PATTERNS & PHENOTYPES

Spatio-Temporal Expression of MRF4Transcripts and Protein During Xenopus laevisEmbryogenesisBruno Della Gaspera,1 Ines Sequeira,1 Frederic Charbonnier,1 Christel Becker,1 De-Li Shi,2 andChristophe Chanoine1*

Whereas there have been extensive studies of the expression of XMyf5 and XMyoD during Xenopusembryogenesis, nothing is known about the spatio-temporal accumulation of XMRF4 transcripts andprotein. In this report, we describe the cloning and characterization of two full-length MRF4 cDNAs and oftheir proximal promoters in Xenopus laevis. The comparison of the relative transcript levels of the XMRF4-aand -b genes in developing and adult muscles is highly suggestive of specific functions for the correspondingXMRF4 proteins. Whole-mount embryo in situ hybridization revealed the first XMRF4 transcripts in themore differentiated anterior myocytes of the embryo when the myosin heavy chain E3 mRNA begins to bedetectable. XMRF4 mRNA accumulation later extended posteriorly but was never detected in the posteriorunsegmented mesoderm, in contrast to XMyoD and XMyf-5. Whole-mount embryo immunohistochemistryrevealed that XMRF4 protein accumulated in somite nuclei slightly after XMRF4 transcripts. DevelopmentalDynamics 235:524–529, 2006. © 2005 Wiley-Liss, Inc.

Key words: MRF4; myogenic regulatory factor; myogenesis; embryogenesis; Xenopus

Accepted 20 September 2005

INTRODUCTION

The four basic helix-loop-helix tran-scription factors of the MyoD family(including MyoD itself, Myf5, myoge-nin, and MRF4) are master genes forskeletal muscle development. In cell-culture transfection experiments, thefour proteins all behave as dominant-acting muscle differentiation factors(or muscle regulatory factors, MRFs)that transactivate muscle structuralgenes and convert non-muscle cells toa muscle phenotype. However, the 4MRFs’ individual roles during in vivocommitment and differentiation of

skeletal muscle cells remain unclear.In mice, gene knockout studies haveshown that MyoD and Myf-5 play par-tially redundant roles in myoblastspecification, whereas differentiationrequires myogenin and either MyoDor MRF4 (Tajbakhsh and Bucking-ham, 2000). Alone, MRF4 did notseem to be required for myogenic com-mitment or muscle differentiationsince the first gene targeting studiesshowed that myogenesis could proceedin the absence of this molecule (Braunand Arnold, 1995; Patapoutian et al.,1995; Zhang et al., 1995). In contrast

to MyoD, myogenin, and Myf5, theMRF4 mRNA continues to accumu-late at a high level after birth and therelative level of MRF4 mRNA is highin adult slow- and fast-twitch muscles,suggesting that MRF4 may regulateskeletal muscle gene expression in theadult (Rhodes and Konieczny, 1989;Braun et al., 1990; Miner and Wold,1990). However, the report of Kassar-Duchossoy et al. (2004), which showedthat MRF4 can determine skeletalmuscle identity in Myf5/MyoD double-mutant mice and drive myogenesis inembryonic trunk and limbs, has re-

1UMR 7060 CNRS, Equipe Biologie du Developpement et de la Differenciation Neuromusculaire, Centre Universitaire des Saints-Peres,Universite Rene Descartes, Paris, France2Groupe de Biologie Experimentale, Laboratoire de Biologie du Developpement, CNRS UMR 7622, Universite Paris, FranceGrant sponsor: Association Francaise contre les Myopathies.*Correspondence to: Prof. C. Chanoine, Laboratoire de Biologie du Developpement et de la Differenciation Neuromusculaire,Centre Universitaire des Saints-Peres, Universite Rene Descartes, 45 rue des Saints-Peres, F-75720 Paris Cedex 06, France.E-mail: chanoine@biomedicale.univ-paris5.fr

DOI 10.1002/dvdy.20628Published online 28 October 2005 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 235:524–529, 2006

© 2005 Wiley-Liss, Inc.

cently revised the views of the epi-static relationship between the MRFsin identifying MRF4 as a determina-tion gene.

Whether an active myogenic regula-tory factor (MRF) can discriminateamong the different muscle gene tar-gets or whether it activates transcrip-tion at all E-box-containing promotersis a relatively unexplored and recur-rent question in the biology of MRFs(reviewed in Chanoine et al., 2004).Our recent findings, using the Xeno-pus embryo developmental model,strengthen the hypothesis of a specificregulation of a subset of muscle genesby a given MRF, thus precluding theiractivation by other members of theMRF family (Charbonnier et al., 2002,2003).

Understanding how these factorsfunction requires a detailed analysisof their expression patterns during de-velopment. Whereas expression ofMyf5 and MyoD has been studied indetail during Xenopus embryogenesis(reviewed in Chanoine and Hardy,2003), nothing is known about thespatio-temporal accumulation ofXMRF4 transcripts and protein.

In this report, we describe the clon-ing and characterization of two full-length MRF4 cDNAs and the pro-moter of each gene in Xenopus. TwocDNAs, designated XMRF4-a andXMRF4-b, code for proteins closely re-lated to each other and are likely to beexpressed from duplicated XenopusMRF4 genes. We examined the tem-poral and spatial pattern of accumu-lation of XMRF4 at the mRNA andprotein levels during embryogenesisand show that the two isoforms aredifferentially expressed during devel-opment and muscle regeneration, sug-gesting a functional difference.

RESULTS AND DISCUSSION

Cloning of the Two Full-Length XMRF4 cDNAs andTheir Proximal PromoterRegions

In Xenopus laevis, two genes werecloned for XMyoD (Hopwood et al.,1989; Harvey, 1990) and Xmyogenin(Charbonnier et al, 2001), and onegene was cloned for XMRF4 (Jen-nings, 1992) and Myf5 (Hopwood etal., 1991), accounting for the

pseudotetraploid genome of Xenopuslaevis. Here, using a PCR-based strat-egy, we report the cloning of the sec-ond full-length XMRF4 cDNA,XMRF4-b, whose existence Ataian etal. (2003) have previously shown evi-dence for. The two cDNAs XMRF4-aand XMRF4-b contained an openreading frame encoding a protein of,respectively, 240 and 241 amino acids(Fig. 1A). XMRF4-b presents an addi-tional serine residue in the serinecluster at position 212. The two aminoacid sequences show 96% sequenceidentity. The isolated cDNAs of, re-spectively, 1,649 and 1,250 bp,present more divergences in the 3�UTR than in the coding and 5� un-translated regions. Moreover, an in-sertion of a repetitive element of 370bp is found in the XMRF4-a 3� UTR.The amino acid identity is higher withtetrapod than fish orthologs, espe-cially outside the bHLH domain (Fig.1A). The amino acid identities of Xe-nopus XMRF4-a and -b compared withtheir tetrapod and fish orthologs were70 and 48%, respectively. XMRF4transcripts begin to be clearly de-tected at stage 18, as previously re-ported by Jennings (1992) using anRNase protection assay. It should benoted that in some RT-PCR experi-ments, XMRF4 transcripts were de-tected as early as stages 13 and 14(Atain et al., 2003; Charbonnier et al.,2003), suggesting that MRF4 genescould be transcribed at faint levels atthese stages. The three probes used inthis study for in situ hybridization ex-periments (Fig. 2) could not discrimi-nate these two XMRF4 transcripts. Inorder to examine the relative abun-dance of the two XMRF4 mRNAs, wehave analyzed their expression by RT-PCR analysis coupled to enzyme re-striction digestion (Fig. 1B), that al-lows for a direct comparison betweenrelative levels of the two transcripts ineach PCR experiment. The resultsshowed a differential expression ofXMRF4-a and XMRF4-b genes. At em-bryonic stage 18, the two XMRF4mRNAs were detected in approxi-mately equal levels. XMRF4-a de-clined at stage 25, and XMRF4-b wasthe main transcript form detectedduring regeneration as well as inadult muscles. These results couldsuggest different functions for the twoXMRF4 proteins. Indeed, we have pre-

viously shown that despite the highdegree of structural homology be-tween the two isoforms of Xmyogenin,i.e., only 11 different amino acid resi-dues, XmyogU1, and XmyogU2 targetdifferent sets of genes, the adult fastisoform of MHC is induced byXmyogU1 and not by XmyogU2 (Char-bonnier et al., 2002). It is also inter-esting and surprising to note that inXenopus, three MRFs—XMyoD,Xmyogenin, and XMRF4—are eachencoded by two non-allelic genes dif-ferentially regulated during develop-ment.

Fragments of 1,755 and 1,383 bpcorresponding to the two XMRF4 pro-moters were isolated using thegenomic walker method. We have notfound the more 5�UTR sequence pub-lished by Jennings (1992) in ourgenomic clones and we decided to de-termine the start transcription site bysmart technology (Fig. 1C, arrow-head). The 5� UTR sequence found inthis way matched with our genomicsequence. In Xenopus, the sequence ofthe more proximal promoter regionsaround the TATA box element (�103,�81), is 64% homologous to the mam-malian counterparts (Fig. 1C), sug-gesting an important role for this re-gion in the control of MRF4expression. Indeed, in mouse, only areporter gene containing 8.5 kb ofMRF4 5� flanking sequence repro-duces a subset of the full expressionpattern in vivo and exhibits transcrip-tion in myotomal cells during somiticdifferentiation (Pin et al., 1997), andMRF4 expression during mouse em-bryogenesis requires at least four ele-ments located in the 140-kb upstreamsequences of the gene (Carvajal et al,2001). However, in vitro studies haveshown that expression of the MRF4gene is controlled by a proximal pro-moter element (�336 to �71) (Blacket al., 1995), which presents a weakbut muscle-specific activity. Two re-gions of the XMRF4 promoterspresent identities of 80% (�1,742 to�1,329 for XMRF4-a and �1,383 to�993 for the XMRF4-b gene) and 90%(�262 to �1 for both genes). Analysisof the promoter sequences of the twoXMRF4 genes has identified sevenand three putative E-box sites(CANNTG) for XMRF4-a andXMRF4-b, respectively, that might beinvolved in controlling their muscle-

EXPRESSION OF MRF4 DURING XENOPUS EMBRYOGENESIS 525

specific expression. In addition to theE-boxes, four putative MEF2 bindingsites (A/T rich element) were identi-fied in the XMRF4 promoters using aMatInspector software tool (http://www.genomatix.de).

Expression of XMRF4 mRNAand Protein

Whole-mount in situ hybridization re-vealed that XMRF4 transcripts began

to be observed at detectable levelsonly in the more anterior differenti-ated myocytes of the embryo at stage18–19 (Fig. 2A) when the embryonicmyosin heavy chain E3 mRNA, a rel-atively late-activating muscle gene,began to be observed in situ and byRT-PCR (data not shown). At thisstage, XMyf5 mRNA strongly accumu-lated in the undifferentiated meso-derm of the caudal region and slightly

in muscle cells initiating their full dif-ferentiation program. XMyoD was de-tected in somites as well as in unseg-mented presomitic mesoderm. In thefollowing stages (stage 21–22),XMRF4 mRNA accumulation ex-tended posteriorly in the embryo (Fig.2B). It is well established that musclemesoderm differentiates in an anteri-or-to-posterior direction. During thisperiod, the pattern of XMRF4 accumu-

Fig. 1. A: Amino acid sequence comparison between the two Xenopus MRF4 proteins either with fish counterparts (Te, Tetraodon nigroviridis; Ta,Takifugu rubripes; Da, Danio rerio) or tetratopod counterparts (Bo, Bos taurus; Ho, Homo sapiens; Mu, Mus musculus; Ra, Rattus norvegicus; Ga,Gallus gallus). Asterisks indicate identical amino acids, while colons and periods represent conservative amino acid changes. The highly conservedbHLH domain is overlined. Residues not conserved between the two Xenopus proteins are shown in gray boxes. B: Expression of the two MRF4 genesduring Xenopus embryogenesis (numbers refer to the developmental stages), in adult muscles (1: tibialis anterior, 2: adductor brevis dorsalis, 3: tibialisposterior) and during brachial muscle regeneration (numbers refer to cardiotoxin postinjection days). In order to perform PCR during the exponentialphase of amplification, 33 and 30 cycles were required in the case of XMRF4 during development and in adult muscles, respectively. In the case ofregenerating muscle, the number of cycles was fixed to 30. Twenty-five cycles were nedeed for EF1-alpha in all cases. The XMRF4 PCR productsdigested with Hind III, allowing for selective discrimination between MRF4-a (top bands) and MRF4-b (bottom bands), were submitted to Southern blotwith a labeled primer complementary to both sequences. PCR products of MRF4-a and -b plasmids were used as controls (Ca and Cb). Because RNAextraction and PCR were not performed under the same conditions, no relevant comparison could be drawn on the basis of the hybridization signalintensity between adult muscle and regenerating or developing muscle. C: Sequence comparison between the more proximal promoter region of thetwo MRF4 genes (Xa, Xb) with human (Ho) and mouse (Mu) counterparts. The nucleotides were numbered beginning with the first nucleotide of thesmart product (black arrowhead, �1). TATA-box motif is underlined. Asterisks indicate identical nucleotides.

526 DELLA GASPERA ET AL.

lation was rather similar to that ofXMyoD transcripts except thatXMyoD mRNA was also detected inthe posterior region of the embryowhere XMyf5 accumulated (Fig. 2B).We also note that the intense stainingof the dorsomedial region forXMRF4 and MyoD evokes the stain-ing observed for MyoD in zebrafishembryo (Pownall et al., 2002). Atstage 27–28, both XMRF4 and theMHC E3 transcripts accumulated inthe whole somitic differentiatedmyotome whereas XMyoD mRNA de-

Fig. 2.

Fig. 3.

Fig. 2. Whole-mount in situ hybridization atstage 18–19 (A) 21–22 (B), and 27–28 (C) withlabeled antisense probes against XMRF4,XMyf5, XMyoD, and embryonic MHC E3 mR-NAs. The same results were obtained using thethree MRF4 probes. The anterior side of theembryos is on the left. Lateral view (C, right inB), dorsal view (A, left in B). Arrowhead: poste-rior unsegmented mesoderm

Fig. 3. A: Control of specificity of the anti-XMRF4 antibody: One nanogram of syntheticFlag-tagged XMRF4-a (1) or -b (2) mRNA wasbilaterally injected into the animal pole of two-cell stage embryos. Ten micrograms of egghomogenates was subjected to sodium dode-cyl sulfate-polyacrylamide gel electrophoresisfollowed by Western blotting. The affinity-puri-fied anti-XMRF4 antibody recognizes the twoXMRF4 proteins in the same way as the anti-Flag antibody. No band was obtained in unin-jected embryos (data not shown). B: Wholemount immunostaining using an affinity-purifiedpolyclonal antibody against Xenopus MRF4 atstage 24 (up), 26 (middle), 28 (down). The an-terior side of the embryos is on the left. Arrow-head, posterior unsegmented mesoderm. C: In-direct immunofluorescent detection of Anti-XMRF4 (green) and 4�,6-diamidine-2-phenylidole-dihydrochloride (DAPI) staining(blue) on longitudinal cryosection of somiticmuscles at stage 28. No labels were detectedwith secondary antibody alone (data notshown).

EXPRESSION OF MRF4 DURING XENOPUS EMBRYOGENESIS 527

clined from the anterior tissue thathad differentiated first (Fig. 2C). Atthis stage, XMyf5 transcripts weredetected in the posterior unseg-mented mesoderm and in the lastformed somites that initiate the fulldifferentiation program (Fig. 2C).

We have previously developed an af-finity-purified polyclonal antibodyagainst Xenopus MRF4 (Becker et al.,2003). This specific antibody recog-nized the two proteins, XMRF4-a andXMRF4-b (Fig. 3A). Whole-mount em-bryo immunohistochemistry revealedthat XMRF4 protein was present inthe more differentiated anterior myo-cytes of the embryo at stage 24,slightly after MRF4 transcripts, andits accumulation extended posteriorlyin the next few hours of development.However, XMRF4 protein was neverdetected in the less differentiated pos-terior region (Fig. 3B). Indirect Immu-nofluorescence analysis performed oncryosections indicated that the accu-mulation of XMRF4 protein was lim-ited to somitic nuclei, as revealed bynuclear 4�,6-diamidine-2-phenylidole-dihydrochloride (DAPI) counterstain-ing (Fig. 3C). In Xenopus, the primarymyogenesis is characterized by the se-quential expression of the three deter-mination factors XMyf5, XMyoD, andXMRF4, the myogenin genes being ac-tivated later during the secondarymyogenesis that takes place at thetime of metamorphosis (Nicolas et al.,1998). During early embryogenesis,XMyf-5 and XMyoD begin to be ex-pressed in presomitic mesoderm, atgastrula stages, whereas XMRF4 ex-pression is strictly associated tosomitic terminal differentiation whenmany muscle gene products alreadypresent are upregulated or some late-activating genes begin to be tran-scribed (Chanoine and Hardy, 2003).XMRF4 expression overlaps with theformation of neuroneuromuscularconnections (Blackshaw and Warner,1976; Kullberg et al., 1977), but recentreports (Ataian et al., 2003; Becker etal., 2003) now fail to support the hy-pothesis that MRF4 gene expressionis triggered or is significantly up-reg-ulated in myogenic cells by signalsfrom motor axons. By using a combi-nation of gain and loss of functionstrategies in Xenopus, we have shownrecently that the precise orchestrationof muscle gene activation by MRF oc-

curs with a target specificity (re-viewed in Chanoine et al., 2004), rein-forcing the concept that an individualMRF controls a subset of genes thateither cannot be activated by the otherMRFs or are activated with a dramat-ically lower efficiency. For XMRF4,the use of dominant-negative protein,antisense, or morpholino oligonucleo-tide strategies will also permit identi-fication of specific target genes.

EXPERIMENTALPROCEDURES

Cloning of the Two Full-Length XMRF4 cDNAs

Full-length cDNA sequence of the twoXMRF4 genes was determined usingMarathon and SMART™ technologiesfrom Clontech (Palo Alto, CA), accord-ing to the manufacturer’s instruc-tions. SMART™ cDNA synthesis usedthe terminal transferase activity ofthe reverse transcriptase at the 5� endof the mRNA that allows for the an-choring of a specific primer to obtain aresulting cDNA that contains the com-plete 5� end of the mRNA. Multipleclones were fully sequenced. The nu-cleotide sequences of cDNA cloneswere deposited into the GenBank da-tabase with accession numbersAY970802 and AY970828 forXMRF4-a and -b, respectively.

Isolation of the Two XMRF4Promoters

Xenopus MRF4 promoters were iso-lated using the GenomeWalker kitfrom Clontech. This method allows forwalking in genomic DNA. The start-ing genomic DNA must be digestedwith restriction enzymes, and is li-gated to adaptator. The PCR amplifi-cation uses adaptor primers (Ap-1 andAp-2) and gene-specific primers. Tworounds of PCR were necessary to iso-late 1,755 and 1,383 bp of proximalpromoter fragments for the XMRF4aand XMRF4b genes, respectively. Themore proximal elements were isolatedusing XMRF4 cDNA-specific primersfor each gene and the adaptor prim-ers. The more distant 3� flanking re-gions were amplified by the nextround of PCR using gene-specificprimers together with Ap-1 and Ap-2.All PCR fragments were cloned into a

pGEM-T cloning vector and used forsequence analysis. The nucleotide se-quences of the two XMRF4 promoterswere deposited into the GenBank da-tabase with accession numbersAY970829 and AY970830 forXMRF4-a and -b genes, respectively

RT-PCR, Western Blot, andEmbryo Injection

RT-PCR and Western blot were per-formed as described previously (Char-bonnier et al., 2002; 2003, Becker etal., 2003). XMRF4 PCR products wereamplified with sense and antisenseprimers 5�-ATGGACCTATTTGAAA-CAAA-3� and 5�-TTAATTCTCTAC-CAGCTCCT-3�, respectively, and werecut with Hind III. EF1-alpha, used asinternal control, was amplified withsense and antisense primers 5�-CCA-GATTGGTGCTGGATATGC-3� and5�-AGTCTGAGAAGCTCTCCACGC-3�, respectively. For expression in Xe-nopus embryos, the full-lengthXMRF4-a and XMRF4-b cDNAs weresubcloned into the expression vectorpSP64T by PCR-directed cloning, amethod developed in our laboratory(see Charbonnier et al., 2003 and firstreported in Chanoine’s AFM grant,2000) and by Geiser et al. (2001). Thissimple PCR protocol, which is an ex-tension of the quick change mutagen-esis method (Stratagene, La Jolla,CA), allows insertion anywhere in aplasmid target. The flag epitope wasadded in the C-terminal position asindicated elsewhere (Charbonnier etal., 2003).

Whole-Mount In SituHybridization,Immunohistochemistry, andCryosection IndirectImmunofluorescence

Fixation of embryos and whole-mountin situ hybridization with digoxige-nin-labeled antisense RNA probeswere carried out as described by Har-land (1991) with some modifications.Probes for whole-mount in situ hy-bridization were prepared as follows.Probes were amplified from cDNA re-verse transcribed from stage 20 or 30total RNA and cloned into the PgemTvector. Total RNA was extracted fromstage 20 and 30 embryos with a mini

528 DELLA GASPERA ET AL.

RNAeasy kit (Quiagen, Chatsworth,CA) and then reverse transcribed withpolydT primer. Targeted cDNAs wereamplified by PCR with the followingprimers (sense and antisense, respec-tively): 5�-GCAGATGAGGAACCCT-TCTC-3� and 5�-CAAGGAACAT-GGGCATTTAG-3� for MRF4 probe 1directed against the C-terminal cod-ing region and almost the full-length3� UTR of XMRF4-b; 5�-GGCTGCCA-GAGCAATCAAGG-3� and 5�-TGGTG-GAGCTAAGACATGTT-3� for MRF4probe 2 directed against 5� UTR andN-terminal coding region of XMRF4-a;5�-GCAGATGAGGAACCCTTGTC-3�and 5�-ATGTCACGTCAGTGCAACA-AG-3� for MRF4 probe 3 directedagainst the C-terminal coding region,and the beginning of 3� UTR ofXMRF4-a; 5�-CCAACTGCTCCGATG-GCATG-3� and 5�-CACCAAGCACAT-AAATAAGGCA-3� for XMyoD; 5�-AC-TACAGTCTCCCAGGACAG-3� and5�-GCATGTTTATTAACACAATAG-3�for XMyf5. Sense strand controls wereprepared from all plasmids and testednegative by in situ hybridization.Whole-mount immunohistochemistrywas performed using an anti-XMRF4affinity-purified antibody (Becker etal., 2003) diluted 1:100. The primaryantibody was detected with an alka-line phosphatase-conjugated anti-guinea pig antibody diluted 1:500 fol-lowed by reaction with BM purple.Indirect immunofluorescence was per-formed as described previously(Becker et al., 2003).

ACKNOWLEDGMENTSWe thank Dr. G.P.Radice for E3cDNA.

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