Notochord grafts do not suppress formation of neural crest cells or commissural neurons.

Grafting experiments previously have established that the notochord affects dorsoventral polarity of the neural tube by inducing the formation of ventral structures such as motor neurons and the floor plate. Here, we examine if the notochord inhibits formation of dorsal structures by grafting a notochord within or adjacent to the dorsal neural tube prior to or shortly after tube closure. In all cases, neural crest cells emigrated from the neural tube adjacent to the ectopic notochord. When analyzed at stages after ganglion formation, the dorsal root ganglia appeared reduced in size and shifted in position in embryos receiving grafts. Another dorsal cell type, commissural neurons, identified by CRABP and neurofilament immunoreactivity, differentiated in the vicinity of the ectopic notochord. Numerous neuronal cell bodies and axonal processes were observed within the induced, but not endogenous, floor plate 1 to 2 days after implantation but appeared to be cleared with time. These results suggest that dorsally implanted notochords cannot prevent the formation of neural crest cells or commissural neurons, but can alter the size and position of neural crest-derived dorsal root ganglia.     

Control of neural crest cell dispersion in the trunk of the avian embryo

Many hypotheses have been advanced to explain the orientation and directional migration of neural crest cells. These include positive and negative chemotaxis, haptotaxis, galvanotaxis, and contact inhibition. To test directly the factors that may control the directional dispersion of the neural crest, I have employed a variety of grafting techniques in living embryos. In addition, time-lapse video microscopy has been used to study neural crest cells in tissue culture. Trunk neural crest cells normally disperse from their origin at the dorsal neural tube along two extracellular pathways. One pathway extends laterally between the ectoderm and somites. When either pigmented neural crest cells or neural crest cells isolated from 24-hr cultures are grafted into the space lateral to the somites, they migrate: (1) medially toward the neural tube in the space between the ectoderm and somites and (2) ventrally along intersomitic blood vessels. Once the grafted cells contact the posterior cardinal vein and dorsal aorta they migrate along both blood vessels for several somite lengths in the anterior-posterior axis. Neural crest cells grafted lateral to the somites do not immediately move laterally into the somatic mesoderm of the body wall or the limb. Dispersion of neural crest cells into the mesoderm occurs only after blood vessels and nerves have first invaded, which the grafted cells then follow. The other neural crest pathway extends ventrally alongside the neural tube in the intersomitic space. When neural crest cells were grafted to a ventral position, between the notochord and dorsal aorta, in this intersomitic pathway at the axial level of the last somite, the grafted cells migrate rapidly within 2 hr in two directions: (1) dorsally, in the intersomitic space, until the grafted cells contact the ventrally moving stream of the host neural crest and (2) laterally, along the dorsal aorta and endoderm. All of the above experiments indicate that neither a preestablished chemotactic nor adhesive (haptotactic) gradient exists in the embryo since the grafted neural crest cells will move in the reverse direction along these pathways toward the dorsal neural tube. For the same reason, these experiments also show that dispersal of the neural crest is not directed passively by other environmental controls, since the cells can clearly move counter to their usual pathway and against such putative passive mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructure of the rostral notochord of the 35-day rhesus monkey (Macaca mulatta) embryo

The notochords of three normal, 35-day Macaca mulatta embryos were examined ultrastructurally. Notochordal cells had numerous polysomes and ribosomes, and some rough endoplasmic reticulum, mitochondria, Golgi complexes, coated vesicles, and secretory granules. A discontinuous basal lamina surrounded the notochord. Intercellular channels and the perinotochordal sheath contained fibrils. It was found that the ultrastructure of the rhesus monkey notochord at stage 17 resembles that of the chick and mouse.     

Biochemical specificity of Xenopus notochord

The biochemical composition and biosynthetic activity of Xenopus notochord were examined and compared with those of chick and mouse notochord. The notochords of all three species contain type-II collagen, and the notochords of Xenopus and chick synthesize a soluble glycoprotein with a molecular mass of 86 kilodaltons (kd). Mouse embryos were not tested for this molecule, because their notochords are too small to be dissected out. Most interestingly, Xenopus and chick notochords share a keratan-sulphate-containing proteoglycan which appears to be absent from mouse notochord. The presence or absence of keratan sulphate in the notochords of the different species reflects its presence or absence in cartilage. Since one role of the notochord in vivo is to stimulate chondrogenesis in the sclerotomes of the somites, this result provides support for the view that cells responding to the extracellular matrix produced by one tissue do so by increasing their production of the same matrix components.     

Absence of neural crest cells from the region surrounding implanted notochords in situ

Avian neural crest cells migrating along the trunk ventral pathway are distributed throughout the rostral half of the sclerotome with the exception of a neural crest cell-free space of approximately 85 microns width surrounding the notochord. To determine if this neural crest cell-free space results from the notochord inhibiting neural crest cell migration, a length of quail notochord was implanted lateral to the neural tube along the neural crest ventral migratory pathway of 2-day chicken embryos. The subsequent distribution of neural crest cells was analyzed in embryos fixed 2 days after grafting. When the donor notochord was isolated using collagenase, neural crest cells avoided the ectopic notochord and were absent from the area immediately surrounding the implant (mean distance of 43 microns). The neural crest cell-free space was significantly less when notochords were isolated using trypsin or chondroitinase digestion and was completely eliminated when notochords were fixed with paraformaldehyde or methanol prior to implantation. The implanted notochords did not appear to affect the overall number of neural crest cells, and therefore were unlikely to exert this effect by altering their viability. These results suggest that the notochord produces a substance that can inhibit neural crest cell migration and that this substance is trypsin and chondroitinase labile.     

Mitogenic effect of muscle on the neuroepithelium of the developing spinal cord

A previous study revealed that segments of bowel grafted between the neural tube and somites of a younger chick host embryo would induce a unilateral increase in cellularity of the host's neural tube. The current experiments were done to test the hypotheses that muscle tissue in the wall of the gut is responsible for this growth-promoting effect and that the spinal cord enlargement is the result of a mitogenic action on the neuroepithelium. Fragments of skeletal (E8-15) or cardiac muscle (E4-14) were removed from quail embryos and grafted between the neural tube and somites of chick host embryos (E2). Both skeletal and cardiac muscle grafts mimicked the effect of bowel and induced an increase in cell number as well as a unilateral enlargement of the region of the host's neural tube immediately adjacent to the grafts. The growth-promoting effect of muscle-containing grafts was restricted to the neural tube itself and was not seen in proximate dorsal root or sympathetic ganglia. The action of the grafts of muscle was neither species- nor class-specific, since enlargement of the neural tube was observed following implantation of fetal mouse skeletal muscle into quail hosts. Grafts of skeletal muscle or gut increased the number of cells taking up [3H]thymidine in the host's neuroepithelium as early as 9 h following implantation of a graft. The increase in the number of cells entering the S phase of the cell cycle preceded the increase in cell number. These observations demonstrate that muscle-containing tissues can increase the rate of proliferation of neuroepithelial cells when these tissues are experimentally placed together.     

Dorsal dermis development depends on a signal from the dorsal neural tube, which can be substituted by Wnt-1

To investigate the origin and nature of the signals responsible for specification of the dermatomal lineage, excised axial organs in 2-day-old chick embryos were replaced by grafts of the dorsal neural tube, or the ventral neural tube plus the notochord, or aggregates of cells engineered to produce Sonic hedgehog (Shh), Noggin, BMP-2, Wnt-1, or Wnt-3a. By E10, grafts of the ventral neural tube plus notochord or of cells producing Shh led to differentiation of cartilage and muscles, and an impaired dermis derived from already segmented somites. In contrast, grafts of the dorsal neural tube, or of cells producing Wnt-1, triggered the formation of a feather-inducing dermis. These results show that the dermatome inducer is produced by the dorsal neural tube. The signal can be Wnt-1 itself, or can be mediated, or at least mimicked by Wnt-1.     

Tissue interactions affecting the migration and differentiation of neural crest cells in the chick embryo.

A series of microsurgical operations was performed in chick embryos to study the factors that control the polarity, position and differentiation of the sympathetic and dorsal root ganglion cells developing from the neural crest. The neural tube, with or without the notochord, was rotated by 180 degrees dorsoventrally to cause the neural crest cells to emerge ventrally. In some embryos, the notochord was ablated, and in others a second notochord was implanted. Sympathetic differentiation was assessed by catecholamine fluorescence after aldehyde fixation. Neural crest cells emerging from an inverted neural tube migrate in a ventral-to-dorsal direction through the sclerotome, where they become segmented by being restricted to the rostral half of each sclerotome. Both motor axons and neural crest cells avoid the notochord and the extracellular matrix that surrounds it, but motor axons appear also to be attracted to the notochord until they reach its immediate vicinity. The dorsal root ganglia always form adjacent to the neural tube and their dorsoventral orientation follows the direction of migration of the neural crest cells. Differentiation of catecholaminergic cells only occurs near the aorta/mesonephros and in addition requires the proximity of either the ventral neural tube (floor plate/ventral root region) or the notochord. Prior migration of presumptive catecholaminergic cells through the sclerotome, however, is neither required nor sufficient for their adrenergic differentiation.     

Opponent activities of Shh and BMP signaling during floor plate induction in vivo.

We performed in vivo experiments in chick embryos that examined whether application of an exogenous source of Shh protein mimics the ability of the notochord to induce ectopic floor plate cells in the neural tube. Shh cannot act alone to induce a floor plate. However, coapplication of Shh and chordin, a BMP antagonist normally coexpressed with Shh in the notochord, results in a marked switch from dorsal to ventral cell fate, including a dramatic and widespread induction of floor plate cells. These data provide in vivo evidence that notochord-derived BMP antagonists may normally generate a permissive environment for the Shh-mediated induction of floor plate. Further experiments performed to address the source of BMPs that are inhibited by the action of chordin suggest that they derive specifically from the surface ectoderm and dorsal-most neuroepithelium. These data indicate that, at neural groove stages, dorsally derived BMPs affect ventral-most regions of the neural plate, suggesting a novel long-range action of BMPs. Together, these studies suggest that the balance of dorsally derived signals and notochord-derived signals determines the extent of floor plate cell induction.     

Induction of an additional floor plate in the neural tube

The role of the notochord in the morphogenesis of the neural tube was investigated by implanting a notochord fragment laterally to the neural wall of a 1.5 day chick embryo. Embryos were sacrificed at 4 days. In the basal part of the neural tube an additional floor plate was induced in the vicinity of the implant. This floor plate was characterized by a low proliferative activity, a thin wall, spindle-like nuclei crowded peripherally and some neuroblast-like cells. It was either blending with the natural floor plate or separated from it, depending on the exact position of the implant. In the latter case neuroblasts were observed in between both floor plates. The additional floor plate was present only when the implanted notochord was less than 25 micron apart from the neural tube; at larger distance an increase of the ventral horn neuroblast area could be seen. It is concluded that the implanted notochord is able to induce a floor plate at 1.5 days of incubation. The specific influence of the notochord on the morphogenesis of the neural tube, its inductive period as well as the presence of the neuroblast-like cells in the additional floor plate are discussed.     

Effect of the notochord on proliferation and differentiation in the neural tube of the chick embryo

After implantation of a notochord fragment lateral to the neural tube in a 2-day chick embryo, at 4 days the ipsilateral neural tube half was increased in size and axons left the neural tube in a broad dorsoventral area (van Straaten et al. 1985). This enlargement appears to coincide with an increased area of AChE-positive basal plate neuroblasts, as determined with scan-cytophotometry. The effect was ipsilateral and local: clear effects were seen only when the implant was localized less than 80 microns from the neural tube and over 120 microns from the ventral notochord. In order to investigate the expected enhancement of proliferation, the mitotic density and the number of cells at the site of the implant at 3 days was determined and the mitotic index calculated. All three parameters showed an increase. It was concluded that the cell cycle was shorter in the implant area relative to the control area, at least during the third day. At 4 days the number of cells was still increased, predominantly in the basal plate. It appeared that the numerical increase was for the larger part due to neuroblasts. The synergism of two notochords thus resulted in enhancement of proliferation and differentiation in the neural tube. It is suggested that the notochord merely regulates and arranges the surrounding sclerenchymal cells, which are the effective cells in the regulation of neural tube development.     

Vital dye labelling demonstrates a sacral neural crest contribution to the enteric nervous system of chick and mouse embryos

We have used the vital dye, DiI, to analyze the contribution of sacral neural crest cells to the enteric nervous system in chick and mouse embryos. In order to label premigratory sacral neural crest cells selectively, DiI was injected into the lumen of the neural tube at the level of the hindlimb. In chick embryos, DiI injections made prior to stage 19 resulted in labelled cells in the gut, which had emerged from the neural tube adjacent to somites 29-37. In mouse embryos, neural crest cells emigrated from the sacral neural tube between E9 and E9.5. In both chick and mouse embryos, DiI-labelled cells were observed in the rostral half of the somitic sclerotome, around the dorsal aorta, in the mesentery surrounding the gut, as well as within the epithelium of the gut. Mouse embryos, however, contained consistently fewer labelled cells than chick embryos. DiI-labelled cells first were observed in the rostral and dorsal portion of the gut. Paralleling the maturation of the embryo, there was a rostral-to-caudal sequence in which neural crest cells populated the gut at the sacral level. In addition, neural crest cells appeared within the gut in a dorsal-to-ventral sequence, suggesting that the cells entered the gut dorsally and moved progressively ventrally. The present results resolve a long-standing discrepancy in the literature by demonstrating that sacral neural crest cells in both the chick and mouse contribute to the enteric nervous system in the postumbilical gut.     

MG160, a Membrane Protein of the Golgi Apparatus Which Is Homologous to a Fibroblast Growth Factor Receptor and to a Ligand for E-selectin, Is Found Only in the Golgi Apparatus and Appears Early in Chicken Embryo Development

While over 20 intrinsic proteins of the Golgi apparatus have been identified and sequenced, there is no information on their developmental history, i.e., whether all Golgi proteins are expressed simultaneously or whether there is a hierarchical or stage-specific order of their expression during embryonic development. In this study we have examined the emergence and distribution of MG160 during the development of chicken embryos. MG160 is a conserved membrane sialoglycoprotein of the Golgi apparatus of most cells displaying over 90% amino acid sequence identities with two apparently unrelated molecules, namely CFR, a chicken fibroblast growth factor receptor, and ESL-1, a ligand for E-selectin (Gonatas et al., J. Biol. Chem. 1989, 264, 646-653; Burrus and Olwin, J. Biol. Chem. 1989, 264, 18647-18653; Burrus et al., Mol. Cell Biol. 1992, 12, 5600-5609; Gonatas et al., J. Cell Sci. 108, 457-467; Steegmaier et al., Nature 1995, 373, 615-620). This study was carried out by in situ hybridization, using a 56-mer antisense probe for the chicken homologue of MG160 which differs only by four bases from the corresponding segment of the rat cDNA and by immunocytochemistry and Western blotting using a polyclonal antiserum against MG160. The protein was ubiquitously and exclusively localized in the Golgi apparatus and appeared early in development within the ectoblast and primitive endoblast prior to the formation of the primitive streak. At 2 to 3 days, MG160 was particularly prominent in the notochord, neural tube, somites, and cartilage cells. In organs with central lumens, such as the neural tube, the Golgi apparatus, visualized by immunostaining for MG160, was elongated and it was located at the apical pole of cells. In 6-day-old embryos, the ongoing physiologic degeneration of the notochord was accompanied by fragmentation of the immunostained Golgi apparatus and decreased labeling of the mRNA for MG160. In order to gain information on possible interactions between MG160 and basic fibroblast growth factor (bFGF), the localization of both molecules was studied by immunocytochemistry in 3-day-old chicken embryos. While MG160 was ubiquitous in the Golgi apparatus of all cells and tissues, endogenous bFGF was not detected while exogenous bFGF bound only to basement membranes. These results indicate that MG160 is a primordial protein of the Golgi apparatus and are consistent with the hypothesis that the binding of MG160 to fibroblast growth factors and E-selectin is not related to the still unknown principal function of MG160 in the Golgi apparatus.     

Notochordal induction of cell wedging in the chick neural plate and its role in neural tube formation

Cells in the median hinge point (MHP) of the bending chick neural plate are tightly apposed to the underlying notochord. These cells differ from those in adjacent lateral neuroepithelial areas (L) in that MHP cells are short and mainly wedge-shaped and line a furrow, whereas L cells are tall and mainly spindle-shaped and do not line a furrow. Cell generation time also differs in these regions. These consistent differences are detectable only after the notochord has formed and established contact with the neural plate; it is unclear whether they result from self-differentiation or induction. Two experiments were performed to evaluate the hypothesis that MHP characteristics develop owing to inductive interactions between the notochord and overlying neuroepithelial cells. First, notochordless chick embryos were generated to determine whether midline neuroepithelial cells still developed typical MHP characteristics. In the absence of the notochord, such characteristics did not develop. Second, isolated segments of quail notochord were transplanted subjacent to L of chick hosts to ascertain whether the notochord is capable of inducing MHP characteristics in L cells. When transplanted notochordal segments established apposition with host L cells, the apposing L cells usually developed typical MHP characteristics. Collectively, these results provide strong evidence that the notochord plays an inductive role in the formation of MHP characteristics. This investigation further revealed that bending can occur in the absence of MHP characteristics, forming a neural tube with an abnormal morphology. Thus, the formation of such characteristics, particularly cell wedging, is not required for bending but plays a major role in generating the normal cross-sectional morphology of the neural tube.     

Mapping of neural crest pathways in Xenopus laevis using inter- and intra-specific cell markers

This study examines the pathways of migration followed by neural crest cells in Xenopus embryos using two recently described cell marking techniques. The first is an interspecific chimera created by grafting Xenopus borealis cells into Xenopus laevis hosts. The cells of these closely related species can be distinguished by their nuclear dimorphism. The second type of marker is created by microinjection of lysinated dextrans into fertilized eggs which can then be used for intraspecific grafting. These recently developed fluorescent dyes are fixable and identifiable in both living and fixed embryos. After grafting labeled donor neural tubes into unlabeled host embryos, the distribution of neural crest cells at various stages after grafting was used to define the pathways of neural crest migration. To control for possible grafting artifacts, fluorescent lysinated dextran was injected into a single blastomere which gives rise to a large number of neural crest cells, thereby labeling the neural crest without grafting. By all three techniques, Xenopus neural crest cells were observed along two predominant pathways in the trunk. The majority of neural crest cells were observed along a "ventral" route, between the neural tube and somite, the notochord and somite, and along the dorsal mesentery. A second group of neural crest cells was observed "dorsally" where they populated the dorsal fin. A third minor "lateral" pathway was observed primarily in borealis/laevis chimerae and in blastomere-injected embryos; some neural crest cells were observed underneath the ectoderm lateral to the neural tube. Along the rostrocaudal axis, neural crest cells were not continuously distributed but were primarily located across from the caudal two-thirds of the somite. Fewer than 3% of the neural crest cells were observed across from the rostral third of each somite. When grafted to ventral locations, neural crest cells were not able to migrate dorsally but migrated laterally along the dorsal mesentery. Labeled neural crest cells gave rise to cells of the spinal, sympathetic, and enteric ganglia as well as to adrenal chromaffin cells, Schwann cells, pigment cells, mesenchymal cells of the dorsal fin, and some cells in the integuments and in the region of the pronephros. These results show that the neural crest migratory pathways in Xenopus differ from those in the avian embryo. In avians NC cells migrate as a closely associated sheet of cells while in Xenopus they migrate as individual cells. Both species exhibit a metamerism in the neural crest cell distribution pattern along the rostrocaudal axis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of a notochordal implant on the early morphogenesis of the neural tube and neuroblasts: histometrical and histological results

The role of the notochord on the early development of ventral horn neuroblasts was investigated in chick embryos by implanting an additional notochord fragment near the right side of the thoracic neural tube. When the implant was located directly lateral to the neural tube, an enlargement of the right half of the neural tube and of the area of neuroblasts occurred, and axons were found to pass through the outer membrane of the neural tube over a broad dorsoventral trajectory. When the notochord was located ventrolaterally a population of neuroblasts including their efferent axons was found at a more dorsal location. It is concluded that a notochordal implant is able to influence the differentiation of neuroblasts.