9506352 Shankland This study will yield novel insights into the formation, regional specification and segmentation of the endodermal germ layer in the glossiphoniid leech, Helobdella. The leech endoderm shows an overt pattern of segmentally periodic differentiation, and the mechanistic basis of this segmental patterning will be investigated through a combination of experimental embryology and molecular biology. The developmental origin of the definitive endodermal tissues will be characterized to ascertain whether particular cells have a stereotyped or variable lineage, the latter being suggestive that segmental organization is not intrinsic to the endoderm at the time of its formation. Another set of experiments will examine the development of the endoderm following either ablation or abnormal juxtaposition of the overlying mesoderm. These experiments will test the hypothesis that segmentation and regionalization are imprinted onto an initially unpatterned endodermal cell layer by local inductive cues from the adjacent mesoderm. A third set of experiments will perturb the segmental pattern of a particular homeobox gene within the endoderm, examining whether that gene is either necessary or sufficient to bring about the segmentation of the endodermal germ layer. The final set of experiments will investigate the derivation of the nuclei that take part in endoderm formation. The leech's endoderm arises from a midgut primordium which is formed by cell fusion early in embryogenesis, and which gives rise to a cellularized gut wall several days later. A nuclear lineage tracer will be used to follow the fate of individual pre- fusion nuclei, with the express purpose of testing whether some or all of these nuclei arise from ectodermal or mesodermal cell lineages. Should this prove tho be the case, it would suggest that the nuclei of ectodermal and mesodermal cells are respecified to an endodermal fate by cytoplasmic determinants encountered following their fusion i nto the midgut primordium. ***
