Towards a cellular and molecular understanding of neurulation.

Neurulation occurs during the early embryogenesis of chordates, and it results in the formation of the neural tube, a dorsal hollow nerve cord that constitutes the rudiment of the entire adult central nervous system. The goal of studies on neurulation is to understand its tissue, cellular and molecular basis, as well as how neurulation is perturbed during the formation of neural tube defects. The tissue basis of neurulation consists of a series of coordinated morphogenetic movements within the primitive streak (e.g., regression of Hensen's node) and nascent primary germ layers formed during gastrulation. Signaling occurs between Hensen's node and the nascent ectoderm, initiating neurulation by inducing the neural plate (i.e., actually, by suppressing development of the epidermal ectoderm). Tissue movements subsequently result in shaping and bending of the neural plate and closure of the neural groove. The cellular basis of the tissue movements of neurulation consists of changes in the behavior of the constituent cells; namely, changes in cell number, position, shape, size and adhesion. Neurulation, like any morphogenetic event, occurs within the milieu of generic biophysical determinants of form present in all living tissues. Such forces govern and to some degree control morphogenesis in a tissue-autonomous manner. The molecular basis of neurulation remains largely unknown, but we suggest that neurulation genes have evolved to work in concert with such determinants, so that appropriate changes occur in the behaviors of the correct populations of cells at the correct time, maximizing the efficiency of neurulation and leading to heritable species- and axial-differences in this process. In this article, we review the tissue and cellular basis of neurulation and provide strategies to determine its molecular basis. We expect that such strategies will lead to the identification in the near future of critical neurulation genes, genes that when mutated perturb neurulation in a highly specific and predictable fashion and cause neurulation defects, thereby contributing to the formation of neural tube defects.

Classification scheme for genes expressed during formation and progression of the avian primitive streak.

We have systematically examined the expression patterns of thirteen genes by in situ hybridization during the formation and progression of the avian primitive streak. Based on common patterns of expression, we classify these genes into three distinct groups. Group 1 genes, subdivided into group 1A (Wnt8c, Slug, Vg1, and Nodal) and group 1B (Fgf8, Brachyury, and Cripto), were expressed first in the epiblast and then, throughout most of the length of the primitive streak. Group 2 genes, namely, cNot1, Sonic hedgehog (Shh), Hnf3 beta and Chordin, were confined to the rostral end of the primitive streak, and then, to Hensen's node. In contrast, Group 3 genes, comprising Goosecoid (GSC) and Crescent, were expressed in the hypoblast. This classification scheme provides a rational basis for categorizing genes expressed during avian gastrulation, and such systematization is likely to provide insight into the relationships among different genes and their potential roles in key events of gastrulation.

Subtractive hybridization identifies chick-cripto, a novel EGF-CFC ortholog expressed during gastrulation, neurulation and early cardiogenesis.

EGF-CFC genes encode a novel class of extracellular, membrane-associated proteins that notably play an important role during vertebrate gastrulation. Whereas the two cysteine-rich domains that characterize these proteins, namely the extracellular EGF-like and the CFC domain, are known to be encoded by two evolutionarily conserved exons, it is generally assumed, based on weak primary sequence identity, that the remaining parts of the protein differ among vertebrates, suggesting that known members of the EGF-CFC family do not represent true orthologs. Here, by characterizing the full cDNA and genomic sequences of a new EGF-CFC gene in chick, and by comparing them with their counterparts in human (CRIPTO), mouse (cripto and cryptic), Xenopus (FRL-1) and zebrafish (one-eyed pinhead), we show that all EGF-CFC genes share an identical genomic organization over the entire coding region. Not only are the central two exons (coding for the EGF-like and CFC motifs) conserved, but also conserved are the total number of exons, their size, their intron phase and their correlation with discrete protein modules, in particular those modules that allow the EGF-CFC motif to become membrane-associated. Therefore, despite apparent divergence between their 5' and 3'-terminal exons, all known CRIPTO-related genes are structurally orthologous. We named this novel ortholog in bird, chick-cripto. We report the mRNA distribution of chick-cripto, which begins in the epiblast of the gastrula, with a pattern similar to EGF-CFC genes of other vertebrates.

Ectodermal markers delineate the neural fold interface during avian neurulation.

The formation and morphogenesis of the neural folds are important processes underlying neurulation. We showed previously that these processes comprise four key events in avian embryos: epithelial ridging, kinking, delamination, and apposition. Collectively, these events establish the paired, bilaminar neural folds, which fuse in the dorsal midline during late neurulation to close the neural groove and to establish the neural tube. Here, we use an antisense riboprobe for a new gene called Plato, as well as an antibody for a previously cloned transcription factor, AP-2, as markers to identify critical subpopulations of ectodermal cells during the formation and morphogenesis of the avian neural folds. Plato antisense riboprobe marks the cranial neural ectoderm and premigratory cranial neural crest cells, whereas AP-2 antibody marks the epidermal ectoderm and the early migratory neural crest. We show that subpopulations of ectodermal cells at the forebrain and midbrain levels undergo considerable rearrangement within the neural fold transition zone, which redistributes incipient neural crest cells from the neural ectodermal side of the forming neural fold interface to the epidermal ectodermal side. Additionally, we show that Plato and AP-2 provide useful markers for delineating the incipient neural fold interface.

Evidence that translation of smooth muscle alpha-actin mRNA is delayed in the chick promyocardium until fusion of the bilateral heart-forming regions.

Heart development in the chick embryo proceeds from bilateral mesodermal primordia established during gastrulation. These primordia migrate to the midline and fuse into a single heart trough. During their migration as a cohesive sheet, the cells of the paired heart fields become epithelial and undergo cardiac differentiation, exhibiting organized myofibrils and rhythmic contractions near the time of their fusion. Between the stages of cardiomyoblast commitment and overt differentiation of cardiomyocytes, a significant time interval exists. Using a new riboprobe (usmaar) for whole-mount in situ hybridization in chick embryos, we report the earliest phases of smooth muscle alpha-actin (smaa) mRNA distribution during the precontractile developmental window. We show that ingressed heart-forming regions express smaa by the head-process stage (Hamburger and Hamilton stage 5). In addition, we used usmaar to study the formation and early morphogenesis of the heart. Consistent with fate mapping studies (Garcia-Martinez and Schoenwolf [1993] Dev. Biol. 159:706-719; Schoenwolf and Garcia-Martinez [1995] Cell Mol. Biol. Res. 41:233-240; Garcia-Martinez et al., in preparation), our results with this probe, combined with detailed histological and SEM analyses of the so-called cardiac crescent, demonstrate unequivocally that the heart arises from separated and paired heart rudiments, rather than from a single crescent-shaped rudiment (that is, prior to fusion of the paired heart rudiments to establish the straight-heart tube, the rostral midline of the cardiac crescent lacks mesodermal cells and consequently fails to label with usmaar). Smaa is also expressed in the splanchnic and somatic mesoderm, marking the earliest step in coelom formation. Consequently, we also used usmaar to describe formation of the pericardium. Finally, we provide evidence of a post-transcriptional level of control of smaa gene expression in the heart fields. Our results suggest that the expression of smaa may mark a primitive mesodermal state from which definitive cell types can be derived through inductive events.

Serotonin synchronises convergent extension of ectoderm with morphogenetic gastrulation movements in Drosophila.

During Drosophila gastrulation, convergent extension of the ectoderm is required for germband extension. Adhesive heterogeneity within ectodermal cells has been proposed to trigger the intercalation of cells responsible for this movement. Segmentation genes would impose this heterogeneity by establishing a pair-rule pattern of cell adhesion properties. We previously reported that the serotonin receptor (5-ht(2Dro)) is expressed in the presumptive ectoderm with a pair-rule pattern. Here, we show that the peaks of 5-ht(2Dro) expression and serotonin synthesis coincide precisely with the onset of convergent extension of the ectoderm. Gastrulae genetically depleted of serotonin or the 5-ht(2Dro) receptor do not extend their germband properly, and the ectodermal movements becomes asynchronous with the morphogenetic movements in the endoderm and mesoderm. Associated with the beginning of this desynchronisation, is an altered subcellular localisation of adherens junctions within the ectoderm. Combined, these data highlight the role of the ectoderm in Drosophila gastrulation and support the notion that serotonin signalling through the 5-HT(2Dro) receptor triggers changes in cell adhesiveness that are necessary for cell intercalation.

Maternal and zygotic control of serotonin biosynthesis are both necessary for Drosophila germband extension.

In the accompanying paper, we report that Drosophila gastrulae genetically depleted for the 5-HT(2Dro) serotonin receptor or for serotonin show abnormal germband extension. In wild-type gastrulae, peaks of both the 5-HT(2Dro) receptor and serotonin coincide precisely with the onset of germband extension. Here, we assessed the genetic requirement for this peak of serotonin. We report the characterisation of the serotonin content of individual Drosophila embryos, progeny from flies heterozygous for mutations in genes that are involved in the serotonin synthesis pathway and include the GTP-cyclohydrolase, tryptophan hydroxylase and DOPA decarboxylase loci. The peak of serotonin synthesis at the beginning of germband extension appears strictly dependent upon the maternal deposition of biopterins, products of GTP-cyclohydrolase and cofactors of tryptophan hydroxylase and upon the zygotic synthesis of both tryptophan hydroxylase and DOPA decarboxylase enzymes. Mutant embryos with an impairment in this peak of serotonin synthesis die with a cuticular organisation which is also observed in embryos deficient for the 5-HT(2Dro) receptor. This characteristic cuticular phenotype is thus the hallmark of desynchronisation of the morphogenetic movements during gastrulation. Together, these findings provide additional support for the notion that serotonin, acting through the 5-HT(2Dro) receptor, is necessary for proper gastrulation.

Mouse 5-HT2B receptor-mediated serotonin trophic functions.

5-HT2B receptors, in addition to phospholipase C stimulation, are able to trigger activation of the proto-oncogene product p21ras. During mouse embryogenesis, a peak of 5-HT2B receptor expression is detected at the neurulation stage; we localized the 5-HT2B expression in neural crest cells, heart myocardium, and somites. The requirement for functional 5-HT2B receptors shortly after gastrulation, is supported by culture of embryos exposed to 5-HT2B-high affinity antagonist such as ritanserin, which induces morphological defects in the cephalic region, heart and neural tube. Functional 5-HT2B receptors are also expressed during the serotonergic differentiation of the mouse F9 teratocarcinoma-derived clonal cell line 1C11. Upon 2 days of induction by cAMP, 5-HT2B receptors become functional, and on day 4, the appearance of 5-HT2A receptors coincides with the onset of active serotonin transporter by these cells. Active serotonin uptake is modulated by serotonin suggesting autoreceptor functions for 5-HT2B receptors.

The mouse 5-HT2B receptor: homologous subtype or species variant?

The recently characterized 5-HT2B subfamily of serotonin receptors has now been reported from three different species: human, rat and mouse. Their genomic structures include 2 introns present at identical positions. Despite this similarity, their respective protein sequences show some diversities. In addition, the pharmacology of these receptors is distantly related, and their sites of expression vary amongst species. Thus, it appears difficult at present to unambiguously classify these receptors into the same subfamily, raising the possibility of the existence of other 5-HT2B-like receptors, yet to be discovered.

Dual organisation of the Drosophila neuropeptide receptor NKD gene promoter.

Neuropeptide function in the peripheral and central nervous systems has been described in mammals as well as in insects. We previously reported the cloning of the neuropeptide receptor NKD, a Drosophila melanogaster homologue of the mammalian tachykinin receptors. This receptor is expressed during Drosophila embryonic development and in the adult fly. Use of the NKD promoter region to drive beta-galactosidase expression in transgenic flies reveals a bipartite promoter organisation: the distal region controls NKD expression in neurosecretory cells of the central nervous system during late embryogenesis, whereas the proximal region is responsible for transient expression in peripheral nervous system during late embryogenesis, whereas the proximal region is responsible for transient expression in peripheral nervous system precursor cells early in development. This early NKD expression, first restricted to the sensory organ precursor cell, an atonal positive cell, is abolished in the ato1 mutant. In addition, we show that the proneural protein atonal, in association with daughterless, transactivates the NKD promoter in Schneider S2 cells via the proximal E box NKDE2. Furthermore, heterodimers of atonal and daughterless interact with this E box in gel shift assay.

Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation.

Serotonin, first described as a neurotransmitter in invertebrates, has been investigated mostly for its functions in the mature central nervous system of higher vertebrates. Serotonin receptor diversity has been described in the mammalian brain and in insects. We report the isolation of a cDNA coding for a Drosophila melanogaster serotonin receptor that displays a sequence, a gene organization, and pharmacological properties typical of the mammalian 5-HT2 serotonin receptor subtype. Its mRNA can be detected in the adult fly; moreover, a high level of expression occurs at 3 hr of Drosophila embryogenesis. This early embryonic expression is surprisingly organized in a seven-stripe pattern that appears at the cellular blastoderm stage. In addition, this pattern is in phase with that of the even-parasegment-expressed pair-rule gene fushi-tarazu and is similarly modified by mutations affecting segmentation genes. Simultaneously with this pair-rule expression, the complete machinery of serotonin synthesis is present and leads to a peak of ligand concomitant with a peak of 5-HT2-specific receptor sites in blastoderm embryos.

The mouse 5-HT2B receptor: possible involvement in trophic functions of serotonin.

The novel serotonin receptor 5-HT2B shows the highest homology to the 5-HT2 family of receptors. The pharmacological profile of membranes from 5-HT2B cDNA stably transfected LMTK- cell line, corresponds to a new 5-HT2-like receptor named 5-HT2B, although some difference exists between the mouse and rat pharmacology. A similar pharmacological profile is detected on the immortalized teratocarcinoma-derived cell line 1C11 upon 2 days of serotoninergic differenciation by cAMP. In both cell lines, the analysis 125I-DOI binding reveals the presence of a single class of sites, the affinity of which is one order of magnitude lower than the one reported for the 5-HT2A receptor. This demonstrates that the 5-HT2B receptor is functionally expressed before the complete serotoninergic differentiation of 1C11 cells. These observations are in good agreement with the presence of 5-HT2B mRNA in early mouse embryonic development. Furthermore, the major sites of 5-HT2B mRNA embryonic expression are in the heart, and in the neural fold before the closure of the neural tube. Therefore, this receptor could account at least in part for the trophic functions attributed to the 5-HT2-like receptors.

New mouse 5-HT2-like receptor. Expression in brain, heart and intestine.

A novel member of the family of G protein-coupled receptors has been isolated from a mouse brain cDNA library by screening with polymerase chain reaction (PCR) generated fragment of mouse genomic DNA amplified using degenerated primers. Sequence comparison demonstrates that the encoded protein sequence shows the highest homology to the 5-HT2 family of receptors. The pharmacological profile of membranes from COS cells transfected with this cDNA, corresponds to a new 5-HT2-like receptor that we propose to call 5-HT2C. Its major sites of expression are in the mouse intestine and heart, also with detectable expression in brain and kidney. We speculate that it could account at least in part for the 'atypical' functions attributed to the 5-HT1C/5-HT2 receptors.

NKD, a developmentally regulated tachykinin receptor in Drosophila.

A number of neuropeptides have been described which are present in the insect nervous system. The physiological role of these neuropeptides has not yet been clarified. We have characterized a Drosophila melanogaster cDNA coding for a protein, NKD, whose sequence resembles that of mammalian G protein-coupled neuropeptide receptors. This protein shows 38% homology with the mammalian tachykinin NK3 receptor within the transmembrane domain region. Stable cell lines expressing this cDNA are responsive to Locusta migratoria tachykinin but not to other peptides of the tachykinin family. The expression of this gene is detected principally in adult fly heads, but also in the adult body and in embryos. Interestingly, NKD mRNA is detected at very early stages of Drosophila embryonic development (3 h) and reaches the highest level of expression at 12-16 h, a time which correlates with the period of major neuronal development. In situ hybridization experiments demonstrate that NKD is expressed in the central nervous system, as well as in subsets of neurons in each segment of the developing ventral ganglia. The cytological localization of this gene is at position 86C on the Drosophila third chromosome.