Extracellular matrix protein expression during mouse detrusor development

Bladder morphogenesis. A and B are E14, C and D are E18, E and F are D1, and G and H are 6wk. A, C, E and G are low-power views, whereas B, D, F and H are high-power views. All sections were stained with Masson's trichrome. Arrows in A, C, and E indicate the anterior aspect of the bladder, whereas stars in A to H indicate the lumen of the organ. Through developmental time, note progressive enlargement of the organ, diminution of width of the lamina propria (1) versus the detrusor (d) layer, organisation of detrusor into smooth muscle bundles, and increasing collagen staining. Bars are 100 μm in A, C, E and G and 15 μm in B, D, F and H.

Elastin expression. A to D represent elastin immunohistochemistry at E14, E18, D1, and 6wk, respectively. All sections were counterstained with hematoxylin with bars representing 15 μm. Stars indicate the lumen of the bladder. There was no significant elastin expression in the detrusor (d) at E14, whereas the protein was detected at this site at later stages. The lamina propria (1) expressed elastin at all stages. E is the Western blot of elastin in dissected detrusor layers, with a representative male (m) and female (f) sample at E14, E18, D1, and 6wk. Note increasing signal over developmental time, as a 55 kDa band. F shows the elastin protein quantification (mean + SEM, arbitrary units) of detrusor layers from male (black, n = 4), female (light gray, n = 4) and combined sexes (dark gray, n = 8) at each stage. The steady increase in expression was significant (P = .001).

Collagen type I expression. A to D represent collagen type I immunohistochemistry at E14, E18, D1, and 6wk, respectively. All sections were counterstained with hematoxylin with bars representing 15 μm and stars indicating the bladder lumen. Note that there was no significant collagen type I expression in the detrusor (d) or the lamina propria (1) at E14, whereas the protein was detected in both layers at later stages. The arrows in D indicate the staining of the margins of the detrusor muscle bundles. E depicts a representative western blot for collagen type I in dissected detrusor layers, with male (m) and female (f) samples at E14, E18, D1, and 6wk and rat tail collagen type I as a control. Note the bands at 110 and 140 kDa detected at all stages and in the positive control. F shows collagen type I protein quantification (mean + SEM, arbitrary units) of detrusor layers from male (black, n = 4), female (light gray, n = 4) and combined sexes (dark gray, n = 8) at each stage. The decrease from E14 to E18 was not significant (P = .161) but the increase from E18 to 6wk was significant (P = .035).

Collagen type III expression. A to D represent collagen type III immunohistochemistry at E14, E18, D1, and 6wk, respectively. All sections were counterstained with hematoxylin with bars representing 15 μm and stars indicating the bladder lumen. Note the staining in the detrusor (d), lamina propria (l), and urothelium layers at all stages. The arrows in A to C indicate cytoplasmic “speckles” of immunostaining. Arrows in D indicate immunostaining of the margins of the detrusor muscle bundles. E depicts a representative western blot for collagen type III in dissected detrusor layers, with male (m) and female (f) samples at E14, E18, D1, and 6wk and human placenta collagen type III as a control. Note the band at 140 kDa detected at all stages and in the positive control. F shows collagen type III protein quantification (mean + SEM, arbitrary units) of detrusor layers from male (black, n = 4), female (light gray, n = 4), and combined sexes (dark gray, n = 8) at each stage. The increase from D1 to 6wk was significant at P less than .001.

Collagen type IV expression. A to D represent collagen type IV immunohistochemistry at E14, E18, D1, and 6wk, respectively. All sections were counterstained with hematoxylin with bars representing 15 μm and stars indicating the bladder lumen. Note the staining in the detrusor (d), lamina propria (l), and urothelium layers at all stages. The arrows in A to C indicate the cytoplasmic “speckles” of staining. The arrows in D indicate the “honeycomb” pattern of staining in the detrusor muscle. E depicts a representative Western blot for collagen type IV in dissected detrusor layers, with male (m) and female (f) samples at E14, E18, D1, and 6wk and Engelbreth-Holm-Swarm mouse sarcoma collagen type IV as a control. Note the band at 240 kDa detected at all stages and in the positive control. F shows collagen type IV protein quantification (mean + SEM, arbitrary units) of detrusor layers from male (black, n = 4), female (light gray, n = 4), and combined sexes (dark gray, n = 8) at each stage. The decrease from E18 to 6wk was significant at P = .019.

Laminin expression. A to D represent laminin immunohistochemistry at E14, E18, D1, and 6wk, respectively. All sections were counterstained with hematoxylin with bars representing 15 μm and stars indicating the bladder lumen. Note the staining in the detrusor (d), lamina propria (l), and urothelium layers at all stages. The arrows in D indicate the “honeycomb” pattern of staining in the detrusor muscle. E depicts a representative Western blot for laminin in dissected detrusor layers, with male (m) and female (f) samples at E14, E18, D1, and 6wk. Note the band at 250 kDa detected at all stages. F shows laminin protein quantification (mean + SEM, arbitrary units) of detrusor layers from male (black, n = 4), female (light gray, n = 4), and combined sexes (dark gray, n = 8) at each stage. The increases from E14 to E18 and from D1 to 6wk were significant at P = .003 and P less than .001, respectively.

Fibronectin expression. A to D represent fibronectin immunohistochemistry at E14, E18, D1, and 6wk, respectively. All sections were counterstained with hematoxylin with bars representing 15 μm and stars indicating the bladder lumen. Note the staining in the detrusor (d), lamina propria (l), and urothelium layers at all stages. E depicts a representative western blot for fibronectin in dissected detrusor layers, with male (m) and female (f) samples at E14, E18, D1, and 6wk. Note the band at 260 kDa detected at all stages. F shows fibronectin protein quantification (mean + SEM, arbitrary units) of detrusor layers from male (black, n = 4), female (light gray, n = 4), and combined sexes (dark gray, n = 8) at each stage. The increase from E14 to D1 was significant (P = .028), as was the decrease from D1 to 6wk (P = .002).

Summary of detrusor layer extracellular matrix protein expression through development. The graphs are superimposed on a background illustrating the general pattern of decreasing proliferation (top sections), as assessed by proliferating cell nuclear antigen expression, and increasing differentiation (bottom sections), as assessed by desmin expression, observed during detrusor muscle development.³ Expression of each of the proteins in the detrusor layer is shown as a percentage of their maximal expression during detrusor morphogenesis, as assessed by Western blot (note that Western blot does not indicate absolute levels of protein). A shows collagen type I (black line), collagen type III (gray line), and collagen type IV (white line). Note the significant increase in collagen type I and III expression from E18 to 6wk and from D1 to 6wk, respectively. Collagen type IV levels decline significantly from E18 to 6wk. B shows elastin (black line), laminin (gray line), and fibronectin (white line). Note the significant increase in elastin and laminin expression through detrusor development. Fibronectin levels increase significantly from E14 to D1 and decline significantly from D1 to 6wk.