Development of c‐kit immunopositive interstitial cells of Cajal in the human stomach

Interstitial cells of Cajal (ICC) include several types of specialized cells within the musculature of the gastrointestinal tract (GIT). Some types of ICC act as pacemakers in the GIT musculature, whereas others are implicated in the modulation of enteric neurotransmission. Kit immunohistochemistry reliably identifies the location of these cells and provides information on changes in ICC distribution and density. Human stomach specimens were obtained from 7 embryos and 28 foetuses without gastrointestinal disorders. The specimens were 7–27 weeks of gestational age, and both sexes are represented in the sample. The specimens were exposed to anti‐c‐kit antibodies to investigate ICC differentiation. Enteric plexuses were immunohistochemically examined by using anti‐neuron specific enolase and the differentiation of smooth muscle cells (SMC) was studied with anti‐α smooth muscle actin and anti‐desmin antibodies. By week 7, c‐kit‐immunopositive precursors formed a layer in the outer stomach wall around myenteric plexus elements. Between 9 and 11 weeks some of these precursors differentiated into ICC. ICC at the myenteric plexus level differentiated first, followed by those within the muscle layer: between SMC, at the circular and longitudinal layers, and within connective tissue septa enveloping muscle bundles. In the fourth month, all subtypes of c‐kit‐immunoreactivity ICC which are necessary for the generation of slow waves and their transfer to SMC have been developed. These results may help elucidate the origin of ICC and the aetiology and pathogenesis of stomach motility disorders in neonates and young children that are associated with absence or decreased number of these cells.     

Development of the enteric nervous system,smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract

The generation of functional neuromuscular activity within the pre-natal gastrointestinal tract requires the coordinated development of enteric neurons and glial cells, concentric layers of smooth muscle and interstitial cells of Cajal (ICC). We investigated the genesis of these different cell types in human embryonic and fetal gut material ranging from weeks 4–14. Neural crest cells (NCC), labelled with antibodies against the neurotrophin receptor p75^NTR, entered the foregut at week 4, and migrated rostrocaudally to reach the terminal hindgut by week 7. Initially, these cells were loosely distributed throughout the gut mesenchyme but later coalesced to form ganglia along a rostrocaudal gradient of maturation; the myenteric plexus developed primarily in the foregut, then in the midgut, and finally in the hindgut. The submucosal plexus formed approximately 2–3 weeks after the myenteric plexus, arising from cells that migrated centripetally through the circular muscle layer from the myenteric region. Smooth muscle differentiation, as evidenced by the expression of -smooth muscle actin, followed NCC colonization of the gut within a few weeks. Gut smooth muscle also matured in a rostrocaudal direction, with a large band of -smooth muscle actin being present in the oesophagus at week 8 and in the hindgut by week 11. Circular muscle developed prior to longitudinal muscle in the intestine and colon. ICC emerged from the developing gut mesenchyme at week 9 to surround and closely appose the myenteric ganglia by week 11. By week 14, the intestine was invested with neural cells, longitudinal, circular and muscularis mucosae muscle layers, and an ICC network, giving the fetal gut a mature appearance.A.S.W. is funded by a PhD studentship awarded to A.J.B. by the Child Health Research Appeal Trust.     

Pivotal role of the interstitial cells of Cajal in the nitric oxide signaling pathway of rat small intestine. Morphological evidence

The nitric oxide (NO) signaling pathway is a major nonadrenergic-noncholinergic transmitter mechanism in the enteric nervous system. Our aim was to localize the enzymes in question, i.e., neuronal nitric oxide synthase (nNOS), soluble guanylate cyclase (sGC), and cGMP-dependent kinase type I (cGK-I) in rat small intestine by indirect immunofluorescence. nNOS staining was found in neurons of the myenteric plexus and in varicose nerve fibers mainly in the circular muscle layer. The cells positive for neurokinin-1 (NK-1) receptor and c-kit (interstitial cells of Cajal, ICC) in the deep muscular plexus (DMP) did not show nNOS reactivity, but nNOS-positive nerve fibers were directly adjacent to them. sGC was found in flattened cells surrounding myenteric ganglia (periganglionic cells, PGC), in ICC of the DMP, faintly in smooth muscle cells (SMC), and in cells perivascularly scattered throughout the circular muscle layer. cGK-I immunoreactivity was found abundantly in PGC (which presumably are ICC), in ICC of DMP, in SMC of the innermost circular and longitudinal muscle layers, but less intensively in the outer circular layer. Weak cGK-I staining occurred in nerve cells within the myenteric and submucosal plexus. Conclusively the key enzymes of the NO signaling pathway are differentially distributed: Occurrence of nNOS exclusively in neurons and the presence of sGC and cGK-I predominantly in ICC suggest a sequence of neuronal NO release, activation of ICC, and consecutive smooth muscle relaxation. ICC of the DMP seem to be the primary targets for neurally released NO.     

Three-dimensional demonstration of the interstitial cells of Cajal associated with the submucosal plexus in guinea-pig caecum

The guinea-pig caecum was studied by using immunohistochemistry for Kit receptors and nerves to clarify whether interstitial cells of Cajal (ICC) were localized in association with the submucosal plexus (ICC-SP). A large area of the guinea-pig caecum was nearly devoid of longitudinal muscles, because they were concentrated into three bundles of the taenia caeci (coli) and this allowed clear observation of the myenteric and submucosal plexus as separate networks in whole-mount stretch preparations. The myenteric plexus was observed as a loose polygonal network consisting in elongated ganglia and long connecting nerve strands, whereas the submucosal plexus was identified as smaller ovoid ganglia connected to much thinner nerve strands in different tissue layers. Three-dimensional reconstruction of confocal images revealed multipolar-shaped ICC-SP located around the submucosal ganglion in a basket formation. Bipolar ICC-SP were also observed along the connecting nerve strands of the submucosal plexus. The functional involvement of ICC-SP in mucosal activity is discussed in relation to fluid transportation. This three-dimensional study of ICC-SP thus provides a candidate for the most suitable material available for functional experiments examining the physiological significance of ICC-SP.     

Appearance of interstitial cells of Cajal in the human midgut

Several subtypes of the interstitial cells of Cajal (ICC) form networks that play a role in gastrointestinal motor control. ICC express c-kit and depend on signaling via Kit receptors for development and phenotype maintenance. At 7-8 weeks of development, c-kit-immunoreactive (c-kit-IR) cells are present in the human oesophagus, stomach and proximal duodenum wall. In the remaining small and large bowel, c-kit-IR cells appear later. The object of the present study is to determine the timing of the appearance of c-kit-IR ICC in the parts of the digestive tube originating from the midgut (distal duodenum, jejunum, ileum and proximal colon). Specimens were obtained from eight human embryos and 11 fetuses at 7-12 weeks of gestational age. The specimens were exposed to anti-c-kit antibodies to investigate ICC differentiation. The differentiation of enteric neurons and smooth muscle cells was immunohistochemically examined by using anti-PGP9,5 and anti-desmin antibodies, respectively. In the distal duodenum, jejunum and ileum, c-kit-IR cells emerged at week 9 at the level of the myenteric plexus in the form of a thin row of cells encircling the inception of the ganglia. These cells were multipolar or spindle-shaped with two long processes and corresponded to the ICC of the myenteric plexus. In the proximal colon, c-kit-IR cells emerged at week 9-10 in the form of two parallel belts of cells extending at the submucosal plexus and the myenteric plexus levels. We conclude that ICC develop following two different patterns in the human midgut.     

In ovo transplantation of enteric nervous system precursors from vagal to sacral neural crest results in extensive hindgut colonisation

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC). Within the embryonic avian gut, vagal NCC migrate in a rostrocaudal direction to form the majority of neurons and glia along the entire length of the gastrointestinal tract, whereas sacral NCC migrate in an opposing caudorostral direction, initially forming the nerve of Remak, and contribute a smaller number of ENS cells primarily to the distal hindgut. In this study, we have investigated the ability of vagal NCC, transplanted to the sacral region of the neuraxis, to colonise the chick hindgut and form the ENS in an experimentally generated hypoganglionic hindgut in ovo model. Results showed that when the vagal NC was transplanted into the sacral region of the neuraxis, vagal-derived ENS precursors immediately migrated away from the neural tube along characteristic pathways, with numerous cells colonising the gut mesenchyme by embryonic day (E) 4. By E7, the colorectum was extensively colonised by transplanted vagal NCC and the migration front had advanced caudorostrally to the level of the umbilicus. By E10, the stage at which sacral NCC begin to colonise the hindgut in large numbers, myenteric and submucosal plexuses in the hindgut almost entirely composed of transplanted vagal NCC, while the migration front had progressed into the pre-umbilical intestine, midway between the stomach and umbilicus. Immunohistochemical staining with the pan-neuronal marker, ANNA-1, revealed that the transplanted vagal NCC differentiated into enteric neurons, and whole-mount staining with NADPH-diaphorase showed that myenteric and submucosal ganglia formed interconnecting plexuses, similar to control animals. Furthermore, using an anti-RET antibody, widespread immunostaining was observed throughout the ENS, within a subpopulation of sacral NC-derived ENS precursors, and in the majority of transplanted vagal-to-sacral NCC. Our results demonstrate that: (1) a cell autonomous difference exists between the migration/signalling mechanisms used by sacral and vagal NCC, as transplanted vagal cells migrated along pathways normally followed by sacral cells, but did so in much larger numbers, earlier in development; (2) vagal NCC transplanted into the sacral neuraxis extensively colonised the hindgut, migrated in a caudorostral direction, differentiated into neuronal phenotypes, and formed enteric plexuses; (3) RET immunostaining occurred in vagal crest-derived ENS cells, the nerve of Remak and a subpopulation of sacral NCC within hindgut enteric ganglia.     

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     

Development of the submucous plexus in the large intestine of the mouse

In the small intestine of both embryonic birds and mammals, neuron precursors aggregrate first at the site of the myenteric plexus, and the submucous plexus develops later. However, in the large intestine of birds, the submucosal region is colonised by neural-crest-derived cells before the myenteric region (Burns and Le Douarin, Development 125:4335-4347, 1998). Using antisera that recognize undifferentiated neural-crest-derived cells (p75NTR) and differentiated neurons (PGP9.5), we examined the colonisation of the murine large intestine by neural-crest-derived cells and the development of the myenteric and submucosal plexuses. At E12.5, when the neural crest cells were migrating through and colonising the hindgut, the hindgut mesenchyme was largely undifferentiated, and a circular muscle layer could not be discerned. Neural-crest-derived cells migrated through, and settled in, the outer half of the mesenchyme. By E14.5, neural-crest-derived cells had colonised the entire hindgut; at this stage the circular muscle layer had started to differentiate. From E14.5 to E16.5, p75NTR- and PGP9.5-positive cells were observed on the serosal side of the circular muscle, in the myenteric region, but not in the submucosal region. Scattered, single neurons were first observed in the submucosal region around E18.5, and groups of neurons forming ganglia were not observed until after birth. The development of the enteric plexuses in the murine large intestine therefore differs from that in the avian large intestine.     

Divergent fate and origin of neurosphere-like bodies from different layers of the gut

Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.     

RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro

The enteric nervous system (ENS) in vertebrate embryos is formed by neural crest-derived cells. During development, these cells undergo extensive migration from the vagal and sacral regions to colonize the entire gut, where they differentiate into neurons and glial cells. Guidance molecules like netrins, semaphorins, slits, and ephrins are known to be involved in neuronal migration and axon guidance. In the CNS, the repulsive guidance molecule (RGMa) has been implicated in neuronal differentiation, migration, and apoptosis. Recently, we described the expression of the subtypes RGMa and RGMb and their receptor neogenin during murine gut development. In the present study, we investigated the influence of RGMa on neurosphere cultures derived from fetal ENS. In functional in vitro assays, RGMa strongly inhibited neurite outgrowth of differentiating progenitors via the receptor neogenin. The repulsive effect of RGMa on processes of differentiated enteric neural progenitors could be demonstrated by collapse assay. The influence of the RGM receptor on ENS was also analyzed in neogenin knockout mice. In the adult large intestine of mutants we observed disturbed ganglia formation in the myenteric plexus. Our data indicate that RGMa may be involved in differentiation processes of enteric neurons in the murine gut.     

Fate determination of neural crest cells by NOTCH-mediated lateral inhibition and asymmetrical cell division during gangliogenesis

Avian trunk neural crest cells give rise to a variety of cell types including neurons and satellite glial cells in peripheral ganglia. It is widely assumed that crest cell fate is regulated by environmental cues from surrounding embryonic tissues. However, it is not clear how such environmental cues could cause both neurons and glial cells to differentiate from crest-derived precursors in the same ganglionic locations. To elucidate this issue, we have examined expression and function of components of the NOTCH signaling pathway in early crest cells and in avian dorsal root ganglia. We have found that Delta1, which encodes a NOTCH ligand, is expressed in early crest-derived neuronal cells, and that NOTCH1 activation in crest cells prevents neuronal differentiation and permits glial differentiation in vitro. We also found that NUMB, a NOTCH antagonist, is asymmetrically segregated when some undifferentiated crest-derived cells in nascent dorsal root ganglia undergo mitosis. We conclude that neuron-glia fate determination of crest cells is regulated, at least in part, by NOTCH-mediated lateral inhibition among crest-derived cells, and by asymmetric cell division.     

Morphology of the interstitial cells of Cajal of the human ileum from foetal to neonatal life

The so-called interstitial cells of Cajal myenteric plexus (ICC-MP), interstitial cells of Cajal intramuscular (ICC-IM) and interstitial cells of Cajal deep muscular plexus (ICC-DMP) are the three types of ICC endowed within the intestinal muscle coat where they play different roles in gut motility. Studies on ICC ontogenesis showed ICC-MP in the human ileum by 7-9 weeks while information on ICC-IM and ICC-DMP in foetuses and newborns are not exhaustive. Functional recordings in the fasting state of prematurely born babies aged 28-37 weeks showed immature ileal motility. To gain more information on the time of appearance of the three ICC types in the human ileum and on the steps of the acquisition of mature features, we studied by c-kit immuno-histochemistry foetuses aged 17-27 weeks and newborns aged 36-41 weeks. In parallel, the maturative steps of enteric plexuses and muscle layers were immunohistochemically examined by using anti-neuron specific enolase (NSE), anti-S-100 and anti-alpha smooth muscle actin (alphaSMA) antibodies. The appearance and differentiation of all the ICC types were seen to occur in concomitance with those of the related nerve plexuses and muscle layers. ICC-MP appeared first, ICC-IM and ICC-DMP later and their differentiation was incomplete at birth. In conclusion, the ICC-MP, the intestinal pacemaker cells, in spite of absence of food intake, are already present during the foetal life and the ICC-IM appear by pre-term life, thus ensuring neurotransmission. The ICC-DMP and their related nerve plexus and smooth muscle cells, i.e. the intestinal stretch receptor, begin to differentiate at birth. These findings might help in predicting neonatal ileal motor behaviour and in interpreting the role of ICC abnormalities in the pathophysiology of intestinal motile disorders of neonates and young children.     

In vivo transplantation of mammalian neural crest cells into chick hosts reveals a new autonomic sublineage restriction.

The study of mammalian neural crest development has been limited by the lack of an accessible system for in vivo transplantation of these cells. We have developed a novel transplantation system to study lineage restriction in the rodent neural crest. Migratory rat neural crest cells (NCCs), transplanted into chicken embryos, can differentiate into sensory, sympathetic, and parasympathetic neurons, as shown by the expression of neuronal subtype-specific and pan-neuronal markers, as well as into Schwann cells and satellite glia. In contrast, an immunopurified population of enteric neural precursors (ENPs) from the fetal gut can also generate neurons in all of these ganglia, but only expresses appropriate neuronal subtype markers in Remak's and associated pelvic parasympathetic ganglia. ENPs also appear restricted in the kinds of glia they can generate in comparison to NCCs. Thus ENPs have parasympathetic and presumably enteric capacities, but not sympathetic or sensory capacities. These results identify a new autonomic lineage restriction in the neural crest, and suggest that this restriction preceeds the choice between neuronal and glial fates.     

Fibronectin Combined with Stem Cell Factor Plays an Important Role in Melanocyte Proliferation,Differentiation and Migration in Cultured Mouse Neural Crest Cells

Stem cell factor (SCF) is essential to the migration and differentiation of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, KIT, result in defects in coat pigmentation in mice. Using a neural crest cell (NCC) primary culture system from wild‐type mice, we previously demonstrated that KIT‐positive and/or L ‐3, 4‐dihydroxyphenylalanine (DOPA)‐positive melanocyte precursors proliferate following the addition of SCF to the culture medium. Extracellular matrix (ECM) proteins are considered to play a role in the migration and differentiation of various cells including melanocytes. We cultured mouse NCCs in the presence of SCF in individual wells coated with ECM; fibronectin (FN), collagen I (CLI), chondroitin sulphate, or dermatan sulphate. More KIT‐positive cells and DOPA‐positive cells were detected in the presence of SCF on ECM‐coated wells than on non‐coated wells. A statistically significant increase in DOPA‐positive cells was evident in FN and CLI wells. In contrast, in the absence of SCF, few DOPA‐positive cells and KIT‐positive cells were detected on either the ECM‐coated or non‐coated wells. We concluded that ECM affect melanocyte proliferation and development in the presence of SCF. To determine the key site of FN function, RGDS peptides in the FN sequence, which supports spreading of NCCs, were added to the NCC culture. The number of DOPA‐positive cells decreased with RGDS concentration in a dose‐dependent fashion. Immunohistochemical staining revealed the presence of integrin a5, a receptor of RGDS, in NCCs. These results suggest the RGDS domain of FN plays a contributory role as an active site in the induction of FN function in NCCs. In addition, we examined the effect of FN with SCF on the NCC migration by measuring cluster size, and found an increase in size following treatment with FN.     

Scanning electron microscopy of the muscle coat of the guinea-pig small intestine

Summary We have studied the layers of the muscular coat of the guinea-pig small intestine after enzymatic and chemical removal of extracellular connective tissue. The cells of the longitudinal muscle layer are wider, have rougher surfaces, more finger-like processes and more complex terminations, but fewer intercellular junctions than cells in the circular muscle layer. A special layer of wide, flat cells with a dense innervation exists at the inner margin of the circular muscle layer, facing the submucosa. The ganglia of the myenteric and submucosal plexuses are covered by a smooth basal lamina, a delicate feltwork of collagen fibrils, and innumerable connective tissue cells. The neuronal and glial cell processes at the surface of ganglia form an interlocking mosaic, which is loosely packed in newborn and young animals, but becomes tightly packed in adults. The arrangement of glial cells becomes progressively looser along finer nerve bundles. Single varicose nerve fibres are rarely exposed, but multiaxonal bundles are common. Fibroblast-like cells of characteristic shape and orientation are found in the serosa; around nerve ganglia; in the intermuscular connective tissue layer and in the circular muscle, where they bridge nerve bundles and muscle cells; at the submucosal face of the special, flattened inner circular muscle layer; and in the submucosa. Some of these fibroblast like cells correspond to interstitial cells of Cajal. Other structures readily visualized by scanning electron microscopy are blood and lymphatic vessels and their periendothelial cells. The relationship of cellular elements to connective tissue was studied with three different preparative procedures: (1) freeze-cracked specimens of intact, undigested intestine; (2) stretch preparations of longitudinal muscle with adhering myenteric plexus; (3) sheets of submucosal collagen bundles from which all cellular elements had been removed by prolonged detergent extraction.     

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.     

Distribution and morphological characteristics of the interstitial cells of Cajal in the ileocaecal junction of the guinea-pig

The guinea-pig ileocaecal junction including the valve was studied by immunohistochemistry to clarify the organization of the muscle bundles, the enteric nerves and the interstitial cells of Cajal (ICC). This region clearly exhibited characteristic features in the distribution patterns of ICC in a proximal to distal direction: (1) the thickened portion of the terminal ileum immediately adjacent to the ileocecal junction contained many ICC throughout the circular (ICC-CM) and longitudinal (ICC-LM) muscle layers, but ICC were few or absent in the rest of the ileum; (2) the ileal side of the valve contained ICC associated with the deep muscular plexus (ICC-DMP) as in the small intestine, whereas ICC-DMP were absent in the caecal side as in the caecum; (3) the valve contained many ICC-CM and ICC-LM in both the ileal and caecal sides; (4) many ICC associated with the myenteric plexus were observed in both the ileal and caecal sides of the valve, whereas they were only sparsely found in the caecum; (5) ICC were also observed around the submucosal plexus in a confined area of the terminal ileum and the ileocaecal valve. These observations provide morphological evidence that the terminal ileum and ileocaecal valve are specially equipped for their active involvement in the movement of the junctional area.     

Origins of the insect enteric nervous system: differentiation of the enteric ganglia from a neurogenic epithelium.

The enteric nervous system (ENS) of the moth Manduca sexta is organized into two distinct cellular domains: an anterior domain that includes several small ganglia on the surface of the foregut, and a more posterior domain consisting of a branching nerve plexus (the enteric plexus) that spans the foregut-midgut boundary. Previously, we showed that the neurons of the posterior domain, the enteric plexus, are generated from a large placode that invaginates from the caudal lip of the foregut; subsequently, the cells become distributed throughout the enteric plexus by a sequence of active migration. We now demonstrate that the neurons of the anterior domain, the cells of the enteric ganglia, arise via a distinct developmental sequence. Shortly after the foregut has begun to form, three neurogenic zones differentiate within the foregut epithelium and give rise to chains of cells that emerge onto the foregut surface. The three zones are not sites of active mitosis, as indicated by the absence of labelling with a thymidine analogue and by clonal analyses using intracellularly injected dyes. Rather, the zones serve as loci through which epithelial cells are recruited into a sequence of delamination and neuronal differentiation. As they emerge from the epithelium, the cells briefly become mitotically active, each cell dividing once or twice. In this manner, they resemble the midline precursor class of neural progenitors in the insect central nervous system more than neuroblast stem cells. The progeny of these zone-derived precursors then gradually coalesce into the ganglia and nerves of the anterior ENS. Although this reorganization results in some variability in the precise configuration of neurons within the ganglia, the overall morphology of the ganglia is highly stereotyped, consisting of cortical layers of cells that surround a ventral neuropil. In addition, a number of the neurons within the frontal and hypocerebral ganglia express identifiable phenotypes in a manner that is similar to many cells of the insect central nervous system. These observations indicate that the differentiation of the enteric ganglia in Manduca involves an unusual combination of features seen during the formation of other regions of the nervous system and, as such, constitutes a distinct program of neurogenesis.