Adrenergic innervation of the developing chick heart: neural crest ablations to produce sympathetically aneural hearts

Ablation of various regions of premigratory trunk neural crest which gives rise to the sympathetic trunks was used to remove sympathetic cardiac innervation. Neuronal uptake of [3H]-norepinephrine was used as an index of neuronal development in the chick atrium. Following ablation of neural crest over somites 10-15 or 15-20, uptake was significantly decreased in the atrium at 16 and 17 days of development. Ablation of neural crest over somites 5-10 and 20-25 caused no decrease in [3H]-norepinephrine uptake. Removal of neural crest over somites 5-25 or 10-20 caused approximately equal depletions of [3H]-norepinephrine uptake in the atrium. Cardiac norepinephrine concentration was significantly depressed following ablation of neural crest over somites 5-25 but not over somites 10-20. Light-microscopic and histofluorescent preparations confirmed the absence of sympathetic trunks in the region of the normal origin of the sympathetic cardiac nerves following neural crest ablation over somites 10-20. The neural tube and dorsal root ganglia were damaged in the area of the neural-crest ablation; however, all of these structures were normal cranial and caudal to the lesioned area. Development of most of the embryos as well as the morphology of all of the hearts was normal following the lesion. These results indicate that it is possible to produce sympathetically aneural hearts by neural-crest ablation; however, sympathetic cardiac nerves account for an insignificant amount of cardiac norepinephrine. The adrenal medulla is the most likely source of cardiac norepinephrine in sympathetically aneural hearts.     

Quantitative distribution of chick neural crest cells during gangliogenesis

Summary We have quantitated the distribution of chick neural crest cells after they have completed early migration and are aggregating into ganglia. Variables tested for an influence on the distribution of cells include stage, level of somites, position in each of the primary body axes, and individual embryo. The 11th–15th cervical somites of embryos at stages 30, 35, and 40 somites (s) incubated for 2.5, 3.0, and 3.5 days were labeled with antibody to HNK-1 to detect neural crest cells, and doubly labeled with antibody to HNK-1 and to the 150 kD neurofilament subunit to detect neural crest-derived neurons. Significantly more neural crest cells appear at older stages, but cells are uniformly distributed among the 11th–15th somites at any given stage. Significant differences in the total number of neural crest cells among three embryos sampled at the same stage indicate that the number of cells is independent of the staging series used. As early as the 35 s stage about one-third of the neural crest cells throughout the somite exhibit NF staining. At the 40 s stage, doubly labeled NF cells, as well as HNK-1 labeled cells, aggregate in a circumscribed portion of the mediolateral axis to form presumptive sensory ganglia in the dorsal region of the somites. Also at 40 s a wave of cell aggregation into sympathetic ganglia proceeds anteroposteriorly along the ventral border of the somitic mesenchyme. The results show a sequence of phenotypic expression beginning with neurofilament antigen, then ganglionic aggregation, and finally, in the case of sympathetic neurons, catecholamine transmitter.     

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.     

Increase in the cholinergic cardiac plexus in sympathetically aneural chick hearts

Summary Embryonic chick hearts were made sympathetically aneural by removal of premigratory neural crest over somites 10–20. The cholinergic cardiac plexus was assessed at 12 to 15 days of incubation using morphological and biochemical techniques. The cholinergic innervation to the heart was increased by 50 to 100% due to both hypertrophy and hyperplasia of the ganglion cells as well as their terminals. The additional cells were an expansion of the normal population of cholinergic neurons in the heart rather than from some extra source. The expanded population of neurons did not express an adrenergic phenotype in response to the absence of adrenergic cardiac innervation.     

Developmental potentialities in the nonneuronal population of quail sensory ganglia

Sensory ganglia taken from quail embryos at E4 to E7 were back-transplanted into the vagal neural crest migration pathway (i.e., at the level of somites 1 to 6) of 8- to 10-somite stage chick embryos. Three types of sensory ganglia were used: (i) proximal ganglia of cranial sensory nerves IX and X forming the jugular-superior ganglionic complex, whose neurons and nonneuronal cells both arise from the neural crest; (ii) distal ganglia of the same nerves, i.e., the petrosal and nodose ganglia in which the neurons originate from epibranchial placodes and the nonneuronal cells from the neural crest; (iii) dorsal root ganglia taken in the truncal region between the fore- and hindlimb levels. The question raised was whether cells from the graft would be able to yield the neural crest derivatives normally arising from the hindbrain and vagal crest, such as carotid body type I and II cells, enteric ganglia, Schwann cells located along the local nerves, and the nonneuronal contingent of cells in the host nodose ganglion. All the grafted cephalic ganglia provided the host with the complete array of these cell types. In contrast, grafted dorsal root ganglion cells gave rise only to carotid body type I and II cells, to the nonneuronal cells of the nodose ganglion, and to Schwann cells; the ganglion-derived cells did not invade the gut and therefore failed to contribute to the host's enteric neuronal system. Coculture on the chorioallantoic membrane of aneural chick gut directly associated with quail sensory ganglia essentially reinforced these results. These data demonstrate that the capacity of peripheral ganglia to provide enteric plexuses varies according to the level of the neuraxis from which they originate.     

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.     

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.     

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.     

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

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

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)     

Pathogenesis of persistent truncus arteriosus induced by nimustine hydrochloride in chick embryos

Nimustine hydrochloride (ACNU) is a nitrosourea derivative anticancer agent which has been shown to cause persistent truncus arteriosus in chick embryos. The objective of this study was to confirm the teratogenic effects of ACNU on the cardiovascular system of chick embryos and to determine whether ACNU induces persistent truncus arteriosus by interfering with neural crest cells. Various doses of ACNU ranging from 10 to 200 micrograms were injected under the chorioallantoic membrane of chick embryos on the third day of incubation. Saline solution was used as the control. After 10 to 11 days of incubation, 242 (46%) survivors of the 524 treated eggs were obtained. The survival rates of the embryos and the frequencies of cardiovascular anomalies were dose dependent. Of 146 embryos with cardiovascular anomalies, 104 (71%) had persistent truncus arteriosus. Ventricular septal defect and double-outlet right ventricle were seen in 37 (25%) and one (1%), respectively. Aortic arch anomalies were seen in 116 embryos (79%). Quail-chick chimeras (chick embryos with quail cardiac neural crest) were treated with 50 micrograms of ACNU and examined histologically 24 hours later. These chimeras showed dying neural crest cells in the pharyngeal arches. Dying cells were also noted in the neural tube, cranial ganglia, retina, and otocyst. These results suggest that persistent truncus arteriosus in chick embryos treated with ACNU is induced by neural crest cell death.     

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.     

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.     

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     

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     

The rostral level of origin of sympathetic neurons in the chick embryo, studied in tissue culture.

Groups of three consecutive somites from the first to the eleventh somite from chick embryos of stages 17-18 were grown in tissue culture for seven days. Sympathetic neurons, identified both by phase contrast microscopy and FIF histochemistry, occurred only in cultures which included the sixth, or more caudal, somites. If it is assumed that sympathetic precursor cells (neural crest cells) have not undergone a caudal shift prior to stages 17-18, and taking into account the loss of one or two rostral somites, then the anterior sympathetic ganglia are derived from neural crest caudal to the sixth or seventh somite. Thus, the vagal zone (level with somites 1-7) contributes little to the sympathetic nervous system.     

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.     

Temporospatial patterns of apoptosis in chick embryos during the morphogenetic period of development

We examined the temporospatial pattern of naturally occurring apoptosis in chick embryos to five days of incubation (H.H. stages 1-25; Hamburger and Hamilton, 1951) using TUNEL labeling. The initial TUNEL-positive structure was the embryonic shield at stage 1. Apoptotic cells became ubiquitously present within embryos by stage 3, which is early in gastrulation. Until stage 6, TUNEL-positive cells were restricted to the headfold region. In embryos of stages 7-8, most cell death was localized at the most anterior neural plate. TUNEL-positive neural plate, notochord and somites appeared at stage 9. Otic and optic regions became TUNEL-positive at stage 11. The aggregation of cells from which the tail bud arises contains apoptotic cells from stage 11 onwards. At stage 16, scattered TUNEL-positive cells appeared in the branchial arches. Three streams of apoptotic neural crest cells in the cranial region became most clearly visible at stage 18. The secondary neural tube from which caudal structures develop contains apoptotic cells at stage 14. Apoptotic cells are present in the branchial arches and lateral body wall for extended periods, stages 16-25 and 25 respectively. At stages 24-25, intense positive regions of cell death were confined to the caudal regions of the arches, to limb and tail buds and to the lateral body wall, the latter in relation to body wall closure. The new findings in this study are discussed along with past studies to provide the temporospatial pattern of cell death during early chick development.     

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.     

Early appearance in neural crest and crest-derived cells of an antigenic determinant present in avian neurons

A monoclonal antibody (“$\text{E}\text{C8}$”) against chicken dorsal root ganglion cells has been produced. The epitope (antigenic determinant) to which this antibody binds appears in neuronal cells—of both the peripheral and central nervous systems—and in a limited number of nonneuronal cell types in avian embryos. The epitope is intracellular and is probably part of a protein as judged by its susceptibility to proteases. This epitope appears very early in neuronal development. It may be detected in brain, spinal cord, and ventral root nerve fibers of Hamburger-Hamilton stage 16 chicken embryos (51–56 hr of incubation). At this same age, $\text{E}\text{C8}\text{-}\text{immunoreactive}$ cells can be found in the neural crest migratory space between the neural tube and the somite about a day before dorsal root ganglia begin to coalesce. Since some cultured neural crest cells (but not somitic mesenchymal cells) also express this epitope, we propose that the $\text{E}\text{C8}$ monoclonal antibody identifies an early differentiating subpopulation of neural crest cells which express this putative neuronal trait soon after the time of cessation of migration in vivo.