Genes that play roles in embryonic pattern formation and developmental signaling pathways are likely to contain many genomic cis-regulatory sequences that must function properly to allow normal development. The study of such genes and regulatory sequences that control their expression will further our understanding of the function of genomic regulatory DNA in human development and of genetic factors underlying congenital defects in humans. Therefore, the PIs propose to analyze the regulatory architecture of the vertebrate Gdf6 genes. Gdf6 is a member of the BMP signaling factor gene family, which encodes many signaling ligands required for vertebrate skeletal patterning and development. In mouse, Gdf6 is important for normal development of limb, ear, skull and spine skeletal joints, the larynx, and potentially other structures including the heart. These structures have been differently modified during evolution of mammals and fish, so it is unclear to what extent Gdf6 cis-regulatory sequences are conserved across species. However, the extent to which sequence conservation generally correlates with conserved function is also unclear, especially with regard to noncoding regulatory DNA. Here we propose to definitively address the expression and regulation of orthologs for Gdf6, an important skeletal patterning gene, during mouse and zebrafish skeletal development. Using transgenic approaches, they will compare the modular structure of functionally similar and dissimilar Gdf6 cis-regulatory elements in both species, and identify novel long-range genomic regulatory elements controlling Gdf6 expression in developing limb joints, skull sutures, jaw and ear bones, and laryngeal structures. The PIs will correlate the degree of cross-species conservation in individual cis-acting regions with potential for regulatory function, by testing a large data set of candidate regulatory sequences. This will provide a comprehensive "scan" of a large, well-characterized genomic locus for regulatory sequences that impact skeletal development. These experiments will provide extensive insights into the conservation of this important signaling pathway gene in different animal models and the role genomic sequences play in normal skeletal patterning.