Smad4 Regulates Ureteral Smooth Muscle Cell Differentiation during Mouse Embryogenesis

Proper formation of ureteral smooth muscle cells (SMCs) during embryogenesis is essential for ureter peristalsis that propels urine from the kidney to the bladder in mammals. Currently the molecular factors that regulate differentiation of ureteral mesenchymal cells into SMCs are incompletely understood. A recent study has reported that Smad4 deficiency reduces the number of ureteral SMCs. However, its precise role in the ureteral smooth muscle development remains largely unknown. Here, we used Tbx18:Cre knock-in mouse line to delete Smad4 to examine its requirement in the development of ureteral mesenchyme and SMC differentiation. We found that mice with specific deletion of Smad4 in Tbx18-expressing ureteral mesenchyme exhibited hydroureter and hydronephrosis at embryonic day (E) 16.5, and the mutant mesenchymal cells failed to differentiate into SMCs with increased apoptosis and decreased proliferation. Molecular markers for SMCs including alpha smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) were absent in the mutant ureters. Moreover, disruption of Smad4 significantly reduced the expression of genes, including Sox9, Tbx18 and Myocardin associated with SMC differentiation. These findings suggest that Smad4 is essential for initiating the SMC differentiation program during ureter development.


Introduction
Congenital malformations of the urinary tract resulted from embryonic ureter obstruction lead to hydroureter and/or hydronephrosis with dilated ureter and/or renal pelvis, and are one of the main causations of renal failure among children and young adults [1][2][3]. Currently the molecular mechanisms underlying these congenital defects are poorly understood.
The urinary tract is composed of three important cell types: the inner epithelial cells (urothelium), the outer smooth muscle cells (SMCs) that provide contractility to evacuate urine from kidney to bladder [4], and fibroblasts. During ureter development (from E14.5 in mice), the SMCs start to differentiate from mesenchymal cells surrounding ureter pelvis [5,6]. Failure in differentiation of ureteral mesenchymal cells into SMCs results in obstruction and hydronephrosis with atrophy of kidney parenchyma [2,7,8]. Therefore, it is important to understand molecular factors regulating ureteral SMC differentiation during embryonic development.
Molecular regulation of ureteral SMC differentiation is extremely complex. Previous studies revealed several genes, including Tbx18, Six1, Sox9, Tszh3 and b-catenin, are essential for SMC differentiation during ureter development [2,[7][8][9][10][11]. TGF-b super family signals play vital roles in ureter development [10,12,13]. In mice, Bmp4 promotes ureter growth and elongation [10,12]. Decreased Bmp4 signaling results in loss of ureteral smooth muscle formation [14]. As a central mediator in TGF-b signaling [15], Smad4 has been found crucial for vascular SMC differentiation and proliferation [16]. Deletion of Smad4 in the ureteral mesenchyme leads to a reduced number of SMCs [17]. However, whether Smad4 is essential for ureteral SMC differentiation remains unclear. In particular, the downstream genes through which Smad4 regulates ureter development are largely unknown. In this study, we generated Tbx18:Cre knock-in mice to ablate Smad4 in the ureteral mesenchyme. Our data revealed that Smad4 acts as upstream of several key genes associated with ureteral SMC differentiation, and plays critical roles for ureter development during mouse embryogenesis.

X-gal staining
Ureters from Tbx18 nlaZ/+ mouse were checked for b-galactosidase activity by X-gal staining. Mouse ureters were treated with fixation solution (4% paraformaldehyde in PBS) for 30 min at 4uC. The fixed ureters were washed twice with PBS and then stained with X-gal solution (5 mM Potassium Ferricyanide, 5 mM Potassium Ferrocyanide, 2 mM MgCl 2 , 1 mg/ml X-gal) for 12 hours at room temperature. Tissues were visualized with a Leica steromicroscope.

Histology
Mouse ureters were washed in PBS and fixed with 4% paraformaldehyde overnight at 4uC, dehydrated in an ascending ethanol series (25%, 50%, 75%, 100%) followed by two changes of 100% xylene. The tissues were then immersed in liquid paraffin for 2 hours and left on a cold plate until wax was solidified. Paraffin blocks were cut into 6 mm in thickness on a microtome. The sections were stained with Hematoxylin and Eosin (H&E) using standard procedures.

Proliferation and apoptosis analysis
For cell proliferation assay, pregnant mice were intraperitoneally injected with 10 mM EdU (Invitrogen) in PBS (5 mg per 100 g body weight). Embryos were harvested 4 hours later and fixed in 4% paraformaldehyde for 30 min at 4uC, and were embedded in OCT compound. Cell proliferation was assessed on 6 mm frozen sections using Click-iT EdU Cell Proliferation Assay Kit (Invitrogen). To quantify cell proliferation, 8 ureter sections were prepared from each embryo, and sections are from comparable locations in the control and mutant. Two embryos of each genotype were analyzed. Proliferation index was calculated as the percentage of EdU positive cells relative to ureteral mesenchymal cells and ureteric epithelial cells, respectively. Apoptosis assay was performed on 6 mm frozen sections using in situ Cell Death Detection Kit (Roche) according to the manufacturer's instructions. Nine representative ureter sections from each of three embryos with control or mutant genotype at comparable locations were used for apoptosis assay. Apoptosis index was calculated as percentage of TUNEL positive cells relative to ureteral mesenchymal cells.

Quantitative real-time PCR
Total RNA was isolated from ureters with Trizol reagent (Invitrogen) according to the manufacturer's instructions. Quanti-Tect Reverse Transcription Kit (Qiagen) was used to synthesize First-strand cDNA from total RNA. Quantitative PCR was performed using StepOnePlus PCR system and SYBR green detector (Qiagen). All transcripts were normalized to b-actin. The relative amounts of mRNA were calculated using the comparative Ct (threshold cycle) method. Primers used in this study are listed in Table 1. Data are presented as mean 6 SD. Statistical analysis was performed by t-test and a value of p,0.05 was considered significant.

Disruption of Smad4 in the ureteral mesenchyme causes hydroureter and hydronephrosis
To investigate the potential role of Smad4 in ureter development, we first performed immunostaining and found that Smad4 is universally expressed in both ureteral mesenchyme and epithelial cells, and is co-expressed with Tbx18 lineages in the mesenchyme and SMCs (Fig. 1E,F, and data not shown for E13.5-E15.5). We examined Tbx18 Cre/+ ureters found they developed normally without defects at birth (P0) when compared to their wild type littermates (Fig. S2A,B). Further examination of Tbx18 mRNA expression in the ureters by quantitative RT-PCR (qRT-PCR) showed no significant difference between the wild type and Tbx18 Cre/+ mice at this stage (Fig. S2C).
We crossed Tbx18 Cre/+ ;Smad4 f/+ double heterozygous mice to Smad4 f/f mice and Tbx18 Cre/+ ; Smad4 f/f mutant animals (denoted as Smad4 CKO ) were collected for analysis. Littermates with Tbx18 Cre/+ ;Smad4 f/+ genotype were utilized as controls. To determine if Tbx18 Cre/+ mediates efficient recombination in the ureteral mesenchyme and SMCs, we performed immunostaining in the mutant tissues. It showed that Smad4 expression was absent in the ureteral mesenchyme and smooth muscles (notched arrows in Fig. 2, data not shown for E14.5), but was unaffected in ureteric epithelium (arrowheads in Fig. 2A-D), suggesting that Smad4 was specifically disrupted in ureteral mesenchyme and SMCs on Smad4 CKO embryos. At E16.5, Smad4 CKO embryos displayed a prominent bilateral hydroureter and hydronephrosis (Fig. 3A, B), and the defects became more severe at E18.5 (Fig. 3C,D). Histological analysis of these embryos showed dilated and thinwalled ureters with renal parenchymal atrophy and dilation of the renal pelvis ( Fig. 3E-H). The mutant mice cannot survive beyond 24 hours after birth, whereas control mice with Tbx18 Cre/+ ; Smad4 f/+ genotype develop normally and can survive to adulthood without any apparent morphogenetic defects.

Smad4 is essential for ureteral mesenchymal cell differentiation
SMCs are essential for ureter peristalsis that propels urine towards bladder. Previous studies suggested that hydronephrosis and hydroureter could primarily result from malformed SMCs [2,7,8]. To determine whether the hydronephrosis and hydroureter defects in Smad4 CKO embryos were due to insufficient differentiation of the ureteral SMCs, we examined mutant ureters with SMC markers. During embryonic development, ureteral SMC differentiation is characterized by increased expression of contractile SMC markers, such as a-SMA and SM-MHC [27], from E14.5 to E16.5. While we found a-SMA and SM-MHC were highly expressed in the ureteral SMCs at E15.5 and E16.5 in the controls, they could be barely detected in the mutant embryos (notched arrows in Fig. 4A-P). Similar observations were made for Myocardin (Myocd), a key regulator and marker of SMC differentiation, on E14.5 mutant ureters (notched arrows in Fig. 4Q,R). Further qRT-PCR showed that Myocd, Acta2 and Myh11 were down-regulated in the Smad4 CKO ureters by 75%, 72% and 65% at E14.5, and by 78%, 81% and 81% at E15.5, respectively (Fig. 4S, p,0.01). Moreover, we investigated whether Smad4 deficiency in the ureteral mesenchyme also affects urothelium formation. Uroplakin, a marker for urothelium, was still expressed in the mutant embryos, suggesting that urothelial development was unaffected in Smad4 CKO mice (arrowheads in Fig. 4A-H).
Increased apoptosis and decreased proliferation in Smad4 CKO ureteral mesenchyme Cell apoptosis and proliferation are highly associated with hydroureter during ureter development [2,5,28,29]. We examined the effect of Smad4 deletion on cell apoptosis and proliferation in the ureteral mesenchyme. TUNEL analysis revealed no change of apoptosis in ureteric epithelium between control and mutant at E14.5. In contrast, we found a increased number of apoptotic cells in the mutant ureteral mesenchyme (Fig. 5A,B,E, control: 1.8860.38%; mutant: 10.160.58%; p,0.01). Moreover, we assessed mesenchymal cell proliferation by EdU incorporation. The ureteral mesenchymal cell proliferation rate was ,15% less in the Smad4 CKO mutants at E14.5 (control: 16.9361.44%; mutant: 14.3261.22%; p,0.05), yet cell proliferation rate in the epithelium was unaffected (Fig. 5C,D,F, p.0.05). Consistent with these observations, expression of Mycn, a pro-proliferative factor, was significantly reduced in Smad4 CKO ureters at E14.5 and E15.5 (Fig. 5G, p,0     assessed the effect of Smad4 deficiency on these genes during mouse embryonic development. qRT-PCR revealed ,38% reduction in Sox9 and ,53% reduction in Tbx18 on the mutant ureters at E14.5, and ,31% reduction in Sox9 and ,50% reduction in Tbx18 at E15.5 (p,0.05), yet Tszh3 and Ptch1 (receptor of Shh) expression was not significantly changed (Fig. 6A, p.0.05). Further immunostaining and RNA in situ hybridization showed that Sox9 was down-regulated in the mutant ureteral mesenchyme as early as E13.5 (Fig. 6B,C), and Tbx18 mRNA was significantly decreased in the mutant mesenchyme at E13.0 ( Fig. 6D,E). These data suggest that Sox9 and Tbx18 may act as downstream of Smad4 to govern ureteral smooth muscle differentiation during mouse embryogenesis.

Discussion
Although a few genes (e.g., Tbx18, Sox9, Tszh3 and b-catenin) have been identified crucial for ureter development [2,[7][8][9], the interaction network of these genes with other factors in regulating ureteral SMC development remains to be determined. In this study, we found loss of Smad4 in the ureteral mesenchyme led to failure of SMC differentiation, resulting in severe hydroureter and hydronephrosis during mouse embryogenesis. Smad4 acts as upstream of Sox9, Tbx18 and Myocardin, and is required for their normal expressions in ureter development [2,8].
Tbx18 Cre/+ mouse model as a robust genetic tool to study ureteral mesenchyme and SMC development The expression analysis of Tbx18 with Tbx18 nlacZ/+ and Tbx18 H2B-GFP/+ mice revealed that Tbx18 was specifically expressed in the ureteral mesenchyme and SMCs (Fig.1A-D), consistent with the RNA in situ hybridization [2]. Lineage tracing experiments with Tbx18 Cre/+ ; Rosa26 tdTomato animals revealed Tbx18 progeny include undifferentiated ureteral mesenchyme and its SMC derivatives, but not ureteric epithelium. Therefore, the Tbx18 Cre/+ animal could be utilized as an excellent genetic tool to dissect functions of genes in the developing ureteral mesenchyme and/or SMCs during mouse embryogenesis with Cre-loxP technology. Comparing to Tbx18 Cre/+ knock-in mice, Pax3-Cre transgenic line introduces recombination in ureteral mesenchyme (including SM layers and the adventitia) and metanephric mesenchyme (including the glomeruli and the proximal and distal tubules) [28]. The BAC transgenic Tbx18-Cre line was also generated [30]. However, it may not fully recapitulate endogenous Tbx18 expression thus it is not clear whether it introduces effective recombination in all the ureteral mesenchymal cells. Evidence in supporting this argument is thatTbx18-Cre mouse seems not able to mediate effective recombination in the somites of mouse embryos [30].

Smad4 is required for ureteral SMC differentiation from mesenchyme
Previous studies demonstrated that Bmp4 plays an important role in ureter development [10,12,31]. Smad4 is a central mediator of TGF-b/Bmp signaling pathway [32,33] and it regulates morphogenesis of various organs during embryonic development [34][35][36]. Our study showed that Smad4 is expressed in both ureteral mesenchyme and epithelium during ureter development. Mice with genetic deletion of Smad4 in ureteral mesenchyme displayed hydroureter and hydronephrosis as early as E16.5, and the mutant mesenchymal cells failed to differentiate into SMCs at E15.5. Myocd, a critical transcription factor required for expression of SMC contractile proteins (aSMA, SM22a and SM-MHC) [37], is dramatically down-regulated in the mutant ureters. Ureter SMC developmental defects are highly associated with defective cell proliferation and survival [9,10,28]. In Smad4 CKO embryos, we also detected less proliferative ureteral mesenchymal cells with apoptosis at E14.5. The increased mesenchymal cell death in Smad4 CKO mice may be attributed to the reduced Myocd expression, given that Myocd is required for cell survival [38]. Furthermore, loss of Smad4 resulted in downregulation of Mycn in the ureter. Mycn (Nmyc) is a Myc transcription factor and it plays critical roles in cell proliferation [16,39,40]. In the developing hearts, Mycn is a direct downstream target of Smad4 and inactivation of Smad4 down-regulates Mycn expression [39]. Mycn may play a key role in Smad4-mediated signaling cascades for the ureteral mesenchymal cell proliferation. Collectively, these observations suggested that Smad4 signals are crucial for SMC differentiation, proliferation and survival during ureter development.
A recent study has attempted to disrupt Smad4 with Tbx18-Cre transgenic line, and the mutant embryos displayed hydronephrosis at E17.5 [17]. The defects in these animals were not as severe as our Smad4 CKO mice: although the ureteral SMC number was reduced, SMC differentiation still occurred and thickness of smooth muscle layer was not affected. Moreover, no significant difference was found in cell survival and proliferation in the mesenchyme or SMCs. The discrepancies may be due to Cre excision efficiency: Tbx18-Cre transgenic line may not able to mediate efficient excision in the ureteral mesenchymal cells [17]. Some of the mesenchymal cells in the mutant ureters may still maintain Smad4 expression and thus develop normally into SMCs. Tbx18 Cre/+ knock-in mouse line used in our study can mediate efficient ablation in almost all the ureteral mesenchymal cells ( Fig. 2A-D), and therefore SMC differentiation does not occur in Smad4 CKO ureters.
Sox9 and Tbx18 act as downstream of Smad4 during ureteral mesenchymal cell development Genetic regulation of ureteral SMC differentiation is a complex process. Pathways underlying this process are still largely to be determined. To date, a few genes, including Sox9 and Tbx18, were identified as key regulators for ureteral SMC differentiation [2,8].
In addition, microRNA-21 (miR-21) has been shown to promote vascular SMC differentiation by inducing expression of SMC contractile proteins including aSMA, calponin and SM22a [51]. MiR-21 also regulates vascular SMC growth and survival by silencing expression of PTEN and increasing expression of BCL2 [52]. TGF-b and Bmps (Bmp4) signaling elevates the expression of mature miR-21 [51,53]. It is not certain whether miR-21 mediates ureteral SMC differentiation as downstream of Smad4, although miR-21 biogenesis in the vascular SMCs is only regulated by ligand-specific Smad proteins [51].
In the future, it will be of interest to determine whether Smad4 directly activates the expression of Sox9/Tbx18/Myocd by binding their regulatory elements, and whether miR-21 is affected by Smad4 during ureteral development. Furthermore, given the critical roles of Smad4 in ureter development, it will be also of interest to perform genetic studies to determine whether Smad4 and its associated downstream genes Sox9, Tbx18, and Myocd are mutated in human patients with congenital urinary tract malformations.