Enteric neurons and glia arise from the neural crest. The phenotype of crest-derived cells was examined as they differentiated into neurons or glia in the mouse small and large intestine. Previous studies have shown that undifferentiated enteric crest-derived cells are Phox2b(+)/Ret(+)/p75(+)/Sox10(+), and at embryonic day (E) 10.5, about 10-15% of the crest-derived cells in the small intestine have started to differentiate into neurons. In the current study, by E12.5 and E14.5, about 25% and 47%, respectively, of Phox2b(+) cells in the small intestine were immunoreactive to the pan-neuronal protein, ubitquitin hydrolase (PGP9.5), and the percentage did not change dramatically from E14.5 onward. The differentiation of crest-derived cells into neurons in the colon lagged behind that in the small intestine by several days. Differentiating enteric neurons showed high Ret, low p75, and undetectable Sox10 immunostaining. Glial precursors were identified by the presence of brain-specific fatty acid binding protein (B-FABP) and detected first in the fore- and rostral midgut at E11.5. Glial precursors appeared to be B-FABP(+)/Sox10(+)/p75(+) but showed low Ret immunostaining. S100b was not detected until E14.5. Adult glial cells were B-FABP(+)/Sox10(+)/p75(+)/S100b(+). A nucleic acid stain (to identify all ganglion cells) was combined with immunostaining for PGP9.5 and S100b to detect neurons and glial cells, respectively, in the postnatal intestine. At postnatal day 0, fewer than 5% and 10% of cells in myenteric ganglia of the small and large intestine, respectively, were neither PGP9.5(+) nor S100b(+). Because some classes of neurons are not present in significant numbers until after birth, the expression of PGP9.5 by developing enteric neurons appeared to precede the expression of neuron type-specific markers.

Neural crest-derived cells that form the enteric nervous system undergo an extensive migration from the caudal hindbrain to colonize the entire gastrointestinal tract. Mice in which the expression of GFP is under the control of the Ret promoter were used to visualize neural crest-derived cell migration in the embryonic mouse gut in organ culture. Time-lapse imaging revealed that GFP(+) crest-derived cells formed chains that displayed complicated patterns of migration, with sudden and frequent changes in migratory speed and trajectories. Some of the leading cells and their processes formed a scaffold along which later cells migrated. To examine the effect of population size on migratory behavior, a small number of the most caudal GFP(+) cells were isolated from the remainder of the population. The isolated cells migrated slower than cells in large control populations, suggesting that migratory behavior is influenced by cell number and cell-cell contact. Previous studies have shown that neurons differentiate among the migrating cell population, but it is unclear whether they migrate. The phenotype of migrating cells was examined. Migrating cells expressed the neural crest cell marker, Sox10, but not neuronal markers, indicating that the majority of migratory cells observed did not have a neuronal phenotype.

Cell therapy has the potential to treat gastrointestinal motility disorders caused by diseases of the enteric nervous system. Many studies have demonstrated that various stem/progenitor cells can give rise to functional neurons in the embryonic gut; however, it is not yet known whether transplanted neural progenitor cells can migrate, proliferate, and generate functional neurons in the postnatal bowel in vivo. We transplanted neurospheres generated from fetal and postnatal intestinal neural crest-derived cells into the colon of postnatal mice. The neurosphere-derived cells migrated, proliferated, and generated neurons and glial cells that formed ganglion-like clusters within the recipient colon. Graft-derived neurons exhibited morphological, neurochemical, and electrophysiological characteristics similar to those of enteric neurons; they received synaptic inputs; and their neurites projected to muscle layers and the enteric ganglia of the recipient mice. These findings show that transplanted enteric neural progenitor cells can generate functional enteric neurons in the postnatal bowel and advances the notion that cell therapy is a promising strategy for enteric neuropathies.

Mutations in genes encoding members of the GDNF and endothelin-3 (Et-3) signaling pathways can cause Hirschsprung's disease, a congenital condition associated with an absence of enteric neurons in the distal gut. GDNF signals through Ret, a receptor tyrosine kinase, and Et-3 signals through endothelin receptor B (Ednrb). The effects of Gdnf, Ret, and ET-3 haploinsufficiency and a null mutation in ET-3 on spontaneous motility patterns in adult and developing mice were investigated. Video recordings were used to construct spatiotemporal maps of spontaneous contractile patterns in colon from postnatal and adult mice in vitro. In Ret(+/-) and ET-3(+/-) mice, which have normal numbers of enteric neurons, colonic migrating motor complexes (CMMCs) displayed similar properties under control conditions and following inhibition of nitric oxide synthase (NOS) activity to wild-type mice. In the colon of Gdnf(+/-) mice and in the ganglionic region of ET-3(-/-) mice, there was a 50-60% reduction in myenteric neuron number. In Gdnf(+/-) mice, CMMCs were present, but abnormal, and the proportion of myenteric neurons containing NOS was not different from that of wild-type mice. In the ganglionic region of postnatal ET-3(-/-) mice, CMMCs were absent, and the proportion of myenteric neurons containing NOS was over 100% higher than in wild-type mice. Thus impairments in spontaneous motility patterns in the colon of Gdnf(+/-) mice and in the ganglionic region of ET-3(-/-) mice are correlated with a reduction in myenteric neuron density.

In mature animals, neurons and interstitial cells of Cajal (ICC) are essential for organized intestinal motility. We investigated motility patterns, and the roles of neurons and myenteric ICC (ICC-MP), in the duodenum and colon of developing mice in vitro. Spatiotemporal mapping revealed regular contractions that propagated in both directions from embryonic day (E)13.5 in the duodenum and E14.5 in the colon. The propagating contractions, which we termed ripples, were unaffected by tetrodotoxin and were present in the intestine of embryonic Ret null mutant mice, which lack enteric neurons. Neurally mediated motility patterns were first observed in the duodenum at E18.5. To examine the possible role of ICC-MP, three approaches were used. First, intracellular recordings from the circular muscle of the duodenum did not detect slow wave activity at E16.5, but regular slow waves were observed in some preparations of E18.5 duodenum. Second, spatiotemporal mapping revealed ripples in the duodenum of E13.5 and E16.5 W /W v embryos, which lack KIT+ ICC-MP and slow waves. Third, KIT-immunoreactive cells with the morphology of ICC-MP were first observed at E18.5. Hence, ripples do not appear to be mediated by ICC-MP and must be myogenic. Ripples in the duodenum and colon were abolished by cobalt chloride (1 mm). The L-type Ca