Current Topics in Developmental Biology, Volume 66 (Current Topics in Developmental Biology)

262 Contents of Previous Volumes Volume 66 1. Stepwise Commitment from Embryonic Stem to Hematopoietic and Endothelia...

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Contents of Previous Volumes

Volume 66 1. Stepwise Commitment from Embryonic Stem to Hematopoietic and Endothelial Cells Changwon Park, Jesse J. Lugus, and Kyunghee Choi

2. Fibroblast Growth Factor Signaling and the Function and Assembly of Basement Membranes Peter Lonai

3. TGF-b Superfamily and Mouse Craniofacial Development: Interplay of Morphogenetic Proteins and Receptor Signaling Controls Normal Formation of the Face Marek Dudas and Vesa Kaartinen

4. The Colors of Autumn Leaves as Symptoms of Cellular Recycling and Defenses Against Environmental Stresses Helen J. Ougham, Phillip Morris, and Howard Thomas

5. Extracellular Proteases: Biological and Behavioral Roles in the Mammalian Central Nervous System Yan Zhang, Kostas Pothakos, and Styliana-Anna (Stella) Tsirka

6. The Genetic Architecture of House Fly Mating Behavior Lisa M. Meffert and Kara L. Hagenbuch

7. Phototropins, Other Photoreceptors, and Associated Signaling: The Lead and Supporting Cast in the Control of Plant Movement Responses Bethany B. Stone, C. Alex Esmon, and Emmanuel Liscum

8. Evolving Concepts in Bone Tissue Engineering Catherine M. Cowan, Chia Soo, Kang Ting, and Benjamin Wu

9. Cranial Suture Biology Kelly A Lenton, Randall P. Nacamuli, Derrick C. Wan, Jill A. Helms, and Michael T. Longaker

Volume 67 1. Deer Antlers as a Model of Mammalian Regeneration Joanna Price, Corrine Faucheux, and Steve Allen

Series Editor Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTAL CENTER Deputy Director, Magee-Women’s Research Institute Professor and Vice-Chair of Ob-Gyn Reproductive Sci. & Cell Biol.-Physiology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15213

Editorial Board Peter Gru¨ss Max-Planck-Institute of Biophysical Chemistry Go¨ttingen, Germany

Philip Ingham University of Sheffield, United Kingdom

Mary Lou King University of Miami, Florida

Story C. Landis National Institutes of Health National Institute of Neurological Disorders and Stroke Bethesda, Maryland

David R. McClay Duke University, Durham, North Carolina

Yoshitaka Nagahama National Institute for Basic Biology, Okazaki, Japan

Susan Strome Indiana University, Bloomington, Indiana

Virginia Walbot Stanford University, Palo Alto, California

Founding Editors A. A. Moscona Alberto Monroy

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

Kyunghee Choi (1), Developmental Biology Program and Molecular Cell Biology Program, Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, Missouri 63110 Catherine M. Cowan (239), Department of Bioengineering, University of California Los Angeles, Los Angeles, California 90095 Marek Dudas (65), Developmental Biology Program at the Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, California 90027 and Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California 90089 C. Alex Esmon (215), University of Missouri–Columbia, Columbia, Missouri 65211 Kara L. Hagenbuch (189), Department of Ecology and Evolutionary Biology, Rice University, Houston, Texas 77251-1892 Jill A. Helms (287), Children’s Surgical Research Program, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148 Vesa Kaartinen (65), Developmental Biology Program at the Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, California 90027 and Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California 90089 Kelly A Lenton (287), Children’s Surgical Research Program, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Standford, California 94305-5148 Emmanuel Liscum (215), University of Missouri–Columbia, Columbia, Missouri 65211 {

{

Peter Lonai (37), Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel 76100

Deceased.

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Contributors

Michael T. Longaker (287), Children’s Surgical Research Program, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148 Jesse J. Lugus (1), Molecular Cell Biology Program, Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, Missouri 63110 Lisa M. Meffert (189), Department of Ecology and Evolutionary Biology, Rice University, Houston, Texas 77251-1892 Phillip Morris (135), Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, Wales, United Kingdom Randall P. Nacamuli (287), Children’s Surgical Research Program, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148 Helen J. Ougham (135), Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, Wales, United Kingdom Changwon Park (1), Developmental Biology Program, Washington University School of Medicine, Department of Pathology and Immunology, St. Louis, Missouri 63110 Kostas Pothakos (161), Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651 Chia Soo (239), Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles, Los Angeles, California 90095 and University of Southern California, Keck School of Medicine, Division of Plastic Surgery, Los Angeles, Calfornia 90053 Bethany B. Stone (215), University of Missouri–Columbia, Columbia, Missouri 65211 Howard Thomas (135), Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, Wales, United Kingdom Kang Ting (239), Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles, Los Angeles, California 90095 Styliana-Anna (Stella) Tsirka (161), Department of Pharmocological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651

Contributors

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Derrick C. Wan (287), Children’s Surgical Research Program, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, Stanford, California 94305-5148 Benjamin Wu (239), Department of Bioengineering, University of California Los Angeles, Los Angeles, California 90095 Yan Zhang (161), Department of Pharmocological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651

In Memorium, Professor Peter Lonai

August 11, 1936–November 6, 2004

Professor Peter Lonai was a scientist with wide knowledge and perspectives in biology. He had the courage and vision to enter into new fields before his peers realized the importance of these topics. He had deep understanding in classical developmental biology, and many scientists from the two faculties of biology at the Weizmann Institute of Science consulted him on these topics. His work on early developmental steps in the mouse embryo was highly appreciated in the field. This includes his studies on epithelial– mesenchymal interactions, the FGF signaling, and most recently, the laminin-dependent mechanisms that regulate endodermal and ectodermal embryonic stem cell fates. Peter was a loved colleague in the Weizmann Institute’s Department of Molecular Genetics, an intellectual, and a real gentleman. He was among the founders of the transgenic/knock-out facilities in the Institute. In fact, his knowledge and expertise initiated this important operation that helped many groups at the Weizmann Institute. Science meant everything to him, and he continued to come to the lab until the last days of his life. His last review is the best documentation of his enormous dedication to science. Professor Adi Kimchi

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Stepwise Commitment from Embryonic Stem to Hematopoietic and Endothelial Cells Changwon Park,* Jesse J. Lugus,{ and Kyunghee Choi*,{ *Developmental Biology Program {

Molecular Cell Biology Program Washington University School of Medicine Department of Pathology and Immunology St. Louis, Missouri 63110

I. Embryonic Stem Cell A. Signaling Pathways Regulating Embryonic Stem Cell Self-Renewal B. Transcriptional Control of Embryonic Stem Cell Self-Renewal II. From ES to Hematopoietic Progenitors A. An Overview of Hematopoietic Development B. Hemangioblast C. The Identification of Blast Colony–Forming Cells from In Vitro DiVerentiated Embryonic Stem Cells D. From Flk-1-Expressing Mesoderm to Hematopoietic and Endothelial Cells E. Hematopoietic Inductive Signals F. Transcriptional Control of Hematopoietic and Endothelial Cell Lineage Commitment G. In Vivo Potential of Embryonic Stem Cell–Derived Hematopoietic Progenitors III. Conclusions and Future Directions Acknowledgments References

There is great excitement in generating diVerent types of somatic cells from in vitro diVerentiated embryonic stem (ES) cells, because they can potentially be utilized for therapies for human diseases for which there are currently no eVective treatments. Successful generation and application of ES-derived somatic cells requires better understanding of molecular mechanisms that regulate self-renewal and lineage commitment. Accordingly, many studies are aimed toward understanding mechanisms for maintaining the stem cell state and pathways leading to lineage specification. In this chapter we discuss recent studies that examine molecules that are critical for ES cell self-renewal, as well as hematopoietic and endothelial cell lineage diVerentiation from ES cells. C 2005, Elsevier Inc.

Current Topics in Developmental Biology, Vol. 66 Copyright 2005, Elsevier Inc. All rights reserved. 0070-2153/05 $35.00

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I. Embryonic Stem Cell In 1981 investigators successfully derived pluripotent embryonic stem (ES) cells from blastocysts, the preimplantation stage of mouse embryos (Evans and Kaufman, 1981; Martin, 1981). Subsequently, ES cell lines from many diVerent species, including human, have been derived (Thomson et al., 1995, 1996, 1998). The derivation of ES cells is quite straightforward in that blastocysts are plated onto a feeder layer of fibroblasts and the inner cell mass of blastocysts ultimately gives rise to colonies of undiVerentiated cells (ES cell colonies), which are isolated and further expanded. Once established, ES cells can be maintained as pluripotent stem cells on a feeder layer of fibroblasts. When introduced back into a blastocyst, ES cells can contribute to all tissues with the exception of extraembryonic endoderm and trophoblast of the developing embryo (Beddington and Robertson, 1989; Bradley et al., 1984). It is this particular trait that makes ES cells a valuable tool for genetic engineering. In addition, ES cells can be diVerentiated in vitro into many diVerent somatic cell types (Bagutti et al., 1996; Bain et al., 1995; Buttery et al., 2001; Dani et al., 1997; Doetschman et al., 1985; Drab et al., 1997; Fraichard et al., 1995; Keller et al., 1993; Kramer et al., 2000; Liu et al., 2000; Maltsev et al., 1993, 1994; Nakano et al., 1994; Potocnik et al., 1994; Risau et al., 1988; Rohwedel et al., 1994; Strubing et al., 1995; Vittet et al., 1996; Wang et al., 1992; Wiles and Keller, 1991; Yamashita et al., 2000), opening up the possibility to utilize ES-derived cells as a potential source for cell transplantation or cell-based therapy (Fig. 1). Thus, ES cells have gained much scientific and general public attention. A. Signaling Pathways Regulating Embryonic Stem Cell Self-Renewal In 1987 Smith and Hooper discovered that buValo rat liver cells (BRLCs) secreted a substance into the media that could maintain ES cells as pluripotent in the absence of a feeder layer (Smith and Hooper, 1987). Previously, when removed from a feeder layer, ES cells diVerentiated into extraembryonic endoderm. However, the BRLC-conditioned media possessed a Self-renewal

Differentiation

ES cells Embryoid bodies Figure 1 Schematic diagram of in vitro diVerentiation of ES cells. Embryoid bodies (EBs) are composed of many diVerent cell types, as indicated by diVerent colors.

1. Hematopoietic and Endothelial Cells

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diVerentiation-inhibiting activity (DIA) that prevented the diVerentiation of ES cells in the absence of a feeder layer. Furthermore, this activity could be dialyzed out from the media, showing that soluble factor(s) could maintain ES cell self-renewal without any interaction with feeder cells. The same year, Moreau and coworkers reported the discovery of a novel human interleukin (IL) that induced the proliferation of the murine DA-1 early myeloid cell line and was given the moniker HILDA for human interleukin DA responsive cytokine (Moreau et al., 1987b). Later work showed that HILDA was also an activator of eosinophils, as well as a potent inducer of burst-promoting activity on human marrow (Moreau et al., 1987a). Leukemia inhibitory factor (LIF) first emerged as a cytokine that was able to suppress the proliferation and drive the diVerentiation of M1 myeloid leukemia cells (Gearing et al., 1987). Subsequently, work emerged that tied LIF, DIA, HILDA, and the maintenance of ES cell pluripotency together. First, Williams and coworkers noted that partially purified DIA and LIF had a number of similarities (Williams et al., 1988). The group discovered that in the absence of feeder cells, LIF associated with the cell membranes of a number of ES and embryonal carcinoma (EC) cell lines and was able to maintain >95% of ES cells in ES cell colonies. Moreover, using ES cells maintained in purified, recombinant LIF for up to 22 passages, chimeric animals were successfully produced and some showed up to 90% chimerism. Smith and colleagues then showed that 10 ng/ml of either DIA or HILDA/ LIF was suYcient to suppress any type of diVerentiation of CP1 ES cells (Smith et al., 1988). The final piece of the puzzle came from Moreau and coworkers when they showed that the genes for LIF, HILDA, and DIA were one and the same (Moreau et al., 1988). LIF is a ligand for a heterodimeric receptor composed of LIF receptor (LIFR ) and the gp130 cytokine receptor (Davis et al., 1993). This ligand–receptor complex then activates Janus-associated tyrosine kinases (Jak), which phosphorylate the receptor chains. The phosphorylated receptors, in turn, serve as docking sites for Srchomology 2 (SH2) domains of additional proteins that may also be phosphorylated by the Jaks. A key substrate of the Jaks is the signal transducer and activator of transcription (Stat) family of transcription factors. Specifically, the LIFR –gp130 complex is an eVector of Stat3 phosphorylation and dimerization, inducing nuclear translocation and subsequent transcription (Boeuf et al., 1997; Niwa et al., 1998). Stat3 recruitment and activiation are integral to ES cell self-renewal, because a dominant negative Stat3 isoform rapidly induces ES cell diVerentiation (see the following). More recent work has given insight into other important mechanisms of ES cell self-renewal (Qi et al., 2004; Ying et al., 2003). Ying and coworkers, noting the antagonist eVects of bone morphogenetic proteins (BMPs) on neural diVerentiation, showed that in the absence of serum, BMP-4 provides a synergistic eVect to LIF in the maintenance of ES cell phenotype, because

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the eVect of BMP-4 was additive to that of LIF alone (Ying et al., 2003). Moreover, E14Tg2a ES cells passaged six times in serum-free N2B27 media with LIF and BMP-4 could generate chimeric animals. The authors went on to show that BMP-4 does not signal through the LIFR –gp130 complex nor does it activate Stat3. Instead, BMP-4 activates the Smad pathway to induce ES cell self-renewal and activate members of the Id family. Additionally, in this study, the authors also showed that BMP-4 activated the Erk pathway. The Id genes encode helix-loop-helix (HLH) factors that antagonize transcriptional activation by basic helix-loop-helix (bHLH) transcription factors. The Ids bind bHLH transcription factors, preventing bHLH heterodimerization and DNA binding to suppress transcriptional activity. Gene expression showed that BMP-4 is capable of inducing up to tenfold increases in both Id1 and Id3 gene expression. The authors posit that the Ids are integral to the suppression of ES cell diVerentiation. For example, the Id-mediated suppression of ES cell diVerentiation can be overcome through expression of supra-physiological levels of E47. The authors theorize that high levels of E47 successfully out-compete the Ids to interact with Neurogenin2 and drive ES cells to a neurogenic fate. Similarly, Qi et al. (2004) reported the synergistic eVect of BMP-4 and LIF on ES cell self-renewal. However, in this study they showed that BMP-4 could keep the pluripotency of ES cells by inhibiting both Erk and p38 mitogen-activated protein kinases (MAPKs). Importantly, in the presence of MAPK inhibitors such as PD98059 and SB203580 (type IA receptor for BMPs), they were able to establish ES cell lines from Alk-3/ blastocysts, which normally fail to expand and form ES colonies. Lastly, work from Anneren et al. has shown the importance of a nonreceptor tyrosine kinase in maintenance of ES cell pluripotency (Anneren et al., 2004). The authors examined the Src tyrosine kinase cYes, found to be expressed at high levels in multiple types of stem cells (Ivanova et al., 2002; Ramalho-Santos et al., 2002), for its role in ES cell self-renewal. The authors found that inhibiting cYes had no antagonistic eVect on Jak, Erk, or Stat3 phosphorylation, but expression of the ES cell marker Nanog was lost. Additionally, in cells treated with both a synthetic Src inhibitor (SU6656) and retinoic acid, an inducer of ES cell diVerentiation, ES cells displayed synergistic diVerentiation cues from the two molecules, demonstrating that cYes is an important molecule in maintaining ES cell self-renewal. B. Transcriptional Control of Embryonic Stem Cell Self-Renewal 1. Stat3 The third mammalian gene cloned of the signal transducers and activators of transcription (Stat) family was originally identified as an IL-6-activated transcription factor. All Stats share a phosphotyrosine-binding, SH2


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domain. Work by Boeuf et al. (1997) and Niwa et al. (1998) has shown that one of the most important ES cell self-renewal cytokines, LIF, signals through the LIFR –gp130 dimer complex to activate both the Shp2–Erk and Jak–Stat pathways. Through generation of chimeric receptors, Niwa et al. (1998) showed that Stat3 docking sites on the cytoplasmic region of gp130 are necessary to maintain ES cell self-renewal, implicating Stat3 in the modulation of transcriptional activity. Additionally, these chimeric receptors showed high levels of Erk activity upon receptor stimulation but that addition of the Mek inhibitor PD098059 did not inhibit stem cell colony formation, demonstrating that Erk pathway activation is not required for ES cell self-renewal. Lastly, use of the dominant negative mutant Stat3F, which has a tyrosine substitution at Tyr705 to phenylalanine (Y705F) and is incapable of phosphorylation, dimerization, and nuclear translocation, indicated that Stat3 is necessary for stem cell colony formation because no colonies could be generated from cells harboring a ‘‘supertransfected’’ episome expressing the Stat3F cDNA. 2. Nanog Nanog, or Enk, was originally identified in a screen for homeobox-containing transcripts via degenerate oligonucleotide polymerase chain reaction in a murine ES cell-derived cDNA library (Wang et al., 2003). The authors sought to identify transcripts of the Nk-2 family, which contains members such as the cardiogenic factor Nkx2–5. In this report the authors mapped the gene locus and determined the genetic architecture and showed that Nanog expression in developing embryos was first detected in compacted morulae, localized to interior cells, the future site of the inner cell mass (ICM). Nanog expression was further specified to the epiblast and absent from the primitive endoderm. Later, work from two groups (Chambers et al., 2003; Mitsui et al., 2003) further characterized the gene under the Nanog name. Several new observations came about from these two papers. Mitsui et al. (2003) used digital diVerential display to identify expressed sequence tags (ESTs) preferentially expressed in ES cells. Herein, they discovered that overexpression of Nanog under control of the chicken actin promoter/CMV-IE enhancer yielded ES cells that did not diVerentiate upon LIF withdrawal. The authors also demonstrated that Nanog expression is lost upon exposure to retinoic acid. Through targeted disruption of Nanog via introduction of a -Gal-Neo fusion gene, the authors demonstrated that Nanog/ ES cells lost their pluripotency and went down an endodermal diVerentiation pathway, expressing markers of the pariental (LamininB1, Dab2) and visceral endoderm (Bmp2, Ihh), as well as endoderm-specific transcription factors (Hnf4, Gata6). The authors generated Nanog heterozygous animals through blastocyst injection of Nanogþ/ ES cells, but Nanog/ embryos were found to be

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lethal by embryonic day (E) 5.5. Lastly, the DNA recognition sequence of Nanog was determined and was shown to be highly divergent from the consensus DNA-recognition sequence of other murine Nk-2 factors, demonstrating that Nanog is indeed distinct from the Nk-2 family. Using a ‘‘supertransfected’’ episomal expression screen, Chambers et al. (2003) identified Nanog in parallel as a self-sustaining factor in ES cells, capable of bypassing the LIF requirement for self-renewal. The relationship between the Jak–Stat pathway and Nanog was examined by using the inhibitor of Jak activity D6665 to show that Nanog-mediated self-renewal of ES cells is not Jak–Stat dependent, and showing that addition of LIF to cells carrying a Nanog episome augmented their ability to form ES cell colonies. Additionally, because LIF signaling is enhanced by inhibition of the Erk mitogenactivated kinase pathway (Burdon et al., 1999), the Ras–Erk pathway was stimulated and there was no change in the Nanog expression upon Erk stimulation. Lastly, Nanog expression was examined in Oct4/ embryos and it was determined that Nanog was expressed even in the absence of Oct4. 3. Oct3/4 To study transcription factors that are active in early mammalian development, Rosner and colleagues (1990) screened a cDNA library generated from F9 EC mRNA with a probe from the homeobox region of Oct2 at low stringency. All cDNAs isolated were from the same murine gene, which was given the name Oct3 due to the presence of an octamer sequence that was originally identified in the gene products of Oct1 and Oct2 (Singh et al., 1986; Staudt et al., 1986). Oct3 also turned out to be the gene that encodes the previously identified nuclear factor NF-A3 (Lenardo et al., 1989). Previously, Oct4 had been identified by Scholer and colleagues (1989) in a screen for octamer binding proteins. It was then determined that the two groups were working on the same gene, and the names Oct3, Oct4, and Oct3/4 have been used to identify the same gene and gene product. Oct3/4 is preferentially expressed in unfertilized oocytes, the ICM of the blastocyst, primitive ectoderm in egg-cylinder-stage embryos, and primordial germ cells, in addition to pluripotent cells in the mouse (Pesce et al., 1998). Rosner and colleagues (1990) showed that upon diVerentiation of embryonal carcinoma cells with retinoic acid, Oct3/4 expression decreases. These data from various expression patterns suggested that Oct3/4 plays an important role in maintaining cellular pluripotency. Further work demonstrated that Oct3/4 encodes a transcription factor that specifically binds an octamer motif and is capable of inducing transcription in in vitro assays. Through targeted gene ablation (Nichols et al., 1998), it was shown that Oct3/4 is required to initiate fetal development, because Oct3/4 deficiency leads to inability for founder cells of the ICM to acquire pluripotency. Instead, these cells become diverted


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into trophectoderm lineage. Conditional expression/repression of Oct3/4 showed that a precise level of Oct3/4 is required, because three diVerent fates are seen to be dependent upon Oct3/4 expression. ES cells require a critical level to maintain stem cell self-renewal, and a more than twofold increase causes diVerentiation into mesoderm and endoderm, whereas a reduction to less than 50% leads to induction of trophectoderm (Niwa et al., 2000). Collectively, models have emerged where ES cell fate (selfrenewal, endoderm/mesoderm, or trophectoderm) is regulated by Oct3/4 dosage, stoichiometric interactions with trans-acting factors, and the presence of LIF (Niwa, 2001). To date, a number of Oct3/4 target genes have been identified. Oct3/4 can bind a number of target sequences, including the consensus octamer motif ATGCAAAT and the AT-rich sequence (Okamoto et al., 1990; Saijoh et al., 1996). The variation in sequence means that Oct3/4 DNA binding is accomplished through both interactions with other sequence-specific trans-acting factors and homo- and heterodimerization (Tomilin et al., 2000). Identified by their stem-cell specific expression as Oct3/4 target genes were Fgf-4 (Yuan et al., 1995), the transcriptional coactivator Utf-1 (Nishimoto et al., 1999), the Zn-finger transcription factor Rex1 (Ben-Shushan et al., 1998), and the platelet-derived growth factor receptor (PDGFR) (Kraft et al., 1996). Other genes have been identified, and the common trait that these genes all have is ES cell-specific expression. Importantly, Oct3/4 has also been shown to demonstrate transcriptional repression activity toward several other transcription factors, including the caudal-related homeobox transcription factor Cdx-2 and the cardiac-specific bHLH transcription factor eHand (Niwa et al., 2000). Surprisingly, very little is known about the function of Oct3/4 target genes. The most heavily studied, Fgf-4, has been shown to be required for peri-implantation development yet is dispensable for stem cell self-renewal (Feldman et al., 1995). Clearly, future work is needed to ascertain the other trans-acting molecules that interact with Oct3/4, as well as the essential self-renewal ES cell genes that it activates.

II. From ES to Hematopoietic Progenitors A. An Overview of Hematopoietic Development The production of blood cells takes place in several distinct anatomical sites during mouse embryogenesis. Morphologically distinct primitive blood cells are first identifiable in the blood islands of the yolk sac at E7.5 of gestation. The liver rudiment is colonized by hematopoietic cells by E10.5 of the 20-day murine gestation period and thereafter becomes the principal fetal hematopoietic organ (Houssaint, 1981). Beginning at birth, bone marrow is

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colonized by hematopoietic stem cells (HSCs) originating from the fetal liver. From birth and throughout adult life, all mature blood cells are produced in the bone marrow. The term primitive hematopoiesis is given to the initial yolk sac-derived erythroid lineage, whereas definitive hematopoiesis is applied to all lineages other than primitive erythroid (Keller et al., 1999). The origin of the HSCs that colonize the fetal liver has remained controversial. There are currently two models concerning the spatial origin of HSCs. The first model is that the shift in the hematopoietic sites reflects the migration of HSCs from the yolk sac to the fetal liver and from the fetal liver to the bone marrow. According to this model, the microenvironment of the yolk sac, fetal liver, or bone marrow will determine the developmental potential of the HSCs to produce primitive versus definitive blood. The second model is that the HSCs that establish fetal liver hematopoiesis develop within the intraembryoic para-aortic-splanchnopleure (PAS)/ aorta-gonad-mesonephros (AGM) region. Support for the first model comes from studies showing that the yolk sac contains multiple definitive hematopoietic progenitors including long-term repopulating cells, even though in situ the yolk sac appears to have limited potential in generating mature blood cells (Cumano et al., 1993; Huang and Auerbach, 1993; Huang et al., 1994; Liu and Auerbach, 1991; Wong et al., 1986). Recent studies by Palis and colleagues (1999) demonstrate that definitive erythroid progenitors develop within the yolk sac even before the circulation is established. In addition, high proliferative potential colony-forming cells (HPP-CFCs), which can generate definitive erythroid cells and macrophages, were first detected exclusively in the yolk sac at early somite stages (E8.25) (Palis et al., 2001). Furthermore, when E7 mouse yolk sac and embryos were cultured separately, hematopoietic cells developed only from the yolk sac (Moore and Metcalf, 1970). Many studies reinforce the notion that the yolk sac contains hematopoietic stem cells. For example, Weissman et al. (1978) transplanted yolk sac cells from E8–10 mouse embryos into the E8–9 yolk sac of recipient embryos. The donor cells could be identified by an H-2 haplotype or Thy-1 marker. In this experiment the authors saw a low level of donor cell contribution to lymphoid cells in the recipients. Huang and Auerbach (1993) have taken AA4.1þWGAbright yolk sac cells and reconstituted the myeloid and lymphoid compartments of lethally irradiated adult mice. In this study, bone marrow cells depleted of long-term repopulating cells were coinjected. Finally, Yoder et al. used busulfan-myeloablated neonatal mice as recipients to demonstrate that E9 or E10 yolk sac cells could successfully reconstitute the hematopoietic system (Yoder and Hiatt, 1997; Yoder et al., 1997a,b). These findings suggest that primitive hematopoietic stem cells require an embryonic environment and that the adult microenvironment may not support the diVerentiation of the primitive HSCs. Consistent with this


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interpretation, yolk sac-derived cells can reconstitute the adult hematopoietic system when precultured on AGM-derived stromal cells (Matsuoka et al., 2001). In vitro progenitor assays and long-term repopulation studies utilizing yolk sac cells clearly suggest that HSCs are present within the yolk sac. However, the timing of the emergence of HSCs within the yolk sac is still unknown. HSC activity can be measured only after the emergence of primitive erythroid populations. Many investigators are actively pursuing the possibility that the primitive erythroid progenitor could develop directly from the mesodermal cells. The notion that the definitive hematopoietic system originates from intraembryonic progenitors initially derives from the studies of avian embryos. When yolk sac chimeras between a quail embryonic body (from the E2 embryo) and the extraembryonic area of a chick (from the E2 embryo) were generated and the chimeras analyzed between E5 and E13, the intraembryonic organs were always of quail origin (Dieterlen-Lievre, 1975; Dieterlen-Lievre and Martin, 1981). Similarly, when chick–chick chimeras were generated and blood cells analyzed, adult hematopoietic cells were shown to be of intraembryonic origin. In these chick–chick chimeras, sex chromosomes, immunoglobulin allotypes, and major histocompatibility complexes were used to distinguish extraembryonic versus intraembryonic origin (Lassila et al., 1978). Accumulating studies now support the notion that HSCs colonizing the fetal liver originate within the mouse embryo, in an area called the PAS/AGM. The AGM gives rise to vascular, excretory, and reproductive tissues of the embryo (reviewed in Dzierzak, 1999). The earliest tissues to form in the AGM of the E8 embryos are the paired dorsal aortas (Kaufman, 1992). The aortas become connected to the yolk sac vasculature via the vitelline (omphalomesenteric) artery by E8.5. The paired aorta will fuse to form a single dorsal aorta by E9 (Garcia-Porrero et al., 1995). The umbilical artery forms the connection between the dorsal aorta and the placenta (Garcia-Porrero et al., 1995). The urogenital and gonadal systems mature within the AGM soon after the vascular system is established (Kaufman, 1992). When the PAS/AGM region from an E8.5–9 embryo is isolated and grafted under the kidney capsule in SCID mice, serum immunoglobulin M (IgM), IgM-secreting plasma cells, and B cells of the B1a phenotype of donor origin can be detected 3–6 months after the engraftment (Godin et al., 1993). Furthermore, the PAS/AGM region gives rise to both myeloid and lymphoid cells when cultured in vitro (Cumano et al., 1996). More importantly, lymphoid potential of the PAS/AGM region is found even before the circulation is established (Godin et al., 1995). Finally, the PAS/AGM region contains spleen colony-forming cells (CFU-S) and the AGM from precirculation stage or E10 embryos contains long-term repopulating HSCs (Cumano et al., 2001; Medvinsky and Dzierzak, 1996;

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Medvinsky et al., 1993). By further dissecting the AGM and testing for longterm repopulating HSC activity, the aorta has been shown to be the initial site of adult HSC emergence (E10.5), followed by the vitelline and umbilical arteries (de Bruijn et al., 2000). These observations strongly argue that cells that colonize the fetal liver originate within the embryo.

B. Hemangioblast The origin of the blood cells that establish the yolk sac blood islands and the AGM is another area of active investigation. Studies over the last 100 years have shown that blood cells within yolk sac blood islands and the AGM develop in close proximity to the vascular system. It has to be noted, however, that the vascular system does not necessarily associate with hematopoiesis. In the yolk sac, mesodermal cells, which have migrated from the primitive streak, form aggregates to establish blood islands at around E7. Over the next 12 hours, the central cells within the blood islands generate primitive blood cells while the peripheral cells diVerentiate into endothelial cells. These blood islands subsequently fuse to form the first extraembryonic vascular network. The close developmental association of the hematopoietic and endothelial cell lineages within the yolk sac blood islands of the developing embryo has led to the hypothesis that they arise from a common precursor, termed the hemangioblast (Murray, 1932; Sabin, 1920; Wagner, 1980). Similarly, blood cells of the embryo proper develop in close association with the endothelium of the dorsal aorta (Dieterlen-Lievre, 1997; Garcia-Porrero et al., 1995; Tavian et al., 1996, 1999). The major arteries such as the vitelline and umbilical arteries of embryos also have been reported to associate with emerging blood cells (de Bruijn et al., 2000; Garcia-Porrero et al., 1995). In contrast to the common progenitor concept in the yolk sac, blood cells in the embryo proper are believed to diVerentiate from the endothelium. For example, in the floor of the dorsal aorta of the chicken or quail (JaVredo et al., 1998), intra-aortic CD45þVEGFR-2 (Flk-1) hematopoietic cells appear to develop from VEGFR-2þ (Flk-1þ) cells that take up DiI-conjugated acetylated low-density lipoprotein (DiI-acLDL). In mice, VE-cadherinþ, CD45, TER119 cells (potentially endothelial cells) from E9.5 mouse embryos could generate hematopoietic cells, including lymphocytes (Nishikawa et al., 1998a). Similarly, the HSC activity in the AGM regions and vitelline and umbilical arteries of E11.5 Runx1þ/Lacz embryos was derived from cells in the endothelium that do not express CD45 but do express Runx1, a critical transcription factor for definitive hematopoietic development (North et al., 2002). Therefore, the term hemogenic endothelium is often used to describe the hematopoietic potential of presumably aortic endothelial cells. The precise relationship between the hemangioblast and hemogenic endothelial cells is currently not known.


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C. The Identification of Blast Colony–Forming Cells from In Vitro Differentiated Embryonic Stem Cells Even though there has been a great interest in identifying the hemangioblast in the developing embryo, the use of embryo-derived cells has proven diYcult because the developmental sequence occurs rapidly, the tissues are diYcult to access, and only a small number of cells can be obtained. An alternate source of embryonic cells for the studies of early embryonic events is the in vitro diVerentiated progeny of ES cells. ES cells diVerentiate eYciently in vitro and give rise to three-dimensional, diVerentiated cell masses called embryoid bodies (EBs) (Fig. 1; reviewed in Choi, 2002; Keller et al., 1999). ES cells can also be diVerentiated on stromal cells or type IV collagen without intermediate formation of the EB structure (Nakano et al., 1994; Nishikawa et al., 1998b). Many diVerent lineages have been reported to develop within EBs, including neuronal, muscle, endothelial, and hematopoietic lineages (reviewed in Choi, 2002). Of these, the hematopoietic lineage has been the most extensively characterized. The development of hematopoietic and endothelial cells within EBs mimics in vivo events such that yolk sac blood island-like structures with vascular channels containing hematopoietic cells can be found within cystic EBs (Doetschman et al., 1985). As in the developing embryo, the primitive erythroid cells develop prior to definitive hematopoietic populations (Keller et al., 1993; Palis et al., 1999). The developmental kinetics of various hematopoietic lineage precursors within EBs and molecular and cellular studies of these cells have demonstrated that the sequence of events leading to the onset of hematopoiesis within EBs is similar to that found within the normal mouse embryo. In addition, EBs provide a large number of cells representing an early or primitive stage of development that is otherwise diYcult to access in an embryo. Therefore, the in vitro diVerentiation model of ES cells is an ideal system for obtaining and studying primitive progenitors of all cell lineages. Using the in vitro ES diVerentiation model system, the blast colonyforming cell (BL-CFC) population present within day 2.5–3.5 EBs has been shown to represent the hemangioblast (Choi et al., 1998). BL-CFCs are transient and develop prior to the primitive erythroid population (Choi et al., 1998; Kennedy et al., 1997). BL-CFCs form blast colonies in response to vascular endothelial growth factor (VEGF), a ligand for the receptor tyrosine kinase, Flk-1 (Matthews et al., 1991; Millauer et al., 1993), in semi-solid media such as methylcellulose cultures. Gene expression analysis indicated that cells within the blast colonies (blast cells) express a number of genes common to both hematopoietic and endothelial lineages, including Scl, CD34, and the VEGF receptor, Flk-1 (Kennedy et al., 1997). Blast cells contain primitive and definitive hematopoietic as well as endothelial cell progenitors (Choi et al., 1998; Kennedy et al., 1997). Most importantly,

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the hematopoietic and endothelial precursors present within the blast colonies are clonal, as demonstrated by cell-mixing studies of two diVerent ES lines (Choi et al., 1998). D. From Flk-1-Expressing Mesoderm to Hematopoietic and Endothelial Cells Great progress has been made in recent years toward delineating the cellular sequence leading to hematopoietic and endothelial cell development from mesoderm (Fig. 2). First, Fehling et al. (2003) examined Brachyuryþ/GFP ES cells and demonstrated that Brachyuryþ mesodermal cells develop first. Flk-1 is turned on within Brachyuryþ cells to form BrachyuryþFlk-1þ mesoderm. Cell-replating studies demonstrated that BrachyuryþFlk-1þ cells contained hemangioblasts. Second, Motoike et al. (2003) performed fatemapping studies of Flk-1þ cells by examining Flk-1þ/Cre knock-in mice. When crossed to Rosa-26 reporter (R26R) mice (Soriano, 1999), and when E8.5 embryos were stained for LacZ expression, Flk-1 expression was seen in all vascular endothelial cells and hematopoietic cells. When LacZ staining of Flk1þ/Cre; R26R to Flk-1þ/LacZ mice was compared, the authors observed that Cre excision occurs precisely at the time of endogenous Flk-1 expression. In addition to vascular and hematopoietic cells, Flk-1 expression was also detected in cardiac and skeletal muscles in E10.5 embryos. Third, Ema et al. (2003) generated Flk-1þ/Scl ES cells and demonstrated that they generated an increased number of blast colonies as compared with wild-type

Figure 2 Flk-1-expressing mesoderm is thought to generate the circulatory system, including blood, endothelial, skeletal, smooth muscle, and cardiac muscle cells.


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/Scl

ES cells. Flk-1 embryos were also generated and examined. Although blast colony generation was rescued in these ES cells, the hematopoietic and endothelial defects in the mice were not, likely due to the migratory defects of cells lacking Flk-1. Furthermore, Flk-1-expressing cells from Flk-1þ/Scl produced predominantly hematopoietic and endothelial cells in culture, whereas Flk-1 expressing cells from Scl/ ES cells could not diVerentiate into endothelial cells. Instead, Scl-deficient Flk-1þ cells readily generated smooth muscle cells in vitro. These studies indicate that Scl expression is critical for hemangioblast development, and also suggest that the coordinated expression of Flk1 and Scl is critical for proper development of hematopoietic, endothelial, and smooth muscle cells. The contribution of Flk-1þ cells to pericytes was also shown by Yamashita et al. (2000). In this study, ES-derived Flk-1þ cells were able to generate endothelial and smooth muscle cells in vitro and in vivo. Finally, Flk-1 and Scl were shown to be molecular determinants of the ES-derived hemangioblast (Chung et al., 2002). In this study, a nonfunctional human CD4 (hCD4) was knocked into the Scl locus and cells expressing Flk-1 and hCD4 were sorted and shown to readily generate blast colonies. Moreover, the kinetic and cell-replating studies of Flk-1- and hCD4-expressing cells demonstrated that hematopoietic and endothelial cells developed via sequential generation of Flk-1 and Scl-expressing cells. Flk-1þ cells first arise in developing EBs, and the Scl gene is turned on within Flk-1þ cells to give rise to Flk-1þhCD4þ cells. Alternatively, a subpopulation of the initial Flk-1þhCD4 cells remains Scl negative. Within Flk-1þhCD4þ cells, Flk-1 is downregulated to generate Flk-1hCD4þ cells. Replating studies demonstrate that hematopoietic progenitors are enriched within Flk-1þhCD4þ and Flk-1hCD4þ cells, whereas endothelial cells develop from Flk-1þhCD4þ and Flk-1þhCD4 cell populations. These studies indicate that there are two populations of endothelial cell progenitors, Scl dependent and independent. The report suggests that Scl-dependent endothelial cells develop from the hemangioblast (Fig. 3).

Figure 3 Schematic diagram of the emergence of Flk-1- and Scl-expressing hemangioblast, angioblast, and hematopoietic progenitors from ES cells. Transcription factors that function at each developmental stage are also indicated.

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E. Hematopoietic Inductive Signals One of the most heavily pursued areas of current research in developmental hematopoiesis has been to identify inductive signals that initiate the hematopoietic program. The fact that only the mesoderm that is situated adjacent to visceral endoderm generates both blood and endothelial cells in the extraembryonic yolk sac indicates that factors/signals from the visceral endoderm are critical for hematopoietic induction. Studies of quail–chick chimeras (Pardanaud et al., 1996) have shown that the endoderm can induce hematopoiesis from the somatopleural mesoderm, which only has angiogenic potential. Similarly, BelaoussoV et al. (1998) have shown that primitive endoderm can induce hematopoiesis from the anterior epiblast, which is prospective neural ectoderm. Xenopus animal cap cultures have been useful in identifying factors/signals that can induce hematopoietic diVerentiation. Animal caps, which normally diVerentiate into ectoderm, can generate mesoderm in the presence of several growth factors such as BMPs, Activin A, and basic fibroblast growth factor (bFGF). Given the fact that blood cells develop from mesoderm, these mesoderm-inducing factors could have a hematopoietic inductive role. Indeed, the formation of erythroid cells from the animal cap can be induced by BMP-4 and bFGF or by BMP-4 and Activin A, and the generation of erythroid cells by exogenously expressed Gata1 can be potentiated by bFGF (Huber et al., 1998). In addition, BMP-4 could induce generation of erythroid cells through upregulation of Gata2 (Maeno et al., 1996). Similarly, the formation of blood islands from quail epiblasts is dependent on bFGF (Flamme and Risau, 1992). In this system, bFGF-mediated blood island formation correlates with the induction of the Flk1 gene (Flamme et al., 1995), suggesting that bFGF is critical for the emergence of the hemangioblast. Furthermore, in quail embryos, bFGF, VEGF, and transforming growth factor (TGF)- 1 can induce hematopoietic diVerentiation from the somatopleural mesoderm (Pardanaud et al., 1996). Similarly in mice, both Activin A and BMP-4 can induce hematopoietic diVerentiation from the anterior headfold region (Kanatsu and Nishikawa, 1996), and Indian hedgehog can promote hematopoietic diVerentiation from the anterior epiblast (Dyer et al., 2001). The ES–EB system has proven valuable for identifying factors involved in hematopoietic induction. Ultimately, this information can be used to manipulate the system such that all the progeny of EB cells take a hematopoietic fate. Several studies have shown that mesoderm-inducing factors can aVect EB diVerentiation. For example, the addition of bFGF or Activin A to diVerentiatin EBs enhances Brachyury gene expression, a marker of mesodermal tissue (Yamada et al., 1994). This observation indicates that cells within EBs can respond to external signals and therefore are useful for


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examining factors involved in hematopoietic specification. Johansson and Wiles (1995) initiated studies investigating the role of mesoderm-inducing factors on hematopoietic development. When ES cells were diVerentiated in serum-free, chemically defined medium (CDM) in the presence of various mesoderm-inducing factors, BMP-4 could induce the expression of the embryonic globin gene, H1. Similarly, studies by Adelman et al. (2002) demonstrated that BMP-4 was a prerequisite for erythroid-lineage-specific gene expression in ES cells. In this study, BMP-4 was added to serum-free medium and the expression of the erythroid cell-specific genes, Eklf and Gata1, was examined to show that BMP-4 was important for Eklf and Gata1 gene induction. Recently, factors that can induce Flk-1- and Scl-expressing cells were examined by utilizing in vitro diVerentiation models of ES cells (Park et al., 2004). In serum-free conditions, BMP-4 was critical for Flk-1 induction. BMP-4 activated the Smad1/5 pathway, whereas inhibition of the Smad1/5 pathway resulted in a reduction of Flk-1þ cell generation. Consistent with the notion that BMP-4 is critical for the generation of Flk-1þ cells, Bmp4deficient mice die between E7.5 and E9.5 with defects in mesoderm formation and patterning. Those that survive up to E9.5 display severe defects in blood islands (Winnier et al., 1995). Additionally, mice lacking the type I BMP receptor (Alk-3), which binds BMP-2 and BMP-4, fail to complete gastrulation, and none survive up to E7.0 (Mishina et al., 1995). Mice deficient in Smad1 or Smad5, downstream signaling molecules of TGF- family members, display varying degrees of defects (or no obvious phenotype) in hematopoietic and vascular development. This variation may be due to overlapping function between Smad1, 5, and 8 (Tremblay et al., 2001). For example, Smad1-deficient mice display early embryonic lethality and die between E9.5 and E10.5 due to failure of chorioallantoic fusion (Lechleider et al., 2001; Tremblay et al., 2001). Smad5-deficient mice die between E9.5 and E11.5. Mutant embryos are anemic and have disorganized vessels, despite formation of the primitive plexus. There seemed to be more primitive blood cells in E8.5 mutant yolk sacs, although E9.5 mutant yolk sacs contained almost no blood cells (Chang et al., 1999). Subsequent studies demonstrated that Smad5-deficient yolk sacs contained a higher frequency of HPP-CFCs, and Smad5-deficient ES cells gave rise to increased hematopoietic progenitors, including blast colonies in vitro (Liu et al., 2003). For proper hematopoietic development, the expansion of Scl-expressing cells requires VEGF-mediated signaling (Park et al., 2004). VEGF has been shown to play a critical role in vasculogenesis, angiogenesis, and hematopoiesis during embryonic development. VEGF expression can be detected as early as E5.5 in the extraembryonic visceral and parietal endoderm during mouse embryogenesis (Miquerol et al., 1999). At E7.0–7.5, VEGF is expressed in the extraembryonic visceral endoderm and extraembryonic

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mesoderm, but not in the embryo proper. VEGF expression within the embryo proper can be detected at E8.0 in the definitive endoderm. The dorsal aorta forms in close proximity to the embryonic endoderm. Mice heterozygous for Vegf (Vegf þ/) are embryonic lethal due to defects in vascular development (Carmeliet et al., 1996; Ferrara et al., 1996). In these mice the production of hematopoietic cells is significantly reduced. Furthermore, mice with slightly higher levels of VEGF expression (two to threefold) display early embryonic lethality due to severe abnormalities in heart development (Miquerol et al., 2000). Finally, hypomorphic Vegf þ/lo animals are viable and normal, but Vegf lo/lo embryos die early due to abnormalities in yolk sac vasculature and from deficiencies in the development of the dorsal aorta (Damert et al., 2002). Recent studies demonstrate that VEGF production from the yolk sac visceral endoderm is suYcient and necessary for blood island formation and for vascular development. In these studies, chimeras between Vegf wild-type tetraploid embryos and diploid Vegf lo/lo embryos showed rescue in blood island formation and in vascular development (Damert et al., 2002). In these chimeras, yolk sac visceral endoderm and trophoblast tissue will develop from the tetraploid embryos. Moreover, the hematopoietic cell population in the embryo proper of these chimeras increased as the contribution of Vegf wildtype tetraploid cells to the yolk sac visceral endoderm was augmented. Importantly, chimeras generated between Vegf lo/lo tetraploid embryos with Vegf þ/þ ES cells showed defects in yolk sac vascular development. Collectively, these studies indicate that tight regulation of VEGF expression is crucial for correct vascular and hematopoietic development in the early embryo. Another important player in hematopoiesis is TGF- 1, which is expressed in yolk sac blood islands, mesodermal cells of the allantois, and cardiogenic mesoderm of the embryo (Akhurst et al., 1990). TGF- 1 binds its cognate receptors TGF- RII and TGF- RI. TGF- receptor II expression largely correlates with that of TGF- 1 (Lawler et al., 1994). Tgf 1/ mice showed two distinct phenotypes; 50% were perinatal lethal, and the rest died around E10.5 (Dickson et al., 1995). The latter displayed yolk sac anemia due to a severe reduction of erythrocytes and a defect in endothelial cell diVerentiation. Mice deficient in Tgf rII showed defects in yolk sac hematopoiesis and vasculogenesis (Oshima et al., 1996). Tgf rI / mice displayed increased numbers of erythroid progenitors, whereas granulocyte-macrophage colony–forming cells (CFU-GM) and mixed colony-forming cells (CFU-Mix) appeared to be normal (Larsson et al., 2001). These studies suggest that TGF- 1 signaling is necessary for normal vascular development, but could be inhibitory to the growth and diVerentiation of hematopoietic progenitors. Consistent with these data, TGF- 1 inhibited BMP-4 and VEGF-mediated hematopoietic induction in the ES–EB system (Park et al., 2004). Hedgehog signaling is important in pattern formation and morphogenesis in a variety of developing embryos (McMahon et al., 2003). In mice, three


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members have been identified so far: Sonic hedgehog (Shh), Desert hedgehog (Dhh), and Indian hedgehog (Ihh). Among these, only Ihh is expressed in the visceral endoderm of the yolk sac (Dyer et al., 2001; Farrington et al., 1997) and its receptor–downstream molecules are expressed in the posterior epiblast, which is destined to form blood and endothelial cells during early gastrulation. Two reports have demonstrated that primitive endoderm or IHH itself can induce hematopoiesis from explanted epiblasts (BelaoussoV et al., 1998; Dyer et al., 2001). In addition, the anterior epiblast, which is fated to give rise to neuroectoderm, can generate hematopoietic cells upon IHH treatment. These findings clearly suggest that IHH-mediated signaling has an important role for hematopoiesis. Furthermore, IHH could also induce the expression of BMP-4 from anterior epiblast (Dyer et al., 2001). Given the role of BMP-4-mediated signaling in the upregulation of Brachyury expression and the generation of Flk-1þ cells from ES cells, it is possible that IHH acts as an upstream regulator of BMP-4. The Notch pathway has been considered a cell fate determinant of multipotent precursor cells (Artavanis-Tsakonas et al., 1999). During hematopoiesis, Notch-mediated signaling is required for T-cell commitment and has also been implicated in modulating the self-renewal capacity of hematopoietic stem cells in adult bone marrow (Ohishi et al., 2003). Recent work by Kumano et al. (2003) demonstrated new roles for the Notch pathway during early hematopoiesis. The PAS culture from Notch1/ embryos displayed severely impaired hematopoiesis, although the yolk sac generated a similar number of hematopoietic colonies as compared with wild type. Notch2/ embryos did not show any significant diVerence from wild-type embryos. However, when transplanted into conditioned neonatal mice, E9.5 Notch1/ PAS or yolk sac-derived cells could not reconstitute the hematopoietic system, indicating an essential role for Notch1 in generating HSCs in the yolk sac and PAS. F. Transcriptional Control of Hematopoietic and Endothelial Cell Lineage Commitment 1. Scl Originally identified as a target of t(1;14) chromosomal translocations in Tcell acute lymphoblastic leukemia (Finger et al., 1989), Scl is a bHLH transcription factor that is required for the generation of all hematopoietic lineages in the mouse (Robb et al., 1995; Shivadasani et al., 1995). In the adult, Scl is expressed in hematopoietic and endothelial cells. In the developing embryo Scl is expressed as early as E7.5, and by E8.5 the expression is localized to the yolk sac blood progenitors and endothelial cells (Green et al., 1991). Scl is also expressed in the developing brain (Green

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et al., 1992). Like many genes important for hematopoiesis, the role of Scl was delineated through generation of Scl-deficient mice. Scl-null animals die by E10.5 due to defective embryonic hematopoiesis (Robb et al., 1995; Shivdasani et al., 1995). Moreover, Scl-null ES cells failed to contribute to any hematopoietic tissues in chimeric animals. Subsequent studies showed that Scl is also required for endothelial cell development, because Scl/ ES cells fail to contribute to the remodeling of the primary vascular plexus in the yolk sac (Visvader et al., 1998). Scl deficiency resembles loss of the erythroid transcription factor GATA-1 or the LIM protein Lmo2. At the molecular level, Scl-null ES cells do not express either major or minor isoforms of globin. Additionally, several other hematopoietic genes are downregulated in Scl/ cells. Myb, Pu.1, and Nf-E2 all have diminished levels of expression as compared with wild type, and Gata1 and Eklf are nearly absent (Robb et al., 1996). Conversely, it has been demonstrated that not only does enforced expression of Scl strongly enhance the blood formation in embryos (Gering et al., 1998), but Scl is capable of converting somitic and pronephric duct tissues into hemangioblasts (Gering et al., 2003), indicating that Scl has a dominant role in the commitment to hemangioblasts, much like the ability for MyoD to commit cells to a myogenic fate. Several studies further support the notion that Scl is critical for hemangioblast specification. First, Scldeficient ES cells failed to give rise to blast colonies (Faloon et al., 2000; Robertson et al., 2000), the progeny of the hemangioblasts. Moreover, enhanced expression of Scl from the Flk-1 locus (Flk-1þ/Scl ES cells) produced a higher number of blast colonies compared with Flk-1þ/þ cells (Ema et al., 2003). The mechanisms of Scl-mediated transcription are still unclear. Studies by Porcher et al. (1999) indicate that mutant Scl unable to bind DNA can still rescue embryonic hematopoiesis and restore definitive hematopoiesis considerably in Scl / ES cells. Because the HLH domain was absolutely required, the emerging model for Scl initiating embryonic hematopoiesis is that it functions as a nucleating factor to bring transcription factors together. For example, Scl will recruit an E protein, such as E2A, to an E-box (CANNTG), bound to a bridging molecule that are LIM domain-containing molecules such as Lmo2 (see later) and Ldb1 (Wadman et al., 1997). This complex then links the E-box to a GATA motif bound by either GATA-1 or GATA-2 (more later). The precise architecture and role of this complex remains an attractive model, albeit needing further inquiry (Cantor and Orkin, 2002). 2. GATA-2 The second member cloned of the six-member GATA binding protein family, GATA-2, shows strong expression in pluripotent cells. All GATA proteins bind the consensus (T/A)GATA(G/A) DNA sequence (Orkin,


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1992). Additionally, each GATA protein contains two Zn-finger motifs. The amino-terminal finger is used for protein–protein interactions with other trans-acting factors, and the carboxy–terminal finger is required for DNA binding. GATA-1, GATA-2, and GATA-3 are considered to be the ‘‘hematopoietic’’ GATAs, whereas GATA-4, GATA-5, and GATA-6 are all considered to be ‘‘endodermal’’, because they are expressed in endodermal tissues and their absence leads to developmental defects in endodermally derived tissues such as the heart, gut, intestines, and lungs. Gata1 and Gata2 have been identified as early acting genes in hematopoiesis. Current models indicate that Gata2 is induced early in the extraembryonic yolk sac and induces expansion of hematopoietic progenitors, whereas Gata1 is induced later, by GATA-2 and functions in erythroid cell maturation (Ohneda and Yamamoto, 2002). Unlike many other hematopoietic transcription factors, GATA-2 is rarely associated with leukemias caused by aberrant expression. Gata2/ animals are embryonically lethal at E10.5 due to severe anemia (Tsai et al., 1994). The Gata2/ animals displayed a severe reduction in the number of primitive erythroid cells. When Gata2/ ES cells were used to generate chimeric animals, there was no contribution to any hematopoietic compartments nor was -globin made by the Gata2/ cells. Thus, early work defined GATA-2 as having a role in the expansion/proliferation of the early, primitive hematopoietic compartment but largely dispensable for the diVerentiation of the majority of hematopoietic lineages. Subsequent work using conditional induction of GATA-2 has helped to show that enforced GATA-2 expression can enhance the production of hematopoietic progenitors (Kitajima et al., 2002). This report showed that forced expression of GATA-2 suppressed expression of other later hematopoietic transcription factors, including Pu.1 and c-Myb. Additionally, from this same report, the link between GATA-2 and the formation of HSCs is examined as the authors see that enforced GATA-2 expression leads to a three-fold increase in the number of hematopoietic colonies and that upon FACS analysis through the presence of an internal ribosomal entry site-enhanced green fluorescent protein (IRES-EGFP) linked to the Gata2 artificial promoter, nearly all EGFPþ cells were also Scaþ and c-Kitþ, showing that GATA-2 directly augments the HSC pool. Lastly, recent work has demonstrated the mechanism by which GATA-2 induces transcription of Gata1 and GATA-1 in turn suppresses Gata2 transcription. Using a quantitative chromatin immunoprecipitative (ChIP) assay, Grass and colleagues (2003) show that GATA-1 binds a highly restricted upstream region of the approximately 70kb Gata2 domain. GATA-1 then rapidly displaces GATA-2, which is coupled to transcriptional repression. GATA-1 also displaces cAMP response element-bind protein (CREB) binding protein (CBP), despite the fact that GATA-1 binds CBP in other contexts. This repression correlates with reduced domain-wide histone acetylation. GATA-1 instigates Gata2

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repression by means of disruption of positive autoregulation, followed by establishment of a domain-wide repressive chromatin structure (Grass et al., 2003). This GATA switch likely accounts for the change to a diVerentiation transcriptional program from a proliferative transcriptional program in hematopoietic progenitors. Collectively, GATA-2 appears to function in the expansion/proliferation of the early, primative hematopoietic compartment. 3. Lmo2 Lmo2 is a Zn-finger, LIM-only protein. Unlike other Zn-finger transcription factors, the LIM-only proteins contain only the -helical structure of other Zn-finger family members, thus enabling protein–protein interactions but making protein–DNA interactions impossible (Perez-Alvarado et al., 1994). Like many other hematopoietic transcription factors, aberrant expression of Lmo2 through chromosomal translocations can lead to leukemias. This occurs because Lmo2 acts like a transcription factor, serving to bridge complexes together to induce gene transcription with the aid of the basal transcription machinery. As described earlier, Lmo2 (and Ldb1) does not physically bind DNA, but directly interacts with transcription factors that do. Lmo2’s tight linkage with Scl-mediated transcription is further evidence that it behaves like a bona fide transcription factor, because loss of Lmo2 leads to impairment of Scl-mediated transcription (Larson et al., 1996; ValgeArcher et al., 1994). Accordingly, Lmo2/ mice die due to failure of yolk sac erythropoiesis (Warren et al., 1994). In addition, because Lmo2 is posited to bridge Scl/E2A and GATA proteins, the phenotype of the Gata1-null mutant animal is similar to the phenotype of the Lmo2-null animal (Weiss et al., 1994). Finally, coexpression of GATA-1 with Scl and Lmo2 in Xenopus organisms at one-cell-stage embryo leads to ventralization, and blood cell formation in these embryos becomes obvious throughout the dorsal–ventral axis. Thus, it appears that a Scl–Lmo2–GATA-1 complex is critical for specifying mesoderm to become blood during Xenopus embryogenesis. 4. Runx1 Also known as Aml1 and Cbf2, Runx1 is a frequent target of translocations in leukemias. Runx1 shows homology to the Drosophila paired-rule gene Runt and binds to the TGT/cGGT DNA sequence (Daga et al., 1992; Meyers et al., 1993). To achieve basal transcriptional activity, Runx1 heterodimerizes with Cbf through an 118 amino acid Runt homology domain (RHD) (Meyers et al., 1993; Ogawa et al., 1993; Wang et al., 1993). The AML– ETO fusion is one of the most frequent translocations seen in acute myeloid leukemia (AML) caused by t(8;21) that fuses the DNA-binding domain of Runx1 (AML) to the activation moiety of the Zn-finger transcription factor


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Eto (Erickson et al., 1992; Miyoshi et al., 1993; Nisson et al., 1992). Understanding of the normal gene product came in 1996 when two groups ablated the gene encoding Runx1 (Okuda et al., 1996; Wang et al., 1996). Homozygous animals died between E12.5 and E13.5. Although the appearance and development of both the embryo and the extraembryonic yolk sac were normal, the null animals showed hemorrhage in the central nervous system. Moreover, despite normal yolk sac hematopoiesis and the presence of primitive nucleated erythrocytes, the animals lacked any definitive fetal-liverproduced hematopoietic tissue. Whereas normal animals will have primitive nucleated erythrocytes, immature granulocytes, and macrophages/monocytes, as well as numerous less diVerentiated cells, Runx1-null animals had only primitive nucleated erythrocytes present in their liver (Wang et al., 1996). Analysis of chimeric animals showed that although Runx1þ/ ES cells were capable of contributing to bone marrow, peripheral blood, thymus, and spleen, Runx1/ ES cells could not contribute to any hematopoietic tissues despite contribution to other tissues (Okuda et al., 1996). Studies with Runx1þ/LacZ mice further support the role of Runx1 in definitive hematopoiesis (North et al., 1999, 2002). Runx1 is expressed in the ventral wall of the dorsal aorta, as well as in the vitelline and umbilical arteries. Its expression then is found in clusters of cells that are closely located to the lumen of these regions. These cells are hematopoietic cells, as shown by their round shape and expression of the pan-leukocyte marker CD45 (JaVredo et al., 1998; Tavian et al., 1996). Taking into account the study that the AGM, vitelline, and umbilical arteries harbor long-term repopulation hematopoietic cells (LTR-HSCs), it raises the possibility that LacZ (Runx1)-expressing round cells in these regions could be HSCs. Recently, North et al. (2002) demonstrated that Runx1þ cells are HSCs. In this study it was demonstrated that transplanted Runx1þ cells, not Runx1 cells from the AGM, vitelline, and umbilical arteries can reconstitute the hematopoietic system of irradiated recipient mice, suggesting that Runx1 is critical for HSC generation. In addition to its role in definitive hematopoiesis, several lines of evidence suggest that Runx1 could be involved in hemangioblast development. Runx1 is first detectable in the extraembryonic mesodermal cells (E7.25) and then in both primitive erythrocytes and endothelial cells in the yolk sac blood islands at E8.0 (Lacaud et al., 2002; North et al., 1999). A study of Runx1 expression kinetics showed that Runx1 is also expressed at the same time that Flk-1 and Scl are expressed. Moreover, blast colonies and BL-CFCs express Runx1, suggesting that Runx1 is an important regulator of hemangioblast formation. Consistent with this idea, Runx1/ ES cells generated a significantly reduced number of blast colonies. Subsequent replating experiments revealed that blast colonies from Runx1/ ES cells normally gave rise to primitive erythroid colonies, but failed to generate definitive hematopoietic

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colonies. Collectively, these results indicate that Runx1 is required for both hemangioblast development and definitive hematopoiesis. Runx1-mediated gene transcription is achieved through cooperation with other trans-acting molecules such as Ets-1. For instance, the T-cell receptor promoter contains adjacent Runx1 and Ets binding motifs. Alone, each is suYcient to bind its cognate DNA. However, mutation of either single site disrupts transcription, although transcriptional activation is independent of the spacing of the sites (Wotton et al., 1993, 1994). This same site synergy is also seen with c-Myb (Hernandez-Munain and Krangel, 1995). More recent data indicate that Runx1 serves as an assembly factor, inducing conformation changes and recruiting additional trans-acting molecules to activate gene transcription, and may possess little trans-activating activity itself (Hernandez-Munain and Krangel, 2002). Yamaguchi and coworkers have shed further light on Runx1’s trans-activating capabilities, showing that acetylation of specific lysine residues (Lys-24 and Lys-43) by p300 is required to obtain maximal DNA binding and transcriptional capability (Yamaguchi et al., 2004).

G. In Vivo Potential of Embryonic Stem Cell–Derived Hematopoietic Progenitors The ability of ES cells to generate many diVerent somatic cells in vitro argues for their usage as a source for cell transplantation, provided that they can function in vivo. Accumulating studies demonstrate that in vitro generated hematopoietic progenitors can function in vivo, although the generation of HSCs from ES cells has not been firmly established. Muller and Dzierzak (1993) have utilized in vitro diVerentiated ES cells as donor cells in cell transfer studies using newborn Wv/Wv and SCID mice as recipients. In these studies, donor-derived cells were found only within the lymphoid cell lineage, although donor-derived cells persisted for longer than 6 months. Day 13 EB cells were found to be the most eYcient in repopulating the hematopoietic system. Because entire EBs without further enrichment of hematopoietic progenitor cells were used in this study, the findings that ES-derived cells had limited lymphoid potential could reflect the rarity of HSCs present within day 13 EB cell populations. Potocnik et al. (1997) have isolated AA4.1þB220þ and AA4.1þB220 cell populations and tested the in vivo reconstitution potential in Rag-1-deficient mice (4–6 weeks old). Both cell populations engrafted the recipients, but AA4.1þB220þ cells had limited life span and limited potential (i.e., they persisted for only up to 8 weeks in the recipients and could only give rise to the B-cell lineage): However, AA4.1þB220 cells survived longer in the recipients and could be found even 24 weeks after the transplantation. AA4.1þB220 cells were found to be

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1. Hematopoietic and Endothelial Cells þ

þ

þ

more primitive compared with AA4.1 B220 cells, because AA4.1 B220 cells could give rise to both B and T cells. Donor-derived B220þc-Kitþ cells were also found in the recipient bone marrow, suggesting that AA4.1þB220 cells contained more primitive hematopoietic progenitors. However, the HSC activity was not examined in this study. The ability for ES-derived cells to repopulate the hematopoietic system was much lower, compared with fetal liver cells, arguing for a need to develop an optimal protocol for utilizing ES-derived cells for hematopoietic reconstitution. It is important to point out that ES cells derived from the 129-mouse strain were used in both studies to reconstitute C57 (i.e., C57B46) BL/6 mice, demonstrating that allogeneic transplantation works well. A study by Hole et al. (1996) showed multilineage (lymphoid and myeloid compartment) reconstitution by in vitro diVerentiated EBs. In this study, ES cells were diVerentiated for 4 days and then transplanted into lethally irradiated adult mice. It should be noted that the authors used the earliest time point at which primitive multilineage hematopoietic precursors can be detected. The recipient mice survived for more than 3 months and contained EB-derived lymphocytes and granulocytes. However, EB-mediated repopulation was lost by 6 months, indicating that the day 4 EB cells that they used could have contained short-term repopulating hematopoietic progenitors or committed multilineage precursors. Most recently, Burt et al. (2004) were able to isolate a c-KitþCD45þ population from diVerentiated ES cells. ES-derived c-KitþCD45þ cells could reconstitute hematopoietic compartments when transplanted into lethally irradiated recipients. The authors showed donor-derived contribution to lymphocytes, monocytes, and granulocytes in the peripheral blood of the recipients. Other hematopoietic organs were not examined in this study. This study showed that intrabone injection generated more eYcient engraftment of the ES-derived cells, compared with intravein injection. In addition to nonmanipulated EB cell transplantation, genetically modified ES cells have been used in several studies. Perlingeiro et al. (2001) have utilized the Bcr–Abl oncogene to transduce a blast cell population, containing both multipotential hematopoietic and endothelial cell progenitors (Choi et al., 1998). Blast cells transformed with the Bcr–Abl oncogene and cultured on OP9 cells, previously shown to support HSCs (Nakano et al., 1994), could repopulate sublethally irradiated, 8-week-old 129Sv–Ev or NODSCID mice. Consistent with previous studies showing that blast cells contain both primitive and definitive erythroid, and myeloid progenitors (Choi et al., 1998; Kennedy et al., 1997), the Bcr–Abl-transduced blast cells generated primitive and definitive erythroid, myeloid, and lymphoid cell lineages in the recipients. The caveat of this study is that the cell population used for transplantation harbors an oncogene. The recipients ultimately developed myeloproliferative disorders between 5 and 9 weeks after transplantation. HoxB4, one of the homeotic genes, is expressed and implicated in

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self-renewal of definitive HSCs (Sauvageau et al., 1995). Kyba et al. (2002) generated ES cells overexpressing a tetracycline-inducible HoxB4. When induced, HoxB4-overexpressing EBs cultured on OP9 cells showed a higher percentage of cell population expressing HSC markers c-kit and CD31. Also, the HoxB4-induced EBs were able to reconstitute the hematopoietic system of irradiated recipients, including myeloid and lymphoid lineages. Importantly, the EB-derived bone marrow cells from the first recipient were detected in secondary recipients.

III. Conclusions and Future Directions During ES diVerentiation, Flk-1-expressing cells initially develop from the mesoderm. Flk-1þ mesoderm generates many types of cells of the circulatory system, including blood, endothelial, smooth muscle, cardiac muscle, and skeletal muscle cells. Scl expression will further specify Flk-1þ cells to the hemangioblast. Pending issues concerning hemangioblasts are as follows. First, ES-derived BL-CFCs fit the description of in vitro equivalent hemangioblasts of the yolk sac blood islands. Nevertheless, there is no definite proof that such a cell exists in the developing embryo, yolk sac, or AGM. Clearly, cell-marking experiments will be necessary to determine the existence of a common progenitor. Second, the existence of the hemangioblast in adults needs to be investigated. Although it is conceptually accepted that hemangioblasts develop during embryogenesis and produce hematopoietic and endothelial cells of adults, recent studies suggest that hemangioblasts exist in adult stages as well. For example, human AC133þ cells from granulocyte colony-stimulating factor mobilized peripheral blood can diVerentiate into both hematopoietic and endothelial cells in cultures. These AC133þ cells can form new blood vessels in vivo (Gehling et al., 2000). In addition, Pelosi et al. (2002) showed that single CD34þFlk-1þ cells from human bone marrow or cord blood can generate both hematopoietic and endothelial cells. These potential postnatal hemangioblasts exhibited long-term proliferative potential in culture. Furthermore, a population of cells enriched for hematopoietic stem cells, such as Sca-1þc-KitþLin cells, could also contribute to new blood vessel formation at the single-cell level (Bailey et al., 2004; Grant et al., 2002). Third, the full developmental potential of the hemangioblast should be determined. BL-CFCs can generate both hematopoietic and endothelial cells, although their full potential has not been carefully examined. Recent studies suggest that smooth muscle cells can also diVerentiate from BL-CFCs (Ema et al., 2003). In this study, Ema and colleagues showed that blast cells generated smooth muscle cells in the absence of VEGF. Lastly, molecular regulatory mechanisms involved in hemangioblast


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specification and diVerentiation should be identified and investigated. This knowledge will be valuable for characterizing hemangioblast self-renewal and diVerentiation, modulating hemangioblast function, and isolating hemangioblast, hematopoietic, and endothelial progenitors for therapeutic purposes.

Acknowledgments We would like to thank many investigators for providing valuable reagents. We apologize to the many authors that we could not cite due to space constraints. This work was supported by grants from the National Institutes of Health, NHLBI, R01s HL63736 and HL55337 (to K.C.).

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Fibroblast Growth Factor Signaling and the Function and Assembly of Basement Membranes Peter Lonai { Department of Molecular Genetics The Weizmann Institute of Science Rehovot, Israel 76100

I. Introduction II. Earlyl Embryogenesis, Growth Factors, Growth Factor Receptors, and the Basement Membrane A. Early Mammalian Development B. Embryoid Bodies: A Model for Early Mammalian Development C. Fibroblast Growth Factor Signaling During Early Embryogenesis D. Relative Localization of Growth Factors, Growth Factor Receptor, and the Basement Membrane E. AYnity of Growth Factors and Their Receptors to Heparin and Heparan Sulfates F. Basement Membranes and Their Network-Forming Elements III. Laminins and Basement Membrane–Mediated Signaling A. Genetic Analysis of Laminins B. Laminin-1 and Epiblast DiVerentiation C. Endoderm and Ectoderm DiVerentiation Follow DiVerent Pathways D. From Stem Cells to Pregastrulation Embryo: A Cascade of Cellular and Molecular Interactions E. Laminin Receptors and Anchorage Sites IV. Current Questions References

I. Introduction The subject of this chapter is the contribution of the extracellular matrix (ECM) to signal transduction during early development and epithelial diVerentiation. Special emphasis is given to basement membranes (BMs) and fibroblast growth factors (FGFs) as studied in embryoid body (EB) cultures. Extensive literature analyses the role of the ECM in cell physiology. This activity gained great impetus from recognizing, already more than 30 years {

Deceased


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ago, that epithelial mesenchymal interactions are required for morphogenesis and that many cells, and especially cell sheets, interact via the ECM (Kallman and Grobstein, 1965; Saxen and ThesleV, 1992; Wessels, 1970). The ensuing studies were summarized in excellent reviews on the protein chemistry and structural biology of BM (Colognato and Yurchenco, 2000; Hohenester et al., 1999a; Timpl and Brown, 1996), and on the biochemistry of intercellular haparan sulfate molecules (Bernfield et al., 1999) and their integrin receptors (Hynes, 2002). The role of the ECM, and especially that of the BM, in tubulogenesis (Hogan and Kolodziej, 2002; Lubarsky and Krasnow, 2003; Sottile, 2004), branching morphogenesis (Hogan and Yingling, 1998), cell migration, metastasis formation (Kalluri, 2003; Patarroyo et al., 2002), tumor angiogenesis (Folkman, 2002; Kalluri, 2003), and in tumor–stroma interactions (Fata et al., 2004) greatly advanced this field. This large body of evidence pointed to the great importance of the ECM in manifold physiological and pathological processes rather than explaining the mechanism of its contributions. Recent progress in understanding the connection between laminin isotypes and signal transduction in the EB system provided a specific if narrow insight into the mechanism of BM-mediated epithelialization in the early embryo. Our discussion pays special attention to these results.

II. Early Embryogenesis, Growth Factors, Growth Factor Receptors, and the Basement Membrane A. Early Mammalian Development One focus of our discussion concentrates on interactions between the BM separating the extraembryonic endoderm and the epiblast of the mammalian embryo. These early stages of development bridge the diVerentiation of the preimplantation blastocyst into the pregastrulation egg-cylinder embryo. Before analyzing the ECM’s role in these early processes of embryogenesis, a brief introduction into early mammalian development and into the relationship of growth factors with the ECM may be useful. Following fertilization, the mammalian embryo goes through a limited number of cleavage divisions during which the blastomers retain their pluripotency. At the eight-cell stage the mouse embryo undergoes compaction, when the hitherto loosely attached blastomers form a tightly packed mass, and the contour of the individual blastomers becomes barely detectable. The first cell fate decisions take place during this stage. At compaction, adhesion structures become expressed (Johnson et al., 1986) and, as shown by Graham and his colleagues, the external blastomers become committed to trophoblast, while the inner blastomers acquire intercellular matrix (ICM) fate (Graham, 1978). Major molecular elements of compaction are

2. Fibroblast Growth Factor Signaling

39

E-cadherin and the connected -catenin-APC-wnt pathway (Ohsugi et al., 1996). The earliest expressed FGF receptor, Fgfr2, is also expressed at this stage, when it is localized to the external blastomers and later to the trophectoderm lineage (HaVner-Krausz et al., 1999). As blastocyst development starts, a fluid-filled cavity forms in the morula and apical–basal polarity is established in the peripheral cells. Cells of the prospective ICM, some 20 in number, are the ancestors of all embryonic tissues. The ICM is localized at the apical pole of the blastocyst, and the rest of the blastocyst is covered by a polarized epithelium, the trophectoderm. An additional cell fate decision takes place in the late blastocyst [at embryonic day (E) 3–3.5 in mouse embryos]; ICM cells bordering the blastocyst cavity transform into primitive endoderm (Gardner, 1982). At this stage the ICM expresses Fgf4 (Niswander and Martin, 1992), the ligand of Fgfr2, which is localized in the adjacent trophectoderm (HaVner-Krausz et al., 1999). Trophoblast proliferation is promoted by Fgf4 and Fgfr2 (Tanaka et al., 1998), which are also required for endoderm diVerentiation (Chen et al., 2000; Feldman et al., 1995; Goldin and Papaioannou, 2003). The first BM components, laminin- 1 and laminin- 1, are expressed at the eight-cell stage. In the blastocyst, laminin-1 and 11 (Fig. 1A) (L. Sorokin, personal communication) and collagen IV (Fig. 1C) are expressed in the BM situated along the basal side of the trophectoderm and later also at the basal side of the primitive endoderm that separates the ICM from the endoderm (Fig. 1A–C). Laminin-1 and laminin-5 (laminin-10–11) are expressed together at this stage (Ekblom et al., 2003; Miner et al., 2004). After implantation, the ICM and the polar trophectoderm covering it undergo fast proliferation and intrude into the former blastocyst cavity, forming a conical structure, the so-called egg-cylinder. The primitive endoderm also proliferates and covers the entire cavity of the late blastocyst. At the eggcylinder stage, the endoderm covering the egg-cylinder becomes the visceral endoderm, whereas the layer associated with the trophectoderm becomes the parietal endoderm. The parietal endoderm synthesizes the thick BM of the Reichert membrane at the maternal–fetal interface, and the visceral endoderm synthesizes the subendodermal embryonic and extraembryonic BM (Fig. 1E). Initially the egg-cylinder contains round stem cells, which are similar to the cells of the ICM and form a solid epithelial bud. Later, close to gastrulation, (between E5.5 and 6.0 in the mouse embryo), a cavity in the proamniotic canal is formed and the stem cells of the primitive endoderm (ICM) surrounding it diVerentiate into a columnar monolayer, the pseudo-stratified columnar epithelium of the epiblast. With epithelialization of the primitive ectoderm into epiblast, the early postimplantation embryo is ready for gastrulation. During gastrulation the definitive germ layers, the neuroectoderm, the mesoderm, and the embryonic endoderm are formed; the segmented body plan is realized; and organ and limb development commences. An excellent short description of early embryogenesis and gastrulation can be found in Manipulating the

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Figure 1 Distribution of basement membrane proteins in the early mouse embryo. (A)–(C) 3.5 days postcoitum (dpc) blastocysts; (D) 4-day-old embryoid body; (E) 7.5 dpc embryo. (A)–(D) Whole mount confocal images according to Li et al. (2001a). (E) Section, immunofluorescence. (A)–(D) Green: fluoresceinated phalloidin to detect fibrillar actin. Red: specific antibody. (A) 1LG4; (B) laminin- 1; (C) Collagen IV; (D) 1LG4; (E) 1LG4. Arrow in (A) and (B): ICM; arrowhead in (A) and (B): primitive endoderm. In (E) arrow: subendodermal BM; double arrow: extraembryonic subendodermal BM; arrowhead: Reichert’s membrane.

Mouse Embryo by Hogan et al. (1994), and a more detailed description is given by Tam and Behringer (1997).

B. Embryoid Bodies: A Model for Early Mammalian Development EB cultures constitute an ideal system for investigating pregastrulation development. Cultured ES cells diVerentiate into EBs when grown on bacteriological plates, a condition that does not support cell adhesion (Martin


41

et al., 1977). On bacteriological plates floating ES cell aggregates form, which diVerentiate into round structures made of two epithelia, the outer endoderm and the inner ectoderm layer. The endoderm synthesizes laminin and type IV collagen isotypes, which form the network of the subendodermal BM and consist of vacuolated polar cells, which at their basal domain face the BM, whereas their external apical domain wears microvilli. The endoderm of the EB, both by its morphology and by virtue of the genes it expresses, resembles the primitive and later visceral endoderm of the late blastocyst and the pregastrulation egg-cylinder embryo. The inner ectoderm of the EB is a pseudo-stratified columnar epithelium. Its basal domain is attached to the subendodermal BM, and its apical domain faces the central cavity and is distinguished by an accumulation of actin fibers. The columnar epithelium of the EB is similar to the embryonic ectoderm or epiblast, an epithelium, which gives rise to all cell lineages of the embryo. Taken together, the EB contains two epithelia, each of which is attached in a reversed polarity to a common subendodermal BM at their basal domains. EBs are considered to be faithful models of the pregastrulation mouse embryo (Coucouvanis and Martin, 1995). Although the pregastrulation mouse embryo is exceedingly small and diYcult to isolate, EBs can be grown in large amounts with considerable ease. This is why EB cultures became the tool for recent research on the role of the BM in epithelial diVerentiation (Henry and Campbell, 1998; Li et al., 2001a, 2002, 2004; Murray and Edgar, 2000).

C. Fibroblast Growth Factor Signaling During Early Embryogenesis Of the 24 FGF genes, three, Fgf4, Fgf3, and Fgf5, are expressed during preimplantation embryogenesis. According to genetic evidence, Fgf5 is a regulator of the hair growth cycle (Hebert et al., 1994), Fgf3 is required for normal ear and tail development (Mansour et al., 1993), while Fgf4 is the only one that is by itself required for trophectoderm and primitive endoderm diVerentiation of the late preimplantation blastocyst and early postimplantation embryo (Feldman et al., 1995; Goldin and Papaioannou, 2003; Wilder et al., 1997), where it is expressed in the ICM (Niswander and Martin, 1992). None of the four FGF receptors (FGFRs) is individually required for early gastrulation or pregastrulation embryogenesis. Fgfr1 is required for development of the posterior mesoderm (Deng et al., 1994; Yamaguchi et al., 1994) by controlling the migration of the prospective mesoderm through the primitive streak (Ciruna and Rossant, 2001). Loss of Fgfr2 is lethal at midgestation due to severe defects of lung diVerentiation and causes the complete loss of limb outgrowth (Arman et al., 1999; Xu et al., 1998). It has

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been shown that this Fgfr2 loss of function phenotype is due to the b transcriptional variant (De Moerlooze et al., 2000), whereas targeted mutagenesis of the Fgfr2c variant causes craniofacial and bone development anomalies due to defective osteoblast diVerentiation (Eswarakumar et al., 2002, 2004). Fgfr3, somewhat in contrast to Fgfr2c, exhibits negative control of chondrogenesis during endochondral bone formation (Colvin et al., 1996; Deng et al., 1996), whereas loss of Fgfr4 exhibits no independent phenotype and cooperates with Fgfr3 in the control of lung alveogenesis (Weinstein et al., 1998). The role of FGF signaling in early embryogenesis is suggested by the requirement of Fgf4 for trophectoderm and endoderm development (Goldin and Papaioannou, 2003) and by the finding that dominant negative FGFR mutation elicited by expressing truncated Fgfr2 cDNA in ES cells abolishes endoderm and ectoderm diVerentiation in the EB, where multiple Fgfr are expressed in synchrony (Chen et al., 2000). This latter result explains why a rearranged targeting vector of Fgfr2 induced peri-implantation lethality and loss of endoderm diVerentiation (Arman et al., 1998). Involvement of FGF signaling at stages preceding endoderm diVerentiation was reported by Chai et al. (1998), who found that dominant negative, truncated Fgfr4 cDNA disrupts embryogenesis in the early blastocyst after the fifth cleavage division. The early pattern of Fgfr2 expression suggests that this receptor, presumably together with other Fgfr, is important for preimplantation embryogenesis. HaVner-Krausz et al. (1999) reported that Fgfr2c is expressed maternally in the unfertilized egg and that both Fgfr2c and b are active in the external cells of the compacted morula and in the trophectoderm lineage that develops from them. According to the present consensus, Fgf4 in the ICM mediates early FGF signaling. Fgf4 signals are transmitted by Fgfr2 and other Fgfr localized in the trophectoderm and later in the primitive endoderm. Better understanding of the exact distribution of FGFR in the early embryo was hindered by the lack of splice variant–specific antibodies that are suitable for immunofluorescence detection; moreover, the resolution of in situ hybridization proved to be less than adequate for the resolution of the very small distances in the preimplantation embryo. New antibodies and double-mutant crosses should provide more definite understanding. Much is still to be learned about the gene network of preimplantation development. As mentioned before, the first cell fate decision is made at compaction, when the outer cells of the eight-cell mouse morula acquire trophectoderm fate while the inner cells acquire ICM fate (Graham, 1978). Compaction is achieved by E-cadherin (Johnson et al., 1986), required for trophectoderm diVerentiation (Larue et al., 1994). The earliest cell fate decisions, namely, the choice between pluripotent, trophectoderm, and endoderm cell fate, is regulated by the concentration of Oct3/4 (Niwa et al.,


43

2000), whereas the pluripotency of ES cells and the primitive ectoderm is maintained by nanog (Chambers et al., 2003). A number of other genes, the expression level of which decreases with ES cell diVerentiation, may serve similar functions (Tanaka et al., 2002). Oct3/4 expression is in part controlled by COUP-TF1 and COUP-TFII (Ben-Shushan et al., 1995), which influence laminin-1 expression in the primitive endoderm (Murray and Edgar, 2001b). Endoderm diVerentiation is controlled by GATA-6 and GATA-4. These transcription factors are under FGF control. They transform ES cells into endoderm-like cells, which, among other endodermspecific proteins, express COUP-TF1 and the three chains of laminin-1 (Fujikura et al., 2002; Li et al., 2004). Akt/PKB of the PI3K pathway also contributes to the control of laminin expression (Li et al., 2001b). This eVect is regulated by FGF signaling (Chen et al., 2000).

D. Relative Localization of Growth Factors, Growth Factor Receptor, and the Basement Membrane Many interactions between growth factors and their receptors take place across BMs that separate them, since growth factors and growth factor receptors are frequently localized to cell layers adjacent to alternate sides of the BM (Lonai, 2003). The growth factor, kit, and its ligand, the stem cell factor, have been localized to neighboring epithelial and mesenchymal cell sheets (Keshet et al., 1991). A similar arrangement was found for the Pdgfra receptor and its PDGF-A ligand (Orr-Urtreger and Lonai, 1992). Outstanding examples are represented by FGFR isotypes. First it was observed that Fgfr1 is localized mainly to mesenchymal cells, whereas a structurally closely related isotype, Fgfr2, is mainly expressed in epithelia (Orr-Urtreger et al., 1991; Peters et al., 1992). More sophisticated regulation was found for localization of the transcriptional alternatives of Ffr2. The C-terminal half of the third immunoglobulin (Ig)-like loop in the ligand-binding domains of FGFR1, FGFR2, and FGFR3 are encoded by one of two mutually exclusive exons conferring diVerent ligand-binding specificity (Givol et al., 2003; Johnson and Williams, 1993). Two splice variants, b and c, are distinguished. It has been shown that Fgfr2b is mostly expressed in epithelia, whereas Fgfr2c is expressed in mesenchymes (Orr-Urtreger et al., 1993). For example, the epithelial Fgfr2b receptor binds mesenchymal FGF isotypes, such as Fgf10 (Ohuchi et al., 1997), whereas the mesenchymal Fgfr2c variant recognizes Fgf9, since it has been demonstrated for epithelia of the developing lung (Arman et al., 1999). General applicability of this mutual regulation was supported by a study of the mitogenicity of FGFR isotypes in BAF3 cells expressing various FGF isotypes. It was found that c- and b-type receptors recognize separate groups of FGF ligands, and, as far as it has

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been determined, those that activate b-type receptors were expressed in mesenchymal tissues, whereas those that recognize c-type receptors were expressed in epithelial tissues (Ornitz et al., 1996). Numerous clinical studies indicate the prevalence of this mutual regulation. For example, misexpression of FGFR2 splice variants leads to defective osteogenesis, as observed in Apert syndrome (Hajihosseini et al., 2001; Ibrahimi et al., 2001), whereas its ligand-independent activation causes various craniosynostosis syndromes (Eswarakumar et al., 2004; Wilkie, 1997). It may not be far-fetched to suggest that this mutual control of transcriptional localization of FGF and FGFR developed to serve epithelial mesenchymal interactions.

E. Affinity of Growth Factors and Their Receptors to Heparin and Heparan Sulfates FGFs are distinguished by their aYnity to heparin and once were denominated as heparin-binding growth factors. By now most growth factors, or at least some of their variants, were shown to have aYnity to heparin or to heparan sulfates (HS) of the ECM (Turnbull et al., 2001). Because HS proteoglycans (HSPGs) are organic components of the ECM, this finding indicates that growth factors may be stored and concentrated by ECM components, such as syndecans and glypicans or the BM. The importance of cell surface HS molecules for FGF signaling was first shown by experiments with cells that carry null mutations of HS synthesis. Yayon et al. (1991) showed that FGFR expressed in mutant cells of this kind were incapable of FGF binding. HS are synthesized by a cascade of sugar transfer enzymes, and the variable sequence of their sugar moieties has the potential to create very high multiplicity (Turnbull et al., 2001). Actually it has been found that diVerent FGF isotypes have selective aYnity to diVerent HS variants (Allen and Rapraeger, 2003; Guimond and Turnbull, 2000; Ostrovsky et al., 2002). Specific contribution of heparin-like molecules to FGF signaling was demonstrated by crystallographic analysis of FGF–FGFR complexes in the presence of heparin. These studies demonstrated that the FGF–FGFR complex creates a groove, which accommodates heparin moieties contributing to the stability of the receptor–ligand complex and enhancing its dimerization (Pellegrini et al., 2000; Plotnikov et al., 1999; Schlessinger et al., 2000). Genetic analysis lent independent support to the structural and biochemical evidence for the role of HSPGs in developmental signaling. Mutant analysis in Drosophila organisms revealed their importance for early development (Perrimon and Bernfield, 2000). It has been shown that HSPG mutations phenocopy FGF and FGFR mutations in Drosophila organisms,


45

which contributes to the importance of FGF–FGFR–HS linkage in FGF signaling (Lin et al., 1999). Mutations in vertebrates, including human orthologues of the genes active in HS synthesis (Bullock et al., 1998; Kurima et al., 1998; Lin et al., 2000), interfere with organogenesis at later stages than in Drosophila organisms, probably due to functional overlap in the complex vertebrate genome. Taken together, these data suggest that HSPGs of the matrix actively contribute to cell-to-cell signaling. Multiple mechanisms may be involved. HS may be instrumental in the storage and concentration of heparin-binding growth factors in the vicinity of cells that bear the relevant signaling receptor (Schlessinger et al., 1995). In the case of FGF signaling, the relationship between receptor tyrosine kinaseses, growth factors, and HS developed into a functional requirement in the sense that the signaling unit requires the contribution of specific HS components (Plotnikov et al., 1999). The specificity of receptor–matrix interactions contributes to the specificity of signal transduction, and the variability of HS molecules may have a decisive role here. Future research will have to clarify to what degree the specificity of growth factor signaling is influenced by the vast polymorphism of HS sequences.

F. Basement Membranes and Their Network-Forming Elements The role of the BM as an activator of epithelialization of the primitive endoderm is a central issue of this article. The BM and its components have been extensively reviewed (Colognato and Yurchenco, 2000; Ekblom et al., 2003; Timpl and Brown, 1996) and are only briefly introduced here. The BM is a thin, blanketlike modification of the ECM, which separates cell layers in most organs. As such it can reach large dimensions such as beneath the germinal layer of the skin, which covers the entire surface of the body. In certain organs the BM fulfills specific functions, such as in the glomerular membrane of the kidney, in synaptic membranes of neuromuscular junctions, and in the Schwann cell layers of myelinated nerves. The network-forming components of the BM are laminin and type IV collagen isotypes. Laminins are cross-shaped molecules that form an independent network (Yurchenco et al., 1992), whereas collagen IV, which is essential for the stability of the BM, is dispensable for its initiation (Poschl et al., 2004). Laminins form a flat polymer by associating through their N-terminal side chains (Cheng et al., 1997), whereas the C-terminal globular domains of the chains provide anchoring sites to the cell membrane (Hohenester et al., 1999b). Laminin globular domains 1–3 (LG1–3) display aYnity to 6 1, 6 4, and 7 1 integrins, whereas the LG4–5 domains of the E3 peptic fragment exhibit aYnity for dystroglycan, heparin, and

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sulfatides. Recent data separate the dystroglycan-binding site from the site that binds heparin and sulfatides (Wizemann et al., 2003). Fifteen laminin isotypes are distinguished. They use of one of five , one of three , and one of three chains (Table I). All chains have cell-binding globular domains, and the globular domains of 2 (Talts et al., 1998) 4 (Talts et al., 2000), and 5 (Nielsen et al., 2000) were shown to have similar structure and binding aYnity. Although laminin-1 is the most characteristic isotype during early development (Ekblom et al., 2003), it is localized to specific sites in the adult as well (Virtanen et al., 2000). The exact localization of laminin isotypes is far from understood, and multiple isotypes are expressed in specific sites of certain organs throughout development and in the adult. Well-illustrated examples are the kidney (Miner et al., 1997) and the neuromuscular junction (Patton et al., 1997), which express multiple laminin isotypes at specific localizations. Inhibition with antibodies to specific peptic fragments of laminin chains revealed that the E3 fragment of the 1 chain, which contains the LG4–5 modules, is required for kidney or salivary gland diVerentiation in organ culture (Kadoya et al., 1995, 2003; Klein et al., 1988, 1990; Sorokin et al., 1992). These results, as well as the protein chemistry of laminin globular domains (Timpl et al., 2000), indicate that BM anchorage and BMmediated signaling resides in the LG modules of laminin chains. Therefore, although many other BM components, such as agrin, perlecan, and Hsp-47, fulfill important functions (Yurchenco et al., 2004), in the following we concentrate on discussing the laminin family.

Table I Laminin Isotypes chain 1 2

3

4

5

and chains

Denomination

1, 1 2, 1 1, 1 2, 1 1, 3 1, 1 2, 1 3, 2 1, 1 2, 1 2, 3 B1, 1 B2, 1 B2, 3

Laminin-1 Laminin-3 Laminin-2 Laminin-4 Laminin-12 Laminin-6 Laminin-7 Laminin-5 Laminin-8 Laminin-9 Laminin-14 Laminin-10 Laminin-11 Laminin-15

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III. Laminins and Basement Membrane–Mediated Signaling A. Genetic Analysis of Laminins Gene targeting revealed the delicate specificity of laminin isotypes. The mutant defects largely follow the specific gene expression patterns of the laminin isotypes (Table II). This is apparent in chain mutations, where disruption of Lama2 (Miyagoe et al., 1997; Timpl et al., 2000) and Lama4 (Patton et al., 2001; Thyboll et al., 2002) expressed in the neuromuscular junction cause peripheral nervous system defects, whereas the Lama5 mutation is distinguished by defects in kidney glomeruli and kidney agenesis (Miner and Li, 2000; Miner et al., 1998). Moreover, in agreement with the early embryonic expression pattern of laminin-1, mutations of laminin-1, laminin- 1 (Miner et al., 2004), laminin- 1 (Smyth et al., 1999), and the deletion of its E3 fragment containing 1LG4–5 (P. Ekblom and S. Scheele, personal communication) aVect diVerentiation of the first epithelia of the

Table II Laminin Mutations AVecting Embryogenesis or Embryoid Body DiVerentiation Gene Lamb2 Lama2 Lama3 Lamc1 Lama5

Lama4

Lamb1 Lama1

1LG4–5

Phenotype Adult lethal. Neuromuscular junction (NMJ) and glomerular defects. Disrupted BM. Lethal around 5 weeks. Muscular dystrophy, peripheral neuropathy, disrupted BM. Neonatal lethal. Epithelial adhesion defects. Defective skin and teeth. Restricted BM defects E5.5 lethal. No epiblast diVerentiation. Endoderm retained. Failure of BM assembly. Late embryonic lethality; exencephaly, syndactyly, placental labyrinth defect, glomerular defects, sporadic kidney agenesis. BM discontinuous. Transient microvascular defect; NMJ defect, small blood vessels BM defective at birth, later becoming normal. Enhancement of vascularisation, enhanced tumor growth and metastasis. E5.5 lethal. Loss of epiblast diVerentiation. Failure of BM assembly. E5.5–6.5 lethal. Loss of epiblast diVerentiation. BM present. 5 chain replaces 1. Embryoid bodies not investigated. E5.5 lethal. Endoderm and BM retained no epiblast diVerentiation. Truncated 1 chain incorporated in BM.

Reference Noakes et al., 1995 Miyagoe et al., 1997 Ryan et al., 1999 Smyth et al., 1999 Miner et al., 1998

Patton et al., 2001; Thyboll et al., 2002; Zhou et al., 2004

Miner et al., 2004 Miner et al., 2004

P. Ekblom and S. Scheele, personal communication

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mammalian embryo. The specificity of localization and severity of these mutant phenotypes suggest that laminin isotypes fulfill specific functions in the organs and tissues of their expression. Localized expression and localized function of individual members of a gene family reflect localized transcriptional regulation, that is, the availability of specific transcription factors at specific locations. Local regulation may or may not be associated with locally specific function. In the latter case an isotype expressed at a specific localization fulfills a unique function and is not replaced by another isotype, even if their localization partially overlaps. There are only few data in the laminin field that shed light on this question. Muscular dystrophy caused by laminin-2 defects is partially rescued by laminin-1 (Gawlik et al., 2004), suggesting that the two chains display similar functions at the neuromuscular junction. In contrast, although the 1 chain of laminin-1 and the 5 chain of laminin-10 or laminin-11 are coexpressed at the subendodermal BM of the early embryo or EB, laminin5 does not rescue the loss of epiblast diVerentiation due to the deletion of 1LG4–5 (P. Ekblom and S. Scheele, personal communication). It follows that although the function of laminin-1 and laminin-2 may be similar at the neuromuscular junction, laminin-1 and laminin-5 fulfill diVerent roles in the subendodermal BM.

B. Laminin-1 and Epiblast Differentiation Genetic analysis of laminin function by gene targeting (see Table II) and especially the targeted disruption of the constituent loci encoding of the earliest expressed laminin isoform, laminin-1, revealed unexpected insights into its role in epithelial diVerentiation. Disruption of Lamc1 encoding laminin- 1, a laminin chain present in 10 out of 15 laminin isoforms (see Table I), resulted in defective blastocyst development and early postimplantation lethality (Smyth et al., 1999). The trophectodermal and subendodermal BM of the early postimplantation Lamc / embryo was absent, although endoderm development was observed and the internal ES cells of the EB did not develop beyond the stem cell stage. This phenotype was similar to the targeted disruption of 1-integrin (Fassler and Meyer, 1995), which is thought to result from defective laminin-1 synthesis (Aumailley et al., 2000). On the other hand, a third gene, dystroglycan, a membranebound protein in communication with dystrophin, was shown to be required for the stability of the BM (Henry and Campbell, 1998; Li et al., 2002). The Lamc1 mutation could be rescued by exogenous laminin-1, suggesting that laminin-1 is required for BM assembly and that the BM and its laminin-1 component are required for the epithelialization of ES cells (Murray and Edgar, 2000). The role of the cell-binding LG4–5 domains of the laminin-1


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chain was dissected by a series of experiments, where the rescue of the Lamc1, 1-integrin, and dystroglycan mutations by exogenous laminin-1 was inhibited by the addition of 1LG4–5 containing E3 fragments (Li et al., 2002). The results demonstrated that the cell-binding domain of laminin-1 is required for epithelialization of the primitive ectoderm and suggested that 1-integrin and dystroglycan, which were thought to act as laminin receptors, do not fulfill this role during epiblast diVerentiation. Independent results obtained while investigating the role of FGF signaling in early development support and extend these conclusions. FGFR monomers truncated at the cytoplasmic domain act as a dominant negative mutation. Heterodimerization among FGFR isotypes with a truncated monomer inactivates most FGFRs synthesized by the same cell. Indeed, ES cells expressing truncated Fgfr2 cDNA (abbreviated as dnFgfr) inhibited the diVerentiation of both epithelia of the EB although, as we have shown, EBs express multiple FGFR isotypes (Chen et al., 2000). Besides defective expression of numerous endoderm- and ectoderm-specific genes, such as Hnf4, vHnf1, Evx1, Gata4, and Gata6, as well as Eomes, the kit ligand and embryonic globin, dnFgfr ES cells failed to transcribe the polypeptide chains of laminin-1 and collagen IV. Significantly, this phenotype could be partially rescued by exogenous laminin-1 (Li et al., 2001a). These results establish the role of laminin-1 in the epithelialization of the primitive ectoderm and demonstrate that EB diVerentiation is initiated by FGF signaling. An important intermediate of FGF signaling was discovered when the GATA-4 and GATA-6 transcription factors were investigated. GATA-6 and GATA-4 have been shown to be required for endoderm diVerentiation (Morrisey et al., 1998) and to transform ES cells into endoderm-like cells (Fujikura et al., 2002). We showed that endoderm-like GATA transformed ES cells, similar to exogenous laminin-1 rescue of both laminin and collagen IV expression and epiblast diVerentiation when cocultivated with the dnFgfr ES cells (Li et al., 2004). Using the Lamc1 / mutation of Smyth et al. (1997), it was demonstrated that the active principle of this physiological cell-to-cell interaction is in fact laminin-1. This result suggested that once expressed, GATA-4 and GATA-6, which are downstream of FGF signaling, are suYcient to activate endoderm diVerentiation and to initiate epiblast diVerentiation independently from FGF signaling (Li et al., 2004).

C. Endoderm and Ectoderm Differentiation Follow Different Pathways Partial rescue of the dnFgfr mutant by exogenously added laminin, or cocultivation with GATA factor-transformed endoderm-like cells, revealed that laminin-1 is a specific inducer of epiblast epithelialization. In appropriate

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concentrations laminin-1, or spent media of GATA-6 or GATA-4-transformed ES cells, induced epiblast diVerentiation without inducing endoderm diVerentiation in ES cells, thus separating endoderm and ectoderm diVerentiation into two consecutive but distinct pathways (Li et al., 2001a, 2004). Taken together, these observations suggest that although endoderm diVerentiation is induced by FGF signaling, epiblast diVerentiation is induced by laminin-1. Endoderm and ectoderm diVerentiation were also separated by their dependence on Lif (Murray and Edgar, 2001a). Murray and Edgar showed that although Lif inhibited the complete diVerentiation of the endoderm, its precursors still produced a BM and could activate epiblast diVerentiation. This result suggested that Lif inhibits only certain aspects of endoderm diVerentiation and does not interfere with epithelialization of the primitive ectoderm, although it failed to characterize the BM-producing primitive endoderm precursor in detail. Further issues separating endoderm and ectoderm diVerentiation were discovered when the downstream elements of laminin-induced epiblast diVerentiation were studied. A dominant negative mutation of the Rho kinase, ROCK, when expressed in ES cells, abolished epiblast epithelialization without aVecting endoderm diVerentiation or the deposition of the basement membrane. Moreover, when laminin binding after the addition of laminin containing spent media from GATA-transformed cultures was assessed by an 1LG4-specific antibody, only the ectoderm series, including undiVerentiated ES cells, exhibited laminin-binding receptors (Li et al., 2004). These results indicate, in addition to separating endoderm and ectoderm diVerentiation, that the two cell lineages originate in one undiVerentiated precursor pool. GATA-4 and GATA-6 activate endoderm diVerentiation when overexpressed in ES cells (Fujikura et al., 2002); moreover, epiblast diVerentiation is induced in pluripotential stem cells (Li et al., 2004). The most likely scenario for EB diVerentiation, therefore, is that external signals activate peripheral cells of the ES cell aggregate, which then undergo endoderm diVerentiation controlled by GATA factors. Early endoderm cells produce laminin and collagen IV chains and deposit the subendodermal BM. Subsequently, the laminin component of the BM induces cytoskeletal rearrangements in the remaining undiVerentiated stem cells through ROCK kinase, which is required for the epithelialization of the primitive ectoderm (Fig. 2). This suggests that neither endoderm nor epiblast diVerentiation requires diVerentiated precursors. Therefore, the most likely explanation is that both the extraembryonic ectoderm and the epiblast directly derive from the same pluripotential stem cells. This signifies that the role of laminin-1 is to confer epithelial cell fate on pluripotential precursors. Future studies will have to answer whether this role extends to the stem cells of other epithelia. Concomitant to epiblast diVerentiation, EBs undergo cavitation, similar to the formation of the so-called proallantoic canal of the later egg-cylinder


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Figure 2 From ES cells to endoderm and epiblast: a scheme of basement membrane-mediated interactions. This scheme depicts the most important cellular interactions and some of the molecular interactions that lead from diVerentiation of the ICM into embryonic ectoderm and and through deposition of the subendodermal basement membrane to the pregastrulation epiblast.

embryo. Cavitation, as suggested by Coucouvanis and Martin (1995), is based on the apoptotic death of cells that do not participate in endoderm or ectoderm diVerentiation. These authors proposed that cavitation is due to a signal emanating from the endoderm and that the diVerentiated ectoderm is rescued by its contact with the BM. Recent results tend to modify and extend this interpretation. Columnar ectoderm diVerentiation is induced independently from endoderm diVerentiation by laminin-1 (Li et al., 2001a) and specifies the endoderm-derived signal as the laminin-1 component of the subendodermal BM. The BM’s role in rescuing the diVerentiated

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ectoderm seems to have little relevance if we consider that the ectoderm develops from pluripotent ES cells, which can survive for weeks in vitro without BM deposition; therefore, the remaining stem cells do not need to be rescued from apoptosis. Moreover, only those stem cells that have made contact with laminin-1 of the BM diVerentiate into columnar ectoderm. It is therefore possible that cavitation is due to mechanical separation between the two diVerentiated epithelia surrounding the BM and the remaining stem cells of the EB. Alternatively and in addition, anoxic necrosis or the apoptotic eVect of BMP-4 produced by the diVerentiating ectoderm (Coucouvanis and Martin, 1999) may be responsible for the local death of stem cells. Future results are expected to decide between these alternatives.

D. From Stem Cells to Pregastrulation Embryo: A Cascade of Cellular and Molecular Interactions Activation of endoderm diVerentiation by FGF signaling, which leads to BM deposition and ROCK-mediated cytoskeletal rearrangement of the epiblast, is only part of the interactions that take place during EB diVerentiation (see Fig. 2). As mentioned before, zygotic Fgfr2 is expressed in the peripheral blastomers of the compacted morula and later in the primitive endoderm (HaVner-Krausz et al., 1999). Its ligand, Fgf4, first appears in the ICM of the blastocyst. The spatial temporal regulation of this receptor– ligand pair coincides with the appearance of the first BMs, in the basal domain of the trophectoderm and at the primitive endoderm–ICM interface. There are few data to interpret the functional aspects of this expression pattern. Chai et al. (1998) reported that dominant negative Fgfr4 cDNA interferes with preimplantation embryogenesis at the fifth cleavage division, coinciding with early stages of blastocyst development. Future research will have to clarify the interactions between Fgf4 and Fgfr2 (and other Fgfr) and the BM, as exhibited during the preimplantation stages. FGF signaling creates multiple interactions with docking proteins, leading to Ras or PI3K activation. A frequent partner of FGF signaling is FRS2 (Lax et al., 2002), which interacts with Grb2. Targeted disruption of FRS2 is lethal at the gastrulation stage (Hadari et al., 2001), whereas Grb2 embryos die at implantation and Grb2 / ES cells form neither endoderm or ectoderm (Table III) (Cheng et al., 1998). Because FRS2 is upstream of Grb2, one could have expected a more severe phenotype. Therefore, it might be interesting to explore early FGF signaling further. Evidence for the involvement of additional signaling pathways in EB diVerentiation was indicated by the loss of Akt/PKB phosphorylation in dnFgfr EBs as compared with wild-type EBs (Chen et al., 2000). A tight connection between PI3K–Akt/PKB signaling and BM assembly was indicated by a

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2. Fibroblast Growth Factor Signaling Table III Mutations AVecting Embryoid Body DiVerentiation Gene

Mutation

Cdh1

Targeted

Itgb1

Targeted

Grb2

Targeted

Dag1

Targeted

dnFgfr

Truncated cDNA; dominant negative mutation Rho binding negative, dominant negative mutation

Rock2

Phenotype

Reference

E-cadherin is maternally expressed. It is required for compaction of the morula; in its absence ES cells do not aggregate and EB diVerentiation is defective. Loss of 1-integrin is lethal in the preimplantation embryo. Interferes with BM assembly and laminin-1 expression. No epiblast epithelialization. Adaptor protein for receptor tyrosine kinase signaling E5.5 lethal. No endoderm or epiblast diVerentiation. Dystroglycan is required for stabilization of the BM. In EBs BM assembly and epiblast diVerentiation takes place. Truncated Fgfr2 cDNA expressed in ES cells. No endoderm or epiblast diVerentiation. Defective PI3K signaling. No laminin-1 or collagen IV synthesis.

Huber et al., 1996; Larue et al., 1994

Rho kinase 2. Mediator of Rho isoforms active in cytoskeletal rearrangements. Dominant negative mutation expressed in ES cells. No epiblast diVerentiation. Endoderm and BM retained.

Aumailley et al., 2000; Fassler and Meyer, 1995 Cheng et al., 1998

Henry and Campbell, 1998

Chen et al., 2000

Li et al., 2004

robust increase in laminin and collagen IV synthesis and BM assembly of EBs that derived from ES cells expressing constitutively active p110 or Akt (Li et al., 2001b). The first cytological change of the diVerentiating EB following ES cell aggregation is the formation of endoderm cells at the periphery of the aggregate. GATA-6 and, under its control, GATA-4 (Morrisey et al., 1998), are necessary and suYcient to induce endoderm diVerentiation (Fujikura et al., 2002). Next, GATA-6 is activated by FGF signaling and then activates both the cytoskeletal rearrangement and BM protein synthesis that are required for endoderm diVerentiation (Li et al., 2004). GATA-6, among other endoderm-specific genes, activates the COUP-TFI and COUPTFII transcription factors, which in turn can activate the expression of laminin isotypes (Murray and Edgar, 2001b). Endoderm diVerentiation takes place in cells along the periphery of the ES cell aggregate, and the BM deposited along their basal domain is not

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required for their survival (Smyth et al., 1999), since these cells do not bind laminin (Li et al., 2004). It follows that BM proteins are synthesized by the endoderm but are bound by the ectoderm and its precursors, and not by the endoderm cells of their origin. BM anchorage defines the direction of BM-mediated signaling as leading from the endoderm to the ectoderm and the definite position of the epiblast. Hence, the BM contributes to the establishment of the simple patterns of the EB and the egg-cylinder embryo. Despite their incompleteness, these data allow us to delineate the main elements of an EB diVerentiation pathway (see Fig. 2). FGF and E-cadherin signaling activates the commitment of peripheral cells in the ES cell aggregate (or in the ICM) to primitive endoderm diVerentiation. An important arm of this interaction follows the PI3K–Akt/PKB pathway and culminates in the synthesis of laminin and collagen chains and in their deposition as the subendodermal BM. Laminin-1 of the BM, through the LG4–5 globular domains of its E3 fragment (Li et al., 2002), activates epithelial transformation of ES cells that form a columnar epithelium requiring the activity of ROCK and RhoC (Li et al., 2004). DiVerentiation of the primitive ectoderm of the ICM into columnar epithelium of the epiblast makes the egg-cylinder embryo ready for gastrulation. Gastrulation by forming the mesoderm and definitive endoderm and by defining the relative extent of the neuroectoderm puts down the basis for the development of the head, trunk, limbs, and visceral organs.

E. Laminin Receptors and Anchorage Sites BMs are anchored to the cell surface through the C-terminal end of their chains containing five globular modules (Andac et al., 1999). Thus, the polymeric laminin network represents an organized lattice of ligands, which requires a complementary array of anchorage sites (Colognato et al., 1999). Recent research revealed that the C-terminal end of laminin 1 chains (or more accurately, their LG4–5 modules), besides anchoring the laminin network to the cell membrane, induces epiblast diVerentiation (Li et al., 2001a, 2002, 2004; Murray and Edgar, 2000). The question therefore arises whether the laminin anchorage and receptor sites are separate or bifunctional. This problem is under extensive investigation, but at the time being there are only few data to be discussed. The globular domains of laminin-1 bind numerous specific cell surface molecules. LG1–3 binds integrin 6 1, 6 4, and 7 1, whereas LG4–5 (or more accurately, the LG4 domain) binds dystroglycan, sulfatides, perlecan, fibulin, and heparin (Timpl et al., 2000). In a penetrating study, Li et al. (2002) investigated whether the E3 fragment of laminin-1 containing the 1LG4–5 modules can inactivate the rescue of EB diVerentiation by


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exogenous laminin-1. In contrast to previous consensus, this research revealed that although dystroglycan and 1-integrin are required for BM maintenance and for the continuous synthesis of the laminin 1 chain, they do not fulfill the role of an E3 receptor that could be responsible for ES cell epithelialization. These results are in good agreement with structural analysis, which separated the dystroglycan-, heparin-, and sulfatide-binding sites of 1LG4 (Wizemann et al., 2003). Although externally added heparin inhibits EB diVerentiation (Li et al., 2002), it is not clear whether LG4–5 behaves as a ligand for anchorage, a ligand for receptor signaling, or both. Deletion of the 1LG4–5 domain by gene targeting contributed to the clarification of this problem. 1LG4–5 / embryos die shortly after implantation, but, in contrast to other mutants of laminin-1, laminin- 1, and laminin- 1 (Miner et al., 2004; Smyth et al., 1999), these mutants develop a subendodermal BM, which exhibits the epitopes of the 1LG1–3 domain, suggesting that laminin-1 can retain its anchorage function in the absence of 1LG4–5 (P. Ekblom and S. Scheele, personal communication). According to this result, 1LG4–5 may be recognized by the signaling receptor that initiates stem cell epithelialization, although it cannot represent the single anchorage site for laminin-1.

IV. Current Questions The significance of 1LG4–5 as an inducer of epithelialization is in providing a definite molecular mechanism for a major ECM component. This insight contributes to a better understanding of the pregastrulation stage of mammalian development. However, it also holds promise for a broader view of epithelial transition and BM-mediated crosstalk in general. In the following section some of the emerging questions and experimental hypotheses are analyzed. The question arises whether, similar to the induction of primitive ectoderm epithelialization by the 1LG4–5 domain, other LG4–5 domains of other laminin isotypes are also involved with epithelial diVerentiation. According to structural analysis, laminin-1 and laminin-2 chains both bind perlecan, heparin, -dystroglycan, and sulfatides (Talts et al., 1999) and similar characteristics also have been established for laminin-4 (Talts et al., 2000) and laminin-5 (Nielsen et al., 2000), indicating that the globular domains of diVerent chain variants display similar structures and similar binding aYnities. As the functional representation of structural similarity, exogenous laminin-1 can rescue the reversed polarity of Madine-Darby canine kidney epithelial cells induced by dominant negative Rac1 (O’Brien et al., 2001), or human mammary epithelia cultured in collagen I gels (Gudjonsson et al., 2002). These results suggest that the polarity-inducing

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capacity of laminin-1 is not restricted to one aspect of early embryogenesis. A step further, Streuli et al. (1995) demonstrated that the E3 fragment of laminin-1 activates -casein expression in human mammary epithelia, and Slade et al. (1999) showed that the polarity of mammary luminal epithelia can be corrected by the E8 and E3 fragments of the laminin -1 chain. In addition, Kadoya et al. (2003) reported similar activity of another isotype, the LG4 module of laminin-5, which could restore salivary gland-branching morphogenesis. We conclude, therefore, that the diVerent G-terminal globular domains of laminin chain isotypes exhibit similar structures and binding aYnities and that they are involved in the establishment of polarity not only in the first embryonic epithelia, but also during organogenesis and epithelialization in general. The most straightforward question regarding laminin-induced epithelial polarization relates to the signaling receptor recognizing the 1LG4–5 module. Taking the binding characteristics of the LG4 module in account (Hohenester et al., 1999b; Timpl et al., 2000), it is reasonable to assume that it should be a heparin or heparan sulfate-binding molecule. Discovery of the receptor will provide the key to unravel the pathway of laminin-activated signaling. Although the BM could anchor to both cell sheets surrounding it, epithelialization of the primitive endoderm is distinguished by its polarity. Although laminin and type IV collagen are synthesized by the endoderm, the 1LG4–5 modules bind exclusively to the ectoderm and its derivatives. Thus, the flow of signals from endoderm to ectoderm is established. Although the polarity and main elements of this pathway are known, it is not clear whether FGFRs are the only receptor tyrosine kinases involved in the process. Neither is the specific role of P13K and Akt/PKB in the control of laminin and collagen IV synthesis (Li et al., 2001b) known, and we know next to nothing, besides the role of Rho kinase (Li et al., 2004), of the pathway that starts with the laminin-induced initiation of diVerentiation and with the formation of the columnar ectoderm of the epiblast. It has been shown by Fujikura et al. (2002) and Li et al. (2004) that GATA-4 and GATA-6 transform ES cells to endoderm-like cell lines. Similarly, laminin-1 can induce epithelialization of pluripotent stem cells into a uniform columnar epithelium (Li et al., 2004). It follows that the target cells of FGF and GATA factor-induced epithelialization of the endoderm, and the subsequent laminin-induced epithelialization of the columnar epithelium, derive from a common stem cell pool. Does this mean that FGF signaling and GATA transcription factors have the propensity to induce endoderm diVerentiation and BM assembly, whereas laminin-1 is a specific inducer of multipotent columnar epithelia? Future experiments with diVerent laminin isotypes in appropriate in vitro systems will have to clarify this problem.


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Assuming that the role of laminin LG4–5 modules is connected to the polarization of epithelia, we also have to consider the role of integrin-binding sites of the LG1–3 and the N-terminal LN-VI domains of the chain. Aumailley et al. (2000) have reported that 1-integrin is required for laminin-1 expression. Another study connects ILK, the integrin-like kinase, with cell spreading and the actin cytoskeleton (Sakai et al., 2003). Additional research is required to clarify the role of laminin-binding integrins, which themselves are signaling molecules (Hynes, 2002). Finally, we have to ask whether all or most BM-associated functions are connected to laminins. The answer must be no. Through their multiple heparin-binding moieties, ECM molecules may indeed store and present haparan sulfates. HS binding is not a unique characteristics of the BM, because intercellular ECM proteins, such as glypicans and syndecans, also bind them. The high sensitivity of developmental and organogenetic processes to loss of the enzymes for HS synthesis (Perrimon and Bernfield, 2000) points to their great importance in maintaining the cell to matrix crosstalk.

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Fujikura, J., Yamato, E., Yonemura, S., Hosoda, K., Masui, S., Nakao, K., Miyazaki Ji, J., and Niwa, H. (2002). DiVerentiation of embryonic stem cells is induced by GATA factors. Genes Dev. 16, 784–789. Gardner, R. L. (1982). Investigations of cell lineage and diVerentiation in the extraembryonic ectoderm of the mouse embryo. J. Embryol. Exp. Morphol. 68, 175–198. Gawlik, K., Miyagoe-Suzuki, Y., Ekblom, P., Takeda, S., and Durbeej, M. (2004). Laminin 1 chain reduces muscular dystrophy in laminin 2 chain deficient mice. Hum. Mol. Genet. 13, 1775–1784. Givol, D., Eswarakumar, V. P., and Lonai, P. (2003). ‘‘Molecular and Cellular Biology of FGF Signaling.’’ Oxford University Press, New York. Goldin, S. N., and Papaioannou, V. E. (2003). Paracrine action of FGF4 during periimplantation development maintains trophectoderm and primitive endoderm. Genesis 36, 40–47. Graham, C. F. (1978). Features of cell lineage in preimplantation mouse development. J. Embryol. Exp. Morphol. 48, 53–72. Gudjonsson, T., Ronnov-Jessen, L., Villadsen, R., Rank, F., Bissell, M. J., and Petersen, O. W. (2002). Normal and tumor-derived myoepithelial cells diVer in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition. J. Cell. Sci. 115, 39–50. Guimond, S. E., and Turnbull, J. E. (2000). Fibroblast growth factor receptor signaling is dictated by specific heparan sulphate saccharides. Curr. Biol. 9, 1343–1346. Hadari, Y. R., Gotoh, N., Kouhara, H., Lax, I., and Schlessinger, J. (2001). Critical role for the docking-protein FRS2 alpha in FGF receptor-mediated signal transduction pathways. Proc. Natl. Acad. Sci. USA 98, 8578–8583. HaVner-Krausz, R., Gorivodsky, M., Chen, Y., and Lonai, P. (1999). Expression of FGFR2 during oogenesis, preimplantation and early postimplantation embryogenesis. Mech. Dev. 85, 167–172. Hajihosseini, M. K., Wilson, S., De Moerlooze, L., and Dickson, C. (2001). A splicing switch and gain-of-function mutation in FgfR2-IIIc hemizygotes causes Apert/PfeiVersyndrome-like phenotypes. Proc. Natl. Acad. Sci. USA 98, 3855–3860. Hebert, J. M., Rosenquist, T., Gotz, J., and Martin, G. R. (1994). FGF5 as a regulator of the hair growth cycle: Evidence from targeted and spontaneous mutations. Cell 78, 1017–1025. Henry, M. D., and Campbell, K. P. (1998). A role for dystroglycan in basement membrane assembly. Cell 95, 859–870. Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994). ‘‘Manipulating the Mouse Embryo: A Laboratory Manual.’’ Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Hogan, B. L., and Kolodziej, P. A. (2002). Organogenesis: Molecular mechanisms of tubulogenesis. Nat. Rev. Genet. 3, 513–523. Hogan, B. L., and Yingling, J. M. (1998). Epithelial/mesenchymal interactions and branching morphogenesis of the lung. Curr. Opin. Genet. Dev. 8, 481–486. Hohenester, E., Tisi, D., Talts, J. F., and Timpl, R. (1999a). The crystal structure of a laminin G-like module reveals the molecular basis of -dystroglycan binding to laminins, perlecan and agrin. Mol. Cell 4, 783–792. Hohenester, E., Tisi, D., Talts, J. F., and Timpl, R. (1999b). The crystal structure of a laminin G-like module reveals the molecular basis of alpha-dystroglycan binding to laminins, perlecan, and agrin. Mol. Cell 4, 783–792. Huber, O., Bierkamp, C., and Kemler, R. (1996). Cadherins and catenins in development. Curr. Opin. Cell Biol. 8, 685–691. Hynes, R. O. (2002). Integrins: Bidirectional, allosteric signaling machines. Cell 110, 673–687.

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TGF- Superfamily and Mouse Craniofacial Development: Interplay of Morphogenetic Proteins and Receptor Signaling Controls Normal Formation of the Face Marek Dudas and Vesa Kaartinen Developmental Biology Program at the Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, California 90027 and Department of Pathology, Keck School of Medicine University of Southern California Los Angeles, California 90089

I. Introduction II. TGF- Superfamily Signaling A. Bone Morphogenetic Proteins and Related Growth Factors B. Structure of TGF- Family Ligands C. Ligand Antagonists and Ligand Heterodimers Increase the Signaling Complexity D. Receptors Do Not Make Our Understanding of the TGF- System Easier E. Signaling Convergence by Type I Receptors and Smad Proteins F. Unconventional Receptors and Alternative TGF- Signaling Pathways III. Craniofacial Phenotypes in Mutants of TGF- Superfamily Ligands and Receptors A. BMP and GDF Signaling B. TGF- Signaling C. Activin and Inhibin Signaling IV. Head Organizers and Early Anterior Development A. Anterior Visceral Endoderm Acts Synergistically with Derivatives of the Gastrula Organizer B. The Future Head Location: Prechordal Plate Mesenchyme with BMP Downregulation/Nodal Upregulation C. Paraxial Mesoderm, Neural Crest Segregation, and Possible Involvement of BMP-4 Signaling V. Neural Crest in Early Craniofacial Development A. Neural Crest Cells Migrate to Multiple Sites of the Developing Embryo B. Cranial Neural Crest Is the Major Player in Head Development C. BMP Signaling and the Induction of Neural Crest D. BMP Signaling and Delamination of Neural Crest Cells VI. Facial Prominences and Formation of the Face A. Facial Development Is Based on Fusion of Several Regions of Tissue VII. Mandibular Development A. Identity of the First Branchial Arch B. TGF- /Smad Signaling Regulates Growth of Meckel’s Cartilage Current Topics in Developmental Biology, Vol. 66 Copyright 2005, Elsevier Inc. All rights reserved. 0070-2153/05 $35.00

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Dudas and Kaartinen C. BMP Signaling Is Critical in the Rostral Part of Developing Lower Jaw D. Intramembranous Ossification of the Mandible

VIII. Palatal Development and Cleft Palate A. Palatogenesis in Mice as a Model for Human Development and Disease B. Apoptosis C. Alternative Fates of the MEE: Migration, Epithelial to Mesenchymal TransdiVerentiation, or Both? D. The Role of TGF- Superfamily Signaling in Palatogenesis E. Epithelial–Mesenchymal Interactions, Interactive Signaling Pathways, and Morphogenesis of the Prefusion Palatal Shelves IX. Clinical Research and Applications A. Craniofacial Fracture Healing B. Prevention of Heterotopic Bone Formation C. Teeth and Periodontal Regeneration X. Conclusions Acknowledgments References

I. Introduction The combination of brain, sensory organs, craniofacial skeleton, and cephalic musculature within the head makes it a uniquely complex structure. The sensory organs of the head are far more intricate than in the rest of the body, and originate from neurogenic placodes, structures found only in the embryonic head region. The head muscles, with the exception of the tongue musculature, are formed from unsegmented paraxial and prechordal mesoderm, in striking contrast to somatic muscles, which are derived from epithelial somites of a segmental nature. Interspecies comparisons show that facial bones are the most variable parts of the skeleton, contributing to formation of such a complex phenotypic feature as facial expression, one of the strongest visual stimuli used for the recognition of individuals, especially among primates (Kendrick et al., 2001). Skeletal components such as teeth, membrane bone, and secondary cartilage are tissue types located exclusively in the head (clavicles being the only exception). One of the greatest discoveries in developmental biology was the finding that the craniofacial skeleton is intimately connected with neural tissue—the vast majority of craniofacial bones and cartilages are, in fact, derivatives of neural crest cells (Northcutt and Gans, 1983). Why ectodermal cells that usually give rise to peripheral nervous system, neural ganglia, neuroendocrine cells, and melanocytes can also form structures that are derived strictly from the mesoderm elsewhere in the body skeleton is a challenging developmental and evolutionary question. The fact that the craniofacial skeleton is a mixture of bones and cartilage originating from the cranial neural crest and those originating from the

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mesoderm brings new insights to craniofacial development, since it is now clear that molecular processes involved in chondrogenesis, osteogenesis, and fracture healing are remarkably diVerent between these two groups of skeletal compounds (Helms and Schneider, 2003). Furthermore, birth defects with a craniofacial component belong to the most frequent malformations in humans, thus representing a considerable health, psychological, and economic burden to aVected families, as well as to society. The treatment of these disorders is often impossible, or represents a painful, stressful, and lengthy multistep procedure. Thus, an understanding of the biological processes underlying craniofacial development and physiology can bring a substantial contribution to current medical knowledge. Recent advances in genetics and molecular biology (e.g., vertebrate genome projects, new bioinformatic tools, microarray assays, gene knockout technology, tissue-specific gene targeting, in vivo imaging and microimaging) have produced an exponential growth of knowledge of embryonic development, and a substantial amount of data on head morphogenesis has accumulated over the past decade. Unlike ever before, the nature of these new techniques has opened windows into cellular and molecular mechanisms underlying the body plan creation, resulting in frequent redefinitions and updates in embryology, as well as in classification of developmental diseases. Inherently, this information boom results in increased branching and complexity in the scientific literature, raising the need to consolidate new data into logical and vital blocks of knowledge. Bone morphogenetic proteins and related peptides from the transforming growth factor beta (TGF- ) superfamily represent a distinct group of growth factors involved in head embryogenesis. They play roles in processes essential for craniofacial development: neural crest formation and migration, and cartilage and bone physiology. Their involvement in embryonic angiogenesis further underlines their importance in normal morphogenesis and related developmental diseases. This chapter is a compilation of the current knowledge of the role of members of the TGF- superfamily and their signaling pathways in facial development.

II. TGF- Superfamily Signaling A. Bone Morphogenetic Proteins and Related Growth Factors The discovery of bone morphogenetic proteins (BMPs) was instigated by the observation that ectopic bone was formed in fascia that had been used during surgery to bridge large gaps in the urinary bladder (Neuhof, 1917). This was followed by the discovery that, in addition to urinary epithelium, demineralized bone also possesses osteogenic capability when transplanted

Table I TGF- Superfamily Ligands, Their Corresponding Type I Receptors with Downstream Smads, and Type II Receptors*


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into connective tissues (Senn, 1989; Urist, 1965; Van de Putte and Urist, 1965). Osteogenic tissues were purported to produce osteogenic factors, which were later isolated and characterized (Reddi, 1997). Independently from this, a growth factor capable of supporting anchorage-independent growth of nonneoplastic fibroblasts in culture was described (Roberts et al., 1981; Tucker et al., 1984). This factor was named transforming growth factor beta (TGF- ) and was later shown to inhibit or promote cell growth, depending on the cell type studied, and on the presence of other growth factors (Massague, 1990; Moses et al., 1987; Sporn and Roberts, 1987). The osteogenic activity that gave BMPs their name is just one specialized application of their broad-spectrum physiological functions, namely, the induction of bone diVerentiation. Many BMPs have nothing to do with osteogenesis at all, but play important morphogenetic roles during the embryonic development of internal organs, skin, and nervous system. Based on sequence and structural homology (Chang et al., 2002; de Caestecker, 2004; Zhao, 2003), BMPs, together with growth and diVerentiation factors, TGF- s, activins, and inhibins, are now united in a group of around 40 evolutionary conserved, small signaling peptides, called the TGF- superfamily (Table I). They share a small group of receptors with common downstream signaling pathways, which directly aVect the regulation of transcription. Resulting signaling eVects diverge in diVerent cell types into a broad spectrum of physiological changes, usually aVecting the cell cycle, cell survival, and cell diVerentiation. It has been shown that the same growth factors may induce opposite eVects in diVerent concentrations, and these eVects diVer in diVerent cell types, which means that we cannot define their function simply in physiological terms. For the purpose of this chapter, we consider TGF- family members to be morphogens used by the developing embryo to transduce the spatial and/or temporal tissue-specific information. This information is usually evaluated together with many other signals and handled in a cell-specific manner, making the dissection of the role of TGF- signaling diYcult. Herein, we review situations where the role of TGF- signaling is so critical that its abrogation enables study of the aVected developmental process, with the focus on the craniofacial structures. We hope that this chapter will provide useful information that contributes to the unfolding story of ontogenesis.

*Type I receptors ALK-1 to ALK-8 have been grouped into three categories based on structural and functional similarity (highlighted with yellow, green, and blue). R-Smads activated by individual type I receptors are marked with a dot (). Bone morphogenetic proteins (BMP) and growth and diVerentiation factors (GDF) that show a remarkable similarity have been indicated with the same color font. Other TGF- superfamily ligands are grouped together by the name. y (‘‘yes’’) indicates known ligand-receptor interaction; n (‘‘no’’) indicates no interaction.

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B. Structure of TGF- Family Ligands All members of the TGF- superfamily share several common features: they are secreted into the extracellular space after intracellular proteolytic cleavage of large inactive dimeric precursor molecules by subtilisin-like pro-protein convertases such as SPC1/Furin, SPC4/PACE4, and SPC6A (Constam and Robertson, 2000; Cui et al., 1998; Sha et al., 1989). The carboxy-terminal products of this cleavage are receptor-binding molecules capable of direct signaling. In the case of TGF- s and growth and diVerentiation factor (GDF)-8, the N-terminal inactive part remains noncovalently attached as latency-associated peptide (LAP) to the C-terminal part, so the secreted products are inactive (latent ligands). An extracellular proteolytic action (e.g., by thrombospondin 1, plasminogen system) is required to release signaling dimers (mature ligands) from these large complexes (Chen et al., 2000; Rifkin et al., 1997, 1999). For example, latent TGF- 1 is stored in large amounts in the secretory granules of circulating platelets and is released during platelet activation, together with thrombospondin 1 (Assoian et al., 1983; Chen et al., 2000). Furthermore, the inactive precursor complexes may interact with specific components of the extracellular matrix [e.g., latent TGF- binding proteins (LTBPs)], and accumulate as an extracellular pool, further complicating our understanding of their life cycle and signaling logic (Kaartinen and Warburton, 2003). TGF- superfamily members are also characterized by six intramolecular disulfide bridges that form the cysteine knot, a folding structure important for interactions with receptors (Sun and Davies, 1995). The seventh conserved cysteine is responsible for covalent dimerization in order to form active ligands, the exceptions being GDF-3, GDF-9, BMP-15, lefty1, and lefty2, which dimerize noncovalently, since the cysteine is substituted with serine (Chang et al., 2002). It should be noted here that BMP-1, unlike the other BMPs, is not a member of the TGF- superfamily, and thus its name is confusing, although still used in the literature (Rattenholl et al., 2002). BMP-1 is a proteinase involved in the processing of many biologically important molecules, including procollagens, laminin, and, interestingly, Chordin, an inhibitor of TGF- signaling (Scott et al., 1999). BMP-1 is a product of the same gene as the proteinase tolloid of the metzincin family and results from alternative mRNA splicing (Takahara et al., 1994).

C. Ligand Antagonists and Ligand Heterodimers Increase the Signaling Complexity Several secreted peptides are known to have an antagonistic eVect on TGF- superfamily signaling. This heterogenous group of signaling modulators is


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still growing, and currently comprises Noggin, Chordin/SOG, Follistatin, Follistatin-related protein (FSRP), DAN/Cerberus protein family, Sclerostin, decorin, 2-macroglobulin, and connective tissue growth factor (CTGF) (Kusu et al., 2003; Shi and Massague, 2003). These antagonists directly interact with ligands and/or with ligand-receptor binding and block signal transmission. Structural studies provide increasing evidence that some of these inhibitors share the same cysteine knot structural feature of TGF- growth factors, and can also form dimers as well, suggesting that they may compete for receptor binding with TGF- growth factors, and/or that they may have evolved from a common ancestor (Groppe et al., 2002; Shi and Massague, 2003). Otherwise, the antagonists share only slight sequence homology, and it is generally assumed that they have evolved independently from each other (Balemans and Van Hul, 2002). Accumulated knowledge shows that nothing is black and white in TGF- signaling. For example, the ‘‘antagonist’’ CTGF interferes with TGF- 1 and BMP-4 signaling, but with opposite eVects—it acts as a signaling activator in one case and as an antagonists in the other (Abreu et al., 2002). Signaling interactions within the TGF- ligand family are even more complex, and not only because some ligands show a partial aYnity to receptors for other ligands, but also because several family members act as antagonists of other ligands (e.g., inhibins vs activins; Lefty vs Nodal; activin, Nodal, or GDF-8 vs BMPs) (de Caestecker, 2004). Furthermore, in addition to forming homodimers, many TGF- members can form heterodimers with other members, resulting in mixed ligands with unexpected aYnities for diVerent receptors. In addition, the same ligand dimer can give rise to diVerent signaling outcomes upon interaction with diVerent receptors, ranging from normal binding with subsequent receptor activation, through binding only with no downstream action, to functioning as a signaling inhibitor (Israel et al., 1996; Sampath et al., 1990). Rather than exceptional, this behavior seems to be typical for the TGF- group of growth factors, and a complete description of new findings with all relevant contradictions in the current literature would be worth a separate extensive review. More details can be found in recent literature (de Caestecker, 2004; Shi and Massague, 2003).

D. Receptors Do Not Make Our Understanding of the TGF- System Easier The ‘‘canonical’’ and mostly studied transduction node of TGF- signaling is the activation of the receptor complex, composed of two diVerent types of transmembrane receptors. Type II receptors (five members known: Tbr2, Actr2, Bmpr1a, Bmpr1b, Misr2) are constitutively active Ser/Thr kinases capable of phosphorylating and thus activating type I receptors (Mehra and Wrana, 2002; Moustakas et al., 1993). Type I receptors (eight members

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Table II Interactions of TGF- Superfamily Ligands with Type I and Type II Receptors or Receptor Complexes, and Subsequent Downstream Signal Transmission*

*Data in Tables I and II have been collected and combined from multiple literature sources (Attisano et al., 1993, 1996; Chapman et al., 2002; Cheng et al., 2003; de Caestecker, 2004; Derynck and Feng, 1997; Ebisawa et al., 1999; Erlacher et al., 1998; Franzen et al., 1993; Gouedard et al., 2000; Hogan, 1996a,b; Ikeda et al., 1996; Jamin et al., 2002; Lebrun and Vale, 1997; Lee and McPherron, 2001; Liu et al., 1995; Macias-Silva et al., 1998; Miettinen et al.,


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identified so far—activin receptor-like kinase ALK-1 to ALK-8) are inactive proteins that must be phosphorylated to acquire Ser/Thr kinase activity, resulting in phosphorylation of specific downstream messengers. Current understanding is that downstream signaling occurs only when two type I receptors form a complex with two type II receptors in the presence of a dimeric ligand. Only in such a complex are type I receptors activated by type II receptors, and the signal is transmitted into the cytoplasm. Everything else is variable: the strength and order of ligand binding (either to type I or first to type II) is diVerent for diVerent ligand–receptor combinations (Hart et al., 2002; Kirsch et al., 2000; Massague, 1998; Shi and Massague, 2003); both type I and type II receptors may or may not form homodimers, which can be ligand-dependent as well as ligand-independent (de Caestecker, 2004; Gilboa et al., 1998; Nohe et al., 2002). In addition, type I or type II heterodimers (or at least their mutual interactions) have been described (de Caestecker, 2004; Goumans et al., 2003; Matsuyama et al., 2003; Oh et al., 2000; ten Dijke and Hill, 2004; Ward et al., 2002). Finally, if the same ligand finds receptors preassembled in dimers, the signaling results may diVer from the situation where dimerization occurs only upon ligand binding (de Caestecker, 2004; Nohe et al., 2002). Ligand–receptor interactions are summarized in Tables I and II. In addition to type I and II receptors, so-called accessory receptors, coreceptors, or type III receptors have been described: betaglycan, endoglin/CD105, Cripto, Cryptic, and Nma/BAMBI (SchiVer et al., 2001; Shen and Schier, 2000; Shi and Massague, 2003; Yeo and Whitman, 2001). These accessory receptors probably do not transfer any signal, but may play important roles in processes such as ligand attraction to type I or II receptors, receptor–ligand complex stabilization or destabilization, type I– type II receptor complex stabilization or destabilization, or interactions with other regulatory molecules, as suggested by several studies and reviews (de Caestecker, 2004; Shen and Schier, 2000).

E. Signaling Convergence by Type I Receptors and Smad Proteins Extensive studies and numerous reviews indicate that type I receptors are the main transmitters of the TGF- signal from the cell surface into the cell. The list of currently known intracellular downstream targets is probably 1994; Moore et al., 2003; Nishitoh et al., 1996; Oh et al., 2000, 2002; Reissmann et al., 2001; Rosenzweig et al., 1995; ten Dijke et al., 1994; Visser et al., 2001; Wiater and Vale, 2003; Wrana et al., 1992; Yamashita et al., 1995; Yeo and Whitman, 2001; Zhao and Hogan, 1996). Observations in live organisms are sometimes diVerent from conclusions based on experiments in vitro. Thus, despite our best eVort, this compilation cannot be perfect, and furthermore, updates occur in the literature practically on a monthly basis.

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incomplete, but, based on the structural homology, it can be assumed that individual type I receptors will use similar downstream signaling mechanisms. The mainstream signaling occurs via phosphorylation of a small set of Smad proteins (Smad 1, 2, 3, 5, and 8, called receptor-regulated, or R-Smads). Smad4 cooperates with all R-Smads after their phosphorylation, and is also called cooperative or common partner Smad (co-Smad). Smad6 and Smad7 bind to intracellular domains of type I receptors and function as inhibitors of R-Smad phosphorylation (I-Smads). Smad9 is in fact Smad8, and Smad10 is a novel amphibian molecule similar to Smad4 (Howell et al., 1999; LeSueur et al., 2002). In addition to I-Smads, downstream signaling is also controlled by intracellular regulation of receptor activation, or by regulation of the receptor access to downstream messengers by regulatory proteins such as FKBP12, SARA, Tob, and others (Attisano and Wrana, 1996; de Caestecker, 2004; Huse et al., 1999; Massague and Chen, 2000; Miyazono et al., 2001; Yoshida et al., 2000). Type I receptors show a strict substrate specificity for their downstream Smads, which is defined by the L45 loop in the kinase domain. According to their sequence homology and kinase activity, type I receptors can be divided into three groups, as distinguished with color in Table I (Chen et al., 1997; de Caestecker, 2004): (1) ALK-1, -2, and -8 phosphorylate Smads 1 and 5 (although in amphibians ALK-2 has also been shown to phosphorylate Smad8); (2) ALK-3 and 6 phosphorylate Smads 1, 5, and 8; (3) ALK-4, -5, and -7 phosphorylate Smad2 and Smad3. All phosphorylated R-Smads form complexes with their common partner, Smad4, and are subsequently translocated to the nucleus, where they act as transcriptional regulators. Taken together, the type I receptors are the key node, where the various actions of multiple ligand/receptor/antagonist/coreceptor combinations (40 dimerizing ligands/8 5 dimerizing receptors, etc.) are finally translated into three phosphorylation patterns of five proteins. Thus, experimental abrogation of type I and II receptors provides a tool to better understand the role of TGF- signaling in developmental processes, and the ability to precisely dissect involved downstream events.

F. Unconventional Receptors and Alternative TGF- Signaling Pathways In addition to Smad pathways, several other downstream signaling mechanisms have been proposed for type I and/or II receptors, including mitogenactivated protein kinase (MAPK) pathways (ERK, JNK, p38 MAPK, MAPKKK TAK1, TAB1), PI3 kinase, protein kinase C (PKC), PP2A, small


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Rho-related guanosine triphosphatases (GTP-ases), XIAP, LIM kinase 1 (LIMK1), and p70 S6K (Bakin et al., 2002; de Caestecker, 2004; Itoh et al., 2003; Mulder, 2000; Petritsch et al., 2000; Takekawa et al., 2002). However, the exact connection of these pathways with TGF- receptors is not fully known, and the possibility of their secondary activation/inhibition by Smaddependent processes is frequently discussed in literature. Existence of Smad-independent pathways is strongly supported by a recent work showing that Smad4 (the universal downstream signaler for all ALK receptors) is dispensable in certain TGF- signaling processes in early embryos (Chu et al., 2004). Also, it has been known that some tumor cells lack functional Smad4, but still respond to TGF- 1 by growth arrest in the same manner as cells that have this tumor suppressor intact (Giehl et al., 2000; Sheppard, 2001; Simeone et al., 2000). Likewise, TGF- was shown to stimulate fibronectin expression in a N-terminal Jun kinasedependent, but Smad4-independent manner in human fibrosarcoma-derived cells (Hocevar et al., 1999). The molecular mechanisms underlying these findings are not clear, but several studies show possible directions for future research. For example, in platelets, the fibrinogen receptor integrin IIb 3 has been shown to respond to TGF- 1, which improves its fibrinogenbinding properties and influences the signaling via PKC (Hoying et al., 1999). Because platelets do not have a nucleus, all these eVects must be based on nontranscriptional interactions. Interestingly, integrin v 6 has been previously known to bind and proteolytically activate latent TGF- 1 through local release of MT1 matrix melalloproteinase (Mu et al., 2002; Munger et al., 1999). These findings bring a new aspect into understanding the local nature of TGF- action in tissues, and raise the possibility that diVerent integrins may functionally cooperate in ligand attraction and activation, resulting in alternative signal transmission. Another interesting finding comes from the search for the molecules interacting with LIMK1, the key negative regulator of actin depolymerization. The cytoplasmic tail of Bmpr2 appeared among the hit clones in a yeast two-hybrid screen, and has been shown to bind and inactivate LIMK1 only in the absence of BMP-4 ligand (Foletta et al., 2003). This work shows for the first time that type II receptors are involved in downstream signaling through physical interaction with downstream targets other than their natural substrates, type I receptors. In summary, all of these alternative pathways act in a nontranscriptional manner (at least in the initial steps), in contrast to the ‘‘canonical’’ pathways employing Smads, which are direct members of transcriptionregulating complexes. None of the alternative signaling has been shown to play a role in embryonic development, thus representing a challenge for future research.

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III. Craniofacial Phenotypes in Mutants of TGF- Superfamily Ligands and Receptors A. BMP and GDF Signaling Based on their abundance and expression pattern, BMPs and GDFs represent a heterogeneous group of growth factors sharing the same receptors in a complicated manner that is poorly understood. Based on the sequence homology and similarities in physiological behavior, these ligands are currently sorted into eight groups, but this sorting should be considered approximate and not final (see Table I) (Hogan, 1996a,b; Zhao and Hogan, 1996). The BMP–GDF signaling logic during development is diYcult to understand, and our present knowledge is very fragmented, composed of hundreds of mutually unconnected observations coming from multiple animal species, developmental stages, and practically all organ systems. The summary overview given in Table III shows that except for BMP-4, the deletion of any single ligand or receptor has practically no eVect on craniofacial development in viable mutants, or else the deletion causes early lethality prior to the beginning of cranial development. On the other hand, expression patterns suggest that the importance of BMP–GDF signaling in the head region should be comparable to the rest of the body (Mina, 2001). New important knowledge in this field comes from conditional inactivation of individual receptors in a tissue-specific manner utilizing the Cre–IoxP system. This allows deletion of early lethal genes later in development, and only in certain cell lineages and tissue types. Thus, vitally important early developmental processes are often not aVected, and the later eVects of every mutation can be studied in detail. Briefly, in this binary transgenic system, two diVerent transgenic strains are crossed with each other. One strain expresses the Cre recombinase under the control of a tissue-specific promoter, and another strain is genetically manipulated to possess a so-called floxed allele, in which two loxP sites flank a functionally essential segment of the gene. When the Cre recombinase is expressed in a cell harboring the floxed allele, a piece of DNA flanked by the loxP sites is spliced oV by Cre-recombinase activity, leading to a loss of an essential function of the targeted gene. Recently, Dudas et al. analyzed the eVect of abrogation of Alk2 in neural crest cells using this powerful system. These Alk2/Wnt1-Cre mice display severe defects both in the calvaria and in facial structures (Dudas et al., 2004b). As can be seen in Fig. 3 frontal bones show poor ossification when compared with a control littermate. Moreover, zygomatic arches display deficient posterior regions. The mandible in Alk2/Wnt1-Cre mice is hypoplastic, and Meckel’s cartilages fail to fuse in the midline. These mice also display cleft palate, which appears to result from the defective elevation of

Table III Craniofacial Phenotypes of Animals with Mutations in TGF- Superfamily Members and Related Signaling Molecules

(Continued )

Table III

Continued

(Continued )

Table III Continued

(Continued )

Table III Continued

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palatal shelves. Some of these findings are described later in paragraphs discussing individual developmental processes in more detail.

B. TGF- Signaling There are three isoforms of TGF- in mammals (TGF- 1, 2, and 3). They all display unique expression patterns during mouse development, both temporally and spatially (Akhurst et al., 1990a,b; Fitzpatrick et al., 1990; Hogan et al., 1994; Pelton et al., 1990a; Wall and Hogan, 1994). Still, it is somehow surprising that mice lacking one particular TGF- isoform do not share phenotypic features with mice lacking another isoform, nor do they demonstrate obvious functional redundancy. Tgf- 1 / mice exhibit two diVerent phenotypes (Shull et al., 1992) without craniofacial involvement (Kulkarni et al., 1993). About 50% of them die around embryonic day 8.5 (E8.5), with severe defects in yolk sac vasculogenesis and hematopoiesis, whereas the remaining mice survive beyond birth, but then develop severe multifocal inflammatory disease. Mice lacking Tgf- 2 display several diVerent developmental defects with variable penetrance, and die soon after birth. These phenotypes include cleft palate. Interestingly, mice deficient in Tgf- 3 display nonsyndromic cleft palate with 100% penetrance. Although the palatal defect in Tgf- 2 / mice is likely caused by poor mesenchymal proliferation leading to a growth retardation of prefusion palatal shelves (Sanford et al., 1997), the palatal shelves in Tgf- 3-null mutants grow normally, become adherent, but still fail to fuse (Kaartinen et al., 1995, 1997; Proetzel et al., 1995; Taya et al., 1999). Mice deficient in Tgfbr2 display a phenotype identical to that seen in the most severely aVected Tgf- 1-null mutants (Oshima et al., 1996), whereas Alk5 / mice die at midgestation with defects in angiogenesis, but not in hematopoiesis (Larsson et al., 2001). Craniofacial defects of mice lacking Tgfbr2 specifically in neural crest cells (NCCs) were recently described (Ito et al., 2003). In these mice, cranial NCCs migrate normally. However, aVected mice display cleft palate, mandibular hypoplasia, and severe defects in calvaria development, which result from defects in cell proliferation, both in the dura mater and in the NCC-derived palatal mesenchyme, respectively.

C. Activin and Inhibin Signaling Activin A and B subunits are widely expressed during development, as well as during adulthood (Feijen et al., 1994; Roberts and Barth, 1994). These proteins can form three dimeric ligands: activin A ( A + A), activin B ( B + B), and activin AB ( A + B). Dimerization with the distantly related inhibin-specific subunit yields two diVerent inhibins, inhibin A ( + A) and


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inhibin B ( + B). Two more activin chains, C and E, have been cloned in mammals (Fang et al., 1997; Schmitt et al., 1996), whereas D has so far been identified only in Xenopus laevis (Oda et al., 1995). Activins C and E seem to be specific for the adult liver with dispensable function, and no role during embryogenesis has been revealed by gene targeting (Lau et al., 2000). Mice deficient in A subunit are viable, but die within 24 hours with partially penetrant hypotrophic, missing, or cleft secondary palate. Mutants always lack lower (mandibular) incisors and mandibular molars, which do not develop beyond a rudimentary bud (Ferguson et al., 1998); all other mandibular and maxillar teeth develop normally, despite the fact that A expression is equal in all tooth types. Act B deficiency leads only to impaired eyelid development and female infertility, and defect of both subunits leads simply to a combined phenotype (Matzuk et al., 1995a,b). Inhibin chain deletion does not cause any craniofacial abnormalities (Matzuk et al., 1992), whereas mice deficient in activin antagonist Follistatin display several pathological phenotypes, including cleft palate (Matzuk et al., 1995c). In conclusion, from the activin family only activin subunit A plays a critical role in craniofacial development. The role of other activin subunits, if any, remains unclear. The possibility that other ligands compensate for their function in activin knockouts without penetrating phenotypes must be experimentally addressed. Combined deletion of receptors Actr2A and Actr2B results in a lethal defect in mesoderm formation, and all embryos die at the gastrulation stage (Song et al., 1999). Keeping in mind that activin double mutants passed this developmental stage normally, it is clear that activin type II receptors must mediate other than activin A, B, or AB signal during this process, which is just one of many examples of receptor sharing in TGF- signaling. Individual inactivation of Actr2A leads to partially penetrant cleft palate and a lack of incisors, a defect similar to those seen in A mutants, whereas 10% of embryos showed mild eyelid defects similar to B mutants (Matzuk et al., 1995a; Song et al., 1999). Around 80% of embryos developed normally with no malformations detected. In contrast to the ligand mutants, Actr2A inactivation also caused mandibular hypoplasia with variable, often severe dysmorphism of Meckel’s cartilage and its derivatives. No craniofacial defects were detected in Actr2B mutants (Oh and Li, 1997), except low-penetrant cleft palate dependent on the genetic background (Ferguson et al., 2001). It is remarkable that deletions of the individual activin type II receptors only partially overlap with ligand-dependent malformations, which can be explained by mutual functional compensation between Actr2A and 2B. Because double mutants do not survive beyond gastrulation, this question must be addressed with appropriate tools (e.g., by utilizing conditional gene knockout technology). The fact that new phenotypical features appeared in Actr2A mutants in comparison to ligand mutants suggests that ligands other than activins must be involved (based on known receptor aYnities, candidates may be inhibins,

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nodal, BMP-2, -6, -7, and GDF-1, -5, -8, -9b, -11). The nature of these defects (hypotrophic mandible, defective Meckel’s cartilage) suggest that they may be connected with neural crest development, which can be addressed in future by tissue-specific deletion. Partial penetrance of defects suggests that this will not be a simple task, because receptor–ligand promiscuity is likely. Another unresolved question is: which type I receptor passes the activin signal during the developmental processes aVected by ligand and type II receptor inactivation? Homozygous Smad2 knockouts die at embryonic day E10 (Nomura and Li, 1998), but heterozygotes survive with a range of variable phenotypes. Around 3% of them lack mandibular incisors and molars, suggesting the involvement of one of the Smad2-signaling receptors. Among them, ALK-4 has been shown to bind activins, but further research is necessary, because Alk4 knockouts die before gastrulation (Gu et al., 1998). Current knowledge suggests that the most typical craniofacial malformations caused by the activin family signaling are a lack of mandibular incisors and molars, and cleft palate. These features depend strongly on the Actr2A receptor and on an unknown type I receptor, and other unidentified receptors and compensating ligands may be involved. Furthermore, the Actr2A receptor is involved in the mandible and Meckel’s cartilage development through an activin-independent pathway.

IV. Head Organizers and Early Anterior Development To keep this review straightforward and focused, we think the best starting point is the formation of anterior identity, prechordal mesoderm (the future source of the most head musculature and of some connective tissue and skeletal elements), and delamination of cranial NCCs (future source of the majority of skeletal and connective tissues of the head).

A. Anterior Visceral Endoderm Acts Synergistically with Derivatives of the Gastrula Organizer In mammals, extraembryonal tissues have been shown to play a key role in establishing the body plan (Beddington and Robertson, 1998). The anterior visceral endoderm (AVE), which overlies the prospective anterior side of the epiblast and defines initial anterior identity of the mouse embryo, acts synergistically with the prechordal plate and paraxial mesoderm (derivatives of the gastrula organizer) to induce development of the head (Tam and Steiner, 1999). Nodal, expressed from the epiblast, has been shown to play an important role in initial establishment of the AVE. During normal gastrulation, the AVE is gradually displaced by the definitive endoderm, whereas in mice lacking the


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BMP type I receptor Alk3 (Bmpr1a) in the epiblast, the AVE appears expanded, suggesting that BMPs provide inhibitory signals for the AVE (Davis et al., 2004). Secreted BMP antagonists Chordin and Noggin, expressed by the mouse node, are not a requisite for establishing the AVE, but are required for the subsequent maintenance and further expansion of the anterior pattern (Bachiller et al., 2000). To conclude, the current view favors a model where (1) anterior identity is first displayed by the AVE and is established before initiation of gastrulation, and (2) the AVE provides signaling activity to promote anterior and suppress posterior patterning (Robb and Tam, 2004).

B. The Future Head Location: Prechordal Plate Mesenchyme with BMP Downregulation/Nodal Upregulation The prechordal plate is a region located immediately rostral to the notochord, just under the developing forebrain. This region is sometimes called the head organizer, and, in fact, in some vertebrates such as amphibians and fish, this region originates in part from the Spemann’s gastrula organizer (Foley et al., 1997; Pera and Kessel, 1997; Zoltewicz and Gerhart, 1997). The formation of the prechordal mesoderm is dependent on intricate changes in TGF- superfamily signaling. First, inhibition of BMP signaling has been shown to be critical for successful prechordal gastrulation. This involves the expression of secreted multifunctional BMP/Nodal/Wnt inhibitor Cerberus (Glinka et al., 1997; Piccolo et al., 1999). Inactivation of other BMP antagonists, Noggin and Chordin, results in forebrain defects and cyclopia (Bachiller et al., 2000). On the other hand, activin and nodal signaling must be switched on to allow formation of the prechordal mesoderm and its subsequent maintenance. Their inactivation leads to similar developmental defects with cyclopia (Agius et al., 2000; Bisgrove et al., 1999; Conlon et al., 1994; Feldman et al., 1998; Gritsman et al., 1999, 2000; Meno et al., 1999; Sampath et al., 1998; Thisse and Thisse, 1999; Zhou et al., 1993). Chimeric embryos composed of cells deficient in Smad2 (downstream of Nodal) and wild-type cells, as well as Nodal–Smad2 heterozygotes, are cyclopic (Heyer et al., 1999; Nomura and Li, 1998), and inactivation of Smad2 in the epiblast has been shown to result in failure to specify prechordal plate progenitors (Vincent et al., 2003). The idea that BMP downregulation and simultaneous Nodal upregulation are both required for prechordal mesoderm specification is further supported by observations of cyclopia in one-eyedpinhead (Oep) factor mutants in fish (Schier et al., 1997; Zhang et al., 1998). This EGF-CFC-related protein has been shown to function as a cofactor of Nodal and inhibitor of BMP signaling (Gritsman et al., 1999; Kiecker et al., 2000; SchiVer et al., 2001; Whitman, 2001; Yeo and Whitman, 2001). The fact that Nodal inhibitor Cerberus, as well as a high level of Nodal itself, are both required for

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proper prechordal mesenchyme development suggests the involvement of complex spatio-temporal relations in this region that await more detailed investigation. The complexity of tissue–tissue interactions will further increase later, after the immigration of NCCs with their own expression pattern of TGF- –BMP ligands and receptors. C. Paraxial Mesoderm, Neural Crest Segregation, and Possible Involvement of BMP-4 Signaling The paraxial mesenchyme represents another important precursor of the head tissues. It is located laterally to the prechordal plate, and ventrally/ laterally from the developing cranial portion of the neural tube. Although this region is frequently described as unsegmented, a transient formation of seven loose aggregations (somitomeres) of its cells has been observed during development (Fig. 1). Cells of these repetitive structures migrate into various parts of the head and contribute to the formation of muscles, skeletal components, and endothelium of arteries of all branchial arches (Jacobson, 1988; Meier and Tam, 1982; Tam et al., 1982). One of the best-documented roles of the paraxial mesoderm in patterning of the head is the influence on the flow of NCCs into specific destinations (see section V). As demonstrated in Fig. 1, cranial NCCs in vertebrates migrate ventrally as three distinct streams: trigeminal stream from rhombomeres 1 and 2 (R1, R2) populating the head, including the first pharyngeal arch; hyoid stream from R4, populating the second branchial arch; and postotic stream from R6 and R7, populating the third and more distal branchial arches. In mice (and chick), there are no streams arising from R3 and R5, although their NCCs originate from these segments. Most of these NCCs die by apoptosis in chickens probably due to increased BMP-4 signaling, whereas the NCCs from the neighboring even-numbered rhombomeres express BMP antagonist Noggin, protecting them from death (Smith and Graham, 2001); so far, no similar mechanism has been identified in mice. The surviving minority of NCCs joins the more rostral or caudal adjacent stream. Migration of NCCs derived from the R4 transplanted to the R3 region copies the migration pattern of original R3-derived NCCs, and pieces of R3 transplanted into the R6 region generated areas with no NCC formation around them (Farlie et al., 1999; Kuratani and Eichele, 1993; Niederlander and Lumsden, 1996). These observations imply that both the rhombomeres and somitomeres carry molecular determinants, and that mutual interaction controls the proper segmental behavior of NCC migration. EphrinB2 and ephrinA5 are involved in attraction–repulsion regulation of cranial NCC migration in mice, and proper segmental guidance has been shown to be

Figure 1 A schematic presentation of cranial neural crest cell (NCC) migration, and contribution of cranial neural crest cells and somitomeres to craniofacial structures. Lateral and dorsal views (left and right) show the approximate positions of the segmented neural tube and the neighboring paraxial mesoderm in the embryo. Blue arrows indicate the origin and the migration direction of cranial NCCs. Final destinations of NCCs and cells derived from the somitomeres of the paraxial mesoderm are indicated with lines. F, Forebrain; ggl., ganglion; MA, anterior midbrain; MP, posterior midbrain; opt., optic vesicle; OV, otic vesicle; pom, periotic mesenchyme; R1 to R7, rhombomeres 1 to 7; S1 to S3, somites 1 to 3; SM1 to SM7, somitomeres 1 to 7.

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critical for the proper patterning of the mandible and pharyngeal arch derivatives (Francis-West et al., 2003).

V. Neural Crest in Early Craniofacial Development A. Neural Crest Cells Migrate to Multiple Sites of the Developing Embryo Described for the first time in 1868 by the Swiss embryologist His as a band of cells between the surface epithelium and the neural tube in chicken embryos, NCCs became one of the most studied cells during embryogenesis (Hall, 1999; Trainor et al., 2003). They appear at the dorsolateral edge of the closing neural folds, along practically the entire length of the vertebrate neuraxis. This is the line generally referred to as the neural plate border, where the surface ectodermal epithelium functionally splits into two areas: neuroectoderm, or neural plate, which forms the neural tube and completely invaginates into the embryonic body, and the rest, which continues to be the body surface epithelium and becomes the epidermis. Cell fate tracing experiments were extraordinarily fruitful in neural crest research and revealed that NCCs originate by delamination from the epithelium, migrate into the mesenchyme, and populate multiple distant sites in the embryo, giving rise to multiple cell types. The cell types described were the neurons of peripheral nerves and neural ganglia, Schwann cells forming the myelin, various chromaYn and neuroendocrine cells, melanocytes, and glial cells.

B. Cranial Neural Crest Is the Major Player in Head Development During the 1890s, Julia Platt demonstrated in the mud puppy Necturus sp. that the visceral cartilages of the head and dentinoblasts also originate from the neural crest (NC) (Platt, 1897; Trainor et al., 2003). This was a very interesting observation opposing the contemporary classical knowledge on the origin of tissue types from the three germ layers. Indeed, future research provided substantial evidence that in the head region, and only there, most bone, cartilage, and connective tissue does not arise from the mesoderm, but from the NC. As demonstrated by grafting experiments, the cranial portion of the NC maintains the osteogenic activity when transplanted into the body trunk and forms ectopic hard-tissue nodules, whereas the trunk NC transplanted to the head region cannot contribute to the formation of the craniofacial bones, meninges, and smooth muscle layer of blood vessels (Etchevers et al., 1999; Le Douarin et al., 1974, 1977; Nakamura, 1982; Nakamura and Ayer-le Lievre, 1982). These and other functional diVerences contributed to the definition of the cranial neural crest (CNC) as a distinct part of the NC.


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It comprises the entire NC from the forebrain to caudal hindbrain, where the trunk NC begins. The segmentation and cell fate of cranial NCCs are summarized in Fig. 1. C. BMP Signaling and the Induction of Neural Crest Contact-dependent interactions between the surface ectoderm and neuroepithelium are a trigger for NCC specification, and both epithelial cell types contribute to the neural crest population (Selleck and Bronner-Fraser, 1995). In preneural avian embryos, the entire surface ectoderm expresses BMP-4 and BMP-7, whereas at later stages this expression ceases. The exception is the dorsal neural tube, where the expression continues, suggesting that BMP signaling plays a role in positioning the neural plate borders (the dorsal part of the tube is formed by fusion of the left and right borders of the neural plate during the final stage of invagination). Cell fate tracing demonstrated that daughter cells of a single cell from this dorsal part of the neural tube are later found in both the neural tube and the NC, suggesting that BMP signaling is involved in generating of at least a part of the NCC population. In in vivo and in vitro experiments with avian embryos, BMP-4 and BMP-7 can substitute for the missing surface epithelium in NC induction assays, and BMP-4 is suYcient to induce the expression of NC-specific zincfinger transcription factor Slug, followed by NC segregation from the neural tube (Liem et al., 1995). Other neural tube genes shown to be expressed under the control of BMP-4 are cadherin 6b, RhoB, Msx1, Msx2, and Pax3. Implantation of cells expressing the Noggin inhibitor into the closing neural tube inhibits NC formation and migration. Taken together, the spatiotemporal expression pattern of BMP-4 correlates well with functional studies showing its role in NC induction by the neural tube. Important questions remain: how important is BMP-4 expression in the surface epithelium, and what role is played by BMP-7? The weak part of the studies described earlier is that serum-enriched culture media were used in some experiments, and several results are not reproducible with chemically defined substrates (Garcia-Castro et al., 2002). Because this topic is not directly related to craniofacial development, we refer the reader to reviews describing this controversial issue in more detail (Gammill and Bronner-Fraser, 2002, 2003; Santagati and Rijli, 2003; Trainor et al., 2003). D. BMP Signaling and Delamination of Neural Crest Cells Cells from the neural plate and the marginal ectoderm that become NCCs undergo a specific process of phenotypical transformation. Cells of epithelial origin transdiVerentiate into mesenchyme, while they completely rebuild

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their cell–cell contacts, acquire motility, and change their interactions with the extracellular matrix (Duband et al., 1995; Monier-Gavelle and Duband, 1995). Transformed cells must destroy or pass the barrier represented by the basal lamina, and migrate in the appropriate direction. This process, known as epithelial-to-mesenchymal transdiVerentiation (EMT), occurs at many places in the developing embryo, and it occurs during tissue remodeling and/or at the fusion of two structures covered by epithelium. In adults, EMT probably does not occur under physiological conditions, but has been implicated as a cancer invasion mechanism (Ellenrieder et al., 2001). Guanosine triphosphate (GTP)-binding protein RhoB from the Ras superfamily is expressed in the dorsal part of the neural tube under the control of BMP-4 (and possibly other BMPs). This protein is also transiently expressed in new NCCs, and its inhibition prevents NC delamination but not the specification of premigratory NCCs (Liu and Jessell, 1998). The exact mechanism of action of RhoB during the NCC delamination remains unclear. However, closer examination reveals that inhibition of RhoB causes a lack of actin stress fibers and prevents morphological changes of prospective NCCs. This is consistent with the known role of Rho proteins in cytoskeletal rearrangements during EMT, especially in reorganization of the actin cytoskeleton (Kaartinen et al., 2002; Nobes and Hall, 1995; Ridley and Hall, 1992). Studies on several cell types suggest that RhoB, but not RhoA or RhoC, is the rapidly inducible source of Rho activity in the cell, which is rapidly and strongly expressed after exposure to growth factors such as BMPs (Jahner and Hunter, 1991; Liu and Jessell, 1998; Nobes et al., 1995). This can explain in part how the TGF- superfamily signaling influences the cell fate of responsive cells.

VI. Facial Prominences and Formation of the Face A. Facial Development Is Based on Fusion of Several Regions of Tissue Early steps of facial development are astonishingly similar between diVerent vertebrate species. In mice, cranial neural crest cells (CNCs) start to migrate soon after gastrulation, and the entire delamination takes place between the stages of 3 to 16 somites (Francis-West et al., 2003). CNCs populate the five facial prominences: the frontonasal prominence, the paired maxillary, and the paired mandibular prominences (Fig. 2), which form superior, inferior, and lateral boundaries of the future oral cavity, stomodeum. These facial primordia are covered by a thin epithelium derived from the ectoderm. As outlined previously, the mesenchyme is largely composed of the NC, whereas the core of the facial prominences contains some mesodermal cells (Francis-West et al., 2003). DiVerential proliferation of facial processes is


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Figure 2 Initial phases of facial formation. (A) Frontal views demonstrate the development of the facial region by fusion of various prominences. The frontonasal mass (FNM, yellow) gives rise to medial and lateral nasal processes (MNP, orange; LNP, red), which fuse together to form the nostrils. In addition, FNM fuses in the midline with maxillary prominences (MXP, green) of the first branchial arch to form the upper lip, maxilla, and palate. The lower portions of the first branchial arches (BA1, blue) fuse in the midline and give rise to the mandible and lower lip. (B) Fate mapping of NCCs using the Wnt1-Cre/R26R reporter assay (Ito et al., 2003) shows that facial bones and cartilage, and frontal bones, stain blue and are derived from the neural crest.

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believed to define the final shape of the embryonic face (Gui et al., 1993; McGonnell et al., 1998; MinkoV, 1980, 1991). As soon as facial processes reach the appropriate form and size, they fuse with neighboring primordia (Johnston and Bronsky, 1995). For instance, shortly after NCC migration, the ectoderm of frontonasal processes forms two thickened epithelial regions, the nasal placodes, which subsequently curl outward to give rise to the lateral and medial nasal processes (LNP and MNP). LNP and MNP grow further and eventually fuse with the maxillary processes of the first branchial arches, forming the upper lip and the primary palate. As delineated earlier, the role of TGF- –BMP signaling during the induction and migration of NCCs has been intensively studied; in contrast, little is known about the role of TGF- s and BMPs in other aspects of facial formation, particularly in fusion of facial primordia. Based on the expression patterns of BMP-2 and BMP-4 in the developing chick face, it has been suggested that these growth factors play a role in outgrowth of the primordia (Francis-West et al., 1994). Consistent with this, Ashique et al. (2002) demonstrated that the BMP antagonist Noggin reduced proliferation and outgrowth of the frontonasal mass and maxillary prominences. Interestingly, it also has been shown in chick embryos that Noggin, in conjunction with retinoids, can induce a duplicate set of frontonasal mass skeletal elements in place of maxillary bones, suggesting that BMP signaling, together with retinoids, is involved in specification of tissue identity, at least in avian facial prominences (Lee et al., 2001). Furthermore, it has been shown that BMPs can both induce and maintain expression of their bona fide eVectors Msx1 and Msx2. Both these genes display very strong and characteristic patterns of epithelial expression in the first pharyngeal arch, and mice deficient in these closely related homeobox genes display severe developmental defects in derivatives of the first pharyngeal arch, including the mandible, palate, and frontal bones (Satokata et al., 2000).

VII. Mandibular Development Jaws, especially teeth, are remarkable anatomical structures. Currently, it is highly controversial regarding which emerged first during phylogeny (Butler, 1995; Smith and Coates, 1998), and also, information about the contribution of the embryonic germ layers to developing teeth is being frequently refined. The largest portion of present knowledge on teeth development comes from studies in rodents that possess monophyodont dentition with continuously growing incisors, and missing canine and premolar teeth. Because this knowledge is not directly applicable as a model for human diseases, and because this topic is already well covered in the present literature, we refer the reader to the latest research and review articles by specialists in this field (Chai et al.,


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2000; Cobourne and Sharpe, 2003; Jernvall and ThesleV, 2000; Jung et al., 2003; Laurikkala et al., 2003; Slavkin et al., 1992; ThesleV, 2003; ThesleV and Mikkola, 2002; Xu et al., 2003; Zhang et al., 2003). Here we concentrate on the lower jaw development, a derivative of the first branchial arch. A. Identity of the First Branchial Arch Branchial arches are metameric, segmental structures of the embryonic neck that give rise to gills in some groups of vertebrates (e.g., fish), while they are transformed into many unsimilar derivatives in others. Similarly, as in other repetitive structures (somites, vertebrae, limb parts), branchial arch identity is given by expression of an unique combination of homeobox genes (GendronMaguire et al., 1993; Mallo and Brandlin, 1997; Rijli et al., 1993; Schneider and Helms, 2003; Trainor and Krumlauf, 2000, 2001). The subepithelial mass of the first branchial arch is populated by NCCs delaminating from the midbrain to the rhombomere 2. These cells do not show homeobox expression (Francis-West et al., 2003; Hunt and Krumlauf, 1991; Hunt et al., 1998; Richman and Lee, 2003). This represents an exception from other branchial arches expressing Hox genes, and may be a result of distinct regulation of the corresponding NC by the isthmus (Hunt et al., 1998; Irving and Mason, 2000; Noden, 1983; Prince and Lumsden, 1994; Trainor et al., 2002). After immigration of NCCs, the first branchial arch consists of the ectodermal epithelium of stomodeum (primitive oral cavity), followed by the primitive gut endoderm after the pharyngeal membrane, surface ectodermal epithelium (future skin) and ectomesenchyme (i.e., cells derived from NCCs that lie peripherally under the epithelium) (Chai et al., 2000; Noden, 1986, 1988; Trainor and Tam, 1995), and a small portion of central mesenchymal core, probably derived from the original mesodermal cells of the first branchial arch (Francis-West et al., 2003). Both mesenchymal components combine during further development of the skeleton, musculature, and other connective tissues, with involvement of many signaling pathways setting the axial polarity and/or segmentation of developing tissues. B. TGF- /Smad Signaling Regulates Growth of Meckel’s Cartilage A key event in the development of derivatives of the first branchial arch is the formation of Meckel’s cartilage, which acts as an axis along which the mandible, nerves, and vessels develop. Meckel’s cartilage appears for the first time in the molar region as a cell condensate. Modern molecular tools for cell lineage labeling provided a new insight into composition of Meckel’s cartilage (Ito et al., 2002): NCCs initiate its formation and form two chondrogenic fronts responsible for anterior and posterior growth.

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Later, middle parts contain a major portion of non-NCCs. Speculations are held about the origin of these non-NCCs; they may be of mesodermal origin from the branchial arch mesenchymal core, or migrate from the neural tube as so-called VENT cells (Chai et al., 2000; Sohal et al., 1999). During further development, Meckel’s cartilage is mostly resorbed, with the exception of the anterior part forming the mandibular symphysis, and the posterior part giving rise to the middle ear ossicles malleus and stapes, and to anterior malleolar and sphenomandibular ligaments. The proliferation and chondrogenic diVerentiation of cells in Meckel’s cartilage is under the direct control of TGF- –Smad2 signaling (Chai et al., 1994). In this study, R-Smads Smad2 and Smad3 have been detected in the cartilage and its perichondrium, where they show phosphorylation and nuclear localization, whereas inhibitory Smad7 is localized predominantly in the perichondrium. Exogenous TGF- 1 selectively increased the proliferation of NC-derived cartilage cells and promoted formation of type II collagen in chondrocytes and type I collagen in the perichondrium. This eVect was diminished in Smad2+/ explants, whereas Smad3 haploinsuYciency had no eVect. The exact role of particular receptors, as well as the involvement of other TGF- superfamily ligands, is still controversial. For example, mice deficient in Tgfbr2 (the canonical partner of ALK-5 in mediation of TGF- signals) in NCCs develop the rostral process of Meckel’s cartilage, and the length of the cartilage seems to be proportional to the length of the lower jaw (Ito et al., 2003). Although that study did not describe whether Meckel’s cartilages fuse normally in the midline, the findings in the Tgfbr2 mutant raise questions about whether TGF- /Smad2 signaling is dominant in regulation of the growth of Meckel’s cartilage as proposed earlier and/or whether the signaling cascade works in a canonical manner. Ongoing experiments suggest that the signaling network is not simple, because inactivation of BMP receptor ALK-2 severely aVects mandibular morphology and growth of the anterior pole of Meckel’s cartilage (see later).

C. BMP Signaling Is Critical in the Rostral Part of Developing Lower Jaw The specific role of TGF- superfamily signaling in NC-derived cells of Meckel’s cartilage has been definitely confirmed by recent experiments using conditional gene knockout techniques. In these studies, the Cre recombinase was driven by the NC-specific Wnt1-Cre promoter, which was used to inactivate several TGF- superfamily receptors. Inactivation of Alk2 (Fig. 3) did not aVect NCC delamination and migration, but resulted in fully penetrant mandibular hypoplasia by approximately 40% (Dudas et al., 2004b). Detailed analysis at E13 revealed that the anterior


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Figure 3 Tissue-specific deletion of Alk2 receptor in neural crest cells leads to numerous developmental defects in the craniofacial region. (A, B) Micro-CT (computerized tomography) of the skull, frontal superior aspect; (C, D) micro-CT, frontal inferior aspect; (E, F) alizarin red/alcian blue staining of the skull, lateral view; (G, H) staining of the mandibles, upper view.

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part of Meckel’s cartilage, containing the chondrogenic front, was missing. Further experiments showed that cell proliferation was noticeably reduced in the anterior region, which can explain shortening of the cartilage. Intermediate parts of Meckel’s cartilage appeared normal in whole-mount stainings, but histological evaluation revealed smaller and rounded diameter of cartilage and lower mitotic index of chondrocytes. This may be a consequence of insuYcient performance of the anterior growth front, or of continuing insuYciency of cartilage cells deficient in ALK-2 to respond to growth factors. Posterior parts of Meckel’s cartilages were not aVected, and their derivatives (middle ear ossicles) developed normally. This can serve as one of many examples of functional regionalization in the first branchial arch, when cells of the same origin and morphology acquire diVerent biological responsiveness, depending on their anteroposterior location. The phenotype of newborn Alk2 mutants in the NC was fully consistent with the previously described findings. Rostral processes of Meckel’s cartilages were missing (i.e., did not reach the rostral midline, and, subsequently, the mental symphysis was completely absent). The mandibles themselves were more or less normally shaped, but substantially smaller than in controls. Because it is known that the mandible is formed from NC-derived as well as non-NC-derived cells (Ito et al., 2002), it is possible that NCCs play a passive, osteogenic role in the developing mandible, without a substantial morphogenetic function. Striking exceptions are secondary cartilages of the condylar and angular processes, which were completely missing (see Fig. 3). It is not clear whether this defect is a consequence of failed chondrogenic induction in the performed mandible or of a failure of NCCs to contribute to these structures. Addressing this question may help to further understand specific aspects of BMP signaling in craniofacial bone development in general, because similar defects were found in several other bones, including complete absence of the temporomandibular joint and calvaria defects. For more details and for a complete phenotype description in all aVected systems, see the original articles (Dudas et al., 2004b; Kaartinen et al., 2004). Almost exactly opposite eVects on mandibular development have been described after inactivation of FGF-8 in the epithelium of the first branchial arch. This deletion resulted in a complete loss of mandibular skeletal structures, with the exception of mandibular midline with incisors and the rostral process of Meckel’s cartilage, which developed normally (Trumpp et al., 1999). Taken together with the ALK-2 study, it is tempting to conclude that the anterior mandible and Meckel’s cartilage represent a separate Micro-CT, as well as bone and cartilage staining (red and blue, respectively), in mutant mouse newborns reveals impaired ossification in frontal bones, short mandible, cleft palate (not shown), and absence of several structures in the temporal region (dotted square in E, F), including completely missing temporomandibular joint (arrow).


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developmental unit. This part of the lower jaw may be under the control of signals from the ectodermal epithelium, such as BMP-4, which is a potential ligand for ALK-2 and is expressed in the rostral tips of mandibular arch epithelium (Bennett et al., 1995; Francis-West et al., 1994). Other possible signaling candidates are BMP-7, expressed also by the ectoderm of the first branchial arch (Lyons et al., 1995), and BMP-2, present in the arch ectomesenchyme (Bennett et al., 1995; Francis-West et al., 1994). Further support for the idea that BMPs are predominantly involved in the development of the anterior part of the lower jaw comes from the observation that abrogation of TGF- signaling (Tgfbr2) in NCCs seems to have little eVect on the rostral process of Meckel’s cartilage (Ito et al., 2003), in contrast to inactivation of the BMP type I receptor ALK-2. On the other hand, the mandible is smaller, similar to Alk2 mutants, and, in addition, the angular process is completely missing. This suggests that both BMP and TGF- signaling act simultaneously and synergistically in the NC-derived cells of the developing mandible, but in some of its regions, one of them plays a more dominant or critical role (BMP signaling in rostral Meckel’s cartilage and secondary processal cartilages, TGF- signaling in the angular process, Fig. 4).

D. Intramembranous Ossification of the Mandible Ossification of the head bones is diVerent from endochondral ossification seen in the body. So-called intramembranous ossification starts as condensations of the mesenchyme into nodules. Some of them turn into capillaries, whereas others give rise to osteoblasts producing osteoid matrix, capable of binding and depositing calcium (Cohen, 2000b). The mandibular bone is formed from the ectomesenchyme, which is derived from NCCs. The head epidermis was proposed as a source of proossification instruction signals; BMP-2, BMP-4, and BMP-7 are the candidate signalers that instruct NCCs to diVerentiate into bone by inducing the expression of CBFA1/RUNX2, and core binding factor A1/runt homeodomain protein 2 (Ducy et al., 1997; Harada et al., 1999). The role of CBFA1 is to upregulate the expression of osteocalcin, osteopontin, and several other extracellular matrix proteins characteristic of bone. Mice homozygous for the knockout CBFA1 allele completely lack bone, whereas the cartilagenous skeleton is fully formed (Komori et al., 1997; Otto et al., 1997). In the mandibular region of newborns, the mandible and teeth are missing, but Meckel’s cartilage is present in an intact form. CBFA1 links BMP signaling to cleidocranial dysplasia in humans, because heterozygous mice showed a very similar phenotype, and all aVected human individuals are heterozygous for mutations in the CBFA1 gene (Lee et al., 1997; Mundlos et al., 1997).

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Figure 4 Early development of the mandible. (A) Ossification of the future mandible starts in mesenchymal condensations around Meckel’s cartilage (blue), that becomes partly embedded into the bony tissue (B), while other parts disappear. The posterior part of Meckel’s cartilage gives rise to the middle ear ossicles malleus and incus, the anterior malleal ligament, and the sphenomandibular ligament (B, C, D). Rostral tip of Meckel’s cartilage persists as the cartilage of the mandibular symphysis (D). In the posterior end, secondary cartilages appear on the condylar and angular processes (green). (C) A schematic representation of diVerential eVects of BMP and TGF- signalings on the mandibular development.

VIII. Palatal Development and Cleft Palate A. Palatogenesis in Mice as a Model for Human Development and Disease Cleft palate is one of the most common congenital birth defects in humans. It aVects approximately 1 out of 700 individuals with some variations in all ethnic groups all over the world. During recent decades, palatal fusion


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(palatogenesis) has been very intensely studied, and, in conjunction with significant improvements in many diVerent disciplines of biomedical research (e.g., molecular biology, mouse and human genetics and bioimaging), has produced a wealth of information about the physiology of normal palatogenesis, as well as information about the pathogenetic mechanism of cleft palate. Because palatogenesis in mice is remarkably similar to that in humans, the mouse has become a laboratory animal of choice for studies on palatal fusion (Abbott and Birnbaum, 1991; Blavier et al., 2001; Kosazuma et al., 2004; Schutte and Murray, 1999; Taya et al., 1999). In mice, the palate is formed around E12 from outgrowths of maxillary processes of the mandibular arch. Proliferation of the mostly NC-derived mesenchyme of palatal shelves leads to their rapid growth, which initially takes place vertically along the sides of the tongue (Fig. 5A, B). Elongation of the lower jaw and other morphogenetic events direct rapid expansion of the oral cavity, which allows descent of the tongue and subsequent elevation of the palatal shelves (Fig. 5C, D). In mice this takes place around the E14. Soon after the elevation, the epithelium in tips of the apposing palatal shelves [so-called medial edge epithelium (MEE)] becomes adherent (Taya et al., 1999). MEE cells intercalate and form the so-called palatal epithelial midline seam (Martinez-Alvarez et al., 2000b), and eventually disappear (Fig. 5E–H). Cleft palate can result from a failure in any one of these steps (see the schemes on Fig. 5). The critical step in palatal fusion is removal of MEE cells from the midline seam. Three diVerent fates have been hypothesized to account for the disappearance of the MEE from the palatal midline: (1) programmed cell death (apoptosis), (2) epithelial to mesenchymal transdiVerentiation, and (3) migration.

B. Apoptosis The original hypothesis of apoptosis of the MEE was presented several decades ago by numerous investigators (Farbman, 1968; Glucksmann, 1965; Pourtois, 1966; Saunders, 1966; Shuler, 1995). During recent years, development of new analytical tools has played an instrumental role in revitalizing studies on the role of apoptosis in midline palatal fusion. First, the TUNEL assay was used to verify that there actually are positively staining cells in the midline seam, particularly in the epithelial triangles (Mori et al., 1994; Taniguchi et al., 1995). These studies were more recently extended by Martinez-Alvarez et al. (2000b), who also suggested that TGF- 3 has a role as an inducer of apoptosis during palatogenesis, and by Cuervo and colleagues, who showed that retinoids play a key role in induction of apoptosis in the MEE (Cuervo et al., 2002; Cuervo and Covarrubias, 2004). Additional in vivo evidence to support an idea that apoptosis plays a key role in defining the fate

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Figure 5 Palatal shelf growth, elevation, and fusion. Schemes on the left side show cross sections through the embryonic palate during various developmental stages. Drawings on the right side demonstrate the corresponding macroscopic views. (A, B) Palatal shelves appear as short protrusions from the maxillary region and grow vertically by the sides of the tongue.


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of the MEE was provided by Yoshida et al. (1998), who demonstrated that mice deficient in Apaf-1 (mammalian homologue of C. elegans CED-4) display a palatal phenotype very similar to that of TGF- 3 / mice, in which fully grown palatal shelves fail to fuse due to a failure of MEE cells to die. An alternative view presented by Nawshad et al. (2004) suggests that only the outer layer of the MEE, called the periderm, undergoes apoptosis, whereas the basal MEE cells undergo EMT. C. Alternative Fates of the MEE: Migration, Epithelial to Mesenchymal Transdifferentiation, or Both? Lineage tracing using membrane-intercalating vital dye, DiI, was originally used to study the fates of migrating NCCs (Serbedzija et al., 1989). Subsequently, this method was used to study MEE fate in mouse palates both in vitro (Carette and Ferguson, 1992; Fitchett and Hay, 1989; Shuler et al., 1991) and in vivo (Shuler et al., 1992). Although Carette and Ferguson concluded that MEE cells migrate to oral and nasal epithelial triangles, Fitchett and Hay, and Shuler et al., showed that a large portion of MEE cells transdiVerentiate to mesenchymal cells during palatal fusion (EMT). These conclusions were also supported by immunostaining for epithelial and mesenchymal markers (Shuler et al., 1991, 1992). Subsequent lineage-tracing studies using green fluorescent protein in conjunction with retroviral or adenoviral gene transduction have been consistent with these original findings, showing that the basal layer of the MEE undergoes EMT (Cuervo et al., 2002; Martinez-Alvarez et al., 2000b). However, neither the molecular mechanism of induction of this process during palatogenesis nor how it is coordinated with apoptotic cell death is currently known. D. The Role of TGF- Superfamily Signaling in Palatogenesis All three mammalian TGF- isoforms are expressed in the palatal region before and during the fusion. TgfF- 3 expression can first be seen in the

Growth defects occurring at this stage in the palatal mesenchyme lead to wide palatal clefts. (C, D) Subsequently, palatal shelves elevate to a horizontal position. Again, defects in palatal growth and/or elevation defects lead to wide clefts. Moreover, unrelated disorders accompanied with reduced size of the oral cavity may mechanically prevent palatal elevation by blocking the descent of the tongue. (E, F) Palatal shelves continue to grow horizontally until they meet and adhere to each other in the midline. In addition to insuYcient mesenchymal growth, epithelial dysfunction preventing disappearance of the midline epithelial seam, and subsequent fusion of mesenchymal palatal masses, can lead to complete, partial, or submucous palatal clefts. (G, H) Under physiological conditions, palatal shelves fuse in the midline forming the secondary palate, the upper wall of the mouth consisting of the hard and soft palate.

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epithelium of tips of vertically growing shelves. Its expression is very strong in the MEE of apposing shelves and in the midline seam, but ceases simultaneously with the disappearance of the midline seam. In contrast, Tgf- 2 is expressed in the palatal mesenchyme during growth of palatal shelves, as well as during elevation and fusion, whereas the pattern of Tgf- 1 is more diVuse both in the mesenchyme and, later, in the epithelium (Fitzpatrick et al., 1990; Pelton et al., 1990a,b). As described before, mice deficient in TGF- 3 suVer from isolated cleft palate without any other craniofacial symptoms (Kaartinen et al., 1995; Proetzel et al., 1995). In these mice, fully grown palatal shelves fail to fuse, and therefore the role of TGF- 3, specifically in the MEE, has been a subject of intense study. Some studies have suggested that TGF- 3 specifically induces EMT (Kaartinen et al., 1997; Sun et al., 1998a,b), and other investigations have shown that TGF- 3 induces specific morphological changes in the MEE (Gato et al., 2002; Martinez-Alvarez et al., 2000a; Taya et al., 1999; Tudela et al., 2002). These include the formation of long filopodia on the apical surface of the apposing epithelia, expression of chondroitin sulfate proteoglycan on the apical surface of the MEE, and emergence of bulging or protruding cells, which were postulated to be critical for palatal adhesion and intercalation of apposing shelves, and for subsequent apoptosis (Gato et al., 2002; Taya et al., 1999; Tudela et al., 2002). Other studies have shown that TGF- 3 regulates expression of matrix metalloproteinases (MMPs), particularly MMP-13, which likely plays an important role in the remodeling of the basement membrane during epithelial fusion (Blavier et al., 2001). Recently, Dudas et al. (2004a) demonstrated that TGF- 3 signaling in the MEE is mediated predominantly by the TGF- type I receptor, ALK-5, which subsequently activates the intracellular signal transducer Smad2. In addition, it was recently reported that TGF- 3 signaling is capable of activating the LEF1 gene in the MEE (Nawshad and Hay, 2003). These authors demonstrated that the activation of LEF1 was Smad2-dependent, but did not involve -catenin. This is rather unexpected, because TCF/LEF1 transcription factors are usually activated by the canonical Wnt/ -catenin pathway, which is the only well-characterized signaling system involved in the induction of EMT at the time of this writing.

E. Epithelial–Mesenchymal Interactions, Interactive Signaling Pathways, and Morphogenesis of the Prefusion Palatal Shelves As outlined earlier, many details are now known about TGF- signaling and its biological role in the MEE. In addition, recent studies have started to address the complex but important questions of how TGF- signaling


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Figure 6 A model of signaling interactions during palatogenesis. A schematic representation of a transversal section through the palatal shelf. Light yellow, nasal (up) and oral (down) epithelium; strong yellow, medial edge epithelium; tan, palatal mesenchyme. TGF- signaling molecules are in green, BMPs in yellow, FGF signaling in orange. Green arrows represent stimulatory or upregulatory eVects; red pointers represent inhibitory eVects or negative regulation. (A) TGF- 3 knockout mice have cleft palate caused by a failure of fully grown palatal shelves to fuse, because the midline epithelium fails to disappear. ALK-5 is the canonical

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interacts with other indispensable signal transduction pathways and how the coordinated reciprocal interactions between the palatal epithelium and the underlying mesenchyme take place during palatal shelf growth, elevation, and fusion. These novel findings have been summarized in the schematic presentation in Fig. 6. Zhang et al. (2002) first showed that the bona fide TGF- –BMP eVector Msx1 is expressed in the palatal mesenchyme and controls a genetic hierarchy of BMPs and sonic hedgehog (Shh). These investigators proposed that in the anterior palate, Msx1 expression induced by BMP-4 is also required to maintain steady levels of BMP-4. Furthermore, BMP-4 is required to induce Shh expression in the anterior palatal epithelium, which in turn signals back to the mesenchyme to induce cell proliferation and palatal growth. Because Alk2/Wnt1-Cre mutants display defective palatal shelf elevation (Dudas et al., 2004b), it is conceivable that at least some of these mesenchymal BMP signals are mediated via ALK-2 (however, mechanical prevention of shelf elevation by reduced size of the oral cavity cannot be excluded, as discussed in the original paper). Rice et al. (2004) recently showed that also Fgf10 (expressed in the mesenchyme), which signals via Fgfr2b (expressed in the epithelium), is required in induction of epithelial expression of Shh and that this network, in conjunction with the BMP signaling described earlier, is needed both in growth and in appropriate morphogenesis (shaping) of palatal shelves. In addition, it was recently reported that Tgf- 3 / mice display high levels of TGF- 1 in the palatal mesenchyme (Martinez-Alvarez et al., 2004). This in turn was postulated to lead to aberrant epithelial expression of the zinc-finger transcriptional repressor Snail and subsequent promotion of cell survival in the MEE. Interestingly, NCC specific ALK-5 abrogation leads to severe facial clefting, including cleft palate, which underlines the importance of mesenchymal TGF- signaling in palatogenesis (M. Dudas and V. Kaartinen, unpublished results).

receptor for TGF- 3 that has been shown to mediate downstream signaling required for successful palatal fusion via Smad2 (Cui et al., 2003; Dudas et al., 2004a; Kaartinen et al., 1995, 1997; Proetzel et al., 1995). (B) It has been suggested that PI-3 kinase is one of the downstream eVectors of TGF- 3 signaling (Kang and Svoboda, 2002, 2003). (C) Snail is normally expressed in a small subgroup of midline epithelial cells during a physiological fusion. However, in the absence of TGF- 3, aberrant activation of epithelial Snail by pathologically elevated levels of TGF- 1 in the mesenchyme promotes cell survival (Martinez-Alvarez et al., 2004). (D) FGF-10 expressed in the palatal mesenchyme has been shown to induce sonic hedgehog (Shh) in the oral epithelium of the developing palate (Rice et al., 2004). Interactions between hedgehog and BMP-2 and BMP-4 signaling are intriguing and may represent important regulatory mechanisms for palatal development (Murray and Schutte, 2004; Rice et al., 2004). (E) Downstream hedgehog signaling through patched (Ptc), smoothened (Smo), and mammalian Gli probably contribute to upregulation of BMP-2 expression in the palatal mesenchyme (Lum and Beachy, 2004; Rice et al., 2004).


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IX. Clinical Research and Applications Germline mutations, somatic mutations, and polymorphisms of genes related to TGF- signaling lead to various diseases in humans. In addition to developmental malformations, a large portion of TGF- superfamily signaling research is devoted to cancer (Siegel and Massague, 2003; Waite and Eng, 2003). Some human ‘‘TGF- ’’ diseases have phenotypic features similar to those described in rodent knockout models, but many others are quite diVerent (Akhurst, 2004; Chang et al., 2002; Kosaki et al., 1999; Mizuguchi et al., 2004). These diVerences often prevent a direct application of research observations into treatment of human diseases. The most promising approach for the practical use in the near future is the delivery of individual signaling ligands or antagonists into tissue in order to induce or facilitate healing and regeneration, or to block excessive production of hard tissues.

A. Craniofacial Fracture Healing Bone morphogenetic proteins have been shown to play a role not only during skeletal development, but also in bone injury healing (Bostrom, 1998). BMP-2 is currently one of the most intensively studied regenerative proteins with the most promising properties. For example, a mesenchymal cell line from calvariae of newborn mice responds to BMP-2 by osteoblastic diVerentiation and was successfully used to repair experimental craniotomy defects (Kadowaki et al., 2004). Similarly, bone marrow cells induced with basic FGF (bFGF) and BMP-2 diVerentiate into mature bone and are capable to heal cranial defects in rats (Akita et al., 2004). Recently, BMP-7 was clinically used in a human patient to induce bone formation from the bone marrow, in order to repair a large defect in the mandible (Gronthos, 2004; Warnke et al., 2004). TGF- signaling is also involved in membranous bone healing and may become important in mandibular fracture repair (Steinbrech et al., 2000). TGF- s also have positive eVects on the joint cartilage—they can promote cartilage anabolism and osteochondrogenesis, and are currently being experimentally tested for the treatment of osteoarthrosis (Grimaud et al., 2002), which may have implications for diseases of the temporomandibular joint.

B. Prevention of Heterotopic Bone Formation BMP-signaling antagonists have been tested in animals for use in prevention of heterotopic ossification, which is a frequent complication in

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patients following head or neck traumas, or after bone transplantations (Weber et al., 2003). Another use for BMP antagonists would be in the prevention of the developmental defects of craniosynostoses—an idea based on recent findings that BMP signaling and BMP inhibitor Noggin are involved in the process of cranial suture ossification (Cohen, 2000a; Levine et al., 1998; Liu et al., 1999; Mavrogiannis et al., 2001; Satokata et al., 2000; Warren et al., 2003).

C. Teeth and Periodontal Regeneration A lot of enthusiasm has recently been expressed in the area of tooth biology after the identification of stem cells in the enamel organ epithelium, dental papilla, and dental pulp mesenchyme, and in late cap-stage and bell-stage tooth organs. Adult odontogenic stem cells are responsive to various biological, or even mechanical, diVerentiation stimuli and can potentially lead to new tooth repair technologies (Chai and Slavkin, 2003) or peridontal tissue regeneration. For example, human recombinant BMP-2 is able to promote osteogenesis as well as cementogenesis and is being investigated for the use in periodontal regeneration (King, 2001).

X. Conclusions During recent years, much has been learned about TGF- superfamily signaling in facial morphogenesis. In this progress, genetically manipulated mice in conjunction with new state-of-the-art methods of developmental, cell, and molecular biology have been critically important. Using these techniques, the function of individual components of specific TGF- signaling pathways in vivo can be analyzed and better understood. Future challenges will be in understanding the role of TGF- signaling in interactions between diVerent cell types, such as epithelial, mesenchymal, and neural crest cells, and particularly the relationship between TGF- and other morphogenetic signaling pathways.

Acknowledgments We thank S. Buckley for comments on the manuscript. This work was supported by CHLA RCDF Award (to M.D.), and the E. Schneider Foundation and the NIH grant DE13085 (to V.K.).


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The Colors of Autumn Leaves as Symptoms of Cellular Recycling and Defenses Against Environmental Stresses Helen J. Ougham, Phillip Morris, and Howard Thomas Institute of Grassland and Environmental Research Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, Wales, United Kingdom

I. Introduction II. The Role of Chlorophyll in Protein Recycling A. The Biochemistry of Chlorophyll Degradation in Senescing Leaves B. Genes and Genetic Variation for Chlorophyll Degradation C. Chlorophyll as a Regulator of Protein Metabolism in Senescing Cells D. Senescence in Relation to Programmed Death of Green Plant Cells III. Non-Green Pigments in Senescing Leaves A. Revelation of Autumn Colors B. Carotenoids C. Anthocyanins and Other Flavonoids IV. Pigments and Stress Defenses in Senescing Leaves A. Color Changes in Senescence as Signals B. Is Autumn Color a Costly Signal? C. Possible Functions of Leaf Color D. Does Dishonesty Pay? E. Insect Preference for Green Leaves F. Visual and Olfactory Signals V. Conclusions Acknowledgments References

The color changes that occur during foliar senescence are directly related to the regulation of nutrient mobilization and resorption from leaf cells, often under conditions of biotic and abiotic stress. Chlorophyll is degraded through a metabolic pathway that becomes specifically activated in senescence. Chlorophyll catabolic enzymes and genes have been identified and characterized and aspects of their regulation analyzed. Particular genetic interventions in the pathway lead to disruptions in protein mobilization and increased sensitivity to light-dependent cell damage and death. The chemistry and metabolism of carotenoid and anthocyanin pigments in senescing leaves are considered. Bright autumn colors observed in the foliage of some woody species have been hypothesized to act as a defense signal to potential insect herbivores. Critical consideration of the biochemical and physiological Current Topics in Developmental Biology, Vol. 66 Copyright 2005, Elsevier Inc. All rights reserved. 0070-2153/05 $35.00

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features of normal leaf senescence leads to the conclusion that accumulating or unmasking compounds with new colors are unlikely to represent a costly investment on the part of the tree. The influences of human evolutionary and social history on our own perception of autumn coloration are discussed. The possibility that insect herbivores may respond to volatiles emitted during leaf senescence, rather than to bright colors, is also presented. Finally, some new approaches to the analysis of protein recycling in senescence are briefly considered. C 2005, Elsevier Inc.

I. Introduction This discussion considers the significance of the striking changes in pigmentation that occur when green plant tissues undergo senescence. The metabolic events underlying the highly visible symptoms of senescence are directly concerned with the functional and structural transdiVerentiation (Thomas et al., 2003) of cells, from units with a primary assimilation role into centers of nutrient mobilization and recovery. Plants, as sessile organisms, experience nonoptimal environments as a way of life. A period of remodeling, such as occurs in senescence, is a potentially vulnerable time for tissues, organs, and the whole plant, and this is reflected in physiological changes accompanying the developing nutrient-recycling function that serve to defend against the intrusion of abiotic and biotic challenges. Here again, pigments are diagnostic of defenses against stress. In this review, we focus on two specific aspects of recycling and stress resistance: recent developments in understanding the molecular and cellular control of chlorophyll degradation, and autumn colors as potential signals in biotic interactions between plants and animals.

II. The Role of Chlorophyll in Protein Recycling A. The Biochemistry of Chlorophyll Degradation in Senescing Leaves Protein mobilization in senescence is regulated by a network of processes (Dangl et al., 2000; Ho¨ rtensteiner and Feller, 2002; Thomas and Donnison, 2000), among which the induction of chlorophyll degradation is an early and, for plastid membrane polypeptides, essential event (Thomas et al., 2002). Net loss of chlorophyll from green tissues during senescence and other terminal developmental events culminates in the accumulation of colorless products (nonfluorescent chlorophyll catabolites, or NCCs) (Mu¨ hlecker and Kra¨ utler, 1996). The enzymic pathway of NCC formation from chlorophyll (Ho¨ rtensteiner, 2004) commences with chlorophyllase, which dephytylates

4. Leaf Senescence, Nutrient Recycling, and Stress Defenses

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chlorophyll a. Magnesium (Mg) is removed from chlorophyllide a by a dechelatase activity. The tetrapyrrole macrocycle of the product of Mg removal is opened oxygenolytically by pheophorbide a oxygenase (PaO), producing a red bilin, RCC. A reductase immediately converts RCC into a colorless fluorescent product, FCC. Further enzymic and nonenzymic reactions metabolize FCC to NCCs in a species-specific manner (Ho¨rtensteiner and Feller, 2002; Thomas et al., 2001). Catabolites of chlorophyll b are not normally observed in senescing tissues, leading to the notion that there is interconversion between chlorophyll(ide) a and b and catabolism exclusively by the a-specific pathway. An enzymic activity capable of converting chlorophyllide b to a has been shown to become elevated during senescence (Scheumann et al., 1999). Terminal catabolites are sequestered in the cell vacuole. There is no evidence that the N of the chlorophyll ring is exported from the cell during senescence. Chlorophyll catabolism is summarized in Fig. 1.

B. Genes and Genetic Variation for Chlorophyll Degradation Genes for most of the steps in the chlorophyll catabolism pathway have been cloned (Gray et al., 2002; Jakob-Wilk et al., 1999; Pruzˇ inska´ et al., 2003; Tanaka et al., 2003; Tommasini et al., 1998; Tsuchiya et al., 1999; Wu¨ thrich et al., 2000). A number of mutations, genetic variants, and transgenics modifying chlorophyll catabolism have been described (Pruzˇ inska´ et al., 2003; Thomas and Howarth, 2000; Thomas et al., 2001). In general, they fall into two main categories: 1. Stay-greens are genetic variants in which the yellowing of senescing leaves is delayed, or slowed, or both, relative to comparable normal genotypes. Stay-greens have been diVerentiated in turn into two kinds—functional and cosmetic (Thomas and Smart, 1993). In functional stay-greens, the link between enhanced stability of chlorophyll, retention of photosynthetic capacity, and delayed protein mobilization is maintained. In cosmetic stay-greens, yellowing is disabled but photosynthetic rate usually declines over a similar time-course to normally senescing yellowing genotypes, and there is partial stabilization of protein. Section II.C discusses this further. 2. Photosensitive genotypes. As described in more detail in Section II.D, variants with deficiencies in particular steps of tetrapyrrole metabolism display pathological symptoms that often mimic disease lesions and are consistent with the accumulation of photodynamic intermediates upstream of the metabolic blockage (Ho¨ rtensteiner, 2004). Interestingly, in those cosmetic stay-greens in which the location of the metabolic

Figure 1 The chlorophyll degradation pathway. 1, Chlorophyllase; 2, magnesium dechelatase; 3, pheophorbide a oxygenase; 4, RCC reductase; 5, species-specific enzymic and nonenzymic conversion of FCC to nonfluorescent terminal catabolites.


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deficiency has been studied, the evidence points to disruption of one or the other step in the chlorophyll catabolic pathway but no corresponding increase in photosensitivity (Bachmann et al., 1994; Roca et al., 2004). Conversely, and contrary to expectation, photosensitive chlorophyll catabolism variants seem not to behave as cosmetic stay-greens when induced to senesce in the absence of light (Tanaka et al., 2003).

C. Chlorophyll as a Regulator of Protein Metabolism in Senescing Cells As long as the nitrogen requirements of growing tissues and organs can be met by uptake from the soil and assimilation in roots and leaves, senescence does not usually make a major contribution to the plant’s internal nitrogen cycle. However, if sink demand cannot be met by current assimilation— as may happen when development switches from the vegetative to reproductive phase, for example—nitrogen reserves become remobilized. First, lowmolecular-weight sources, such as free amino acids and vacuolar nitrate, are drained from the system, then polymers begin to be catabolized. Chloroplasts are protein storage bodies as well as photosynthetic organelles. The onset of senescence marks the functional transition of plastids from assimilation to remobilization, of which chlorophyll catabolism is the visible symptom. Yellowing and protein nitrogen remobilization are generally quite well correlated (Thomas et al., 2002). Genetic and environmental factors that interfere with chlorophyll degradation during senescence also modify protein degradation. For example, a mutant gene that confers cosmetic staygreenness in Festuca and Lolium species has the eVect of stabilizing chloroplast membrane proteins during senescence (Roca et al., 2004; Thomas et al., 2002). On the evidence of mutants and in vitro reconstitution experiments, pigment-binding proteins must be properly complexed with chlorophyll if they are to fold correctly, otherwise they are vulnerable to proteolytic attack (Thomas, 1997). Because pigment proteolipids have both a photosynthetic function and a role in thylakoid structure (Allen and Forsberg, 2001), stabilizing chlorophyll–protein complexes in senescence confers durability on chloroplast membranes and reduces the lability of membrane-associated components that are not themselves directly stabilized by chlorophyll, such as cytochrome f (Bachmann et al., 1994; Davies et al., 1990). Conversely, an analysis of the behavior of the light-harvesting chlorophyll complex of photosystem 2 during senescence of a stay-green Festuca mutant revealed that part of an otherwise deeply-buried thylakoid intrinsic protein that extends into the stroma may be subject to proteolysis, just like soluble-phase plastid proteins (Thomas and Howarth, 2000).

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D. Senescence in Relation to Programmed Death of Green Plant Cells In the present discussion, the term senescence is used in the specialized context of the controlled recovery of nutrients from green tissues and is associated with the transdiVerentiation of cells and organelles from centers of primary photoassimilation into remobilizing storage structures. Senescence, as the name implies, usually occurs at the end of the life of the leaf and is often classified as an aspect of programmed cell death (Dangl et al., 2000; van Doorn and Woltering, 2004). Nevertheless, senescence in the context of green cell transdiVerentiation has features that distinguish it from other cell death processes in a fundamental way. Particularly significant is its reversibility. In many, perhaps most, species it is possible to induce regreening of senescent leaves by interfering with source-sink or hormonal status, or both. Zavaleta-Mancera et al. (1999a,b) showed that during regreening of tobacco leaves, gerontoplasts were rediVerentiated into chloroplasts, senescence-enhanced genes and their products were turned oV, and components of the plastid assembly machinery were reactivated. Thomas et al. (2003) argued that the reversibility of senescence, among other characteristics, classifies the process as a diVerentiation event and not an aspect of programmed cell death. In some ways, it is unfortunate that history has left us with the term senescence to describe a phenomenon that, mechanistically, is better understood in developmental rather than deteriorative terms. It may be significant that scientists in the field of animal aging and cell death have moved away from employing the word senescence in recent years because of its imprecise, ambiguous associations (Gordon Lithgow, personal communication). We suggest that we accept the inappropriate etymology and choose to define senescence in our own pragmatic way (in the words of Humpty Dumpty, ‘‘When I use a word . . . it means just what I choose it to mean— neither more nor less.’’) (Carroll, 1872), thereby avoiding the distractions of semantics (van Doorn and Woltering, 2004). If senescence is accepted as being functionally distinct from, rather than a form of, programmed cell death, a fruitful area of study opens up concerned with the relationship between the two phenomena in plant development and survival. Ho¨ rtensteiner (2004) has expressed this most dramatically in terms of the obligate requirement for correct expression of the senescence syndrome to avoid the pathological consequences of cell death. In other words, programmed senescence and (programmed) cell death are mutually antagonistic. This has become increasingly clear from recent studies in which the molecular genetics of lesion formation and programmed cell death has converged with the identification and cloning of genes for the enzymes of the chlorophyll catabolism pathway. Mach et al. (2001) cloned the ACD2 (accelerated cell death 2) locus of Arabidopsis and found it to be identical with the gene encoding the chlorophyll catabolic enzyme RCC reductase


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(Wu¨ thrich et al., 2000). The knockout has a phenotype that takes the form of light-dependent spreading lesions. Subsequently, the ACD1 (accelerated cell death 1) gene of Arabidopsis, a mutation of which also gives a photosensitive cell death phenotype, was shown to encode PaO, the enzyme that opens the chlorophyll macrocycle during senescence (Pruzˇ inska´ et al., 2003; Tanaka et al., 2003). Abnormalities of tetrapyrrole metabolism are well known to lead to pathological photosensitivity, and not just in plants (Ho¨ rtensteiner, 2004). There remains the enigma of the contrasting senescence and lightresponse behavior of photosensitive mutants and cosmetic stay-greens as described earlier in Section II.B. It will be necessary to understand in much greater detail the mechanisms by which senescing leaves respond to light and other abiotic stresses, and the control mechanisms by which these stresses are resisted or avoided, before this paradox can be resolved.

III. Non-Green Pigments in Senescing Leaves A. Revelation of Autumn Colors Removal of chlorophyll is a defining feature of leaf senescence in all higher plant species. In contrast, the coloration remaining, after the chlorophyll has been catabolized and before tissue death, is much more variable, depending on both genetic background—diVerences can be found within as well as between species—and environmental factors, particularly stresses due to low temperature, high light, drought, and so on. Optical brighteners synthesized in senescing leaves can enhance the color contributed by other pigments; a striking example is the compound 6-hydroxykynurenic acid, which, by reinforcing carotenoid coloration, imparts the brilliant golden shade characteristic of senescent Ginkgo biloba leaves (Matile, 1994). However, the range of leaf colors from yellow through orange to red, pink, and purple is mainly due to two classes of compound: carotenoids and anthocyanins (Matile, 2000).

B. Carotenoids Leaf carotenoids are highly hydrophobic, and most are yellow or orange in color. The most abundant carotenoids in the chloroplast of a green leaf are typically beta-carotene and alpha-carotene, and the xanthophylls (oxygenated carotenoids) violaxanthin, neoxanthin, antheraxanthin, zeaxanthin, and lutein. The proportions vary with species, leaf age, and environmental conditions (reviewed in Biswal, 1995). In green mesophyll cells in the light, carotenoids function as accessory pigments in the photosynthetic apparatus

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and as protectants against photooxidative damage. During leaf senescence, disappearance of the chlorophyll reveals these previously masked compounds. Thus, in the many plant species that do not accumulate hydrophilic pigments such as anthocyanins, the senescing leaf appears yellow or orangish. Although red carotenoids do exist, they are more common in reproductive and dispersal structures, the best-known example being lycopene, which is synthesized in solanaceous fruits as they ripen. One unusual example of red carotenoids in tree leaves was observed by Hormaetxe et al. (2004), who found eschscholtzxanthin and derivatives in the foliage of box trees under photoinhibitory conditions encountered during winter acclimation. Like senescent chloroplasts (see Section II.D), and in contrast to fruit chromoplasts, the red plastids in these leaves are able to rediVerentiate to green chloroplasts when environmental conditions change; unlike many of the phenylpropanoid compounds described in the next section, the red carotenoids are not terminal metabolites. In general, remobilization of carotenoids during leaf senescence does not appear to be a consistently high priority for the plant. Being composed almost entirely of carbon and hydrogen (Fig. 2), they do not contain elements that are in short supply at this stage. Chlorophyll and its derivatives, the breakdown products of which are also not exported from the senescing leaf, present a potential hazard to leaf cells as photosynthesis declines and are therefore catabolized by the detoxification process described in Section II, but carotenoids represent no such threat. Rather, as antioxidants they may have a role to play in protecting against photooxidative damage during a vulnerable phase of the life of the leaf. For example, in leaves of the mastic tree Pistacia lentiscus, lutein and neaxanthin levels remained constant during the early stages of senescence (up to 20% chlorophyll loss) while beta-carotene levels increased by 9%; only once chlorophyll had largely disappeared did carotenoid levels decline (neoxanthin by approximately 20%; lutein and betacarotene by approximately 35%) (Munne-Bosch and Penuelas, 2003). Taken in conjunction with similar behavior by other antioxidant compounds, including alpha-tocopherol and ascorbate, the authors inferred a photoprotective role for carotenoids during the chlorophyll catabolism phase of leaf senescence. Similarly, Merzlyak and Gitelson (1995) considered that the retention of carotenoids responsible for the intense yellow color of Acer platanoides leaves in autumn was required for protection against blue light irradiation. Whatever the extent of their participation in protection against light damage, it is certainly the case that in many plant species, ranging from beech trees (Garcia-Plazaola and Becerril, 2001) to temperate grasses (Biswal et al., 1994), the disappearance of carotenoids is retarded relative to that of chlorophylls during leaf senescence (reviewed in Biswal, 1995). In green leaves, the carotenoids are almost exclusively localized in the thylakoid membranes, where they form part of the photosynthetic pigment–protein


Figure 2 leaves.

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Some of the carotenoid pigments that contribute to autumn colors in senescing

complexes. During senescence, they relocate mainly to the lipid-rich spherical bodies known as plastoglobuli, which are a striking feature of the plastids in senescent tissues (Steinmu¨ller and Tevini, 1985; Tevini and Steinmu¨ ller, 1985). In this respect, senescing leaf chloroplasts resemble the specialized chromoplasts of flower petals and fruit, in which plastoglobuli also often contain the carotenoid pigments. In addition to the change in cellular compartmentation, the complement of carotenoid compounds also often alters during leaf senescence. Frequently, the proportion of esterified carotenoids increases at the expense of unesterified forms (Biswal et al., 1994; Garcia-Plazaola and Becerril, 2001; Tevini and Steinmuller, 1985; Young et al., 1991). Some or all of the xanthophylls zeaxanthin, violaxanthin, antheraxanthin, and lutein often become more abundant relative to other carotenoids (Afitlhile et al., 1993; Garcia-Plazaola

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et al., 2003). Both esterification and changes in relative proportions of these pigments can result in subtle alterations to the yellow-orange coloration of leaves during senescence. Carotenoids, therefore, can undergo degradation, relocation, and chemical modification during leaf senescence, but there is little evidence for de novo synthesis of carotenoids in senescing leaves; genes encoding enzymes of the carotenoid biosynthesis pathway, such as geranyl-geranyl pyrophosphate synthase, phytoene synthase, and phytoene desaturase, are not reported among the wide range of genes whose expression is upregulated during leaf senescence (Andersson et al., 2004; Buchanan-Wollaston et al., 2003). It appears, therefore, that any contribution that carotenoids make to the colors of senescent leaves depends mainly on their preexistence in those leaves before the onset of senescence.

C. Anthocyanins and Other Flavonoids The major classes of flavonoid polyphenols contributing to the color of flowers, leaves, and fruits are the anthocyanins, flavonols, chalcones, and aurones (Fig. 3). The anthocyanins are the most widespread and recognizable group of plant pigments after chlorophyll, because these water-soluble compounds are responsible for nearly all of the red, pink, mauve, violet, blue, and purple colors in the petals, leaves, stems, and fruits of plants. Anthocyanins are present in nature as heterosides whose aglycone (or anthocyanidin) is a derivative of the flavylium ion. This combination with sugars is important in the case of flower pigments in providing solubility and stability to light and may be important in leaf development as a way of keeping potentially toxic metabolites in an inactive form within the cell. The three most common anthocyanidins are cyanidin (magenta), pelargonidin (orange-red, with one less hydroxyl group than cyanidin), and delphinidin (purple-blue, with one more hydroxyl than cyanidin). Ji et al. (1992) surveyed the leaf pigments of 119 taxa within the aceraceae and found that cyanidin and delphinidin glycosides accounted for most of the anthocyanins in the highly-colored autumnal foliage of these species. Cyanidin, pelagonidin, and delphinidin correspond to the three main flavonols (quercetin, kaempferol, and myricetin) in order of increasing B-ring hydroxylation. Three anthocyanidin methyl esters are also quite common, peonidin derived from cyanidin and petunidin and malvidin derived from delphinidin. Each of these anthocyanidins occurs in plants with various sugar attachments as a range of O-glycosides (anthocyanins) rather than as the aglycones (anthocyanidins). The main variation is in the type of sugar (glucose, galactose, rhamnose, xylose, or arabinose), the number of sugars (mono, di, or triglycosides) and the attachment of the sugar (usually at the 3 OH or the


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Figure 3 Generalized biosynthetic pathway of flavonoids leading to anthocyanin and related pigments in plants. Key enzymes: CHI, chalcone isomerase; CHS, chalcone synthase; DFR, dihydroflavonol reductase; F3OH, flavanone-3-hydroxylase; PAL, phenylalanine ammonia lyase.

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3-and 5-OH). Anthocyanins with acyl attachments are also quite common in some genera such as the solanaceae. The anthocyanins are therefore all based on the single aromatic structure of cyanidin (see Fig. 3), and their structural diversity is derived by modification of hydroxyl groups either by methylation or by glycosylation, or by acylation with diVerent organic acids. These compounds are nearly universal in vascular plants except for the centrospermae, where they are replaced by a chemically distinct group of pigments, the betacyanins, derived from the amino acid l-dopa, and typified by the major pigment of beetroot (Beta vulgaris). In summary, anthocyanin biosynthesis proceeds via p-coumaroyl-CoA, derived from l-phenylalanine in general phenylpropanoid metabolism, which enters a condensation reaction with three molecules of malonyl-CoA to form a C15 tetrahydroxychalcone intermediate. In the subsequent flavonoid pathway (see Fig. 3), this cyclizes to the corresponding flavanone and is hydroxylated and then reduced to produce the flavan-3-4-cis diol precursors of anthocyanins and also of condensed tannins. The flavan3-4-cis diols then undergo dehydration to form the flavylium cation, although the details of the latter steps of anthocyanin biosynthesis are still incomplete. Important regulatory steps in the metabolic sequence leading to anthocyanidin synthesis occur at phenylalanine ammonia lyase, chalcone synthase, and dihydroflavonol reductase. In general, these activities, and transcription of the genes that encode them, are sustained or even increased with organ age, but there seems to be no clear obligate relationship with leaf senescence. For example, Romero-Puertas and Delledonne (2003) have described how nitrous oxide delays leaf senescence but also activates the expression of phenylalanine ammonia lyase and chalcone synthase genes as part of a disease resistance/cell death mechanism. On the other hand, Kannangara and Hansson (1998) observed an interaction between anthocyanin and chlorophyll metabolism in young Euphorbia leaves, where several enzymes of chlorophyll biosynthesis sharply decreased in abundance at the onset of red anthocyanin accumulation. It would be interesting to know if a comparable metabolic relationship exists to enhance chlorophyll catabolism in leaves that turn red during senescence. In considering the metabolic costs of biosynthesising anthocyanins de novo, it should be kept in mind that the fluxes through the phenylpropanoid pathway do not need to be particularly large to result in a significant observable change. For example, even in Acer rubrum, which had the most intensely colored leaves analyzed by Lee et al. (2003), anthocyanins accounted for less than 6 g cm2; by contrast, chlorophyll in presenescent leaves of this species amounted to more than five times this value. The final step in anthocyanin biosynthesis is glycosylation, which has the eVect of stabilizing the molecule. Further modifications then include additional methylations, glycosylations, and acylations of hydroxyl groups to


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produce the great range of anthocyanin colours present in the plant kingdom. One of the largest monomeric anthocyanins known is the heavenly blue complex from the petals of Ipomoea tricolor (morning glory) and Commelina (Hondo et al., 1992). This has a molecular weight for the flavylium cation of 1759 (Goto et al., 1987), comprises peonidin with six molecules of glucose and three molecules of caVeic acid, and serves to illustrate the complexity that can arise in naturally occurring anthocyanins. Anthocyanins are mainly found in flower petals and in developing fruits, where they impart a broad spectrum of colors to these tissues. However, they may also accumulate in roots, leaves, bracts, seeds, and stems of both developing seedlings and mature plants. Anthocyanin accumulation is very sensitive to climatic conditions and is often associated with stress responses, particularly to low temperatures, and this is generally under tight genetic control, often mediated by MYB domain and basic helix-loop-helix transcription factors (Koes et al., 1994; Weisshaar and Jenkins, 1998). Anthocyanins accumulate in the cell vacuole (Alfenito et al., 1998), within which they are often located in spherical organelles known as anthocyanoplasts (Pecket and Small, 1980). There is a suggestive parallel here with aspects of chlorophyll metabolism in senescence, where conjugation and translocation of glycosides across the tonoplast is the ultimate metabolic fate, followed in some cases by nonenzymic chemical modifications within the acid milieu of the vacuolar sap (Thomas et al., 2001). The tissue distribution of anthocyanins in the autumnal foliage of a range of woody species has been comprehensively surveyed by Lee et al. (2003). With a few exceptions, in which the pigment was partly or entirely confined to cells of the adaxial epidermis (e.g., Acer spicatum, Euonymus atropurporeus, some Prunus spp.), anthocyanin concentrates in palisade parenchyma cells. This observation, taken with the complete absence of pigmentation from lower layers of mesophyll and the abaxial epidermis, is consistent with a function in light interception. Anthocyanins are compounds that readily alter their structures and, hence, color through the action of diVerent agents. The stability of anthocyanins increases with the number of methoxyl groups on the B ring and decreases with decreasing hydroxylation, and in general they are less labile at acid pH. In aqueous solution, anthocyanins are found equilibrated in four basic structures (the flavylium cation, quinonoidal base, carbinol base, and chalcone base), and the proportions of these forms (and hence the color) are determined principally by pH, with the red flavylium ion predominating in acid solution (Strack and Wray, 1989). At higher pH the color changes to anthocyanic forms that may be colored (bluish in the case of the quinonoidal base and yellow to orange for the chalcone base) or colorless (in the case of the carbinol base), depending on whether the A and B rings are conjugated. Hence, the relative amounts of the structural forms that coexist in equilibrium is a function of pH and the extent of addition of functional groups to the

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basic anthocyanin structure. In acid solution, the colors range from orangered (pelargonidin) through magenta (cyanidin) to mauve (delphinidin). If the pH is raised to near pH 7.0, solutions become colorless due to the formation of the pseudobase, and above pH 7.0 the bluer anhydrobases are formed, whereas at very high pH irreversible changes occur following ionization of the phenolic hydroxyls. Age-related acidification of the vacuole may well account for intensification of the red color of preexisting blue or leuko molecular species. Although glycosylation of anthocyanidins at C3 to produce anthocyanins results in a marked shift in color, the amount of anthocyanin present in the tissue, which can vary from 0.01% to 15.0% of dry weight, has a much more marked eVect. For example, in normal blue cornflowers the anthocyanin concentration is 0.05%, whereas in the deep purple varieties it is 13–14% (Goodwin and Mercer, 1972). Blueness of flowers can also be due to copigmentation between an anthocyanin and a flavonol. An example of this is in maroon and mauve Primula species, where the anthocyanin is malvidin-3glucoside in both cases, the diVerence in color being due to copigmentation with high concentrations of kempferol glucosides in the mauve variety. Spectral shifts due to copigmentation occur at pH values of 1–7, but these are not limited to polyphenolics and can also occur with purines and alkaloids, resulting in bathochromic shifts in the visible region of the adsorption spectra. Anthocyanins can also form complexes with several divalent or trivalent metals such as copper (Cu), aluminum (AI), or iron (Fe), leading to changes in color and varying as a function of pH. For example, Alþþþ ions bond with anthocyanins with ortho-dihydroxyl groups on the B ring, causing a bathochromic shift to give more blue coloration. This is best illustrated by comparing the blue color of cornflowers (Centaurea cyanus) with the red color of roses: the anthocyanin is cyanidin in both cases, but the blue color of cornflowers is due to a combined eVect of metal chelation with iron and copigmentation with apigenin diglycoside to form procyanin, a blue crystalline iron complex. It is also well known that if the mineral balance of Hydrangea species is correct, aluminum is easily accumulated and the petals turn blue, otherwise they are red. Many mineral elements, including Fe, are extensively mobilized from senescing leaves (Himelblau and Amasino, 2001), making it unlikely that concentration of metal ions by normal physiological mechanisms plays a part in color intensification, although toxic accumulation may be significant under some circumstances. The brown color in some petals (e.g., wallflowers) is due to a combination of the magenta anthocyanin in the vacuole with the yellow carotenoids in the chromoplast. The yellows and browns of autumn foliage occurring at the end of the senescence period are accounted for mainly by the presence of carotenoids (see Section III.B.), and by the formation of dark oxidation


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products of polyphenols such as condensed or hydrolyzable tannins as subcellular compartmentation collapses. Loss of anthocyanin pigments during this period may also occur by intramolecular rearrangements, resulting in the formation of the colorless pseudo-bases. A recently discovered fate for anthocyanins is via reduction to colorless 2,3-cis-flavan-3-ols (e.g., epicatechin), mediated by a newly discovered enzyme anthocyanin reductase, which has been proposed to be involved in synthesis of condensed tannins (Xie et al., 2003). The factors aVecting the determination of the color of plant tissues as a result of anthocyanin accumulation are therefore a combination of the extent of glycosylation and acylation, the pH in the vacuole, and the presence of metal ions and copigments such as flavonols and flavones (Mol et al., 1998). The perception of anthocyanin color in vivo can also be appreciably altered by cell structure. For example, Noda et al. (1994) showed that a transcription factor regulating the intensity of Antirrhinum flower color does so via control of cell shape. It is clear, therefore, that the progression of autumn colors shown by leaves of many trees may not be due solely to the loss of chlorophyll revealing the underlying colors of anthocyanins. Senescence-induced changes in vacuolar pH, increased or decreased levels of metal ions, the degradation of copigments such as flavonols and carotenoids, and the polymerization and oxidation of condensed and hydrolyzable tannins may well combine to produce the progression of color changes, from reds, oranges, yellows, and finally browns, independent of levels of de novo synthesis of anthocyanins. Evidence for de novo synthesis of anthocyanins in leaves during senescence is currently weak, and, in fact, in senescing leaves of the copper varieties of beech and hazel, senescence is preceded by the loss of anthocyanin so that, for a while, the foliage turns as green as in wild-type trees (Matile, 2000). Furthermore, although condensed tannins (or proanthocyanidins) give rise to anthocyanidins on acid hydrolysis, there is no evidence that this occurs in planta even during senescence. The wide range of potential modifications to which anthocyanins are subject cautions against assumptions that enhanced coloration in autumn must be the result solely of net synthesis of these compounds and therefore metabolically costly.

IV. Pigments and Stress Defenses in Senescing Leaves A. Color Changes in Senescence as Signals We have seen that the recycling function of leaf senescence is potentially vulnerable to disruption by light stress, and that pigment metabolism is normally organized and controlled in senescence to minimize photodamage. Moreover, the relationship between, on the one hand, pigment metabolism

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and, on the other, the formation of lesions mimicking the symptoms of fungal and bacterial disease, emphasizes the importance of senescence processes in plant responses to biotic stress. Developing the theme of pigments and biotic interactions further, the case of herbivorous predation on plant tissues illustrates the ecological significance of cellular events in foliar senescence. Archetti (2000) and Hamilton and Brown (2001) have elaborated the autumn signaling hypothesis, which raises interesting questions about the origins and functions of plant pigments and the way the animal eye responds to them. The hypothesis proposes that the autumn coloration observed in many tree species acts as an honest (handicap) signal to potential insect predators about the tree’s investment in defense—defensively committed and vigorous trees should produce the most intense coloration and, hence, the greatest deterrent to insects. It is suggested that the signaling mechanism in trees, and the insects’ avoidance response, are features that have coevolved. Although some subsequent publications have lent support to the hypothesis (e.g., Hagen et al., 2003), others have provided experimental evidence (Holopainen and Peltonen, 2002) or theoretical grounds (Wilkinson et al., 2002) to refute it. The majority of work in this area has so far been published by ecologists. Here we examine some aspects of the hypothesis and the results presented to date in the context of our knowledge about leaf senescence and plant physiology.

B. Is Autumn Color a Costly Signal? A concept widely used in the autumn signaling theory is that autumn coloration is costly for the tree to produce (and, by implication, must therefore have some definite purpose). This idea probably arises by analogy with animal metabolism, in which any biosynthetic process has requirements for both energy and carbon that must be met from food intake or by breaking down some of the animal’s own tissue to generate fuel and raw materials. However, plants are autotrophic, and, provided they are exposed to adequate light, air, and water, carbon and energy are not limiting factors as they are for heterotrophs. Indeed, it has been argued that terrestrial plants have evolved physically and physiologically to dump excess carbon captured through promiscuous assimilation (Thomas and Sadras, 2001). According to this proposition, the general overabundance of carbon and energy with which plants are cursed has resulted in proliferation of the huge range of carbon-rich secondary compounds that are unique to the plant kingdom (Hadacek, 2002; Pichersky and Gang, 2000) as a consequence of a kind of speculative metabolic doodling that occasionally pays oV in terms of improved fitness. In any event, it is questionable to assume that activities requiring carbon and energy are necessarily costly to a plant in the same


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sense that they would be for an animal or other heterotroph. This is particularly true of autumnal processes occurring in vegetation. Growth in plants is more sensitive to reduced temperature than is photosynthesis (Hjelm and Ogren, 2003); hence, as the temperature declines in autumn, the tree stops growing, further reducing the need for carbon and energy, which it can easily obtain again when required. The recycling function of autumnal senescence is part of a strategy to safeguard winter survival and resumption of growth in spring and culminates in the discarding of foliar skeletons consisting largely of carbon, oxygen, and hydrogen in various combinations. This throw-away residue may include two classes of pigment containing no elements of great reclamation value: carotenoids, which, as we have seen, can be unmasked, transformed, or relocated, but not in general synthesized de novo during senescence; and anthocyanins, which often are newly synthesized during the senescence process (Ishikura, 1972), although, as discussed, the argument that to do so is metabolically costly is at best questionable.

C. Possible Functions of Leaf Color The question as to why trees produce bright colors in autumn, if they are not an honest signal about defense capability, has been reviewed by Wilkinson et al. (2002). Briefly, plant biologists have two main hypotheses to explain the synthesis of anthocyanins. They may have a role in defense against abiotic stress (Steyn et al., 2002), protecting against potentially damaging forms of oxygen and chemical radicals. As photosynthesis declines during foliar senescence, light energy must be dissipated in alternative ways, some of which lead to the generation of reactive oxygen species (Feild et al., 2001). EYcient recapture of nutrients exported from the senescing leaf requires protection against photooxidative damage. Hoch et al. (2003) demonstrated the capacity of anthocyanins to facilitate nutrient recovery during leaf senescence in three deciduous woody species; Lee et al. (2003) similarly inferred a correlation between anthocyanin production and eYciency of nitrogen resorption in a number of deciduous forest species. It is noteworthy that many plant species also synthesize anthocyanins in the leaf in response to stresses such as cold, drought, or very high light intensity, when again carbon assimilation and demand are unmatched and there may be an increased risk of free radical production (Hoch et al., 2001). The coevolution theory suggests that vigorous and defensively committed trees can ‘‘aVord’’ the loss of photosynthate resulting from early senescence, whereas less vigorous trees need to continue photosynthesizing for longer—but, as pointed out earlier, during the autumn period it is not carbon and energy that are at a premium, but nitrogen and other nutrients. A tree lacking vigor because of nutrient deficiency or abiotic stress cannot make productive use

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of photosynthesis and is more, rather than less, likely to undergo early senescence. Interestingly, Schaberg et al. (2003) found that the extent and earliness of onset of red coloration in maple leaves was positively correlated with foliar nitrogen deficiency, an observation that contradicts the hypothesis that it is vigorous and healthy trees that initiate leaf senescence early but is entirely in accord with plant scientists’ observations of many species under many conditions. Other suggested functions of phenylpropanoid pigments in protecting against abiotic stresses include roles as antioxidants (Tsuda et al., 1994) and osmolytes (Chalker-Scott, 2002). An additional role not often considered is suggested by the striking fact that intense pigmentation is a characteristic of deciduous species. It may be that colored secondary compounds benefit the plant by contributing to the allelopathic properties of leaf litter (Wardle et al., 1998). Alternatively, the anthocyanins may simply represent a convenient dumping ground for excess carbon, in a form that is not metabolizable by, or attractive to, potential predators and pathogens. The fact that leaves are colored may be coincidental; our own evolutionary and social history has led human beings to attribute great significance to pigments in the wavelength range we can perceive, but a compound’s color may not have any particular correlation with its function (there is, for example, no particular reason why the human gall bladder needs to be green!). The comparative cell and molecular biology of foliar senescence supports the view that the senescing leaf is the evolutionary progenitor of brightly colored floral and reproductive structures attractive to animal pollinators and dispersers (Matile et al., 1999; Thomas et al., 2003). It follows that the heightened physiological and psychological sensitivity of humans to the colors of autumn foliage may not have any direct biological meaning. It may, rather, be a secondary consequence of spectral tuning by fruit and leaf color during evolution of the trichromatic primate visual system (Surridge et al., 2003). The connection between the colors of fruits and autumn leaves has been considered by Stiles (1982), who suggested that trees bearing colored fruit in fall may have evolved synchronization between fruit ripening and leaf coloration as an additional signal to seed-dispersing birds.

D. Does Dishonesty Pay? Even if the red coloration is not costly to produce, could the signaling hypothesis still hold good? Subsequent authors (Lachmann et al., 2001; Wilkinson et al., 2002) have pointed out that it is not essential that an honest signal be costly provided that dishonesty is penalized. In the case of autumn colors, dishonesty would consist in a poorly defended tree producing bright colors as a misleading deterrent. However, as indicated earlier, it is precisely


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stressed, and therefore generally poorly defended, trees that do produce more of the bright autumn colors. If the coloration were indeed a deterrent, far from being a costly misdirection, it could in fact act in the tree’s favor. Therefore, neither is the ‘‘signal’’ costly to produce, nor does its dishonest production penalize the tree. However, an alternative explanation should also be considered. The signaling theory would still be valid if trees that are poor in nutrients in the autumn subsequently defend their (limited) resources more heavily in the following spring, compared to ‘‘richer’’ individuals that may simply outgrow their pests. In this scenario, the signal becomes an honest and unfakeable indicator of resources, since early bright leaves represent low resources. If, for a given plant species, low nonrenewable nutrient resource is associated with increased defenses in spring, then insects would be expected to make appropriate evasive action in autumn. Future experimental work on the relationships between resource status and leaf coloration in autumn, insect responses, and defensive commitment the following spring will be necessary to clarify this issue.

E. Insect Preference for Green Leaves Moving from the reason why trees develop the coloration in the first place to a consideration of the reasons why insect predators avoid brightly colored leaves, the composition of these leaves in comparison with green foliage should be taken into account. By the time a leaf is orange or red, it will have broken down and exported a high proportion of its total protein. Photosynthesis will have ceased, remaining low-molecular-weight carbohydrates will have been removed, and, in general, it will have a much lower content of nutrients that an insect could digest than would a green leaf on the same tree. Anthocyanins that have accumulated will not be digestible by insects; they, or other, colorless, secondary products accumulating at the same time, may even be unpalatable and act as antifeedants. Furthermore, antinutritional factors such as inhibitors of digestive tract proteases are prominent among the genes and gene products upregulated in senescence and cell death (e.g., Huang et al., 2001). It is therefore quite feasible that insects initially land on green and red/yellow leaves in equal numbers but quickly vacate the latter after an initial sampling—to assess this possibility would require more detailed observation of insect behavior than has been presented in any of the studies to date. Alternatively, the predators may indeed be responding to the visual signal, but its significance is not ‘‘this is a vigorous tree able to withstand your attack’’ but simply ‘‘this is a leaf with poor nutritional quality, possibly unpalatable.’’ Wilkinson et al. (2002) point out that because an individual tree may simultaneously bear green, yellow, and red leaves, the hypothesis that leaf color signals the overall vigor or defensive

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capability of the tree is suspect. However, it could feasibly signal the nutritional value of the individual leaf; because studies to date (Hagen et al., 2003; Holopainen and Peltonen, 2002) have measured insect colonization or insect damage relative to the mean proportion of brightly colored leaves on a tree, rather than on an individual leaf basis, the question remains open. To complicate the issue further, Holopainen and Peltonen (2002) point out that birch aphids preferentially land on yellow rather than on green or red leaves, and propose that such leaves, which are actively exporting lowmolecular-weight nitrogenous compounds such as amino acids during early senescence, are a rich source of accessible nutrient for the insects. A diYculty in interpreting the data on insect behavior as a whole is that most studies (e.g., Hagen et al., 2003) did not look at red and yellow leaves separately; it may indeed be the case that some insect species are attracted to yellowing leaves more than green leaves, but to red leaves least of all. This possibility would resolve some of the inconsistencies in the story so far, including the fact pointed out by Wilkinson et al. (2002) that yellow is normally an attractive color to aphids. An interesting perspective on this issue is provided by the common observation that the proportion of infertile individuals in a plant population increases under stress. In species with strongly expressed monocarpic or reproduction-associated senescence patterns, barren plants may remain green while fertile individuals degrade their chlorophyll normally. Such barren plants have been reported to benefit the population by acting as decoys, reducing herbivory pressures on individuals destined to produce the next generation (Thomas and Sadras, 2001).

F. Visual and Olfactory Signals The color changes observed in tree leaves in autumn can be so spectacular that it is easy for humans to overlook the possibility that the predators may be responding to diVerent signals entirely, arising from other biochemical changes that the tree may be undergoing at the same time. It is possible that the insects are deterred not by color at all, but by the volatile substances emanating from plant leaves during senescence. Most land plants emit significant amounts of volatiles such as aldehydes and isoprenes during natural or wound-induced senescence (de Gouw et al., 1999; Fall et al., 1999), and it is known that insects can perceive and respond to quite low concentrations of these compounds (Ruther et al., 2002). There is also evidence that the volatiles may act as defense agents by attracting insect parasitoids (Hoballah and Turlings, 2001). Peak emission of such volatiles would be expected to coincide with the phase when the leaf was yellow or red but still alive. It would be informative to measure volatile emissions during the color change period and correlate the results with levels of insect colonization.


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V. Conclusions In recent years, a fairly complete picture has emerged of the metabolic molecular and subcellular networks responsible for pigmentation changes during the growth and senescence of foliage. This has allowed the development of a model of leaf senescence in which pigmentation changes partially set the pace for proteolysis and nitrogen recycling at the same time as they play a critical role in sustaining cell viability under conditions of abiotic and biotic challenge. Although rapid progress has been made in understanding pigment metabolism in senescence, the mechanism of protein degradation and its control remains poorly understood. The interconversions and relocations of the amino acid products of proteolysis in leaf tissues are quite well established (Dangl et al., 2000), but the step between the intact protein and its hydrolysis products continues to elude definitive analysis. Many senescenceassociated and upregulated protease genes have been described (Bhalerao et al., 2003; Buchanan-Wollaston, 1997; Buchanan-Wollaston et al., 2003), but it is unclear how many, if any at all, are necessary for normal protein breakdown. Some new developments may help to introduce much-needed innovative ideas into the field of proteolysis and its control in senescence. Improved microscopy techniques are beginning to provide evidence for traYc between plastids (which contain most of the mobilizable protein in senescing cells) and vacuoles, which have long been considered to be the main sites of intracellular proteolysis (Chiba et al., 2003; Guiame´ t et al., 1999). Cascades of caspase proteases are characteristic of programmed cell death in animal systems, but plant genomes seem not to include orthologues of caspase genes; nevertheless, recent sequence searches and functional analyses have revealed families of so-called metacaspases in plants that may fulfil various signaling and proteolytic roles in terminal and pathological plant processes, including senescence (Watanabe and Lam, 2004). Another potentially fruitful development is the application of quantitative trait mapping to test the relative map positions of genetic loci for, on the one hand, nitrogen assimilation and reallocation traits in crop development and, on the other, protease enzyme activities and gene sequences (Andreas Fischer, unpublished results). Much work needs to be done before protein recycling in plant senescence could be said to be a well-understood process, but new tools and approaches are being applied and rapid progress can be expected in the near future.

Acknowledgments The authors’ research on pigment metabolism and leaf senescence is supported by the UK Biotechnology and Biological Sciences Research Council and the Department of Environment, Food, and Rural AVairs.

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Extracellular Proteases: Biological and Behavioral Roles in the Mammalian Central Nervous System Yan Zhang, Kostas Pothakos, and Styliana-Anna (Stella) Tsirka Department of Pharmacological Sciences State University of New York at Stony Brook Stony Brook, New York 11794-8651

I. Introduction II. Plasminogen Activators A. Tissue-Type Plasminogen Activator B. Urokinase-Type Plasminogen Activator III. IV. V. VI. VII.

Plasmin(ogen) Inhibitors of Plasminogen Activators and Plasmin tPA EVects on Rodent Behavior uPA and Plasminogen EVects on Rodent Behavior Conclusions Acknowledgments References

Extracellular proteases and their inhibitors have been implicated in both physiological and pathological states in the central nervous system (CNS). Given the presence of several classes of proteases, it is believed that each enzyme may undertake distinct biological roles. Some are indispensable for neuronal migration, neurite outgrowth and pathfinding, and synaptic plasticity. Others are required for neuronal death and tumor growth and invasion. Furthermore, studies from transgenic animals lacking or overexpressing one or more of the proteases have suggested that functional compensation and redundance among diVerent members do exist. Normally, protease activity is tightly regulated by specific inhibitors to prevent disastrous proteolysis. Various insults can disrupt the fine control of proteolysis and cause pathological changes. Novel strategies have been attempted to maintain or restore proteaseinhibitor homeostasis, thus minimizing damages to the CNS. They may provide us with eVective therapeutic tools for fighting certain neurological disorders. C 2005, Elsevier Inc.


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I. Introduction There are several major classes of proteases present in the CNS, which are grouped according to diVerences in the composition of their catalytic sites. These are the families of (1) serine proteases, with the most well-known members such as thrombin, tissue-type plasminogen activator (tPA), and plasmin; (2) matrix metalloproteinases (MMPs), which consist of more than 20 members identified to date that all require Zn2þ for their enzymatic activity; (3) cysteine proteases, which include 14 caspases involved in distinct steps of the apoptotic pathway; and (4) aspartic proteases, such as the lysosomal peptidases cathepsins. Proteases are considered to be key players in the maintenance of CNS homeostasis. They are implicated in almost every aspect of normal developmental processes, such as cell proliferation, cell migration, apoptosis, axonal growth, and synaptogenesis. They also contribute to a wide variety of neuropathology, including neurodegenerative diseases (e.g., Alzheimer’s disease, Parkinson’s disease), demyelination diseases (e.g., multiple sclerosis), brain tumors, and cerebral ischemia. For each family of proteases, there exist specific endogenous inhibitors whose basic function is to restrain protease activities. Imbalance between the strength of proteases and their inhibitors compromises neural homeostasis and results in neuropathological changes. In this chapter, we provide an overview of recent research progresses on plasminogen activators, plasmin(ogen), and their inhibitors in the CNS with specific emphasis on (1) their unique roles in neurophysiology and behavior, (2) factors that regulate their functions, and (3) what is known about the potential mechanisms through which these proteases and their inhibitors contribute to several CNS diseases.

II. Plasminogen Activators Plasminogen activators (PAs) are best known as thrombolytics, because they dissolve blood clots in the vasculature. They specifically cleave the Arg–Val bond in the zymogen plasminogen to generate the active protease plasmin. Plasmin is then able to digest the fibrin polymers in the blood clots. In the last few decades, with the generation of gene-targeted mice, deficient in one or more components of the fibrinolytic system (Bugge et al., 1995a,b, 1996; Carmeliet et al., 1994; Dewerchin et al., 1996; Ploplis et al., 1995), a rapidly growing list of cellular functions outside of the bloodstream has been attributed to these proteins, especially in the CNS. For example, in addition to its traditional substrate fibrin, plasmin has a wide spectrum of substrates, including most of the extracellular matrix (ECM) proteins in the CNS, such as fibronectin and laminin. Therefore, activation of plasminogen by PAs serves to initiate a potent proteolytic cascade leading to ECM degradation

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and remodeling. There are two types of immunologically distinct PAs, tPA, and urokinase-type plasminogen activator (uPA). They are encoded by diVerent genes and share similar enzymology, but diVer in their domain organization and properties of the noncatalytical regions (Vassalli et al., 1991). It has been shown by others and us that tPA is the predominant PA constitutively expressed in normal CNS (Carroll et al., 1994; Qian, 1993; Sappino et al., 1993; Tsirka et al., 1997; Ware et al., 1995), whereas uPA in the CNS may be more relevant in the context of brain tumor biology (Levicar et al., 2003; Mohanam et al., 1994).

A. Tissue-Type Plasminogen Activator tPA belongs to the serine protease family of the ECM proteases. It has a catalytic site consisting of a serine, histidine, and an aspartic acid residue, and uses the serine residue for nucleophilic catalysis. Structurally, tPA starts with an N-terminal finger domain homologous to the fibrin-binding fingers of fibronectin, followed by a domain homologous to the epidermal growth factor domain (EGF) and two triple-disulfide structures called kringle domains, and ends with a C-terminal catalytic domain homologous to trypsin-like proteases (Dobrovolsky and Titaeva, 2002) (Fig. 1). The presence of multiple noncatalytic domains of tPA is critical in mediating

Figure 1 Domain structure of tPA, uPA, and plasminogen. tPA consists of the finger-like domain of fibronectin, epidermal growth factor domain, two kringles, and the catalytic domain. uPA consists of the epidermal growth factor domain, followed by the kringle and catalytic domains. Plasminogen consists of five kringle domains and the catalytic domain. Human plasminogen is cleaved at the Arg561–Val562 peptide bond by tPA or uPA to generate the active protease plasmin.

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protein–protein and cell–cell interactions. In the mature brain parenchyma, tPA is produced by neurons and microglia with the most prominent expression in the hippocampus and hypothalamus (Carroll et al., 1994; Qian, 1993; Sappino et al., 1993; Tsirka et al., 1997; Ware et al., 1995); it is stored intracellularly and can be secreted upon membrane depolarization (Gualandris et al., 1996). tPA has been implicated in normal CNS physiology, such as neuronal migration, long-term potentiation associated with learning and memory, and neurite outgrowth. In 1981, Krystosek and Seeds first reported the implication of t-PA in neurite growth using diVerentiated lines of neuroblastoma cells. A fibrin overlay assay revealed that the predominant site of t-PA activity was on the growth cones. During embryogenesis, increased t-PA expression coincides with extensive cell migration, proliferation, and tissue remodeling in the CNS (Friedman and Seeds, 1995). The generation of mice in which the tPA gene (tPA/) was rendered nonfunctional has opened the door to new studies on the tPA-dependent physiological and pathological events in the CNS (Carmeliet et al., 1994). tPA/ mice showed delayed migration of cerebral granule neurons in the developing cerebellum (Seeds et al., 1999). Later, when these mice reached adulthood, they exhibited significantly impaired cerebellar motor learning similar to that caused by specific tPA inhibitors such as endogenous plasminogen activator inhibitor (PAI), type 1 plasminogen activator inhibitor (PAI-1), or a synthetic t-PA inhibitor, t-PA stop (Seeds et al., 2003). tPA/ mice also displayed a selective reduction in the late phase of the phenomenon of long-term potentiation (Huang et al., 1996). Similarly, the administration of inhibitors of t-PA proteolytic activity inhibits the long-term potentiation induced in the mossy fibers of the hippocampus (Baranes et al., 1998). A recent report from our group described the direct evidence of tPA’s involvement in hippocampal mossy fiber outgrowth in vivo, a commonly used animal model for human temporal lobe epilepsy (Wu et al., 2000). tPA/ mice presented with decreased and disarrayed sprouts, due to the lack of processing of an ECM protein called DSD-1-PG/phosphacan. This protein is a potent regulator of neurite outgrowth in the CNS. The accumulation of uncleaved DSD-1-PG/ phosphacan in tPA/ mice interferes with the appropriate neurite extension and termination. In a PC12 cell culture model, it was demonstrated that the expression level of neuroserpin, a serine protease inhibitor widely expressed in developing and mature brain, inversely correlates with the number and length of neurites extending from the cells upon growth factor treatment (Parmar et al., 2002). In summary, the available evidence indicates that t-PA is central to the regulation of neuroplasticity in both the developing and adult brain. The actions of tPA could be aVecting several pathways, such as degradation of matrix and cell adhesions via the plasmin–metalloproteases cascade to facilitate movement, activation of a cell-signaling event via


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ligand–receptor binding, activation of pro-growth factors, liberation of matrix-associated cytokines, and promotion of cell adhesion mediated via PA–PAI interactions and integrin (Seeds et al., 1997). On the other hand, exaggerated tPA activity contributes to pathological processes such as neurodegeneration and inflammation (Siao and Tsirka, 2002b). Expression of tPA is quickly upregulated in the CNS in response to excitotoxic insults that mimic several clinical conditions, including stroke, Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis. tPA/ mice are more resistant to excitotoxin-induced neuronal death in the hippocampus (Tsirka et al., 1995), and the neuronal sensitivity can be restored when exogenous tPA protein is infused back into the brains of these mice prior to the delivery of excitotoxin (Tsirka et al., 1997). Plasminogen-deficient (plg/) mice exhibit resistance to excitotoxicity similar to that of tPA/ mice (Tsirka et al., 1997), suggesting that the tPA–plasmin proteolytic cascade promotes neuronal death acutely. Destruction of the supportive ECM substratum appears as one potential mechanism, because plasmin has been shown to degrade ECM laminin in a time-dependent manner preceding neuronal death (Chen and Strickland, 1997) (Fig. 2). Another mechanism is proposed as tPA potentiates signaling through glutamatergic receptors by proteolytically regulating the function of N-methyl-D-aspartate (NMDA) receptor and thus increasing calcium influx (Nicole et al., 2001) (see Fig. 2). tPA-mediated neurodegeneration also occurs through mechanisms independent of its proteolytic activity, such as through microglial activation (Tsirka, 2002). Microglia are CNS immunocompetent cells of the monocyte lineage. Upon insults, they are stimulated from their usual ramified-shaped resting state to undergo the process of activation (Giulian and Baker, 1986). The state of microglial activation is presented by morphological changes to ameboid shape, cell migration toward injury site followed by local proliferation, and upregulation of gene expression, all of which lead to increased capacity of antigen presentation and phagocytosis. Activated microglia increase the production of cytokines, proteases, reactive oxygen species, and nitric oxide (Kreutzberg, 1996; Lipton and Rosenberg, 1994). Blocking excessive microglial activation can confer protection against neurotoxicity in diVerent injury models (Rogove and Tsirka, 1998; Thanos et al., 1993), suggesting that overly activated microglia are detrimental to neuronal wellbeing. tPA/ mice exhibit attenuated microglial activation in response to excitotoxic stimuli (Tsirka et al., 1997); lipopolysaccharide-induced activation of cultured tPA/ microglia is dampened as well (Siao and Tsirka, 2002a). However, microglial activation in plg/ mice is comparable to that of wild-type animals (Tsirka et al., 1997). In addition, catalytically inactive tPA (S478A tPA, recombinant human tPA with a S478 ) A478 mutation at the active site) can activate microglia just as the wild-type protein does,

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Figure 2 Pro-survival and anti-survival properties of tPA. tPA modulates neuronal well-being via distinct but intercalated pathways. It promotes neuronal survival via adequate microglial activation, and counteracting zinc toxicity. Meanwhile, it precipitates neuronal death via disruption of ECM substratum, potentiation of glutamatergic toxicity, and exaggerated microglial activation. Balance between the two opposing forces determines the final fate of neurons.

indicating that tPA activates microglia via a nonproteolytic mechanism (Rogove et al., 1999). It turned out that tPA mediates microglial activation via its finger domain through interaction with annexin II on the microglial cell surface (Siao and Tsirka, 2002a). How this interaction is translated into intracellular signaling events is currently under intensive investigation. Because tPA comes from both neurons and microglia, one important issue is to assess how tPA produced from distinct cellular origins is coordinated in the context of microglial activation and neurodegeneration. By introducing


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tPA transgenes under the control of neuronal- or microglial-specific promoters into tPA/ mice, Siao et al. (2003) compared the outcome of excitotoxic damage in transgenic mice with selective tPA expression in neurons or microglia only. The neuronal tPA-expressing mice exhibit accelerated microglial activation but slower progression of neuronal death, whereas microglial tPA-expressing mice exhibit greater neurodegeneration. Based on these findings, a working hypothesis was put forward that tPA, initially released from injured neurons, acts as a cytokine to activate microglia at the site of injury, perhaps with the intention to restrain and clear tissue damage. These activated microglia then secrete additional tPA, which promotes microglial activation and ECM degradation beyond controllable/ regulatable level, and eventually promotes neurodegeneration (Siao et al., 2003) (see Fig. 2). Surprisingly, the story between tPA and neuronal integrity did not end with tPA’s characterization as the accomplice of neuron killing. Using a cell culture system, Kim et al. (1999) reported a neuroprotective role of tPA against zinc-induced toxicity through a nonproteolytic mechanism. Later, this eVect was confirmed in vivo as well (Siddiq and Tsirka, 2004). Zinc is an abundant trace element in the CNS, required by many proteins for their normal functions (such as MMPs). It exists in two interconvertible states, a protein-bound form and a free form. High levels of free zinc can cause neurotoxicity via modulating postsynaptic glutamate receptors (Choi and Koh, 1998), which is similar to that caused by excitotoxins (Olney, 1986). Despite the fact that tPA and zinc both contribute to neurodegeneration when acting separately, they attenuate each other’s toxicity when working together (Kim et al., 1999; Siddiq and Tsirka, 2004). Further exploration of this complex relationship was attempted (Siddiq and Tsirka, 2004). Zinc has been shown to inhibit tPA’s enzymatic activity. On the other hand, tPA can prevent the accumulation of extracellular free zinc by two ways. When zinc concentration is still low, tPA can bind to it and counteract its toxicity. When there is not enough tPA to buVer the rising level of zinc, tPA then facilitates zinc import into the cells, where zinc can be sequestered, although little is known about the intracellular destination of zinc. Considering the coexistence of tPA and zinc in both normal and diseased brains, there seems to be a point at which the balance between the two eVectors of cell death is finely tuned to optimize neuronal survival. Approaches to keep this delicate balance intact or restore its normal function may be of great benefit in preventing pathological complications after CNS injury (see Fig. 2). Recently, one plasminogen activator isolated from the saliva of vampire bat Desmodus rotundus (DSPA1) (Kratzschmar et al., 1991), which shares over 72% amino acid sequence identity with human tPA, was found to be free of neurotoxicity while retaining full strength of fibrinolytic capacity

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(Liberatore et al., 2003). When DSPA1 was intracerebrally infused into tPA/ mice, no excitotoxin-induced neurodegeneration was observed as seen in tPA-infused animals, even when DSPA1 was used at 10-fold the concentration of infused tPA. DSPA1 did not restore microglial activation in tPA/ mice either. In addition, DSPA1 did not exacerbate NMDAmediated neuronal death in wild-type mice like tPA did. The unique property of DSPA1 is likely due to its peculiar dependence on fibrin (Bringmann et al., 1995; Toschi et al., 1998). Its catalytic eYciency increases 102,000-fold in the presence of fibrin yet only 8-fold by fibrinogen, whereas the catalytic eYciency of tPA is specifically enhanced only 72-fold by fibrin (Bringmann et al., 1995). Therefore, DSPA1 will be specifically activated at the site of fibrin deposition without causing generalized proteolytic tissue damage. Because of this major advantage, there is an ongoing clinical trial in Europe using DSPA1 in patients suVering from acute cerebral ischemia (Liberatore et al., 2003).

B. Urokinase-Type Plasminogen Activator uPA is a trypsin-like protease first isolated from the urine. It exists either in a 54-kD single-chain form or as a two-chained protein linked by an interchain disulfide bond. The domain structure of uPA is quite similar to that of tPA, consisting of an N-terminal EGF-like domain followed by a kringle domain, and a C-terminal domain homologous to trypsin-like proteases (Dobrovolsky and Titaeva, 2002) (see Fig. 1). Histidine, aspartic acid, and serine residues form the catalytic site of uPA. Nascently synthesized and secreted uPA is a proenzyme with little or no activity, which can be activated after being cleaved by various proteases, including its immediate substrate plasmin, thereby generating a positive feedback loop of selfactivation (Levicar et al., 2003). In contrast to the ambiguous identity of a cell surface-binding partner for tPA, it has been clearly shown that a specific receptor for uPA (uPAR) is expressed on the surface of many cell types (Roldan et al., 1990). uPAR is a 65-kD glycoprotein consisting of three homologous extracellular domains (D1–3) and is covalently bound to the cell membrane at the C-terminus via a glycosylphosphatidylinositol (GPI) anchor (Mondino et al., 1999). The uPA–uPAR interaction occurs between the EGF-like domain of uPA and the N-terminus of the receptor with high specificity and aYnity. This ligand–receptor binding induces conformational changes in uPAR, which is then able to interact with integrins and ECM protein vitronectin. Therefore, uPA–uPAR interaction results in the formation of a multiprotein complex that facilitates local proteolysis, cell adhesion, and migration. Considering the necessity of cell movement and tissue invasion during tumor progression, it is not surprising to see that uPA–uPAR is closely associated with the growth and dissemination of


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various tumors, including those that originate in the brain (Mohanam et al., 1994; Schmitt et al., 1997). It was first noted that mRNA and protein levels of uPA and uPAR are dramatically elevated in malignant brain tumors compared with normal brain tissues and low-grade tumors (Caccamo et al., 1994; Kinder et al., 1993; Landau et al., 1994; Yamamoto et al., 1994a). The expression is localized in tumor cells and in endothelial cells of the surrounding neovasculature. In addition, higher expression is consistently present at the invasive edge of tumors. Later, a positive correlation was observed between uPA–uPAR expression levels and tumor invasiveness and recurrence, which are associated with worse prognosis (Hsu et al., 1995; Zhang et al., 2000). The causal relationship between high uPA–uPAR level and tumor invasiveness was confirmed in culture using several brain tumor cell lines of diVerent grades (MacDonald et al., 1998). The cells were evaluated for surface uPAR expression, endogenous uPA activity, and capacity to degrade ECM judged by migration on Transwell membranes and invasion of Matrigel. High levels of uPAR and uPA activity correlate with cellular degradation of ECM, cell migration, and Matrigel invasion. Cell migration and invasion were enhanced by exogenously added uPA in a dose-dependent manner. These eVects can be abolished by disruption of uPA–uPAR interaction at the cell surface with removal of membrane-bound uPAR. A working model was therefore formed, because uPA–uPAR binding on the surface of malignant cells is directly involved in the activation of ECM proteolytic cascades responsible for the invasiveness of those cells. Thereafter, blocking uPA–uPAR interaction is being actively pursued as a novel strategy to inhibit growth and spread of malignant brain tumors that are refractory to conventional therapies (Reuning et al., 2003). Downregulation of uPAR levels by antisense RNA inhibits glioblastoma cell migration and invasion in vitro (Mohan et al., 1999; Mohanam et al., 1997); it also reduces glioblastoma formation and causes regression of preexisting tumor in vivo (Mohan et al., 1999). Ligands that specifically target the overexpressed uPAR on glioblastoma multiforme can cause regression in tumor growth by blocking angiogenesis, decreasing tumor cell proliferation, and increasing tumor cell apoptosis (Bu et al., 2004; Mohanam et al., 2002; Rustamzadeh et al., 2003). These seemingly promising strategies need yet to be tested in a larger scale of animals and examined for their long-term eYcacy and side eVects. Given the fact that uPA and uPAR are synthesized by many cell types throughout the body and are critical in maintaining ECM turnover, it is worrisome that nonspecific interruption of their physiological functions might cause harmful consequences. In addition, information about tumor behaviors in mice deficient in uPA, uPAR, or both is not yet available; a thorough evaluation of these knockout mice will enrich our knowledge of the uPA–uPAR system in tumorigenesis and may disclose compensatory mechanisms in promoting tumor growth, invasion, and metastasis in the absence of uPA and uPAR.

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III. Plasmin(ogen) Plasminogen is the precursor of serine protease plasmin. It is a 92-kD glycoprotein consisting of seven structural domains: a N-terminal preactivation peptide, five kringle domains, and a C-terminal catalytic domain (see Fig. 1). The multiple kringle domains are important for substrate recognition, membrane association, and inhibitor binding (Syrovets and Simmet, 2004). Plasminogen can be activated by both tPA and uPA, as well as by kallikrein and several coagulation factors. The activation of plasminogen involves the cleavage of the Arg561–Val562 peptide bond, generating plasmin in the form of a two-chain protein linked by a disulfide bond (Dobrovolsky and Titaeva, 2002). In contrast to the dual cellular sources of tPA in the CNS, plasminogen is exclusively synthesized by neurons, with high expression in the hippocampus, cerebellum, and cerebral cortex (Sappino et al., 1993; Tsirka et al., 1997; Zhang et al., 2002a). The separate origins of tPA and plasminogen are considered to be a self-protecting mechanism to avoid intracellular proteolysis (Sandgren et al., 1991; Tsirka et al., 1997). Plasmin has a broad spectrum of substrates, including the ECM components laminin and fibronectin. More importantly, plasmin also activates MMPs, which can degrade other ECM proteins. Therefore, the two protease systems work in concert to promote ECM proteolytic degradation and remodeling (Lijnen, 2001a). Plasmin has been shown to play an important role in both physiological and pathological processes in the CNS (Syrovets and Simmet, 2004). Plasmin is one major prosecutor in tPA-mediated neurodegeneration. Plg/ mice are resistant to excitotoxic injuries, because in these mice ECM substratum for neuronal survival is not disturbed due to the lack of plasmin activity (Chen and Strickland, 1997; Tsirka et al., 1997). Some recent studies on prion diseases demonstrate that the pathological form of prion protein can bind to both tPA and plasminogen, and it stimulates tPA-catalyzed plasmin generation (Epple et al., 2004; Fischer et al., 2000); in turn, tPA accelerates the cleavage of prion protein by plasmin (Kornblatt et al., 2003). The significance of these interactions is still under investigation. It was speculated that ECM degradation initiated by the tPA–plasmin system may contribute to the pathogenesis of this group of degenerative diseases (Bass and Ellis, 2002), or elimination of the infectious moiety of prion protein via plasmin cleavage may prevent disease propagation (Kornblatt et al., 2003). On the other hand, plasmin appears to be neuroprotective in the case of Alzheimer’s disease (AD). Plasmin has been shown to degrade amyloid protein and block its neurotoxicity, and there are decreased levels of plasmin in the brain of AD patients (Ledesma et al., 2000; Melchor et al., 2003; Tucker et al., 2000). Plasmin enriched in lipid rafts of neurons can clear excessive amyloid deposition. However, such rafts are disorganized in AD


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patients; therefore, the plasmin-mediated amyloid clearance pathway is crippled (Ledesma et al., 2003). The seemingly opposing roles of plasmin in neuronal survival versus death emphasize the functional complexity of this protease that is determined by particular cellular settings and various stimuli. Plasmin has also been shown to work as a downstream eVector of tPA to promote neurite outgrowth. This function is attributed to the remodeling of ECM substratum by tPA–plasmin-initiated proteolytic cascade. Previously we reported that the tPA/plasmin system is involved in the regulation of hippocampal mossy fiber outgrowth after excitotoxin-stimulation of amygdala (Wu et al., 2000). This regulation consists of two diVerent events: neurite pathfinding through the supragranular layer, which is independent of the activation of plasminogen by tPA; and the termination of neurite outgrowth, which is mediated by proteolyic cleavage of the chondroitin sulfate proteoglycan phosphacan by plasmin. Unprocessed phosphacan is a potent repellent of mossy fiber outgrowth in culture, presumably by opposing cell–cell and cell–matrix interactions mediated by neural cell adhesion molecules. Recent studies showed that another chrondroitin sulfate proteoglycan, NG2, binds to the kringle domains of plasminogen and enhances plasmin generation by uPA (Goretzki et al., 2000) and tPA (our unpublished observations). NG2 is inhibitory to neurite outgrowth both in vivo and in vitro. By analogy with phosphacan cleavage by plasmin, one can speculate that plasmin may process NG2 to alter its suppressive eVect on neurite outgrowth as well.

IV. Inhibitors of Plasminogen Activators and Plasmin Serpins (serine protease inhibitor) are suicide substrate-like inhibitors of serine proteases (Silverman et al., 2001). The family includes PAIs, nexin-1 (PN-1), neuroserpin, and 2 antiplasmin. They act by binding to the active site of target proteases, which involves docking of the inhibitor to the target protease, cleavage of the reactive center loop, and rapid translocation of the protease to the opposite pole of the inhibitor (Silverman et al., 2001). PN-1 is the first identified neural serpin, expressed by both neurons and glia (Gloor et al., 1986; Guenther et al., 1985; Mansuy et al., 1993; Reinhard et al., 1988). It is the most potent inhibitor of thrombin, but it can also inhibit tPA, uPA, and plasmin to a lesser extent. Transgenic mice with neuronal PN-1 overexpression show increased long-term potentiation (LTP) in the hippocampus and develop progressive disturbances of motor behavior and sensorimotor integration, whereas PN-1-deficient mice have decreased hippocampal LTP. Both overexpression and lack of PN-1 cause epileptic activity in vivo and in vitro due to an imbalance between the

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excitatory and inhibitory synaptic transmission (Luthi et al., 1997; Meins et al., 2001). Neuroserpin is an axonally secreted inhibitor used preferentially against tPA, and less eVectively toward uPA and plasmin (Hastings et al., 1997; Osterwalder et al., 1996, 1998). Intracranial distribution of neuroserpin coincides well with that of tPA, suggesting that it is likely to be a critical regulator of tPA activity-mediated events in the CNS (Hastings et al., 1997). Mice with neuroserpin overexpression have decreased tPA enzymatic activity (Cinelli et al., 2001), which remains unchanged in neuroserpin-deficient mice (Madani et al., 2003). This phenotype could be explained by compensation from other tPA inhibitors for the lack of neuroserpin or undetected subtle changes of tPA activity at the synaptic level. Just as in the on-demand secretion of intracellularly stored tPA (Gualandris et al., 1996), neuroserpin is also rapidly released from the cells upon depolarization (Berger et al., 1999), making the pair of protease and inhibitor readily available in response to neuronal activity. Compared to the extensively studied functions of tPA in CNS physiology and pathology, there is limited information regarding the role of neuroserpin as the endogenous regulator of tPA’s activity in these processes (Yepes and Lawrence, 2004). Expression of neuroserpin is most prominent during neurogenesis in embryos and correlates with synaptic activity in adult brains (Muller and Griesinger, 1998; Osterwalder et al., 1996). It decreases the overall length and number of extending neurites during neuronal diVerentiation in culture (Parmar et al., 2002), suggesting that neuroserpin may modulate neuroplasticity by counterbalancing the action of tPA. Mice overexpressing neuroserpin and mice lacking neuroserpin both exhibit neophobic phenotype in explorative behaviors, suggesting that neuroserpin may modify emotional behaviors independent of tPA’s catalytic activity (Madani et al., 2003). Neuroserpin overexpression has been detected by DNA microarray in patients with chronic schizophrenia (Hakak et al., 2001), a condition often associated with emotional instabilities and behavioral disturbances (Lewis and Lieberman, 2000). On the other hand, mice overexpressing neuroserpin suVer from smaller infarcts after induction of ischemic stroke, along with an attenuation of microglial activation (Cinelli et al., 2001). In addition, treatment with neuroserpin alone or in combination with tPA significantly reduces brain lesions associated with solo tPA treatment (Yepes et al., 2000; Zhang et al., 2002b). This is consistent with earlier reports on decreased infarct volumes in tPA/ mice subjected to cerebral ischemia (Nagai et al., 1999; Wang et al., 1998). Therefore, neuroserpin assumes a neuroprotective role against ischemiainduced brain damage by blocking extravascular tPA proteolytic activity and microglial activation. Neuroserpin is also implicated in the prevention of seizure propagation (Yepes et al., 2002). Intrahippocampal delivery of neuroserpin markedly delays seizure progression to a similar level as seen

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/

in tPA mice, whereas plg mice develop seizure comparable to that seen in wild-type animals. This suggests that neuroserpin regulates tPAmediated seizure spreading via mechanisms independent of plasminogen. Neuroserpin’s interference with tPA-modulated glutamatergic synaptic transmission is certainly one interpretation for its antiseizure activity, because NR1 subunit of NMDA receptor is a newly discovered nonplasminogen substrate for tPA (Nicole et al., 2001). Mutations of the human neuroserpin gene have been linked to a newly recognized familial encephalopathy with neuroserpin inclusion bodies, an autosomal-dominantly inherited progressive dementia accompanied by myoclonus epilepsy (Davis et al., 1999; Takao et al., 2000). These mutations not only confer abnormal polymerization of neuroserpin but also compromise its inhibition of tPA (Belorgey et al., 2002). However, absence of seizure or any other signs of altered excitability in neuroserpin-null mice suggests that other tPA inhibitors may be involved and compensate for the defect, which is in line with the normal level of tPA activity in these animals (Madani et al., 2003). In summary, many roles of tPA within the CNS seem to bypass the activation of plasminogen, and neuroserpin is a key tPA opponent in these events. However, neuroserpin does share functional redundancy with other tPA inhibitors in certain context. There are three distinct PAIs, PAI-1 (previously known as endothelial inhibitor), PAI-2 (placental or monocyte/macrophage-derived inhibitor), and PAI-3 (urine-derived inhibitor) (Sprengers and Kluft, 1987). PAI-1 and PAI-2 both are specific inhibitors for tPA and uPA, but PAI-1 is the primary physiological regulator of tPA and uPA activity (Wind et al., 2002). Besides the inhibition of tPA and uPA, PAI-1 also binds to the ECM protein vitronectin with high aYnity, whereas PA-complexed PAI-1 does not. Lowdensity lipoprotein receptor-related protein (LRP) is another binding partner for PAI-1. However, the high-aYnity binding site for LRP is cryptic in free PAI-1, and it can be exposed upon PA–PAI-1 interaction. Formation of PA–PAI-1–LRP multiprotein complex is believed to facilitate endocytosis and degradation of PA and PAI-1 (Nykjaer et al., 1992; Stefansson et al., 1998). It is interesting to note that there is an upregulation of PAI-1 mRNA after restraint stress (Yamamoto et al., 2002), which may function to prevent anxiety development by blocking tPA activity, suggesting a role for PAI-1 in the regulation of stress-induced neuronal rewiring (Pawlak et al., 2003). Working against tPA-mediated neurodegeneration, PAI-1’s neuroprotective role was best studied in the context of transforming growth factor beta 1 (TGF 1) signaling (Buisson et al., 2003). It has been shown that TGF 1 protects cultured neurons from tPA-mediated NMDA excitotoxicity via a mechanism involving upregulation of astrocyte-derived PAI-1 driven by transcription factor Smad3 and ERK kinase activation (Buisson et al., 1998; Docagne et al., 2002; Gabriel et al., 2003). The same mechanism was

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proven to be eVective in rescuing neuronal damage in animal models of cerebral ischemia as well (Docagne et al., 1999; Zhu et al., 2002); whereas deficiency in the PAI-1 gene was associated with a significant increase in infarct size (Nagai et al., 1999). Thus, restoration of tPA–PAI-1 balance merits further attention in battling excitotoxic neuronal injury. There is also a growing body of evidence that suggests a causal role for PAI-1 in tumor growth, vascularization, and metastasis (Wind et al., 2002). It is known that pericellular plasmin activity generated by the uPA–uPAR system is decisive for the degradation of ECM during tumor invasion (Levicar et al., 2003). It was therefore puzzling to see the presence of high PAI-1 amounts in human malignant astrocytic tumors rather than in the corresponding normal tissues (Sandstrom et al., 1999; Yamamoto et al., 1994b). In addition, high levels of tumor-associated PAI-1 were found to be correlated with a poor prognosis (Schmitt et al., 1997). This unexpected phenomenon led to a debate on whether PAI-1 is proinvasive or antiinvasive during tumorigenesis (Andreasen et al., 2000; Rakic et al., 2003; Wind et al., 2002). Accumulating evidence suggests that it may function both ways, being antiinvasive by blocking uPA–PAR-mediated ECM degradation, and being proinvasive by antiproteolytic and nonproteolytic mechanisms (Rakic et al., 2003; Wind et al., 2002). It has been shown that malignant cell invasion and angiogenesis are severely impaired in PAI-1/ mice, which can be restored to control levels by applying exogenous PAI-1 (Bajou et al., 1998), whereas angiogenesis is increased in mice overexpressing PAI-1 (McMahon et al., 2001). However, these eVects are critically dependent on the concentration of PAI1, which is proangiogenesis at low concentrations, but antiangiogenesis at supraphysiological concentrations (Bajou et al., 2004). The eVects also vary depending on the cellular source of PAI-1 (tumor cells vs host cells); tumorderived PAI-1 even at high levels cannot change the course of angiogenesis and tumor progression in PAI-1/ mice (Bajou et al., 2004). Several mechanisms underlying the multiple functionality of PAI-1 have been put forward (Levicar et al., 2003; Rakic et al., 2003; Wind et al., 2002). By antagonizing the PA–plasmin system-mediated matrix degradation, PAI-1 may preserve ECM integrity that serves as a supporting substratum for colonization by vascular endothelial cell and subsequent neovasculature assembly. Meanwhile, tumor cells may be trapped by the intact basement barrier in the ECM, leading to failure of migration. On the other hand, complex interactions between PAI-1 and multiple ECM proteins (vitronectin and integrins) control cell migration through a nonproteolytic pathway. Binding of PAI-1 and vitronectin blocks cell-attachment sites between vitronectin and its receptor integrin that are required for cell motility (Stefansson and Lawrence, 1996). uPA forms a complex with PAI-1, making vitronectin accessible to integrin, and cell migration is restored (Deng et al., 1996; Kjoller et al., 1997). Thereby, PAI-1 can control cell–matrix


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interaction by regulating the accessibility of specific cell-adhesion sites. Together, these findings suggest that a precise balance between proteases and inhibitors may be essential for tumor growth and invasion. 2 Antiplasmin is the main physiological inhibitor of plasmin activity. It is secreted as a 70-kDa single-chain protein by hepatocytes (Dobrovolsky and Titaeva, 2002). It rapidly scavenges free uncomplexed plasmin by formation of an inactive 1:1 stoichiometric complex, ensuring a short half-life of plasmin in blood and tissue (Lijnen, 2001b). Although 2 antiplasmin expression in the CNS has not been documented, it is likely to be produced solely by brain resident cells in the absence of a compromised blood–brain barrier (BBB). With BBB breakdown, there may be an influx from the bloodstream. The main role of 2 antiplasmin in the CNS is in the regulation of plasmin activity to modulate neuronal survival in various injury paradigms. It has been shown that intracerebral infusion of 2 antiplasmin confers neuroprotection to excitotoxic damage in the mouse hippocampus (Tsirka et al., 1997) and rat striatum (Campbell et al., 2004). Infiltration of inflammatory cells into the lesioned site is greatly attenuated in the presence of 2 antiplasmin (Campbell et al., 2004). Paradoxically, it was reported that 2 antiplasmin-deficient mice display decreased infarct volume after focal cerebral ischemia, whereas plg/ mice suVer from larger infarcts (Nagai et al., 1999). Despite the excitotoxic modality in ischemic cell damage, it was speculated that these phenotypes are the result of an alternative pathway other than tPA–plasmin-initiated ECM degradation. This pathway involves exacerbated vascular occlusion due to lack of plasmin-mediated fibrin clearance, leading to expansion of the infarct area (Nagai et al., 1999).

V. tPA Effects on Rodent Behavior Following the initial reports that tPA is expressed in the mammalian rodent brain, and that neuronal activity in the rat and mouse hippocampus and cerebellum induces tPA mRNA expression (Carroll et al., 1994; Qian et al., 1993; Seeds et al., 1995), studies were initiated to investigate the role of tPA in tasks associated with hippocampal or cerebellar function, namely, spatial learning, and consolidation of memory and motor learning, respectively. The results do not delineate an undisputed role for tPA as far as higher cognitive functions are concerned (learning and memory), but associate tPA to structural and electrophysiological changes related to learning and occurring in relevant brain areas (Table I). Furthermore, little attention has been paid to more basic behavioral attributes of the tPA/ mouse. Specifically, it has been shown that tPA aVects the learning of tasks dependent on the cerebellum and the striatum. Following the learning of a complex motor task where the rats needed to transverse a runway by placing

Table I

Studies That Have Examined the Relationship of tPA to Learning, LTP, and mRNA Levels tPA/ Performance

tPAþ/þ Performance

LTP

Centonze et al., 2002 #

Qian et al., 1993 Increased mRNA 9 Baranes et al., 1998> = Frey et al., 1996 " > Huang et al., 1996 ; L-LTP

Morris water maze

Huang et al., 1996 No diVerences Huang et al., 1996 No diVerences Horwood et al., 2004 No diVerences Pawlak et al., 2002 #

Tasks and LTP

Barnes maze Radial arm maze (spatial version) Step down inhibitory avoidance Context conditioning

Striatum-related tasks Operant conditioning tasks

Madani et al., 1999 "

Calabresi et al., 2000 # Huang et al., 1996 No diVerences

Cerebellum-related tasks

tPA Overexpression Performance

Seeds et al., 1995 Increased mRNA Calabresi et al., 2000 # Huang et al., 1996 Horwood et al., 2001 # Horwood et al., 2004 Ripley et al., 2001

Arrow pointing down signifies decrease; arrow pointing up signifies increase.


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their paws on pegs (a cerebellum-dependent task), tPA mRNA levels were increased in the Purkinje neurons of the cerebellum (Seeds et al., 1995). When a two-way active avoidance task was used (a striatum-dependent task where the mouse learns to move from one chamber to another to avoid a mild shock) tPA/ mice were shown to have impaired performance (Calabresi et al., 2000; Huang et al., 1996). tPA/ mice have also been found to have impaired inhibitory behavior in operant conditioning tasks where the mouse learns that a certain behavior on its part will be followed by a reward after varying intervals (Horwood et al., 2001, 2004; Ripley et al., 2001). Interestingly PAI-1/ mice showed deficits in acquiring the operant task similar to the ones of the tPA/ mice (Horwood et al., 2001). Furthermore, activation of the mossy fiber pathway in the hippocampal region of tPA/ mice resulted in aberrant mossy fiber outgrowth (Wu et al., 2000). Treatment of hippocampal cells with tPA aVects their morphological characteristics. When tPA inhibitors were used, the formation of perforated synapses (via an increased activation of NMDA receptors) was significantly impaired (NeuhoV et al., 1999). Treatment with tPA was also responsible for elongated mossy fibers, as well as the formation of synaptic varicosities in hippocampal cell cultures (Baranes et al., 1998). The same researchers also found that tPA enhances the late phase of long-term potentiation (L-LTP, a molecular phenomenon long believed to underlie learning and memory) in the hippocampal mossy fiber pathway. Similar eVects on the L-LTP of the hippocampal SchaVer collateral pathway were reported earlier in other studies (Frey et al., 1996; Huang et al., 1996). Impaired LTP has also been reported in the corticostriatal pathway of tPA/ mice (Centonze et al., 2002). Mice with a disruption in the tPA gene also appear to have impaired performance in learning tasks that are hippocampus related. For example, when such mice were trained in a context conditioning task (where the mouse is exposed to a mild shock in an experimental chamber on the first day of testing followed by the measurement of time, and the mouse remains ‘‘frozen’’ in the same environment 24 h later), their performance was inferior compared with that of wild-type mice (Calabresi et al., 2000). Notably, the same task did not result in significant diVerences in the hands of another group (Huang et al., 1996). It is possible that the discrepancy is due to genetic background diVerences between the two strains of mice. Another study used the step-down inhibitory avoidance task (where the mouse learns to associate the floor of the chamber with a mild shock as it steps down from a platform, and, following a delay, it is measured how long it takes for the mouse to step down from the same platform) to find that the tPA/ mice performance was significantly worse than that of the wild-type ones, as early as 90 min after training, and 1, 2, and 7 days later (Pawlak et al., 2002). In a study that used mice that were overexpressing tPA, the results

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were consistent with those of the tPA mouse studies. Specifically, these mice performed significantly better in the Morris water maze (where the mouse learns to find a hidden platform underneath the opaque water surface of a pool) and the homing holeboard task (where the mouse learns to find an escape hole on a maze that leads to its home cage) compared with wild-type mice; furthermore, the same animals exhibited increased hippocampal LTP potentiation at levels proportional to the tPA overexpression (Madani et al., 1999). On the other hand, two studies found no diVerences between tPA/ and wild-type mice in tasks testing spatial learning. The first study reported that tPA/ mice did not diVer from wild-type mice in the Morris water maze and the Barnes maze (where the mouse finds a hole out of 18 that leads to a safe box) tasks (Huang et al., 1996). However, in this case the extent to which the animals were back-crossed has been uncertain (and the variability of genetic material may contribute to the results). The second study using a spatial version of the radial arm maze (where the mouse learns to explore and consume a reward in each of the eight maze arms by visiting them for the fewest possible times) found no diVerences between tPA/ mice and wild-type mice (Horwood et al., 2004). Nonetheless, it seems that the increased interest shown for the learning and memory tasks resulted in the sporadic and nonsystematic testing of the fundamental motor, exploratory, and anxiety-related behaviors of tPA/ mice. The degree of exploration of an open field was shown to be the same between tPA/ mice and controls, except for the females of both groups, which had a lower number of movements (Huang et al., 1996). To determine if there were any biases present in the step-down avoidance task, one study examined the overall locomotion of the mice in their cage and anxiety-like behaviors in the elevated-plus maze (where the amount of time the mouse spends in open unprotected arms as opposed to closed ones is a measure of its anxiety) and found no diVerences between the two groups (Pawlak et al., 2002). A more detailed characterization of the tPA/ mice was made when horizontal and vertical activities in an open field were found to be similar and less than that of the wild-type mice, respectively (Calabresi et al., 2000). The same study reported that the tPA/ mice showed lower habituation and reactivity to spatial change rates in an open field, but did not diVer from wild-type mice when the reaction to change of an object was measured. Another notable finding was that following the application of chronic restraint stress, tPA/ mice exhibited less anxiety-like behaviors on the elevated-plus maze compared with the wild-type mice (Pawlak et al., 2003). It is becoming apparent from the previous studies that tPA has a role in learning and memory, but probably not a central one. Its eVects have been more prominent in tasks associated with the striatum and the cerebellum, but not the hippocampus. The tPA mRNA levels are increased following the


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learning of diVerent tasks, indicating that tPA is released according to its characterization as an immediate early gene and the process for it to be replenished is getting underway. It is possible that tPA facilitates, via its substrates, the changes in the extracellular matrix necessary to accommodate the formation of new long-term memories. In this context, its facilitation of LTP would serve the same purpose. Specifically, the impaired L-LTP in the SchaVer collateral synapse of the hippocampus has been associated with deficits in hippocampus-related tasks. That would contradict the unimpaired performance of tPA/ in hippocampus-related tasks reported in the same studies (e.g., Baranes et al., 1998; Frey et al., 1996; Huang et al., 1996). However, first it is possible that experimental conditions (such as the temperature in an intact animal could compensate for such a defect) could have influenced the electrophysiological results. Second, L-LTP was not completely absent in the tPA/ slices, which could lead to the conclusion that it is present in the intact animal at a threshold high enough to produce the needed eVects for the formation of memories (Huang et al., 1996). Finally, the findings that tPA/ mice exhibit impaired behavioral inhibition while learning an operational task (Ripley et al., 2001) and limited anxietylike behavior following chronic constraint (Pawlak et al., 2003) warrant further study.

VI. uPA and Plasminogen Effects on Rodent Behavior Research examining a possible role for uPA and plasminogen in mouse behavior has been even more sporadic than that examining tPA. It has been reported that mice overexpressing uPA consume less food, weigh less, and live longer (Miskin and Masos, 1997). The same research group had reported earlier that these transgenic animals were impaired in their learning of the Morris water maze, as well as in a conditioned taste aversion task, where the mice learn to associate a novel taste with malaise (Meiri et al., 1994). uPA/ mice have been reported to not diVer from wild-type mice in acquiring an operant conditioning task (Horwood et al., 2001). Plasminogendeficient mice exhibit increased stress response of grooming in an open field task and impaired response in the acoustic startle response task, where the mouse’s flinch in response to a loud noise is measured (Hoover-Plow et al., 2001). The same study did not find any diVerences between the groups in the acquisition of the Morris water maze task. A study that examined mice that either overexpress or are deficient of neuroserpin, an inhibitor of tPA and uPA, reported that both groups displayed anxiety-like behavior in an open field task and neophobic responses toward novel objects (Madani et al., 2003). Clearly, more studies are warranted that will examine in a systematic manner the behavioral phenotypes of uPA and plasminogen-deficient mice,

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as well as those of their inhibitors. The previously mentioned studies are hard to interpret in their isolation, but even with them the theme that seems to emerge is that performance in learning and memory tasks related to the hippocampus is not impaired.

VII. Conclusions It is evident that proteases and their inhibitors play a critical role in mammalian neurophysiology and neuropathology. Tightly regulated balance between proteases and their inhibitors is vital to normal CNS function and survival, whereas pathological conditions are inevitable when control is lost over various CNS injuries. A thorough understanding of regulatory mechanisms that maintain or disrupt this delicate balance will provide us with codes to decipher its role in CNS activity and novel therapeutic strategies for related CNS diseases.

Acknowledgments The authors thank members of the Tsirka laboratory for helpful discussions. This work was supported by an NIH grant to S. E. T.

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The Genetic Architecture of House Fly Mating Behavior Lisa M. Meffert and Kara L. Hagenbuch Department of Ecology and Evolutionary Biology Rice University Houston, Texas 77251-1892

I. Introduction II. Evolutionary Dynamics of Quantitative Genetic Interactions A. Dominance B. Epistasis C. Genotype-by-Environment Interactions and Learning D. Pleiotropy III. Courtship in the House Fly IV. Evidence of Quantitative Genetic Interactions in House Flies A. Assays of Additive Genetic Variances in Bottlenecked Populations B. Line Cross Analyses C. Repeatability Assays V. Prevalence of Nonadditive Genetic EVects in Animal Behavior VI. Future Directions VII. Summary Acknowledgments References

This chapter summarizes several experimental approaches used to identify the eVects of dominance, epistasis, and genotype-by-environment interactions in the genetic architecture of the mating behavior of the common house fly (Musca domestica L.). Quantitative genetic investigations of mating behavior hold special intrigue for unraveling the complexities of fitness traits, with applications to theory on sexual selection and speciation. Besides being well suited to large-scale quantitative genetic protocols, the house fly has a remarkably complex courtship repertoire, aVording special opportunities for studies on communication, social interactions, and learning. Increased additive genetic variances for the courtship repertoire of experimentally bottlenecked populations provided evidence for the presence of dominance and/or epistasis. Negative genetic variances in these populations suggested genotype-by-environment interactions, where the environment is the mating partner. Line cross assays of populations that had been subjected to selection for divergent courtship repertoire confirmed that both dominance and epistasis have significant eVects. These crosses also showed more directly that Current Topics in Developmental Biology, Vol. 66 Copyright 2005, Elsevier Inc. All rights reserved. 0070-2153/05 $35.00

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the expression of the male’s genotype is dependent upon the preferences of his mating partner. Repeatability studies also detailed how males alter their courtship performances with successive encounters within and across females, such that the males learn to improve their techniques in securing copulations. A review of 41 animal behavior studies found that a wide range of traits and taxa have dominance, epistasis, and genotype-y-environment interactions, although house fly courtship may remain a unique model where learning is an intersexually selected trait. Future development of more sophisticated molecular techniques for the M. domestica genome will help unravel the underlying biochemical and developmental pathways of these quantitative genetic interactions for a more complete understanding of the processes of inbreeding depression, outbreeding depression, and pleiotropy. C 2005, Elsevier Inc.

I. Introduction The most fundamental motive for the genetic dissection of house fly mating behavior is exploiting a model experimental system for understanding the architecture of fitness traits. Mating behavior, in particular, is intrinsically tied to overall reproductive success and thus serves as a major fitness component. Natural selection (along with genetic drift) is expected to erode the additive genetic variance of such fitness traits, resulting in characteristically low heritabilities (Fisher, 1958). Additionally, dominance and epistasis are expected to camouflage the residual components of additive genetic variance (Goodnight, 1988; Willis and Orr, 1993; and see later). Thus, the level of additive genetic variation can serve as a measure of historical selection pressure and future evolutionary potential (RoV and Mousseau, 1987). Moreover, the relative contributions of dominance and epistasis indicate the potential for the release of sequestered additive genetic variance by bottlenecks (see MeVert, 2000 for a review). The house fly is especially amenable to the quantitative genetic manipulations required for investigating the architecture of fitness traits. It shares many of the advantages of the well-studied Drosophila melanogaster, such as fast generation length, large family sizes, and simple culturing techniques. Its comparatively large body size, however, facilitates the large-scale parent– oVspring analyses, selection experiments, and repeatability assays necessary to partition genetic variance components. For example, the technical eVorts to sex virgins, videotape courtship, and collect oVspring from individual male– female pairs are less demanding when stereoscopes and transportation pooters are unnecessary. More importantly, the intricate male–female interactions in house fly courtship (see later) provide excellent opportunities for evaluating the genetics of particularly complex fitness traits, especially in terms of the evolutionary roots of communication, social behavior, and learning.

6. Quantitative Genetic Interactions in House Fly Courtship

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House fly courtship also serves as a model system for more specific experimental tests of sexual selection theory. Sexual selection theory seeks to explain the evolution of traits that confer mating advantages even when the traits have negative consequences for other aspects of fitness (e.g., see Andersson, 1994; Price et al., 1993). In intersexual selection, male traits evolve when females discriminate among potential mates, driving the coordinated evolution of the female preferences themselves (e.g., Fisher, 1958; Lande, 1981). In this basic model, a genetic correlation generated as a daughter with its mother’s preference carries alleles for the desired male trait, while a son with the trait also carries alleles for the female preference (Lande, 1981). The reinforcing coevolution of preferences and male traits then proceeds until the additive genetic variance is exhausted or some equilibrium is achieved with antagonistic natural selection pressures (Lande, 1981). Much of the intrigue of intersexual selection involves the evolution of sexual dimorphisms, whereby the genome component expressed in one gender (typically the female) acts as a selective pressure on the other. Whether the sexual selection process is cooperative or antagonistic (termed run-away and chase-away, respectively) is very controversial, especially since both features can operate in the same system (e.g., Hicks et al., 2004). Again, the experimental advantages of the house fly oVer special opportunities to unravel the genetic underpinnings and evolutionary consequences of sexual selection. Theoretical work suggests that sexual selection can drive the formation of new species. In his landmark paper, Lande (1981) examined how divergent sexual selection responses (sensu Fisher, 1958) can generate reproductive isolation, and thus form new species. Moreover, founder-flush events have been proposed to open evolutionary avenues for such divergence by releasing additive genetic variance from nonadditive genetic structure (see MeVert, 1999, 2000 for reviews). Part of the extensive species radiation of the Hawaiian Drosophila, for example, has been attributed to founder-induced stimulation of divergent sexual selection process (e.g., Kaneshiro, 1980). There has been some debate about the likelihood and stability of such behavioral divergence (e.g., see Iwasa and Pomiankowski, 1995; Price, 1998). For example, Boake et al. (1997) noted that the species recognition pattern found in the D. heteroneura–D. silvestris system was unrelated to a sexually selected head-width trait. They contended that this was negative evidence for a continuum in the processes of sexual selection and speciation. Aspi (2000), however, held that the sexual selection–species recognition continuum does occur within the D. montana–D. littoralis system (see also Aspi and Hoikkala, 2000). These kinds of sexual selection studies rely on post-hoc interpretations of extant species, but the experimental accessibility of the house fly permits investigations of the initial stages of speciation via sexual selection and founder events (e.g., MeVert, 1999; MeVert and Regan, 2002).

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In this chapter, we first discuss the evolutionary significance of the quantitative genetic interactions of dominance, epistasis, genotype-by-environment interactions, and pleiotropy. We then detail the courtship repertoire of the house fly and summarize various experiments that have identified all of these dynamics, including a special form of genotype-by-environment interaction: learning. We also summarize the animal behavior literature to show that such complexities are common in a wide range of taxa and traits. Finally, we provide predictions for future explorations with this model system.

II. Evolutionary Dynamics of Quantitative Genetic Interactions Figure 1 depicts phenotypic deviations for the quantitative genetic models of pure additivity, pure dominance, additive-by-additive epistasis, and genotype-by-environment interactions. In each case, two representative loci are modeled (i.e., ‘‘X’’ and ‘‘Y’’), with each locus having two alleles (i.e., X–x and Y–y, respectively). The surfaces represent the phenotypic values for the nine possible genotypes under this simplified two-locus, two-allele system. Under pure additivity (i.e., no interaction), the phenotypic values simply summate across alleles and across loci (Fig. 1A). With pure dominance (i.e., one allele completely masks the eVects of another allele at the same locus), phenotypic plateaus are apparent across the wide range of genotypic values, compounded by the additive eVects across loci (Fig. 2B). Under epistasis, the interlocus eVects act synergistically, yielding like phenotypes for disparate genetic combinations (i.e., compare genotypes XXYY and xxyy in Fig. 1C). Figure 1C depicts additive-by-additive epistasis, which is the simplest form of the three major components of digenic epistasis (i.e., additive-by-additive, additive-by-dominant, and dominant-by-dominant). The representative genotype-by-environment case here (Fig. 1D) simply reverses the eVects of the second locus from the additive model in Fig. 1A, due to reversed expression in a diVerent environment. Pleiotropy occurs when one or more loci influence the expression of more than one trait. Most experimental studies oVer univariate analyses, but the coordination of elements of courtship repertoire are especially illuminating under multivariate analyses.

A. Dominance Natural selection should work eYciently in culling out detrimental dominant alleles, but detrimental recessive alleles will remain in large populations, hidden in the pool of heterozygotes (Falconer, 1989). Mutation contributes to this deleterious recessive load (Frankham, 1995; Lande, 1994, 1995; Lynch


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Figure 1 Phenotypic values for four models on two-locus genetic interactions: (A) pure additivity (i.e., no interaction), (B) pure dominance, (C) additive-by-additive epistasis, and (D) genotype-by environment interaction. For each panel, the surfaces represent the phenotypic eVects for the nine possible genotypes for two loci (denoted ‘‘X’’ and ‘‘Y’’), each with two alleles (i.e., X–x and Y–y, respectively). For the additive (A) and dominance (B) panels, the eVects of the two loci are summed to obtain the resultant phenotypic value. In the epistatic panel (C), the eVects are multiplicative (by definition). The representative genotype-by-environment panel (D) depicts the additive model (A) where the influence of the ‘‘B’’ locus is reversed, due to an interaction with a diVerent environment.

et al., 1995; Schultz and Lynch, 1997), and severe population bottlenecks can promote the fixation of old mutations, simulating many generations of input by new deleterious mutations (Lande, 1994). More generally, inbreeding can expose the additive genetic variance that is hidden by dominance, as rare recessive alleles increase in frequency (Hill and Caballero, 1992; Lo´pez-Fanjul and Villaverde, 1989; Whitlock et al., 1993; Willis and Orr, 1993). The widespread occurrence of inbreeding depression attests to the ubiquity of deleterious load and, thus, the importance of dominance (e.g., see reviews by Charlesworth and Charlesworth, 1987; Frankham, 1995;

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Thornhill, 1993). The reduction in average heterozygosity for traits with heterozygote advantage is also a factor in inbreeding depression (e.g., Brewer et al., 1990), although simple dominance has been suggested to be more common (Charlesworth and Charlesworth, 1987). With such structuring, dominance can be responsible for heterosis (i.e., the increased fitness of hybrids), along with the decreased fitness of inbred individuals (Falconer, 1989). Thus, dominance structures the ability of populations to withstand population bottlenecks and to recover from extinction threats. Multiple experiments have identified the house fly as being an excellent model for investigating the consequences of inbreeding and the evolutionary significance of dominance (see later).

B. Epistasis Epistasis plays a central role in Wright’s shifting balance theory (Wright, 1969). In contrast to the Fisherian view of large panmictic populations with negligible epistasis, Wright’s model relies on epistasis to generate the amongdeme variation of structured populations (Goodnight and Wade, 2000). In particular, the sorting of adaptive among-locus interactions within a deme can create incompatibility of the genomes of diVerent demes (Lo´ pez-Fanjul et al., 2000; Lynch, 1991; Wolf et al., 2000). More specifically, outbreeding depression can result when interdemic hybrids suVer from the reshuZing of coadapted epistatic complexes (Aspi, 2000; Lynch, 1991; Wolf et al., 2000). Consequently, epistasis is thought to drive the evolution of reproductive incompatibility and, thus, the process of speciation (Aspi, 2000). The prevalence of epistatic variation within species remains controversial (e.g., see Coyne et al., 1997; Goodnight and Wade, 2000), although both the Wrightian and Fisherian views hold that epistasis critically influences reproductive isolation. Within populations, traits structured by some forms of epistasis can also manifest inbreeding depression (Charlesworth, 1998; Falconer, 1989). Thus, even though the cascading eVects of interlocus interactions are commonly recognized in developmental biology, the relative importance in quantitative genetic architecture needs to be explored. Importantly, the logistical demands of identifying epistasis relegate the work to relatively few study systems (see later)—the house fly being one.

C. Genotype-by-Environment Interactions and Learning Genotype-by-environment interactions can distort the mapping of genotype onto phenotype, and thus diminish the ability to predict evolutionary trajectories through heritability coeYcients (Via and Lande, 1985, 1987).


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Indeed, genotype-by-environment eVects can result in negative selection responses (Via and Lande, 1987). Behavior traits, in particular, are susceptible to two special forms of genotype-by-environment eVects: learning and social interactions, which are not mutually exclusive. Learning constitutes a general form of genotype-by-environment interaction. With social interactions, however, the ‘‘environment’’ is the interacting conspecific (Boake and Hoikkala, 1995), creating genotype-by-genotype interactions as a special form of epistasis (MeVert, 1995; Wolf et al., 1998). These kinds of behavioral modifications assess the relative sensitivity or canalization of the genetic architecture to environmental pressures and risks (Lynch and Walsh, 1998). In a behavioral ecology sense, the relative influence of genotype-byenvironment interactions dictate the level of adaptive plasticity available for optimal expression, such as in house fly mating strategies.

D. Pleiotropy Pleiotropy is also a critical influence on development and evolution, and is thought to be very widespread due to the complexity of biochemical and developmental webs (Lynch and Walsh, 1998). The genetic and phenotypic intercorrelations generated by pleiotropy can directly influence selection responses and thus dictate the amount of evolutionarily accessible space (Gromko, 1987; Lynch and Walsh, 1998). Importantly, inbreeding or selection can alter the pleiotropic architecture of traits structured by dominance or epistasis (Cheverud and Routman, 1996; Goodnight, 1988; Hansen and Wagner, 2001). In particular, the conversion of additive genetic variance from the nonadditive components (Cheverud and Routman, 1996; Willis and Orr, 1993; see MeVert, 2000 for a review) can restructure genetic covariances/correlations across traits (Bryant and MeVert, 1988; Shaw et al., 1995). This restructuring can open up or close down evolutionary pathways (e.g., see Regan et al., 2003). Dipteran mating behavior is controlled by the numerous aspects of ambulatory activity and sensory capabilities that are integrated through metabolic and neurological webs (Faugeres et al., 1971; Markow, 1981; Sharp, 1984; Taylor, 1975). Thus, the influences of dominance and epistasis found for house fly courtship (e.g., MeVert, 2000 and later) need to be explored on the level of the multivariate phenotype.

III. Courtship in the House Fly There are two basic stages in housefly courtship: a stalking type of behavior by the male, followed by his mounted displays and attempted copulation. In the stalking phase, the male makes creeping movements toward the female,

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at which point he might stand next to her for brief episodes (see MeVert and Bryant, 1991). During this premounting phase, the male often taps the female with his forelegs, often stimulating a fending-oV behavior from the female (MeVert and Bryant, 1991). Figure 2A–E depicts five stereotypical displays that are performed once the male mounts the female (male performance of BUZZ, LUNGE, HOLD, and LIFT and the female’s WING OUT). At mounting, the male will buzz his wings (BUZZ), ostensibly producing auditory and/or tactile cues (see arrow in Fig. 2A). The male then orients toward the head of the female and lunges over her head (LUNGE, Fig. 2B). During LUNGE, the male periodically stops BUZZ and holds his wings either in their normal resting position (horizontally along the dorsal plane) or over the female’s head (HOLD, Fig. 2B, C). During LUNGE, the male can also periodically attempt to lift the female’s forelegs with his own forelegs (LIFT; see arrow in Fig. 2B). At any point during mounting, the female thrusts her wings out perpendicular to her body to kick her hind legs up and over her wings (WING OUT; see arrow in Fig. 2C). The male then attempts copulation (Fig. 2E), during which time the female stops WING OUT (see arrow in Fig. 2D). Figure 2F–J shows the same relative sequences for a mating pair that omits HOLD, LIFT, and WING OUT, using characteristically less LUNGE. Table I quantifies the intercorrelations among the five courtship traits (see MeVert and Regan, 2002). LUNGE, HOLD, LIFT, and WING OUT are positively intercorrelated, with BUZZ being negatively correlated with HOLD. This general relationship is also identified in the principal component solution (Table II). In standard morphometrics, the first principal component, which summarizes the major positive intercorrelations, is termed the size axis (Pimentel, 1979). For these behavior analyses, the first principal component (PC1) explains the gradation from more complex (i.e., larger ‘‘size’’) to simpler (i.e., smaller) courtships. Figure 2A–E thus portrays a characteristically complex courtship, with a strong positive loading on this axis, whereas Fig. 2F–J portrays the characteristically simple repertoire: one with a negative loading. This classification of the continuum of courtship complexity largely reflects the level of female reluctance to mate, in that populations with negative loadings have females that are significantly more receptive than those with positive loadings (MeVert and Bryant, 1991; MeVert and Regan, 2002). To a lesser extent, this size continuum also explains diVerences in the males’ ability to successfully acquire copulations, such that the more aggressive males are found in populations with positive loadings on this size axis (MeVert and Bryant, 1991; MeVert and Regan, 2002). Earlier studies suggested that the male forces the execution of WING OUT (Tobin and StoVolano, 1978), but we have found that the female is directly involved in the display. Sacca (1964) reported that the female leg kick serves to mutilate the male’s wings, but we

Figure 2 Qualitative comparison of the complex (A–E) and simple (F–J) courtship repertoires of the house fly. See ‘‘Courtship in the House Fly’’ for a description of the figure.

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Table I Pearson Correlation CoeYcients Among the Five Courtship Displays Measured in 160 Matings

BUZZ LUNGE HOLD LIFT

BUZZ

LUNGE

HOLD

1.000 — — —

0.245{ (0.002) 1.000 — —

0.370{ (

Current Topics in Developmental Biology, Volume 66 (Current Topics in Developmental Biology)

Current Topics Developmental Biology (Current Topics in Developmental Biology)

Current Topics Developmental Biology (Current Topics in Developmental Biology)