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The neural crest, a multipotent embryonic cell type, originates at the border between neural and nonneural ectoderm. After neural tube closure, these cells undergo an epithelial--mesenchymal transition, migrate to precise, often distant locations, and differentiate into diverse derivatives. Analyses of expression and function of signaling and transcription factors in higher vertebrates has led to the proposal that a neural crest gene regulatory network (NC-GRN) orchestrates neural crest formation. Here, we interrogate the NC-GRN in the lamprey, taking advantage of its slow development and basal phylogenetic position to resolve early inductive events, 1 regulatory step at the time. To establish regulatory relationships at the neural plate border, we assess relative expression of 6 neural crest network genes and effects of individually perturbing each on the remaining 5. The results refine an upstream portion of the NC-GRN and reveal unexpected order and linkages therein; e.g., lamprey AP-2 appears to function early as a neural plate border rather than a neural crest specifier and in a pathway linked to MsxA but independent of ZicA. These findings provide an ancestral framework for performing comparative tests in higher vertebrates in which network linkages may be more difficult to resolve because of their rapid development. neural plate border | transcription factor | agnathan

Transcription factors regulate tissue patterning and cell fate determination during development; however, expression of early regulators frequently abates upon differentiation, suggesting that they may also play a role in maintaining an undifferentiated phenotype. The transcription factor paired box 3 (Pax3) is expressed by multipotent neural crest precursors and is implicated in neural crest disorders in humans such as Waardenburg syndrome. Pax3 is required for development of multiple neural crest lineages and for activation of lineage-specific programs, yet expression is generally extinguished once neural crest cells migrate from the dorsal neural tube and differentiate. Using a murine Cre-inducible system, we asked whether persistent Pax3 expression in neural crest derivatives would affect development or patterning. We found that persistent expression of Pax3 in cranial neural crest cells resulted in cleft palate, ocular defects, malformation of the sphenoid bone, and perinatal lethality. Furthermore, we demonstrated that Pax3 directly regulates expression of Sostdc1, a soluble inhibitor of bone morphogenetic protein (BMP) signaling. Persistent Pax3 expression renders the cranial crest resistant to BMP-induced osteogenesis. Thus, one mechanism by which Pax3 maintains the undifferentiated state of neural crest mesenchyme may be to block responsiveness to differentiation signals from the environment. These studies provide in vivo evidence for the importance of Pax3 downregulation during differentiation of multipotent neural crest precursors and cranial development., Introduction Among the most intriguing discoveries in developmental biology in the last century has been the identification of powerful developmentally regulated transcription factors that regulate patterning and cell-fate determination during [...]

The Noelin-4 extracellular protein's neural precursor expansion activity is reversed by Noelin-1 co-expression, indicating that the interaction of Noelin isoforms may help regulate timing of differentiation.

Cranial neural crest cells (NCCs) migrate into the pharyngeal arches in three primary streams separated by two cranial neural crest (NC)-free zones. Multiple tissues have been implicated in the guidance of cranial NCC migration; however, the signals provided by these tissues have remained elusive. We investigate the function of semaphorins (semas) and their receptors, neuropilins (nrps), in cranial NCC migration in zebrafish. We fred that genes of the sema3F and sema3G class are expressed in the cranial NC-free zones, while nrp2a and nrp2b are expressed in the migrating NCCs. sema3F/3G expression is expanded homogeneously in the head periphery through which the cranial NCCs migrate in lzr/pbx4 mutants, in which the cranial NC streams are fused. Antisense morpholino knockdown of Sema3F/3G or Nrp2 suppresses the abnormal cranial NC phenotype of lzr/pbx4 mutants, demonstrating that aberrant Sema3F/3G-Nrp2 signaling is responsible for this phenotype and suggesting that repulsive Sema3F/3G-Npn2 signaling normally contributes to the guidance of migrating cranial NCCs. Furthermore, global over-expression of sema3Gb phenocopies the aberrant cranial NC phenotype of lzr/pbx4 mutants when endogenous Sema3 ligands are knocked down, consistent with a model in which the patterned expression of Sema3 ligands in the head periphery coordinates the migration of Nrp-expressing cranial NCCs. Keywords: Cranial neural crest migration; Pbx; Neuropilin; Semaphorin

Cranial suture development involves a complex interaction of genes and tissues derived from neural crest cells (NCC) and paraxial mesoderm. In mice, the posterior frontal (PF) suture closes during the first month of life while other sutures remain patent throughout the life of the animal. Given the unique NCC origin of PF suture complex (analogous to metopic suture in humans), we performed quantitative real-time PCR and immunohistochemistry to study the expression pattern of the NCC determinant gene Sox9 and select markers of extracellular matrix. Our results indicated a unique up-regulated expression of Sox9, a regulator of chondrogenesis, during initiation of PF suture closure, along with the expression of specific cartilage markers (Type II Collagen and Type X Collagen), as well as cartilage tissue formation in the PF suture. This process was followed by expression of bone markers (Type I Collagen and Osteocalcin), suggesting endochondral ossification. Moreover, we studied the effect of haploinsufficiency of the NCC determinant gene Sox9 in the NCC derived PF suture complex. A decrease in dosage of Sox9 by haploinsufficiency in NCC-derived tissues resulted in delayed PF suture closure. These results demonstrate a unique development of the PF suture complex and the role of Sox9 as an important contributor to timely and proper closure of the PF suture through endochondral ossification. Keywords: Neural crest cells; Sox9; Chondrogenesis; Endochondral ossification; Cranial suture patterning; Posterior frontal suture

The neural crest is a multipotent population of migratory cells unique to the vertebrate embryo. Neural crest arises at the lateral edge of the neural plate and migrates throughout the embryo to give rise to a wide variety of cell types including peripheral and enteric neurons and glia, craniofacial cartilage and bone, smooth muscle, and pigment cells. Here we review recent studies that have addressed the role of several signaling pathways in the induction of the neural crest. Work in the mouse, chick, Xenopus, and zebrafish have shown that a complex network of genes is activated at the neural plate border in response to neural crest-inducing signals. We also summarize some of these findings and discuss how the differential activation of these genes may contribute to the establishment of neural crest diversity. Keyword: Neural crest; Induction; Bmp; Wnt; Fgf; Notch/Delta; Transcriptional regulators

The AP-2[alpha] transcription factor is required for multiple aspects of vertebrate development and mice lacking the AP-2[alpha] gene (tcfap2a) die at birth from severe defects affecting the head and trunk. Several of the defects associated with the tcfap2a-null mutation affect neural crest cell (NCC) derivatives including the craniofacial skeleton, cranial ganglia, and heart outflow tract. Consequently, there is considerable interest in the role of AP-2[alpha] in neural crest cell function in development and evolution. In addition, the expression of the AP-2[alpha] gene is utilized as a marker for premigratory and migratory neural crest cells in many vertebrate species. Here, we have specifically addressed how the presence of AP-2[alpha] in neural crest cells affects development by creating a conditional (floxed) version of tcfap2a which has subsequently been intercrossed with mice expressing Cre recombinase under the control of Wnt1 cis-regulatory sequences. Neural crest-specific disruption of tcfap2a results in frequent perinatal lethality associated with neural tube closure defects and cleft secondary palate. A small but significant fraction of mutant mice can survive into adulthood, but have retarded craniofacial growth, abnormal middle car development, and defects in pigmentation. The phenotypes obtained confirm that AP-2[alpha] directs important aspects of neural crest cell function. At the same time, we did not observe several neurocristopathies affecting the head and heart that might be expected based on the phenotype of the AP-2[alpha]-null mouse. These results have important implications for the evolution and function of the AP-2 gene family in both the neural crest and the vertebrate embryo. Keywords: tcfap2a; AP-2[alpha]; Neural crest cell

The chick ciliary ganglion is a neural crest-derived parasympathetic ganglion that innervates the eye. Here, we examine its axial level of origin and developmental relationship to other ganglia and nerves of the head. Using small, focal injections of DiI, we show that neural crest cells arising from both the caudal half of the midbrain and the rostral hindbrain contribute to the ciliary as well as the trigeminal ganglion. Precursors to both ganglia have overlapping migration patterns, moving first ventrolaterally and then rostrally toward the optic vesicle. At the level of the midbrain/forebrain junction, precursors to the ciliary ganglion separate from the main migratory stream, turn ventromedially, and condense in the vicinity of the rostral aorta and Rathke's pouch. Ciliary neuroblasts first exit the cell cycle at early E2, prior to and during ganglionic condensation, and neurogenesis continues through E5.5. By E3, markers of neuronal differentiation begin to appear in this population. By labeling the ectoderm with DiI, we discovered a new placode, caudal to the eye and possibly contiguous to the trigeminal placode, that contributes a few early differentiating neurons to the ciliary ganglion, oculomotor nerve, and connecting branches to the ophthalmic nerve. These results suggest for the first time a dual neural crest and placodal contribution to the ciliary ganglion and associated nerves. Keywords: Chick; DiI; Migration; Neuronal differentiation; Neurogenesis; Ophthalmic nerve; Trigeminal ganglion

Neural crest precursors to the autonomic nervous system form different derivatives depending upon their axial level of origin; for example, vagal, but not trunk, neural crest cells form the enteric ganglia of the gut. Here, we show that Slit2 is expressed at the entrance of the gut, which is selectively invaded by vagal, but not trunk, neural crest. Accordingly, only trunk neural crest cells express Robo receptors. In vivo and in vitro experiments demonstrate that trunk, not vagal, crest cells avoid cells or cell membranes expressing Slit2, thereby contributing to the differential ability of neural crest populations to invade and innervate the gut. Conversely, exposure to soluble Slit2 significantly increases the distance traversed by trunk neural crest cells. These results suggest that Slit2 can act bifunctionally, both repulsing and stimulating the motility of trunk neural crest cells.

Endothelin-A (ETA) is a G-protein-coupled receptor expressed in the neural crest-derived mesenchyme of the pharyngeal arches during craniofacial development. Targeted deletion of the [ET.sub.A] receptor or its ligand endothelin-1 (ET-1) causes cleft palate and hypoplasia of the mandible, otic cup, and tympanic ring. Previously we showed that G[[alpha].sub.q]/G[[alpha].sub.11]-null mice die around E 11.0, whereas G[[alpha].sub.q.sup.(-/-)] G[[alpha].sub.11.sup.(+/-)] mice survive to birth with hypomorphic phenotypes similar to, but less severe than, ETA or ET-1-null mice. To determine whether ET-1 signaling is transduced by G[[alpha].sub.q]/G[[alpha].sub.11] proteins, we examined the expression patterns of several ET-1 dependent and independent transcription factors in G[alpha].sub.q]/G[[alpha].sub.11]-deficient embryos. Expression of genes encoding the ET-1-dependent transcription factors D1x3, D1x6, dHAND, and eHAND was specifically downregulated in the pharyngeal arches of G[[alpha].sub.q]/G[alpha].sub.11]-deficient mice. In contrast, pharyngeal arch expression of the homeobox gene Msx1, which is not regulated by ET-1 signaling, was maintained in these embryos. We conclude that the G[[alpha].sub.q] and G[[alpha].sub.11] proteins serve as the intracellular mediators of ET-1 signaling in the pharyngeal arch mesenchyme. Keywords: G-protein; Neural crest; Pharyngeal arches; Endothelin; G[[alpha].sub.q]/G[[alpha].sub.11]; D1x3; D1x6; dHAND/HAND2; eHAND/HAND1; Craniofacial development

Clinical expression of Hirschsprung disease (HSCR) requires the interaction of multiple susceptibility genes. Molecular genetic analyses have revealed that interactions between mutations in the genes encoding the RET receptor tyrosine kinase and the endothelin receptor type B (EDNRB) are central to the genesis of HSCR. We have established two locus noncomplementation assays in mice, using allelic series at Ednrb in the context of Ret kinase-null heterozygotes, to understand the clinical presentation, incomplete penetrance, variation in length of aganglionic segment, and sex bias observed in human HSCR patients. Titration of Ednrb in the presence of half the genetic dose of Ret determines the presentation of an enteric phenotype in these strains, revealing or abrogating a sex bias in disease expression depending on the genotype at Ednrb. RET and EDNRB signaling pathways are also critical for the normal development of other tissues, including the kidneys and neural crest-derived melanocytes. Our data demonstrate that interaction between these genes is restricted to the enteric nervous system and does not affect renal, coat color, and retinal choroid development.

Research has been conducted on fibroblast growth factor signaling. The functions of this signaling during pharyngeal region development have been investigated, and the results demonstrate that these factors mediated by tyrosine kinase-type receptors are required for the neural crest cell entry into the second branchial arch.

Ascidians are simple invertebrate chordates whose lineage diverged from that of vertebrates at the base of the chordate tree. Their larvae display a typical chordate body plan, but are composed of a remarkably small number of cells. Ascidians develop with an invariant cell lineage, and their embryos can be easily experimentally manipulated during the cleavage stages. Their larval nervous system is organised in a similar way as in vertebrates but is composed of less than 130 neurones and around 230 glial cells. This remarkable simplicity offers an opportunity to understand, at the cellular and molecular levels, the ontogeny and function of each neural cell. Here, we first review the organisation of the ascidian nervous system and its lineage. We then focus on the current understanding of the processes of neural specification and patterning before and during gastrulation. We discuss these advances in the context of what is currently known in vertebrates. Key Words: ascidians; chordates; evolution; nervous system; neural induction; embryo; development; neurone; Otx; BMP; FGF; proteases; CNS; PNS; placodes; neural crest.

The complex and dynamic pattern of Hoxb3 expression in the developing hindbrain and the associated neural crest of mouse embryos is controlled by three separate cis-regulatory elements: element I (region A), element IIIa, and the r5 enhancer (element IVa). We have examined the cis-regulatory element IIIa by transgenic and mutational analysis to determine the upstream trans-acting factors and mechanisms that are involved in controlling the expression of the mouse Hoxb3 gene in the anterior spinal cord and hindbrain up to the r5/r6 boundary, as well as the associated neural crest which migrate to the third and posterior branchial arches and to the gut. By deletion analysis, we have identified the sequence requirements within a 482-bp element III482. Two Hox binding sites are identified in element III482 and we have shown that in vitro both Hoxb3 and Hoxb4 proteins can interact with these Hox binding sites, suggesting that auto/cross-regulation is required for establishing the expression of Hoxb3 in the neural tube domain. Interestingly, we have identified a novel GCCAGGC sequence motif within element III482, which is also required to direct gene expression to a subset of the expression domains except for rhombomere 6 and the associated neural crest migrating to the third and posterior branchial arches. Element III482 can direct a higher level of reporter gene expression in r6, which led us to investigate whether kreisler is involved in regulating Hoxb3 expression in r6 through this element. However, our transgenic and mutational analysis has demonstrated that, although kreisler binding sites are present, they are not required for the establishment or maintenance of reporter gene expression in r6. Our results have provided evidence that the expression of Hoxb3 in the neural tube up to the r5/r6 boundary is auto/cross-regulated by Hox genes and expression of Hoxb3 in r6 does not require kreisler. Key Words: Hoxb3; hindbrain; rhombomere; kreisler; neural crest; cis-regulation.

In the chick ciliary ganglion, neuronal number is kept constant between St. 29 and St. 34 (E6-E8) despite a large amount of cell death. Here, we characterize the source of neurogenic cells in the ganglion as undifferentiated neural crest-derived cells. At St. 29, neurons and nonneuronal cells in the ciliary ganglion expressed the neural crest markers HNK-1 and [p75.sup.NTR]. Over 50% of the cells were neurons at St. 29; of the nonneuronal cells, a small population expressed glial markers, whereas the majority was undifferentiated. When placed in culture, nonneuronal cells acquired immunoreactivity for HuD, suggesting that they had commenced neuronal differentiation. The newly differentiated neurons arose from precursors that did not incorporate bromodeoxyuridine. To test whether these precursors could undergo neural differentiation in vivo, purified nonneuronal cells from St. 29 quail ganglia were transplanted into chick embryos at St. 9-14. Subsequently, quail cells expressing neuronal markers were found in the chick ciliary ganglion. The existence of this precursor pool was transient because nonneuronal cells isolated from St. 38 ganglia failed to form neurons. Since all ciliary ganglion neurons are born prior to St. 29, these results demonstrate that there are postmitotic neural crest-derived precursors in the developing ciliary ganglion that can differentiate into neurons in the appropriate environment. Key Words: ciliary ganglion; parasympathetic; neuronal differentiation; quail; Islet-1; HuD; transplantation; neurogenesis; neural crest.

The velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a genetic disorder characterized by phenotypic abnormalities of the derivatives of the pharyngeal arches, including cardiac outflow tract defects. Neural crest cells play a major role in the development of the pharyngeal arches, and defects in these cells are likely responsible for the syndrome. Most patients are hemizygous for a 1.5- to 3.0-Mb region of 22q11, that is suspected to be critical for normal pharyngeal arch development. Mice hemizygous for a 1.5-Mb homologous region of chromosome 16 (Lgdel/+) exhibit conotruncal cardiac defects similar to those seen in affected VCFS/DGS patients. To investigate the role of Lgdel genes in neural crest development, we fate mapped neural crest cells in Lgdel/+ mice and we performed hemizygous neural crest-specific inactivation of Lgdel. Hemizygosity of the Lgdel region does not eliminate cardiac neural crest migration to the forming aortic arches. However, neural crest cells do not differentiate appropriately into smooth muscle in both fourth and sixth aortic arches and the affected aortic arch segments develop abnormally. Tissue-specific hemizygous inactivation of Lgdel genes in neural crest results in normal cardiovascular development. Based on our studies, we propose that Lgdel genes are required for the expression of soluble signals that regulate neural crest cell differentiation. Key Words: Tbx1; pharyngeal arches; aortic arches; smooth muscle; DiGeorge syndrome.

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.

This review examines the evolution of the cardiovascular system along distinct pathways based upon developmental biology and clinical cardiology. While the former relies upon genetics based mechanism, the latter emphasizes physiology and an integrative approach toward mechanism. Research is discussed with reference to cardiovascular development and disease.

The pharyngeal arches are one of the defining features of the vertebrates, with the first arch forming the mandibles of the jaw and the second forming jaw support structures. The cartilaginous elements of each arch are formed from separate migratory neural crest cell streams, which derive from the dorsal aspect of the neural tube. The second and more posterior crest streams are characterized by specific Hox gene expression. The zebrafish has a larger overall number of Hox genes than the tetrapod vertebrates, as the result of a duplication event in its lineage. However, in both zebrafish and mouse, there are just two members of Hox paralogue group 2 (PG2): Hoxa2 and Hoxb2. Here, we show that morpholino-mediated 'knock-down' of both zebrafish Hox PG2 genes results in major defects in second pharyngeal arch cartilages, involving replacement of ventral elements with a mirror-image duplication of first arch structures, and accompanying changes to pharyngeal musculature. In the mouse, null mutants of Hoxa2 have revealed that this single Hox gene is required for normal second arch patterning. By contrast, loss-of-function of either zebrafish Hox PG2 gene individually has no phenotypic consequence, showing that these two genes function redundantly to confer proper pattern to the second pharyngeal arch. We have also used hoxb1a mis-expression to induce localized ectopic expression of zebrafish Hox PG2 genes in the first arch; using this strategy, we find that ectopic expression of either Hox PG2 gene can confer second arch identity onto first arch structures, suggesting that the zebrafish Hox PG2 genes act as 'selector genes.' Key Words: zebrafish; Hox; hindbrain; pharyngeal arches; morpholino; knock-down; selector genes; redundancy; Meckel's cartilage.

The Hoxa2 and Hoxb2 genes are members of paralogy group II and display segmental patterns of expression in the developing vertebrate hindbrain and cranial neural crest cells. Functional analyses have demonstrated that these genes play critical roles in regulating morphogenetic pathways that direct the regional identity and anteroposterior character of hindbrain rhombomeres and neural crest-derived structures. Transgenic regulatory studies have also begun to characterize enhancers and cis-elements for those mouse and chicken genes that direct restricted patterns of expression in the hindbrain and neural crest. In light of the conserved role of Hoxa2 in neural crest patterning in vertebrates and the similarities between paralogs, it is important to understand the extent to which common regulatory networks and elements have been preserved between species and between paralogs. To investigate this problem, we have cloned and sequenced the intergenic region between Hoxa2 and Hoxa3 in the chick HoxA complex and used it for making comparative analyses with the respective human, mouse, and horn shark regions. We have also used transgenic assays in mouse and chick embryos to test the functional activity of Hoxa2 enhancers in heterologous species. Our analysis reveals that three of the critical individual components of the Hoxa2 enhancer region from mouse necessary for hindbrain expression (Krox20, BoxA, and TCT motifs) have been partially conserved. However, their number and organization are highly varied for the same gene in different species and between paralogs within a species. Other essential mouse elements appear to have diverged or are absent in chick and shark. We find the mouse r3/r5 enhancer fails to work in chick embryos and the chick enhancer works poorly in mice. This implies that new motifs have been recruited or utilized to mediate restricted activity of the enhancer in other species. With respect to neural crest regulation, cis-components are embedded among the hindbrain control elements and are highly diverged between species. Hence, there has been no widespread conservation of sequence identity over the entire enhancer domain from shark to humans, despite the common function of these genes in head patterning. This provides insight into how apparently equivalent regulatory regions from the same gene in different species have evolved different components to potentiate their activity in combination with a selection of core components. Key Words: Hoxa2; Hoxb2; hindbrain; neural crest cells; segmentation; regulation; conservation; gene expression; vertebrates; evolution.

Sox10, a transcription factor, has been shown to be a vital peripheral glial development regulator. Sox 10 also controls expression of ErbB3 in neural crest cells. Haploinsufficiency of Sox10 can cause pigmentation and megacolon defects also observed in Sox10(super.Dom/+) mice and in Waardenburg-Hirschsprung patients if the disease is caused by heterozygous SOX10 mutations.

Signal transduction and transcription in the melanocyte lineage are discussed in this review article. Gene expression and how it is controlled in neural crest-derived melanocytes by interaction of specific transcription factors and signal transduction pathways are emphasized along with regulation of the expression and function of the microphthalmia-associated transcription factor, Mitf. Mouse studies and zerbrafish studies will be valuable in the study of melanocyte development. It will give novel insights into the integration of gene expression and signal transduction pathways and how they control development of a specific cell lineage.

We have shown that cultured mouse neural crest (NC) cells exhibit transient increases in intracellular calcium. Up to 50% of the cultured NC-derived cells exhibited calcium transients during the period of neuronal differentiation. As neurogenic activity declined, so did the percentage of active NC-derived cells and their calcium spiking frequency. The decrease in calcium transient activity correlated with a decreased sensitivity to thimerosal, which sensitizes inositol 1,4,5-triphosphate receptors. Thimerosal increased the frequency of oscillations in active NC-derived cells and induced them in a subpopulation of quiescent cells. As neurogenesis ended, NC-derived cells became nonresponsive to thimerosal. Using the expression of time-dependent neuronal traits, we determined that neurons exhibited spontaneous calcium transients as early as a neuronal phenotype could be detected and continued through the acquisition of caffeine sensitivity, soon after which calcium transient activity stopped. A subpopulation of nonneuronal NC-derived cells exhibited calcium transient activity within the same time frame as neurogenesis in culture. Exposing NC-derived cells to 20 mM Mg(super 2+) blocked calcium transient activity and reduced neuronal number without affecting the survival of differentiated neurons. Using lineagetracing analysis, we found that 50% of active NC-derived cells gave rise to clones containing neurons, while inactive cells did not. We hypothesize that calcium transient activity establishes a neuronal competence for undifferentiated NC cells. Key Words: differentiation; lineage analysis; cell autonomous; IP(sub 3); neuronal progenitor

With cDNA microarrays, it is now possible to compare the expression of many genes simultaneously. To maximize the likelihood of finding genes whose expression is altered under the experimental conditions, it would be advantageous to be able to select clones for tissue-appropriate cDNA sets. We have taken advantage of the extensive sequence information in the dbEST expressed sequence tag (EST) database to identify a neural crest-derived melanocyte cDNA set for microarray analysis. Analysis of characterized genes with dbEST identified one library that contained ESTs representing 21 neural crest-expressed genes (library 198). The distribution of the ESTs corresponding to these genes was biased toward being derived from library 198. This is in contrast to the EST distribution profile for a set of control genes, characterized to be more ubiquitously expressed in multiple tissues (P < 1 x [10.sup.-9]). From library 198, a subset of 852 clustered ESTs were selected that have a library distribution profile similar to that of the 21 neural crest-expressed genes. Microarray analysis demonstrated the majority of the neural crest-selected 852 ESTs (Mell array) were differentially expressed in melanoma cell lines compared with a non-neural crest kidney epithelial cell line (P < 1 x [10.sup.-8]). This was not observed with an array of 1,238 ESTs that was selected without library origin bias (P = 0.204). This study presents an approach for selecting tissue-appropriate cDNAs that can be used to examine the expression profiles of developmental processes and diseases.

The Slug gene encodes a zinc finger protein, homologous to the product of the Drosophila Snail gene, that is implicated in the generation and migration of both mesoderm and neural crest cells in several vertebrate species. We describe here the cloning and genetic analysis of the mouse Slug (Slugh) gene. Slugh encodes a 269-amino-acid protein that shares 92% amino acid identity with the product of the chicken Slug gene. We have characterized Slugh gene expression during early mouse embryogenesis by whole mount in situ hybridization of Slugh mRNA and through detection of [Beta]-galactosidase expression from an in-frame [Slugh.sup.1acZ] allele generated through homologous recombination. Slugh expression is first detected in extraembryonic mesoderm and is later detected in many mesodermal subsets, although it is not detected in the primitive streak. In contrast to many other vertebrates, the mouse Slug gene is not expressed in premigratory neural crest cells but is expressed in migratory neural crest cells. Analysis of a targeted null mutation that deleted all Slugh coding sequences revealed that Slugh is not required for mesoderm formation or for neural crest generation, migration, or development in mice. These results indicate that neither the expression pattern nor the biological function of the Slug gene is conserved among all vertebrates. These data also raise interesting questions about the regulation of neural crest generation, which is one of the distinguishing characteristics of the vertebrate subphylum.

Connexin 43 (Cx43) knockout mice and transgenic mice (CMV43) overexpressing the Cx43 gap junction gene exhibit heart defects involving the conotruncus and right ventricle. based on the heart phenotype and Cx43 gene and transgene expression pattern, we previously proposed that the heart defects may reflect a role for gap junctions in the modulation of cardiac neural crest development. To further elucidate the mechanism by which these heart defects may arise, fetal heart structure and function in these transgenic and knockout mice were examined by magnetic resonance microscopy and Doppler echocardiography. Magnetic resonance microscopy of E14.5 fetuses revealed an enlargement of the right ventricular chamber in the heterozygous Cx43 knockout and CMV43 transgenic mice. This was accompanied by thinning of the chamber wall. In the homozygous Cx43 knockout mouse, heart malformation was also restricted to the right ventricle. This was generally characterized by two pouches at the base of the pulmonary outflow tract, but occasionally hearts with a single pouch were found. Magnetic resonance microscopy showed in some of the CMV43 and Cx43 knockout mice an attenuation of the ductus arteriosus, a phenotype which may be indicative of outflow tract obstruction. This was confirmed by the in utero Doppler echocardiography, which showed increased outflow velocity in E12.5 to 14.5 CMV43 and Cx43 knockout fetuses. In some of these fetuses, Doppler analysis also revealed arrhythmia and absence of isovolemic contraction time. Further examination of these hearts by histology and immunohistochemistry showed abnormal myocardial development in the conotruncus. Particularly interesting was the presence of abundant subendocardial fibrous tissue expressing smooth muscle actin. In the developing heart, such mesenchyme in the outflow tract is usually considered neural crest-derived tissue. Together, these results confirm the importance of Cx43 gene dosage in conotruncal heart development and suggest that this likely involves a role for Cx43 gap junctions in cardiac crest development. In future studies, these transgenic mice may serve as valuable animal models for further studying the role of gap junctions and cardiac crest cells in conotruncal heart development. Key Words: connexin 43; gap junctions; transgenic mice; heart defects; outflow tract; neural crest; Doppler echocardiography; magnetic resonance microscopy.

The Dlx homeobox gene family is expressed in a complex pattern within the embryonic craniofacial ectoderm and ectomes-enchyme. A previous study established that Dlx-2 is essential for development of proximal regions of the murine first and second branchial arches. Here we describe the craniofacial phenotype of mice with mutations in Dlx-1 and Dlx-1 and -2. The skeletal and soft tissue analyses of mice with Dlx-1 and Dlx-1 and -2 mutations provide additional evidence that the Dlx genes regulate proximodistal patterning of the branchial arches. This analysis also elucidates distinct and overlapping roles for Dlx-1 and Dlx-2 in craniofacial development. Furthermore, mice lacking both Dlx-1 and -2 have unique abnormalities, including the absence of maxillary molars. Dlx-1 and -2 are expressed in the proximal and distal first and second arches, yet only the proximal regions are abnormal. The nested expression patterns of Dlx-1, -2, -3, -5, and -6 provide evidence for a model that predicts the region-specific requirements for each gene. Finally, the Dlx-2 and Dlx-1 and -2 mutants have ectopic skull components that resemble bones and cartilages found in phylogenetically more primitive vertebrates.

Mammalian cadherin-6 (K-cadherin, cad6) was originally identified by means of the polymerase chain reaction, but its biological functions have not yet been determined. We analyzed the expression pattern of the mouse homologue of this cadherin during development and found that it was transiently expressed in restricted rhombomeres and in other subdivisions of the neural plate and tube. In the midbrain and anterior hindbrain of E8.0-8.5 embryos, cad6 was expressed only in neural crest-generating regions. In contrast, in the posterior hindbrain and contiguous spinal cord of these embryos, cad6 occurred throughout the neural plate, forming a sharp anterior limit at the future rhombomere 4 and 5 boundary. Subsequently, this neural plate expression became confined to rhombomere 6, although most of the neural crest-generating areas remained positive throughout the body. Neural crest cells expressing cad6 migrated out of the neural tube, and subsequently accumulated mainly along peripheral nerves. We then studied the effect of Hoxa-1 mutation on the expression of cad6, as their expressions spatiotemporally overlapped with each other in the early posterior hindbrain. In E8.0-8.5 Hoxa-1 mutants, cad6 expression was suppressed in the region of rhombomeres 4 to 6, although that in the other regions was not essentially affected. At later stages, however, cad6-positive crest cells appeared and migrated out of rhombomeres 4 to 6, indicating that the suppression of cad6 expression was transient and restricted to early stages. Importantly, this effect of the Hoxa-1 mutation concurred with the timing of the expression of this gene. We also studied Hoxa-3 mutants, but found no effect of this mutation on the cad6 expression pattern. These findings suggest that cad6 may contribute to the formation of the segmental structure of the early brain through its ability to confer specific adhesiveness on cells and that Hoxa-1 may be required for early cad6 expression in the posterior hindbrain.

The Patch (Ph) mutation in the mouse, a deletion that includes the gene for PDGFR[Alpha], is a recessive lethal that exhibits a dominant pigment phenotype in heterozygotes. To assess whether the Ph mutation acts cell-autonomously or non-autonomously on melanocyte development, we have examined the melanogenic potential of neural crest populations from normal and mutant crest cells in vitro and the pattern of dispersal and survival of melanocyte precursors (MPs) in vivo. We report that trunk neural crest cells from homozygous Ph embryos give rise to pigmented melanocytes in vitro in response to Steel factor (SlF). In vivo, homozygous Ph embryos contain a subpopulation of crest-derived cells that express c-kit and tyrosinase-related protein-2 characteristic of MPs. These cells begin to migrate normally on the lateral crest migration pathway, but then fail to disperse in the dermal mesenchyme and subsequently disappear. Although dermal mesenchyme is adversely affected in Ph homozygotes, SlF mRNA expression by the cells of the dermatome is normal in Ph embryos when neural crest-derived MPs start to migrate on the lateral pathway. In contrast, mRNA for the SlF receptor, c-kit, was observed to be ectopically expressed in somites and lateral mesenchyme in embryos carrying the Ph mutation. Based on this ectopic expression of c-kit in Ph mutant embryos, and the observed distribution of SlF protein in normal and mutant embryos, we suggest that competition for limited amounts of SlF localized on the lateral neural crest migration pathway alters melanocyte dispersal and survival.

Dominant megacolon (Dom) is a mutation in an uncharacterized murine gene which is associated with intestinal aganglionosis and the focal absence of melanocytes in heterozygous animals. The phenotype of Dom/+ heterozygotes is similar to the lethal spotted and piebald lethal mutations, which are due to defects in endothelin-mediated intercellular signals. In this study, the D[Beta]H-n1acZ transgenic marker for enteric neural crest cells is used to study the distribution of enteric neurons and their precursors in Dom/+ mice and embryos. Vagal neural crest-derived cells in wild-type embryos colonize the gut in a cranial-to-caudal progression. In Dom/+ embryos, colonization was retarded from the earliest stages examined (embryonic Day 11.0), including progression through the small intestine. The early onset of this defect contrasts with impaired neural crest colonization associated with the lethal spotted and piebald lethal mutations which manifest only in the large intestine. Analysis of Dom/+ [equivalence] +/+ aggregation chimeras indicated that defective colonization is not an autonomous (intrinsic) property of Dom/+ neuroblasts, but like lethal spotted and piebald lethal, the Dominant megacolon mutation directly or indirectly affects microenvironmental signals which influence the migration, proliferation, and/or survival of enteric neural crest cells.

The neural crest is a transient tissue of the vertebrate embryo that gives rise to most primary sensory neurons and pigment cells in the adult organism, among other cell types and tissues. Many neural crest cells are pluripotent in the sense that their progeny can generate more than one phenotype. The presence of pluripotent neural crest cell-derived cells at sites of terminal differentiation suggests that location-specific cues from the embryonic environment, such as growth factors, are involved in directing their survival, proliferation, and cell type specification. We have therefore examined the influences of one pertinent growth factor, stem cell factor (SCF), on neural crest cell development by in vitro colony assay in a serum-free culture medium. SCF showed three major effects. (1) SCF is trophic for early neural crest ceils, that is, either pluripotent cells and/or their more mature progeny. This effect occurs only if SCF is present throughout the culture period, and it is not observed when a neurotrophin is present in addition to SCF. (2) More colonies contain sensory neuron precursors in the presence of SCF. This effect is neutralized by NGF and neurotrophin-3 (NT-3), but not by brain-derived neurotrophic factor (BDNF). (3) The combination of SCF and any neurotrophin tested (NGF, BDNF, NT-3) is trophic for melanogenic cells, whereas SCF alone does not detectably affect melanogenesis. This suggests either that both types of factor are required for melanotrophic action or that melanogenic cells become dependent on neurotrophins after exposure to SCF. Our observation that SCF is required during the first half of the culture period only, and NGF during the second half only, indicates the latter possibility. Whereas coat color changes in the mouse mutants W (c-kit defect) and Steel (SCF defect) and several in vivo and in vitro studies by other investigators have shown previously that SCF is melanotrophic, they also indicated the requirement of an additional factor, or factors, in melanogenesis. Our data suggest that SCF affects neural crest cell development at multiple levels and that survival of melanogenic cells is mediated by a combination of SCF and a neurotrophin, rather than by SCF alone.

The expression pattern of eHAND, a novel protein gene in mice, is described. The expression pattern is different from that of other class B helix-loop-helix (bHLH) genes, and the gene may contribute to the formation of extraembryonic tissues, heart and neural crest derivatives. eHAND may express a new subclass of class B bHLH proteins because of the low sequence identity of eHAND with other bHLH family members as well as its unique expression pattern during development.

Researchers used in vitro studies to analyze the developmental potential of mesenchymal cells obtained from explants of trunk epidermal ectoderm of the quail embryo. A significant fraction of the neural crest cells coming early in the ectoderm are pluripotent cells that can lead to the development of pigment cells, sympathoadrenal cells, primary sensory neuron precursors and probably other cells still not discussed.

Mutations in regulatory genes containing homeobox genes in vertebrates is implicated in the evolution of ontogenetic patterns resulting in anatomical features different from those of invertebrates. This was inferred from comparative studies of the sequences and expression patterns of homeobox genes in representative vertebrates and invertebrates, in particular those controlling the formation of the neural crest, organogenesis and craniofacial morphogenesis.

Migratory neural crest cells (NCC) are required for normal cardiovascular development. Recently Sonic hedgehog (Shh) has been implicated as a critical signaling molecule for neural crest survival. We have therefore examined the cardiovascular development of Shh homozygous null ([Shh.sup.-/-]) mouse embryos and have identified severe patterning defects in the branchial arch arteries (BAA) as well as the outflow tract and chambers of the heart. Normally NCC populate and maintain the BAA as well as the aorticopulmonary septum that divides the single outflow vessel of the heart into the pulmonic and Aortic arteries. In [Shh.sup.-/-] mutant embryos, the aorticopulmonary septum does not form properly resulting in a single outflow tract. In addition, the fourth and sixth BAA are markedly reduced or absent. Marker analysis in [Shh.sup.-/-] embryos demonstrates that NCC are specified and migrate into the branchial arches but then undergo massive apoptosis resulting in the failure of outflow tract septation and of BAA maintenance. We examined the position of NCC relative to the expression of Shh and the receptor Patched1 (Ptch). Strikingly, the expression patterns of Shh and Ptch are complimentary to the position of NCC around the BAA and the outflow tract. This suggests that SHH does not signal directly to NCC but rather might act to restrict the domains that NCC can populate. The apoptosis of NCC that occurs in [Shh.sup.-/-] embryos may therefore be secondary to mislocalization of the NCC or, alternatively, to the spread of other factors into normal NCC domains in the absence of SHH.

The AP-2[Alpha] transcription factor is critical for proper craniofacial morphogenesis, and it is expressed in the cranial neural crest and mesenchyme of the developing facial prominences. Targeted disruption of AP-2[Alpha] results in a severe midline facial cleft in homozygous-null embryos. Chimeras, generated by combining wild-type and mutant cells, have clefts specifically in the lip and palate. In addition, genetic mapping studies indicate that the human AP-2[Alpha] locus may have a role in orofacial clefting. These findings suggest that AP-2[Alpha] may be required for growth, morphogenesis, and/or fusion of the facial primordia. To understand how AP-2[Alpha] influences craniofacial development, we are comparing gene expression patterns between facial mesenchyme samples microdissected from wild-type and null mouse embryos. We have taken several approaches to uncover differentially expressed transcripts, including RT-PCR, suppression subtractive hybridization (SSH), and microarray analyses. Candidate genes have been isolated and are currently being analyzed for expression in wild-type and mutant embryos. Characterization of these molecules will provide insight into the roles of AP-2[Alpha] in facial morphogenesis and identify putative targets of its transcriptional regulation.

Three members of the vertebrate Distal-less genes family, DLx3,5,6, are transcribed in early gastrula embroys of Xenopus laevis. This expresion is confined to ectoderm and is excluded from the presumptive neural plate region. Consistent with this ecpression pattern, expression of all three genes is dependent upon BMP signaling. However, there are significant differences in how the three genes respond to the BMP antagonist chordin. This correlates with the different expression domain boundaries for Dlx3 compared to Dlx5 and 6. Furthermore, overexpression of Dlx factors has both negative and positive effects on genes expressed in cement gland, neural plate, and cranial neural crest, in a manner that correlated with expression domains in vivo. Thus differential responsiveness to BMP signaling by Dlx genes may be a mechanism whereby Xenopus embryonic ectodermal cells interpret BMP-based morphogenetic gradients, leading to spatially restricted specification during gastrulation.

Expression profiling using cDNA microarrays has allowed for the analysis of gene expression data on a genome size scale. We have generated a neural crest-melanocyte (NC-M)-enriched cDNA microarray containing 4356 ESTs that are expressed at various stages of neural crest development. RNA from 15 cell lines was compared including 11 melanoma lines with varying levels of NC-M differentiation. Hierarchical clustering analysis identified over 72 genes with an expression profile that correlated expression with pigmentation level. Within this cluster are 41 previously characterized genes, 8 which are known to be involved in NC-M development and/or function. The remaining 31 ESTs represent novel genes that have not yet been assessed in neural crest development. This collection of genes will be evaluated for their role in neural crest development and as candidate genes for neural crest diseases.

Neural crest cells that emerge from the developing neural tube represent a population of pluripotent cells that differentiate and migrate to adopt diverse fates including sensory and enteric neurons, pigment cells, and cells that contribute to the craniofacial skeleton. The transcription factor Sox10 controls the development of a subset of neural crest derivatives that form pigment cells and the peripheral nervous system. In zebrafish disruption of Sox10 function by colourless mutations results in premature apoptosis of neural crest cells. The resulting phenotype combines pigment cell loss with enteric agangliosis and is an appropriate model of human Waardenburgh-Shah syndrome. Although we are beginning to understand how Sox10 expression controls the fate of some neural crest derivatives little is known about the regulation of Sox10 expression. To learn more about how Sox10 gene expression is controlled in different animals we are combining a comparative approach with detailed molecular manipulation of the zebrafish Sox10 promoter. We have begun a comparison of the known Sox10 promoter sequences to identify conserved regulatory elements. The consequences of their disruption will be recorded using a GFP reporter driven by a Sox10 promoter that reproduces a Soxl0 expression pattern in zebrafish. This approach, combined with a precise deletion analysis of the zebra fish Sox10 promoter, is designed to uncover elements that together regulate Sox 10 expression in tile developing neural crest.

Neural crest cells migrate from the dorsal neural tube to give rise to a variety of structures throughout development. In birds and mammals, cardiac neural crest (CNC) contributes to cardiac outflow tract septation, conotruncal cushion formation, and aortic arch artery remodeling. The absence of these neural-crest-dependent processes in zebrafish raises the question of whether neural crest contributes to the development of the prototypical heart in zebrafish. The goal of this study is to examine whether neural crest cells contribute to cardiovascular development in zebrafish. Detailed fate-map analysis of premigratory neural crest was performed by laser activation of caged-fluorescein lineage label, with three surprising results. First, CNC that contribute to the heart originate both from neural tube regions analogous to those found in birds and mammals and from a novel region that is rostral to the otic vesicle. Second, CNC cells migrate to structures such as atrioventricular cushion, pharyngeal arches, and outflow tract as in other vertebrates, but also invade the muscular walls of the atrium and ventricle in zebra fish. Third, some CNC cells in the walls of the heart transform into cardiomyocytes and express MF20 (sarcomeric myosin heavy chain), suggesting a novel developmental source for cardiac muscle. Fate-map analysis identified three distinct subgroups of CNC along the rostrocaudal axis of the neural tube that have differential propensities to contribute to structures in the heart. In situ hybridization analysis indicated that wnt1, wnt3a, and lef1 are differentially expressed in these subgroups before migration, suggesting that the three distinct groups of CNC are defined before reaching their targets in the heart. Laser ablation studies will assess the roles of each CNC subgroup. We conclude that zebrafish utilize neural crest in cardiovascular development and will serve as a useful model to investigate the evolution and development of complex cardiovascular structures.

The enteric nervous system (ENS) is the largest and most complicated subdivison of the peripheral nervous system and is completely derived from the neural crest. In zebrafish the entire ENS appears to be derived only from postotic neural crest cells. We are undertaking a genetic screen to identify zebrafish mutants that have defects in the development of the ENS. We have been using an antibody that recognizes differentiated neurons to screen embryos of ENU-mutagenized fish. To date we have identified 2 zebrafish ENS mutants enema (enm) and lessen (lsn). Both mutants have a significant reduction in the number of enteric neurons at 4 days postfertilization, enm and lsn mutants also have defects in other structures that are believed to be derived, at least in part, from the postotic neural crest. Alcian blue staining reveals that both mutants have severe defects in the gill arch cartilages. In addition, rag-1 in situ hybridization reveals that enm mutants completely lack a functional thymus while lsn mutants have a major reduction in thymus size. To determine if these mutations affect the initial specification or migration of the postotic neural crest we examined the expression pattern of dlx-2 and crestin. Expression of both these markers appears unchanged in both mutants. These mutations along with a third previously identified ENS mutant, flat top, define a group of genes that affect cell fate specification of postotic neural crest derivatives. We are currently mapping the enm and 1sn mutations.

A subset of trunk neural crest cells form the metamerically organized dorsal root ganglia (DRG) which develop adjacent to the spinal cord. To investigate mechanisms underlying this specification event, we are characterizing mutations affecting DRG development in zebrafish. Disruption of shh signaling leads to interesting effects on DRG; most segments lack DRG neurons but some segments contain a large excess of neurons. These phenotypes are not due to effects on the initial establishment of neural crest since expression of foxd3, a premigratory neural crest marker, is normal. However, expression of neurogeninl, an early marker for DRG precursors, is missing in mutant embryos. To determine where shh signaling is required for normal DRG development, we have transplanted cells between wild-type and mutant embryos defective in the gli2 gene, which encodes a transcriptional effector of shh signaling. Normal DRG development is restored in a mutant background only when neurons originate from wild-type donors, indicating that shh directly affects DRG precursors. However, preliminary experiments suggest the excess neuron phenotype is a non-cell-autonomous defect. Treatment of embryos during somitogenesis with cyclopamine, an inhibitor of shh signaling, results in a temporal separation of the two DRG phenotypes. Taken together, our results suggest that shh has differential effects on distinct DRG precursors.

We are interested in genetic regulation of cell fate choice in the vertebrate nervous system. We have evidence that a domain of precursor cells in the zebrafish lateral neural plate gives rise to both Rohon-Beard sensory neurons and neural crest, neurogenin1 is the zebrafish homologue of Neurogenin1, required in mouse for formation of sensory neurons. We show that zebrafish embryos with reduced neurogenin1 function lack many types of sensory neurons, including Rohon-Beard neurons. In embryos with reduced Delta signaling, there are supernumerary Rohon-Beard neurons and a virtual absence of premigratory neural crest cells. However, when neurogenin1 function is reduced in zebrafish embryos that also have reduced Delta signaling, trunk neural crest is restored. These data imply (A) that Neurogenin1 directs lateral neural plate precursor cells toward the Rohon-Beard neuron fate and away from the neural crest fate and (B) that the role of Delta signaling in trunk neural crest formation is to down-regulate Neurogenin1 in responding cells, rather than to instruct the neural crest fate. {Supported by NIH NS10119 and HD22486.)

The zebrafish [colgate.sup.b382] mutant was isolated in a screen for mutations affecting neural crest development based on its abnormal melanocyte pattern. Further analysis has revealed abnormal patterning of neural-crest-derived iridiphores and xanthophores, a severe reduction in crest-derived median fin fold mesenchyme, absence of sympathetic neurons, and the lack of crest-derived craniofacial elements. In contrast, neural-crest-derived dorsal root ganglion sensory neurons are present although abnormally patterned dorsoventrally. Using sublineage-specific markers at earlier developmental stages in mutant embryos, we found that the number and pattern of precursors for melanocytes, iridiphores, xanthophores, and branchial arches predicted the subsequent phenotypes of differentiated derivatives. Similarly, analysis of neural crest morphogenesis using the panneural crest marker crestin revealed both a decrease in the number of neural crest cells and a retardation of migration. These results suggest that colgate is required for the production of specific subsets of neural crest cells and may be generally required for appropriate migration. We have also found abnormalities in the development some other nonneural crest cells in colgate mutants. For example, we have observed axonal path finding errors in primary motor neurons, the lateral line nerve, and axons from trigeminal ganglion neurons. In addition, we see an absence of most neurons in a portion of the hindbrain and an apparent absence of a subset of reticulospinal neurons. Finally, colgate mutants entirely lack pectoral fins. Consistent with this observation, we have found that the pectoral fin primordium, defined by statement of shh, bmp-2, and dlx2, is completely absent in mutant embryos. We are currently undertaking a more detailed characterization of colgate mutants that will more fully elucidate its role in these developmental processes.

The border of the neural plate marks the division between the neural ectoderm and presumptive epidermis and is the site of neural crest formation. The molecular mechanisms required for formation of a sharp border between the neural and nonneural ectoderm are not well understood. Recent genetic studies from our lab and others implicates the homeodomain transcription factor Dlx3 in neural nonneural ectoderm border formation. A member of the highly conserved distal-less family, Dlx3 is expressed in the ventral ectoderm in Xenopus laevis at the onset of gastrulation and throughout the nonneural ectoderm during neurulation. To determine the function of Dlx3, we constructed activating and repressing versions by fusing the homeodomain to the VP16 transcriptional activation domain or the engrailed repressor domain. Expression of activated Dlx3 caused a loss of neural plate and neural plate border markers. Conversely, expression of the inhibitory Dlx3 caused the expansion of the neural plate and, importantly, an outward shift of the neural plate border. Strikingly, the correct pattern of lateral neurons, neural crest, and placodal cells was recapitulated at the new border. We conclude that the formation of the neural plate border, including neural crest, may require the juxtaposition of Dlx3-negative neural plate and Dlx3 positive nonneural ectoderm. Specifically, Dlx3 appears to function by preventing expansion of the neural plate into nonneural ectoderm.

Neural crest cells migrate from the neural tube to differentiate as neurons and glia in the peripheral nervous system, craniofacial cartilages, and pigment cells. Fate of the neural crest is, in part, determined by its premigratory location: i.e., neural crest cells from different regions of the embryos anterior-posterior axis form characteristic structures. We have identified a mutation, flat [top.sup.b369], that shows defects in derivatives formed by neural crest cells that migrate from a region between the ear and the first few somites. In mutant embryos homozygous for flat top, the posterior gill arch cartilages do not form. Wild-type cells transplanted into mutant embryos can form proper cartilages, demonstrating that environmental factors are still present to support this process. Early markers of neural crest formation, such as d1x2, or axial-specific neural crest markers like hoxa3 are apparently normal. Other arch-associated segmental structures not derived from neural crest are also normal. However, thymus formation is defective, heart valves are abnormal, and enteric neurons are drastically reduced. Molecular analysis of the flat top mutation may lead to better understanding of the complex tissue interactions involved in postotic neural crest specification.

Neural crest cells (NCCs) are a population of pluripotent cells that migrate from their birthplace at the dorsal neural tube to a wide number of destinations in the developing embryo. The NCCs that become the glia of the PNS, unlike other crest, colocalize with embryonic structures that are also quickly moving during development, namely, the axon bundles they ensheath. It is however unclear whether the axons determine the final positions of the glia or vice versa. By making transgenic zebrafish lines expressing GFP under the control of the zFoxD3 promoter we have been able to follow the migration of one subset of NC-derived glia in living embryos. These glia migrate as chains with the leading cells acting as 'pioneers' during pathfinding. DiI labeling of their associated axons shows that glia migration is tightly coupled to axon extension, with the pioneer glial cell apparently 'riding' on the axon growth cone itself. We show that these GFP-labeled glia always colocalize with extending axons even in mutants that strongly affect axon pathfinding, showing that the axons provide instructive guidance cues necessary for the migration of this NCC lineage. We have isolated a group of mutants showing defects in neural crest morphogenesis and we are currently investigating these phenotypes in living mutant embryos expressing the zFoxD3 transgene. This approach has allowed us to identify a mutant in which NCC-derived glia are no longer able to respond to axon-derived guidance cues.

Eph receptors and ephrins have been implicated in guiding the migration of hindbrain neural crest cells. However, questions of how the receptors and ligands function in guiding migration remain unresolved. RT-PCR with redundant primers and in situ RNA localization indicate that in avian embryos, streams of neural crest destined for branchial arches II and III express the ephB1 receptor. Localization of ligands with chimeric ephB1 receptors indicates that the pathways for branchial arch II and III neural crest cells have lower levels of ligand than adjacent areas. In genetic mosaic embryos, in which neural crest cells destined for branchial arch II or III express a dominant negative form of ephB1, cells migrate into areas cranial or caudal to the normal pathway. To further test the avoidance model, we produced genetic mosaic embryos in which ectoderm and mesenchyme express elevated levels of ephrin B2 in the pathways to branchial arch II and III. In these embryos, HNK-l-expressing cells deviate from the normal pathway, avoiding regions expressing this ephB1 ligand. From these experiments, we conclude that ephB1 on neural crest cells alters cell behavior to exclude neural crest from regions in which head mesenchyme express the ephrin ligands.

In a screen for genes involved in neural crest development, we identified DBHR (DBH-Related), a putative monooxygenase with low homology to dopamine [Beta]-hydroxylase (DBH). Here, we describe novel expression patterns for DBHR in the developing embryo and particularly the neural crest. DBHR is an early marker for prospective neural crest, with earliest expression at the neural plate border where neural crest is induced. Furthermore, DBHR expression persists in migrating neural crest and in many, though not all, crest derivatives. DBHR is also expressed in the myotome, from the earliest stages of its formation, and in distinct regions of the neural tube, including even-numbered rhombomeres of the hindbrain. In order to investigate the signals that regulate its segmented pattern in the hindbrain, we microsurgically rotated the rostrocaudal positions of rhombomeres 3/4. Despite their ectopic position, both rhombomeres continued to express DBHR at the level appropriate for their original location, indicating that DBHR is regulated autonomously within rhombomeres. We conclude that DBHR is a divergent member of a growing family of DBH-related genes; thus, DBHR represents a completely new type of neural crest marker, expressed throughout the development of the neural crest, with possible functions in cell-cell signaling. Key Words: DBHR; MOX; monooxygenase; dopamine-[Beta]-hydroxylase (DBH); chick development; neural crest; hindbrain.

The neural crest is a distinct population of embryonic cells found only in craniates. Several genes, including BMP-4and the zinc-finger transcription factor Slug, have been shown to mediate neural crest development in both chickens and mice. We examined the function of Slug in the frog Xenopus laevisby injecting antisense RNA (ASR) targeted to the 3' UTR of Xenopus Slug (XSlug). Injection of XSlugASR inhibits neural crest-cell migration and leads to reduction or loss of many neural crest derivatives, including pigment cells and cranial cartilages. Moreover, regions injected with XSlug ASR show significantly elevated levels of cell death, and expanded regions of the BMP-4antagonist, Chordin. In addition to its important role in neural crest migration, XSlug may mediate the cell-death-signaling pathway and other significant developmental pathways in Xenopus laevis. Supported by NSF.