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 f actor (GDNF) and its receptor(s), and endothelin-3 (ET-3) and its rece ptor, are crucial for enteric nervous system development. Mutations in terrupting either of these signaling pathways cause aganglionosis in t he 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 vag al level neural crest of E1.7 quail embryos prior to entry into the gu t and from the developing midgut at stages corresponding to migrating (E4.7) and longer resident differentiating cells (E7) using HNK-1 immu noaffinity and magnetic beads. These cells were tested for their respo nse to GDNF and ET-3 in culture. ET-3 and GDNF had little effect in vi tro 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 E 7 gut. Also, many more neurons and neurites developed in cultures trea ted with GDNF, disproportionately greater than the effect on cell numb ers. 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 cultur e condition, the stimulatory effect of GDNF on neuron numbers was stri kingly diminished by the simultaneous presence of ET-3. We propose fir st that GDNF promotes the proliferation in the migratory enteric neura l precursor cell population once the cells have entered the gut and is especially crucial for the differentiation of these cells into nonmig rating, 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 nu mbers to construct the entire enteric nervous system. Third, we sugges t that generalized defects in enteric neural precursor cell numbers an d differentiation due to mutations in the ET-3 and GDNF systems are co nverted to distal gut neural deficiencies by the rostrocaudal migratio n pattern of the precursors. Fourth, we suggest that additional factor s such as those found in serum and produced by the enteric neural cell s themselves are likely also to be involved in enteric nervous system development and consequently in Hirschsprung's disease. (C) 1998 Acade mic Press.