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Pattern formation
Reproductive Toxicology, Vol. 11, Nos. Z/3, pp. 339-344, 1997 Copyright 0 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0890-6238/97 $17.00 + .OO ELSEVIER
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PATTERN FORMATION RICK WOYCHIK,* BRIGID HOGAN,? SUSAN BRYANT,+ GREGOREICHELE,~ DAVID KIMELMAN,~ DREW NODEN,# GARY SCHOENWOLF,** and CHRISTOPHER
WRIGHT-/-
*Oak Ridge National Laboratory, Mammalian Genetics and Development Section, Oak Ridge, Tennessee tvanderbilt University Medical School, Department of Cell Biology, Nashville, Tennessee $University of California Irvine, Developmental Biology Center, Irvine, California §Baylor College of Medicine, Department of Biochemistry, Houston, Texas IUniversity of Washington, Department of Biochemistry, Seattle, Washington #Cornell University, College of Veterinary Medicine, Department of Anatomy, Ithaca, New York **University of Utah School of Medicine, Department Neurobiology and Anatomy, Salt Lake City, Utah
spreads over the egg cylinder to displace the primitive endoderm. Fate mapping experiments in the mouse have provided a general framework for understanding cell movements and lineage during gastrulation of the mammalian embryo. They support the idea that the node acts not only as an organizing center for mesoderm cells moving into it, but also as a localized region of proliferation for a resident population of stem cells. In situ hybridization studies have identified a set of mouse genes that are expressed in the primitive streak [e.g., T (Brachyury), FGF family members, FGF receptors], in the node (the TGFP-related gene, nodal) and in the notochord, presumptive somites, and more lateral mesoderm Cforkhead family members, mouse lim-1). Current conceptual models for how cellular diversity is established during mammalian gastrulation are almost entirely dominated by studies of mesoderm induction and specification in amphibian embryos, and of gastrulation movements and fate mapping in avian embryos. Comparative studies at the molecular level support the idea that the basic mechanisms underlying gastrulation and pattern formation have been highly conserved among vertebrates. Such studies have been extremely valuable in catalyzing new ideas and lines of investigation in the mouse. However, it is important not to lose sight of the fact that unique mechanisms may have evolved in the mammalian embryo, providing targets for teratogens that cannot be revealed in studies with other vertebrates. Moreover, the mouse provides the only opportunity to apply experimentally the ultimate genetic tests (deletion and misexpression) to hypotheses about gene function during mammalian embryogenesis. In view of the central importance of gastrulation to the establishment of the basic body plan of the mammalian embryo, and the great need for a molecular description of the underlying mechanisms to understand how
AXIS FORMATION IN MAMMALIAN EMBRYOS If we are to understand how toxic chemicals and teratogens disrupt embryonic development, it is crucial that we have a better description of the molecules and genes that control the normal program of growth, differentiation, and pattern formation. Fundamental to development is the establishment of the primary body axes (dorsalventral, anterior-posterior, and proximal-distal), yet surprisingly little is known about the underlying mechanisms in mammalian embryos. In the mouse, it is thought that the primary axes are set up some time around implantation and are first revealed in the process of gastrulation, which begins at approximately 6.5 d postcoitum (p.c.). Cells delaminate from a restricted region of the embryonic ectoderm, at what will be the posterior of the embryo, and form a layer of mesoderm between the ectoderm and the primitive endoderm. Over the next 12 h or so this region of mesodermal delamination, known as the primitive streak, extends along the anterior-posterior axis of the embryo to the distal tip of the cup-shaped ectoderm (the egg cylinder). By about 7 d p.c. the anterior of the primitive streak is morphologically distinguishable as the “node.” This structure is thought to be analogus to the dorsal lip of the amphibian blastopore and to Hensen’s node of the chick. Cells that emerge anteriorly in the midline from the node differentiate into notochord, while cells lateral to this develop into the presumptive somites and intermediate mesoderm. The definitive endoderm is also generated at the node, and it
Participants: Kathy Sulik, George D&on, Phil Mirkes, Waldy Generoso, Roger Pedersen, &role Kimmel. Address correspondence to Rick Woychik, Ph.D., Oak Ridge National Laboratory, Biology Division, P.O. Box 2009, Oak Ridge, TN 378314077. 339
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they are affected by teratogens, new genes that are expressed in different germ layers and regions of the gastrulation stage mouse embryo need to be identified. Such genes could throw light on cellular interactions and inductive processes, as well as provide molecular markers for different mesodermal lineages. A variety of strategies could be used to identify such new genes including 1) the search for mouse homologs of genes known to be expressed during Drosophila development or during gastrulation in amphibian and avian embryos, 2) differential screening of cDNA libraries made from different stages and cell populations of mouse embryos, and 3) promoter and gene trap experiments in ES cells using a reporter gene such as 1acZ for revealing gene expression in chimerit embryos. Positional cloning might be used to isolate defective genes in a number of preexisting mouse mutants that become disorganized around the time of gastrulation and during primary axis formation. A panel of insertional mutants that become disorganized around the time of gastrulation might also be generated. A good example of the power of this approach is the retroviral insertion mutant, 413-d, which disrupts the nodal gene and blocks mesoderm differentiation and axis formation. Finally, more lineage analysis in the mouse and chick embryo would be helpful in understanding relationships between cell position with respect to the node and specification of mesodermal phenotype. HOMEOBOX GENES AND PATTERN FORMATION The discovery that a cluster of Antennapedia class homeobox genes, expressed in a linear sequence along the anterior-posterior axis of the embryo, has been highly conserved throughout a large part of metazoan evolution represents one of the highlights of modern developmental biology. The temporal and spatial expression of these genes in highly organized structures such as the limb bud and genital tubercule also reflects their central role in pattern formation during development. Studies on the expression of the four Hox gene clusters in the mouse embryo, the effects of misexpressing specific genes in transgenic embryos, and the study of mutants in which specific genes have been inactivated, all support a model in which Hox genes provide a combinatorial code for positional information along the anterior-posterior axis. There is very good evidence that the homeotic selector genes of Drosophila (HOM genes), and the four clusters of vertebrate homeobox genes (Hox genes), are truly homologous complexes encoding region-specific transcription factors. The Hox and HOM clusters not only have similar organization, but in both, gene order is colinear with the spatiotemporal expression patterns in the anteroposterior (A-P) axis. Most comprehensively
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studied in the early mouse embryo, Hox genes are expressed in overlapping domains, differing by increments at their anterior border. This arrangement is thought to constitute a positional code for region-specific differentiation. The presence of four homologous clusters in vertebrates is presumably to allow for greater embryonic complexity, especially in the highly specialized central nervous system and the pluripotent neural crest. What controls the expression of the Hox genes in order down the A-P axis? In human teratocarcinoma cells, increased dose and time of exposure to retinoic acid (RA) results in sequential expression of the Hox genes. Linked to this observation are the findings that pulsed RA application causes gross phenotypic defects in the early body plan of Xenopus and zebrafish embryos (especially anterior truncations), and mice (including skeletal abnormalities and cleft palate), which have been correlated with changes in Hox gene expression patterns. Nested patterns of Hox cluster expression, similar to those seen in the main A-P embryonic axis, are also observed in the developing limb bud. Manipulations that disturb normal limb morphogenesis, including localized RA applications, can be correlated with, and preempted by, predictable changes in the Hox gene expression pattern, suggesting that they underlie region-specific differentiation within the limb bud. Many other more diverged homeobox genes are present in the vertebrate genome, lying separate from the Hox clusters. These include engruiled (en), paired @rd), orthodenticle (otd), empty spirucles (ems), buttonheud, distul-less (dlx), NK, cuudul (cdx), and even skipped (Ev,K) (named after their Drosophila genes in which specific homeodomain sequences were initially found). Furthermore, while the overlapping Hox gene expression patterns cover the hindbrain and structures posterior to it, some of these genes (en, otd, ems, dlx) are expressed in the most anterior regions of the embryo, including the forebrain, midbrain, and associated structures. Homeobox genes have also been isolated that probably control axial specification during and just after gastrulation. In Xenopus embryos, goosecoid, Xlim-I, Xnot, Siumois, Xotx2, and Xlub are all expressed in the dorsal lip of the blastopore, the organizing centre of the embryo that is homologous to Hensen’s node of other vertebrates. Microinjection of goosecoid mRNA can duplicate the embryonic axis, suggesting strongly that goosecoid is one of the central molecules in axial determination. Strong connections exist between peptide growth factor-induced embryonic induction (e.g., BFGF, activin) and homeobox gene activation. For example, activin treatment of presumptive ectodermal cells switches them to a mesodermal fate and activin-treated cells can organize a fairly complete A-P axis after transplantation into another embryo. One would predict that genes control-
Pattern formation
ling the first steps of mesodermal specification are activated as a primary response to the growth factor signal (i.e., in the presence of protein synthesis inhibitors), and this is indeed the case for goosecoid and Xlim. Xlim appears to be unique in that it is also activated by RA alone. With respect to homeobox genes, the basic observation from which various assumptions and hypotheses evolved is the colinearity of homeobox gene position within the gene cluster in the chromosome and their expression along the body axis. It has been taken for a fact that this colinearity must have mechanistic significance. However, while this presumption has been a key rationale in many studies of the role of Hox genes in pattern formation, the significance, if any, of this colinearity has not really been satisfactorily clarified. Several gaps in our knowledge of how homeobox genes control development remain to be filled. What are the targets of homeodomain proteins in vivo-genes encoding other general transcription factors, or structural genes, or both? Do they act as part of a multifactor complex, and is redundancy of function built into the system? More directly related to the developmental toxicology problem are questions such as: How does each gene become activated and inactivated at the right time and place? What agents can adversely affect this program, especially for the anteriorly expressed genes, and how subtle or drastic are the effects? What is the best way to rapidly, reliably, and accurately assay these changes? Correlating morphogenetic defects with expression patterns using in situ hybridization may be one way. It will also be useful to undertake higher resolution immunohistochemical studies. Comparative molecular embryologic studies led to the conclusion that the basic principles and molecules involved in early vertebrate embryogenesis are extremely conserved from frogs to birds to mammals. Rapidly testing different agents on amphibian embryos, produced at will in biochemical quantities, could be a feasible approach. Also, the use of amphibian embryos would allow the testing at precise stages of development. A considerable amount of detailed work needs to be done to answer outstanding questions about the evolution and significance of the four Hox clusters in mammals. In addition, it is necessary to ask whether other genes and gene families encoding DNA-binding proteins play a role in encoding positional information in parts of the embryo where homeobox genes are not expressed. Studies of the expression of different members of the homeobox gene families in the mouse embryo at the level of the individual cell could be undertaken with emphasis placed on parts of the mouse embryo where extensive and complex morphogenesis is taking place, for example, in the craniofacial region and in developing
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limbs. This would necessitate generating a panel of polyclonal and monoclonal antibodies to homeodomain proteins or fusion proteins. In addition, it would be useful to have lines of transgenic mice expressing reporter genes in identical patterns to the endogenous homeobox genes. In many cases these could be generated by homologous recombination in ES cells, inserting 1acZ in place of the coding sequence of the homeobox gene. The antibodies and transgenic reporter lines could be used to rapidly assay for teratogenic chemicals that alter the anteriorposterior pattern of the embryo. Similar studies might also be carried out with homeobox genes and genes encoding other families of transcription factors expressed in the very anterior (and possibly posterior) section of the embryo, outside the regions where the four Hox gene clusters are normally expressed. A coordinated and interdisciplinary effort could also be made to analyze morphologic defects in homeobox gene knock-out mice. For example, it would be interesting to analyze all members of one Hox cluster and paralogs within all four clusters. Such analyses would be optimized if antibodies and 1acZ reporter mice were available as described above. THE ROLE OF GROWTH VERTEBRATE PATTERN
FACTORS IN FORMATION
Seventy years ago the landmark experiments of Spemann and Mangold on amphibian embryos established intercellular signaling as a major element in vertebrate development, challenging the long-held view that the development of the egg required only prelocalized determinants. Spemann further recognized that a small region of the embryonic mesoderm, which he named the organizer, was able to regulate most of the early development of the amphibian embryo through a process termed induction. For the ensuing 65 years, the identity of the signaling molecules utilized in this inductive process have been a major mystery, but recent efforts have identified several of these factors. The factors identified to date are small secreted proteins often referred to as growth factors, and the retinoids. The finding that purified polypeptide growth factors can induce specific kinds of mesoderm from the pluripotential cells of the early amphibian embryo has had a powerful catalyzing effect on the study of early vertebrate development. It is now known that a number of growth factor gene families are expressed in amphibian, mouse, avian, and zebrafish embryos, and considerable effort is being devoted to identifying new members of these families and to defining the precise role that each member plays in the overall developmental program. The growth factors fall principally into two groups: factors that can induce ectoderm to differentiate as a variety of mesodermal tissues, and factors that can regu-
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late the type of mesoderm that forms but cannot cause mesodermal differentiation. The two members of the mesoderm-inducing group are fibroblast growth factor (FGF) and activin, which belong to the transforming growth factor-p (TGFB) superfamily. Members of the regulatory group are various related factors called wnts, BMP-4, chordin, and noggin. Recently it has been shown that chordin and noggin act by binding and inhibiting BMP4. BMP4 can also induce mesodetm, but this may be an effect of high level overexpression. Through a yet poorly understood synergistic process these factors are thought to induce the formation of the various mesoderma1 cell types at the equator of the amphibian embryo in a defined pattern that includes the specification of the organizer at the most dorsal part of the equator. The role of these various factors in amphibian development is being studied by several means. Purified factors can be added to ectodermal explants of the early embryo, which serve as a model system for mesoderm induction. Alternatively, RNA encoding these factors can be injected into the early embryo to study their role in embryogenesis. Another approach has been to create dominant-negative mutants of the receptors for these factors, which, by blocking the endogenous pathway of these factors, reveals their in vivo role. These approaches have been limited by the inability to recognize the results of various perturbations because the early embryo does not have many clear morphologic features or differentiated tissues. However, a number of marker genes have been identified that permit the analysis of various perturbations while the embryo is still in the very early stages. It is likely that some teratogens will disrupt normal development by interfering with specific steps in the pathway between synthesis and secretion of polypeptide factors, and with their binding to receptors and the generation of intracellular signals. It is, therefore, important to learn as much as possible about the various proteins that act as morphogens in the early embryo and about their receptors and binding factors. New members of known polypeptide growth factor families need to be identified, for example, by low stringency hybridization to cDNA libraries made from early embryos and/or specific tissues, or by RT-PCR with degenerate primers to conserved regions. New growth factor receptors and binding proteins may also be identified using similar strategies or alternative strategies based on “rescue” of experimentally dorsalized or ventralized amphibian embryos. Elucidation of the intracellular signalling pathways activated during embryonic induction is also important as are investigations of the effects of adding combinations of growth factors to embryonic cells rather than (or in addition to) the effects of single factors. It is likely that in viva pluripotent cells will be exposed to a
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combination of inducing factors. Varying the relative amount of each different factor in a model induction system may reveal underlying rules about intracellular signalling pathways and thresholds for switching between alternate phenotypes. THE ROLE OF RETINOIC ACID AND ITS DERIVATIVES IN EMBRYONIC PATTERN FORMATION Retinoids are small hydrophobic molecules that are found endogenously in vertebrate embryos [reviewed in (1,2)]. Retinoid signaling is extraordinarily versatile and very complex [reviewed in (3,4)]. Retinoids signal by binding to specific nuclear receptors that are members of a superfamily of eukaryotic transcription factors. There are two types of retinoid receptors: the retinoic acid receptors and the retinoid X receptors. Three isoforms of each receptor type are known, and referred to as retinoic acid receptor (Y, B, y (RARo, B, y) and retinoid X receptor (Y, l3, y (RXRo, B, y). Receptors form homodimers (RXR-RXR) or heterodimers (RXR-RAR), which bind to specific DNA sequences known as retinoid response elements (RAREs, RXREs). Response elements are either direct repeats, inverted repeats, or everted repeats of the hexameric core motif (AGGTCA) separated by a varying number of nucleotide spacers (5). It is proposed that a nuclear receptor corepressor protein is bound to unliganded RAR, thereby inhibiting activation of transcription. A ligand-induced conformational change releases the corepressor from the receptor and allows the binding of coactivator proteins, which then leads to the activation of the target gene (3). Retinoids function as extracellular signals that enter a cell and activate a signaling cascade through binding to their receptors. An elegant bioassay demonstrating this mechanism of action is provided by indicator cells that express retinoid receptors and, in addition, a 1acZ gene under the control of a retinoic acid responsive element (6). When these cells are exposed to exogenous RA or placed in contact with a tissue that releases retinoids, they produce B-galactosidase, which can be revealed by a color reaction. The retinoid signaling system is thus suitable to translate an extracellular signal, retinoic acid, or related retinoids into the expression of specific genes. This type of mechanism in which cells respond to long or medium range cues, is thought to be important in pattern formation. Retinoids have striking effects on morphogenesis and can function as powerful teratogens during development. For example, the exogenous application of alltrans-retinoic acid (atRA) to avian and mammalian embryos has profound effects on axial structures and has been the basis for a considerable number of experiments
Pattern formation 0 R. in vertebrate pattern formation [for a review see (1,2)]. The local application of atRA to the chick limb bud will induce digit pattern duplications, mimicking the effect of grafts of the zone of polarizing activity (7,8). This observation in conjunction with the presence of endogenous atRA in the limb bud (9) has led to the idea that retinoids are, or at last mimic, the morphogen thought to be released by the zone of polarizing activity (10,ll). It has recently been shown that digit pattern duplications are also evoked by ectopically produced Sonic hedgehog protein, a secreted signaling molecule expressed in the polarizing region (12,13). Interestingly, atRA induces Sonic hedgehog expression (12,14), implicating retinoids in the regulation of this gene. Further evidence for this hypothesis was recently provided by Helms et al. (15), who showed that blocking the retinoid signaling pathway with retinoid receptor antagonists abolishes Sonic hedgehog expression and results in patterning defects. Taken together, these observations support a model in which the retinoid signaling is upsteam of Sonic hedgehog. This model is also consistent with the observation that atRA can induce an ectopic polarizing region (16,17). Whether retinoids also operate directly as morphogens in the limb bud remains to be determined, but the availability of retinoid antagonists should make it possible to address this question. With respect to the role of retinoic acid during early development, it has been shown that retinoids are present during gastrulation and that they are synthesized by the cells of the avian Hensen’s node and the dorsal lip of the amphibian blastopore [reviewed in (1,2)]. It is possible that atRA produced by Hensen’s node signals to the cells of the gastrulating embryo and thereby divides the embryo into developmental units, including a retinoic acidpositive trunk region, and a retinoic acid-negative head region. This subdivision may be mediated by members of the family of Hox genes and by 0tx2 (a vertebrate homolog of the Drosophila head-specific homeobox gene, orthodenticle) [see (2) for more details about this model]. Thus, in the early embryo retinoids may act as morphogenetic agents that regulate the expression of genes involved in the establishment of the anteroposterior body axis. An important approach to determining which developmental processes require retinoid signaling is provided by inactivation of retinoid receptors in mice through homologous recombination. This line of investigation revealed the enormous complexity of retinoid-dependent signal transduction in vivo. Morphologic defects in almost any developmental process can be observed, if the appropriate compound mutations are generated [see (4) for a review]. Such gene targeting studies have also demonstrated that a particular retinoid receptor type mediates retinoid teratogenicity. For example, in the case of limb
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malformations, mice lacking RXR~Y are resistant to retinoic acid-induced limb defects (18). In summary, there are now several powerful tools available to determine the physiologic and toxicologic effects of retinoids during the development of the vertebrate embryo. These include mutations in the genes encoding the components of the retinoid signaling pathway (4), receptor-selective retinoid agonists (19), retinoid receptor antagonists (15,19), and a wide spectrum of genes, the expression of which is regulated by retinoids. REFERENCES I. Hofmann C, Eichele G. Retinoids in development. In: Spom MB, Roberts AB, Goodman DS, eds. The retinoids, biology, chemistry and medicine. New York: Raven Press; 1994:387441. 2. Conlon RA. Retinoic acid and pattern formation in vertebrates. Trends Genet. 1995;11:314-9. 3. Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell. 1995;83:841-50. 4 Kastner P, Mark M, Chamhon P. Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell. 1995:83:859%69. 5 Umesono K, Murakami KK, Thompson CC, Evans RM. Direct repeats as response elements for the thyroid hormone, retinoic acid, vitamin D3 receptors. Cell. 1991;65:125546. 6 Wagner M, Han B, Jesse11 TM. Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay. Development. 19921 lti55-66. 7 Tickle C, Alberts B, Wolpert L, Lee J. Local application of retinoic acid to the limb bud mimics the action of the polarizing region. Nature. 1982;296:564-6. 8 Summerbell D. The effect of local application of retinoic acid to the anterior margin of the developing chick limb. J Embryo1 Exp Morphol. 1983;78:269-89. 9 Thaller C, Eichele G. Identification and spatial distribution of retinoids in the developing chick limb bud. Nature. 1987;327:625-8. 10 Saunders JW, Gasseling MT. Ectodermal-mesenchymal interactions in the origin of limb symmetry. In: Fleischmajer R, Billingham E, eds. Epithelial-mesenchymal interactions. Baltimore, MD: Williams and Wilkins; 1968:78-97. 11 Wolpert L. Positional information and pattern formation. Curr Top Dev. 1971;6:183-224. 12 Riddle RD, Johnson RL, Laufer E, Tabin C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell. 1993;75:1401-16. 13. L6pez-Martinez A, Chang DT, Chiang C, Porter JA, Ros MA, Simandl BK, Beachy PA, Fallon J. Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage. Curr Biol. 1995;5:791-6. 14. Helms J, Thaller C, Eichele G. Relationship between retinoic acid and sonic hedgehog, two polarizing signals in the chick wing bud. Development. 1994; 120:3267-74. 15. Helms JA, Kim CH, Eichele G, Thaller C. Retinoic acid signaling is required for chick limb development. Development. 1996; 122: 1385-94. 16. Noji S, Nohno T, Koyama E, Muto K, Ohyama K, Yoshinobu A, Tamura K, Ohsugi K, Ide H, Taniguchi S, Saito T. Retinoic acid induces polarizing activity but is unlikely to be a morphogen in the chick limb bud. Nature. 1991;350:83-6. 17. Wanek N, Gardiner DM, Muneoka K, Bryant SV. Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing bud. Nature. 1991;350:81-3.
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18. Sucov HM, Izpisua-Belmonte J-C, Gaiian Y, Evans RM. Mouse embryos lacking RXRo are resistant to retinoic-acid-induced limb defects. Development. 1995; 121:3997-4003. 19. Chen JY, Clifford J, Zusi C, Starrett J, Tortolani D, Ostrowski J, Reczek PR, Chambon P, Gronemeyer H. Two distinct actions of retinoid-receptor ligands. Nature. 1995;382:819-22.
OTHER REFERENCES Beato M. Gene regulation
by steroid hormones.
Cell. 1989;56:33544.
Boncinelli E, Simeone A, Acampora D, Mavlio F. HOX gene activation by retinoic acid. Trends Genet. 1991;7:329-34. Chisaka 0, Capecchi M. Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene Hox-1.5. Nature. 1991;350:473-9. Chisaka 0, Musci TR, Capecchi MR. Targeted disruption of the mouse homeobox gene Hox-1.6 results in developmental defects of the ear, cranial nerves and hindbrain. Nature. 1992;355:516-20. Evans RM. The steroid and thyroid Science. 1988;240:889-95.
hormone
receptor
superfamily.
Giguere V, Ong ES, Segui P, Evans RM. Identification of a receptor for the morphogen retinoic acid. Nature. 1987;330:624-9. Heyman RA, Mangelsdorf DJ, Dyck JA, Stein R, Eichele G, Evans RM, Thaller C. Y-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell. 1992;68:397+%06. Hogan BL, Thaher C, Eichele G. Evidence that Hensen’ node is a site of retinoic acid synthesis. Nature. 1992;359:23741. Kessel M, Gruss P. Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell. 1991;67:89-104. Levin AA, Sturzenbecker LJ, Kazmer S, Bosakowski T, Huselton C, Allenby G, Speck J, Krataeisen C, Rosenberger M, Lovey A, Grippo JF. 9.cis-retinoic acid stereoisomer binds and activates the nuclear receptor RXRa. Nature. 1992;355:359-6 1. Lulkin T, Dierich A, Lemeur M, Mark M, Chambon P. Disruption of the Hox-1.6 homeobox gene results in defects in a region corresponding to its rostra] domain of expression. Cell. 1991;66:1105-19.
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Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM. Nuclear receptor that identifies a novel retinoic acid response pathway. Nature. 1990;345: 224-9. McCafferey P, Lee MO, Wagner MA, Sladek NE, Drager UC. Asymetrical retinoic acid synthesis in the dorsoventral axis of the retina. Development. 1992;115:371-82. Morris-Kay GM, Murphy P, Hill RE, Davidson DR. Effects of retinoic acid excess on expression of Hox 2.9 and krox-20 and on morphological segmentation in the hindbrain of mouse embryos. EMBO J. 1991;10:2985-95. Petkovich M, Brand NJ, Krust A, Chambon P. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature. 1987;330:444-50. Ruiz i Altaba A, Jesse11 TM. Retinoic acid modifies mesodermal patterning in early Xenopus embryos. Genes Dev. 1991;5:175-87. Ruiz i Altaba A, Jesse11 TM. Retinoic acid modifies the pattern of cell differentiation in the central nervous system of neurula stage Xenopus embryos. Development. 199 1; 112:94558. Sharpe CR. Retinoic acid can mimic endogenous signals involved in transformation of the Xenopus nervous system. Neuron. 1991;7:23947. Simeone A, Acampora D, Nigro V, Faiella A, D’Esposito M, Stomaiuolo A, Mavilio F, Boncinelli E. Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells. Mech Dev. 1991;33:215-28. Simeone A, Acampora D, Arcioni L, Andrew PW, Boncinelli E, Mavilio F. Sequential activation of HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells. Nature. 1990;346:763-6. Sive HL, Draper BW, Harland RL, Weintraub H. Identification of a retinoic acid-sensitive period during primary axis formation in Xenopus Levis. Genes Dev. 1990;4:93242. Sive HL, Cheng PF. Retinoic acid perturbs the expression of Xhox.lab genes and alters mesodermal determination in Xenapu.s laevis. Genes Dev 1991;5:1321-32. Thaller C, Eichele G. Isolation of 3,4_didehydroretinoic acid, a novel morphogenetic signal in the chick wing bud. Nature. 1990;345: 815-9. Wagner M, Thaller C, Jesse11 T, Eichele G. Polarizing activity and retinoid synthesis in the floor plate of the neural tube. Nature. 1990; 345:819-22.
PI1SOS90-6238(96)00217-l
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WORKSHOPS
PATTERN FORMATION RICK WOYCHIK,* BRIGID HOGAN,? SUSAN BRYANT,+ GREGOREICHELE,~ DAVID KIMELMAN,~ DREW NODEN,# GARY SCHOENWOLF,** and CHRISTOPHER
WRIGHT-/-
*Oak Ridge National Laboratory, Mammalian Genetics and Development Section, Oak Ridge, Tennessee tvanderbilt University Medical School, Department of Cell Biology, Nashville, Tennessee $University of California Irvine, Developmental Biology Center, Irvine, California §Baylor College of Medicine, Department of Biochemistry, Houston, Texas IUniversity of Washington, Department of Biochemistry, Seattle, Washington #Cornell University, College of Veterinary Medicine, Department of Anatomy, Ithaca, New York **University of Utah School of Medicine, Department Neurobiology and Anatomy, Salt Lake City, Utah
spreads over the egg cylinder to displace the primitive endoderm. Fate mapping experiments in the mouse have provided a general framework for understanding cell movements and lineage during gastrulation of the mammalian embryo. They support the idea that the node acts not only as an organizing center for mesoderm cells moving into it, but also as a localized region of proliferation for a resident population of stem cells. In situ hybridization studies have identified a set of mouse genes that are expressed in the primitive streak [e.g., T (Brachyury), FGF family members, FGF receptors], in the node (the TGFP-related gene, nodal) and in the notochord, presumptive somites, and more lateral mesoderm Cforkhead family members, mouse lim-1). Current conceptual models for how cellular diversity is established during mammalian gastrulation are almost entirely dominated by studies of mesoderm induction and specification in amphibian embryos, and of gastrulation movements and fate mapping in avian embryos. Comparative studies at the molecular level support the idea that the basic mechanisms underlying gastrulation and pattern formation have been highly conserved among vertebrates. Such studies have been extremely valuable in catalyzing new ideas and lines of investigation in the mouse. However, it is important not to lose sight of the fact that unique mechanisms may have evolved in the mammalian embryo, providing targets for teratogens that cannot be revealed in studies with other vertebrates. Moreover, the mouse provides the only opportunity to apply experimentally the ultimate genetic tests (deletion and misexpression) to hypotheses about gene function during mammalian embryogenesis. In view of the central importance of gastrulation to the establishment of the basic body plan of the mammalian embryo, and the great need for a molecular description of the underlying mechanisms to understand how
AXIS FORMATION IN MAMMALIAN EMBRYOS If we are to understand how toxic chemicals and teratogens disrupt embryonic development, it is crucial that we have a better description of the molecules and genes that control the normal program of growth, differentiation, and pattern formation. Fundamental to development is the establishment of the primary body axes (dorsalventral, anterior-posterior, and proximal-distal), yet surprisingly little is known about the underlying mechanisms in mammalian embryos. In the mouse, it is thought that the primary axes are set up some time around implantation and are first revealed in the process of gastrulation, which begins at approximately 6.5 d postcoitum (p.c.). Cells delaminate from a restricted region of the embryonic ectoderm, at what will be the posterior of the embryo, and form a layer of mesoderm between the ectoderm and the primitive endoderm. Over the next 12 h or so this region of mesodermal delamination, known as the primitive streak, extends along the anterior-posterior axis of the embryo to the distal tip of the cup-shaped ectoderm (the egg cylinder). By about 7 d p.c. the anterior of the primitive streak is morphologically distinguishable as the “node.” This structure is thought to be analogus to the dorsal lip of the amphibian blastopore and to Hensen’s node of the chick. Cells that emerge anteriorly in the midline from the node differentiate into notochord, while cells lateral to this develop into the presumptive somites and intermediate mesoderm. The definitive endoderm is also generated at the node, and it
Participants: Kathy Sulik, George D&on, Phil Mirkes, Waldy Generoso, Roger Pedersen, &role Kimmel. Address correspondence to Rick Woychik, Ph.D., Oak Ridge National Laboratory, Biology Division, P.O. Box 2009, Oak Ridge, TN 378314077. 339
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they are affected by teratogens, new genes that are expressed in different germ layers and regions of the gastrulation stage mouse embryo need to be identified. Such genes could throw light on cellular interactions and inductive processes, as well as provide molecular markers for different mesodermal lineages. A variety of strategies could be used to identify such new genes including 1) the search for mouse homologs of genes known to be expressed during Drosophila development or during gastrulation in amphibian and avian embryos, 2) differential screening of cDNA libraries made from different stages and cell populations of mouse embryos, and 3) promoter and gene trap experiments in ES cells using a reporter gene such as 1acZ for revealing gene expression in chimerit embryos. Positional cloning might be used to isolate defective genes in a number of preexisting mouse mutants that become disorganized around the time of gastrulation and during primary axis formation. A panel of insertional mutants that become disorganized around the time of gastrulation might also be generated. A good example of the power of this approach is the retroviral insertion mutant, 413-d, which disrupts the nodal gene and blocks mesoderm differentiation and axis formation. Finally, more lineage analysis in the mouse and chick embryo would be helpful in understanding relationships between cell position with respect to the node and specification of mesodermal phenotype. HOMEOBOX GENES AND PATTERN FORMATION The discovery that a cluster of Antennapedia class homeobox genes, expressed in a linear sequence along the anterior-posterior axis of the embryo, has been highly conserved throughout a large part of metazoan evolution represents one of the highlights of modern developmental biology. The temporal and spatial expression of these genes in highly organized structures such as the limb bud and genital tubercule also reflects their central role in pattern formation during development. Studies on the expression of the four Hox gene clusters in the mouse embryo, the effects of misexpressing specific genes in transgenic embryos, and the study of mutants in which specific genes have been inactivated, all support a model in which Hox genes provide a combinatorial code for positional information along the anterior-posterior axis. There is very good evidence that the homeotic selector genes of Drosophila (HOM genes), and the four clusters of vertebrate homeobox genes (Hox genes), are truly homologous complexes encoding region-specific transcription factors. The Hox and HOM clusters not only have similar organization, but in both, gene order is colinear with the spatiotemporal expression patterns in the anteroposterior (A-P) axis. Most comprehensively
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studied in the early mouse embryo, Hox genes are expressed in overlapping domains, differing by increments at their anterior border. This arrangement is thought to constitute a positional code for region-specific differentiation. The presence of four homologous clusters in vertebrates is presumably to allow for greater embryonic complexity, especially in the highly specialized central nervous system and the pluripotent neural crest. What controls the expression of the Hox genes in order down the A-P axis? In human teratocarcinoma cells, increased dose and time of exposure to retinoic acid (RA) results in sequential expression of the Hox genes. Linked to this observation are the findings that pulsed RA application causes gross phenotypic defects in the early body plan of Xenopus and zebrafish embryos (especially anterior truncations), and mice (including skeletal abnormalities and cleft palate), which have been correlated with changes in Hox gene expression patterns. Nested patterns of Hox cluster expression, similar to those seen in the main A-P embryonic axis, are also observed in the developing limb bud. Manipulations that disturb normal limb morphogenesis, including localized RA applications, can be correlated with, and preempted by, predictable changes in the Hox gene expression pattern, suggesting that they underlie region-specific differentiation within the limb bud. Many other more diverged homeobox genes are present in the vertebrate genome, lying separate from the Hox clusters. These include engruiled (en), paired @rd), orthodenticle (otd), empty spirucles (ems), buttonheud, distul-less (dlx), NK, cuudul (cdx), and even skipped (Ev,K) (named after their Drosophila genes in which specific homeodomain sequences were initially found). Furthermore, while the overlapping Hox gene expression patterns cover the hindbrain and structures posterior to it, some of these genes (en, otd, ems, dlx) are expressed in the most anterior regions of the embryo, including the forebrain, midbrain, and associated structures. Homeobox genes have also been isolated that probably control axial specification during and just after gastrulation. In Xenopus embryos, goosecoid, Xlim-I, Xnot, Siumois, Xotx2, and Xlub are all expressed in the dorsal lip of the blastopore, the organizing centre of the embryo that is homologous to Hensen’s node of other vertebrates. Microinjection of goosecoid mRNA can duplicate the embryonic axis, suggesting strongly that goosecoid is one of the central molecules in axial determination. Strong connections exist between peptide growth factor-induced embryonic induction (e.g., BFGF, activin) and homeobox gene activation. For example, activin treatment of presumptive ectodermal cells switches them to a mesodermal fate and activin-treated cells can organize a fairly complete A-P axis after transplantation into another embryo. One would predict that genes control-
Pattern formation
ling the first steps of mesodermal specification are activated as a primary response to the growth factor signal (i.e., in the presence of protein synthesis inhibitors), and this is indeed the case for goosecoid and Xlim. Xlim appears to be unique in that it is also activated by RA alone. With respect to homeobox genes, the basic observation from which various assumptions and hypotheses evolved is the colinearity of homeobox gene position within the gene cluster in the chromosome and their expression along the body axis. It has been taken for a fact that this colinearity must have mechanistic significance. However, while this presumption has been a key rationale in many studies of the role of Hox genes in pattern formation, the significance, if any, of this colinearity has not really been satisfactorily clarified. Several gaps in our knowledge of how homeobox genes control development remain to be filled. What are the targets of homeodomain proteins in vivo-genes encoding other general transcription factors, or structural genes, or both? Do they act as part of a multifactor complex, and is redundancy of function built into the system? More directly related to the developmental toxicology problem are questions such as: How does each gene become activated and inactivated at the right time and place? What agents can adversely affect this program, especially for the anteriorly expressed genes, and how subtle or drastic are the effects? What is the best way to rapidly, reliably, and accurately assay these changes? Correlating morphogenetic defects with expression patterns using in situ hybridization may be one way. It will also be useful to undertake higher resolution immunohistochemical studies. Comparative molecular embryologic studies led to the conclusion that the basic principles and molecules involved in early vertebrate embryogenesis are extremely conserved from frogs to birds to mammals. Rapidly testing different agents on amphibian embryos, produced at will in biochemical quantities, could be a feasible approach. Also, the use of amphibian embryos would allow the testing at precise stages of development. A considerable amount of detailed work needs to be done to answer outstanding questions about the evolution and significance of the four Hox clusters in mammals. In addition, it is necessary to ask whether other genes and gene families encoding DNA-binding proteins play a role in encoding positional information in parts of the embryo where homeobox genes are not expressed. Studies of the expression of different members of the homeobox gene families in the mouse embryo at the level of the individual cell could be undertaken with emphasis placed on parts of the mouse embryo where extensive and complex morphogenesis is taking place, for example, in the craniofacial region and in developing
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limbs. This would necessitate generating a panel of polyclonal and monoclonal antibodies to homeodomain proteins or fusion proteins. In addition, it would be useful to have lines of transgenic mice expressing reporter genes in identical patterns to the endogenous homeobox genes. In many cases these could be generated by homologous recombination in ES cells, inserting 1acZ in place of the coding sequence of the homeobox gene. The antibodies and transgenic reporter lines could be used to rapidly assay for teratogenic chemicals that alter the anteriorposterior pattern of the embryo. Similar studies might also be carried out with homeobox genes and genes encoding other families of transcription factors expressed in the very anterior (and possibly posterior) section of the embryo, outside the regions where the four Hox gene clusters are normally expressed. A coordinated and interdisciplinary effort could also be made to analyze morphologic defects in homeobox gene knock-out mice. For example, it would be interesting to analyze all members of one Hox cluster and paralogs within all four clusters. Such analyses would be optimized if antibodies and 1acZ reporter mice were available as described above. THE ROLE OF GROWTH VERTEBRATE PATTERN
FACTORS IN FORMATION
Seventy years ago the landmark experiments of Spemann and Mangold on amphibian embryos established intercellular signaling as a major element in vertebrate development, challenging the long-held view that the development of the egg required only prelocalized determinants. Spemann further recognized that a small region of the embryonic mesoderm, which he named the organizer, was able to regulate most of the early development of the amphibian embryo through a process termed induction. For the ensuing 65 years, the identity of the signaling molecules utilized in this inductive process have been a major mystery, but recent efforts have identified several of these factors. The factors identified to date are small secreted proteins often referred to as growth factors, and the retinoids. The finding that purified polypeptide growth factors can induce specific kinds of mesoderm from the pluripotential cells of the early amphibian embryo has had a powerful catalyzing effect on the study of early vertebrate development. It is now known that a number of growth factor gene families are expressed in amphibian, mouse, avian, and zebrafish embryos, and considerable effort is being devoted to identifying new members of these families and to defining the precise role that each member plays in the overall developmental program. The growth factors fall principally into two groups: factors that can induce ectoderm to differentiate as a variety of mesodermal tissues, and factors that can regu-
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late the type of mesoderm that forms but cannot cause mesodermal differentiation. The two members of the mesoderm-inducing group are fibroblast growth factor (FGF) and activin, which belong to the transforming growth factor-p (TGFB) superfamily. Members of the regulatory group are various related factors called wnts, BMP-4, chordin, and noggin. Recently it has been shown that chordin and noggin act by binding and inhibiting BMP4. BMP4 can also induce mesodetm, but this may be an effect of high level overexpression. Through a yet poorly understood synergistic process these factors are thought to induce the formation of the various mesoderma1 cell types at the equator of the amphibian embryo in a defined pattern that includes the specification of the organizer at the most dorsal part of the equator. The role of these various factors in amphibian development is being studied by several means. Purified factors can be added to ectodermal explants of the early embryo, which serve as a model system for mesoderm induction. Alternatively, RNA encoding these factors can be injected into the early embryo to study their role in embryogenesis. Another approach has been to create dominant-negative mutants of the receptors for these factors, which, by blocking the endogenous pathway of these factors, reveals their in vivo role. These approaches have been limited by the inability to recognize the results of various perturbations because the early embryo does not have many clear morphologic features or differentiated tissues. However, a number of marker genes have been identified that permit the analysis of various perturbations while the embryo is still in the very early stages. It is likely that some teratogens will disrupt normal development by interfering with specific steps in the pathway between synthesis and secretion of polypeptide factors, and with their binding to receptors and the generation of intracellular signals. It is, therefore, important to learn as much as possible about the various proteins that act as morphogens in the early embryo and about their receptors and binding factors. New members of known polypeptide growth factor families need to be identified, for example, by low stringency hybridization to cDNA libraries made from early embryos and/or specific tissues, or by RT-PCR with degenerate primers to conserved regions. New growth factor receptors and binding proteins may also be identified using similar strategies or alternative strategies based on “rescue” of experimentally dorsalized or ventralized amphibian embryos. Elucidation of the intracellular signalling pathways activated during embryonic induction is also important as are investigations of the effects of adding combinations of growth factors to embryonic cells rather than (or in addition to) the effects of single factors. It is likely that in viva pluripotent cells will be exposed to a
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combination of inducing factors. Varying the relative amount of each different factor in a model induction system may reveal underlying rules about intracellular signalling pathways and thresholds for switching between alternate phenotypes. THE ROLE OF RETINOIC ACID AND ITS DERIVATIVES IN EMBRYONIC PATTERN FORMATION Retinoids are small hydrophobic molecules that are found endogenously in vertebrate embryos [reviewed in (1,2)]. Retinoid signaling is extraordinarily versatile and very complex [reviewed in (3,4)]. Retinoids signal by binding to specific nuclear receptors that are members of a superfamily of eukaryotic transcription factors. There are two types of retinoid receptors: the retinoic acid receptors and the retinoid X receptors. Three isoforms of each receptor type are known, and referred to as retinoic acid receptor (Y, B, y (RARo, B, y) and retinoid X receptor (Y, l3, y (RXRo, B, y). Receptors form homodimers (RXR-RXR) or heterodimers (RXR-RAR), which bind to specific DNA sequences known as retinoid response elements (RAREs, RXREs). Response elements are either direct repeats, inverted repeats, or everted repeats of the hexameric core motif (AGGTCA) separated by a varying number of nucleotide spacers (5). It is proposed that a nuclear receptor corepressor protein is bound to unliganded RAR, thereby inhibiting activation of transcription. A ligand-induced conformational change releases the corepressor from the receptor and allows the binding of coactivator proteins, which then leads to the activation of the target gene (3). Retinoids function as extracellular signals that enter a cell and activate a signaling cascade through binding to their receptors. An elegant bioassay demonstrating this mechanism of action is provided by indicator cells that express retinoid receptors and, in addition, a 1acZ gene under the control of a retinoic acid responsive element (6). When these cells are exposed to exogenous RA or placed in contact with a tissue that releases retinoids, they produce B-galactosidase, which can be revealed by a color reaction. The retinoid signaling system is thus suitable to translate an extracellular signal, retinoic acid, or related retinoids into the expression of specific genes. This type of mechanism in which cells respond to long or medium range cues, is thought to be important in pattern formation. Retinoids have striking effects on morphogenesis and can function as powerful teratogens during development. For example, the exogenous application of alltrans-retinoic acid (atRA) to avian and mammalian embryos has profound effects on axial structures and has been the basis for a considerable number of experiments
Pattern formation 0 R. in vertebrate pattern formation [for a review see (1,2)]. The local application of atRA to the chick limb bud will induce digit pattern duplications, mimicking the effect of grafts of the zone of polarizing activity (7,8). This observation in conjunction with the presence of endogenous atRA in the limb bud (9) has led to the idea that retinoids are, or at last mimic, the morphogen thought to be released by the zone of polarizing activity (10,ll). It has recently been shown that digit pattern duplications are also evoked by ectopically produced Sonic hedgehog protein, a secreted signaling molecule expressed in the polarizing region (12,13). Interestingly, atRA induces Sonic hedgehog expression (12,14), implicating retinoids in the regulation of this gene. Further evidence for this hypothesis was recently provided by Helms et al. (15), who showed that blocking the retinoid signaling pathway with retinoid receptor antagonists abolishes Sonic hedgehog expression and results in patterning defects. Taken together, these observations support a model in which the retinoid signaling is upsteam of Sonic hedgehog. This model is also consistent with the observation that atRA can induce an ectopic polarizing region (16,17). Whether retinoids also operate directly as morphogens in the limb bud remains to be determined, but the availability of retinoid antagonists should make it possible to address this question. With respect to the role of retinoic acid during early development, it has been shown that retinoids are present during gastrulation and that they are synthesized by the cells of the avian Hensen’s node and the dorsal lip of the amphibian blastopore [reviewed in (1,2)]. It is possible that atRA produced by Hensen’s node signals to the cells of the gastrulating embryo and thereby divides the embryo into developmental units, including a retinoic acidpositive trunk region, and a retinoic acid-negative head region. This subdivision may be mediated by members of the family of Hox genes and by 0tx2 (a vertebrate homolog of the Drosophila head-specific homeobox gene, orthodenticle) [see (2) for more details about this model]. Thus, in the early embryo retinoids may act as morphogenetic agents that regulate the expression of genes involved in the establishment of the anteroposterior body axis. An important approach to determining which developmental processes require retinoid signaling is provided by inactivation of retinoid receptors in mice through homologous recombination. This line of investigation revealed the enormous complexity of retinoid-dependent signal transduction in vivo. Morphologic defects in almost any developmental process can be observed, if the appropriate compound mutations are generated [see (4) for a review]. Such gene targeting studies have also demonstrated that a particular retinoid receptor type mediates retinoid teratogenicity. For example, in the case of limb
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malformations, mice lacking RXR~Y are resistant to retinoic acid-induced limb defects (18). In summary, there are now several powerful tools available to determine the physiologic and toxicologic effects of retinoids during the development of the vertebrate embryo. These include mutations in the genes encoding the components of the retinoid signaling pathway (4), receptor-selective retinoid agonists (19), retinoid receptor antagonists (15,19), and a wide spectrum of genes, the expression of which is regulated by retinoids. REFERENCES I. Hofmann C, Eichele G. Retinoids in development. In: Spom MB, Roberts AB, Goodman DS, eds. The retinoids, biology, chemistry and medicine. New York: Raven Press; 1994:387441. 2. Conlon RA. Retinoic acid and pattern formation in vertebrates. Trends Genet. 1995;11:314-9. 3. Mangelsdorf DJ, Evans RM. The RXR heterodimers and orphan receptors. Cell. 1995;83:841-50. 4 Kastner P, Mark M, Chamhon P. Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell. 1995:83:859%69. 5 Umesono K, Murakami KK, Thompson CC, Evans RM. Direct repeats as response elements for the thyroid hormone, retinoic acid, vitamin D3 receptors. Cell. 1991;65:125546. 6 Wagner M, Han B, Jesse11 TM. Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay. Development. 19921 lti55-66. 7 Tickle C, Alberts B, Wolpert L, Lee J. Local application of retinoic acid to the limb bud mimics the action of the polarizing region. Nature. 1982;296:564-6. 8 Summerbell D. The effect of local application of retinoic acid to the anterior margin of the developing chick limb. J Embryo1 Exp Morphol. 1983;78:269-89. 9 Thaller C, Eichele G. Identification and spatial distribution of retinoids in the developing chick limb bud. Nature. 1987;327:625-8. 10 Saunders JW, Gasseling MT. Ectodermal-mesenchymal interactions in the origin of limb symmetry. In: Fleischmajer R, Billingham E, eds. Epithelial-mesenchymal interactions. Baltimore, MD: Williams and Wilkins; 1968:78-97. 11 Wolpert L. Positional information and pattern formation. Curr Top Dev. 1971;6:183-224. 12 Riddle RD, Johnson RL, Laufer E, Tabin C. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell. 1993;75:1401-16. 13. L6pez-Martinez A, Chang DT, Chiang C, Porter JA, Ros MA, Simandl BK, Beachy PA, Fallon J. Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage. Curr Biol. 1995;5:791-6. 14. Helms J, Thaller C, Eichele G. Relationship between retinoic acid and sonic hedgehog, two polarizing signals in the chick wing bud. Development. 1994; 120:3267-74. 15. Helms JA, Kim CH, Eichele G, Thaller C. Retinoic acid signaling is required for chick limb development. Development. 1996; 122: 1385-94. 16. Noji S, Nohno T, Koyama E, Muto K, Ohyama K, Yoshinobu A, Tamura K, Ohsugi K, Ide H, Taniguchi S, Saito T. Retinoic acid induces polarizing activity but is unlikely to be a morphogen in the chick limb bud. Nature. 1991;350:83-6. 17. Wanek N, Gardiner DM, Muneoka K, Bryant SV. Conversion by retinoic acid of anterior cells into ZPA cells in the chick wing bud. Nature. 1991;350:81-3.
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18. Sucov HM, Izpisua-Belmonte J-C, Gaiian Y, Evans RM. Mouse embryos lacking RXRo are resistant to retinoic-acid-induced limb defects. Development. 1995; 121:3997-4003. 19. Chen JY, Clifford J, Zusi C, Starrett J, Tortolani D, Ostrowski J, Reczek PR, Chambon P, Gronemeyer H. Two distinct actions of retinoid-receptor ligands. Nature. 1995;382:819-22.
OTHER REFERENCES Beato M. Gene regulation
by steroid hormones.
Cell. 1989;56:33544.
Boncinelli E, Simeone A, Acampora D, Mavlio F. HOX gene activation by retinoic acid. Trends Genet. 1991;7:329-34. Chisaka 0, Capecchi M. Regionally restricted developmental defects resulting from targeted disruption of the mouse homeobox gene Hox-1.5. Nature. 1991;350:473-9. Chisaka 0, Musci TR, Capecchi MR. Targeted disruption of the mouse homeobox gene Hox-1.6 results in developmental defects of the ear, cranial nerves and hindbrain. Nature. 1992;355:516-20. Evans RM. The steroid and thyroid Science. 1988;240:889-95.
hormone
receptor
superfamily.
Giguere V, Ong ES, Segui P, Evans RM. Identification of a receptor for the morphogen retinoic acid. Nature. 1987;330:624-9. Heyman RA, Mangelsdorf DJ, Dyck JA, Stein R, Eichele G, Evans RM, Thaller C. Y-cis retinoic acid is a high affinity ligand for the retinoid X receptor. Cell. 1992;68:397+%06. Hogan BL, Thaher C, Eichele G. Evidence that Hensen’ node is a site of retinoic acid synthesis. Nature. 1992;359:23741. Kessel M, Gruss P. Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell. 1991;67:89-104. Levin AA, Sturzenbecker LJ, Kazmer S, Bosakowski T, Huselton C, Allenby G, Speck J, Krataeisen C, Rosenberger M, Lovey A, Grippo JF. 9.cis-retinoic acid stereoisomer binds and activates the nuclear receptor RXRa. Nature. 1992;355:359-6 1. Lulkin T, Dierich A, Lemeur M, Mark M, Chambon P. Disruption of the Hox-1.6 homeobox gene results in defects in a region corresponding to its rostra] domain of expression. Cell. 1991;66:1105-19.
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2/3, 1997
Mangelsdorf DJ, Ong ES, Dyck JA, Evans RM. Nuclear receptor that identifies a novel retinoic acid response pathway. Nature. 1990;345: 224-9. McCafferey P, Lee MO, Wagner MA, Sladek NE, Drager UC. Asymetrical retinoic acid synthesis in the dorsoventral axis of the retina. Development. 1992;115:371-82. Morris-Kay GM, Murphy P, Hill RE, Davidson DR. Effects of retinoic acid excess on expression of Hox 2.9 and krox-20 and on morphological segmentation in the hindbrain of mouse embryos. EMBO J. 1991;10:2985-95. Petkovich M, Brand NJ, Krust A, Chambon P. A human retinoic acid receptor which belongs to the family of nuclear receptors. Nature. 1987;330:444-50. Ruiz i Altaba A, Jesse11 TM. Retinoic acid modifies mesodermal patterning in early Xenopus embryos. Genes Dev. 1991;5:175-87. Ruiz i Altaba A, Jesse11 TM. Retinoic acid modifies the pattern of cell differentiation in the central nervous system of neurula stage Xenopus embryos. Development. 199 1; 112:94558. Sharpe CR. Retinoic acid can mimic endogenous signals involved in transformation of the Xenopus nervous system. Neuron. 1991;7:23947. Simeone A, Acampora D, Nigro V, Faiella A, D’Esposito M, Stomaiuolo A, Mavilio F, Boncinelli E. Differential regulation by retinoic acid of the homeobox genes of the four HOX loci in human embryonal carcinoma cells. Mech Dev. 1991;33:215-28. Simeone A, Acampora D, Arcioni L, Andrew PW, Boncinelli E, Mavilio F. Sequential activation of HOX2 homeobox genes by retinoic acid in human embryonal carcinoma cells. Nature. 1990;346:763-6. Sive HL, Draper BW, Harland RL, Weintraub H. Identification of a retinoic acid-sensitive period during primary axis formation in Xenopus Levis. Genes Dev. 1990;4:93242. Sive HL, Cheng PF. Retinoic acid perturbs the expression of Xhox.lab genes and alters mesodermal determination in Xenapu.s laevis. Genes Dev 1991;5:1321-32. Thaller C, Eichele G. Isolation of 3,4_didehydroretinoic acid, a novel morphogenetic signal in the chick wing bud. Nature. 1990;345: 815-9. Wagner M, Thaller C, Jesse11 T, Eichele G. Polarizing activity and retinoid synthesis in the floor plate of the neural tube. Nature. 1990; 345:819-22.