Neural crest (NC) cells are a dynamic population of embryonic stem cells that create various adult tissues in vertebrate species including craniofacial bone and cartilage and the peripheral and enteric nervous systems. NC development is thought to be a conserved and complex process that is controlled by a tightly-regulated gene regulatory network (GRN) of morphogens, transcription factors, and cell adhesion proteins. While multiple studies have characterized the expression of several GRN factors in single species, a comprehensive protein analysis that directly compares expression across development is lacking. To address this lack in information, we used three closely related avian models, Gallus gallus (chicken), Coturnix japonica (Japanese quail), and Pavo cristatus (Indian peafowl), to compare the localization and timing of four GRN transcription factors, PAX7, SNAI2, SOX9, and SOX10, from the onset of neurulation to migration. While the spatial expression of these factors is largely conserved, we find that quail NC cells express SNAI2, SOX9, and SOX10 proteins at the equivalent of earlier developmental stages than chick and peafowl. In addition, quail NC cells migrate farther and more rapidly than the larger organisms. These data suggest that despite a conservation of NC GRN players, differences in the timing of NC development between species remain a significant frontier to be explored with functional studies.

Zic family zinc-finger proteins play various roles in animal development. In mice, five Zic genes (Zic1-5) have been reported. Despite the partly overlapping expression profiles of these genes, mouse mutants for each Zic show distinct phenotypes. To uncover possible redundant roles, we characterized Zic2/Zic3 compound mutant mice. Zic2 and Zic3 are both expressed in presomitic mesoderm, forming and newly generated somites with differential spatiotemporal accentuation. Mice heterozygous for the hypomorphic Zic2 allele together with null Zic3 allele generally showed severe malformations of the axial skeleton, including asymmetric or rostro-caudally bridged vertebrae, and reduction of the number of caudal vertebral bones, that are not obvious in single mutants. These defects were preceded by perturbed somitic marker expression, and reduced paraxial mesoderm progenitors in the primitive streak. These results suggest that Zic2 and Zic3 cooperatively control the segmentation of paraxial mesoderm at multiple stages. In addition to the segmentation abnormality, the compound mutant also showed neural tube defects that ran the entire rostro-caudal extent (craniorachischisis), suggesting that neurulation is another developmental process where Zic2 and Zic3 have redundant functions.

A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.

How neural progenitor cell (NPC) behaviors are temporally controlled in early developing embryos remains undefined. The in vivo functions of microRNAs (miRNAs) in early mammalian development remain largely unknown. Mir-302/367 is a miRNA cluster that encodes miR-367 and four miR-302 members (miR302a-d). We show that miR-302b is highly expressed in early neuroepithelium and its expression decline as development progresses. We generated a mir-302/367 knockout mouse model and found that deletion of mir-302/367 results in an early embryonic lethality and open neural tube defect (NTD). NPCs exhibit enhanced proliferation, precocious differentiation, and decreased cell survival in mutant embryos. Furthermore, we identified Fgf15, Cyclin D1, and D2 as direct targets of miR-302 in NPCs in vivo, and their expression is enhanced in mutant NPCs. Ectopic expression of Cyclin D1 and D2 increases NPC proliferation, while FGF19 (human ortholog of Fgf15) overexpression leads to an increase of NPC differentiation. Thus, these findings reveal essential roles of miR-302/367 in orchestrating gene expression and NPC behaviors in neurulation; they also point to miRNAs as critical genetic components associated with neural tube formation.

Numb is an adaptor protein implicated in diverse basic cellular processes. Using the yeast-two hybrid system we isolated a novel Numb interactor in zebrafish called NBP which is an ortholog of human renal tumor suppressor Kank. NBP interacts with the PTB domain of Numb through a region well conserved among vertebrate Kanks containing the NGGY sequence. Similar NBP and Numb morphant phenotype such as impaired convergence and extension movements during gastrulation, neurulation and epidermis defects and enhanced phenotypic aberrations in double morphants suggest that the genes interact genetically. We demonstrate that the expression of NBP undergoes quantitative and qualitative changes during embryogenesis and that the protein accumulates at the cell periphery to sites of cell-cell contact during gastrulation and later in development it concentrates at the basal poles of differentiated cells. These findings imply a possible role of NBP in establishing and maintaining cell adhesion and tissue integrity.

Dpysl2 (CRMP2) and Dpysl3 (CRMP4) are involved in neuronal polarity and axon elongation in cultured neurons. These proteins are expressed in various regions of the developing nervous system, but their roles in vivo are largely unknown. In dpysl2 and dpysl3 double morphants, Rohon-Beard (RB) primary sensory neurons that were originally located bilaterally along the midline shifted their position to a more medial location in the dorsal-most part of spinal cord. A similar phenotype was observed in the cdk5 and dyrk2 double morphants. Dpysl2 and Dpysl3 phosphorylation mimics recovered this phenotype. Cell transplantation analysis demonstrated that this ectopic RB cell positioning was non-cell autonomous and correlated with the abnormal position of neural crest cells (NCCs), which also occupied the dorsal-most part of the spinal cord during the neural rod formation stage. The cell position of other interneuron and motor neurons within the central nervous system was normal in these morphants. These results suggest that the phosphorylation of Dpysl2 and Dpysl3 by Cdk5 and DYRK2 is required for the proper positioning of RB neurons and NCCs during neurulation in zebrafish embryos.

One of the earliest stages of brain morphogenesis is the establishment of the neural tube during neurulation. While some of the cellular mechanisms responsible for neurulation have been described in a number of vertebrate species, the underlying molecular processes are not fully understood. We have identified the zebrafish homolog of protocadherin-19, a member of the cadherin superfamily, which is expressed in the anterior neural plate and is required for brain morphogenesis. Interference with Protocadherin-19 function with antisense morpholino oligonucleotides leads to a severe disruption in early brain morphogenesis. Despite these pronounced effects on neurulation, axial patterning of the neural tube appears normal, as assessed by in situ hybridization for otx2, pax2.1 and krox20. Characterization of embryos early in development by in vivo 2-photon timelapse microscopy reveals that the observed disruption of morphogenesis results from an arrest of cell convergence in the anterior neural plate. These results provide the first functional data for protocadherin-19, demonstrating an essential role in early brain development.

Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood. In this report, we show that Xenopus Ena (Xena) is localized to apical adhesive junctions of neuroepithelial cells during neurulation and that Xena knockdown disrupts cell behaviors integral to neural tube closure. Changes in the shape of the neural plate as well as apical constriction within the neural plate are perturbed in Xena knockdown embryos. Additionally, we demonstrate that Xena is essential for cell-cell adhesion. These results demonstrate that Xena plays an integral role in coordinating the regulation of cytoskeletal dynamics and cellular adhesion during neurulation in Xenopus.

Failure of neural tube closure leads to neural tube defects (NTDs), common congenital abnormalities in humans. Among the genes whose loss of function causes NTDs in mice, Grainyhead-like3 (Grhl3) is essential for spinal neural tube closure, with null mutants exhibiting fully penetrant spina bifida. During spinal neurulation Grhl3 is initially expressed in the surface (non-neural) ectoderm, subsequently in the neuroepithelial component of the neural folds and at the node-streak border, and finally in the hindgut endoderm. Here, we show that endoderm-specific knockout of Grhl3 causes late-arising spinal NTDs, preceded by increased ventral curvature of the caudal region which was shown previously to suppress closure of the spinal neural folds. This finding supports the hypothesis that diminished Grhl3 expression in the hindgut is the cause of spinal NTDs in the curly tail, carrying a hypomorphic Grhl3 allele. Complete loss of Grhl3 function produces a more severe phenotype in which closure fails earlier in neurulation, before the stage of onset of expression in the hindgut of wild-type embryos. This implicates additional tissues and NTD mechanisms in Grhl3 null embryos. Conditional knockout of Grhl3 in the neural plate and node-streak border has minimal effect on closure, suggesting that abnormal function of surface ectoderm, where Grhl3 transcripts are first detected, is primarily responsible for early failure of spinal neurulation in Grhl3 null embryos.

In vertebrates, the nervous system arises from a flat sheet of epithelial cells, the neural plate, that gradually transforms into a hollow neural tube. This process, called neurulation, involves sequential changes in cellular interactions that are precisely coordinated both spatially and temporally by the combined actions of morphogens. To gain further insight into the molecular events regulating cell adhesion during neurulation, we investigated whether the adhesive and migratory capacities of neuroepithelial cells might be modulated by Sonic hedgehog (Shh), a signaling molecule involved in the control of cell differentiation in the ventral neural tube. When deposited onto extracellular matrix components in vitro, neural plates explanted from avian embryos at early neurulation readily dispersed into monolayers of spread cells, thereby revealing their intrinsic ability to migrate. In the presence of Shh added in solution to the culture medium, the explants still exhibited the same propensity to disperse. In contrast, when Shh was immobilized to the substrate or produced by neuroepithelial cells themselves after transfection, neural plate explants failed to disperse and instead formed compact structures. Changes in the adhesive capacities of neuroepithelial cells caused by Shh could be accounted for by inactivation of surface beta1-integrins combined with an increase in N-cadherin-mediated cell adhesion. Furthermore, immobilized Shh promoted differentiation of neuroepithelial cells into motor neurons and floor plate cells with the same potency as soluble Shh. However, the effect of Shh on the neuroepithelial cell adhesion was discernible and apparently independent from its differentiation effect and was not mediated by the signaling cascade elicited by the Patched-Smoothened receptor and involving the Gli transcription factors. Thus, our experiments indicate that Shh is able to control sequentially adhesion and differentiation of neuroepithelial cells through different mechanisms, leading to a coordinated regulation of the various cell interactions essential for neural tube morphogenesis.

The mesoderm, comprising the tissues that come to lie entirely in the deep layer, originates in both the superficial epithelial and the deep mesenchymal layers of the early amphibian embryo. Here, we characterize the mechanisms by which the superficial component of the presumptive mesoderm ingresses into the underlying deep mesenchymal layer in Xenopus tropicalis and extend our previous findings for Xenopus laevis. Fate mapping the superficial epithelium of pregastrula stage embryos demonstrates ingression of surface cells into both paraxial and axial mesoderm (including hypochord), in similar patterns and amounts in both species. Superficial presumptive notochord lies medially, flanked by presumptive hypochord and both overlie the deep region of the presumptive notochord. These tissues are flanked laterally by superficial presumptive somitic mesoderm, the anterior tip of which also appears to overlay the presumptive deep notochord. Time-lapse recordings show that presumptive somitic and notochordal cells move out of the roof of the gastrocoel and into the deep region during neurulation, whereas hypochordal cells ingress after neurulation. Scanning electron microscopy at the stage and position where ingression occurs suggests that superficial presumptive somitic cells in X. laevis ingress into the deep region as bottle cells whereas those in X. tropicalis ingress by "relamination" (e.g., [Dev. Biol. 174 (1996) 92]). In both species, the superficially derived presumptive somitic cells come to lie in the medial region of the presumptive somites during neurulation. By the early tailbud stages, these cells lie at the horizontal myoseptum of the somites. The morphogenic pathway of these cells strongly resembles that of the primary slow muscle pioneer cells of the zebrafish. We present a revised fate map of Xenopus, and we discuss the conservation of superficial mesoderm within amphibians and across the chordates and its implications for the role of this tissue in patterning the mesoderm.

The axial midline is an important source of patterning and morphogenesis cues in the vertebrate embryo. The midline derives from a small group of cells in the gastrulating embryo, known as "the organizer" in recognition of its ability to organize an entire body plan. The mammalian organizer, the node, gives rise to axial midline structures: the notochord, dorsal foregut, and part of the floor plate of the neural tube. Only some of the genes that direct midline development are known. In this study, we present the complete coding sequence for a novel gene, cordon-bleu (cobl), expressed specifically in the node and its derivatives until organogenesis stages. The deduced sequence does not resemble any gene of known function. However, cobl is widely conserved: apparent orthologs and paralogs are found in many vertebrate species, with several sequence domains of high conservation but unknown function. We find that chicken cordon-bleu is similarly expressed in the node and its derivatives, suggesting functional conservation. We also report the sequence and nonoverlapping expression of a related mouse gene, Coblr1. Finally, we show that cobl interacts with the neurulation gene Vangl2 to facilitate midbrain neural tube closure, demonstrating roles for both cobl and Vangl2 in midbrain neurulation.

Development of the central nervous system requires orchestration of morphogenetic processes which drive elevation and apposition of the neural folds and their fusion into a neural tube. The newly formed tube gives rise to the brain in anterior regions and continues to develop into the spinal cord posteriorly. Conspicuous differences between the anterior and posterior neural tube become visible already during neural tube closure (NTC). Planar cell polarity (PCP)-mediated convergent extension (CE) movements are restricted to the posterior neural plate, i.e. hindbrain and spinal cord, where they propagate neural fold apposition. The lack of CE in the anterior neural plate correlates with a much slower mode of neural fold apposition anteriorly. The morphogenetic processes driving anterior NTC have not been addressed in detail. Here, we report a novel role for the breast cancer susceptibility gene and microtubule (MT) binding protein Hmmr (Hyaluronan-mediated motility receptor, RHAMM) in anterior neurulation and forebrain development in Xenopus laevis. Loss of hmmr function resulted in a lack of telencephalic hemisphere separation, arising from defective roof plate formation, which in turn was caused by impaired neural tissue narrowing. hmmr regulated polarization of neural cells, a function which was dependent on the MT binding domains. hmmr cooperated with the core PCP component vangl2 in regulating cell polarity and neural morphogenesis. Disrupted cell polarization and elongation in hmmr and vangl2 morphants prevented radial intercalation (RI), a cell behavior essential for neural morphogenesis. Our results pinpoint a novel role of hmmr in anterior neural development and support the notion that RI is a major driving force for anterior neurulation and forebrain morphogenesis.

Early development in Xenopus is characterized by dramatic changes in the organization of the microtubule cytoskeleton. We have used whole-mount immunocytochemistry to follow the expression of the acetylated form of alpha-tubulin during early Xenopus development. In the egg and early embryo, the monoclonal anti-acetylated tubulin antibody 6-11B-1 stained meiotic and mitotic spindles, midbody microtubules, and what appears to be the central region of the sperm aster; the antibody did not stain the sperm aster itself or the cortical microtubule system associated with the rotation of the fertilized egg. Following gastrulation, acetylated tubulin disappeared from all but mitotic midbody microtubules. During the course of neurulation high levels of acetylated tubulin reappeared in the precursors of the ciliated epidermal cells (stage 15), transiently in neural folds (stage 16/17), in neuronal processes (stage 18/19), and in somas (stage 21). The changing pattern of anti-acetylated tubulin staining during Xenopus development raises intriguing questions as to the physiological significance of tubulin acetylation.

Morphogenesis of the vertebrate neural tube occurs by elongation and bending of the neural plate, tissue shape changes that are driven at the cellular level by polarized cell intercalation and cell shape changes, notably apical constriction and cell wedging. Coordinated cell intercalation, apical constriction, and wedging undoubtedly require complex underlying cytoskeletal dynamics and remodeling of adhesions. Mutations of the gene encoding Scribble result in neural tube defects in mice, however the cellular and molecular mechanisms by which Scrib regulates neural cell behavior remain unknown. Analysis of Scribble mutants revealed defects in neural tissue shape changes, and live cell imaging of mouse embryos showed that the Scrib mutation results in defects in polarized cell intercalation, particularly in rosette resolution, and failure of both cell apical constriction and cell wedging. Scrib mutant embryos displayed aberrant expression of the junctional proteins ZO-1, Par3, Par6, E- and N-cadherins, and the cytoskeletal proteins actin and myosin. These findings show that Scribble has a central role in organizing the molecular complexes regulating the morphomechanical neural cell behaviors underlying vertebrate neurulation, and they advance our understanding of the molecular mechanisms involved in mammalian neural tube closure.

Ascidian larvae develop after an invariant pattern of embryonic cleavage. Fewer than 400 cells constitute the larval central nervous system (CNS), which forms without either extensive migration or cell death. We catalogue the mitotic history of these cells in Ciona intestinalis, using confocal microscopy of whole-mount embryos at stages from neurulation until hatching. The positions of cells contributing to the CNS were reconstructed from confocal image stacks of embryonic nuclei, and maps of successive stages were used to chart the mitotic descent, thereby creating a cell lineage for each cell. The entire CNS is formed from 10th- to 14th-generation cells. Although minor differences exist in cell position, lineage is invariant in cells derived from A-line blastomeres, which form the caudal nerve cord and visceral ganglion. We document the lineage of five pairs of presumed motor neurons within the visceral ganglion: one pair arises from A/A 10.57, and four from progeny of A/A 9.30. The remaining cells of the visceral ganglion are in their 13th and 14th generations at hatching, with most mitotic activity ceasing around 85% of embryonic development. Of the approximately 330 larval cells previously reported in the CNS of Ciona, we document the lineage of 226 that derive predominantly from A-line blastomeres.

The cranial neural crest (CNC) arises within the developing central nervous system, but then migrates away from the neural tube in three consecutive streams termed mandibular, hyoid and branchial, respectively, according to the order along the anteroposterior axis. While the process of neural crest emigration generally follows a conserved anterior to posterior sequence across vertebrates, we find that ray-finned fishes (bichir, sterlet, gar, and pike) exhibit several heterochronies in the timing and order of CNC emergence that influences their subsequent migratory patterns. First, emigration of the cranial neural crest in these fishes occurs prematurely compared to other vertebrates, already initiating during early neurulation and well before neural tube closure. Second, delamination of the hyoid stream occurs prior to the more anterior mandibular stream; this is associated with early morphogenesis of key hyoid structures like external gills (bichir), a large opercular flap (gar) or first forming cartilage (pike). In sterlet, the hyoid and branchial CNC cells form a single hyobranchial sheet, which later segregates in concert with second pharyngeal pouch morphogenesis. Taken together, the results show that despite generally conserved migratory patterns, heterochronic alterations in the timing of emigration and pattern of migration of CNC cells accompanies morphological diversity of ray-finned fishes.

Cell shape changes are critical for morphogenetic events such as gastrulation, neurulation, and organogenesis. However, the cell biology driving cell shape changes is poorly understood, especially in vertebrates. The beginning of Xenopus laevis gastrulation is marked by the apical constriction of bottle cells in the dorsal marginal zone, which bends the tissue and creates a crevice at the blastopore lip. We found that bottle cells contribute significantly to gastrulation, as their shape change can generate the force required for initial blastopore formation. As actin and myosin are often implicated in contraction, we examined their localization and function in bottle cells. F-actin and activated myosin accumulate apically in bottle cells, and actin and myosin inhibitors either prevent or severely perturb bottle cell formation, showing that actomyosin contractility is required for apical constriction. Microtubules were localized in apicobasally directed arrays in bottle cells, emanating from the apical surface. Surprisingly, apical constriction was inhibited in the presence of nocodazole but not taxol, suggesting that intact, but not dynamic, microtubules are required for apical constriction. Our results indicate that actomyosin contractility is required for bottle cell morphogenesis and further suggest a novel and unpredicted role for microtubules during apical constriction.

The neural crest (NC) is a transient multipotent cell population that migrates extensively to produce a remarkable array of vertebrate cell types. NC cell specification progresses in an anterior to posterior fashion, resulting in distinct, axial-restricted subpopulations. The anterior-most, cranial, population of NC is specified as gastrulation concludes and neurulation begins, while more posterior populations become specified as the body elongates. The mechanisms that govern development of the more posterior NC cells remain incompletely understood. Here, we report a key role for zebrafish Cdx4, a homeodomain transcription factor, in the development of posterior NC cells. We demonstrate that cdx4 is expressed in trunk NC cell progenitors, directly binds NC cell-specific enhancers in the NC GRN, and regulates expression of the key NC development gene foxd3 in the posterior body. Moreover, cdx4 mutants show disruptions to the segmental pattern of trunk NC cell migration due to loss of normal leader/follower cell dynamics. Finally, using cell transplantation to generate chimeric specimens, we show that Cdx4 does not function in the paraxial mesoderm-the environment adjacent to which crest migrates-to influence migratory behaviors. We conclude that cdx4 plays a critical, and likely tissue autonomous, role in the establishment of trunk NC migratory behaviors. Together, our results indicate that cdx4 functions as an early NC specifier gene in the posterior body of zebrafish embryos.

Geminin is a nucleoprotein that can directly bind chromatin regulatory complexes to modulate gene expression during development. Geminin knockout mouse embryos are preimplantation lethal by the 32-cell stage, precluding in vivo study of Geminin's role in neural development. Therefore, here we used a conditional Geminin allele in combination with several Cre-driver lines to define an essential role for Geminin during mammalian neural tube (NT) formation and patterning. Geminin was required in the NT within a critical developmental time window (embryonic day 8.5-10.5), when NT patterning and closure occurs. Geminin excision at these stages resulted in strongly diminished expression of genes that mark and promote dorsal NT identities and decreased differentiation of ventral motor neurons, resulting in completely penetrant NT defects, while excision after embryonic day 10.5 did not result in NT defects. When Geminin was deleted specifically in the spinal NT, both NT defects and axial skeleton defects were observed, but neither defect occurred when Geminin was excised in paraxial mesenchyme, indicating a tissue autonomous requirement for Geminin in developing neuroectoderm. Despite a potential role for Geminin in cell cycle control, we found no evidence of proliferation defects or altered apoptosis. Comparisons of gene expression in the NT of Geminin mutant versus wild-type siblings at embryonic day 10.5 revealed decreased expression of key regulators of neurogenesis, including neurogenic bHLH transcription factors and dorsal interneuron progenitor markers. Together, these data demonstrate a requirement for Geminin for NT patterning and neuronal differentiation during mammalian neurulation in vivo.