PATTERNS & PHENOTYPES

Mohawk Is a Novel Homeobox Gene Expressedin the Developing Mouse EmbryoDouglas M. Anderson,1,2 Jennifer Arredondo,1,3 Katherine Hahn,1,2 Graziela Valente,4

James F. Martin,5 Jeanne Wilson-Rawls,1 and Alan Rawls1,4*

Homeodomain-containing proteins comprise a superfamily of transcription factors that participate in theregulation of almost all aspects of embryonic development. Here, we describe the mouse embryonicexpression pattern of Mohawk, a new member of the TALE superclass of atypical homeobox genes that ismost-closely related to the Iroquois class. During mouse development, Mohawk was transcribed in celllineages derived from the somites. As early as embryonic day 9.0, Mohawk was expressed in an anterior toposterior gradient in the dorsomedial and ventrolateral lips of the dermomyotome of the somites thatnormally give rise to skeletal muscle. Mohawk transcription in the dorsomedial region required theexpression of the transcription factor paraxis. As somites matured, Mohawk transcription was observed inthe tendon-specific syndetome and the sclerotome-derived condensing mesenchyme that prefigures theproximal ribs and vertebral bodies. In the limbs, Mohawk was expressed in a pattern consistent with thedeveloping tendons that form along the dorsal and ventral aspect of the phalanges. Finally, Mohawk wasdetectable in the tips of the ureteric buds in the metanephric kidneys and the testis cords of the male gonad.Together, these observations suggest that Mohawk is an important regulator of vertebrate development.Developmental Dynamics 235:792–801, 2006. © 2006 Wiley-Liss, Inc.

Key words: Iroquois; Mohawk; Homeobox; Somite; gonad; kidney; tendon; myogenesis; chondrogenesis

Accepted 22 November 2005

INTRODUCTION

Homeobox genes are a superfamily oftranscription factors that regulate thespatial organization of the embryonicbody plan, cellular identity, prolifera-tion, and differentiation during orga-nogenesis (Carroll et al., 2001). Typi-cal homeobox genes contain a con-served 180 base pair sequence thatencodes a 60 amino acid DNA bindingdomain, called the homeodomain. X-ray crystallography and NMR studieshave shown that the homeodomain

forms three alpha-helices, with thefirst two helices in parallel and thethird at an angle that allows it to fitinto the major groove of DNA. Fiveamino acids, tryptophan 48 (W48),phenylalanine 49 (F49), asparagine 51(N51), arginine 53 (R53), and lysine/arginine 55 (K/R55) found in helix III,are conserved among all known ho-meobox genes and are predicted to beimportant in DNA binding (Burglin,1994). Greater than 200 homeodo-main-containing genes have been

identified in the mammalian genomeand are divided into seven subgroups:Hox, extended Hox, Paired, NK, LIM,Pou, and atypical, based on sequencesimilarity within the homeodomain(Burglin, 1994; Banerjee-Basu andBaxevanis, 2001; Tupler et al., 2001).Identifying new homeobox genes andtheir function is essential to unravel-ing the regulation of basic develop-mental processes.

The Iroquois class (IRO) of genesbelong to the three–amino-acid loop

The Supplementary Material referred to in this article can be found at http://interscience.wiley.com/jpages/1058-8388/suppmat1School of Life Sciences, Arizona State University, Tempe, Arizona2Molecular and Cellular Biology Graduate Program, Arizona State University, Tempe, Arizona3MARC program, Arizona State University, Tempe, Arizona4Center for Evolutionary Functional Genomics, Arizona Biodesign Institute, Tempe, Arizona5Alkek Institute for BioTechnology, Texas A&M University, College Station, Texas*Correspondence to: Alan Rawls, School of Life Sciences, Life Sciences C Bldg, Room 544, Arizona State University, Tempe,AZ, 85287-4501. E-mail: jrawls@imap4.asu.edu

DOI 10.1002/dvdy.20671Published online 11 January 2006 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 235:792–801, 2006

© 2006 Wiley-Liss, Inc.

extension (TALE) superclass of atypi-cal homeobox genes, characterized bythree additional amino acids in theloop region between helix I and helixII of the homeodomain. The IRO geneswere first identified in Drosophila as acomplex of three clustered and highlyrelated genes, araucan, caupolican,and mirror, that control the transcrip-tion of the proneural genes, specifywing identity, and act as dorsal selec-tor genes in the eye (Leyns et al.,1996; Gomez-Skarmeta et al., 1996;Grillenzoni et al., 1998; Kehl et al.,1998; Cavodeassi et al., 1999; Diez del

Fig. 1.

Fig. 2.

Fig. 1. Evolutionary classification of mouse Mo-hawk (Mkx) as a new three–amino-acid loopextension (TALE) atypical homeobox gene. A:Comparison of the 63 amino acids of the ho-meodomains of mouse Mkx and other mouseTALE genes: Irx1, 2, 4, Pbx1, Meis1, and Tgif.Residues that are conserved among all home-odomains are highlighted in blue. The predictedorganization of the homeodomain into three al-pha-helices is displayed above the sequence. A“.” marks identical residues compared withMkx. B: A neighbor-joining tree of homeodo-mains of Mkx and representative genes of theanimal TALE classes (Irx1–6, Pbx1, Meis1, andTgif) constructed using the evolutionary dis-tances estimated under the Jones-Taylor-Thornton’s (JTT) model in MEGA3 (Kumar et al.,2003). C: Schematic of the genomic organiza-tion of Mkx on mouse chromosome 18. Exonsand introns are represented by boxes and lines,respectively. The coding regions are denotedby dark green shading and the 5� and 3� non-coding regions are denoted by light greenshading. D: Schematic of the predicted 353amino acid Mkx protein with the positions of theN terminal homeodomain (HD) and a putativenuclear localization signal displayed above(NLS). Roman numerals mark the predictedcontribution of individual exons to the protein.

Fig. 2. Examination of Mkx transcript expres-sion during mouse development by whole-mount in situ hybridization. A: In embryonic day(E) 9.0 embryos, Mkx mRNA was detected inthe dorsomedial lip (DML) of the dermomyo-tome in the anterior somites. B: In E10.5 em-bryos, Mkx mRNA was present in the DML ofhindlimb-level somites as well as the ventrolat-eral lip (VLL) of the interlimb somites. MkxmRNA was also detected in the frontonasalmass (fnm), forelimb (fl) and hindlimb (hl).C,D: At E11.5, Mkx expression is maintained inthe dorsal and ventral most aspects of the bodysomites and in the DML and VLL of tail somites.Mkx transcription was expressed throughoutthe dermomyotome of somites 0, �1, and �2 atE11.5. E: A transverse cryosection through theinterlimb somites of an E10.5 embryo showedrestriction to the epithelial DML and VLL of thedermomyotome. lpm, lateral plate mesoderm;mg, midgut; hl, hindlimb; nt, neural tube. Scalebars � 0.5 mm.

MKX HOMEOBOX GENE DEVELOPMENTAL EXPRESSION 793

Corral et al., 1999). Murine orthologs,Iroquois 1–6 (Irx1–6), are expressedbroadly during development, includ-ing the central nervous system, ven-tricles of the heart, somites, lungs, go-nads, and cartilage (Bosse et al., 1997;Bellefroid et al., 1998; Bao et al., 1999;Cohen et al., 2000; Houweling et al.,2001). There is evidence that they pro-mote proneural gene transcription,patterning of the neural tube and ven-tricular-specific transcription in theheart (Bao et al., 1999; Gomez-Skarmeta and Modolell, 2002). Aunique feature of the Irx homeodo-main is an alanine at position 50(Ala50) of the recognition helix (helixIII), which is believed to participate indetermining DNA binding specificity(Hanes and Brent, 1989; Stepchenkoet al., 1997; Duan and Nilsson, 2002).All Irx proteins also possess a highlyconserved acidic motif (IRO Box) intheir C-terminal region (Burglin,1997; McNeill et al., 1997).

In these studies, we identify Mo-hawk (Mkx) as novel homeobox genethat is most-closely related to the Irxgenes. Divergence within the home-odomain and the absence of an IROBox suggest that Mkx should beconsidered a new atypical TALEhomeobox class. During embryonicmouse development, Mkx is tran-scribed in the discrete premyogeniccell populations of the somite, the con-densing prechondrogenic mesenchy-mal cells of the axial skeleton, the pre-tendenous cells of the tail and limbs,the testis cords of the developing malegonad, and the metanephric kidney.On the basis of the specificity in thetissues in which Mkx is expressed andthe high degree of overlap in expres-sion with transcription factorsparaxis, Pax3, Sox9, and scleraxis,Mkx is predicted to play an importantregulatory role during development.

RESULTS AND DISCUSSION

Identification andClassification of the MohawkHomeobox Gene

To identify related members of the Irxgenes, the amino acid sequence of theIrx2 homeodomain was used to searchthe mouse genome in the GenBankDatabase using tblastn (Gish andStates, 1993). The outcome of this

search included a previously unde-scribed mouse gene (GenBank mRNAaccession no. NM_177595 and proteinaccession no. NP_808263) that wehave subsequently named Mohawk(Mkx). The nucleotide sequence ofMkx was confirmed by sequencingcDNA generated from embryonic day(E) 10.5 mouse embryos. A tblastnsearch of the mouse genome using thefull-length Mkx protein as the queryshowed that members of the IRO classwere the most-closely related genes.Alignment of the predicted amino acidsequence of the Mkx homeodomainwith the homeodomains of Irx1, 2, and4, and representative murine mem-bers of the TALE class PBC (Pbx1;Nourse et al., 1990), MEIS (Meis1;Moskow et al., 1995), and TGIF (Tgif;Bertolino et al., 1995) revealed thatthe greatest sequence homology laywithin helix III of the Irx genes (82%identity, 14/17 residues), includingthe Ala50 (Fig. 1A). Over the entireMkx homeodomain, however, thehighest sequence homology was only56% (35 identical residues with Irx2).Furthermore, Mkx shared less se-quence similarity outside the home-odomain with the Irx genes, and didnot contain the IRO Box found in allmembers of the Irx genes (data notshown).

The PBC class of genes has beenshown previously to be the next clos-est relatives to the IRO genes (Bur-glin, 1997). A phylogenetic analysis ofthe homeodomain amino acid se-quences revealed a closer evolutionaryrelationship of the Mkx homeodomainwith the Irx homeodomains (evolu-tionary distance less than one substi-tution per site) as compared withPbx1, Meis1, and Tgif, all of whichshow higher sequence similarity witheach other than with the Irx and Mkxhomeodomains (Fig. 1A,B). In anycase, the Mkx homeodomain is dis-tantly related to the Irx homeodo-mains, because it shows an evolution-ary distance �0.7 substitutions persite when compared with Irx home-odomains. Therefore, it may be consid-ered to be in its own TALE class,which we refer to as the MKX class.

The genomic organization of Mkx inthe mouse genome was examined us-ing the University of Santa Clara Ge-nome Browser (Karolchik et al., 2003).The gene is located at qA1 on chromo-

some 18, which is distinct from thetwo Irx gene clusters, IrxA and IrxB,found on chromosomes 13 and 8, re-spectively (Houweling et al., 2001).Mkx consists of seven exons that span69,755 bases and code for a 353 aminoacid protein (Fig. 1C). The homeodo-main is encoded by exon III and IV(Fig. 1D). Exon II and III encode aputative bipartite nuclear localizationsignal.

Mkx Orthologs

A tblastn search of the Ensembl andGenBank databases, using the mouseMkx homeodomain sequence, re-vealed predicted orthologs in both ver-tebrate and invertebrate species. Thehomeodomain was completely con-served at the amino acid level acrossall the vertebrate orthologs examined(Table 1; for protein sequence align-ment, see Supplementary Material,which can be viewed at http://www.interscience.wiley.com/jpages/1058-8388/suppmat). Among the whole pro-tein, the degree of sequence conserva-tion varied from 87% identity in Rat-tus norvegicus to 47% identity inDanio rerio. Regression of sequenceidentity with time of species diver-gence (between mouse and other spe-cies listed in Table 1; see Hedges andKumar, 2003) indicates that sequenceidentity has decayed at a rate of fiveamino acids per 100 million years invertebrates, when the whole protein isconsidered. Among invertebrates, wefound proteins with weak sequencehomology (46% identity) in the home-odomain in the Anopheles gambiaeand Drosophila melanogaster ge-nomes (Table 1). This finding is alower percent similarity than is nor-mally seen between invertebrate andvertebrate orthologs of homeoboxgenes (Burglin, 1994). However, whenusing the invertebrate homeodomainsequences as the queries in a tblastnsearch of the mouse genome, Mkx wasfound to be the most-related murineprotein.

Dynamic Mkx Expression inthe Somites

Whole-mount in situ hybridization(WISH) analysis with an Mkx-specificdigoxigenin-labeled antisense RNAprobe was used to examine Mkx tran-

794 ANDERSON ET AL.

scription in developing mouse em-bryos between E9.0 to E11.5. A domi-nant feature of Mkx transcription wasa dynamic pattern in the somites ofembryos beginning at E9.0 (Fig. 2).Mkx was transcribed in the dorsal re-gion of the dermomyotome of themost-anterior somites of E9.0 em-bryos (Fig. 2A). This transcription ex-tended to the tail somites by E10.5(Fig. 2B). A second ventral domain ofsomite expression was noted in E10.5embryos, with the strongest stainingin the interlimb region (Fig. 2B). InE11.5 embryos, Mkx transcriptionpersisted in these discrete populationsin the dorsal and ventral aspects ofthe dermomyotome of body and tailsomites (Fig. 2C,D). Transverse sec-tions through the interlimb region ofan E10.5 embryo revealed that ex-pression was limited to the epithelialdorsomedial lip (DML) and ventrolat-eral lip (VLL) of the dermomyotome(Fig. 2E).

The DML and VLL of the dermo-myotome in the maturing somiteshave been characterized as the sites ofself-renewing premyogenic cell popu-lations that act as a source of cells forthe expanding myotome (Christ andOrdahl, 1995; Cossu et al., 1996;Denetclaw et al., 1997; Venters andOrdahl, 2002). Cells at the VLL arealso the source of a highly migratorycell population that establishes theappendicular, hypoglossal, and dia-phragm muscles (Ordahl and Le

Douarin, 1992; Denetclaw and Or-dahl, 2001). To examine the associa-tion of the Mkx transcription domainto myoblasts and myocytes of the myo-tome, WISH was performed on mouseembryos carrying the muscle-spe-cific �1565Myogenin/lacZ transgene(Cheng et al., 1992). In the thoracicregion of E10.5 embryos, Mkx tran-scription in the DML was closely as-sociated with the dorsal aspect of themyotome (Fig. 3A). Transverse sec-tions revealed that Mkx-expressingcells in the epithelial DML are adja-cent to the myotome but are not foundwithin the myotome (Fig. 3B). It isinteresting to note that the BerkeleyDrosophila Genome Project (BDGP)found that the Drosophila melano-gaster ortholog of Mkx (Table 1) is ex-pressed in the embryonic/larval mus-cle system (Tomancak et al., 2002).This finding suggests a conserved rolefor Mkx in regulating myogenesis invertebrates and invertebrates.

Little is known about the signalsinvolved in specifying cells to the pre-myogenic fate or regulating the mor-phological events associated with theepithelial to mesenchymal transitionand migration into the myotome. Thetranscription factors Pax3 and paraxishave been demonstrated to regulatethe specification of premyogenic cells,proliferation, migration into the limbsand the regulation of their epithelialstate (Franz et al., 1993; Goulding etal., 1994; Williams and Ordahl, 1994;

Daston et al., 1996; Burgess et al.,1996; Tajbakhsh et al., 1997; Wilson-Rawls et al., 1999; Schubert et al.,2001; Wiggan et al., 2002). A compar-ison of Mkx transcription with paraxisand Pax3 revealed an overlapping ex-pression pattern in the DML of thesomites of E10.5 embryos, while theVLL expressed only Mkx and Pax3(Fig. 3C,D). In E10.5 embryos defi-cient for paraxis, Mkx transcriptionwas absent in the DML but was un-changed in the VLL and nonsomiticregions such as the forelimb bud (Fig.3E,F). We have shown previously thatparaxis differentially regulates pre-myogenic populations in the DML andVLL of the dermomyotome (Wilson-Rawls et al., 1999). Pax3 transcriptionin the DML was dependent onparaxis, whereas transcription in thesubpopulation of cells in the VLL thatmigrate to give rise to the hypaxialmuscles in the limbs, tongue, dia-phragm, and ventral midline, wereunaffected. Because the pattern ofMkx in the paraxis mutant was simi-lar to that of Pax3, this draws someintriguing parallels between the twogenes in their regulation by paraxis.

The maturation of the tail somitesin E12.5 embryos is associated withthe remodeling of the myotome from acontinuous sheet of myocytes into dis-tinct dorsal and ventral musclemasses. These muscle groups becomethe short intrinsic and extrinsic bicip-ital muscles that span adjacent verte-

TABLE 1. Predicted Protein Sequence Identity Comparing Mouse Mkx to its Orthologs

Species Protein Accession No. Homeodomaina Whole Proteinb

VertebratesRattus norvegicus ENSRNOP00000025623 63/63 (100%) 314/357 (87%)Pan troglodytes ENSPTRP00000004041 63/63 (100%) 290/353 (82%)Homo sapiens AAH36207 63/63 (100%) 288/353 (81%)Xenopus tropicalis ENSXETP00000009890 63/63 (100%) 247/354 (69%)Fugu rubripes SINFRUP00000151281 63/63 (100%) 216/411 (52%)Danio rerio XP_683366 63/63 (100%) 172/287 (59%)InvertebratesAnopheles gambiae ENSANGP00000012403 29/63 (46%) 38/84 (45%)Drosophila

melanogasterCG11617-PA 29/63 (46%) 52/171 (30%)

a A pairwise alignment between the predicted amino acid sequence of the homeodomain from the mouse Mkx and each ortholog,using ClustalW v1.82 (Chenna et al., 2003). Identity is expressed as the number of identical amino acids over the total number ofamino acids in the homeodomain.

b A pairwise alignment between the whole predicted amino acid sequences of mouse Mkx and each ortholog, using bl2seq usingblastp (Tatusova and Madden, 1999). Identity is expressed as the number of identical amino acids over the total number of aminoacids in high-scoring segment pairs.

MKX HOMEOBOX GENE DEVELOPMENTAL EXPRESSION 795

brae of the tail (Shinohara, 1999). Be-fore the remodeling of the myotome(E11.5), Mkx and Pax3 were tran-scribed in the DML and VLL in thedermomyotome (Fig. 3). In E12.5 tails,Mkx expression shifted from the dor-sal and ventral aspects of the somiteto two distinct domains along the pos-terior edge of the somite (Fig. 4A).This shift occurred over six somites,with a somite in the middle expressingequally in both domains. The new do-main of expression overlapped withthe transcription of the tendon-spe-cific scleraxis gene that defines thesyndetome compartment of the somite(Cserjesi et al., 1995; Schweitzer etal., 2001) and not with the dermomyo-tome-specific gene Pax3 (Fig. 4B,C).The Mkx transcription domain be-came long and thin and crossed seg-mental boundaries in E13.5 embryos

Fig. 3.

Fig. 4.

Fig. 3. Mkx is expressed in the premyogeniccells of the somite and is downstream ofparaxis. A: Embryonic day (E) 10.5 embryoswere doubly stained for the expression of the�1565Myogenin/lacZ transgene and Mkx tran-scription. B: A transverse cryosection throughthe interlimb region of the embryo shows theposition of Mkx in the lip region of the dermo-myotome adjacent to the developing myotome.C–F: Whole-mount in situ hybridization (WISH)of E10.5 embryos with probes specific for Pax3(C) and paraxis (D). Mkx was transcribed nor-mally in E10.5 paraxis�/� embryos (E) but ab-sent in the dml of paraxis�/� embryos (F). Scalebars � 0.5 mm.

Fig. 4. A–F: Transcription shifts to the syn-detome in the mature tail somites. Whole-mount in situ hybridization (WISH) was per-formed on the tail region of embryonic day (E)12.5 and E13.5 embryos using probes specificfor (A,D) Mkx, or (B,E) Scx, or (C,F) Pax3. A: AtE12.5, the domain of Mkx transcription in thecaudal somites shifted from the entire dml (yel-low arrowhead) to the syndetome (blue arrow-head). In the intervening somites, Mkx tran-scription was present in both the dml and thesyndetome (green arrowhead). B: Scx was tran-scribed in the syndetome, characterized as dor-sal and ventral domains along the posterior as-pect of each somite in the tail. C: Pax3transcription was maintained throughout thedermomyotome at this stage. D,E: At E13.5, thedorsal and ventral (D) Mkx and (E) Scx transcrip-tion domains elongated and moved anteriorly ina similar manner. F: The tail of a E13.5 embryowas co-stained for expression of the�1565Myogenin/lacZ transgene and Mkx tran-scripts. The Mkx transcription domain remainedclosely associated with myotomal cells as theyremodeled into the bicipital tail muscles. nt,neural tube; dmm, dorsal muscle mass; vmm,ventral muscle mass. Scale bars � 0.3 mm.

796 ANDERSON ET AL.

(Fig. 4D). The same morphologicalchanges were observed in the scleraxistranscription domain, suggesting thatthese cells are forming the tendonsthat connect to the bicipital tail mus-cles (Fig. 4E). This suggestion wassupported by the alternating patternof �1565Myogenin/lacZ expression inthe myotome and Mkx transcription(Fig. 4F).

The expression of Mkx in the syn-detome during somite maturation pre-dicts a second role for the gene in reg-ulating the tendons that formjunctions with the fetal muscles. Ithas been demonstrated previouslythat FGFs secreted from the myotomeare essential to stimulate scleraxistranscription in the syndetome (Brentet al., 2003). The expression of Mkx inboth the premyogenic populations ofthe dermomytome and syndetomeraises the possibility of additional reg-ulatory links between the myogenicand tendon lineages.

Mkx transcription was examined inthe body wall of E11.5 embryos thathad been bisected midsagittally andeviscerated. Under these conditions,Mkx mRNA was detected in the verte-bral bodies and notochord, ventral tothe neural tube (Fig. 5A). Stripes oftranscripts were observed in the con-densing mesenchyme of the proximalribs that form from the posterior scle-rotome (Fig. 5A). This was confirmedby examining the expression of Sox9,a prechondrogenic mesenchymalmarker at the same developmentalstage, which is transcribed in all ofthese tissues (Wright et al., 1995; Fig.5B). Furthermore, coronal sectionsthrough an embryo doubly stained for�1565Myogenin/lacZ expression andMkx showed that Mkx was expressedthroughout the discrete condensingmesenchyme of the proximal ribs andnot the muscle or tendons (Fig. 5C).Mkx transcripts were also found in thefrontonasal mass beginning at E10.5(Fig. 2B). Cells in this region give riseto skeletal elements of the face, in-cluding the forehead, nasal cartilage,and philtrum (Richman and Tickle,1989). Thus, Mkx is expressed in thecondensing prechondrogenic mesen-chyme of the axial skeleton, predictinga role for this gene in regulating theearly events in chondrogenesis.

Expression of Mkx in theLimb Bud

Based on the expression of Mkx in thetendons and prechondrogenic cells de-rived from the somites, transcriptionwas examined in the limbs. At E12.5,Mkx was strongly expressed in the au-topod in a pattern that overlaps withthe forming phalanges and metacar-pals, in a pattern similar to scleraxisand Sox9 (Fig. 6A–C). Transverse sec-tions revealed that Mkx transcriptionwas superficial to the condensing mes-enchyme, marked by Sox9 transcrip-tion, which was different than whatwas observed in the developing axialskeleton (Fig. 6D,F). The pattern ofMkx transcription was more similar toscleraxis, suggesting that Mkx-posi-tive cells in the limbs at this stagemay be in the tendon lineage (Fig. 6E).However, the broader expression pat-tern of Mkx predicts that the genemay be involved in other developmen-tal processes in this region, includingchondrogenesis or myogenesis.

Mkx Transcription in theGonads and Kidney

The gonads are derived from bipoten-tial cells that appear along the ventro-medial surface of the urogenital ridgearound E10.5 in the mouse. Sexualdetermination occurs through themale-specific transcription of Sry (sex-determining region, Y chromosome)and Sox9 in the gonadal ridge, whichleads to differentiation of Sertoli cellsand the production of anti-Mullerianhormone (Gubbay et al., 1990; Sinclairet al., 1990; Kent et al., 1996; Moraisde Silva et al., 1996). We examinedthe transcription of Mkx in male andfemale indifferent urogenital ridgesisolated from E11.5 embryos usingWISH (Fig. 7A–D). Mkx transcriptionwas present throughout the male go-nadal ridge and absent in the femalegonadal ridge (Fig. 7A,C), this expres-sion pattern overlapped that of Sox9(Fig. 7B,D). In E13.5 gonads, Mkxtranscription was restricted to the tes-tis cords of the male gonad, similar tothat of Sox9 (Fig. 7E,F). The testiscords are formed from the aggregationof cells that will differentiate into Ser-toli cells and are the site of the primor-dial germ cells. The expression of Mkxin these cells and its absence in the

female gonad suggest that the genemay play a role in regulating Sertolicell differentiation and/or sex determi-nation.

The metanephric kidney developsfrom the ureteric bud of the meso-nephric duct and the adjacent meta-nephric mesenchyme. The uretericbud invades the mesenchyme and un-dergoes repeated rounds of branchingin response to mesenchymal signals.Mesenchyme at the tip of the uretericbud condenses to give rise to nephronsthat will drain into the collectingducts generated through branching.At E11.5, Mkx was expressed diffuselyin the newly forming kidney of bothmales and females (Fig. 7A,C). Ex-pression became restricted to the tipsof the ureteric buds by E13.5 (Fig. 7G).Similar to the gonads, the expressionpattern overlapped with Sox9, whichhas been reported to be expressed inthe epithelium at the distal tip of theureteric buds (Pepicelli et al., 1997;Fig. 7H).

In summary, Mohawk defines a newclass of TALE atypical homeoboxgenes that is highly conserved amongvertebrates. Analysis of the Mkx ex-pression in the mouse embryo re-vealed transcription in developmen-tally important regions that give riseto skeletal muscle, tendons, cartilage,male gonads, and the ureteric buds ofthe kidney. In each of these cell types,the expression of Mkx preceded differ-entiation, suggesting that Mkx partic-ipates in the early events that lead todifferentiation. Changes in cell mor-phology associated with an increase incell–cell contact are common in thesetissues. Cell aggregation occurs dur-ing the condensation of prechondro-genic mesenchyme, formation of themale sex cords, and the tendons(Wezeman, 1998; Delise et al., 2002;Moreno-Mendoza et al., 2003). In thedermomyotome and ureteric buds,Mkx-positive cells are maintained inan epithelial state associated withgrowth and differentiation of the lipsof the dermomyotome and branchingof the ureter (Christ and Ordahl,1995; Davies et al., 1999). This findingraises the possibility that Mkx mayact as a morphogenic regulator of celladhesion. This possibility is supportedby the observation that Mkx lay down-stream of paraxis, a regulator of themesenchyme-to-epithelia transition of

MKX HOMEOBOX GENE DEVELOPMENTAL EXPRESSION 797

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 5. Mkx was transcribed in the condensingmesenchyme that prefigures the axial skeleton.A,B: Whole-mount in situ hybridization (WISH)was performed on embryonic day (E) 11.5mouse embryos after a midsagittal cut, usingantisense RNA probes specific to either Mkx (A)or Sox9 (B). Both were expressed in the devel-oping proximal ribs (pr), the vertebral body (vb)and the notochord (n). C: A frontal sectionthrough an embryo co-stained for Mkx and the�1565Myogenin/lacZ transgene showed thatMkx is expressed throughout the condensedmesenchyme positioned between the hypaxialmyotome. nt, neural tube. Scale bars � 0.5 mm.

Fig. 6. Mkx transcription in the limbs. A–F:Whole-mount in situ hybridization (WISH) wasperformed on embryonic day (E) 12.5 limbs withprobes specific for Mkx (A,D), Scx (B,E), andSox9 (C,F). Mkx was transcribed in the region ofthe phalanges, similar to Scx and Sox9. Trans-verse sections through the limbs showed thatMkx (D) was excluded from the cartilaginousdigits that expressed Sox9 (E). F: Mkx over-lapped with Scx, which marks cells dorsal andventral to the phalanges that are committed tothe tendon lineage. Scale bars � 0.5 mm.

Fig. 7. Transcription of Mkx in the urogenitalridge. A–F: Whole-mount in situ hybridization(WISH) was performed on embryonic day (E)11.5 indifferent gonads (A–D) and E13.5 gonads(E,F), using antisense RNA probes specific toeither Mkx (A,C,E) or Sox9 (B,D,F). Mkx wasselectively transcribed in the male gonadalridge (gnr) of E11.5 embryos and the testiscords (tc) of E13.5 embryos. Mkx was tran-scribed in the metanephric kidney (K) of bothsexes. G: Mkx was expressed in discrete re-gions of the metanephric kidney at E13.5. Thispattern was similar to (H) Sox9, which has beenreported to be expressed in the epithelial cellsat the tip of the ureteric buds (ub tips) in thekidney. m, mesonephros. Scale bars � 0.1 mm

the somite. We also note a strikingspatial and temporal overlap in tran-scription between Mkx and Sox9 inprechondrogenic cells, the testis cords,and the ureteric buds of the kidneys.Sox9 is required for differentiation ofchondrocytes and is a critical regula-tor of sexual differentiation of themale gonad (Sinclair et al., 1990; Kentet al., 1996; Morais de Silva et al.,1996; de Crombrugghe et al., 2000;Akiyama et al., 2002). Similarly, Mkxtranscription overlaps with Scleraxis,which is expressed in the tendon pre-cursors, and Sertoli cells (Brown et al.,1999; Muir et al., 2005). Based onthese observations, Mkx is predictedto participate in these regulatorypathways. However, further mecha-nistic studies will be needed to under-stand the potential contribution ofMkx to the existing regulatory path-ways in these cells.

EXPERIMENTALPROCEDURES

Intercrosses and Genotyping

Mice carrying the �1565Myogenin/lacZtransgene (Cheng et al., 1992) weremaintained as a homozygous colony.Paraxis null mice were described previ-ously (Burgess et al., 1996) and weremaintained as a heterozygous breedingstock for the null allele (paraxis�/�).Paraxis�/� and paraxis�/� embryoswere generated from heterozygouscrosses. The genotype was determinedby a polymerase chain reaction (PCR)strategy using primers specific to the 3�recombination arm (upstream primer,5�-ACCACCAAGCGAAAACATC-3�;downstream primer, 5�-CAAGAGGAA-GGAACCAGA-3�). To determine thesex of the embryos from which E11.5urogenital ridges were isolated,genomic DNA isolated from the em-bryo’s head was used to screen for thepresence (male) or absence (female) ofthe Y chromosome by PCR amplifica-tion of the Sry gene locus (Hogan et al.,1994).

WISH

WISH on tissues were performed inthe automated InsituPro (Intavis,LLC, San Marcos, CA), as describedpreviously (Belo et al., 1997). In exper-iments where tissues were examined

for both Mkx transcription and �-galactosidase expression from the�1565Myogenin/lacZ transgene, the�-galactosidase staining was per-formed first. Tissues were dissected inDPBS and fixed in 0.8% paraformal-dehyde, 0.2% glutaraldehyde in DPBSat 4°C. Tissues were washed in DPBSand then incubated in lacZ stain (5mM KFerrocyanide, 5 mM KFerricya-nide, 2 mM MgCl2, 1 mg/ml X-Gal, inDPBS) at 37°C. This was followed bywashing in DPBS and postfixation in4% formaldehyde in DPBS at 4°C.

Antisense digoxigenin-labeled RNAprobes were generated using a PCRproduct specific for each gene. Gene-specific templates were amplified byRT-PCR from total RNA extractedfrom E9.5 mouse embryos (Rhee et al.,2003). Gene-specific primers were de-signed using Primer3 (Rozen and Ska-letsky, 2000) and then modified byadding the T7 RNA polymerase bind-ing site sequence (5�-CTAATACGA-CTCACTAT AGGGAGA-3�) to the 5� endof the downstream primer. Mouse Mkxupstream primer 5�-GAGCCGTGCTTT-TTGAAGAC-3� and downstream primer5�-TACTTGGGCGGTGACACATA-3�;mouse paraxis upstream primer 5�-ACCTTCTGTCTCAGCAACCA-3� anddownstream primer 5�-CCCCGATTT-GCTCACATACT-3�. A tenth of the25-�l PCR reaction product was di-rectly added to an in vitro transcrip-tion reaction with digoxigenin RNAlabeling mix (Roche Applied Sciences,Indianapolis, IN) as described inJohnson et al. (2001). The Pax3 probewas generated as described previously(Wilson-Rawls et al., 2000).

Cryosections on WISHStained Tissues

Tissues were washed in DPBS andthen equilibrated in 5% sucrose inDPBS at room temperature and then15% sucrose in DPBS at 4°C. Tissueswere then equilibrated in a solution of15% sucrose and 7.5% gelatin inDPBS at 37°C overnight. The tissueswere embedded in the same solutionand frozen in liquid nitrogen. Theblock was sectioned on a cryostat keptat �30°C at a thickness of 25 �m andcollected on gelatin-subbed slides. Theslides were incubated in warm DPBSto remove the gelatin and then dehy-

drated in an ethanol series andmounted with coverslips.

ACKNOWLEDGMENTSWe thank the Evolutionary and Func-tional Genomics Center of the ArizonaBiodesign Institute for the contribu-tion of the InsituPro, Dr. MilesOrchinik for his assistance in the gen-eration and visualization of the cryo-sections, and Sarah Parsons andLeann Chavez for their technical as-sistance. We also thank Dr. SudhirKumar for his valuable discussion.J.A.R. and J.W.R. are supported bygrants from the National ScienceFoundation and the Muscular Dystro-phy Association (0131726).

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