The long term aim of this work is to understand the molecular mechanisms of gene regulation in the early vertebrate embryo. The cis-acting sequences essential for embryonic gene expression will be determined by microinjection of cloned genes into fertilized frog eggs. Following identification of the cis-acting elements, gel retardation and DNase footprinting techniques will be used to detect and characterize the DNA-binding proteins that interact with the regulatory elements. The frog system is ideal for these studies, not only because of the ease of microinjection, but also because the availability of very large numbers of embryos will provide an ample source of material for the isolation of DNA- binding factors. Using this approach, the experiments outlined in this proposal will investigate gene regulation at two important stages in embryonic development. Firstly, the regulation of the first genes to be expressed from the zygotic genome will be examined. The trans-acting factors that regulate these genes are expected to be maternal and the experiments in this proposal seek to isolate and characterize these maternal regulatory factors. It is important to understand how maternal factors regulate zygotic gene transcription because this step is critical for the success of all subsequent embryonic development. Secondly, the regulation of genes expressed during neurogenesis will be examined. Neural induction is one of the classic problems of developmental biology but almost nothing is known of its mechanism. The experiments in this proposal will identify the cis and trans-acting factors responsible for the temporal and tissue-specific regulation of the neural cell adhesion molecule, (N-CAM), which is one of the first genes to be transcribed in response to neural induction. These studies will lead to a better understanding of transcriptional regulation in vertebrate embryos. In particular, basic information on the regulation of neural genes will contribute to a complete picture of neurogenesis, and this in turn will be important for understanding the neurogenic defects that result in anencephaly and spina bifida.
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