GENES INDUCING BLADDER SMOOTH MUSCLE FORMATION The goal of this proposal is to investigate the mechanism of bladder smooth muscle formation. Previously, we have shown that the induction of bladder smooth muscle is dependent on an epithelial signal to the undifferentiated bladder mesenchyme. The key question is what are the signals or gene products from the urothelium that control bladder smooth muscle formation. The hypothesis of this proposal is that Sonic Hedgehog (Shh) signaling from bladder urothelium acts as a morphogenetic switch, inducing as well as inhibiting bladder smooth muscle formation as a function of Shh concentration. We propose that Shh signals the adjacent undifferentiated bladder mesenchyme to activate the Shh receptor Patched (Ptc). In turn through the downstream Shh signaling pathway, Serum Response Factor (SRF) is activated localizing to the periphery of the bladder. Subsequently the activated SRF pathway induces smooth muscle formation in the periphery of the bladder away from the Shh inducing epithelium. This would explain the two fundamental features of embryonic bladder development: 1) epithelium is necessary for bladder smooth muscle formation and 2) smooth muscle formation occurs at a defined distance in the peripheral bladder mesenchvme away from the inducing epithelium.
Specific Aim # 1 and #2 will define the ontogeny of smooth muscle structural proteins and regulatory proteins in the Shh and Serum Response Factor pathways during bladder development in wild- type and Shh null mouse embryos.
Specific Aim #3 (Blocking Experiments) will disrupt Shh signaling during murine bladder development which should impair smooth muscle formation.
Specific Aim #4 (Gain of function) will induce bladder smooth muscle in undifferentiated bladder mesenchyme with A) recombinant Shh impregnated microbeads and B) an over-expressing Shh cell line. Tissue recombination experiments will be performed in specific aim #5 to control smooth muscle patterning as a function of normal and ectopic urothelial (Shh) signaling. In summary, we have designed experiments to explore the role of Shh both as an inducer and inhibitor of bladder smooth muscle formation via activation of the SRF pathway. Understanding the mechanism of bladder smooth muscle formation has the potential to facilitate development of new therapeutics to prevent bladder fibrosis, the sequelae of congenital hydronephrosis and the treatment of urinary incontinence.
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