Animal studies and tissue preparationAll animal experiments were performed in accordance with the guidelines of theinstitutional committee for the use of animals for research. Mice were killed by a lethaldose of anaesthesia. In vivo luciferase imaging and doxycycline treatment were performedas previously described11. Animals were injected intraperitoneally (i.p.) with 20 ng humanrecombinant TNFa (Roche) and killed after 1 h for liver p65 immunostaining and RNAand protein extraction. For anti-TNFa treatment, mice were injected with 22mg of goatanti-mouse TNFa neutralizing antibodies i.p. (R&D Systems; AF-410-NA) for 3consecutive days, or with control goat IgG. All mice were injected i.p. with BrdU(Amersham; 100 ml per 10 g body weight) 3 and 24 h before they were killed. In someanimals, liver samples were removed and snap-frozen for protein and RNA analyses. Theanimal was then perfused through the left ventricle with 10ml of cold heparinized PBS,followed with 25ml of 4% buffered formalin. Livers were removed, weighed,photographed and fixed in formalin over night. The next day the entire liver wassubmitted for paraffin embedding in three to five cassettes. 5-mM sections were stainedwith H&E and evaluated by a pathologist to whom the genetic makeup or treatment groupwere not known.

MRI analysisMRI was performed on a horizontal 4.7T Biospec spectrometer (Bruker Medical), using abirdcage coil. Mice were anaesthetized (30mg kg21 pentobarbital, i.p.) and placed supinewith the liver located at the centre of the coil. Liver and tumour volumes were determinedfrom multi-slice coronal and axial T1 weighted fast spin echo images covering the entireliver both coronally and axially (repetition time, 400ms; echo time, 17ms; slice thickness,1mm; field of view, 5 cm (coronal) and 3.4 cm (axial) using a 256 £ 256 matrix). Toestimate tumour and liver volumes, the liver and tumour boundaries visualized in eachslice were outlined by using image processing software (NIH image), by an observer towhom the genetic makeup was not known. The number of pixels (for both liver andtumour) were converted to an area by multiplying by the factor ((field ofview)2 £ (matrix)2).

ImmunohistochemistryFor p65 immunostains, 5-mM sections, cut on the same day, were de-waxed and hydratedthrough graded ethanols, cooked in 25mM citrate buffer pH 6.0 in a pressure cooker at115 8C for 3min (decloaking chamber, Biocare Medical), transferred into boilingdeinoized water and left to cool down for 20min. After 5min treatment in 3%H2O2, slideswere incubated with rabbit polyclonal p65 antibodies diluted 1:100 in CAS-Block (Zymed)for 3 h at room temperature, washed three times with Optimax (Biogenex), incubated for30min with anti rabbit Envisionþ (DAKO) and developed with DAB for 15min. Antigenretrieval for TNFa and p65 double immunostaining was performed in 100mM glycinebuffer pH 9.0. After 5min treatment in 3% H2O2, slides were incubated with goatpolyclonal anti-TNFa antibodies diluted 1:50 in CAS-Block for 1 h at room temperature,washed in Optimax, incubated with (1:100) rabbit anti-goat antibodies (JacksonLaboratories) for 30min, washed again with Optimax and incubated with anti-rabbitEnvisionþ (DAKO) and developed with AEC for 15min at 37 8C. Following this, slideswere reboiled for 7min in 100mM glycine buffer in a microwave oven and cooled down toroom temperature. Slides were incubated with rabbit polyclonal anti-p65 antibodiesdiluted 1:100 in CAS-Block for 3 h at room temperature, washed three times withOptimax, post-fixed for 5min in 4% formaldehyde in Tris buffered saline, washed withOptimax, incubated for 30minwith alkaline phosphatase-conjugated polymer anti-rabbitIgG (Zymed) and developed with BCIP/NBT. Slides were counterstained with nuclear fastred. Antigen retrieval for CD3, cJun, BrdU, activated caspase-3, luciferase, MPO and Ki-67were performed in 25mMcitrate buffer pH 6.0. Antigen retrieval for TNFawas performedin 100mM glycine buffer pH 9.0. Detailed protocols for immunostaining with eachantibody are available on request.

Received 14 June; accepted 10 August 2004; doi:10.1038/nature02924.

Published online 25 August 2004.

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animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis.Am.

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12. Gupta, S. Hepatic polyploidy and liver growth control. Semin. Cancer Biol. 10, 161–171 (2000).

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(2004).

14. Chaisson, M. L., Brooling, J. T., Ladiges, W., Tsai, S. & Fausto, N. Hepatocyte-specific inhibition of

NF-kB leads to apoptosis after TNF treatment, but not after partial hepatectomy. J. Clin. Invest. 110,

193–202 (2002).

15. Maeda, S. et al. IKKb is required for prevention of apoptosis mediated by cell-bound but not by

circulating TNFa. Immunity 19, 725–737 (2003).

16. Beg, A. A., Sha, W. C., Bronson, R. T., Ghosh, S. & Baltimore, D. Embryonic lethality and liver

degeneration in mice lacking the RelA component of NF-kB. Nature 376, 167–170 (1995).

17. Lavon, I. et al. Nuclear factor-kB protects the liver against genotoxic stress and functions

independently of p53. Cancer Res. 63, 25–30 (2003).

18. Friedberg, E. C.,McDaniel, L. D. & Schultz, R. A. The role of endogenous and exogenousDNAdamage

and mutagenesis. Curr. Opin. Genet. Dev. 14, 5–10 (2004).

19. Wang, J. S. & Groopman, J. D. DNA damage by mycotoxins. Mutat. Res. 424, 167–181 (1999).

20. Eferl, R. et al. Liver tumor development. c-Jun antagonizes the proapoptotic activity of p53. Cell 112,

181–192 (2003).

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TRAIL-induced apoptosis mediated by NF-kB. Oncogene 22, 3842–3852 (2003).

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Supplementary Information accompanies the paper on www.nature.com/nature.

Acknowledgements Mdr2-knockout mice were a gift from R. P. Oude Elferink and the TALAP1

mice were received fromH. Bujard.We are grateful to N. Berger, T. Golub andN. Kidess-Bassir for

technical assistance, to A. Hatzubai, O. Mandlboim, N. Lieberman, G. Kojekaro, M. Davis and

H.Harel for helping withMRI, FACS,mRNAand protein analysis.We thankK.Meir andM.Oren

for discussions and a critical reading of the manuscript. This research was supported by grants

from the Israel Science Foundation funded by the Israel Academy for Sciences and Humanities

(Center of Excellence Program), Prostate Cancer Foundation Israel—Center of Excellence,

German-Israeli Foundation for Scientific Research and Development (GIF, in collaboration with

H. Bujard), a grant in memory of H. and F. Brody from H. M. Krueger as trustee of a charitable

trust and the Israel Cancer Research Foundation (ICRF). I.S. is supported by the Lady Davis

Fellowship Trust.

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests formaterials should be addressed to E.P. (peli@hadassah.org.il) and

Y.B.-N. (yinon@cc.huji.ac.il).

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Mrf4 determines skeletalmuscle identity in Myf5:Myoddouble-mutant miceLina Kassar-Duchossoy1, Barbara Gayraud-Morel1*, Danielle Gomes1,Didier Rocancourt2, Margaret Buckingham2, Vasily Shinin1*& Shahragim Tajbakhsh1

1Stem Cells and Development, 2Molecular Genetics of Development,Department of Developmental Biology, CNRS URA 2578, 25 rue du Dr Roux,75724 Paris Cedex 15, France

* These authors contributed equally to the work

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In vertebrates, skeletal muscle is a model for the acquisition ofcell fate from stem cells1. Two determination factors of the basichelix–loop–helix myogenic regulatory factor (MRF) family, Myf5and Myod, are thought to direct this transition because double-mutant mice totally lack skeletal muscle fibres and myoblasts2–4.In the absence of these factors, progenitor cells remain multi-potent and can change their fate5,6. Gene targeting studies haverevealed hierarchical relationships between these and the otherMRF genes, Mrf4 and myogenin, where the latter are regarded asdifferentiation genes7. Here we show, using an allelic series ofthree Myf5 mutants that differentially affect the expression of thegenetically linkedMrf4 gene, that skeletal muscle is present in the

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Figure 1 Allelic series of Myf5 mutants. a, Both Myf5 nlacZ and Myf5 GFP-P contain a

deletion of the N terminus and bHLH domain. Mrf4 was targeted with an nlacZ reporter

gene (Methods). b–i, Early myotome is perturbed in Myf5-null embryos. Myotome (My) is

present in somites of heterozygous E10.5 embryos (b, e) and is perturbed in null mutants

(c, d, f). Null mutants display mislocalized MPCs along the epaxial (dorsal, red arrowhead)

and hypaxial (ventral, white arrowhead) somites. Arrows indicate branchial arch muscles

(b–f). Tails of adult wild-type (g), Myf5 loxP/loxP (h) and Myf5 GFP-P/GFP-P (i) animals were

sectioned on a cryostat and stained with anti-MyHC. Note that dorsal tail muscles are

lacking in the mutants (arrows). Asterisk, dorsal vein; kbp, kilobase pairs; TK DTA,

diphtheria toxin gene with thymidine kinase promoter; pA, SV40 polyA; Puro-TKpA,

Puromycin-thymidine kinase fusion with TK promoter and bovine polyA; V, vertebra. Scale

bar, 300mm.

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new Myf5:Myod double-null mice only when Mrf4 expression isnot compromised. This finding contradicts the widely held viewthat myogenic identity is conferred solely by Myf5 and Myod, andidentifies Mrf4 as a determination gene. We revise the epistaticrelationship of the MRFs, in which both Myf5 and Mrf4 actupstream of Myod to direct embryonic multipotent cells into themyogenic lineage.Segmented structures called somites provide muscle progenitor

cells (MPCs) for the skeletal muscles of the trunk, limbs and tail,whereas head muscles are derived predominantly from paraxialhead and prechordal mesoderm7. As somites mature in an anterior–posterior gradient, they transiently retain a dorsal dermomyotomeepithelium, which is the source of resident skeletal muscle cellsconstituting the myotome and of migratory MPCs1. Myf5 andMyod are thought to initiate muscle identity in MPCs.Our analyses of several Myf5 mutants generated by homologous

recombination indicated that Mrf4 is expressed in MPCs, and itsexpression was differentially affected in an allelic series comprisingthe previously described Myf5nlacZ allele5 and two newly generatedMyf5GFP-P and Myf5 loxP alleles (Fig. 1a). The null status of these

alleles was examined in vitro and in vivo. Myf5nlacZ and Myf5GFP-P

both lack the basic helix–loop–helix (bHLH) domain required forDNA binding and dimerization and are therefore null8. TheMyf5 loxP allele is also null because it lacks the amino-terminaldomain required for transactivation (Supplementary Figs 1, 2). Aswith all Myf5-null embryos3,5,9,10, these mutants lack an earlymyotome until at least embryonic day 10 (E10; Fig. 1c, d, f; seebelow), compared with normal embryos (Fig. 1b, e). Anothercharacteristic of Myf5-null embryos is a mislocalization of MPCsalong the epaxial-most (dorsal) and hypaxial-most (ventral) der-momyotome lips, particularly evident in interlimb somites(Fig. 1c, d, f). In addition, Myf5-null mutants lack epaxial (dorsal)muscles7,11 (Fig. 1g–i; data not shown).

To determine which MRF rescues the myotome inMyf5mutants,we examined Mrf4 and Myod expression by whole-mount in situhybridization (WM-ISH). Mrf4 expression initiates from E9.5 inwild-type embryos, and by E9.75Mrf4 transcripts accumulate in theepaxial and hypaxial somite. Epaxial somite expression markspredominantly differentiating cells in the myotome that werepreviously programmed by Myf5. By contrast, hypaxial Mrf4

Figure 2 Myod expression depends on Mrf4 in Myf5 mutants. a–c, WM-ISH of E9.75

(27–28 somites) wild-type embryos withMyf5 (a),Myod (b) andMrf4 (c) riboprobes.Myf5

and Mrf4 are expressed before Myod in the epaxial and hypaxial somite. d–f, Mrf4

transcripts in somites of E10.5 wild-type (d), Myf5 nlacZ/þ (e) and Myf5 nlacZ/nlacZ (f )

embryos. g, Mrf4 ISH on E10 Myf5 loxP/loxP embryo. h–j, The myogenin gene is not

expressed at E9.75 in Myf5 loxP/loxP and Myf5 GFP-P/GFP-P compared with wild type. Note

start of hypaxial expression inMyf5 loxP/loxP (arrowheads). k–p,Mrf4 (k, n),Myod (l, o) and

myogenin (m, p) ISHs on E10.5 Myf5 loxP/loxP (k–m) and Myf5 GFP-P/GFP-P (n–p) embryos.

Note thatMyod andmyogenin gene expression appear afterMrf4; the central somite lacks

signal owing to misplaced MPCs (see Fig. 1d, f). Mrf4 and Myod expression in interlimb

somites begins in the hypaxial somite, unlike Myf5, and later in the epaxial somite.

q–t, Myod transcripts are absent at E10.5 in Myf5 nlacZ/nlacZ (r) and Mrf4 nlacZ-P/nlacZ-

P:Myf5 loxP/loxP (t) mutants compared with controls (q, s). u–v, Perturbations ofMrf4 in cis

(u) correlate with perturbations of Myod in trans (v). The curved line thickness in u and v

reflects the relative intensity of inhibition or activation, such that the greatest reduction in

Mrf4 expression (u, v) is observed withMyf5 nlacZ/nlacZ, which correlates with the greatest

delay in Myod expression (v). FL, forelimb; ki, Myf5 knock-ins; so, somite; red

arrowheads, epaxial (dorsal); black arrowheads, hypaxial (ventral).

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expression was observed concomitantly with Myf5 in the dermo-myotome12, before myotome formation and before Myodexpression (Figs 2a–c, 3g, h; data not shown), suggesting thatMrf4 might have a function in MPCs. Strikingly, examination ofMyf5 mutants demonstrated that Mrf4 expression was directlydependent on the nature and size of the knock-in generated at theMyf5 locus, suggesting that these perturbations are in cis (Fig. 2u).In the most severe case, Mrf4 expression was not detected inMyf5nlacZ/nlacZ embryos at E10.5, compared with robust expressionin wild-type embryos (Fig. 2d, f). Consistent with this, Mrf4expression in Myf5nlacZ/þ embryos was about half of that observedin the wild type (Fig. 2d–f). These observations suggest thatMyf5nlacZ/nlacZ mutants correspond to a loss of function of bothMyf5 andMrf4 at these early stages. By contrast,Mrf4was expressedin both Myf5 loxP/loxP and Myf5GFP-P/GFP-P embryos (Fig. 2g, k, n).The onset of Mrf4 expression was not appreciably affected in thehypaxial somite ofMyf5 loxP/loxP embryos at E10 (Fig. 2c, g), whereas

Mrf4 was not detected at this stage in Myf5GFP-P/GFP-P embryos butwas observed at later stages (Fig. 2n; data not shown). As inMyf5nlacZ/nlacZ embryos, differentiated cells were lacking in thesemutants at these early stages (Fig. 2i, j).We showed previously that Myod expression in somites was

delayed until after E10.5 in Myf5nlacZ/nlacZ embryos, and proposedthat Myf5 acts upstream of Myod13 (Fig. 2q, r; n ¼ 10). We nowshow that in Myf5 mutants in which Mrf4 expression is notcompromised, Myod expression is detectable before E10.5 (Fig. 2l,o, s; n ¼ 18). Furthermore, as in wild-type embryos, comparativeWM-ISH analysis showed that Mrf4 expression precedes that ofMyod in thesemutants (Fig. 2k, l, n, o; n ¼ 12), suggesting thatMrf4as well asMyf5might participate in the activation ofMyod. Similarresults were obtained with a fourth Myf5-null allele (Myf5dCT/dCT;data not shown). Given these findings, we would predict that thedirect inactivation of both Mrf4 and Myf5 would phenocopyMyf5nlacZ/nlacZ with respect to Myod expression. To test this,and to distinguish between effects in cis and in trans, we examinedMyod expression in mutants lacking functional Mrf4 as well asMyf5 (Fig. 1a). Myod expression was not detected at E10.5 inMyf5nlacZ/nlacZ or Mrf4nlacZ-P/nlacZ-P :Myf5 loxP/loxP embryos com-pared with controls (Fig. 2q–t). Furthermore, Myod expressionlevels in Mrf4nlacZ-P/þ:Myf5 loxP/þ mutants were about half thoseobserved in the wild type (data not shown). In addition, theexpression of desmin (which marks undifferentiated myoblasts aswell as differentiated muscle), myogenin, and differentiation mar-kers was lacking before E10.5 inMrf4nlacZ-P/nlacZ-P:Myf5 loxP/loxP andMyf5nlacZ/nlacZ embryos (Fig. 3d–i; data not shown). In contrast,desmin andmyogeninwere expressed by this stage inMyf5GFP-P/GFP-P andMyf5 loxP/loxP embyros (Figs 2m, p, 3b, c). Notably, desmin andmyogenin expression were absent before E9.5 in Myf5 loxP/loxP andMyf5GFP-P/GFP-P mutants (Fig. 2h–j; data not shown; n ¼ 8). Theirexpression appeared after that of Mrf4 transcript and protein, andbefore the expression ofMyod (Figs 2g, k, l, n, o, 3a–c; n ¼ 12, datanot shown). A graded delay in Myod expression and myotomeformation was therefore observed such that Myf5nlacZ/nlacZ .

Myf5GFP-P/GFP-P

. Myf5 loxP/loxP, and this was correlated with Mrf4expression. These results suggest that Mrf4 can determine muscleidentity and acts upstream of Myod (Fig. 2v). To test whether Mrf4can determine muscle identity autonomously, we examined Myf5:Myod double mutants.Extensive analysis of Myf5nlacZ/nlacZ:Myod2/2 mutants failed to

demonstrate skeletal myogenesis with a number of musclemarkers at all developmental stages4 (data not shown). However,robust skeletal muscle differentiation was observed at E12.5 inMyf5 loxP/loxP:Myod2/2 and Myf5GFP-P/GFP-P:Myod2/2 embryos inwhich Mrf4 expression was not fully compromised (Fig. 4a–d).Muscle differentiation was detected in both the epaxial and hypaxialregions (Fig. 4b, d, arrowheads), which was consistent with theobservation that MPCs of both express Mrf4 (Figs 2g, 3g, h). Thedesmin, myogenin and troponin T proteins were also observed inthese differentiated cells (Fig. 4e–i; data not shown). Skeletal musclewas not detected in the limbs of these double mutants at this stage(Fig. 4d; data not shown), in keeping with previous observationsthat skeletal muscle formation is delayed until E13.5 in limbs ofMyod2/2 embryos11. Although some MyHCþ cells and skeletalmuscle fibres were detected in the limbs at E14.5, fetal myogenesiswas severely compromised so that the new Myf5:Myod doublemutants were born essentially devoid of skeletal muscle. It is notablethat skeletal myogenesis in the head was absent in the new doublemutants (data not shown).Previous work demonstrated that Myf5:Myod double mutants,

and those carrying only one functional allele ofMyf5 orMyod, diedpostnatally2. Our examination of the new Myf5:Myod mutants inwhich Mrf4 was functional revealed that only the homozygousdouble mutant was lethal, whereas viable mice were generated fromall other allelic combinations (data not shown). Taken together,

Figure 3 Myotome formation and desmin staining in Myf5-null and Mrf4:Myf5 double-

mutant embyros. a–c, Progressively less anti-desmin staining in whole mount of E10.5

wild-type (a),Myf5 loxP/loxP (b) andMyf5 GFP-P/GFP-P (c) embryos. Note mislocalized muscle

precursors along the epaxial and hypaxial somite in the mutants. d, X-Gal staining of

E10.5 Mrf4 nlacZ-P/þ:Myf5 loxP/þ embryo. e, Phase-contrast image of transverse cryostat

section of d. f, Anti-desmin (red) staining of section in e showing correct myotome

formation. g, X-Gal staining of E10.5 Mrf4 nlacZ-P/nlacZ-P:Myf5 loxP/loxP embryo showing

MPCs mislocalized along the epaxial and hypaxial somite. Note that Myf5 is not essential

forMrf4 activation. h, Phase contrast image of section in g showingMrf4 expression in the

dermomyotome epithelium (inset). i, absence of desmin corresponds to lack of myotome.

dm, dermomyotome; fl, forelimb; my, myotome; nt, neural tube; white arrowheads,

epaxial; black arrowheads, hypaxial; arrow, Mrf4þ cells in limb.

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these findings reinforce the notion that both the nature of themyogenic factor and the total MRF levels are crucial for ensuringpostnatal viability.We also examined whether myogenesis would be enhanced or

deregulated in culture, as with myogenin null embryos in whichdifferentiated fibres are made in vitro but not in the embryo14,15.Monodisperse micromass cultures were made from all threedouble-mutant embyros at E11.5: Myf5 nlacZ/nlacZ:Myod2/2,Myf5GFP-P/GFP-P:Myod2/2 and Myf5 loxP/loxP:Myod2/2. Althoughskeletal myogenesis was readily observed with both Myf5GFP-P/GFP-P:Myod2/2 and Myf5 loxP/loxP:Myod2/2 by using various markers,neither desminþ cells nor skeletal muscle differentiation wasdetected in cultures of Myf5nlacZ/nlacZ:Myod2/2 double-mutantembryos (Supplementary Fig. 3a–e; data not shown; n ¼ 7),which is consistent with the in vivo observations2–4. Examinationby time-lapsemicroscopy and imaging ofMyf5GFP-P/GFP-P:Myod2/2

double mutants showed no detectable difference from controls inthe ability of these cells to migrate on the dish or in their ability to

divide (Supplementary Fig. 3f–h).Perturbations in cis, resulting from the insertion of foreign

sequences, have been reported for several genes, including Mrf4(refs 16–18), where Myf5 expression is affected. This locus isregulated by a complex array of proximal and distal regulatoryelements19,20. We now show that gene manipulations in Myf5 canalter the regulated expression of Mrf4. We showed previously thatPax3 and Myf5 act upstream of Myod because body muscles arelacking in Pax3Sp/Sp:Myf5nlacZ/nlacZ embryos13. Our present studiesshow that bothMrf4 andMyf5 act genetically upstream ofMyod forembryonic skeletal myogenesis in the body. In the absence of thesefactors, Myod expression is delayed, probably until Pax3-mediatedevents activate this gene13,21. Furthermore, our demonstration thatMyf5nlacZ/nlacZ mutants correspond to double Myf5:Mrf4 geneinactivations at early stages suggests that, in Pax3:Myf5 loxP/loxP

double mutants, which allow for Mrf4 expression, Myod shoulddrive skeletal myogenesis in the trunk (Fig. 5). Our preliminaryobservations suggest that this is so. Our finding that Mrf4 does notdirect skeletal muscle identity in the head points to a molecularheterogeneity in skeletal muscle stem cells at different anatomicallocations. The recent finding that MyoR:capsulin double mutantslack a subset of head muscles also supports this notion22.

In a recently described Myf5Dlox/Dlox:Myod2/2 double mutant,skeletal muscle was not detected at birth3; it is possible thatembyronic myogenesis is rescued in this mutant. Our findingsalso suggest that the insertion of a myogenin cDNA into the Myf5locus would permit Mrf4 expression23. Indeed, the muscle pertur-bations in Myf5myg-ki/myg-ki:Myod2/2 resemble those of the newMyf5:Myod double mutants described here. We also propose thatMrf4 or Myod is necessary to activate the myogenin gene, becauseMrf4:Myod double mutants phenocopy the myogenin null pheno-type15.

Finally, our findings provide in vivo evidence consistent withpreviously unexplained molecular observations showing that thechromatin-remodelling domain of Mrf4, but not that of myogenin,can substitute for Myod24. Those studies in vitro, and ours in vivonow support the notion that the formation of skeletal muscledepends on three determination factors which direct multipotentprogenitors into the myogenic lineage. A

Figure 4 Myf5:Myod double mutants make muscle. a–d, Transverse cryostat sections of

E12.5 control Myf5 GFP-P/GFP-P:Myod þ/2 (a), Myf5 loxP/þ:Myod þ/2 (c), and double-null

mutant Myf5 GFP-P/GFP-P:Myod 2/2 (b), Myf5 loxP/loxP:Myod 2/2 (d) embryos stained with

anti-MyHC antibody. Note lack of limb muscles in d compared with c at this stage.

e–j, High magnification of anti-MyHC (e, h) anti-desmin (f, i) and anti-myogenin (g, j)

antibody stainings of control (e–g) and double-null (h–j) embryos. Arrowheads indicate

epaxial (red) and hypaxial (white) muscles. FL, forelimb; NT, neural tube. Scale bar,

300mm (a–d); 30 mm (e–j).

Figure 5 Genetic hierarchy governing the acquisition of skeletal muscle identity in the

mouse. Pax3, Myf5 and Mrf4 act upstream of Myod for skeletal myogenesis in the trunk.

Mrf4 can direct embryonic, but not fetal, skeletal muscle identity and differentiation in

the absence of Myf5 and Myod. Myod is initially activated by Myf5 and Mrf4, and later

through Pax3 (ref. 13). Mrf4 drives myogenesis in the embryonic trunk and limbs but not

in the head or the fetus. Myf5, Myod and Mrf4 are determination genes. Arrows depict

genetic relationships, and do not necessarily imply direct interactions.

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MethodsGene targeting, generation of mutant mice, and genotypingEmbryonic stem cells (CK35, provided by C. Kress25) were targeted with the constructsindicated in Fig. 1a as described previously5. Diphtheria toxin5, or PGK-DTA-bpA (a giftfrom P. Soriano) and PGK-puromycin-bpA (a gift from A. Bradley) was used for selectionin embryonic stem cells. The enhanced green fluorescent protein (EGFP) contains aVal164Ala mutation in pEGFP-C1 (Clontech; a gift from H. LeMouellic) and is followedby an SV40pA sequence. BothMyf5nlacZ andMyf5GFP-P comprise a ScaI–BstYI deletion inexon 1 (122 amino acids) and thus lack the bHLH domain, whereas the floxedNeo–Hygrocassette inMyf5NH was an insertion at the ScaI site in exon 1 (residue 13). The remainingstructure ofMyf5 is intact in all alleles. The two initiator ATG codons ofMyf5 inMyf5NH

andMyf5GFP-Pwere mutated (ATG to GTG). The first ATG codons suitable for translationin Myf5 loxP (residue 69, non-Kozak; residue 82, Kozak) are located in the chromatinremodelling (amino acids 39–78) and basic domains. Myf5GFP-P/GFP-P, Myf5loxP/loxP andMyf5nlacZ/loxP were viable and were reduced in size.Myod (provided by M. Rudnicki2) andMyf5 (129sv/C57BL6/DBA2) mutant mice were interbred to obtain double-mutant mice.A ubiquituously expressing PGKCre deleter strain (provided by Y. Lallemand26) was usedto generate Myf5 loxP and Mrf4nlacZ-P/þ:Myf5 loxP/þ. Mrf4 was targeted with an nlacZreporter in embryonic stem cells previously targeted with, and in cis to,Myf5NH (data notshown). All embryos and mice were genotyped by Southern blotting or polymerase chainreaction (Supplementary Methods), and double mutants were genotyped again aftersectioning. Myf5nlacZ and Myf5GFP-P allelic combinations were also phenotyped formislocalized MPCs by staining with 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside(X-Gal)27 and GFP epifluorescence, respectively.

Processing of embryos and detection of transcripts and proteinEmbryos were processed for WM-ISH or immunohistochemistry13. Riboprobes forMyf5,Mrf4 and Myod were as described previously13,28. An Mrf4 riboprobe spanning 421 basepairs (KpnI–NdeI) was also used. For comparative WM-ISH experiments, age-matchedand litter-matched embryos were used with independent probe sets and litters, and theISH reactions were stopped at the same time. For protein detection by western blotting,the head and heart of E10.75 embryos were removed and lysates were prepared29. Primarypolyclonal anti-Myf5 antibodies (dilution 1:500, carboxy-terminal specific; Santa Cruz)and secondary anti-rabbit peroxidase-conjugated secondary antibodies (dilution1:40,000; Pierce) were applied and filters were treated with SuperSignal WestPicochemiluminescent substrate (Pierce).

Explant cultures and immunofluorescenceMicromass cultures were made as described previously13, and time-lapse studies aredescribed in Supplementary Methods. For immunofluorescence27, two antibodies wereused for Myf5 (polyclonal C-terminal29, and a polyclonal, dilution 1:800; Santa Cruz),myogenin (F5D, monoclonal, dilution 1:5; Developmental Studies Hybridoma Bank,NICHD), Myod (monoclonal, dilution 1:100; Dako), myosin heavy chain (polyclonal,dilution 1:400; provided by G. Cossu) and desmin (monoclonal, dilution 1:100; Dako).Secondary antibodies were AlexaFluor 488 and 594 (Molecular Probes). For whole mountantibody staining see Supplementary Methods and http://www.pasteur.fr/recherche/unites/Csd. Images were taken either with a Zeiss Axiocam or Nikon numerical cameraand a Zeiss fluorescent SZ11 or Axiophot microscope. Images were assembled in AdobePhotoshop and Indesign.

Received 13 March; accepted 23 July 2004; doi:10.1038/nature02876.

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Supplementary Information accompanies the paper on www.nature.com/nature.

Acknowledgements We thank S. Shorte, E. Perret and P. Roux at the Pasteur Dynamic Imaging

Center, andC. Cimper for assistance. The laboratories of S.T. andM.B. were funded by the Pasteur

Institute, CNRS, AFM, ACI Integrative Biology programme of the French Ministry. M.B. was

funded by the European Union. S.T. was also funded by grants from the HFSPO, Association pour

la Recherche sur le Cancer. B.G.-M. received a fellowship from the ARC, and V.S. a grant from the

Fondation pour la Recherche Medicale and Pasteur Institute. L.K.-D. was funded by the HFSPO,

the EU and Pasteur Institute.

Competing interests statement The authors declare that they have no competing financial

interests.

Correspondence and requests for materials should be addressed to S.T. (shaht@pasteur.fr).

..............................................................

Exogenous control of mammaliangene expression throughmodulation of RNA self-cleavageLaising Yen1, Jennifer Svendsen1, Jeng-Shin Lee1, John T. Gray1,Maxime Magnier1, Takashi Baba2, Robert J. D’Amato2

& Richard C. Mulligan1

1Department of Genetics, Harvard Institute of HumanGenetics, HarvardMedicalSchool, and Division of Molecular Medicine and2Department of Surgery, Children’s Hospital, Boston, Massachusetts 02115, USA.............................................................................................................................................................................

Recent studies on the control of specific metabolic pathways inbacteria have documented the existence of entirely RNA-basedmechanisms for controlling gene expression. These mechanismsinvolve the modulation of translation, transcription terminationor RNA self-cleavage through the direct interaction of specificintracellular metabolites and RNA sequences1–4. Here we showthat an analogous RNA-based gene regulation system can effec-tively be designed for mammalian cells via the incorporation ofsequences encoding self-cleaving RNA motifs5 into the transcrip-tional unit of a gene or vector. When correctly positioned, thesequences lead to potent inhibition of gene or vector expression,

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