Non-muscle Myosin II Deletion in the Developing Kidney Causes Ureter-bladder Misconnection and Apical Extrusion of the Nephric Duct Lineage Epithelia

Abstract

In kidney development, connection of the nephric duct (ND) to the cloaca and subsequent sprouting of the ureteric bud (UB) from the ND are important for urinary exit tract formation. Although the roles of Ret signaling are well established, it remains unclear how intracellular cytoskeletal proteins regulate these morphogenetic processes. Myh9 and Myh10 encode two different non-muscle myosin II heavy chains, and Myh9 mutations in humans are implicated in congenital kidney diseases. Here we report that ND/UB lineage-specific deletion of Myh9/Myh10 in mice caused severe hydroureter/hydronephrosis at birth. At mid-gestation, the mutant ND/UB epithelia exhibited aberrant basal protrusion and ectopic UB formation, which likely led to misconnection of the ureter to the bladder. In addition, the mutant epithelia exhibited apical extrusion followed by massive apoptosis in the lumen, which could be explained by reduced apical constriction and intercellular adhesion mediated by E-cadherin. These phenotypes were not ameliorated by genetic reduction of the tyrosine kinase receptor Ret. In contrast, ERK was activated in the mutant cells and its chemical inhibition partially ameliorated the phenotypes. Thus, myosin II is essential for maintaining the apicobasal integrity of the developing kidney epithelia independently of Ret signaling.

Introduction

The kidney develops by reciprocal induction between two precursor tissues: the metanephric mesenchyme and the ureteric bud (UB). The former gives rise to the upper part of the nephron (glomeruli and renal tubules), while the latter contributes to the lower part of the nephron and urinary exit tract (collecting ducts and ureters). At embryonic day (E) 9.5 in the mouse, the nephric duct (ND; Wolffian duct) elongates and reaches the cloaca. At E10.5–11.5, the UB sprouts from the ND and invades the metanephric mesenchyme, thereby inducing the mesenchyme to transform into the nephron epithelia. Simultaneously, the UB branches extensively and forms a tree-like structure consisting of collecting ducts and ureters. The initial sprouting site of the UB and its branching patterns are strictly controlled, and its main regulator is Glial cell line-derived neurotrophic factor (GDNF)-Ret signaling [1]. GDNF secreted from the metanephric mesenchyme acts on the Ret tyrosine kinase receptor on the ND/UB epithelia. Ret signaling leads to activation of a phosphorylation cascade including ERK and stimulates many downstream target genes, such as Etv4/5, Wnt11, and Ret itself [2]. Thus, Ret or Gdnf deficiency results in reduced UB branching and eventually kidney agenesis [1,3]. In contrast, deletion of negative regulators, such as Robo2 and Spry1 [4,5], and a hyperactive Ret mutant [6] lead to excessive ERK activation, positional shift of the UB sprouting site, and ectopic UB budding. The position of the UB sprouting site is critical for the subsequent proper connection between the ureter and the bladder. In normal development, the ND region caudal to the UB sprouting site, which is called the common nephric duct (CND), undergoes physiological apoptosis, and the distal end of the UB eventually connects to the bladder directly [7,8]. The above-described disorders leading to excessive Ret activation impair this process, leading to misconnection of the ureter and the bladder, and eventually to dilatation of the ureters (hydroureter), and in more severe cases, dilatation of the urinary tract in the kidney (hydronephrosis) [4–6].

While the roles of Ret signaling are well established, it remains unclear how intracellular cytoskeletal proteins regulate the morphogenetic processes of UB sprouting and branching. Non-muscle myosin II is a motor protein family that modulates cell adhesion and polarity together with actin filaments [9,10]. Actomyosin anchors cadherins at adherens junctions via β and α catenins. Adherens junctions, as well as subsequently formed tight junctions, separate the apical and basal domains of the epithelia, thus establishing apicobasal polarity. Actomyosin also interacts with integrin, a receptor for the extracellular matrix, and regulates epithelial attachment to the basement membrane [9]. In vertebrates, there are three isoforms of non-muscle myosin heavy chains (NMHCs), IIA, IIB, and IIC, encoded by three different genes, Myh9, Myh10, and Myh14, respectively [9]. Myh9-deficient mice exhibit loss of cell adhesion in the visceral endoderm at the peri-implantation period, while Myh10 deficiency result in heart and brain defects during mid-gestation [11,12]. Mice lacking Myh14 alone show no apparent phenotypes [13], suggesting predominant roles of Myh9 and Myh10 over Myh14 in embryonic development. In humans, Myh9 mutations cause infantile disorders, such as Fechtner and Epstein syndromes, which are characterized by platelet and kidney abnormalities [14–16]. We previously reported that Myh9 and Myh10 encode the major myosin II proteins in mouse kidney development [17]. We further showed that Myh9 deletion in the metanephric mesenchyme causes tubular dilatation leading to renal failure in the adult stage, while deletion of both Myh9 and Myh10 affects nascent nephron formation resulting in perinatal death, suggesting redundant roles of the two genes [17]. In contrast, Myh9 deletion in the ND/UB lineage shows no apparent phenotypes [17]. In this study, we deleted both Myh9 and Myh10 in the ND/UB lineage and found unexpected phenotypes, thereby demonstrating important roles of non-muscle myosin II in this lineage in vivo.

Results

ND/UB lineage-specific deletion of Myh10 causes no apparent phenotypes

During kidney development, both NMHC IIA (encoded by Myh9) and NMHC IIB (encoded by Myh10) were ubiquitously expressed, including expression in the ND and UB epithelia (Fig. 1A). We previously reported ND/UB lineage-specific deletion of Myh9 in mice, which showed no apparent phenotypes [17]. In the present study, we deleted Myh10 by crossing the floxed allele of Myh10 with the Hoxb7Cre mouse strain expressing Cre recombinase specifically in the ND/UB lineage. Myh10 disappeared only in the E-cadherin+ ND/UB epithelia, indicating successful deletion of Myh10, although deletion was not complete because of the mosaic Cre activity of the Hoxb7Cre mouse strain (Fig. 1B). The mutant mice showed no obvious defects in their kidney morphology. Immunostaining of cytokeratin (UB marker), Wt1 (glomerular marker), and LTL (proximal tubule marker) revealed similar patterns of expression in both the control and mutant mice (Fig. 1B). The levels of blood urea nitrogen, creatinine, and albumin in sera from 13-week old mice did not differ significantly between the two groups (Fig. 1C). Thus, deleting only one of the two genes in the ND/UB lineage did not affect kidney development.

Fig. 1. ND/UB lineage-specific deletion of Myh10 causes no apparent phenotypes,

(A) Expression of NMHC IIA (Myh9) and NMHC IIB (Myh10) at E11.5 and E14.5. Both Myh9 (red) and Myh10 (green) are expressed in the ND and UB epithelia. (B) Histological analysis of control kidneys (left column) and ND/UB-specific Myh10 mutant kidneys (right column) at birth. Top two rows: immunostaining of Myh10. Myh10 is absent in the E-cadherin⁺ mutant epithelia. The presence of Myh10 expression in some of the mutant epithelia indicates the mosaic Cre activity of Hoxb7Cre mice. Third row: HE staining. Fourth, fifth, and sixth rows: immunostaining of cytokeratin (CK), Wt1 (glomerular marker), and LTL (proximal tubule marker). Dark purple indicates a positive signal. Sections are counterstained with nuclear fast red. (C) The levels of blood urea nitrogen (BUN), creatinine, and albumin in sera from 13-week old mice show no significant differences between control and ND/UB-specific Myh10 mutant mice. Scale bars: 50 μm in A; 25μm in the first and second rows of B; 500 μm in the third row of B; 50 μm in the fourth, fifth and sixth rows of B.

ND/UB lineage-specific Myh9/Myh10 deletion causes hydroureter/hydronephrosis at birth

Next, we generated mice lacking both Myh9 and Myh10 in the ND/UB lineage. The double-mutant mice (Hoxb7Cre; Myh9^flox/flox Myh10^flox/flox) were born, but many of them showed hydroureter and/or hydronephrosis (Fig. 2A, B). Ink injected in the mutant renal pelvis failed to flow into the urinary bladder, indicating physical blockade between the ureter and the bladder. Furthermore, we did not detect the ureter opening in these mutant mice by histological examination of serial sections (Fig. 2C). In severe cases (4 of 22 ureters examined), the dilated ureters were blind-ended and completely detached from the urinary bladder (Fig. 2D). Thus, deletion of both Myh9 and Myh10 caused misconnection of the ureter and the bladder, resulting in hydroureter/hydronephrosis.

Fig. 2. ND/UB lineage-specific Myh9/Myh10 deletion causes hydroureter/hydronephrosis at birth,

(A) Frequencies of ND/UB-specific Myh9/Myh10 mutant kidneys with hydronephrosis and/or hydroureter at birth. (B) Hydronephrosis and hydroureter in a newborn Myh9/Myh10 mutant mouse. Left panel: control mouse. (C) Physical blockade of the ureter and the bladder in Myh9/Myh10 mutant mice at birth. Urine cannot flow through the ureter to the bladder, which eventually leads to hydroureter and hydronephrosis. Top row: ink injected into the renal pelvis flows through the ureter into the bladder in the control mouse, while ink is trapped in the ureter and fails to flow into the bladder in the mutant mouse (no ink stain). Second and third rows: HE staining shows that the mutant ureter lacks the ureter opening (black arrowheads). Third row: magnified views of portions from the second row. (D) Shortened ureters that are completely detached from the bladder in severe cases of Myh9/Myh10 mutant mouse at birth. Both kidneys exhibit hydronephrosis and both ureters are blind-ended. Upper rows: injected ink does not reach the bladder. Second and third rows: without ink injection. The third row shows a magnified view of the mutant kidney in the second row. Abbreviations: b: bladder; k: kidney; u: ureter. Scale bars: 1 mm in all the panels except for the third row of C (100 μm).

Myh9/Myh10 mutant epithelia exhibit aberrant basal protrusion and ectopic budding at mid-gestation

At E10.5–11.5, the UB sprouts from the ND, and the more caudal part of the ND connected to the cloaca is known as the CND. Subsequently, the CND undergoes physiological apoptosis, leading to a direct connection between the caudal end of the UB (future ureter) and the cloaca (future urinary bladder) [18,19]. Thus, it is well established that an abnormal UB budding site at this early developmental stage, as well as malformation of the CND, causes misconnection of the ureter and the bladder later in development [7,8]. We observed various abnormalities in the Myh9/Myh10 double-mutant mice at E11.5 (Fig. 3A and Fig. S1A). In the control kidneys (n = 6), the UB (Pax2⁺/E-cadherin⁺) sprouted from the ND and invaded into the metanephric mesenchyme (Pax2⁺/E-cadherin⁻), while the thick CND was connected to the cloaca (Pax2⁻/E-cadherin⁺). In the mutant mice, 6 of 10 kidneys (60%) showed ectopic, and thus multiple, UB formation. The caudal part of the CND, which was in proximity to the cloaca, was apparently narrowed (6 kidneys; 60%). This region exhibited physiological apoptosis in the control mice, which was significantly reduced in the mutant mice (Fig. 3B and Fig. S1B). In addition, tail-like structures were observed in some of the mutant CNDs (3 kidneys; 30%). These phenotypes likely represented incomplete CND attachments to the cloaca. Furthermore, we observed that the mutant ND was barely attached to the cloaca and seemingly passed through the site in one of the E10.5 mutant mice (Fig. S1C). Taken together, the mutant kidneys exhibited various degrees of abnormal UB/CND development, which subsequently resulted in ureter-bladder misconnection and hydroureter/hydronephrosis.

Fig. 3. Mutant epithelia exhibit aberrant basal protrusion and ectopic budding at mid-gestation,

(A) Whole-mount immunostaining of Pax2/E-cadherin (Ecad) at E11.5. Left panels: control kidneys, in which a thick CND (Pax2⁺/E-cadherin⁺; cyan arrowheads) is connected to the cloaca (Pax2⁻/E-cadherin⁺; yellow arrowheads). Middle and right columns: Myh9/Myh10 mutant kidneys show various abnormalities: dilation at ND/UB junctions (asterisks), narrowing of caudal part of the CND (cyan arrowheads), multiple UBs (white arrowheads), and tail-like structures (white arrows). The second and third rows show magnified views of the first row. (B) Reduced physiological apoptosis in the narrowed part of the CND in the Myh9/Myh10 mutant kidney (right panel) compared with the control kidney (left panel). Images show immunostaining of cleaved caspase-3 (Casp3; apoptosis marker) and E-cadherin in E11.5 sections. (C) Protrusions from the basal domain of epithelia at E11.5, shown by immunostaining of fibronectin (FN; red) and E-cadherin (green). In the control kidney (left panel), the basal domain of the epithelia is surrounded by fibronectin. In the Myh9/Myh10 mutant kidney (right panel), fibronectin is discontinuous and aligned with the protrusions (white arrowheads). Scale bars: 100 μm in A; 25 μm in B, C.

Next, we examined the cause of the abnormalities and found numerous protrusions from the basal domain of the epithelia (Fig. 3C). While fibronectin surrounded the basal domain of the epithelia in the control mice, it was discontinuous in the mutant mice and aligned with the protrusions. Because actomyosin interacts with a fibronectin receptor, integrin [9], the absence of myosin II may affect the activity of integrins located at the cell-basement membrane interface, which would normally maintain the basal integrity of the epithelia. The resultant promiscuous basal protrusions and reduced recognition of the basally-contacted epithelia may lead to ectopic UB budding and weaker attachment of the CND to the cloaca, respectively.

Myh9/Myh10 mutant epithelial cells extrude apically and undergo apoptosis

Another marked abnormality of the mutant mice was dilation of the UB and ND, which was shown to be most prominent at the ND/UB junctions by whole-mount staining (Fig. 3A). Histological sections confirmed these findings in the mutant mice (Fig. 4A). In addition, mutant cells in the epithelial layer apically extruded en masse into the lumen, and underwent massive apoptosis, as detected by cleaved caspase-3 staining (Fig. 4A). In contrast, we did not observe significant differences in the proliferation of the mutant epithelia (Fig. S2A). While atypical protein kinase C (aPKC) was expressed continuously in the apical domain facing the lumen in the control mice, it was discontinuous in the mutant mice (Fig. 4B, C). In the control mice, E-cadherin accumulated as dots in most of the apical intercellular regions, which likely represented adherens junctions, but this accumulation was not evident in some areas of the mutant mice, which coincided with the absence of aPKC. In these areas, the width of the apical domain was enlarged, compared with the tightly-constricted apical domains in the control mice (Fig. 4B, C). These features were observed in the mutant cells residing in the epithelial layer or at the forefront of the extruding cell mass (Fig. 4B, C). Interestingly, the cells inside the extruding mass still retained E-cadherin expression, but lacked the apicobasally oriented E-cadherin distribution (Fig. 4C). Given the well-established interaction between actomyosin and E-cadherin at adherens junctions, our results suggest that the absence of myosin II led to a reduction in E-cadherin-mediated intercellular adhesion and apical constriction, which likely resulted in the apical extrusion. We also noticed that continuous fibronectin staining was often disrupted in the regions where apical extrusion occurred (Fig. 3C and Fig. S1D), suggesting that the impaired basal attachment may also play a role in the apical extrusion. UB dilatation and luminal apoptosis were persistent at E14.5, a stage before hydronephrosis occurred (Fig. S2B, C).

Fig. 4. Myh9/Myh10 mutant epithelial cells extrude apically and undergo apoptosis,

(A) HE staining and immunostaining of cleaved caspase-3 (Casp3; red)/E-cadherin (Ecad; green) in E11.5 sections. Top two rows: UB tips and dilated ND with cells extrude en masse into the lumen. Bottom two rows: massive apoptosis of the cells in the lumen. Left panels: sections from control mice. (B) Immunostaining of aPKC (red; apical marker) and E-cadherin (green) in E11.5 sections. Left panel: section from control mouse. aPKC is continuously expressed in the apical domain facing the lumen. E-cadherin accumulates as dots at the apical intracellular regions. Right panel: section from Myh9/Myh10 mutant mouse. aPKC expression is not continuous (white arrowheads), dot-like E-cadherin accumulation is not evident at these portions, and the apical domains are widened. (C) Immunostaining of another mutant ND at E11.5. Left panel: aPKC expression is discontinuous (white arrowheads) and cells extrude en masse into the lumen. i (magnified portion from left panel): a cell at the forefront of the extruding mass lacks aPKC and dot-like E-cadherin accumulation, and its apical domain is widened. ii (magnified portion from left panel): cells inside the extruding mass retain E-cadherin, but lack the apicobasally oriented distribution. Scale bars: 25 μm in A; 5 μm in B, C.

The phenotypes in Myh9/Myh10 mutant mice are caused by a Ret-independent mechanism

Ret, which is expressed in the ND/UB, is a critical regulator of UB budding and branching, and Ret hyperactivation leads to ectopic UB budding and subsequent hydroureter/hydronephrosis [4–6], resembling the phenotypes of the Myh9/Myh10 mutant mice to some extent. Therefore, we examined the involvement of Ret hyperactivation in our mutant mice. Wnt11 and Ret itself serve as specific targets in the ND/UB downstream of Ret signaling. Etv4 functions downstream of Ret, although it is also activated in the metanephric mesenchyme downstream of fibroblast growth factor (FGF) signaling. However, in situ hybridization of these genes did not show any increased expression in the mutant ND/UB (Fig. 5A, B). We further crossed the Myh9/Myh10 mutant mice with Ret mutant mice to generate Myh9/Myh10-null/Ret heterozygous mice, which were used to examine whether genetic dosage reduction of Ret could ameliorate the phenotypes. However, the resulting mice showed no significant reduction in the frequencies of hydroureter/hydronephrosis at birth (Fig. 5C, D). These data suggest that the phenotypes in the Myh9/Myh10 mutant mice are unlikely to be caused by Ret hyperactivation. This conclusion is also supported by the fact that narrowing or tail-like structures of the CND, as well as ND/UB dilatation and apical extrusion, have not been reported in any mice exhibiting a hyperactive state of Gdnf/Ret signaling.

Fig. 5: The phenotypes in Myh9/Myh10 mutant mice are caused by a Ret-independent mechanism,

(A) In situ hybridization of Wnt11 in the UB and ND (white dotted lines) at E11.5. Wnt11 is expressed in the UB tips, but not in the ND, in both the control and mutant mice. (B) In situ hybridization of Ret and Etv4 in the ND (dotted whites line in right column) at E10.5, showing no difference in expression. Etv4 is also expressed in the metanephric mesenchyme (white asterisks). (C) Both Myh10/Myh10 mutant mice (left panel) and Myh9/Myh10-null/Ret heterozygous mice (right panel) exhibit hydronephrosis/hydroureter at birth. (D) The frequencies of hydronephrosis/hydroureter in Myh9/Myh10 mutant mice and Myh9/Myh10-null/Ret heterozygous mice at birth are similar. Scale bars: 50 μm in A, B; 1 mm in C.

Meanwhile, similar phenotypes were observed after ND/UB-specific deletion of Yap/Taz [20]. These mutant mice showed narrowing and occasionally complete detachment of the CND, as well as tail-like structures of the CND, reportedly resulting from improper ND insertion into the cloaca. UB dilatation and apoptosis were also mentioned in the cited report, but were not described in detail. These phenotypic similarities led us to examine the expression of Yap/Taz in our mutants. However, we did not observe that Yap/Taz were absent, significantly excluded from the nuclei, or accumulated in the nuclei (Fig. S3). These results suggest, at least, that the phenotypes of the Myh9/Myh10 mutant mice were unlikely to be explained by loss-of-function of Yap/Taz.

Inhibition of hyperactivated ERK partially ameliorates the phenotypes

Ret regulates UB budding and branching by activating ERK, and its excessive activation causes ectopic UB budding [1][6]. Although the above data suggested that Ret was unlikely to be involved in the Myh9/Myh10 mutant mouse phenotypes, it remained possible that ERK activation was involved in the process. We observed enhanced staining of phosphorylated ERK in the mutant mice, in areas where basal protrusion or apical extrusion occurred (Fig. 6A–C). Triple-staining revealed that most of the phosphorylated ERK signals were detected in Myh9-deficient epithelia, indicating that myosin II deficiency led to ERK activation in the mutant cells (Fig. 6D).

Fig. 6: Inhibition of hyperactivated ERK partially ameliorates the phenotypes,

(A) Immunostaining of phosphorylated ERK (pERK; red) and E-cadherin (green) in the ND at E10.5. ERK is activated in basally protruding cells in the Myh9/Myh10 mutant mouse. Left panel: control mouse. (B) Immunostaining of pERK (red) and E-cadherin (green) in the ND at E11.5. ERK is activated in apically extruding cells in the mutant mouse (right panel). Left panel: control mouse. (C) Quantitative analysis of pERK⁺ cells in the ND at E10.5 and E11.5. Significant increases are observed in the mutant mice at both stages (p<0.01). (D) Triple-immunostaining of pERK (green), Myh9 (red), and E-cadherin (magenta) in the ND at E11.5 (dotted white lines). ERK is activated in the mutant epithelial cells (white asterisks; right panels), while cells retaining Myh9 do not show ERK activation (diamonds; right panels). Left panels: control mouse. The top and bottom rows show separate images of the same triple-stained sections. (E) Whole-mount immunostaining of Pax2 (red) and E-cadherin (green) in kidney rudiments cultured for 48 h starting from E10.75. Top row: multiple UB budding (white arrowheads) from the ND in Myh9/Myh10 mutant kidney rudiments (right panel) cultured in the presence of vehicle (DMSO). Bottom row: suppressed budding in Myh9/Myh10 mutant kidney rudiments cultured in the presence of ERK inhibitor U0126. Left panels: wild-type kidneys. Yellow arrowheads indicate metanephric mesenchyme. Scale bars: 20 μm in A, B, D; 200 μm in E.

Finally, we cultured isolated kidney rudiments at E10.75 in the presence of the ERK inhibitor U0126. The concentration of U0126 was optimized to show the minimal or partial suppression of UB sprouting in wild-type embryos (Fig. 6E and Fig. S4). While the mutant kidney rudiments showed multiple and dilated UB budding from the ND, the ERK inhibitor suppressed the multiple budding (Fig. 6E and Fig. S4). UB dilatation was not ameliorated, probably because the UB was already dilated at the start of the organ culture. Harvesting at earlier developmental stages resulted in variable development of kidney tissues. These results indicate that ERK activation had, at least partially, an important role in causing the mutant mouse phenotypes. Taken together, myosin II deficiency in the ND/UB epithelia causes Ret-independent ERK activation that possibly leads to basal protrusion and apical extrusion, resulting in hydroureter/hydronephrosis and dilation/luminal apoptosis, respectively.

Discussion

In the present study, we investigated the role of non-muscle myosin II in the kidney by tissue-specific deletion of myosin heavy chain genes. NMHC IIA and IIB (encoded by Myh9 and Myh10, respectively) have redundant functions in the ND/UB lineage. We have shown that non-muscle myosin II is essential for maintenance of epithelial apicobasal integrity, and that its deletion leads to promiscuous apical extrusion and basal protrusion, resulting in luminal apoptosis and urinary tract malformation, respectively. In contrast, deletion of myosin II in the metanephric mesenchyme, another essential component of the kidney, does not exhibit these phenotypes, but affects nascent nephron morphogenesis, especially lumen elongation [17]. In the metanephric mesenchyme, epithelial tubes are newly formed through mesenchymal-to-epithelial transition and subsequently convolute to form nephrons, while epithelial tubes with a pre-existing lumen elongate and branch in the ND/UB lineage. These differences in epithelial nature may underlie the distinct roles of myosin II in the two components of the kidney. However, apical constriction is likely to be involved in both types of epithelia, suggesting that at least this role of myosin II is conserved. It will be interesting to examine the roles of myosin II in epithelia of other organs. Embryonic lung explants treated with the myosin inhibitor blebbistatin showed impaired cell shape and arrangement [21], but myosin deletion in vivo was not performed. Intestinal epithelia-specific Myh9-deficient mice show intestinal barrier disruption, resulting in increased permeability [22]. In these mice, expression of some of the adherens junction proteins is decreased, but the gross morphology is unaffected. Meanwhile, Myh9 deletion in mouse tongue epithelia resulted in spontaneous squamous cell carcinoma [23], the cause of which remains controversial. To the best of our knowledge, deletion of both Myh9 and Myh10 in vivo has not been reported in organ epithelia other than the kidney.

Myosin II is involved in cadherin-mediated intercellular adhesion, as well as apical constriction. It also plays an important role in integrin-mediated adhesion to the basement membrane. The apical extrusion and basal protrusion observed after myosin II deletion suggest that myosin maintains epithelial integrity by preventing the cells from escaping from the epithelial layer. Reduced apical intercellular adhesion and broadened apical domain could evoke apical extrusion followed by apoptosis, while impaired basal recognition may lead to promiscuous basal protrusion and reduced basal adhesion to the neighboring cloaca epithelia. These phenotypes are rarely observed in other mutant mice in which kidney development is affected.

Ret is a major regulator of ND/UB development, and its excessive activation leads to ectopic UB budding through ERK activation [6]. However, our data suggest that Ret hyperactivation is unlikely to be involved in the phenotypes caused by myosin II deletion. Nevertheless, ERK activation can at least partly explain the phenotypes, based on our chemical inhibition experiments. While it remains unclear how ERK is activated upon myosin II deletion, our preliminary experiments showed that inhibition of myosin functions in MDCK cells using the chemical inhibitor blebbistatin led to ERK activation within 5 min (Fig. S5). Although this finding may not be directly related to the situation in vivo, a mechanism by which myosin-mediated adhesion suppresses excessive ERK activation and contributes to epithelial integrity could exist.

ND/UB-specific deletion of Yap and Yap/Taz in mice produces marked similarities to the phenotypes of our myosin mutant mice, including hydronephrosis and ERK activation [20]. To the best of our knowledge, blind-ended ureters completely detached from the bladder are only observed in Yap, Yap/Taz, and Myh9/Myh10 mutant mice among many mutant mouse strains with impaired UB development. In E11.5 Yap or Yap/Taz mutant mice, the CND is narrowed or already detached from the cloaca, with the former phenotype also observed in our Myh9/Myh10 mutant mice, possibly leading to the later detachment of the ureter from the bladder. It is also noteworthy that metanephric mesenchyme-specific deletion of Yap/Taz in mice shows similar phenotypes to those of Myh9/Myh10 deficiency (morphology defects in nascent nephrons) [17,24]. Yap/Taz are effectors downstream of tension, as well as upstream tension regulators [25,26]. The unaltered Yap/Taz distribution in our myosin mutant mice suggests that Yap/Taz are not downstream but possibly upstream of myosin. However, expression of Etv4, a Ret activity indicator, is increased in Yap-deficient mice and their phenotypes are largely rescued by genetic dosage reduction of Ret [20]. Thus, myosin may play a distinct role from Yap/Taz, or may have a possible role in Ret-independent molecular events downstream of Yap/Taz. Further studies are needed to clarify the relationships between Yap/Taz and myosin II in kidney epithelia.

Another possible mechanism leading to apical extrusion and basal protrusion is cell competition, which occurs when mutant cells are juxtaposed with wild-type cells [27–29]. MDCK cell lines harboring impaired polarity or activated oncogenic signaling, such as Ras/ERK signaling, are often used in two-dimensional culture [29,30]. As Hoxb7Cre deletes myosin in a mosaic manner, cell competition may be involved in the three-dimensional situation in vivo. However, evidence for non-cell-autonomous effects is required to prove this tempting hypothesis.

Finally, Myh9 mutations cause hereditary kidney diseases in humans [14–16]. Although glomerular abnormalities are their major manifestations, involvement of the ND/UB lineage would worthy of examination in patients. In addition, several groups, including ours, recently reported the generation of human kidney tissues from induced pluripotent stem (iPS) cells [31–33]. If a complete ND/UB induction protocol can be established, we will be able to examine whether the described phenotypes in the mutant mice are also found in humans by using genetically modified iPS cells.

In summary, we have revealed the essential roles of non-muscle myosin II in maintaining renal epithelial integrity. Further studies will provide important information on the complex three-dimensional structures of the kidney, as well as on human diseases.

Materials and Methods

Generation of the mutant mice

Myh9^flox/flox and Myh10^flox/flox mice (MMRRC #32096 and #16981, respectively) were described elsewhere [34,35]. The Hoxb7Cre mouse strain was obtained from the Jackson Laboratory [36]. The RetCreER mouse strain [37,38], which was used for genetic crosses for Ret heterozygosity (without tamoxifen treatment), was kindly provided by Dr. Hideki Enomoto at Kobe University, Japan (deposited as CDB0518K, http://www2.clst.riken.jp/arg/mutant%20mice%20list.html). These mouse strains were backcrossed to the C57BL/6 mouse strain for at least three generations. The following primer were used for genotyping: Hoxb7Cre-F2 (5′-TGGGCCGGGGTCACGTGGTCAGA-3′) and Hoxb7Cre-R2 (5′-CGACGATGAAGCATGTTAGCTG-3′) for the Hoxb7Cre allele (500 bp); and hER-1 (5′-TGGAGATCTTCGACATGCTG-3′) and hER-2 (5′-GCCATCAGGTGGATCAAAGT-3′) for the RetCreER allele (209 bp). The primer sequences for the Myh9 and Myh10 alleles, as well as PCR reaction conditions, were described previously [17]. Blood urea nitrogen, creatinine, and albumin levels in serum samples were analyzed by standard methods using ultraviolet, hydroxyl triiodobenzoic acid, and bromocresol green, respectively. All animal experiments were approved by the Animal Care and Use Committee of Kumamoto University (#A27–018), and were performed in accordance with the institutional guidelines of Kumamoto University.

Section immunostaining

Mouse embryos or kidneys were fixed with 10% formalin or 4% paraformaldehyde, and embedded in paraffin or OCT compound, respectively. Immunostaining was carried out using a BlueMap Kit and automated Discovery System (Roche) or manually for immunofluorescence staining. The following primary antibodies were used: anti-Myh9 (HPA001644; Sigma); LTL (B1325; Vector Laboratories); anti-Wt1 (sc192; Santa Cruz Biotechnology); anti-cytokeratin (c2562; Sigma); anti-cleaved caspase-3 (9661; Cell Signaling Technology); anti-p-aPKC (ab62372; Abcam); anti-pHH3 (06–570; Millipore); anti-E-cadherin (610181; BD Transduction Laboratories); anti-pERK (4370; Cell Signaling Technology); anti-fibronectin (ab2413; Abcam); and anti-Pax2 (901001; Biolegend). Immunofluorescence was visualized with an LSM780 confocal microscope (Zeiss). Seven mutant and seven control embryos were analyzed at E11.5, while four mutant and four control embryos were analyzed at E10.5, with consistent results.

In some experiments, an ImmPress Reagent Kit (MP-7401 or MP-7402; Vector Laboratories) and Alexa Fluor tyramide (T20948; Thermo Fisher Scientific) were used to enhance the signal or perform double-staining with antibodies from the same host species. Sections were incubated with the primary antibodies and anti-rabbit or anti-mouse secondary antibodies conjugated with peroxidase polymers, according to the manufacturers’ instructions. The sections were then incubated with Alexa Fluor tyramide (diluted in amplification buffer containing hydrogen peroxidase) for 15 min at room temperature. Subsequently, the sections were subjected to a second round of staining in a standard manner. The following primary antibodies were used: anti-Myh10 (DSHB, CH11 23–5; Developmental Studies Hybridoma Bank) in Fig. 1; anti-YAP (sc101199; Santa Cruz Biotechnology) and anti-Yap/Taz (8418; Cell Signaling Technology) in Fig. S3; and anti-pERK (4370; Cell Signaling Technology) in Fig. 6C, the latter of which allowed triple-staining of pERK (rabbit), Myh9 (rabbit), and E-cadherin (mouse) with minimal cross-reactivity. The specificity of the anti-Yap and anti-Yap/Taz antibodies were previously demonstrated in Yap-null and Yap/Taz-null mutant mice, respectively [20].

Whole-mount immunostaining

Immunostaining of dissected or cultured tissues was carried out as described [6]. Briefly, tissues were fixed with 4% paraformaldehyde for 30 min at 4°C, washed with PBS-Tr (0.3% Triton X-100 in PBS), blocked with PBS-BB (1% BSA, 0.2% skim milk, 0.3% Triton X-100 in PBS) overnight at 4°C, and incubated with anti-Pax2/E-cadherin primary antibodies (diluted in PBS-BB) overnight at 4°C. After washing with PBS-Tr, the samples were incubated with Alexa Fluor secondary antibodies (diluted in PBS-BB) overnight at 4°C, followed by washing with PBS-Tr. Some of the stained samples were cleared with SeeDB [39] for better visualization and embedded in agarose. Immunofluorescence was visualized with an SZX16 stereo microscope (Olympus). Six kidneys from control mice and 10 kidneys from mutant mice were analyzed, with consistent results.

Organ culture

Kidney tissues were dissected from E10.75 mice (tail somite numbers: 35–40) and cultured in DMEM/F12 (Thermo Fisher Scientific) containing 10% serum on Transwell filters (0.4 μm; Corning) [6][40]. Left and right kidneys were cultured separately, one in the presence of ERK inhibitor U0126 (3 μM, Sigma) and the other in the presence of vehicle (DMSO). The concentration of U0126 was optimized by using the wild-type embryos (Fig. S4). After culture for 48 h, the specimens were subjected to whole-mount immunostaining for Pax2 and E-cadherin. Three independent experiments were performed, and representative data are shown.

In situ hybridization

In situ hybridization of 10% formalin-fixed paraffin sections was performed as described [40], using an automated Discovery System (Roche) equipped with an OmniMap anti-rabbit HRP Amplification HQ Kit, Anti-HQ Alkaline Phosphatase Multimer Kit, and ChromoMap Blue Kit (all from Roche), according to the manufacturer’s protocols. The probes for Ret, Wnt11, and Etv4 were cloned by PCR, and labeled with digoxigenin using RNA polymerase.

MDCK cell culture

MDCK cells were cultured on collagen-coated plates as described previously [29]. Briefly, type-IA collagen was prepared according to the manufacturer’s protocol (Collagen Gel Culture Kit; Nitta Gelatin) and each well of a 12-well plate was plated with 0.6 ml of collagen. Cells were seeded at a concentration of 5 × 10⁵ cells/well and cultured in DMEM containing 10% serum. Upon reaching confluence, the cells were treated with 50 or 100 μM blebbistatin (Wako) for short time periods (5 or 10 min). Total cell lysates were harvested using NuPAGE LDS sample buffer (Thermo Fisher). Western blotting was performed as described previously [41]. The following primary antibodies were used: anti-pERK (4370; Cell Signaling Technology); and anti-β-Actin (3700; Cell Signaling Technology). The experiments were repeated three times, with consistent results. Images were taken and quantified by densitometry using Image J software [42]. After the band intensities were measured, relative intensities were calculated and normalized by the intensities of β-actin (loading control). Statistical analysis was performed with Student’s t-test.

Statistical analyses

Six fields in stained sections (two fields/section for three mice) were counted for pHH3⁺/E-cadherin⁺ cells from both control and Myh9/Myh10 mutant mice. The percentage of pHH3⁺/E-cadherin⁺ cells in each field was calculated and analyzed by Student’s t-test. Data were presented mean ± standard deviation. The percentages of Casp3⁺/E-cadherin⁺ cells and pERK⁺/E-cadherin⁺ cells were calculated and analyzed in a similar manner.

Supplementary Material

1

Fig. S1. Variations of UB/CND abnormalities in Myh9/Myh10 mutant mice. (A) Whole-mount immunostaining of Pax2 (red) and E-cadherin (green) at E11.5. Left column: the UB/ND junctions are dilated (white arrow), but UB branching is observed (white arrowhead). Middle column: the UB/ND junctions are dilated (white arrow) and there is no apparent UB branching. Right column: double UB budding (white arrowheads) from adjacent points of the ND that are also dilated. Two separate kidneys are formed (asterisks). (B) Quantitative analysis of apoptotic cells in the CND at E11.5. A significant decrease in physiological apoptosis is observed in the mutant mice (p<0.05). (C) Section immunostaining of Pax2 (red) and E-cadherin (green) in the CND (Pax2⁺/E-cadherin⁺)/cloaca (Pax2⁻/E-cadherin⁺) junction at E10.5. While the control CND attaches to the cloaca through a wider area (left panel), the ^Myh9/Myh10 mutant CND barely attaches to the cloaca and seems to pass away from the site (right panel, white arrow). (D) Discontinuous fibronectin expression (red) in areas where apical extrusion occurs (marked by dashed white line). Scale bars: 100 μm in A; 25 μm in C, D.

2

Fig. S2. Proliferation is not affected and persistent luminal apoptosis occurs at E14.5. (A) Immunostaining of mitosis marker pHH3 (red) and E-cadherin (green) at E11.5. Left panels: mitotic cells (red) in the UB/ND of Myh9/Myh10 mutant epithelia (green). Right panel: there is no significant difference in the percentages of proliferation (pHH3-positive/E-cadherin-positive cells) between control and Myh9/Myh10 mutant epithelia (p=0.42). (B) HE staining of control and Myh10/Myh10 mutant kidneys at E14.5. Dilated UB tips with pyknotic cells in the lumen are observed. (C) Immunostaining of Casp3 (red) and E-cadherin (green) at E14.5. Apoptotic cells are detected in the lumen. Scale bars: 100 μm in A, C; 50 μm in B.

3

Fig. S3. Yap and Yap/Taz shows a similar nuclear-cytoplasmic distribution, (A)(B) Immunostaining of Yap (A) or Yap/Taz (B) at E11.5. The nuclear-cytoplasmic distribution of Yap or Yap/Taz (green) in the E-cadherin⁺-epithelial cells (red) of the UB tip/stalk and ND is not significantly affected in the Myh9/Myh10 mutant mice. Yap or Yap/Taz is distributed ubiquitously in the nuclei and cytoplasm of most cells. A few cells lack nuclear Yap (asterisks) or Yap/Taz (asterisks) but retain cytoplasmic Yap or Yap/Taz. Second rows in A and B: magnified views of the first rows. Fourth rows in A and B: magnified views of the third rows. Scale bar: 25 μm.

4

Fig. S4. ERK inhibitor suppresses the ureteric budding of kidney explants cultured in vitro, (A) Whole-mount immunostaining of Pax2 (red) and E-cadherin (green) in kidney rudiments cultured for 48 h starting from E10.75 in the presence of vehicle (DMSO) or ERK inhibitor U0126 at the indicated concentrations. The images show minimal suppression of UB budding at 3 μM U0126, but complete suppression of UB budding at 7 μM and 10 μM U0126. White arrowheads indicate UB budding, and yellow arrowheads indicate the metanephric mesenchyme. (B) Whole-mount immunostaining of Pax2 (red) and E-cadherin (green) in the mutant kidney rudiments cultured for 48 h starting from E10.5. Top row: multiple and dilated UB budding (white arrowheads) from the ND in Myh9/Myh10 mutant kidney rudiments cultured in the presence of vehicle (DMSO). Bottom row: suppressed budding in Myh9/Myh10 mutant kidney rudiments cultured in the presence of ERK inhibitor U0126. A single UB sprouts, but fails to reach the metanephric mesenchyme (yellow arrowheads). Scale bars: 200 μm.

5

Fig. S5. ERK activation in MDCK cells upon blebbistatin treatment, (A and B) MDCK cells were treated with two different concentrations of blebbistatin (50 or 100 μM) for 5 or 10 min. For both concentrations, ERK is activated within 5 min of treatment. As a control, MDCK cells were treated with vehicle (DMSO). The data at 10 min were obtained from distant lanes of the same membranes, and the intervening lanes were deleted. (C) Quantitative analysis of the ERK activation in panels A and B. Data represent the relative intensities of pERK normalized by the intensities of the loading control (β-actin). Significant ERK activation is observed upon blebbistatin treatment for 5 min (*p<0.05; **p<0.01).

Highlights.

• Myosin II deletion in the developing kidney causes hydronephrosis.

• Nephric duct dilatations and ectopic ureteric budding occur at mid-gestation.

• Apical extrusion and basal protrusion of the epithelia underlie the defects.

• The phenotypes are caused in a Ret-independent mechanism.