Gene targeting of the transcription factor Mohawk inrats causes heterotopic ossification of Achillestendon via failed tenogenesisHidetsugu Suzukia,b, Yoshiaki Itoa, Masahiro Shinoharaa,c, Satoshi Yamashitaa, Shizuko Ichinosed, Akio Kishidae,Takuya Oyaizub, Tomohiro Kayamaa, Ryo Nakamichia, Naoki Kodaa, Kazuyoshi Yagishitab,f, Martin K. Lotzg,Atsushi Okawab, and Hiroshi Asaharaa,g,h,1

aDepartment of Systems BioMedicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan; bDepartment of Orthopaedic Surgery,Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan; cPrecursory Research for Embryonic Science and Technology, Japan Science andTechnology Agency, Kawaguchi, Saitama 332-0012, Japan; dResearch Center for Medical and Dental Sciences, Tokyo Medical and Dental University,Bunkyo-ku, Tokyo 113-8510, Japan; eDepartment of Material-Based Medical Engineering, Institute of Biomaterials and Bioengineering, Tokyo Medical andDental University, Chiyoda-ku, Tokyo 101-0062, Japan; fSports Science Organization, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510,Japan; gCore Research for the Evolutionary Science and Technology, Japan Agency for Medical Research and Development, Chiyoda-ku, Tokyo100-0004, Japan; and hDepartment of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037

Edited by Dennis A. Carson, University of California at San Diego, La Jolla, CA, and approved May 25, 2016 (received for review November 8, 2015)

Cell-based or pharmacological approaches for promoting tendonrepair are currently not available because the molecular mecha-nisms of tendon development and healing are not well under-stood. Although analysis of knockout mice provides many criticalinsights, small animals such as mice have some limitations. Inparticular, precise physiological examination for mechanical loadand the ability to obtain a sufficient number of primary tendoncells for molecular biology studies are challenging using mice. Here,we generated Mohawk (Mkx)−/− rats by using CRISPR/Cas9, whichshowed not only systemic hypoplasia of tendons similar to Mkx−/−

mice, but also earlier heterotopic ossification of the Achilles tendoncompared with Mkx−/− mice. Analysis of tendon-derived cells(TDCs) revealed that Mkx deficiency accelerated chondrogenic andosteogenic differentiation, whereas Mkx overexpression sup-pressed chondrogenic, osteogenic, and adipogenic differentiation.Furthermore, mechanical stretch stimulation of Mkx−/− TDCs led tochondrogenic differentiation, whereas the same stimulation inMkx+/+ TDCs led to formation of tenocytes. ChIP-seq of Mkx over-expressing TDCs revealed significant peaks in tenogenic-relatedgenes, such as collagen type (Col)1a1 and Col3a1, and chondrogenicdifferentiation-related genes, such as SRY-box (Sox)5, Sox6, andSox9. Our results demonstrate that Mkx has a dual role, includingaccelerating tendon differentiation and preventing chondrogenic/osteogenic differentiation. This molecular network of Mkx providesa basis for tendon physiology and tissue engineering.

tendon development | Mkx | knockout rat | CRISPR-Cas9 |Achilles tendon ossification

Tendons play a critical role in the musculoskeletal system byconnecting muscle to bone to transmit mechanical loads and

enable movement. Tendon injuries and damage are repaired slowlyand incompletely because of poor intrinsic healing capacity, whichin part results from tissue hypocellularity and hypovascularity (1).Even after surgical tendon repair, a standard treatment for tendonrupture, clinical outcomes are not satisfactory because of recurrentrupture or adhesions (2). To develop cell-based or pharmacologicalapproaches for promoting tendon repair, the molecular mechanismof tendon development and regeneration must be determined;however, the key genome network for tendon differentiation andhomeostasis has not been well characterized.We, along with other researchers, recently reported the ten-

don-specific expression and functions of the transcription factorMohawk (Mkx), which regulates tendon-related gene expression (3,4). Mkx knockout mice showed general tendon hypoplasia (5, 6),suggesting that Mkx plays an important role during tendon develop-ment. Moreover, overexpression of Mkx in mesenchymal stem cells

(MSC) elevates tendon-related markers, and transplantation ofthese cells increases the diameter of collagen fibers in tendons(7, 8), suggesting the potential application of Mkx in cell therapy fortendon injury.Although the results from analysis of Mkx knockout mice have

provided critical information about tendon development, theutility of mice as an animal model has some limitations. In par-ticular, regenerative experiments for tendon repair with precisesurgical interventions are challenging, and most reports of celltherapy for tendon repair used animals that were larger than mice(7, 9). For physiological experiments, such as treadmill exercise totest the effect of mechanical load on tendons/ligaments, rats arepreferable for analyzing the exact responses to the stress becausethey are physiologically more similar to humans than mice (10). Itis also difficult to obtain a sufficient number of primary tenocytesfrom mice for tendon/ligament research.To overcome these limitations, rats are frequently used in

musculoskeletal research. However, technical challenges relatedto the isolation and culture of ES cells posed difficulties in gen-erating genetically modified rats (10, 11). Recent developments ingene-editing technologies, such as zinc-finger nuclease (ZFN)(12), transcription activator-like effector nuclease (TALEN) (13),and clustered regularly interspaced short palindromic repeats/CRISPR associated proteins (CRISPR/Cas9) facilitate the

Significance

Molecular mechanisms of tendon development and homeo-stasis are not well understood. Generation and analysis ofMkx−/− rats revealed new functions of Mohawk (Mkx) in me-diating cellular responses to mechanical stress. An Mkx-ChIPassay in rat tendon-derived cells with Mkx expression sug-gested that this factor may associate with both tendon- andcartilage-related genes to orchestrate tendon cell differentiationand maintenance. These findings advance our understanding oftendon physiology and pathology.

Author contributions: H.S. and H.A. designed research; H.S., M.S., S.Y., S.I., and T.O.performed research; H.S., S.Y., S.I., A.K., T.O., and T.K. contributed new reagents/analytictools; H.S., Y.I., M.S., S.Y., S.I., A.K., T.O., T.K., R.N., N.K., K.Y., M.K.L., A.O., and H.A.analyzed data; and H.S., T.K., M.K.L., and H.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The ChIP-seq data have been deposited in the DNA Database in Japan,www.ddbj.nig.ac.jp/ (accession no. DRA004354).1To whom correspondence should be addressed. Email: asahara@scripps.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1522054113/-/DCSupplemental.

7840–7845 | PNAS | July 12, 2016 | vol. 113 | no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1522054113

generation of genetically modified rats in a one-step injection(14). The objective of this study was to investigate the function ofMkx in vitro and in vivo, including developmental phenotype andmolecular targets, through the generation of Mkx knockout rats.

ResultsEmbryonic and Adult Rat Tendons Express Mkx. Mouse tendonsexpress Mkx in the embryonic and adult stages (3, 5, 15). Toconfirm that the same is true in rats, the expression of Mkx wasanalyzed at several developmental stages using whole-mount insitu hybridization. Embryonic day (E) 11.5 embryos showed ex-pression of Mkx mRNA in the dermomyotome dorsomedial lip,forelimb, and hind limb (Fig. S1A). In E15.5 embryos, the ex-pression of Mkx was identical to the tendon tract (15). In thepostnatal stage, quantitative RT-PCR (RT-qPCR) revealed thattendon showed significantly higher expression of Mkx than othertissues (Fig. S1B).

Mkx Knockout Rats Show “Wavy Tails” and Systemic Hypoplasia ofTendons.Expression ofMkx correlating with tendon developmentsuggested that Mkx is a tenogenesis-related transcription factornot only in mice but also in rats. To evaluate the function of Mkx,we generated Mkx knockout rats using a CRISPR/Cas9 system.The mRNA of hCas9 and guide RNA (gRNA), which targets thesecond exon of the Mkx gene, were injected into 96 rat zygotes(Fig. 1A) (14). Twenty-six rats were obtained; direct sequencingof genomic DNA revealed that nine rats (34%) contained

mutations at the target site. Of these nine chimeric rats (F0),three were crossed with Wistar rats and germ-line transmissionwas confirmed. Three lines of F1 rats were obtained. Putativeoff-target sites (16) of CRISPR/Cas9 were evaluated by directsequencing, which showed no mutations (Table S1). Two primerpairs were designed to analyze the mRNA expression of Mkx inthe patellar tendon. RT-qPCR with primers that flanked thetarget site showed significantly decreased Mkx expression. Whenone primer was designed precisely to be on the target site, theMkx expression was reduced in Mkx−/− rats, and these primerpairs were used for subsequent experiments (Fig. S1 C and D).Mkx−/− mice are known to have a “wavy tail” phenotype, which

becomes more apparent when the mice are running (6), butMkx−/− rats have a more profound wavy tail phenotype withoutrunning (Fig. S2). Mkx−/− rats manifest general hypoplasia oftendons, such as the flexor and extensor tendons of limbs, tailtendons, and patellar tendons while maintaining collagen ori-entation (Fig. 1 B and C). The tensile strength of the patellartendons was decreased in the Mkx−/− rats even after normaliza-tion with their cross-sectional area (Fig. 1D). These wavy tailphenotypes and general hypoplasia of the tendon were con-firmed in all three knockout lines (Fig. S2). One of these lines(14-deletion) was used for subsequent experiments. mRNA lev-els of tendon-related markers, such as tenomodulin (Tnmd),collagen type 1 α 1 (Col1a1), fibromodulin (Fmod), decorin (Dcn),and tenascin XB (Tnxb), were decreased in the patellar tendons ofMkx−/− rats (Fig. 1E). Scleraxis, known as a tenogenesis-relatedtranscription factor (17), was also decreased inMkx−/− rats but thedifference was smaller compared with the other tendon markers.Osteogenesis- and chondrogenesis-related genes, such as Col2a1,Acan, Runx2, Alpl, and IBSP, were elevated in Mkx−/− rats.Transmission electron microscopy (TEM) showed that the colla-gen fibril diameter in the tail tendon of Mkx−/− rats was uniformlysmaller than that in Mkx+/+ rats (Fig. S3). These phenotypes ofMkx−/− rats were similar to those of Mkx−/− mice (5, 6), but weresevere and more readily observed because of their larger sizes.

Heterotopic Ossification in Achilles Tendon of Mkx−/− Rats. Mkx−/−

rats showed elevated osteogenic and chondrogenic markers inthe patellar tendon, suggesting heterotopic ossification in thetendon. To check general ossification of Mkx− /− rats, micro-computed tomography (microCT) was performed, which revealedearly heterotopic ossification in the Achilles tendon ofMkx−/− rats(Fig. S4A). Heterotopic ossification occurred in 20% of 3-wk-oldMkx−/− rats and in all 5-wk-old Mkx−/− rats (Fig. S4B). The cross-sectional area of the ossifications gradually increased until the ageof 15 weeks in Mkx−/− rats (Fig. S4C). No heterotopic ossificationin other tendons or ligaments was observed in 15-wk-old Mkx−/−

rats (Fig. S5). Histochemistry of the Achilles tendon revealed thatpostnatal day 9 (P0) Mkx−/− rats already had chondral lesions intheir Achilles tendon (Fig. 2A). Endochondral ossification oc-curred from the middle of the chondral lesion in 3- to 4-wk-oldanimals. To compare rats and mice, Venus knockin homozygousmutant mice (5) were also analyzed. P0 and 4-wk-oldMkx−/− micehad no chondral lesion or ossification in the Achilles tendon (Fig.S4D). RT-qPCR on RNA from the Achilles tendon revealed thatexpression of chondrogenic genes increased in the 2-wk-oldMkx−/−

rat Achilles tendon (Fig. 2B). The mRNA levels of osteogenic-related genes increased in Mkx−/− rats from 2 to 4 wk of age.The expression of bone morphogenetic protein (BMP) pathway-related genes, such as BMPr1a, BMPr2, Smad1, and Smad5 wasalso elevated in Mkx−/− rats. Immunohistochemistry also showedexpression of chondrogenic/osteogenic genes at the site of het-erotopic ossification in Mkx−/− rats (Fig. S4E). To evaluate theinfluence of heterotopic ossification on the physiological func-tion, gait analysis was performed, which revealed a significantdecrease in the maximum ankle plantar flexion of Mkx−/− rats(Fig. S6 and Movies S1 and S2).

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Fig. 1. Generation of Mkx−/− rats. (A) Target site of gRNA and result ofdirect sequencing. (B) Tendons of Mkx+/+ or Mkx−/− rats (8-wk-old). (C) H&Estaining (Upper) and Picrosirius red staining (Lower) of the patellar tendons(black arrowhead) in 2-wk-old Mkx+/+ or Mkx−/− rats. (D) Tensile strength ofthe patellar tendon in Mkx+/+ or Mkx−/− rats. Absolute value of tensilestrength (Left) and tensile strength per unit area (Right). Error bars, SEM (n =3). (E) RT-qPCR analysis in the patellar tendon of 3-wk-old Mkx+/+ or Mkx−/−

rats. GAPDH was used as an internal control. Error bars, SEM (n = 3). **P <0.01; ***P < 0.005.

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Mkx Deficiency Accelerates Chondrogenic and Osteogenic Differentiationof Tendon-Derived Cells. Endochondral ossification involves mes-enchymal stem cell condensation and chondrocytic differentiation(18). Endochondral ossification in the Achilles tendon of Mkx−/−

rats prompted us to test whether Mkx is critical for restricting mes-enchymal cell differentiation into tenocytes and for preventing chon-drocytic differentiation. It was previously reported that a stemcell population, known as tendon stem/progenitor cells (TSPCs),were enriched in tendon tissues (19).We isolated and cultured tendon-derived cells (TDCs) from the

patellar tendons of 3-wk-old Mkx+/+ and Mkx−/− rats (Fig. 3A).There were no differences in the morphology of Mkx+/+ andMkx−/− TDCs (Fig. S7A) and FACS analysis revealed that thesecell preparations did not contain hematopoietic stem cells (Fig.S7B). These TDCs were capable of osteogenic, chondrogenic, andadipogenic differentiation, supporting the idea that the TDCscontained stem/progenitor populations.Under chondrogenic differentiation conditions, the pellets of

Mkx−/− TDCs were larger than those of Mkx+/+ TDCs (Fig. 3 Band C). RT-qPCR revealed that the expression of chondrogenicmarkers was higher in Mkx−/− TDCs after 1 wk of chondrogenicdifferentiation (Fig. S7C). Under osteogenic differentiation,Alizarin red staining revealed greater ossification in Mkx−/−

TDCs than in Mkx+/+ TDCs at 14 d (Fig. 3 D and E). The dif-ference in ossification between Mkx+/+ and Mkx−/− decreased at21 d, but remained significant. RT-qPCR showed higher ex-pression of osteogenic genes in Mkx−/− TDCs (Fig. S7D) andthere were no differences in adipogenic differentiation betweenMkx+/+ andMkx−/− TDCs (Fig. S7 E and F). These data suggestthat Mkx deficiency leads to enhanced osteogenic and chon-drogenic differentiation of TSPCs.

Mkx Overexpression Suppressed Chondrogenic, Osteogenic, andAdipogenic Differentiation of Mkx−/− TDCs. To rescue the loss ofMkx, Mkx−/− TDCs were retrovirally transduced with the Mkxcoding sequence containing a FLAG tag. Retrovirus-encodingVenus protein was also used as a control. Induction of FLAG-tagged Mkx was confirmed by RT-qPCR, Western blotting, andimmunocytochemistry (Fig. S7 G–I).Mkx-transduced Mkx−/− TDCs showed lower expression of

chondrogenic markers after chondrogenic differentiation (Fig. S7J).

In the osteogenic differentiation condition, Mkx-transduced TDCshad fewer calcium deposits (Fig. S7 K and L) and reduced osteo-genic markers (Fig. S7M). Mkx-transduced TDCs lost the ability todifferentiate into adipocytes (Fig. S7 N and O).

Mechanical Stretch Stimulation of Mkx−/− TDCs Leads to ChondrogenicDifferentiation.Tendons respond to appropriate mechanical strainsby increasing collagen production in tenocytes (20) and mechan-ical strains promote MSC differentiation into tenocytes (21). Asshown above, Mkx−/− TDCs showed a strong ability to differentiateinto osteocytes and chondrocytes, suggesting that the loss of Mkxaffects the response to mechanical stress of TDCs. To investigate thistheory, TDCs were subjected to mechanical stretch stimulation (Fig.4). After 4% monoaxial cyclic elongation for 6 h, Mkx+/+ TDCsshowed elevated levels of tendon-related genes, such asMkx,Col1a1,and Col3a1, indicating the tenogenic differentiation. However, thesame mechanical stimulation of Mkx−/− tendon-derived cells in-creased chondrogenic markers, such as SRY-box (Sox)6, Sox9,and Acan, rather than tendon-related genes.

Both Tendon-Related and Chondrogenic Differentiation-Related GenesAre Putative Targets of Mkx. Although Mkx appears to be a crit-ical transcription factor for tendon development and homeo-stasis, a genome-wide approach to identify the direct targets ofMkx in tenocytes has not yet been reported, partly because of thedifficulty of assembling a sufficient number of samples in mice. Inthe experiments described above (Fig. 3 and Fig. S7 G–O), wesuccessfully rescued theMkx−/− TDC phenotype by overexpressionof Mkx. Because a ChIP-grade antibody for mice and rats is notyet available, we attempted to perform ChIP-sequencing usingthe Mkx−/− TDCs overexpressing tagged Mkx. The hemagglutinin(HA) tag-fused Mkx binding region inMkx−/− TDCs was analyzedby ChIP-seq in the next-generation sequencer MiSeq (Fig. 5A).We obtained 6,356,463 sequence reads with the anti-HA antibodyChIP sample and 7,541,415 sequence reads from input DNA. Thesequencing data were aligned with the rat genome (rn6) usingbowtie software (22), resulting in 4,177,212 read maps of ChIP and4,961,690 read maps of input samples. By using the mapped se-quence reads, Mkx binding regions were detected by model-basedanalysis of ChIP-seq (MACS) (23) using the default parameters.The following analysis revealed 6,000 peaks of putative Mkx

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Fig. 2. Heterotopic ossification in the Achilles tendon of Mkx−/− rats. (A) H&E staining, Safranin O-Fast green staining, and Alizarin red staining of theAchilles tendons in P0, 3-wk-old, and 4-wk-old Mkx+/+ or Mkx−/− rats. (B) RT-qPCR analysis in the Achilles tendon of 2-wk-old and 4-wk-old Mkx+/+ or Mkx−/−

rats. Gapdh was used as an internal control. Error bars, SEM (n = 3); ns, not significant. *P < 0.05; **P < 0.01; ***P < 0.005.

7842 | www.pnas.org/cgi/doi/10.1073/pnas.1522054113 Suzuki et al.

binding sites. Within these peaks, protein-coding genes were se-lected and functional annotation was performed using DAVIDv6.7 (https://david.ncifcrf.gov/home.jsp). Among the functionalannotation chart, “skeletal system development (GO: 0001501)”was listed with a P value 7.6E-11, which included tendon-relatedgenes, such as Col1a1, Col3a1, and Mkx. Interestingly, chondro-genic differentiation-related genes, such as Sox5, Sox6, and Sox9were also included in the ontology (Fig. 5B). Moreover, othertendon-related genes, such as tenascin C (Tnc) and Fmod, werealso included within these peaks. De novo motif discovery of thesepeaks using MEME-ChIP software (24) revealed three bindingmotifs, and two of them contained A-C-A, which is the putativebinding site of mouse Mkx (25) (Fig. 5C). To support the physicalinteraction between Mkx and promoters, peaks in the promoterregions of Mkx, Col1a1, and Col3a1 were inserted into a thymidinekinase (TK) minimal promoter luciferase vector, and luciferaseassays were performed after coexpression of Mkx tagged with theVP16 effector. As a result, higher luciferase activity was observedwith VP16-Mkx expression compared with the control (Fig. S7P).This result indicates that these promoter regions may interact withMkx either directly or indirectly.

DiscussionAlthough the rat is a preferable experimental animal comparedwith the mouse in several medical and biological research fields,including that of the musculoskeletal system, limited information isavailable regarding the use of knockout rats. This limitation is be-cause of the technical difficulty in manipulating rat ES cells (26)and germ-line stem cells (27) for targeted genome deletion com-pared with that in the widely used mouse ES cells (11). In thisregard, recent genome-editing technologies, such as ZFN, TALEN,and CRISPR/Cas9, are powerful strategies by which gene knock-outs can be generated in various species of animals without usingES cells and homologous recombination (10). Here, we successfullygenerated genetically modified rats with deletion of Mkx withCRISPR/Cas9. The analysis of Mkx−/− rats not only confirms butalso extends our knowledge on Mkx-dependent tendon differenti-ation and regulation, by allowing us to observe a more severephenotype of heterotopic ossification in the Achilles tendon, toperform the physiological assessment of the ankle joint angle dur-ing ambulation, and to collect sufficient amounts of primary teno-cytes from knockout rats for mechanistic analyses and ChIP-seq.

In human, heterotopic ossification is a substantial medicalproblem because it is associated with pain and dysfunction (28).In systemic heterotopic ossification, a fibrodysplasia ossificansprogressiva-like phenotype (29) and ossification of the posteriorlongitudinal ligament of the spine (30) are important hereditarydiseases, although these ossifications occur only several yearsafter birth. BMP4 transgenic mice (31) and Npps−/− mice, re-ferred to as tip-toe-walking mice (32), have been considered asanimal models of these human diseases, with a fibrodysplasiaossificans progressiva-like phenotype and posterior longitudinalligament of the spine, respectively. Biglycan (Bgn)- and Fmod-deficient mice showed heterotopic ossification in not only theAchilles tendon, but also around the knee joint (33). These Bgn/Fmod-deficient mice also showed decreased diameter of collagenfibrils, similar to that observed in Mkx knockout mice and rats.Our data also indicate that Fmod expression in the patellartendons of Mkx−/− rats was decreased. Furthermore, ChIP-seq ofMkx showed significant peaks around Fmod, which suggest aninteraction between Fmod and Mkx.Although many patients with Achilles tendon heterotopic os-

sification have a history of trauma or surgery, hereditary factorsare thought to be involved in the etiology of this disorder (34).Excessive stress on the Achilles tendon, as well as surgical in-tervention or injection of growth factors (35–37) have beenshown to cause heterotopic ossification of the tendon.Here, we observed Achilles tendon ectopic ossification in

neonatal Mkx−/− rats. This ectopic ossification was related to anendochondral ossification program; at birth, chondrogenesis oc-curs in the center of the Achilles tendon and then cartilage tissuesare replaced by bone tissues. The precise molecular mechanismsof this heterotopic ossification of the tendon are not fully un-derstood; however, our data support that idea that mesenchymalcells (i.e., TSPCs), which should differentiate into tenocytes, maylose their fate without Mkx and can differentiate into chondrocytesduring embryogenesis. Several factors, such as Indian hedgehog,insulin-like growth factors, and BMPs, regulate the behavior ofchondrocytes during endochondral ossification (38, 39). The up-regulation of BMP pathway-related genes in the Achilles tendonof Mkx−/− rats suggests that Mkx regulates the BMP pathway.Mechanical stress affects tendon development before and af-

ter birth (40), and excessive mechanical stress can lead to ossi-fication of the tendon (28, 41). It is also reported that mechanicalstrain promotes the differentiation of MSCs into tenocytesin vitro (21, 42). In this regard, we recently found that Gtf2ird1translocates into the nucleus in response to mechanical strainand activates the Mkx promoter through chromatin regulation(43). Here, our mechanical stretch experiments with TDCs

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Fig. 3. Mkx regulates chondrogenic, osteogenic, and adipogenic differen-tiation in TDCs. (A) Protocol for isolation of TDCs. (B) Appearance of pelletsof Mkx+/+ or Mkx−/− TDCs after chondrogenic differentiation (Alcian bluestaining). (C) Diameter of the pellets of Mkx+/+ or Mkx−/− TDCs. Error bars,SEM (n = 3). (D) Appearance of the wells of Mkx+/+ or Mkx−/− TDCs afterosteogenic differentiation (Alizarin red staining). (E) Relative absorbance at450 nm of Alizarin red dye elution. Error bars, SEM (n = 3). *P < 0.05; **P <0.01; ***P < 0.005.

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support the idea that mechanical stimulation causes Mkx−/−

TDCs but not wild-type cells to undergo chondrogenic differ-entiation. The reason why rats showed a more severe phenotypethan mice with Achilles tendon ossification may be explained asfollows. Rats are larger than mice, which may increase me-chanical stimulation to the Achilles tendon during embryogen-esis and more readily stimulate Mkx−/− TSPCs to undergochondrogenic differentiation. In Mkx−/− rats, ectopic chondro-genesis in the Achilles tendon was observed in the embryonicand neonatal stages; however, increased chondrogenic markergene expression was terminated by 4 wk, suggesting that addi-tional chondrogenic changes may not occur in the Achilles ten-don after birth. The discrepancy of cartilaginous changephenotypes between the embryonic stage and after birth mayreflect the pluripotency of TSPCs in embryos and adults. In thisregard, it is of great interest to examine whether there would be adifference in TSPCs from Mkx−/− rat embryos and adults withmechanical loading. In addition, whether excessive exercisepromotes ectopic ossification in other tendons in mature Mkx−/−

rats should be examined in the future.Overexpression of Mkx has been shown to promote tendon-

related gene expression and to repress gene expression charac-teristic of other cell lineages (7, 8, 25, 44). Consistent with theseprevious reports, our study also showed that osteogenic andchondrogenic differentiation occurs more readily in TDCs from anMkx−/− background than in those from an Mkx+/+ background(Fig. 3 B–E). Regulation of Mkx expression was shown to affectexpression of essential extracellular matrix genes of tendon tissues,such as Col1a1, decorin, and Tnc, and chondrogenesis mastergenes. These in vitro observations, as well as the in vivo ectopicossification phenotype, indicate that the potential function ofMkx is to regulate cell fate of TSPCs via repressing chon-drogenenic factors. Our ChIP-seq data revealed that Mkx in-teracts with both extracellular matrix genes and chondrogenicgenes. Further detailed experiments are needed to clarify theprecise molecular mechanisms of how Mkx coordinates ex-pression of this diverse set of genes.

Therefore, we show in Mkx knockout rats that Mkx plays acritical role in TSPC differentiation to tenocytes and develop-ment of tendon tissues. These findings indicate that Mkx can beapplied as a therapeutic target for tendon repair or tissue engi-neering. In addition, the Mkx knockout rats represent a powerfulanimal model for further research on musculoskeletal tissuesand diseases.

Materials and MethodsDetailed materials and methods were described in SI Materials and Methods.The list of primer sequences for RT-PCR is shown in Table S2.

Preparation of hCas9 and gRNA. The preparation of hCas9 and gRNA has beendescribed previously (45). Briefly, the gRNA, the target of which wasdesigned to the second exon of the Mkx gene, was constructed by inversePCR. In vitro RNA synthesis and purification were performed.

Generation of Mkx Knockout Rats and Genotyping. All animal experimentswere approved by the Institutional Animal Care and Use Committee at theTokyo Medical and Dental University. The gRNA and hCas9 mixture RNA wasmicroinjected into the cytoplasm of Wistar rat zygotes by the UNITECHCorporation. The resulting chimeric offspring were crossed with Wistar ratsand germ-line transmission was confirmed by sequencing.

Tensile Testing. Patellar tendons from 6-mo-old wild-type or Mkx−/− rats (n =3) were pulled at a constant strain rate of 0.05 mm/s by a uniaxial materialstesting system (5).

Isolation of Rat TDCs. Patellar tendons of 3-wk-old wild-type or Mkx−/− ratswere dissected. The samples were cut into small pieces and digested withcollagenase (Sigma). After filtration with a nylon filter, digested cells werecultured. All experiments were performed until passage 5.

Adipogenic/Osteogenic Differentiation. TDCs were plated into 24-well plates at37 °C. After 24 h, the mediumwas changed to Adipogenesis Induction Medium(Lonza) and incubated for 7 d for adipogenic differentiation. The medium waschanged to Osteogenesis Induction Medium (Lonza) and incubated for 14 d,and Alizarin red staining was performed for osteogenic differentiation.

Chondrogenic Differentiation and Alcian Blue Staining. TDCs were suspended inChondrogenic Incomplete Medium (Lonza). After centrifuging and changingthe medium to Chondrogenic Complete Medium (Lonza), the pellets wereincubated for 21 d and Alcian blue staining was performed.

Retrovirus Infection. Venus, FLAG-Mkx, or HA-Mkx was inserted to the MIGRvector (Addgene). The plasmids were transfected into PLAT-E cells. After 24 h, thefiltered supernatant was used to infect TDCs (P1) derived from an Mkx−/− rat.

Mechanical Stretch Stimulation. Cells were seeded into the elastic siliconrubber chambers 12 h before stretching. The chambers were set on amonoaxial stretching device (STB-140, Strex) and monoaxial cyclic strain wasapplied for 6 h.

ChIP. After fixation, the cells were washed with cell lysis buffer containingprotease inhibitors, and resuspended in nuclear lysis buffer containingprotease inhibitors. Chromatin was fragmented to 100–400 base pairsusing sonication. The solution was then incubated with HA or normalrabbit IgG antibodies bound to beads. The immunoprecipitates wereeluted from the beads, incubated to reverse the cross-linking, and purifiedfor DNA analysis.

ChIP-seq Library Preparation and Data Analysis. DNA libraries for next-gen-eration sequencing were prepared using the TruSeq ChIP Sample Preparationkit (Illumina) from 3-ng ChIP DNA or input DNA and sequenced on a MiSeq(Illumina). ChIP DNA-enriched regions were detected by MACS v1.4.2 withdefault parameters. De novomotif discovery was performedwithMEME-ChIP(24) using the default parameter.

Luciferase Reporter Assays. HEK293FT cells were seeded in 96-well plates at30% confluence and were transfected with pcDNA-VP16-Mkx or controlpcDNA-Venus along with firefly and Renilla luciferase reporters. Thirty-sixhours after transfection, luciferase activity was measured. The results werenormalized to Renilla luciferase activity.

Sox6

157kb 186kb 433kb

52kb293 kb

Sox5

Sox914kb51kb

Col1a116kb13kb

350 bpCol3a1

Mkx2.5kb 9kb

Fmod53kb 52 kb

(E-value 8.2e-035)

bits

1 2 3 4 5 6 7 8

2

1

0

bits bits

(E-value 8.7e-002)(E-value 2.9e-013)

Mkx-/- TDCs

HA-Mkx induced TDCs

Retrovirus encoding HA-Mkx

HA-Mkx

Y

MkxHA

anti-HA antibody

ChIP

Next-Generation Sequencer analysis

1kb

1kb

1kb

1kb

10kb

10kb

1kb

Mkx binding motif

2

1

0

2

1

01 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

A

C

B

Fig. 5. ChIP-seq of HA-Mkx–overexpressing TDCs revealed that Sox5, Sox6,and Sox9 are putative targets of Mkx. (A) Protocols of ChIP-seq in HA-Mkx–overexpressing TDCs. (B) ChIP-seq peaks of Mkx. Red bars indicate the peak.The distance from the transcription start site is shown above each peak.(C) De novo motif analysis of ChIP-seq.

7844 | www.pnas.org/cgi/doi/10.1073/pnas.1522054113 Suzuki et al.

Statistical Analysis. The two-tailed independent Student’s t test was used tocalculate the P values.

ACKNOWLEDGMENTS. We thank Dr. Tomoki Chiba, Dr. Masaki Mori,Dr. Masafumi Inui, Dr. Masashi Naito, Dr. Yusuke Mochizuki, and all otherlaboratory members for the helpful discussions; Dr. Mari Uomizu forproviding helpful advice for tensile testing; Dr. Takaaki Kubota and Dr. ZhangYongwei for their help with tensile testing; and Dr. Mitsuhiro Enomoto for

providing helpful advice for gait analysis. This work was supported by the CoreResearch for the Evolutionary Science and Technology funding from the JapanAgency forMedical Research and Development; JSPS KAKENHI (Grants 26113008,15H02560, and 15K15544); grants from the NIH (AR050631, AR065379, andAG007996); the Takeda science foundation; a Bristol-Myers K.K. RA ClinicalInvestigation grant (to H.A.); the Japan Aerospace Exploration Agency (Grant14YPTK-005512); and “Creation of Life Innovation Materials for Interdisciplinaryand International Researcher Development” project, Ministry of Education.

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