The bladder trigone, ureter and renal collecting ducts arise from directional migration and morphogenesis of the ureteric bud (UB). Perhaps the central question in collecting system development is what factors regulate UB migration and morphogenesis. The goal of this proposal is to purify and characterize soluble factors involved in UB migration and morphogenesis. The source is a conditioned medium produced by a metanephric mesenchyme (BSN) cell line developed and extensively characterized by us. We ultimately expect to focus on two activities. Purification of the first activity appears to have been achieved; pleiotrophin has preliminary been identified as a UB cell migration stimulation factor. The second activity is a UB morphogenesis- stimulating factor that is likely to be necessary for tubule and lumen formation of UB cells and may also induce the isolated ureteric bud to undergo impressive morphogenesis. These two systems (in vitro UB cell tubulogenesis assay and isolated UB culture assay) were recently developed by our lab and are unique and highly reproducible. Thus, we believe they are the best systems currently available to assay and functionally analyze reproducible. Thus, we believe they are the best systems currently available to assay and functionally analyze complex UB morphogenesis. The systems/assays have remarkable similarities in their requirements for complex morphogenesis, including a virtually absolute requirement for BSN cell conditioned medium, from which the factors are being purified in SA1 (techniques: cell culture, column chromatography, SDS-PAGE, microsequencing). Among morphogenetic factors, we will focus on the non-HGF, non-EGF receptor ligand activity that induces UB cells to form tubules with lumens. Purification of the tubule factor is the goal of SA1. SA2 deals with the characterization of pleiotrophin (which appears to be the UB cell migration factor) and the tubulogenic factor (when purified) to unambiguously determine what role they plan in collecting system development using our assays for UB migration and morphogenesis (techniques: 3-D cell and tissue culture, organ culture, isolated UB culture, cloning and sequencing, northern analysis, in situ hybridization antisense and antibody inhibition). The experiments proposed should address key questions in kidney and urinary tract development.
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