The goal of this COMPETITIVE RENEWAL application is to provide a molecular explanation of the role of soluble factors and mesenchymal contact in development of the urinary collecting system from the ureteric bud (UB). The UB undergoes repetitive branching tubulogenesis during formation of the collecting system. In the previous project period, we developed two novel systems to study branching tubulogenesis of the UB: l) an in vitro UB cell culture model for branching tubulogenesis in which UB cell morphogenesis is stimulated by soluble factors produced from a metanephric mesenchymal cell line; and 2) a system to induce branching tubulogenesis in isolated UB tissue in the presence of soluble factors (which can be extended to study UB-mesenchyme interactions by recombining the cultured UB with mesenchyme). These are powerful systems in which to examine the function of genes involved in collecting system development. For reasons detailed in the proposal, novel and known receptor tyrosine kinases (RTKs) are likely to play a key role in both early and late UB morphogenesis. Using a method pioneered by us known as Codon Optimized Differential Display (CODD),, we targeted RTKs that might be involved in UB branching tubulogenesis. In addition to identifying at least 10 novel gene sequences, some of which are likely to be novel RTKs, we obtained the surprising result that more that 80% of the known RTKs upregulated during UB cell branching tubulogenesis belong to the Eph subfamily. Based on extensive preliminary data, we hypothesize that: l) As yet unexamined or new RTKs are involved in branching tubulogenesis of the UB specifically mediated by soluble factors (SA2); 2) Eph RTKs and their ephrin ligands play a role in branch elongation and guidance induced by direct contact of UB tips with the metanephric mesenchyme (SA1). Thus, we postulate (and provide data in support of) separate roles of soluble factors (UB branching morphogenesis) and mesenchymal contact (UB branch elongation and guidance) in collecting system development and propose that these are mediated by separate subfamilies of RTKs.
We aim to characterize their: 1) spatiotemporal patterns of expression during development of the ureter and collecting ducts (Northerns, in situ hybridization, immunohistochemistry); 2) clone and sequence any novel RTKs of interest; and 3) perform a careful functional analysis of the role of these genes (using soluble monomeric ephrins and Ephs, antisense technology) in the unique model systems we established during the previous project period.
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