Kruppel-like factor 5 is required for formation and differentiation of the bladder urothelium

Abstract

Kruppel-like transcription factor 5 (Klf5) was detected in the developing and mature murine bladder urothelium. Herein we report a critical role of KLF5 in the formation and terminal differentiation of the urothelium. The Shh(GfpCre) transgene was used to delete the Klf5(floxed) alleles from bladder epithelial cells causing prenatal hydronephrosis, hydroureter, and vesicoureteric reflux. The bladder urothelium failed to stratify and did not express terminal differentiation markers characteristic of basal, intermediate, and umbrella cells including keratins 20, 14, and 5, and the uroplakins. The effects of Klf5 deletion were unique to the developing bladder epithelium since maturation of the epithelium comprising the bladder neck and urethra was unaffected by the lack of KLF5. mRNA analysis identified reductions in Pparγ, Grhl3, Elf3, and Ovol1expression in Klf5 deficient fetal bladders supporting their participation in a transcriptional network regulating bladder urothelial differentiation. KLF5 regulated expression of the mGrhl3 promoter in transient transfection assays. The absence of urothelial Klf5 altered epithelial-mesenchymal signaling leading to the formation of an ectopic alpha smooth muscle actin positive layer of cells subjacent to the epithelium and a thinner detrusor muscle that was not attributable to disruption of SHH signaling, a known mediator of detrusor morphogenesis. Deletion of Klf5 from the developing bladder urothelium blocked epithelial cell differentiation, impaired bladder morphogenesis and function causing hydroureter and hydronephrosis at birth.

Figure 1. KLF5 expression and efficiency of recombination of the Klf5^floxed allele by Shh^GfpCre during bladder and ureter development

KLF5 immunohistochemistry was performed at the indicated embryonic time points. (A-C) control embryos. (D-F) Klf5^Δ/Δ embryos. Klf5 is efficiently deleted throughout the bladder lumen epithelium (arrowheads) and bladder neck but not from the epithelium of the ureter. B’ and E’ insets are 10x magnification of the lumen epithelium shown in B and E. Note absence of KLf5 expression and presence of a single cell layer in E’ vs 3-4 cells comprise the control (B’). Small region between arrowheads in F shows a segment containing KLF5 positive cells that was observed in the bladders of a few embryos where deletion did not occur. (G) β-galactosidase whole mount staining of an isolated Shh^GfpCreRosa26^+/- urinary tract. Note most staining is restricted to the proximal ureter with little to no staining in the mid and distal ureter. (H-I) IHC staining of sectioned ureters with anti-β-galactosidase. (H) Proximal region indicated in (G) closest to renal pelvis exhibits varying β-galactosidase expression. (I) Within the middle segment of the ureter as indicated in (G) only a few scattered cells express β-galactosidase.

Figure 2. Vesicoureteric reflux, hydroureters, and ureteral maturation in Klf5^Δ/Δ fetuses

(A-B) India ink injection into bladder lumen followed by palpation. (B) Ink is present in ureters and renal pelvi. (C-D) Dissected out urinary tracts. Note hydroureter in D vs C (arrows) and opaqueness of bladder in (D). (E-L) Immunohistochemical staining for the indicated markers. (E-F) Some dilation of the ureter is evident as early as E16.5 in Klf5^Δ/Δ fetuses and the ureteric urothelium appears slightly thinner. Insets are higher magnification of the indicated region. (G-H) The Klf5^Δ/Δ ureteric urothelium expresses KLF5 but is only 1-2 cells thick vs the 2-3 cell layers observed in controls (arrowheads). (I-J) Hydronephrosis and hydroureter in Klf5 deficient fetuses. Smooth muscle lines the renal pelvis and surrounds the ureter in the Klf5^Δ/Δ fetus and in controls (arrows). (K-L) Ureter cross sections, arrows indicate UPK1B staining on lumenal cells. (A-L) All scale bars represent 10 microns. (M-N) Micro-CT scans of fetuses. (M) Control, (N) Klf5^Δ/Δ. A distended bladder, hydronephrosis of the renal pelvis, and a patent hydroureter (arrowheads) are shown. Kidney (K), Bladder (Bl), Renal pelvis (RP).

Figure 3. Decreased expression of transcription factors associated with urothelial maturation in the absence of KLF5

(A-F) Immunohistochemical analysis at E16.5 of the indicated proteins. (A’-D’) higher magnification of bladder urothelium. (A’’-D’’) higher magnification of adjacent bladder neck/urethra. The bladder neck/urethra stratified normally in Klf5^Δ/Δ fetuses, whereas the bladder urothelium remained single layered. (E-F) Arrows indicate expression of cyclin D1 within the basal cells of the stratified bladder neck and urethral epithelium. (G-H) E14.5 embryos. Note demarcation in SOX2 positive staining at bottom edge of bladder vs bladder neck region in both control and Klf5^Δ/Δ embryos.

Figure 4. KLF5 is required for maturation of the bladder urothelium

Immunohistochemical staining of control (A,C,E) and Klf5^Δ/Δ (B,D,F) E18.5 fetuses with the antibodies indicated. UPK1B, KRT20, and KRT14 are not expressed by the single layered epithelium lining the Klf5^Δ/Δ bladder. Insets in E and F are higher magnification images of bladder urothelium. Note the normal maturation of bladder neck and urethra that are also Klf5 deficient. The muscle wall is abnormally thin in the Klf5^Δ/Δ bladder (F). Scale bar in D represents 10 microns for A-D.

Figure 5. Candidate transcriptional targets of KLF5

(A) Normalized RT-qPCR gene expression of whole bladder cDNAs at E14.5. Asterisks indicate statistically significant differences by Student t-test (p≤ 0.05). (B) Regulation of pGL4mGrhl3Luc by KLF5 in HEK293T cells. (C) T24 cells transfected with Klf5 siRNA exhibited diminished pGL4mGrhl3Luc activity. (D) Western blot analysis of cell lysates from T24 cells transfected with the indicated SiRNA for the presence of KLF5.

Figure 6. KLF5 is required for normal formation of the bladder mesenchyme

(A-D) Immunohistochemical analysis of E18.5 fetuses for α-smooth muscle actin and smoothelin reveals both markers are expressed by the detrusor muscle (dm). Klf5^Δ/Δ fetuses possess an ectopic α-SMA positive (arrow in B), smoothelin negative (arrowhead in D) region subjacent to the bladder epithelium/urothelium (u) as indicated at higher magnification in insets (A, B). Green arrowhead in (C) indicates thick lateral detrusor muscle compared to diminished bladder wall thickness, green arrowhead in (D). (E) Normalized RT-qPCR gene expression of E14.5 and E16.5 whole bladders suggests SHH signaling is not disrupted in Klf5^Δ/Δ bladders. Bars indicate s.e.m. between individual samples (E14.5 n=3, E16.5 n=5). No statistically significant differences were observed (p<.05).