Lats1/2 Regulate Yap/Taz to Control Nephron Progenitor Epithelialization and Inhibit Myoﬁbroblast Formation

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† † Helen McNeill* and Antoine Reginensi

*Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; and †Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada

ABSTRACT In the kidney, formation of the functional filtration units, the nephrons, is essential for postnatal life. During development, mesenchymal progenitors tightly regulate the balance between self-renewal and differentiation to give rise to all nephron epithelia. Here, we investigated the functions of the Hippo pathway serine/ threonine-protein kinases Lats1 and Lats2, which phosphorylate and inhibit the transcriptional coactivators Yap and Taz, in nephron progenitor cells. Genetic deletion of Lats1 and Lats2 in nephron progenitors of mice led to disruption of nephrogenesis, with an accumulation of spindle-shaped cells in both cortical and medullary regions of the kidney. Lineage-tracing experiments revealed that the cells that accumulated in the interstitium derived from nephron progenitor cells and expressed E-cadherin as well as vimentin, a myofibroblastic marker not usually detected after mesenchymal-to-epithelial transition. The accumulation of these interstitial cells associated with collagen deposition and ectopic expression of the myofibroblastic markers vimentin and a-smooth-muscle actin in developing kidneys. Although these myofibroblastic cells had high Yap and Taz accumulation in the nucleus concomitant with a loss of phosphorylated Yap, reduction of Yap and/or Taz expression levels completely rescued the Lats1/2 phenotype. Taken together, our results demonstrate that Lats1/2 kinases restrict Yap/Taz activities to promote nephron progenitor cell differentiation in the mammalian kidney. Notably, our data also show that myofibroblastic cells can differentiate from nephron progenitors.

J Am Soc Nephrol 28: ccc–ccc, 2016. doi: 10.1681/ASN.2016060611

Kidney organogenesis is a remarkably orchestrated, during embryonic development and have been im- reiteratedprocessthatdependsonreciprocalsignaling plicated in the growth of metastatic tumors.8 During between the epithelial ureteric bud (UB) and the MET, mesenchymal cells alter their shape and motile surrounding condensing mesenchyme (CM).1–4 Sig- behavior as they differentiate into epithelial cells by naling from the mesenchyme induces successive acquiring an apical-basal polarity, a basement mem- rounds of UB branching, generating the collecting brane, and adhesion with neighboring cells. Such duct (CD) of the kidney. Surrounding the UB are events of cellular transition and movements were un- self-renewing mesenchymal progenitor cells (called covered by means of lineage-tracing experiments in the CM or nephron progenitor cells [NPCs]) that express Six2 and Cited1.5,6 A subset of CM cells is reciprocally induced by the UB to form a pretubular Received June 2, 2016. Accepted August 2, 2016. aggregate (PA), which subsequently undergoes Published online ahead of print. Publication date available at mesenchymal-to-epithelial transition (MET) to www.jasn.org. form the renal vesicle (RV). The RV will then undergo Correspondence: Dr. Helen McNeill or Dr. Antoine Reginensi, morphogenesis to form the comma-shaped body Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, (CSB), followed by the S-shaped body (SSB) that 600 University Avenue, Room 881, Toronto, ON, M5G 1X5, will elongate to form the nephron.7 Both MET and Canada. Email: [email protected] or [email protected] epithelial-to-mesenchymal transition are essential Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 28: ccc–ccc, 2016 ISSN : 1046-6673/2803-ccc 1 BASIC RESEARCH www.jasn.org the developing kidney using, among others, the Hoxb7 (UB), medullary regions (Figure 1, D–I). In control kidneys, higher Six2 (CM), and Foxd1-reporter models that label the CDs, neph- magnification views identified an active nephrogenic zone, a rons, and stromal derivatives, respectively.9–11 cortex with numerous glomeruli, proximal and distal tubules, The Hippo pathway is a conserved kinase cassette that and a medulla filled with stromal cells and CDs. Remarkably, 2 2 controls tissue growth through the activities of the Yap and Taz neither glomeruli nor tubules were observed in Lats1/2CM / effectors in both flies and mammals.12–14 These closely related kidneys at E18.5, and both the cortex and the medulla showed transcriptional coactivators promote the expression of pro- an accumulation of cells in the interstitium with a dense, proliferative and antiapoptotic genes. Upstream of Yap and glycoprotein-rich (periodic acid–Schiff [PAS]-positive) extra- Taz are the Hippo kinases Mst1/2 and Lats1/2, which nega- cellular matrix, surrounded by dense stroma (Figure 1, F–I). tively regulate Yap and Taz by causing their exclusion from the CM2/2 nuclear compartment. Loss of Hippo signaling (Mst1/2 or Accumulation of NP-Derived Cells in the Lats1/2 Lats1/2 inactivation) leads to unrestricted nuclear accumula- Kidney tion of Yap and Taz and has been linked to a variety of de- Nephrogenesis occurs in a repetitive manner, with new velopmental abnormalities and cancers.15–17 nephrons being formed throughout development in the In the developing mammalian kidney, Yap and Taz play nephrogenic zone. NPCs surrounding the UB tips concurrently different roles: Yap promotes nephron formation within the self-renew to replenish the pool of NPCs and undergo MET to NPC population18 and Taz prevents renal cyst formation.19,20 form early nephrons. To determine whether cells that accu- 2 2 Yap and Taz are also essential in the UB lineage for lower mulate in Lats1/2CM / kidney interstitium were derived from urinary tract development21 and branching morphogene- the NPCs, we performed lineage-tracing experiments using the sis.22 Finally, increased Yap and Taz have also been found to Rosa-mTomato/mGFP reporter mouse line (mTmG24) which, correlate with kidney fibrosis, using a unilateral ureteral ob- combined with the Six2:Cre system, produces a membrane- struction model.23 From these published data, it is clear that bound green fluorescent protein (GFP) that permanently labels loss of Yap and Taz are detrimental to kidney development and the NPCs and their daughter cells. We generated double con- fl fl function in the adult. However, the roles of the core Hippo ditional knockout Six2:Cre Lats1/2 ox/ ox mTmG mice, wherein fl kinases in NPCs remain to be investigated. compound mutants (Six2:Cre Lats1/2 ox/+ mTmG–double het- fl fl fl Here we uncover a role for the Hippo kinases, Lats1 and erozygous knockout, Six2:Cre Lats1 ox/+ Lats2 ox/ ox mTmG,and fl fl fl fl fl Lats2, in nephron progenitor (NP) differentiation and dem- Six2:Cre Lats1 ox/ ox Lats2 ox/+ mTmG) and Lats1/2 ox/ ox onstrate that Lats1 and Lats2 activities are critical during neph- mTmG (no Cre) were used as controls. We performed GFP ron formation. Removal of Lats1/2 from NPCs causes loss of and calbindin staining to mark the NPCs and daughter cells, nephron formation, accompanied with a change in cell fate and and the UB compartment, respectively, at E18.5. As expected, accumulation of myofibroblasts. The interstitial myofibroblas- no GFP staining was observed in any Cre controls (Figure 2A). tic cells also show high nuclear Yap and Taz accumulation with In both double heterozygous knockout animals and mutants loss of phospho-Yap. Remarkably, the conditional loss of Lats1/ with loss of three out of four Lats alleles, we observed GFP 2 in the NPC population is successfully rescued by depletion of expression in all NPCs throughout the nephrogenic zone and Yap and/or Taz. Taken together, these data demonstrate an es- in their epithelial descendants (both early and more mature sential role for the core Hippo kinases (Lats1 and Lats2) in nephrons [Figure 2, B and C, and higher magnification in Fig- restricting Yap/Taz activity during the self-renewal of NPCs ure 2E]). Thus, loss of three out of the four Lats alleles does not and MET and nephron formation in the mammalian kidney. affect nephrogenesis (Figure 2C). In contrast, nephrogenesis 2 2 does not occur in Lats1/2CM / kidneys, and all cells that ac- 2 2 cumulate in the Lats1/2CM / interstitium are GFP-positive, RESULTS indicating that they were derived from the NPCs (Figure 2, D and F). Lats1 and Lats2 Dual Deletion Results in Loss of Nephrogenesis Lats1/2 Deletion Impaired Maintenance and To investigate the role of Lats1 and Lats2 kinases in NPCs, both Differentiation of NPCs genes were conditionally inactivated using the Six2:CreTGC/+ To determine the developmental origin of the abnormal 2 2 allele.10 This system depletes Lats1 and Lats2 expression from nephrogenesis observed in Lats1/2CM / kidneys, we exam- fl fl NPCs and their epithelial derivatives. Six2:CreTGC/+ Lats1 ox/ ox ined earlier time points. In E14.5 control kidneys, 2–3 layers of fl fl 2 2 Lats2 ox/ ox (called Lats1/2CM / ) mice died within 24 hours Six2-positive self-renewing NPCs surround the dorsal side of after birth. Gross anatomic examination revealed that embry- the UB, with early nephron structures (PA, RV, CSB and SSB) 2 2 onic day 18.5 (E18.5) Lats1/2CM / embryos had a dramatic forming on the ventral side of the UB. Six2 immunofluores- 2 2 decrease in kidney size compared with controls (Lats1/2CM / : cence (Figure 3, A–G) and lineage tracing (Figure 2F) 2.360.1 mm2, n=6; controls: 4.560.2 mm2, n=8; Figure 1, revealed a reduced pool of Six2-positive cells capping UB A–C). Histologic examination of E18.5 kidneys revealed loss of tips (quantification in Figure 3G), indicating that Lats1/2 de- nephrogenesis and accumulation of cells in both cortical and letion leads to poor maintenance of the NPCs. Moreover,

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Figure 1. Lats1/2 deletion leads to loss of nephrogenesis. (A and B) Macroscopic views of the urogenital system at E18.5 show severe 2 2 2 2 kidney hypoplasia in Lats1/2CM / and control embryos. (C) Quantiﬁcation reveals a 49% decrease in kidney size in Lats1/2CM / 2 2 embryos compared with controls at E18.5. (D and E) PAS staining of wild-type and Lats1/2CM / kidneys at E18.5. (F–I) Higher 2 2 magniﬁcation views reveal loss of glomeruli and tubules in Lats1/2CM / mutants, and accumulation of cells in the interstitium (*). A, adrenal; B, bladder; G, glomerulus; K, kidney; NZ, nephrogenic zone; PT, proximal tubule; ST, stroma; T, testis. Scale bars represent 1mm(AandB),200mm (D and E), and 50 mm(F–I).

Lats1/2 deletion also abolished expression of the NPC marker (Supplementary Figure 1, A–B’ and E–F’). Discontinuous Cited1, suggesting loss of progenitor potential (Figure 3, H E-cadherin staining was observed in accumulated interstitial 2 2 2 2 and I). The reduced maintenance of NPCs in Lats1/2CM / is cells in Lats1/2CM / kidneys and, consistent with the loss of accompanied by a complete absence of early nephron struc- tubule formation, we observed loss of the apical marker tures. No RV, CSB, SSB, or glomerulus were observed in Lats1/ Crumbs3 and reduced expression of the basement membrane 2 2 2CM / E14.5 kidneys compared with controls as seen by his- marker laminin (Supplemental Figure 1, C, D, G, and H). tology or Sox9 staining, a marker for UB tips and early neph- Expression of E-cadherin, NCAM, and b-catenin gradually rons25 (Figure 3, J–O and the absence of SSB in Figure 3D). reduced as cells located further away from the nephrogenic zone (boxes in Supplemental Figure 1), and by E18.5, the level Lats1 and Lats2 Deletion Induces Loss of Epithelial of E-cadherin expression in the cells that accumulate in the Characteristics interstitium was greatly reduced (Figure 3F). We next characterized the accumulated interstitial cells ob- During MET, expression of vimentin, a type-III interme- 2 2 fl fl served in Lats1/2CM / kidneys. Since controls (Lats1/2 ox/ ox diate filament protein normally expressed in mesenchymal mTmG) do not have the Six2:Cre allele, we used either Neural cells, becomes downregulated as cells adopt epithelial charac- Cell Adhesion Molecule (NCAM) or E-cadherin to mark the teristics.26 In control kidneys, vimentin protein is strongly ex- CM and/or early nephron structures and GFP staining for NPs pressed in stromal cells and, to a smaller extent, in NPCs, but is 2 2 and their epithelial derivatives in Lats1/2CM / kidneys (Six2: absent from all epithelial structures (UB and CM-derived PA, fl fl Cre Lats1/2 ox/ ox mTmG). At E14.5, the accumulated intersti- RV, CSB, and SSB; Figure 4, A and A’). Surprisingly, Six2- 2 2 2 2 tial cells in Lats1/2CM / still expressed Six2 (Figure 3D’). In derived cells (GFP-positive) in Lats1/2CM / mTmG kidneys 2 2 addition, cells that accumulate in Lats1/2CM / kidneys ex- showed high vimentin expression (Figure 4, B and B’)while pressed NCAM and b-catenin, as in control early nephrons still expressing E-cadherin (Figure 3, A and B), suggesting that

J Am Soc Nephrol 28: ccc–ccc, 2016 Hippo in Nephron Progenitor Fate 3 BASIC RESEARCH www.jasn.org

2 2 Figure 2. Accumulation of NP-derived cells in the Lats1/2CM / kidney. (A–D) GFP and calbindin staining in (A) negative control; (B) Six2:Cre Lats1/2flox/+ mTmG;(C)Six2:Cre Lats1flox/+ Lats2flox/flox mTmG; and (D) Six2:Cre Lats1/2flox/flox mTmG E18.5 kidneys, where GFP staining labels the Six2 expressing NPCs and their progeny, and calbindin the UB-derived structures (purple arrows). Note that calbindin is also expressed in distal tubules. (E) High magnification views of the cortical zone of double heterozygous knockout showed GFP staining is observed in NPCs (arrows), early nephrons (white arrowheads) and differentiated tubules (yellow arrowheads). (F) In 2 2 contrast, nephrogenesis does not occur in Lats1/2CM / kidneys, and all cells that accumulate in the interstitium are GFP-positive (*), 2 2 indicating they were derived from NPCs. Note the reduced pool of NPCs in Lats1/2CM / kidneys (white arrows). Co, cortex; M, medulla. Scale bars represent 500 mm(A–D) and 200 mm(EandF). they do not undergo complete MET. Accordingly, Masson tri- Altogether, these data suggest that Lats1/2 deletion converts 2 2 chrome staining revealed collagen deposition in Lats1/2CM / NPCs into myofibroblasts. kidneys (further validated by collagen IV expression at E14.5 and E18.5), suggesting that Lats1/2 deletion led to accumula- Unrestricted Yap/Taz Activity Accounts for tion of cells with fibrotic characteristics (Figure 4, C–L). Myofibroblast Differentiation Observed in CM2/2 Moreover, in control kidneys, no expression of the myofibro- Lats1/2 Embryos blast marker a-smooth muscle actin (a-SMA) could be de- To investigate whether Lats1/2 functions upstream of Yap/Taz 2 2 tected (apart from the vascular smooth-muscle cells [Figure 4, in NPCs, we stained E14.5 control and Lats1/2CM / mTmG M and O]). In contrast, a fraction of the more medullary kidneys using phospho-Yap, Yap, and Taz antibodies. Immu- 2 2 accumulated interstitial cells in Lats1/2CM / kidneys ectop- nostaining showed loss of phospho-Yap and strong nuclear Yap 2 2 ically expressed a-SMA at E14.5 (Figure 4, N and P), whereas and Taz in NP-derived cells of Lats1/2CM / kidneys compared most of them expressed a-SMA at E18.5 (Figure 4, Q and R). with early nephron structures in controls (Figure 5, A–F’).

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Figure 3. Lats1/2 deletion affect NPC maintenance and nephron formation. (A–F) Six2 and E-cadherin staining at E14.5 and E18.5 2 2 showed poor maintenance of the population of NPCs throughout kidney development in Lats1/2CM / compared with controls. (C–D’) 2 2 Note that early nephron structures (SSB) are absent in Lats1/2CM / mutants and cells that accumulate in the interstitium still express 2 2 Six2. (D) Note the discontinuous E-cadherin staining in accumulated interstitial cells in Lats1/2CM / kidneys (inset in D). (G) Quan- tification of all Six2 cells (E-cadherin–negative) per entire kidney section at E14.5 and E18.5 in both genotypes. Quantification of the total number of Six2 cells was made in six kidneys, except for the control at E18.5 where only one kidney section was used to quantify the number of Six2 cells. (H and I) Lats1/2 deletion leads to loss of Cited1 expression from the NPCs (arrows) and accumulation of 2 2 E-cadherin–positive cells in the interstitium (*). (J–M) PAS staining of E14.5 kidneys from wild-type and Lats1/2CM / kidneys. Higher 2 2 magnification views reveal absence of early nephrons in Lats1/2CM / kidneys (compare M to L). Arrows point to the UB tips. (N and O) 2 2 Sox9 and E-cadherin staining at E14.5 showed loss of early nephron structures in Lats1/2CM / kidneys compared with controls (arrows point to the UB tips, white arrowheads to early nephrons). G, glomerulus; ST, stroma. Asterisk points to accumulated cells. Scale bars represent 100 mm(A–D’,H–O) and 200 mm (E and F).

To test whether unrestricted Yap/Taz expression is respon- DISCUSSION 2 2 sible for the cell fate change observed in Lats1/2CM / kidneys, 2 2 we attempted to rescue the Lats1/2CM / phenotype by Here, we demonstrate that Lats1/2 kinases regulate Yap/Taz in reducing Yap and/or Taz levels by generating Six2:CreTGC/+ NPCs, as Lats1/2 deletion leads to increased nuclear Yap and fl fl fl fl fl fl fl fl Lats1 ox/ ox Lats2 ox/ ox Yap ox/( ox/+) Taz ox/( ox/+) embryos. Taz and loss of phosphorylated Yap. Moreover, increased Yap/ We then performed PAS and Six2/E-cadherin staining in all Taz activity is responsible for the Lats1/2 phenotype, as a com- genotypes. Strikingly, the abnormal nephrogenesis seen in plete rescue is observed upon genetic ablation of a single allele 2 2 Lats1/2CM / kidneys was largely rescued by reducing Yap of Yap or Taz, or by the deletion of one copy of both Yap and and/or Taz levels (Figure 6). These data indicate that Lats1/2 Taz. Ablation of Lats1/2 impaired MET, which is essential for kinases inhibit Yap/Taz in NPCs to promote epithelialization nephron formation, and induced a phenotypic change from and nephron formation. epithelial (nephron) to myofibroblast. These findings

J Am Soc Nephrol 28: ccc–ccc, 2016 Hippo in Nephron Progenitor Fate 5 BASIC RESEARCH www.jasn.org

Figure 4. Lats1/2 deletion leads to myofibroblast formation. (A and A’) In controls, vimentin staining at E14.5 shows that NP and 2 2 derived cells (marked by NCAM) are vimentin-negative. (B and B’)InLats1/2CM / mTmG kidneys, NP-derived cells (*, marked by GFP) are vimentin-positive. White arrowheads point to early nephrons. White arrows point to NPCs. (C and D) Masson trichrome staining at 2 2 E18.5 reveals collagen deposition in Lats1/2CM / kidneys. (E–L) Immunohistochemistry for collagen IV staining demonstrates collagen 2 2 deposition at E14.5 (E–H) and E18.5 (I–L). (M–P) a-SMA staining at E14.5 identifies myofibroblasts in Lats1/2CM / kidneys, whereas a-SMA expression is only observed in smooth-muscle vascular cells (arrows) in controls. (Q and R) At E18.5, all NP-derived cells in 2 2 Lats1/2CM / kidneys are a-SMA–positive. Asterisk points to accumulated cells. G, glomerulus; ST, stroma. Scale bars represent 100 mm(A–B’,E–H, and K–R) and 500 mm (C, D, I, and J). demonstrate that myofibroblasts can derive from NPCs and of Yap/Taz activation. Altogether, these results strongly suggest bring new insights into the regulation of self-renewal and dif- that increased Yap/Taz activities in the NPCs as a consequence ferentiation of NPCs. of Lats1/2 deletion is sufficient to directly drive epithelial-to- During development, NPCs self-renew and undergo MET myofibroblast cell fate change. to form nephrons. During this process, cells decrease in Upon Lats1/2 deletion, the pool of NPs is greatly reduced. motility and acquire epithelial characteristics. However, in Loss of NPCs could not be explained by increased cell death, as 2 2 2 2 Lats1/2CM / kidneys, NP-derived cells never become fully little or no apoptosis is observed in Lats1/2CM / NPCs as seen epithelial, as seen by the absence of Crumbs3, strong vimentin in controls (Figure 5, G and H). We found increased prolifera- 2 2 expression, and the absence of tubule organization. Proximal tion of NPCs in Lats1/2CM / compared with controls, consis- 2 2 NP-derived cells in Lats1/2CM / kidneys express b-catenin, tent with the overall increased nuclear Yap and Taz (Figure 5, NCAM, discontinued E-cadherin, and reduced laminin. This I–K). An appealing hypothesis is that NPCs with higher nuclear expression profile suggests that accumulated interstitial cells Yap/Taz (Hippo inactive) are more likely to exit the NP niche and 2 2 in Lats1/2CM / might be in a transient state. Nevertheless, as accumulate in the cortical and medullary parts of the kidney. NP descendants localize further away from the nephrogenic Consistent with this model is our finding that NPCs depleted for 2 2 zone in Lats1/2CM / kidneys, expression of NCAM, Lats1/2 show loss of the progenitor marker Cited1. NPCs treated b-catenin, and E-cadherin is gradually lost, and they ectopi- with Bmp7 also lose Cited1, which results in a population of cally express the myofibroblast marker a-SMA. We speculate ‘Six2-only’ progenitors more likely to respond to Wnt signaling, that complete myofibroblast conversion, observed in the more and p-Smad signaling has been shown to promote the transition medullary accumulated interstitial cells, depends on the extent of progenitors out of the Cited1 positive compartment.27

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Figure 5. Lats1/2 restricts Yap/Taz expression in NPC. (A–F’) Lats1/2 deletion leads to loss of phospho-Yap (A–B’), increased nuclear 2 2 Yap (C–D’), and increased nuclear Taz (E–F’)inLats1/2CM / mTmG NP-derived cells compared with early nephrons in controls. 2 2 Staining with E-cadherin and GFP marks early nephron structures in controls and NP-derived cells in Lats1/2CM / mTmG kidneys, 2 2 respectively. (G and H) Cleaved caspase 3 staining in control and Lats1/2CM / kidneys at E14.5. (I and J) EdU incorporation in Lats1/ 2 2 2 2 2CM / and control NPCs at E14.5 shows an overall increase in the percentage of proliferating Six2 cells in Lats1/2CM / kidneys compared with controls. (See quantiﬁcation in [K]. For the quantiﬁcation, 3611 and 875 Six2Pos-E-cadherinNeg cells were counted in 2 2 total in controls and Lats1/2CM / embryos, respectively [n=5].) G, glomerulus; PT, proximal tubule; ST, stroma. Asterisk points to accumulated cells. Scale bars represent 50 mm.

Consistent with this, we found increased p-Smad1/5 staining in Yap/Taz nuclear accumulation in Lats1/2-deﬁcient NPCs leads 2 2 Lats1/2CM / kidneys (Supplemental Figure 2). Interestingly, to rapid exhaustion of the NPCs that differentiate into myoﬁ- Yap depletion from the NPCs also led to the loss of Cited1 ex- broblasts. It is possible that Lats1/2 deletion may lead to a stron- pression,18 suggesting that Yap levels in NPCs need to be tightly ger level of Yap activation compared with Fat4 deletion, possibly controlled for commitment to the nephron lineage. explaining the opposite observations. Alternatively, Yap might Fat4 mutants have an enlarged CM.28–30 It has been proposed not be the primary cause of increased CM in Fat4 mutants, as that increased nuclear Yap is responsible for the increased CM suggested by the lack of suppression of the expanded CM in both 2 2 2 2 2 2 2 2 thickness observed in Fat4 mutants.31 This implies that in- Fat4 / Yap CM / and Fat4 / Taz / mutants.29,30 The differ- creased nuclear Yapwould favor self-renewal, rather than different outcomes observed upon Yap activation are inconsistent entiation of NPCs. In contrast, our data demonstrate that and will therefore require further analysis.

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2 2 Figure 6. Rescue of the Lats1/2CM / phenotype by loss of Yap and/or Taz. PAS and Six2/E-cadherin staining of kidneys from (A–A”) 2 2 2 2 2 2 2 2 2 2 2 control, Lats1/2CM / (n=6 kidneys) (B–B”), Lats1/2CM / YapCM+/ (n=2) (C–C”), Lats1/2CM / TazCM / (n=2) (D–D”), Lats1/2CM / 2 2 2 2 2 2 Yap/TazCM+/ (n=4) (E–E”), and Lats1/2CM / YapCM / TazCM+/ (n=2) (F–F”) at E17.5. Scale bars represent 250 mm(A–F), 100 mm (A’–F’ and A”–F”), and 500 mm (insets in A”–F”).

Is the sole function of Lats1 and Lats2 kinases in the NPCs ofYapandTazinNPCsareimportanttodriveproper to restrict Yap/Taz activities? Our rescue experiments suggest nephrogenesis. that this is the case, since lowering the level of Yap, Taz, or In conclusion, our data indicates that Lats1/2 function is both is enough to rescue the nephrogenesis defects ob- necessary to inhibit Yap/Taz activity during nephron forma- 2 2 served in Lats1/2CM / kidneys. Intriguingly, we also tion and to allow self-renewal and differentiation of NPCs. found that loss of Lats1/2 suppresses the cyst formation nor- Remarkably, the effects of deletion of the core Hippo kinases mally observed in Taz mutants,18–20 as there were no cysts in Lats1 and Lats2 is to impair epithelialization of NPCs, efﬁ- 2 2 2 2 Lats1/2CM / Taz CM / compound mutants. Thus, the levels ciently converting all NPC into myoﬁbroblasts.

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CONCISE METHODS wild-type versus mutant). All statistical analyses were conducted using GraphPad Prism 5.0a software (GraphPad Software, La Jolla, Mouse Lines CA). fl fl fl fl The Six2:CreTGC/+, Lats1 ox, Lats2 ox Taz ox, Yap ox,andmTmG Each experiment was performed using littermate control and mouse strains were generously provided and have been described mutant embryos, and the observations are representative of n$4 elsewhere.10,18,24,32 Allmicewerebredonamixedgeneticback- embryos (8 kidneys), except for the rescue experiments were n values ground. Husbandry and ethical handling of mice were conducted are mentioned in the figure legend (Figure 6). according to guidelines approved by the Canadian Council on Animal Care. Genotyping was done by PCR using genomic DNA prepared from mouse-ear punches. ACKNOWLEDGMENTS

Histologic and Immunologic Analyses We thank Randy Johnson and Jeff Wrana for kindly providing the Embryonic samples from timed matings (day of vaginal plug = E0.5) Lats1/2 flox and Yap/Taz flox mouse strains. were collected, fixed in 4% paraformaldehyde overnight at 4°C, seri- H.M. is a Tier 1 Canada Research Chair in Coordinating Growth ally dehydrated, and then embedded in paraffin. Microtome sections and Polarity. of 7 mm thickness were examined histologically via PAS staining. For A.R. designed, performed, and analyzed all experiments. A.R. and immunofluorescent analysis, paraffin sections were dewaxed and re- H.M. wrote the manuscript. hydrated via ethanol series. Antigen retrieval was performed for This work was supported by grants from the Canadian Institutes of 20 minutes in boiling Antigen Unmasking Solution (H-3300; Vector Health Research (MOP 136924, 84468), March of Dimes (1-FY11- Laboratories, Burlingame, CA). Sections were incubated for 1 hour in 506), and US Department of Defense (W81XWH-15-1-0461) to H.M. blocking solution (3% BSA, 10% goat serum, and 0.1% Tween20 in PBS) at room temperature. Blocking solution was replaced with a solution of primary antibodies diluted in 3% BSA, 3% goat serum, and 0.1% Tween20 in PBS. Relevant conjugated secondary antibodies DISCLOSURES (Jackson ImmunoResearch Laboratories, West Grove, PA) were used None for primary antibody detection. Slides were mounted using Vectashield with or without 49,6-diamidino-2-phenylindole (DAPI; Vector Labo- ratories). The following primary antibodies (1:300 dilution if not REFERENCES specified) were used: b-Catenin (#9562S; Cell Signaling Technology, Danvers, MA), calbindin (PC253C; Calbiochem, San Diego, CA), 1. Saxén L, Sariola H, Lehtonen E: Sequential cell and tissue interactions Cited1 (RB-9219-P0; Neomarkers), cleaved caspase 3 (#9661; Cell governing organogenesis of the kidney. Anat Embryol (Berl) 175: 1–6, 1986 Signaling Technology), Collagen IV (#681241; MP Biomedicals), 2. Schedl A: Renal abnormalities and their developmental origin. Nat Rev Genet 8: 791–802, 2007 Crumbs3 (HPA013835; Sigma-Aldrich, St. Louis, MO), E-cadherin 3. Little MH, McMahon AP: Mammalian kidney development: principles, (610181, BD Transduction Laboratories; #3195, Cell Signaling Tech- progress, and projections. Cold Spring Harb Perspect Biol 2012. nology), GFP (ab13970; Abcam Inc., Cambridge, MA), HA (11 867 Available at http://cshperspectives.cshlp.org/content/4/5/a008300. 423 001; Roche Diagnostics, Indianapolis, IN), laminin (L9393; Accessed 2012 Sigma-Aldrich), NCAM (C9672; Sigma-Aldrich), Pax2 (PRB-276P; 4. Costantini F, Kopan R: Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development. Dev Covance), Phospho-Smad1/5 (#9516; Cell Signaling Technology), Cell 18: 698–712, 2010 Phospho-Yap (#4911, Cell Signaling Technology; 1:150 dilution), 5. Self M, Lagutin OV, Bowling B, Hendrix J, Cai Y, Dressler GR, Oliver G: Six2 (11562–1-AP; Proteintech), a-SMA (A2547, Sigma-Aldrich; Six2 is required for suppression of nephrogenesis and progenitor re- 1:500 dilution), Sox9 (AB5535; Chemicon), Taz (560235, BD Trans- newal in the developing kidney. EMBO J 25: 5214–5228, 2006 duction; 1:200 dilution), Vimentin (ab92547; Abcam), Yap (sc- 6. Boyle S, Misfeldt A, Chandler KJ, Deal KK, Southard-Smith EM, Mortlock DP, Baldwin HS, de Caestecker M: Fate mapping using 101199, Santa Cruz Biotechnology, Santa Cruz, CA; 1:150 dilution). Cited1-CreERT2 mice demonstrates that the cap mesenchyme contains self-renewing progenitor cells and gives rise exclusively to 5-Ethynyl-2’-Deoxyuridine Incorporation nephronic epithelia. Dev Biol 313: 234–245, 2008 A solution containing 5-ethynyl-2’-deoxyuridine (EdU) (10 mg/ml) 7. Kopan R, Chen S, Little M: Nephron progenitor cells: shifting the balance – was injected intraperitoneally into pregnant mice (50 mg EdU/kg of of self-renewal and differentiation. Curr Top Dev Biol 107: 293 331, 2014 – 8. Larue L, Bellacosa A: Epithelial-mesenchymal transition in develop- mice) 10 15 minutes before embryonic dissection. The samples were ment and cancer: role of phosphatidylinositol 39 kinase/AKT pathways. prepared and sectioned as described above before using the Click-iT Oncogene 24: 7443–7454, 2005 EdU Alexa Fluor 488/455 Imaging Kit (Life Technologies, Carlsbad, 9. Yu J, Carroll TJ, McMahon AP: Sonic hedgehog regulates proliferation CA). and differentiation of mesenchymal cells in the mouse metanephric kidney. Development 129: 5301–5312, 2002 10. Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, Oliver G, Statistical Analyses McMahon AP: Six2 defines and regulates a multipotent self-renewing All data are expressed as meanvalues with SD. An unpaired two-tailed nephron progenitor population throughout mammalian kidney devel- t test was used to determine differences between two groups (e.g., opment. Cell Stem Cell 3: 169–181, 2008

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