RESEARCH ARTICLE

Loss ofMob1a/b in mice results in chondrodysplasia due to YAP1/TAZ-TEAD-dependent repression of SOX9Hiroki 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,‡

ABSTRACTHippo signaling is modulated in response to cell density, externalmechanical forces, and rigidity of the extracellular matrix (ECM). TheMps one binder kinase activator (MOB) adaptor proteins are corecomponents of Hippo signaling and influence Yes-associated protein 1(YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ),which are potent transcriptional regulators. YAP1/TAZ are keycontributors to cartilage and bone development but the molecularmechanisms by which the Hippo pathway controls chondrogenesisare largely unknown. Cartilage is rich in ECM and also subject tostrong external forces – two upstream factors regulating Hippo signaling.Chondrogenesis and endochondral ossification are tightly controlledby growth factors, morphogens, hormones, and transcriptional factorsthat engage in crosstalk with Hippo-YAP1/TAZ signaling. Here, wegenerated tamoxifen-inducible, chondrocyte-specific Mob1a/b-deficientmice and show that hyperactivation of endogenous YAP1/TAZ impairschondrocyte proliferation and differentiation/maturation, leading tochondrodysplasia. These defects were linked to suppression of SOX9,a master regulator of chondrogenesis, the expression of which ismediated by TEAD transcription factors. Our data indicate that a MOB1-dependent YAP1/TAZ-TEAD complex functions as a transcriptionalrepressor of SOX9 and thereby negatively regulates chondrogenesis.

KEY WORDS: MOB1, YAP1, TAZ, WWTR1, SOX9, Chondrocytes,Chondrodysplasia, Mouse

INTRODUCTIONChondrogenesis and endochondral ossification are key contributorsto the development of the vertebral skeleton. During chondrogenesis,mesenchymal stem cells (MSCs) undergo condensation and

differentiation into resting chondrocytes, followed by theproliferation of the chondrocytes and their maturation intoprehyperplastic and finally hypertrophic chondrocytes (Kronenberg,2003; Michigami, 2013). Eventually, these terminally differentiatedchondrocytes undergo apoptosis during endochondral ossification,leaving a cartilaginous matrix that becomes mineralized and replacedwith bone. The processes of chondrogenesis and endochondralossification are tightly regulated by multiple entities, includingtranscription factors, growth factors, morphogens and hormones(Melrose et al., 2016).

Among the transcription factors involved in chondrogenesis andendochondral ossification is SOX9. In fact, SOX9, which is amember of the Sry-related high mobility group box (SOX) family, isan indispensable master regulator of chondrogenesis (Bi et al., 1999;Akiyama, 2008; Lefebvre et al., 1998). In humans, heterozygousmutations of the SOX9 gene lead to campomelic dysplasia, which ischaracterized by severe skeletal malformation (Wagner et al., 1994).Supporting evidence provided by mouse models has revealed thatloss of Sox9 results in hypoplastic cartilage (Akiyama et al., 2002).At the molecular level, SOX9 interacts cooperatively with SOX5and SOX6 to drive chondrocyte proliferation and differentiation(Lefebvre et al., 1998; Akiyama et al., 2002; Smits et al., 2001;Ikeda et al., 2004). Other signaling pathways involving fibroblastgrowth factors (FGFs) (Ornitz, 2005), bone morphogenetic proteins(BMPs) (Tsuji et al., 2006), parathyroid hormone (Ellegaard et al.,2010), Indian hedgehog (IHH) (Vortkamp et al., 1996) andWNT/β-catenin (Huang et al., 2012) are also key players in chondrocytedifferentiation during skeletal development.

Hippo signaling is modulated in response to cell density, externalmechanical forces, and rigidity of the extracellular matrix (ECM)(Edgar, 2006; Nishio et al., 2013). The core components of the Hippopathway are the mammalian STE20-like protein (MST) kinases(Creasy and Chernoff, 1995), the large tumor suppressor homolog(LATS) kinases (Tao et al., 1999), and the adaptor proteins salvadorhomolog 1 (SAV1) (Valverde, 2000) and Mps one binder kinaseactivator 1 (MOB1) (Moreno et al., 2001). MOB1A/B are the adaptorproteins for the LATS kinases. By binding to LATS kinases,MOB1A/B strongly increase the kinase activities of these enzymes(Moreno et al., 2001). Activated LATS kinases in turn phosphorylateYes-associated protein 1 (YAP1) and transcriptional co-activator withPDZ-binding motif (TAZ; also known as WWTR1) (Sudol, 1994;Kanai et al., 2000). YAP1/TAZ are key downstream transcriptionalco-factors that act mainly on TEA domain transcription factors(TEADs) to regulate numerous target genes involved in cell growthand differentiation (Zhao et al., 2008). After phosphorylationby LATS kinases, YAP1/TAZ are excluded from the nucleusand retained in the cytoplasm, where they are ubiquitylated byE3-ubiquitin ligase SCFβTRCP (also known as BTRC) and subjectedto proteasome-mediated degradation (Zhao et al., 2010). Thus, inReceived 13 September 2017; Accepted 19 February 2018

1Division of Cancer Genetics, Medical Institute of Bioregulation, Kyushu University,Fukuoka 812-8582, Japan. 2Division of Molecular and Cellular Biology, KobeUniversity Graduate School of Medicine, Kobe, Hyogo 650-0017, Japan.3Department of Developmental and Regenerative Biology, Medical ResearchInstitute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan.4Department of Orthopaedic Surgery, Gifu University School of Medicine, Gifu 501-1194, Japan. 5Campbell Family Institute for Breast Cancer Research at the PrincessMargaret Cancer Centre, University Health Network, Toronto M5G 2C1, Canada;Department of Medical Biophysics, University of Toronto, University HealthNetwork, Toronto M5G 2C1, Canada. 6Department of Sensory and Motor SystemMedicine, Faculty of Medicine, University of Tokyo, Tokyo 113-8655, Japan.7Department of Diabetes, Endocrinology and Nutrition, Kyoto University GraduateSchool of Medicine, Kyoto 606-8501, Japan. 8Department of Cell Growth andDifferentiation, Center for iPS Cell Research and Application, Kyoto University,Kyoto 606-8507, Japan.*These authors contributed equally to this work

‡Author for correspondence (suzuki@med.kobe-u.ac.jp)

Y.T., 0000-0002-7980-7186; T.O., 0000-0002-0371-1866; A.S., 0000-0002-5950-8808

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most cell types, YAP1/TAZ are essentially positive regulators of cellproliferation that are negatively controlled by upstream Hippo corecomponents.YAP1/TAZ are considered to be key factors in the regulation of

MSC lineage commitment. Under the control of SOX2, YAP1maintains MSC self-renewal and inhibits osteogenic differentiation(Seo et al., 2013). However, some studies have reported that lowTAZ expression promotes adipogenesis, whereas high TAZ levelsdrive osteogenesis (Hong et al., 2005; Cui et al., 2003; Yang et al.,2013). Thus, the functions and molecular mechanisms by whichYAP1/TAZ influence mesenchymal cells are complicated andremain largely unknown. It is clear that cartilage is rich in ECM andalso subject to strong external forces, both of which are importantupstream regulators of Hippo-YAP1/TAZ signaling. In addition,Hippo-YAP1/TAZ signaling has been shown to engage in crosstalkwith FGFs (Rizvi et al., 2016), BMPs (Alarcón et al., 2009), IHH(Wang et al., 2016), WNT/β-catenin (Varelas et al., 2010), SOX2(Lian et al., 2010) and SOX9 (Song et al., 2014), all of whichare crucial for chondrogenesis. Nevertheless, the molecularmechanisms by which Hippo-YAP1/TAZ signaling controlschondrocyte generation and homeostasis remain unclear.Col2a1-Yap1 transgenic mice were recently reported to display

increased early chondrocyte proliferation driven by YAP1/TEAD-dependent SOX6 activation, but also exhibited YAP1/RUNX2-dependent COL10A1 inhibition, impaired chondrocyte maturationand reduced skeleton size (Deng et al., 2016). However, chondrocyte-specific Yap1-deficient mice are slightly larger than age-matchedwild-type (WT) littermates (Deng et al., 2016). In contrast to YAP1,TAZ overexpression was found to accelerate chondrocyte maturationand promote RUNX2-dependent COL10A1 expression (Deng et al.,2016). It appears that TAZ competes with YAP1 for interaction withRUNX2 in order to modulate COL10A1 expression and controlchondrocyte maturation.We previously reported thatMob1a/b null mutant mice succumb

to embryonic lethality at embryonic day (E) 6.5 (Nishio et al., 2013).We have also demonstrated that Mob1a/b loss induces extremehyperactivation of endogenous YAP1/TAZ, resulting in the mostsevere phenotypes reported among mice mutated in Hippo corecomponents in various tissues (Nishio et al., 2017). Thus, MOB1A/B is a crucial hub in the Hippo signaling pathway. In this study, wegenerated chondrocyte-specific Mob1a/b-deficient mice and foundthat hyperactivation of endogenous YAP1/TAZ induced by loss ofMob1a/b impaired chondrocyte proliferation and differentiation andled to the onset of chondrodysplasia. Our data indicate that thesephenotypes occur because a YAP1/TAZ-TEAD complex functionsas a transcriptional repressor of SOX9, a master regulator ofchondrogenesis.

RESULTSLoss of Mob1a/b in murine chondrocytes results inchondrodysplasiaTo analyze the functions of endogenous YAP1/TAZ inchondrogenesis in vivo, we generated chondrocyte-specificMob1a/b double-knockout mice (Col2a1-CreERT; Mob1aflox/flox;Mob1b−/−; hereafter cMob1 DKO) by mating Col2a1-CreERTtransgenic mice with Mob1aflox/flox and Mob1b−/− mice.Administration of 4-hydroxytamoxifen (tamoxifen) at P0 activatesCre expression, deleting the floxed Mob1a gene. We confirmedefficient Mob1a deletion in Mob1b null chondrocytes by PCRanalysis of DNA from chondrocytes isolated from control andcMob1 DKO mice (Fig. S1). cMob1 DKO mice were born at theexpected Mendelian ratio but developed slowly, showing an overall

reduction in body size at postnatal day (P) 84 compared withlittermate controls (Mob1aflox/flox; Mob1b−/−) (Fig. 1A).

To study the roles of MOB1A/B during postnatalchondrogenesis, we measured the lengths of the long bones andthe size of the cartilaginous growth plates in control and cMob1DKO mice at P84. Compared with controls, mutants withMob1a/bdeficiency in chondrocytes showed significant decreases in totalbody length as well as in the length of the femur, tibia, humerus andforelimb (Fig. 1B). The size of the articular cartilage layer was alsodecreased in the mutants at P12 (Fig. 1C). Close histologicalexamination of growth plates at P21 revealed that each chondrocytezone (resting, proliferative, and hypertrophic) was present in cMob1DKO mice but proportionally reduced in size compared with that incontrol animals (Fig. 1D). Thus, loss of Mob1a/b in chondrocytesresults in chondrodysplasia.

MOB1A/B deficiency in chondrocytes impairs theirproliferation and differentiationBecause cMob1 DKO mice exhibited abnormal histology in theirgrowth plate cartilage, we analyzed the proliferation anddifferentiation of chondrocytes. Histological examination of controland mutant growth plates at P21 using PCNA staining to identifyproliferating cells revealed that many PCNA-positive cells werepresent in control growth plates (as expected), especially in theproliferative zone. However, numbers of PCNA-positive cells ingrowth plates of P21 cMob1 DKO mice were significantly decreasedcompared with controls (Fig. 2A). Notably, the percentage ofTUNEL-positive apoptotic cells in control and cMob1 DKO growthplates was not significantly different (Fig. S2). To confirm ourobservations at the molecular level in vitro, we performed siRNA-mediated knockdown of MOB1A/B in the human chondrocyte cellline H-EMC-SS. Depletion of MOB1A/B significantly reducedthe proliferation of these cells in vitro (Fig. 2B), indicating that lossof Mob1a/b negatively affects chondrocyte proliferation in vivo andin vitro.

We next examined the expression levels of several genes requiredfor the establishment and maintenance of ECM. We isolatedchondrocytes from control and cMob1 DKO mice and used qRT-PCR to assess mRNA levels of Col2a1, Col9a1, Col9a2, Comp,Col11a1 and aggrecan (Acan). In all cases, relative mRNA expressionwas significantly downregulated in cMob1 DKO chondrocytescompared with controls (Fig. 2C). To evaluate chondrocytematuration, we used immunohistochemistry to detect stage-specificmarkers in chondrocytes in growth plates of control and cMob1 DKOmice. Type 2 collagen (Col II) is expressed in all chondrocyte layers,whereas osterix (also known as SP7) and IHH are markers of theprehypertrophic layer, and Col X is specific to the hypertrophicchondrocyte layer. Numbers of chondrocytes expressing thesemarkerswere significantly decreased in cMob1 DKO mice (Fig. 2D, Fig. S3),indicating that MOB1 plays a fundamental role in supportingchondrocyte differentiation/maturation.

Lastly, we examined endochondral ossification by assessing thebone volume, osteoid volume, trabecular thickness, trabecularnumber and osteoblast number of the proximal tibia of control andcMob1 DKO mice (Fig. S4A) at P12, as well as the longitudinalgrowth rate in the primary spongiosa of this limb (Fig. 2E). We alsosubjected proximal tibia tissue to Col I staining (Fig. S4B). All ofthese properties were significantly reduced in the mutant micecompared with controls. As a further corroborating approach, wecrossed cMob1 DKO mice with Rosa26-LSL-YFP reporter mice togenerate Col2a1-CreERT; Mob1aflox/flox; Mob1b−/−; Rosa26-LSL-YFP (mutant) reporter animals. Examination of YFP expression

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(green) by osteoblasts around bone ECM containing Col I (red)beneath the growth plate from P21 mice confirmed that none of theosteoblasts in the mutant tissue was YFP positive, suggesting thatMOB1-deficient chondrocytes did not differentiate into osteoblasts(Fig. S5). Thus, loss of Mob1a/b in chondrocytes inhibitstheir proliferation, differentiation/maturation, and endochondralossification, resulting in the chondrodysplasia phenotype observedin cMob1 DKO mice.

Mob1a/b deletion activates YAP1/TAZ and downregulatesSOX9 expressionTo investigate the effects of chondrocyte-specific Mob1a/b loss onHippo pathway components and downstream effectors, primarychondrocytes isolated from Col2a1-CreERT; Mob1a+/+; Mob1b−/−;Rosa26-LSL-YFP (control) and Col2a1-CreERT; Mob1aflox/flox;Mob1b−/−; Rosa26-LSL-YFP (mutant) reporter mice were treatedwith/without 0.1 μM tamoxifen for 96 h in vitro, and YFP+

chondrocytes were sorted and used as control and cMob1 DKOreporter chondrocytes. Chondrocytes lacking MOB1A/B showedreduced YAP1 (Ser127) and LATS1 (Thr1079) phosphorylation, anda modest increase in total YAP1 and TAZ proteins (Fig. 3A). Nodifferences were detected in phosphorylated (T138/T180) MST1/2(also known as STK4/3), total MST1, SAV1 or LATS1. Notably, lossofMob1a/b significantly downregulated mRNA levels of Sox9, Sox5and Sox6 compared with controls (Fig. 3B), and SOX9 protein wasmarkedly reduced inMob1a/b-deficient chondrocytes (Fig. 3C). Thislatter result was confirmed by immunohistochemical examination ofthe growth plates from control versus mutant mice at P21 (Fig. 3D).As noted above, SOX9 is a master transcription factor that acts ongenes involved in cartilage development and cooperates with SOX5

and SOX6 to regulate chondrogenesis (Akiyama, 2008; Ikeda et al.,2004). In control mice at P21, YAP1 was strongly expressed in thenucleus of prehypertrophic chondrocytes but less so in hypertrophicchondrocytes (Fig. 3D). However, proliferative and hypertrophicchondrocytes from cMob1 DKO mice showed both enhanced YAP1activation and reduced levels of SOX5, SOX6 and SOX9 (Fig. 3D).Thus, the defect in chondrodysplasia caused by loss of Mob1a/b isvery likely to be due (at least in part) to a decrease in expression ofSOX9.

Hyperactivated YAP1/TAZ suppresses chondrogenesis incMob1 DKO miceTo investigate the role of YAP1 in chondrocyte proliferation anddifferentiation, we generated H-EMC-SS cells that conditionallyexpressed YAP1(5SA), a constitutively active form controlledusing doxycycline (Dox) and a Tet-On system. Overexpressionof YAP1(5SA) significantly decreased the proliferation ofchondrocytes as determined by the MTS assay (Fig. 4A). Inaccordance with this result, qRT-PCR revealed that mRNA levels offive genes involved in the establishment and maintenance of ECMwere also reduced (Fig. 4B).

To determine the dependence of the chondrodysplasia phenotypeof cMob1 DKO mice on YAP1/TAZ, we generated two tripleknockout (TKO) mouse strains: TKO(YAP) mice, which wereMob1a/b homozygous deficient plus Yap1 homozygous deficient(Col2a1-CreERT; Mob1aflox/flox; Mob1b−/−; Yap1flox/flox); andTKO(TAZ) mice, which were Mob1a/b homozygous deficientplus Taz homozygous deficient (Col2a1-CreERT; Mob1aflox/flox;Mob1b−/−; Tazflox/flox). The defects in the lengths of the growthplates and the long bones and overall body size were all significantly

Fig. 1. Chondrocyte-specific Mob1a/b double-knockout (cMob1 DKO) mice exhibit hypochondroplasia. (A) Representative littermate control and cMob1DKO 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 thearticular 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 ofthe articular cartilaginous zone. (D) Representative H&E staining of the growth plate (arrow) in the proximal tibia of control and DKOmice at P21, as quantified onthe right (n=5). ***P<0.001, Student’s t-test. Scale bars: 50 μm in C; 100 μm in D.

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rescued by either Yap1 or Taz deletion (Fig. 4C,D). These resultsindicate that, in WT mice, YAP1 functions to inhibit chondrocyteproliferation and maturation, and that the cartilage abnormalitiesobserved in cMob1 DKO mice result from hyperactivation of YAPand/or TAZ activity.

AMOB1-YAP1/TAZ-TEAD axis regulates SOX9 expression viatranscriptional repressionTo clarify the links between MOB1A/B, YAP1/TAZ and SOX9, wecarried out siRNA-mediated knockdown of MOB1A/B proteins inhuman H-EMC-SS or mouse ATDC5 cells (Fig. 5A, Fig. S6A).Levels of phosphoYAP1 (Ser127) and SOX9 were decreasedwhereas total YAP1 and TAZ proteins were increased in theseMOB1A/B-depleted cells, as compared with cells transfected withsi-scramble control. Transfection of H-EMC-SS chondrocyteswith vectors overexpressing human WT YAP [YAP(WT)] orconstitutively active YAP1(5SA), or with human WT TAZ[TAZ(WT)] or constitutively active TAZ [TAZ(SA)], showed thatoverexpression of YAP1 (WT or 5SA) or TAZ (WT or SA)significantly decreased SOX9 protein (Fig. 5B,C). Similarly,knockdown of MOB1A/B (Fig. 5D, Fig. S6B) or overexpression ofeither YAP1(5SA) (Fig. 5E) or TAZ(SA) (Fig. 5F) resulted in

reduced SOX9, SOX5 and SOX6mRNA levels (Fig. 5E,F, Fig. S6B).Thus, the Hippo-YAP1/TAZ pathway regulates the expression ofSOX9, SOX5 and SOX6 mRNAs and thus influences their proteinlevels.

It is primarily the TEAD family of transcription factors that ismodulated by binding to the YAP1/TAZ co-factors. To clarifywhether the decrease in the expression of SOX mRNAs dependedon TEADs, we analyzed mRNA levels of SOX9, SOX5 and SOX6after siRNA-mediated knockdown of TEAD1-4. Application ofsiTEAD1-4 to H-EMC-SS chondrocytes efficiently inhibited theexpression of TEAD proteins (Fig. 6A) and led to upregulatedproduction of SOX9, SOX5 and SOX6 mRNAs (Fig. 6B). Thesedata implied that a YAP1/TEAD complex might function as atranscriptional repressor tasked with controlling SOX mRNAexpression. To investigate this hypothesis, we engineered H-EMC-SS chondrocytes to express Dox-inducible YAP1(5SA/S94A),which has an additional mutation (S94A) in the TEAD-bindingdomain that prevents binding to TEADs (Zhao et al., 2008;Shimomura et al., 2014). Expression of YAP1(5SA/S94A) tendedto bolster SOX9 and SOX6 mRNA expression and induced amodest but statistically significant increase in SOX5 mRNA(Fig. 6C,D).

Fig. 2. Loss ofMob1a/b in chondrocytes inhibits their proliferation, differentiation and endochondral ossification. (A) Representative immunostaining todetect 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 ofPCNA+ 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/Bfor 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 indicatedgenes in primary chondrocytes isolated from control and cMob1 DKO mice at P2 (n=3). (D) Representative immunohistochemistry to detect the indicatedstage-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 andIHH, 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 DKOmice at P12. The longitudinal growthrate of primary spongiosa (arrow) is quantified on the right (n=4). Calcein (green) was subcutaneously administered 24 h before themicewere sacrificed. *P<0.05,**P<0.01, ***P<0.001, Student’s t-test. Scale bars: 50 μm.

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We next applied ChIP assays to nuclear extracts of H-EMC-SScells to determine YAP1(5SA) binding to the SOX9 promoter.Indeed, YAP1(5SA) was strongly recruited to the TEAD bindingsite of the SOX9 promoter in these H-EMC-SS cells (Fig. 6E,F).These results show that a YAP1/TEAD complex directly binds tothe SOX9 promoter, allowing this complex to act as a transcriptionalrepressor of the SOX9 gene and so exert a profound negativeregulatory effect on chondrogenesis.

YAP1/TAZ signaling is not triggered by FGFR3 activationIn humans, achondroplasia (ACH), which is classified as short-limbed dwarfism, is frequently caused by a hereditary autosomaldominant mutation in the proximal tyrosine kinase domain ofFGFR3 (Shiang et al., 1994). In mice overexpressing Fgfr3with thecorresponding ACH mutation (Fgfr3ach), the growth plate cartilageshows reductions in height of both the proliferative chondrocytezone and the hypertrophic chondrocyte zone (Naski et al., 1998).Because Hippo-YAP1/TAZ signaling has been shown to engage incrosstalk with FGFs (Rizvi et al., 2016), we investigated whetherHippo-YAP1/TAZ signaling might be triggered downstreamof FGFR3 engagement. We overexpressed FGFR3 G380R, aconstitutively active form of the humanmutant protein (Bellus et al.,1995), in H-EMC-SS cells and confirmed high levels of FGFR3mRNA in the altered cells (Fig. 7A). However, examination of thesecells by western blotting showed that this increase in FGFR3G380Rexpression did not activate YAP1 or TAZ in either H-EMC-SSor ATDC5 cells (Fig. 7B,C). This was confirmed byimmunohistochemical assays to detect YAP1 in the proximal tibiaof P14 control mice compared with Fgfr3ach mice (Naski et al.,1998) (Fig. 7D). Finally, constitutive FGFR3 activation in H-EMC-SS and ATDC5 cells did not increase the mRNA levels of YAP1/

TAZ target genes such as CTGF and CYR61 (Fig. 7E,F). These datasuggest that Hippo-YAP1/TAZ signaling may contribute to theonset of dwarfism in humans via an independent pathway that doesnot function downstream of FGFR3.

DISCUSSIONAlthough the regulation of YAP1/TAZ is crucial for the commitmentof MSCs to differentiate into osteoblasts or adipocytes (Seo et al.,2013; Hong et al., 2005), there have been few studies analyzing theeffects of the Hippo signaling pathway on chondrocytes. A recentreport showed that homozygous Col2a1-Yap1Tg/Tg transgenic miceexhibit a chondrodysplasia phenotype (Deng et al., 2016), and wehave demonstrated here that MOB1 deletion also results in YAP1/TAZ-dependent chondrodysplasia. Thus, the Hippo-YAP1/TAZaxis must be important for chondrogenesis. Considering that thechondrocyte defects observed in MOB1-deficient mice are morepronounced than those of heterozygous Col2a1-Yap1Tg/+ transgenicmice, and that MOB1 deletion in other organs consistently results inthe most severe phenotypes reported among animals with conditionaldeletions of Hippo core components (Nishio et al., 2016), the MOB1adaptors must constitute the most important hub in Hippo signaling.

With respect to the mechanism driving chondrodysplasia in theabsence of MOB1, in vitro studies have shown that increased YAP1activity under conditions of elevated matrix rigidity or high fluid-flow shear stress leads to impaired chondrocyte maturation, whereasdownregulation of YAP1 in response to a soft substrate maintainschondrogenic marker expression (Zhong et al., 2013a,b). Otherwork has demonstrated that overexpression of YAP1 in murineC3H10T1/2 mesenchymal-like cells can inhibit chondrogenicdifferentiation in vitro (Karystinou et al., 2015). Similarly, in vivo,overexpression of YAP1 in mice attenuates endochondral

Fig. 3. MOB1-dependent Hippo signaling controls SOX9. (A) Western blot to detect the indicated Hippo pathway proteins (p, phosphorylated) in primarychondrocytes from control and cMob1 DKO mice at P2. GAPDH, loading control. (B) qRT-PCR determination of relative Sox9, Sox5 and Sox6 mRNA levelsin 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 andimmunostaining to detect SOX5, SOX6, SOX9 or YAP1 protein in primary chondrocytes in the indicated zones of the growth plates of the proximal tibia of controland 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.

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maturation and inhibits the formation of cartilaginous callus tissueafter bone fracture (Deng et al., 2016). We have shown here thatMOB1 deletion leading to hyperactive YAP1 expression impairsECM production (Fig. 2C). All these reports are consistent in theirconclusion that increased YAP1 activation blocks chondrocytedifferentiation/maturation. However, the function of YAP1 inchondrocyte proliferation, as well as the base functions of TAZin chondrocytes, remain controversial. Although YAP1overexpression reportedly increases the proliferation both of theATDC5 chondrocyte cell line in vitro and murine chondrocytesin vivo (Deng et al., 2016), the expected effects on immaturechondrocytes have been difficult to document. In contrast to Denget al. (2016), our data show that MOB1 deletion leading to YAP1/TAZ activation decreases chondrocyte proliferation, as establishedby the MTS assay in vitro (Fig. 4A) and by PCNA staining in vivo(Fig. 2A). The fact that both the proliferative and hypertrophicchondrocyte layers in our mutant mice were decreased in sizesupports our contention that chondrocyte proliferation is impairedwhen YAP1 signaling is excessive. Deng et al. (2016) reported thatTAZ competes with YAP1 for RUNX2 activation and promoteschondrocyte maturation. However, we show here that thephenotypes of MOB1-deficient chondrocytes can be mostlyrescued by additional deficiency of YAP1 or TAZ (Fig. 4C,D). Inaddition, we demonstrate that either constitutively activated YAP1or activated TAZ can suppress the expression of SOX factors(Fig. 5B,C,E,F), leading us to conclude that there are no functional

differences between YAP1 and TAZ in this context. The reasonsunderlying the discrepancies between our work and that of Denget al. (2016) are currently unknown.

SOX9 is an indispensable initiator and master regulator ofchondrogenesis (Bi et al., 1999; Akiyama, 2008; Lefebvre et al.,1998). As noted above, SOX9 interacts cooperatively with SOX5and SOX6 to regulate cartilage matrix genes, including Col2a1,Col9a1, Col11a1 and Acan, to drive chondrocyte proliferation anddifferentiation (Oh et al., 2014). Accordingly, neither Sox5 nor Sox6expression can be detected in Col2a1-Cre; Sox9flox/flox conditionalknockout mice (Lefebvre et al., 1998; Akiyama et al., 2002; Ikedaet al., 2004). Loss of Sox9 specifically in murine chondrocytesresults in severely hypoplastic cartilage (Akiyama et al., 2002), andheterozygous mutations in the human SOX9 gene cause hereditarycampomelic dysplasia (Wagner et al., 1994). We found that loss ofMOB1 in murine chondrocytes significantly suppressed bothprotein and mRNA expression of Sox9, Sox5 and Sox6 in aYAP1/TAZ-TEAD-dependent manner (Fig. 3B-D), and thatoverexpression of YAP1 or TAZ also downregulated SOX9, SOX5and SOX6 expression levels (Fig. 5B,C,E,F). Deletion of SOX9 inmouse limb buds reportedly abolishes the expression of both SOX5and SOX6, confirming that SOX9 is the master regulator ofchondrogenesis and is necessary for SOX5 and SOX6 generationduring chondrocyte differentiation (Akiyama et al., 2002). Withinenhancer regions, SOX5 and SOX6 bind to recognition sites nearthat bound by SOX9, thereby consolidating SOX9 binding to DNA

Fig. 4. YAP1/TAZ hyperactivation inhibits chondrocyte proliferation and the expression of ECM genes. (A) MTS assay of the proliferation of H-EMC-SScells 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 relativemRNA 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) RepresentativeH&E staining to reveal length of growth plate (arrow) in the proximal tibia of control, cMob1DKO, or cMob1 TKO (cMob1DKO plusYap1 or Taz homozygous) miceat P28, as quantified on the right (n=5). Scale bar: 100 μm. (D) Length of total body, femur or humerus in control, cMob1DKO, and cMob1 TKOmice at P28 (n=5).*P<0.05, **P<0.01, ***P<0.001, Student’s t-test.

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and potentiating SOX9 activity (Liu and Lefebvre, 2015). Theseobservations imply that the reduced expression of SOX5 and SOX6that accompanies the decrease in SOX9 caused by MOB1 deletioncontributes to the chondrodysplasia observed in our mutant mice.Song et al. (2014) reported that YAP1 directly regulates SOX9

transcription through a conserved TEAD binding site in the SOX9promoter, and that YAP1 maintains the cancer stem cell propertiesof esophageal tumor cells by upregulating SOX9 expression (Songet al., 2014). However, we show here that, although YAP1 is indeedrecruited to the SOX9 promoter, it induces repression of SOX9expression in a TEAD-dependent manner (Fig. 6A-F). Thissituation of context-dependent opposing effects has beenfrequently observed for various transcription factors (Fry andFarnham, 1999; Kim et al., 2015). In addition, although YAP1/TAZmost often form complexes with TEADs that enhance their activity,recent studies have revealed that YAP1/TAZ can also act astranscriptional co-repressors (Kim et al., 2015; Zaidi et al., 2004;Valencia-Sama et al., 2015). Thus, it is not unreasonable toconclude that a YAP1/TAZ-TEAD complex may function as eithera transcriptional activator or repressor of SOX9 expression,depending on the tissue-specific context.As noted above, ACH is caused by a hereditary autosomal

dominant mutation in FGFR3 (Shiang et al., 1994). However, weshow here that overexpression of the constitutively activated FGFR3G380R mutant protein does not activate YAP1/TAZ in vitro(Fig. 7B,C) or in vivo (Fig. 7D). Furthermore, mRNA levels of

YAP1/TAZ target genes such as CTGF and CYR61 were not alteredin two chondrocyte cell lines (H-EMC-SS and ATDC5)overexpressing FGFR3 G380R (Fig. 7E,F). Our finding that FGFsignaling does not activate YAP1/TAZ is in line with a report byanother group (Yu et al., 2012) that examined human embryonickidney cells (HEK293A). Additional studies are required todetermine the nature and extent of the crosstalk between theHippo and FGF signaling pathways.

In conclusion, we have clarified the physiological functions ofMOB1-YAP1/TAZ signaling in chondrocytes, and have shown thatinappropriate hyperactivation of a YAP1/TAZ-TEAD complex thatfunctions as a transcriptional repressor of SOX9 can lead to the onsetof chondrodysplasia in mice. In this light, it would be interesting toanalyze the frequency of YAP1/TAZ hyperactivation in dwarfismpatients. Our results increase our molecular understanding of theeffects of the Hippo signaling pathway in vivo, and might providenew insights into potential therapeutic strategies for dwarfismpatients.

MATERIALS AND METHODSMiceMouse strains used in this study were Col2a1-CreERT Tg (The JacksonLaboratory), Mob1aflox/flox; Mob1b−/− (Nishio et al., 2012, 2016), Rosa26-LSL-YFP reporter (Srinivas et al., 2001), Yap1flox/flox (Knockout MouseProject Repository, UC Davis, CA, USA), Tazflox/flox (kindly provided byDr J. Wrana), and Fgfr3ach (kindly provided by Dr H. Akiyama).

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-SScells 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-SScells 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 ofrelative 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-mediatedinduction of (E) YAP1(5SA) or (F) TAZ(SA) (n=3). *P<0.05, **P<0.01, ***P<0.001, Student’s t-test.

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Mice were kept in pathogen-free facilities at Kyushu and KobeUniversities. Protocols for animal experiments were approved by theAnimal Research Committees of Kyushu and Kobe Universities.

Generation of cMob1 DKO mice and related strainsChondrocyte-specificMob1a/b homozygous double-mutant mice (Col2a1-CreERT; Mob1aflox/flox; Mob1b−/−) were generated by mating Col2a1-CreERT Tg with Mob1aflox/flox; Mob1b−/− mice. Col2a1-CreERT Tg micewere of the C57BL/6 background, and Mob1aflox/flox; Mob1b−/− mice werebackcrossed to C57BL/6 for more than six generations. Mob1aflox/flox;Mob1b−/− mice without the Col2a1-CreERT transgene were usually chosento serve as controls because no significant differences in total body length orlengths of long bones and cartilaginous zones were observed betweenMob1aflox/flox; Mob1b−/− and Col2a1-CreERT; Mob1aflox/flox; Mob1b−/−

mice that were injected with 4-hydroxytamoxifen (Sigma-Aldrich). Todelete the floxed Mob1a gene, a single dose of tamoxifen (0.1 mg) wasinjected into Col2a1-CreERT; Mob1aflox/flox; Mob1b−/− and control pups atP0. Primers used for genotyping PCR are listed in Table S1.

Cell cultureThe human chondrocyte cell line H-EMC-SS (Riken Cell Bank, Tsukuba,Japan) was maintained in MEMα medium (Wako) supplemented with 10%heat-inactivated fetal calf serum (FCS), penicillin (100 U/ml) andstreptomycin (100 μg/ml) and cultured in a humidified incubator at 37°Cand 5% CO2. The mouse embryonal carcinoma-derived chondrogenic cellline ATDC5 (Riken Cell Bank) was maintained in a 1:1 mixture of DMEM

and Ham’s F-12 medium (Wako) containing 5% FCS in a humidifiedatmosphere at 37°C and 5% CO2.

Preparation of mouse primary chondrocytesThe ventral parts of rib cages of P2 mice were digested with collagenaseD and chondrocytes were isolated as described previously (Beier et al., 1999).Chondrocytes isolated from control mice (Col2a1-CreERT; Mob1a+/+;Mob1b−/−), or fromCol2a1-CreERT; Mob1aflox/flox; Mob1b−/−mice carryingthe Rosa26-LSL-YFP reporter allele, were plated in 6-well dishes at 5000cells/cm2 and grown to confluence in DMEM containing 10% FCS. Platedchondrocytes were treated with 0.1 μM tamoxifen for 96 h. YFP+ cells werecollected using an SH800 cell sorter (Sony).

Dimethylthiazol carboxymethoxyphenyl sulfophenyl (MTS)assayCell proliferation was measured by the MTS method (Cory et al., 1991).MTS assays were performed using the CellTiter 96 assay (Promega)according to the manufacturer’s instructions.

ImmunohistochemistryMouse tissues were fixed in 4% paraformaldehyde in PBS, decalcified in10% EDTA, embedded in paraffin, and sectioned. Deparaffinized sectionswere antigen-retrieved using Immunosaver (Nissin EM, Tokyo, Japan), andthen incubated with primary antibodies at 4°C overnight. Primary antibodieswere against SOX5 (ab94396, Abcam; 1:200), SOX6 (sc-393314, SantaCruz Biotechnology; 1:100), SOX9 (sc-20095, Santa Cruz Biotechnology;

Fig. 6. Regulation ofSOX9 expression byYAP1-TEAD-mediated transcription. (A)Western blot to detect pan-TEAD inH-EMC-SS cells that were treatedwithsi-scramble or si-TEAD1-4 #1 or #2 for 48 h. (B) qRT-PCR determination of relative SOX9, SOX5 and SOX6mRNA levels in the cells in A (n=3). (C) Western blotto 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/mlDox 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.

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1:100), PCNA (610664, BD Transduction Laboratories; 1:200), Col I(ab34710, Abcam; 1:100), Col II (LB-1297, LSL, Tokyo, Japan; 1:400), ColX (LB-0092, LSL; 1:200), IHH (ab39634, Abcam; 1:100), osterix (SP7)(ab22552, Abcam; 1:100), YFP/GFP (ab6673, Abcam; 1:500) or YAP1(WH0010413M1, Sigma-Aldrich; 1:500). Anti-rabbit/mouse-HRP (Dako)was used for DAB staining. Secondary antibodies were tagged with AlexaFluor 488 or Alexa Fluor 568 (Molecular Probes). In some slides, nuclei werevisualized using Hematoxylin and Eosin (H&E) or DAPI.

TUNEL stainingApoptosis of chondrocytes was analyzed by TUNEL staining using the InSitu Cell Death Detection Kit (Roche) according to the manufacturer’sinstructions. Nuclei were visualized with DAPI.

HistomorphometryEthanol-fixed tibiae from P12 mice were fixed in 70% ethanol, embedded inglycolmethacrylate resin, and sectioned into 5 μmslices. For histomorphometricanalyses, an area (1.62-2.34 mm2) 1.2 mm below the growth plate in theproximal tibia was evaluated. Histomorphometric parameters, such as trabecularbone volume/tissue volume (BV/TV), osteoid bone volume/tissue volume (OV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and osteoblastnumber/bone surface (N.Ob/BS), were calculated.

Measurement of longitudinal growth rate in primary spongiosaMice received subcutaneous administration of 20 mg/kg calcein. Thelongitudinal growth rate of the primary spongiosawas measured 24 h later inhistological tissue sections as previously described (Pass et al., 2012).

Quantitative reverse-transcription PCR (qRT-PCR)Total RNA was isolated from cells using RNAiso Plus (Takara Bio)according to the manufacturer’s instructions. Real-time qRT-PCR analysis

was carried out with THUNDERBIRD SYBR qPCR Mix (Toyobo)following the manufacturer’s instructions, and with the primers listed inTable S2. PCR amplifications were performed using the StepOne real-timePCR system (Applied Biosystems). Ct values for each gene amplificationwere normalized by subtracting the Ct value calculated for Gapdh/GAPDH.Normalized gene expression values report the relative quantity of mRNA.

Western blottingWestern blotting was carried out using a standard protocol and primaryantibodies recognizing MOB1 (3863, Cell Signaling; 1:1000), MST1 (3682,Cell Signaling; 1:1000), phosphoMST1/2 (3681, Cell Signaling; 1:1000),SAV1 (13301, Cell Signaling; 1:1000), LATS1 (3477, Cell Signaling;1:1000), phosphoLATS1 (9159, Cell Signaling; 1:1000), YAP1 (4912, CellSignaling; 1:1000), phosphoYAP1 (4911, Cell Signaling; 1:1000), TAZ(V386) (4883, Cell Signaling; 1:1000), SOX9 (sc-20095, Santa CruzBiotechnology; 1:500), FGFR3 (sc-13121, Santa Cruz Biotechnology;1:500), GAPDH (sc-25778, Santa Cruz Biotechnology; 1:1000) and actin(A2066, Sigma-Aldrich; 1:1000). Primary antibodies were detected usingHRP-conjugated secondary antibodies (Cell Signaling).

Transfection of siRNA or cDNAsiRNAs targeting MOB1A/B or TEAD1-4 expression are listed in Table S3.Transfection of siRNA oligonucleotides (30 nM) into H-EMC-SS cells wasperformed using Lipofectamine RNAiMAX (Invitrogen) following themanufacturer’s protocol. Transfection of empty pcDNA3.1 vector, orpcDNA3.1 vector expressing FGFR3 G380R (Bellus et al., 1995), intoH-EMC-SS cells was performed using Lipofectamine 2000 (Invitrogen)following the manufacturer’s protocol. Transfection of empty pcDNA3.1vector, pcDNA3.1 vector expressing FGFR3 G380R, or siRNAoligonucleotides (30 nM) into ATDC5 cells was performed using FuGENEHD Transfection Reagent (Promega) following the manufacturer’s protocol.

Fig. 7. Hippo-YAP1/TAZ signaling does not function downstream of FGFR3. (A) qRT-PCR determination of relative FGFR3mRNA 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 todetect 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 Fgfr3G380R).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 FGFR3G380R (n=3). *P<0.05, **P<0.01, ***P<0.001, Student’s t-test.

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After 24 h growth to achieve confluence, siRNA-transfected or cDNA-transfected ATDC5 cells were cultured for 6 days in a 1:1 mixture of DMEMand Ham’s F-12 medium containing 5% FCS and 10 µg/ml insulin.

Lentiviruses expressing YAP1(WT), YAP1(5SA), YAP1(5SA/S94A),TAZ(WT) or TAZ(SA) were produced by transient transfection of HEK293Tcells with pMDLg/pRRE, pRSV-Rev, pMD2.G, and either pSLIK-Flag-Myc-YAP1(WT), pSLIK-Flag-Myc-YAP1(5SA), pSLIK-Flag-Myc-YAP1(5SA/S94A), pSLIK-Flag-His-TAZ(WT) or pSLIK-Flag-His-TAZ(SA) using Lipofectamine 2000 (Invitrogen) (Otsubo et al., 2017). At48 h post-transfection, lentivirus-containing supernatant was collected.Cultured H-EMC-SS cells were incubated with lentivirus supernatant for24 h and then transferred to growth medium containing G418 to select forstable transfectants.

Chromatin immunoprecipitation (ChIP) assayChIP assays were performed as described (Goto et al., 2015). Briefly, cellswere cross-linked with formaldehyde and homogenized by sonication.Precleared chromatin was incubated with either anti-DYKDDDDK (FLAG)tag antibody beads (Wako) or mouse IgG, followed by precipitation withProtein G Sepharose 4 Fast Flow resin (Amersham Biosciences). Semi-quantitative PCR analysis was performed using KAPA Taq polymerase(Kapa Biosystems). Quantitative PCR analysis was carried out usingTHUNDERBIRD SYBR qPCR Mix (Toyobo). Primers used for PCR inChIP assays are listed in Table S4.

Statistical analysisData are presented as mean±s.d. Statistical significance of differencesbetween experimental groups was determined using Student’s t-test. P<0.05was considered statistically significant.

AcknowledgementsWe thank H. Akiyama (Gifu University) and J. Wrana (Lunenfeld-TanenbaumResearch Institute) for the Fgfr3ach mutant and Tazflox/flox mice, respectively; A. Ito(Ito Bone Histomorphometry Institute), A. Fujimoto, M. Kamihashi and M. Suzuki (allof Kyushu University) for expert technical assistance; and K. Nakao (KyotoUniversity) for critical discussions.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: H.G., M.N., Y.T., T.O., T.W.M., A.Y., N.T., A.S.; Methodology: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.,M.N., T.O., T.W.M., A.Y., N.T., A.S.; Formal analysis: H.G., M.N., Y.T., T.O.,Y. Miyachi, A.S.; Investigation: H.G., M.N., Y.T., T.O., Y. Miyachi, A.S.; Resources:M.N., H.N., H.A., Y. Makii, T.S., A.Y., N.T., A.S.; Data curation: A.S.; Writing - originaldraft: H.G., A.S.; Writing - review & editing: H.G., M.N., T.M., T.W.M., T.S., A.Y., N.T.,A.S.; Visualization: H.G., M.N., T.O., A.S.; Supervision: T.M., H.N., H.A., T.W.M.,T.S., A.Y., N.T., A.S.; Project administration: A.S.; Funding acquisition: H.G., T.M.,A.S.

FundingWe are grateful for the funding provided by Ministry of Education, Culture, Sports,Science and Technology (MEXT; grant 15K19026 to H.G.); Japan Society for thePromotion of Science (JSPS; grants 17H01400 and 26114005 to A.S.); theCooperative Research Project Program of the Medical Institute of Bioregulation,Kyushu University; Nanken-Kyoten, Tokyo Medical and Dental University (TMDU);Project for Development of Innovative Research on Cancer Therapeutics(P-DIRECT; grant 11088019 to A.S.); Japan Agency for Medical Research andDevelopment (AMED; grant 16770279 to A.S. and H.G.); the Uehara MemorialFoundation (to A.S.); the Shinnihon Advanced Medical Research Foundation (toA.S.); the Smoking Research Foundation (to T.M.); and the Daiichi-SankyoScholarship Donation Program (to A.S.).

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.159244.supplemental

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RESEARCH ARTICLE Development (2018) 145, dev159244. doi:10.1242/dev.159244

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