Urothelium-derived Sonic hedgehog promotes mesenchymal proliferation and induces bladder smooth muscle differentiation.
ABSTRACT Induction of smooth muscle differentiation from bladder mesenchyme depends on signals that originate from the urothelium. We hypothesize Sonic hedgehog (Shh) is the urothelial signal that promotes bladder mesenchymal proliferation and induces bladder smooth muscle differentiation. Pregnant FVB mice were euthanized on embryonic day (E) 12.5 and fetal bladders were harvested. Two experimental protocols were utilized: Specimens were sized by serial sectioning. Cell counts were performed after trypsin digestion. Immunohistochemistry was performed to detect smooth muscle-specific protein expression. alpha-Actin expression was quantified using Western blot. All specimens were viable at 72h. BLM cultured without Shh survived but did not grow or undergo smooth muscle differentiation. IB cultured without Shh and BLM cultured with Shh grew and expressed smooth muscle proteins at 72h. IB cultured with Shh were larger and contained more cells than IB cultured without Shh (all p<0.05). Increasing Shh concentration from 48 to 480nM did not change bladder size, cell counts, or the level of alpha-actin expression. Prior to culture, IB did not express alpha-actin. After culture of IB in Shh-deficient media, alpha-actin was detected throughout the mesenchyme except in the submucosal layer. The IB submucosa was thinner after culture with 48nM Shh and smooth muscle completely obliterated the submucosa after culture with 480nM Shh. In fetal mouse bladders, urothelium-derived Shh is necessary for mesenchymal proliferation and smooth muscle differentiation. Shh concentration affects mesenchymal proliferation and patterning of bladder smooth muscle.
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ABSTRACT: Subtotal cystectomy (STC; surgical removal of ∼75% of the rat urinary bladder) elicits a robust proliferative response resulting in complete structural and functional bladder regeneration within 8-weeks. The goal of these studies was to characterize the early cellular response that mediates this regenerative phenomenon, which is unique among mammalian organ systems. STC was performed on eighteen 12-week-old female Fischer F344 rats. At 1, 3, 5 and 7-days post-STC, the bladder was harvested 2-hours after intraperitoneal injection of bromodeoxyuridine (BrdU). Fluorescent BrdU labeling was quantified in cells within the urothelium, lamina propria (LP), muscularis propria (MP) and serosa. Cell location was confirmed with fluorescently co-labeled cytokeratin, vimentin or smooth muscle actin (SMA), to identify urothelial, interstitial and smooth muscle cells, respectively. Expression of sonic hedgehog (Shh), Gli-1 and bone morphogenic factor-4 (BMP-4) were evaluated with immunochemistry. Three non-operated rats injected with BrdU served as controls. Less than 1% of cells in the bladder wall were labeled with BrdU in control bladders, but this percentage significantly increased by 5-8-fold at all time points post-STC. The spatiotemporal characteristics of the proliferative response were defined by a significantly higher percentage of BrdU-labeled cells within the urothelium at 1-day than in the MP and LP. A time-dependent shift at 3 and 5-days post-STC revealed significantly fewer BrdU-labeled cells in the MP than LP or urothelium. By 7-days the percentage of BrdU-labeled cells was similar among urothelium, LP and MP. STC also caused an increase in immunostaining for Shh, Gli-1 and BMP-4. In summary, the early stages of functional bladder regeneration are characterized by time-dependent changes in the location of the proliferating cell population, and expression of several evolutionarily conserved developmental signaling proteins. This report extends previous observations and further establishes the rodent bladder as an excellent model for studying novel aspects of mammalian organ regeneration.PLoS ONE 10/2012; 7(10):e47414. DOI:10.1371/journal.pone.0047414 · 3.53 Impact Factor
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ABSTRACT: In this study, we examined the expression of Sonic Hedgehog, Patched, Gli1, Gli2, Gli3 and Myocardin in the developing bladders of male and female normal and megabladder (mgb-/-) mutant mice at embryonic days 12 through 16 by in situ hybridization. This analysis indicated that each member of the Sonic Hedgehog signaling pathway as well as Myocardin displayed distinct temporal and spatial patterns of expression during normal bladder development. In contrast, mgb-/- bladders showed both temporal and spatial changes in the expression of Patched, Gli1 and Gli3 as well as a complete lack of Myocardin expression. These changes occurred primarily in the outer mesenchyme of developing mgb-/- bladders consistent with the development of an amuscular bladder phenotype in these animals. These results provide the first comprehensive analysis of the Sonic Hedgehog signaling pathway during normal bladder development and provide strong evidence that this key signaling cascade is critical in establishing radial patterning in the developing bladder. In addition, the lack of detrusor smooth muscle development observed in mgb-/- mice is associated with bladder-specific temporospatial changes in Sonic Hedgehog signaling coupled with a lack of Myocardin expression that appears to result in altered patterning of the outer mesenchyme and poor initiation and differentiation of smooth muscle cells within this region of the developing bladder.PLoS ONE 01/2013; 8(1):e53675. DOI:10.1371/journal.pone.0053675 · 3.53 Impact Factor
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ABSTRACT: Background: While urothelial signals, including sonic hedgehog (Shh), drive bladder mesenchyme differentiation, it is unclear which pathways within the mesenchyme are critical for its development. Studies have shown fibroblast growth factor receptor 2 (Fgfr2) is necessary for kidney and ureter mesenchymal development. Objective: To determine the role of Fgfr2 in bladder mesenchyme.. Methods: We used Tbx18cre mice to delete Fgfr2 in bladder mesenchyme (Fgfr2(BM-/-)). We performed three dimensional (3D) reconstructions, real time PCR (qPCR), in situ hybridization, immunolabeling, ELISAs, immunoblotting, void stain on paper (VSOP), ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Results: Compared to controls, embryonic day (E) 16.5 Fgfr2(BM-/-) bladders have thin muscle layers with less alpha smooth muscle actin (aSMA) and thickened lamina propria with increased collagen Ia and IIIa that intrude into the muscle. The reciprocal changes in mutant layer thicknesses appear due partly to a cell fate switch. P1 to P30, Fgfr2(BM-/-) bladders demonstrate progressive muscle loss and increased collagen expression. Postnatal Fgfr2(BM-/-) bladder sheets exhibit decreased agonist mediated contractility and increased passive stretch tension vs. controls. Cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2(BM-/-) bladders vs. controls. Mechanistically, while Shh expression appears normal, mRNA and protein readouts of hedgehog activity are increased in E16.5 Fgfr2(BM-/-) bladders vs. controls. Moreover, E16.5 Fgfr2(BM-/-) bladders exhibit higher levels of Cdo and Boc, hedgehog co-receptors that enhance sensitivity to Shh, compared with controls. Conclusion: Loss of Fgfr2 in bladder mesenchyme leads to abnormal bladder morphology and decreased compliance and contractility. Copyright © 2014, American Journal of Physiology - Renal Physiology.American journal of physiology. Renal physiology 02/2015; 308(8):ajprenal.00624.2014. DOI:10.1152/ajprenal.00624.2014 · 3.30 Impact Factor