496 Cryba1, cataracts and, 497 CRYBB2, cataracts and, 497 Crybb2, cataracts and, 497 Cryg cataracts and, 497–498, 498f mutations of, plate 38 CRYGC, cataracts and, 498 CRYGD, cataracts and, 498 CRYGD-P23T mutation, cataracts and, 498 Crystallins expression in cornea, 705 in lens, 703–704, 704f, 711–713, 716, 716f

posttranslational modification of, 715, 716 lens development and, 272 α-Crystallins αA-crystallin structure and, plate 67 cataracts and, 496, 496f expression in lens, 703–704, 712f, 712–713 β-Crystallins cataracts and, 497 expression in lens, 704, 713 τ-Crystallins cataracts and, 497–498, 498f expression in lens, 704, 713 Cued and contextual fear conditioning, 27 Cued platform task, for measuring visual perception, 110 Curare, retinal wave blocking by, 345 Current-clamp recordings, for retinal wave measurement, 343 Cx36 expression, by photoreceptors, plate 5 Cyclooxygenase-2 (COX-2) inhibitors, herpes simplex virus infection and, 506 CYP1B1 primary congenital glaucoma and, 483 primary open-angle glaucoma and, 482 Cytomegalovirus infections, murine, 509 Cytoskeletal proteins, cataracts and, 498–499

D DBA/2HaSmn strain, auditory evoked brainstem response in, 22 DBA, DBA/2, and DBA/2J strain, 13 anxiety-related behavior tasks and, 29 auditory evoked brainstem response in, 22 conditioned odor preference in, 28 cued and contextual fear conditioning in, 27 as glaucoma model, pattern electroretinogram application in, 122–124, 123f hearing and visual abilities in, 15t, 16 learning and memory in, 27 mutation and effect in, 62t pain sensitivity in, 25 pattern discrimination in, 18 pigment dispersion syndrome in, 483–485, 484f melanosome contribution to, plate 32 tactile sensitivity in, 24 taste ability in, 24 visual acuity of, 20 DBA/2NCrlBR strain conditioned odor preference in, 28 pattern discrimination in, 18 db/db mice diabetes in, 550 diabetes-induced retinal neurodegeneration in, 551 Delayed-type hypersensitivity (DTH) responses, 505

Dendritic arborization, Cre-lox recombinase system for study of, 601 Dendritic cells, antigen-pulsed, experimental autoimmune uveitis induced by injection of, 516 Dendritic outgrowth, of horizontal cells, 150, 150f Depolarizing bipolar cells (DBCs), 136 synaptic transmission in outer retina and, 176f, 176–179, 177t Depth of field, measurements of, 80, 80f Developmental glaucoma, 483 Diabetes, adherence in retinal microvasculature of diabetic mouse, plate 47 Diabetic retinopathy, 547–554 biochemical changes in mouse retinal contributing to development of, 551–553, 552f in humans, 547 in mice, 547–548, 548f nonproliferative, mouse models of vascular lesions of, 548–551 chemically induced diabetes and, 548–549, 549f nondiabetic, developing diabetic-like retinopathy, 550–551 spontaneous diabetes and, 549–550 nonproliferative stage of, 547 proliferative, mouse models of, 553–554 insulin-like growth factor-1 and, 554 oxygen-induced retinopathy and, 553–554 VEGF overexpression and, 554 proliferative stage of, 547 retinal neurodegeneration and, mouse models for study of, 551 Dickkopf1, eye field formation and, 301 Diffusers, deprivation myopia from, 82 Dopaminergic amacrine cells genetic labeling of, 597–598 mosaic architecture of, 151–153 Dorsal terminal nuclei (DTN), retinal projections to, 41 Dorsal visual stream, 373f, 373–374 Dorsolateral geniculate nucleus (dLGN), 219–229 intrinsic physiology of neurons of, 219–221 calcium currents and, 220 hyperpolarization-activated currents and, 221 oscillations and, 221 potassium currents and, 220–221 sodium currents and, 220, 220t modulation of retinal signal by, 225–228 burst and tonic responses and, 227–228, 228f of intrinsic thalamocortical relay cell physiology, 226–227 local geniculate inhibition and, 225–226, 226f presynaptic, 225 output of, 221–225 retinogeniculate inputs and spatial information and, 221–223, 222f–224f

retinogeniculate inputs and temporal information and, 223–225 retinal projections to, 40, 44–45 Dräger, Ursula, 245 Drug-controlled gene expression Cre-lox recombinase system for study of, 600f, 600–601 reversible, 601

E Early-life stimulation, neuronal and behavioral development and, 450–451 Efnb2, retinal vascularization and, 288 Egr-1 knockout, refractive errors in, 83 Egr-1 mRNA, regulation of, 82 Eicosanoids, diabetic retinopathy and, 553 Eight-arm radial maze, 18 Electrical stimulation, retinal ganglion cell axon growth and, 404–405 Electroporation, 621–631 controlling expression following, 626–630 cell type–specific regulation using specific promoters and, 626–627, 628t, 628f–630f, 629 conditional RNAi and, 629–630, 632f temporal and cell type–specific regulation and, 629, 631f temporal regulation and, 626, 627f future applications of, 630–631 method of, 621–626 in vitro, retinal, 623, 625f, 625–626, plate 55, plate 56 in vivo, comparison to other in vivo gene delivery methods, 621 in vivo, brain, 626 in vivo, retinal, 621–623, 622f–624f, plate 50, plate 51, plate 52, plate 53, plate 54 Electroretinogram (ERG) with cone phototransduction gene inactivation, 138, 138f functional features of cones and, 138, 138f Elevated plus maze, 29 Elevated zero maze, 29 ELOVL4 gene, age-related eye diseases and, 582, 584–588 ELOVL4 mice, 613, 638 Embryonic stem cells, in vitro generation of retinal ganglion cells from, 329–330 Encephalitis, viral, 509 Endostatin, anti-angiogenic properties of, 610 Endothelial cells, apoptosis of, oxygeninduced retinopathy and, 293 Endothelial tip cell guidance, retinal vascularization and, 287 Endotoxin-induced uveitis (EIU), 513–515, 514f histology of, plate 39 Environmental enrichment plasticity and, 449–459

early-life stimulation effects on neuronal and behavioral development and, 450–451 neural consequences of environmental enrichment and, 450 visual system development and, 451–459, 452f, 454f, 455f, 457f, 459f, plate 28, plate 29, plate 30 temporal phases during development controlled by environmental richness and, 458, 459f Enzyme replacement therapy, 639 EphB2, targeting of retinal ganglion cell axons to optic disc and exit from eye and, 383f, 383–385 EphB3, targeting of retinal ganglion cell axons to optic disc and exit from eye and, 383f, 383–385 Ephb4, retinal vascularization and, 288 Ephrin(s), targeting of retinal ganglion cell axons to optic disc and exit from eye and, 383f, 383–385 Ephrin ligands, retinal vascularization and, 288 Ephrin-B1/ephrin-B2, retinal ganglion cell axon divergence at chiasm and, 393, 394f, 395 Erythropoietin, proliferative retinopathy and, 292–293 Esi-1/Elf3, in cornea and lens development, 703 Ethylnitrosourea (ENU), as mutagenic agent, 493 Evans, Martin J., 35 Evolution centrifugal model of, 6, 7f of house mice, 6–8, 7f sequential (linear) model of, 6–7, 7f Excitatory postsynaptic potentials (EPSPs), in lateral geniculate nucleus, 420, 421f, 422, 424 Experience-dependent visual cortical plasticity, 465–473 adult ocular dominance plasticity and, 472–473, 474f mechanisms of ocular dominance shift in mice and, 468–471 monocular deprivation in juvenile mice and, 468, 469f, 470f stimulus-selective response potentiation and, 471–472, 472f visually evoked potential recording technique and, 466, 467f, 468 Experimental autoimmune uveitis (EAU) associated with anti-tumor response to melanoma antigen, 517 histology of, plate 39 in human lymphocyte antigen transgenic mice, 516 induced by active immunization with retinal antigen, 515, 516t, 517f induced by adoptive transfer of antigenspecific T cells, 515 induced by injection of antigen-pulsed dendritic cells, 516

index

741

Experimental autoimmune uveitis (continued) in mice expressing a transgenic target antigen in the retina, 515–516 pathogenesis of, critical checkpoints in, plate 39 spontaneous, 516–517 Expressed sequence tags (ESTs), 662–663 ey1 mutation, 269 ey2 modifier genes, 269 Eye, of mouse compared to other mammalian species, 35 Eye field transcription factors (EFTFs), 299–302 expression in retinal progenitor cells, 299, 302–303 interaction of, 300 regulation of, 300–301 transcription targets of, 301–302 Eye movements, 87–102 after-nystagmus and velocity storage integrator and, 94–96, 96f angular vestibulo-ocular reflex and, 88–91, 89f, 90f compensatory, 87, 88 adaptive properties of, 96–99, 97t, 98f, 99f control of, importance of, 87 in foveate and afoveate mammals, 87 gaze holding and velocity-to-position neural integrator and, 96 gaze-shifting, 87–88 nystagmus and, fast phases of, 99–100, 100f off-vertical axis stimulation and, 93–94, 94f optokinetic reflex and, 91, 91f otolith-ocular reflexes and, 91–93, 92f rapid, saccade-like, 233 electrically induced, in mice, 235–238, 237f measurement of, 233–234, 234f spontaneous, in mice, 234f, 234–235, 236f reasons for studying, 87 recordings of, applications of, 100–101 visual-vestibulo-ocular reflex and, 89f, 89–91, 90f Eyeless. See Pax6

F Fear conditioning, cued and contextual, 27 Fetal loss, in Ndph knockout mice, 534–535 Fgfr signaling, lens development and, 276–277 Field mice, 5 Filensin, cataracts and, 498–499 Fixation mechanism, 88 Fluorescence resonance energy transfer (FRET), 597 Fluorescent reporters under control of Thy-1 promoter, 598 subcellular spatial resolution using, 597

742

index

Fontana’s spaces, 130 Force-choice apparatus, two-alternative, for visual acuity measurement, 18, 20 Forced choice discrimination task, for measuring visual perception, 109 Formalin test, 25 Forward genetics approach, 66–67, 67f Fox, cataracts and, 494 Foxd1, chiasm patterning and, 396 FOXE3, in cornea and lens development, 701 Foxe3, lens development and, 277 Foxg1, chiasm patterning and, 396 Foxn4, Math5 repression in retinal progenitor cells and, 326 Frs2α, lens development and, 277 Fungal infections, mouse models of, 508 FVB/N strain, 15 FVB/NJ anxiety-related behavior tasks and, 29 auditory evoked brainstem response in, 22 avoidance learning in, 28 hearing and visual abilities in, 15t pain sensitivity in, 25 pattern discrimination in, 18 visual acuity of, 20 mutation and effect in, 62t FVBS/Ant strain, 15

G GABAC null mice, refractive errors in, 83 GABACR, inhibition mediated by alteration of visually and electrically evoked responses of ON-center retinal ganglion cells by, 183–184, 184f control of output of bipolar cell synaptic output by, 182–183 GABAergic neurons, of superior colliculus, 239 GAD67 (Gad)-GFP knock-in mice, GABAergic neurons in, 239 Galactose feeding, diabetes and, 550 α(1,3)-Galactosyltransferase, cataracts and, 499 Ganglion cell(s), gap junctions of, 169, 170f, 171 functional role of, 171 Ganglion cell layer (GCL), 635, 636f GAP-43, retinal ganglion cell axon guidance through visual pathway and, 392f, 393 Gap junctions, 165–171 of amacrine cells, 167 of bipolar cells, 167 transgenic mice to study, 596–597 ganglion cell coupling and, 169, 170f, 171 functional role of, 171 of horizontal cells, 166f, 166–167 of photoreceptors, 165–166, 166f in proximal rod pathways, 167–169, 168f retinal waves and, 346–347 Gaze holding, oculomotor neural integrator and, 96

Gaze-shifting movements, 87, 88 GC1−/− mice, as model for Leber congenital amaurosis, 611 Gdf8/Myostatin, control of retinal progenitor cell proliferation by, 328 Geminin, eye field transcription factor regulation and, 302 Gene(s). See also specific genes controlling vision, finding, 62–63, 64f, 65f epistatic interaction between, 651–653, 652t Gene deletion, to define role of cholinergic signaling by starburst amacrine cells, 598 Gene expression, 685–694, 686f analysis of correlation of retinal pigment epithelium gene expression and retinal vascularization and, 692–693 isolation of retinal pigment epithelium and, 688, 689f, 690f, 690–691 isolation of retinal vasculature and subsequent gene expression studies and, 688, 689f, 691–692 study methods for, 686–687 transgenics in, 692 in cornea and lens, 697–706 cornea and lens development and, 697, 698f large scale analyses of, 698–699 regulation of specific genes and, 703–705 transcription factors and, 699f, 699–703, plate 66 degeneration networks by expression profiling of photoreceptors from rd mice and, 679–680 drug-controlled Cre-lox recombinase system for study of, 600f, 600–601 reversible, 601 expression profile of mRNA from whole mouse retinas at different postnatal developmental stages and, plate 65 inducible, in differentiated Müller glia, plate 61 mapping of, for ocular dominance plasticity assessment, 442–443 in mouse eye, 662 in retinal pigment epithelium correlation of retinal pigment epithelium gene expression and retinal vascularization and, 692–693 isolation of retinal pigment epithelium and, 688, 689f, 690f, 690–691 in retinal vasculature correlation of retinal pigment epithelium gene expression and retinal vascularization and, 692–693 isolation of retinal vasculature and subsequent gene expression studies and, 688, 689f, 691–692 spatial regulation of, using cell type– specific promoters, plate 58 Gene expression profiling, 654–655

Gene targeting methods, 594–595, 595f Gene therapy antiangiogenic, 607 neurotrophic factor, 613 for retinal degeneration, 675 vectors for, 605 adenoassociated virus. See Adenoassociated virus (AAV) gene therapy Gene transfer methods, 593–594, 594f. See also Electroporation Genetic modifiers as cause of phenotypic variation, 651–653, 652t retinal degeneration and, 652–653 cloning of, strategies for, 653–655 chromosomal localization of modified traits and, 653–654 gene expression profiling and, 654–655 haplotype analysis and, 654 microarray analysis and, 654–655 modifier screens using mutagenesis and, 655 segregating crosses with multiple strains and, 654 Genetic networks in mouse eye, 667f, 667–668 microarray methods and, 669 Genetic tagging technique, to ablate starburst amacrine cells, 598 Genomics, glaucoma therapy and, 488 GFP, expression in rods, plate 63 Ggta1, cataracts and, 499 Gja3, cataracts and, 495–496 Gja8 cataracts and, 496, 496f, 588 mutations of, plate 37 Glaucoma future prospects for new therapies for, 487–488 genomics and proteomics and, 488 neuroprotection as, 487–488, 488f genetic component in, 479 human clinical research on, pattern electroretinography in, 124 mouse models of, 479–489 accuracy of intraocular pressure measurements in, 480 advantages of, 479 of developmental glaucoma, 483 experimentally induced glaucoma and, 481 pattern electroretinogram application in, 122–124, 123f of pigmentary glaucoma, 483–485, 484f of primary congenital glaucoma, 483 of primary open-angle glaucoma, 481–482 similarity between mouse and human structures relevant to, 479–480 mutagenesis and new models of, 488–489 neurodegeneration and, 485, 485f optic nerve excavation in, prevention by radiation treatment, plate 35 overview of, 479

primary open-angle models of, 489 mouse model of, 481–482 retinal ganglion cell death in regional patterns of, 485–486, plate 33 soma death and axon degeneration and, 486–487, 487f treatment of, 644 future prospects for new therapies for, 487–488 Glaucomatous optic neuropathy, retinal ganglion cell axon regeneration in, 409 Gli family, control of retinal progenitor cell proliferation by, 329 Glial cell(s), in optic nerve, 201–202 Glial cell line-derived neurotrophic factor (GDNF), 613 retinal ganglion cell axon growth and, 401 Glial fibrillary acidic protein-green fluorescent protein (GFAP-GFP) transgenic mice, 287 Glucagon amacrine cells, in mouse versus chicken eye, 82–83 GluR6, signaling through, 596 Glycine receptor α2, photoreceptor development and, 305 Gnao1tm1Lbi mice, control of depolarizing bipolar mGluR6 signaling cascade in, 178–179, 179f GpnmbR150X, pigmentary glaucoma and, 483 Grm6nob4 mice control of depolarizing bipolar mGluR6 signaling cascade in, 178–179, 179f retinal ganglion cell responses in, 181 Grm6Tm1Nak mice control of depolarizing bipolar mGluR6 signaling cascade in, 178–179, 179f retinal ganglion cell responses in, 181 Growth, of retinal ganglion cell axons. See Retinal ganglion cells (RGCs), axon growth and regeneration of Growth factors. See also specific growth factors lens and, 273 retinal vascularization and, 286–287 Growth of mouse eye, 74 Guidance molecules, retinal vascularization and, 288

H Habituation-discrimination test, 22–23 Habituation-dishabituation test, 22–23 Hamilton Eye Institute Mouse Database (HEIMED), 664 BXD Genetic Reference Panel of Mice and, 664–665, 666t, 667 genetic networks in eye and, 667, 667f quantitative trait locus mapping in, 668–669, 670 Hargreaves test, 24, 25 Hartmann-Shack aberrometry optical aberration measurement using, 79f, 79–80 spherical aberration found on, 75–76 Head tilt mice, 101

Hearing, in mice, 15t Hearing ability, measurement of, 20, 22 Hearing loss, in Ndph knockout mice, 533–534 Heat shock transcription factors cataracts and, 496–497 in cornea and lens development, 702 Hematopoietic stem cells (HSCs), retinal vascularization and, 289 Herpes simplex virus (HSV) infections, 505 Herpes stromal keratitis (HSK), 505–506 Hes1 eye field formation and, 301 retinal development and, 324 Hes5, eye field formation and, 301 Hind limb denervation, as model of anesthesia dolorosa, 25 Hmgn1, in cornea and lens development, 701 Horizontal cells development of, Rb and, 318 gap junctions of, 166f, 166–167 mosaic architecture of, 148–150, 149f, 150f Hot-plate test, 24–25 House mice, 5 Hsf4, cataracts and, 496–497 Hspg2, cataracts and, 499–500 Human genome, 660 Human leukocyte antigen (HLA) transgenic mice, experimental autoimmune uveitis in, 516 Hyperoxia, depletion-induced, lack of oxygen supply to outer retina and, 565–566 Hyperpolarizing bipolar cells (HBCs), 136 synaptic transmission in outer retina and, 176f, 176–179, 177t Hypopigmented mice, 61

I I-κB-ζ, in cornea and lens development, 702 I/LnJ mice, auditory evoked brainstem response in, 22 Immunoprecipitation (IP), visual cycle protein interactions and, 729 Impact-rebound (I/R) tonometry, 480 Inbred strains genetic variability in, 668 power of, 668 used for research, 13 on lens, 271–272 Inducible nitric acid synthase (iNOS), diabetic retinopathy and, 553 Inducible RNAi, in retina, plate 62 Infantile neuronal ceroid lipofuscinosis (INCL), gene replacement therapy for, 612 Infectious eye diseases, 505–509 bacterial, 506–508 fungal, 508 herpetic, 505–506 parasitic, 508–509 retinal, mouse models of, 509

index

743

Inferometry, low-coherence, 77–79, 78f Infrared photoretinoscopy, 74–76, 76f, 77 Inhibitory postsynaptic potentials (IPSPs), in lateral geniculate nucleus, 420, 421f Inner plexiform layer (IPL) dendrites in, 194 lateral inhibition in, 175 Insulin-like growth factor (IGF) cell death and, 336 retinal ganglion cell axon growth and, 401 Insulin-like growth factor-1 (IGF-1) eye field formation and, 301 overexpression of, 554 visual cortical plasticity and, 456 Interferometer, Michelson, 79 Interferon-τ (IFN-τ) experimental autoimmune uveitis and, 520 Pseudomonas aeruginosa infections and, 507 Intergeniculate leaflet (IGL), retinal projections to, 40, 41 Interlaminar connections, in superior colliculus, 239, 240f Interleukin-1β (IL-1β), diabetic retinopathy and, 553 Interleukin-8 (IL-8) proliferative retinopathy and, 292, 293 Pseudomonas aeruginosa infections and, 507 Interphotoreceptor retinoid-binding protein (IRBP) experimental autoimmune uveitis induced by, 515 in visual cycle, 726 Intracellular adhesion molecule-1 (ICAM-1), diabetic retinopathy and, 553 Intraocular pressure (IOP) aqueous humor outflow and, 131 measurement of, accuracy of, 480 Ischemic optic neuropathy, retinal ganglion cell axon regeneration in, 409 Islet2 retinal ganglion cell axon divergence at chiasm and, 395 as retinal ganglion cell marker, 193 Isoelectric focusing (IEF), in proteomics, 713–714 Isomerohydrolase, in visual cycle, 723–724

J Jackson Laboratory, 13, 581–582 Junctional adhesion molecule-B (JAM-B), as retinal ganglion cell marker, 193 Juxtacanalicular connective tissue (JCT), 131, 131f

K Keratins, expression in cornea, 705 Keratitis, stromal, herpes, 505–506 Keratocan, expression in cornea, 705 Keratomycosis, 508 Kessler, Paul Leonhard, 270 Kinases, cell death and, 338 KKAY mouse diabetes in, 550

744

index

diabetes-induced retinal neurodegeneration in, 551 Knockout mouse models, 65, 635–645. See also specific models anterior chamber cell types and, 644–645 developers of, 35 for lens research, 273–274 P7, plate 42 refractive errors in, 83 retinal cell types and, 635, 636f, 637 retinal pigment epithelium and, 637–639, 639f vascular cell types and, 639–643, 641f, 643f

L Lac repressor-lac operator system, for study of retinal ganglion cells, 599 Lamina cribrosa (LC), in glaucoma, 485–486 Lamina cribrosa region, of optic nerve, 201, 202f Lang, Richard, 275 Lateral geniculate nucleus (LGN) cellular composition of, 419 differentiation of neurons of, 419 dorsal. See Dorsolateral geniculate nucleus (dLGN) excitatory responses in, 420, 422, 424 eye-specific segregation in, development of, 415–416, 416f, 417f, 418 inhibitory responses in, 422 retinal ganglion cell projections to, 192 synaptic connectivity patterns in, 422, 423f, 424 synaptic remodeling in, 429–430, 430f, 430–431, 431f activity and, 431–432 comparison with remodeling in other regions of visual system, 433–434 cortical critical period in, 433 genes involved in, 434–435 plasticity in lateral geniculate nucleus and, 432–433 superior colliculus and, 433–434 vision-sensitive phase of, 433 Lateral terminal nuclei (LTN), retinal projections to, 41 Lateromedial area, extrastriate, intracortical connections of, plate 9, plate 10 Lathrop, Abbie, 13 Learning tasks, 25–29 visually dependent, 25–28 visually independent, 28–29 Leber congenital amaurosis (LCA), gene replacement therapy for, 611–612 Lecithin:retinol acyltransferase (LRAT), in visual cycle, 723 Le-Cre strain, Cre expression in, plate 12 Lens, 267–279 cell proliferation in, 270 connexins in, cataracts and, 495–496, 496f crystallins in, 703–704, 704f, 711–713, 716, 716f posttranslational modification of, 716

posttranslational modifications and, 715 development of, 267, 268f, 697, 698f transcription factors involved in, 699f, 699–703, plate 36 epithelial cells of, 267 fiber cells of, 267 function of, 267–268 gene expression in. See Gene expression, in cornea and lens lens transplantation experiments and, 270 membranes of, mutations affecting, 495–496 morphological descriptors of, 270 mutations affecting structural proteins of, cataracts and, 496–499 proteomics of, 711–718, 716f crystallins and, 711–713, 716 posttranslational modifications and, 715 refractive power of, 73 research on development of conditional gene alterations using Cre recombinase and, 274–275, 275f customizing mouse genome for, 273–274 differences among mouse strains and, 271–272 historical background of, 268–269 lens complementation system for, 274 lens fiber differentiation and, 278 molecular biology and, 272 mouse and human lenses compared and, 270–271 mouse lens induction and, 275–278, 276f murine models for, 269–270 nonmurine model systems for, 268–269 transgenic mice for, 272–273 structure of, 267 Lens complementation system, 274 Leukemia inhibitory factor (LIF), retinal vascularization and, 287 Leukocytes adherence in retinal microvasculature of diabetic mouse, plate 47 mouse model of oxygen-induced retinopathy and, 293–294 Lhx2, as eye field transcription factor, 300 Lid suture, deprivation myopia from, 82 Liegéois, Nanette, 274 Light acceleration of photoreceptor degeneration by, lack of oxygen supply to outer retina and, 566 continuous, refractive development and, 82 raising of oxygen levels in outer retina by, 565 responses to exposure to, Rpe65 alleles and, 650–651 retinal degeneration induced by, 16, 573 mouse strains with, 16 Light-dark transition test, 29 Lim2, cataracts and, 495 Lineage tracing, Cre-lox recombinase system for study of, 601

Linear control systems analysis, 88n Linear model of evolution, 6–7, 7f Linear vestibulo-ocular reflex (lVOR), 88 Lip12, cataracts and, 588 Lipofuscin, in retinal pigment epithelium, 539–544, 540f, 638 autofluorescence of, plate 44 inhibiting formation of fluorophores and, targeting visual cycle for, 543f, 543–544 Leu450Met variant in murine Rpe65 associated with, 542–543, 543f Rpe65−/− mice and, 539–540, 540f Stargardt macular degeneration and Abca4/Abcr null mutant mice and, 540–542, 541f Liquid chromatography, in proteomics, 714 Listeria monocytogenes infections, 508 Little, Clarence Cook, 13, 269 Long-term depression (LTD) AMPA receptors and, 470 aVOR gain and, 100 ocular dominance shift and, 468 Long-term potentiation (LTP) mechanism for, 471 ocular dominance shift and, 468 retinoic acid and, 366 Lop10, cataracts and, 588 Low-coherence inferometry, 77–79, 78f Lrat−/− mice, 723 Lucentis (ranibizumab), for age-related macular degeneration, 607 Lurcher mice, 101 Lysosomal storage disease, gene replacement therapy for, 612

M Mab21/2, lens development and, 277 MacDowell, E. Carlton, 13 macrophage inhibitory protein-2 (MIP-2), herpes simplex virus infection and, 506 Macrophages, mouse model of oxygeninduced retinopathy and, 294 Macugen (pegaptanib), for age-related macular degeneration, 607 Macular degeneration age-related, gene therapy for, 607 Stargardt, in Abca4/Abcr null mutant mice, 540–542, 541f Maf, cataracts and, 494 Maf transcription factors, in cornea and lens development, 702 Magnetic search coil oculography, 89 Major histocompatibility complex I (MHC I) antigen, lateral geniculate nucleus synaptic remodeling and, 434–435 MA/MyJ strain, auditory evoked brainstem response in, 22 Mann, Ida, 269 Mass spectrometry in proteomics, 714 visual cycle protein interactions and, 729

Maternal care, neuronal and behavioral development and, 451, 455–456, 458 Math5 circadian photoentrainment and, 210, 211–212 regulation of expression of, 325–327 of duration of expression, 325–326, 326f math5 activation in retinal progenitor cells and, 325 repressing expression in other retinal progenitor cells and, 326–327 retinal ganglion cell development and, 324–325 activation of retinal ganglion cell fate and, 325 cell cycle progression and, 324 retinal ganglion cell competence and, 324 suppression of non-retinal ganglion cell fates and, 324–325 retinal ganglion cell differentiation downstream of, regulation of, 327–329 control of retinal progenitor cell proliferation by retinal ganglion cellsecreted signaling molecules and, 328–329 genes regulated by Pou4f2 and, 327–328 regulation of retinal ganglion cell axon outgrowth and pathfinding and, 328 transition for retinal ganglion cell competence to retinal ganglion cell commitment and, 327 Math5 expression in retinogenesis, plate 16 retinal ganglion cell development and, 194 Matrix metalloproteinase-9 (MMP-9), corneal neovascularization and, 506 Mechanical sensitivity, von Frey filament test of, 24 Medial terminal nucleus (MTN), retinal projections to, 41 Melanoma antigen, anti-tumor response to, uveitis associated with, 516 Melanopsin, 213–214 phototransduction mechanism and, 213–214 Melanosomes, DBA/2J pigment dispersion and, plate 32 Membrane currents, in dorsolateral geniculate nucleus, 220f, 220–221 Memory tasks visually dependent, 25–28 visually independent, 28–29 Mesencephalon, visual cortex connections with, 46 Michelson interferometer, 79 Microarray analysis, 654–655, 663–667 BXD Genetic Reference Panel of Mice and, 664–665, 666t, 667 defining genetic networks in, 669 expression profiling using, 675–676 gene expression and, 686–687

Hamilton Eye Institute Mouse Database and, 664 Microarray-based technologies, glaucoma therapy and, 488 Microglia, mouse model of oxygen-induced retinopathy and, 293–294 Microphthalmia, in C57BL mice, 271 Mip, cataracts and, 495 MLR10 mice, 274–275 Cre expression in, plate 12 MLR39 mice, 275 Cre expression in, plate 12 MOLF/EiJ strain anxiety-related behavior tasks and, 29 hearing and visual abilities in, 15t pattern discrimination in, 18 visual acuity of, 20 Monocular deprivation (MD) in juvenile mice, effects of, 468, 469f, 470f ocular dominance shift and, 470–471 ocular dominance studies and. See Ocular dominance (OD) plasticity M-opsin, cone, expression of, 136, 138–139 Morris water maze, 25–26, 26f for measuring visual perception, 109–110 Mosaic architecture, of retina, 147–153 cholinergic amacrine cells and, 150–151, 151f development of, 147–148, 148f dopaminergic amacrine cells and, 151–153 horizontal cells and, 148–150, 149f, 150f Mouse genome, 660–662 sequence differences among mice and, 660–662, 661f, 662t Mouse hepatitis virus (MHV) infections, 509 Mouse models. See also specific models advantages of, 363 historical background of, 61–62, 62t mutant. See also specific models eye movements and, 100–101 of synaptic transmission in outer retina, 176f, 176–179, 177t Mox-Cre;RbLox/Lox mice, rod development in, 314 Mucopolysaccharidosis type VII (MPS VII), gene replacement therapy for, 612 Müller glia, differentiated, inducible expression in, plate 61 Multielectrode arrays (MEAs), for retinal wave measurement, 343 Murine coronavirus infections, 509 Murine cytomegalovirus infections (MCMV), 509 Mus, 3–8 phylogenetic relationships in genus, 8 subgenera of, 3–6, 4f Mus booduga, 5, 8 Mus caroli, 8 Mus cervicolor, 5, 8 Mus Coelomys, 3–5, 8 Mus cookii, 8 Mus crociduroides, 5 Mus cypriacus, 6 Mus famulus, 5, 8 Mus fernandoni, 5

index

745

Mus fragulicauda, 5, 8 Mus lepidoides, 5–6, 8 Mus macedonicus, 6, 8 Mus mayori, 5 Mus Mus, 3–4, 8 Mus mus castaneus, 6, 7, 8 Mus mus domesticus, 6–7, 8 Mus mus gentilulus, 6, 7, 8 Mus mus musticus, 6, 7, 8 Mus musculus, 3, 5, 6, 8 evolutionary history of, 6–8, 7f Mus Nannomys, 3, 5, 8 Mus nitidulus, 5, 8 Mus pahari, 5 Mus phillipsi, 5 Mus plathythrix, 5 Mus Pyromys, 3–4, 5, 8 Mus Sensu stricto, 5 Mus shortridgei, 5 Mus spicilegus, 6, 8 Mus spretus, 6, 8 Mus terricolor, 8 Mus vulcani, 5 Mutagenesis, modifier screens using, 655 Mutant mouse models, 14. See also specific models eye movements and, 100–101 of synaptic transmission in outer retina, 176f, 176–179, 177t postsynaptic mouse mutants and, 177t, 178–179, 179f presynaptic mouse mutants and, 177t, 177–178 Muthukkaruppan, V., 269–270 Myelinated portion, of optic nerve, 201, 202f Myelinogenesis, of optic nerve, 201–202 MYO7A, age-related eye diseases and, 584 Myocilin (MYOC), in primary open-angle glaucoma, 481–482 Myopia, deprivation induced by frosted diffusers, 82 from lid suture, 82

N nAChR subunits, retinal waves and, 345–346 Nakano cataract, 501 Nance-Horan syndrome (NHS), 499 Nbn gene, senile cataracts and, 500 NDP, 528 evolutionary conservation and expression pattern of, 528, 529f Ndph, 529–530 Ndst1, lens development and, 277 Negative masking, circadian photoentrainment and, 210 Neo-antigen-driven experimental autoimmune uveitis, 515–516 Neovascularization choroidal, gene therapy for, 610 corneal, matrix metalloproteinase-9 and, 506 retinal gene therapy for, 607, 609–610 limitation by laser burns, 565

746

index

protection against, by degradation of photoreceptors, 565 Netrin-1 regulation of retinal ganglion cell axon exit from eye by, 383f, 384 retinal ganglion cell axon guidance through visual pathway and, 392, 392f NEUBank, 663 Neurodegeneration, glaucoma and, 485, 485f Neurofilament H, antibodies against, as retinal ganglion cell marker, 193 Neuroglobin, retinal oxygen consumption and, 562 Neuronal plasticity, RALDH3 colocalization with markers for, 368, 369f Neuronal tissue, in optic nerve, 201 Neuron-specific enolase (NSE), cell death and, 337 Neuropathic pain, measurement, 25 Neuroprotection, for glaucoma treatment, 487–488, 488f Neurotrophic factor gene therapy, 613 Neurotrophin(s), cell death and, 336 Neurotrophin-3 postnatal cortical maturation patterns defined by, 369–370, 370f RALDH3 relationship with, 368–369, 369f Neutrophils, Pseudomonas aeruginosa infections and, 507 Nhs1 protein, cataracts and, 499 Night blindness, stationary, congenital, 650 NMDA, excitatory lateral geniculate nucleus responses and, 420 NMDA receptors (NMDARs), 223–225 ocular dominance shift and, 469 in reticulogeniculate synapses, 431, 439 NMDA-type glutamate receptor antagonist, stratum griseum intermediale burst generation and, 240 nob knockout mice, refractive errors in, 83 NOD strain, diabetes in, 550 NOD/LtJ strain, auditory evoked brainstem response in, 22 Noggin, eye field formation and, 301 Nonpigmented ciliary epithelium (NPE), 129 Nop, cataracts and, 497 NOR/LtJ strain, auditory evoked brainstem response in, 22 Norrie, Gordon, 527 Norrie, function of, 528–529 Norrie disease, 527–535 clinical background of, 527 extraocular features of, 527 knockout mouse model for, 529–530 abnormal retinal vascular development in, 530–531, 531f, 532f ectopic norrin expression in lens and, 534 fetal loss in homozygous females and, 534–535 hearing loss and abnormal cochlear vasculature in, 533–534

hyaloid vessel regression versus angiogenesis of retinal blood vessels in, 532, 533f phases of, 532–533 retinal physiology of, 530 sprouting angiogenesis failure in, 531–532 molecular basis of, 527–528 NDP gene conservation and expression pattern and, 528, 529f norrin and, 528–529 transgenic mouse line with norrin overexpression in lens and, 534 Norrin, 528 ectopic expression in lens, rescue of knockout phenotype by, 534 overexpression in lens, transgenic mouse line with, 534 Norrin knockout mice, hematoxylin-eosinstained sections of whole eyes from, plate 43 Notch pathway eye field formation and, 301 retinogenesis regulation and, 303–304 Npp, 501 Nr-CAM, retinal ganglion cell axon divergence at chiasm and, 394f, 395 Nr2e3, rod development and, 316 Nr2e3, photoreceptor development and, 304–305 NRL, gene profiling of purified photoreceptors and, 676, 677f Nrl, photoreceptor development and, 304–305 Nrl, rod development and, 316 Nrl −/− knockout mice as model for central field loss, 613 photoreceptors of caveats for investigations of, 142 cones as, 139, 141–144 cone-specific genes and their molecular function and, 143–144 differentiation of, 143 mouse cone protein biochemistry and, 144 prospects for studies using, 143–144 Nuclear posterior polar opacity, 501 Nucleus of the optic tract (NOT), retinal projections to, 41 Null mutation. See Knockout mouse models Nystagmus fast phases of, 99–100, 100f off-vertical axis rotation engendering, 94 optokinetic, velocity storage integrator and, 95f, 95–96 optokinetic after-nystagmus and, 94 postrotatory, 94 Nyxnob mice control of depolarizing bipolar mGluR6 signaling cascade in, 178–179, 179f retinal ganglion cell responses in, 181 NZB/BINJ strain, taste ability in, 23 NZW strain, mutation and effect in, 62t NZW/LacJ strain, photoreceptor response damage by light and, 575, 577

O ob/ob mice, diabetes in, 550 Ocular dominance (OD), mapping of, plate 26 Ocular dominance (OD) plasticity, 439–446. See also Visual cortical plasticity, experience-dependent adult, 443, 444f, plate 27 enhancement by prior experience, 445f, 445–446, 446f mechanisms of, 443–445 behavioral correlates of, 447 chondroitin sulfate proteoglycans and, 454–455 extended duration visually evoked potentials for assessment of, plate 31 juvenile, mechanisms of, 443–445 methods for assessing, 439–443 calcium imaging with two-photon microscopy as, 441–442, 442f mapping of gene expression as, 442–443 optical imaging as, 440f, 440–441, 441f single-unit recordings as, 439 visually evoked potentials as, 439–440 ocular dominance column plasticity and, 465–466 Ocular hypertension, treatment of, 644 Oculography, applications of, 101 Oculomotor control, importance of, 87 Oculomotor neural integrator, 96 Odor discrimination ability, measurement of, 22–23 Odor preference, conditioned, 28 Off-vertical axis rotation (AVOR), 93–94, 94f Olfactory ability, measurement of, 22–23 Olfactory tubing maze, 28 Oligodendrocytes, in optic nerve, 202 Olivary pretectal nuclei (OPT), retinal projections to, 41 ON and OFF pathways in mouse retina, 353–360 cellular and synaptic structure of, 353–354, 354f segregation of mechanisms of, 358–360, 359f, 360f retinal synaptic circuitry development and, 354–357, 355f, 356f synaptic activity and, 357–358 Onchocerca volvulus infections, 508 129/J. See 129P3/J strain 129P3/J strain auditory evoked brainstem response in, 22 mutation and effect in, 62t 129P3/PJ, mutation and effect in, 62t pain sensitivity in, 25 tactile sensitivity in, 24 129/ReJ strain, auditory evoked brainstem response in, 22 129S1/SvImJ strain hearing and visual abilities in, 15t 129/S1, olfactory ability in, 22, 23 pattern discrimination in, 18 photoreceptor response damage by light and, 575, 577 visual acuity of, 20

129S2/Sv strain, anxiety-related behavior tasks and, 29 129/SvEms strain, learning and memory in, 27 129/SvEvTac strain avoidance learning in, 28 pattern discrimination in, 18 129SvJ. See 129X1/SvJ 129/SvPasCrl strain, conditioned odor preference in, 28 129Sv-ter/C57BL/6 mice, olfactory ability in, 22, 23 129X1/SvJ strain auditory evoked brainstem response in, 22 pain sensitivity in, 25 Open field test, 29 Operant olfactometer, 22, 23 Opn4 −1− mice, circadian photoentrainment and, 210, 211 Opsins of cones, expression of, 136, 138–139 development of, 305 signaling molecules responsible for expression of, 304–306 Optic chiasm regulatory genes in retina and chiasm important for patterning, 396–397 retinal ganglion cell axon divergence at, 393–396 crossed projection and, 395–396 uncrossed projection and, 393–395 retinal ganglion cell axon guidance toward, 392f, 392–393 Optic disc directed growth of retinal ganglion cell axons toward, 382–384, 383f targeting of retinal ganglion cell axons to, 383f, 383–384 Optic disc astrocyte precursor cells (ODAPs), 390, 390f Optic fiber layer (OFL) control of initial retinal ganglion cell axon outgrowth polarity in, 381–382, 382f retinal ganglion cell axon restriction to, 379–381, 380f Optic nerve, 201–204 astrocytes and astrocyte progenitors in, 390, 390f connective tissue in, 203, 203f development of, 201–202 glaucomatous excavation of, prevention by radiation treatment, plate 35 morphological features of, 201, 202f neuronal and glial tissue in, 202–203, 203f retinal ganglion cell axon projection through, 391–392, 392f vascular supply of, 203–204, 204f Optic nerve head, 201, 202f Optic neuritis, retinal ganglion cell axon regeneration in, 409 Optic neuropathy glaucomatous, retinal ganglion cell axon regeneration in, 409 ischemic, retinal ganglion cell axon regeneration in, 409

traumatic, retinal ganglion cell axon regeneration in, 409, 409f Optic vesicle (OV), 267 Optical imaging, for ocular dominance plasticity assessment, 440f, 440–441, 441f Optical interferometry tonometry (OIT), 480 Optics, of mouse eye, 74 Optineurin (OPTN), primary open-angle glaucoma and, 482 Optokinetic after-nystagmus (OKAN), velocity storage integrator and, 95f, 95–96 Optokinetic drum, 20 Optokinetic nystagmus (OKN), velocity storage integrator and, 95f, 95–96 Optokinetic reflex (OKR), 88, 91, 91f adaptation of, 98f, 98–99, 99f measurement of, 112–113, 114f Optomotor response, “whole body,” spatial vision and, 81–82 Optomotor test apparatus, 17f Organelles, loss during lens differentiation, 278 Orienting vestibulo-ocular reflex, 92 Otolith-ocular reflexes, 91–92, 92f Outer nuclear layer (ONL), 635, 636f Outer plexiform layer (OPL), 637 lateral inhibition in, 175 Oxygen, 559–569 delivery to retina, 559–561 choroidal circulation and, 560–561 retinal circulation and, 559–560 inhaled blocking of vasogenesis by, 563 killing of photoreceptors by, 562–563, 564f, plate 48 lack of oxygen supply to outer retina and, 562–563, 563f lack of supply to outer retina, 562–568 acceleration of photoreceptor degeneration by light and, 566 degeneration of edge of normal retina and, 566–568, 567f depletion-induced hyperoxia and, 565–566 inhaled oxygen and, 562–563, 563f reduction in photoreceptor metabolism and, 564–565 retinal detachment and, 564 oxygen stress during development and, 568, plate 49 retinal consumption of, 561–562 in adults, 561f, 561–562 developmental onset of, 562 neuroglobin and, 562 retinal degeneration and, 568–569 Oxygen-induced retinopathy (OIR), 285, 553–554 epithelial cell death and, 293 mouse model of, 289–294. See also Retinopathy, of prematurity (ROP) characterization of, 289–291, 291f, 292f cytokines and, 292–293 endothelial cell apoptosis and, 293 microglia and leukocytes and, 293–294

index

747

Oxygen-induced retinopathy (continued) retinal flat-mount preparation from, plate 13 VEGF-A and, 291–292

P P7 knockout mice, plate 42 P7 wild type mice, plate 42 P21 knockout mice, central retina from, plate 41 P21 wild type mice, central retina from, plate 41 p107 retinal development and, 313 rod development and, 316 p130, retinal development and, 313 Pac1r, circadian photoentrainment and, 214 Pain sensitivity, measurement of, 24–25 Paramedian pontine reticular formation (PPRF), 238 Parasitic infections, 508–509 Pars plicata region, 129 Pathogen-associated molecular patterns (PAMPs), 519 Pattern electroretinogram (PERG), retinal ganglion cell activity measurement by, 119–124, 120f correlates of visual behaviors and, 120–121, 121f in human clinical research, 124 limitations of, 124 methods for, 120 in mouse models of glaucoma, 122–124, 123f in study of central nervous system plasticity and degeneration, 121–122 Pax6 in cornea and lens development, 699–700 eye field formation and, 301 Notch1 retinal development and, 323–324 Pax6 cataracts and, 493 as eye field transcription factor, 299 Le-Cre mice and, 274 lens development and, 275–277, 276f retinal ganglion cell development and, 194 retinal development and, 322–323 Pax6αCre line, 598 Pax6-Cre;RbLox/Lox mice, rod development in, 314, 315 Pde6b, age-related eye diseases and, 584 Pde6b gene, 14, 63 rd mutation of, 61 rd1 mutation of, 26 Pdeb6b mutations, 650 Pde6brd1 mutation, 271 Pearlman, Alan, 245 PEDF, in gene therapy for retinal neovascularization, 607, 609 Pentraxins, lateral geniculate nucleus synaptic remodeling and, 435 Per genes, circadian photoentrainment and, 214, 214f

748

index

Pericyte(s), retinal vascularization and, 288, 290–291 Pericyte recruitment, 642 Pericyte-derived angiopoietin-1, retinal vascularization and, 288 Perlecan, cataracts and, 499–500 Phakinin, cataracts and, 498 Phenotypic variation alleles as cause of, 649–651 genetic modifiers as cause of, 651–653, 652t retinal degeneration and, 652–653 Photokeratometry, 76–77, 78f Photoreceptors, 635, 636f, 637. See also Cone(s); Rod(s) candidates for light damage quantitative trait loci and, 577–579, 578t cell death of, pathways of, 678–679, 679f Cx36 expression by, plate 5 degeneration of by light, 566 Pde6b mutations leading to, 650 Rpe65 alleles and, 650–651 Rpgrip 1 mutations leading to, 650 degradation of, protection against retinal neovascularization by, 565 depletion of, raising of oxygen levels in outer retina by, 565 development of, 304–306 biological network characteristics and relevance to photoreceptors and, 676–677 critical period in, 568 hierarchy in regulatory networks underlying photoreceptor development and, 677–678, 678f NRL and gene profiling of purified photoreceptors and, 676, 677f expression profiling of, degeneration networks and, 679–680 gap junctions of, 165–166, 166f killing of, by inhaled oxygen, 562–563, 564f, plate 48 by light damage of, 566 light damage of, 573–579 gene modifier having strong influence on, 573–575, 576f, 577, 577f of mouse compared to other mammalian species, 36 at peripheral margin of retina, stress from early postnatal life and, plate 49 phagocytosis of outer segments of, 638 promoter transgenes and, 595 reduction in metabolism of, lack of oxygen supply to outer retina and, 564–565 retinal pigment epithelium relationship with, 639 targeted ablation to assess role of, 595–596 toxicity of depletion-induced hyperoxia to, 565–566 Photoretinoscopy, infrared, 74–76, 76f, 77 Photosensitivity, circadian, 209 Physical contact, plasticity and, 455f, 455–456

Pigment dispersion syndrome (PDS), 483–485, 484f Pineal melatonin rhythm, circadian photoentrainment and, 210 Pitx2, in cornea and lens development, 701 Pitx3, cataracts and, 493–494, 494f Pitx3, in cornea and lens development, 701 Placental growth factor (PGF), retinal vascularization and, 286–287 Plasmids, delivery using electroporation. See Electroporation Plasminogen activator inhibitor-1 (PAI-1), diabetes and, 550–551 Plasticity environmental enrichment and. See Visual cortical plasticity in lateral geniculate nucleus, mouse compared to other species and, 432–433 ocular dominance. See Ocular dominance (OD) plasticity reticulogeniculate synapses remodeling and, 431–432 Platelet-derived growth factor-A (PDGF-A), retinal vascularization and, 287 Platelet-derived growth factor-B (PDGF-B) diabetes and, 551 retinal vascularization and, 288 Postrotatory nystagmus (PRN), 94 Posttranslational modifications, of proteins, 715 Potassium currents, in dorsolateral geniculate nucleus, 220–221 POU homeodomain transcription factors, in cornea and lens development, 701 POU4f1, retinal ganglion cell development and, 194 Pou4f2 expression in retinogenesis, plate 16 genes regulated by, 327–328 POU4f2, retinal ganglion cell development and, 194 POU4f3, retinal ganglion cell development and, 194 PPT1, 612 P/Q mutants, 101 Presumptive lens ectoderm (PLE), 267 Primary congenital glaucoma (PCG), 483 Primary open-angle glaucoma (POAG) models of, 489 mouse model of, 481–482 Prior experience, enhancement of ocular dominance plasticity by, 445f, 445–446, 446f Promoter transgenes, and photoreceptors, 595 Prostaglandin analogues, for intraocular hypertension and glaucoma, 644 Protein(s). See also specific proteins posttranslational modifications of, 715 proteome-wide identification of species of, 715 Rb family of, retinal development and, 311–319 intrinsic genetic compensation and redundancy and, 312f, 312–313

Protein(s) (continued) rod development and, 216f, 313–317, 315f species-specific differences in Rb family compensation and, 313 synaptogenesis defects in Rb-deficient retinas and, 317–318, 318f Protein-bound carbohydrates, cataracts and, 499–500 Proteomics definition of, 711 glaucoma therapy and, 488 of lens, 711–718 of visual cycle, 727–729 of interacting components, 727–729 Prox1, lens fiber differentiation and, 278 Prox1, in cornea and lens development, 700–701 Prph2Rd2/Rd2 mouse, gene replacement therapy for, for retinal degeneration in, 610–611 prRdh−/− mice, 723 Pseudomonas aeruginosa infections, 506–507, 508 Ptf1a, Math5 repression in retinal progenitor cells and, 326–327 Purkinje cell degeneration mice, 101

Q Quantitative trait genes (QTGs), for light damage, 577–579, 578t Quantitative trait locus (QTL) analysis, 63, 64f, 65f Quantitative trait locus (QTL) mapping, 668–669, 670

R Radial arm maze, 26–27 Radial glia, midline, in retinal ganglion cell axon projection pathway, 390–391 Rafferty, Nancy, 270 RALDH3 band, medial, anatomical locations of, plate 20 Ramón y Cajal, S., 635 Rapid eye movements, saccade-like, 233 electrically induced, in mice, 235–238, 237f measurement of, 233–234, 234f spontaneous, in mice, 234f, 234–235, 236f Rb family of proteins, retinal development and, 311–319 intrinsic genetic compensation and redundancy and, 312f, 312–313 rod development and, 216f, 313–317, 315f species-specific differences in Rb family compensation and, 313 synaptogenesis defects in Rb-deficient retinas and, 317–318, 318f RBP receptor, in visual cycle, 725 RDH5 gene, 725 RDH12 mutations, 723 Receptive fields (RFs), of dorsolateral geniculate nucleus cells, 221–224, 223f

Refractive errors induction of, by frosted diffusers or spectacle lenses, 82–83 in knockout mouse models, 83 Regeneration, of retinal ganglion cell axons. See Retinal ganglion cells (RGCs), axon growth and regeneration of Regulatory networks degeneration and, expression profiling of photoreceptors from rd mice and, 679–680 model for retinal ganglion cell development, plate 15 photoreceptors and, 676–677 underlying photoreceptor development, hierarchy in, 677–678, 678f Reinforcement-based tasks, for measuring visual perception, 109–111, 111f–113f Reticulogeniculate synapses. See Lateral geniculate nucleus (LGN), synaptic remodeling in Retina cell types in, 635, 636f, 637 developing, plate 14 electroporation and. See Electroporation gap junctions in. See Gap junctions inducible RNAi in, plate 62 layers of, plate 64 lineage tracing in, using cell type–specific promoters, plate 60 mature, plate 14 mosaic architecture of, 147–153 cholinergic amacrine cells and, 150–151, 151f development of, 147–148, 148f dopaminergic amacrine cells and, 151–153 horizontal cells and, 148–150, 149f, 150f of mouse compared to other mammalian species, 35–39 multicolor labeling of, using cell type– specific promoters, plate 59 normal, degeneration of edge of, lack of oxygen supply to outer retina and, 566–568, 567f outer, lack of oxygen supply to, 562–568 acceleration of photoreceptor degeneration by light and, 566 degeneration of edge of normal retina and, 566–568, 567f depletion-induced hyperoxia and, 565–566 inhaled oxygen and, 562–563, 563f reduction in photoreceptor metabolism and, 564–565 retinal detachment and, 564 outer, raising of oxygen levels in, by light, 565 oxygen consumption of, 561–562 in adults, 561f, 561–562 developmental onset of, 562 neuroglobin and, 562 oxygen delivery to, 559–561 retinal circulation and, 559–560

physiology of, in norrin knockout mice, 530 retinal ganglion cell axon exit from, 391 vascular development of, in norrin knockout mice, 530–531, 531f, 532f whole mount, confocal image of, plate 4 Retinal antigen, experimental autoimmune uveitis induced by, 515, 516t, 517f Retinal circulation, oxygen delivery to retina and, 559–560 Retinal degeneration, 675–680 age-related, in human and mouse eyes, 582, 584–588 expression profiling of photoreceptors from rd mice and, 679–680 expression profiling using microarrays and, 575–676 gene replacement therapy for, 610–611 gene therapy for, 675 genetic modification of, 652–653 light-induced, 16 mouse strains with, 16 mouse models of, 678–679, 679f synaptic reorganization due to, 317–318 Retinal detachment lack of oxygen supply to outer retina and, 564 retinopathy of, oxygen to relieve, 564 Retinal G protein-coupled receptor (RGR), in visual cycle, 724–725 Retinal ganglion cells (RGCs) activation of, Math5 and, 325 axon growth and regeneration of, 401–410 area of action of extracellular signals and, 402–403, 403f control of neuronal responsiveness to trophic peptides and, 404f, 404–405 dendrite growth ability and, 406–407, 407f developmental regulation of intrinsic growth and, 405–406, 406f extracellular signals inducing growth and, 401–402, 402t glaucoma and, 486–487, 487f intrinsic and extrinsic control of regeneration and, 409–410 mechanism of axon induction by extracellular signals and, 403–404 molecular changes underlying developmental loss in rapid axon growth ability and, 407–408 need for specific extrinsic signals for growth and, 401, 402f regeneration in mature visual system, 408–409 regulation of, 409–410 study of, 409, 409f study of regeneration and, 409, 409f axon outgrowth and pathfinding and, 379–386, 380f control of initial polarity of outgrowth within optic fiber layer and, 381–382, 382f

index

749

Retinal ganglion cells (continued) directed growth toward optic disc, 382–383, 383f dorsal-ventral differences in mechanisms regulating, 385–386 growth cones and guidance cues and, 379, plate 21 regulation of, 328 restriction of axons to optic fiber layer and, 379–381, 380f targeting to optic disc and exit from eye, 383f, 383–385 axonal exit from retina and, 391 cell death of, 333, 334 in glaucoma, 479, 481, 485–487, 487f, plate 33, plate 34 matching sizes of interconnected neuronal populations and, 335, 335f molecules involved in signaling, regulating, and executing, 337, 338 neurotrophic hypothesis of, 336 classes of, distinguishing, 189–190, 190f, 191f, 192 competence of, Math5 and, 324 crush injury of, plate 23 dendrites of, growth ability of, axon growth ability and, 406–407, 407f development of, 321–330, 322f developmental regulation of retinal ganglion cell subtypes and, 329 early regulatory events in, 322–324 gene regulatory network for, 321–322, 323f Math5 and, 324–325 regulation of Math5 expression and, 325–327 regulation of retinal ganglion cell differentiation downstream of Math5 and, 327–329 in vitro generation of retinal ganglion cells from embryonic stem cells and, 329–330 developmental timing and, 261 differentiation of, downstream of Math5, regulation of, 327–329 direction-selective calcium imaging of responses of, 598 genetic tagging to study responses of, 598 functional properties of, 192 development of, 194–197 generation and differentiation of, 193–194 generation from embryonic stem cells, in vitro, 329–330 genetic approaches for study of, 599 intrinsically photosensitive, 192, 211–212 morphology, ontogeny, and functional diversity of, 211–212, 212f phototransduction mechanism and, 213–214 target brain regions innervated by, 212f, 212–213 melanopsin-expressing, circadian photoentrainment and, 210–211 molecular markers for, 192–193

750

index

morphological properties of, plate 6 development of, 194, 195f, 196f of mouse compared to other mammalian species, 35, 37–38 ON and OFF pathway segregation and, 353–354, 355–360 mechanisms of, 358–360, 359f, 360f retinal synaptic circuitry development and, 354–357, 355f, 356f synaptic activity and, 357–358 synaptic circuitry development and, 355f, 355–357, 356f ON-center, alteration of visually and electrically evoked responses of, by GABACR-mediated feedback inhibition, 183–184, 184f pattern electroretinogram measurement of function of, 119–124, 120f correlates of visual behaviors and, 120–121, 121f in human clinical research, 124 limitations of, 124 methods for, 120 in mouse models of glaucoma, 122–124, 123f in study of central nervous system plasticity and degeneration, 121–122 phenotypes of, complex, altered outer retinal neurotransmission resulting in, 179 “pioneer axons” of, 389 projections of, 192, plate 6 albinism and, 397 axon divergence at chiasm and, 393–396 early development of, 389, 390f glia in projection pathway and, 390–391 guidance cues and, 391f, 391–393, plate 21 late phase of development of, 390, 390f middle phase of development of, 389–390, 390f new approaches for study of, 397, 398f regulatory genes in retina and chiasm important for chiasm patterning and, 396–397 techniques for studying, plate 22 retinal waves and. See Retinal waves spontaneous burst activities of. See Retinal waves subtypes of, developmental regulation of, 329 survival after lesion, 122, 123f YFP expression, dendritic ramification patterns of, plate 19 Retinal infections, mouse models of, 509 Retinal neovascularization Bcl-2 and, 293 gene therapy for, 607, 609–610 limitation by laser burns, 565 protection against, by degradation of photoreceptors, 565 Retinal network, of mouse compared to other mammalian species, 36f, 36–37

Retinal pigment epithelium (RPE), 637–639, 639f, plate 64 function of, 638 gene expression in correlation of retinal pigment epithelium gene expression and retinal vascularization and, 692–693 isolation of retinal pigment epithelium and, 688, 689f, 690f, 690–691 lipofuscin of, 539–544, 540f, 638 autofluorescence of, plate 44 inhibiting formation of fluorophores and, targeting visual cycle for, 543f, 543–544 Leu450Met variant in murine Rpe65 associated with, 542–543, 543f Rpe65−/− mice and, 539–540, 540f Stargardt macular degeneration and Abca4/Abcr null mutant mice and, 540–542, 541f relationship with photoreceptors, 639 visual cycle and, 721–722 Retinal progenitor cells (RPCs) differentiation of, 302 eye field transcription factor expression in, 299, 302–303 Math5 activation in, 325 Math5 repression in, 326–327 proliferation of control by retinal ganglion cell-secreted signaling molecules, 328–329 regulation of, 311 Retinal projections, ipsilateral and contralateral, plate 1 Retinal synaptic transmission, 175–184, 176f control of synaptic output of bipolar cells by GABACR-mediated inhibition and, 182–183 evoked response alteration of ON-center retinal ganglion cells by GABACRmediated inhibition and, 183–184, 184f in outer retina, 175–179 altered, complex retinal ganglion cell phenotypes resulting from, 179 postsynaptic mutants and, 177t, 178–179, 179f presynaptic mutants and, 177t, 177–178 retinal ganglion cell responses in mutants with outer plexiform layer presynaptic defects and, 179–181, 180f retinal ganglion cell responses in mutants with postsynaptic defects in depolarizing bipolar cell signaling and, 181 Retinal vasculature, plate 45 degenerate capillaries in, plate 46 development of, 285–289, 286f, 289f gene expression in correlation of retinal pigment epithelium gene expression and retinal vascularization and, 692–693

Retinal vasculature (continued) isolation of retinal vasculature and subsequent gene expression studies and, 688, 689f, 691–692 leukocyte adherence in retinal microvasculature of diabetic mouse and, plate 47 Retinal waves, 343–348 cellular mechanisms underlying, retinal development and, 343–344, 344f circuits generating, functional stages of, plate 17 gap junctions and, 346–347 measurement techniques for, 343 mediation of, 357 stage I, properties of, 347, plate 18 stage II mechanisms of, 345f, 345–346 properties of, plate 18 spatiotemporal properties of, 344–345, 345f stage III, properties of, 347 visual circuit development and, 348, 349f Retinaldehyde, 364 Retinaldehyde dehydrogenases (RALDHs), 364 RALDH2, 367, 367f RALDH3, 367, 368f colocalization with markers for neuronal plasticity, 368, 369f maturation patterns in postnatal cortex defined by, 369–370, 370f relationship between neurotrophin-3 and, 368–369, 369f retinoic acid colocalization with sites of expression of, 365f, 365–366 Retinitis, viral, 509 Retinoblastoma human susceptibility to, 311, 313 species-specific differences in susceptibility to, 313 Retinofugal projections, of mouse compared to other mammalian species, 39t, 39–40, 40f, 41f Retinogenesis, early regulatory events in, 322–324 Retinogeniculate pathway. See also Lateral geniculate nucleus (LGN) axon segregation in, in developing mouse, plate 24 development of, 415–426, 425f functional composition of developing pathway and, 419–420, 421f, 422 mechanisms underlying remodeling of connections and, 424 structural composition of developing pathway and, 418–419 Retinohypothalamic tract (RHT), circadian photoentrainment and, 209–210 Retinoic acid (RA), 363–374 actions in brain, 364–365 data bank searches and, 370–372 Allen Brain Atlas, 370–371, 371f Brain Gene Expression Map, 370 evaluation of, 372 dorsal telencephalon function and, 366–367

dorsal visual stream and, 373f, 373–374 photoreceptor development and, 304, 305 RALDH2 and, 367, 367f RALDH3 and, 367, 368f colocalization with markers for neuronal plasticity, 368, 369f maturation patterns in postnatal cortex defined by, 369–370, 370f relationship between neurotrophin-3 and, 368–369, 369f reporter mice and, 365f, 365–366 retinoid system and, 364 Retinoid flow in visual cycle, in visual cycle, 726–727 11-cis-Retinol dehydrogenase, in visual cycle, 725 Retinol-binding protein, in visual cycle, 725 Retinopathy of detachment, oxygen to relieve, 564 diabetic. See Diabetic retinopathy oxygen-induced, 553–554 of prematurity (ROP), 285–294, 642 mouse model for study of, 285, 289 scoring system for, 291 Retinopathy scoring system, 291 Retinorecipient nuclei, of mouse compared to other mammalian species, 40–45 Retinoscopy, streak, 75 RF/J strain, hearing and visual abilities in, 16 Rgr−/− mice, 724, 725 Rhodopsin gene, 63 Rhodopsin gene mutations, 651 RIIIS/J strain, tactile sensitivity in, 24 Rlbp−/− mice, 726 RNAi, inducible, in retina, plate 62 Rod(s). See also Photoreceptors bipolar cells of, ON and OFF pathway segregation and, 354 development of, Rb and, 313–317, 315f, 316f dysfunction of, cone damage due to, 566 gap junctions of, 167 GFP expression in, plate 63 of mouse compared to other mammalian species, 36 scaling of, 263 spatial vision and, 81–82 Rod monochromatism, gene replacement therapy for, 612–613 Rod pathways, proximal, gap junctions in, 167–169, 168f Rod visual cycle. See Visual cycle, rod Roscoe B. Jackson Memorial Laboratory, 13 Rpe65, Leu450Met variant in, reduced levels of retinal pigment epithelial lipofuscin associated with, 542–543, 543f Rpe65, photoreceptor response to light and, 574–575 RPE65 alleles, 638–639, 639f Rpe65 alleles, retinal responses to light exposure and, 650–651 Rpe65 knockout mice, as model for Leber congenital amaurosis, 611–612 Rpe65−/− mice, photosensitivity in, 214

Rpe65−/− mice, retinal pigment epithelium lipofuscin in, 539–540, 540f Rpgrip knockout mice, as model for Leber congenital amaurosis, 612 Rpgrip 1 mutations, 650 RPR65, 637 isomerohydrolase reaction and, 723–724 Rupture of lens cataract, 501 Rx, as eye field transcription factor, 299–300 Rx-Cre transgenic mice, 274 RXR-τ, opsins and, 305 Rx/rax homeobox transcription factor, 269

S Saccade(s), 87, 88, 99 Saccade-like rapid eye movements (SLREMs), 233 electrically induced, in mice, 235–238, 237f measurement of, 233–234, 234f spontaneous, in mice, 234f, 234–235, 236f Saccadic eye movements, 233–242 study methods for, 233 superior colliculus and, 233. See also Superior colliculus (SC) SB strain, mutation and effect in, 62t Scaling, of eye and cortex, 263–264 Schematic eye modeling, 73–74, 74f Schlemm’s canal aqueous humor outflow and, 131 inner wall cells of, 132, 132f Scotopic signal transmission, of mouse compared to other mammalian species, 36f, 36–37 Scotopic threshold response (STR), pattern electroretinography and, 124 Second messengers, retinal ganglion cell axon growth ability and, 408 Self-complementary AAV vectors (scAAV), 613–614 Senescence-accelerated mouse (SAM), 500 Senile cataracts, mouse models for, 500 Sequential model of evolution, 6–7, 7f Serial analysis of gene expression (SAGE), 662, 663, 687 Severe combined immunodeficiency (SCID), 506 Shh, control of retinal progenitor cell proliferation by, 328 SHIRPA test battery, 30 Shrew mice, 3–5, 8 Single-cell recordings, cones and, 139, 140f–142f Single-unit recordings, for ocular dominance plasticity assessment, 439 Sinusoidal stimuli, optokinetic reflex gain in response to, 91 Sip 1, lens development and, 277 Sip1, in cornea and lens development, 701 Site-specific recombination, Cre-lox recombinase system for study of, 599–600 Six3, in cornea and lens development, 700 Six homeodomain genes, as eye field transcription factors, 300

index

751

SJL/J strain anxiety-related behavior tasks and, 29 conditioned taste aversion in, 29 cued and contextual fear conditioning in, 27 hearing and visual abilities in, 15t, 16 mutation and effect in, 62t pattern discrimination in, 18 visual acuity of, 20 SKH2/J strain, auditory evoked brainstem response in, 22 Slit1, retinal ganglion cell axon guidance through visual pathway and, 392f, 392–393 Slit2, retinal ganglion cell axon guidance toward optic chiasm and, 392f, 392–393 Slit signaling, control of initial retinal ganglion cell axon outgrowth polarity in, 381–382, 382f Small eye artifact, 74 Smithies, Oliver, 35 SM/J strain pattern discrimination in, 18 tactile sensitivity in, 24 taste ability in, 23 visual acuity of, 20 Smooth pursuit movements, 87, 88 Snell, George D., 13 Sodium currents, in dorsolateral geniculate nucleus, 220, 220f Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), in proteomics, 713–714 Sonic hedgehog. See also Shh directed growth of retinal ganglion cell axons toward optic disc and, 383 eye field formation and, 301 optic nerve development and, 201 S-opsin, cone, expression of, 136, 138–139 Sorsby’s fundus dystrophy, gene therapy for, 609–610 Sox, cataracts and, 494 Sox1, lens fiber differentiation and, 278 Sox1, in cornea and lens development, 701 Sox2 in cornea and lens development, 701 lens development and, 276 Spacing rule, in horizontal cell mosaic, 148–150, 149f, 150f Spatial acuity, primary visual cortex and, 51–52 Spatial frequency (SF) threshold, of optokinetic reflex, measurement of, 113–115, 114f, 115f Spatial information, retinogeniculate inputs and, 221–223, 222f–224f Spatial regulation, of gene expression, using cell type–specific promoters, plate 58 Spatial resolution of mouse compared to other mammalian species, 38–39 in mouse eye, 73

752

index

Spatial vision illuminances and, 81, 81f rods and cones and, 81–82 Spectacle lenses, refractive errors induced by, 82 Spemann, Hans, 268 Spiny mice, 3–4, 5, 8 Sp1/Krüppel-like transcription factors, in cornea and lens development, 702 Spontaneous activity, developmental, postnatal retinal ganglion cell development and, 121–122, 122f SPRET/EiJ strain hearing and visual abilities in, 15t pattern discrimination in, 18 visual acuity of, 20 Staggerer mutant mice, olfactory ability in, 22, 23 Staphylococcus aureus infections, 507–508 Starburst amacrine cells (SACs) retinal waves and, 346 study approaches for, 598 Stargardt disease, 582, 723 in Abca4/Abcr null mutant mice, 540–542, 541f Stimulation. See also Environmental enrichment early-life, neuronal and behavioral development and, 450–451 electrical, retinal ganglion cell axon growth and, 404–405 tactile, plasticity and, 455, 456 Stimulus-selective response potentiation (SRP), 471–472, 472f, 473 Strain background, 65 Stratification patterns, Cre-lox recombinase system for study of, 601 Stratum album intermediale (SAI), 235 Stratum album profundum (SAP), 235 Stratum griseum intermediale (SGI), 235 burst generation in, 240–241, 241f cholinergic innervation of, 242 Stratum griseum profundum (SGP), 235 Stratum griseum superficiale (SGS), 43, 235 Stratum opticum (SO), 43, 235 Stratum zonale (SZ), 235 Streak infrared, 75 Streak retinoscopy, 75 Streptozotocin, diabetes induced by, 548–549, 549f Strong, Leonell C., 13 Sugar-induced cataracts, in mouse, 499 Sulik, Kathleen, 270 Superior colliculus (SC), 233 electrically induced saccade-like rapid eye movements and, 235–238, 237f lateral geniculate nucleus synaptic remodeling and, 433–434 layers of, 235 local circuits of, 238–242 burst generation mechanism and, 239–241, 241f cellular properties of, 238, 238f GABAergic, 239 interlaminar connections and, 239, 240f

neuromodulator actions and, 241–242 retinal projections to, 42f, 42–44, 43f Superoxide dismutase, diabetic retinopathy and, 553 Suprachiasmatic nucleus (SCN) circadian photoentrainment and, 209–210, 211, 212f, 212–213 retinal projections to, 40, 41, 212f, 212–213 SvEvTac. See 129/SvEvTac strain SWR/J strain, hearing and visual abilities in, 16 Synaptic circuitry, retinal, development of, ON and OFF pathway segregation and, 354–357, 355f, 356f Synaptic remodeling, 429–435 description of, 429 in lateral geniculate nucleus, 430f, 430–431, 431f activity and, 431–432 comparison with remodeling in other regions of visual system, 433–434 genes involved in, 434–435 plasticity in lateral geniculate nucleus and, 432–433 lateral geniculate nucleus synapses and, 429–430 Synaptic transmission, retinal. See Retinal synaptic transmission Synaptogenesis, defects in Rb-deficient retinas, 317–318, 318f

T T cells antigen-specific experimental autoimmune uveitis induced by adoptive transfer of, 515 migration to eye, 521 defects in selection of repertoire of, susceptibility to experimental autoimmune uveitis and, 518 deficient peripheral tolerance and, susceptibility to experimental autoimmune uveitis and, 518–519 Pseudomonas aeruginosa infections and, 507 Tactile sensitivity, measurement, 24 Tactile stimulation, plasticity and, 455, 456 Tail flick test, 24 TALE homeobox proteins, eye field transcription factor regulation by, 301 Targeted ablation, to assess role of photoreceptors, 595–596 Targeted gene manipulations, 594–595, 595f Targeted mutagenesis, 63, 65–66 Taste ability, measurement of, 23–24 Taste aversion, conditioned, 28–29 Tau-lacZ reporter, for study of retinal ganglion cells, 599 Taurine, photoreceptor development and, 304, 305 Tcm mutant, 501

Telencephalon, dorsal, retinoic acid and, 366–367 Temporal information, retinogeniculate inputs and, 223–225 Temporal resolution, primary visual cortex and, 51–52 Tetrodotoxin (TTX), lateral geniculate nucleus synaptic remodeling and, 434 Th1, experimental autoimmune uveitis and, 520 Th17, experimental autoimmune uveitis and, 520 Thalamocortical relay cells (TCs) calcium current of, 220 hyperpolarization-activated currents of, 221 intrinsic conductances of, modulation of, 226–227 potassium current of, 220–221 Thalamus, dorsal and ventral, visual cortex connections with, 46 Thanatins, cell death and, 337 Thermal nociception, measurement of, 24–25 Thy-1 fluorescent reporters under control of, 598 as retinal ganglion cell marker, 193 Thy-1-GFP-H line, for study of retinal ganglion cells, 599 Thymidine birth dating analysis, in retina, 302 Thyroid hormone receptor-β2 (TRβ2), opsins and, 305 Tilt, static, mouse response to, 92f, 92–93 Tilt vestibulo-ocular reflex (titlVOR), 92 Tilted mice, 101 TIMP3, gene therapy for Sorsby’s fundus dystrophy using, 610 Tissue inhibitor of matrix metalloproteinase1, herpes simplex virus infection and, 506 Toll-like receptors (TLRs) autoimmunity triggered by, 519–520 infections and, 508 Tonometry, 480 Tono-Pen tonometer, 480 Toxoplasma gondii infections, 509 Trabecular meshwork (TM), 131f, 131–132, 132f in pigment dispersion syndrome, 484 in primary open-angle glaucoma, 484 Transcallosal cells, binocularity and, 50–51 Transcription factors. See also specific factors cell death and, 338 in cornea and lens development, 699f, 699–703, plate 36, plate 66 homeobox, 699–701 non-homeobox, 702–703 photoreceptor development and, 304–305 retinal ganglion cell development and, 194 Transforming growth factor β (TGF-β) cell death and, 336 retinal vascularization and, 290–291 Transforming growth factor β1 (TGF- β1), retinal vascularization and, 288 Transgene insertion, 593–594, 594f

Transgenic mice, 14–15. See also specific transgenic mice in gene expression analysis, 692 for lens research, 272–273 location of transgene, 65–66 study of amacrine cells using, 597–598 study of role of gap junctions in bipolar cells using, 596–597 temporal control of gene expression in, Cre recombinases for, 626, 627f, plate 57 uses of, 65 Translational vestibulo-ocular reflex (transVOR), 92 Traumatic optic neuropathy, retinal ganglion cell axon regeneration in, 409, 409f Tα1–3RT τTG mice, olfactory ability in, 22, 23 Two-bottle test, of taste ability, 23–24 Two-dimensional electrophoresis, in proteomics, 714–715 Tyrosine hydroxylase (TH), primary congenital glaucoma and, 483 Tyrp1b, pigmentary glaucoma and, 483

U Unmyelinated region, of optic nerve, 201, 202f Usher’s syndrome, type 1b, 584 Uveitis, 513–523 endotoxin-induced, 513–515, 514f experimental autoimmune uveitis, pathogenesis of, critical checkpoints in, plate 40 experimental autoimmune uveoretinitis, 515–517 associated with anti-tumor response to melanoma antigen, 517 histology of, plate 39 in human lymphocyte antigen transgenic mice, 516 induced by active immunization with retinal antigen, 515, 516t, 517f induced by adoptive transfer of antigenspecific T cells, 515 induced by injection of antigen-pulsed dendritic cells, 516 in mice expressing a transgenic target antigen in the retina, 515–516 spontaneous, 516–517 immunotherapeutic approaches for, 522–523 infectious. See Infectious eye diseases mechanisms involved in, 517–522 antigen presentation to lymphocytes and, 519 deficient peripheral tolerance as, 518–519 effector cell migration, infiltration, and inflammatory cell recruitment as, 521 innate immune signals and, 519–520 natural resolution mechanisms as, 521–522

pathogenic effectors and, 520 T cell repertoire selection and, 518 Uveoscleral outflow pathways, 132–133

V Vacuolated lens mutant, 500 V-amacrine cells, 597 Vascular cell types, 639–643, 641f, 643f Vascular endothelial growth factor (VEGF) gene therapy for retinal neovascularization and, 609 herpes simplex virus infection and, 506 overexpression of, diabetic retinopathy and, 554 Vascular endothelial growth factor-A (VEGF-A) in angiogenesis, 640–641 oxygen-induced retinopathy and, 290, 291–292 retinal vascularization and, 286, 287 Vascular patterning, 642 Vascularization, 640, 641f Vasculogenesis, 285, 286 Vasogenesis, blocking of, by inhaled oxygen, 563 Vax1, optic nerve development and, 201 Vax1/2, chiasm patterning and, 396 Vegf, control of retinal progenitor cell proliferation by, 328, 329 Velocity storage integrator, 94–96, 95f Ventrolateral geniculate nucleus (vLGN), retinal projections to, 40, 41 Vergence movements, 87, 88 Vestibular compensation, 99 Vestibulo-ocular reflex (VOR) angular, 88–91, 89f, 90f gain and response axis of, adaptation of, 96–98, 97t linear, 88 tile (orienting), 92 translational, 92 visual, 89f, 89–90 Vibrissae sensitivity, measurement, 24 Vimentin, cataracts and, 498 Viral retinitis, 509 Virtual optokinetic reflex system, 112–113, 114f Virtual optokinetic system, 20 Vision lack of, environmental enrichment and, 453–455, 454f in tasks evaluating higher order cognitive functions, 25 Visual ability measures of of brightness/visual detection, 16, 17f, 18 gross, 16 of pattern discrimination, 17f, 18, 19f of visual acuity, 18, 20, 21f in mice, 15t, 16–20 Visual acuity of mouse compared to other mammalian species, 35 operational definition of, 108 of small vertebrate eyes, 73

index

753

Visual behavior, correlates of ocular dominance plasticity and, 447 Visual circuits, development of, retinal waves and, 348, 349f Visual cortex, 245–252 binocular mapping ocular dominance in, plate 26 retinotopic mapping of, plate 25 callosal connections of, 46–47, 47f connections of, 248–251 areal hierarchy of, 251, 251f consolidation of, environmental enrichment and, plate 29 between different cortical areas, 249, 250f, 251 with dorsal thalamus, 46 subcortical inputs and, 248 within V1, 248–249 with ventral thalamus and mesencephalon, 46 Vi, topographic maps of, plate 8 corticotectal cells of, physiology of, 47–48 cytoarchitectonic fields of, 245, 246f extrastriate, in mouse compared with other mammalian species, 51–52, 52f extrastriate lateromedial area intracortical connections in, plate 9, plate 10 interareal feedforward and feedback connections in, laminar organization of, plate 11 intrinsic associational corticocortical connections of, 46 long-range associational corticocortical connections of, 46 maps of, plate 7 of mouse compared to other mammalian species, 45–52 anatomy and, 45f, 45–47 binocularity and, 49–51, 50f physiology and, 47–59, 48t spatial acuity and temporal resolution and, 51–52 nonoriented cells of, physiology of, 47 orientation selectivity of, 48–49 relationship between cytoarchitectonic areas and topographic maps of, 248 topographically defined areas of, 246f, 246–248, 247f visuotopic organization of, 45–46 Visual cortical plasticity environmental enrichment and, 449–459 early-life stimulation effects on neuronal and behavioral development and, 450–451 neural consequences of environmental enrichment and, 450 visual system development and, 451–459, 452f, 454f, 455f, 457f, 459f

754

index

experience-dependent, 465–473 adult ocular dominance plasticity and, 472–473, 474f mechanisms of ocular dominance shift in mice and, 468–471 monocular deprivation in juvenile mice and, 468, 469f, 470f stimulus-selective response potentiation and, 471–472, 472f visually evoked potential recording technique and, 466, 467f, 468 Visual cycle, 721–729 cone, 727, 728f, plate 69 inhibition of formation of retinal pigment epithelial lipofuscin fluorophores and, 543f, 543–544 proteomic analysis of, 727–729 of interacting components, 727–729 retinoid, 637 rod, genetic analysis of, 721–727, 722f, plate 68 all-trans-retinol dehydrogenase and, 722–723 ATP-binding cassette A4 and, 723 cellular retinaldehyde-binding protein and, 726 cellular retinol-binding protein type 1 and, 725 11-cis-retinol dehydrogenase and, 725 interphotoreceptor retinoid-binding protein and, 726 isomerohydrolase and, 723–724 lecithin:retinol acyltransferase and, 723 retinal G protein-coupled receptor and, 724–725 retinoid flow in visual cycle and, 726–727 retinol-binding protein and RBP receptor and, 725 Visual detection, measurement of, 16, 17f, 18 Visual evoked potentials (VEPs) for ocular dominance plasticity assessment, 439–440, plate 31 pattern electroretinographic measurement of, 120–121, 121f recording of, for assessing visual cortical plasticity, 466, 467f, 468 Visual field, of mouse compared to other mammalian species, 39t, 39–40, 40f, 41f Visual function, measuring gains and losses of, behavioral methods for, 116, 116f Visual pathways, organization of, plate 1 Visual phenotypes, screening of, behavioral methods for, 115–116 Visual stimulation, ON and OFF pathway segregation and, 357–358

Visual system development of acceleration by environmental enrichment, plate 28 consolidation of visual cortical connections and, plate 29 environmental enrichment and, 451–459, 452f, 454f, 455f, 457f, 459f, plate 29, plate 30 special features in mouse affecting research, 66–67 Visual thalamus. See Lateral geniculate nucleus (LGN) Visual vestibulo-ocular reflex (VVOR), 89f, 89–90 Visual water box, 16, 17f, 18, 20 Visual water task, for measuring visual perception, 110–111, 111f, 112f Visual-somatosensory interaction, 24 Vitamin A. See also Retinoic acid (RA) deficiency of, 364 excess of, 364 role in vision, 363 uptake from RBP, 725 Vogt-Koyanagi-Harada disease, 516 von Frey filament test, 24 Vpac2r, circadian photoentrainment and, 214

W Water T-maze, modified, 16, 17f Whole-cell voltage-clamp recordings, for retinal wave measurement, 343 Williams syndrome, visual cortical functions in, 245 Wnt signaling, eye field transcription factor regulation by, 300–301 Wolbachia, 508

X X-linked juvenile retinoschisis (XLRS), gene replacement therapy for, 611 X-linked recessive familial exudative vitreoretinopathy (XLEVR), 528

Y Yamamoto, Yuhiro, 270 Yerkes apparatus, 16, 17f

Z Zernike coefficients, 80 Zic2 retinal ganglion cell axon divergence at chiasm and, 394f, 395 as retinal ganglion cell marker, 193 Zonular apparatus, 129, 130f ZRDCT strain, 269

17 (M)

18b

17 (B) 18a

dLGN

vLGN

IGL

1 mm

V/T

V/T

D/N

D/N

Monocular visual field

Monocular visual field

Binocular field

Plate 1 Schematic diagram showing the organization of the ipsilateral and contralateral visual pathways in mice. Blue and red indicate fibers and regions representing the left and right eyes, respectively. Purple indicates binocular regions. Ipsilateral projections arising from the ventrotemporal retina terminate in dorsomedial dLGN. Contralateral retinal projections fill the rest of the dLGN. The locations of other retinorecipient nuclei in the dorsal

thalamus, the intergeniculate leaflet (IGL), and ventral LGN (vLGN) are also shown. The dLGN projects topographically to primary visual cortex (area 17). The medial two-thirds of area 17 receives monocular input from the contralateral eye (17M). The lateral one-third receives binocular inputs (17B). Adjacent to area 17 laterally is area 18a; area 18b is medial. (See figure 3.3.)

Plate 2 Disposition and numerosity of mouse cones. The image superimposes confocal DIC and fluorescence image of a frozen section of the photoreceptor and RPE cell layer of a mouse retina.

The mouse from whose eye the section was made expressed EGFP under the human M/L cone promoter (Fei and Hughes, 2001). (See figure 10.1.)

Plate 3 Dim-flash responses of single mouse cones. Average dimflash response data of S-dominant (A and C) and M-dominant (B and D) cones, stimulated with either UV (361 nm) or midwave (501 nm) flashes, are shown. In each case, analysis of the action spectrum reveals that the UV flash activates only the S-opsin in the cone, while the midwave flash activates M-opsin (the two being coexpressed). F, The averaged responses in A and B are compared

with those of the cones of Gta −/− and Nrl −/− mice and with responses from wild-type mouse rods recorded under the same conditions (Nikonov et al., 2006). The midwave background light required to suppress rod responses in the wild-type mouse is not needed in experiments with Gta −/− and Nrl −/− cones and is at least partially responsible for the faster response kinetics of the wild-type cones. (See figure 10.5.)

Plate 4 Confocal image of a whole mount mouse retina in which horizontal cells are revealed with antibodies against calbindin D (red signal), while their axonal complexes are labeled with antineurofilament antibodies (green staining). (See figure 12.5.)

A

B

C

Plate 5 A, β-Gal reporter in transgenic mouse retina indicates that Cx36 is expressed by photoreceptors in the ONL and by bipolar cells and amacrine cells in the INL. Small arrowheads indicate photoreceptor somata; large arrowheads indicate somata of an AII amacrine cell and bipolar cells. B, Transverse view of immunolabeling of the wild-type mouse retina for Cx36. Labeling is confined

to the plexiform layers, consistent with the known locations of gap junctions between retinal neurons. C, Immunolabeling for Cx36 is absent in the Cx36 KO mouse retina. (See figure 13.1.) (A, Adopted from Deans et al., 2002, with permission. B and C, Adopted from Deans et al., 2001, with permission.)

Plate 6 Morphology and projections of mRGCs in mouse. A, Retina flat mount stained with a polyclonal antimouse melanopsin antibody showing specific staining of dendrites, somata, and axons of a small subset of retinal ganglion cells. B, Schematic drawing showing direct axonal projections of mRGCs to several brain regions. (Results from Hattar et al., 2006, are redrawn here.) Brain regions receiving significant projections are represented in large, bold letters. AH, anterior hypothalamic nucleus; IGL, inter-

geniculate leaflet; LGd, lateral geniculate nucleus, dorsal division; LGv, lateral geniculate nucleus, ventral division; LH, lateral hypothalamus; LHb, lateral habenula; MA, median amygdaloid nucleus; OPN, olivary pretectal nucleus; PAG, periaqueductal gray; PO, preoptic; pSON, perisupraoptic nucleus; SC, superior colliculus; SCN, suprachiasmatic nucleus; SPZ, subparaventricular zone. (See figure 17.2.)

Plate 7 Different maps of mouse visual cortex. A, Flat map of cytoarchitectonic areas (black outlines) in the left hemisphere of mouse cerebral cortex published by Caviness and Frost (1980). Red shaded regions represent schematic outlines of visuotopically organized areas, identified by Wang and Burkhalter (2006). Note that areas 18a and 18b contain multiple visuotopic areas. B, Flat map of cytoarchitectonic areas of the left mouse cerebral cortex constructed by David C. Van Essen by unfolding coronal sections taken from the atlas of Paxinos and Franklin (2001). Red outlines represent schematic borders of visuotopically defined areas identified by Wang and Burkhalter (2007). Note that areas V2L and

V2ML contain multiple visuotopically organized areas. C, Visuotopic organization of the left mouse visual cortex derived by mapping of receptive fields, published by Wagor et al. (1980). In this map, cytoarchitectonic area 18a contains areas V2 and V3 and cytoarchitectonic area 18b contains the rostral and caudal medial visual areas, Vm-r and Vm-c. D, Area map of left mouse visual cortex derived by topographic mapping of V1 connections and receptive field mapping (Wang and Burkhalter, 2007). Blue shading represents the distribution of callosal connections in superficial layers. (See figure 20.1.)

Plate 8 Topographic maps of V1 connections in mouse visual cortex. Representation of azimuth in extrastriate visual cortex is shown in horizontal sections of left occipital cortex. The maps were generated by making three simultaneous injections of fluororuby (FR, red), fluoroemerald (FE, green), and biotinylated dextran amine (BDA, yellow) into V1, followed by triple-anterograde tracing of intracortical connections. A, Darkfield image showing heavy myelination in primary visual cortex (V1) and the barrel field of primary somatosensory cortex (S1). Arrowheads indicate myeloarchitectonic borders. Arrows indicate injection sites in V1.

B, Fluorescently labeled axonal connections after injections of FR, FE, and BDA at different nasotemporal locations (azimuth) of the upper visual field representation in V1. Dashed lines indicate areal borders, which were determined by mapping inputs from the perimeter of V1. Solid lines indicate myeloarchitectonic borders. C, Overlay of BDA-labeled axonal projections shown in B and bisbenzimide-labeled callosally projecting neurons (blue). D, Higher magnification image of axonal labeling shown in area A (inset in B). A, anterior; L, lateral; M, medial; P, posterior. Scale bar = 1 mm (A–C), 0.1 mm (B, inset), and 0.3 mm (D). (See figure 20.2.)

Plate 9 Intracortical connections of the extrastriate lateromedial area (LM) in mouse visual cortex, shown in tangential sections through the flattened posterior cerebral cortex. A, Fluorescence image showing the distribution of retrogradely labeled callosal connections. Dashed lines represent the myeloarchitectonic borders of V1, S1, and RSA. B, Darkfield image of biotinylated dextran amine

(BDA)–labeled axonal connections of area LM. Asterisk indicates the injection site. Note the strong connections to areas POR and 36p. C, Superimposition of BDA-labeled LM connections (gold) with callosal connections (blue somata). A, anterior; L, lateral; M, medial; P, posterior. Scale bar = 1 mm in all images. (See figure 20.3.)

Plate 10 Intracortical connections of the extrastriate lateromedial area (AL) in mouse visual cortex, shown in tangential sections through the flattened posterior cerebral cortex. A, Fluorescence image showing the distribution of retrogradely labeled callosal connections. Dashed lines represent the myeloarchitectonic borders of V1, S1, and RSA. B, Darkfield image of biotinylated dextran amine

(BDA)–labeled axonal connections of area AL. Asterisk indicates injection site. Note the strong connections to areas PL, A, and Cg/RS. C, Superimposition of BDA-labeled LM connections (gold) with callosal connections (blue somata). A, anterior; L, lateral; M, medial; P, posterior. Scale bar = 1 mm in all images. (See figure 20.4.)

Plate 11 Laminar organization of inter-areal feedforward and feedback connections in mouse visual cortex labeled by anterograde tracing with BDA. A, Coronal section showing feedforward axons that originate from the lower area V1 and terminate in the higher extrastriate area LM. The projection column includes layers

2/3 to 6, and inputs to layer 1 are sparse. B, Coronal section showing feedback axons that originate from area LM and terminate in V1. The projections to layer 1, 2/3, and 5 are dense, whereas inputs to layer 4 are sparse. Scale bar = 0.2 mm. (See figure 20.5.)

Plate 12 Cre expression patterns in three different transgenic strains: Le-Cre (A–C), MLR10 (D–F ), and MLR39 (G–I ). Cre expression in whole-mount (A and B ) or tissue sections (C–I ) is indicated by blue (B and D–I ) or purple (A and C) staining following histochemical detection of a Cre-activated reporter allele. Arrows in

A and B indicate Cre expression in the developing pancreas of LeCre mice. Developmental time points are indicated. (See figure 22.2.) (Adapted from Ashery-Padan et al., 2000, and Zhao et al., 2004.)

Plate 13 Visualization of vessels in P17 retinal flat-mount preparations stained with GS-lectin from a mouse model of OIR. A, Low-power (magnification ×25) image of the retina shows areas of central vaso-obliteration, as well as neovascular tufts. B, Higher

magnification (×200) shows neovascular tuft formation at the transition zone between vascular and central avascular retina. C, At even greater magnification (×400), multiple neovascular tufts are evident. (See figure 23.4.)

Plate 14 Developing and mature mouse retinas. A, An embryonic retina at E12.5. B, A postnatal retina at P16. Nuclei are labeled in red with propidium iodide and represent the positions of the cell soma. Neurite processes are labeled in green and blue with antineurofilament antibody and anticholine acetyltransferase antibody, respectively, and represent axons and dendrites. C, Schematic representation of the mature retina. RGCs and displaced

amacrine cells are found within the ganglion cell layer (GCL). Intermediate neurons (horizontal, bipolar, and amacrine) are found within the inner nuclear layer (INL), and rod and cone photoreceptor cells are found within the outer nuclear layer (ONL). IPL and OPL are inner plexiform and outer plexiform layers where axons and dendrites synapse. RGC, retinal ganglion cell. (See figure 26.1.)

Plate 15 A gene regulatory network model for RGC development. Genes are depicted such that the right side of the bent arrow for each gene indicates the gene product and the left side indicates the transcriptional control region. Solid lines connecting genes indicate established upstream-downstream relationships, and dashed lines suggest inferred relationships. Downward-pointing arrows indicate gene activation, and downward perpendicular lines indicate gene repression. In most cases the connections have not been shown to be direct. Genes boxed in yellow represent regulators of general retinal competence and RPC proliferation; genes boxed in orange are proneural bHLH genes associated with establishing competence in

RPCs for specific retinal cell fates. The light brown-purple box represents the general repression of RGC genes hypothesized to be mediated by the neural differentiation transcriptional repressor NSRF/REST (Mu et al., 2005b); the blue and purple-blue boxes represent genes encoding RGC-specific upstream (blue) and downstream (purple-blue) transcription factors. Genes boxed in green are those that encode proteins associated with RGC maturation and function. Genes boxed in purple encode secreted signaling molecules. In some cases only representative examples are shown for each box. A more detailed description can be found in Mu et al., 2005b. (See figure 26.2.)

Plate 16 Segregated expression of Math5 and Pou4f2 in retinogenesis. The image represents an E14.5 retina from a knock-in mouse in which a gene encoding an HA-epitope-tagged Math5 replaces the endogenous Math5 allele. Math5-HA expression is

detected by immunostaining with an anti-HA antibody (red). Pou4f2 expression is detected by immunostaining with an antiPou4f2 antibody (green). Yellow staining shows the overlap in expression of Math5 and Pou4f2. (See figure 26.3.)

Plate 17 Three functional stages of retinal wave–generating circuits. A, Schematic of circuits that mediate retinal waves. (Modified from Catsicas and Mobbs, 1995.) B, Summary of development of the synaptic circuitry that mediates waves in mice. Each color corresponds to a different wave-generating circuit. Yellow corresponds to non-nAChR circuitry, which mediates the nonpropagating events in embryonic mice. There is pharmacological evidence that stage I waves in other species are mediated by gap junctions, but this has not been directly demonstrated in mouse retina. In addi-

tion, it is not known which gap junction–coupled networks mediate stage I waves. Red corresponds to stage II circuits, which require activation of nAChRs. Stage II waves are initiated and propagate through a network of starburst amacrine cells. Blue corresponds to stage III circuits, which require activation of ionotropic glutamate receptors. The source of stage III wave initiation and the location of the horizontal coupling that drives coordinated release of glutamate during this stage are not yet known. (See figure 28.1.) (Modified from Bansal et al., 2000.)

Plate 18 Calcium imaging reveals spatial and temporal properties of stage I and stage II waves. A, Time evolution of a single stage II retinal wave visualized with fluorescence imaging of the calcium indicator fura-2. Decreases in fura-2 fluorescence associated with the increased calcium evoked by waves are shown at successive 0.5 s intervals. The final frame represents the total area of tissue covered by a single wave. B, Retinal waves of embryonic day 17 (E17) and postnatal day 2 (P2) retinas. Each frame summa-

rizes 90 s of activity in control ACSF (top row) and in 100 μM d-tubocurarine, a general nAChR antagonist (bottom row). Gray background represents the total retinal surface labeled with fura2AM. Each color corresponds to individual domains, with a colorcoded time bar below each frame to indicate the time of occurrence of each wave. Scale bar = 100 μm. (See figure 28.2.) (Modified from Bansal et al., 2000.)

Plate 19 Dendritic ramification patterns of YFP expressing RGCs in the IPL can be determined using confocal microscopy from Thy1-YFP transgenic mouse retina. A, View from vitreal side of a flat-mounted retina harvested from a Thy1-YFP-expressing mouse. B, Enlarged view of the area inside the box in A. Axons from individual RGCs cross the retina from each soma to the optic nerve head. C, Four frames taken from a representative stack of confocal images of a bistratified RGC showing the soma and axon, the dendrites ramified in sublamina b (blue), the dendrites ramified in sublamina a (green) of the RGC, and immunolabeling of dopami-

nergic amacrine cells (red). D, A stacked image of the same cell as shown in panel C. E, The 90° rotation view of the cell in D. Three dashed lines indicate the inner border of the IPL, the boundary of sublaminae a and b, and the outer border of the IPL. F, Normalized pixel intensity of the dendrites of each frame (open circles) plotted as a function of IPL depth of the cell in panel C. The data were fitted with two Gaussian distributions (green and blue lines). Doublearrow lines indicate widths. Single arrows indicate the locations of the two peaks of dendritic density. (See figure 29.3.)

Plate 20 The anatomical locations of the medial RALDH3 band are indicated for P15 in the form of a color code, in which prefrontal includes the prelimbic and medial orbital cortices; cingulate includes areas 1 and 2 of the cingulate cortex; motor designates mostly the secondary motor cortex, in addition to a small caudal part of the

primary motor cortex; parietal includes the medial and lateral parietal association cortices; and medial extrastriate extends from a location where the rostral extrastriate region abuts the parietal association cortex all the way caudally, an elongated region also called area 18b or V2M (Wagner et al., 2006). (See figure 30.4.)

RGC cell bodies and axons Netrin-1/EphA4 EphB2/B3 and BMPR1 CSPG Slit1 Slit2 Sema5A Hs2st Hs6st1 Ephrin-B2–expressing radial glia cells

Plate 21 Cues that guide RGC axons through the visual pathway. Top, RGC axons (blue) express many receptors for a number of cues in the retina that guide them toward the optic disc and out of the retina. Bottom, A variety of cues surrounding the optic nerve,

chiasm, and tract help to funnel RGC axons properly through the ventral diencephalon toward their final targets, the LGN and SC. ODAPs, optic disc astrocyte precursor cells. (See figure 32.2.)

Plate 22 Important techniques for studying RGC projections in the developing mouse visual system. A, DT and VT retinal explants plated near a border of 0.5 μg/mL ephrin-B2 (red). Axons from DT explants project into the ephrin-B2 region, while VT axons are clearly inhibited. B, Time-lapse imaging can be used to study RGC growth cone behavior in real time as they encounter a border, in this case ephrin-B2. Arrowheads highlight two growth cones, one of which projects into the ephrin-B2 region and the other of which is repelled and travels parallel to the ephrin-B2 border. C, Diagram

depicting in utero retinal electroporation. Briefly, DNA is injected into embryonic retina. Current is passed through electroporation paddles, and embryos are allowed to survive for several days. Embryos are perfused, the retina is collected, and heads are cryosectioned. In this case, GFP-positive cells can be seen in the retina, and many GFP-positive RGC projections are visible through the optic nerve, chiasm, and tract. Red denotes neurofilament. (See figure 32.5.) (All images courtesy of T. Petros and C. Mason.)

Plate 23 After an optic nerve crush injury, RGC axons were labeled with an intravitreal injection of fluorophore-conjugated cholera toxin B. The bright axons (green) failed to regrow across

the lesion site (arrows) after 8 days in vivo. Optic nerve nuclei are counterstained with a nuclear dye, DAPI. (See figure 33.6.) (Y. Duan and J. L. Goldberg. [2008]. Unpublished data.)

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Plate 24 Retinogeniculate axon segregation in the developing mouse. A, Anterograde transport of CTβ conjugated to Alexa Fluor 594 (red) labels contralateral eye projections, and anterograde transport of CTβ conjugated to Alexa Fluor 488 (green) labels ipsilateral eye projections. Panels from left to right depict red and green fluorescence labeling of the same section of LGN. Adjacent to these are the superimposed fluorescent pattern and corresponding pseudo-colored image, where pixel intensity is assigned a single value above a defined threshold. Pixels that contain both red and green fluorescence are considered areas of overlap and are represented in yellow. Scale bar = 100 μm. B, Pixel intensity analysis reveals degree of eye-specific segregation in the developing LGN. Top, Scatterplots of pixel intensity for a single section through LGN at P3 and P28. Each point represents a pixel in which the fluorescence intensity of the ipsilateral projection is plotted against the intensity of the contralateral projection. At P3, when projections overlap, pixel intensities show a positive correlation. At P28, pro-

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jections are segregated and pixel intensities show a negative correlation. Bottom, Corresponding R distributions of pixel intensity. For each pixel the logarithm of the intensity ratio (R = log10 FI/FC) is plotted as a frequency histogram (bin size = 0.1 log units). A narrow R distribution (P3) shows an unsegregated pattern; a wide one (P28) shows a segregated pattern. C, Summary plots showing the spatial extent of retinal projections (left) and the variance of R distributions (right) at different ages. Left, Percent area in LGN occupied by contralateral, ipsilateral, and overlapping terminal fields at different ages. Each point represents the mean and SEM for a group of same-aged animals. Note that ipsilateral projections and the degree of overlap recede between P3 and P12. Right, Mean and SEM variance values obtained from R distributions. Changes in spatial extent are accompanied by a parallel increase in variance and reflect a progressive increase in the degree of eye-specific segregation between P3 and P12. (See figure 34.2.) (Adapted from JaubertMiazza et al., 2005.)

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Plate 25 Retinotopic mapping of mouse binocular visual cortex using optical imaging of intrinsic signals. A, Schematic illustration of primary visual pathway in the mouse, depicting the location of the dorsal lateral geniculate nucleus (dLGN) and the primary visual cortex (V1), with the binocular zone (bV1) located laterally. B, Top, Cortical blood vessel pattern as imaged through the skull. Bottom, Arrangement of grating stimuli used to map the central visual field. Color denotes stimulus position. C, Individual activity maps dis-

playing responses to the 3 × 3 stimulus grid depicted in B are presented separately to the two eyes. The coordinates of the yellow cross are fixed in each map. D and E, Color-coded maps of the combined responses to contralateral (D) and ipsilateral (E) eye stimulation superimposed on the cortical blood vessel pattern, revealing the extent of the binocular zone. Color indicates stimulus position eliciting the strongest response at each pixel. Scale bars = 0.5 mm. (See figure 36.1.)

F

Plate 26 Mapping OD in mouse binocular visual cortex by twophoton calcium imaging. A, Examples of visually evoked calcium transients (fluorescent change, ΔF/F) recorded from two different neurons in the binocular visual cortex loaded with the calcium indicator dye OGB-1AM. Thin traces show individual responses to stimulation of each eye; thick traces are average responses to eight stimulus presentations. Stimulation periods are indicated by gray bars. B, Response maps (ΔF) for the contralateral (left panel) and ipsilateral (right panel) eye 230 μm below the pial surface in a normal mouse. C, A cell-based OD map computed from the single-eye

response maps in B. Individual neurons are color coded by the OD score, as indicated by the labels in D. OD score of 1 or 0 denotes exclusive response to contralateral or ipsilateral eye stimulation, respectively, and a value of 0.5 indicates an equal response to both eyes. D, Overlay of cell-based OD maps at different depths for a normal mouse (left, four depths, 190–290 μm), after a 5-day contralateral MD (center, six depths, 195–410 μm), and after a 5-day ipsilateral MD (right, two depths, 200 and 225 μm). Note weak clustering of cells with similar OD values in the normal mouse. Scale bars = 50 μm. (See figure 36.3.)

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Plate 27 OD plasticity in adult visual cortex assessed with intrinsic signal imaging and multielectrode recordings. A and B, Ipsilateral and contralateral eye responses in the same mouse before (A, P80, contralateral/ipsilateral ratio = 2.21) and after 6 days of MD (B, P92, contralateral/ipsilateral ratio = 1.09). Stimulus arrangement as in figure 36.2A. C, Ratio of contralateral to ipsilateral eye response strength after 6–7 days of MD in normal adult mice plotted against age shows no significant correlation. R2 = 0.12, P = 0.24. D, Contralateral/ipsilateral ratio values from repeated experiments in adult control animals (gray, interimaging period

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1–3 weeks) and in deprived adult animals before and after 6 days of MD in the contralateral eye (black). E, Functional map of the binocular region (green corresponds to the ipsilateral eye representation), used for targeting electrode penetrations (circles). Scale bar = 0.5 mm. F, Distribution of contralateral/ipsilateral ratio values from all recording sites in adult control (2.99 ± 0.28, mean ± SEM, 324 recording sites, 4 mice) and deprived (1.05 ± 0.12, 260 recording sites, 5 mice) animals, showing strong OD shifts in response to MD. Each color represents a different animal. Horizontal lines indicate mean group values. (See figure 36.4.)

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Plate 28 Acceleration of visual system development by environmental enrichment. A, EE accelerates the maturation of visual acuity. Visual acuity of non-EE (green) and EE (red) rats has been assessed by means of visual evoked potentials (VEPs) recorded from the binocular portion of the visual cortex at different ages during postnatal development. VEP acuity is normalized to the acuity value at P44–P45 and is plotted as a function of age for each experimental group to show the leftward shift of the curve for EE animals. B, Higher levels of BDNF and GAD65/67 expression in EE pups. ELISA and Western blot analysis have been used to measure, respectively, BDNF and GAD65/67 protein levels in the visual cortex of EE and non-EE animals at different postnatal ages. Data are plotted as percentage of variation between the two groups,

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with positive values indicating higher levels in EE mice. C, Accelerated CRE-mediated gene expression development in the visual cortex of EE mice. C1, Examples of brains at different ages (P10–P30) from CRE-lacZ transgenic mice reared in non-EE or EE conditions. X-gal histochemistry has been used to reveal the occurrence of CRE-mediated gene expression (blue precipitate). C2, Quantification of the density of X-gal-positive cells for non-EE (green) and EE (red) mice at the indicated ages. Fields were chosen to sample layers II–VI of the binocular visual cortex. CREmediated gene expression is developmentally regulated in both groups, but its peak is accelerated in EE mice. (See figure 37.1.) (A, Modified from Landi et al., 2007b. B and C, Modified from Cancedda et al., 2004.)

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Plate 29 Environmental enrichment promotes consolidation of visual cortical connections in dark-reared rats. A, Schematic diagram of the dark-rearing (DR) protocol combined with EE. Newborn rats were reared in complete darkness from P0 until P50. Together with DR, they were maintained, starting from P18, in either non-EE or EE conditions. Rats were then subjected to 1 week of MD, in normal light conditions. B and C, Normal closure of the critical period for MD in EE rats. B, Summary of MD effects in all DR animals. The OD distribution of each animal has been summarized with the contralateral bias index (CBI = {[N(1) − N(7)] + 1/2[N(2/3) − N(5/6)] + N(Tot)}/2N(Tot), where N(tot) is the total number of recorded cells and N(i) is the number of cells in class (i) (open diamonds denote individual data; circles denote mean of the group ± SE). CBI scores in DR-non-EE + MD rats differ from those in DR-EE + MD rats, which do not differ from those in

normal adults (shaded rectangle). C, Cumulative fractions for OD scores. For each cell, an OD score was computed as {[Peak(ipsi) − baseline(ipsi)] − [Peak(contra) − baseline(contra)]}/{[Peak(ipsi) − baseline(ipsi)] + [Peak(contra) − baseline(contra)]} (Rittenhouse et al., 1999). This score is −1 for class 1 cells, +1 for class 7 cells, and around 0 for class 4 cells. Only the curve for DR + non-EE MD animals significantly differs from that in normal rats. D, Examples of staining for Wisteria floribunda agglutinin (which labels perineural nets, green) and NeuN (neuronal marker, red) in Oc1B of a normal rat, a DR-non-EE rat, and a DR-EE rat at P50. The decrease caused by DR in the number of perineural net– surrounded neurons is reduced in EE-DR animals. Calibration bar = 100 μm. (See figure 37.2.) (Modified from Bartoletti et al., 2004.)

EE and visual system development: A three-phase interpretive model

I) Prenatal maternal influence (embryonic life) Placental exchanges II) Postnatal maternal influence (birth-weaning) Possible factors: IGF-I Observed changes: increased IGF-I levels in the RGC layer Accelerated dynamics of natural RGCs’ death

Increased maternal care levels

Possible factors: IGF-I, BDNF and Inhibitory system development Observed changes: increased cortical BDNF and GAD expression; increased retinal IGF-I and BDNF expression; accelerated RGC dendritic stratification; trigger for later events (accelerated development of retinal acuity, partial acceleration of visual acuity development)

Plate 30 Model of environmental enrichment effects on visual system development and plasticity. Shown is an interpretive framework for understanding the data on EE influence on the developing visual system. The effects elicited by EE on visual system development and plasticity are due not only to changes in the levels of sensory visual stimulation but also to very early factors activated even in the absence of vision. The available data support a model in which three distinct temporal phases during pup development are differently controlled by the richness of the environment: a

III) Direct environmental influence (weaning-adult age) Enhanced sensory-motor stimulation

Possible factors: IGF-I, BDNF, PNN and inhibitory system Observed changes: increased cortical IGF-I expression; prevention of dark rearing effects; precious closure of the CP for OD plasticity; acceleration of visual acuity development; increase of adult visual cortical plasticity

prenatal phase in which the mother mediates the influence of the environment through placental exchanges with the fetus, an early postnatal phase in which higher levels of maternal care in EE stimulate the expression of experience-dependent factors in the visual system, and a third (and final) phase in which the autonomous interaction of the developing pup with the enriched environment further promotes the maturation of visual functions (see the text for details). (See figure 37.5.)

Plate 31 The extended duration VEP recording technique is a reliable method to assess OD plasticity in mice. A, Representation of VEP recording setup. A mouse previously implanted with recording electrodes in Oc1 is placed in a restraint apparatus in front of a computer monitor displaying visual stimuli. Mice are fully awake and alert during all recording sessions. B, Schematic diagram of the mouse visual system. Input from the stronger contralateral eye arrives via the LGN to the monocular and binocular zones of V1, whereas input from the weaker ipsilateral eye projects only to the small binocular zone. C, Current source density profile of VEPs for an adult mouse. Binocular VEP depth profile is shown in the left panel, with the corresponding cortical layers designated by arrowheads. Middle panel shows the CSD profile. Current sinks are nega-

tive-going and shaded black. The color image plot in the right panel is an interpolation of CSD traces. The earliest latency current sink is observed in layer 4, followed by longer latency sinks in layers 2/3 and 5. Cold and warm colors represent current sinks and sources, respectively. D, Typical VEP responses obtained during the three viewing conditions used in all experiments. Each trace is an average of 100 presentations of a reversing (1 Hz) sinusoidal grating. E, VEPs recorded from awake mice are stable over several days. Displayed in the left panel are the average VEP amplitude (n = 6 mice) in response to contralateral eye (blue bars) and ipsilateral eye (yellow bars) stimulation at baseline (day 0) and after 5 days of normal visual experience. Right panel shows the stability of contralateral/ ipsilateral ratios over days. (See figure 38.1.)

Plate 32 Melanosomes contribute to DBA/2J pigment dispersion. A–D, Mutations in genes encoding the melanosomal proteins TYRP1 and GPNMB cause iris disease. DBA/2J mice that are homozygous mutant for both genes have the most severe disease (D). All images are from 24-month-old mice. E, Evidence of an immune contribution to DBA/2J pigment dispersion. Bone marrow genotype has an important effect on the phenotype. (See

figure 39.1.) (Modified from John, 2005. A–D, Reproduced from Mo et al., 2003 [ Journal of Experimental Medicine, 2003, 197:1335– 1344. Copyright 2003, Rockefeller University Press]. E, Reproduced from John, 2005 [Investigative Ophthalmology and Visual Science, 2005, 46:2650–2661. Copyright 2005, Association for Research in Vision and Ophthalmology].)

sup nas

tem inf

Plate 33 Whole-mounted glaucomatous retina showing fanshaped patterns of RGC loss and survival. A, High-resolution survey of a whole-mounted retina from a moderately affected eye stained for axons (green, Smi32) and amacrine cells (red, ChAT) and nuclei counterstained with TOPRO (blue). The axon bundles entering the optic nerve head are markedly reduced and show

sectors of relatively high axon densities alternating with sectors with almost no persisting axons. Scale bar = 500 μm. B–E, High-power views of the boxed areas outlined in A. Scale bar = 100 μm. (See figure 39.3.) (Reproduced from Jacobs et al., 2005 [ Journal of Cell Biology, 2005, 171:313–325. Copyright © 2005, Rockefeller University Press].)

Plate 34 Bax deficiency prevents glaucomatous RGC death but is not required for optic nerve degeneration. RGC layers (A, C, E, and G) and optic nerves (B, D, F, and H) were analyzed in Bax-sufficient and Bax-deficient DBA/2J mice (genotype indi-

cated). Mice were analyzed at preglaucomatous (A–D) and severe glaucomatous (E–H) stages. Bax-deficient DBA/2J mice had severe optic nerve damage but no RGC death. (See figure 39.4.) (Modified from Libby et al., 2005c.)

Plate 35 Radiation treatment prevents glaucomatous optic nerve excavation. A–F, Hematoxylin-eosin-stained sections of untreated and treated DBA/2J mouse eyes (A and B). Optic nerve head (ONH) (A) and retina (B) from nonglaucomatous DBA/2J mice showing large numbers of axons as evidenced by a thick nerve fiber layer (NFL) (arrowheads). ONH (C) and retina (D) of a treated DBA/2J mouse (14 months old) is indistinguishable from nonglaucomatous controls. E and F, In contrast, untreated DBA/2J mice show severe excavation of ONH (*) and no NFL. Cross sections of

the optic nerve from treated (G) and untreated (H) DBA/2J mice stained with PPD, a stain that labels myelin sheath and sick or dying axons. The severe axon loss and scarring in the untreated eye are absent in the treated eye. The vast majority (>96%) of eyes from radiation-treated DBA/2J mice were completely rescued from glaucoma. (See figure 39.5.) (Modified from Anderson et al., 2005. Reproduced from Proceedings of the National Academy of Sciences of the USA, 2005, 102:4566–4571. Copyright © 2005 National Academy of Sciences.)

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Plate 36 Mutations in transcription factors and their effect on lens development. A, Histological section through a developing eye of a Pax6 mutant (Pax6Aey11; E17.5) clearly demonstrates the persisting lens stalk (arrow), the connection between the lens and the cornea. B, Histological section of the developing eye of the aphakia

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Plate 37 Mutations in Gja8 and Cryaa. Gross appearance of lenses of the Gja8Aey5 mutant mouse (A) compared with lenses from a CryaaAey7 mutant mouse (B). The upper row indicates the pheno-

mouse (deletions in the Pitx3 promoter) shows absence of the lens at E18.5; the eyeball is filled with retinal derivatives. C, cornea; R, retina. (See figure 40.1.) (A, From Graw et al., 2005. B, From Semina et al., 2000.)

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type for the heterozygotes and the lower row that for the homozygous mutants. In both cases, a gene dose effect can be observed. (See figure 40.2.)

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Plate 38 Mutations in Cryg. Histological sections of cataractous lenses from two different mutant lines are shown. The mutations have been induced by ENU in the Cryga gene (A) or in the Crygd

gene (B). Inset in the overview marks the magnification, which is given below. The differences in severity are apparent. Bars = 100 μm. (See figure 40.3.) (From Graw et al., 2004.)

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Plate 39 Typical histology of EIU and EAU in the mouse. Healthy compared to diseased eye tissue in EIU (A and B) and in EAU (C and D). A, Healthy anterior chamber. Shown is the angle where the iris and ciliary body connect to the sclera. C, cornea; CB, ciliary body; I, iris; S, sclera. B, EIU in a C3H mouse induced by subcutaneous injection of LPS. Note infiltration of inflammatory cells in the angle and around the iris and ciliary processes. Original magnification ×1200. C, Healthy mouse retina.

C, choroid; G, ganglion cell layer; P, photoreceptor cell layer; R, retinal pigment epithelium; S, sclera; V, vitreous. D, EAU in the B10.RIII mouse induced by immunization with IRBP. Note structural disorganization and loss of nuclei in the ganglion and photoreceptor cell layers, retinal folds, subretinal exudate, vasculitis, damage to the retinal pigment epithelium, and choroid inflammation. Original magnification ×1000. (See figure 42.2.) (Photomicrographs courtesy of Dr. Chi-Chao Chan, NEI, NIH.)

(This figure was previously published in Caspi, 2006. Copyright does not apply.)

Plate 40 Critical checkpoints in EAU pathogenesis. Schematic representation of critical events and checkpoints in the pathogenesis of EAU as revealed by studies in mouse models. (See figure 42.3.)

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Plate 41 Sections of the central retina from wild-type and knockout mice at P21 after staining with a collagen IV antibody (red), which detects the extracellular matrix of endothelial cells, and diamidinophenylindol/DAPI (blue), which labels cell nuclei. Blood vessels (red) in wild-type mice are present in three layers, indicating completed development of the superficial (ganglion cell layer [GCL]), deep (outer plexiform layer [OPL]), and intermediate (border between the inner plexiform layer [IPL] and inner nuclear

50 μm

layer [INL]) vessel networks. In contrast, age-matched knockout mice show only enlarged superficial vessels, while the deeper and intermediate networks failed to develop. In addition, the presence of nuclei in the IPL (white arrowheads) may reflect the characteristic disorganization in the GCL. (See figure 43.2.) (Images acquired and provided courtesy of Nikolaus Schäfer, Division of Medical Molecular Genetics, Institute of Medical Genetics, University of Zurich, Switzerland.)

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Plate 42 Three-dimensional microscopy of retinal flat mounts stained with anticollagen IV from wild-type and knockout mice at P7. Top panels show an area of approximately 300 × 400 μm from wild-type and knockout mice. Vessels in the knockout are dilated, and the capillary network is less dense than in the wild-type control. Bottom panels (z-axis) show how blood vessels enter the deeper retinal

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layers in wild-type mice by angiogenic sprouting. In contrast, this process is abolished in knockout mice, and the vessels do not invade the deeper retinal layers. (See figure 43.3.) (Images acquired using the Zeiss ApoTome technology and provided courtesy of Nikolaus Schäfer, Division of Medical Molecular Genetics, Institute of Medical Genetics, University of Zurich, Switzerland.)

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Plate 43 Hematoxylin-eosin-stained sections of whole eyes from wild-type (wt) and norrin knockout (ko) mice at P5, P10, P15, and P21. The hyaloid vessel system (H) in the vitreous body (V) is clearly present in wild-type mice at P5 and P10, and remnants are evident at P15. In knockout mice, the hyaloid vasculature persists through

P21 and beyond, and regression of this vascular network is profoundly delayed. L, lens; O, optic nerve; R, retina. (See figure 43.4.) (Images acquired and provided courtesy of Dr. Ulrich Luhmann, Division of Medical Molecular Genetics, Institute of Medical Genetics, University of Zurich, Switzerland.)

Plate 45 The retinal vasculature of the mouse, following isolation by proteolytic digestion of the formalin-fixed retina. (See figure 45.1.)

Plate 44 Autofluorescence of RPE lipofusin in Abca4/Abcr−/− mouse retina. Cross section of mouse retina viewed by epifluorescence microscopy (excitation 425 ± 45 nm, emission 510 nm long pass). Nuclei are stained with DAPI. The yellow-gold autofluorescence in RPE cells is associated with lipofuscin in RPE cells. The less pronounced autofluorescence in photoreceptor outer segments (POS) is attributable to lipofuscin precursors that form in this location. The albino mouse was 8 months old and homozygous for leucine at amino acid 450 of Rpe65. GC, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium. (See figure 44.3.)

Plate 46 Degenerate (acellular) capillaries (arrows) in the diabetic mouse retina. The degenerate vessels lack vascular cell nuclei and have an irregular or shrunken diameter compared to surrounding healthy capillaries. (See figure 45.2.)

Plate 47

Leukocyte adherence, or leukostasis (arrows), in the retinal microvasculature of the diabetic mouse. (See figure 45.4.)

Plate 48 Oxygen is selectively toxic to photoreceptors. A–D, Sections across the retina of C57BL/6 mice exposed to high oxygen levels in inhaled air. Before exposure, dying (red, TUNEL-labeled) cells were rare. Exposure to hyperoxia for 14–35 days (B–D) increased the frequency of dying cells, selectively in the ONL (o). By 35 days, the ONL had thinned and TUNEL+ debris appeared

in the inner nuclear layer (i), probably ingested by Müller cells. The blue dye is a DNA label (bisbenzamide). E and F, When the sheaths of cones were labeled with PNA lectin, the nuclei of some were TUNEL labeled (red), indicating that cones as well as rods were oxygen sensitive. (See figure 46.3.) (From Geller et al., 2006.)

μ μ

μ 2.5

21% O2 10% O2 70% O2

4

P14 P16 P18

2

3.5 3 2.5

1.5

2 1

1.5 1

0.5 0.5 0

0 0 to 80 μm

80 to 160 μm

160 to 240 μm

240 to 320 μm

Plate 49 Photoreceptors at the most peripheral margin of the retina are subject to stress from early postnatal life. A–C, The edge of the retina of C57BL/6 mouse retina, with dying cells (bright red, TUNEL labeled) clustering at the edge of P14, P16, and P18. All are in the ONL. D and E, This cluster of dying cells colocalizes with the upregulation of two stress-induced proteins. GFAP is normally expressed (red) only at the inner surface of the retina, by astrocytes (E and G). At the edge, GFAP is upregulated also in Müller cells. FGF-2 is strongly upregulated (green) in photorecep-

0 to 80 μm

80 to 160 μm

160 to 240 μm

240 to 320 μm

tors, in a gradient that is maximal at the end. H, Quantification of the frequency of dying cells in the peripheral-most 100 μm of retina. I, Oxygen influences this distribution of dying cells at the edge of the early postnatal retina. Hypoxia increases photoreceptor death throughout most of the retina; at the edge, however, hyperoxia reduces death, suggesting that the edge-related death is driven by oxygen stress (see the text). Hyperoxia causes a small increase in the frequency of dying cells at the edge of the retina. (See figure 46.4.) (From Mervin and Stone, 2002a, 2002b.)

Plate 50 Strategy for in vivo electroporation A, Electroporation to the scleral (RPE) side of the retina. B, Electroporation to the vitreal side of the retina. (See figure 51.1.)

Plate 51 Electrodes and procedure for in vivo electroporation. Tweezer-type electrodes (A) are placed to hold the head of newborn (P0) rat or mouse (B). (See figure 51.2.)

Plate 52 Whole-mount preparation of rat retina in vivo electroporated at P0 with CAG-GFP (Matsuda and Cepko, 2004), a GFP expression vector driven by the CAG (chicken β-actin promoter

with cytomegalovirus enhancer) promoter and harvested at P21. Images are from the scleral side. Bright-field (A), GFP (B), and merged (C ) images are shown. (See figure 51.3.)

Plate 53 In vivo electroporated retina (P0 electroporation, section). Rat retinas were in vivo electroporated with CAG-GFP at P0 and harvested at P2 (top panel) or P20 (bottom panel). At P2, most of the GFP-positive cells have the morphology of progenitor/precursor cells, suggesting that DNAs are preferentially transfected to progenitor/precursor cells. Retinogenesis is completed within the

first 2 weeks after birth. At P20, GFP is observed in four differentiated cell types: rod photoreceptors, bipolar cells, amacrine cells, and Müller glia. Early-born cell types (cone, horizontal, and ganglion cells) are not labeled by P0 electroporation. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment; VZ, ventricular zone. (See figure 51.4.)

Plate 54 In vivo electroporated retina (E14 electroporation, section). Mouse embryonic retinas were electroporated with UBGFP (Matsuda and Cepko, 2004), a GFP expression vector driven by the human ubiquitin promoter, at E14 in utero, and harvested at P20. Early-born cell types (cone, amacrine, horizontal, and ganglion cells) are clearly labeled with GFP, while late-born cell types (bipolar and Müller glial cells), which are generated from E14

RPCs after several rounds of cell division, are poorly labeled. This is probably due to dilution of introduced plasmids. Red arrowheads indicate the labeled cone photoreceptors. Yellow arrowhead indicates the labeled horizontal cell. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment. (See figure 51.6.)

Plate 55 A, Microchamber for in vitro electroporation. B, Orientation of the retina in the chamber. Maximum transduction efficiency can be obtained when the scleral side is facing the minus electrode. (See figure 51.7.)

Plate 56 In vitro electroporated retinal explant (whole mount). Mouse retinas of P0 CD1 (A and B), adult CD1 (C and D), or adult Swiss Webster mice with a retinal degeneration mutation (E and F) were in vitro electroporated with CAG-GFP from the scleral side (A, C, and E) or from the vitreal side (B, D, and F) and cultured for

5 days. Images A, C, and E are from the scleral side and images B, D, and F are from the vitreal side. Note that only the scleral side of developing retina or of degenerated retina is highly transfectable. In E, most of the GFP-positive cells are Müller glial cells. (See figure 51.8.)

Plate 57 Temporal regulation of gene expression in the retina using inducible Cre recombinases. A, CAG-CreERT2: Fusion protein (CreERT2) between Cre recombinase and the mutated ligand-binding domain (ERT2) of the human estrogen receptor is expressed under the control of the CAG promoter. CAG-ERT2 CreERT2: Fusion protein (ERT2CreERT2) composed of Cre and two ERT2 domains is expressed under the control of the CAG promoter. CreERT2 and ERT2CreERT2 are conditionally activated in response to 4-OHT. B, CALNL-DsRed: Cre/loxP-dependent inducible expression vector. DsRed is expressed only in the presence of Cre. C, A scheme of the experiment. D–I, P0 rat retinas were coelectroporated with three plasmids: CAG-GFP (transfection control),

CALNL-DsRed (recombination indicator), and CAG-CreERT2 (D) or CAG-ERT2CreERT2 (E and F). The retinas were stimulated without 4-OHT (D and E) or with 4-OHT (F) by IP injection at P20 and then harvested at P21. Whole-mount preparations of the harvested retinas are shown. G–I, Sections of the retinas shown in D–F. Cell nuclei were stained with DAPI. When CreERT2 was used, significant background recombination was observed even in the absence of 4-OHT. On the other hand, ERT2CreERT2 had no detectable basal activity in the absence of 4-OHT. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (See figure 51.9.)

Plate 58 Spatial regulation of gene expression in the retina using cell type–specific promoters. Retinal cell type–specific promoters were fused to DsRed cDNA and electroporated into P0 rat retinas. The retinas were harvested at P20 (A–F and H) or P2 (G), sectioned,

and stained with DAPI. Promoters of rhodopsin (A), Nrl (B), Cabp5 (C ), Ndrg4 (D), Cralbp (E), clusterin (F), and Hes1 (G and H) were used to express DsRed. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (See figure 51.10.)

Plate 59 Multicolor labeling of the retina using cell type–specific promoters. P0 rat retina was electroporated with three reporter constructs: rhodopsin promoter 2.2K-CFP (specific for rods), Cabp5 promoter 4.7K-YFP (specific for a subset of bipolar cells),

and Cralbp promoter 4.0K-DsRed (specific for Müller glia). The retina was harvested at P20, sectioned, and stained with DAPI. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment. (See figure 51.11.)

Plate 60 Lineage tracing experiments in the retina using the Cre/loxP system and cell type–specific promoters. P0 rat retinas were coelectroporated with retinal cell type–specific promoter Cre and CALNL-DsRed (recombination indicator). DsRed expression, induced by Cre/loxP-mediated recombination, was driven by the ubiquitous CAG promoter. The retinas were harvested at P20,

sectioned, and stained with DAPI. Promoters of rhodopsin (A), Nrl (B), Cabp5 (C ), Ndrg4 (D), Cralbp (E), clusterin (F), Hes1 (G), and Rax (H) were used to express Cre. Yellow arrowheads indicate the labeled rods. Blue arrowhead indicates the labeled bipolar cell. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. (See figure 51.12.)

Plate 61 Inducible expression in the differentiated Müller glia (A). Clusterin promoter was fused to ERT2CreERT2 cDNA and coelectroporated into P0 rat retinas with CALNL-DsRed (recombination indicator) and CAG-GFP (transfection control). Retinas were stimulated with or without 4-OHT at P14 by IP injection, then harvested at P16. B, Whole-mount preparation of the trans-

fected retina harvested at P16 without 4-OHT stimulation. No DsRed expression was detected. C, Whole-mount preparation of the transfected retina stimulated with 4-OHT at P14 and harvested at P16. D, The retina shown in C was sectioned and stained with DAPI. Only Müller glial cells are labeled with DsRed. (See figure 51.13.)

Plate 62 Inducible RNAi in the retina. A, CAG-mir30: The mir30 expression cassette is expressed under the control of the CAG promoter. The mir30 expression cassette has the hairpin stem composed of siRNA sense and antisense strands (22nt each), a loop derived from human mir30 (19nt), and 125nt mir30 flanking sequences on both sides of the hairpin. The mir30 primary transcript is processed to generate the mature shRNA. CALSLmir30: Cre-dependent inducible shRNA expression vector carrying a floxed transcriptional stop cassette (3xpolyA signal sequences).

Only in the presence of Cre, the mir30 expression cassette is expressed under the control of the CAG promoter. B and C, Conditional GFP knockdown in the retina. CAG-GFP and CAGDsRed and CALSL-mir30(GFPshRNA) expressing an shRNA against GFP were coelectroporated without (B) or with (C ) the rhodopsin promoter Cre into P0 rat retinas. The retinas were harvested at P20, sectioned, and stained with DAPI. Rod-specific GFP knockdown was observed in the presence of the rhodopsin promoter Cre. (See figure 51.14.)

OS

IS

B

A C

D

50 μm

Plate 63 A, A single rod photoreceptor isolated from adult wt-Gfp mice shows the expression of GFP. B, The same cell shows staining for rhodpsin (red) in the outer segment (OS) and inner segment (IS) regions and bis-benzimide staining (blue) for the nucleus.

C, Samples of dissociated cells are viewed under the microscope before (shown) and after flow sorting. D, Flow cytometry allows the GFP-positive population of cells to be gated (R3) and sorted separately. (See figure 55.1.)

superficial

GCL IPL

intermediate

INL deep

OPL ONL

photoreceptor outer segments

Light Absorption OS

Epithelial Transport

Visual Cycle

Glia

Phagocytosis

Secretion

k+

h·γ

11-cis-retinal

MV

PEDF

photoreceptor outer segments RPE Bruch’s membrane choriocapillaris

Fenestrated Capillary Bed Glucose Vitamin A

CI –

H2O

Plate 64 Top, The retina is a complex, striated tissue with highly organized neuronal layers. The inner retinal vasculature forms three distinct plexuses: The superficial plexus forms within the ganglion cell layer (GCL), the intermediate plexus forms at the inner edge of the inner nuclear layer (INL), and the deep plexus

VEGF

forms at the outer edge of the INL. Bottom, Posterior to the retina are the RPE, Bruch’s membrane, and the choriocapillaris. The RPE performs many functions that are critical to retinal function. (See figure 56.1.) (Top, From Dorrell and Friedlander, 2006. Bottom, From Strauss, 2005.)

A No correlation with retinal developmental functions

Expression profile correlates with visual functions

Expression profile correlates with neuronal development

100

100

100

10

10

10

1

1

1

0.1

0.1

0.1

49.8 %

0.01 P0

P4

2.5 %

0.01

P8 P10 P12 P14 P21 Adult

P0

P4

P8 P10 P12 P14 P21 Adult

0.01

100

100

100

10

10

10

1

1

1

0.1

0.1

0.1

5.5 %

0.01 P0

2,500

P8 P10 P12 P14 P21 Adult

R-cadherin gene expression values

P0

P4

C

P4

P8 P10 P12 P14 P21 Adult

D Control

R-cadherin

1,000

P8 P10 P12 P14 P21 Adult

7.7 % P0

2,000 1,500

P4

0.01

P8 P10 P12 P14 P21 Adult

Control

Raw expression values

B

P4

1.5 %

0.01

2.5 % P0

INL PRL ONL

IPL GCL

500 0 P0

P4 P8 P10 P12 P14 P21 Adult

Timepoint

R-cadherin blocked

R-cadherin blocked

PRL ONL

Superficial vascular plexus formation

Plate 65 A, Gene expression profile clustering of mRNA from whole mouse retinas at different postnatal developmental stages. Based on expression profile, gene function could be classified as having probable involvement in the onset of vision (mouse retinal development nears completion and vision begins around P14), neuronal development and differentiation (nears completion around P14), or potential involvement in postnatal retinal vascular development (a process that undergoes multiple changes during these

INL IPL GCL Deep vascular plexus formation

times, and thus gene expression would vary throughout). B, The expression profile of R-cadherin was determined to have a potential relationship to retinal vascular development. When R-cadherin function was blocked, the retinal vasculature failed to form normally in the superficial plexus (C ), and guidance to the normal deep vascular plexuses was disrupted (D). Vessels migrated through the photoreceptor layer and into the subretinal space. (See figure 56.2.) (Adapted from Dorrell et al., 2002, 2004.)

Surface Ectoderm

Development of Lens E7.5

Meis 1 & 2

E8.5

Presumptive Lens Ectoderm

E10

Lens Placode

E11

Development of Cornea Six3

Pax6 + pRb, Maf, Mitf, Sox2, Sox3 or AP2α

Foxe3

Maf + Pax6, or Sox2 Sip1 Foxc1 Sox1 Foxc2 Lens Foxe3

Pitx2 Prox1 Sox2 AP1 Pitx3 Sp1 Sox3 Klf4 Presumptive AP2α Elf3 corneal IκBζ epithelium Klf6

Mesenchymal cell migration between lens vesicle and corneal epithelium

Vesicle

Hsf4

E12.5 Primary fiber cells

Expression of crystallins and other genes

E15

E10

E11

E12.5 Condensation of mesenchymal cells begins Mesenchymal cells E15 form corneal stroma and endothelium

Secondary fiber cells

Maturation PN15

Lens Plate 66 Transcription factors regulating the development of mouse lens and cornea. The network of transcription factors regulating the development of lens (red), or cornea (blue), or both (green)

Cornea

Proliferation and PN15 stratification of corneal epithelial cells

is shown, along with the embryonic age and the developmental stages of lens and cornea. Many of these transcription factors remain active in the mature lens and cornea. (See figure 57.2.)

Plate 67 Model of αA-crystallin secondary and tertiary structure, colored according to the hydrophobicity of its amino acid residues. The most hydrophobic residues are colored dark blue; the least hydrophobic residues are colored red. The Rosetta algorithm on the HMMSTR server (www.bioinfo.rpi.edu/~bystrc/hmmstr/ server.php) (Bystroff and Shao, 2002) available on the ExPASY (http://au.expasy.org/) home page was used for molecular modeling. Full-length αA-crystallin secondary structure was calculated at 29.5% α helix and 32% β sheet content. The N-terminal globular domain is organized into three helices, displayed as ribbons with hydrophobic side chains buried. Structure-function regions identified earlier (Smith et al., 1996; Pasta et al., 2003) make up the first two of these N-terminal α helices. The highly conserved residues 102–117 of the “α crystallin domain” (Caspers et al., 1995), containing the substantial first part of a DNA-binding motif (Singh

et al., 1998), as well as an arginine residue 116 shown to be critical for molecular integrity (Bera et al., 2002), are predicted to have αhelical conformation and are displayed as ribbons. This is consistent with an older 3D model (Farnsworth et al., 1998) and makes the α-helical prediction that is somewhat higher than previous calculations (Farnsworth et al., 1997; Horwitz et al., 1998; Bova et al., 2000) seem plausible. However, it is inconsistent with sitedirected spin label studies that demonstrate β sheet conformation for residues 109–120 (Berengian et al., 1997). The model confirms the β sheet secondary structure of residues 67–101, determined to be an alcohol dehydrogenase (ADH) and 1,1′-bi (4-anilino) naphtalene-5,5′-disulfonic acid (bis-ANS) binding site and to exhibit extensive chaperone activity (Farnsworth and Singh, 2004). (See figure 58.1.)

Plate 68 Hypothetical schematic of the rod photoreceptor visual cycle. Transcellular diffusion and intracellular enzymatic processing of visual cycle retinoids are shown. A retinal pigmented epithelial (RPE) cell is depicted with apical process extending toward a rod outer segment, with one disc membrane. Reactions and processes are as discussed in the text. Circular insets depict putative roles of components in RPE apical processes. cis-RDH, 11-cis-retinal dehydrogenase; CRALBP, cellular retinaldehyde-binding protein; CRBPI, cellular retinol-binding protein type I; EBP50, ERM-

binding phosphoprotein of 50 kd, also known as NHERF-1 (sodium hydrogen exchanger regulatory factor type 1); IRBP, interphotoreceptor retinoid-binding protein; ISOMERO, isomerohydrolase, recently identified as RPE65; LRAT, lecithin : retinol acyltransferase; PDZ, a scaffold domain protein; RGR, retinal G protein– coupled receptor; RDH, all-trans-retinol dehydrogenase; Rho, rhodopsin; Rho*, activated rhodopsin (metarhodopsin II); RPE65, retinal pigmented epithelial protein of 65 kd. (See figure 59.1.)

Plate 69 Hypothetical schematic of the cone photoreceptor visual cycle. Transcellular diffusion and intracellular enzymatic processing of cone visual cycle retinoids are shown. Structure at left depicts a portion of the inner and outer segments of a cone photoreceptor cell. Structure at right depicts the apical end of a Müller

cell. The cells are joined by structures of the external limiting membrane (short wavy line). ARAT, acylCoA : retinol acyltransferase; HYDROLASE, 11-cis-retinyl ester hydrolase; ISOM, isomerase. See also the abbreviation key for figure 59.1. (See Figure 59.2.) (After Mata et al., 2002.)