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.

Fig. 1

Urothelium and Shh support BLM growth. After 72 h, (A) intact bladders incubated without Shh (375 μm) were larger than (B) BLM incubated without Shh (200 μm). In contrast, (C) BLM incubated with 480 nM of Shh (350 μm) were over twice as large as BLM incubated without Shh and were comparable in size to intact bladders. All images at 10 × magnification.

Fig. 2

The E12.5 fetal mouse bladder does not express smooth muscle. Note α-actin staining of umbilical artery and absence of staining of bladder mesenchyme.

Fig. 3

Urothelium and Shh regulates BLM smooth muscle differentiation. Immunohistostaining of explants after 72 h of incubation in serum free DMEM (20 ×). (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 480 nM 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.

Fig. 4

Urothelium and Shh induce expression of smooth muscle markers in BLM. Markers of smooth muscle differentiation were expressed in intact bladders cultured without Shh (top panel) and in BLM cultured in media containing 480 nM of Shh (bottom panel) after 72 h of incubation (10 ×). MHC=myosin heavy chain, calponin, L-Cald=caldesmon, SM22 = smooth muscle actin-22.

Fig. 5

Shh regulates bladder mesenchyme proliferation. Light microscopy (10 ×) images of (A) E12.5 intact bladder prior to organ culture. (B) Intact bladder after 72 h of incubation in serum-free DMEM without Shh. The bladders incubated without Shh were smaller than (C) the bladders incubated with 48 nM of Shh and (D) the bladders incubated with 480 nM of Shh. (E) Box plot representation of the number of sections obtained from intact bladders at harvest and after 72 h of incubation in 0, 4.8, and 480 nM of Shh. The box is the graphical representation of the distribution of data between the first and third quartiles and the line within the box represents the median bladder size. The “whiskers” represent 1.5 times the interquartile range of the pertinent data set. There were no outliers. (F) Cell counts (× 10⁵) of intact bladders at harvest (E12.5) and after 72 h of incubation with increasing concentrations of Shh. After 72 h, the cell counts of intact bladders incubated without Shh nearly tripled relative to preculture levels. Intact bladders cultured with Shh had a greater number of cells than bladders cultured without Shh although there was no difference in the cell counts of intact bladders incubated in 48 nM versus those incubated in 480 nM of Shh.

Fig. 6

Shh concentration affects bladder smooth muscle patterning. Immunohistostaining of intact mouse fetal bladders after 72 h of incubation in serum free DMEM (20 ×). (A, B, C) Intact bladders incubated without Shh express (A) α-actin which is separated from the urothelium, stained with (B) UPK, by a thin submucosal layer. (D, E, F) Intact bladders incubated with 48 nM of Shh demonstrate similar staining patterns as the bladder incubated in Shh-free media with the exception that the submucosal layer is thinner. (G, H, I) Intact bladders incubated in 480 nM of Shh demonstrated robust α-actin expression, which obliterated the submucosal layer. Specimens were viable as indicated by homogenous Ki67 expression. α-actin=smooth muscle α-actin, UPK=uroplakin, Ki67=nuclear proliferation marker.

Fig. 7

Shh concentration affects expression of α-actin in fetal mouse bladders: (A) Western blot analysis of whole mouse fetal bladders after 72 h of incubation in serum free DMEM and increasing concentrations of Shh. (B) Expression of smooth muscle α-actin in intact bladders relative to β-actin incubated with various concentration of Shh after 72 h. There was no significant difference in the level of α-actin expression between bladders cultured in 480 nM of Shh and those cultured in 48 nM of Shh. SMAA=smooth muscle α-actin, β-actin=control.