This proposal describes a program utilizing didactic and research experience for the development of an independent investigator in the fields of extracellular matrix biochemistry, cell biology, and renal development. Phase I (years 1-2) of this proposal will consist of graduate course work with a strong emphasis on biochemistry, developmental cell biology, and advanced techniques in molecular biology. Research projects during phase I will be supervised by an experienced and recognized scientist in the field of extracellular matrix. Mechanisms of transcriptional regulation of the alpha1(I) collagen gene will be identified in fetal calf ligament fibroblasts and in bovine aortic endothelial cells exhibiting angiogenesis in vitro. Cis-regulatory regions will be identified by transfection with plasmids containing fusion genes driven by alpha1(I) collagen 5' flanking and intronic sequences and by DNA-protein binding analyses including mobility shift assays and DNase I footprinting. Phase I research will also include development of expertise in techniques of murine metanephric explantation and transfilter induction. These culture systems will be used to analyze the temporal-spatial expression of type I collagen, thrombospondin, and SPARC (osteonectin) during induction and differentiation of the murine metanephric mesenchyme. This analysis will be performed by immunohistochemistry, metabolic labeling and SDS-PAGE analysis of secreted proteins, and Northern blot analysis of mRNA. Specific cellular levels of gene regulation will be identified. Phase II of this program will involve independent work by the candidate in collaboration with the sponsor and will concentrate on the mechanisms which regulate type I collagen expression after metanephric induction. Techniques, reagents, and results from Phase I will be used to formulate an approach to transfection of transfilter-induced mesenchyme. The long term objectives of this work are to identify DNA regulatory sequences and transcriptional factors which promote metanephric differentiation. These objectives are of direct relevance to the understanding of abnormal renal development, the major cause of kidney failure in childhood.
| Fouser, L; Avner, E D (1993) Normal and abnormal nephrogenesis. Am J Kidney Dis 21:64-70 |
