Spatio-Temporal Distribution of Smads and Role of Smads/TGF-β/BMP-4 in the Regulation of Mouse Bladder Organogenesis

Abstract

Although Shh, TGF-β and BMP-4 regulate radial patterning of the bladder mesenchyme and smooth muscle differentiation, it is not known what transcription factors, local environmental cues or signaling cascades mediate bladder smooth muscle differentiation. We investigated the expression patterns of signaling mediated by Smad2 and Smad3 in the mouse embryonic bladder from E12.5 to E16.5 by using qRT-PCR, in situ hybridization and antibodies specifically recognizing individual Smad proteins. The role of Smad2 and Smad3 during smooth muscle formation was examined by disrupting the Smad2/3 signaling pathway using TβR1 inhibitor SB-431542 in organ culture system. qRT-PCR results showed that R-Smads, Co-Smad and I-Smads were all expressed during bladder development. RNA ISH for BMP-4 and immunostaining of TGF-β1 showed that BMP-4 and TGF-β1 were expressed in the transitional epithelium, lamina propia and muscularis mucosa. Smad1, Smad5 and Smad8 were first expressed in the bladder epithelium and continued to be expressed in the transitional epithelium, muscularis mesenchyme and lamina propia as the bladder developed. Smad2, Smad3 and Smad4 were first detected in the bladder epithelium and subsequently were expressed in the muscularis mesenchyme and lamina propia. Smad6 and Smad7 showed overlapping expression with R-Smads, which are critical for bladder development. In bladder explants (E12.5 to E16.5) culture, Smad2 and Smad3 were found localized within the nuclei, suggesting critical transcriptional regulatory effects during bladder development. E12.5 to E16.5 bladders were cultured with and without TβR1 inhibitor SB-431542 and assessed by qRT-PCR and immunofluorescence. After three days in culture in SB-431542, α-SMA, Smad2 and Smad3 expressions were significantly decreased compared with controls, however, with no significant changes in the expression of smooth muscle myosin heavy chain (SM-Myh. Based on the Smad expression patterns, we suggest that individual or combinations of Smads may be necessary during mouse bladder organogenesis and may be critical mediators for bladder smooth muscle differentiation.

Migration pathways of sacral neural crest during development of lower urogenital tract innervation

“…Another possibility is that there are molecular signals forming a gradient that induces differentiation from the serosal surface inward. Clearly, additional investigation is required, particularly in light of a previous study implicating signaling gradients of Smad family members initiating at the urothelium and extending through bladder mesenchyme as essential for smooth muscle differentiation (Islam et al, 2013). …”

Fgfr2 is integral for bladder mesenchyme patterning and function

“…To our knowledge, Fgfr2 is now only the third known signaling pathway critical for bladder mesenchyme patterning, outside of Shh and the transforming growth factor-␤ superfamily signaling (5,7,8,14,20). Interestingly, previous studies (17,20) have indicated that both Shh and transforming growth factor-␤ signaling families, in particular Bmp4, are tightly associated (17,20).…”

The Interactivity between TGFβ and BMP Signaling in Organogenesis, Fibrosis, and Cancer

“…TGFβ counteracts the action of BMP2,4,6,7,9 on differentiation of mesenchymal stem cells (MSCs) into osteocytes and adipocytes, although TGFβ1 and TGFβ3 can cooperate with BMP2 to induce the differentiation of MSCs into chondrocytes [28,29,30,31]. TGFβ and BMP4 coordinately initiate and modulate a spatiotemporal pattern of activation of canonical SMADs (SMAD1, 2, 4, 5, 8) to control bladder development in mouse [32]. The degree of structural affinity shared by members of the TGFβ superfamily results in the binding of some BMPs to TGFβ receptors, and the consequent activation of SMAD2/3.…”