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Differential expression of two cell adhesion molecules, Ephrin-A5 and Integrin alpha6, during cranial neurulation in the chick embryo.

The formation of the neural tube (neurulation) depends on the physical properties of the cells and tissues both inside and outside the neural plate. One such important physical property is cell adhesion. Theoretical and biological evidence support a role for cell adhesion in neurulation, but few specific cell adhesion molecules have been identified during this process. Ephrin-A5 and Integrin alpha6 are two of the known genes encoding cell adhesion molecules that are likely to be directly involved in neurulation because neural tube defects result when they are knocked out in mice. Yet it remains unclear how they can act on the cell and tissue behaviors of neurulation, because their domains of expression in neurulating tissues have not been reported. We report here the detailed pattern of expression of these two cell adhesion molecules in the chick embryo throughout the stages of neurulation at the mRNA and protein level. We show that Ephrin-A5 and Integrin alpha6 are differentially expressed in the ectoderm, outside and inside the neural plate, respectively, and that they are both restricted to neurulation at cranial (brain) levels. We discuss the potential contribution of this differential expression to the cell adhesion mechanisms involved in cranial neurulation and anencephaly.

Animals↗

Morphogenetic pattern formation during ascidian notochord formation is regulative and highly robust.

The ascidian notochord forms through simultaneous invagination and convergent extension of a monolayer epithelial plate. Here we combine micromanipulation with time lapse and confocal microscopy to examine how notochord-intrinsic morphogenetic behaviors and interactions with surrounding tissues, determine these global patterns of movement. We show that notochord rudiments isolated at the 64-cell stage divide and become motile with normal timing; but, in the absence of interactions with non-notochordal tissues, they neither invaginate nor converge and extend. We find that notochord formation is robust in the sense that no particular neighboring tissue is required for notochord formation. Basal contact with either neural plate or anterior endoderm/lateral mesenchyme or posterior mesoderm are each alone sufficient to ensure that the notochord plate forms and extends a cylindrical rod. Surprisingly, the axis of convergent extension depends on the specific tissues that contact the notochord, as do other patterns of cell shape change, movement and tissue deformation that accompany notochord formation. We characterize one case in detail, namely, embryos lacking neural plates, in which a normal notochord forms but by an entirely different trajectory. Our results show ascidian notochord formation to be regulative in a fashion and to a degree never before appreciated. They suggest this regulative behavior depends on a complex interplay between morphogenetic tendencies intrinsic to the notochord plate and instructive and permissive interactions with surrounding tissues. We discuss mechanisms that could account for these data and what they imply about notochord morphogenesis and its evolution within the chordate phylum.

Animals↗

The homeodomain-containing gene Xdbx inhibits neuronal differentiation in the developing embryo.

The development of the vertebrate nervous system depends upon striking a balance between differentiating neurons and neural progenitors in the early embryo. Our findings suggest that the homeodomain-containing gene Xdbx regulates this balance by maintaining neural progenitor populations within specific regions of the neuroectoderm. In posterior regions of the Xenopus embryo, Xdbx is expressed in a bilaterally symmetric stripe that lies at the middle of the mediolateral axis of the neural plate. This stripe of Xdbx expression overlaps the expression domain of the proneural basic/helix-loop-helix-containing gene, Xash3, and is juxtaposed to the expression domains of Xenopus Neurogenin related 1 and N-tubulin, markers of early neurogenesis in the embryo. Xdbx overexpression inhibits neuronal differentiation in the embryo and when co-injected with Xash3, Xdbx inhibits the ability of Xash3 to induce ectopic neurogenesis. One role of Xdbx during normal development may therefore be to restrict spatially neuronal differentiation within the neural plate, possibly by altering the neuronal differentiation function of Xash3.

Amino Acid Sequence↗

Conserved regulatory elements establish the dynamic expression of Rpx/HesxI in early vertebrate development.

TheRpx/Hesx1 homeobox gene is expressed during gastrulation in the anterior visceral and definitive endoderm and the cephalic neural plate. At later stages of development, its expression is restricted to Rathke's pouch, the primordium of the pituitary gland. This expression pattern suggests the presence of at least two distinct regulatory regions that control early and late Rpx transcription. Using transgenic mice, we have demonstrated that regulatory sequences in the 5' upstream region of Rpx are important for early expression in the anterior endoderm and neural plate and regulatory elements in the 3' region are required for late expression in Rathke's pouch. We have found that the genetically required LIM homeodomain-containing proteins Lim1/Lhx1 and Lhx3 are directly involved in the regulation of Rpx transcription. They bind two LIM protein-binding sites in the 5' upstream region of Rpx, which are required for Rpx promoter activity in both mice and Xenopus. Furthermore, we have found that a conserved enhancer in the 3' regulatory sequences of Rpx is not only required, but is also sufficient for the expression of Rpx transgenes in the developing Rathke's pouch.

Animals↗

Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling.

Cells at the anterior boundary of the neural plate (ANB) can induce telencephalic gene expression when transplanted to more posterior regions. Here, we identify a secreted Frizzled-related Wnt antagonist, Tlc, that is expressed in ANB cells and can cell nonautonomously promote telencephalic gene expression in a concentration-dependent manner. Moreover, abrogation of Tlc function compromises telencephalic development. We also identify Wnt8b as a locally acting modulator of regional fate in the anterior neural plate and a likely target for antagonism by Tlc. Finally, we show that tlc expression is regulated by signals that establish early antero-posterior and dorso-ventral ectodermal pattern. From these studies, we propose that local antagonism of Wnt activity within the anterior ectoderm is required to establish the telencephalon.

Animals↗

Experimental evidence for the early commitment of the presumptive adenohypophysis.

To investigate the commitment potentialities of the adenohypophyseal tissue, its presumptive territory, localized in the anterior ridge of the neural plate in the chick embryo, was surgically removed from 2- to 4-somite stages of development. At later stages, operated embryos were progressively collected to study the extent (if any) of the adenohypophyseal tissue. The results revealed that Rathke's pouch and later the adenohypophysis were completely absent in 33 out of the 45 studied embryos. The surrounding tissues in the operated embryos developed in a comparable manner to those in normal embryos. The head morphogenesis was not affected by the surgical ablation. Strikingly, no regeneration of adenohypophyseal tissue was observed in the operated embryos. At younger stages, a very small invagination was observed close to the diencephalon of 9 out of the 45 studied embryos. Its extent was reduced to less than 20% when compared to normal embryos at the same stages. This epithelial invagination is believed therefore to be a residuum of the stomodeal ectoderm and/or the stomodeo-adenohypophyseal channel which normally regresses at later stages of development. Therefore, the anterior ridge of the neural plate seems irrevocably destined to form adenohypophyseal tissue even as early as 2- to 4-somite stages. This suggests that the adenohypophysis presumptive territory is already committed prior to neural tube closure and also seems to confirm its origin from the neural primordium in avian embryos. Since close topological relationships link the presumptive adenohypophysis territory with both those of the hypothalamus and ectoderm of nasal cavity, possible developmental influences through such intimate contacts cannot be totally excluded.

Animals↗

Neural tube closure in the chick embryo is multiphasic.

Progression of neurulation in the chick embryo has not been well documented. To provide a detailed description, chick embryos were stained in ovo after the least manipulation possible to avoid distortion of the neural plate and folds. This allowed a morphological and morphometric description of the process of neurulation in relatively undisturbed chick embryos. Neurulation comprises several specific phases with distinct closure patterns and closure rates. The first closure event occurs, de novo, in the future mesencephalon at the 4-6 somite stage (sst 4-6). Soon afterwards, at sst 6-7, de novo closure is seen at the rhombocervical level in the form of multisite contacts of the neural folds. These contacts occur in register with the somites, suggesting that the somites may play a role in forcing elevation and apposition of the neural folds. The mesencephalic] and rhombocervical closure events define an intervening rhombencephalic neuropore, which is present for a brief period before it closes. The remaining pear-shaped posterior neuropore (PNP) narrows and displaces caudally, but its length remains constant in embryos with seven to ten somites, indicating that the caudal extension of the rhombocervical closure point and elongation of the caudal neural plate are keeping pace with each other. From sst 10 onward, the tapered cranial portion of the PNP closes fast in a zipper-like manner, and, subsequently, the wide caudal portion of the PNP closes rapidly as a result of the parallel alignment of its folds, with numerous button-like temporary contact points. A role for convergent extension in this closure event is suggested. The final remnant of the PNP closes at sst 18. Thus, as in mammals, chick neurulation involves multisite closure and probably results form several different development mechanisms at varying levels of the body axis.

Animals↗

Patterns of cell movement in early organ primordia of the chick embryo.

Purse-string constriction of the cytoskeleton at cell poles is generally accepted as the causal mechanism for invagination during early stages of organ formation. However, it is known that other cell movements, including intercalation, play a role in the organotypic shape changes that occur during gastrulation and neurulation. Such cell movements have not been investigated in pouching and branching epithelial primordia. There is reason to suspect that cells within these organ primordia might exchange their neighbors for others, that is, intercalate or translocate, at sites of sharp folding such as borders with the surrounding epithelial sheet or where a bend occurs within the primordium. The greatest difficulty in identifying these movements has been the need to use intact embryos so that the processes are not distorted. This study explores the possibility of using time-lapse video recording to identify cell movement at these locations. Three organ primordia were tested: otic and thyroid placodes, which had not been tested previously, and neural plate as a control, where movements of this sort have been documented. Embryos or parts containing the primordia were immobilized and cell apices visualized with Hoffman modulation contrast optics. Recordings to an optical memory disc recorder were transferred to a microcomputer for image analysis. The viewing procedure allows reasonably clear visualization of cell apices, and image analysis permits tracking of a number of adjacent cell apices over an extended time period. Several types of movement were found to occur within cell sheets, and the relative abundance of each type depends on the specific primordium. In the neural plate, some cells move many cell diameters from their neighbors. In the other two primordia, most cells show limited shifts in position relative to their neighbors except at regions where folds are formed. In other regions, adjacent cells move as a unit. Knowledge of the movements which occur in any particular primordium is essential to an understanding of the mechanisms controlling its formation.

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Induction from posterior hypothalamus is essential for the development of the pituitary proopiomelacortin (POMC) cells of the toad (Bufo japonicus).

The role of the posterior hypothalamus in the development of the epithelial hypophysis was studied in Bufo embryos. In animals from which the central part of the neural plate (NP) had been surgically removed at the open neurula stage, the infundibulum did not develop, and the epithelial hypophysis was formed away from the normal site without morphological connection with the brain. Immunoreactive MSH cells and ACTH cells, i.e., the pituitary POMC cells, were not detected in any of the surgically treated animals, while other types of secretory cells (PRL, GH, TSH and GTH cells) were invariably present. In view of the fact that POMC cells originate in the anterior neural ridge, and not in the neural plate, the embryonic brain seems to exert an inductive influence upon the primordial pituitary POMC cells. Since these cells differentiate in a tail graft, isolated from the brain at a later stage (tail-bud stage), the inductive stimuli must be conveyed from/via the posterior hypothalamus to the pituitary anlage between the open neurula and the tail-bud stages.

Adrenocorticotropic Hormone↗

The homeodomain-containing transcription factor X-nkx-5.1 inhibits expression of the homeobox gene Xanf-1 during the Xenopus laevis forebrain development.

Expression of the homeobox gene Xanf-1 starts within the presumptive forebrain primordium of the Xenopus embryo at the midgastrula stage and is inhibited by the late neurula. Such stage-specific inhibition is essential for the normal development as the experimental prolongation of the Xanf-1 expression elicits severe brain abnormalities. To identify transcriptional regulators that are responsible for the Xanf-1 inhibition, we have used the yeast one-hybrid system and identified a novel Xenopus homeobox gene X-nkx-5.1 that belongs to a family of Nkx-5.1 transcription factors. In terms of gene expression, X-nkx-5.1 shares many common features with its orthologs in other species, including expression in the embryonic brain and in the ciliated cells of the otic and lateral line placodes. However, we have also observed several features specific for X-nkx-5.1, such as expression in precursors of the epidermal ciliated cells that may indicate a possible common evolutionary origin of all ciliated cells derived from the embryonic ectoderm. Another specific feature is that the X-nkx-5.1 expression in the anterior neural plate starts early, within the area overlapping the Xanf-1 expression territory at the midneurula stage, and it correlates with the beginning of the Xanf-1 inhibition. Using various loss and gain-of-function techniques, including microinjections of antisense morpholino oligonucleotides and mRNA encoding for the X-nkx-5.1 and its dominant repressor and activator versions, we have shown that X-nkx-5.1 can indeed play a role of stage-specific inhibitor of Xanf-1 in the anterior neural plate during the Xenopus development.

Amino Acid Sequence↗

Variation in development of rat embryos at the presomite period.

Rat embryos at a single gestational time in the presomite period were studied for their variation in development and their fate after culture. They were explanted at 8 A.M. on day 9 of gestation from timed-pregnant Sprague-Dawley rats which were obtained by mating between 8 and 10 A.M. (plug day = day 0). In the first experiment, a total of 203 embryos from 20 litters were examined for their variation in development. Several dimensions of embryo/egg cylinder were measured and development of various embryonic/extraembryonic structures were assessed using a scoring system that we developed for the present study. Embryos were then divided into different stages of development based on their scores using the staging system that we developed previously. A large variation in developmental stage was demonstrated; the youngest embryo was at the early primitive streak stage with no signs of amniotic folds and the oldest one was at the late neural plate stage with a foregut pocket but without visible somites. No strong correlation was demonstrated between developmental stage and size of embryo/egg cylinder, nor between developmental stage and development of the proamniotic tube, ectoplacental cavity, or allantois. In the second experiment, embryos were explanted at the same time and those at different stages were cultured separately in rotating bottles and their outcomes were compared after 49 hours. The difference in mean somites number of embryos cultured from the mid primitive streak and late neural plate stages was 6.1. This difference corresponds to approximately 10 hours based on the known linear increase of somites number on day 11 of approximately 0.6 somites per hour. These results indicate a large variation in development of presomite period embryos supposedly of the same gestational age and suggest the importance of careful staging at the time of explantation if precision is needed for whole embryo culture experiments.

Animals↗

Specification of the vertebrate eye by a network of eye field transcription factors.

Several eye-field transcription factors (EFTFs) are expressed in the anterior region of the vertebrate neural plate and are essential for eye formation. The Xenopus EFTFs ET, Rx1, Pax6, Six3, Lhx2, tll and Optx2 are expressed in a dynamic, overlapping pattern in the presumptive eye field. Expression of an EFTF cocktail with Otx2 is sufficient to induce ectopic eyes outside the nervous system at high frequency. Using both cocktail subsets and functional (inductive) analysis of individual EFTFs, we have revealed a genetic network regulating vertebrate eye field specification. Our results support a model of progressive tissue specification in which neural induction then Otx2-driven neural patterning primes the anterior neural plate for eye field formation. Next, the EFTFs form a self-regulating feedback network that specifies the vertebrate eye field. We find striking similarities and differences to the network of homologous Drosophila genes that specify the eye imaginal disc, a finding that is consistent with the idea of a partial evolutionary conservation of eye formation.

Animals↗

Distribution and functional role of laminin during induction of the embryonic axis in the chick embryo.

Laminin is a major glycoprotein of basement membranes and has been shown to promote cell adhesion, and movement of various nonepithelial cells and tumour cells. Using antibodies to laminin in paraffin sections and cultured embryos, we have studied the distribution of laminin and its involvement in the first morphogenetic events, beginning with the first extensive cellular migrations and interactions that result in the induction of the primitive streak (PS) and of the neural plate in the early chick embryo. Laminin immunogold labeling was not detected in the blastoderm at stage X. At stage XIII, laminin immunoreactivity was detected at the ventral surface of the epiblast and in the entire hypoblast. The intense labeling of the hypoblast indicated that these cells are active in laminin synthesis. Extracellular matrix (ECM) started accumulating as the first embryonic spaces were forming, before the morphogenetic movements of gastrulation were initiated. Immunogold labeling revealed a punctate pattern of laminin distribution in the ECM in the blastocoele, and in the space below the neural plate. Laminin, which is a multidomain molecule known to interact with other molecules of the ECM and with the cell surface, could serve as the scaffold for highly specific contact points of migrating cells and for the folding of epithelial sheets during this time in the developing embryo. We incubated blastoderms at stages X and XIII with laminin antibodies (1:30 dilution) for 4 h, then cultured the blastoderms further in plain egg albumin. The laminin antibodies did not interfere with triggering of PS cell movements, but perturbed the normal migration pattern of these cells. A normal PS did not form and, as a consequence, the embryonic axis was not induced.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

The Xfeb gene is directly upregulated by Zic1 during early neural development.

The transcription factor Zic1 plays important roles in patterning the neural plate in early vertebrate development. However, few genes that are regulated by Zic1 are known. We have identified a new direct downstream target gene of Zic1 that we have named Xfeb. Xfeb is a member of the pathogenesis-related (PR) protein superfamily and contains five tandem SCP domains. The sequence of Xfeb suggests that it may possess serine protease activity. Xfeb is expressed in the presumptive hindbrain region during neurula stages and in somite tissues later in development. Xfeb represses the hindbrain gene hoxB1 and the anterior neural gene otx2, suggesting that Xfeb is involved in regionalizing the neural plate, possibly by ensuring a posterior expression limit for otx2.

Amino Acid Sequence↗

[Immunohistochemical study of developing rat embryo--localization of vimentin, GFAP, neurofilament protein within rat embryo central nervous system].

The identification of intermediate filaments within immature cells is valuable as a means to investigate neuronal and glial differentiation. The purpose of this study is to investigate the time of appearance of neuron and glia within rat embryo by identifying cell-specific intermediate filaments. Pregnant rat were daily sacrificed from day 9-20 of pregnancy. Rat embryos were fixed with 100% ethanol and then we have made the paraffin section. We have stained the specimens for vimentin in accordance with ABC method and for GFAP in accordance with PAP method. We have performed Bodian stain. The neural plate of day 9 rat embryo is composed with vimentin positive immature cells which are disposed with pseudostratified arrangement. In midbrain of day 13 rat embryo, immature cells and their fibers are vimentin positive which are spanning from ventricular surface to marginal layer. NFP is detectable in immature cells and fibers of marginal layer. But GFAP positive cells and fibers are not identified in the same region. In anterior column of day 18 rat embryo, GFAP positive fibers are detectable. Summarizing of these results, we can display first: immature cells composing of neural plate are vimentin positive at day 9, second: NFP positive fibers and cells appear at day 13, third: NFP positive cells are vimentin positive, too, forth: GFAP positive fibers appear at day 18 and they are vimentin positive, too.

Animals↗

Churchill, a zinc finger transcriptional activator, regulates the transition between gastrulation and neurulation.

Gastrulation generates mesoderm and endoderm from embryonic epiblast; soon after, the neural plate is established within the epiblast-both events require FGF signaling. We describe a zinc finger transcriptional activator, Churchill (ChCh), which acts as a switch between different roles of FGF. FGF induces ChCh slowly; this activates Smad-interacting-protein-1 (Sip1), which blocks further induction of the mesoderm markers brachyury and Tbx6L by FGF. ChCh is first expressed as cells stop migrating through the primitive streak, and we show that it regulates cell ingression. We propose a simple mechanism by which FGF sensitizes cells to BMP signals. These results reveal that neural induction requires cessation of mesoderm formation at the midline in addition to the decision between epidermis and neural plate.

Amino Acid Sequence↗

Her5 acts as a prepattern factor that blocks neurogenin1 and coe2 expression upstream of Notch to inhibit neurogenesis at the midbrain-hindbrain boundary.

Neurogenesis in both vertebrates and invertebrates is tightly controlled in time and space involving both positive and negative regulators. We report here that the bHLH factor Her5 acts as a prepattern gene to prevent neurogenesis in the anlage of the midbrain/hindbrain boundary in the zebrafish neural plate. This involves selective suppression of both neurogenin1 (ngn1) and coe2 mRNA expression in a process that is independent of Notch signalling, and where inhibition of either ngn1 or coe2 expression is sufficient to prevent neuronal differentiation across the midbrain-hindbrain boundary. A ngn1 transgene faithfully responds to Her5 and deletion analysis of the transgene identifies an E-box in a ngn1 upstream enhancer to be required for repression by Her5. Together our data demonstrate a role of Her5 as a prepattern factor in the spatial definition of proneural domains in the zebrafish neural plate, in a manner similar to its Drosophila homologue Hairy.

Animals↗

Combinatorial expression of zebrafish Brn-1- and Brn-2-related POU genes in the embryonic brain, pronephric primordium, and pharyngeal arches.

Vertebrate class III POU genes are widely expressed in the embryonic and adult central nervous system, where they act as transcriptional regulators of cell- and/or region-specific gene expression. We isolated four zebrafish class III POU genes, named zp-12, zp-23, zp-47 and zp-50. In this study, we examined the developmental expression patterns of the Brn-1- and Brn-2-related zp-12, zp-23 and zp-47 genes by means of whole-mount in situ hybridization. Similarly to their mammalian orthologues, the major expression site of all zebrafish zp genes is the CNS. Neurectodermal expression was first detected at the beginning of somitogenesis in spatially restricted segment-like domains in different parts of the neural plate. During somitogenesis transcript distributions changed from highly restricted to widespread but nevertheless distinct patterns found in all major subdivisions of the CNS. While zp-47 expression was detected exclusively in the CNS, localized expression of zp-12 and zp-23 was also found in the pronephric primordium and in cell clusters within the mandibular and hyoid arches. Furthermore, zp-23 transcripts were transiently detected in a restricted region of the paraxial mesendoderm and, at late embryogenesis stages, in the auditory vesicles. The early regionalized expression of all three zp genes is compatible with roles in regional specification of the neural plate. Comparison of the distinct yet overlapping expression of zp-12, zp-23, zp-47 and the previously characterized zp-50 gene implies both unique, as well as redundant functions for each family member. We propose that coordinate expression of particular combinations of class III POU genes contribute to pattern formation or cell fate determination in the developing CNS and other structures.

Animals↗