Hand2 regulates chondrogenesis in vitro and in vivo

Bone 46 (2010) 1359–1368

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Bone j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b o n e

Hand2 regulates chondrogenesis in vitro and in vivo Makoto Abe a,⁎, Ikumi Michikami a,c, Toshiya Fukushi a, Akiko Abe b, Yoshinobu Maeda b, Takashi Ooshima c, Satoshi Wakisaka a a b c

Department of Oral Anatomy and Developmental Biology, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Removable Prosthodontics, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Pediatric Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan

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Article history: Received 19 June 2009 Revised 10 November 2009 Accepted 16 November 2009 Available online 22 November 2009 Edited by: M. Noda Keywords: Hand2 Chondrogenesis Collagen Transgenic Basic helix-loop-helix

a b s t r a c t Hand2 is a transcription factor of the basic helix-loop-helix family that plays essential roles during development. Hand2 determines the anterior–posterior axis during limb development and there is also substantial evidence that Hand2 regulates limb skeletogenesis. However, little is known about how Hand2 might regulate skeletogenesis. Here we show that, in a limb bud micromass culture system, over-expression of Hand2 represses chondrogenesis and the expression of chondrocytic genes, Sox9, type II collagen and aggrecan. Furthermore, we show that Hand2 is induced by the activation of canonical Wnt signaling, which strongly represses chondrogenesis. Surprisingly, Hand2 repressed chondrogenesis in a DNA binding- and dimer formation-independent manner. To examine the in vivo role of Hand2 in mice, we targeted the expression of Hand2 to the cartilage using regulatory elements from the collagen II gene. The resulting transgenic mice displayed a dwarf phenotype, with axial, appendicular and craniofacial skeletal deformities. Hand2 strongly inhibited chondrogenesis in the axial and cranial base skeleton. In the sternum, Hand2 inhibited endochondral ossification by slowing chondrocyte maturation. These data support a model of Hand2 regulating endochondral ossification via at least two steps: (1) determination of the site of chondrogenesis by outlining the region of the future cartilage template and (2) regulation of the rate of chondrocyte maturation. © 2009 Elsevier Inc. All rights reserved.

Introduction Most of the vertebrate skeleton is comprised of endochondral bones [1]. In the process of endochondral ossification, chondrogenesis begins as an aggregation of mesenchymal cells. This cell condensation differentiates into chondrocytes, which express type II collagen and aggrecan, and these condensations form the templates of the future skeleton. The cartilage enlarges through chondrocyte proliferation and matrix production. Chondrocytes in the center of the condensation stop proliferating and enlarge to become hypertrophic chondrocytes which leads to vascular invasion followed by recruitment of bone-forming osteoblasts and bone-resorbing osteoclasts. Formation of the proper size and shape of each skeletal element relies on appropriate chondroblast differentiation. Sox genes (L-5, -6 and -9), which contain a high mobility group domain and are homologous to Sry (sex-determining region of Y chromosome), are considered to be intimately involved in this initial step [2,3]. However, the regulation of Sox gene expression and of Sox protein activities is poorly understood.

⁎ Corresponding author. Fax: +81 6 6879 2875. E-mail address: [email protected] (M. Abe). 8756-3282/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2009.11.022

Cell-specific basic helix-loop-helix (bHLH) transcription factors function as regulators of lineage commitment and differentiation [4-6]. Hand2 is expressed in the limb bud, pharyngeal arches and heart in a highly restricted manner. Previous reports have shown that it plays crucial roles for normal development in various vertebrate species [7-13]. In the mouse limb bud, Hand2 is expressed in the posterior mesenchymal cells after embryonic day (E) 9 and is known to determine the anterior–posterior (A–P) axis by regulating Shh expression at the zone of polarizing activity (ZPA) [14-16]. Widespread ectopic expression of Hand2 in the limb mesenchyme results in preaxial polydactyly due to ectopic Shh expression outside the ZPA [14,15]. Abnormal endochondral ossifications were also observed following ectopic Hand2 expression in limbs, in addition to digit patterning abnormalities. An essential role for Hand2 in limb A–P axis determination has been suggested; however, a direct role of Hand2 on skeletal development is still unknown. Canonical Wnt signaling plays crucial roles during skeletal development. Canonical Wnts transduce signal by binding to the membrane bound receptor, Frizzled, and to a co-receptor, low-density lipoprotein receptor-related protein (Lrp) 5 and 6. Intracellular signaling inhibits the activity of glycogen synthase kinase 3 (GSK3), which ultimately stabilizes and accumulates β-catenin in the cytoplasm. Accumulated β-catenin enters the nucleus and, together with Lef/Tcf family molecules, activates target genes [17]. Canonical

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Wnt signals regulate the lineage specification between osteoblasts and chondrocytes, which differentiate from common progenitor cells [18-21]. Recent evidence indicates that Wnt signals, together with fibroblast growth factors (Fgfs), expand the undifferentiated cell population in the limb bud mesenchyme while maintaining the chondrogenic potential of these cells [22]. At the later stage of chondrocyte development, canonical Wnt signaling strongly induces the maturation of chondrocytes toward hypertrophy and calcification [23]. During postnatal growth Lrp5 regulates the peak bone mass [24]. Thus, Wnt signaling regulates development and also homeostasis of the skeleton. In this report, we show that Hand2 regulates endochondral ossification by showing that (1) Hand2 is expressed in the region surrounding the nascent cartilage, (2) Hand2 is induced by activating canonical Wnt signals, and (3) Hand2 represses chondrogenesis in vitro and in vivo. Furthermore, our results indicate that Hand2 regulates chondrocyte maturation. Materials and methods Constructs and reagents A Hand2-Cre construct, which contains a 7.4-kb mouse Hand2 promoter and NLS-Cre recombinase, was used to generate a Hand2 luciferase reporter. A HindIII/KpnI fragment, including the cardiac enhancer [25], was removed from the 7.4-kb mouse Hand2 promoter [26] to generate a Hand2 promoter without the cardiac enhancer. An approximately 5-kb fragment, including the Hand2 transcription start site, was released by XhoI/NotI digestion, filled-in with klenow (Invitrogen, Carlsbad, CA) and subcloned into pGL3-basic (Promega, Madison, WI) to generate the Hand2 luciferase reporter (Hand2-luc). To generate a type II collagen luciferase vector (Col2-luc), the firefly luciferase cDNA from pGL3-basic (Promega) was excised by HindIII/ SalI digestion and subcloned into the HindIII/SalI site of p3000i3020Col2a1 [27]. Myc-epitope-tagged mouse Hand2 cDNA (generously supplied by A. Firulli, Indiana Univ. School of Medicine), β-catenin S45A cDNA (generously supplied by D. Rimm, Yale Univ. School of Medicine), TCF4/dN-TCF4 (generously supplied by B. Vogelstein through Addgene Inc., Cambridge, MA, #16512, #16513) [28] and Axin2-luciferase vector (generously supplied by F. Constantini through Addgene Inc., #21275) were also used. LiCl (Sigma, St. Louis, MO), SB216763 (Tocris Bioscience, Ellisville, MO), Wnt3A (R&D Systems, Inc., Minneapolis, MN) were used for the study. Mutant Hand2 clones were generated by PCR-based mutagenesis and the mutations were confirmed by sequencing. The primer sequences are available upon request. Adenoviral constructs were generated as described previously [29]. Cell culture C3H10T1/2 and ATDC5 (both obtained from ATCC, Manassas, VA) and N1511 cells (generously provided by T. Maeda, Osaka Univ. School of Dentistry) were grown in MEM alpha or DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), penicillin G (100 units/ml), and streptomycin (100 μg/ml) at 37 °C in a humidified, 5% CO2 incubator. Limb-bud mesenchyme culture and recombinant adenovirus transduction were performed as previously described [30]. In situ hybridization (ISH), immunohistochemistry, and Western blot analysis ISH was performed as previously described [31]. Col2a1 (Genbank accession NM_031163; nt. 4201–4531), Col10a1 (Genbank accession NM_009925, nt.2427-2991), Runx2 (Genbank accession AF010284, nt.922-1746), Mmp13 (generously provided by P. Angel, Deutsches

Krebsforschungszentrum, Signal Transduction and Growth Control) [32], Twist1 (Genbank accession NM_011658, nt.746-1224), Axin2 (generously provided by F. Constantini through Addgene Inc., #21277) [33] and Hand2 [34] probes were used. Immunohistochemistry was performed as previously described using rabbit polyclonal anti-c-Myc epitope Tag antibody (Affinity BioReagents, Golden, CO, USA) [34]. Western blot analysis was performed as described previously using mouse monoclonal anti-Myc antibody (Invitrogen) or rabbit polyclonal ant-ERK2 antibody (sc-154, Santa Cruz Biotechnology, Inc., CA, USA) [29]. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) Limb-bud mesenchyme cultures were maintained until the indicated time points for each assay. Seven spots of micromass culture were used for each treatment and combined as one sample for further analysis. Total RNA preparation was performed with Trizol (Invitrogen) according to the recommended protocol and DNA was eliminated by RQ1 DNase treatment (Promega). 1 μg of the total RNA was reverse transcribed using an RT kit (New England Biolabs, Ipswich, MA). Real-time PCR was performed using iQ SYBR Green Supermix (BIO-RAD Laboratories, Hercules, CA) and the Mini Opticon Real-Time PCR System (BIO-RAD Laboratories). The primers used were as follows; ubiquitin, 5′-CGGTCTTTCTGTGAGGGTGT-3′ and 5′-TCACTGGGCTCCACCTCTA-3′; Sox9, 5′-CGGCTCCAGCAAGAACAAG-3′ and 5′-TGCGCCCACACCATGA-3′; type II collagen (Col2a1), 5′-GAAGGTGGAAAGCAAGGTGA-3′ and 5′-CATCAGTACCAGGAGTGCCA-3′; aggrecan (Acan), GGAATCCCTCGATGCTTCG-3′ and 5′-ACTGCAGCGATGACCCTC-3′; type I collagen (Col1a1), 5′TCCCGGTCAGAGAGGAGAAAG-3′ and 5′-GGAGACCAGAGAAGCCACGAT-3′; Axin2 (conductin), 5′-AAAACGGATTCAGGTCCTTCAA-3′ and 5′-GTCAGTGCGTCGCTGGATAAC-3′; Hand2, 5′-TACCAGCTACATCGCCTACCT-3′ and 5′-TCACTGCTTGAGCTCCAGGG-3′. DNA transfections and luciferase assays C3H10T1/2 and ATDC5 cells, seeded on 12-well plates, were transfected with 0.5 μg of reporter luciferase vector, 10 ng of pRL-TK or pRL-CMV, and 0.5 μg of each expression vector using Lipofectamine 2000 reagent (Invitrogen). Cells were transfected in triplicate for each experiment. After the indicated time points, cells were harvested and firefly luciferase activity, generated by the experimental reporter, and Renilla luciferase activity, generated by the normalizing reporter, were simultaneously measured with the Dual-Luciferase Reporter Assay System (Promega) using a Lumat LB 9507 (Berthold, Wildbad, Germany). Reporter gene activity was calculated by dividing firefly luciferase activity by Renilla luciferase activity, and the result was expressed as relative luciferase activity. For the Axin2-luciferase experiment, similar amount of the protein was used to measure firefly luciferase activity since combination of β-catenin S45A and TCF4 strongly activated the value of normalizing Renilla luciferase activity. The lysate was used for anti-ERK2 Western blotting to further confirm the accuracy of the protein amount measurement. Each experiment was performed at least three times. Generation and genotyping of transgenic animals To target Hand2 to resting and proliferating chondrocytes, the MYC-Hand2 cDNA was cloned into a transgenic expression vector (p3000i3020Col2a1) containing the promoter and enhancer sequence from the mouse type II collagen gene [35]. The murine Hand2 cDNA with the N-terminal 6xMYC epitope was excised from pCS2-MYC Hand2 (described above) by ClaI and KpnI digestion and subsequently cloned into the same restriction sites of p3000i3020Col2a1. The targeting construct was excised with NotI and injected into zygotes obtained from an F1 hybrid cross (BDF1 × BDF1). Embryos were then

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transferred into the oviducts of 0.5-dpc ICR pseudopregnant female mice. F0 embryos were collected at E17.5 and skin samples were retained for genotyping. Presence of the transgene was determined by PCR using following primer pair; 5′-TACCAGCTACATCGCCTACCT-3′ and 5′-TCACTGCTTGAGCTCCAGGG-3′ (PCR amplification produced a 221 bp transgene-specific product). The expression of Hand2 and the localization of Hand2 protein were determined by in situ hybridization and immunohistochemistry, respectively, as described above.

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within the site of chondrogenesis. At E12.5, Hand2 expression in the autopod was observed in the region surrounding the forming digit anlagen (Figs. 1C–F). High magnification images showed that Hand2 expressing cells are observed in close vicinity to the type II collagen expressing cells; however, they were excluded from the limb forming chondrocytes. At E16.5, Hand2 was expressed in the prehypertrophic and hypertrophic chondrocytes but was not or only weakly detected in the resting and proliferating chondrocytes (Supplementary Fig. 5B). Hand2 was also observed in the bone cells (Supplementary Fig. 5B).

Statistical analysis Hand2 represses chondrogenesis in a limb-bud mesenchyme assay All statistical significance was determined using Student's t-test (⁎ in the figures indicate p b 0.05). Results Hand2 expression is excluded from the site of initial chondrogenesis in the limb bud To examine the precise expression of Hand2 during chondrogenesis, we performed sectional in situ hybridization of Hand2 on the developing forelimb. At E11.5, Hand2 expression was weak in the anterior and strong in the posterior limb mesenchyme (Fig. 1A). Type II collagen (Col2a1) was expressed in the cartilage primordium of the future limb skeleton (Fig. 1B). Hand2 was strongly expressed in the region closely juxtaposing the aggregating chondrocytes of the zeugopod and autopod, a region which expressed type II collagen. However, Hand2 expression was weak or below the detection level

Fig. 1. Expression of Hand2 and type II collagen (Col2a1) in the limb bud. Hand2 (A, C, E) and Col2a1 (B, D, F) expression in E11.5 (A, B) and E12.5 (C–F) mouse embryos. The expression was detected using serial sections. (A, B) At E11.5, strong Hand2 expression (A) is observed at the region where Col2a1 expression is very low (B). (C–F) At E12.5, Hand2 is expressed in the mesenchyme surrounding the forming digit (C, E), while type II collagen is expressed in the forming digit anlagen (D, F). E and F are magnified views of C and D, respectively. Red dashed lines in E and F indicate the margin of the cartilage primordium of the digit.

In situ analysis showed that nascent cartilage develops at the site where Hand2 is not expressed. We, therefore, hypothesized that Hand2 might repress the initial stage of chondrogenesis. To directly investigate whether Hand2 regulates chondrogenesis, we transduced adenovirus producing Hand2 into limb-bud mesenchyme in culture. Mesenchyme from E11.5 mouse embryos was prepared and plated at high density to initiate cartilage differentiation. Limb-bud mesenchyme cells were infected with recombinant adenovirus expressing Hand2 (AdHand2) or β-galactosidase (Adβgal). Chondrogenesis was repressed by Hand2 after 4 and 6 days in culture, indicated by the reduced number of Alcian blue-positive nodules (Fig. 2A and data not shown). Although the number of nodules was reduced at both days 4 and 6, the difference in nodule number between Hand2- and βgaltransduced cells was much smaller at day 6 compared with day 4 (day 4, βgal 16.25 ± 1.70 and Hand2 7.5 ± 1.29, p b 0.0005; day 6, βgal 71.6 ± 7.19 and Hand2 53.2 ± 4.76, p b 0.005). The Hand2 inhibition of chondrogenesis, as inferred from culture morphology,

Fig. 2. Hand2 represses chondrogenesis and chondrocyte gene expression. (A) Alcian blue-stained cartilage nodules in limb-bud mesenchyme transduced with the indicated recombinant adenovirus after 4 days in culture. The image is representative of 21 micromass cultures from three independent experiments. (B) Real-time quantitative PCR determination of Hand2, type II collagen (Col2), aggrecan (Acan), Sox9 and type I collagen (Col1) expression after 3 days in culture. Expression levels were normalized to an unaffected control, ubiquitin. The results are representative of three independent experiments.

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was corroborated by gene expression studies, which showed that expression levels of type II collagen, aggrecan and Sox9 (Fig. 2B) were significantly repressed at day 3. Hand2 did not globally repress gene expression since type I collagen was not repressed by Hand2 (Fig. 2B). Hand2 is induced by canonical Wnt signaling via β-catenin stabilization in limb-bud mesenchyme and in N1511 cells In cultured limb-bud mesenchyme, canonical Wnt signaling strongly represses chondrogenesis and chondrocyte gene expression

[36]. Alcian blue staining and real-time PCR analysis showed that LiCl or SB216763 treatment, which both inhibit GSK3 activity but via different pathways, strongly repressed chondrogenesis and chondrocyte gene expression, as previously reported (Figs. 3A–D) [36]. Expression of Axin2 (conductin), a direct target of canonical Wnt signaling, was induced by almost 8-fold over control in both lithium and SB216763-treated cultures (Supplementary Figs. 1A and B). Significantly, both treatments induced higher levels of Hand2 expression when compared with the control treatment (1.8-fold by LiCl and 1.4-fold by SB216763; Figs. 3C and D). In both treatments, type I collagen expression was repressed to a lesser extent compared

Fig. 3. LiCl, SB216763 or Wnt3A protein application represses chondrogenesis and induces Hand2 expression. (A, B) Alcian blue-stained cartilage nodules from limb-bud mesenchyme treated with 20 mM LiCl (A) or 20 μM SB216763 (B) after 4 days in culture. (C, D) Real-time quantitative PCR determination of Hand2, type II collagen (Col2), aggrecan (Acan), and type I collagen (Col1) 24 h following LiCl (C) or SB216763 (D) treatment. Expression levels were normalized to an unaffected control, ubiquitin. (E) Real-time quantitative PCR determination of Hand2, type II collagen (Col2), aggrecan (Acan) 24 h following Wnt3A protein application to N1511 chondrocytic cells. Expression levels were normalized to an unaffected control, ubiquitin. (F) Co-transfection of an undegradable form of β-catenin (S45A) and TCF4 stimulates the Hand2 luciferase construct (Hand2-luc) in C3H10T1/2 cells. Cells were transiently transfected with Hand2-luc and β-catenin with or without TCF4 or δN-TCF4 expression vector. The total amount of DNA for each transfection was normalized with the β-galactosidase expression vector. After 48 h, cells were lysed and luciferase activity assayed. Firefly luciferase activity was normalized to protein amount. Inset in F shows the representative blot of anti-ERK2 using each lysate. The sample of each lane coincides with the luciferase figure. (G) Real-time quantitative PCR determination of Hand2, type II collagen (Col2a1), aggrecan (Acan) expression during limb-bud chondrogenesis at the indicated days after plating. Expression levels were normalized to an unaffected control, ubiquitin. Relative expression at day 1 was set to 1 for each reaction. All results show representative results of three independent experiments.

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with that of type II collagen or aggrecan (Figs. 3C and D). Addition of Wnt3A protein, which signals through canonical Wnt pathways, to chondrocytic N1511 cells strongly induced Axin2 expression by 15fold over control culture (Supplementary Fig. 1C). This treatment repressed type II collagen expression and induced Hand2 expression (Fig. 3E). Aggrecan expression was not affected by Wnt3A in N1511 cells. The reason why Aggrecan gene was not repressed by Wnt3A in N1511 cells is currently unclear. It is possible that the stage of differentiation differs significantly between N1511 cells and micromass culture, namely the Aggrecan expression might be at its low basal level in N1511 cells. If sufficient amount of Aggrecan is expressed in N1511 cells, we would expect to see its repression by Wnt3A treatment since Sox9 expression was repressed by Wnt3A in N1511 cells (data not shown). To determine the effect of β-catenin on the Hand2 promoter, C3H10T1/2 cells were transfected with a luciferase reporter containing 7.4-kb of the proximal Hand2 promoter from which the known cardiac enhancer had been removed [25]. Significantly, the Hand2 promoter was activated by a stabilized form of β-catenin (S45A β-catenin) and co-transfection of TCF4 synergistically augmented the activity (Fig. 3F). The synergistic activation with S45A β-catenin was not observed when δN-TCF4, which lack the βcatenin-binding motif, was used (Fig. 3F). TCF4 or δN-TCF4 slightly reduced the luciferase activity when transfected to the cells alone (Fig. 3F). The weak repression observed by transfected TCF4 is possibly caused by foreign TCF4 scavenging the free β-catenin in the cytosol. These data support the conclusion that inhibition of GSK3 induces Hand2 expression through up-regulation of β-catenin protein levels. Significantly, the expression of Hand2 in the cultured limb-bud mesenchyme rapidly diminished as chondrocyte differentiation progressed (Fig. 3G). To investigate the mechanism of Hand2 chondrogenesis repression, we analyzed the effects of two Hand2 mutants on type II collagen promoter/enhancer sequences. bHLH transcription factors form homo- or heterodimers with other bHLH molecules via their HLH motifs and thereafter bind to a canonical consensus site, termed the Ebox (CANNTG) [5]. Three arginine residues located in the basic region of Hand2 were exchanged to acidic amino acid residues to generate RRR N EDE Hand2 (referred to hereafter as EDE). This mutant protein cannot bind to the E-box under the conditions of the gel shift assay [15]. In another mutant Hand2, a phenylalanine residue in the helix1 region of the HLH motif was mutated to a proline residue to generate F119P Hand2 (referred to hereafter as F119P). This protein cannot heterodimerize with E protein and is unable to bind to the E-box [15]. C3H10T1/2 and ATDC5 cells were co-transfected with the type II collagen reporter construct (Col2-luc) and the Hand2 expression vectors. In both cell lines, wild-type and Hand2 mutant clones repressed type II collagen transcription to a similar extent (Supplementary Fig. 2A). Immunoblot analysis of total lysate showed similar levels of wild-type and mutant Hand2 proteins (Supplementary Fig. 2B). We generated adenovirus producing the mutant Hand2 proteins (AdEDE and AdF119P) and transduced them into limb-mesenchyme cells. In these experiments, wild-type Hand2 virus was infected at 2fold MOI (referred to as 2 MOI hereafter) compared to the AdEDE and AdF119P (1 MOI) to induce similar levels of exogenous Hand2 expression by each infection (Supplementary Fig. 3). The difference in the expression level is possibly due to the difference in stability of mRNA between wild-type and mutant Hand2. After 4 days in culture, numbers of Alcian blue-positive nodules were repressed more strongly by AdEDE and AdF119P compared with the repression observed by wild-type Hand2 (Supplementary Fig. 2C). Expression of type II collagen and aggrecan were strongly repressed in wild-type AdHand2, AdEDE and AdF119P transduced cells at day 3 (Supplementary Fig. 2D). Type I collagen expression was not affected significantly by either wild-type or mutant Hand2, indicating the specific effect of Hand2 on chondrocytic gene expression (Supplementary Fig. 2D). The number of alcian blue-positive nodules was

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repressed to a similar extent in the cells infected with 1 MOI wildtype AdHand2 compared to 2 MOI wild-type AdHand2-infected culture (1 MOI, 2.42 ± 1.61; 2 MOI, 2.14 ± 1.06, p = 0.56). However, the repression of type II collagen and aggrecan expression were significantly weaker in 1 MOI wild-type AdHand2 compared to 2 MOI (data not shown). Hand2 transgenic mice show multiple skeletal defects To ask whether Hand2 regulates chondrogenesis in vivo, we generated transgenic mice that express Hand2 under a murine type II collagen promoter/enhancer (Supplementary Fig. 4A) [27]. The construct was injected into oocytes, which were then transferred into the oviducts of pseudopregnant females. Three out of six pups from a natural delivery showed severe difficulty in breathing and died immediately after birth. Later, the dead pups were confirmed to be positive for the transgene. Since it was possible that we would fail to obtain viable founders, we dissected and analyzed the F0 embryos at E17.5. We obtained a total of 37 embryos and 14 embryos were positive for the transgene (hereafter referred to as Col2-Hand2 embryos). We analyzed Hand2 expression in the limb skeleton by in situ hybridization using the Hand2 coding sequence probe. The color development was terminated before the endogenous Hand2 signals were detected. Hand2 was expressed in the limb cartilage in Col2Hand2 embryos, while the transgene-negative embryos (hereafter referred to as control embryos) did not show such staining (Supplementary Figs. 4B–F and Figs. 6C and D). To assess the ectopic protein localization in the Col2-Hand2 embryos, we performed αMYC immunohistochemistry. Distinct immunostaining was observed in the cartilage primordium of the ulna (Supplementary Fig. 4F) and in the autopod (Supplementary Fig. 4H) in Col2-Hand2 embryos. In the control embryos, background staining was observed in the mineralized region in the ulna but signal was not detected in chondrocytes (Supplementary Figs. 4G and I). The Col2-Hand2 embryos showed variable phenotypes, possibly due to differences in Hand2 expression levels among the embryos. We will describe the phenotype of embryos that showed severe skeletal defects in multiple regions and that also showed specific expression of Hand2 mRNA (detected by ISH) and Hand2 protein (detected by αMYC immunostaining) in cartilage. As expected, transgenic embryos showed severe skeletal defects in every skeleton which develops through endochondral ossification. Skeletal defects were also observed in regions where endogenous Hand2 is not usually expressed (Supplementary Fig. 5A) since we used the general cartilage enhancer to generate these transgenic mice. Col2-Hand2 embryos showed severe dwarfism compared with control littermates (Fig. 4A and Supplementary Fig. 6). The lateral view of the craniofacial region of Col2-Hand2 embryos showed an abnormally domed-shape skull but the size and shape of other skeletal elements were comparable to those of the control embryos (Figs. 4B and C). Meckel's cartilage, which is normally observed in the lower jaw and posteriorly forms malleus (the most anterior middle ear ossicle) (Figs. 4D and H), was blunted at its posterior end (Figs. 4E and I). Furthermore, the cartilage primordia of the middle ear ossicles were extremely abnormal in shape, although several cartilaginous fragments were observed in this region (Figs. 4D and E). The lateral view of the Col2-Hand2 cervical region showed poorly formed occipital cartilage and the basi- and exoccipital bone formations were essentially missing (Figs. 4F and G). The cartilage primordium of the cervical vertebrae was very small in both the body and the neural arch, and mineralization of the atlas was not observed (Figs. 4F and G). The poor development of the occipital arch and atlas resulted in a large spacing of the cervical skeleton in Col2-Hand2 embryos. The formation of the primordia of the hyoid, thyroid and cricoid cartilage were also very poor (Figs. 4F and G). The caudal view of the cranial base showed normal formation of the palatine, basisphenoid, basioccipital and exoccipital bones in the

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control embryo (Fig. 4H). In the Col2-Hand2 embryos (Fig. 4I), most of the cranial base cartilage was missing and only a small portion of the basisphenoid bone (pterygoid process) was observed. The abnormal shape of the cranium is possibly due to the developmental defect observed at the cranial base since this region contributes to the enlargement of the neurocranium [37]. Development of the fore- (Fig. 5A) and hindlimbs (Fig. 5B) were also affected in Col2-Hand2 embryos. The scapula, humerus, radius and ulna in the forelimb and the femur, tibia and fibula in the hindlimb were considerably shorter in the transgenic embryos (Supplementary Fig. 6 and data not shown). The mineralized site

indicated by the alizarin red-positive domain was much shorter in the stylopod and zeugopod of both fore- and hindlimb (Supplementary Fig. 6 and data not shown). However, the digit patterning was normal and no polydactyly was observed. Detailed analysis of the autopod region showed under-mineralization of the cartilage primordia of the metacarpal and metatarsal bones in the Col2-Hand2 embryos (Fig. 5D and data not shown) compared with the control embryos (Fig. 5C and data not shown). The rib cage was much smaller in the transgenic embryos (Fig. 5F) compared with the control embryos (Fig. 5E) and mineralization of the sternum was not observed (compare inset in Figs. 5E and F). Also some mispatterning of the costal cartilage, such as blunting (Fig. 5F) and bifurcation (data not shown) was observed. In the lumbar vertebrae of the transgenic embryo, the body and neural arch of the vertebrae were mostly not formed and only the lateral portion of the neural arch was observed (Figs. 5G and H). Surprisingly, abnormal lumbar vertebrae in the transgenic embryos showed a small region of mineralization at the neural arch (Fig. 5H), as seen in the control embryos (Fig. 5F). Histological sections showed well-organized epiphyseal cartilage in the distal ulna of Col2-Hand2 embryos (Fig. 6B), which was similar to the control growth plate (Fig. 6A), even though in situ hybridization detected broad ectopic Hand2 expression in chondrocytes (Figs. 6C and D). The domain of type II collagen (Col2a1; Figs. 6E and F) and type X collagen (Col10a1; Figs. 6G and H) was not altered in the transgenic embryos, indicating that the maturation process, at least in the distal epiphyseal cartilage of ulna at E17.5, is not disturbed by Hand2. Also, proliferation of cartilage, detected by the expression of Histone 1 H4c (H4c), did not show a detectable difference between transgenic (Fig. 6J) and control (Fig. 6I) embryos. We further analyzed the expression of genes known to be regulated by Hand2 in Col2-Hand2 embryos. Runx2, a master gene for osteoblast differentiation and chondrocyte maturation, was expressed in hypertrophic chondrocytes and bone cells in both control (Fig. 7A) and transgenic limbs (Fig. 7B). Consequently, Mmp13 (Collagenase-3), a direct target of Runx2 [32], was expressed in similar domain as Runx2 in both control (Fig. 7C) and transgenic embryos (Fig. 7D). Twist1 is known to physically and functionally associate with Hand2 during limb development [38]. As previously reported, Twist1 was mainly detected in the bone cells [39] in control embryos (Fig. 7E) and similar expression was observed in the transgenic limbs (Fig. 7F). Next, we asked whether β-catenin-mediated signals are altered in Col2-Hand2 embryos. Again, we examined Axin2 expression, a direct target of canonical Wnt signaling. Axin2 was detected in prehypertrophic and hypertrophic chondrocytes [40] in control embryos (Fig. 7G). There was some variation of Axin2 expression in the transgenic limbs. One out of three transgenic embryos showed severely down-regulated Axin2 expression (Fig. 7H). One showed

Fig. 4. Transgenic Col2-Hand2 embryos show abnormal endochondral ossification in the head and neck. Bone and cartilage stained E17.5 embryos. (A) Lateral view of the whole body of control (left) and Col2-Hand2 (right) embryos. The Col2-Hand2 embryo has a dwarf phenotype and shows skeletal deformity of the axial, appendicular and craniofacial skeleton. (B–G) Lateral view of the cranial (B, C), middle ear (D, E) and cervical (F, G) region of control (B, D, F) and Col2-Hand2 (C, E, G) embryos. The frontal bones of the Col2-Hand2 embryo (C) are dome-shaped (arrowhead in C) compared with the control embryo (B). Distinct cartilage primordia of the malleus, incus and stapes are observed in the control embryo (D) while abnormally shaped cartilage fragments are detected in this region in the mutant embryo (E). Hyoid, thyroid, cricoid and tracheal cartilages are observed in the anterior portion in the control embryo (F). In contrast, the cartilage primordia formed in the mutant embryo in the anterior cervical region are very poor and tracheal cartilages are missing (G). Also, in the posterior region, some of the cervical vertebrae are missing in the mutant embryo (G) compared with the control embryo (F). (H, I) Ventral view of the cranial base of control (H) and Col2-Hand2 (I) embryos. Palatine, basisphenoid, basioccipital and exooccipital bones are formed in the control embryo (H), while most bones and cartilages are not formed in the cranial base of mutant embryos (I). Key: at, atlas; ax, axis; bo, basioccipital bone; bs, basisphenoid bone; cc, cricoid cartilage; eo, exooccipital bone; fm, foramen magnum; hy, hyoid; inc, incus; mal, malleus; md, mandible; pl, palatine bone; pp, pterygoid process; stp, stapes; tc, tracheal cartilage; th, thyroid cartilage; ty, tympanic ring.

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Fig. 5. Col2-Hand2 embryos show abnormal endochondral ossification in the limb, thoracic region and lumbar vertebrae. Bone and cartilage stained tissues at E17.5. Forelimb (A) and hindlimb (B) of control (upper) and mutant (lower) embryos. The size of the mineralized region is reduced in both fore- and hindlimbs of the mutant. (C, D) Magnified view of the autopod of the forelimb. The digit patterning appears normal in both control (C) and mutant (D) embryos. Mineralization of the metacarpus is observed in the control embryo (arrowhead in C) while the metacarpus is entirely cartilaginous in the mutant embryo (D). (E, F) Frontal view of the rib cage of control (E) and mutant (F) embryos. The rib cage is much smaller in the mutant (F) compared with the control embryo (E). Furthermore, mineralization of the sternum is not observed in the mutant (inset in F) compared with the control embryo (inset in E). (G, H) Ventral view of the lumbar vertebrae of control (G) and mutant (H) embryos. Body and neural arch of the lumbar vertebrae are formed and three independent mineralized regions are observed in each vertebra in the control embryo (G). In the mutant (H), the central part of each vertebrae is completely missing while the lateral portion of the neural arch is observed. Key: fe, femur; fi, fibula; h, humerus; r; radius; sc, scapula; t, tibia; u, ulna.

slight down-regulation compared to the control embryos and another showed equivalent level of Axin2 expression in prehypertrophic and hypertrophic chondrocytes (data not shown). We performed an in vitro assay to further ask whether Hand2 could regulate the proximal promoter of the Axin2 gene [33]. To this end, we analyzed the effect of Hand2 and β-catenin on Axin2-luciferase vector by co-transfecting into C3H10T1/2 cells. β-catenin strongly activated Axin2 transcription (Supplementary Fig. 7) as previously reported [33]. Interestingly, cotransfection of Hand2 significantly repressed the effect of β-catenin on Axin2 promoter (Supplementary Fig. 7). Discussion In the present study, we showed that Hand2 represses chondrogenesis and chondrocyte gene expression in the limb-bud mesenchyme

Fig. 6. Normal expression of Col2a1, Col10a1 and H4c in E17.5 ulnar epiphyseal cartilage. Sections of control (A, C, E, G, I) and mutant (B, D, F, H, J) embryos. (A, B) Control (A) and mutant (B) sections stained with hematoxylin and eosin. (C, D) In situ hybridization for Hand2 on control (C) and mutant (D) embryos. The color development was terminated before the endogenous Hand2 signal appeared. (E–J) In situ hybridization for type II collagen (Col2a1) (E, F), type X collagen (Col10a1) (G, H) and histone 1 H4c (H4c) (I, J) in control (E, G, I) and mutant (F, H, J) embryos.

assay. Consistent with this observation, Hand2 was not expressed at the site of initial chondrogenesis. Interestingly, activating β-catenin signals, which strongly repress chondrogenesis, induced Hand2 in the limb-bud mesenchyme and chondrogenic cells. To study the role of Hand2 in skeletogenesis in vivo, without affecting the A–P patterning of the limb, we targeted Hand2 specifically to chondrocytes. Transgenic expression of Hand2 in chondrocytes strongly affected endochondral ossification,

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Fig. 7. Normal expression of Runx2, Mmp13, Twist1 and repression of Axin2 expression in E17.5 ulnar epiphyseal cartilage. Section of control (A, C, E, G) and transgenic (B, D, F, H) embryos. In situ hybridization for Runx2 (A, B), Mmp13 (C, D), Twist1 (E, F) and Axin2 (G, H).

with differing severity depending on the site of chondrogenesis. The most severe effect was agenesis of cartilage primordia (observed in the vertebrae and cranial base). A chondrocyte maturation defect was observed in the sternum and the autopod skeleton. The growth plate architecture of the zeugopod skeleton was histologically and molecularly quite similar between the control and mutant embryos; however, the overall size of each skeletal element was extremely small in the transgenic embryos. Although some variation among different transgenic embryos was observed, Axin2, a direct target of canonical Wnt signaling, was down-regulated in the growth plate of Col2-Hand2 embryos compared to the control embryos. During the initial stage of chondrogenesis, β-catenin-mediated signals strongly repress chondrogenesis and chondrocytic gene expressions [36,41]. Since β-catenin induces Hand2 in various chondrocytic culture models and Hand2 strongly repressed chondrogenesis, it is likely that Hand2 functions under the control of Wnt signaling to regulate endochondral ossification. Intriguingly, Twist1, which is a bHLH transcription factor and a dimerization partner of Hand2 [38], is also induced by canonical Wnt signals and represses chondrogenesis [36]. These observations indicate that Hand2 might cooperate with Twist1 to repress chondrogenesis. This scenario is

possible during the very early stage of chondrogenesis, but not at later stages after the cartilage primordium has developed since (1) deletion of Twist1 in cartilage show no defects in the skeletal development [39] and (2) expression of Twist1 is excluded from the chondrocytes. However, our analysis revealed that Hand2 represses chondrogenesis in a dimer formation-independent manner. Furthermore, the repression of chondrogenesis was observed in a DNA binding-independent fashion. Since the expression patterns of Hand2 and Twist1 overlap in various developmental sites [38,42,43], and both genes are considered to be involved in genetic skeletal defects [44-46], it will be important to further investigate whether there is any relationship between Hand2 and Twist1 with respect to Wnt signaling. The Hand2−/− mouse is embryonic lethal around E10 due to a severe cardiac defect [9,12]. The early lethality of the knockout has hampered studying the potential role played by Hand2 in later limb skeletal development. Although various animal models have been generated, the exact role played by Hand2 in in vivo skeletogenesis remains unresolved. This is partly because over-expression of Hand2 affects A–P patterning in the limb [14,15,47], which might disrupt various parameters of normal limb skeletal development (such as cell number in the limb mesenchyme). To circumvent this issue, we analyzed transgenic mice that express Hand2 in chondrocytes. The resulting embryo had a dwarf phenotype and showed multiple skeletal defects, including agenesis of skeletal elements. Agenesis was mainly observed in the axial region of the embryo. We never observed agenesis of the limb skeleton, although transgenic embryos showed severe reduction in skeleton size. It is known that the timing of Collagen II promoter/enhancer activity depends on each developmental site [48]. In the sclerotome, from which axial skeletons are derived, the enhancer activity is detected before the initiation of chondrogenesis (starting around E9.5). There is also low-level enhancer activity detectable in the notochord, which functions as one of the signaling centers during development. However, in the lateral plate mesoderm, from which the appendicular skeleton is derived, the enhancer activity is observed only after mesenchymal condensation has occurred. Thus the difference in the severity of the defects observed between axial and appendicular skeleton is presumably due to the earlier expression of the transgene in the sclerotome, which severely reduced the size of the cartilage template. However, we cannot exclude the possibility that cartilage primordia of the axial and appendicular skeleton express different Hand2 partner(s), which resulted in different outcomes. We observed defects of chondrocyte maturation in the sternum and autopod skeletons. Also, even though the limb skeleton was significantly shortened in transgenic embryos, the height of the epiphyseal growth plate was quite similar. This is presumably due to the reduced proportion of mineralized region in the transgenic limbs (Supplementary Fig. 6). Chondrocyte maturation is regulated by a number of molecules [1]. Interestingly, Hand2 has been shown to repress Runx2 activity and control osteoblast differentiation [49]. Runx2 is one of the factors intimately involved in chondrocyte hypertrophy [50-52]. Hand2 is expressed in prehypertrophic chondrocytes and in the perichondrium in the epiphyseal growth plate [14]. Since Hand2 expression clearly overlaps with that of Runx2 in the growth plate [50], it is possible that Hand2 possess physiological roles during chondrocyte maturation. However, normal expression of Mmp13 was detected in Col2-Hand2 limbs suggesting that regulating Runx2-mediated transcription might not be the role played by Hand2 in vivo. Activation of canonical Wnt signaling strongly augments chondrocyte maturation at a later stage of cartilage development. This is evidenced by various in vitro and in vivo studies [23,40].We observed significant down-regulation of Axin2 expression in some transgenic embryos. Since Axin2 is induced by β-catenin-mediated signal and known to play a role in negative feedback regulation, it would be important to identify whether (1) β-catenin signaling is hyperactivated by down-regulation of Axin2 by Hand2 in the growth plate or (2) hypo-activation of the β-catenin signaling by Hand2 resulted in

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lower Axin2 expression in our Col2-Hand2 embryos. In either case, other target molecules of β-catenin signaling, such as Lef-1, should be either up-regulated (if hyper-activated) or down-regulated (if hypoactivated). However, we did not detect significant difference in Lef-1 expression in the transgenic limbs (data not shown). It is possible that down-regulation of Axin2 affects signaling cascades other than βcatenin-mediated ones, since Axin2 was reported to regulate Tgf beta signaling and other molecules which regulate skeletal development [53-56]. This point is currently under investigation. Acknowledgments We gratefully acknowledge Satoshi Ueno, Ryo Hamanaka and Satoru Toyosawa for technical assistance, Weiguo Feng for probes and Peter Angel, Frank Constantini, Bert Vogelstein, Anthony Firulli, David Clouthier and David Rimm for constructs. M.A. is supported by grantsin-aid (19791344, 21791875, 21st Century COE Program) from MEXT.

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used to prepare cDNA sample. Each cDNA was amplified with Hand2, Col2a1 and Gapdh specific primers. (B) In situ hybridization for Hand2 in zeugopod skeleton of the wild-type mouse embryo at E16.5. Supplementary Figure 6. Measurement of forelimb skeletons in control and transgenic embryos at E17.5. The images of each skeletal element were captured and the length was analyzed using ImageJ software. 3 embryos from each genotype were used for the measurement except for ulna measurement (n = 2) since one transgenic embryo showed extremely short ulna resulting in huge S.D.. Supplementary Figure 7. Axin2 transcription is repressed by wildtype Hand2 in C3H10T1/2 cells. Cells were transfected with the Axin2 reporter vector, β-catenin, and Hand2 expression vectors. After 48 h, cells were lysed and luciferase activity assayed. Firefly luciferase activity was normalized to Renilla luciferase activity derived from a constitutive co-transfected plasmid. Results are representative of two independent experiments. Note: The supplementary material accompanying this article is available at (doi:10.1016/j.bone.2009.11.022).

Appendix A. Supplementary data Supplementary Figure 1. Relative expression of Axin2 by lithium, SB216763 and Wnt3A treatment. (A, B) Real-time PCR results of Axin2 expression in limb-bud culture treated with 20 mM LiCl (A) or 20 μM SB216763 (B). The samples were harvested after 3 days in culture. (C) Real time PCR results of Axin2 expression in N1511 cells treated with 100 ng/ml Wnt3A. Sample was harvested 24 h after Wnt3A addition. Expression levels were normalized to an unaffected control, ubiquitin. Supplementary Figure 2. EDE and F119P Hand2 mutants repress chondrogenesis and chondrocyte gene expression. (A) Type II collagen transcription is repressed by wild-type or mutant Hand2s in C3H10T1/2 and ATDC5 cells. Cells were transfected with the type II collagen reporter vector and various Hand2 expression vectors. After 36 h, cells were lysed and luciferase activity assayed. Firefly luciferase activity was normalized to Renilla luciferase activity derived from a constitutive co-transfected plasmid. Results are representative of three independent experiments. (B) Equal amounts of the cell lysate used for the assay in (A) were further used to detect the wild-type and mutant Hand2 protein levels by Western blot analysis. The result is representative of two independent experiments. (C, D) Wild-type and mutant Hand2s repress chondrogenesis and chondrocyte gene expression. (C) Alcian blue-stained cartilage nodules from limb-bud mesenchyme transduced with the indicated recombinant adenovirus after 4 days in culture. The images are representative of 21 spots from three independent experiments. The numbers below each image shows the quantified nodule number (n = 7). (D) Real-time quantitative PCR determination of type II collagen (Col2), aggrecan (Acan), type I collagen (Col1) expression after 3 days in culture. Expression levels were normalized to an unaffected control, ubiquitin. The results are representative of two independent experiments. Supplementary Figure 3. Relative expression of Hand2 after infection of wild-type and mutant Hand2 adenovirus. Real-time PCR result of Hand2 expression after 3days in culture. Control culture was infected with βgal producing virus. Expression levels were normalized to an unaffected control, ubiquitin. Supplementary Figure 4. Targeted expression of Hand2 in the cartilage of Col2-Hand2 embryos. (A) Construct for targeting Hand2 in chondrocytes. (B, C) E17.5 forelimb section of control (C) and transgenic (B) embryos stained with hematoxylin and eosin. (D, E) In situ analysis for Hand2 in control (E) and transgenic (D) embryos at E17.5. Magnified view of the ulna is shown in Figs. 6C and D. (F-I) E17.5 forelimb section stained with anti-MYC antibody to detect MYCHand2 protein in control (G, I) and transgenic (F, H) embryos. Key: A, autopod; Z, zuegopod. Supplementary Figure 5. Expression of Hand2 in skeletal tissues in vivo. (A) Skeletal tissue from autopod, scapula, vertebra, and ribs region were dissected out from postnatal day 2 wild-type mouse and

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