GDNF and ET-3 Differentially Modulate the Numbers of Avian Enteric Neural Crest Cells and Enteric Neuronsin Vitro

Abstract

Vagal (hindbrain) neural crest cells migrate rostrocaudally in the gut to establish the enteric nervous system. Glial-derived neurotrophic factor (GDNF) and its receptor(s), and endothelin-3 (ET-3) and its receptor, are crucial for enteric nervous system development. Mutations interrupting either of these signaling pathways cause aganglionosis in the gut, termed Hirschsprung's disease in humans. However, the precise functions of GDNF and ET-3 in enteric neurogenesis are still unknown. We isolated precursor cells of the enteric nervous system from the vagal level neural crest of E1.7 quail embryos prior to entry into the gut and from the developing midgut at stages corresponding to migrating (E4.7) and longer resident differentiating cells (E7) using HNK-1 immunoaffinity and magnetic beads. These cells were tested for their response to GDNF and ET-3 in culture. ET-3 and GDNF had little effect in vitro on the growth, survival, migration, or neurogenesis of E1.7 vagal neural crest cells. In contrast, GDNF increased the proliferation rate and numbers of enteric neural precursors isolated from the E4.7 and E7 gut. Also, many more neurons and neurites developed in cultures treated with GDNF, disproportionately greater than the effect on cell numbers. At high cell density and in the presence of serum, ET-3, and GDNF had an additive effect on proliferation of neuron precursor cells. In defined medium, or low cell density, ET-3 reduced cell proliferation, overriding the proliferative effect of GDNF. Regardless of the culture condition, the stimulatory effect of GDNF on neuron numbers was strikingly diminished by the simultaneous presence of ET-3. We propose first that GDNF promotes the proliferation in the migratory enteric neural precursor cell population once the cells have entered the gut and is especially crucial for the differentiation of these cells into nonmigrating, nonproliferating enteric neurons. Second, we suggest that ET-3 modulates the action of GDNF, inhibiting neuronal differentiation to maintain the precursor cell pool, so ensuring sufficient population numbers to construct the entire enteric nervous system. Third, we suggest that generalized defects in enteric neural precursor cell numbers and differentiation due to mutations in the ET-3 and GDNF systems are converted to distal gut neural deficiencies by the rostrocaudal migration pattern of the precursors. Fourth, we suggest that additional factors such as those found in serum and produced by the enteric neural cells themselves are likely also to be involved in enteric nervous system development and consequently in Hirschsprung's disease.

Spatiotemporal regulation of endothelin receptor-B by SOX10 in neural crest–derived enteric neuron precursors

“…The amount of signaling mediated by the receptor tyrosine kinase RET, another locus associated with HSCR, is also crucial for the colonization of the hindgut by ENS precursors and seems to modulate EDNRB function [21][22][23] . and both pathways may regulate pluripotency or migration of the neural crest progenitors [24][25][26][27][28][29] . Future studies will determine if the individual SOX10 sites identified in this study are required for both tissue-specific and spatiotemporal regulation of the endogenous Ednrb.…”

The migratory behavior of immature enteric neurons

“…As endothelin-3 signaling inhibits neuronal differentiation in vitro (Hearn et al, 1998;Wu et al, 1999;Bondurand et al, 2006;Nagy and Goldstein, 2006), and as there is a delayed migration of ENCCs along the gut in mice lacking endothelin-3 (Barlow et al, 2003), it has been proposed that endothelin-3 prevents premature neuronal differentiation and facilitates the persistence of a precursor pool for sufficient time to colonize the entire gut (Hearn et al, 1998;Wu et al, 1999;Nagy and Goldstein, 2006). It is implicit in this argument that enteric neuronal differentiation directly inhibits migration, or indirectly inhibits migration by reducing proliferation; proliferation has recently been shown to be essential for migration (Simpson et al, 2007).…”

“…In mice lacking endothelin-3 or its receptor Ednrb, the migration of ENCCs along the gut is delayed (Cass et al, 1992;Kapur et al, 1992Kapur et al, , 1993Kapur et al, , 1995Barlow et al, 2003;Kruger et al, 2003). In vitro and in vivo studies have shown that endothelin-3 inhibits the differentiation of ENCCs (Hearn et al, 1998;Wu et al, 1999;Bondurand et al, 2006;Nagy and Goldstein, 2006). However, the mechanisms by which differentiation might affect ENCC migration rate have yet to be established.…”

“…If neuronal differentiation retards the migration of ENCCs directly, endothelin-3 might promote ENCC migration by inhibiting differentiation. As proliferation is essential for ENCC migration (Simpson et al, 2007), endothelin-3 might promote ENCC migration indirectly by increasing the rate of proliferation (Hearn et al, 1998;Bondurand et al, 2006;Nagy and Goldstein, 2006). It is therefore of interest to determine whether immature neurons migrate, and if so, whether they migrate at the same speed as undifferentiated ENCCs.…”

Neural Precursor Death Is Central to the Pathogenesis of Intestinal Aganglionosis inRetHypomorphic Mice

“…RET signaling regulates multiple aspects of the development of the enteric nervous system (ENS), including cell proliferation (Chalazonitis et al, 1998;Hearn et al, 1998;Taraviras et al, 1999;Gianino et al, 2003), migration (Young et al, 2001;Natarajan et al, 2002), differentiation (Taraviras et al, 1999), and survival (Taraviras et al, 1999). Mice homozygous for Ret deletion show total intestinal aganglionosis (Schuchardt et al, 1994) caused by impaired migration and apoptosis of immature enteric neural crest-derived cell (ENCCs).…”