LETTER

Irxl1 Mutant Mice Show Reduced Tendon Differentiationand No Patterning Defects in MusculoskeletalSystem Development

Wataru Kimura,1 Masashi Machii,1 XiaoDong Xue,1 Nishat Sultana,1 Keisuke Hikosaka,1

Mohammad T.K. Sharkar,1 Tadayoshi Uezato,1 Masashi Matsuda,2 Haruhiko Koseki,2 andNaoyuki Miura1*

1Department of Biochemistry, Hamamatsu University School of Medicine, 1-20-1 Handa-yama,Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan

2Developmental Genetics Group, RIKEN Center for Allergy and Immunology, 1-7-22 Suehiro,Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan

Received 9 April 2010; Accepted 21 October 2010

Summary: Irxl1 (Iroquois-related homeobox like-1) is anewly identified three amino-acid loop extension (TALE)homeobox gene, which is expressed in various meso-derm-derived tissues, particularly in the progenitors ofthe musculoskeletal system. To analyze the roles ofIrxl1 during embryonic development, we generatedmice carrying a null allele of Irxl1. Mice homozygous forthe targeted allele were viable, fertile, and showedreduced tendon differentiation. Skeletal morphologyand skeletal muscle weight in Irxl1-knockout miceappeared normal. Expression patterns of severalmarker genes for cartilage, tendon, and muscle progen-itors in homozygous mutant embryos were unchanged.These results suggest that Irxl1 is required for the ten-don differentiation but dispensable for the patterning ofthe musculoskeletal system in development. genesis49:2–9, 2011. VVC 2010 Wiley-Liss, Inc.

Key words: TALE homeodomain; tendon; muscle;skeleton; knockout mice

Irxl1 (Iroquois-related homeobox like-1, also known asMohawk homeobox) is a newly identified homeodo-main-containing transcription factor that belongs to thethree amino-acid loop extension (TALE) superclass ofatypical homeodomain proteins, which have three extraamino acid residues between the first and second heli-ces of their homeodomains (Anderson et al., 2006; Liu

et al., 2006; Takeuchi and Bruneau, 2007). The homeo-domain of Irxl1 is most closely similar to that of the Irxproteins, but different domain structures outside of thehomeodomains distinguish the Irxl1 protein from theIrx family proteins; Irxl1 lacks Irx-specific domains,‘‘IRO-A’’ and ‘‘IRO-BOX,’’ and contains other Irxl1-spe-cific domains some of which possess transcriptionalrepression activities (Anderson et al., 2006; Kerneret al., 2009; Liu et al., 2006; Mukherjee and Burglin,2007; Takeuchi and Bruneau, 2007).

During mouse embryonic development, Irxl1 isexpressed in various mesoderm-derived tissues, such asthe dorsomedial lip and the ventrolateral lip of somites,limb buds, endocardia, kidneys, and male gonads(Anderson et al., 2006; Liu et al., 2006; Takeuchi andBruneau, 2007). Expression of Irxl1 in the somite andlimb bud is restricted to the progenitors of skeletalmuscle, tendon, and cartilage (Anderson et al., 2006).Interestingly, the fruit fly orthologue of Irxl1 is alsoexpressed in larval muscle progenitor cells (Tomancaket al., 2002). It has been reported that, in cultured cells,the Irxl1 protein represses transcription and inhibits

* Correspondence to: Naoyuki Miura, Department of Biochemistry,

Hamamatsu University School of Medicine, 1-20-1 Handa-yama, Higashi-ku,

Hamamatsu, Shizuoka 431-3192, Japan. E-mail: nmiura@hama-med.ac.jp

Contract grant sponsor: Ministry of Education, Science, Sports, Culture

and Technology of Japan.

Published online 4 November 2010 in

Wiley Online Library (wileyonlinelibrary.com).

DOI: 10.1002/dvg.20688

' 2010 Wiley-Liss, Inc. genesis 49:2–9 (2011)

MyoD-dependent myogenic conversion of 10T1/2 fibro-blasts (Anderson et al., 2009). The zebrafish homolog ofthe Irxl1 gene is also expressed in the mesenchymal

cells in the embryonic head region and required for thebrain, pharyngeal arch development (Chuang et al.,2009). These evolutionarily conserved embryonic

FIG. 1. Generation of Irxl1-null mice. (a) Schematic representation of the Irxl1 genomic locus, gene targeting construct, and the targeted al-lele. The top line shows the wild-type 50 Irxl1 locus with exons 1–5 denoted as E1–E5. The middle line shows the Irxl1 targeting constructdesigned for the replacement of exon 3 with the neo cassette. The 50 and 30 homologous regions of the targeting construct were 1.9 and12.1 kb, respectively. On the bottom line, the Irxl1 mutant allele is illustrated. TK, thymidine kinase; Ba, BamHI; Bg, BglII; Xb, XbaI; closedboxes, untranslated sequences; open boxes, coding sequences; red boxes, homeodomain-coding sequences. Only relevant restrictionenzyme sites are shown. The probe used for the Southern blot analysis and the primers used for PCR are indicated by the bar and arrow-heads, respectively. (b) The upper panel shows the Southern blot analysis of tail DNA from mice that were WT (1/1), heterozygous (1/2),and homozygous (2/2) for the Irxl1 null allele. The probe shown in (a) detected a 10.9-kb BamHI fragment in the wild-type allele and a 3.4-kb BamHI fragment in the targeted allele. The lower panel shows PCR analysis with primers 1, 2, and 3 under the conditions described inthe Methods section. The size of the PCR product for the wild-type allele is 333 bp and that for the targeted allele is 171 bp. (c) RT-PCR anal-ysis of Irxl1 gene expression. Expression of Irxl1 was observed in the wild-type (1/1) and Irxl1 mutant heterozygous (1/2) embryos, but notfor the Exon3 primer set (upper panel) or significantly reduced for the Primer4/5 set (middle panel) in the homozygous (2/2) embryos.Exon3 primers lie inside the 3rd exon. EF-1a expression was used as an internal control. [Color figure can be viewed in the online issue,which is available at wileyonlinelibrary.com.]

3CHARACTERIZATION OF IRXL1-KNOCKOUT MICE

expression patterns and biochemical characterizationsuggest that Irxl1 plays a role in the mesoderm-derivedtissue development, particularly, in the musculoskeletalsystem.

To investigate the roles of Irxl1 in mammalian devel-opment, we generated mice carrying a null allele of theIrxl1 gene using homologous recombination in ES cells.A targeting vector was designed to replace the thirdexon of the Irxl1 gene, which encodes helix 1 and helix2 of the homeodomain with a neo cassette (Fig. 1a). Inaddition, the removal of the third exon will also resultin a frameshift and create a stop codon in exon 4 and istherefore highly likely to produce a functionally nullallele of the Irxl1 gene. We identified three independenthomologously recombined ES cell lines by Southernblot analysis (data not shown) and generated three chi-meric founder mice. The chimeric mice were matedwith C57BL/6 mice to produce heterozygous progeny,

and, subsequently, heterozygotes were interbred to pro-duce homozygous Irxl1-knockout mice (Fig. 1b). Theexpression of Irxl1 mRNA in the wild type (WT), and inheterozygous and homozygous mice for the targeted al-lele, was assessed by reverse transcription-polymerasechain reaction (RT-PCR). Amplifications with two differ-ent sets of primers (Exon3 primers and Primer4/Primer5) showed that the Irxl1 mRNA was absent fromhomozygous mutant mice (Fig. 1c), which means thatthe replacement of exon 3 with a Neo cassette resultedin the absence of normally processed mRNA in exons 4and 5.

We analyzed 332 F2 mice produced by the intercrossof heterozygous mice, of which 90 (27%) were WT, 154(46%) were heterozygous, and 88 (27%) were homozy-gous mutants. The distribution of these three genotypeswas not significantly different from the expected Men-delian ratios, and all the mice grew without lethality to

FIG. 2. Irxl1-knockout mice show reduced tendon formation. (a) A kinky tail with an incomplete penetrance in the Irxl1 null mice. An upperIrxl1 mutant homozygous mouse presents a kinky tail, and a lower sibling heterozygous mouse does not. (b–i) Tendons of Irxl1 null miceshow reduced differentiation compared with those of littermate wild type. Tendons of erector spinae muscles (b, c), forelimb (d, e), and dor-sal sacrococcygeal muscles (f–i) were hypoplastic. (h) and (i) are higher magnification of (f) and (g), respectively.

4 KIMURA ET AL.

at least 6 months of age, showing that the mutant micewere viable. To test whether the loss of Irxl1 functionaffects fertility, Irxl1 homozygous males and femaleswere intercrossed. Homozygous pups were born andnursed by their mothers, suggesting that the deletion ofIrxl1 has no significant effect on fertility. It has beenreported that Irxl1 is expressed in the endocardium ofthe heart, ureteric bud of the kidney, and testis cord ofthe male gonad during embryonic development(Anderson et al., 2006; Liu et al., 2006; Takeuchi andBruneau, 2007). Histological analysis of renal architec-ture with H&E-stained paraffin sections did not showany difference between mutant and control kidneys,hearts and testes of 3-month-old mice, and newbornpups (data not shown). Whole-mount in situ hybridiza-tion (WISH) analysis of Sox9 expression in the malegonads and testes in E13.5 embryos failed to reveal anydefect in the testis cord of the male gonad and the ure-teric buds in the kidney in Irxl1 mutant embryos (datanot shown).

Notably, roughly one third of Irxl1 homozygous mice(14 of 43) presented with a kinky tail (Fig. 2a). To iden-tify and explore the cause of this morphological defi-ciency, we further examined musculoskeletal apparatusin the Irxl1-knockout mice. Compared to littermate, allthe Irxl1-knockout mice examined (n 5 6) showedreduced tendon formation throughout the body includ-ing that in the head (data not shown), trunk (Fig. 2b,c),forelimb (Fig. 2d,e), hindlimb (data not shown), and tail(Fig. 2f–i).

To examine skeletal development in the Irxl1-defi-cient mice, we performed alcian blue and alizarin redstaining of newborn mice. The staining of bone and car-tilage was similar for Irxl1 mutant homozygotes (n 5 6)and sibling heterozygotes or WT (Fig. 3a). Abnormalitiesin the axial skeleton (Fig. 3b,c,f,g) or the limb skeleton(Fig. 3d,e) were carefully examined, but we did not findany differences. Next, we investigated the skeletal mus-cle to determine whether the lack of Irxl1 resulted inany changes in skeletal muscle formation. The weightsof the gastrocnemius and the greater pectral muscleswere similar in the WT and Irxl1 KO mice (Table 1).These results indicate that Irxl1 is required for theproper tendon formation but not for the broad pattern-ing of the skeletal and muscular development.

We next examined the effect of the Irxl1 deficiencyon musculoskeletal system development during theembryogenesis. Because Irxl1 is expressed in somite asearly as the beginning of the somite compartmentaliza-tion (Anderson et al., 2006; Liu et al., 2006; Takeuchiet al., 2007), we hypothesized that the Irxl1 mutant

FIG. 3. Skeletal morphology of wildtype (1/1), Irxl1 heterozygous (1/2), and homozygous (2/2) mutant mice were apparently indistin-guishable. (a) A lateral view of whole skeletons. (b, c) Enlarged dorsal views of Irxl1 mutant heterozygous (b) or homozygous (c) newborns.(d, e) Forelimbs isolated from the Irxl1 heterozygous (d) and homozygous (e) newborns. (f, g) Lateral views of isolated tails from the wild-type (f) and homozygous (g) newborns.

Table 1Muscle Weights of Wild Type and Littermate Irxl1

Mutant Homozygous Mice

Greater pectral (mg) Gastrocnemius (mg)

# WT (n5 11) 133.36 4.2 80.46 3.9KO (n 5 8) 132.96 4.6 83.36 3.2

$ WT (n5 10) 93.06 4.1 76.56 3.2KO (n5 12) 94.86 4.1 80.26 3.8

Results are presented as the mean6 standard error of the mean.

5CHARACTERIZATION OF IRXL1-KNOCKOUT MICE

mice would exhibit defects in the differentiation ofsomitic mesoderm into dermomyotome, sclerotome,and syndetome and compared the expression of eachsomitic compartment marker between WT and Irxl1-de-ficient embryos. Scleraxis is expressed in the tendonprogenitor, syndetome (Brent et al., 2003; Cserjesiet al., 1995); Myogenin and MyoD are expressed in themyotome (Sassoon et al., 1989; Wright et al., 1989);Sox9 is expressed in the sclerotome (Wright et al.,1995); and Pax3 is expressed in the dermomyotome(Goulding et al., 1994; Williams and Ordahl, 1994).WISH of E10.5 whole embryos and the tail region ofE13.5 embryos revealed that the expression of Scleraxis(Fig. 4a–d, n 5 5), Myogenin (Fig. 4e–h, n 5 6), MyoD

(data not shown, n 5 3), Sox9 (Fig. 4i–l, n 5 5), andPax3 (Fig. 4m–p, n 5 5) were unchanged in Irxl1

homozygous embryos, suggesting that the somitic mes-oderm was correctly differentiated into dermomyo-tome, myotome, sclerotome, and syndetome in the

Irxl1 null embryo. We examined neural tube and noto-chord formation both of which have influences on theproper differentiation of the somites. Morphology andexpression pattern of Shh in the neural tube and noto-chord were normal in the Irxl1-knockout embryos atE9.5, E10.5, and E11.5 (data not shown).

To further investigate whether the somitic compart-mentalization is correctly organized in the Irxl1-knock-out embryos, we demonstrated two-color in situ hybrid-ization. In Irxl1-knockout embryos, expression of Myo-

genin and Scleraxis in the rib cartilages and abdominalmuscle primorda at E11.5 (Fig. 5a,b, n 5 4) or in thetendon and muscle primordia in the tail at E13.5 (Fig.5c–f, n 5 3) did not show any difference compared tothose in WT littermates.

To examine the patterning and differentiation of thelimb bud mesenchyme where appandicular skeletonsand tendons are derived from and Irxl1 is expressed in,we assessed the gene expression in the forelimb of

FIG. 4. Whole-mount in situ hybridization analysis with the markers of somite differentiation. The trunk at E10.5 (a, b, e, f, i, j, m, and n)and tail at E13.5 (c, d, g, h, k, l, o, and p) were examined with whole-mount in situ hybridization. The expression patterns of markers for thetendon precursor, syndetome (Scleraxis, a–d), myotome (Myogenin, e–h), sclerotome (Sox9, i–l), and dermomyotome (Pax3, m–p) areunchanged in the mutant homozygous embryos. Scale bars: 500 lm.

6 KIMURA ET AL.

Irxl1 heterozygous and homozygous mutant embryos.At E13.5, the expression of the tendon lineage markerScleraxis (Fig. 6a,b, n 5 5) or condensing mesenchymalmarker Sox9 (Fig. 6c,d, n 5 5) was indistinguishablebetween the heterozygotes and homozygotes.

These results indicate that Irxl1plays an importantrole in tendon differentiation but is not essential for thepatterning of tendon, cartilage, and skeletal muscleprecursors during early development. This suggests thatIrxl1 is dispensable for the initial lineage specificationbut required for the later maturation of the tendon. As ithas been revealed that Irxl1 protein functions as a tran-scription repressor and inhibits the myoblast differentia-tion in the context of MyoD-induced myogenic conver-sion of fibroblasts (Anderson et al., 2009), it is possiblethat Irxl1 protein stabilizes the tendon differentiationby suppressing genes, which induce myogenic differen-tiation in the tendon progenitor cells. Because Irxl1

genes are widely conserved amongst the metazoanphyla, the role of Irxl1 gene that we present here in ten-don differentiation may also well be evolutionally con-

served. Our findings therefore can provide new insightinto what molecular processes underly the tendondifferentiation in mammals. Identification of Irxl1 targetgenes may be an intriguing challenge to reveal thetranscriptional network that regulates proper formationof musculoskeletal system development and/orregeneration.

During the revision of this work, similar results werereported independently by two groups (Ito et al., 2010;Liu et al., 2010). They did not report patterning of themusculoskeletal system during early embryonic devel-opment in detail; instead, they examined molecular andphysiological characteristics of Irxl1-knockout tendonand showed that Irxl1 is crucial for the proper tendonformation. Their results further support our conclusionthat Irxl1 is involved in the later tendon maturation.

METHODS

Generation of Irxl1 Null Mice

Genomic DNA containing the Irxl1 locus was isolatedfrom a Lambda FixII mouse 129/SvJ genomic library(Stratagene, CA). The BglII-XbaI fragment containingthe third exon of the Irxl1 gene was replaced by the ne-

omycin resistant gene flanked by a 50 BglII-BglII frag-ment and a 30 XbaI-XbaI partial digestion fragment. Theherpes simplex thymidine kinase gene was connectedat the 50 end of the targeting construct (Fig. 1a). The lin-earized targeting vector was electroporated into 129/B6embryonic stem cells to generate homologous recombi-nants. The targeted cell lines were enriched by G418and gancyclovir selection and identified by Southernblot analysis using the 0.9 kb fragment as a probe(Fig. 1a), hybridized with the BamHI-digested genomicDNA prepared from ES cells.

FIG. 5. Double in situ hybridizations with Myogenin in orange (INT/BCIP substrate, indicated by orange arrowhead) and Scleraxis (Scx)in dark-blue (NBT/BCIP substrate, indicated by dark-blue arrow-head) in the wild-type and Irxl1-null embryos. (a, b) Expression in adistal trunk of E11.5 embryos. (c–f) Expression in a tail of E13.5embryos. Lateral view (c, d) and dorsal view (e, f) are presented.

FIG. 6. Cartilage and tendon differentiations in the forelimb atE13.5. Dorsal views showing the expression of a tendon markerScleraxis (Scx) in the Irxl1 heterozygous (a) and homozygous (b)embryos, and a cartilage marker Sox9 in the Irxl1 mutant heterozy-gous (c) and homozygous (d) embryos. Scale bars: 500 lm.

7CHARACTERIZATION OF IRXL1-KNOCKOUT MICE

Genotyping of Progeny

The genotyping of progeny mice was determined bySouthern blotting and PCR (Fig. 1b) using a tail or yolksac DNA. The PCR reaction was performed under the fol-lowing condition: denaturation at 948C for 5 min fol-lowed by 30 cycles of denaturation at 948C for 30 s,annealing at 638C for 30 s, and extension at 728C for 30s. Primers were as follows: primer 1, 50-TTCACAGAACC-TAGGCAGCC-30; primer 2, 50-TGAATAGCCTGCTGGC-CACG-30; primer 3, 50-GCGGTGGGCTCTATGGCTTC-30

(Fig. 1a).

RT-PCR Analysis

Total RNA was extracted from the E13.5 wholeembryos of WT, heterozygous, and homozygousmutants using the RNeasy Protection Mini Kit (Quagen,Hilden, Germany) according to the manufacturer’s rec-ommendation. Reverse transcription was performedwith PrimeScript 1st strand cDNA synthesis kit (TaKaRa,Kyoto, Japan) with an oligo d(T) primer according tothe specifications of the manufacturer. PCR was per-formed with ExTaq DNA polymerase (TaKaRa) with thefollowing primers: exon 3: forward, 50-CGGCAGAATGGAGGGAAGGT-30, reverse, 50-TGCAC-TAGCGTCATCTGCGA-30; primer 4: 50-AGTGTGAGCAGC-GATGGCGA-30; primer 5: 50-CCGATGTCCGGGGTGTCTGT-30; EF-1a: forward, 50-AACATGCTGGAGCCAAGTGC-30, reverse, 50-TATAGACATCCTGGAGGGGC30.

Skeletal Preparation, Histological Examination,and Statistical Analysis

Skeletal preparation. Alcian blue and alizarin redstaining was performed as previously described (Kesseland Gruss, 1991).

Muscle weight. Both left and right gastrocnemiusand greater pectral muscles were carefully dissectedfrom 3-month-old WT mice and homozygous mutant lit-termates. Dissected muscles were washed in PBS,trimmed of fat, and weighed.

Whole-Mount In Situ Hybridization

Whole-mount in situ hybridization (WISH) was per-formed as described in a manual (Nagy et al., 2003).Two-color in situ hybridization was performed asdescribed in the previous report (Takahashi and Osumi,2008). Templates for WISH probes were cloned by RT-PCR with cDNA synthesized using total RNA extractedfrom E9.5 whole mouse embryos. Primer sequences forRT-PCR are as follows: Irxl1: forward, 50-CAGGACA-GAAGCCAATCGCC-30, reverse, 50-GCTGCACGAGAAGAACCCAA-30. Sox9: forward, 50-TCCTCCGGCATGAGTGAGGT-30, reverse, 50-CCAGGAGTGCTGTCCGTCTG-30. Scler-axis: forward, 50-CTGGCGGCCCCACTCCAGTC-30,reverse, 50-CCCAGTGGGCCTGGGTCAGT-30. Myogenin:

forward, 50- AGTGGCAGGAACAAGCCTTT-30, reverse, 50-CAGGTCAGGG CACTCATGTC-30. Pax3: forward, 50-CGCTGGAAGTGTCCAC CCCT-30, reverse, 50-CTGGCTGA-CACCGTGGTCGT-30. MyoD: forward, 50-TGAAACCGGAG-GAGCACGCA-30, reverse, 50-GTG CCAGGAGGGCTCCAGAA-30.

WISH probes were synthesized with DIG-RNA label-ing mix (Roche) or fluorescein-RNA labeling mix(Roche) following the manufacturer’s instructions.

ACKNOWLEDGMENTS

We thank Professor Hideaki Hayashi for initial supportand Masumi Tsujimura for secretarial assistance. Wealso thank Drs. Victoria A. Barron and Melissa N. Hin-man, Department of Genetics, School of Medicine, CaseWestern Reserve University, for critical reading of themanuscript.

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9CHARACTERIZATION OF IRXL1-KNOCKOUT MICE