GDNF and ET-3 differentially modulate the numbers of avian enteric neural crest cells and enteric neurons in vitro.

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.