INTRODUCTION

In vertebrate embryos, skeletal muscle is derived from twocomponents: a connective tissue substratum that develops insitu and provides the form and anchorage to the skeleton(Chevalier and Kieny, 1982; Köntges and Lumsden, 1996), andthe myoblasts, which migrate into position, differentiate intomyotubes and provide the bulk of the muscle and itscharacteristic histotype (Chevalier et al., 1977; Christ et al.,1977). We are interested primarily in the commitment ofmyoblasts, which derive from the paraxial mesoderm thatforms immediately adjacent to the neural tube duringgastrulation. In the trunk, the segmental units, the somites,which are located either side of the neural tube, formsequentially from the anterior end of the presomitic mesodermsuch that the most cranial pair was the first to be born (review:Christ et al., 1992). The somites produce the myoblasts of thetrunk, limbs and tongue. The remaining head myoblasts arederived from the anterior paraxial mesoderm, which is notovertly segmented (Noden, 1986; Trainor and Tam, 1995).

It is generally assumed that the developmental processes ofsomitic myogenesis in the mouse will be essentially the sameas those defined using direct cell marking experiments in chickand quail embryos (review: Christ and Ordahl, 1995). Theepaxial myoblasts (which migrate into the prospective musclesdorsal to the transverse processes of the vertebrae) appear first,originating at the dorsomedial lip of the dermomyotome. Thecells involute from the epithelial edge and accumulate onthe inner surface of the dermomyotome to form the dorsalcomponent of the myotome. The behaviour of hypaxialmyoblasts depends on their position along the craniocaudalaxis. In most trunk somites, some hypaxial myoblasts involutefrom the ventrolateral lip of the dermomyotome andaccumulate on its inner surface forming the ventral componentof the myotome. However, other presumptive myoblastsremain attached to the epithelial dermomyotome forming thesomitic bud, which invades the lateral body wall by blastemalgrowth and then differentiates in situ. At the appropriatesomitic levels, cells also detach from the ventraldermomyotomal lip and migrate into their final location in the

3745Development 127, 3745-3757 (2000)Printed in Great Britain © The Company of Biologists Limited 2000DEV2592

The development of skeletal muscle in vertebrate embryosis controlled by a transcriptional cascade that includes thefour myogenic regulatory factors Myf5, Myogenin, MRF4and MyoD. In the mouse embryo, Myf5 is the first of thesefactors to be expressed and mutational analyses suggestthat this protein acts early in the process of commitment tothe skeletal muscle fate. We have therefore analysed theregulation of Myf5 gene expression using transgenictechnology and find that its control is markedly differentfrom that of the other two myogenic regulatory factor genespreviously analysed, Myogenin and MyoD. We show thatMyf5 is regulated through a number of distinct and discreteenhancers, dispersed throughout 14 kb spanning theMRF4/Myf5 locus, each of which drives reporter geneexpression in a particular subset of skeletal muscle

precursors. This region includes four separate enhancerscontrolling expression in the epaxial muscle precursors ofthe body, some hypaxial precursors of the body, some facialmuscles and the central nervous system. These elementsseparately or together are unable to drive expression in thecells that migrate to the limb buds and in some othermuscle subsets and to correctly maintain expression at latetimes. We suggest that this complex mechanism of controlhas evolved because different inductive signals operate ineach population of muscle precursors and thus distinctenhancers, and cognate transcription factors, are requiredto interpret them.

Key words: Myf5, Myogenic regulatory factor, MRF, Muscle,Branchial arch, Epaxial, Hypaxial

SUMMARY

The expression of Myf5 in the developing mouse embryo is controlled by

discrete and dispersed enhancers specific for particular populations of

skeletal muscle precursors

Dennis Summerbell*, Peter R. Ashby ‡, Oliver Coutelle, David Cox*, Siu-Pok Yee § and Peter W. J. Rigby* ,¶

Division of Eukaryotic Molecular Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK*Present address: Section of Gene Function and Regulation, Institute of Cancer Research, Chester Beatty Laboratories, 237, Fulham Road, London, SW3 6JB, UK‡Present address: Wellcome Trust Building, University of Dundee, WTB/MSI Complex, Dow Street, Dundee DD1 5EH, UK§Present address: Cancer Research Laboratories, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, N6A 4L6, Canada¶Author for correspondence (e-mail: p.rigby@icr.ac.uk)

Accepted 20 June; published on WWW 9 August 2000

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forelimbs and hindlimbs and the thoracic and pelvicdiaphragms. Head muscle formation is less well understood butexperiments in both mouse and chick suggest that myoblastsfrom the occipital somites and head mesoderm follow acomplicated program of cell migration in which myoblastsmigrate first into the hypoglossal cord (Noden, 1983;Mackenzie et al., 1998) or into the branchial arches (Hackerand Guthrie, 1998) and then back out into appropriate locationsin the tongue and head.

While each of these groups of cells from the somites oranterior paraxial mesoderm differentiate into myoblasts, theyfollow very different paths and presumably respond to differentenvironmental signals that govern their behaviour.Manipulative embryological and explant experiments, in bothchick and mouse, have shown that the environmental signalsthat instruct cells as to their skeletal muscle fate emanate fromaxial structures including the notochord (Pourquie et al., 1993)and neural tube (Teillet and Le Douarin, 1983; Rong et al.,1992; Buffinger and Stockdale, 1994; Munsterberg and Lassar,1995), from the overlying surface ectoderm and from lateralplate mesoderm (Pourquie et al., 1995; Munsterberg et al.,1995; Dietrich et al., 1998). The effects of some of thesesignals can be mimicked by well-characterised molecules, forexample Sonic hedgehog (Shh) and various Wnt proteins(Cossu et al., 1996a). The signals induce a cascade oftranscription factors that involves the four myogenic regulatoryfactors (MRFs): Myf5 (Braun et al., 1989), Myogenin (Myog;Edmondson and Olson, 1990; Wright et al., 1989), MRF4(Rhodes and Konieczny, 1989; Braun et al., 1990; Miner andWold, 1990) and MyoD (Davis et al., 1987), which aremembers of the basic helix-loop-helix (bHLH) superfamily(Weintraub et al., 1989). In the mouse, Myf5 is the first suchgene to be expressed at 8.0 dpc (Ott et al., 1991), followedwithin half a day by Myog (Sassoon et al., 1989). It has beenshown that Myogexpression depends on an E-box, theconsensus binding site for the bHLH factors themselves andthe available data are all consistent with that binding proteinbeing Myf5 (Cheng et al., 1993; Yee and Rigby, 1993). InMyf5-null mice, somitogenesis is delayed by several days, asis the onset of Myog expression, although thereafter muscledevelopment proceeds normally, presumably because of aMyoD-dependent pathway (Braun et al., 1994). In Myog-nullmice, myoblasts accumulate in normal numbers but terminaldifferentiation is drastically reduced and most muscles do notform (Venuti et al., 1995). This indicates that Myogis involvedin cytodifferentiation. MyoD acts preferentially in limb andbranchial arch myogenesis (Kablar et al., 1997; Mankoo et al.,1999) and plays a central role in regenerative pathways in adultanimals (Megeney et al., 1996), while the function of MRF4remains unclear (Olson et al., 1996) although it may overlapwith that of MyoD(Rawls et al., 1998). The simplest view ofskeletal muscle development in the mouse would thus have theextracellular signals inducing the expression of Myf5, whichthen directly activates the Myog gene, and is also likely to beinvolved in chromatin remodelling at other loci expressed inskeletal muscle (Gerber et al., 1997). Myogenin in turnactivates the genes encoding the terminal differentiationproducts. According to this view, Myf5 is both the initiator andthe coordinator of the myogenic cascade.

If we are to understand the control of skeletal muscledetermination, we need to identify all of the transcription

factors that interact with theMyf5 promoter and enhancers.Such knowledge would open the way to elucidation of thebiochemical mechanisms by which the inductive signalsregulate the activities of these factors. We have therefore usedreporter gene assays in transgenic mice to identify the elementsthat regulate Myf5expression. Here we provide evidence thatthe control of the expression of Myf5 is extremely complex,with discrete elements responsible for driving expression indifferent anatomical locations. Our data show that the controlmechanisms for Myf5are distinct from those that operate foreither Myogor MyoD.

MATERIALS AND METHODS

Isolation of a genomic clone containing the murine Myf5locusA cosmid (MF5.2) was isolated by screening a genomic libraryderived from a T-cell clone of (CBA × B10)F1 origin in the vectorcos202 (gift of D. Kioussis, NIMR) with a mouse Myf5 cDNA probe(gift of A. Buonnano, NIH, accession number: X56182). All of thereporter constructs used to produce transgenic mice were derived fromthis cosmid using standard recombinant DNA techniques (Sambrooket al., 1989). Full construction details and maps of all constructs areavailable on request.

Preparation of Myf5-lacZ fusion constructsFor further manipulations, a 14 kb KpnI-EcoRI cosmid fragmentcontaining Myf5 was subcloned into pBluescript KS(+) (Stratagene)with a modified polylinker. PCR was used to mutate the ATG of thetranslational start codon of Myf5into a BamHI restriction site (Yeeand Rigby, 1993), and a BamHI cassette containing the lacZ reporter,which included a nuclear localisation signal and an SV40polyadenylation sequence (gift of R. Krumlauf, NIMR) was insertedat this site. This construct (#1) was used to generate a deletion series(constructs #1-7). Construct #4 was prepared similarly to constructs#1-3 and 5-7 but the reporter cassette came from pD16.43 (Fire et al.,1990). Construct #4 utilises the endogenous Myf5 polyadenylationsequences. Constructs #10-13 contained the appropriate Myf5sequences cloned upstream of a β-globin minimal promoter driving acytoplasmic lacZ reporter with an SV40 polyadenylation sequence inplasmid BGZA, a pBluescript KS(+) based derivative of BGZ40 (Yeeand Rigby, 1993). Construct #14 contained the appropriate PCRgenerated Myf5 fragment (primer pair: IN1f [5′-ctgagggaacaggtgg-agaac-3′] and UTRr [5′-catgctgtataattgcacct-3′]) cloned upstream ofthe hsp68-lacZ-SV40poly(A) reporter gene (Whiting et al., 1991).

Prior to injection, novel reporter cassettes were tested for functionby transfection of 16 µg of plasmid DNA into mouse C2C12(myoblast), 10T1/2 (fibroblast) or Neuro2a (neuroblastoma) cellsusing the calcium phosphate method (Sambrook et al., 1989).

Analysis of transgenic animals, in situ hybridisation andhistologyTransgenic mice were produced as previously described (Yee andRigby, 1993). Transgenic mice were diagnosed using the primers:LZD (5′-gtttttcccgatttggctac-3′) and STD (5′-ggacaaaccacaactaga-atgc-3′) which span the lacZ-SV40 poly(A) junction; or in thecase of construct #4, Myf-140f (5′-caggcatctgtccttgttaattacag-3′) andNLSr (5′-ttgaaacgctgggcaatatc-3′), which span the Myf5-nuclearlocalisation signal boundary. Embryos for lacZ whole mounts andsections were fixed in Mirsky’s fixative (National Diagnostics) andstained using 500 µg/ml of X-gal in 5 mM K3Fe(CN)6, 5 mMK4Fe(CN)6, 2 mM MgCl2 in PBS with 0.1% NP40 at 37°C in thedark. In some cases, such stained embryos were lightly counterstainedin 0.1% aqueous Acid Fuchsin so as to enhance contrast betweensomites and other tissues. Whole-mount in situ hybridisation was

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3747Elements controlling Myf5 expression

carried out using digoxigenin-labelled riboprobes (Wilkinson, 1992)with modifications provided by D. Henrique and D. Ish-Horowicz(ICRF, Lincoln’s Inn Fields, London, UK). Riboprobes were preparedusing T7 RNA polymerase on an XbaI linearised 1063 bp (BbrPI-MscI) genomic subclone of Myf5. Only 354 bp of the 504 bp riboprobeform duplexes with Myf5mRNA. Some of the whole-mount-stainedembryos were embedded in paraffin wax or agarose and sectioned.

RESULTS

The expression pattern of the mouse Myf5 gene has previouslybeen studied using both radioactive in situ hybridisation (Ottet al., 1991) and histochemistry on embryos in which a lacZreporter gene has been knocked-in to one of the Myf5 alleles(Tajbakhsh and Buckingham, 1994). Whilst very informative,the published data do not provide a complete control set forour experiments. We therefore reanalysed the pattern ofMyf5 expression using digoxigenin whole-mount in situhybridisation.

Myf5 in situ expression patternFig. 1 shows a 9.5 dpc (25-somite) embryo that was hybridisedwith a digoxigenin-labelled Myf5 antisense probe and thenserially sectioned. Myf5transcripts were not detectable in thepresomitic mesoderm (psm) and at this stage first appeared incells at the dorsal lip of the dermomyotome of somite I. Thesecells upregulate expression of the gene as they involute to formthe myotome (m) in somite II. This expression domain liesclose to dorsal neural tube and ectoderm, but distant fromthe notochord/floorplate and lateral plate mesoderm.Approximately 4 hours later, Myf5 was strongly expressed inthe ventral myotome of somite IV, a domain close to ectoderm

and lateral plate but distant from neural tube or notochord.Note also some Myf5-expressing cells that do not obviouslybelong to either the dorsal or ventral domains (red arrows,somites II and IV). By somite VI, Myf5 was expressedthroughout the myotome and the somitic bud (sb) had formedin thoracic somites. It is now widely assumed that these dorsaland ventral domains of expression equate to the presumptiveepaxial and hypaxial muscle masses (Ordahl and Williams,1998). The hypaxial cells from somite XIV (corresponding tothoracic somite 1), which migrate into the thoracic diaphragm(td) expressed Myf5while the equivalent cells in the adjacentsomite XV (cervical somite 8), which later migrate into thelimb bud, did not (arrowhead). The Myf5-expressing cellspreviously described in the neural tube and midbrain (Ott etal., 1991; Tajbakhsh et al., 1994) appeared very faint and werenormally not visible in intact whole-mount embryos.

Expression of a 14 kb Myf5 transgeneConstruct #1 contains sequences from 8.6 kb upstream (KpnI)to 5.3 kb downstream (EcoRI) of the Myf5 transcriptioninitiation site (Fig. 2). The upstream end-point of this constructis within the first exon of the adjacent MRF4 gene, so as toremove the MRF4promoter and avoid the possibility thatexpression of MRF4 from the transgene altered normal muscledevelopment.

Expression began well before 8.5 dpc in the domain of thesomites (Figs 2a, 3a) which corresponds to the normal onset ofMyf5 expression (Ott et al., 1991; Fig. 1). At this stage, six ofthe nine somites were clearly expressing. In older embryos(Fig. 2b, 9.5 dpc), the transgene was expressed in the dorsallip of somite I as soon as it formed from the presomiticmesoderm (Fig. 2b, side panel arrow). This is precisely the

Fig. 1.The expression of Myf5in a 9.5 dpc (25 somite) mouseembryo. The centre panel showsan embryo that has beensubjected to whole-mount in situhybridisation with a Myf5probetogether with the anatomicallevel through which vibratometransverse sections were cut.Sections occupy a thickness ofabout one third of a somite. Notethat, in addition to the somites,there is expression in thebranchial arches. The apparentstaining in the head is a trappingartefact. The side panelstravelling clockwise show: psm,presomitic mesoderm with noexpression; somite I, newly bornsomite with strong expression in cells that haveinvoluted into the dorsal myotome (m) and faintexpression in the dorsalmost dermomyotome (dm)cells; somite II, much stronger staining in the dorsalmyotome and weak staining ventrally (red arrowsindicate Myf5-expressing cells that are not obviouslylocated in either the epaxial or hypaxial domains);somites IV and VI, the intensity and completeness of staining in the myotome (m) increases progressively as the somites mature, there isexpression in the somitic bud (sb); somite XIV, ventral myotome cells from thoracic somite 1 migrate in the direction of the presumptivethoracic diaphragm (td); somite XV, cells from the ventral myotome that will migrate into the limbs do not express Myf5 (arrowhead).

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same timing and location of Myf5 expression as shown in Fig.1.

At 10.5 dpc, expression in the hypaxial domain had begunat thoracic and lumbar levels and included the somitic bud(Figs 2c, 3c, arrow). At all times, expression in the hypaxialdomain was inappropriately weak relative to that in the epaxialdomain. This was particularly apparent at 11.5 dpc in thehypaxially derived intercostal muscles (Fig. 3d). Expression inthe relatively immature somites of the tail appeared normalexcept at the extreme ventral margin (Figs 2d, side panel, 3d,e,arrow).

While the transgene in construct #1 lines activated correctlyin the epaxial region of newly born somites, it was apparent,by comparison with the whole-mount in situ data, that in moremature somites the expression pattern was inexact. In thedorsal half of the somites, expression corresponded to thenormal pattern, but progressively extended down the posteriorrather than anterior margin of the somite (Figs 2b, 3b, arrow).The phase of inappropriate expression in the somites wastransient. Transverse sections at interlimb level of a 10.5 dpcembryo (Fig. 2c, side panel XVI) showed that, in youngsomites, the transgene was correctly expressed in the myotome(m) but also incorrectly expressed in the posteriordermomyotome (dm). Transverse sections through moremature rostral somites (Fig. 2c, side panel XXV) showed thatexpression became restricted to the myotome (m). The early,strong dermomyotomal expression was not characteristicof either whole-mount in situ hybridisation (Fig. 1) orheterozygous lacZ knock-in embryos (Tajbakhsh andBuckingham, 1994; Tajbakhsh et al., 1997), suggesting thatconstruct #1 lacks an element that normally repressesexpression in the dermomyotome. By 11.5 dpc, allinappropriate somitic expression had disappeared and lacZwasrestricted to the normal Myf5-positive population (Fig. 2d),except in the dermomyotome of the distal tail somites (Fig. 2d,side panel).

Expression in the branchial arches commenced at around 9.0dpc (20 somites; Figs 2b, 3b). While expression in the secondarch was constant and reliable, expression in arches 1 and 3was often faint and variable, even between siblings of the sameline. By 10.5 dpc, there was strong expression in facial muscleprecursors (Figs 2c, 3c), which continued at 11.5 dpc (Figs 2d,3d) but was downregulated by 12.5 dpc (Fig. 3e).

From 12.5 dpc onwards, expression was markedlydownregulated in the epaxial and facial muscle precursors (Fig.3e) and, by 13.5 dpc, the only muscle components stillexpressing were in the epaxial somitic compartment (Fig. 3f).This downregulation is not seen in heterozygous knock-inembryos (Tajbakhsh et al., 1997), indicating that construct #1lacks an element(s) required for the maintenance of expression.

We have never observed staining that parallels the knownexpression pattern of Myf5 in the limbs and thus conclude that

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Fig. 2.Whole-mount β-galactosidase histochemistry and serialsections of transgenic mouse embryos containing construct #1. (a) 8.5 dpc (9 somite): there is strong expression in 6 of the 9 somitesand in the head mesoderm (side panel, arrow). (b) 9.5 dpc (25 somite): the transgene expresses in the epaxial domain as soon asthe somite is born. Note also that the earliest expressing cells in thebranchial arches are seen around this stage in the second arch. Theside panel shows no expression in the presomitic mesoderm, it isbeginning to appear in somite I (arrow) and clearly visible in somiteII. (c) 10.5 dpc: whole-mount embryo showing anatomical levelsthrough which vibratome transverse sections were cut. Note stainingin the somites, branchial arches and ectopic expression in the headmesoderm. The section through somite XVI shows appropriateexpression in the myotome (m) but also ectopic expression in thedermomyotome (dm) while, in the more mature somite XXV,expression is restricted to the correct location in the myotome (m).(d) 11.5 dpc: while transgene expression persists strongly in theepaxial domain, it is inappropriately weak in the hypaxial domain.The side panel shows that, in tail somites, there is no expression inthe most ventral region. The diagram illustrates the structure ofconstruct #1.

MRF4 Myf5

3749Elements controlling Myf5 expression

construct #1 lacks at least one necessary element required forexpression in the limb, confirming the data of Zweigerdt et al.(1997).

Expression in the neural tube was inconsistent and weak andin whole mounts normally masked by strong expressionelsewhere. We were unable to detect neural expression in theappropriate location in the midbrain.

We routinely observed unexpected expression in headmesoderm, which was strong from 8.5 (Fig. 2a, side panel,arrow) until 12.5 dpc. Towards the end of this period theexpression started to concentrate in the meninges in the regionof the midbrain flexure (data not shown), presumably formingpart of the dura, which is usually considered to be ofmesodermal origin. It is noteworthy that this ectopic expression

Fig. 3.Time course of expression in a line ofmice carrying construct #1. (a) 8.5 dpc (9 somite): strong expression in the epaxialdomain of the somites and ectopically in headmesoderm. (b) 9.5 dpc (23 somite): expressionspreads ventrally into the hypaxial domain ofmore mature somites, expression begins in thebranchial arches. (c) 10.5 dpc: somiticexpression extends into tail, in mature somiteslocalises in myotome and in the thoracic regionextends into the presumptive intercostals. (d) 11.5 dpc: expression strong in all previouslypositive domains except intercostals whichremain weak, no indication of expression inlimbs. (e) 12.5 dpc: expression in bothintercostals and face downregulated. (f) 13.5dpc: expression only in presumptive backmucles of epaxial domain. Note the lack of limbexpression at all times.

Table 1. Percentage of expressing embryos with β-galactosidase-positive cells in identified anatomical domainsTransient expressors Line expressors Non-expressing Epaxial domain Hypaxial domain Branchial arches Neural tube1 Head mesoderm

Construct (n) (n) (n) (%) (%) (%) (%) (%)

Deletion series#1 8 8 10 83 100 83 17 72#2 9 5 78 56 67 11 56#3, 4 17 4 11 57 86#52 11 4 1 100 83 58#6,7 12 9 83 17#8 14 2 4 100#93 7 13 14 14 28

Enhancer seriesDorsal element #10 7 1 4 100 100Arch element #11 5 3 100Ventral element #12,13,14 32 6 18 100 24

1Cord expression was always difficult to diagnose as it was almost always weak. It was therefore difficult to see in weakly expressing embryos, and easilymasked by more superficial staining in strongly expressing embryos.

2A few placentae were not tested for transgenesis.3Five of the seven blue embryos had only light inconsistent ectopic expression patterns. Most sites of expression were outside the normal Myf5 expression

domains.

3750 D. Summerbell and others

Fig. 4. Deletion mapping of Myf5 regulatory elements. Details of all constructs used are shown on the right; constructs in grey are notillustrated. (a) Construct #1 (10.0 dpc): starting point of the deletion series. Immature somites initially express correctly along the dorsal lip (dl) of the dermomyotome but later have ectopic expression in the posterior part of the dermomyotome (dm). Correct myotomal (m)expression can be seen in the mature somites. Expression is also faintly visible in the neural tube (nt) beneath the more cranial somites. Theconsistently strong expression in the head mesoderm is ectopic. (b) Construct #2 (9.5 dpc): the expression pattern is unchanged.(c) Construct #3 (9.75 dpc): expression in the epaxial domain of the somites and in the head mesoderm is abolished; the same pattern ofexpression is observed with construct #4 (data not shown). (d) Construct #5 (10.0 dpc): the pattern of expression is very similar to theprevious two constructs but consistently gave stronger somitic and weaker arch expression. (e) Construct #7 (11.0 dpc): the remainingsomitic expression is abolished. The same pattern of expression is observed with construct #6 (data not shown). (f) Construct #8 (10.5 dpc):expression in the arches is abolished leaving only neural expression, seen as a double column from the dorsal surface. In 4 cases out of 14,there was strong ectopic expression at the caudal end of the embryo. (Not shown: construct #9, 294 bp upstream of the translation start siteis insufficient to drive neural tube expression.)

3751Elements controlling Myf5 expression

domain is also observed in transgenicmice carrying YAC constructs containingclose to 600 kb of DNA (Zweigerdt et al.,1997). Nonetheless we have no evidencefrom our in situ hybridisation studies thatthis head mesoderm is a normal site ofMyf5 expression.

Deletion mapping of Myf5regulatory elementsTo map control elements within construct#1, we generated a deletion series. Thenumbers of transgenic embryos and thepercentage expressing in differentanatomical domains are shown in Table 1and key members of the series areillustrated in Fig. 4. Removal of 2 kb fromthe 5′-end deleted the main body of theMRF4 gene (construct #2, Fig. 4b) butproduced no obvious changes in theexpression pattern. Removal of a further 3kb (construct #3, Fig. 4c) abolishedexpression in the epaxial domain of thesomites and the ectopic expression in thehead mesoderm. Following deletion of 1.4kb from the 5′-end and 1.8 kb from the 3′-end (construct #5, Fig. 4d), the patternremained unchanged except that construct#3 produced consistent and strongexpression in the branchial arches andweak expression in the somites, whileconstruct #5 showed the conversetendency. In constructs #3-5, where thereis no epaxial somitic expression, it is clear(Fig. 4c,d) that in cervical, brachial andtail somites there is little or no hypaxialexpression, while in the somites betweenthe limbs such expression is moreextensive. Removal of the remainder ofthe DNA 3′to the reporter deleted most ofthe Myf5gene (construct #6 or #7, Fig. 4e)and abolished expression in the hypaxialdomain leaving only branchial arch andneural tube expression. When seen intransverse section, the latter coincidedprecisely with the location described byTajbakhsh et al. (1995, Fig. 5b; our datanot shown). Removal of a further 1 kbfrom the 5′ intergenic region (construct#8, Fig. 4f) abolished arch expression andleft expression only in the neural tube.This neural element on the homologouspromoter driving lacZ had a highincidence (28%) of strong ectopicexpression, particularly at the caudal endof the embryo (data not shown). Finally,removal of a further 420 bp down to a 294bp minimal promoter (construct #9, datanot shown) abolished the remainingexpression. These results delineated fourseparate elements which, in the context of

Fig. 5.Enhancer test forsufficiency. (a,d) Construct#10 (9.5 dpc, epaxial element):a 485 bp XmnI-BamHIfragment. The initialexpression along the dorsal lip(dl) is correct. In more maturesomites there is correctexpression in the dorsalmyotome but also ectopicexpression throughout the dermomyotome (dm) which persists until this structuredisperses to become the dermis. The transverse section through a mature somite of thesame embryo shows appropriate expression in the dorsal part (epaxial domain) of themyotome (m) but widespread ectopic expression in the dermomyotome (dm). b) Construct#11 (10.5 dpc, arch element): a 1111bp NheI-BsaBI fragment driving the β-globinpromoter expresses in the branchial arch domain of the somites. c, e, f, g) Construct #14(9.5 dpc, hypaxial element) a 2.9 kb fragment comprising the Myf5 introns and 3′ UTR onthe hsp68 promoter drives expression only in the ventral half of the somites. The close-up(which has been counterstained) shows clear expression in ventral somite IV (arrow),coinciding with the normal onset of hypaxial expression as shown in Fig. 1. The timing ofonset of expression is correct even though labelled cells appear first in the dermomyotomerather than the myotome. The coronal section (f) through immature somites of the embryoin panel c) shows that the initial expression pattern is mainly ectopic along the posterioredge of the dermomyotome (dm). The transverse section (g) through a more mature somiteof the same embryo shows that expression subsequently appears in the ventral myotome(m) but ectopic expression persists in the dermomyotome (dm). Expression in thenotochord is ectopic. Details of all constructs used are shown below; constructs in grey arenot illustrated.

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construct #1, are required for expression in the epaxial andhypaxial domains of the somites, the branchial arches and theneural tube.

The somite and arch elements act as enhancersComparison between constructs #2 and #3 (Fig. 4b,c)delineated a putative element required for expression in theepaxial domain of the somite. Analysis of subfragments of thisregion on the β-globin promoter drivinglacZ identified a 484bp XmnI-BamHI fragment sufficient for expression of thereporter in the epaxial domain (construct #10, Fig. 5a). In earlysomites (I to VII), the expression pattern appeared normal, butlater the dermomyotomal expression persisted and spreadventrally. Transverse sections (Fig. 5d) showed a transitionalphase in which there was expression in both dorsal myotomeand dermomyotome. This expression domain coincided withthe location of the precursors of the epaxial muscles and thusthe enhancer may represent the epaxial control element. Therewas also consistent expression in the head mesoderm. Strongectopic staining in the dermomyotome (dm) again suggested amissing element that acts negatively in the dermomyotome.

This enhancer never gave expression in the hypaxial myotome,in the arches or in the neural tube.

Comparison between constructs #7 and #8 (Fig. 4e,f)delineated a putative element required for branchial archexpression. Analysis of subfragments of this region on the β-globin promoter driving lacZ identified a 1111 bp NheI-BsaBIfragment (construct #11) that was sufficient to give theexpression pattern (Fig. 5b) previously described byPatapoutian et al. (1993). This expression domain coincidedwith the location of the branchial arch muscle precursors andthus the enhancer may represent an arch control element.

Comparison between constructs #5 and #6 (Fig. 4d,e)delineated a putative enhancer element required for expressionin the ventral half of the somites. Analysis of subfragments ofthis region on the β-globin or hsp68 promoters driving lacZshowed that the introns and 3′ UTR of Myf5 were sufficient togive this pattern (construct #12-14, Fig. 5c). The expressionpattern was anomalous in that it appeared in the ventral-posterior quadrant of the dermomyotome. While the timing ofexpression in the ventral half of the somite was correct, thelocation was inaccurate, spreading along the posterior rather

D. Summerbell and others

Fig. 6.The arch element and late maintenance of expression. (a-d) Constructs containing the upstream arch element maintain expressionthroughout the normal time course. Construct #5 expresses in the arches starting from 9.5 dpc, and is maintained through to 12.5 dpc (arrows).Note the well-developed facial muscles at 12.5 dpc. (e,f) Construct #14, (hypaxial element), a 2.9 kb fragment comprising the Myf5 introns and3′ UTR also drives weak expression in the arches (arrows). The expression is transitory, down regulating before 10.5 dpc.

3753Elements controlling Myf5 expression

than the anterior margin from somite IV (Fig. 5e). Coronalsections through immature somites showed that this posteriorexpression domain was located primarily in thedermomyotome (Fig. 5f). In more mature somites, transgeneexpression converged on the normal Myf5pattern, appearing inboth myotome and somitic bud (Fig. 5g). It later extended intothe intercostal muscles of thoracic somites (data not shown).In some cases, these constructs also gave weak transientexpression in the branchial arches (see below). They alsotended to express ectopically in dorsal root ganglia. While thisenhancer activated expression in the ventral part of the somiteand is complementary to the epaxial control element describedabove, it was insufficient to drive the full hypaxial expressiondomain.

Late maintenance activityWe have already drawn attention to a number of differencesbetween the pattern of expression produced by our largestconstruct and the pattern as shown by the lacZ knock-in(Tajbakhsh and Buckingham, 1994; Tajbakhsh et al., 1997).These are (1) posterior rather than anterior expression inventral somites, (2) missing limb expression, (3) ectopicexpression in head mesoderm, (4) persistent expression indermomyotome, and (5) premature downregulation in severalpopulations of muscle precursors. We do not know how manyadditional elements will be required to correct these defects.While the premature loss of expression, most obvious in theventral somites, indicates a requirement for a distal element(s)that maintains expression at late times (Figs 2d, 3d-f), latemaintenance of expression is also influenced by elementsproximal to Myf5.

Construct #11 was sufficient to drive expression in thebranchial arches (5 out of 5 transient embryos, Table 1; Fig.5b). We have no lines from this construct; however, all 8 linesfrom constructs #3 and #5 (which contain the arch controlelements defined by construct #11) expressed from 9.5-12.5dpc (Fig. 6a-d, arrows). This pattern of expression was alsoconsistent with other transient data (e.g. Fig. 4e, construct #7)and with the description of Patapoutian et al. (1993). Incontrast, construct #1 downregulated arch expression from11.5 dpc (8/8 lines, compare Fig. 2d and e with Fig. 6c and d).This suggests that there are sequences within construct #1 butnot in construct #3 which block the late maintenance of archexpression. This block is itself suppressed in the wider contextof the lacZ knock-in (Tajbakhsh and Buckingham, 1994;Tajbakhsh et al., 1997). Note that the late arch maintenanceelement in construct #11 was required from 10.0 dpc (seebelow) but was suppressed in construct #1 from 11.0 dpc.

Constructs #12-14 were sufficient to drive expression inventral somites (32/32 transient embryos and 6/6 lines) butwere also able to drive expression in the branchial arches (5/32transient embryos and 4/6 lines; Table 1; Fig. 5c). In isolation,the intragenic sequences downregulated their expression in thehypaxial domain from 10.5 dpc and in the arch domain frombefore 10.0 dpc (Fig. 6e,f; arrows). In the transient embryos,the five that expressed within the arches all lay within the 9.0-10.0 dpc time window indicated by the lines, while most of thenon-expressing transient embryos were older (10.5 dpc). Inboth domains, downregulation was earlier than in otherconstructs. In the context of constructs #1-#5 the characteristicmodified hypaxial expression was maintained until 11.5 dpc(Figs 3d, 6c; black arrow). In the context of constructs #1-#7

Nhe

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b cFig. 7.Summary of knownexpression domains and theircontrol elements. (a) Identifiedcontrol elements are colourcoded for the region of theembryo (b) in which they affectthe expression pattern. (c) Thelocation of the expressiondomains is then shown intransverse section relative toregions which have beenproposed as sources of signalsthat may lie upstream of themyogenic cascade.

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and #11 branchial arch expression was maintained until at least11.5 dpc (e.g. Figs 3d, 4e, 6c,d; arrows).

DISCUSSION

Our data show that the transcription of Myf5 is controlled bya number of elements dispersed throughout the MRF4/Myf5locus (Fig. 7). Each of the three elements that we haveexamined in detail functions as a classical enhancer; it iscapable of imposing on a heterologous promoter a discretesubset of the overall Myf5expression pattern, and there is,thus far, no evidence of overlap or synergy between theseenhancers. It appears that the transcription of Myf5 in theepaxial, hypaxial and facial muscle precursors is independentlycontrolled and, furthermore, the data indicate that there areseparate regulatory circuits operating in subsets of the hypaxialand facial precursors. The facts that even our largest constructgives no expression in the limbs, fails to maintain expressionproperly after 12.5 dpc and allows ectopic expression in thedermomyotome, indicate that further control elements mustexist either 5′ of MRF4 or 3′ of Myf5. It is thus clear that theregulation of Myf5is markedly more complex than that ofMyoD or Myog, genes that are known to be downstream ofMyf5 in the MRF transcriptional hierarchy. We conclude thatthe regulation of Myf5is tailored to its role as the determinationgene for skeletal muscle. The initiation of the transcriptionalcascade that leads to terminally differentiated skeletal musclecells requires that Myf5 transcription be initiated in a varietyof distinct progenitor cell populations; each population islocated in a different signalling environment and the gene (orthe locus) has evolved to have a distinct enhancer capable ofinterpreting each signalling environment.

The epaxial enhancer that we have characterised, locatedclose to the MRF4 gene, is capable of activating transcriptionin the dorsomedial lip of the dermomyotome of newbornsomites and is thus likely to be responsible for the initalactivation of Myf5 transcription. We cannot exclude thepossibility that there are other, more remote enhancers that alsofunction in the epaxial domain, and it is clear from the presentdata that this enhancer is normally constrained by a negativelyacting element that prevents expression in the dermomyotome.In the enhancer tests, this element downregulated sharplybetween 11.5 dpc and 12.5 dpc but, in the context of our largestconstruct, retained levels of expression at later stages thatwere similar to the Myf5 heterozygous lacZ knock-in mice(Tajbakhsh et al., 1997). It may therefore require a latemaintenance element, not yet identified, which lies within ourlargest construct.

In agreement with the previous data of Patapoutian et al.(1993), we find that sequences from the intergenic region actto initiate transcription in the cells that have migrated from theanterior paraxial mesoderm into the branchial arches, andwhich subsequently give rise to facial muscles. However,examination of a large number of transgenic embryos leads usto the conclusion that this intergenic enhancer does not operatewith equal efficiency in all facial muscle precursors. It is mostefficient in arch 2 where expression is constant and reliable. Itis less reliable in arch 1 and even less so in arch 3; however,expression is very variable even within the same line. Despitethis variability, where there is expression, it appears on time at

9.5 dpc and is maintained through to 12.5 dpc. However in thecontext of our largest construct, late expression driven by thiselement is blocked by a second element lying in or near theepaxial enhancer region, which is itself blocked by a missingdistal element in the context of the entire locus. This blockingaction affects maintenance but not initiation.

The intragenic enhancer is particularly complex. It clearlyfunctions in the hypaxial, as opposed to the epaxial, domainbut it is not sufficient to direct all aspects of the hypaxialexpression pattern. In isolation, it drives reporter geneexpression in only the posterior half of each somite and it doesnot appear to operate in the most ventral regions of the somiticbud. Like the epaxial enhancer, this element is normallyconstrained by a negatively acting element that functions in thedermomyotome and, like the intergenic arch element, it has acontext-dependent requirement for maintenance. In isolation,the hypaxial enhancer downregulates in the somites from 10.5dpc. In all constructs containing intergenic sequence (includingboth the arch element and the Myf5 promoter), expressionpersists in the somites until 11.5 dpc and then downregulates.In the Myf5heterozygous lacZknock-in mice, hypaxial somiticexpression persists until at least 14.5 dpc (Tajbakhsh et al.,1997). The intragenic enhancer also drives weak transientexpression in the arches but, because of the strength ofexpression, it is not possible to tell if the pattern mirrors thatof the intergenic arch element. Only the strongest expressinglines showed this activity and it downregulated from 10.0 dpc.The onset of downregulation in the arches significantlypreceded downregulation in the somites.

We have further shown that the expression in the neural tube,which has been extensively documented by Tajbakhsh et al.(1994), is controlled by an element close to the Myf5 minimalpromoter. This minimal promoter, defined in this analysis asthe 294 bp upstream of the transcriptional start site, is, inisolation, incapable of directing any discrete aspect of theoverall Myf5 expression pattern.

In summary, our data show that the regulation of the mouseMyf5 gene is much more complex than might have beenexpected from comparison to other MRFs, and that expressionof Myf5 requires multiple discrete enhancer elements that areresponsible for driving or suppressing expression in at least fivedifferent anatomical expression domains.

Myf5 regulation is different from that of Myog andMyoDThe complex and highly dispersed arrangement of the Myf5transcriptional control elements contrasts with the relativesimplicity of the elements that control the transcription of theclosely related genes Myog and MyoD, both of which actdownstream of Myf5 in the transcriptional hierarchy. In thecase of MyoD, there are two identified enhancers. A 258 bpelement that lies 20 kb upstream of the transcriptional start siteappears to recapitulate the full expression pattern (Goldhameret al., 1992, 1995) while a proximal enhancer approx. 5 kbupstream of the promoter, although widely expressed, givesdelayed expression in limb buds and branchial arches (Tapscottet al., 1992; Asakura et al., 1995). Linker scanning mutagenesisof the distal element identified sequences that are not requiredin head muscle precursors or in the migratory precursors of thelimb and diaphragm but are essential in most body muscleprecursors (Kucharczuk et al., 1999).

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3755Elements controlling Myf5 expression

In the case of Myog, all of the cis-acting sequences requiredfor correct spatial and temporal expression in the embryo liewithin 133 bp upstream of the transcriptional start site,although there are additional elements within 1 kb of thepromoter that increase the level of expression (Cheng et al.,1993; Yee and Rigby, 1993). Mutation of the MEF2-bindingsite within the minimal control sequences specifically affectsexpression in the epaxial domain, while mutation of theadjacent MEF3 binding-site affects expression in the epaxialdomain and in the muscle progenitors that migrate to thelimb and head (Spitz et al., 1998; P. R. A. and P. W. J. R.,unpublished data).

Thus, for both MyoDand Myog, there are different upstreamregulatory circuits in different muscle precursor cellpopulations. However, in neither of these cases are therediscrete enhancer elements that are specific for particularprecursor cell populations arising at distinct locations in theembryo. We believe that the more complex arrangement of theMyf5 control sequences reflects the fact that it alone initiatesthe myogenic cascade by responding to diverse sets ofinductive signals that vary depending on the location of theparticular population of myoblasts: epaxial somite, hypaxialsomite, face and limb. It has been argued that there is functionalredundancy between Myf5 and MyoD(Rudnicki et al., 1993;Braun et al., 1994). However, while MyoD can initiatemyogenesis in the absence of Myf5, it does not normally do so.At least in mice, Myf5 initiates the myogenic cascade beforeMyoD first appears and, indeed, in Myf5null knock-ins, theexpression of MyoD and Myog is significantly delayed(Tajbakhsh et al., 1997). In normal mice both genes liedownstream of Myf5: the fact that Myf5 has to respond todiverse inductive signals necessitates the complex controlsystem that we have revealed.

Signals regulating Myf5 expressionThe identification of signalling molecules that might act in theprocess of myogenesis has generally depended on explantculture systems that are not capable of distinguishing betweensignals that operate in particular domains of the somite.However, the diversity of signals and their suggested sites ofsynthesis (Fig. 7) suggest complicated graded distributionswithin the responding tissues: e.g. Shh in the notochord andfloorplate (Fan and Tessier-Lavigne, 1994); Wnts in the dorsalneural tube (Munsterberg et al., 1995; Stern and Hauschka,1995), Wnts and BMPs in the surface ectoderm and BMP4 inthe lateral plate mesoderm (Pourquie et al., 1996; Dietrich etal., 1998). Along the dorsoventral axis, different concentrationsof signals secreted ventrally from the notochord, the floorplateand the lateral plate mesoderm and dorsally from the surfaceectoderm and dorsal neural tube could potentially inducehypaxial and epaxial muscle precursors, respectively. Shh forexample has short- and long-range signalling functions (Lee etal., 1994; Marti et al., 1995; Roelink et al., 1995). A long-rangefunction of Shh is to induce competence in chick somites I-IIIto respond to Wnt signals from the dorsal neural tube. Indeedpresomitic mesoderm requires temporary exposure to Shh toenable it to subsequently respond to Wnt signals and initiatemyogenesis (Münsterberg et al., 1995). BMP4 has beenimplicated as a repressor of lateral Myf5 expression (Cossu etal., 1996b) and the BMP4 antagonist Noggin is expressed in

dorsal somites (Ikeya and Takada, 1998) which could permitactivation of Myf5epaxially but not hypaxially.

While it is entirely possible that there are unidentifiedmyogenic signals, combinatorial and dose-dependentsignalling are sufficient to allow those molecules alreadyimplicated in myogenic induction to uniquely specify thevarious populations of muscle precursor cells. We do not wishto imply that the signalling molecules that act on each myoblastpopulation are necessarily distinct, only that each populationis exposed to a particular combination of signals acting at aparticular concentration. According to this view, the initiationof Myf5 transcription in each cell population would depend onan enhancer that binds a particular set of transcription factorsand thus has distinct sequence motifs. Indeed, we have foundno evidence for recurrent sequence motifs in the variousenhancer regions examined (D. S., C. Halai and P. W. J. R.,unpublished data).

In the mouse, direct evidence on the relationship betweenputative signalling molecules upstream of Myf5 is becomingavailable as knock-outs of more of these genes are produced.In 9.5 dpc Wnt1 and Wnt3a double null embryos, thedorsomedial lip of the dermomyotome is lost and thus Myf5expression in the epaxial domain is abolished (Ikeya andTakada, 1998). In shh-null embryos, which fail to expressepaxial Myf5 properly, the dorsal lip of the dermomyotomeappears to form initially in young somites but soondisintegrates (Borycki et al., 1999). These data support a rolefor Shh and Wnt signalling in myogenesis but cannot saywhether such signals act directly on Myf5transcription. Thereare thus numerous candidates in the different anatomicaldomains that may be involved in the regulation of Myf5. Thenext step in our research will be the identification of requiredbinding sites within the enhancer elements.

Know your neighboursThree independent null mutations of the MRF4gene have beengenerated in mice (Braun and Arnold, 1995; Patapoutian et al.,1995; Zhang et al., 1995). Although the three alleles were allobtained by disruption of the MRF4gene through insertion ofa neomycin cassette involving different deletions of the locus,the phenotypes were surprisingly variable. Despite thisvariation, all three alleles have resemblances to the Myf5knockout. We have previously suggested that the two mostlikely, but not mutually exclusive, possibilities were thattranscription of the inserted neomycin selection cassette in thesame direction as Myf5 interferes with the expression of thelatter, or that the insertion/deletion removed essential Myf5regulatory elements lying close to the MRF4 gene (Olson etal., 1996).

In the Olson allele (Zhang et al., 1995), neomycin is insertedin the opposite transcriptional orientation to Myf5 and thedeletion is more extensive than in the Arnold (Braun andArnold, 1995) or Wold (Patapoutian et al., 1995) mutations.The Olson deletion extended from the PstI site in the first exonof MRF4 to the BamHI site in the intergenic region, spanningnoncoding regions within MRF4 and the adjacent intergenicregion. In this paper, we show that this allele deletes the entireepaxial control element of Myf5 (construct #10, Fig. 5) and thisis the most likely cause for the apparent Myf5phenotype.Nevertheless, it is possible that insertion of neomycin mightalso interfere with Myf5 transcription.

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Unlike the Olson allele, the Arnold and Wold alleles carrythe neomycin gene inserted in the same transcriptionalorientation as Myf5and, while both delete part of the first exonof MRF4and some adjacent sequences, most of the introns anduntranslated region remain intact. Our data show that removalof MRF4sequences deleted in the Wold allele does not producean obvious change in the expression pattern of Myf5 intransgenic mice (compare constructs #1 and #2, Fig. 3a,b).Therefore, the most-likely explanation for the Myf5phenotypesis interference of neomycin transcription with Myf5 expression.However, in the case of the Arnold allele, we cannot excludethe possibility that deletion of an additional 102 bp of 5′ non-coding sequences could also interfere with Myf5transcription.It is plausible that such elements exist since synteny betweenMRF4and Myf5 is conserved in one of the earliest vertebrates,the teleost Fugu rubripes, suggesting a functional requirementfor the two genes to remain linked as a pair throughoutvertebrate evolution (O. C., D. S. and P. W. J. R., unpublisheddata). We are currently investigating whether any of theelements that we have identified regulates MRF4 as well asMyf5.

ConclusionMyf5 is regulated very differently to the other MRFs in themyogenic cascade. We have defined discrete enhancers for theepaxial somite, hypaxial somite and branchial arches. Inaddition, we have mapped a neural element and providedindirect evidence for a missing dermomyotomal repressor, foran element required for correct hypaxial expression and for latemaintenance elements. We speculate that this discrete butdispersed arrangement is due to Myf5 having evolved so as torespond to the disparate developmental signals or combinationsof signals in different anatomical regions of the embryo and toinitiate the myogenic cascade appropriately in these regions.

We are grateful to the staff of the Biological Services Division,particularly Hannah Boyes, Jane Sealby and Zoe Webster for expertanimal husbandry; to Chandrika Halai for expert technical assistanceand to the members of the Division of Eukaryotic Molecular Genetics,past and present, for their critical reading of the manuscript. Duringpart of this project, P. R. A. was supported by a Fellowship from theInternational Human Frontier Science Program Organisation. O. C.held a Graduate Studentship from the Medical Research Council,which also paid for this work.

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