Fgfr2 is integral for bladder mesenchyme patterning and function

Abstract

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 that fibroblast growth factor receptor 2 (Fgfr2) is necessary for kidney and ureter mesenchymal development. Our objective was to determine the role of Fgfr2 in bladder mesenchyme. We used Tbx18cre mice to delete Fgfr2 in bladder mesenchyme (Fgfr2^BM-/-). We performed three-dimensional reconstructions, quantitative real-time PCR, in situ hybridization, immunolabeling, ELISAs, immunoblotting, void stain on paper, ex vivo bladder sheet assays, and in vivo decerebrated cystometry. Compared with controls, embryonic (E) day 16.5 (E16.5) Fgfr2^BM-/- bladders have thin muscle layers with reduced α-smooth muscle actin levels and thickened lamina propria with increased collagen expression that intrudes into muscle. From postnatal (P) day 1 (P1) to P30, Fgfr2^BM-/- bladders demonstrate progressive muscle loss and increased collagen expression. Postnatal Fgfr2^BM-/- bladder sheets exhibit decreased contractility and increased passive stretch tension compared with controls. In vivo cystometry revealed high baseline and threshold pressures and shortened intercontractile intervals in Fgfr2^BM-/- bladders compared with controls. Mechanistically, while Shh expression appears normal, mRNA and protein readouts of hedgehog activity are increased in E16.5 Fgfr2^BM-/- bladders compared with controls. Moreover, E16.5Fgfr2^BM-/- bladders exhibit higher levels of Cdo and Boc, hedgehog coreceptors that enhance sensitivity to Shh, than controls. Fgfr2 is critical for bladder mesenchyme patterning by virtue of its role in modulation of hedgehog signaling.

Keywords: bladder development; bladder dysfunction; fibroblast growth factor receptor 2.

Fig. 1.

Tbx18cre drives fibroblast growth factor (Fgf) receptor 2 (Fgfr2) deletion in developing bladder mesenchyme. A: hematoxylin-and-eosin (H&E)-stained cross section of a Tbx18cre;CAG bladder at embryonic (E) day 13.5 (E13.5). B: fluorescence image from a section adjacent to A illustrates red fluorescent protein (cre) expression in bladder mesenchyme, but not urothelium. C and D: in situ hybridization for Fgfr2 shows expression in bladder mesenchyme (arrowhead) and urothelial tissue layers (arrow) in control bladder, but not mesenchyme, of Fgfr2^BM−/− bladder. Dotted lines indicate outer boundary of developing bladder. *, Bladder lumen; BM, bladder mesenchyme; U, urothelium; UA, umbilical artery. Scale bars = 150 μm. E: Western blot demonstrates reduced Erk1/2 phosphorylation (pErk1/2) with comparable total Erk1/2 (tErk1/2) in E16.5 Fgfr2^BM−/− vs. age-matched control bladder (cropped blots are shown as indicated by lines; these blots were run under the same experimental conditions). a/b-Tubulin, α/β-tubulin loading control.

Fig. 2.

E13.5 control and Fgfr2^BM−/− bladders appear comparable. A and B: H&E-stained sections from E13.5 control and Fgfr2^BM−/− bladders demonstrate similar histology. Scale bars = 150 μm. C and D: 3-dimensional (3D) reconstructions reveal similar-appearing urothelial volume (purple), lamina propria volume (red), and muscle volume (brown) in E13.5 Fgfr2^BM−/− and control bladders. *, Lumen; U, urothelium; LP, lamina propria; Mus, muscle. E: quantification of 3D bladder reconstructions confirms comparable total bladder volume (BV) and muscle (Mus), lamina propria (LP), and urothelial (Uro) mean volumes in E13.5 controls and mutants. F: when normalized to total bladder volumes, mean muscle, lamina propria, and urothelial volume percentages are comparable in E13.5 Fgfr2^BM−/− and age-matched control bladders. G and H: graphs of 3D bladder “dome” reconstructions confirm comparable bladder, muscle, lamina propria, and urothelial mean volumes and normalized dome muscle, lamina propria, and urothelial volume percentages in E13.5 controls and mutants. I and J: graphs of 3D bladder neck reconstructions confirm comparable bladder, muscle, lamina propria, and urothelial mean volumes and normalized neck muscle, lamina propria, and urothelial volume percentages in E13.5 controls and mutants. Values are means ± SD.

Fig. 3.

E16.5 Fgfr2^BM−/− bladders have reduced muscle and increased lamina propria. A and B: representative H&E-stained E16.5 Fgfr2^BM−/− bladder section appears to have thinner muscle regions (arrowheads) and an expanded lamina propria compared with age-matched control. Scale bars = 300 μm. C and D: 3D reconstructions appear to show an expanded volume of lamina propria (red) and reduced volume of muscle (brown) in E16.5 Fgfr2^BM−/− bladder compared with control. *, Lumen; U, urothelium; LP, lamina propria; Mus, muscle. E: graph of 3D bladder reconstructions illustrates comparable total bladder (BV), muscle (Mus), and urothelial (Uro) mean volumes in E16.5 mutants and controls but a significant increase in lamina propria (LP) mean volume in Fgfr2^BM−/− compared with control bladders. F: when normalized to total bladder volume, mean muscle volume percentage is reduced, lamina propria percentage is increased, and urothelial percentage is equivalent in E16.5 Fgfr2^BM−/− compared with control bladders. G: graph of 3D bladder dome reconstructions illustrates comparable bladder, muscle, and urothelial mean volumes in E16.5 mutants and controls but a significant increase in lamina propria mean volume in Fgfr2^BM−/− compared with control bladders. H: when normalized to bladder dome volume, mean muscle volume percentage is reduced, lamina propria percentage is increased, and urothelial percentage is equivalent in E16.5 Fgfr2^BM−/− compared with control bladders. I: graph of 3D bladder neck reconstructions illustrates comparable bladder, lamina propria, and urothelial mean volumes in E16.5 mutants and controls but a significant decrease in muscle mean volume in Fgfr2^BM−/− compared with control bladders. J: when normalized to bladder neck volume, mean muscle volume percentage is reduced, lamina propria percentage is increased, and urothelial percentage is equivalent in E16.5 Fgfr2^BM−/− compared with control bladders. *P < 0.05, **P < 0.01. Values are means ± SD.

Fig. 4.

E16.5 Fgfr2^BM−/− bladders have decreased smooth muscle and increased collagen levels. A and B: representative α-smooth muscle actin (αSMA) immunofluorescence (red, arrows) reveals regions of thinner muscle in Fgfr2^BM−/− (arrowheads) than control bladder. Blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. C: quantitative PCR (qPCR) shows equivalent αSMA mRNA expression in E16.5 Fgfr2^BM−/− and control bladders. D: Western blot demonstrates reduced αSMA protein expression in whole E16.5 Fgfr2^BM−/− compared with age-matched control bladder (cropped blots are shown as indicated by lines; these blots were run under the same experimental conditions). Tub,  α/β-tubulin loading control. E and F: representative collagen type Ia (Col1a) immunofluorescence (green) reveals more intense staining in lamina propria (below dotted lines) and regions of collagen infiltration in smooth muscle layer (arrowheads) of E16.5 Fgfr2^BM−/− than control bladder. G: qPCR illustrates significantly increased Col1a mRNA expression in E16.5 Fgfr2^BM−/− compared with control whole bladders. H and I: collagen type IIIa (Col3a) immunofluorescence illustrates brighter staining (below dotted lines) and collagen infiltration into smooth muscle layer (arrowheads) of E16.5 Fgfr2^BM−/− compared with control bladder. J: qPCR illustrates significantly increased Col3a mRNA expression in E16.5 Fgfr2^BM−/− compared with control bladders. K: graph showing increased collagen protein by ELISAs in whole E16.5 Fgfr2^BM−/− bladder lysates compared with age-matched controls. Scale bars = 300 μm. *P < 0.05, **P < 0.01. Values are means ± SD.

Fig. 5.

E16.5 Fgfr2^BM−/− bladders have increased muscle apoptosis, decreased muscle cell proliferation, and increased number of lamina propria cells. A and B: representative bladder cross sections reveal more TUNEL-positive cells (green, arrowheads) in mutant than control detrusor muscle layer, with little staining in mutant and control lamina propria and urothelium. Blue, DAPI nuclear staining. C and D: representative bladder cross sections illustrate decreased phosphorylated histone H3 (pH3)-positive proliferating cells (red, arrowheads) in E16.5 Fgfr2^BM−/− muscle layers compared with control, with minimal staining in mutant and control lamina propria and urothelium. Scale bars = 300 µm. E: increased apoptosis and decreased cell proliferation in Fgfr2^BM−/− compared with age-matched control bladders. F and G: increased mean number of cells and mean cell density per cross section within lamina propria (LP, cells/mm²) in Fgfr2^BM−/− compared with age-matched control bladders. *P < 0.05. Values are means ± SD.

Fig. 6.

Postnatal (P) day 1 (P1) Fgfr2^BM−/− bladders are smaller, with muscle and lamina propria mispatterning. A and B: representative H&E-stained sections reveal a decrease in bladder size and regions of thin muscle in P1 Fgfr2^BM−/− compared with control bladder. *, Lumen; U, urothelium; LP, lamina propria; Mus, muscle. Scale bars = 500 μm. C: graph of 3D bladder reconstructions demonstrates significant reductions in total bladder volume (BV) and muscle volume (Mus) in Fgfr2^BM−/− compared with age-matched control bladders. D: when normalized to total bladder volumes, mean muscle (Mus) percentage is reduced, lamina propria (LP) percentage is increased, and urothelial (Uro) percentage is equivalent in P1 Fgfr2^BM−/− compared with age-matched control bladders. *P < 0.05. Values are means ± SD.

Fig. 7.

P1 Fgfr2^BM−/− bladders have decreased smooth muscle and increased collagen. A and B: representative αSMA immunostaining (red) appears less compact in P1 Fgfr2^BM−/− than control bladder. Blue, DAPI nuclear staining. C: qPCR illustrates reduced aSMA mRNA expression in P1 Fgfr2^BM−/− compared with control bladder. D: Western blot demonstrates reduced αSMA protein in P1 Fgfr2^BM−/− compared with age-matched control bladders (cropped blots are shown as indicated by lines; these blots were run under the same experimental conditions). Tub, α/β-tubulin loading control. E and F: Col1a immunolabeling (green) reveals regions of apparently increased collagen deposition (arrowheads) in P1 Fgfr2^BM−/− compared with control bladder muscle layer. G: qPCR illustrates equivalent Col1a mRNA expression in P1 Fgfr2^BM−/− and control bladders. H and I: Col3a immunostaining (green, arrowheads) appears similar in P1 Fgfr2^BM−/− and control bladder smooth muscle layers. J: qPCR illustrates equivalent Col3a mRNA expression in P1 Fgfr2^BM−/− and control bladders. K: increased collagen protein in whole P1 Fgfr2^BM−/− compared with age-matched control bladder lysates. In E, F, H, and I, dotted lines represent boundary of lamina propria and smooth muscle layer. Scale bars = 300 μm. *P < 0.05, **P < 0.01. Values are means ± SD.

Fig. 8.

P30 Fgfr2^BM−/− bladders have reduced muscle and increased collagen. A, A′, B, and B′: representative H&E staining illustrates thinner and less compact muscle layer in P30 Fgfr2^BM−/− (arrowheads) than age-matched control bladder. *, Lumen; U, urothelium; LP, lamina propria; Mus, muscle. C and C′: trichrome staining shows increased collagen deposition (blue stain) in detrusor layer of P1 Fgfr2^BM−/− compared with age-matched control bladder. Scale bars = 700 μm (A and A′), 500 μm (B and B′), and 300 μm (C and C′). D: qPCR illustrates significantly reduced aSMA mRNA expression in P30 Fgfr2^BM−/− compared with control bladders. E: Western blot demonstrates reduced αSMA protein in P30 Fgfr2^BM−/− compared with age-matched control bladders (cropped blots are shown as indicated by lines; these blots were run under the same experimental conditions). Tub, α/β-tubulin loading control. F and G: qPCR illustrates equivalent Col1a in P30 Fgfr2^BM−/− and control bladders and increased Col3a mRNA expression in P30 Fgfr2^BM−/− compared with age-matched control bladders. H: graph of ELISAs showing increased collagen in whole P30 Fgfr2^BM−/− compared with age-matched control bladder lysates. *P < 0.05, ***P < 0.001. Values are means ± SD.

Fig. 9.

Fgfr2^BM−/− mice have bladder and voiding dysfunction. A: ex vivo bladder sheets from P1 and P30 Fgfr2^BM−/− mice exhibit attenuated contractile force generation when stimulated via α,β-methylene ATP (ABMA, purinergic) or carbachol (muscarinic) agonists compared with age-matched controls. *P < 0.05, **P < 0.01. Values are means ± SD. B: representative electrical field stimulation profiles of P30 bladder sheets demonstrate attenuated responses to all frequencies in Fgfr2^BM−/− compared with age-matched control bladders. C: passive tension profiles in response to stretch show a leftward shift in P1 and P30 Fgfr2^BM−/− compared with age-matched control bladder sheets. D: representative void-stain-on-paper assays from P30 control and Fgfr2^BM−/− mice illustrate differences in frequency and location of voids (arrows) relative to sources of food and water (◇) within the cage. E: representative 20-min in vivo cystometry traces indicate higher baseline and threshold pressures and shortened intercontraction intervals in P30 Fgfr2^BM−/− compared with control mouse. Arrows, voiding responses.

Fig. 10.

P1 Fgfr2^BM−/− mice have no urethral obstruction. H&E-stained serial sections from most distal (A) to proximal (X) portion of urethra in P1 Fgfr2^BM−/− male show lumen (arrowheads) at all levels with no evidence of obstruction. Scale bar = 150 µm.

Fig. 11.

E16.5 Fgfr2^BM−/− bladders have augmented sonic hedgehog (Shh) signaling. A and A′: in situ hybridization reveals comparable urothelial Shh mRNA expression (arrowheads) in E16.5 control and Fgfr2^BM−/− bladders. B, B′, C, C′, D, and D′: in situ hybridization reveals more intense Ptch1, Hhip, and Bmp4 mRNA expression (arrowheads) in suburothelial lamina propria in E16.5 Fgfr2^BM−/− (B′–D′) than age-matched control (B–D) bladders. E and E′: Gli1 mRNA expression is more robust in suburothelial (arrowheads) and muscle (arrows) layers in E16.5 Fgfr2^BM−/− than control bladder. Scale bars = 300 μm. F: qPCR confirms no change in Shh mRNA but increased expression of hedgehog readouts, Ptch1, Hhip, Bmp4, and Gli1, in E16.5 Fgfr2^BM−/− compared with control bladders. G: Western blot illustrates increased Gli1 protein in E16.5 Fgfr2^BM−/− compared with age-matched control bladder (cropped blots are shown as indicated by lines; these blots were run under the same experimental conditions). Tub, α/β-tubulin loading control. *P < 0.05, **P < 0.01. Values are means ± SD.

Fig. 12.

E16.5 Fgfr2^BM−/− bladders have increased Boc and Cdo expression. A: qPCR illustrates comparable Gas1 mRNA expression but elevated Boc and trends for elevated Cdo mRNA expression in E16.5 Fgfr2^BM−/− compared with age-matched control bladders. B, B′, C, and C′: in situ hybridization reveals more intense Boc expression in suburothelial (arrowheads) and muscle layers adjacent to lamina propria (arrows) in E16.5 Fgfr2^BM−/− (B′ and C′) than control (B and C) bladders. D, D′, E, and E′: in situ hybridization reveals that Cdo mRNA expression is confined to muscle adjacent to lamina propria (arrowheads) and outer serosal layer (arrows) of E16.5 control bladder (D and E), whereas in the E16.5 mutant (D’ and E’), it is strongly expressed throughout the entire muscle layer (concave arrowheads) up to the serosal layer. *P < 0.05. Values are means ± SD. Scale bars = 150 μm (B, B′, C, C′, E, and E′) and 300 μm (D and D′).