Development of the urinary collecting system depends on appropriate tubulogenesis and branching morphogenesis of the ureteric bud. Abnormal development of these structures and their connections with the bladder (e.g. ectopic ureteric orifice, intravesical ureterocele) are associated with considerable morbidity. A number of these anomalies predominate in women (e.g. intravesical ureterocele, Ask-Upmark syndrome). However, little is known about molecular mechanisms of tubulogenesis and branching in the ureteric tree, a complex process that is difficult to study in the intact embryo or by organ culture alone. For the purpose of identifying """"""""tubulogenic"""""""" and """"""""branching morphogenetic"""""""" gene products and elucidating their mechanism of action during development of the urinary collecting system, perhaps the best cell culture systems currently available utilize MDCK and IMCD cells in grown in collagen gels. When exposed to hepatocyte growth factor, the cells form branching tubules; the morphogenetic events are very similar to those in the developing ureteric tree. Considerable evidence indicating these models are highly relevant to development of the urinary collecting system has been obtained in the PI's lab. By applying a combination of biochemical, cellular and molecular approaches to these models, it may be possible to identify gene products critical for ureteric bud morphogenesis; abnormal expression or function of these gene products is likely to be responsible for developmental disorders of the urinary tract. Based on preliminary data from the PI's lab, it is hypothesized that such gene products include: 1) extracellular matrix proteins and their integrin receptors (tubulogenesis-promoting and inhibiting); and 2) extracellular proteases (e.g. collagenase, urokinase). Furthermore, data from the PI's lab indicates that multiple phosphorylation mechanisms modulate tubulogenesis and branching. This proposal seeks to: a) identify these candidate """"""""tubulogenic/branching morphogenetic"""""""" gene products and understand their regulation by phosphorylation (Northern analysis of ECM proteins, integrins and proteases, substrate gel assays for proteases, immunoprecipitation from 32P labeled cells); b) by perturbation of expression or function, establish whether these gene products are critical for tubulogenesis and branching in the MDCK and IMCD cell models (antisera and antisense oligonucleotides); and c) by perturbing expression or function (antisera and antisense oligonucleotides), determine if these gene products are critical for ureteric tree development in embryonic organ culture (histological, immunocytochemical and in situ analysis). Thus, the strengths of different in vitro models will be utilized to identify gene products essential for development of the urinary collecting system.
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