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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 ShhGfpCre transgene was used to delete the Klf5floxed 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 were 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.
Congenital disorders of the kidney and urinary tract include renal dysplasia, hydroureter, vesicoureteric reflux (VUR), and bladder outlet obstruction (Kerecuk et al., 2008). VUR involves the retrograde flow of urine from the bladder to the kidneys, placing affected individuals at risk for upper urinary tract infections leading to renal scarring and ultimately renal failure. VUR presents in 1-2% of all children and ~8% of patients with VUR develop end-stage renal failure (Murer et al., 2007; Kerecuk et al., 2008). The embryological origin of VUR has been attributed to several different mechanisms. In humans and mice, Pax2 heterozygosity delays the disassociation of the ureter from the Wolffian duct resulting in a shortened intravesicle ureter with an aberrant angle of entry into the bladder causing VUR (Murawski et al., 2007). Incorrectly positioned distal ureters resulting in ureteric obstruction, hydronephrosis, and megaureters are observed in Rar α β double mutants and Ret-/- animals (Mendelsohn et al., 1994; Srinivas et al., 1999). In contrast, over expression of Ret results in malformed kidneys and VUR (Yu et al., 2004). VUR also occurs in humans and mice with duplicate ureters due to inappropriate positioning of the ureters into the bladder wall (Murer et al., 2007; Murawski and Gupta, 2008). A delay in the timing of ureteric bud outgrowth and distal ureter contact with the UGS is observed in mice in the absence of Lim1 resulting in VUR, renal hypoplasia, and dilated ureters (Pedersen et al., 2005). Animals deficient in either uroplakin 3a (Upk3a) or UpkII exhibit VUR and hydronephrosis indicating that a lack of normal cytoskeletal organization within urothelial cells can contribute to VUR (Hu et al., 2000; Kong et al., 2004).
Although derived from different embryological lineages, the epithelium lining in both the ureters and bladder differentiates into a stratified urothelium with well defined morphological and cellular characteristics. The mature urothelium consists of three distinct cell layers: basal, intermediate and superficial. Cell proliferation is highest in the basal cell layer and is sometimes observed in the intermediate cells (Ayres et al., 1985). The superficial cells, also termed umbrella cells, are highly adapted to maintain a permeability barrier between urine and blood. The surface area of the urothelium changes dynamically with the cycles of contraction and distension of the bladder as it empties and fills. These functions involve a unique apical membrane specialization of the umbrella cells that produces an asymmetrical unit membrane comprised of integral membrane proteins belonging to the uroplakin (Upk) gene family (Wu et al., 2009). With the exception of Upk3b, Upks are expressed exclusively in urothelial cells during advanced stages of differentiation. The paracellular barrier of the urothelium is mediated by differential expression of tight junction proteins including the claudins (Cldn). The expression and localization of several CLDN family members has been associated with specific urothelial cell types and stages of urothelial maturation (Acharya et al., 2004; Varley et al., 2006). Urothelial maturation also involves alterations in cytokeratin (Krt) gene expression (Baskin et al., 1996). The expression of Krts 5 and 14 is initiated and co-localized with Krts 7 and 19 in basal and intermediate cells (Smith et al., 2002). Fully differentiated umbrella cells uniquely express Krt 20 (Erman et al., 2006). Although the morphology and cell types comprising the bladder urothelium are well characterized, little is known of the transcription factors and pathways that mediate urothelial maturation.
Kruppel-like transcription factor 5 (KLF5) is expressed in highly proliferative epithelial cell types during embryogenesis and in the adult including the skin, gut, prostate, lung, mammary gland, and bladder, as well as in immortalized epithelial cell lines and proliferating primary cultures (McConnell et al., 2007). Embryonic stem cell studies indicate an important role for KLF5 in self-renewal and the maintenance of pluripotency (Nandan and Yang, 2009). We previously demonstrated that KLF5 is required for terminal maturation of lung epithelial cells (Wan et al., 2008). In the present study, we demonstrate that KLF5 is required for normal maturation of the murine bladder urothelium and suggest that Grhl3 and Pparγ are candidate transcriptional downstream targets that mediate this process. Deletion of Klf5 from the developing bladder urothelium causes prenatal VUR, hydroureter, and hydronephrosis.
Animal protocols were approved by the Institutional Animal Care and Use Committee in accordance with NIH guidelines. Animals harboring the ShhGfpCre allele were purchased from Jackson Laboratories (Bar Harbor, ME) and mated with Klf5flox/flox animals. Klf5flox/flox females were time mated to Klf5flox/+ShhGfpCre males overnight and the presence of a vaginal plug defined as embryonic day 0.5 (E0.5). For immunohistochemistry, embryos were fixed in paraformaldehyde (4%) in PBS and subsequently dehydrated in graded ethanols for paraffin embedding or equilibrated in 30% sucrose prior to embedding in OCT. Genomic DNA isolated from fetal tails was used for genotyping.
For whole mount visualization, dissected urinary tracts were fixed 2 hours in 2% PFA, 0.25% gluteraldehyde in PBS, rinsed 3 times in PBS, and developed for 8 hours in 5 mM K3Fe(CN6), 5 mM K4Fe(CN6), 2 mM MgCl2, 0.01% Sodium deoxycholate, 0.2% NP-40, and 1 mg/ml X-Gal. Stained tissues were equilibrated in sucrose and embedded in OCT prior to sectioning.
E18.5 whole fetuses were fixed and stored in 4% paraformaldehyde in PBS. Forty-eight hours prior to imaging, fetuses were rinsed in PBS and placed in isotonic 25% Iodine solution as described by Degenhardt et al. (2010). Images were obtained using a microCAT II micro CT using a kVp of 80 and an anode current of 225 micro-amps. 776 projections were collected through 194 degrees of gantry rotation using an x-ray exposure time of 2.30 sec and a bin factor of 2. The set of projections was reconstructed using COBRA (cone beam reconstruction algorithm, Exxim Computing Corporation, Pleasanton, CA USA). The reconstructed image voxel size was 19.4 microns isotropic. The raw CT slice data were loaded in to Amira (Visage Imaging, San Diego, CA USA) for viewing and conversion to DICOM format that have been viewed and processed in OsiriX, an open-source image processing software (Pixmeo, Geneva, Switzerland).
Paraffin embedded tissue was sectioned at 6 μm, dewaxed, rehydrated in graded ethanols, subjected to antigen retrieval (as necessary using 0.1M citrate buffer, pH 6.0, and heat), and endogenous peroxidase activity was quenched in methanol and H2O2 for 15 minutes. Frozen sections were cut at 7 μm, dried overnight and fixed in 4% PFA for 10 minutes prior to antigen retrieval and quenching. Primary antibodies requiring antigen retrieval were guinea pig anti-KLF5 (in house, 1:1500; Wan et al., 2008), mouse anti-Keratin14 (Neomarkers LL002, 1:300), rabbit anti-Keratin 5 (in house, 1:1000), rabbit anti-FOXA1 (Seven Hills Bioreagents, 1:1000), rabbit anti-SOX2 (Seven Hills Bioreagents, 1:2500), rabbit anti-SOX9 (Millipore, AB5535, 1:800), rabbit anti-PhosphoHistone H3 (SantaCruz, SC8656R, 1:500), rat anti-Ki67 (Dako, TEC-3 clone M7249, 1:1000), p63 (Santa Cruz, SC71827, 1:500), mouse anti-smoothelin (Millipore, MAB3242, 1:200), chicken anti-Beta Galactosidase (Abcam, ab9361, 1:3000), rat anti-cyclin D1 (Millipore, 04-1151, 1:1000) and rabbit anti-PAX2 (Invitrogen, 716000, 1:1000). Other antibodies included rabbit anti-Desmin (Abcam, 152001, 1:400), mouse anti-α-smooth muscle actin (Sigma, monoclonal 1A4, 1:6,000); goat anti-UPK1B (Santa Cruz, Ni20, 1:100), and anti-KRT20 (Dako, monoclonal KS20.8 M7019, 1:25). Biotinylated secondary antibodies (1:200, Vector Laboratories) were detected using an avidin-biotin-horseradish-peroxidase detection system (ABC reagent, Vector Laboratories, Burlingame, CA). Sections were counterstained with Nuclear Fast Red. As a negative control, primary antibody was omitted on some slides. At least three animals of each genotype were evaluated at each time point using the antibodies indicated.
Total RNA was isolated from E14.5 or E16.5 embryonic bladders dissected free from the ureters and urethra using the QIAGEN microRNA isolation kit. Klf5wt/wtShhGfpCre+ embryos were used for the control sample. cDNA was generated using the Versa RT-kit (ThermoScientific). At least three independent cDNA samples were used per genotype. cDNA samples were amplified in triplicate on a StepOne Plus Real Time PCR system (Applied Biosystems). All values were normalized to ribosomal protein 18s within the sample and values calculated using the 2-ΔΔCT (Livak) method. The Applied Biosystems TAQMAN primer pairs used were Klf5 (Mm00456521_m1), Pparγ (Mm01184322_m1), Grhl3 (Mm01193339_m1), p63 (Mm00495788_m1), Ovol1 (Mm00498263_m1), Elf3 (Mm01295975_m1), Shh (Mm00436527_m1), Ptch1 (Mm00436026_m1), Gli1 (Mm00494645_m1), Gli2 (Mm01293116_m1), and Bmp4 (Mm00432087_m1).
The bladders of E18.5 and P0 mice were surgically exposed following removal of the gastrointestinal tract. Klf5flox/floxShhGfpCre-, Klf5flox/wtShhGfpCre-, and Klf5flox/wtShhGfpCre+ littermates were used as the controls. A 1 cc syringe filled with India ink was attached to a 30-gauge needle. The needle was inserted into the bladder lumen and a small volume of ink was delivered followed by palpation of the external surface of the bladder wall. Each fetus was evaluated for the appearance of ink in the ureters, renal pelvi, and evacuation out the urethra.
Bladders from 4 Klf5Δ/Δ and 4 controls (either Klf5+/+ShhGfpCre+ or Klf5flox/floxShhGfpCre-) embryos were sectioned. Three sections per E16.5 bladder were taken approximately 100 μM apart and stained for phospho-Histone H3 (pHH3). The entire luminal surface of each bladder section (except the region containing and distal to the trigone) was photographed using a 20X objective. The total number of pHH3 positive cells and the total number of urothelial cells were determined using MetaMorph software (Molecular Devices; Sunnyvale, CA). For each embryo, the number of pHH3 positive nuclei and total nuclei were summed from the three sections to determine the percentage of pHH3 positive nuclei/bladder. These values were subjected to a 2-tailed paired Student t-test.
Total RNA from E14.5 whole bladders isolated from Klf5Δ/Δ and Klf5+/+ShhGfpCre+ embryos (n=3 samples/genotype, each sample a pool of two bladders) was isolated using the Qiagen MicroRNA Kit. The cDNA was then hybridized according to the manufacturer's protocol to the Affymetrix Mouse Gene 1.0 ST Array, covering 28,853 genes with approximately 27 probes per gene spread across the full length of each gene. The RNA quality and quantity assessment, probe preparation, labeling, hybridization and image scan were carried out in the CCHMC Affymetrix Core using standard procedures. Hybridization data were sequentially subjected to normalization, transformation, filtration, functional classification and pathway analysis as previously described (Xu et al., 2009). Data analysis was performed with Genespring GX11. All probe sets on the array were pre-filtered based on their signal intensity such that a probe was kept for further analysis if the probe signal intensity was > 35th percentile in at least 2 of 3 samples. Differentially expressed genes in response to Klf5 deletion vs control mice were identified using an unpaired Student t-test. Changes in gene expression were considered statistically significant if their p value was less than 0.05 and fold-change was greater than 1.5. Gene Ontology Analysis was performed using the publicly available web-based tool DAVID (database for annotation, visualization, and integrated discovery)(Huang da et al., 2009). A gene ontology term is considered to be overly-represented when a Fisher's exact test P value is ≤ 0.001 and gene hits are ≥ 5. The complete dataset can be accessed from vog.hin.mln.ibcn@oeg by the accession number GSE27014.
The mGrhl3 promoter was isolated by PCR using the primers 5’-GCATAAAGAAGGCTTGGCACG-3’ and 5’GGCTGGAAGCACAGGTGCCGACTG-3’ to amplify a 1744 bp product which was sequence verified and subcloned into pGL4 (Promega) to create GL4mGrhl3Luc. T24 (human bladder epithelial transitional carcinoma, ATCC HTB-4) and HEK293T (human embryonic kidney epithelia) cells were grown to 40% confluence in six well plates and transfected with plasmid DNA (0.5 μg) using FuGene 6 (Roche). Promoter activity was determined by measurement of luciferase activity normalized to β-galactosidase activity 48 hours after transfection. All experiments were done in triplicate in 3 independent experiments. Lipofectamine 2000 (Invitrogen) was used to transfect 100 pmoles of either hKlf5 (Am16708) or control (Am4611) siRNA (Ambion). Part of each lysate was utilized for Western Blot analysis. Blots were hybridized with a guinea pig anti-KLF5 antibody (Wan et al., 2008; 1:4000) and rabbit anti-GAPDH (Abcam, ab9485, 1:5000) as a loading control.
Immunohistochemical (IHC) analysis revealed that KLF5 was expressed in the invaginating cloacal epithelium beginning on E10.5. At E14.5, E16.5, and E18.5, KLF5 was detected in the epithelium lining the bladder, bladder neck, urethra, ureter and kidney (Fig.1 A-C, data not shown). In the fetal and adult bladder urothelium KLF5 was detected in all cell layers (Fig. 1B’), with the lowest levels of expression in the terminally differentiated umbrella cells.
To begin to understand the role of KLF5 in maturation of the bladder urothelium, Klf5floxed animals were mated with animals harboring the ShhGfpCre allele in which an EGFPCre-recombinase fusion cassette was inserted into the start site of the Shh locus resulting in Cre expression under control of the endogenous Shh promoter (Harfe et al., 2004; Seifert et al., 2008). Shh expression begins as early as E10 in the lung and invaginating cloacal epithelium and is expressed throughout the bladder epithelium until at least E14.5 (Harris et al., 2006; Haraguchi et al., 2007). KLF5 IHC revealed an almost complete loss of KLF5 in the bladder, bladder neck, and urethral epithelium of the urinary tract, as well as the lung epithelium of E14.5 (n=3), E16.5 (n=5), and E18.5 (n=6) Klf5flox/floxShhGfpCre+ (Klf5Δ/Δ) fetuses (Fig. 1D-F, data not shown). Consistent with our previous studies deleting Klf5 using lung specific drivers, all Klf5Δ/Δ fetuses died shortly after birth of respiratory distress (Wan et al., 2008). Previous reports indicated that Shh is expressed in the developing ureter and kidney (Yu et al., 2002). KLF5 expression was maintained in the ureter and renal pelvis Klf5Δ/Δ mice (Fig. 1E, ,2H,2H, data not shown). ShhGfpCre dependent recombination at the Rosa26 locus was evaluated by whole mount β-galactosidase staining of dissected urogenital tracts and/or IHC for β-galactosidase. Both analyses indicated inefficient levels of recombination within the ureters with the highest levels of recombination present in the most proximal third of the ureters (Fig. 1 G-H). Little to no recombination occurred in the middle and distal regions of the ureters (Fig. 1G, I). Combined these observations indicate that Klf5 is not efficiently removed from the ureteric epithelium by the ShhGfpCre allele.
At E18.5 and P0, the ureters and renal pelvi of all Klf5Δ/Δ fetuses were dilated. Hydroureter and hydronephrosis were confirmed by visualization, serial sectioning, and high resolution micro-computerized tomography (CT)(Fig. 2). Differences in size or structure of the kidneys at E14.5 and E16.5 were not detected by IHC staining for PAX2 or SOX9 (Fig. 2E,F, data not shown). Injection of India ink into the bladder lumen followed by palpation of the bladder resulted in vesicoureteric reflux in 6/6 Klf5Δ/Δ fetuses that occurred bilaterally in 4 of the 6 fetuses (Fig. 2A vs B). In contrast, of the 10 nonmutants evaluated, only 1 exhibited reflux and it was unilateral. In previous studies, VUR was attributed to defects in formation of the kidneys and/or ureters (Chang et al., 2004; Airik et al., 2006, 2010; Iizuka-Kogo et al., 2007; Murawski et al., 2007; Nie et al., 2010). In all Klf5Δ/Δ fetuses a single ureter and kidney were observed bilaterally, indicating that VUR is not attributable to duplications of the kidneys and/or ureters.
The ShhCre transgene was highly active in the bladder urothelium resulting in efficient early deletion of Klf5 that inhibited maturation of the bladder urothelium. At E14.5, a single cell layered epithelium lined the forming bladder in Klf5Δ/Δ embryos, whereas epithelial stratification was evident in control embryos (Fig. 1A,D). As urothelial maturation proceeded, three distinct cell layers: basal, intermediate, and umbrella were first evident in control fetuses at E16.5 (Fig. 1B’) and persisted at E18.5. As seen at E14.5, a single cell layered epithelium lined the bladder of E16.5 and E18.5 Klf5Δ/Δ fetuses (Fig 1D-F). At E16.5, p63 expression was predominantly localized to basal cells in control mice and was dramatically reduced or undetectable (Fig. 3A,A’ vs B,B’) in the single layer of cells lining the bladder of Klf5Δ/Δ fetuses. Unlike p63, expression of FOXA1 persisted in most cells comprising the single layered epithelium lining the E16.5 and E18.5 Klf5Δ/Δ bladders while in control mice a gradient of FOXA1 expression was established with the highest levels of expression present in the terminally differentiated umbrella cell layer (Fig. 3C,C’,D,D’). At E18.5, umbrella cells within the superficial layer of control fetuses expressed KRT20 and UPK1B (Fig. 4A,C). KRT14 and KRT5 were expressed in the basal and intermediate cell layers of the bladder urothelium (Fig. 4E, data not shown). UPK1B, KRT20, KRT14 or KRT5 were not detected in the simple epithelium lining the bladder of Klf5Δ/Δ fetuses indicating the absence of terminally differentiated basal and umbrella cells (Fig. 4B,D,F, data not shown).
In contrast, stratification of the urothelium of control and Klf5Δ/Δ ureters was initiated by E16.5 (Fig. 2E, F inserts) although fewer cell layers were noted in Klf5Δ/Δ fetuses. At E18.5, KLF5 was detected in most cells lining the ureteric epithelium of Klf5Δ/Δ fetuses (Fig. 2H). However, the urothelium of control fetuses possessed 2-3 cell layers whereas only 1-2 cell layers were evident in the dilated ureters of Klf5Δ/Δ fetuses Fig. 2G, H). Whether this difference in appearance is attributable to the dilated state of the ureter or partly to incomplete recombination of the Klf5floxed allele within this tissue is not known. Ureters from Klf5Δ/Δ E18.5 fetuses expressed UPK1B (Fig. 2K,L) and Keratin 20 (KRT20)(data not shown) indicating that the ureteric urothelium expresses mature urothelial markers. Alpha smooth muscle actin (αSMA) was detected in smooth muscle surrounding the dilated renal pelvi and proximal ureters of Klf5Δ/Δ mice (Fig. 2I-J). Taken together these data indicate that in contrast to the profound affect on morphogenesis of the bladder urothelium, the ureteric urothelium undergoes normal terminal differentiation suggesting that the observed hydroureters are not attributable to deficiencies in ureter urothelial maturation.
The bladder, bladder neck, and urethral epithelium are all derived from the cloacal epithelium (Seifert et al., 2008). In sharp contrast to the findings in the bladder, the absence of KLF5 in the bladder neck and urethra did not prohibit their normal differentiation. In the normal bladder, bladder neck and urethral epithelium; KLF5, p63, FOXA1, KRT14 and KRT5 were expressed by cells within all three compartments, whereas expression of the uroplakins was restricted to the bladder urothelium (Figs. 1C, 3A-A”, 3C-C”, ,4E,4E, data not shown)(Romih et al., 2005). Distinction between the epithelium destined to form the bladder versus the bladder neck/urethra was demarcated by E14.5 in both control and Klf5Δ/Δ embryos by regionalized expression of SOX2 (Fig. 3G,H). Normal expression of p63, FOXA1, KRT14, KRT5, and cyclin D1 was observed in the bladder neck and urethral epithelium of Klf5Δ/Δ fetuses Fig. 3B, 3B”, 3D, 3D”, 3F, ,4F,4F, and data not shown). These observations reveal a critical role of KLF5 in formation and differentiation of the bladder urothelium but not the adjacent bladder neck and urethral epithelium.
Phosphohistone H3 (pHH3) and Ki67 stains were similar in control and Klf5Δ/Δ mice at E14.5 and E16.5. At E16.5 the proliferative rate was quantified by determining the percentage of phosphohistone H3 positive cells within the epithelial population of the bladder lumen in control and Klf5Δ/Δ fetuses. A statistically significant difference was not observed between cellular proliferation in KLF5 deficient (24.07 ± 4.76 s.e.m.) and control (28.02 ± 4.61 s.e.m.)(p = 0.572) bladder epithelium. In TSU-Pr1 human bladder cancer cells and other tissues, KLF5 induced cellular proliferation by activating cyclin D1 expression (Sun et al., 2001; Nandan et al., 2005; Chen et al., 2006; Yang et al., 2007; Ema et al., 2008). Cyclin D1 staining was absent in the maturing bladder urothelium at E14.5, E15.5 and E16.5 in both control and Klf5Δ/Δ bladder epithelium (Fig. 3E-F, and data not shown). A few cells stained positively for cyclin D1 in the bladder epithelium of controls and Klf5Δ/Δ fetuses at E18.5. In contrast, cyclin D1 was detected in basal cells of the bladder neck at E16.5 and E18.5 in both control and Klf5 deficient epithelium. Due to the absence of effects on epithelial cellular proliferation in the Klf5Δ/Δ bladder epithelium, we evaluated cell death. Tunnel assay detected few dying cells in E16.5 control or Klf5Δ/Δ epithelia (data not shown). Combined these data indicate that cellular proliferation within the bladder epithelium and bladder neck are not altered by the absence of KLF5 and suggest that within the bladder neck epithelium cyclin D1 expression is not dependent upon KLF5.
mRNA microarray analysis was performed on cDNA from whole bladder total RNA isolated from E14.5 Klf5Δ/Δ and Klf5wt/wtShhGfp-Cre+ embryos. Of the 363 genes differentially expressed ≥ 1.5 fold with a p≤.05, GO analysis identified two major subgroupings, epithelial cell development/differentiation (GO:007398, GO:0030855, GO:0060429 GO:0008544) and cell adhesion (GO:0016337 and GO:0022610). As early as E14.5, Klf5Δ/Δ tissue expressed reduced levels of urothelial terminal differentiation markers including Upk1a, Upk1b, Upk3a, Ivl, Sprr1a, Sprr2a, Krt8, Krt4, Krt18, Krt7, Cldn8, Cldn4, Cldn7, and Emp1 (Table I) (Smith et al., 2002; Acharya et al., 2004; Erman et al., 2006; Wu et al., 2009). These observations are consistent with our previous developmental profiling of the urothelium that indicated that expression of many terminal differentiation markers was initiated as early as E13.5 (www.GUDMAP.org). As indicated in Table 1, a number of genes previously identified as KLF5 targets following over expression in ES cells were oppositely affected by deletion of Klf5 in maturing bladder epithelial cells.
Decreased expression of only 11 transcription factors was observed after deletion of Klf5: Ehf (-3.46, p<.0044), Pparγ (-3.33, p<.0011), Elf3 (-2.85, p<.00026), Grhl3 (-2.56, p< .0018), Msx2 (-2.2 p<.0003), Foxq1 (-2.19, p<.0041), Foxa1 (-1.85, p<.022), Zfp750 (-1.77, p<.00067), Ovol1 (-1.62, p<.00082), Elf5 (-1.6, p<.031), and Tcfap2c (-1.53, p<.0099). Three of these genes were previously implicated in urothelial terminal differentiation: Pparγ (peroxisome proliferator-activated receptor γ), Grhl3 (grainyhead-like 3) and FoxA1 (Varley et al., 2009; Yu et al., 2009). Quantitative real-time PCR of E14.5 whole bladder cDNA confirmed reductions in Klf5, Pparγ, Grhl3, Elf3, and Ovol1 expression (Fig. 5A). Although p63 staining was decreased at E16.5, p63 mRNA levels were not altered at E14.5. Taken together, deletion of Klf5 in the bladder urothelium arrested urothelial maturation as early as E14.5.
Since the defect observed in urothelial maturation in Grhl3-/- fetuses was less severe than that observed in the Klf5Δ/Δ embryos, we postulated that Grhl3 is a downstream target of KLF5 (Yu et al., 2009). We created the reporter construct GL4mGrhl3Luc containing 1.7 kb of the mouse Grhl3 promoter driving luciferase gene expression. MatInspector analysis of the mGrhl3 promoter identified 8 putative KLF binding sites located between -1250 and -1 (Cartharius et al., 2005). GL4mGrhl3Luc was active in T24 bladder and HEK293T cells (Fig. 5B, C). Co-transfection of the Klf5 expression construct, pKlf5, induced GL4mGrhl3Luc activity in HEK293T cells while cotransfection into T24 cells did not potentiate activity of the mGrhl3 promoter, perhaps related to the high levels of endogenous KLF5 in these cells. Co-transfection of Klf5 siRNA into T24 cells reduced GL4mGrhl3Luc activity (p≤ .00467) that was accompanied by a reduction in the levels of KLF5 (Fig. 5D). The decrease in Grhl3 mRNA seen in Klf5Δ/Δ bladders in vivo coupled with these in vitro data support the concept that Grhl3 is a downstream target of KLF5.
The profound effect of Klf5 deficiency on maturation of the urothelium was accompanied by alterations in development of the surrounding mesenchyme that forms the smooth muscle and stromal layers of the bladder. The musculature of the bladder wall was frequently thinner in Klf5Δ/Δ fetuses compared to controls (Figs. 1C vs F, 3C vs D, 4E vs F, 6A-D). A prominent layer of cells was seen in all E18.5 Klf5Δ/Δ fetuses that stained positively for αSMA and desmin subjacent to the luminal epithelium that was absent or represented by a few cells in the controls (Klf5flox/floxShhGfpCre- and Klf5flox/+ShhGfpCre+)(Fig. 6A vs B, data not shown). The ectopic αSMA positive layer of cells was first detected at E16.5 in the bladder dome of Klf5Δ/Δ fetuses. Smoothelin staining was only detected in the detrusor muscle of control and Klf5Δ/Δ E18.5 fetuses, indicating that the ectopic αSMA staining population of cells was not likely mature smooth muscle (Fig. 6C vs D).
Maturation of the bladder musculature is influenced by SHH signaling from the epithelium to the subjacent mesenchyme (Tasian et al., 2010). At E14.5 no alterations in the expression of αSMA, serum response factor, Shh, or any of the SHH downstream targets Ptch1, Gli1, Gli2, or Bmp4 implicated in formation of the detrusor were detected in the mRNA microarray analysis or by RT-qPCR of E14.5 and E16.5 whole bladders. At both developmental time points, the levels of expression of the components of the SHH signaling pathway were highly variable in both control and Klf5Δ/Δ tissues consistent with the dynamic regulation of SHH signaling during this window of development (Fig. 6E) (Tasian et al., 2010). Such variability was not seen in the same samples when RT-qPCR was performed in the transcription factor analysis (Fig. 5A, data not shown). These observations indicate that formation of the ectopic α-SMA positive cell layer is not likely attributable to alterations in known components of the SHH signaling pathway and is perhaps dependent on the alteration of other urothelium derived signals.
Klf5 deficiency in the developing bladder urothelium provides a unique model of congenital functional obstructive uropathy presenting with the prenatal occurrence of VUR, hydroureter, and hydronephrosis. In contrast to other mouse models, hydroureter and hydronephrosis seen after deletion of Klf5 were related to epithelial differentiation or morphogenetic abnormalities in the bladder rather than to physical obstruction, altered peristalsis or defects in ureteric budding resulting in VUR due to inappropriate positioning of the ureters into the bladder wall (Chang et al., 2004; Airik et al., 2006, 2010; Iizuka-Kogo et al., 2007; Murawski et al., 2007; Nie et al., 2010). Since Klf5 expression was maintained in the developing kidney and ureter in the present model, we propose that VUR, hydroureters, and hydronephrosis are likely secondary consequences of bladder dysmorphogenesis. Serial sectioning of Klf5ΔΔ E18.5 fetuses indicated that the bladders of many fetuses were distended and the smooth muscle layer was thin when compared to controls. These findings are similar to those in the human syndromes megacystis-microcolon-intestinal hypoperistalsis (OMIM 249210) and Eagle-Barrett (Denes et al., 2004) also characterized by bladder distention, hydroureters, and hydronephrosis at birth but for which genetic causes have not been identified. Megacystis followed by hydroureter and hydronephrosis is also observed in the Megabladder (Mgb) mouse (Singh et al., 2007; Ingraham et al., 2010). Although the gene responsible for the Mgb phenotype has not been identified, the urothelium is normal and the primary defect is thought to be intrinsic to the maturing smooth muscle progenitor cells of the developing bladder. A known regulator of bladder smooth muscle maturation is SHH signaling from the epithelium to the underlying mesenchyme (Tasian et al., 2010). Shh-/- and Gli2-/- fetuses exhibit bladder hypoplasia (Haraguchi et al., 2007). Like Klf5Δ/Δ fetuses, Gli2-/- fetuses also possess an ectopic αSMA positive cell layer subjacent to the bladder epithelium whose formation was attributed to a reduction in Bmp4 expression by the mesenchyme (Cheng et al., 2008). Our examination of Shh, Ptc1, Gli2, Gli1 and Bmp4 mRNAs indicated that SHH signaling from the urothelium to the PTC1 receptor in the mesenchyme was likely not altered in the Klf5Δ/Δ bladder, suggesting that other KLF5-dependent signals made by the maturing urothelium mediate normal radial patterning of the bladder mesenchyme. Prior studies in rats, demonstrated that at postnatal day 10 an α-SMA, desmin, vimentin, and vinculin positive cell layer was first detected subjacent to the urothelium (Baskin et al., 1996). In the adult human bladder, suburothelial cells that express α-SMA, desmin, and vimentin but not smoothelin are referred to as muscularis mucosa (Council and Hameed, 2009). In humans and guinea pigs the suburothelial cells termed myofibroblasts/intersitial cells of the bladder are characterized by their expression of α-SMA, desmin, vimentin, c-kit, and Cx43 and are currently thought to relay signals from the urothelium to the detrusor (Drake et al., 2006; Fry et al., 2007; McCloskey, 2010). In the present study, the absence of staining for smoothelin in the suburothelial region suggests that the α-SMA positive layer of cells observed in Klf5Δ/Δ fetuses at E18.5 may represent precocious formation of the interstitial cells of the lamina propria that may contribute to fetal bladder dysfunction in the present model.
Abnormalities in urothelial maturation have been reported in only a few mouse models. Upk3a and UpkII are expressed exclusively by urothelial cells during advanced stages of differentiation. The ureteric urothelium becomes thickened leading to occlusion and VUR in adult mice lacking the uroplakins (Hu et al., 2000; Kong et al., 2004). Formation of a multilayered urothelium is dependent on FGF7 signaling from the mesenchyme to the overlying epithelium (Tash et al., 2001). The intermediate cell layer is absent and basal and umbrella cells are intermixed within the single cell layered urothelium of Fgf7-/- mice; in contrast to the single layered epithelium observed in Klf5Δ/Δ fetuses that lacked terminally differentiated basal and umbrella cells. The mRNA microarray analysis of E14.5 Klf5-deficient bladders did not detect alterations in expression of Fgf7 or its receptor, Fgfr2 (Finch et al., 1995). In the absence of p63 a single urothelial cell layer expressing Upks forms. Although p63 is expressed by basal cells that give rise to the intermediate and umbrella cells, p63 expression is not a prerequisite for maturation of umbrella-like cells (Signoretti et al., 2005; Cheng et al., 2006). Therefore, the lack of p63 in Klf5Δ/Δ urothelium does not explain the failure of terminal differentiation. The absence of p63, KRT5, and KRT14 expression in the single layer of cells lining the Klf5Δ/Δ bladder suggests these cells are not basal cells. The persistent uniform expression of FOXA1, and the lack of expression of terminal differentiation markers of basal, intermediate, and umbrella cells indicates that in the absence of KLF5, urothelial precursor cells remain in an undifferentiated state.
Little is known of the transcriptional hierarchy regulating urothelial maturation. Pparγ, Grhl3, Ovol1, Foxa1, Elf3 and Ehf are coordinately expressed with Klf5 in the developing bladder and other embryonic epithelial tissues (http//:genepaint.org) (Auden et al., 2006; Feldman et al., 2003; Li et al., 2006; Oettgen et al., 1997). We have determined that KLF5 regulates transcription of the Grhl3 gene in vitro. Although several putative “KLF” binding sites were identified by MatInspector analysis (Cartharius et al., 2005), three distinct KLF5 consensus binding sites were recently proposed based on genes commonly identified in samples subjected to CHIP Seq and mRNA Microarray analysis (Parisi et al., 2010). An examination of 1.7 kb of the mGrhl3 promoter with these sequences identified 7 putative KLF5 binding sites. Thus Grhl3 may be a direct downstream target of KLF5 during urothelial maturation. Consistent with this concept, Grhl3-/- fetuses form a stratified urothelium with a defect in formation of the terminally differentiated umbrella cells (Yu et al., 2009). Transglutaminase I (Tgm1), a key enzyme in epithelial barrier formation, and UpkII are direct downstream targets of GRHL3 (Ting et al., 2005; Yu et al., 2009). In addition to Tgm1 and UpkII, previous studies indicated that the expression of Sprr1a, Snx31, Fmo5, Tmprss13, and Adh1 normally increase during the course of bladder maturation. The expression of all of these genes was down regulated in both E14.5 Klf5Δ/Δ and in E18.5 Grhl3-/- bladders (Table I)(Yu et al., 2009).
Several lines of evidence also suggest that Pparγ plays a critical role in regulating urothelial maturation. Pparγ expression increases as the mouse urothelium matures (Jain et al., 1998) and PPARγ activation by the agonist troglitazone induced expression of the terminal differentiation markers, Upks la, 1b, II and IIIa, Krt13, Krt20 and Cldn3 in cultured human urothelial cells (Varley et al., 2004; Varley et al., 2006; Varley et al., 2009). In committed urothelial cells, PPARγ induced FoxA1 and Irf1 that directly interacted with cis –regulatory elements in the Upk1a, 2, and 3a gene promoters (Varley et al., 2009). In the differentiated E18.5 urothelium, FOXA1 was expressed at the highest levels in the umbrella cell layer suggesting that this signaling paradigm occurs during initial maturation of the mouse urothelium. By E14.5, a dramatic reduction in Pparγ expression occurred in Klf5Δ/Δ bladders accompanied by greater reductions in the expression of Upk1a, 2, and 3a and the absence of terminally differentiated urothelial cells. KLF5 directly regulates Pparγ expression during the differentiation of adipocytes (Oishi et al., 2005). The present observations suggest that KLF5 may also regulate Pparγ during the process of urothelial maturation.
Developmental roles for Elf3 and Ovol1 in maturation of the bladder have not been previously proposed. Prior studies of Elf3 deficient mice revealed that ELF3 is required for normal development of the intestinal epithelium (Ng et al., 2002), morphogenesis of the urothelium was not evaluated. Elf3 expression was dramatically increased in adult urothelium in response to infection and has been shown to directly regulate claudin 7 expression (Mysorekar et al., 2002), Kohno et al., 2006). In the dermis, Ovol1 is required for committed epidermal progenitor cells to exit from proliferation (Nair et al., 2006). Ovol1-deficient mice are unable to properly form the skin barrier and develop cystic kidneys (Teng et al., 2007). A Kruppel-like binding site was previously identified at position -167 within the mOvol1 promoter (Li et al., 2002). Further study of the regulatory mechanisms controlling the expression of these genes will provide new insights into the urothelial differentiation program and provide new paradigms for understanding differentiation and perhaps repair in other epithelial tissues.
Several lines of evidence have suggested that cyclin D1 is regulated by KLF5 (Bateman et al., 2004; Nandan et al., 2004, 2005). Cyclin D1 was increased in bladder cancer cells over expressing KLF5 that showed an increase in the rate of proliferation (Chen et al., 2006). In the present study, changes in the proliferation rate in the bladder urothelium were not noted between controls and Klf5Δ/Δ mice. We also observed that between E14.5 and E16.5, cyclin D1 protein was not detectable in the normal urothelium indicating that proliferation is not dependent on KLF5 or cyclin D1 during this period of development. In contrast to the bladder urothelium, epithelial cells within the fetal bladder neck were proliferative and expressed cyclin D1 even in the absence of KLF5. KLF5 deficient embryonic stem cells (ESC) exhibit a decreased rate of cellular proliferation associated with altered regulation of the cyclin-dependent kinase inhibitor, p21, and the Akt co-activator Tcl1 (Ema et al., 2008). Alterations in these genes were also not observed in the microarray analysis of mRNA isolated from E14.5 whole bladders. In ESC, KLF5 deficiency can be compensated for by KLF2 and KLF4 (Ema et al., 2008; Jiang et al., 2008; Nandan and Yang, 2009). Compensation by KLF2 or KLF4 is unlikely in the Klf5Δ/Δ bladder epithelium since Klf4 mRNA expression was restricted to the bladder mesenchyme and Klf2 expression was not detectable (http://www.genepaint.org).
The present findings support the concept that activities of KLF5 are highly dependent on developmental context, being critical for embryonic urothelial maturation rather than maintenance of proliferation. These studies demonstrate a novel role for KLF5 as a critical regulator of urothelial maturation during late embryonic development. We propose that Pparγ and Grhl3 may participate in a KLF5 dependent gene network regulating maturation of the urothelium.
KLF5 is required for stratification and terminal differentiation of urothelial cells.
Prenatal defects in maturation of the bladder urothelium can induce hydronephrosis, hydroureter, and vesicoureteral reflux.
KLF5 is not required for maturation and stratification of the bladder neck and urethral epithelium.
Grhl3 and Pparγ are candidate downstream targets of KLF5 that mediate urothelial maturation.
KLF5 dependent signals regulate morphogenesis of the bladder mesenchyme.
This work was funded in part by NIH R01:HL-090156 (J.A.W.). The authors would like to thank Gail Macke for technical assistance and Dr. Ron Pratt from the Imaging Research Center at Cincinnati Children's Hospital for obtaining and reconstructing the original Micro CT scan images. H.M.H. is a visiting scholar at the University of Cincinnati College of Medicine and a clinician funded by the Medical Research Council, London, UK.
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