Brg1 determines urothelial cell fate during ureter development

Abstract

Developing and adult ureters express the epigenetic regulator Brg1, but the role of Brg1 in ureter development is not well understood. We conditionally ablated Brg1 in the developing ureter using Hoxb7-Cre and found that Brg1 expression is upstream of p63, Pparγ, and sonic hedgehog (Shh) expression in the ureteral epithelium. In addition, epithelial stratification in the basal cells required Brg1-dependent p63 expression, whereas terminal differentiation of the umbrella cells required Brg1-dependent Pparγ expression. Furthermore, the loss of ureteric Brg1 resulted in failure of Shh expression, which correlated with reduced smooth muscle cell development and hydroureter. Taken together, we conclude that Brg1 expression unifies three aspects of ureter development: maintenance of the basal cell population, guidance for terminal differentiation of urothelial cells, and proper investment of ureteral smooth muscle cells.

Figure 1.

Conditional ablation of Brg1 leads to hydroureter. (A) Fluorescent immunostaining analyses of Brg1 and p63 on cross-sections of wild-type ureters from five different ages are shown. All pictures are taken under the same exposure scheme. (B) Double immunostaining for Brg1 and GFP (expression from the Hoxb7-Cre-IRES-GFP) in ureteral cross-sections of E16.5 embryos. Arrow in h points to a cell that is GFP⁻/Brg1⁺. (C) Photograph demonstrating the normal (a and c) and hydroureter (b and d) in E16.5 embryos (a and b) and P1 pups (c and d). Arrow in b points to the initial site of ureteral dilation. Bladders in c and d are filled with urine before tissue dissection. (D) Histology of proximal ureters of E16.5 and P1 controls and mutants. Whereas control urothelium has multiple cell layers, the mutant urothelium remains as a monolayer (b, e, and f); d and f are close-up view of boxed areas in c and e, respectively. con, control; Brg1 cKO, conditional knockout of Brg1 using the Hoxb7-Cre-IRES-GFP mouse; UE, ureteral epithelial (urothelium); SMC, smooth muscle cell; GFP, green fluorescent protein. Original magnification, ×400 in A and B.

Figure 2.

p63 is downstream of Brg1 and plays indispensable roles in ureter development. (A) Quantification of p63 expression in E13.5 ureters. (B) p63 protein is almost not detectable in Brg1 null E14.5 ureters. (C) Analysis of the mutant phenotype by fluorescent immunostaining on E16.5 proximal ureters using anti-E-cadherin and anti-p63 antibodies. All anti-p63 immunostaining images presented in this article are acquired using the antibody that detects the ∆Np63 isoform. (D) Quantification of p63⁺ basal cells per ureter cross-section (n=7 and n=9 for con and Brg1 cKO, respectively). (E) Histologic comparison of wild-type, heterozygote, and homozygous GFP knock-in (p63 KO) of E16.5 ∆Np63 GFP knock-in embryonic ureters. Whereas the controls (wt and het) have formed multiple urothelial cell layers, the KO contains a mostly monolayer urothelium (arrow) with extensive folding. The green asterisk points to the smooth muscle layer. (F) Expression of Brg1 is not affected by loss of ∆Np63. The GFP signal comes from the knock-in construct, which labels the urothelial cells affected by the knock-in construct. *** P<0.001. (G) Identification of proliferative cells in urothelium by Ki-67. White dashed line on the Ki-67 images demarcates the boundary of urothelium with its outside mesenchymal compartment. GFP signal comes from the knock-in construct. *P<0.05; ^#P<0.001. con, control; Brg1 cKO, conditional knockout of Brg1 using the Hoxb7-Cre-IRES-GFP mouse; GFP, green fluorescent protein; wt, wild-type; het, heterozygote. Original magnification, ×400.

Figure 3.

Shh is downstream of Brg1 and not affected by loss of p63 in developing ureter. (A) Analysis of Shh expression in E16.5 control and Brg1 null ureters. (B) Detection of SMA expression in E16.5 control and Brg1 null ureters. (C) Quantitative analysis of Shh, Tshz3, MyoCD, and αSma expressions in E16.5 ureters. n=6 each. (D) Shh expression in E16.5 control and p63 KO ureters. (E) SMA expression in E16.5 control and p63 KO ureters. *P<0.05. MyoCD, myocardin; NS, not significant. Original magnification, ×400.

Figure 4.

Pparγ is downstream of Brg1 and required for the expression of uroplakins. (A) Expression levels of uroplakins and cytokeratin 20 (Krt20) are significantly reduced in E16.5 Brg1 null ureters, whereas changes in E-cadherin levels were not significant. n=5 and n=3 for con and Brg1 cKO, respectively. (B) Quantification of Pparg expression in E13.5 ureters. n=8 each. (C) Analysis of Pparγ protein expression in E14.5 control and Brg1 null ureters. (D) Quantification of Pparg mRNA levels in E16.5 ureters. n=9 and n=7 for con and Brg1 cKO, respectively. (E) Analysis of Pparg expression in E16.5 ureters. *P<0.05; ***P<0.001; ^#P<0.005. con, control; Brg1 cKO, Brg1 conditional knockout. Original magnification, ×400.

Figure 5.

PPARγ is essential for terminal differentiation of urothelial cells in vivo. (A) Histology of P1 (a and b) and adult ureters (c–e). Green asterisks in controls (a and c) point to the pink staining of uroplakins plaques, whereas the arrow in d points to the absence of plaque staining in the knockout ureters (Pparg cKO). (B) Fluorescent immunostaining of Hoxb7-Cre⁺;Pparγ^fl/fl P1 ureters: Pparg and p63 (a–h), Shh and Upk2 (i–p), and αSma and p63 (q–x). Dashed white lines in i and red lines in j demarcate the umbrella (apical) cells with lowest Shh expression in i and strongest uroplakin 2 in j, in sharp contrast to that in m and n. (C) Counting of p63+ basal cells per P1 ureter cross-sections. n=10 and n=17 for con and Pparg cKO, respectively. (D) BrdU labeling of P1 ureter. White arrows in e–h point to one example of the p63⁺/BrdU⁺ basal cells. (E) Manual counting of proliferative basal cells. n=13 and n=14 for con and Pparg cKO, respectively. (F) Identification of Brg1 and ∆Np63 expression in control (con) and Pparg null ureters (Pparg cKO) in P1 pups. *P<0.05. Original magnification, ×400 in Aa–Ad, Ae inset, and F; 100× in Ae.

Figure 6.

A working model of the Brg1 regulatory network in ureter development. (A) A simplified view of the different cell layers of a relatively developed ureter (E16.5 and later). The type size of each gene roughly correlates to its levels of expression. (B) A working hypothesis suggesting that Brg1 is upstream of p63, Pparg, and Shh, although existing data cannot confirm whether these are the direct targets of Brg1.