Distribution of integrins and their ligands in the trunk of Xenopus laevis during neural crest cell migration

We have examined the distribution in Xenopus embryos of beta 1 subunits of integrin, as recognized by cross-reactive antibodies against the avian integrin beta 1 subunit. These antibodies recognize a doublet of bands of approximately 120 kD in Xenopus embryos. The distribution pattern of these integrin cell surface receptors was compared with that of two possible ligands, fibronectin and laminin, in the extracellular matrix during the time of neural crest cell migration. Integrin immunoreactivity in the early neurula was observed lightly outlining somite and epidermal cells and the notochord. The integrin immunostaining increased with developmental age and was observed on most cell types in the embryo but was particularly notable in the intersomitic clefts through which motoraxons grow. The immunoreactivity in this region was not, however, wholly on the axon surfaces, since intersomitic integrin remained detectable in embryos in which the neural tube had been ablated. Fibronectin and laminin were more extensively distributed than integrin at all stages examined. Immunoreactivity for both was observed around the neural tube, notochord, somites, epidermis, dorsal mesentery, and lateral plate mesoderm. The distribution of laminin and fibronectin around the somites was particularly interesting since it was non-uniform and similar to that of integrin. Strongest staining was observed in the intersomitic clefts, and weakest staining was observed on the medial surface of the somites, which faces the neural tube and notochord. The major differences in distribution pattern between the fibronectin and laminin immunoreactivities were that only fibronectin was detected in the mesenchyme of the dorsal fin. Our results demonstrate that a molecule homologous to avian integrin is present in Xenopus embryos during neural crest cell migration and motoraxon outgrowth. Its presence in the intersomitic clefts and on the surface of many embryonic cell types together with the abundant distribution of its ligands are consistent with a potentially important developmental function in neurite outgrowth and/or muscle development.     

Retention and structure of extracellular matrix in early chick embryos after quick-freezing and freeze-substitution

Early chick embryos, stages 11 to 14, were isolated, quick-frozen by immersion in isopentane/propane cryogen (-185 degrees C) and freeze-substituted for study by scanning electron microscopy. Emphasis was placed on the extracellular matrix (ECM) in the axial region of the segmental plate and developing somites. Ultrarapid freezing, followed by delicate freeze-substitution, immobilizes and retains much more ECM than chemical fixatives that include tannic acid (TA). The matrix on the dorsal surface of the neural tube is preserved as delicate filaments which are expressed bilaterally over the tube in a dorso-ventral orientation. These parallel primary ridges of ECM have a spacing of 1 to 3 micron, forming grooves on the wall of the neural tube. Interrupting this pattern are funnel-shaped ridges about 80 to 100 micron apart along the neural tube. The ridges become decorated with cross-bridges creating a dense lattice in the region of somite development, to the extent that a basal lamina composed of dense fibrillar network and amorphous mats of matrix accumulates on the lateral wall of the neural tube. Heavy strands and fenestrated lamellae of ECM interconnect the neural tube, notochord and somites, and attach the overlying epithelium to the upper surface of the somites. The pattern of ECM is complimentary to the migratory pathways of ventrally migrating neural crest cells and is the basis for suggesting that a physical substratum influencing the direction of neural crest cell migration is an idea that should be revived.     

The Hectd1 ubiquitin ligase is required for development of the head mesenchyme and neural tube closure

Closure of the cranial neural tube depends on normal development of the head mesenchyme. Homozygous-mutant embryos for the ENU-induced open mind (opm) mutation exhibit exencephaly associated with defects in head mesenchyme development and dorsal-lateral hinge point formation. The head mesenchyme in opm mutant embryos is denser than in wildtype embryos and displays an abnormal cellular organization. Since cells that originate from both the cephalic paraxial mesoderm and the neural crest populate the head mesenchyme, we explored the origin of the abnormal head mesenchyme. opm mutant embryos show apparently normal development of neural crest-derived structures. Furthermore, the abnormal head mesenchyme in opm mutant embryos is not derived from the neural crest, but instead expresses molecular markers of cephalic mesoderm. We also report the identification of the opm mutation in the ubiquitously expressed Hectd1 E3 ubiquitin ligase. Two different Hectd1 alleles cause incompletely penetrant neural tube defects in heterozygous animals, indicating that Hectd1 function is required at a critical threshold for neural tube closure. This low penetrance of neural tube defects in embryos heterozygous for Hectd1 mutations suggests that Hectd1 should be considered as candidate susceptibility gene in human neural tube defects.     

Morphogenesis of sclerotome and neural crest in avian embryos

Summary The distribution of sclerotome and neural crest cells of avian embryos was studied by light and electron microscopy. Sclerotome cells radiated from the somites towards the notochord, to occupy the perichordal space. Neural crest cells, at least initially, also entered cell-free spaces. At the cranial somitic levels they moved chiefly dorsal to the somites, favouring the rostral part of each somite. These cells did not approach the perichordal space. More caudally (i.e. trunk levels), neural crest cells initially moved ventrally between the somites and neural tube. Adjacent to the caudal half of each somite, these cells penetrated no further than the myosclerotomal border, but opposite the rostral somite half, they were found next to the sclerotome almost as far ventrally as the notochord. However, they did not appear to enter the perichordal space, in contrast to sclerotome cells.When tested in vitro, sclerotome cells migrated towards notochords co-cultured on fibronectin-rich extracellular material, and on collagen gels. In contrast, neural crest cells avoided co-cultured notochords. This avoidance was abolished by inclusion of testicular hyaluronidase and chondroitinase ABC in the culture medium, but not by hyaluronidase from Streptomyces hyalurolyticus. The results suggest that sclerotome and neural crest mesenchyme cells have a different distribution with respect to the notochord, and that differential responses to notochordal extracellular material, possibly chondroitin sulphate proteoglycan, may be responsible for this.     

The distribution of fibronectin and tenascin along migratory pathways of the neural crest in the trunk of amphibian embryos

It is generally assumed that in amphibian embryos neural crest cells migrate dorsally, where they form the mesenchyme of the dorsal fin, laterally (between somites and epidermis), where they give rise to pigment cells, and ventromedially (between somites and neural tube), where they form the elements of the peripheral nervous system. While there is agreement about the crest migratory routes in the axolotl (Ambystoma mexicanum), different opinions exist about the lateral pathway in Xenopus. We investigated neural crest cell migration in Xenopus (stages 23, 32, 35/36 and 41) using the X. laevis-X. borealis nuclear marker system and could not find evidence for cells migrating laterally. We have also used immunohistochemistry to study the distribution of the extracellular matrix (ECM) glycoproteins fibronectin (FN) and tenascin (TN), which have been implicated in directing neural crest cells during their migrations in avian and mammalian embryos, in the neural crest migratory pathways of Xenopus and the axolotl. In premigratory stages of the crest, both in Xenopus (stage 22) and the axolotl (stage 25), FN was found subepidermally and in extracellular spaces around the neural tube, notochord and somites. The staining was particularly intense in the dorsal part of the embryo, but it was also present along the visceral and parietal layers of the lateral plate mesoderm. TN, in contrast, was found only in the anterior trunk mesoderm in Xenopus; in the axolotl, it was absent. During neural crest cell migration in Xenopus (stages 25-33) and the axolotl (stages 28-35), anti-FN stained the ECM throughout the embryo, whereas anti-TN staining was limited to dorsal regions. There it was particularly intense medially, i.e. in the dorsal fin, around the neural tube, notochord, dorsal aorta and at the medial surface of the somites (stage 35 in both species). During postmigratory stages in Xenopus (stage 40), anti-FN staining was less intense than anti-TN staining. In culture, axolotl neural crest cells spread differently on FN- and TN-coated substrata. On TN, the onset of cellular outgrowth was delayed for about 1 day, but after 3 days the extent of outgrowth was indistinguishable from cultures grown on FN. However, neural crest cells in 3-day-old cultures were much more flattened on FN than on TN. We conclude that both FN and TN are present in the ECM that lines the neural crest migratory pathways of amphibian embryos at the time when the neural crest cells are actively migrating. FN is present in the embryonic ECM before the onset of neural crest migration.(ABSTRACT TRUNCATED AT 400 WORDS)     

The influence of axial structures on chick somite formation

The influence of the axial structures on somite formation was investigated by culturing, on a nutritive agar substrate, segmental plates from chick embryos having 8 to 20 pairs of somites. In the first set of experiments, segmental plate was explanted together with adjacent notochord and approximately the lateral halves of the neural tube and node region. These explants formed 18 to 20 somites within 30 hr. In a second series of experiments, the notochord and neural tube were included as before, but further regression movements in the explants were prevented by removing the node region. These explants formed only 11.9 ± 1.1 somites. Finally, explants of segmental plate that included no neural tube, notochord, or node region were made. These explants had formed 10.7 ± 1.1 somites 14 to 17 hr later. When such explants were cultured for periods longer than 17 hr, there was a marked tendency for the more posterior somites to disperse and for all of the somites to develop a peculiar “hollow” morphology. It was concluded from these results that during the period of development when chick embryos possess 8 to 20 pairs of somites, the segmental plate mesoderm (1) represents about 12 prospective somites, (2) may segment into its full complement of somites without further contact with the axial structures, but (3) requires continued intimate contact with the axial structures for normal somite morphologic differentiation and stability.     

A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus

We have developed a whole-mount immunocytochemical method for Xenopus and used it to map the expression of the intermediate filament protein vimentin during early embryogenesis. We used two monoclonal antibodies, 14h7 and RV202. Both label vimentin filaments in Xenopus A6 cells, RV202 reacts specifically with vimentin (Mr, 55 x 10(3] on Western blots of A6 cells and embryos. 14h7 reacts with vimentin and a second, insoluble polypeptide of 57 x 10(3) Mr found in A6 cells. The 57 x 10(3) Mr polypeptide appears to be an intermediate filament protein immunochemically related to vimentin. In the whole-mount embryo, we first found vimentin at the time of neural tube closure (stage 19) in cells located at the lateral margins of the neural tube. By stage 26, these cells, which are presumably radial glia, are present along the entire length of the neural tube and in the tail bud. Cells in the optic vesicles express vimentin by stage 24. Vimentin-expressing mesenchymal cells appear on the surface of the somites at stage 22/23; these cells appear first on anterior somites and on progressively more posterior somites as development continues. Beginning at stage 24, vimentin appears in mesenchymal cells located ventral to the somites and associated with the pronephric ducts; these ventral cells first appear below the anterior somites and later appear below more posterior somites. The dorsal fin mesenchyme expresses vimentin at stage 26. In the head, both mesodermally-derived and neural-crest-derived mesenchymal tissues express vimentin by stage 26. These include the mesenchyme of the branchial arches, the mandibular arch, the corneal epithelium, the eye, the meninges and mesenchyme surrounding the otic vesicle. By stage 33, vimentin-expressing mesenchymal cells are present in the pericardial cavity and line the vitelline veins. Vimentin expression appears to be a marker for the differentiation of a subset of central nervous system cells and of head and body mesenchyme in the early Xenopus embryo.     

Expression pattern of the Brachyury gene in whole-mount TWis/TWis mutant embryos.

The murine Brachyury (T) gene is required in mesoderm formation. Mutants carrying different T alleles show a graded severity of defects correlated with gene dosage along the body axis. The phenotypes range from shortening of the tail to the malformation of sacral vertebrae in heterozygotes, and to disruption of trunk development and embryonic death in homozygotes. Defects include a severe disturbance of the primitive streak, an early cessation of mesoderm formation and absence of the allantois and notochord, the latter resulting in an abnormality of the neural tube and somites. The T gene is expressed in nascent mesoderm and in the notochord of wild-type embryos. Here the expression of T in whole-mount mutant embryos homozygous for the T allele TWis is described. The TWis gene product is altered, but the TWis/TWis phenotype is very similar to that of T/T embryos which lack T. In early TWis/TWis embryos T expression is normal, but ceases prematurely during early organogenesis coincident with a cessation of mesoderm formation. The archenteron/node region is disrupted and the extension of the notochord precursor comes to a halt, followed by a decrease and finally a complete loss of T gene expression in the primitive streak and the head process/notochord precursor. It appears that the primary defect of the mutant embryo is the disruption of the notochord precursor in the node region which is required for axis elongation. Thus the T gene product is directly or indirectly involved in the organization of axial development.     

HrzicN,a new Zic family gene of ascidians,plays essential roles in the neural tube and notochord development

Two axial structures, a neural tube and a notochord, are key structures in the chordate body plan and in understanding the origin of chordates. To expand our knowledge on mechanisms of development of the neural tube in lower chordates, we have undertaken isolation and characterization of HrzicN, a new member of the Zic family gene of the ascidian, Halocynthia roretzi. HrzicN expression was detected by whole-mount in situ hybridization in all neural tube precursors, all notochord precursors, anterior mesenchyme precursors and a part of the primary muscle precursors. Expression of HrzicN in a- and b-line neural tube precursors was detected from early gastrula stage to the neural plate stage, while expression in other lineages was observed between the 32-cell and the 110-cell stages. HrzicN function was investigated by disturbing translation using a morpholino antisense oligonucleotide. Embryos injected with HrzicN morpholino ('HrzicN knockdown embryos') exhibited failure of neurulation and tail elongation, and developed into larvae without a neural tube and notochord. Analysis of neural marker gene expression in HrzicN knockdown embryos revealed that HrzicN plays critical roles in distinct steps of neural tube formation in the a-line- and A-line precursors. In particular HrzicN is required for early specification of the neural tube fate in A-line precursors. Involvement of HrzicN in the neural tube development was also suggested by an overexpression experiment. However, analysis of mesodermal marker gene expression in HrzicN knockdown embryos revealed unexpected roles of this gene in the development of mesodermal tissues. HrzicN knockdown led to loss of HrBra (Halocynthia roretzi Brachyury) expression in all of the notochord precursors, which may be the cause for notochord deficiency. Hrsna (Halocynthia roretzi snail) expression was also lost from all the notochord and anterior mesenchyme precurosrs. By contrast, expression of Hrsna and the actin gene was unchanged in the primary muscle precursors. These results suggest that HrzicN is responsible for specification of the notochord and anterior mesenchyme. Finally, regulation of HrzicN expression by FGF-like signaling was investigated, which has been shown to be involved in induction of the a- and b-line neural tube, the notochord and the mesenchyme cells in Halocynthia embryos. Using an inhibitor of FGF-like signaling, we showed that HrzicN expression in the a- and b-line neural tube, but not in the A-line lineage and mesodermal lineage, depends on FGF-like signaling. Based on these data, we discussed roles of HrzicN as a key gene in the development of the neural tube and the notochord.     

The distribution of tenascin coincides with pathways of neural crest cell migration

The distribution of the extracellular matrix (ECM) glycoprotein, tenascin, has been compared with that of fibronectin in neural crest migration pathways of Xenopus laevis, quail and rat embryos. In all species studied, the distribution of tenascin, examined by immunohistochemistry, was more closely correlated with pathways of migration than that of fibronectin, which is known to be important for neural crest migration. In Xenopus laevis embryos, anti-tenascin stained the dorsal fin matrix and ECM along the ventral route of migration, but not the ECM found laterally between the ectoderma and somites where neural crest cells do not migrate. In quail embryos, the appearance of tenascin in neural crest pathways was well correlated with the anterior-to-posterior wave of migration. The distribution of tenascin within somites was compared with that of the neural crest marker, HNK-1, in quail embryos. In the dorsal halves of quail somites which contained migrating neural crest cells, the predominant tenascin staining was in the anterior halves of the somites, codistributed with the migrating cells. In rat embryos, tenascin was detectable in the somites only in the anterior halves. Tenascin was not detectable in the matrix of cultured quail neural crest cells, but was in the matrix surrounding somite and notochord cells in vitro. Neural crest cells cultured on a substratum of tenascin did not spread and were rounded. We propose that tenascin is an important factor controlling neural crest morphogenesis, perhaps by modifying the interaction of neural crest cells with fibronectin.     

SOMITE CHONDROGENESIS : A Structural Analysis

Light and electron microscopy are used in this study to compare chondrogenesis in cultured somites with vertebral chondrogenesis These studies have also characterized some of the effects of inducer tissues (notochord and spinal cord), and different nutrient media, on chondrogenesis in cultured somites Somites from stage 17 (54–60 h) chick embryos were cultured, with or without inducer tissues, and were fed nutrient medium containing either horse serum (HS) and embryo extract (EE), or fetal calf serum (FCS) and F12X Amino acid analyses were also utilized to determine the collagen content of vertebral body cartilage in which the fibrils are homogeneously thin (ca. 150 Å) and unbanded. These analyses provide strong evidence that the thin unbanded fibrils in embryonic cartilage matrix are collagen. These thin unbanded collagen fibrils, and prominent 200–800 Å protein polysaccharide granules, constitute the structured matrix components of both developing vertebral cartilage and the cartilage formed in cultured somites Similar matrix components accumulate around the inducer tissues notochord and spinal cord. These matrix components are structurally distinct from those in embryonic fibrous tissue The synthesis of matrix by the inducer tissues is associated with the inductive interaction of these tissues with somitic mesenchyme. Due to the deleterious effects of tissue isolation and culture procedures many cells die in somitic mesenchyme during the first 24 h in culture. In spite of this cell death, chondrogenic areas are recognized after 12 h in induced cultures, and through the first 2 days in all cultures there are larger accumulations of structured matrix than are present in equivalently aged somitic mesenchyme in vivo. Surviving chondrogenic areas develop into nodules of hyaline cartilage in all induced cultures, and in most non-induced cultures fed medium containing FCS and F12X There is more cell death, less matrix accumulation, and less cartilage formed in cultures fed medium containing HS and EE. The inducer tissues, as well as nutrient medium containing FCS and F12X, facilitate cell survival, the synthesis and accumulation of cartilage matrix, and the formation of cartilage nodules in cultured somites.     

Notochordal and foregut abnormalities correlate with elevated neural crest apoptosis in Patch embryos

Although Patch mutants show severe abnormalities in many neural crest-derived structures including the face and the heart, there is a paucity of information characterizing the mechanisms underlying these congenital defects. Via manipulating the genetic background to circumvent early embryonic lethality, our results revealed that Patch phenotypes are most likely due to a significant decrease in migratory neural crest lineage due to diminished neural crest survival and elevated apoptosis. Homozygous mutant neural crest precursors can undergo typical expansion within the neural tube, epithelial-to-mesenchymal transformation, and initiate normal neural crest emigration. Moreover, in vitro explant culture demonstrated that when isolated from the surrounding mesenchyme, Patch mutant neural crest cells (NCCs) can migrate appropriately. Additionally, Patch foregut, notochord and somitic morphogenesis, and Sonic hedgehog expression profiles were all perturbed. Significantly, the timing of lethality and extent of apoptosis correlated with the degree of severity of Patch mutant foregut, notochord, and somite dysfunction. Finally, analysis of Balb/c-enriched surviving Patch mutants revealed that not all the neural crest subpopulations are affected and that Patch mutant neural crest-derived sympathetic ganglia and dorsal root ganglia were unaffected. We hypothesize that loss of normal coordinated signaling from the notochord, foregut, and somites underlies the diminished survival of the neural crest lineage within Patch mutants resulting in subsequent neural crest-deficient phenotypes.     

Regulation of Cell Number in Formation of the Dorsal Root Ganglion Revealed by Transplantation of Quail Neural Crest Cells into Chick Embryos

Neural crest cells appear transiently in early embryogenesis on the dorsal surface of the neural tube and subsequently migrate along specific pathways. Some migrate to between the neural tube and somites, aggregating to form the rudiments of dorsal root ganglia (DRG). The size of DRG at a given somite level is almost constant in all chick embryos. To determine the mechanisms controlling the size of DRG, we transplanted neural crest cells of 2.5-day-old quail embryos into 2.5-day-old chick embryos between the neural tube and the somites, and examined the size of DRG in these chimeric embryos with extra neural crest cells 2 days after the operation, when natural cell death in DRG had not yet occurred. The DRG on the operated side were composed of both chick and quail cells in various proportions. The cell numbers of these chimeric DRG were almost the same as those of the normal DRG on the opposite side. That is, there were significantly fewer chick cells in chimeric DRG than in DRG composed of only chick cells on the opposite unoperated side. This finding indicates that the size of DRG is not determined in migrating neural crest cells but is regulated by the circumstances.     

Apoptosis regulates notochord development in Xenopus

The notochord is the defining characteristic of the chordate embryo and plays critical roles as a signaling center and as the primitive skeleton. In this study we show that early notochord development in Xenopus embryos is regulated by apoptosis. We find apoptotic cells in the notochord beginning at the neural groove stage and increasing in number as the embryo develops. These dying cells are distributed in an anterior to posterior pattern that is correlated with notochord extension through vacuolization. In axial mesoderm explants, inhibition of this apoptosis causes the length of the notochord to approximately double compared to controls. In embryos, however, inhibition of apoptosis decreases the length of the notochord and it is severely kinked. This kinking also spreads from the anterior with developmental stage such that, by the tadpole stage, the notochord lacks any recognizable structure, although notochord markers are expressed in a normal temporal pattern. Extension of the somites and neural plate mirrors that of the notochord in these embryos, and the somites are severely disorganized. These data indicate that apoptosis is required for normal notochord development during the formation of the anterior-posterior axis, and its role in this process is discussed.     

The neural tube/notochord complex is necessary for vertebral but not limb and body wall striated muscle differentiation.

The aim of this work was to investigate the role played by the axial organs, neural tube and notochord, on the differentiation of muscle cells from the somites in the avian embryo. Two of us have previously shown that neuralectomy and notochordectomy is followed by necrosis of the somites and consecutive absence of vertebrae and of most muscle cells derived from the myotomes while the limbs develop normally with muscles. Here we have focused our attention on muscle cell differentiation by using the 13F4 mAb that recognizes a cytoplasmic antigen specific of all types of muscle cells. We show that differentiation of muscle cells of myotomes can occur in the absence of notochord and neural tube provided that the somites from which they are derived have been in contact with the axial organs for a defined period of time, about 10 hours for the first somites formed at the cervical level, a duration that progressively reduces caudalward (i.e. for thoracic and lumbar somites). Either one or the other of the two axial organs, the neural tube or the notochord can prevent somitic cell death and fulfill the requirements for myotomal muscle cell differentiation. Separation of the neural tube/notochord complex from the somites by a surgical slit on one side of the embryo gave the same results as extirpation of these organs and provided a perfect control on the non-operated side. A striking finding was that limb and body wall muscles, although derived from the somites, differentiated in the absence of the axial organs. However, limb muscles that develop after excision of the neural tube started to degenerate from E10 onward due to lack of innervation. In vitro explantation of somites from different axial levels confirmed and defined precisely the chronology of muscle cell commitment in the myotomes as revealed by the in vivo experiments.     

Inhibition of sonic hedgehog signaling in vivo results in craniofacial neural crest cell death

BACKGROUND: Sonic hedgehog (Shh) is well known for its role in patterning tissues, including structures of the head. Haploinsufficiency for SHH in humans results in holoprosencephaly, a syndrome characterized by facial and forebrain abnormalities. Shh null mice have cyclopia and loss of branchial arch structures. It is unclear, however, whether these phenotypes arise solely from the early function of Shh in patterning midline structures, or whether Shh plays other roles in head development. RESULTS: To address the role of Shh after floorplate induction, we inhibited Shh signaling by injecting hybridoma cells that secrete a function-blocking anti-Shh antibody into the chick cranial mesenchyme. The antibody subsequently bound to Shh in the floorplate, notochord, and the pharyngeal endoderm. Perturbation of Shh signaling at this stage resulted in a significant reduction in head size after 1 day, loss of branchial arch structures after 2 days, and embryos with smaller heads after 7 days. Cell death was significantly increased in the neural tube and neural crest after 1 day, and neural crest cell death was not secondary to the loss of neural tube cells. CONCLUSIONS: Reduction of Shh signaling after neural tube closure resulted in a transient decrease in neural tube cell proliferation and an extensive increase in cell death in the neural tube and neural crest, which in turn resulted in decreased head size. The phenotypes observed after reduction of Shh are similar to those observed after cranial neural crest ablation. Thus, our results demonstrate a role for Shh in coordinating the proliferation and survival of cells of the neural tube and cranial neural crest.     

Pax3 functions in cell survival and in pax7 regulation

In developing vertebrate embryos, Pax3 is expressed in the neural tube and in the paraxial mesoderm that gives rise to skeletal muscles. Pax3 mutants develop muscular and neural tube defects; furthermore, Pax3 is essential for the proper activation of the myogenic determination factor gene, MyoD, during early muscle development and PAX3 chromosomal translocations result in muscle tumors, providing evidence that Pax3 has diverse functions in myogenesis. To investigate the specific functions of Pax3 in development, we have examined cell survival and gene expression in presomitic mesoderm, somites and neural tube of developing wild-type and Pax3 mutant (Splotch) mouse embryos. Disruption of Pax3 expression by antisense oligonucleotides significantly impairs MyoD activation by signals from neural tube/notochord and surface ectoderm in cultured presomitic mesoderm (PSM), and is accompanied by a marked increase in programmed cell death. In Pax3 mutant (Splotch) embryos, MyoD is activated normally in the hypaxial somite, but MyoD-expressing cells are disorganized and apoptosis is prevalent in newly formed somites, but not in the neural tube or mature somites. In neural tube and somite regions where cell survival is maintained, the closely related Pax7 gene is upregulated, and its expression becomes expanded into the dorsal neural tube and somites, where Pax3 would normally be expressed. These results establish that Pax3 has complementary functions in MyoD activation and inhibition of apoptosis in the somitic mesoderm and in repression of Pax7 during neural tube and somite development.     

Consequences of neural tube and notochord excision on the development of the peripheral nervous system in the chick embryo

Notochordectomy and neuralectomy were carried out either in one- or in two-step experiments on the chick embryo. The aim of this operation was to study the influence of the axial organs (notochord and neural tube) on the development of the ganglia of the peripheral nervous system. The neural crest cells from which most peripheral ganglion cells arise were labeled through the quail-chick marker system and their fate was followed under various experimental conditions. It appeared that the development of the dorsal root and sympathetic ganglia depends on survival and differentiation of somite-derived structures. In the absence of neural tube and notochord, somitic cells die rapidly, and so do the neural crest cells that are present in the somitic mesenchyme at that time. In contrast, those crest cells which can reach the mesenchymal wall of the aorta, the suprarenal glands, or the gut survive and develop normally into nerve and paraganglion cells. Differentiation of the neural crest- and placode-derived sensory ganglia of the head which develop in the cephalic mesenchyme is not affected by removal of notochord and encephalic vesicles. These results show that the peripheral ganglia are differentially sensitive to the presence of the neural tube and the notochord. Among the various ganglia of the peripheral nervous system, spinal and sympathetic ganglia are the only ones which require the presence of these axial structures. The neural tube allows both the spinal and the sympathetic ganglia to develop in the absence of the notochord. In contrast, if the notochord is left in situ and the neural tube removed, the spinal ganglia fail to differentiate and only sympathetic ganglia can develop.     

Immunocytochemical localization of epidermal growth factor receptor in early embryos of the Japanese medaka fish (Oryzias latipes)

This study was undertaken to localize epidermal growth factor receptor (EGFR) during early development of Japanese medaka embryos using immunocytochemistry. Specific staining was observed in all stages studied. All of the cells of the embryonic disc from the germinal disc (1 cell) through the late high blastula stages stained moderately for EGFR. Beginning with the flat blastula stage, the surface and lateral cells of the embryonic disc and the cells migrating around the yolk stained intensely for EGFR, and this continued throughout the study period. The presence of the keel at the late gastrula stage did not affect the moderate staining of the majority of the embryonic disc cells. When somites first appeared, the keel region stained less intensely than before, but scattered individual cells stained intensely for EGFR. Embryos with 12 somites had a neural tube that was lightly stained except for a few intensely stained individual cells. The neural tube, notochord and somites in 24-somite embryos lacked immunostaining. However, the surface epithelium, aorta, intestinal epithelium and pronephric duct demonstrated EGFR immunostaining. This study demonstrates that EGFR is present during medaka development and supports the hypothesis that EGFR ligands are important during cleavage, gastrulation and early organogenesis.     

Histological comparison of the effects of the splotch gene and retinoic acid on the closure of the mouse neural tube

The splotch gene (Sp) and all-trans retinoic acid (RA) interact to cause spina bifida in mouse embryos. To investigate the mechanisms of action of the two, the spinal regions of Sp homozygotes, RA-treated wild-type, and control wild-type embryos were examined histologically by light microscopy on day 9 of gestation. The mean numbers of cells per section in the neural tube, mesoderm, and notochord were determined, along with the percentages of mitotic and pyknotic nuclei and the numbers of migrating neural crest cells. As well, the effect of Sp and RA on the extracellular matrix was studied histochemically with Alcian blue staining for glycosaminoglycans. The main defect in Sp homozygotes was a marked reduction in the number of migrating neural crest cells and the amount of extracellular matrix around the neural tube. Retinoic acid, on the other hand, caused a number of disruptions in the embryo, including abnormalities in the position of the notochord and the shape of the neural tube. Sp and RA delay neural tube closure and thus cause neural tube defects, through different mechanisms. However, the combined effects of the gene and teratogen on the embryo lead to a greater inhibition of neural tube closure than when either is present separately.