Transcription factor ZEB2 is essential for ureteral smooth muscle cell differentiation

Sudhir Kumar; Xueping Fan; Harshita Pattam; Kun Yan; Easton J. Liaw; Jiayi Ji; Emily Zaltz; Paul Song; Yuqiao Jiang; Yuriko Nishizaki; Yujiro Higashi; Chen-Leng Cai; Weining Lu

doi:10.1371/journal.pgen.1012028

Abstract

Mowat-Wilson Syndrome (MWS) is an autosomal dominant genetic disorder caused by heterozygous mutations or deletions in the Zinc finger E-box-binding homeobox 2 (ZEB2) gene. Congenital anomalies of the kidney and urinary tract (CAKUT), including hydroureter and hydronephrosis, have been reported in patients with MWS. However, the role of the ZEB2 gene in urinary tract development and the cellular and molecular mechanisms underlying the CAKUT phenotypes in MWS remain unknown. In this study, we examined ZEB2 expression in the developing mouse ureter and generated Zeb2 ureteral mesenchyme-specific conditional knockout mice (Zeb2 cKO) by crossing Zeb2 floxed mice with Tbx18Cre⁺ mice. The urinary tract of Zeb2 cKO mice and their wild-type littermates was analyzed for morphological and histological changes. Our results show that ZEB2 is expressed in TBX18⁺ ureteral mesenchymal cells during mouse ureter development. Deleting Zeb2 in these cells caused hydroureter and hydronephrosis, indicating obstructive uropathy. Cellular and molecular marker analysis revealed that the TAGLN⁺ACTA2⁺ ureteral smooth muscle cell (SMC) layer was absent in Zeb2 cKO mice. In contrast, the tunica adventitia cell layer was significantly expanded compared to controls. At the molecular level, Zeb2 cKO mice had significantly decreased TBX18 expression but increased SOX9 expression in the developing ureter compared to wild-type controls. Our findings demonstrate that ZEB2 is crucial for normal ureteral SMC differentiation during ureter development. Additionally, our study suggests that MWS patients may have abnormal ureteral SMC development, which contributes to the abnormalities of the urinary tract.

Author summary

Hydroureter and hydronephrosis are common congenital anomalies with a high incidence (1:100–1:500) that can lead to obstructive uropathy and renal failure in the pediatric population. Mowat-Wilson Syndrome (MWS), caused by heterozygous mutations in the ZEB2 gene, is a genetic disorder characterized by multiple congenital developmental defects, including hydroureter and hydronephrosis. However, the molecular function of ZEB2 in ureter development and the pathogenesis of hydroureter and hydronephrosis remain unknown. Here, we demonstrate that ZEB2 is expressed in developing ureteral mesenchymal cells, and the deletion of ZEB2 in these cells leads to the loss of ureteral smooth muscle cells, which are replaced by tunica adventitia cells, resulting in a hydroureter and hydronephrosis phenotype. Hence, our work not only demonstrates the critical role of ZEB2 in ureter development but also provides insight into the molecular mechanisms underlying urinary tract anomalies in MWS patients.

Citation: Kumar S, Fan X, Pattam H, Yan K, Liaw EJ, Ji J, et al. (2026) Transcription factor ZEB2 is essential for ureteral smooth muscle cell differentiation. PLoS Genet 22(1): e1012028. https://doi.org/10.1371/journal.pgen.1012028

Editor: Bruce A. Hamilton, University of California San Diego, UNITED STATES OF AMERICA

Received: March 14, 2025; Accepted: January 11, 2026; Published: January 23, 2026

Copyright: © 2026 Kumar et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All data underlying the findings in this work are fully available without restriction. All data are in the manuscript and/or supporting information files.

Funding: This work is supported by research grants from NIH/NIDDK grants R01DK133940 (WL), R01DK078226 (WL), and a DOD grant E01 HT9425-23-1-1058 (WL). This work is also supported by the Career Development Award from the American Heart Association (SK, https://doi.org/10.58275/AHA.24CDA1272560.pc.gr.193662), a BU CTSI Integrated Pilot Grant funded by the Department of Medicine (SK, WL), the Department of Medicine Career Investment Award (SK), and the Boston University Undergraduate Research Opportunities Program (HP, EJL, JJ, EZ, YJ). This publication is also supported in part by the National Center for Advancing Translational Sciences, National Institutes of Health, through BU-CTSI Grant Number 1UL1TR001430. Its contents are solely the authors’ responsibility and do not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The mammalian ureters consist of two tissue compartments, each containing three cell types. The outer mesenchymal compartment has an outer layer of fibrocytes of the tunica adventitia, the middle smooth muscle cell (SMC) layer, and the inner lamina propria layer. The inner compartment is composed of epithelial cell layers [1–4]. The three cell layers of the mesenchymal compartment are derived from the TBX18⁺ ureteric mesenchymal progenitors [4]. Between E15.5 and E16.5, a subset of TBX18⁺ ureteric mesenchymal cells differentiates into the middle SMC layer [4,5]. Any abnormality in ureteral SMC development impairs the formation of a functional ureter, leading to ureter dilation, hydroureter, hydronephrosis, and obstructive uropathy [1–3]. Hydroureter and hydronephrosis have a high incidence (1:100–1:500) in the pediatric population and are a leading cause of obstructive uropathy and renal failure [6,7].

Mowat-Wilson Syndrome (OMIM #235730; MWS), caused by heterozygous mutations or deletions in the Zinc finger E-box-binding homeobox 2 (ZEB2) gene, is a multiple congenital anomaly syndrome characterized by an atypical face, moderate-to-severe intellectual retardation, epilepsy, and variable congenital malformations, including Hirschsprung disease (HSCR), congenital heart defects, agenesis of the corpus callosum, and eye anomalies [8–14]. Over 50% of MWS patients have urogenital/renal anomalies, including duplex kidney, pelvic kidney, vesicoureteral reflux (VUR), hydroureter, and hydronephrosis [15–18]. Although many MWS patients develop hydroureter and hydronephrosis, the molecular function of ZEB2 in ureter development and the pathogenesis of CAKUT have not been studied.

ZEB2 is a transcription factor and a member of the zinc-finger E-box–binding (ZEB) protein family. ZEB2 protein plays a crucial role in determining cell fate and the differentiation of embryonic stem cells, including hematopoietic progenitors and neural progenitor cells [19–28]. Previously, we demonstrated that loss of the Zeb2 gene in nephron progenitors leads to glomerulocystic kidney and renal failure [29]. We also found that the Zeb2 gene controls the differentiation of FOXD1 + stromal progenitor cells, and the loss of ZEB2 in kidney stromal progenitors leads to kidney fibrosis [30]. In this study, we hypothesize that ZEB2 also plays a crucial role in ureter development, and the loss of the Zeb2 gene in the developing ureter results in abnormal ureter morphogenesis, hydroureter, and hydronephrosis. To test this hypothesis, we analyzed ZEB2 expression in the developing mouse ureter and generated ureter-specific Zeb2 conditional knockout mice to investigate its role in ureter development.

Our results show that ZEB2 is highly expressed in the TBX18⁺ ureteral mesenchymal cells. Deletion of the Zeb2 gene in mouse ureteral mesenchymal cells using Tbx18Cre⁺ led to hydroureter and hydronephrosis. Cellular marker analyses showed that ureteral mesenchyme-specific Zeb2 conditional knockout mice (also called Zeb2 cKO in this paper) had no TAGLN⁺ACTA2⁺ ureteral smooth muscle cells (SMCs) but instead had an expanded layer of FOXD1⁺POSTN⁺ ureteral tunica adventitia cells during early ureter development. At the molecular level, we found that Zeb2 knockout ureters had reduced TBX18 expression with an upregulated SOX9 expression, which are critical transcriptional factors for ureteral SMC development. Our results indicate that ZEB2 is essential for the differentiation of ureteral SMCs, and loss of ZEB2 leads to depletion in the ureteral SMC layer that is replaced by the tunica adventitia layer, eventually leading to hydroureter, hydronephrosis, obstructive uropathy, and renal failure.

Result

ZEB2 is expressed in TBX18⁺ ureteral mesenchymal cells in the developing ureter

To elucidate the role of Zeb2 during ureter development, we first examined the expression of ZEB2 using immunostaining. We found that ZEB2 is highly expressed in the undifferentiated mesenchyme cells in the developing mouse ureters (Fig 1A-1B). We also confirmed Zeb2 expression in the ureter by in situ hybridization (RNAscope) (Fig 1C). To determine the cell-specific expression of ZEB2, we performed marker analysis by double immunostaining of ZEB2 with ureteral mesenchymal cell marker TBX18. Immunostaining and RNAscope analysis showed that ZEB2 is colocalized with TBX18 (Fig 1C-1D). We also examined ZEB2 expression in urothelial cells by performing double immunostaining of ZEB2 and E-cadherin-1 (CDH1, urothelial cell marker) and found that ZEB2 was not expressed in these cells (Fig 1E). These results suggest that ZEB2 is expressed mainly in TBX18⁺ ureteral mesenchymal cells during ureter development.

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Fig 1. ZEB2 is expressed in TBX18⁺ ureteral mesenchymal cells in the developing ureter.

(A) ZEB2 (green) expression in the proximal section of the developing ureter at the stages (E13.5, E14.5, E15.5) in the wild-type mouse. (B) The panel shows ZEB2 expression at E18.5 and P0. (C) Analysis of Zeb2 expression at E14.5 and co-localization with Tbx18 by RNAscope in situ hybridization. (D) ZEB2 (green) is co-localized with ureteral mesenchymal progenitor cell marker TBX18 (red) in the mouse developing ureter at E14.5. (E) ZEB2 (green) is not co-localized with CDH1 (urothelial cell marker) in the developing mouse ureter. Dotted lines encircle the ureter. Dotted box regions in panel C show an enlarged inset in panel C. IF: immunofluorescence. Scale bars: 25 µm (A-E). Number of animals analyzed: n = 3 per group.

https://doi.org/10.1371/journal.pgen.1012028.g001

Loss of Zeb2 in TBX18⁺ ureteral mesenchymal cells leads to hydroureter, hydronephrosis, and obstructive uropathy

To understand the function of ZEB2 in TBX18⁺ ureteral mesenchymal cells, we generated Zeb2 conditional knockout mice, Zeb2^flox/flox;Tbx18Cre⁺ (Zeb2 cKO), by crossing Zeb2 floxed mice [31] with Tbx18Cre⁺ mice [32]. The deletion of Zeb2 in ureteral mesenchymal cells was confirmed by immunostaining, which revealed the absence of ZEB2 signals in the ureter of Zeb2 cKO mice compared to wild-type controls (Fig 2A). Zeb2 cKO mice were born in the Mendelian ratio (Table 1), but most homozygous Zeb2 cKO mice died within a few hours after birth, and only a few survived up to 3 weeks of age (Table 1). The 3-week-old Zeb2 cKO mice were runted and hunched (Fig 2B) and developed hydroureter and hydronephrosis phenotypes (Fig 2C-2D). Some Zeb2 cKO mice were found to have high-grade hydroureter, which may be classified as megaureters. We also analyzed newborn mice (P0) and found that homozygous Zeb2 cKO newborn pups also developed hydroureter and hydronephrosis phenotypes as well (Fig 2E-2G). The high-grade hydroureter in Zeb2 cKO mice prompted us to investigate whether the ureterovesical junction (UVJ) is similarly affected. We examined the UVJ in newborn Zeb2 cKO mice and wild-type controls. In morphological and histological analyses, we found that Zeb2 cKO mice exhibited partial UVJ obstruction compared to wild-type mice (S1A-D Fig). We also analyzed whether hydronephrosis leads to tubule dilations in the kidneys; we found that 3-week-old Zeb2 cKO mice had significantly dilated tubules (S2 Fig). Taken together, our data suggest that in the absence of ZEB2, ureters do not develop properly, leading to hydroureter and hydronephrosis.

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Table 1. Zeb2 cKO mice were born in a Mendelian ratio but died after birth.

https://doi.org/10.1371/journal.pgen.1012028.t001

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Fig 2. Loss of Zeb2 in TBX18⁺ ureteral mesenchymal cells leads to hydroureter, hydronephrosis, and obstructive uropathy.

(A) ZEB2 (green) expression is absent in Zeb2^flox/flox;Tbx18Cre⁺ knockout mice (Zeb2 cKO) as compared to wild-type controls. (B) Three-week-old Zeb2 cKO mice were runted with smaller body sizes compared to wild-type control littermates. (C) 3-week-old Zeb2 cKO mice developed hydroureter and hydronephrosis. (D) H&E staining of the kidney of a 3-week-old Zeb2 cKO mouse showing a dysplastic kidney with a dilated ureter. (E) Newborn (P0) Zeb2 cKO mice develop hydroureter and hydronephrosis. (F) Histology of a newborn (P0) Zeb2 cKO mouse ureter with hydroureter as compared to wild-type littermate controls. (G) Histology of a newborn (P0) Zeb2 cKO mouse kidney with hydronephrosis (*). u: ureter, k: kidney, UB: urinary bladder. Dotted lines encircle the ureter. Scale bars: 25 µm (A); 1000 µm (D and G). Number of animals analyzed: n = 5 per group.

https://doi.org/10.1371/journal.pgen.1012028.g002

Ureteral mesenchymal progenitors fail to differentiate into smooth muscle cells in the absence of ZEB2

During ureter development, a subset of TBX18 + ureteral mesenchymal cells differentiate into smooth muscle cells (SMCs), which are crucial for functional ureter formation and prevent ureter dilation after urine production [2,5]. To check the SMC formation in the developing ureter of Zeb2 cKO mice, we examined two established SMC markers, the actin alpha 2, smooth muscle (ACTA2, Gene ID: 59), and transgelin (TAGLN, Gene ID: 6876) [4,33]. Pan-cytokeratin was used to demarcate the urothelial cells [34]. Analysis at the proximal ureter level revealed that wild-type mice formed a normal ACTA2⁺TAGLN⁺ SMC layer at E15.5; however, this layer was absent in age-matched Zeb2 cKO mice (Fig 3A-3B, S1 Data). TBX18⁺ ureteral mesenchymal cells also give rise to the tunica adventitia layer that expresses marker genes forkhead box D1 (FOXD1, Gene ID: 2297) and periostin (POSTN, Gene ID: 10631) [4]. We, therefore, asked if there is any change in the development of the tunica adventitia ureter layer in the absence of ZEB2. We examined the tunica adventitia cells by specific antibody immunostaining and found an expanded FOXD1⁺POSTN⁺ tunica adventitia layer in the developing ureter of Zeb2 cKO mice as compared to their wild-type littermate controls (Fig 3C-3D, S1 Data). To determine if there is delayed SMC differentiation in Zeb2 cKO mice, we analyzed the SMC layer development at later stages (E16.5 and P0). We found that the SMC layer did not develop in Zeb2 cKO mice at these stages either (Fig 4A-4B). However, the adventitia cell layer was found to be expanded in Zeb2 cKO mice, as evidenced by POSTN immunostaining at E16.5 (Fig 4C). We also analyzed two additional markers for the tunica adventitia, collagen type 1 alpha 2 (COL1A2) and BMP-binding endothelial regulator (BMPER) [33]. In the wild-type mice, expression of BMPER was restricted to tunica adventitia and urothelial cells; however, there was an expansion of BMPER expression in the Zeb2 cKO mice, and a significant upregulation was observed in the mesenchymal cells (S3A Fig.). This data suggests that ZEB2 may regulate BMP signaling during ureter development [35], as has been reported in neural crest formation. Adventitia marker COL1A2 was also found to be upregulated in Zeb2 cKO mice compared to wild-type controls (S3B Fig), We also analyzed the lamina propria development in Zeb2 cKO mice with ALDH1A2 immunostaining [36]. We found that there is absence of ALDH1A2 expression in the ureters of newborn Zeb2 CKO mice as compared to controls (Fig 4D), suggesting that the lamina propria did not differentiate properly in these mice.

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Fig 3. Ureteral mesenchymal progenitors fail to differentiate into smooth muscle cells in the absence of ZEB2.

(A, B) Ureteral smooth muscle cells are formed in the wild-type mice at E15.5, specified by smooth muscle markers TAGLN (A) and ACTA2 (B). There are no TAGLN⁺ and ACTA2⁺ ureteral smooth muscle cells in Zeb2 cKO mice. Pan-cytokeratin (Pan-cyto) is used to mark urothelial cells. (C, D) Compared to wild-type littermate controls, ureteral tunica adventitia cell layers labeled by POSTN⁺ and FOXD1⁺ markers are expanded in Zeb2 cKO mice. Scale bars: 10 µm (A-D). Number of animals analyzed: n = 3 per group. Sections of the proximal ureter region were analyzed. Values are expressed as mean ± SEM. Student’s t-test is used for statistical significance. **P < 0.01, ***P < 0.001.

https://doi.org/10.1371/journal.pgen.1012028.g003

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Fig 4. Zeb2 cKO mice did not show delayed SMC development.

(A-B) Immunofluorescent staining of ACTA2 (smooth muscle cell marker) at the proximal ureter sections at E16.5 and P0 in the Zeb2 cKO mice and wild-type controls. Pan-cytokeratin (Pan-cyto) and uroplakin III (UPKIII) were used to mark urothelial cells. No SMC layer was found in Zeb2 cKO mice as compared to the wild-type controls. (C) Immunofluorescent staining of POSTN (tunica adventitia cell marker) at the proximal ureter sections at E16.5 in the Zeb2 cKO mice and wild-type controls. (D) Immunofluorescent staining of ALDH1A2 (lamina propria cell marker, marked by arrows) at the proximal ureter sections at P0 in the Zeb2 cKO mice and wild-type controls. Scale bars: 25 µm (A-C), 50 µm (D). Number of animals analyzed: n = 4 per group.

https://doi.org/10.1371/journal.pgen.1012028.g004

To check if urothelial cell differentiation was also disturbed in Zeb2 cKO developing ureter, we analyzed the expression of E-cadherin (CDH1, urothelial cell stratification marker), KRT5 (basal cell marker), UPK1B (intermediate and superficial cell marker), and ∆NP63 (basal and intermediate cell marker) [4]. We found that Zeb2 cKO mice had significantly reduced expression of CDH1 and ∆NP63 in the urothelial cells (Fig 5A-5B). However, other urothelial cell markers (KRT5 and UPK1B) were found to be unaffected in the ureters of Zeb2 cKO mice, suggesting KRT5⁺ and UPK1B⁺ urothelial cells were present in these mice (Fig 5C-5D). These data indicate that urothelial cell stratification was affected in Zeb2 cKO mice. The defective stratification of the urothelial cells may be due to abnormal ureteral mesenchymal differentiation, as the ureteral mesenchymal cells crosstalk with the urothelial cells during ureter development [37]. Taken together, our data suggest that ZEB2 is essential for the normal differentiation of the ureteral mesenchymal cells into SMCs to form functional ureters during development, and loss of ZEB2 causes the absence of SMCs, which leads to hydroureter and hydronephrosis.

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Fig 5. Analysis of urothelium cell differentiation on proximal ureters in Zeb2 cKO mice.

(A) CDH1⁺ urothelium cells are underdeveloped in Zeb2 cKO mice compared to wild-type littermate controls. (B-D) Immunofluorescent staining of ∆NP63 (basal and intermediate cell marker), KRT5 (basal cell marker), and UPK1B (intermediate and superficial cell marker), at the proximal ureter sections in P0 Zeb2 cKO mice and wild-type controls. Urothelial cell differentiation was found to be unaffected in the ureters of Zeb2 cKO mice. Scale bars: 10 µm (A), 50 µm (B-D). Number of animals analyzed: n = 4 per group.

https://doi.org/10.1371/journal.pgen.1012028.g005

Loss of ZEB2 alters the expression of key transcription factors required for proper ureteral SMC differentiation and development

Transcription factor Tbx18 is the earliest marker for the ureteric mesenchyme, which specifies it and gives rise to all the mesenchymal cells of the ureters [5,38]. Previously, it has been shown that TBX18 (Gene ID: 9096) and SOX9 (Gene ID: 6662) are essential for ureteral SMC layer development, and loss of TBX18 or SOX9 leads to the absence of ureteral SMCs, resulting in hydroureter [5,39]. We performed TBX18 and SOX9 immunostaining on the proximal ureter sections and examined their expressions to understand the molecular and phenotypic changes in the Zeb2 cKO ureters. We found a significant reduction in TBX18 expression in the Zeb2 cKO mouse ureter at E14.5 and E15.5 (Fig 6A-6B, S2 Data). Interestingly, SOX9 expression was significantly upregulated in the E15.5 Zeb2 cKO mouse ureter as compared to their wild-type littermate controls (Fig 6C-D, S2 Data). However, SOX9 was significantly lower in epithelial cells at E15.5 in Zeb2 cKO mice as compared to controls, suggesting that epithelial cells did not differentiate properly as we mentioned before. We also examined the expression of these markers using RNAscope (in situ hybridization) and confirmed a reduction in Tbx18 expression, and an increase in Sox9 expression in the Zeb2 cKO mouse ureter compared to the wild-type control (Fig 7A-7B). These data suggest that loss of ZEB2 perturbs the expression of key transcription factors such as TBX18 and SOX9, which are critical for proper ureteral SMC differentiation and ureter development.

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Fig 6. Loss of ZEB2 altered the expression of key factors required for proper ureteral SMC differentiation and development.

(A-B) Immunostaining of TBX18 on the proximal ureter sections shows decreased TBX18 (green) expression in ureteral mesenchymal progenitors at E14.5 and E15.5 in Zeb2 cKO mice compared to wild-type controls. We excluded artifact staining in panel B in wildtype sections (marked by *) in the analysis. (C-D) Immunostaining of SOX9 on the proximal ureter sections shows increased SOX9 (green) expression in ureteral mesenchymal progenitors at E14.5 and E15.5 in Zeb2 cKO mice compared to wild-type controls. Scale bars: 10 µm (A-D). Number of animals analyzed: n = 3 per group. Values are expressed as mean ± SEM. Student’s t-test is used for statistical significance. **P < 0.01, ***P < 0.001.

https://doi.org/10.1371/journal.pgen.1012028.g006

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Fig 7. RNAscope (in situ hybridization) analysis on proximal ureters for Tbx18 and Sox9 expression in Zeb2 cKO mice.

(A) RNAscope of Tbx18 mRNA on the proximal ureter sections shows decreased Tbx18 (red) expression in ureteral mesenchymal progenitors at E14.5 in Zeb2 cKO mice compared to wild-type controls. (B) RNAscope of Sox9 mRNA on the proximal ureter sections shows increased Sox9 (red) expression in ureteral mesenchymal progenitors at E15.5 in Zeb2 cKO mice compared to wild-type controls. Scale bars: 25 µm (A-B). Number of animals analyzed: n = 3 per group.

https://doi.org/10.1371/journal.pgen.1012028.g007

As Zeb2 cKO mice had an expanded FOXD1⁺POSTN⁺ tunica adventitia layer and increased SOX9 expression. We examined whether markers of tunica adventitia (FOXD1 and POSTN) colocalize with SOX9. We performed RNAscope and immunostaining at the proximal ureter level at different embryonic stages and found that Foxd1 and Sox9 colocalize at the same level on the outer side in Zeb2 cKO mice compared to wild-type controls. However, Zeb2 cKO mice had expanded POSTN expression that colocalized with SOX9 as compared to wild-type controls (S4A-C Fig). To determine if these expression alterations affect the proliferation of ureteral cells, we examined the cell proliferation by performing phosphohistone H3 (PHH3) immunostaining on the proximal ureter sections at E14.5 and E15.5. We did not observe any changes in the cell proliferation in Zeb2 cKO mice as compared to wild-type control (S5A-B Fig, S3 Data). We also investigated whether mesenchymal cell condensation was disturbed in Zeb2 cKO mice, as TBX18 is required for ureteral mesenchymal cell condensation [5]. Histological examination showed that ureteral mesenchymal cells were abnormally condensed, compacted, and disorganized in Zeb2 cKO pups as compared to the wild-type controls (S6 Fig). These data suggest that loss of ZEB2 leads to mesenchymal patterning defects, which cause SMC loss in Zeb2 cKO mice ureters.

Discussion

Mowat-Wilson Syndrome (OMIM #235730; MWS) is caused by heterozygous mutations or deletions in the ZEB2 gene, and more than half of MWS patients have urinary tract anomalies, including duplex kidney, pelvic kidney, VUR, and hydronephrosis [8–18,27]. In this report, we found that ZEB2 is expressed in the TBX18⁺ ureteral mesenchymal cells, and deletion of Zeb2 by Tbx18Cre⁺ leads to hydroureter, hydronephrosis, and obstructive uropathy. We found that Zeb2 cKO mice do not develop the ureteral SMC layer but have an expanded layer of tunica adventitia cells. At the molecular level, we found that loss of ZEB2 leads to a reduced expression of transcription factor TBX18 but an increased expression of SOX9 in the ureteral mesenchymal cells, both of which are critical for ureteral SMC formation (Fig 8). These findings, hence, provide new molecular insights into the novel role of ZEB2 in the differentiation and development of ureteral SMCs.

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Fig 8. Proposed Model: Transcription factor ZEB2 is essential for ureteral smooth muscle cell differentiation during ureter development.

https://doi.org/10.1371/journal.pgen.1012028.g008

In recent years, progress has been made in understanding the molecular signals regulating ureteral SMC development [2,3,40]. The factors controlling ureter development have been reported in a recent review and some are major factor are Sonic Hedgehog signaling, WNT signaling, Tbx18, Sox9, Bmp4, Six1, Foxf1, Smo, and Smad4 [1–3,5,33,37,40–46]. Tbx18 regulates the specification and development of the ureteric mesenchyme, and Tbx18^-/- knockout mice do not develop the SMC layer [5]. SOX9 is also crucial for ureteral mesenchymal cell differentiation, and loss of SOX9 also leads to the absence of SMCs [39]. Interestingly, Zeb2 cKO mice also failed to develop SMCs and exhibited decreased expression of TBX18, but increased expression of SOX9 in the developing ureteral mesenchymal cells. Previously, it has been demonstrated that ZEB2 antagonizes the inhibitory effect of SOX2, a member of the family of SOX proteins, on Schwann cell differentiation and myelination [47,48]. ZEB2 also controls SMC phenotypic transition through chromatin remodeling, and loss of ZEB2 leads to differentially open chromatin for mature SMC regulatory markers such as Cnn1, Myh11, and Myocd, and chondrogenesis-related genes such as Spp1, Sox9, and Col2a1 [49]. Our findings in this study discover ZEB2 as a novel ureter regulatory gene and suggest that ZEB2 might antagonize the inhibitory effect of SOX9 during ureteral SMC differentiation and development by altering chromatin accessibility. Further studies are needed to elucidate how ZEB2 interacts with TBX18 and SOX9 during ureter development as well as the downstream genes that ZEB2 regulates in the developing ureter.

We also observed an expansion of the ureteral tunica adventitia cells in the Zeb2 cKO mice in addition to the loss of SMCs in the developing ureter. TBX18⁺ mesenchymal progenitors can subdivide into the inner and outer mesenchymal cells during ureter development. The inner mesenchymal cells further differentiate into the functional SMC layer to support ureter unidirectional peristalsis and prevent ureter dilation; in contrast, the outer compartment of TBX18⁺ mesenchymal cells gives rise to tunica adventitia cells [4]. Expansion of tunica adventitia cells in Zeb2 cKO mice might be due to failure of the TBX18⁺ mesenchymal progenitors to separate into inner and outer compartments in the absence of ZEB2. As a result, all TBX18⁺ mesenchymal progenitors differentiate into tunica adventitia cells. Alternatively, loss of the SMC layer in the Zeb2 cKO may stimulate the expansion of the ureteral tunica adventitia layer. Thirdly, this unexpected cellular phenotype of expanded ureteral tunica adventitia layer could also be driven by upregulated SOX9 expression in the ureteral mesenchymal cells, as prolonged expression of SOX9 in the ureteral mesenchyme reduces smooth muscle gene expression and increases the expression of extracellular matrix (ECM) components, as previously reported [39]. Lastly, a recent study also suggests that SMC-specific loss of ZEB2 results in an inability to turn off contractile programming, leading to SMCs transitioning into a fibroblast-like phenotype [49]. Further studies are needed to confirm if ZEB2 specifically regulates early ureteral mesenchymal cell specification during ureter development and if the expanded ureteral tunica adventitia layer phenotype is merely a compensatory response to SMC loss in Zeb2 cKO mice.

In past years, much progress has been made in understanding the role of ZEB2 during nervous system development [22,50]. However, the role of ZEB2 in the urinary system is poorly understood, despite more than 50% of MWS patients being reported to have urinary tract anomalies. Our current work not only demonstrates the critical role of ZEB2 in ureter development but also provides molecular genetic insights into some aspects of MWS-associated phenotypes in the urinary tract.

In summary, we found that loss of ZEB2 in ureteral mesenchymal cells leads to ureteral SMC loss, which eventually causes hydroureter, hydronephrosis, and obstructive uropathy (Fig 8). Our data suggest that ZEB2 is essential for ureteral SMC differentiation and development during ureter development. Our study also indicates that urinary tract anomalies are the primary phenotype in MWS patients and that MWS patients should be evaluated for urinary tract disorders such as hydroureter, hydronephrosis, and obstructive uropathy.

Materials and methods

Ethics statement

All animal experiments were performed according to the guidelines issued by the Institutional Animal Care and Use Committee of Boston University Medical Campus at Boston University (approved IACUC protocol number: PROTO201800056_TR01).

Animals

Zeb2 floxed mice [31] and Tbx18Cre⁺ mice [32] were genotyped using primers previously reported. The ZEB2-EGFP knock-in reporter mouse was studied previously [51]. Eight-week-old Zeb2^flox/+;Tbx18Cre⁺ mice were bred with Zeb2^flox/flox mice to generate Zeb2^flox/flox;Tbx18Cre⁺ homozygous conditional knockout mice. All mice had free access to drinking water and a standard rodent diet ad libitum. Animals of both sexes with mixed C57BL/6 x 129 genetic backgrounds were used in this study. Wild-type littermates were used as controls. Timed-pregnant females were confirmed by a semen plug and designated at embryonic day 0.5 (E0.5) of gestation and were separated from the breeding cage.

Tissue preparation and histology

Gross urinary tracts of newborn Zeb2 cKO mice and wild-type littermates were dissected and imaged with an Olympus inverted dissecting microscope (Olympus, USA). For histological analysis, urinary tracts were fixed in 4% paraformaldehyde or 10% neutral buffered formalin at 4°C overnight and then processed for paraffin embedding using standard protocols. Serial kidney sections were cut and stained using a Periodic acid Schiff (PAS) stain kit (# 24200, Polysciences, Inc., USA) and Hematoxylin and Eosin (H&E) Stain Kit (H-3502, Vector Labs, USA), according to the manufacturer’s instructions. For immunohistochemistry, paraffin-embedded tissue sections were deparaffinized and steamed for 30 minutes in an antigen retrieval solution (IHC-Tek antigen retrieval solution, IW-1100, IHCworld LLC, USA), or antigen retrieval buffer (100X EDTA Buffer, ab93680, pH 8.0, abcam, USA). Slides were examined with an Olympus upright light microscope and photographed using an Olympus DP72 digital camera.

Immunofluorescence staining and confocal microscopy

Embryos were harvested from timed-pregnant females at embryonic day (E) 13.5, 14.5, 15.5, 16.5, E18.5, and P0. Whole embryos were fixed in 4% formaldehyde overnight at 4°C followed by incubation in 30% sucrose overnight at 4°C, embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura Finetek), and cryosectioned at 8–10 μm. Frozen sections were permeabilized with PBS containing 0.1% Triton X-100 for 10 minutes and blocked in 5% serum-blocking buffer or background buster (NB306, Innovax biosciences, USA) for 1 hour at room temperature. Primary antibodies were incubated overnight at 4°C, followed by secondary antibodies at room temperature for 1 hour. Following primary antibodies were used: ZEB2 (1:100, HPA003456; Sigma-Aldrich, USA or SC-271984, Santa Cruz Biotechnology, USA), TBX18 (1:100, ab115262, Abcam, USA), E-cadherin (1:50, Clone 36/610181; BD Biosciences, USA), ACTA2 (1:200, A2547; Sigma-Aldrich, USA), Uroplakin III (1:20, 610108, Progen, Germany), Cytokeratin (1:100, C2562, Sigma-Aldrich, USA), TAGLN (1:200, ab14106, Abcam, USA), ALDH1A2 (1:200, ab75674, Abcam, USA), Cytokeratin 5 (1:100, ab52635, Abcam, USA), UPK1B (1:100, WH0007348M2; Sigma-Aldrich, USA) ∆NP63 (1:100, ab735, Abcam, USA), BMPER (1:100, AF2299, R&D systems, USA), COL1A2 (1:100, 14695–1-AP, Proteintech, USA), PHH3 (1:100, 9701, Cell Signaling Technology, USA), FOXD1 (1:100 dilution, sc-47585; Santa Cruz Biotechnology USA), POSTN (1:100, ab14041, Abcam, USA), SOX9 (1:200, AB5535, Millipore, USA), GFP (GFP-1020, 1:100, Aves Labs, USA or A11122, 1:250; Invitrogen, Carlsbad, CA). Secondary antibodies were goat anti-mouse IgG2a AF594, goat anti-mouse IgG1 AF488, goat anti-rabbit IgG AF488, goat anti-rabbit IgG AF494, and donkey anti-rabbit IgG AF594. Sections were stained with DAPI (4´,6´-´diamidino-2-phenylindole) and mounted in Vectashield antifade mounting medium (H-1000; Vector Labs, USA). Images were captured by a Zeiss LSM 700 confocal microscope (Zeiss, Germany) and analyzed with ImageJ software (National Institutes of Health, USA).

In situ hybridization (RNAscope) assay

In situ hybridization (RNAscope) assay was performed according to the manufacturer’s instructions using the RNAscope multiplex fluorescent detection kit V2 (Cat No. 323110, Advanced Cell Diagnostics USA) as previously described [30]. Briefly, ureters fixed in 4% paraformaldehyde (PFA) were re-fixed in 4% PFA in 1 × PBS at 4°C for 15 min and rinsed twice in 1 × PBS. The slides were then dehydrated in 50%, 70%, and 100% ethanol for 5 min/each. After that, the ureter tissues were incubated in hydrogen peroxide for 10 min at room temperature, rinsed in water, and then incubated in RNAscope protease IV for 30 min at room temperature, followed by two washes in 1 × PBS. The tissues were then incubated with probes of Zeb2 (RNAscope Probe- Mm-Zeb2, Cat No. 436391; Advanced Cell Diagnostics USA), Tbx18 (RNAscope Probe- Mm-Tbx18-C2, Cat No. 515221-C2; Advanced Cell Diagnostics USA), Sox9 (RNAscope Probe- Mm-Sox9-C2, Cat No. 401051-C2; Advanced Cell Diagnostics USA), and Foxd1 (RNAscope Probe- Mm-Foxd1-C3, Cat No. 495501-C3; Advanced Cell Diagnostics USA) for 2 hours at 40°C, followed by two washes in 1 × PBS. The probe signal was amplified by incubating with RNAscope multiplex FL V2 Amp 1 for 30 min at 40°C, then twice rinsed in 1 × PBS. Finally, the signal was developed by incubating with RNAscope multiplex FL V2 C1, and subsequently with Opal 570 (FP1488001KT, 1:1000 dilution; Akoya Biosciences, USA). After rinsing twice in 1 × PBS, the slides were counterstained with DAPI and mounted in Prolong Diamond antifade mounting medium (Cat No. P36970; Invitrogen USA). Images were captured using a Zeiss 700 confocal microscope (Zeiss, Germany) and analyzed with the NIH ImageJ software.

Image quantification

Image quantification was performed in ImageJ on random images (n = 3 per group). Cell counter or particle analysis plug-in tools were used to quantify the cell numbers. For intensity quantification, the percentage of total pixel area was measured.

Statistics

All statistical analyses were performed using GraphPad Prism statistical software (version 7.0, GraphPad, San Diego, CA) with P < 0.05 considered statistically significant. Wild-type and Zeb2 cKO mice group data were compared using a two-tailed unpaired Student’s t-test. Data are represented as mean ± SEM. A minimum of three mice were used for each analysis group unless stated otherwise.

Disclosures

All authors have nothing to disclose.

Supporting information

S1 Fig. Morphological and histological analysis of ureterovesical junction (UVJ) in Zeb2 cKO mice.

(A) Whole urinary tract system of P0 Zeb2 cKO mice and wild-type controls. (B) Highlighted area in panel A. (C) H&E staining of UVJ in P0 Zeb2 cKO mice and wild-type controls. Zeb2 cKO mice. (D) Highlighted area in panel C. Zeb2 cKO mice had partial obstruction of the UVJ as compared with wild-type control mice. Number of animals analyzed: n = 3 per group. U = ureter, UB = Urinary bladder.

https://doi.org/10.1371/journal.pgen.1012028.s001

(JPG)

S2 Fig. Analysis of kidney tubule dilation in Zeb2 cKO mice: H&E staining of kidneys of 3-week-old Zeb2 cKO mice and wild-type controls.

Zeb2 cKO mice had significantly dilated tubules due to hydronephrosis. Number of animals analyzed: n = 3 per group.

https://doi.org/10.1371/journal.pgen.1012028.s002

(JPG)

S3 Fig. Analysis of additional tunica adventitia cell markers.

(A) Immunofluorescent staining of BMP-binding endothelial regulator (BMPER, green) and collagen 1a2 (COL1A2, red) at the proximal ureter sections at E15.5 in the Zeb2 cKO mice and wild-type controls. (B) Immunofluorescent staining of BMP-binding endothelial regulator (BMPER, green) and collagen 1a2 (COL1A2, red) at the proximal ureter sections at E16.5 in the Zeb2 cKO mice and wild-type controls. Scale bars: 25 µm (A-B). Number of animals analyzed: n = 3 per group.

https://doi.org/10.1371/journal.pgen.1012028.s003

(JPG)

S4 Fig. Co-localization of SOX9 and tunica adventitia cell markers.

(A) RNAscope of Foxd1 (green) and Sox9 (red) at the proximal ureter sections at E14.5 in the Zeb2 cKO mice and wild-type controls. (B) RNAscope of Foxd1 (green) and Sox9 (red) at the proximal ureter sections at E15.5 in the Zeb2 cKO mice and wild-type controls. (C) Immunofluorescent staining of SOX9 (green) and POSTN (red) at the proximal ureter sections at E15.5 in the Zeb2 cKO mice and wild-type controls. The expanded SOX9 expression was marked with arrows in panel C. Scale bars: 25 µm (A-C). Number of animals analyzed: n = 3 per group.

https://doi.org/10.1371/journal.pgen.1012028.s004

(JPG)

S5 Fig. Proliferation analysis in Zeb2 cKO mice.

(A) Immunofluorescent staining of PHH3 at the proximal ureter sections at E14.5 in the Zeb2 cKO mice and wild-type controls. (B) Immunofluorescent staining of PHH3 at the proximal ureter sections at E15.5 in the Zeb2 cKO mice and wild-type controls. Scale bars: 25 µm (A-B). Number of animals analyzed: n = 3 per group. Values are expressed as mean ± SEM. Student’s t-test is used for statistical significance. N.S. = not significant.

https://doi.org/10.1371/journal.pgen.1012028.s005

(JPG)

S6 Fig. Ureteral mesenchymal cells were not condensed adequately in the absence of ZEB2.

(A) H&E staining showing normal condensation of ureteral mesenchymal cells in the wild-type mice at E14.5, but it was compacted and disorganized in Zeb2 cKO mice. Scale bars: 50 µm. Number of animals analyzed: n = 3 per group.

https://doi.org/10.1371/journal.pgen.1012028.s006

(JPG)

S1 Data. Minimal Data set for Figure 3: Ureteral mesenchymal progenitors fail to differentiate into smooth muscle cells in the absence of ZEB2.

https://doi.org/10.1371/journal.pgen.1012028.s007

(XLSX)

S2 Data. Minimal data set for Figure 6: Loss of ZEB2 altered the expression of key factors required for proper ureteral SMC differentiation and development.

https://doi.org/10.1371/journal.pgen.1012028.s008

(XLSX)

S3 Data. Minimal data set for Supplementary Figure 5: Proliferation analysis in Zeb2 cKO mice.

https://doi.org/10.1371/journal.pgen.1012028.s009

(XLSX)

Acknowledgments

We thank Drs. David J. Salant, Laurence Beck, and Hila Milo Rasouly for their helpful discussion, as well as comments from colleagues of the Boston University Academic Writing program and the Kidney Research Program of the Nephrology Section. We thank Mingi Park, Sarayu Chandiri, and Ria Mantri for their help with genotyping Zeb2-floxed mice. We thank Simon Zhang for his help with RNAscope. We thank Boston University’s Analytical Instrumentation Core, Cellular Imaging Core, and Confocal Microscopy Facility for their support in sample and tissue analysis.

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Figures

Abstract

Author summary

Introduction

Result

ZEB2 is expressed in TBX18+ ureteral mesenchymal cells in the developing ureter

Loss of Zeb2 in TBX18+ ureteral mesenchymal cells leads to hydroureter, hydronephrosis, and obstructive uropathy

Ureteral mesenchymal progenitors fail to differentiate into smooth muscle cells in the absence of ZEB2

Loss of ZEB2 alters the expression of key transcription factors required for proper ureteral SMC differentiation and development

Discussion

Materials and methods

Ethics statement

Animals

Tissue preparation and histology

Immunofluorescence staining and confocal microscopy

In situ hybridization (RNAscope) assay

Image quantification

Statistics

Disclosures

Supporting information

S1 Fig. Morphological and histological analysis of ureterovesical junction (UVJ) in Zeb2 cKO mice.

S2 Fig. Analysis of kidney tubule dilation in Zeb2 cKO mice: H&E staining of kidneys of 3-week-old Zeb2 cKO mice and wild-type controls.

S3 Fig. Analysis of additional tunica adventitia cell markers.

S4 Fig. Co-localization of SOX9 and tunica adventitia cell markers.

S5 Fig. Proliferation analysis in Zeb2 cKO mice.

S6 Fig. Ureteral mesenchymal cells were not condensed adequately in the absence of ZEB2.

S1 Data. Minimal Data set for Figure 3: Ureteral mesenchymal progenitors fail to differentiate into smooth muscle cells in the absence of ZEB2.

S2 Data. Minimal data set for Figure 6: Loss of ZEB2 altered the expression of key factors required for proper ureteral SMC differentiation and development.

S3 Data. Minimal data set for Supplementary Figure 5: Proliferation analysis in Zeb2 cKO mice.

Acknowledgments

References

ZEB2 is expressed in TBX18⁺ ureteral mesenchymal cells in the developing ureter

Loss of Zeb2 in TBX18⁺ ureteral mesenchymal cells leads to hydroureter, hydronephrosis, and obstructive uropathy