How does the genome encode instructions that guide embryonic development? Our research uses genes that are expressed during vertebrate development as systems for investigating this question. We have two long-term goals. The first is to shed light on regulatory events driving bone and cartilage development. This is relevant to understanding birth defects, osteoporosis and arthritis. The second is to locate and understand the function of long-range genomic sequences that control gene regulation. These sequences can act across hundreds of kilobases and are often well conserved. We study these elements using tools such as BAC (Bacterial Artificial Chromosome) transgenesis and genomic sequence comparisons.
Currently, we are studying three BMP (Bone Morphogenetic Protein) family genes. All are transcribed in complex patterns during development. Precise regulation of these genes is controlled by multiple, distant cis-regulatory elements. Using transgenic assays in mice and zebrafish, we are charting out the location of many cis-regulatory elements that are spread across hundreds of kilobases around these genes. These genes are: Gdf6, which is required for patterning a subset of limb, skull and other skeletal joints during embryonic development; and Bmp2 and Bmp4, both of which are important in bone formation and many sites of organogenesis. Interestingly, each gene is flanked by large ?gene deserts? that contain strongly conserved noncoding sequences, some of which are long-distant regulatory elements. These projects are relevant to understanding the role of noncoding sequences in development and for evolution of skeletal morphology. Ongoing projects include: (1) Transgenic survey of regulatory function of the mouse Gdf6 3? ?gene desert? which may contain cis-regulatory elements for Gdf6 function in ear, skull, limb or spine joints. (2) Identifying cis-elements of the zebrafish Gdf6a (Radar) gene, using Tol2 transoposon and BAC transgenesis in fish, which will allow evolutionary comparisons to mammalian Gdf6. (3) Identifying function(s) of distant, mammal/fish conserved noncoding elements for the Bmp4 gene, which is crucial for many aspects of organogenesis and mesoderm patterning. (4) Dissecting the molecular mechanisms of a conserved osteoblast cis-enhancer for the Bmp2 gene. This element lies 156 kb 3? to Bmp2 and is required for its expression in bone-forming cells, which may be critical for its crucial role as a signaling factor in osteoblast differentiation. We will dissect the factors that bind this sequence by mutagenesis, gel-shift, chromatin immunoprecipitation and other methods.