MOLECULAR MECHANISMS OF HOMEOBOX GENE INVOLVEMENT IN LIMB PATTERN FORMATION

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The broad goal of this project is to investigate the molecular regulation of the homeobox-related genes Msx-2 (and D1x-5 and/or Msx-1) in the developing vertebrate limb. Msx-2 and msx-1, which are member of the msh- like family of genes, have been implicated in the regulation of limb morphogenesis and pattern formation. Msx-2 and msx-1 are reciprocally expressed by the apical ectodermal ridge (AER) and the underlying subridge mesodermal cells of the developing limb bud, and a variety of studies have suggested that these genes are involved in the reciprocal interactions between the AER and limb mesoderm that are required for limb outgrowth and patterning. In addition, Msx-2 and Msx-1 are co-expressed in the anterior mesoderm of the limb bud, where they may be involved in specification of the anterior non=-skeletal-forming region of limb mesoderm, and in discrete regions of the limb bud in which programmed cell death is occurring that is involved in shaping the contours of the limb. D1x-5 is co-expressed in all Msx-2 domains and is also expressed in the condensing mesenchyme of the limb undergoing chondrogenesis. Studies on tahe altered expression patterns of Msx-2 in mutant limb buds have revealed that the AER and mesodermal domains of its expression are independently regulated. The primary initial goal of this project is to identify the regulatory elements of the Msx-2 gene responsible for its spatially specific domains of expression and for its response to other regulatory molecules involved in limb patterning. To this end, genomic clones containing the full chicken Msx-2 gene plus 15 kb of 5' flanking sequence and 11 kb of 3' flanking sequence have been isolated, and studies on the regulation of Msx- 2 gene expression in transgenic mice have been initiated. These studies have identified a 350 bp region of the msx-2 gene required for its AER domain of expression in transgenic mice, and additional cis-acting regions which define the regulatory regions of the Msx-2 gene expression will be identified. Sites of nucleic acid regulatory factor interactions within spatially-specific cis-acting regions will be identified and characterized. Regulatory elements within the Msx-2 gene will also be identified by DNase hypersensitivity analyses using nuclear extracts from different limb regions. Similar approaches will be used to characterize regulatory elements in the D1x-5 and/or Msx-1 genes. These studies should provide insight into how the Msx-2 (and D1x-5 and Msx-1) genes are regulated and how the regulatory network is involved in pattern formation in the developing limb.