The objective of this proposal is to determine the function of insulin-like growth factor 11 (IGF-II) in the developing and mature nervous system and in developing muscle. The function of IGF-II is not known, but there are intriguing suggestions that it may play an important role in normal development of brain and muscle. Aberrant expression of IGF-II has been associated with some neuropathologic states, including megalencephaly. To evaluate the effects of manipulating IGF-II expression in a development nervous system, transgenic mice carrying IGF-II genes adjacent to sequences which are likely to lead to the expression of IGF-II in specific cell types will be produced. To determine the effects of removing IGF-II during different times in development, antibodies directed against IGF-II will be injected into the mature and developing nervous system. Sequences encoding IGF-II will be used to determine the specific regions in the developing and mature brain that express IGF-II and whether this regional expression changes during development. In situ hybridization to sections of fetal mouse brain will be used to determine the regional distribution of IGF-II at different developmental times. RNA from primary cultures of cells derived from the CNS and PNS will be examined by hybridization and nuclease protection assays yo determine which cells are capable of expressing IGF-II. IGF-II will be added to cultures of isolated cell types from both CNS and PNS to determine its effects on survival, growth and differentiation. If IGF-II affects growth or survival of particular cells, the IGF-II gene will be introduced into the cells to see if endogenous production of IGF-II has the same effect as exogenous IGF-II. To determine whether IGF-II expression is necessary for myoblast differentiation, the IGF-II gene will be introduced into myoblasts under control of a constitutive promoter. Experiments will be done to determine if prevention of IGF-II synthesis prevents myoblast differentiation. The tissue-specific regulatory element responsible for the rapid induction of IGF-II in differentiating myotubes will be identified by attaching portions of the IGF-II gene to a reporter, introducing the constructs into myoblasts and looking for induction of the reporter when the myoblasts are stimulated to differentiate into myotubes.
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