CONGENITAL MEGACOLON: TISSUE INTERACTIONS IN DEVELOPMENT

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The enteric nervous system is formed by precursors that migrate to the bowel from the neural crest. In order to determine why crest-derived cells stop migrating and begin to differentiate in the gut, we have studied a mutant mouse, the lethal spotted (ls/ls), in which the terminal bowel is congenitally aganglionic. We have shown that the defect in these animals arises, not because their crest-derived cells are abnormal, but because neither these, nor any other source of crest-derived cells, is able to colonize the presumptive aganglionic ls/ls gut. Within this tissue, molecular components of basal laminae, including laminin, are overabundant and/or maldistributed. We have proposed that crest-derived cells, when they enter the bowel, acquire a nerve-related 110 kDa laminin receptor which, when activated, induces these cells to withdraw from the cell cycle, extend processes and cease migrating. The 110 kDa laminin binding site is not found on pre- or early migrating crest cells. We thus wish to test the hypothesis that the ls/ls defect is due to the abnormal extracellular matrix (ECM) of the presumptive aganglionic zone. We propose that migrating crest-derived cells stop, cease to proliferate, and extend processes prematurely when they encounter this ECM. As a result, the distal bowel does not become colonized by crest-derived precursors of neurons and glia, and there is no accumulation of neurons at the periphery of the abnormal zone. We will now test this hypothesis by using a purified population of crest-derived cells isolated from within the gut by magnetic immunoselection (employing antibodies to cell surface molecules expressed in the gut only by crest-derived cells). The effects of laminin and a variety of other defined substrates on the behavior of these cells in vitro will be ascertained. Parameters to be studied will include adhesion, migration, proliferation, neurite extension, and phenotypic expression. In addition to observation, quantitative measurements will be made of neural and glial expression, using immunocytochemical markers. Immunoselected enteric crest-derived cells will also be cultured on cyostat sections of control and presumptive aganglionic ls/ls bowel, in order to determine whether laminin or other molecules mimic the action of the abnormal ECM of the ls/ls gut on each the measured parameters of cell behavior. The effects on these parameters of synthetic peptides, which encompass neurite-promoting domains of laminin, antibodies to these domains, and antibodies to the 110 kDa protein will then be tested on immunoselected crest-derived cells cultured on laminin or cryostat sections of presumptive aganglionic ls/ls and control gut. Finally, the behavior of crest-derived cells and the effects of potential laminin antagonists will be analyzed by direct visualization using confocal fluorescence microscopy to observe the cells as they migrate in vitro within explants of fetal bowel from ls/ls and control mice.