Loss of Mob1a/B in Mice Results in Chondrodysplasia Due to YAP1/TAZ

RESEARCH ARTICLE Loss of Mob1a/b in mice results in chondrodysplasia due to YAP1/ TAZ-TEAD-dependent repression of SOX9 Hiroki Goto1,2,*, Miki Nishio1,2,*, Yoko To1, Tatsuya Oishi1, Yosuke Miyachi1,2, Tomohiko Maehama2, Hiroshi Nishina3, Haruhiko Akiyama4, Tak Wah Mak5, Yuma Makii6, Taku Saito6, Akihiro Yasoda7, Noriyuki Tsumaki8 and Akira Suzuki1,2,‡

ABSTRACT differentiation into resting chondrocytes, followed by the Hippo signaling is modulated in response to cell density, external proliferation of the chondrocytes and their maturation into mechanical forces, and rigidity of the extracellular matrix (ECM). The prehyperplastic and finally hypertrophic chondrocytes (Kronenberg, Mps one binder kinase activator (MOB) adaptor proteins are core 2003; Michigami, 2013). Eventually, these terminally differentiated components of Hippo signaling and influence Yes-associated protein 1 chondrocytes undergo apoptosis during endochondral ossification, (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ), leaving a cartilaginous matrix that becomes mineralized and replaced which are potent transcriptional regulators. YAP1/TAZ are key with bone. The processes of chondrogenesis and endochondral contributors to cartilage and bone development but the molecular ossification are tightly regulated by multiple entities, including mechanisms by which the Hippo pathway controls chondrogenesis transcription factors, growth factors, morphogens and hormones are largely unknown. Cartilage is rich in ECM and also subject to (Melrose et al., 2016). strong external forces – two upstream factors regulating Hippo signaling. Among the transcription factors involved in chondrogenesis and Chondrogenesis and endochondral ossification are tightly controlled endochondral ossification is SOX9. In fact, SOX9, which is a by growth factors, morphogens, hormones, and transcriptional factors member of the Sry-related high mobility group box (SOX) family, is that engage in crosstalk with Hippo-YAP1/TAZ signaling. Here, we an indispensable master regulator of chondrogenesis (Bi et al., 1999; generated tamoxifen-inducible, chondrocyte-specific Mob1a/b-deficient Akiyama, 2008; Lefebvre et al., 1998). In humans, heterozygous mice and show that hyperactivation of endogenous YAP1/TAZ impairs mutations of the SOX9 gene lead to campomelic dysplasia, which is chondrocyte proliferation and differentiation/maturation, leading to characterized by severe skeletal malformation (Wagner et al., 1994). chondrodysplasia. These defects were linked to suppression of SOX9, Supporting evidence provided by mouse models has revealed that a master regulator of chondrogenesis, the expression of which is loss of Sox9 results in hypoplastic cartilage (Akiyama et al., 2002). mediated by TEAD transcription factors. Our data indicate that a MOB1- At the molecular level, SOX9 interacts cooperatively with SOX5 dependent YAP1/TAZ-TEAD complex functions as a transcriptional and SOX6 to drive chondrocyte proliferation and differentiation repressor of SOX9 and thereby negatively regulates chondrogenesis. (Lefebvre et al., 1998; Akiyama et al., 2002; Smits et al., 2001; Ikeda et al., 2004). Other signaling pathways involving fibroblast KEY WORDS: MOB1, YAP1, TAZ, WWTR1, SOX9, Chondrocytes, growth factors (FGFs) (Ornitz, 2005), bone morphogenetic proteins Chondrodysplasia, Mouse (BMPs) (Tsuji et al., 2006), parathyroid hormone (Ellegaard et al., 2010), Indian hedgehog (IHH) (Vortkamp et al., 1996) and WNT/β- INTRODUCTION catenin (Huang et al., 2012) are also key players in chondrocyte Chondrogenesis and endochondral ossification are key contributors differentiation during skeletal development. to the development of the vertebral skeleton. During chondrogenesis, Hippo signaling is modulated in response to cell density, external mesenchymal stem cells (MSCs) undergo condensation and mechanical forces, and rigidity of the extracellular matrix (ECM) (Edgar, 2006; Nishio et al., 2013). The core components of the Hippo pathway are the mammalian STE20-like protein (MST) kinases 1Division of Cancer Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan. 2Division of Molecular and Cellular Biology, Kobe (Creasy and Chernoff, 1995), the large tumor suppressor homolog University Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan. (LATS) kinases (Tao et al., 1999), and the adaptor proteins salvador 3Department of Developmental and Regenerative Biology, Medical Research homolog 1 (SAV1) (Valverde, 2000) and Mps one binder kinase Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan. 4Department of Orthopaedic Surgery, Gifu University School of Medicine, Gifu 501- activator 1 (MOB1) (Moreno et al., 2001). MOB1A/B are the adaptor 1194, Japan. 5Campbell Family Institute for Breast Cancer Research at the Princess proteins for the LATS kinases. By binding to LATS kinases, Margaret Cancer Centre, University Health Network, Toronto M5G 2C1, Canada; Department of Medical Biophysics, University of Toronto, University Health MOB1A/B strongly increase the kinase activities of these enzymes Network, Toronto M5G 2C1, Canada. 6Department of Sensory and Motor System (Moreno et al., 2001). Activated LATS kinases in turn phosphorylate Medicine, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan. Yes-associated protein 1 (YAP1) and transcriptional co-activator with 7Department of Diabetes, Endocrinology and Nutrition, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan. 8Department of Cell Growth and PDZ-binding motif (TAZ; also known as WWTR1) (Sudol, 1994; Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kanai et al., 2000). YAP1/TAZ are key downstream transcriptional Kyoto 606-8507, Japan. co-factors that act mainly on TEA domain transcription factors *These authors contributed equally to this work (TEADs) to regulate numerous target genes involved in cell growth ‡Author for correspondence ([email protected]) and differentiation (Zhao et al., 2008). After phosphorylation by LATS kinases, YAP1/TAZ are excluded from the nucleus Y.T., 0000-0002-7980-7186; T.O., 0000-0002-0371-1866; A.S., 0000-0002- 5950-8808 and retained in the cytoplasm, where they are ubiquitylated by E3-ubiquitin ligase SCFβTRCP (also known as BTRC) and subjected

Received 13 September 2017; Accepted 19 February 2018 to proteasome-mediated degradation (Zhao et al., 2010). Thus, in DEVELOPMENT

1 RESEARCH ARTICLE Development (2018) 145, dev159244. doi:10.1242/dev.159244 most cell types, YAP1/TAZ are essentially positive regulators of cell reduction in body size at postnatal day (P) 84 compared with proliferation that are negatively controlled by upstream Hippo core littermate controls (Mob1aflox/flox; Mob1b−/−) (Fig. 1A). components. To study the roles of MOB1A/B during postnatal YAP1/TAZ are considered to be key factors in the regulation of chondrogenesis, we measured the lengths of the long bones and MSC lineage commitment. Under the control of SOX2, YAP1 the size of the cartilaginous growth plates in control and cMob1 maintains MSC self-renewal and inhibits osteogenic differentiation DKO mice at P84. Compared with controls, mutants with Mob1a/b (Seo et al., 2013). However, some studies have reported that low deficiency in chondrocytes showed significant decreases in total TAZ expression promotes adipogenesis, whereas high TAZ levels body length as well as in the length of the femur, tibia, humerus and drive osteogenesis (Hong et al., 2005; Cui et al., 2003; Yang et al., forelimb (Fig. 1B). The size of the articular cartilage layer was also 2013). Thus, the functions and molecular mechanisms by which decreased in the mutants at P12 (Fig. 1C). Close histological YAP1/TAZ influence mesenchymal cells are complicated and examination of growth plates at P21 revealed that each chondrocyte remain largely unknown. It is clear that cartilage is rich in ECM and zone (resting, proliferative, and hypertrophic) was present in cMob1 also subject to strong external forces, both of which are important DKO mice but proportionally reduced in size compared with that in upstream regulators of Hippo-YAP1/TAZ signaling. In addition, control animals (Fig. 1D). Thus, loss of Mob1a/b in chondrocytes Hippo-YAP1/TAZ signaling has been shown to engage in crosstalk results in chondrodysplasia. with FGFs (Rizvi et al., 2016), BMPs (Alarcón et al., 2009), IHH (Wang et al., 2016), WNT/β-catenin (Varelas et al., 2010), SOX2 MOB1A/B deficiency in chondrocytes impairs their (Lian et al., 2010) and SOX9 (Song et al., 2014), all of which proliferation and differentiation are crucial for chondrogenesis. Nevertheless, the molecular Because cMob1 DKO mice exhibited abnormal histology in their mechanisms by which Hippo-YAP1/TAZ signaling controls growth plate cartilage, we analyzed the proliferation and chondrocyte generation and homeostasis remain unclear. differentiation of chondrocytes. Histological examination of control Col2a1-Yap1 transgenic mice were recently reported to display and mutant growth plates at P21 using PCNA staining to identify increased early chondrocyte proliferation driven by YAP1/TEAD- proliferating cells revealed that many PCNA-positive cells were dependent SOX6 activation, but also exhibited YAP1/RUNX2- present in control growth plates (as expected), especially in the dependent COL10A1 inhibition, impaired chondrocyte maturation proliferative zone. However, numbers of PCNA-positive cells in and reduced skeleton size (Deng et al., 2016). However, chondrocyte- growth plates of P21 cMob1 DKO mice were significantly decreased specific Yap1-deficient mice are slightly larger than age-matched compared with controls (Fig. 2A). Notably, the percentage of wild-type (WT) littermates (Deng et al., 2016). In contrast to YAP1, TUNEL-positive apoptotic cells in control and cMob1 DKO growth TAZ overexpression was found to accelerate chondrocyte maturation plates was not significantly different (Fig. S2). To confirm our and promote RUNX2-dependent COL10A1 expression (Deng et al., observations at the molecular level in vitro, we performed siRNA- 2016). It appears that TAZ competes with YAP1 for interaction with mediated knockdown of MOB1A/B in the human chondrocyte cell RUNX2 in order to modulate COL10A1 expression and control line H-EMC-SS. Depletion of MOB1A/B significantly reduced chondrocyte maturation. the proliferation of these cells in vitro (Fig. 2B), indicating that loss We previously reported that Mob1a/b null mutant mice succumb of Mob1a/b negatively affects chondrocyte proliferation in vivo and to embryonic lethality at embryonic day (E) 6.5 (Nishio et al., 2013). in vitro. We have also demonstrated that Mob1a/b loss induces extreme We next examined the expression levels of several genes required hyperactivation of endogenous YAP1/TAZ, resulting in the most for the establishment and maintenance of ECM. We isolated severe phenotypes reported among mice mutated in Hippo core chondrocytes from control and cMob1 DKO mice and used qRT- components in various tissues (Nishio et al., 2017). Thus, MOB1A/ PCR to assess mRNA levels of Col2a1, Col9a1, Col9a2, Comp, B is a crucial hub in the Hippo signaling pathway. In this study, we Col11a1 and aggrecan (Acan). In all cases, relative mRNA expression generated chondrocyte-specific Mob1a/b-deficient mice and found was significantly downregulated in cMob1 DKO chondrocytes that hyperactivation of endogenous YAP1/TAZ induced by loss of compared with controls (Fig. 2C). To evaluate chondrocyte Mob1a/b impaired chondrocyte proliferation and differentiation and maturation, we used immunohistochemistry to detect stage-specific led to the onset of chondrodysplasia. Our data indicate that these markers in chondrocytes in growth plates of control and cMob1 DKO phenotypes occur because a YAP1/TAZ-TEAD complex functions mice. Type 2 collagen (Col II) is expressed in all chondrocyte layers, as a transcriptional repressor of SOX9, a master regulator of whereas osterix (also known as SP7) and IHH are markers of the chondrogenesis. prehypertrophic layer, and Col X is specific to the hypertrophic chondrocyte layer. Numbers of chondrocytes expressing these markers RESULTS were significantly decreased in cMob1 DKO mice (Fig. 2D, Fig. S3), Loss of Mob1a/b in murine chondrocytes results in indicating that MOB1 plays a fundamental role in supporting chondrodysplasia chondrocyte differentiation/maturation. To analyze the functions of endogenous YAP1/TAZ in Lastly, we examined endochondral ossification by assessing the chondrogenesis in vivo, we generated chondrocyte-specific bone volume, osteoid volume, trabecular thickness, trabecular Mob1a/b double-knockout mice (Col2a1-CreERT; Mob1aflox/flox; number and osteoblast number of the proximal tibia of control and Mob1b−/−; hereafter cMob1 DKO) by mating Col2a1-CreERT cMob1 DKO mice (Fig. S4A) at P12, as well as the longitudinal transgenic mice with Mob1aflox/flox and Mob1b−/− mice. growth rate in the primary spongiosa of this limb (Fig. 2E). We also Administration of 4-hydroxytamoxifen (tamoxifen) at P0 activates subjected proximal tibia tissue to Col I staining (Fig. S4B). All of Cre expression, deleting the floxed Mob1a gene. We confirmed these properties were significantly reduced in the mutant mice efficient Mob1a deletion in Mob1b null chondrocytes by PCR compared with controls. As a further corroborating approach, we analysis of DNA from chondrocytes isolated from control and crossed cMob1 DKO mice with Rosa26-LSL-YFP reporter mice to cMob1 DKO mice (Fig. S1). cMob1 DKO mice were born at the generate Col2a1-CreERT; Mob1aflox/flox; Mob1b−/−; Rosa26-LSL- expected Mendelian ratio but developed slowly, showing an overall YFP (mutant) reporter animals. Examination of YFP expression DEVELOPMENT

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Fig. 1. Chondrocyte-specific Mob1a/b double-knockout (cMob1 DKO) mice exhibit hypochondroplasia. (A) Representative littermate control and cMob1 DKO mice at P84. (B) Lengths of total body, femur, tibia, forelimb and humerus in control and cMob1 DKO mice at P84 (n=5/group). (C) Length of the articular cartilaginous zone (arrow) in the distal femur of control and cMob1 DKO mice at P12, as quantified beneath (n=4). The dotted line delineates the end of the articular cartilaginous zone. (D) Representative H&E staining of the growth plate (arrow) in the proximal tibia of control and DKO mice at P21, as quantified on the right (n=5). ***P<0.001, Student’s t-test. Scale bars: 50 μm in C; 100 μminD.

(green) by osteoblasts around bone ECM containing Col I (red) and SOX6 to regulate chondrogenesis (Akiyama, 2008; Ikeda et al., beneath the growth plate from P21 mice confirmed that none of the 2004). In control mice at P21, YAP1 was strongly expressed in the osteoblasts in the mutant tissue was YFP positive, suggesting that nucleus of prehypertrophic chondrocytes but less so in hypertrophic MOB1-deficient chondrocytes did not differentiate into osteoblasts chondrocytes (Fig. 3D). However, proliferative and hypertrophic (Fig. S5). Thus, loss of Mob1a/b in chondrocytes inhibits chondrocytes from cMob1 DKO mice showed both enhanced YAP1 their proliferation, differentiation/maturation, and endochondral activation and reduced levels of SOX5, SOX6 and SOX9 (Fig. 3D). ossification, resulting in the chondrodysplasia phenotype observed Thus, the defect in chondrodysplasia caused by loss of Mob1a/b is in cMob1 DKO mice. very likely to be due (at least in part) to a decrease in expression of SOX9. Mob1a/b deletion activates YAP1/TAZ and downregulates SOX9 expression Hyperactivated YAP1/TAZ suppresses chondrogenesis in To investigate the effects of chondrocyte-specific Mob1a/b loss on cMob1 DKO mice Hippo pathway components and downstream effectors, primary To investigate the role of YAP1 in chondrocyte proliferation and chondrocytes isolated from Col2a1-CreERT; Mob1a+/+;Mob1b−/−; differentiation, we generated H-EMC-SS cells that conditionally Rosa26-LSL-YFP (control) and Col2a1-CreERT; Mob1aflox/flox; expressed YAP1(5SA), a constitutively active form controlled Mob1b−/−; Rosa26-LSL-YFP (mutant) reporter mice were treated using doxycycline (Dox) and a Tet-On system. Overexpression with/without 0.1 μMtamoxifenfor96hin vitro, and YFP+ of YAP1(5SA) significantly decreased the proliferation of chondrocytes were sorted and used as control and cMob1 DKO chondrocytes as determined by the MTS assay (Fig. 4A). In reporter chondrocytes. Chondrocytes lacking MOB1A/B showed accordance with this result, qRT-PCR revealed that mRNA levels of reduced YAP1 (Ser127) and LATS1 (Thr1079) phosphorylation, and five genes involved in the establishment and maintenance of ECM a modest increase in total YAP1 and TAZ proteins (Fig. 3A). No were also reduced (Fig. 4B). differences were detected in phosphorylated (T138/T180) MST1/2 To determine the dependence of the chondrodysplasia phenotype (also known as STK4/3), total MST1, SAV1or LATS1. Notably, loss of cMob1 DKO mice on YAP1/TAZ, we generated two triple of Mob1a/b significantly downregulated mRNA levels of Sox9, Sox5 knockout (TKO) mouse strains: TKO(YAP) mice, which were and Sox6 compared with controls (Fig. 3B), and SOX9 protein was Mob1a/b homozygous deficient plus Yap1 homozygous deficient markedly reduced in Mob1a/b-deficient chondrocytes (Fig. 3C). This (Col2a1-CreERT; Mob1aflox/flox; Mob1b−/−; Yap1flox/flox); and latter result was confirmed by immunohistochemical examination of TKO(TAZ) mice, which were Mob1a/b homozygous deficient the growth plates from control versus mutant mice at P21 (Fig. 3D). plus Taz homozygous deficient (Col2a1-CreERT; Mob1aflox/flox; As noted above, SOX9 is a master transcription factor that acts on Mob1b−/−;Tazflox/flox). The defects in the lengths of the growth genes involved in cartilage development and cooperates with SOX5 plates and the long bones and overall body size were all significantly DEVELOPMENT

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Fig. 2. Loss of Mob1a/b in chondrocytes inhibits their proliferation, differentiation and endochondral ossification. (A) Representative immunostaining to detect PCNA+ cells in the growth plate of the proximal tibia of control and cMob1 DKO mice at P21, as quantified on the right in terms of percentage of PCNA+ cells in the proliferation zone (n=3). (B) MTS assay of the proliferation of H-EMC-SS cells that were treated with si-scramble (si-scr) control or si-MOB1A/B for 72 h (n=3). The efficiency of si-MOB1A/B-mediated knockdown is shown in Fig. 5A. (C) qRT-PCR determination of relative mRNA levels of the indicated genes in primary chondrocytes isolated from control and cMob1 DKO mice at P2 (n=3). (D) Representative immunohistochemistry to detect the indicated stage-specific markers in the proximal tibia of control and cMob1 DKO mice at P21. Arrows indicate the stained layer: Col II, all chondrocyte layers; osterix and IHH, prehypertrophic chondrocyte layer; Col X, hypertrophic chondrocyte layer. Results shown are representative of at least three independent trials. (E) Representative fluorescence microscopy images of the primary spongiosa in the distal femur of control and cMob1 DKO mice at P12. The longitudinal growth rate of primary spongiosa (arrow) is quantified on the right (n=4). Calcein (green) was subcutaneously administered 24 h before the mice were sacrificed. *P<0.05, **P<0.01, ***P<0.001, Student’s t-test. Scale bars: 50 μm. rescued by either Yap1 or Taz deletion (Fig. 4C,D). These results reduced SOX9, SOX5 and SOX6 mRNA levels (Fig. 5E,F, Fig. S6B). indicate that, in WT mice, YAP1 functions to inhibit chondrocyte Thus, the Hippo-YAP1/TAZ pathway regulates the expression of proliferation and maturation, and that the cartilage abnormalities SOX9, SOX5 and SOX6 mRNAs and thus influences their protein observed in cMob1 DKO mice result from hyperactivation of YAP levels. and/or TAZ activity. It is primarily the TEAD family of transcription factors that is modulated by binding to the YAP1/TAZ co-factors. To clarify A MOB1-YAP1/TAZ-TEAD axis regulates SOX9 expression via whether the decrease in the expression of SOX mRNAs depended transcriptional repression on TEADs, we analyzed mRNA levels of SOX9, SOX5 and SOX6 To clarify the links between MOB1A/B, YAP1/TAZ and SOX9, we after siRNA-mediated knockdown of TEAD1-4. Application of carried out siRNA-mediated knockdown of MOB1A/B proteins in siTEAD1-4 to H-EMC-SS chondrocytes efficiently inhibited the human H-EMC-SS or mouse ATDC5 cells (Fig. 5A, Fig. S6A). expression of TEAD proteins (Fig. 6A) and led to upregulated Levels of phosphoYAP1 (Ser127) and SOX9 were decreased production of SOX9, SOX5 and SOX6 mRNAs (Fig. 6B). These whereas total YAP1 and TAZ proteins were increased in these data implied that a YAP1/TEAD complex might function as a MOB1A/B-depleted cells, as compared with cells transfected with transcriptional repressor tasked with controlling SOX mRNA si-scramble control. Transfection of H-EMC-SS chondrocytes expression. To investigate this hypothesis, we engineered H-EMC- with vectors overexpressing human WT YAP [YAP(WT)] or SS chondrocytes to express Dox-inducible YAP1(5SA/S94A), constitutively active YAP1(5SA), or with human WT TAZ which has an additional mutation (S94A) in the TEAD-binding [TAZ(WT)] or constitutively active TAZ [TAZ(SA)], showed that domain that prevents binding to TEADs (Zhao et al., 2008; overexpression of YAP1 (WT or 5SA) or TAZ (WT or SA) Shimomura et al., 2014). Expression of YAP1(5SA/S94A) tended significantly decreased SOX9 protein (Fig. 5B,C). Similarly, to bolster SOX9 and SOX6 mRNA expression and induced a knockdown of MOB1A/B (Fig. 5D, Fig. S6B) or overexpression of modest but statistically significant increase in SOX5 mRNA either YAP1(5SA) (Fig. 5E) or TAZ(SA) (Fig. 5F) resulted in (Fig. 6C,D). DEVELOPMENT

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Fig. 3. MOB1-dependent Hippo signaling controls SOX9. (A) Western blot to detect the indicated Hippo pathway proteins (p, phosphorylated) in primary chondrocytes from control and cMob1 DKO mice at P2. GAPDH, loading control. (B) qRT-PCR determination of relative Sox9, Sox5 and Sox6 mRNA levels in the primary chondrocytes in A (n=3). (C) Western blot to detect SOX9 protein in the primary chondrocytes in A. (D) Representative H&E staining and immunostaining to detect SOX5, SOX6, SOX9 or YAP1 protein in primary chondrocytes in the indicated zones of the growth plates of the proximal tibia of control and cMob1 DKO mice at P21 (n=3). *P<0.05, ***P<0.001, Student’s t-test. Scale bars: 50 μm, except 100 μm in H&E.

We next applied ChIP assays to nuclear extracts of H-EMC-SS TAZ target genes such as CTGF and CYR61 (Fig. 7E,F). These data cells to determine YAP1(5SA) binding to the SOX9 promoter. suggest that Hippo-YAP1/TAZ signaling may contribute to the Indeed, YAP1(5SA) was strongly recruited to the TEAD binding onset of dwarfism in humans via an independent pathway that does site of the SOX9 promoter in these H-EMC-SS cells (Fig. 6E,F). not function downstream of FGFR3. These results show that a YAP1/TEAD complex directly binds to the SOX9 promoter, allowing this complex to act as a transcriptional DISCUSSION repressor of the SOX9 gene and so exert a profound negative Although the regulation of YAP1/TAZ is crucial for the commitment regulatory effect on chondrogenesis. of MSCs to differentiate into osteoblasts or adipocytes (Seo et al., 2013; Hong et al., 2005), there have been few studies analyzing the YAP1/TAZ signaling is not triggered by FGFR3 activation effects of the Hippo signaling pathway on chondrocytes. A recent In humans, achondroplasia (ACH), which is classified as short- report showed that homozygous Col2a1-Yap1Tg/Tg transgenic mice limbed dwarfism, is frequently caused by a hereditary autosomal exhibit a chondrodysplasia phenotype (Deng et al., 2016), and we dominant mutation in the proximal tyrosine kinase domain of have demonstrated here that MOB1 deletion also results in YAP1/ FGFR3 (Shiang et al., 1994). In mice overexpressing Fgfr3 with the TAZ-dependent chondrodysplasia. Thus, the Hippo-YAP1/TAZ corresponding ACH mutation (Fgfr3ach), the growth plate cartilage axis must be important for chondrogenesis. Considering that the shows reductions in height of both the proliferative chondrocyte chondrocyte defects observed in MOB1-deficient mice are more zone and the hypertrophic chondrocyte zone (Naski et al., 1998). pronounced than those of heterozygous Col2a1-Yap1Tg/+ transgenic Because Hippo-YAP1/TAZ signaling has been shown to engage in mice, and that MOB1 deletion in other organs consistently results in crosstalk with FGFs (Rizvi et al., 2016), we investigated whether the most severe phenotypes reported among animals with conditional Hippo-YAP1/TAZ signaling might be triggered downstream deletions of Hippo core components (Nishio et al., 2016), the MOB1 of FGFR3 engagement. We overexpressed FGFR3 G380R, a adaptors must constitute the most important hub in Hippo signaling. constitutively active form of the human mutant protein (Bellus et al., With respect to the mechanism driving chondrodysplasia in the 1995), in H-EMC-SS cells and confirmed high levels of FGFR3 absence of MOB1, in vitro studies have shown that increased YAP1 mRNA in the altered cells (Fig. 7A). However, examination of these activity under conditions of elevated matrix rigidity or high fluid- cells by western blotting showed that this increase in FGFR3 G380R flow shear stress leads to impaired chondrocyte maturation, whereas expression did not activate YAP1 or TAZ in either H-EMC-SS downregulation of YAP1 in response to a soft substrate maintains or ATDC5 cells (Fig. 7B,C). This was confirmed by chondrogenic marker expression (Zhong et al., 2013a,b). Other immunohistochemical assays to detect YAP1 in the proximal tibia work has demonstrated that overexpression of YAP1 in murine of P14 control mice compared with Fgfr3ach mice (Naski et al., C3H10T1/2 mesenchymal-like cells can inhibit chondrogenic 1998) (Fig. 7D). Finally, constitutive FGFR3 activation in H-EMC- differentiation in vitro (Karystinou et al., 2015). Similarly, in vivo,

SS and ATDC5 cells did not increase the mRNA levels of YAP1/ overexpression of YAP1 in mice attenuates endochondral DEVELOPMENT

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Fig. 4. YAP1/TAZ hyperactivation inhibits chondrocyte proliferation and the expression of ECM genes. (A) MTS assay of the proliferation of H-EMC-SS cells that overexpress Dox-inducible YAP1(5SA) and that were treated with (or without) 1.0 μg/ml Dox for 72 h (n=3). (B) qRT-PCR determination of relative mRNA levels of the indicated genes in YAP1(5SA)-expressing H-EMC-SS cells that were treated with (or without) Dox for 48 h (n=3). (C) Representative H&E staining to reveal length of growth plate (arrow) in the proximal tibia of control, cMob1 DKO, or cMob1 TKO (cMob1 DKO plus Yap1 or Taz homozygous) mice at P28, as quantified on the right (n=5). Scale bar: 100 μm. (D) Length of total body, femur or humerus in control, cMob1 DKO, and cMob1 TKO mice at P28 (n=5). *P<0.05, **P<0.01, ***P<0.001, Student’s t-test. maturation and inhibits the formation of cartilaginous callus tissue differences between YAP1 and TAZ in this context. The reasons after bone fracture (Deng et al., 2016). We have shown here that underlying the discrepancies between our work and that of Deng MOB1 deletion leading to hyperactive YAP1 expression impairs et al. (2016) are currently unknown. ECM production (Fig. 2C). All these reports are consistent in their SOX9 is an indispensable initiator and master regulator of conclusion that increased YAP1 activation blocks chondrocyte chondrogenesis (Bi et al., 1999; Akiyama, 2008; Lefebvre et al., differentiation/maturation. However, the function of YAP1 in 1998). As noted above, SOX9 interacts cooperatively with SOX5 chondrocyte proliferation, as well as the base functions of TAZ and SOX6 to regulate cartilage matrix genes, including Col2a1, in chondrocytes, remain controversial. Although YAP1 Col9a1, Col11a1 and Acan, to drive chondrocyte proliferation and overexpression reportedly increases the proliferation both of the differentiation (Oh et al., 2014). Accordingly, neither Sox5 nor Sox6 ATDC5 chondrocyte cell line in vitro and murine chondrocytes expression can be detected in Col2a1-Cre; Sox9flox/flox conditional in vivo (Deng et al., 2016), the expected effects on immature knockout mice (Lefebvre et al., 1998; Akiyama et al., 2002; Ikeda chondrocytes have been difficult to document. In contrast to Deng et al., 2004). Loss of Sox9 specifically in murine chondrocytes et al. (2016), our data show that MOB1 deletion leading to YAP1/ results in severely hypoplastic cartilage (Akiyama et al., 2002), and TAZ activation decreases chondrocyte proliferation, as established heterozygous mutations in the human SOX9 gene cause hereditary by the MTS assay in vitro (Fig. 4A) and by PCNA staining in vivo campomelic dysplasia (Wagner et al., 1994). We found that loss of (Fig. 2A). The fact that both the proliferative and hypertrophic MOB1 in murine chondrocytes significantly suppressed both chondrocyte layers in our mutant mice were decreased in size protein and mRNA expression of Sox9, Sox5 and Sox6 in a supports our contention that chondrocyte proliferation is impaired YAP1/TAZ-TEAD-dependent manner (Fig. 3B-D), and that when YAP1 signaling is excessive. Deng et al. (2016) reported that overexpression of YAP1 or TAZ also downregulated SOX9, SOX5 TAZ competes with YAP1 for RUNX2 activation and promotes and SOX6 expression levels (Fig. 5B,C,E,F). Deletion of SOX9 in chondrocyte maturation. However, we show here that the mouse limb buds reportedly abolishes the expression of both SOX5 phenotypes of MOB1-deficient chondrocytes can be mostly and SOX6, confirming that SOX9 is the master regulator of rescued by additional deficiency of YAP1 or TAZ (Fig. 4C,D). In chondrogenesis and is necessary for SOX5 and SOX6 generation addition, we demonstrate that either constitutively activated YAP1 during chondrocyte differentiation (Akiyama et al., 2002). Within or activated TAZ can suppress the expression of SOX factors enhancer regions, SOX5 and SOX6 bind to recognition sites near

(Fig. 5B,C,E,F), leading us to conclude that there are no functional that bound by SOX9, thereby consolidating SOX9 binding to DNA DEVELOPMENT

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Fig. 5. Regulation of SOX9, SOX5 and SOX6 expression by MOB1-YAP1/TAZ. (A) Western blot to detect the indicated Hippo pathway proteins in H-EMC-SS cells that were treated with si-scramble or si-MOB1A/B for 48 h. Actin, loading control. (B,C) Western blots to detect the indicated proteins in H-EMC-SS cells that expressed Dox-inducible forms of human (B) YAP1(WT) or YAP1(5SA) or (C) TAZ(WT) or TAZ(SA), treated with (or without) 1.0 μg/ml Dox for 48 h. SOX9 protein levels relative to actin were quantified by determination of band intensities using ImageJ, as shown beneath. (D-F) qRT-PCR determination of relative SOX9, SOX5 and SOX6 mRNA levels in H-EMC-SS cells that were (D) treated with si-scramble or si-MOB1A/B, or were subjected to Dox-mediated induction of (E) YAP1(5SA) or (F) TAZ(SA) (n=3). *P<0.05, **P<0.01, ***P<0.001, Student’s t-test. and potentiating SOX9 activity (Liu and Lefebvre, 2015). These YAP1/TAZ target genes such as CTGF and CYR61 were not altered observations imply that the reduced expression of SOX5 and SOX6 in two chondrocyte cell lines (H-EMC-SS and ATDC5) that accompanies the decrease in SOX9 caused by MOB1 deletion overexpressing FGFR3 G380R (Fig. 7E,F). Our finding that FGF contributes to the chondrodysplasia observed in our mutant mice. signaling does not activate YAP1/TAZ is in line with a report by Song et al. (2014) reported that YAP1 directly regulates SOX9 another group (Yu et al., 2012) that examined human embryonic transcription through a conserved TEAD binding site in the SOX9 kidney cells (HEK293A). Additional studies are required to promoter, and that YAP1 maintains the cancer stem cell properties determine the nature and extent of the crosstalk between the of esophageal tumor cells by upregulating SOX9 expression (Song Hippo and FGF signaling pathways. et al., 2014). However, we show here that, although YAP1 is indeed In conclusion, we have clarified the physiological functions of recruited to the SOX9 promoter, it induces repression of SOX9 MOB1-YAP1/TAZ signaling in chondrocytes, and have shown that expression in a TEAD-dependent manner (Fig. 6A-F). This inappropriate hyperactivation of a YAP1/TAZ-TEAD complex that situation of context-dependent opposing effects has been functions as a transcriptional repressor of SOX9 can lead to the onset frequently observed for various transcription factors (Fry and of chondrodysplasia in mice. In this light, it would be interesting to Farnham, 1999; Kim et al., 2015). In addition, although YAP1/TAZ analyze the frequency of YAP1/TAZ hyperactivation in dwarfism most often form complexes with TEADs that enhance their activity, patients. Our results increase our molecular understanding of the recent studies have revealed that YAP1/TAZ can also act as effects of the Hippo signaling pathway in vivo, and might provide transcriptional co-repressors (Kim et al., 2015; Zaidi et al., 2004; new insights into potential therapeutic strategies for dwarfism Valencia-Sama et al., 2015). Thus, it is not unreasonable to patients. conclude that a YAP1/TAZ-TEAD complex may function as either a transcriptional activator or repressor of SOX9 expression, MATERIALS AND METHODS depending on the tissue-specific context. Mice As noted above, ACH is caused by a hereditary autosomal Mouse strains used in this study were Col2a1-CreERT Tg (The Jackson dominant mutation in FGFR3 (Shiang et al., 1994). However, we Laboratory), Mob1aflox/flox; Mob1b−/− (Nishio et al., 2012, 2016), Rosa26- show here that overexpression of the constitutively activated FGFR3 LSL-YFP reporter (Srinivas et al., 2001), Yap1flox/flox (Knockout Mouse G380R mutant protein does not activate YAP1/TAZ in vitro Project Repository, UC Davis, CA, USA), Tazflox/flox (kindly provided by

(Fig. 7B,C) or in vivo (Fig. 7D). Furthermore, mRNA levels of Dr J. Wrana), and Fgfr3ach (kindly provided by Dr H. Akiyama). DEVELOPMENT

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Fig. 6. Regulation of SOX9 expression by YAP1-TEAD-mediated transcription. (A) Western blot to detect pan-TEAD in H-EMC-SS cells that were treated with si-scramble or si-TEAD1-4 #1 or #2 for 48 h. (B) qRT-PCR determination of relative SOX9, SOX5 and SOX6 mRNA levels in the cells in A (n=3). (C) Western blot to detect YAP1, pYAP1 and SOX9 proteins in H-EMC-SS cells that expressed Dox-inducible YAP1(5SA/S94A) and were treated with (or without) 1.0 μg/ml Dox for 48 h. (D) qRT-PCR determination of relative SOX9, SOX5 and SOX6 mRNA levels in the cells in C (n=3). (E,F) Semi-quantitative (E) and quantitative (F) ChIP assays to detect YAP1 binding to the SOX9 promoter in nuclear extracts of H-EMC-SS cells expressing Dox-inducible Flag-tagged YAP1(5SA) (n=3). Negative control, non-TEAD binding site; positive control, CTGF promoter. *P<0.05, ***P<0.001, Student’s t-test.

Mice were kept in pathogen-free facilities at Kyushu and Kobe and Ham’s F-12 medium (Wako) containing 5% FCS in a humidified Universities. Protocols for animal experiments were approved by the atmosphere at 37°C and 5% CO2. Animal Research Committees of Kyushu and Kobe Universities. Preparation of mouse primary chondrocytes Generation of cMob1 DKO mice and related strains The ventral parts of rib cages of P2 mice were digested with collagenase Chondrocyte-specific Mob1a/b homozygous double-mutant mice (Col2a1- D and chondrocytes were isolated as described previously (Beier et al., 1999). CreERT; Mob1aflox/flox; Mob1b−/−) were generated by mating Col2a1- Chondrocytes isolated from control mice (Col2a1-CreERT; Mob1a+/+; CreERT Tg with Mob1aflox/flox; Mob1b−/− mice. Col2a1-CreERT Tg mice Mob1b−/−), or from Col2a1-CreERT; Mob1aflox/flox;Mob1b−/− mice carrying were of the C57BL/6 background, and Mob1aflox/flox; Mob1b−/− mice were the Rosa26-LSL-YFP reporter allele, were plated in 6-well dishes at 5000 backcrossed to C57BL/6 for more than six generations. Mob1aflox/flox; cells/cm2 and grown to confluence in DMEM containing 10% FCS. Plated Mob1b−/− mice without the Col2a1-CreERT transgene were usually chosen chondrocytes were treated with 0.1 μM tamoxifen for 96 h. YFP+ cells were to serve as controls because no significant differences in total body length or collected using an SH800 cell sorter (Sony). lengths of long bones and cartilaginous zones were observed between − − − − Mob1aflox/flox; Mob1b / and Col2a1-CreERT; Mob1aflox/flox; Mob1b / Dimethylthiazol carboxymethoxyphenyl sulfophenyl (MTS) mice that were injected with 4-hydroxytamoxifen (Sigma-Aldrich). To assay delete the floxed Mob1a gene, a single dose of tamoxifen (0.1 mg) was Cell proliferation was measured by the MTS method (Cory et al., 1991). flox/flox −/− injected into Col2a1-CreERT; Mob1a ; Mob1b and control pups at MTS assays were performed using the CellTiter 96 assay (Promega) P0. Primers used for genotyping PCR are listed in Table S1. according to the manufacturer’s instructions.

Cell culture Immunohistochemistry The human chondrocyte cell line H-EMC-SS (Riken Cell Bank, Tsukuba, Mouse tissues were fixed in 4% paraformaldehyde in PBS, decalcified in Japan) was maintained in MEMα medium (Wako) supplemented with 10% 10% EDTA, embedded in paraffin, and sectioned. Deparaffinized sections heat-inactivated fetal calf serum (FCS), penicillin (100 U/ml) and were antigen-retrieved using Immunosaver (Nissin EM, Tokyo, Japan), and streptomycin (100 μg/ml) and cultured in a humidified incubator at 37°C then incubated with primary antibodies at 4°C overnight. Primary antibodies and 5% CO2. The mouse embryonal carcinoma-derived chondrogenic cell were against SOX5 (ab94396, Abcam; 1:200), SOX6 (sc-393314, Santa line ATDC5 (Riken Cell Bank) was maintained in a 1:1 mixture of DMEM Cruz Biotechnology; 1:100), SOX9 (sc-20095, Santa Cruz Biotechnology; DEVELOPMENT

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Fig. 7. Hippo-YAP1/TAZ signaling does not function downstream of FGFR3. (A) qRT-PCR determination of relative FGFR3 mRNA levels in unmodified H- EMC-SS cells (control) or in H-EMC-SS cells overexpressing FGFR3 G380R, a constitutively active form of human FGFR3 (n=3). (B) Western blot to detect the indicated proteins in the H-EMC-SS cells in A. (C) Western blot to detect the indicated proteins in ATDC5 cells overexpressing FGFR3 G380R. (D) Representative immunohistochemistry to detect YAP1 in the proximal tibia of P14 control and Fgfr3ach transgenic mice (which express murine Fgfr3 G380R). Scale bar: 50 μm. (E,F) qRT-PCR determinations of relative CTGF and CYR61 mRNA levels in H-EMC-SS cells (E) or ATDC5 cells (F) overexpressing FGFR3 G380R (n=3). *P<0.05, **P<0.01, ***P<0.001, Student’s t-test.

1:100), PCNA (610664, BD Transduction Laboratories; 1:200), Col I was carried out with THUNDERBIRD SYBR qPCR Mix (Toyobo) (ab34710, Abcam; 1:100), Col II (LB-1297, LSL, Tokyo, Japan; 1:400), Col following the manufacturer’s instructions, and with the primers listed in X (LB-0092, LSL; 1:200), IHH (ab39634, Abcam; 1:100), osterix (SP7) Table S2. PCR amplifications were performed using the StepOne real-time (ab22552, Abcam; 1:100), YFP/GFP (ab6673, Abcam; 1:500) or YAP1 PCR system (Applied Biosystems). Ct values for each gene amplification (WH0010413M1, Sigma-Aldrich; 1:500). Anti-rabbit/mouse-HRP (Dako) were normalized by subtracting the Ct value calculated for Gapdh/GAPDH. was used for DAB staining. Secondary antibodies were tagged with Alexa Normalized gene expression values report the relative quantity of mRNA. Fluor 488 or Alexa Fluor 568 (Molecular Probes). In some slides, nuclei were visualized using Hematoxylin and Eosin (H&E) or DAPI. Western blotting Western blotting was carried out using a standard protocol and primary TUNEL staining antibodies recognizing MOB1 (3863, Cell Signaling; 1:1000), MST1 (3682, Apoptosis of chondrocytes was analyzed by TUNEL staining using the In Cell Signaling; 1:1000), phosphoMST1/2 (3681, Cell Signaling; 1:1000), Situ Cell Death Detection Kit (Roche) according to the manufacturer’s SAV1 (13301, Cell Signaling; 1:1000), LATS1 (3477, Cell Signaling; instructions. Nuclei were visualized with DAPI. 1:1000), phosphoLATS1 (9159, Cell Signaling; 1:1000), YAP1 (4912, Cell Signaling; 1:1000), phosphoYAP1 (4911, Cell Signaling; 1:1000), TAZ Histomorphometry (V386) (4883, Cell Signaling; 1:1000), SOX9 (sc-20095, Santa Cruz Ethanol-fixed tibiae from P12 mice were fixed in 70% ethanol, embedded in Biotechnology; 1:500), FGFR3 (sc-13121, Santa Cruz Biotechnology; glycol methacrylate resin, and sectioned into 5 μm slices. For histomorphometric 1:500), GAPDH (sc-25778, Santa Cruz Biotechnology; 1:1000) and actin analyses, an area (1.62-2.34 mm2) 1.2 mm below the growth plate in the (A2066, Sigma-Aldrich; 1:1000). Primary antibodies were detected using proximal tibia was evaluated. Histomorphometric parameters, such as trabecular HRP-conjugated secondary antibodies (Cell Signaling). bone volume/tissue volume (BV/TV), osteoid bone volume/tissue volume (OV/ TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and osteoblast Transfection of siRNA or cDNA number/bone surface (N.Ob/BS), were calculated. siRNAs targeting MOB1A/B or TEAD1-4 expression are listed in Table S3. Transfection of siRNA oligonucleotides (30 nM) into H-EMC-SS cells was Measurement of longitudinal growth rate in primary spongiosa performed using Lipofectamine RNAiMAX (Invitrogen) following the Mice received subcutaneous administration of 20 mg/kg calcein. The manufacturer’s protocol. Transfection of empty pcDNA3.1 vector, or longitudinal growth rate of the primary spongiosa was measured 24 h later in pcDNA3.1 vector expressing FGFR3 G380R (Bellus et al., 1995), into histological tissue sections as previously described (Pass et al., 2012). H-EMC-SS cells was performed using Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. Transfection of empty pcDNA3.1 Quantitative reverse-transcription PCR (qRT-PCR) vector, pcDNA3.1 vector expressing FGFR3 G380R, or siRNA Total RNA was isolated from cells using RNAiso Plus (Takara Bio) oligonucleotides (30 nM) into ATDC5 cells was performed using FuGENE according to the manufacturer’s instructions. Real-time qRT-PCR analysis HD Transfection Reagent (Promega) following the manufacturer’s protocol. DEVELOPMENT

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After 24 h growth to achieve confluence, siRNA-transfected or cDNA- Akiyama, H., Chaboissier, M. C., Martin, J. F., Schedl, A. and de Crombrugghe, transfected ATDC5 cells were cultured for 6 days in a 1:1 mixture of DMEM B. (2002). The transcription factor Sox9 has essential roles in successive steps of and Ham’s F-12 medium containing 5% FCS and 10 µg/ml insulin. the chondrocyte differentiation pathway and is required for expression of Sox5 and Lentiviruses expressing YAP1(WT), YAP1(5SA), YAP1(5SA/S94A), Sox6. Genes Dev. 16, 2813-2828. Alarcón, C., Zaromytidou, A.-I., Xi, Q., Gao, S., Yu, J., Fujisawa, S., Barlas, A., TAZ(WT) or TAZ(SA) were produced by transient transfection of HEK293T Miller, A. N., Manova-Todorova, K., Macias, M. J. et al. (2009). Nuclear CDKs cells with pMDLg/pRRE, pRSV-Rev, pMD2.G, and either pSLIK-Flag- drive Smad transcriptional activation and turnover in BMP and TGF-beta Myc-YAP1(WT), pSLIK-Flag-Myc-YAP1(5SA), pSLIK-Flag-Myc- pathways. Cell 139, 757-769. YAP1(5SA/S94A), pSLIK-Flag-His-TAZ(WT) or pSLIK-Flag-His- Beier, F., Lee, R. J., Taylor, A. C., Pestell, R. G. and LuValle, P. (1999). TAZ(SA) using Lipofectamine 2000 (Invitrogen) (Otsubo et al., 2017). At Identification of the cyclin D1 gene as a target of activating transcription factor 2 in 48 h post-transfection, lentivirus-containing supernatant was collected. chondrocytes. Proc. Natl. Acad. Sci. USA 96, 1433-1438. Bellus, G. A., Hefferon, T. W., Ortiz de Luna, R. I., Hecht, J. T., Horton, W. A., Cultured H-EMC-SS cells were incubated with lentivirus supernatant for Machado, M., Kaitila, I., McIntosh, I. and Francomano, C. A. (1995). 24 h and then transferred to growth medium containing G418 to select for Achondroplasia is defined by recurrent G380R mutations of FGFR3. stable transfectants. Am. J. Hum. Genet. 56, 368-373. Bi, W., Deng, J. M., Zhang, Z., Behringer, R. R. and de Crombrugghe, B. (1999). Chromatin immunoprecipitation (ChIP) assay Sox9 is required for cartilage formation. Nat. Genet. 22, 85-89. ChIP assays were performed as described (Goto et al., 2015). Briefly, cells Cory, A. H., Owen, T. C., Barltrop, J. A. and Cory, J. G. (1991). Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer were cross-linked with formaldehyde and homogenized by sonication. Commun. 3, 207-212. Precleared chromatin was incubated with either anti-DYKDDDDK (FLAG) Creasy, C. L. and Chernoff, J. (1995). Cloning and characterization of a human tag antibody beads (Wako) or mouse IgG, followed by precipitation with protein kinase with homology to Ste20. J. Biol. Chem. 270, 21695-21700. Protein G Sepharose 4 Fast Flow resin (Amersham Biosciences). Semi- Cui, C. B., Cooper, L. F., Yang, X., Karsenty, G. and Aukhil, I. (2003). quantitative PCR analysis was performed using KAPA Taq polymerase Transcriptional coactivation of bone-specific transcription factor Cbfa1 by TAZ. (Kapa Biosystems). Quantitative PCR analysis was carried out using Mol. Cell. Biol. 23, 1004-1013. THUNDERBIRD SYBR qPCR Mix (Toyobo). Primers used for PCR in Deng, Y., Wu, A., Li, P., Li, G., Qin, L., Song, H. and Mak, K. K. (2016). Yap1 regulates multiple steps of chondrocyte differentiation during skeletal ChIP assays are listed in Table S4. development and bone repair. Cell Rep. 14, 2224-2237. Edgar, B. A. (2006). From cell structure to transcription: Hippo forges a new path. Statistical analysis Cell 124, 267-273. Data are presented as mean±s.d. Statistical significance of differences Ellegaard, M., Jørgensen, N. R. and Schwarz, P. (2010). Parathyroid hormone and between experimental groups was determined using Student’s t-test. P<0.05 bone healing. Calcif. Tissue Int. 87, 1-13. Fry, C. J. and Farnham, P. J. (1999). Context-dependent transcriptional regulation. was considered statistically significant. J. Biol. Chem. 274, 29583-29586. Goto, H., Kudo, E., Kariya, R., Taura, M., Katano, H. and Okada, S. (2015). Acknowledgements Targeting VEGF and interleukin-6 for controlling malignant effusion of primary We thank H. Akiyama (Gifu University) and J. Wrana (Lunenfeld-Tanenbaum effusion lymphoma. J. Cancer Res. Clin. Oncol. 141, 465-474. Research Institute) for the Fgfr3ach mutant and Tazflox/flox mice, respectively; A. Ito Hong, J.-H., Hwang, E. S., McManus, M. T., Amsterdam, A., Tian, Y., Kalmukova, (Ito Bone Histomorphometry Institute), A. Fujimoto, M. Kamihashi and M. Suzuki (all R., Mueller, E., Benjamin, T., Spiegelman, B. M., Sharp, P. A. et al. (2005). TAZ, of Kyushu University) for expert technical assistance; and K. Nakao (Kyoto a transcriptional modulator of mesenchymal stem cell differentiation. Science 309, University) for critical discussions. 1074-1078. Huang, Y., Zhang, X., Du, K., Yang, F., Shi, Y., Huang, J., Tang, T., Chen, D. and Competing interests Dai, K. (2012). Inhibition of beta-catenin signaling in chondrocytes induces The authors declare no competing or financial interests. delayed fracture healing in mice. J. Orthop. Res. 30, 304-310. Ikeda, T., Kamekura, S., Mabuchi, A., Kou, I., Seki, S., Takato, T., Nakamura, K., Author contributions Kawaguchi, H., Ikegawa, S. and Chung, U. I. (2004). The combination of SOX5, SOX6, and SOX9 (the SOX trio) provides signals sufficient for induction of Conceptualization: H.G., M.N., Y.T., T.O., T.W.M., A.Y., N.T., A.S.; Methodology: permanent cartilage. Arthritis. Rheum. 50, 3561-3573. H.G., M.N., Y.T., T.O., T.M., H.N., Y. Makii, T.S., A.Y., N.T., A.S.; Validation: H.G., Kanai, F., Marignani, P. A., Sarbassova, D., Yagi, R., Hall, R. A., Donowitz, M., M.N., T.O., T.W.M., A.Y., N.T., A.S.; Formal analysis: H.G., M.N., Y.T., T.O., Hisaminato, A., Fujiwara, T., Ito, Y., Cantley, L. C. et al. (2000). TAZ: a novel Y. Miyachi, A.S.; Investigation: H.G., M.N., Y.T., T.O., Y. Miyachi, A.S.; Resources: transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain M.N., H.N., H.A., Y. Makii, T.S., A.Y., N.T., A.S.; Data curation: A.S.; Writing - original proteins. EMBO J. 19, 6778-6791. draft: H.G., A.S.; Writing - review & editing: H.G., M.N., T.M., T.W.M., T.S., A.Y., N.T., Karystinou, A., Roelofs, A. J., Neve, A., Cantatore, F. P., Wackerhage, H. and De A.S.; Visualization: H.G., M.N., T.O., A.S.; Supervision: T.M., H.N., H.A., T.W.M., Bari, C. (2015). Yes-associated protein (YAP) is a negative regulator of T.S., A.Y., N.T., A.S.; Project administration: A.S.; Funding acquisition: H.G., T.M., chondrogenesis in mesenchymal stem cells. Arthritis Res. Ther. 17, 147. A.S. Kim, M., Kim, T., Johnson, R. L. and Lim, D.-S. (2015). Transcriptional co- repressor function of the hippo pathway transducers YAP and TAZ. Cell Rep. 11, Funding 270-282. We are grateful for the funding provided by Ministry of Education, Culture, Sports, Kronenberg, H. M. (2003). Developmental regulation of the growth plate. Nature Science and Technology (MEXT; grant 15K19026 to H.G.); Japan Society for the 423, 332-336. Promotion of Science (JSPS; grants 17H01400 and 26114005 to A.S.); the Lefebvre, V., Li, P. and de Crombrugghe, B. (1998). A new long form of Sox5 (L- Cooperative Research Project Program of the Medical Institute of Bioregulation, Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively Kyushu University; Nanken-Kyoten, Tokyo Medical and Dental University (TMDU); activate the type II collagen gene. EMBO J. 17, 5718-5733. Project for Development of Innovative Research on Cancer Therapeutics Lian, I., Kim, J., Okazawa, H., Zhao, J., Zhao, B., Yu, J., Chinnaiyan, A., Israel, (P-DIRECT; grant 11088019 to A.S.); Japan Agency for Medical Research and M. A., Goldstein, L. S. B., Abujarour, R. et al. (2010). The role of YAP Development (AMED; grant 16770279 to A.S. and H.G.); the Uehara Memorial transcription coactivator in regulating stem cell self-renewal and differentiation. Foundation (to A.S.); the Shinnihon Advanced Medical Research Foundation (to Genes Dev. 24, 1106-1118. A.S.); the Smoking Research Foundation (to T.M.); and the Daiichi-Sankyo Liu, C.-F. and Lefebvre, V. (2015). The transcription factors SOX9 and SOX5/SOX6 Scholarship Donation Program (to A.S.). cooperate genome-wide through super-enhancers to drive chondrogenesis. Nucleic Acids Res. 43, 8183-8203. Melrose, J., Shu, C., Whitelock, J. M. and Lord, M. S. (2016). The cartilage Supplementary information extracellular matrix as a transient developmental scaffold for growth plate Supplementary information available online at maturation. Matrix Biol. 52-54, 363-383. http://dev.biologists.org/lookup/doi/10.1242/dev.159244.supplemental Michigami, T. (2013). 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