Neural Folds Research Articles

Expression of Xenopus snail in mesoderm and prospective neural fold ectoderm

Expression of the Xsna gene during Xenopus laevis embryogenesis has been analysed by in situ hybridisation. Like its homologue snail in Drosophila, Xsna is expressed zygotically in all early mesoderm. Expression starts during stage 9 in the dorsal marginal zone and spreads to the ventral side by stage 10. During gastrulation, each cell begins to express as it involutes so that cells newly expressing Xsna are added to the forming mesoderm mantle in an anterior-to-posterior progression. Xsna expression is then down-regulated in a tissue-specific fashion that reveals the subdivision of the mesoderm before its derivatives are overtly differentiated; e.g., the appearance of the notochord, myotomes, and pronephroi are preceded by the disappearance of Xsna mRNA, while undifferentiated mesoderm remains labelled, even into tadpole stages. Xsna is expressed in the suprablastoporal endoderm during gastrulation and in its derivatives, the prechordal and sub-notochordal endoderm, during neurulation. Relationships between Xbra, Xtwi, and Xsna expression are examined. Xsna is also expressed in the prospective neural fold ectoderm from stage 11 in a low arc above the dorsal marginal zone, precisely identifying a distinct band of cells that surrounds the prospective neural plate that we designate the neural plate border. The anterior transverse neural fold, which becomes forebrain, ceases Xsna expression during neurulation. In the longitudinal neural folds, the deep and superficial ectoderm compartments labelled by Xsna expression are the prospective neural crest and prospective roof of the neural tube, respectively. Xsna expression persists in the neural crest during migration and in some derivatives at least until metamorphosis but ceases in the roof of the neural tube soon after neurulation.

Calcium Regulation of Neural Fold Formation: Visualization of the Actin Cytoskeleton in Living Chick Embryos

The involvement of calcium ions during chick neurulation was studied by treating neural plate stage embryos with agonists and antagonists of calcium transport and with inhibitors of calmodulin activity. Organotypic shape changes were examined by light and scanning electron microscopy. Changes in size of cell apices were quantitated by computer image analysis of actin filaments labeled with fluorescent phallicidin in time lapse recordings of living embryos. Both ionomycin and A23187 caused precocious fold elevation around the median hinge point and convergence by bending at the lateral furrows only when Ca2+ was in the external medium. As judged by decreased perimeters of 100 fluorescent apical polygons, cell apices constricted medial to the lateral furrows but did not change significantly within the median hinge point. Pretreatment with dihydrocytochalasin B or cytochalasin D prevented precocious folding and apical constriction. Papaverine and verapamil prevented folding but could be reversed by subsequent ionophore treatment. Calmidazolium and trifluoperazine irreversibly blocked folding. The demonstration in living embryos of constriction of lateral cell apices in a calcium-dependent manner is consistent with a contractile process operating during the formation of neural folds.

Raphe of the posterior neural tube in the chick embryo: its closure and reopening as studied in living embryos with a high definition light microscope.

Chick embryos cultured on a curved substratum show a transient enlargement of the posterior neuropore (PN), mimicking the temporary delay of PN closure as seen in the curly tail (ct) mouse mutant (van Straaten et al. [1993] Development 117:1163-1172). In the present study the PN enlargement in the chick embryo was investigated further with a high definition light microscope (HDmic), allowing high resolution viewing of living embryos in vitro. The temporary PN enlargement appeared due to considerable reopening of the raphe of the posterior neural tube, which was followed by reclosure after several hours. The raphe was subsequently studied in detail. It appeared very irregular, with small zones of apposed, open and fused neural folds. During closure, these raphe features shifted posteriorly. A distinct fusion sequence between surface epithelium and neuroepithelium was not seen. During experimental reopening of the raphe in vitro, small bridges temporarily arose, broke and disappeared quickly; they likely represented the first adhesion sites between the neural folds. More prominent adhesion sites partly detached, resulting in bridging filopodia-like connections; they probably represented the first anteroposterior locations of neural fold fusion. Our observations in the living chick embryo in vitro thus show that the caudal neural tube has an irregular raphe with few adhesion sites, which can be readily reopened. As a result of the irregularity, the PN does not close zipper-like, but button-like by forming multiple closure sites.

Tracing neuroepithelial cells of the mesencephalic and metencephalic alar plates during cerebellar ontogeny in quail-chick chimaeras.

The quail-chick chimaera system was used to investigate the origin of the various neuronal cell types of the cerebellum from the mesencephalic and metencephalic brain vesicles at the 11- to 14-somite stage in the avian embryo. We have already demonstrated that the cerebellum is derived from both the mesencephalic and metencephalic brain vesicles. The mesencephalic contribution to the cerebellum is restricted to a mediodorsal territory inserted as a V-shaped area into the primitive metencephalic vesicle through complex morphogenetic movements taking place from day 2 to day 4 of embryonic development. Here we report that the cerebellar presumptive territory extends along the anteroposterior axis, over the caudal half of the mesencephalon and the rostral half of the metencephalon. Along the dorsoventral axis, the cerebellar anlage is located in the alar plates at the exclusion of the roof and basal plates, i.e. lies in the lateral walls of the neuroepithelium in the area included between approximately 25 and 120 degrees with respect to the sagittal plane. We also report that the neuroepithelium corresponding to the cerebellar presumptive territory also yields other brain structures (e.g. part of the optic tectum in the mesencephalon). The external granular layer (EGL) arises only from the rostral metencephalon, undergoing extensive tangential movements which we have analysed in detail: the more ventral the position of cells in the metencephalic alar plates the more rostral and lateral is their position in the EGL. Finally, we discuss the fact that the cerebellar cortex, an integrative structure of the brain, arises from the alar plates of the neural tube. This is consistent with the general spatial organization of the neural anlage of the vertebrate embryo, in which this part of the neuroepithelium is devoted to the production of interneurons, whereas the basal plate and the neural folds yield motor structures and primary sensory neurons respectively.

Natural wound formation: Endodermal responses in experimental primary neural induction in the chick embryo

Natural wound formation in experimental primary neural induction has been studied by SEM and in paraffin wax sections in embryos from 0 minutes to 10 hours of re-incubation. Stage 4 host and graft embryos were removed from hen's eggs and mounted as for New culture. Graft Hensen's nodes were transplanted into "pockets" created in the host area pellucida and re-incubated for up to 10 hours. Initially the cut edges of the graft establish contact with the host ectoderm layer. After 4 hours the cut edges of the graft move from the host ectoderm to the host endoderm layer. Several small openings form in the host endoderm over the graft tissue. By 6 hours, these openings join to form a single natural wound through which the underlying graft is exposed to the external environment. At 8 hours the graft forms a head-fold and neural folds are evident. During 8 to 10 hours of re-incubation the edges of the graft which attach to the edges of the host endoderm meet in the midline and close the opening in the host endoderm; simultaneously, the graft forms a neural tube. The endodermal wounds form by cell re-arrangement and by a minor contribution from cell loss.

Recovery of viability in triploid hybrids between Japanese and European water frogs, and their reproductive capacity

Diploid and triploid hybrids, consisting of the various combinations of chromosome set and cytoplasm between Rana nigromaculata and Rana lessonae, were generated in order to study their viability and reproductive capacity. Reciprocal diploid hybrids and allotriploids, which had two lessonae genomes and one nigromaculata genome in nigromaculata cytoplasm, all died without closing their neural folds. Allotriploids, which had two nigromaculata genomes and one lessonae genome in nigromaculata cytoplasm, all grew into healthy mature males in 12.4% (21 frogs) of zygotes. Following the elimination of lessonae chromosomes, ten of these males produced many spermatozoa containing a haploid set of nigromaculata chromosomes. Other allotriploids, which had either one nigromaculata genome and two lessonae genomes or two nigromaculata genomes and one lessonae genome in lessonae cytoplasm, exhibited slightly improved viability.

Bilateral eye formation in the eyeless mutant Mexican salamander following unilateral, partial excision of neural fold tissues: A quantitative study

AbstractPrevious results showed that ectopic neural fold grafts, performed unilaterally during neurulation on eyeless mutant axolotl embryos, frequently stimulated eye formation bilaterally. The analysis of the eyeless mutant was expanded in order to obtain quantitative data on the effect of various types of neural fold grafts. While performing these experiments, significant eye formation occurred in controls. Larvae in which neural fold tissues were not grafted but only partially removed at neurula stages formed eyes in some experiments, but not in others. In those cases that eyes did form, partial removal of the posterior neural fold of the prospective ear region stimulated eyes to differentiate more frequently than partial removal of the anterior neural fold of the prospective nose area. In both cases, unilateral neural fold ectomy frequently resulted in bilateral eye formation. There was a positive correlation between the amount of posterior neural fold tissues excised and the frequency of cases in which eyes formed. This increase was roughly 10% if the posterior neural fold excision was performed bilaterally instead of unilaterally.A comparison of eye formation between neural fold excision experiments and ectopic neural fold grafts was performed. There was a statistically significant different between anterior and posterior neural fold operations, but not between grafting and excision experiments. I interpret these results to suggest that there is a signal in the posterior neural fold of the eyeless mutant that prevents eye formation. This signal can be neutralized most efficiently by wounding of the posterior neural folds. In addition, the results provide strong evidence that the effect of wounding propagates from posterior to anterior regions, as well as from one side of the developing neuroepithelium to the other. © 1993 Wiley‐Liss, Inc.

Organometallic complexes in ascidian embryonic development: II. Effects on different stages and larvae

AbstractThe effects of the organometallic compounds Bu2Sn‐D‐(−;)sorbitol, Bu2Sn‐D‐(+)glucose, Bu2Sn‐D‐(−)fructose and Bu2Sn‐D‐(+)glyceraldehyde were tested in vivo on different stages of Ascidian development, larval movement and metamorphosis. Organotin(IV) complexes are organometallic compounds widely used as industrial biocides, antifouling agents and agricultural fungicides and are toxic to a range of organisms. Two‐cell stage embryos, if incubated for one hour in the organotin (IV) solutions, stopped the cleavage, which was restored when they were transferred into normal sea water. The gastrula stage was seriously affected in 10−4mol dm−3 solutions of the above‐mentioned complexes: 85% of the embryos were anomalous neurulae with open neural folds, 5% were twisted larvae. The gastrulae, when incubated for 1 h in 10−5mol dm−3 solutions, developed twisted larvae in ovular envelopes and immobile larvae with twisted tails. Larvae treated with 10−4mol dm−3 and 10−5 mol dm−3 Bu2Sn‐D‐(−)sorbitol, Bu2Sn‐D‐(+)glucose and Bu2Sn‐D‐(+)glyceraldehyde solutions stopped swimming, did not metamorphose and afterwards underwent cytolysis. An initial hyperactivity of circular movements, followed by immobility, was observed in the larvae incubated in Bu2Sn‐D‐(−;)fructose.

Cyclosporin A-Induced Embryopathy in Embryo Culture Is Mediated through Inhibition of the Arachidonic Acid Pathway

Embryos from Swiss Webster mice were grown in culture for 24 hr starting at Day 8.5 of gestation to study the effects of cyclosporin A (CsA) on the developing embryo. The embryos exposed to concentrations of CsA from 0.1 microgram/ml to 10.0 micrograms/ml developed a significant increase in the incidence of malformations from 28.6% to 78.6%, as compared with the 6.8% incidence of malformations in the control embryos. These malformations included defects in the neural tubes, head folds, and facial arches. In addition, inhibition of embryonic growth in CsA-exposed embryos was shown by a lower somite number, crown-rump length, and protein content than those of the control embryos. Supplementation of the culture medium with arachidonic acid or prostaglandin E2 decreased the incidence of CsA-induced malformations by 50% to 70% and prevented the CsA-induced inhibition of growth. We conclude that CsA causes abnormal embryonic development in mouse embryo culture and that the mechanism of CsA-induced embryopathy involves inhibition of the arachidonic acid pathway.

Antisense attenuation of Wnt‐1 and Wnt‐3a expression in whole embryo culture reveals roles for these genes in craniofacial, spinal cord, and cardiac morphogenesis

Wnt-1 and Wnt-3a proto-oncogenes have been implicated in the development of midbrain and hindbrain structures. Evidence for such a role has been derived from in situ hybridization studies showing Wnt-1 and -3a expression in developing cranial and spinal cord regions and from studies of mutant mice whose Wnt-1 genes have undergone targeted disruption by homologous recombination. Wnt-1 null mutants exhibit cranial defects but no spinal cord abnormalities, despite expression of the gene in these regions. The absence of spinal cord abnormalities is thought to be due to a functional compensation of the Wnt-1 deficiency by related genes, a problem that has complicated the analysis of null mutants of other developmental genes as well. Herein, we describe the attenuation of Wnt-1 expression using antisense oligonucleotide inhibition in mouse embryos grown in culture. We induce similar mid- and hindbrain abnormalities as those seen in the Wnt-1 null mutant mice. Attenuation of Wnt-1 expression was also associated with cardiomegaly resulting in hemostasis. These findings are consistent with the possibility that a subset of Wnt-1 expressing cells include neural crest cells known to contribute to septation of the truncus arteriosus and to formation of the visceral arches. Antisense knockout of Wnt-3a, a gene structurally related to Wnt-1, targeted the forebrain and midbrain region, which were hypoplastic and failed to expand, and the spinal cord, which exhibited lateral outpocketings at the level of the forelimb buds. Dual antisense knockouts of Wnt-1 and Wnt-3a targeted all brain regions leading to incomplete closure of the cranial neural folds, and an increase in the number and severity of outpocketings along the spinal cord, suggesting that these genes complement one another to produce normal patterning of the spinal cord. The short time required to assess the mutant phenotype (2 days) and the need for limited sequence information of the target gene (20-25 nucleotides) make this antisense oligonucleotide/whole embryo culture system ideal for testing the importance of specific genes and their interactions in murine embryonic development.

Vital dye analysis of cranial neural crest cell migration in the mouse embryo.

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the forebrain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of antibodies against N-cadherin and N-CAM on the cranial neural crest and neural tube

We have examined the distribution and function of the defined cell adhesion molecules, N-cadherin and N-CAM, in the emigration of cranial neural crest cells from the neural tube in vivo. By immunocytochemical analysis, both N-cadherin and N-CAM were detected on the cranial neural folds prior to neural tube closure. After closure of the neural tube, presumptive cranial neural crest cells within the dorsal aspect of the neural tube had bright N-CAM and weak N-cadherin immunoreactivity. By the 10- to 11-somite stage, N-cadherin was prominent on all neural tube cells with the exception of the dorsal-most cells, which had little or no detectable immunoreactivity. N-CAM, but not N-cadherin, was observed on some migrating neural crest cells after their departure from the cranial neural tube. To examine the functional significance of these molecules, perturbation experiments were performed by injecting antibodies against N-CAM or N-cadherin into the cranial mesenchyme adjacent to the midbrain. Fab' fragments or whole IgGs of monoclonal and polyclonal antibodies against N-CAM caused abnormalities in the cranial neural tube and neural crest. Predominantly observed defects included neural crest cells in ectopic locations, both within and external to the neural tube, and mildly deformed neural tubes containing some dissociating cells. A monoclonal antibody against N-cadherin also disrupted cranial development, with the major defect being grossly distorted neural tubes and some ectopic neural crest cells outside of the neural tube. In contrast, nonblocking N-CAM antibodies and control IgGs had few effects. Embryos appeared to be sensitive to the N-CAM and N-cadherin antibodies for a limited developmental period from the neural fold to the 9-somite stage, with older embryos no longer displaying defects after antibody injection. These results suggest that the cell adhesion molecules N-CAM and N-cadherin are important for the normal integrity of the cranial neural tube and for the emigration of neural crest cells. Because cell-matrix interactions also are required for proper emigration of cranial neural crest cells, the results suggest that the balance between cell-cell and cell-matrix adhesion may be critical for this process.

Exogenous transferrin is taken up and localized by the neurulation-stage mouse embryo in vitro

We have screened neurulation-stage mouse embryos for regional differences in protein distribution, by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The screen has revealed an 83-kD protein (p I 6.8) that is present in embryo regions where neurulation is in progress but not in regions where neurulation is complete. The 83-kD protein is not synthesized in the neurulation-stage embryo or in the yolk sac, but is taken up from the culture serum in vitro and, probably, from the maternal serum in utero. The 83-kD protein has been identified as transferrin on the basis of its electrophoretic migration and recognition on Western blots by an antitransferrin antibody. Culture of embryos in serum containing 125I-transferring, followed by autoradiography of embryo sections, shows that transferrin is taken up and localized in the gut beneath the closing neural folds at several levels of the body axis in 8.5- and 9.5-day embryos. In situ hybridization studies show that the transferrin receptor mRNA is expressed in all cells of the 9.5-day embryo, including the gut endoderm. These findings are consistent with a role for transferrin in development of the gut and perhaps, indirectly, in completion of neurulation during early mouse embryogenesis.

An antisense oligodeoxynucleotide inhibits proopiomelanocortin translation in AtT-20 cell line

The production of peptide hormones through proteolytic cleavage of prohormones, e.g., proopiomelanocortin (POMC), involves a number of regulated secretory proteins, such as prohormone convertase PC1, PC2 and granin family members, that are co-expressed with the prohormone. Although the expression of these proteins has been well-studied in adult animals, data on their expression during development are limited. We used whole-mount in situ hybridization to visualize POMC mRNA expression in the intermediate and anterior pituitary of Xenopus tadpoles. A more sensitive analysis, namely semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) on total RNA isolated from Xenopus developmental stages, revealed that the expression of POMC, PC1 and PC2 mRNA commenced at stages 13 (neural plate stage), 15 (neural fold stage) and 19 (neural tube stage), respectively, with a gradual increase in their expression levels during further development. Surprisingly, and in contrast to what holds for POMC and the convertases, mRNAs for secretogranin II and III (SgII, SgIII) and 7B2 were not only expressed during neural development, but could already be detected in unfertilized mature oocytes, the first cleavage stages and in blastula-stage embryos. These granins are thus maternally present in Xenopus embryos suggesting that they may have a role during oogenesis and/or early embryonic development.

Comparison of staging systems for the gastrulation and early neurulation period in rodents: a proposed new system.

Because there is no standard developmental staging system for the early postimplantation period of rodent embryos, investigators must now choose between a variety of systems that differ significantly. We have reviewed many of these staging systems and have summarized the ambiguities within them and the inconsistencies among them. In order to compare systems, we first obtained a consensus of the order of developmental events from the literature, and then attempted to fit existing systems into this order taking into account inconsistencies in terminology and blurred borderlines between stages. We were able to do this for most systems but not all because some were too divergent. We found that inconsistencies in definition of some terms, such as "primitive streak stage" and those used to describe the early neurulation process (neural plate, neural groove, neural folds, and head fold) cause much confusion. In order to develop an unambiguous system which can be used by all investigators, we propose to modify Theiler's system, which is one of the most commonly used systems but is not defined precisely during the early postimplantation period. We suggest making subdivisions of the original stages as follows: 1) stage 8 into 8a and 8b, by the degree of extension of the proamniotic cavity into the extraembryonic region; 2) stage 10 into 10a and 10b, by the completion of amnion formation; 3) stage 11 into 11a, 11b, and 11c, by the appearance of neural folds and foregut pocket. After Stage 12, the number of somite pairs can be used to precisely stage embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of ethanol-induced cell death in the developing nervous system of mice; Neural fold states through the time of anterior neural tube closure

Vital staining and routine histological analyses of mouse embryos 12 h after acute maternal ethanol administration (2.9 g/kg) illustrated that selected neuronal cell populations are killed. At the time of treatment, embryos had 5–15 somite pairs, corresponding to the developmental stages occurring in humans during the fourth week of post-fertilization; i.e. when neural folds are present and neural tube fusion begins. Affected cell populations in embryos having 6–26 somite pairs (up to the stage of anterior neuropore closure) were in discrete locations in the alar and basal plates of the rhombencephalon, in the otic placode/vesicle, and in the regions of the epibranchial placodes, olfactory placodes and trigeminal ganglion. The potential basis for the vulnerability of these cell populations to ethanol-induced cell death is discussed. Our understanding of the scope of ethanol-induced CNS damage is dependent upon further defining ethanol-sensitive cell populations at all stages of CNS development.

A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo

To obtain gene sequences controlling the early steps of amphibian neurogenesis, we have performed differential screening of a subtractive cDNA library prepared by a novel PCR-based method from a single presumptive neural plate of a Xenopus laevis late-gastrula embryo. As a result we have isolated a fragment of a novel homeobox gene (named XANF-1, for Xenopus anterior neural folds). This gene is expressed predominantly in the anterior part of the developing nervous system. Such preferential localization of XANF-1 mRNA is established from its initially homogenous distribution in ectoderm of early gastrula. This change in the expression pattern is conditioned by a differential influence of various mesoderm regions on ectoderm: anterior mesoderm activates XANF-1 expression in the overlying ectoderm, whereas posterior axial and ventral mesoderm areas inhibit it. The data obtained demonstrate for the first time that selection of genes for specific expression in the CNS of the early vertebrate embryo is affected not only by chordamesoderm (a neural inductor) but also by ventral mesoderm.

Fluorescence-activated sorting of totipotent embryonic stem cells expressing developmentally regulated lacZ fusion genes.

Murine embryonic stem (ES) cells were infected with a retrovirus promoter trap vector, and clones expressing lacZ fusion genes (LacZ+) were isolated by fluorescence-activated cell sorting (FACS). Of 12 fusion genes tested, 1 was repressed when ES cells were allowed to differentiate in vitro. Two of three lacZ fusion genes tested were passed into the germ line, indicating that FACS does not significantly affect stem cell totipotency. The pattern of lacZ expression observed in vivo was consistent with that seen in vitro. Both fusion genes were expressed in preimplantation blastulas. However, a fusion gene whose expression was unaffected by in vitro differentiation was ubiquitously expressed in day-10 embryos, while the other, which showed regulated expression in vitro, was restricted to cells located along the posterior neural fold, the optic chiasm, and within the fourth ventricle. These results demonstrate the utility of using promoter trap vectors in conjunction with fluorescence sorting to disrupt developmentally regulated genes in mice.

Embryonic development of the house shrew (Suncus murinus)

The embryonic development during the period from 1 to 12 pairs of somites was observed in an insectivore species, the house shrew (Suncus murinus), which has been bred within a closed colony. Embryos were staged by the number of somite pairs. Each stage was punctuated at every addition of three pairs of somites and numbered after the Carnegie system. The first somite became apparent between 8 and 9.0 days after fertilization, and the 12th somite appeared between 9.5 and 10.0 days. The rate of somite formation was one pair in every 3-4 h on average. The embryonic events during this period were as follows: 1. From the beginning of stage 9, the embryonic body consistently displayed a kyphosis, and as development progressed, the caudal portion of the embryo spiralled clockwise. 2. The first and second pharyngeal arches formed; their development was precocious among mammalian embryos in relation to somitic count. 3. The segmental pattern of the neural fold was similar to that of laboratory rodents and primates. The first fusion of the cranial neural folds took place in the occipital somite region, the second fusion in the diencephalic region, and the third at the end of the neural plate, thus leaving two neuropores in the cephalic region. 4. The timing of appearance of the optic sulcus was similar to that of human embryos but was delayed in comparison with that of laboratory rodents. 5. The heart always showed a more advanced state than that of other mammalian embryos. From the beginning of stage 9, an unpaired endocardial tube was seen in the bulbo-ventricular region, and deflection from a symmetrical appearance soon took place. 6. The differentiation of foregut was also precocious, and the thyroid and respiratory primordia appeared earlier than in other mammals. The present study emphasizes that there are considerable variations in timing and manner of morphogenesis among early mammalian embryos.

Evaluation of neural fold fusion and coincident initiation of spinal cord occlusion in the chick embryo.

Although it is known that rapid expansion of the vertebrate brain begins near the time that the spinal neurocoel is occluded, it still remains unknown when occlusion occurs in relation to neurulation. Since both morphogenetic events are critical for normal brain growth, it is important to decipher the temporal relationship between the two processes. This study assessed the temporal relationship of the two events with the rationale that if it could be demonstrated that occlusion occurs coincident with the completion of neurulation, then it could be argued that factors shown to direct neurulation could also initiate occlusion. Nearly 600 chick embryos (stages 9- through 12+) were cultured atop egg-agar, the caudal extent of neurulation determined, the cranial five pairs of somites removed and the neurocoels assessed for occlusion. In stage 9- through 10- chicks, neurulation of the spinal cord is incomplete. Stages 10 through 12+ exhibit neurulation and occlusion from the 8th to 19th somites. When lateral tissues were removed in embryos 8 through 10-, the neural folds became dysraphic whereas in embryos stage 10 and older, the folds remained fused dorsomedially and occluded. The only surgical manipulation that was found to prevent occlusion was elimination of the lateral tissues responsible for elevation and closure of the neural folds. Analysis of particular components of the lateral tissues essential for convergence, by treating embryos (n = 75) with chemicals known to degrade tissue-tissue bonds or specific components of the perineural matrix, indicated that more than 75% of the embryos treated with EDTA, EDTA plus Ca2+, trypsin, collagenase, or hyaluronidase exhibited little or no effect on convergence, dorsomedial fusion, and concomitant occlusion.