Smooth muscle differentiation and patterning in the urinary bladder

Abstract

Smooth muscle differentiation and patterning is a fundamental process in urinary bladder development that involves a complex array of local environmental factors, epithelial-mesenchymal interaction, and signaling pathways. An epithelial signal is necessary to induce smooth muscle differentiation in the adjacent bladder mesenchyme. The bladder epithelium (urothelium) also influences the spatial organization of the bladder wall. Sonic hedgehog (Shh), which is expressed by the urothelium, promotes mesenchymal proliferation and induces differentiation of smooth muscle from embryonic bladder mesenchyme. Shh, whose signal is mediated through various transcription factors including Gli2 and BMP4, is likely also important in the patterning of bladder smooth muscle. However, it is not known to what extent early mediators of mesenchymal migration, other Shh-associated transcription factors, and crosstalk between the Shh signaling cascade and other pathways are involved in the patterning of bladder smooth muscle. Here we review the role of epithelial-mesenchymal interaction and Shh signaling in smooth muscle differentiation and patterning in the bladder. We also discuss emerging signaling molecules, transcription factors, and mesenchyme properties that might be fruitful areas of future research in the process of smooth muscle formation in the bladder.

Figure 1. Bladder embryogenesis

A) In humans, the division of the cloaca into the urogenital sinus (UGS) and the rectum occurs during the 6^th week of gestation. The mesodermal-derived urorectal fold consists of two structures: the upper fold (Tourneux) divides the cloaca in a cranial-caudal direction and fuses in the midline with the lower fold (Rathke), which partitions the UGS and hindgut in a lateral-medial direction. B) The bladder then forms from the cranial aspect of the UGS. The allantois becomes the urachus and, in the adult, the median umbilical ligament. Reproduced with permission from Developmental Biology, Eighth Edition, 2006, Sinauer Associates Inc.

Figure 2. Time course of bladder organogenesis and smooth muscle differentiation in the mouse

Gross dissections of mouse embryos: whole embryo (upper panels), genital tracts (middle panels), and bladder with umbilical arteries (lower panels). The white arrows indicate the urinary bladder. The black arrowheads indicate the umbilical arteries. Top panel: Ruler = 1 mm. Bars in the middle panels are 1 mm. Bars in the lower panels are 100 mm. Smooth muscle (SM) actin staining of the embryonic bladder. (A–D) Smooth muscle α-actin (SMAA), (E–H) smooth muscle γ-actin (SMGA). E12.5 (A and E), E13.5 (B and F), E14.5 (C and G), and E15.5 (D and H). Note the positive staining of the umbilical arteries. Final magnifcation 100X A-H. Reproduced with permission from Shiroyanagi et al., Differentiation 2007. [7]

Figure 3. Epithelium patterns bladder smooth muscle

Schematic and results of urothelial recombination with bladder mesenchyme in the orthotopic location. Histologic serial sections: (A) Color triple florescent stain, GFP is green, alpha-smooth muscle actin is pink and Hoescht dye is blue representing the zone of smooth muscle inhibition or submucosa; (B) H&E dbff hematoxylin and eosin; immunohistochemistry: (C) GFP = green fluorescent protein; (D) UPK= uroplakin and (E) α-actin = smooth muscle alpha-actin. (magnification bar = 100 μm). Reproduced with permission Cao et al., Pediatric Research 2008. [6]

Figure 4. Proposed Shh signaling pathway in bladder mesenchymal cells

Upon binding Shh, Ptc1 releases the inhibition of Smo. The effect of Shh on smooth muscle differentiation and patterning is mediated by Gli2, which, upon translocation into the nucleus, regulates downstream genes including BMP4. BMP4 may inhibit cellular proliferation and is a prime candidate for mediating the effect of Shh on smooth muscle patterning during bladder development. Other signaling molecules, such as TGF-β and SRF, likely have a role in bladder smooth muscle differentiation and may interact with the Shh pathway. The mechanism through which Shh affects cellular proliferation in the bladder is uncertain but MPF has been shown to be a Shh-responsive complex that regulates mitosis in other systems.

Figure 5. Shh and Ptc expression in the fetal bladder

In situ hybridization of the embryonic bladder. Sonic hedgehog (Shh) (A and B), Patched (Ptc) (C and D). E12.5 bladder (A and C), E13.5 bladder (B and D). Final magnification 100x. Reproduced with permission from Shiroyanagi et al., Differentiation 2007. [7]

Figure 6. Urothelium and Shh regulates BLM smooth muscle differentiation

Immunohistostaining of explants after 72 hours of incubation in serum free DMEM (20X). (A, D, G = intact bladders incubated without Shh) (A) Intact bladders incubated without Shh demonstrated strong α-actin staining of smooth muscle. (D) The urothelium, which stained for UPK, is seen at the center of the section (G) Diffuse expression of Ki67 was detected, indicating specimen viability. (B, E, H = BLM incubated in Shh-free media) (B) Neither smooth muscle nor (E) urothelium were detected. (H) K67 expression was detected, indicating viability. (C, F, I = BLM cultured with 480nM of Shh) (C) α-actin was expressed homogenously throughout the explant, indicating robust smooth muscle differentiation. (F) Uroplakin was not detected, demonstrating an absence of urothelial contamination of the specimen. (I) Ki67 expression was present. α-actin=smooth muscle α-actin, UPK=uroplakin, Ki67=nuclear proliferation marker. Final magnification 100x. Reproduced with permission from Cao et al., Differentiation 2010. [42]

Figure 7. mRNA expression levels of Shh-pathway and smooth muscle genes in the fetal bladder

Percent RNA gene expression normalized to GAPDH mRNA by real-time reverse transcription polymerase chain reaction (RT-PCR) in embryonic bladders from E12.5 to E15.5 mice. (Y-axis: % mRNA gene expression against GAPDH by real-time RT-PCR). Reproduced with permission from Shiroyanagi et al., Differentiation 2007. [7]

Figure 8. Differential spatial expression of Shh-pathway and smooth muscle genes in the fetal bladder

A) Real time RT-PCR analysis of mRNA encoding Ptc1, Gli1 and Bmp4 in laser-captured mesenchymal components from each gestational stage. Expression of Ptc1, Gli1 and Bmp4 was restricted to a thin layer of mesenchymal cells in the submucosa immediately adjacent to the epithelium. Expression of both Ptc1 and Bmp4 genes was significantly decreased in all mesenchymal cells in the E13.5 bladder. Ptc1 remained at a low level in contrast to Bmp4 whose expression was increased at E15 and E16. Gli1 gene expression was up regulated in the submucosa at E13.5. At E15 the expression changed to the smooth muscle layer (Mann-Whitney test, p<0.001). a = serosal zone; a1 = smooth muscle layer; a2 = intermediate zone; b = submucosal zone. B Real time RT-PCR analysis of mRNA encoding SMAA and SM-MHC in laser captured mesenchymal components from each gestational stage. Expression of SMAA mRNA was detectable in the peripheral cellular population at E13.5 and increased to significantly high levels in smooth muscle cells at E15 and E16. SM-MHC was detectable temporal slightly later than SMAA and increasing in expression at E15 and E16 (Mann-Whitney test, p<0.001). a = serosal zone. a1 = smooth muscle layer; a2 = intermediate zone; b = submucosal zone. Reproduced with permission from Liu et al., Int J Dev Biol., 2010. [47]

Figure 8. Differential spatial expression of Shh-pathway and smooth muscle genes in the fetal bladder

A) Real time RT-PCR analysis of mRNA encoding Ptc1, Gli1 and Bmp4 in laser-captured mesenchymal components from each gestational stage. Expression of Ptc1, Gli1 and Bmp4 was restricted to a thin layer of mesenchymal cells in the submucosa immediately adjacent to the epithelium. Expression of both Ptc1 and Bmp4 genes was significantly decreased in all mesenchymal cells in the E13.5 bladder. Ptc1 remained at a low level in contrast to Bmp4 whose expression was increased at E15 and E16. Gli1 gene expression was up regulated in the submucosa at E13.5. At E15 the expression changed to the smooth muscle layer (Mann-Whitney test, p<0.001). a = serosal zone; a1 = smooth muscle layer; a2 = intermediate zone; b = submucosal zone. B Real time RT-PCR analysis of mRNA encoding SMAA and SM-MHC in laser captured mesenchymal components from each gestational stage. Expression of SMAA mRNA was detectable in the peripheral cellular population at E13.5 and increased to significantly high levels in smooth muscle cells at E15 and E16. SM-MHC was detectable temporal slightly later than SMAA and increasing in expression at E15 and E16 (Mann-Whitney test, p<0.001). a = serosal zone. a1 = smooth muscle layer; a2 = intermediate zone; b = submucosal zone. Reproduced with permission from Liu et al., Int J Dev Biol., 2010. [47]