| Studies presented in this thesis are aimed at elucidating genetic programs directing vertebrate skeletal development, using the distal limb as a model. Towards this goal, I have exploited a brachydactyly mouse mutant ( bdyTg; generated by transgenesis) which shows fore- and hind-limb digit loss. To identify the brachydactyly gene, we cloned genomic sequences flanking the transgene insertion site, mapped these sequences to chromosome 3, and used them to determine that the transgene had integrated into the 5′ region of the type IB bone morphogenetic protein receptor, BmprIB, gene. Upon transgene insertion, ∼105kb of the 330kb BmprIB locus were deleted, resulting in a regulatory allele (BmprIBTg) in which BmprIB expression is absent in limb mesenchyme but maintained in neuroepithelium. Analyses of BmprIBTg mice revealed that BMPrIB is the physiologic transducer mediating development of the cartilage template through which digit skeletal elements form. Also required for proper digit formation is GDF5, a BMP-related molecule. Genetic analyses incorporating both BmprIBTg and Gdf5bp-J alleles revealed that BMPrIB regulates chondrogenesis and segmentation of distal limb cartilage elements through both GDF5-dependent and -independent processes, and that, reciprocally, GDF5 acts through both BMPrIB and other receptors. Analyses of BmprIB gene structure and expression in wild-type mice revealed four mRNA isoforms arising from three distinct promoters: two which initiate expression in the developing limb, and one which drives expression in neuroepithelium. Because BmprIB mRNA in the developing limb is first detected in prechondrogenic mesenchyme and BMPrIB signaling is required for these same cells to differentiate, it is likely that onset of BMPrIB activity in prechondrocytes is regulated, at least in part, at the transcriptional level. Therefore, in order to lay the foundation for uncovering transcriptional regulators that orchestrate the early steps of cartilage differentiation, we used computational and mouse transgenic approaches to identify a 3kb DNA stretch capable of recapitulating aspects of BmprIB expression in skeletal mesenchyme. Within this stretch is an evolutionarily conserved 112bp domain harboring putative regulatory elements relevant to driving skeletal expression of BmprIB . Thus, genetic, molecular and histological analyses of the bdyTg mouse mutant have increased our understanding of the genetic mechanisms underlying skeletal development. |
