Do Peanut Agglutinin Receptors on Somites Control the Behavior of Neural Cells?

Peanut agglutinin (PNA) receptors are expressed in the caudal halves of sclerotomes in chick embryos after 3 days of incubation (stages 19–20 of Hamburger & Hamilton). The neural crest cells forming dorsal root ganglia (DRG) and motor nerves appear to avoid PNA positive regions and concentrate into rostral halves of sclerotomes. To investigate the role of PNA receptors in gangliogenesis and nerve growth, we examined PNA binding ability in quail sclerotomes and in chick-quail chimeric embryos made by transplanting quail somites to chick embryos, comparing the development of DRG, motor nerves and sclerotomes. PNA did not bind to any part of the somites of 4.5-day quail embryos, although dorsal root ganglia and motor nerves appeared only in the rostral halves of sclerotomes as in chick embryos. Moreover, in spite of no PNA binding ability of the transplanted quail somite in 4.5-day chick-quail chimeric embryos, DRG and motor nerves derived from chick tissues appeared only in the rostral halves of the sclerotomes derived from these somites. Thus, both quail and chick neural crest cells and motor nerves recognized the difference between the rostral and caudal halves of sclerotomes of quail embryos in the absence of PNA binding ability, indicating that PNA binding site on somite cells does not support the selective neural crest migration and nerve growth.     

Formation of the dorsal root ganglia in the avian embryo: Segmental origin and migratory behavior of neural crest progenitor cells

The segmental origin and migratory pattern of neural crest cells at the trunk level of avian embryos was studied, with special emphasis on the formation of the dorsal root ganglia (DRG) which organize in the anterior half of each somite. Neural crest cells were visualized using the quail-chick marker and HNK-1 immunofluorescence. The migratory process turned out to be closely correlated with somitic development: when the somites are epithelial in structure few labeled cells were found in a dorsolateral position on the neural tube, uniformly distributed along the craniocaudal axis. Following somitic dissociation into dermomyotome and sclerotome labeled cells follow defined migratory pathways restricted to each anterior somitic half. In contrast, opposite the posterior half of the somites, cells remain grouped in a dorsolateral position on the neural tube. The fate of crest cells originating at the level of the posterior somitic half was investigated by grafting into chick hosts short segments of quail neural primordium, which ended at mid-somitic or at intersomitic levels. It was found that neural crest cells arising opposite the posterior somitic half participate in the formation of the DRG and Schwann cells lining the dorsal and ventral root fibers of the same somitic level as well as of the subsequent one, whereas those cells originating from levels facing the anterior half of a somite participate in the formation of the corresponding DRG. Moreover, crest cells from both segmental halves segregate within each ganglion in a distinct topographical arrangement which reflects their segmental origin on the neural primordium. Labeled cells which relocate from posterior into anterior somitic regions migrate longitudinally along the neural tube. Longitudinal migration of neural crest cells was first observed when the somites are epithelial in structure and is completed after the disappearance of the last cells from the posterior somitic region at a stage corresponding to the organogenesis of the DRG.     

Consequences of somite manipulation on the pattern of dorsal root ganglion development

We have investigated dorsal root ganglion formation, in the avian embryo, as a function of the composition of the paraxial somitic mesoderm. Three or four contiguous young somites were unilaterally removed from chick embryos and replaced by multiple cranial or caudal half-somites from quail embryos. Migration of neural crest cells and formation of DRG were subsequently visualized both by the HNK-1 antibody and the Feulgen nuclear stain. At advanced migratory stages (as defined by Teillet et al. Devl Biol. 120, 329-347 1987), neural crest cells apposed to the dorsolateral faces of the neural tube were distributed in a continuous, nonsegmented pattern that was indistinguishable on unoperated sides and on sides into which either half of the somites had been grafted. In contrast, ventrolaterally, neural crest cells were distributed segmentally close to the neural tube and within the cranial part of each normal sclerotome, whereas they displayed a nonsegmental distribution when the graft involved multiple cranial half-somites or were virtually absent when multiple caudal half-somites had been implanted. In spite of the identical dorsal distribution of neural crest cells in all embryos, profound differences in the size and segmentation of DRG were observed during gangliogenesis (E4-9) according to the type of graft that had been performed. Thus when the implant consisted of compound cranial half-somites, giant, coalesced ganglia developed, encompassing the entire length of the graft. On the other hand, very small, dorsally located ganglia with irregular segmentation were seen at the level corresponding to the graft of multiple caudal half-somites. We conclude that normal morphogenesis of dorsal root ganglia depends upon the craniocaudal integrity of the somites.     

Requirement of a neural tube signal for the differentiation of neural crest cells into dorsal root ganglia

The influence of the neural tube on early development of neural crest cells into sensory ganglia was studied in the chick embryo. Silastic membranes were implanted between the neural tube and the somites in 30-somite-stage embryos at the level of somites 21-24, thus separating the early migrated population of neural crest cells from the neural tube. Neural crest cells and peripheral ganglia were visualized by immunofluorescence using the HNK-1 monoclonal antibody and several histochemical techniques. Separation of crest cells from the neural tube caused the selective death of the neural crest cells from which dorsal root ganglia (DRG) would have developed. Complete disappearance of HNK-1 positive cells was evident already 10 hr after silastic implantation, before early differentiation sensory neurons could have reached their peripheral targets. In older embryos, DRG were absent at the level of implantation. In contrast, the development of ventral roots, sympathetic ganglia and adrenal gland was normal, and so was somitic differentiation into cartilage and muscle, while morphogenesis of the vertebrae was perturbed. To overcome the experimentally induced crest cell death, the silastic membranes were impregnated with a 3-day-old embryonic chick neural tube extract. Under these conditions, crest cells which were separated from the tube survived for a period of 30 hr after operation, compared to less than 10 hr in respective controls. The extract of another tissue, the liver, did not protract survival of DRG progenitor cells. Among the cells which survived with neural tube extract, some even succeeded in extending neurites; nevertheless, in absence of normal connections with the central nervous system (CNS) they finally died. Treatment of silastic implanted embryos with nerve growth factor (NGF) did not prevent the experimentally induced crest cell death. These results demonstrate that DRG develop from a population of neural crest cells which depends for its survival and probably for its differentiation upon a signal arising from the CNS, needed as early as the first hours after initiation of migration. Recovery experiments suggest that the subpopulation of crest cells which will develop along the sensory pathway probably depends for its survival and/or differentiation upon a factor contained in the neural tube, which is different from NGF.     

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.     

Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neurectodermal mesenchymal derivatives, using a biological cell marking technique

By isotopic and isochronic transplantations of fragments of quail neural tube into chick, it has been previously shown that enteric ganglion cells arise from the “vagal” (somites 1–7) and the “lumbo-sacral” (behind somite 28) levels of the neural crest, while the trunk region (somites 8–28) gives rise to orthosympathetic ganglion chain and adrenomedullary cells. The latter originate precisely from the neural crest corresponding to somites 18–24 (i.e., “adrenomedullary” level of the crest). Heterotopic transplantations of fragments of quail neural tube into chick have been carried out in the present work. When the “adrenomedullary” level of the quail neural tube is grafted into the “vagal” region of a chick, the crest cells colonize the gut and differentiate into enteric ganglia of Auerbach's and Meissner's plexi. If quail cephalic neural crest is transplanted in the “adrenomedullary” level of a chick, quail cells migrate into the suprarenal glands and differentiate into adrenomedullary cells. Mesectodermal cells migrate laterally, and differentiate into cartilage, dermis and connective tissues. Thus it appears that preferential pathways located at precise levels of the embryo lead crest cells to their definitive sites. On the other hand the differentiation of the autonomic neuroblasts is controlled by the environment in which crest cells are localized at the end of their migration. On the contrary, mesenchymal derivatives of the cephalic neural crest appear to be early determined since they differentiate according to their presumptive fate when transplanted into the trunk.     

Distribution and migration pathways of HNK-1-immunoreactive neural crest cells in teleost fish embryos.

Whole mounts and cross-sections of embryos from three species of teleost fish were immunostained with the HNK-1 monoclonal antibody, which recognizes an epitope on migrating neural crest cells. A similar distribution and migration was found in all three species. The crest cells in the head express the HNK-1 epitope after they have segregated from the neural keel. The truncal neural crest cells begin to express the epitope while they still reside in the dorsal region of the neural keel; this has not been observed in other vertebrates. The cephalic and anterior truncal neural crest cells migrate under the ectoderm; the cephalic cells then enter into the gill arches and the anterior truncal cells into the mesentery of the digestive tract where they cease migration. These cephalic and anterior trunk pathways are similar to those described in Xenopus and chick. The neural crest cells of the trunk, after segregation, accumulate in the dorsal wedges between the somites, however, unlike in chick and rat, they do not migrate in the anterior halves of the somites but predominantly between the neural tube and the somites, the major pathway observed in carp and amphibians; some cells migrate over the somites. The HNK-1 staining of whole-mount embryos revealed a structure resembling the Rohon-Beard and extramedullary cells, the primary sensory system in amphibians. Such a system has not been described in fish.     

Difference in the expression of asymmetric acetylcholinesterase molecular forms during myogenesis in early avian dermomyotomes and limb buds in ovo and in vitro

The A12 (asymmetric) form of acetylcholinesterase (AChE) is generally considered to be synthesized in leg muscle tissues by myotubes under neural influence, but not by myoblasts. We have examined the expression of the different molecular forms of AChE in explants of developing limb buds and dermomyotomes (the myogenic part of the somites) obtained from 3-day-old chick and quail embryos, either directly after removal or during in vitro culture. We describe a muscular differentiation of both territories in vitro, leading to the formation of myotubes which are morphologically similar to the class of early muscle cells described by Bonner and Hauschka (1974). In vivo the A12 form is present in quail dermomyotomes which are almost entirely composed of mononucleated poorly differentiated cells; in contrast, it is absent from similar cells in chick dermomyotomes and from limb buds in both species. This shows that in the case of quail embryos the appearance of the A12 form precedes the fusion of myoblasts into myotubes. In both species, dermomyotome explants express asymmetric and globular forms of the enzyme during muscular differentiation in vitro, whereas limb buds synthesize only globular forms. After surgical removal of neural tube and/or neural crest at 2 days in ovo, the biosynthesis of the A forms in quail dermomyotomes is not suppressed and is consequently not dependent upon prior connection of the dermomyotomes to central neurons or upon the presence of autonomic precursors. Since limb bud muscle cells derive from somites our results raise questions concerning the differentiation of migrating somitic cells in this territory where a neural influence appears necessary to induce the biosynthesis of asymmetric AChE forms.     

In vivo effect of brain-derived neurotrophic factor on the survival of developing dorsal root ganglion cells.

Implantation of silastic membranes between neural tube and somites at somitic levels 20-24 in 30-somite-stage chick embryos results in separation of early migrated neural crest cells of the dorsal root ganglion (DRG) anlage from the neural tube and their death within a few hours [Kalcheim and Le Douarin, (1986) Dev. Biol., 116, 451-460]. The in vivo effects of brain-derived neutrotrophic factor (BNDF) on survival of HNK-1 immunoreactive DRG cells separated from the tube were examined by implantation of laminin-treated silastic membranes (controls) or BDNF/laminin-treated membranes. In the presence of BDNF/laminin-treated membranes, 20/25 grafted embryos fixed 10 h after implantation, contained many rescued cells on the operated side. In contrast, only a few rescued cells on the operated side. In contrast, only a few rescued cells were observed in sections on the operated in 2/11 embryos implanted with laminin-treated silastic membranes, and no rescued cells at all could be detected in embryos implanted with NGF/laminin-treated (seven embryos) or untreated silastic membranes (12 embryos). The data presented support the hypothesis that early survival and differentiation of neural crest-derived sensory cells depend on central nervous system-derived factor(s). Moreover, this is the first evidence for the in vivo activity of BDNF on survival of developing DRG cells.     

A positive correlation between permissiveness of mesoderm to neural crest migration and early DRG growth.

The microenvironment created by grafting rostral somitic halves in place of normal somites leads to the formation of nonsegmented peripheral ganglia (Kalcheim and Teillet, 1989; Goldstein and Kalcheim, 1991) and is mitogenic for neural crest (NC) cells that become dorsal root ganglia (DRG) (Goldstein et al., 1990). We have now extended these studies by using three surgical manipulations to determine how additional mesodermal tissues affected DRG growth in chick embryos. The following experimental manipulations were performed: (1) unilateral deletion of epithelial somites, similar deletions followed by replacing the somites with (2) a three-dimensional collagen matrix, or (3) fragments of quail lateral plate mesoderm. When somites were absent or replaced by collagen matrix, ganglia were unsegmented, and their volumes were decreased by 21% and 12%, respectively, compared to contralateral intact DRG. In contrast, when lateral plate mesoderm was transplanted in place of somitic mesoderm, NC cells migrated into the grafted mesoderm and formed unsegmented DRG whose volumes were increased by 62.6% compared to the contralateral ganglia. These results suggest that although DRG precursors do not require sclerotome to begin migration and condensation processes, DRG size is modulated by the properties of the mesoderm. Permissiveness to migration is positively correlated with an increase in DRG volume. This volume increase observed in grafts of lateral plate mesoderm is likely to result from enhanced proliferation of neural crest progenitors, previously demonstrated for DRG cells in rostral somitic grafts.     

The Timing of Appearance and Pathway of Migration of Cranial Neural Crest-derived Precursors of Melanocytes in Chick Embryos

The timing of appearance and pathway of migration of precursors of melanocytes in cranial regions of chick embryos were examined by the monoclonal antibody MEBL-1, which can identify precursors of melanocytes soon after their emigration from the neural tube (7). Precursors of melanocytes were first detected on the dorsal side of the mesencephalic neural tube at stage 16, when other neural crest cells had already left the dorsal side of the neural tube. Then precursors of melanocytes at more rostral and caudal levels appeared. After the first appearance on the neural tube, precursors of melanocytes migrated along a dorsolateral pathway under the superficial ectoderm, which followed other neural crest cells. These results indicate that precursors of melanocytes migrate along spatially the same pathway as other neural crest cells, but temporally the different time as considered previously.     

Differentiation of peptidergic neurones in quail-chick chimaeric embryos

The problem raised in this work was whether peptidergic neurones with vasoactive intestinal peptide (VIP)-and substance P-like immunoreactivity could develop in chimaeric embryos in which quail neural crest cells had been implanted into chick at an early developmental stage. Differentiation of peptide-containing nerve somas was looked for in different situations: i) when the quail neural primordium had been grafted orthotopically and isochronically into the chick host either at the adrenomedullary (level of somites 18-24) or at the vagal (level of somites 1-7) levels of the neural axis; ii) when the quail adrenomedullary neural primordium had been heterotopically implanted at the vagal level of the chick host. In all conditions, VIP- and substance P-like immunoreactivity were observed in a number of quail neurones located either in the peripheral ganglia of the trunk at the level of the graft (in orthotopic grafts of the adrenomedullary neural primordium) or in the enteric ganglia of the chick gut (in the other types of grafts). The developmental stage at which the first neurones become detectable in the host conforms to the genetic characteristics of the effector cells, i.e. they differentiate at the same stage in normal quail neuroblasts and in quail neuroblasts transplanted into the chick host. In contrast, the distribution of the peptidergic neurones in the host depends on the tissue into which the neural crest cells migrate and not on their origin in the neural axis and their fate in normal 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.     

Developmental potential of neural crest-derived cells migrating from segments of developing quail bowel back-grafted into younger chick host embryos

The technique of back-transplantation was used to investigate the developmental potential of neural crest-derived cells that have migrated to and colonized the avian bowel. Segments of quail bowel (removed at E4) were grafted between the somites and neural tube of younger (E2) chick host embryos. Grafts were placed at a truncal level, adjacent to somites 14-24. Initial experiments, done in vitro, confirmed that crest-derived cells are capable of migrating out of segments of foregut explanted at E4. The foregut, which at E4 has been colonized by cells derived from the vagal crest, served as the donor tissue. Comparative observations were made following grafts of control tissues, which included hindgut, lung primordia, mesonephros and limb bud. Additional experiments were done with chimeric bowel in which only the crest-derived cells were of quail origin. Targets in the host embryos colonized by crest-derived cells from the foregut grafts included the neural tube, spinal roots and ganglia, peripheral nerves, sympathetic ganglia and the adrenals, but not the gut. Donor cells in these target organs were immunostained by the monoclonal antibody, NC-1, indicating that they were crest-derived and developing along neural or glial lineages. Some of the crest-derived cells (NC-1-immunoreactive) that left the bowel and reached sympathetic ganglia, but not peripheral nerves or dorsal root ganglia, co-expressed tyrosine hydroxylase immunoreactivity, a neural characteristic never expressed by crest-derived cells in the avian gut. None of the cells leaving enteric back-grafts produced pigment. Cells of mesodermal origin were also found to leave donor explants and aggregate in dermis and feather germs near the grafts. These observations indicate that crest-derived cells, having previously migrated to the bowel, retain the ability to migrate to distant sites in a younger embryo. The routes taken by these cells appear to reflect, not their previous migratory experience, but the level of the host embryo into which the graft is placed. Some of the population of crest-derived cells that leave the back-transplanted gut remain capable of expressing phenotypes that they do not express within the bowel in situ, but which are appropriate for the site in the host embryo to which they migrate.     

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)     

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)     

Chicken protocadherin-1 functions to localize neural crest cells to the dorsal root ganglia during PNS formation

In vertebrate embryos, neural crest cells emerge from the dorsal neural tube and migrate along well defined pathways to form a wide diversity of tissues, including the majority of the peripheral nervous system (PNS). Members of the cadherin family of cell adhesion molecules play key roles during the initiation of migration, mediating the delamination of cells from the neural tube. However, a role for cadherins in the sorting and re-aggregation of the neural crest to form the PNS has not been established. We report the requirement for a protocadherin, chicken protocadherin-1 (Pcdh1), in neural crest cell sorting during the formation of the dorsal root ganglia (DRG). In embryos, cPcdh1 is highly expressed in the developing DRG, where it co-localizes with the undifferentiated and mitotically active cells along the perimeter. Pcdh1 can promote cell adhesion in vivo and disrupting Pcdh1 function in embryos results in fewer neural crest cells localizing to the DRG, with a concomitant increase in cells that migrate to the sympathetic ganglia. Furthermore, those cells that still localize to the DRG, when Pcdh1 is inhibited, are no longer found at the perimeter, but are instead dispersed throughout the DRG and are now more likely to differentiate along the sensory neuron pathway. These results demonstrate that Pcdh1-mediated cell adhesion plays an important role as neural crest cells coalesce to form the DRG, where it serves to sort cells to the mitotically active perimeter.     

Axolotls with an under- or oversupply of neural crest can regulate the sizes of their dorsal root ganglia to normal levels

How animals adjust the size of their organs is a fundamental, enduring question in biology. Here we manipulate the amount of neural crest (NC) precursors for the dorsal root ganglia (DRG) in axolotl. We produce embryos with an under- or over-supply of pre-migratory NC in order to find out if DRG can regulate their sizes during development. Axolotl embryos are perfectly suitable for this research. Firstly, they are optimal for microsurgical manipulations and tissue repair. Secondly, they possess, unlike most other vertebrates, only one neural crest string located on top of the neural tube. This condition and position enables NC cells to migrate to either side of the embryo and participate in the regulation of NC cell distribution. We show that size compensation of DRG in axolotl occurs in 2 cm juveniles after undersupply of NC (up-regulation) and in 5 cm juveniles after oversupply of NC (down-regulation). The size of DRG is likely to be regulated locally within the DRG and not via adaptations of the pre-migratory NC or during NC cell migration. Ipsi- and contralateral NC cell migration occurs both in embryos with one and two neural folds, and contralateral migration of NC is the only source for contralateral DRG formation in embryos with only one neural fold. Compensatory size increase is accompanied by an increase in cell division of a DRG precursor pool (PCNA+/SOX2−), rather than by DRG neurons or glial cells. During compensatory size decrease, increased apoptosis and reduced proliferation of DRG cells are observed.     

Determination of somite cells: independence of cell differentiation and morphogenesis

Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2.5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9.5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.     

Neural crest cell migratory pathways in the trunk of the chick embryo

Neural crest cells migrate during embryogenesis to give rise to segmented structures of the vertebrate peripheral nervous system: namely, the dorsal root ganglia and the sympathetic chain. However, neural crest cell arise from the dorsal neural tube where they are apparently unsegmented. It is generally agreed that the somites impose segmentation on migrating crest cells, but there is a disagreement about two basic questions: exactly pathways do neural crest cells use to move through or around somites, and do neural crest cells actively migrate or are they passively dispersed by the movement of somite cells? The answers to both questions are critically important to any further understanding of the mechanisms underlying the precise distribution of the neural crest cells that develop into ganglia. We have done an exhaustive study of the locations of neural crest cells in chick embryos during early stages of their movement, using antibodies to neural crest cells (HNK-1), to neural filament-associated protein in growing nerve processes (E/C8), and to the extracellular matrix molecule laminin. Our results show that Some neural crest cells invade the extracellular space between adjacent somites, but the apparent majority move into the somites themselves along the border between the dermatome/myotome (DM) and the sclerotome. Neural crest cells remain closely associated with the anterior half of the DM of developing somites as they travel, suggesting that the basal lamina of the DM may be used as a migratory substratum. Supporting this idea is our observation that the development of the DM basal lamina coincides in time and location with the onset of crest migration through the somite. The leading front of neural crest cells advance through the somite while the length of the DM pathway remains constant, suggesting active locomotion, at least in this early phase of development. Neural crest cells leave the DM at a later stage of development to associate with the dorsal aorta, where sympathetic ganglia form, and to associate with newly emerging fibers of the ventral root nerve, where they presumably give rise to neuronal supportive cells. Thus we propose that the establishment of the segmental pattern of the peripheral ganglia and nerves depends on the timely development of appropriate substrata to guide and distribute migrating neural crest cells during the early stages of embryogenesis.