Advances in Genetics Volume 38

Volume 38

Advances in Genetics

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Advances in Genetics Edited by Jeffery C. Hall

Jay C. Dunlap

Department of Biology Brandeis University Waltham, Massachusetts

Department of Biochemistry Dartmouth Medical School Hanover, New Hampshire

Theodore Friedmann

Francesco Giannelli

Department of Pediatrics Center for Molecular Genetics School of Medicine University of California, San Diego La Jolla, California

Division of Medical and Molecular Genetics United Medical and Dental Schools of Guy’s and St. Thomas’ Hospital London Bridge, London, United Kingdom

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Copyright 0 1998 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-1998 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-2660198 $25.00

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Contents Contributors

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Body Plan Genes and Human Malformation

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Edoardo Boncinelli, Antonio Mallamaci, and Vania Broccoli I. Introduction 1 11. Establishing the Body Axes 3 111. Patterning the Limb 16 IV. Patterning the Ocular Anlage 21 V. Patterning the Tooth Anlage 22 References 24

Molecular Genetics of the Hereditary Ataxias

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Massimo Pandolfo and Laura Montermini I. Introduction 32 11. The Autosomal Dominant Progressive Ataxias 111. Friedreich Ataxia 49 References 60

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The Minute Genes in Drosophila and Their 69 Molecular Functions Andrew Lambertsson I. Introduction

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11. Historical Review 76 111. Minutes as Genetic Tools in Studies of Growth and Development 84 88 IV. Minutes and the Protein Synthesis Theory 122 V. Conclusions and Prospects References 124 V

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4 Genetics of Biological Rhythms in Drosophria Jeffrey C. Hall I. Introduction 135 11. Chronogenetics 136 111. Chronogenetic Biology 163 IV. A Final Chronogenetic Thought References

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5 DNA Breakage and Repair

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P. A. Jeggo I. Introduction 186 11. Formation of DNA Strand Breaks 186 111. Mechanisms for the Repair of DNA DSBs 188 191 IV. Identification of Genes Required for NHEJ V. Characterization of Proteins Involved in NHEJ by Biochemical, Molecular, and Genetic Analysis 196 VI. DNA-PK as a Protein Kinase 200 VII. The Mechanism of NHEJ 201 VIII. The Contribution of NHEJ and HR to DSB Rejoining in Yeast versus Mammalian Cells 205 206 IX. DNA-PK-Defective Mice 207 X. Radiosensitivity and Immune Deficiency XI. A Model for NHEJ 208 XII. Summary 210 References 2 11 Index

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Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Edoardo Boncinelli DIBIT, Instituto Scientifico H San Raffaele; and Center for Cellular and Molecular Pharmacology, CNR, 20132 Milan, Italy (1) Vania Broccoli DIBIT, Instituto Scientifico H San Raffaele, 20132 Milan, Italy (1)

Jeffrey C. Hall Department of Biology and NSF Center for Biological Xming, Brandeis University, Waltham, Massachusetts 02254 ( 135) Penny A. Jeggo MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR, United Kingdom (185) Andrew Lambertsson Department of Biology, Division of General Genetics, University of Oslo, N-0315 Oslo, Norway (69) Antonio Mallamaci DIBIT, Instituto Scientifico H San Raffaele, 20132 Milan, Italy (1) Laura Montermlni Centre de Recherche Louis-Charles Simard, McGill University, Montreal, Quebec, H2L 4M1 Canada (31) Massimo Pandolfo Centre de Recherche Louis-Charles Simard; Departement de Medecine, Universite de Montreal; and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, H2L 4M1 Canada

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Malformation Edoardo Boncinelli,*lt Antonio Mallamaci,* and Vania Broccoli* *DIBIT, Istituto Scientific0 H San Raffaele, 20132 Milan, Italy; C e n t e r for Cellular and Molecular Pharmacology, CNR, 20132 Milan, Italy

1. Introduction 1 3 11. Establishing the body axes A. Rostrocaudal patterning 3 B. Dorsoventral patterning 14 111. Patterning the limb 16 A. Limb development 16 16 B. Genes encoding secreted molecules 18 C. Homeobox genes and limb development 21 IV. Patterning the ocular anlage A. Eye formation 21 21 B. Homeobox genes and the patterning of the eye anlage 22 V. Patterning the tooth anlage A. Tooth development 22 23 B. Homeobox genes and patterning the tooth anlage References 24

I. INTRODUCTION Development initiates with a series of symmetry-breaking events leading to the establishment of fundamental polarities along the rostrocaudal and dorsoventral body axes and, subsequently, along the proximodistal axes of limbs and appenAdvances in Genetics, Vol. 38 Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved. 0065-2660/98 $25.00

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dages. These polarities have to be maintained for some time and subsequently translated into positional values and regional specification, ultimately leading to cell differentiation and maturation. All of these events are genetically controlled by developmental genes and the identification of these genes is a major task of current biology. Many of these genes act in circuits and there is a plenty of redundancy or better, overdetermination, in this genetic control. A key role is played by the so-called regulatory genes, genes acting through the control of the expression of other genes laying hierarchically downstream from them and sometimes termed target genes. Regulatory genes generally code for transcription factors; that is, nuclear proteins able to recognize specific DNA sequences, bind to them, and modulate, through this specific binding, the level of expression of the corresponding target genes. A relatively large proportion of regulatory genes are the homeobox genes (McGinnis and Krumlauf, 1992). Homeobox genes are regulatory genes, originally discovered in Drosophila, characterized by the presence of a specific, evolutionarily conserved DNA sequence termed homeobox and able to code for a protein domain of some 60 amino acid residues, termed homeodomain. It is through the action of their homeodomain that the protein products of the homeobox genes, the homeoproteins, bind to the regulatory regions of specific genes and control their expression. Vertebrate homeobox genes occur in families (Stein et al., 1996). Most of these gene families have counterparts in Drosophila and are often termed according to this similarity. Thus, for example, vertebrate homeobox genes similar to the fruit fly homeotic genes are termed Hox genes; those related to the Drosophila paired ( p d ) gene are termed Pax; those related to the Drosophila engrailed (en) gene are termed En, and so on. A relevant role is also played in development by genes coding for growth factors and, in general, secreted proteins. Also these genes occur in families; they play a major role the Wnt gene family, related to the Drosophila wingless (wg) developmental gene and genes coding for various FGFs (fibroblast growth factors) and BMPs (bone morphogenetic proteins). Recently, the expression patterns of all these genes have been extensively studied in the mouse and have provided useful suggestions for the function they exert during mammalian development. Moreover, their function has been often assayed in vivo by so-called reverse genetics. A large subset of these genes have been knocked-out via homologous recombination in embryonic stem (ES) cells and animals bearing homozygous null mutations have been generated (recently reviewed in St. Jacques and McMahon, 1996). The analysis of these animals enabled us to understand some major mechanisms controlling vertebrate development, with special emphasis on the identification of genes involved in managing positional information and establishing the body plan. But, despite the recent incredible expansion of our knowledge in molecular biology of development, there has been a relative lack of reports about involvement of body plan

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genes in inherited human disease, but the situation is now changing (reviewed in Engelkamp and van Heyningen, 1996 and in Boncinelli, 1997). O n one hand, the increasing number of gene disruption experiments in transgenic mice (St. Jacques and McMahon, 1996) provides a number of suggestions for possible spontaneous inherited pathologies; on the other, the two converging strategies of candidate disease-genes and positional cloning lead to new discoveries. The field has been recently reviewed (Engelkamp and van Heyningen, 1996; Boncinelli, 1997). In this chapter we focus on recent progress and analyze the problem of patterning the embryo along the major body axes and in well-studied morphogenetic fields, namely the developing limb, eye, and tooth anlagen.

II. ESTABLISHING THE BODY AXES A. Rostrocaudal patterning 1. Three genetic domains O n the sole basis of the regional expression of different families of regulatory genes, the central nervous system, if not the entire body, of vertebrates can be subdivided into at least three broad domains along its rostrocaudal axis (Figure 1.1). Each domain also appears to follow a specific developmental pathway for its specification and regionalization (Bally-Cuif and Boncinelli, 1997 for a recent review). Domain 1, including the fore- and midbrain, represents the domain of action of the homeobox genes of the Otx and Emx families. Domain 3 , corresponding to the rhombencephalic regions posterior to rhombomere 1 ( r l ) and spinal cord, is the domain of the homeobox genes of the Hox family. Domain 2, essentially r l and the so-called met-mesencephalic boundary region, remains outside the domains of action of both the OtxlEmx gene families and the Hox gene family: Pax and En genes play a key role in its developmental regulation and extend their influence also into the posterior region of the adjacent mesencephalon. Domain 2 appears to represent something different from all the rest of the neuraxis and to follow unique developmental criteria, possibly owing to its relative evolutionary novelty (Bally and Wassef, 1995; Joyner, 1996; Bally-Cuif and Boncinelli, 1997; Boncinelli, 1997); the met-mesencephalic boundary included in it could be a source of long-range active morphogen(s) involved in patterning the adjacent fields.

2. The fore- and midbrain region Two couples of homeobox genes, Emxl and Emx2 (Simeone et al., 1992a,b), and Otxl and Otx2 (Simeone e t al., 1992a, 1993; Finkelstein and Boncinelli, 1994), respectively, are related to Drosophila head gap genes empty spiracles (ems) and

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Hox

Figure 1.1. Schematic subdivision of the vertebrate CNS on the basis of the expression of specific families of regulatory genes. The vertebrate CNS can be subdivided along its anterior-posterior axis into at least three domains. Domain 1, comprisedof fore- and midbrain, represents the domain of action of Otx and Emx genes families (Fig. 1.2). Domain 2 includes essentiallyrhombomere 1 ( r l ) and the so-called met-mesencephalicboundary region: Pax, Gbx, and En genes play a key role in its development. Domain 3, corresponding to the rhombencephalon posterior to rhombomere 1 ( r l ) and spinal cord, is the realm of Hox genes (Fig. 1.3). In particular, 3' Hox genes belonging to groups 1-4 control the development of the rhombencephalon and its regionalization into a number of neural segments,termed rhombomeres.Central Hox genes of groups 5-8 and 5' Hox genes of groups 9-13 control the cervicothoracicand the lumbosacralregionsof the spinal cord, respectively (see also Fig. 1.4).

orthodentick (otd), and seem to play a relevant role in patterning the vertebrate fore- and midbrain. In the developing central nervous system of mouse embryos at day 10.5 of development (E10.5) most of the specific differentiative events have not yet occurred and at this stage all four Emx and Otx genes are expressed. Their expression domains (Simeone et al., 1992a) are continuous regions of the developing brain contained within each other in the sequence Emxl < Emx2 < Otxl < Otx2 (Figure 1.2). The Emxf expression domain includes the dorsal telencephalon. Emx2 is expressed in the dorsal and ventral forebrain with an anterior boundary slightly anterior to that of Emxl and a posterior boundary within the roof of presumptive diencephalon. The Otxl expression domain contains the Emx2 domain: it covers a continuous region including part of the telencephalon,

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Figure 1.2. Nested expression domains of Otx and Emx homeobox genes in the developing brain of a El0 mouse embryo. T h e Emxl expression domain includes the dorsal telencephalon. EmxZ is expressed in dorsal and ventral forebrain with a posterior boundary within the roof of presumptive diencephalon. T h e Otxl expression domain contains the Emx2 domain: it covers a continuous region including part of the telencephalon, diencephalon, and mesencephalon. Finally, the O t x Z expression domain contains the Otxl domain, both dorsally and ventrally, and practically covers the entire fore- and midbrain, to the exclusion of the early optic area. Te, telencephalon; Di, diencephalon; Mes, mesencephalon; Met, metencephalon; Mye, myelencephalon; os, optic stalk.

the diencephalon, and the mesencephalon. Finally, the Otx2 expression domain contains the Otxl domain, both dorsally and ventrally, and practically covers the entire fore- and midbrain, to the exclusion of the early optic area. Expression of Emx and Otx genes identifies several regions in the forebrain. Some of these regions seem to correspond to presumptive anatomical subdivisions, whereas the significance of others remains to be assessed. The first appearance of the products of the four genes is also sequential during development: Otx2 is already expressed at least as early as at E5.5 (Simeone et al., 1993), followed by Otxl and Emx2 at E8-8.5 and finally by Emxl at E9.5 (Simeone et al., 1992a). Thus, it seems reasonable to postulate a role for the four homeobox genes in establishing the identity of the various embryonic brain regions. In this line, the regionalization of the early rostral brain seems to be a centripetal process progressing through discrete steps and ultimately leading to the specification of dorsal telencephalon. Studies on mouse, frog, chick, zebrafish, and sea urchin Otx2 (reviewed in Boncinelli and Mallamaci, 1995) imply a role of this gene in the early establishment of the head and rostral brain. An Otx2-like gene is also present in planaria with a comparable role in head development (our unpublished data). Transgenic mice bearing null mutations for all four genes have been produced and analyzed. Otx2 is the first of these genes to be expressed during development and

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it was expected to play an essential role in anterior head formation and probably axis determination (Boncinelli and Mallamaci, 1995). Analysis of mice bearing null mutations in Otx2 produced in three different laboratories confirms these expectations (Acampora et al., 1995; Matsuo et al., 1995; Ang et al., 1996). These mice fail to gastrulate and stop developing at early midgestation. The most conspicuous phenotype of these midgestation embryos is the deletion of rostra1 brain regions, including forebrain and midbrain, anterior to rhombomere 3 (1-3).It is highly likely that the deletion of anterior neural structures is a consequence of the defective formation and migration of anterior axial mesendoderm cells. A similar early phenotype is exhibited by embryos lacking L i d , another homeobox gene expressed almost as early in development (Shawlot and Behringer, 1995). This could implicate these genes in cases of mouse or human anencephaly, but so far no evidence for this connection is available. Otxl null mutants also showed some interesting features that may suggest Otxl as a candidate locus for certain congenital malformations in man. In fact, Otxl null mutants show multiple abnormalities affecting several areas of the cerebral cortex, hippocampus, mesencephalon, and cerebellum, as well as special sense organs (Acampora et al., 1996; Suda et al., 1996). All of these localizations are in agreement with the expression of this gene during embryogenesis. Conversely, it was probably not easy to anticipate that homozygous Otxl null mice are affected by epileptic seizures. Epilepsy is a phenotypically and genotypically heterogeneous disorder; many different forms of epilepsy are known and for some of them a genetic component has been suggested. A number of transgenic mouse models are known for this disease (Noebles, 1996) and Otxl-deficient mice represent a promising new addition to this catalog. Emx2 null mutants have also been analyzed (Pellegrini e t al., 1996; Yoshida et al., 1997). O n the basis of its expression domain, this gene was proposed to play a role in the control of proliferation and migration of cortical neurons (Gulisano et al., 1996). Emx2 null mice are born but do not survive for long since they lack kidneys. Inspection of the brain of late-gestation embryos or newborn animals shows a generalized reduction of the cerebral cortex, both in extension and in thickness, severe malformations of the hippocampus and medial limbic cortex, and complete absence of dentate gyrus. It is not clear why the latter anatomical structures are particularly affected in these mutant mice. It is conceivable that the products of the gene Emx2 play specific roles in defining the identity of these structures. On the other hand, it is known that the hippocampus and dentate gyrus formation requires a prolonged cell proliferative period: so it is reasonable that these structures could suffer in a more intense way detrimental effects deriving from the missing EMX2 homeoprotein. Emxl null mutants do not show any striking cerebral phenotype other than occasional absence of corpus callosum (Yoshida et al., 1997;Qiu e t al., 1996). Although developmental and phylogenetic data suggest a very important role for

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Emxl , we have at present no hints for its function. Perhaps, Emxl may be more relevant in later developmental stages and the analysis of phenotypes deriving from the absence of its products may require refined experiments. Mutations in the human homolog of Emx2, that is EMXZ, have also been associated with a human congenital malformation termed schizencephaly (Brunelli et al., 1996; Granata et al., 1997). Heterozygous de n o w mutations in this gene have been found in sporadic cases of this extremely rare developmental defect of the cerebral cortex associated with full-thickness clefts of the cerebral hemispheres with consequent communication between the ventricle and pericerebral subarachnoid spaces. Based on the space separating the walls of the fissure, an open-lip form and a closed-lip form can be distinguished (Guerrini et al., 1996). The molecular nature of the various mutations detected in these patients is fairly heterogeneous. Even if the number of cases studied is still very restricted, a case may be made for a correlation between the molecular nature of these mutations and the observed clinical severity. In fact, predictably deleterious molecular defects, like frameshift mutations or mutations affecting the splicing pattern are invariably associated with severe, open-lip, bilateral schizencephaly, whereas subtle or leaky mutations are associated with mild, closed-lip, seemingly unilateral schizencephaly (Granata et al. , 1997). This may be ultimately useful for prognosis, given the difficulties associated with the differential diagnosis for this particular pathology and its extreme clinical heterogeneity.

3. The isthmic region As proven by heterotopic homochronic transplants in chickenkquail embryos, the boundary between rhombencephalon and mesencephalon acts as an organizing center. If the midbrain-hindbrain junction region is included in inverted reimplants or transplanted to the diencephalon or rhombencephalon, the tissue maintains its developmental fate and also induces the surrounding cells to form mesencephalic or metencephalic (mes-met) structures (reviewed in Puelles et al., 1996). The growth factor FGF8 is expressed at the mes-met boundary and its application to caudal diencephalon can mimic the effects of a mes-met graft (Crossley et al., 1996). Genes belonging to the Wnt, Pax, En, and Gbx families, namely W n t l , Pax2, P a d , Pax8, En1 , En2, and Gbx2, are specificallyexpressed around this boundary in an highly dynamic way (reviewed in Bally-Cuif and Wassef, 1995; and in Joyner, 1996); it is also noteworthy that the Drosophila homologs, namely wingless, paired, and engrailed, are together involved in estabilishing embryonic intersegmental boundaries in the fruit fly. W n t l , En1 , En2, Pax2, and Pax5 have been knocked-out in the mouse and mutant embryos display various anomalies and deletions in the area of cerebellum and/or midbrain (reviewed in Joyner, 1996). Recently, the phenotype shown by mice homozygous for a naturally occurring mutation in the gene Pax2

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has been analyzed (Favor e t al., 1996). The mutated allele Pax21Neudiffers from the wild-type by a I-nucleotide insertion, which is identical to a previously described mutation in a human family with renal-coloboma syndrome (Sanyanusin et al., 1995). In homozygous mutant embryos, the development of the optic nerve as well as of the metanephric kidney is severely affected, as already described for other Pax2-1- embryos (Torres et al., 1995; Keller et al., 1994). In addition, Pax21Neuhomozygous animals display a deletion of cerebellum and posterior mesencephalon, which was not described for either Pax2-1- embryos (Torres et al., 1995;Keller et al., 1994) or reported in mutant humans (Sanyanusin et al., 1995). These mice should provide an ideal animal model for future studies on the congenital abnormalities associated with human PAX2 mutations.

4. The rhombospinal region a. The Hox genes clusters: Structure and expression A key role in patterning the anatomical regions along the body axis, from the branchial area through the tail, is played by Hox genes. They are homeobox-containing genes which encode transcription factors involved in modeling the definitive embryo shape and represent the true vertebrate homologs of the Drosophila homeotic genes. In the fruit fly embryo, homeotic genes provide biological information for specifying the identity of the various body segments; their mutations result in bizarre phenotypes wherein one body segment gives rise to structures appropriate for another one. Eight of these genes are located in two contiguous chromosomal loci called Antennupedia (ANT-C) and Bithorax (BX-C) complexes, col-

Figure 1.3. Alignment of the four vertebrate Hox loci. Paralogy groups are shown as well as a comparison with Drosophila homeotic (HOM) genes. The grey boxes represent Drosophila homeotic genes: labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), and Antennapedia (Antp), all belonging to the Antennapedia complex ( A N T C ) , and Ulmabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B), forming the Bithorax complex (BX-C). The black boxes represent the 39 vertebrate Hox genes; above each of them is the current nomenclature (Scott, 1992). They are ordered in 13 paralogy groups, labeled at the bottom; at the top, their homology relationships with Drosophila HOM genes are represented. The horizontal arrow indicates direction of transcription of Hox genes. Under it, HOMIHox modulators are listed, which seem to act by regulating the chromatin structure of the loci. Th e Drosophih gene nithorax and its vertebrate homolog MLL should allow an open chromatin conformation; conversely, Drosophila Polycomb genes and their vertebrate relatives h i - 1 , mel-18, and eed would promote heterochromatin formation. Hox genes belonging to groups 1 4 are expressed first, are necessary for hindbrain patterning and display the most prompt and efficient response to retinoic acid in vim0 (reviewed in Boncinelli et al., 1991). Hox genes of groups 5-8 are expressed later, control the patterning of the thoracic region and display an intermediate response to retinoic acid. Hox genes belonging to groups 9-13 are expressed last, control the lumbosacral region, and are the most refractary to retinoic acid stimulation.

DrosoDhila ANlr-C

Vertebrates Hox-a

HOX-c Hox-d Paralogygroups

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Anterior Early 3'

High RA response

4 ' 5 hindbrain I

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8 ' 9 10 11 12 13 thoracic I lumbo-sacral regron I regron 6

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~~ ~ ~ p a~e a n 0 bmtl mel-I8 eed

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lectively called HOM-C, for Homeotic complex. They have arisen from a single gene array that was split during insect radiation. Conversely, along the phyletic line leading to vertebrates, this common ancestral cluster has undergone two duplications giving rise to Hox gene clusters (reviewed in Kenyon, 1994). In mouse and man these complexes, approximately 120 kb in length, were analyzed in detail. Their 39 genes are oriented in the same 5’40-3’ direction of transcription (Figure 1.3). The four loci are highly homologous and can be easily aligned. Corresponding genes in different Hox clusters share the highest sequence similarity; they belong to 13 different sets of genes, which are termed paralogy groups, and are numbered from 1 to 13, starting from the 3‘ end of the various clusters. Most of the Hox genes can be correlated with specific Drosophila homeotic genes. Moreover, a unique feature of both Hox and HOM clusters is the correlation between the physical order of genes along the chromosomes and their anterior boundaries of expression along the rostrocaudal axis of the embryo (reviewed in McGinnis and Krumlauf, 1992). In vertebrates, 3’ Hox genes are expressed early in development and control anterior regions, whereas progressively more 5’ genes are activated later and control more posterior regions; members of the same paralogy group often show coincident expression boundaries. This spatial-temporal colinearity is followed by all 39 Hox genes. Such a characteristic pattern is detectable in several developing embryonic structures, namely the central nervous system, paraxial mesoderm, neural crest, limbs, and genitalia, providing a coordinate system of axial signals involved in generating different regional identities (reviewed in Krumlauf, 1994). Figure 1.4 show an updated map of the expression domains of several developmental genes along the developing central nervous system.

b. Mutations in Hox genes So far, only three inheritable deseases, namely mouse hypodactyly (Hd), human synpolydactyly, and hand-foot-genitalia (HFG) syndrome, affecting limb development, have been associated to naturally occuring mutations in Hox genes (see Section 111,C).In addition, it can be hypothesized that HOX genes, or some of their target, could be good candidates for mutations causing segmental defects of hindbrain or spinal cord development. In humans, this might include Moebius syndrome (OMIM 157900), Arthrogryposis multiplex congenita (OMIM 208100), Axenfeld-Rieger anomaly (OMIM 10920), or segmental spinal muscular atrophy (OMIM 183020).l Here, we will summarize some relevant results obtained by gene targeting strategies in mice. Such artificial mutations help us to circumvent the lack of naturally occurring mutants and provide useful cues for possible spontaneous human inherited pathologies. A large body of evidence have been accumulated on ‘OMIM (&Line Mendelian Inheritance in Man) can be contacted at the URL: http://www3.ncbi.nlm.nih.gov/ornim/

Telencephalon

Diencephalon

Mesencephalon Isthmus Rhomboncephalon

Spinal cord I

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Hoxl Figure 1.4. Expression domains of some major developmental genes in the developing central nervous system (Shimamura et al., 1995 and references therein). Telencephalon and diencephalon are shown as subdivided into six prosomeres according to the model of Puelles and Rubenstein (Shimamura et al., 1995).

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the functions played by Hox genes in controlling the regional identity of axial skeleton. Generally speaking, phenotypes derived from loss-of-function experiments show repeated structures like vertebrae or ribs to undergo anterior transformations; that is, for example, the case of Hoxc8 gene inactivation, which leads to the homeotic transformation of the first lumbar vertebra into the seventh toracic one, which bears an extra pair of ribs (LeMouellic et al., 1992).These phenotypes resemble the changes in body segments displayed by homeotic mutant flies, even though they never show a full transformation as it happens in Drosophila. Conversely, mice bearing complementary gain-of-function mutations display posterior transformations, as is the case for gain-of-function mutant Drosophila embryos. For example, Hoxd4 and Hoxa7 ectopic expression cause posterior transformations of skull occipital bones and cervical vertebrae, respectively (Lufkin et al., 1992; Kessel et al., 1990). This data support the concept of functional homology between Hox and HOM genes (reviewed in Boncinelli et d., 1994).However, this is not an invariant rule, because gain-of-function mutations in Hoxc6 and Hoxc8 lead to anterior transformations (Jegalian and De Robertis, 1992; Pollock et al., 1992). Hox genes are involved not only in patterning the axial skeleton. They also provide molecular cues for specifying the regional identity of other embryonic structures, like hindbrain, branchial arches, genitalia, and the various regions of the digestive tract. In the hindbrain, Hoxbf and Hoxb2 mutant mice fail to form the somatic motor nucleus of the VIIth nerve, which controls the muscles responsible for facial expression, leading to a facial paralysis (Studer et al., 1996; Barrow and Capecchi, 1996; Goddard et al., 1996). Features of this phenotype closely resemble the clinical signs associated with Bell's Palsy (OMIM 134100) and Moebius syndrome in humans. In the branchial area of Hoxa2-1mutant embryos, the identity of the mesenchymal neural crest of the second arch is changed into that of the first arch, resulting in homeotic transformations of second- to first-arch skeletal elements (Rijili et al., 1993). In female genitalia, the Hoxaf 0 mutation causes the homeotic transformation of the proximal part of uterus into oviducts; a parallel transformation is observed in mutant males at the juction between the epididimus and ductus deferens (Benson et d., 1996). Finally, the organization of the smooth layers of the rectum, the most caudal part of the digestive tube, is severely perturbed in Hoxdf 2 and Hoxdl3 mutant embryos (Kondo et al., 1996). In general, single Hox mutant mice display phenotypes which are much less severe than expected on the basis of their expression patterns. These defects correlate with the sites where a single Hox gene is expressed. This can be partially explained by compensatory mechanisms among different Hox genes. A n outstanding example is the axial skeletal abnormalities found in Hoxa9/Hoxd9 double mutants. Here, a homeotic transformation more extended than just the sum of the phenotypes of the individual mutations was observed and this finding may indicate a functional redundancy between the two genes in regions where

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they are coexpressed (Fromental-Ramain et al., 1996). This kind of interaction probably takes place not only between members of the same paralogy group, but also between the various Hox genes of the same cluster. Hoxb5 and Hoxb6 compound homozygous mutants display an anterior homeotic transformation of some cervicothoracic vertebrae, which is absent in mice lacking either one or the other of these genes (Rancourt et al., 1995). In all double mutant combinations and in the triple mutant generated though the knockout of Hoxa4, Hoxb4, and Hoxd4, progressively more dramatic homeotic transformations of the axial skeleton were observed and their severity increased with the total number of mutated alleles (Horan et al., 1994). This also suggests a dose-dependent requirement for functionally interchangeable gene products and that these proteins may interact on a quantitative basis. The malformations derived from targeted Hox mutations cannot always be explained as simple homeotic transformation. In particular, the complete absence of the atlas in Hoxa3/Hoxd3 compound mutant mice supports the hypothesis that Hox genes can be also necessary for the very existence of some embryonal structures and not only for their later specification (Condie and Capecchi, 1994). In keeping with that, suppression of the Hoxal function results in the deletion of rhombomere r5, reduction of r4, and loss of specific rhombencephalic nuclei (Chisaka et al., 1992).

c. Regulators of the Hox genes In Drosophila, members of the Polycomb group gene family (Pc-G) act as negative regulators of the homeotic genes by binding to sites in the HOM-C complex and inducing the formation of heterochromatin (reviewed in Lawrence and Morata, 1994). Three vertebrate homologs of these genes have been discovered: h i - 1 , mel-18, and eed. The first two show high homology and their targeted mutations offer similar results. Changes of vertebral identities occur that lead to a general posterior transformation of the axial skeleton (van der Lugt et al., 1994;Akasaka et al., 1996).That is attributable to an anterior shift of the expression domains of some Hox genes, so altering positional values in the sclerotome precursors (van der Lugt et al., 1996;Akasaka et al., 1996). In humans, h i - 1 was found to act as a proto-oncogene, whereas mel-18 appears to display tumor suppressor activity (Kanno et al., 1995). The third vertebrate homolog of Pc-G Drosophila genes, namely eed (for embryonic ectoderm development), has been identified via positional cloning of the corresponding classical mouse mutation (Shumacher et al., 1996). Mice carrying a hypomorphic eed allele exhibit posterior transformations along the axial skeleton. Moreover, mice homozygous for an eed null mutation show disruption of the primitive streak during gastrulation, which might reflect very early eed involvement in regulating anteroposterior patterning (Shumacher et al., 1996).

The Drosophila gene crithorax ( t r x ) and its vertebrate homolog MLL can

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activate HOM and Hox gene transcription, respectively, by acting on their chromatin genomic environment. Mll null mice are embryonic lethal and fail to express several Hox genes, whereas MU+/- individuals have aberrations of segment identity and corresponding caudal shifts in the anterior Hox expression boundaries (Yu et al., 1995). MLL is disrupted by chromosomal translocation in some patients affected by acute leukemias, often displaying a mixed lymphoid-myeloid phenotype. In the developing nervous system, kreiskr and krox20 genes seem to act as Hox regulators. Spontaneous kreisler mutant mice are deaf and lack presumptive r5 and r6 territories. The early absence of krox20 and group 4 Hox gene expressions in these areas suggest a possible involvement of Kreisler in their functional regulation, even if a direct interaction has yet to be shown (Frohman et al., 1993). In contrast, Krox20 is a direct modulator of the r3/r5 expression of both Hoxu2 and Hoxb2 (Nonchev et al., 1996). A key role in regulating the overall Hox gene expression is played by retinoic acid (RA), a molecule belonging to the group of retinoids that are vitamin A (retinol) derivatives. RA has been known for long time to produce teratogenic effects in humans (Sporn et al., 1994). The first hint about the functional relationship between RA and Hox genes came from the sequential induction of their expression observed in embryonal carcinoma cell lines upon treatment with exogenous RA (reviewed in Boncinelli et al., 1991). Hox genes located at the 3' end of the four loci are activated first and respond to relatively low concentrations, whereas progressively more 5' Hox genes are activated later and require higher concentrations of RA (Figure 1.3).In vivo experiments subsequently confirmed the importance of its role in normal development of several embryonic structures (reviewed in Conlon, 1995; Kastner et al., 1995). Exposure of murine embryos to teratogenic doses of different retinoids can induce anterior shifts of Hox gene expression domains in paraxial mesoderm that elicits, later in development, posterior transformations of several vertebrae along the axial skeleton (Kessel and Gruss, 1991). Similar manipulations also lead to complex malformations in the central nervous system. In the hindbrain, exogenous RA causes transformation of rhombomere r2 into an r4 identity, a phenomenon associated with changes in Hoxbl expression (Marshall et al., 1992). This findings provide cues about the mechanisms by which RA might induce teratogenesis in the human embryo.

B. Dorsoventral patterning

1. Role of notochord and floor plate The fate of cells located a t different dorsoventral positions within the neural tube and the paraxial mesoderm depends upon signals that derive initially from adja-

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cent axial mesodermal cells forming a notochord. These signals induce the differentiation of ventral cell types within the neural tube and the paraxial mesoderm and suppress the differentiation of dorsal fates. Floor plate cells differentiate in the neuroepithelium immediately adjacent to the notochord, motor neurons in the ventral lateral neuroepithelium, and sclerotome cells in the ventralmost paraxial mesoderm. Induced floor plate cells now share with the notochord its patterning activities, so that a source of ventralizing signals is available in close proximity to the neuroepithelium and paraxial mesoderm even after the ventral displacement of the notochord, which occurs as development proceeds (reviewed in Placzek, 1995).

2. Sonic hedgehog A secreted protein encoded by a gene termed Sonic hedgehog (Shh) is expressed specifically in the notochord and in the floor plate as well as at the posterior edge of limb buds (reviewed in Ingham, 1995, and Roelink, 1996): this suggests it can mediate dorsoventral patterning properties exhibited by these structures. Support for this suggestion comes from experimental expression of the Shh gene or placement of SHH protein-releasing beads at ectopic locations in developing embryos, as well as from treatment of explanted embryonic target tissue with the purified protein. In the first case, SHH mimics notochord and floor plate by promoting expression of ventral markers in neural tube, brain, eye and somites (reviewed in Ingham, 1995; and Roelink, 1996; Ericson et al., 1995; McDonald et al., 1995). In the second, purified SHH protein induces ventral cell-specific genes, with floor plate markers induced at higher concentration and motor neurons and sclerotome markers at lower (Roelink et al. , 1995; Fan et al., 1995). Knock-out mice confirm Shh involvement in dorsoventral patterning of vertebrate embryonic tissues, including brain, spinal cord, and axial skeleton. Early defects are detectable in the establishment or maintenance of midline structures, like notochord and floor plate; later defects include the absence of spinal column and most of the ribs, the absence of ventral cell types in the neural tube, cyclopia, and defects in distal limbs (Chiang et al., 1996). Heterozygous alterations in the Shh locus are specifically associated with the occurrence of some cases of holoprosencephaly in our species. Holoprosencephaly (HPE) is a common developmental defect of the forebrain and the midface in humans, involving incomplete development and septation of midline structures in the central nervous system with a broad range of clinical severity. Alobar HPE, the most severe form, involves complete lack of division of the forebrain into two hemispheres and is associated with facial anomalies including cyclopia, a primitive nasal structure (the so-called proboscis) and/or midface clefting. A t the mild end of the spectrum there are microcephaly, mild hypertelorism, single maxillary central incisor, and other defects (Cohen, 1989a,b; Cohen and Sulik, 1992).The phe-

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notypic variability is common also between affected members of the same family. A t least four types of familial forms have been described with autosomal dominant or recessive inheritance. Cases of autosomal dominant holoprosencephaly have been reported to be associated to different types of mutations at the HPE3 locus (Roessler et al., 1996, Belloni et d., 1996).In humans, loss of one SHH allele is sufficient to cause HPE, whereas both alleles need to be lost in Shh-/- mice to produce a similar phenotype (Chiang et d., 1996). The presence of only one copy of SHH in humans, though able to disturb ventral midline neurogenesis, is not sufficient to cause defects stemming from somite ventralization or limb abnormalities characteristic of Shh-1- mice (Belloni et al., 1996; Roessler et al., 1996).

111. PATTERNING THE LIMB A. Limb development The limb is one of the best characterized morphogenetic fields in the developing mammalian embryo, thanks to its accessibility to experimental manipulations as well as to the possibility of extrapolating to mammals the large body of evidence accumulated in chicken. In the limb bud, the apical ectodermal ridge (AER), namely a thickening of the distal ectoderm of the bud, stimulates the proliferation of mesenchyme present in the underlying progress zone (PZ). The interaction between ridge and mesenchyme is reciprocal and a signal from the latter maintains the former. During limb bud elongation, as cells leave the PZ, they lay down structures along the proximodistal axis of the limb in sequence, starting with proximal structures and progressing distally. A region of the posterior bud mesenchyme, called the zone of polarizing activity (ZPA), has the ability to direct the formation of mirror-image duplications when transplanted to the anterior margin of a host limb bud. This has been proposed to be a source of a morphogen, the concentration of which could provide anteroposterior positional information to limb bud cells. Finally, ectodermal signals control the dorsoventral patterning of mesenchyme, which determines the disposition of tendons and muscles (reviewed in Johnson et al., 1994; Xckle, 1996).

B. Genes encoding secreted molecules Candidate molecules have been identified which could mediate the patterning function played by the AER, ZPA, and dorsal ectoderm (DE) (see Figure 1.5A). Shh is expressed in the ZPA and mimics some properties of the ZPA when applied to the anterior edge of the limb bud (reviewed in Tickle, 1996). Retinoic acid also mimics the ZPA and is present in the limb bud where it has been suggested to act

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Figure 1.5. Genes involved in patterning the vertebrate developing limb. In A the expression of genes encoding secreted molecules in the limb bud and their cross-talk are represented. Sonic hedgehog (SM) is expressed at the posterior edge, while various FGFs (including FGFZ, FGF4, and FGF8) are present along the distal margin of the bud; the dorsal ectoderm is marked by Wnt7a. A positive feedback loop takes place between SM and Fgf4, whereas Wnt7a stimulates Shh expression (bent arrows). In B the expression domains of some homeobox genes in mid-late developing limb are summarized. Lmxl transcripts are in the dorsal mesenchyme, whereas En1 products are in the ectoderm lining the ventral half of the limb. Genes belonging to the 5' end of the Horn cluster are restricted in a proximal4istal way: Howl 3 transcripts are in the autopod anlage, Horn11 in the developing zeugopod, HoxulO is expressed throughout the field. Conversely, genes of the 5' region of Hoxd (d13, d12, dl I , dIO, and d9) are restricted along the anterior-posterior axis. Their Russian doll-shaped domains share the posterior margin, at the caudal edge of the limb, and have anterior boundaries colinear with the position of the gene within the cluster: the rostralmost being d9 and the caudalmost being d13.

upstream of Shh (Helms et al., 1996), but its function in this field remains to be clarified. Molecules belonging to the FGF family, namely FGFZ, FGF4, and FGF8, are variously expressed in the AER: the application of them to a limb bud, from

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which the AER has been previously removed, leads to limb outgrowth. Wnt7a is specifically expressed in the DE and it has been suggested to mediate DE action. It has been shown that the various sources of patterning signals cross-talk between each other: positive feedback loops take place between Shh and Fgf4 and Wnt7a sustains Shh expression (reviewed in Tickle, 1996; Cohn and Tickle, 1996). Mice lacking Wnt7a activity have been generated and their limb mesoderm displays dorsal to ventral transformations in cell fate. Many mutant animals also lack posterior digits (Parr and McMahon, 1995). This may be attributed to the interruption of the positive feedback loop normally occurring between Wnt7u and Shh and to the physiological role played by the latter in promoting the development of posterior digits. In Shh-1- mice the distal structures are most affected, with a complete absence or fusion of the zeugopod bones: in the hindlimb, the most severely affected, the femur, is formed, but tibia and fibula are completely absent; in the forelimb, a bony extension of the humerus may represent either a fused ulna/radius or a long, bent humerus. A reasonable link between these distal truncations and loss of Shh function could be the apparent role of FGFs in promoting limb outgrowth and the requirement for Shh in the maintenance of Fgf4 expression in the posterior AER (Chiang et al., 1996). Patients bearing one SHH mutated allele and affected by holoprosencephaly, like Shh+/- mice, don’t display any limb phenotype (Roessler et al., 1996; Belloni et al., 1996). Finally, retinoids have been described to cause teratogenic effects in limbs, like digit truncations and reductions in long bones (Sucov et al., 1995).

C. Homeobox genes and limb development Positional information within this field is probably first conveyed as a graded concentration of diffusible molecules along the three main limb axes and is subsequently translated into the expression levels of some homeobox genes. Lmxl is expressed in dorsal mesenchyme in response to Wnt7a signaling and appears to mediate the dorsalizing effects acted by it (Riddle et al., 1995; Vogel et al., 1995). En2 is expressed in ventral limb ectoderm (see Figure 1.5B): it is necessary for the proper ventral limb patterning and acts in part by repressing dorsal differentiation induced by Wnt7a. Enl-1- mice display dorsal transformations of ventral paw structures and dorsal duplications of ventrodistal structures (Loomis et al., 1996). The Horn and Hoxd expression patterns are highly dynamic: in the midlate developing limb, genes belonging to the 5’ end of the Hoxa cluster (a23, al2, and a20) are restricted in a proximodistal way; genes at the 5‘ of Hoxd (d23 , d2 2 , d2 2 , d20, and d9) are restricted along the anteroposterior axis (see Figure 1.5B for more details) (reviewed in Johnson et al., 1994). It has been proposed that Horn and Hoxd genes can simply pattern the limb along these two axes, respectively, by giving rise to combinatorial code units able to specify the identity of all major morphogenetic subfields within the limb itself. The mirror-image duplication of the H o d expression pattern, which can be obtained together with the analogous

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duplication of the digital pattern by applying RA-releasing beads to the anterior border of the limb (Izpisua-Belmonte et al., 1991), seems to be a good support for that. However, this model is an oversimplification and must be reconsidered more carefully. All Hox genes involved in limb patterning have been knocked out one by one and, via genetic crosses, in different combinations; in addition, among these genes are the only two Hox genes that have been demonstrated to be affected in naturally occurring mutants, namely Hornl3, involved in murine hypodactyly and human HFG syndrome, and HOXD13, involved in human s p polydactyly (see below). The phenotype displayed by some of these mutants casts some doubts on the simple patterning model described above. Functional redundancy and extensive expression overlapping between genes belonging to the same paralogy group could explain some discrepancies between our expectations and the actual situation. That is the case for Hoxa genes: their knock-outs yield roughly the expected phenotypes, but only if associated to the ablation of the corresponding Hoxd paralogs. For example, the zeugopod derivatives, radius and ulna, still present in Hoxal J -1- mice, even if abnormal (Small and Potter, 1993), and are completely absent in the double-mutants Hoxal 1 -/-/Hoxdll-/- (Davis et al., 1995). In a similar way, Hoxal3-/-/Hoxdl3-/- double knock-outs lack almost any chondrified condensation in the autopods (Fromental-Ramain et al., 1996), at variance with hypodactyly Hd/Hd mice (Mortlock et al., 1996) and artificially generated Hoxal3-l- mice (Fromental-Ramain e t al., 1996), which display one or more fingers. However, relevant difficulties remain about the function postulated for Hoxd genes. In chicken tulpid mutants, for example, all 5' Hoxd genes are expressed uniformly along the anteroposterior axis of the limb and a large number of morphologically similar digits are present. However, they look like third digits and not fourth, as predicted by the model. In addition, Hoxdl3-1mice (Doll6 et al., 1993) don't display any digit V-to-digit IV homeotic transformation, as expected, but simply a reduction in size of the digits and delay in their development. It has been proposed (Doll6 e t al.,1993) that the main role of Hoxd genes in the developing limb is not in specifying positional identities of already formed blastemas, but rather in molding their shape by regulating their proliferation, growth, and differentiation. The model of Shubin and Alberch (1986) can possibly help in resolving these difficulties. In this model, the autopod is a skewing of the topological proximodistal axis of the limb, which runs from the ulna through the ulnare and then bends anteriorly through the distal carpals. According to Shubin and Alberch, the five digits share all the same anteroposterior positional value, which is in good agreement with the late expression of Hoxdl3, spreading up to the digit I anlage. For this reason, Hoxd genes, acting in limb as selectors of anteroposterior identity, cannot pattern the digital anlagen as identity selectors. They might do it only through different mechanisms: for example, by modulating cell proliferation and growth, as suggested by Doll6 et al. (1993). Hypodactyly (Hd) is a semidominant mutation in mice that maps in a genetic interval overlapping the Horn cluster. Hd/+ animals have a shortened dig-

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it I on all four limbs but are otherwise normal and fertile; they also display alterations in timing of ossification of specific cartilagineous elements of the autopod. Limbs of Hd/Hd animals usually have a single digit (possibly IV) and are affected by loss of some carpal and tarsal bones; the few retained carpal and tarsal elements are small. In Hd mutants a 50-bp deletion has been specifically detected in the first exon of the Horn13 gene, and it probably originated through heterologous recombination between triplet repeats. The mutation could lead to the production of aberrant proteins able to act in a dominant-negative manner by interfering with the function of other homeoproteins and/or by altering directly the expression of target genes (Mortlock et al., 1996). Remarkably, the phenotype displayed by Hd/Hd mutants is similar but not identical to the one shown by artificially generated Hoxa13-/- mice, which lack only one digit, the first (Fromental-Ramain et al., 1996). The human HOXA13 gene quite recently had been found to be mutated in a family with hand-foot-genital syndrome, an autosomal dominant, fully penetrant disorder implying hand and foot anomalies remarkably similar to those of hypodactyly in the mouse (Mortlock and Innis, 1997). These include short first metacarpals, small distal phalanges of the thumbs, short middle phalanges of the fifth fingers, and fusion or delayed ossification of wrist bones. Similarly, in the feet, the great toe is shorter due to a short first metatarsal and a small, pointed distal phalanx. Associated to these limb anomalies are also uterine anomalies typically involving a partially divided (bicomate) or completely divided (didelphic) uterus, represeting defects of Mullerian duct fusion. The nonsense mutation found in this family causes a truncation of the corresponding HOXA13 protein before the end of the homeodomain. Mutations in HOXD13 have been found in five families affected by synpolydactyly, an autosomal, dominantly inherited human abnormality of the hands and feet (Muragaky et al., 1996; Akarsu et al., 1996). Heterozygotes display proper synpolydactyly, namely a malformation involving both webbing between fingers and duplication of them. The homozygous phenotype includes the transformation of metacarpal and metatarsal bones to short carpal- and tarsal-like bones. Affected people of these families were found to contain a HOXD13 gene mutated in a novel way. Many HOX homeoproteins are known to contain in their amino-terminal portion a number of short sequences of repeated amino acid residues, mostly alanine and serine, but sometimes also glutamine and proline, that are believed to participate in the function of gene repression, or activation, exerted by this region. In particular, in the HOXD13 homeoprotein there are two serine stretches and one alanine stretch consisting of 15 residues in a row. Affected individuals were found to be heterozygous or homozygous for an expansion of this alanine stretch. In the various families analyzed, this expansion ranges between 7 and 10 additional alanine residues as compared to the wild-type situation (Mugaraky et d., 1996; Akarsu et d., 1996). All mutated chromosomes within a given family exhibit the same type of expansion, 7, 8, 9, or 10 additional residues,

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showing that the situation is relatively stable through several generations. These mutations are not likely to act through a mere loss of function mechanism. In fact, mice lacking the corresponding Hoxdl3 gene show a similar but distinct phenotype (Doll6 et al., 1993). Conversely, the authors propose that the observed expansions of the alanine tract may alter the function of the protein. For example, the mutated protein may still be able to interact with DNA but may not be able to interact with other proteins. The hypothesis of gain of abnormal function is potentially supported by the observation that mice simultaneously lacking Hoxdl3, Hoxdl2, and Hoxdl 1 genes show a synpolydactyly phenotype (Zakany and Duboule, 1996).

IV. PATTERNING THE OCULAR ANLAGE A. Eye formation The vertebrate eye originates through the reciprocal interaction between two tissues early in the development. At the late headfold stage, the optic sulcus is formed by an evagination of the presumptive forebrain neuroectoderm: this grows out to form the optic vesicle, which approaches the surface ectoderm and remains connected to forebrain through the so-called optic stalk. When the optic vescicle reaches the surface ectoderm, a thickening of the surface ectoderm, also called the lens placode, becomes apparent; later on during development, this placode invaginates forming the lens vesicle and finally the lens. The optic vesicle invaginates to form the two-layered optic cup surrounding the developing lens: the outer layer of the cup forms the pigmented epithelium, the inner layer differentiates into the neuroretina. Finally, the ectoderm overlying the lens and some associated mesenchyme gives rise to the cornea. Two key points in this process are the refinement of the positional information along the proximodistal axis of the developing optic vesicle, which preceeds its subdivision into optic stalk, pigmented retina and neuroretina, and the restriction of lens forming properties to the overlying placodal ectoderm. Plenty of genes have been demonstrated to be expressed specifically in the developing eye: among them at least three homeobox genes, namely P a d , Pax6, and Chxl 0, are likely to play a key role in these patterning processes.

B. Homeobox genes and the patterning of the eye anlage As reported elsewhere, Pax2 and Pax6 display a wide and complex expression pattern (reviewed in Chalepakis and Gruss, 1996).Among the optic vesicle derivatives, Pax2 expression is confined to cells within the optic vesicle that contribute to the optic stalk and parts of the ventral retina around the choroid fissure, which is the domain of retina where ventral nasal and ventral temporal retina fuse to create the closed optic cup. Conversely, Pax6 transcripts are restricted to the op-

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tic cup, being absent in the optic stalk (reviewed in McDonald and Wilson, 1996). In zebrafish cyclops mutants, where SM expression is reduced, Pax6 expression expands toward the medial portion of the optic recess and these cells acquire some properties characteristic of retina. Conversely, in wild-type embryos microinjected with Shh synthetic mRNA complementary alterations occur. The Pax2 expression domain expands and there are more cells with features typical of optic stalk derivatives. These findings are in keeping with the idea that Pax2 and Pax6 could promote the differentiative programs specific for derivatives of optic stalk and optic cup, respectively (McDonald et al. 1995).Chxf 0 RNA is detectable in neuroblasts of the inner layer of the optic cup and in part of their derivatives (Burmeister et al., 1996). In addition, Pax6 products, originally detectable throughout the anterior ectoderm, become subsequently confined to the lens and corneal anlage (reviewed in McDonald and Wilson, 1996). A mutational analysis of PAX2 has been conducted in a human family with optic nerve colobomas, renal hypoplasia, mild proteinuria, and vesicouretral reflux, and a heterozygous point mutation has been found, causing a frame-shift of the PAX2 coding region in the octapeptide domain (Sanyanusin et d., 1995). Noticeably, optic nerve colobomas, in which the choroid fissure fails to close, occur also in mice lacking Pax2 (Torres et al., 1995; Keller et al., 1994). PAX6 has been shown to be involved in semidominant developmental disorders implying eye defects, like Aniridia in man and S d eye (Sey) in mice and rats. Heterozygous Sey-’+ mice show defects in both optic cup and placodal derivatives, like reduced eye, iris hypoplasia, and lens vacuolization and dislocation; humans affected by heterozygous Aniridia mutation display iris hypoplasia, cataract formation, and corneal vascularization (reviewed in Hanson and Van Heynigen 1995).Homozygous Sey mice lack eyes and nasal cavities and exhibit brain abnormalities (Schmal et d., 1993);in a similar way, humans compound heterozygous for mutant PAX6 alleles display bilateral anophthalmia with fused eyelids, a small malformed nose, and severe forebrain abnormalities (Glaser et d.l 1994). Blocking the expression of the homeobox gene ChxlO in zebrafish embryos results in delayed development and reduction of neuroretina and in microphthalmia (Barabino et d., 1997). Recently, an involvement of ChxlO in ocular retardation in the mouse has been reported: nonsense mutations within its homeodomain affect proliferation of retinal progenitors and differentiation of bipolar cells in the retina (Burmeister et al., 1996).

V. PATTERNING THE TOOTH ANLAGE A. Tooth development Odontogenesis takes place on both maxillary and mandibular processes of the first branchial arch. It requires the correct positioning of the odontogenetic field along

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the lingual-buccal axis of the developing branchial arch processes as well as a finer specification of medial-lateral positional values inside this field, related to the capability to give rise to different dental types (incisors, canines, premolars, and molars). The epithelium derived from the oral ectoderm forms the enamel organ, while the remainder of the tooth and its supporting tissues are derived from the mesenchyme of the first branchial arch (Lumsden, 1988). The process encompasses a complex series of epithelial-mesenchymal interactions by which the hystogenetic properties of the two forming tissues are refined and information concerning positional identities is reciprocally transferred.

B. Homeobox genes and patterning the tooth anlage It is reasonable to hypothesize that some of the homeobox genes expressed in the first branchial arch could bear positional information necessary to set up the correct onset and progression of the odontogenetic process: Otlx2/RIEG and Msxl are among them. Otlx2IRIEG expression, originally distinguishing stomodeum from other ectoderm, becomes subsequently restricted to the teeth forming areas (Mitsiadis,personal communication). The distal ectomesenchymeof first branchial arch processes is patterned by Msxl and Msx2: at the lingual border of their domains, a stripe of Msxl +/Msx2- mesenchymal cells corresponds exactly to the overlying primary epithelial thickening, namely the area from which tooth buds will develop (reviewed in Sharpe, 1995). It has been suggested that lesions in any of these genes could interfere with the onset and/or the progression of tooth formation. RIEG, the human homolog of murine RieglOtlx2, has been identified and shown to be mutated in cases of Rieger syndrome, an autosomal dominant human complex disorder that essentially includes three cardinal features, namely hypodontia (both oligo- and microdontia), developmental problems in the anterior chamber of the eye, and umbilical anomalies (Semina et d., 1996).In a humanfamily with autosomal dominant tooth agenesis mapped to chromosome 4~16.1,a missense mutation within the MSX 1 homeodomain was found to segregate at high penetrance with the defect: all affected individuals lacked both maxillary and mandibular second premolars and third molars; some of them lacked other teeth too (Vastardis et d., 1996). These findings are in keeping with data from phenotypic analysis of MsxI-/- mice (Satokata and Maas, 1994). Finally, it was suggested that Dlxl and Gsc,by patterning the mandibular ectomesenchyme along the medial-latera1 direction, could specify the type of tooth which will develop in each area (molar in Dlxl +/Gsc-,canine in Dlxl +/Gsc+,and incisor in Dlxl-/Gsc+ areas, respectively) (Sharpe, 1995). This prediction has not yet been confirmed.

Acknowledgments We thank Thimios Mitsiadis, who provided us data before their publication; we are also indebted to Wolfgang Wurst for interesting and helpful discussions. Work of our group is supported by grants from

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Telethon-Italia Programme, the EU BIOMED and BIOTECH Programmeand the Italian Association for Cancer Research, AIRC.

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Hereditary Ataxias Massimo Pandolfo*st**and Laura Montermini* *Centre de Recherche Louis-Charles Simard tDepartement de Medecine, Universite de Montreal $Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, H2L 4M1 Canada

I. Introduction 32 II. The autosomal dominant progressive ataxias 34 A. Genetic classification of the autosomal dominant ataxias 35 35 B. Triplet-repeat expansions in the dominant ataxias C. Polyglutamine proteins in the dominant ataxias 39 D. Cloned dominant ataxia genes 40 E. Mapped dominant ataxia genes 47 F. Epidemiology of the autosomal dominant ataxias 49 111. Friedreich ataxia 49 A. Pathology 50 B. Clinical features 51 C. Friedreich ataxia gene 52 D. Frataxin 53 E. Point mutations in the frataxin gene 54 55 F. FRDA-associated GAA triplet repeat expansion G. Molecular mechanisms of disease in Friedreich ataxia 57 H. Phenotype-genotype correlations in Friedreich ataxia 58 60 References One of us (MP) learned about the mapping of Huntington disease gene to chromosome 4 from the late Dr. Anita Harding. She got the news over the phone from her London office during a visit to Italy for a meeting on hereditary ataxias. In Advances In Genetics, Vol. 38 Copyright 0 1998 by Academic Press All rights of reproduction in any form reserved. @@65-266@/98 $25.00

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Britain, they receive Nature at least a week earlier than us. Dr. Harding was very excited, and she immediately said that that was the way to go if we wanted to understand the causes of hereditary ataxias, classify these diseases in a rational way, and eventually find a treatment. At that time, the challenge seemed, and indeed was, formidable. No clue was then available about the genetic basis of what Dr. Harding aptly called “hereditary ataxias of unknown cause,” their classification was confused and controversial, and all attempts to find specific biochemical abnormalities had failed. Fourteen years later, the success of the molecular genetic studies is astounding. The defective genes have been identified for Friedreich ataxia, the major recessive “hereditary ataxia of unknown cause,” and for five dominantly inherited “hereditary ataxias of unknown cause.” Three more dominant ataxia genes have been mapped. The molecular pathogenesis of the dominant ataxias begins to be unraveled and animal models have been and are being developed. Information is also quickly accumulating about the defective protein in Friedreich ataxia. Direct molecular diagnosis is now possible. Classification has been revolutionized. Diagnostic criteria are being redefined in the light of the molecular discoveries. The goal of this review, dedicated to the memory of the late Dr. Harding, is to offer a concise summary of current knowledge about the molecular genetics of some of the hereditary ataxias that used to be classified as of “unknown cause.”

The hereditary ataxias are a heterogeneous group of inherited neurodegenerative diseases whose clinical presentation is characterized by loss of coordination of movements (ataxia) as a main feature. Ataxia may be part of the picture in many neurological disorders, but it may be a late, variable, or minor problem compared to other symptoms. Conversely, in the hereditary ataxias, it is a cardinal feature appearing early in the course of the disease. The common pathological substrate of the inherited ataxias consists in the atrophy of parts of the neural system formed by the cerebellum and its connections. Many clinical and pathological variations occur on these common themes. As a consequence, classification of the hereditary ataxias has long been a problem, raising endless controversies. Many older classifications focused on pathological findings, difficult to univocally relate to clinical manifestations and impossible to substantiate in the living patient, at least before the development of the modern imaging techniques. Harding’s classification ( 1983) represented a major effort to systematize the field (Table 2.1). In her scheme, hereditary ataxias are classified according to the genetic defect when known or according to the salient clinical features. Four groups are defined: congenital ataxias, ataxias due to known metabolic defect; early-onset (20 in most cases) ataxias of unknown cause. The great value of this classification is that it is based on data that can be obtained by clinical examination and laboratory testing, so it can be applied to the living patient. Furthermore, it is a dynamic classification,designed to change as our understanding of these diseases increases as a consequence of the progress of molecular genetic research. In this review, Harding’s classification will be utilized as reference framework, relating to it all the genetic entities that are being defined. The focus will be on some diseases that changed status from “ataxia of unknown cause” to diseases whose genetic defect has been identified. These include several dominantly inherited, progressive, usually late-onset ataxias and Friedreich ataxia, generally an early-onset disorder. Congenital, metabolic and intermittent ataxias, as well as ataxia-teleangiectasia, will not be discussed here.

II. THE AUTOSOMAL DOMINANT PROGRESSIVE ATAXIAS The dominantly inherited degenerative ataxias are probably the group whose classification has been most confused and controversial. Harding (1982, 1983, 1984) used the acronym ADCA (autosomal dominant cerebellar ataxia) to label these disorders and distinguished several types according to clinical features that are consistently observed within families. This choice led to the gathering of several genetically distinct entities under the same label, but generally prevented the opposite from happening; that is, to classify the same disease under different headings. Consequently, as ataxia genes are being mapped and cloned, it becomes possible to single-out specific genetic diseases from the each of the broader clinically defined groups identified in the Harding classification. Most dominant ataxias belong to the ADCA I group. Patients with ADCA I may show, in addition to progressive ataxia usually developing after age 20, ophtalmoplegia, optic atrophy, extrapyramidal signs, and dementia. Machado-Joseph disease was explicitly included in this group. The association of pigmentary retinopathy defines ADCA 11. This feature is either present or absent in all affected members of a family, appearing therefore to be a specific and universal manifestation of the gene defect of ADCA 11. ADCA 111is a “pure” cerebellar ataxia; that is, not associated with other neurological signs or symptoms that may indicate involvement of other structures, usually appearing after age 50. This is again a syndrome showing consistency within affected families. Other ADCAs are rare disorders, among which the intermittent ataxias (ADCA V) are notable for their clinical peculiarity of presenting as recurring acute episodes of imbalance and incoordination. The diseases included in the ADCA V group are due to mutations in specific ion channels and will not be discussed here. For dominant ataxias, it is useful to also briefly summarize other classifications, particularly those based on neuropathological findings, as they are still

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commonly referred to in the literature. One problem with these classifications, in addition to the practical inapplicability in the living patient, is the frequent lumping of genetic cases with sporadic, probably nongenetic cases and sometimes the lack of consideration of the type of inheritance of the genetic cases. The main neuropathological distinction is made between degenerative processes involving the cerebellar cortex and the inferior olives (cerebellar cortical or cerebelloolivary atrophy, CCA) and multisystem degenerations involving cerebellum, brain stem, pyramidal tracts, spinocerebellar tracts, and posterior columns of the spinal cord (olivopontocerebellar atrophy, OPCA) (Critchley and Greenfield, 1948). In addition, degenerative processes involving pontine nuclei, pyramidal tracts, spinocerebellar tracts, and posterior columns of the spinal cord but sparing the cerebellar cortex have been recognized (spinopontine atrophy, SPA). Each of these groups has been further subdivided. A much followed classification of OPCAs is that of Koenigsmark and Wiener (1970), who identified five subtypes (OPCA I to OPCA V). OPCA I1 is autosomal recessive, OPCA 111 corresponds to Harding’s ADCA 11, while the other hereditary OPCAs fit in the ADCA I group.

A. Genetic classification of the autosomal dominant ataxias We now know the mutated genes in several ADCAs and the chromosomal localization of a few more. These discoveries have been accompanied by the development of a system of classification in which each entity is defined on the basis of the involved gene, and it is often difficult to relate this genetic nomenclature to the previous clinicopathological classifications. Most ADCA genes are named SCA (for spinocerebellar ataxia), followed by a progressive number indicating the various involved loci, approximately in the order by which they were mapped. So, SCAl is the first ADCA gene that was localized to a specific chromosome, SCA2 the second, and so on, with the exception of SCA6, which was mapped after SCA7. Somewhat confusingly, SCA8 is instead a recessive ataxia gene. Some ADCA genes have also retained the name given to the disease when it was originally described, as is the case for Machado-Joseph disease (MJD), which coincides with SCA3. At the time of writing of this chapter, four ADCA genes have been identified (SCA1, SCA2, SCA3, and SCA6), and three more have been mapped to specific chromosomal regions (SCA4, SCA5, and SCA7).

6. Triplet repeat expansions in the dominant ataxias All so-far identified genes for ADCAs show the same type of mutation (the unstable expansion of a CAG trinucleotide repeat polymorphism localized in the protein-coding region of the respective gene). In this regard, ADCAs are akin to other neurodegenerative disorders such as Huntington disease (HD), dentatorubropallidoluysian atrophy (DRPLA), and spinobulbar muscular atrophy

36

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(CAG)n

-rl

-

SCA 1

+

SCA 2

(CAG)n

SCA 3

-1Kb

Figure 2.1. Position of CAG trinucleotide repeats within three cDNAs from genes involved in autosomal dominant degenarative ataxias. The protein coding region in each cDNA is shown as a box, the 5‘ and 3‘ untranslated regions are shown as lines. In all three cases, the CAG repeat is localized within the protein coding region.

(SBMA, Kennedy disease) (Willems, 1994). Figure 2.1 shows the location of the SCA1, SCA2, and SCA3 repeats within the respective genes. The normal and disease-associated size ranges for each of these repeats are shown in Table 2.2. The CAG triplet repeats associated with dominant ataxias share many common features. In normal chromosomes the repeats are polymorphic, although less so in SCA2 (see below). Normal alleles are transmitted from parent to child without changes in size (i.e., they are meiotically stable). Analysis of different tissues from the same individual shows that normal alleles are of the same size in all samples (i.e., they are mitotically stable). Polymorphism in normal alleles likely originated from rare events in which, during replication of the DNA segment corresponding to the repeat, the newly synthesized strand momentarily dissociated from the template strand and reassociated out of register. This “slippage” mechanism has probably generated all simple sequence repeat polymorphisms (Wells, 1996). Disease-associated repeats contain more triplets then normal ones, from a few more to 2-3 times as many, up to about 10 times in some SCA3 cases. They are highly unstable during parent-to-child transmission, so much that size changes are seen in essentially all parent-child pairs. Mitotic instability has also been observed, so far for SCAl and SCA3, resulting in heterogeneity in the size of the expanded repeat in most tissues (Chong e t al., 1995; Lopez-Cendes et al., 1996). Although the size of disease-associated repeats varies among SCAl, SCA2, and SCA3, in these and in the other CAG repeat-containing disease genes (HD, SBMA, and DRPLA), normal alleles always have less than 35-40 triplets, suggesting that a general threshold exists beyond which CAG repeats become unstable and are liable to larger expansions. It has been proposed that such a threshold coincides with the acquisition of a secondary structure, possibly a hairpin, by the DNA strand containing a repeat of that length (Gacy et al., 1995; Wells, 1996). When this occurs in the newly synthesized strand during DNA replication, it causes a large slippage of the polymerase and reiterative DNA synthesis result-

Table 2.2. Autosomal Dominant Ataxias with Identified Triplet Repeat Expansions ~~~~

~~

~~

~

Repeats (CAG) Name

Harding classification

Chromosomal localization

Normal

Expansion

Interruptions in normal alleles

SCAl

AKA1

6p23

Ataxin 1

50% difference in reduction of RpS3 transcript between the two alleles this cannot be a likely explanation. Nor would it seem possible that the 3' penultimate uAUG with its premature terminator codon is responsible for the reduced RpS3 mRNA in RpS3pv9.

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Instead, it is more likely that it is the inserts per se that cause the reduction in transcript amount; the longer the insert the stronger the phenotype/suppression. We are presently analyzing nuclear and cytoplasmic RNA to try to pinpoint at what level these inserts affect gene expression.

4. RpS5 [M(1)15D] a. Gene and mutant phenotype In a genetic and molecular analysis of region 15E on the X chromosome, the gene encoding ribosomal protein RPS5 was identified as M(1)15D (McKim e t al., 1996). The two alleles investigated, M(f)f5D’ and M(1)f5D2, are associated with a 6-kb insertion of unknown DNA and a breakpoint rearrangement, respectively, that affect the genomic region that produces the 0.8 kb RpS5 transcript. A third allele, M(f ) 15DR28t”, was EMS-induced. Since RpS5 was mapped to 15E, perhaps the correct name of M( I ) 15D should be M( I ) 15E. The Drosophila S5 protein shows a very high conservation, with 86% sequence identity to the rat protein (McKim et d., 1996). Ribosomal protein S5 has homologs both in the eubacterial and archaebacterial kingdom and thus belongs to the Group I ribosomal proteins (Wool et al., 1995). McKim et al. (1996) also studied X-ray-induced lethal mutations in region 14 and two of the lethal complementation groups defined genetically separable Minute loci, M(1)14C andM(1)fqE; the M(f)14C locus most likelycorresponds to a Minute locus mapped to this region by Schalet (1986) and Stanewsky et al. (1993). Some interesting observations were made regarding combinations with these three Minutes. Whereas double-mutant heterozygous females with the genotype M(1)14E +/+ M(1)15D (both point mutations according to McKim et al., 1996) were fully viable, the M(1) f4C +/+ M(1) f4E heterozygotes were lethal. However, M(1) 14Cas well asM(1)14E were X-ray induced (McKim etal., 1996) and may therefore have overlapping deletions which could explain the lethality ofM(f)14C M(1)14E. With respect to M(1) 14E and M(f ) 15D, heterozygosity for either a single deficiency uncovering both Minutes [Df(l )BK14/+; no breakpoint was given for this deficiency] or two deficiencies affecting them separately [Df(1) I9 +/+ Df(1 )BK28] were lethal. Since Df( f ) f9/M(f) f4E female heterozygotes are viable and Minute (the deficiency uncovers M(f ) 14C, at least, but should also uncover M(f)14E according to Kim e t al., 1996; no data are presented on the status of Df(l)BK28/+ heterozygotes), Df(l)BKI4 and Df(I)BK28 may both uncover an essential gene. It is therefore noteworthy that yet another rp gene has been mapped to this region, namely RpS19 in 15A (Baumgartner et al., 1993).No Minute has been mapped to 15A,and this may be due to the deficiency uncovering 14E and/or 15D, which at the same time uncovers a Minute locus at 15A. Alternatively, it is pos-

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sible (but not likely) that a mutation in RpS19 may not be hemizygous lethal or even show a Minute phenotype. This is a reminder of the danger of using deficiencies when mapping Minutes. It should also be mentioned that an RPS18 cDNA clone was previously mapped by in situ hybridization to 15B (Bums et al., 1984). This was later contradicted by Ganvood and Lepesant (1994), who mapped RpS18 to region 56F on the second chromosome. These contradictory results do not exclude the possibility that 15B harbors an rp gene. To explain the survival of M(1) 14E +/+ M(1) 15D heterozygotes, as opposed to the lethality of the deficiency heterozygotes, the authors suggested that this might be due to one of the Minute alleles being a hypomorph (McKim et d., 1996). While hypomorphy has been shown to be true in some developmental stages for P-element-induced Minutes encoding ribosomal proteins (transcript variation between 30 and 80% of wild type; Szbpre-Larssenet al., 1997), we may assume that a 50% reduction (null allele) in expression of an rp gene is sufficient to produce a Minute phenotype (weak, medium, or strong). We may also assume that the sensitivity to transcript reduction varies among rp genes and we cannot rule out the possibility that two Minute mutations have a synergistic effect, as suggested by the lethality ofM(1)14C +/+ M(1)14E heterozygotes (McKim et al., 1996). However, since deficiencies were involved in this analysis some caution should be observed when interpreting these results. Interaction between two Minutes was first reported by Morgan (1929, who showed that M(2)d/+ ;M(3)d/+ heterozygotes were Minute, whereas M(2)d/+ and M(3)d/+ were wild type. Much later, Minute interaction had been reported to occur between M(3)65F and M(3)69E (Del Prado and Ripoll, 1983). These Minutes were first believed to be alleles, because they did not complement for lethality. By using a series of duplications in the region they showed that these Minutes were separated by approximately four subdivisions (65F-69E). These cases of Minute interaction should be investigated further.

5. RpS6 [M( 1)7C?] a. Gene and mutant phenotype The Drosophila RpS6 gene had previously been sequenced and shown to have the potential to encode two isoforms of the RPS6 protein via differential splicing (Stewart and Denell, 1993a). In P-element mutagenesis experiments, four lethal mutations (hen', hen2, WG1288, and aid) were isolated that affect the larval hemocytes, mediators of the insect immune response (Watson et al., 1992; Stewart and Denell, 1993b). Mutant larvae show melanotic tumors, and dying animals develop grossly hypertrophied hematopoietic organs because of overproliferation and aberrant development of hemocytes. Surprisingly, it was found that the Pelement had inserted in the 5' regulatory region of the RpS6 gene, which maps to position 20.45 on the standard genetic map and to chromosome position 7C5-Dl.

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This region harbors another ribosomal protein gene, namely RpS14 (Brown et al., 1988). This gene is, as far as we know, the only duplicated ribosomal protein gene in Drosophila. Haploinsufficiencyfor RpS14 was suggested to cause the Minute phenotype of M( J)7C (Anderson and Lambertsson, 1990), but this was later shown to be wrong (Doreretal., 1991). It wassuggested that M(1)7C is adigenic Minute, which can only be produced by the interaction between two Minutes or one Minute and another gene (Dorer et al., 1991). Such an interaction was reported by Morgan et al. (1925) for M(2)d and M(3)d (both lost), which produced a Minute phenotype only when the flies were heterozygous for both mutations. Heterozygotesfor either one alone were wild type, whereas homozygotes for either one were lethal. Interactions between Minutes have been suggested to take place also between M(1)14C and M(1)J4D (McKim et al., 1996) and M(3)65F and M(3)69E (Shellenbarger and Duttagupta, 1978).However, in the former case the Minutes were deficiencies, and therefore we cannot rule out the possibility that they uncovered more than one Minute or some other essential gene. Therefore, these results should be interpreted with caution. All hemizygous RpS6 mutant males are developmentally delayed at all stages and can remain living as third instar larvae for up to 3 weeks (Stewart and Denell, 199313).Lethality has been observed at all developmental stages but generally occurs during the extended third instar period. In air8 hemizygotes the ring gland, salivary glands, and most imaginal disks fail to reach normal size, and in the late-larval lethal phase large melanotic tumors can be seen in the body cavity (Watson et al., 1992). The fact that RpS6 mutants fail to pupate but can live as larvae for up to 3 weeks may suggest that the ring gland fails to produce enough ecdysone or that the tissues are unable to react to the hormone (cf., Brehme, 1939).It was recently shown by Herrmann et al. (1995) that mutation of the gene encoding RPS2l in Drosophila also causes tissue overgrowth of the hematopoietic organs and considerably delayed larval development. Therefore, RpS2 I may also be classified as a tumor-suppressor gene. Northern analysis of hemizygous hen’ and hen2 third instar larvae revealed that the RpS6 transcript is quantitatively reduced (apparently 0 > mRNA < 50%, but no quantitative analysis was made) in these animals (Stewart and Denell, 1993b). The level of RpS6 transcript is restored to wild-type levels in hen’ and hen2 revertant male larvae, indicating that the P-element insertions disrupt the gene. Northern analysis of mRNA from air8 hemizygotes revealed an almostcomplete absence of RpS6 transcript (Watson et al., 1992; no quantitative analysis was made). As for the hen revertants the level of RpS6 mRNA was restored to a level indistinguishable from wild-type in air8 revertants. The difference between the alleles in the expression of RpS6 may be due to the fact that the P-element in air8 inserted 10 bp from the potential transcription start site, whereas the Pelement inserted 10-20 bp further downstream in the hen alleles (Watson et al., 1992; Stewart and Denell, 1993b). The difference is quite small but emphasizes

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the importance of analyzing more than one mutant allele in studies of gene regulation and effect on phenotype. What is notable about the RpS6 insertion alleles is that the P-element insertions do not completely knock out RpS6 gene activity. The explanation for this is very likely that the P-element inserts 5’ to the transcription start site, which may allow a reduced rate of transcription. Similar results were observed in M(3)66D (Szebae-Larssen et al., 1997) and M(2)30D/E (sop; Cramton and Laski, 1994) where the P-element inserted in the same region. In contrast, insertion of the P-element in the 5’ UTR resulted in complete disruption of gene expression; that is, M(2)32A and M(3)95A (Szebae-Larssen and Lambertsson, 1996; Anderson et al., 1994). Therefore, the low rate of transcription of RpS6 in hemizygous mutant animals makes them survive up to and including the late third larval instar. Surprisingly, though, no mutant male develops to the adult stage (cf., RpS3 revertants below). It is also noteworthy that none of the mutant heterozygous females were reported to have a Minute phenotype (Watson et al., 1992, Stewart and Denell, 199313). Admittedly, weak Minutes may be difficult to classif y (and the classification is more often than not biased), but after having examined the alleles I have come to the conclusion that all heterozygous mutant females are weak Minutes (A. Lambertsson, unpublished results). Considering that the P-element mutations are not null alleles, the amount of RpS6 mRNA in heterozygous females could be around 75% of wild type, a level that may result in a weak Minute phenotype, if not wild type (cf., M(3)95A revertants below). Transcriptional control of rp genes in higher organisms is generally executed by sequences in the first intron but also by sequences both upstream and downstream of the mRNA start site (Atchison et al., 1989; Hariharan and Perry, 1990). RpS6 has a CAGCCAAC sequence at position +21 to 28 which is identical at 6 of 8 bases to the internal promoter sequence of the mouse RpL32 gene (Atchison et al., 1989). In addition, RpS6 also has a number of motifs in the 5’ region that are known parts of internal promoters of developmentally regulated Drosophila genes lacking functional TATA boxes; for example, Ultrabithorax, Deformed, Antennapedia, engaikd, white, and zeste (Arkhipova and Ilyin, 1991). The cDNA sequence for Drosophila RpS6 was also reported by Spencer and Mackie (1993). Transcription initiation of mammalian and several Drosophila ribosomal protein genes typically starts in a pyrimidine-rich tract (Mager, 1988; Stewart and Denell, 199313; Anderson et al., 1994). As already mentioned, the presence of a polypyrimidine tract has been found to be important for both promoter function and translational regulation (Chuang and Perry, 1989; Moura-Net0 et al., 1989; Hariharan and Perry, 1990; Levy e t al., 1991). It should be noted, though, that not all Drosophila ribosomal protein genes have this motif (Mager, 1988). RpS6 has three transcription start sites, two of which have the pyrimidine rich tract, and there are no TATA or CAAT motifs present in the promoter region. These

+

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transcription start sites contain the typical CTTTT sequence where transcription usually starts at the C (Andersson et al., 1994; Saebgie-Larssen and Lambertsson, 1996; Schmidt et al., 1996). Of 75 rat rp cDNAs analyzed, 40 have the pyrimidine-rich tract, which range from 4 to 20 nucleotides (Wool et al., 1995). Ten of the 40 have the sequence CTTTCC and a variant (CT, or C , or 2 ) in the others. This motif may function as a promoter that binds a warn-acting factor that regulates and coordinates translation of rp mRNAs (Wool et al., 1990; Perry and Meyuhas, 1990; Levy et al., 1991). It is interesting to note that the Drosophila RpS6 gene has the capacity to encode two isoforms of the RPS6 protein via alternative splicing (Stewart and Denell, 1993a). The predicted -0.75-kb alternate transcript was never detected on Northern blots (Stewart and Denell, 1993b), suggesting that its abundance is low or its expression is temporally and/or spatially restricted or that differential splicing does not take place. It should be recalled that RpS3 produces two equally abundant transcripts differing by about 50 nt (Andersson et al., 1994). In this case the origin of the second transcript is still not known and nothing is known about whether two proteins are produced (M. Lyamouri and A. Lambertsson, work in progress).

b. The RPS6 protein S6 is the major, but not the only, phosphorylated eukaryotic ribosomal protein (Wool, 1979; Leader, 1980); the others are PO, P1, and P2 (Tsurugi et al., 1978; Towbin et al., 1982). In the rat S6 protein there are five serines at the carboxyl end that are known to be phosphorylated (Gressner and Wool, 1974a; Chan and Wool, 1988). There is an extraordinary number and variety of agents and of alterations in the cellular environment that affect the phosphorylation of S6 (Chan and Wool, 1988). These include hormones, viral infection, serum treatment, fertilization, heat-shock, and more. This response to mitogenic stimulation is highly conserved and occurs in all systems examined so far (Sturgill and Wu, 1991). The phosphorylation of S6 is supposed to trigger or facilitate the high rates of protein synthesis required for cell division (Pardee, 1989) and to alter the patterns of translation (Palen and Traugh, 1987; Thomas et al., 1981). Unfortunately, the function(s) of S6 is not known, and it is therefore not possible to foresee how its function(s) may be altered by phosphorylation. Even though there is a general correlation between an increase in the phosphorylation of S6 and an increase in protein synthesis, known inhibitors of protein synthesis such as cycloheximide and puromycin are very effective stimuli for the phosphorylation of S6 (Gressner and Wool, 1974b). Two suggestions for the consequences of the phosphorylation of S6 have been put forward. The first one proposes that S6 forms part of the mRNAbinding domain in the 40s subunit and that the attachment of certain mRNAs is favored by phosphorylation (Wool, 1979; Terao and Ogata, 1979; Duncan and

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McConkey, 1982; Takahashi and Ogata, 1981; Thomas et al., 1982). However, there is no substantial evidence that phosphorylation of S6 in any way affects the activity of the ribosome (Leader et al., 1981). The second proposal is that the phosphorylation of S6 has no purpose at all and that S6 is just a fortuitous substrate for protein kinases that have other and more specific substrates (Chan and Wool, 1988). Indirect evidence supports this negative hypothesis. The yeast homolog YS6 has only two phosphorylation sites instead of the five present in the mammalian S6, but the phosphorylation of YS6 is affected by the same stimuli; for example, growth, sporulation, heat-shock, and other conditions (Johnson and Warner, 1987).After inactivating the two genes encoding YS6 cells were transfected with a plasmid containing a mutant YS6 cDNA in which the two serines that are phosphorylated were replaced with alanines (Johnson and Warner, 1987). The mutant YS6 was not phosphorylated but was nonetheless incorporated into ribosomes. The mutant strain grew and responded normally to the induction of sporulation and heat-shock. In a competitive growth experiment equal numbers of mutant and wild-type cells were mixed together and diluted into fresh medium, which is a strong stimulus for growth and phosphorylation of YS6, when growth became stationary (Johnson and Warner, 1987). Wild-type YS6 is phosphorylated and dephosphorylated during each cell cycle; after 70 generations the fraction of mutant and wild-type cells was unchanged. This experiment demonstrated that there was no selection against cells with the mutated YS6 that could not be phosphorylated, and that the phosphorylation of the two serines in YS6 serves no observable purpose. Thus, our knowledge of the functions of S6 is meager, and the somewhat obscure role of S6 phosphorylation in physiological processes is puzzling. The observation that mutations in the Dosophila S6 gene cause hypertrophy of the larval hematopoietic organs may therefore be important in our efforts to gain more information about this protein.

6. RpS13 [M(2)32A] a. Gene and mutant phenotype Using the P{aCWI mutagenesis procedure described by Bier et al. (1989),we have induced and cloned two previously undescribed Minutes, one on the second and one on the third chromosome. The second-chromosome Minute, PflacWM(2)32A1, is characterized as an intermediate-level defective phenotype, with respect to abnormal bristle formation and retardation of larval development (Szb@e-Larssen and Lambertsson, 1996).The one on the third chromosome, P{IacWM(3)66D1, is a severe Minute (Saebae-Larssen et al., 1997;see below). P{aCW)M(2)32A1 was mapped to region 32A on the second chromo-

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some by in situ hybridization to salivary gland chromosomes and by recombination and deficiency mapping. Remobilization of the P-element restored the phenotype to wild type, showing that the Minute phenotype was caused by the P-element insertion. After cloning the Minute gene via plasmid rescue, M(2)32A was then characterized and sequenced. Comparisons of M(2)32A genomic and cDNA clones with the sequences in the EMBL and GeneBank databases revealed a very high level of identity (98.9% in a 564-bp overlap) to the D. rnelanoguster RPZ7 cDNA (McNabb and Ashbumer, 1993) and to RPSZ3 cDNA sequences from Musca domestzca (76%; 2. H. Zhou and M. Syvanen, unpublished results), Rattus rattus (73%; Suzukietal., 1990), andHomosapiens (71%;Chadeneauetal., 1993). The very high level of identity between the RPZ 7 cDNA and the one reported by our laboratory and the fact that both cDNAs derived from single-copy genes suggested that both originated from the same gene. However, the Rp17 gene was cytologically mapped to 29A (McNabb and Ashbumer, 1993), a region not containing a Minute locus (Lindsley and Zimm, 1992). Based on our genetic data and the high level of identity to RPSZ 3 cDNA sequences from various species, we concluded that our cDNA corresponds to the eukaryotic RpSl3. Given the high identity between RPSI3 and RPI7, we also believe that RP17 corresponds to this locus. Comparing the RPS 13 cDNA sequence with the corresponding genomic sequence M(2)32A revealed two introns of 226 and 62 bp, respectively. There is one major transcription start site at position -30 relative to the open reading frame, with additional minor transcription start sites at positions -31, -33, and -34. Transcription is initiated in a polypyrimidine tract, typical for mammalian and several Drosophila ribosomal protein gene cap sites (Mager, 1988; Stewart and Denell, 1993; Andersson et al., 1994).The presence of a polypyrimidine tract has been found to be important for both promoter function and translational regulation (Chung and Perry, 1990;Moura-Net0 et al., 1989; Hariharan and Perry, 1990; Levy et al., 1991). Also, the promoter of M(2)32A has no TATA or CAAT motifs typically found in promoters of genes transcribed by RNA polymerase 11. The exact location of the inserted P-element was found to be in the middle of the 5’ untranslated leader between nucleotides - 15 and - 16 relative to the translation initiation codon. Northem blot analysis, using a single stranded cDNA probe (antisense), revealed an abundantly and in all developmental stages expressed transcript of 0.6 kb. This transcript is -3.5 times more abundant in females (ovaries) than in males when compared with total poly(A)+ RNA. The abundance of this transcript is reduced by -50% in P[IacWm(2)32A1 heterozygotes, implying that the Pelement insertion completely disrupts the M(2)32A locus. The amount of transcript is restored to the wild-type level in homozygous male and female revertants (Szbfle-Larssen and Lambertsson, 1996).

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b. The RPS13 protein The Drosophila RPSl3 cDNA is 629 bp long, including the poly(A) tail, and contains 17 nucleotides of untranslated leader, a 453-bp open reading frame encoding a protein of 151 amino acids and a 123-bp untranslated trailer (poly(A) tail not included) containing two polyadenylation signals ( AATAAA). Conceptual translation of the open reading frame predicts a protein with 151 amino acids and a MW of =17 kDa. As a comparison, rat S13 has 150 amino acids and a MW of 17,080 Da (Wool et al., 1995). Drosophila S13 is well conserved and shows 87% identity to both human and rat S13 (Suzuki et al., 1990; Chadeneau 1993), 78% to the parasitic nematode B. pahngi S13 (Ellenberger et al., 1989), 69% to the fission yeast Schizosacchromyces pombe S13 (Marks and Simanis, 1992), 71% to the monocot plant 2. mays S13 (Joanin et al., 1993), and 32% identity to the archaebacterium Haloarcula marismortui S15 (Scholzen and Amdt, 1992). Consistent with the need for transportation to the nucleolus, Drosophila S13 has the bipartite nuclear targeting signal IKK{ll}RKDKD in position 92-1 10 (Saebae-Larssen and Lambertsson, 1996; Dingwall and Laskey, 1991).

7. oho23B [RpS21; M(2)23B] a. Gene and mutant phenotype The RpS2I gene was isolated in a large-scale P-element mutagenesis screen of the second chromosome as a lethal with overgrown hematopoietic organs; the mutant is called oho23B or l(2)kl68-14 (Herrmann et al., 1995; I. Toeroek, D. Herrmann, I. Kiss, and B. Mechler, personal communication). The P-element had inserted in the promoter region, causing a 30- to 40-fold reduction of the RpS2l transcript in homozygous mutant larvae. Heterozygous flies show a weak Minute bristle phenotype. This phenotype is more severe (severe Minute) in an allele where the P-element deleted the RpS21 promoter region upon excision. Pelement-mediated transformation of a 2.5-kb genomic DNA fragment containg the RpS21 gene rescues both mutant phenotypes (I. Toeroek, D. Herrmann, I. Kiss, and B. Mechler, personal communication). The development of mutant animals is arrested at the larval to pupal transition and the larvae may survive for 2 weeks after the pupation of the heterozygous siblings. The most striking phenotype is the massive overgrowth of the hematopoietic organs and particularly of the most anterior lobes, which become as big as the brain. It is interesting to note there is no release of hemocytes from the hematopoietic organs into the hemolymph; all cells remain in the glands. At the time of normal pupation the imaginal disks of the mutant larvae are smaller than normal, but in older mutant larvae the disks become much larger with duplicated structures and the leg and wing disks tend to fuse together, although they keep a more or less apparent folding pattern.

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The RpS2I gene consists of four exons of 41,60,192, and 75 bp in length separated by three relatively small introns of 71, 71, and 63 bp. The major form of the RPSZI transcript has a size of about 0.4 bp. There is also a minor 0.7-kb transcript representing about 5% of the total level. Investigation of a cDNA corresponding to this transcript revealed that it contains an extension of the 3' UTR without any additional intron (Herrmann et al., 1995; I. Toeroek, D. Herrmann, I. Kiss, and B. Mechler, personal communication). Using the yeast two-hybrid system it was shown that the S21 protein strongly interacts with the ribosome-associated protein p40 encoded by the stubarista ( s t a ) gene (I. Toeroek, D. Herrmann, I. Kiss, and B. Mechler, personal communication). This result is certainly encouraging in that it very likely will allow future detection of interactions between other ribosomal proteins or between ribosomal and nonribosomal proteins.

8. RpL9 [M(2)32D] a. Gene and mutant phenotype In experiments analyzing male-sterile mutations caused by the integration of the PfhcWl enhancer trap P-element, Schmidt et al. (1996) stumbled on a new Minute locus at 32D on the second chromosome. Analyzing the cloned region they found, among others, an abundantly expressed 0.8-kb transcript 5' to the Pelement insertion site. Searching the data bases the authors found significant matches to eukaryotic ribosomal protein L9 and the corresponding L6 in bacteria and organelles. No Minute had been mapped to 32D before (Lindsley and Zimm, 1992; the M(2)32A paper was published at about the same time); so to find out whether this region harbored a Minute locus the P-element was remobilized to generate deletions in that area. Among the white-eyed male offspring one was isolated that had the typical Minute phenotype: short and thin bristles and slightly rough eyes. The mutant line, M(2)32D, also showed delayed development, and the females, but not the males, had severely impaired fertility (Schmidt et al., 1996).The mutation also enhanced the wing vein defects of Delta mutants, as was also shown for M(2)60E (Hart et al., 1993). Southern analysis indicated that an imprecise excision of the P-element had truncated the 3' end of the RpL9 gene. Since the remobilization of the P-element caused the Minute phenotype it was, of course, impossible to use this approach to restore the normal one. Therefore, the authors had to perform transformation rescue experiments. By introducing a genomic DNA fragment (3.9 kb) containing the RpL9 transcription unit the wild-type phenotype was restored, showing that the M(2)32D gene encodes RPL9. A more detailed genetic and molecular analysis of the 32D region has been made by Schmidt (1996). Interestingly, there is a lethal PZ element insertion, l(2)01501 (Karpen

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and Spradling, 1992), about 900 bp upstream of M(2)32D (Schmidt et al., 1996). Since M(2)32D/l(2)01501 flies are fully viable these mutations are not alleles, and the lethality associated with l(2)01501 must be caused by a mutation in a gene 5’ to the M(2)32D gene. Remobilizing the PZ element generated yet another M(2)32D allele, M(2)32D2, which was lethal in trans with the original M(2)32D mutation (Schmidt et al., 1992). The authors also used the Df(2L)/39 deficiency with breakpoints in 3 1AB and 32D-E and showed that it uncovers the male-sterile mutation associated with the P{lacW} insertion at 32D, the M(2)32D gene as well as the l(2)01501 mutation. The Minute phenotype of Df(2L)]39 has been ascribed to another Minute, namely M(2)30A, and introducing the 3.9-kb genomic fragment into Df(2L)J39 flies did not restore the wild-type phenotype. We now know that there is at least one other Minute in this region, namely M(2)32A (Szbae-Larssen and Lambertsson, 1996). This emphatically shows why it has been impossible to use Minutes caused by deficiencies in fine genetic and molecular genetic/biology studies. Whether there is a Minute at 30A remains to be shown. The RpL9 gene is relatively complex consisting of two short exons (18 and 50 nucleotides) and two long ones (25 1 and 398 nucleotides) separated by three introns of 73, 243, and 275 nucleotides (Schmidt et al., 1996). Drosophila ribosomal protein genes usually have one or two introns. The estimated length of the RpL9 mRNA, including the poly(A) tail, is 800 nucleotides.

b. The RPL9 protein Conceptual translation of the RpL9 cDNA generates a polypeptide of 190 amino acids and a molecular weight of 21406 Da. The RPLB is homologous to rat L9, and this protein has been conserved during evolution, having homologs both in archaebacteria and eubacteria (Wool et al., 1995). Together with more than 30 other eukaryotic ribosomal proteins L9 phylogenetically belongs to Group I (Wool et al., 1995) that have homologs in the eubacterial and archaebacterial kingdom. It should be noted that in cultured human cells RPLB has been implicated in the delivery and binding of the ricin A chain to the ribosome where the toxin attacks the large rRNA (Vater et al., 1995).

9. RpL14 [M(3)66D] a. Gene and mutant phenotype In the same P-element mutagenesis experiment described above we isolated P{IaCWM(3)66DJ,a severe Minute with respect to defective bristle formation and retardation of larval development; fertility and viability of heterozygotes are good (Szbae-Larssen et al., 1997). The bristles of P(lacW)(M3)66DJ heterozygous flies are extremely thin and short, and in the two to three generations after the mutant was isolated the Minute flies also very often had notched wings, missing or

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Figure 3.3. Scanning electron micrograph of Drosophila head (frontal view) from (A) wild-type and (B)P/hcWIM(3)66D'/+ flies. T h e malformed and undeveloped antennae (arrowheads), the missing maxillary palp (open short arrow) and the malformed right side of the head (open short arrow) with the much smaller right eye. (a2-a3) Second and third antenna1 segment; (ar) arista; (ce) compound eye; (mpl) maxillary palp.

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malformed antennae, maxillary palps, ocelli, eyes, and legs (Figures 3.3 and 3.4). However, after some more generations these extreme characters disappeared in the balanced stock and only reappear when P{kxWm/i(3)66D1flies are mated with unrelated flies. The M(3)66D gene was cloned, sequenced, and characterized in a fashion analogous to that described above for M(2)32A. Sequence comparisons performed by Dr. A. Gluck (personal communication) revealed that the Drosophila sequence was homologous to the rat L14 sequence (Chan et al., 1996). The exact location of the inserted P-element was found to be in the promoter region 52 nucleotides upstream of the M(3)66D transcription start site (cf., RpS6 and sopp). The M(3)66D gene contains 5 nucleotides of untranslated leader, a 498bp long open reading frame, and a 92-bp untranslated trailer with a polyadenylation signal (AATAAA) located 27 bp upstream of the polyadenylation site (Szbpe-Larssen et al., 1997). There are three introns of 276,232, and 260 bp (5‘ to 3’).The most upstream intron intersects the open reading frame immediately after the ATG start codon, making the first intron only 19 bp long. There is one major transcription start site within a pyrimidine-rich tract at position - 16 relative to the open reading frame and the transcript is -650 nucleotides. The M(3)66D promoter region contains putative upstream elements in the form of two consensus Drosophila CAAT motifs, AGCAACA and TGCAACT [consensus: (A/T)GCA(A/T)N(A/T); O’Connell and Rosbash, 19841. Similarly positioned CAAT boxes are also present in the promoters of the rp genes encoding RPS17, RPL7, and RPL32 (Szbpe-Larssen et al., 1997). There is, however, no apparent TATA box present in the promoter region. The latter is usually not present in rp genes. The insertion of the P-element in P{kxWIM(3)66DJ 52 bp upstream of the transcription start site causes an overall reduction RpLI 4 mRNA abundance. There are, however, significant differences in percentage reduction between the various developmental stages. The decrease is most manifest in third-instar larvae, 54% of wild type, and substantial reductions are also noticed in 1 to 3-dayold pupae and adult females with 60-70% and 63% of wild-type, respectively. The reduction is less apparent in second-instar larvae and adult males, each with -80% of wild-type RpS6 mRNA. These results may indicate that the P-element is interfering with a promoter function that is employed with different efficiency at various developmental stages.

b. The RPL14 protein The M(3)66D cDNA encodes a 19-kDa protein with a large excess of positively charged amino acids. The protein has a calculated net charge of +30 at physiological pH and is increasingly polar toward the C-terminal end (Szbpe-Larssen et al., 1997). The Drosophila RPL14 shares 42% identity with rat and human ribosomal protein L14 (Chan et al., 1996; Li et al., 1993; Aoki et al., 1996), al-

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Figure 3.4. Scanning electron microscopy of DTosophila head (viewed from above) from (A) wildtype and (B) PlhcWlM(3)66D’/+ flies. Notice the small and thin bristles, the deformed and misplaced ocellus (arrowhead), and the undeveloped antenna (open short arrow) lacking segments 3-5 and the arista in the mutant (B). (a2) Second antenna1 segment; (oc) ocelli; (ocb) ocellar bristles; (orb) orbital bristles; (pvb) postvertical bristles; (vb) vertical bristles.

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though the fly protein has 165 residues compared to 214 in rat L14 (Chan et al., 1996); most of the missing amino acids are at the carboxyl terminus. The yeast L14 hasonly138residuescompared to the214ofratL14 (Chanetal., 1996);yeast L14 lacks the carboxylterminal84 amino acids. The large variation in the number of residues in homologous eukaryotic L14 is unusual among ribosomal proteins. It appears that amino acids have been added to the C-terminal end during evolution (Chan et al., 1996). The eukaryote RPL14 has no counterpart in prokaryotes and thus belongs to the Group I11 ribosomal proteins (Wool et al. , 1995).

10. A homozygous viable RpL14 allele As mentioned above, M(3)66D is the middle gene in a tight cluster of three genes at 66D, very closely linked to hairy ( h , 111-26.5; Szbae-Larssen et al., 1997). In a remobilizationexperiment several revertants were isolated (A. Lambertsson,unpublished results). Among them was one white-eyed female that had therefore lost the P-element marker and showed a weak Minute phenotype, P{IacW&l(3)66DJPU3. The next generation revealed homozygous white-eyed flies with a more severe Minute phenotype. These flies were also fertile and can be maintained as a homozygous stock. Sequencing the region of the insertion revealed that the P-element had excised imperfectly and left 30 nucleotides. This small insertion in the promoter is thus capable of upsetting the expression of the RpL14 gene, suggesting that this region is sensitive to small sequence changes.

11. RpL19 [M(2)60E] a. Gene and mutant phenotype M(2)60E was first isolated as a recessive lethal P-element insertion line called E(DlJKP135 (Klein and Campos-Ortega, 1992; Hart et al., 1993) with a dominant enhancing effect on the phenotype of Delta, a gene encoding a surface transmembrane protein with EGF repeats in its extracellular domain (e.g., Vassin et al., 1987; Kopczynski et al. , 1988). Being a P-element induced allele the correct name should be PlhcWhl(2) 60E (Flybase “Genetic Nomenclature for Drosophila melunogaster,” March 23, 1995). The gene appears to have the typical polypyrimidine tract, where transcription usually starts in rp genes, and the Pelement inserted between this stretch of pyrimidines and the tentative translation start codon (Hart et al., 1993). Having three exons and two introns the RpL19 gene should produce a transcript of about 700 nt, including the 3’ trailer (Hart et al., 1993). The L19 ribosomal protein has a homolog in archaebacteria and thus belongs to Group I1 ribosomal protein (Wool et al., 1995). E(Dl)KP135 [M(3)60E] also interacts with the if mutation of the PS2a (position-specific) integrin gene. The PS integrin family contains a-and P-genes, and heterodimers are formed between the common PSP subunit and either PSla

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or PS2ol. The integrins function as cell-adhesion molecules throughout development and are probably involved in the adhesion of the two epithelial surfaces of the developing wing blade (Wilcox e t al., 1989). Most likely, the if mutation causes wing blistering by impairing the mutual adhesion of the cell layers that form the wing (Brower and Jaffe, 1989; Wilcox et al., 1989; Zusman e t al., 1990; Brabant and Brower, 1993). P{hcWb4(2)60E is characterized as a severe Minute. The P-element insertion in the 5' UTR of the RpL19 gene probably reduces gene expression by about 50% (see SaebQe-Larssenand Lambertsson, 1996). It is therefore interesting to note that the Minute phenotype of the two deficiences, Df(2R)ESI and M(2)60E, which are haploinsufficient for RpLl9 and therefore should have no more than 50% of wild-type RpLI9 expression, is slightly less severe than that of PIlacWN(2)60E (Hart et al., 1993). This is also reflected in the interaction with nd' and if, where P{lacWb4(2)60E strongly enhances the notched and blistered phenotypes, respectively; M(2)60E less so and Df(2R)ESI only marginally. Thus, the severity of the Minute alleles appears to correlate with the effects on wing development. The authors offer several explanations for this, and although it seems implausible that any of them can apply, alone or in combination, the differences between the alleles may be ascribed to different genetic backgrounds. Many, but not all, Minutes are known to affect wing morphology (Lindsley and Zimm, 1992). Several Minutes enhance dominance of such venation characters as px and net as well as the notched phenotypes (see above) and bristle characters as sc. In addition, dominant lethal effects are frequent in combination with Delta, Jammed, and in some cases withDichaete.One explanation for this would be that the reduced rates of protein synthesis in Minutes result in reduced levels of these gene products. Normal development is dependent on a critical threshold level of many proteins, and in Minutes the levels of these proteins may fall below this absolute minimum resulting in the described effects, and the more severe the Minute, the stronger the effect.

12. Stuburista ( s t a ) The D. mehmgmter gene stubarista ( s t a ) , which maps to cytological position 2A3B2 on the X chromosome, was shown to encode the highly conserved ribosome associated protein p40 [D-p40; Melnick et al., 1993). Using immunochemistry (McCafferey et al., 1990) as well as sucrose-gradient analysis (Auth and Brawerman, 1992) it was revealed that this protein is associated with polyribosomes in the cytoplasm. After ribosome dissociation p40 remains bound to the 40s ribosomal small subunit. A n association between p40 and the cytoskeleton has also been revealed (Keppel and Schaller, 1991), and this is consistent with studies showing an association between polyribosomes and the cytoskeleton (Cervera et al., 1981; Howe and Hershey, 1984).

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The viable stubarista allele staJ is associated with a number of phenotypes that qualifies stu as a Minute; these include malformed antennae, all bristles short and thin, and female sterility (Melnick et al., 1993). This mutant phenotype and the fact that D-p40 is required during oogenesis and imaginal development (Melnick et al., 1993) suggests that this protein has basic cellular function reminiscent of a true ribosomal protein. The s t a gene produces a single l-kb transcript expressed maternally and throughout development (Melnick et al., 1993). There is a single intron of 192 bp and an open reading frame of 270 amino acids. The Drosophila p40 shows a high degree of identity with p40 proteins from the two evolutionary distant species human (63%) and hydra (58%) and a 27% homology to E. coli S2 (Melnick et d.,1993). E. coli S2 corresponds to mammalian ribosomal protein Sa (Wool et al., 1995), is found on the surface of the small subunit where it helps stabilize conformation (Marion and Marion, 1988), and is also involved in tRNA binding (Shimizu and Craven, 1976). It is possible that D-p40 corresponds to mammalian Sa.

C. Recessive Minutes The dominant visible phenotypes and the recessive lethality have been associated with the Minute mutations since their discovery. In addition, several Minutes have low fertility, especially the females. The dominant Minute phenotype has made it relatively easy to isolate these mutations. In theory, the reduction in mRNA abundance needed to produce a detectable a Minute phenotype will be different for different rp genes, and we would therefore expect to find recessive Minutes. In addition, mutations in some rp genes may not result in a Minute phenotype. In describing the first Minutes, Bridges and Morgan (1923) mentioned that “also a few recessive small-bristle mutants were found and recovered in F,, notably tiny bristles.” And later Mohr (1925) described what he called a “Minutelike recessive.” Unfortunately, it is not clear to what extent these recessive mutants exhibited the other effects typical of the dominant Minute phenotype. These older observations are important, because we now know that recessive or homozygous viable Minutes do exist (see below). These Minutes are interesting, because they lack the characteristic haploinsufficiency but show the dominant Minute traits (small and thin bristles and delayed development) in both heterozygotes and homozygotes, with a clear synergistic effect in the latter; both fertile and sterile recessive Minutes have been isolated. The molecular explanation is that they are not null alleles and that the M/+ heterozygotes thus produce >50 but