The gut requires the presence of intrinsic neurons in order for motility and absorption/secretion to occur. The intrinsic or enteric neurons develop from neural crest cells that leave the neural tube and enter the gut. Within the gut these crest cells migrate, aggregate, proliferate, and differentiate into neurons which form the circuitry that regulates gut motility. The long-term objective of this study is to elucidate the mechanisms which regulate the formation of the enteric nervous system. The placement of the neural crest cells along the gut is critical to the subsequent development of neurons and gut motility. Failure of crest cells to occupy regions of gut results in motility disorders. Congenital megacolon (Hirschsprung's disease) results from the absence of crest cells in the terminal bowel and pseudoobstruction may result from a marginal or inadequate number of crest cells in the midgut or hindgut. Crest cells enter the gut from two levels of the neuroaxis: the hindbrain (vagal neural crest) and the sacral neural tube (sacral neural crest). Vagal neural crest cells enter the foregut and migrate caudally to the hindgut, while the sacral crest cells enter the hindgut and move rostrally to the midgut. The disposition of the crest cells along the gut depends on a number of factors. The objectives of this proposal are to evaluate these factors systematically: where the crest cells come from, where they enter the gut, where they go in the gut, and what they form in the gut. Crest cells will be marked by injection of a replication-incompetent retrovirus which carries the Lac Z gene and will be detected histochemically. Injection of retrovirus in combination with immunostaining for neural markers will be used to establish the contribution of the sacral crest to the enteric nervous system in the hindgut. The contribution of the vagal crest to the hindgut will be determined by surgically ablating the sacral crest. To gain insight into the control of cell migration, the distribution of vagal crest cells along the gut will be mapped with respect to their origin in the neuroaxis. The effect of reducing the number of vagal crest entering the gut will be evaluated by ablating portions of the vagal crest, and determining the length of gut innervated. The role of the vagus nerve in the immigration of vagal crest will be determined by ablation of the vagus nerve. To learn about the process of migration, the motile behavior of living crest cells will be characterized as they move in situ through the gut. This will be done by labeling cells with the fluorescent lipophilic vital dye DiI, and following their movements with a fluorescent microscope and sensitive camera.
