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.     

The early development of cranial sensory ganglia and the potentialities of their component cells studied in quail-chick chimeras

The quail-chick marker system has been used to study the early developmental stages of the ganglia located along cranial nerves VII, IX, and X. The streams of neural crest cells arising from the rhombencephalic-vagal neural crest were followed from the onset of their migration up to the localization of crest cells in the trunk and root ganglia of these nerves. It was shown that two different populations of crest cells are segregated early as a result of morphogenetic movements in the hypobranchial region. The dorsal population gives rise to the root ganglia of nerves IX and X located close to the encephalic vesicles, where the crest cells differentiate both into neurons and into glia. In contrast, the ventral stream of neural crest cells contributes together with cells from epibranchial placodes to the trunk ganglia (geniculate, petrous, and nodose ganglia) of cranial nerves VII, IX, and X. The successive steps of the invasion of the placodal anlage by crest cells can be followed owing to the selective labeling of the neural crest cells. It appears that the latter give rise to the satellite cells of the geniculate, petrous, and nodose ganglia while the large sensory neurons originate from the placodes. The nodose ganglion has been the subject of further studies aimed to investigate whether neuronal potentialities can be elicited in the neural crest-derived cells that it contains. The ability to label selectively either the neurons or the glia by the quail nuclear marker made this investigation possible in the particular case of the nodose ganglion whose neurons and satellite cells have a different embryonic origin. By the technique already described (N. M. Le Douarin, M. A. Teillet, C. Ziller, and J. Smith, 1978, Proc. Nat. Acad. Sci. USA75, 2030–2034) of back-transplantation into the neural crest migration pathway of a younger host, it was shown that the presumptive glial cells of the nodose ganglion are able to remigrate when transplanted into a 2-day chick host and to differentiate into autonomic structures (sympathetic ganglion cells, adrenomedullary cells, and enteric ganglia). It is proposed as a working hypothesis that neuronal potentialities contained in the neural crest cells which invade the placodal primordium of the nodose ganglion are repressed through cell-cell interactions occurring between placodal and crest cells.     

Contributions of placodal and neural crest cells to avian cranial peripheral ganglia

The method of embryonic tissue transplantation was used to confirm the dual origin of avian cranial sensory ganglia, to map precise locations of the anlagen of these sensory neurons, and to identify placodal and neural crest-derived neurons within ganglia. Segments of neural crest or strips of presumptive placodal ectoderm were excised from chick embryos and replaced with homologous tissues from quail embryos, whose cells contain a heterochromatin marker. Placode-derived neurons associated with cranial nerves V, VII, IX, and X are located distal to crest-derived neurons. The generally larger, embryonic placodal neurons are found in the distal portions of both lobes of the trigeminal ganglion, and in the geniculate, petrosal and nodose ganglia. Crest-derived neurons are found in the proximal trigeminal ganglion and in the combined proximal ganglion of cranial nerves IX and X. Neurons in the vestibular and acoustic ganglia of cranial nerve VIII derive from placodal ectoderm with the exception of a few neural crest-derived neurons localized to regions within the vestibular ganglion. Schwann sheath cells and satellite cells associated with all these ganglia originate from neural crest. The ganglionic anlagen are arranged in cranial to caudal sequence from the level of the mesencephalon through the third somite. Presumptive placodal ectoderm for the VIIIth, the Vth, and the VIIth, IXth, and Xth ganglia are located in a medial to lateral fashion during early stages of development reflecting, respectively, the dorsolateral, intermediate, and epibranchial positions of these neurogenic placodes.     

Development of VIP in the peripheral nervous system of avian embryo

The distribution of the VIP containing structures was studied in the gut and in the paravertebral sympathetic ganglia of the quail and chick embryos by immunocytochemistry. In the gut, development of peptidergic nerves followed a craniocaudal gradient. Immunoreactive fibres were first visible in the oesophagus at day 9 in the quail and day 10 in the chick, at 12 days they extended over the whole length of the gut. Cell bodies were localized at day 9 in the foregut and observed in the mid- and hind-gut just before hatching. Transplantations on the chorioallantoic membrane of fragments of various parts of the digestive tract clearly demonstrated that VIP nerve cell bodies belonged to the intrinsic innervation of the gut. Besides the gut, sympathetic paravertebral ganglia contained cells with VIP immunoreactivity detected at day 9 and 10 in quail and chick respectively. In order to find out whether VIP containing neurons differentiated normally in chick embryos in which quail neural crest cells had been implanted at an early stage of development we looked for the appearance of peptidergic neurones in the following situations: when the quail neural primordium had been grafted orthotopically and isochronically into chick host (1) at the adrenomedullary (somites 18-24) and (2) at the vagal (somites 1-7) levels of the neural axis. In all conditions VIP immunoreactivity was observed in quail cells located either in the sympathetic paravertebral ganglia of the trunk at the level of the graft or in the enteric ganglia according to the graft was made at the adrenomedullary and vagal levels respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryonic origin of substance P containing neurons in cranial and spinal sensory ganglia of the avian embryo

The ontogeny of the neurons exhibiting substance P-like immunoreactivity (SPLI) was examined in the spinal and cranial sensory ganglia of chick and quail embryos. It was shown that in dorsal root ganglia (DRG) virtually all neuronal somas occupying the mediodorsal (MD) region of the ganglia are SPLI-positive while the larger neurons of the lateroventral (LV) area are SPLI-negative. In the cranial nerve ganglia, both types of neurons coexist in the trigeminal ganglion but with a different distribution: small neurons with SPLI are proximal while large neurons without SPLI occupy the maxillomandibular and ophthalmic lobes. The distal ganglia of nerves VII and IX (i.e., geniculate, petrosal) do not show cell bodies with SPLI in the two species considered. A few of them only (about 12%) are found in the nodose (distal ganglion of nerve X). The proximal ganglia of nerves IX and X (i.e., superior-jugular complex) are composed of small neurons which virtually all exhibit SPLI. Chimaeric cranial sensory ganglia were constructed by grafting the quail hind-brain primordium into chick embryos. Revelation of SPLI was combined with acridine orange staining on the same sections in order to ascertain the placodal (chick host) or neural crest (quail donor) origin of the SP-positive neurons in each type of ganglion. We found that all the neurons showing SPLI are derived from the neural crest in the trigeminal and in the superior and jugular ganglia. In the geniculate, petrosal, and nodose all the neurons are derived from the placodal ectoderm. The small number of SPLI-positive cells of the nodose ganglia are not an exception to this rule. Therefore, generally speaking, the sensory neurons of the cranial ganglia that express the SP phenotype are derived from the crest, with the exception of some neurons present in the nodose of both quail and chick embryos and which are of placodal origin. The vast majority of placode-derived neurons do not have amounts of SP that can be detected under the conditions of the present study.     

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.     

Sacral neural crest cells colonise aganglionic hindgut in vivo but fail to compensate for lack of enteric ganglia

The vagal neural crest is the origin of majority of neurons and glia that constitute the enteric nervous system, the intrinsic innervation of the gut. We have recently confirmed that a second region of the neuraxis, the sacral neural crest, also contributes to the enteric neuronal and glial populations of both the myenteric and the submucosal plexuses in the chick, caudal to the level of the umbilicus. Results from this previous study showed that sacral neural crest-derived precursors colonised the gut in significant numbers only 4 days after vagal-derived cells had completed their migration along the entire length of the gut. This observation suggested that in order to migrate into the hindgut and differentiate into enteric neurons and glia, sacral neural crest cells may require an interaction with vagal-derived cells or with factors or signalling molecules released by them or their progeny. This interdependence may also explain the inability of sacral neural crest cells to compensate for the lack of ganglia in the terminal hindgut of Hirschsprung's disease in humans or aganglionic megacolon in animals. To investigate the possible interrelationship between sacral and vagal-derived neural crest cells within the hindgut, we mapped the contribution of various vagal neural crest regions to the gut and then ablated appropriate sections of chick vagal neural crest to interrupt the migration of enteric nervous system precursor cells and thus create an aganglionic hindgut model in vivo. In these same ablated animals, the sacral level neural axis was removed and replaced with the equivalent tissue from quail embryos, thus enabling us to document, using cell-specific antibodies, the migration and differentiation of sacral crest-derived cells. Results showed that the vagal neural crest contributed precursors to the enteric nervous system in a regionalised manner. When quail-chick grafts of the neural tube adjacent to somites 1-2 were performed, neural crest cells were found in enteric ganglia throughout the preumbilical gut. These cells were most numerous in the esophagus, sparse in the preumbilical intestine, and absent in the postumbilical gut. When similar grafts adjacent to somites 3-5 or 3-6 were carried out, crest cells were found within enteric ganglia along the entire gut, from the proximal esophagus to the distal colon. Vagal neural crest grafts adjacent to somites 6-7 showed that crest cells from this region were distributed along a caudal-rostral gradient, being most numerous in the hindgut, less so in the intestine, and absent in the proximal foregut. In order to generate aneural hindgut in vivo, it was necessary to ablate the vagal neural crest adjacent to somites 3-6, prior to the 13-somite stage of development. When such ablations were performed, the hindgut, and in some cases also the cecal region, lacked enteric ganglionated plexuses. Sacral neural crest grafting in these vagal neural crest ablated chicks showed that sacral cells migrated along normal, previously described hindgut pathways and formed isolated ganglia containing neurons and glia at the levels of the presumptive myenteric and submucosal plexuses. Comparison between vagal neural crest-ablated and nonablated control animals demonstrated that sacral-derived cells migrated into the gut and differentiated into neurons in higher numbers in the ablated animals than in controls. However, the increase in numbers of sacral neural crest-derived neurons within the hindgut did not appear to be sufficiently high to compensate for the lack of vagal-derived enteric plexuses, as ganglia containing sacral neural crest-derived neurons and glia were small and infrequent. Our findings suggest that the neuronal fate of a relatively fixed subpopulation of sacral neural crest cells may be predetermined as these cells neither require the presence of vagal-derived enteric precursors in order to colonise the hindgut, nor are capable of dramatically altering their proliferation or d     

Colonization of the murine hindgut by sacral crest-derived neural precursors: experimental support for an evolutionarily conserved model

Enteric ganglia in the hindgut are derived from separate vagal and sacral neural crest populations. Two conflicting models, based primarily on avian data, have been proposed to describe the contribution of sacral neural crest cells. One hypothesizes early colonization of the hindgut shortly after neurulation, and the other states that sacral crest cells reside transiently in the extraenteric ganglion of Remak and colonize the hindgut much later, after vagal crest-derived neural precursors arrive. In this study, I show that Wnt1-lacZ-transgene expression, an "early" marker of murine neural crest cells, is inconsistent with the "early-colonization" model. Although Wnt1-lacZ-positive sacral crest cells populate pelvic ganglia in the mesenchyme surrounding the hindgut, they are not found in the gut prior to the arrival of vagal crest cells. Similarly, segments of murine hindgut harvested prior to the arrival of vagal crest cells and grafted under the renal capsule fail to develop enteric neurons, unless adjacent pelvic mesenchyme is included in the graft. When pelvic mesenchyme from DbetaH-nlacZ transgenic embryos is apposed with nontransgenic hindgut, neural precursors from the mesenchyme colonize the hindgut and form intramural ganglion cells that express the transgenic marker. Contribution of sacral crest-derived cells to the enteric nervous system is not affected by cocolonization of grafts by vagal crest-derived neuroglial precursors. The findings complement recent studies of avian chimeras and support an evolutionarily conserved model in which sacral crest cells first colonize the extramural ganglion and secondarily enter the hindgut mesenchyme.     

Neuronal differentiation from postmitotic precursors in the ciliary ganglion

In the chick ciliary ganglion, neuronal number is kept constant between St. 29 and St. 34 (E6-E8) despite a large amount of cell death. Here, we characterize the source of neurogenic cells in the ganglion as undifferentiated neural crest-derived cells. At St. 29, neurons and nonneuronal cells in the ciliary ganglion expressed the neural crest markers HNK-1 and p75(NTR). Over 50% of the cells were neurons at St. 29; of the nonneuronal cells, a small population expressed glial markers, whereas the majority was undifferentiated. When placed in culture, nonneuronal cells acquired immunoreactivity for HuD, suggesting that they had commenced neuronal differentiation. The newly differentiated neurons arose from precursors that did not incorporate bromodeoxyuridine. To test whether these precursors could undergo neural differentiation in vivo, purified nonneuronal cells from St. 29 quail ganglia were transplanted into chick embryos at St. 9-14. Subsequently, quail cells expressing neuronal markers were found in the chick ciliary ganglion. The existence of this precursor pool was transient because nonneuronal cells isolated from St. 38 ganglia failed to form neurons. Since all ciliary ganglion neurons are born prior to St. 29, these results demonstrate that there are postmitotic neural crest-derived precursors in the developing ciliary ganglion that can differentiate into neurons in the appropriate environment.     

Development of the monoaminergic innervation of the avian gut: transient and permanent expression of phenotypic markers

Specific cellular accumulation of [3H]5-hydroxytryptamine ([3H]5-HT) occurs during development of the avian gut. This accumulation is transient in extraganglionic mesenchymal cells (TES cells) but is a permanent characteristic of enteric serotonergic neurons (ESN). Species-specific differences were found in the location of TES cells and ESN. In chicks TES cells surrounded myenteric ganglia and ESN were restricted to the myenteric plexus. In quails TES cells surrounded submucosal ganglia and [3H]5-HT-labeled submucosal as well as myenteric neurons. [3H]Norepinephrine accumulated only in noradrenergic terminals and not in TES cells or ESN. The origins of TES cells and ESN were studied in chimeras, in which neuraxis from appropriate or inappropriate axial levels was grafted from quail to chick. Both types of chimeric bowel contained TES cells and ESN. Most TES cells in chimeras were chick in origin and distributed as in chicks (around myenteric ganglia); however, some TES cells and all ESN were quail cells. To test whether crest cells are required for development of TES cells and ESN, aneuronal chick hindgut was explanted and grown alone, or with quail neuraxis, as chorioallantoic membrane (CAM) grafts. TES cells appeared in CAM grafts whether or not crest cells were present; however ESN only appeared in explants when quail neuraxis was included. In addition, an ectopic [3H]5-HT-labeled chromaffin-like cell, also of quail origin, was found in enteric plexuses in these combined explants of crest and gut. Most TES cells, therefore, are neither derived from nor dependent on the presence of crest cells in the gut wall. Since even an inappropriate axial level of crest was found to produce ESN when it was experimentally induced to colonize the bowel the enteric microenvironment probably plays a critical role in serotonergic neural development. The species-specific location of TES cells and ESN is consistent with the hypothesis that TES cells constitute an important component of this microenvironment.     

Evolution of the lumbo-sacral neural crest in the avian embryo: Origin and differentiation of the ganglionated nerve of Remak studied in interspecific quail-chick chimaerae

Summary Isotopic and isochronic transplantation of fragments of quail neural tube into chick demonstrates that neural and glial cells of the entire ganglion of Remak (RG) arise from the lumbo-sacral level of the neural crest.The ganglioblasts first accumulate in the mesorectum (stage 24 of Hamburger and Hamilton, in the chick and I8 of Zacchei in the quail) and subsequently migrate cranially.Histochemical studies have been carried out on the rectal and cloacal parts of the quail RG at various stages of development. Cholinesterase activity can be detected as soon as the primordium is in place and the intensity of the reaction increases rapidly. During morphogenesis of the cloacal region the RG and the pelvic plexus become intimately associated. Catecholamine-containing cells are found first in the pelvic plexus, then in the cloacal part of the RG. Fluorescent cells are often grouped close to blood vessels and associated with non-fluorescent ganglia. Cranial to the level of the bursa of Fabricius, the RG is composed only of non-fluorescent neurons whatever the developmental stage considered (up to 1 day after hatching).The developmental capabilities of the RG of the 5-day quail have been tested by transplanting various parts of the hind-gut with the dorsal mesentery onto the chorio-allantoic membrane. Catecholamine-containing cells develop only in grafts including the cloacal region.By using quail-chick chimaerae in which the RG belongs to the quail while mesentery and gut are of chick origin, it was possible to show that neurons which develop in the graft (i.e. in the absence of preganglionic innervation), send nerve fibres into the gut wall. Moreover some neuroblasts located in the primordium of the RG migrate into the gut wall and give rise to some enteric ganglion cells. The contribution of the lumbo-sacral neural crest to the enteric ganglia, by this route, is discussed.List of Abbreviations in Text FIF formol-induced fluorescence - H & H Hamburger and Hamilton - Z Zacchei - CAM chorio-allantoic membrane - SIF small intensely fluorescent (cells)     

The effect of back-transplants of the embryonic gut wall on growth of the neural tube

Experiments in which the developing gut of avian embryos was back-transplanted to permit the bowel to interact with the developing neural tube were undertaken. Segments of intestine from 4-day quail embryos were implanted between the somites and neural tubes of chick embryos of 7 to 24 somites. The spinal cord responded to the presence of the bowel by enlarging unilaterally on the side of the graft. This effect encompassed both gray and white matter and was accompanied by the extension of neuritic projections from the spinal cord into the enteric grafts. The growth-promoting effect of enteric transplants was manifest at all levels of the neural tube where the grafts were made and led to enlargement of the brain as well as the spinal cord; however, truncal neural crest derivatives in the region of the grafts, such as developing sympathetic and spinal ganglia, were unaffected. Neither sham operations nor grafts of ciliary ganglion, lung, pancreas, mesonephros, or rudiment of the eye mimicked the action of the gut. The effect of the bowel was manifest as early as 24 hr following back-transplantation and was found to be due to an increase in the number of cells in the neuroepithelium. The cell responsible for the ability of the gut wall to enhance neuroepithelial proliferation was not identified, but the effect lacked species specificity and could be elicited in the absence of endoderm or neural crest derivatives in the explant. We propose that the musculoconnective tissue of the gut produces a short-range diffusible factor that induces mitogenic activity in the neuroepithelial cells of the neural tube, but not in the crest cells that form sympathetic or sensory ganglia. Since the gut is not normally in apposition to the neural tube, we suggest that the physiological targets of this factor are the specialized crest cells that colonize the bowel and give rise to the enteric nervous system.     

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.     

Compensatory responses and development of the nodose ganglion following ablation of placodal precursors in the embryonic chick (Gallus domesticus)

The nodose ganglion is the distal cranial ganglion of the vagus nerve which provides sensory innervation to the heart and other viscera. In this study, removal of the neuronal precursors which normally populate the right nodose ganglion was accomplished by ablating the right nodese placode in stage 9 chick embryos. Subsequent histological evaluation showed that in 54% of lesioned embryos surviving to day 6, the right ganglion was absent. Most embryos surviving to day 12, however, had identifiable right ganglia. In day 12 embryos, the right ganglion which developed was abnormal, with ganglion volume and ganglion cell diameter reduced by 50% and 20%, respectively, compared to control ganglia. To investigate the source of the neuron population in the regenerated ganglion, we combined nodose placode ablation with bilateral replacement of chick with quail cardiac neural crest (from mid-otic placode to somite 3). These cells normally provide only non-neuronal cells to the nodose ganglion, but produce neurons in other regions. At day 9, quail-derived neurons were identified in the right nodose ganglia of these chimeras, indicating that cardiac neural crest cells can generate neurons in the ganglion when placode-derived neurons are absent or reduced in number. On the other hand, we found that sympathetic neural crest (from somites 10 to 20) does not support ganglion development, suggesting that only neural crest cells normally present in the ganglion participate in reconstituting its neuronal population. Our previous work has shown that right nodose placode ablation produces abnormal cardiac function, which mimics a life-threatening human heart condition known as long QT syndrome. The present results suggest that the presence of neural crest-derived neurons in the developing right nodose ganglion may contribute to the functional abnormality in long QT syndrome.This work was supported by grant PO1 HL 36059     

Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate

The cutaneous sensory neurons of the ophthalmic lobe of the trigeminal ganglion are derived from two embryonic cell populations, the neural crest and the paired ophthalmic trigeminal (opV) placodes. Pax3 is the earliest known marker of opV placode ectoderm in the chick. Pax3 is also expressed transiently by neural crest cells as they emigrate from the neural tube, and it is reexpressed in neural crest cells as they condense to form dorsal root ganglia and certain cranial ganglia, including the trigeminal ganglion. Here, we examined whether Pax3+ opV placode-derived cells behave like Pax3+ neural crest cells when they are grafted into the trunk. Pax3+ quail opV ectoderm cells associate with host neural crest migratory streams and form Pax3+ neurons that populate the dorsal root and sympathetic ganglia and several ectopic sites, including the ventral root. Pax3 expression is subsequently downregulated, and at E8, all opV ectoderm-derived neurons in all locations are large in diameter, and virtually all express TrkB. At least some of these neurons project to the lateral region of the dorsal horn, and peripheral quail neurites are seen in the dermis, suggesting that they are cutaneous sensory neurons. Hence, although they are able to incorporate into neural crest-derived ganglia in the trunk, Pax3+ opV ectoderm cells are committed to forming cutaneous sensory neurons, their normal fate in the trigeminal ganglion. In contrast, Pax3 is not expressed in neural crest-derived neurons in the dorsal root and trigeminal ganglia at any stage, suggesting either that Pax3 is expressed in glial cells or that it is completely downregulated before neuronal differentiation. Since Pax3 is maintained in opV placode-derived neurons for some considerable time after neuronal differentiation, these data suggest that Pax3 may play different roles in opV placode cells and neural crest cells.     

Electrophysiological properties of rat nodose ganglion neurons co-transplanted with carotid bodies into the chick chorioallantoic membrane

The electrophysiological properties of nodose ganglion neurons were evaluated immediately after removing nodose ganglia from young adult rats and 3 to 10 days after nodose ganglia implantation -either alone or co-implanted with carotid bodies- onto the chick chorioallantoic membrane. Implanted and co-implanted nodose neurons were less excitable than acutely recorded nodose neurons. Co-implanted neurons also showed reduced amplitudes for both action potentials and spike after-hyperpolarizations relative to those found in. acutely recorded nodose ganglion neurons and a smaller time constant (T) than that found in implanted neurons. In addition, no spontaneous activity was recorded from nodose ganglion neurons co-implanted with carotid bodies during 3-9 days, which suggests that functional synapses between carotid glomus cells and nodose neurons were not yet established. Results indicate the feasibility of obtaining viable nodose neurons for up to 10 days grafted onto the chick chorioallantoic membrane, where they can conserve most of their passive and active membrane properties and also are susceptible to carotid bodies trophic influences. They also suggest that nodose neurons would need more time for the development of functional synapses when grafted with carotid body glomus cells.     

Embryonic origin of avian corneal sensory nerves.

Sensory nerves play a vital role in maintaining corneal transparency. They originate in the trigeminal ganglion, which is derived from two embryonic cell populations (cranial neural crest and ectodermal placode). Nonetheless, it is unclear whether corneal nerves arise from neural crest, from placode, or from both. Quail-chick chimeras and species-specific antibodies allowed tracing quail-derived neural crest or placode cells during trigeminal ganglion and corneal development, and after ablation of either neural crest or placode. Neural crest chimeras showed quail nuclei in the proximal part of the trigeminal ganglion, and quail nerves in the pericorneal nerve ring and in the cornea. In sharp contrast, placode chimeras showed quail nuclei in the distal part of the trigeminal ganglion, but no quail nerves in the cornea or in the pericorneal nerve ring. Quail placode-derived nerves were present, however, in the eyelids. Neural crest ablation between stages 8 and 9 resulted in diminished trigeminal ganglia and absence of corneal innervation. Ablation of placode after stage 11 resulted in loss of the ophthalmic branch of the trigeminal ganglion and reduced corneal innervation. Noninnervated corneas still became transparent. These results indicate for the first time that although both neural crest and placode contribute to the trigeminal ganglion, corneal innervation is entirely neural crest-derived. Nonetheless, proper corneal innervation requires presence of both cell types in the embryonic trigeminal ganglion. Also, complete lack of innervation has no discernible effect on development of corneal transparency or cell densities.     

Trigeminal ganglion cells cocultured with gut express vasoactive intestinal peptide

The plasticity of neural crest cells for the expression of adrenergic and cholinergic transmitter phenotypes has been well studied. The object of this study was to determine if cells of a sensory ganglion are capable of neuropeptide transmitter plasticity. We studied whether cells of the trigeminal ganglion, which do not express the neuropeptide vasoactive intestinal peptide (VIP) in vivo, would express this peptide when grown with a tissue the gut, that contains large numbers of VIP neurons. Embryonic aneural chick rectum was explanted with the embryonic quail trigeminal ganglion on the chorioallantoic membrane of chick hosts for 7-8 days. The explants were fixed, sectioned, and stained for VIP immunoreactivity (IR), for neurofilament protein immunoreactivity, and for the quail nucleolar marker. In sections of the explants we observed two populations of quail neurons: small (10-13 microns) VIP-IR cells and large (25-32 microns) cells lacking VIP-IR and resembling native trigeminal neurons. Trigeminal ganglia explanted with embryonic heart or trigeminal ganglia explanted alone lacked small VIP-IR cells but contained large VIP-negative neurons. These results show that cells of the trigeminal ganglion grown with the gut can express a neuropeptide they do not express in the absence of the gut or in vivo. Thus the embryonic trigeminal ganglion contains cells that are plastic with respect to neuropeptide expression.     

The location and distribution of neural crest-derived Schwann cells in developing peripheral nerves in the chick forelimb.

The location and distribution of neural crest-derived Schwann cells during development of the peripheral nerves of chick forelimbs were examined using chick-quail chimeras. Neural crest cells were labeled by transplantation of the dorsal part of the neural tube from a quail donor to a chick host at levels of the neural tube destined to give rise to brachial innervation. The ventral roots, spinal nerves, and peripheral nerves innervating the chick forelimb were examined for the presence of quail-derived neural crest cells at several stages of embryonic development. These quail cells are likely to be Schwann cells or their precursors. Quail-derived Schwann cells were present in ventral roots and spinal nerves, and were distributed along previously described neural crest migratory pathways or along the peripheral nerve fibers at all stages of development examined. During early stages of wing innervation, quail-derived Schwann cells were not evenly distributed, but were concentrated in the ventral root and at the brachial plexus. The density of neural crest-derived Schwann cells decreased distal to the plexus, and no Schwann cells were ever seen in advance of the growing nerve front. When the characteristic peripheral nerve branching pattern was first formed, Schwann cells were clustered where muscle nerves diverged from common nerve trunks. In still older embryos, neural crest-derived Schwann cells were evenly distributed along the length of the peripheral nerves from the ventral root to the distal nerve terminations within the musculature of the forelimb. These observations indicate that Schwann cells accompany axons into the developing limb, but they do not appear to lead or direct axons to their targets. The transient clustering of neural crest-derived Schwann cells in the ventral root and at places where axon trajectories diverge from one another may reflect a response to some environmental feature within these regions.     

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.