CONGENITAL MEGACOLON: TISSUE INTERACTIONS IN DEVELOPMENT

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The lethal spotted mutant mouse (1S/1S) develops megacolon proximal to a segment of aganglionic terminal bowel. The aganglionic tissue appears to receive an innervation of axons from neurons whose cell bodies lie outside the gut. The cell bodies and processes of intrinsic enteric neurons, however, are excluded from the aganglionic region. We have previously used organotypic tissue cultures to reveal the distribution of neuronal precursors in the normal and 1S/1S fetal mouse gut. Neurons develop in culture if viable precursors are present in the gut at the time of explanation. These experiments have revealed that the terminal 2 mm of bowel from mutant mice at any age will never give rise to neuralized explants in culture, although the entire gut of normal mice will do so as early as day E9. we propose, as a hypothesis to account for the derivation of aganglionosis in 1S/1S animals, that the microenvironment of the terminal 2mm of bowel is abnormal in 1S/1S mice and does not permit the colonization of that region with enteric neuronal precursor cells. This hypothesis is to be tested and, if confirmed, we will attempt to identify the defect. The following questions will be answered by the proposed research. (1) Do neuronal precursors fail to enter the terminal 2 mm of 1S/1S bowel or do they enter this region and die? Neurofilament immunoreactivity will be used as a marker for enteric neuronal precursor cells. (2) Do precursors of enteric glial cells fail to enter (or survive in) the terminal 2 mm of 1S/1S bowel? Immunoreactivity of glial fibrillary acid protein will be used as a glial marker. (3) Is the structure of the terminal 2 mm of 1S/1S bowel abnormal? Are extracellular matrix proteins abnormal in the terminal 2 mm of 1S/1S bowel? Immunocytochemical examination of tissues will be done to determine the distribution of fibronectin, laminin, and types I and IV collagen. These experiments are designed to exploit the defect of the mutant mouse to gain insight into the role of tissue interactions in the development of derivatives of the neural crest.