MOLECULAR ANALYSIS OF HINDBRAIN FORMATION

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The formation of the vertebrate central nervous system (CNS) during embryonic development is thought to be initiated by two processes; first, the induction of dorsal ectoderm to a rostral neural fate by signals from mesoderm and second, the subsequent 're-programming' of some of this tissue to more caudal fates, also by signals from the mesoderm. Together these two processes create a CNS that contains specific structures (forebrain, midbrain, hindbrain and spinalcord) along its rostrocaudal axis. As embryogenesis proceeds, each of these structures becomes refined by further developmental processes until they carry out distinct functions in the adult. Soluble molecules secreted by the mesoderm appear to mediate both the neuralizing (e.g. noggin and chordin) and caudalizing (e.g. fibroblast growth factor, and retinoic acid) functions. We reasoned that the further development of each structure would be mediated by genes activated by these factors and therefore initiated a subtractive hybridization-based molecular screen to isolate such genes. We initially isolated genes activated in the presumptive hindbrain of the zebrafish embryo. Five genes (Meis1, niz, HoxA1, NH7, NH13) isolated in the screen are the subject of this proposal. Meis1, niz, HoxA1, NH7 and NH13 were selected for analysis because they are all expressed in the region of the ectoderm that give rise to the hindbrain. NH7 and Meis1 appear restricted to the hindbrain, and its underlying mesoderm, during gastrulation, while the other genes are expressed in broader domains. Meis1, HoxA1 and niz all appear to encode transcription factors, while the type of protein encoded by NH7 and NH13 is unclear at this point. We will analyze the regulation of these genes in the embryo as well as address their function in hindbrain development. The latter will be accomplished both by gain of function experiments, where the genes are expressed ectopically to determine their effect, and by loss of function experiments, where the genes, or there products, are inactivated genetically or by dominant interfering proteins. These experiments will have an impact on human health in two ways. First, the dysregulation of both Meis1 an Hox genes have been implicated in murine and human myeloid leukemias and understanding their normal function will therefore shed light on oncogenesis. Second, since neural tissue is still regenerating early in development, genes that regulate the process may, in the longer term, be used to repair neural tissue damaged by trauma or degenerative diseases in the adult.