The specific aims during the last grant-period were to examine the roles of the integrin alpha4beta1 and its cell surface ligand VCAM-1 in muscle differentiation, and to determine how expression of the proteins are regulated in this process. We have made significant progress on each of the original specific aims as shown in detail in the appended manuscripts. During the course of our studies we found that a zinc finger/homeodomain protein (ZEB) acts as a transcriptional repressor and regulates expression of alpha4 during muscle differentiation. In addition to alpha4, ZEB also regulates a number of other muscle genes. The present proposal is to further characterize ZEB and a Drosophila homologue, zfh-1. Results in cell culture and with transgenic mice suggest that myogenesis is under negative regulation by ZEB and the release of this inhibition has a role in regulating the timing of muscle gene expression and thus myogenic differentiation. ZEB is also expressed in tissues in addition to muscle, and experiments with transgenic mice suggest that it plays an important role very early in development. Studies are proposed in cell culture and in transgenic mice to analyze the structure/function of ZEB. We have identified the Drosophila protein zfh-1 as a homologue of ZEB. Zfh-1 represses transcription and regulates myogenesis in Drosophila-overexpression of the protein selectively blocks somatic muscle formation in Drosophila embryos without affecting visceral or heart muscle differentiation. One target of zfh-1 is the myogenic protein MEF2, which is activated directly by twist in somatic muscle cells and is essential for somatic myogenesis. A model is proposed where zfh-1 inhibits somatic myogenesis by blocking twist function and thus MEF2 expression. We suggest that it is a release of this zfh-1 inhibition in somatic muscle cells (by downregulation of expression and loss of function) that defines the onset of somatic myogenesis. Studies are proposed to examine the regions of zfh-1 required for its activity in somatic muscle, and to understand its relationship to other regulators of myogenesis such as twist and MEF2.
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