Hirschsprung's disease (congenital megacolon), one of several congenital neuromuscular disorders of gastrointestinal development, is a common and serious birth defect in children. It is proposed that congenital disorders, such as Hirschsprung's disease, arise because precursors of enteric neurons and glia migrating from the neural crest fail to reach their final destinations within the bowel. Congenital megacolon, proximal to an aganglionic terminal segment of gut, similar to that seen in Hirschsprung's disease, appears in the lethal spotted (ls/ls) mutant mouse. It was shown during the previous project period that the terminal intestine becomes aganglionic in ls/ls mice because non-neuronal cells in the presumptive aganglionic region of the fetal gut, rather than the crest-derived cells themselves, are abnormal. Accumulation of molecular components of basal laminae (including collagen type IV and laminin), distal to the front of migrating crest cells, was demonstrated to occur in the abnormal bowel. In contrast to normal gut, this tissue cannot be colonized by control or mutant sources of crest cells. The hypothesis that the accumulation of components of basal laminae is causally related to the development of aganglionosis will now be tested. Initial studies will be done to determine whether there is an increase in pro alpha 1 (or alpha 2) type IV collagen and B1 chain (or other chains) of laminin mRNAs in the aganglionic tissue. This will be accomplished using Northern hybridization, RNase protection assays, and in situ hybridization (which may also reveal which cell type is defective) with appropriate radiolabeled probes. The recent observation that segments of presumptive aganglionic ls/ls gut, backgrafted to crest cell migration pathways of younger quail embryos, block migration of host crest cells, will be analyzed further to determine whether the effect of the abnormal bowel is associated with the deposition of components of basal laminae. These experiments will combine embryonic surgery with immunocytochemical and electron microscopic analyses. In addition, the capacity of substrates produced and/or conditioned by mesenchyme from the aganglionic bowel of ls/ls mice to interfere with the migration of enteric crest-derived cells in vitro will be evaluated. Antibodies to components of basal laminae will then be applied to try to block effects of ls/ls-conditioned matrices. Finally, laminin receptors (those promoting adhesion and the newly observed 110 kDa neural laminin receptor) on the surface of crest-derived cells in the bowel will be analyzed and compared with those on the surfaces of pre-migratory and early migratory crest cells. As part of these analyses, the ability of antibodies to these receptors, or specific peptides that compete with laminin for binding to the receptors, to affect the migration of enteric crest-derived cells will be evaluated.
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