1979Development 121, 1979-1988 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

Mutations in a novel gene, myoblast city, provide evidence in support of the

founder cell hypothesis for Drosophila muscle development

Emma Rushton1,*, Rachel Drysdale1,†, Susan M. Abmayr2, Alan M. Michelson3 and Michael Bate1

1Department of Zoology, University of Cambridge, Downing St, Cambridge CB2 3EJ, UK2Department of Biochemistry and Molecular Biology, The Pennsylvania State University, 459 N. Frear Laboratory, University Park,PA 16802-4500, USA3Howard Hughes Medical Institute Research Laboratories, Brigham and Women’s Hospital, Thorn Research Building, 9th Floor,20 Shattuck Street, Boston, MA 02115, USA

*Author for correspondence†Present address: Department of Genetics, University of Cambridge, Downing St, Cambridge CB2 3EH, UK

We have used mutations in the newly identified genemyoblast city to investigate the founder cell hypothesis ofmuscle development in Drosophila melanogaster. Inembryos mutant for myoblast city the fusion of myoblastsinto multinucleate muscles is virtually abolished. Never-theless, a subset of the myoblasts develop specific muscle-like characteristics, including gene expression appropriateto particular muscles, migration to the appropriate part of

the segment, correct position and orientation, and contactby motor neurons. We suggest that this subset of myoblastsrepresents the proposed muscle founder cells and we drawan analogy between these founder cells and the musclepioneers described for grasshopper muscle development.

Key words: Drosophila, embryo, muscle, myogenesis, founder cell,cell fusion, myoblast city

SUMMARY

INTRODUCTION

The larval muscles of Drosophila melanogaster form acomplex but highly regular array on the body wall of the larva.The muscles are formed during embryogenesis by the fusionof myoblasts, cells of the somatic mesoderm. Midway throughdevelopment, small syncytia containing two or three nucleiappear at defined points in the somatic mesoderm (Bate, 1990).These syncytia are termed ‘muscle precursors’, as each onegives rise, by a continuing process of fusion andextension/migration, to a larval muscle, whose identity isdefined by its size, position, orientation on the body wall of thelarva and pattern of innervation.

Interestingly, fusion does not take place randomly amongmyoblasts, but always begins at particular points in themesoderm in close contact with the ectoderm (Bate, 1990). Amodel for muscle development has been proposed in whichmuscle differentiation begins with the segregation of foundercells – single cells in the somatic mesoderm – which seed thefusion process and which may in some way confer identity onthe developing muscle (Bate, 1990; Dohrmann et al., 1990;Bate et al., 1993).

This view is supported by the observation that genes suchas S59, apterous (ap) and vestigial (vg) are each expressed ina small number of larval muscles, and that earlier, myoblastsexpressing these genes behave like the predicted founder cells.Expression of these genes begins in the mesoderm in a smallnumber of cells before fusion. These cells fuse with neigh-

bouring cells, which then begin to express S59, ap or vg them-selves, and the resulting fused groups go on to form specificmuscles (Dohrmann et al., 1990; Bourgouin et al., 1992; Bate,1993; Bate et al., 1993). S59 and ap contain homeoboxsequences and so are implicated in DNA binding and tran-scriptional control (Dohrmann et al., 1990; Cohen et al., 1992).vg is related to a family of proteins involved in protein-proteininteractions (Williams et al., 1991). All three proteins arelocalised to the nucleus. The function of such genes in muscledevelopment is not known, but it is possible that they areinvolved in specifying the identity of the muscle precursors inwhich they are expressed. The best evidence for such afunction is provided by ap. In the absence of ap function thereis variable loss of one or more of the ap expressing musclesand strikingly, when ap is expressed ubiquitously under heat-shock control, extra muscles develop in a similar location andposition to some of the wild-type ap-expressing muscles, whilethe rest of the muscle pattern is apparently normal (Bourgouinet al., 1992).

The idea of the muscle founder cell in Drosophila waspreceded by the observation of pioneering muscle cells in thegrasshopper embryo. Muscle pioneers in the grasshopperstretch out and span the future territory of the muscle theyprefigure. The pioneers form a scaffold with which mesoder-mal cells fuse to form the mature musculature (Ho et al.,1983). Superficially, the muscle pioneers of the grasshopperare different from the muscle founder cells of Drosophila,because the pioneers extend processes to span their territory

1980 E. Rushton and others

before fusion takes place, while the proposed founder cellsfirst initiate fusion and form multinucleate muscle precursors.It is these precursors in Drosophila that resemble the musclepioneers of the grasshopper in that they start to explore theirterritory and form attachments to the epidermis. This differ-ence in timing may simply reflect the more rapid process ofembryogenesis in Drosophila as compared with the grass-hopper. While the existence of founder cells in Drosophilahas yet to be demonstrated, they have been proposed as amodel to explain early events in the formation of the musclepattern.

Here we describe the embryonic phenotype of lethalmutations in a gene called myoblast city (mbc). The principalcharacteristic of these mutations is lack of myoblast fusion,and we use this phenotype to examine the founder cell hypoth-esis in Drosophila. We present evidence in support of thefounder cell hypothesis and show that, in the absence offusion, two classes of myoblasts exist. The first classresembles muscle pioneers in that these cells stretch out andspan muscle territories, while the second class appears to con-stitute a pool of myoblasts with which the pioneers wouldnormally fuse.

MATERIALS AND METHODS

Genetic methodsAll marker mutations and balancer chromosomes used are describedin FlyBase (1994).

(a) Deficiencies in the 95 region were created as follows: TheP{ry+, lacZ}A189.2F3 line (Bellen et al., 1989) was obtained fromthe Bloomington Stock Center. It carries an enhancer trap element at95A, and homozygotes are phenotypically wild type. Males homozy-gous for P{ry+, lacZ}A189.2F3 were irradiated with 4000 rads of Xrays. Approximately 26,000 chromosomes were screened for the lossof the ry+ marker. A single deficiency, Df(3R)mbc-R1, with break-points in 95A5-7 and 95D6-11 was recovered in this screen.

A parallel mutagenesis utilised an insertion of the plasmidH{ry+har1} at 95A (Blackman et al., 1989). This hobo-associated ry+

insertion (referred to as TnM2) is homozygous lethal, with no obviousmuscle abnormalities. TnM2-bearing males balanced with TM6B(ry+) were irradiated with 4000 rads of X-rays and crossed to MKRS(ry)/TM2(ry) virgin females. Approximately 44,000 chromosomeswere examined for loss of the ry+ insertion. A single deficiency,Df(3R) mbc-30, with breakpoints in 95A5-7 and 95C10-11 wasrecovered.

(b) Four alleles of mbc were identified in several screens, on thebasis of failing to complement mbc deletions for embryonic viability:Homozygous red e males were fed 25 mM EMS in 5% sucrose (Lewisand Bacher, 1968). Mutagenized third chromosomes were recoveredover the TM3 Sb balancer chromosome and test crossed against theDf(3R)mbc-R1 chromosome. Three independent lethal alleles of mbc(mbcC1, mbcC2 and mbcC3) were recovered from a total of 1,440 chro-mosomes tested.

In a parallel analysis, isogenic ru st e males were fed 30 mM EMSin 10% sucrose. Mutagenized third chromosomes were recovered overa TM3 (Sb, ry) balancer and tested for complementation of Df(3R)mbc-30. One lethal allele of mbc (mbcS4) was recovered from a totalof approximately 2400 chromosomes tested. Independent recombina-tion mapping localised this allele to cytological region 95.

Immunohistochemical methodsEggs were laid and allowed to develop on yeasted apple-juice agarplates at 25°C. Embryos were fixed for 20 minutes in heptane and 4%

paraformaldehyde in phosphate buffer pH7.0, and devitellinized in a1:1 mixture of heptane and methanol with vigorous shaking.Embryos were blocked prior to incubation with antibody, inphosphate-buffered saline, 0.3% Triton-X (PBS-TX) + 0.5% bovineserum albumin (Sigma). Anti-myosin, used at a dilution of 1:1000 wasa gift from Dan Kiehart (Kiehart and Feghali, 1986). Anti-S59 wasproduced in collaboration with Fernando Jimenez and Mary Baylies,using an S59 cDNA clone kindly sent by Manfred Frasch (Dohrmannet al., 1990). Rabbit anti-S59 was used at a dilution of 1:5000. Anti-VG was provided by Sean Carroll and used at a dilution of 1:100(Williams et al., 1991). Anti-Connectin was provided by Rob Whiteand used at a dilution of 1:10 (Meadows et al., 1994). Anti-Groovinwas provided by Talila Volk and used at a dilution of 1:3. Anti-Per-oxidasin was provided by J. Fessler and L. Fessler and used at adilution of 1:1000.

Incubation with antibody took place overnight at 4°C. All washeswere in PBS-TX. Incubation in biotinylated secondary antibody(Vector Labs) was followed by Vectastain ABC Elite enhancement,and the staining was developed using 0.5% diaminobenzidene, 0.03%nickel chloride and 0.02% hydrogen peroxide. For the double stains,embryos were incubated first in anti-Peroxidasin, anti-Groovin, anti-S59 or anti-VG and developed in the presence of nickel chloride(giving a blue-black stain), then incubated in anti-myosin anddeveloped in the absence of nickel chloride (for a pale brown stain).

The anti-myosin-stained embryos shown in Fig. 1 were aged bymaking one-hour egg-lays and allowing the embryos to develop forthe appropriate length of time at 25°C. All other embryos were stagedusing morphological criteria.

Embryos were examined and photographed using a Zeiss Axiophotmicroscope.

RESULTS

Mutations in a new gene, mbc, severely affectmuscle developmentAlleles of mbc were recovered in a screen designed to makemutations in the 95 region of the third chromosome. Thisregion includes the gene nautilus (nau), a Drosophilahomologue of vertebrate MyoD. nau is expressed from an earlystage in developing muscles (Michelson et al., 1990). Resultsfor nau will be published elsewhere; here we concentrate onthe phenotype of mbc.

A total of 4 alleles of the mbc locus were isolated: mbcC1,mbcC2, mbcC3 and mbcS4. Deletion mapping using deficiencychromosomes (described in Materials and Methods) maps thembc locus between 95A5-7 and 95C10-11 on the right arm ofthe third chromosome. Deficiencies that remove the nau codingsequence complement the mbc mutations (Erickson andAbmayr, unpublished results), thus mbc is not nautilus.

All known mbc alleles are recessive and cause embryoniclethality. Epidermal development and differentiation appearnormal: cuticle preparations of mbc mutant embryos reveal noabnormalities in segmentation or patterning (data not shown).The embryos lie motionless in the vitelline membrane and failto hatch. Examination of the mbc mutant embryos with apolarised light microscope (Drysdale et al., 1993) shows astriking lack of differentiated muscle.

The same muscle phenotype was consistently found inembryos homozygous for Df(3R)mbc-R.1, Df(3R)mbc-30 andall four mbc alleles, and also in embryos carrying any combi-nation of deficiency and point mutation. This suggests that allmutations generated so far have a null phenotype.

1981Founder cells and Drosophila myogenesis

Fig. 1. Wild-type and mbcC1 mutant embryos stained using an antibody to muscle myosin. Staged embryos were obtained by making hour-longegg collections and allowing them to develop to the appropriate age at 25°C. (A) 13- to 14-hour old wild-type embryo showing mature musclepattern. White arrow points to muscle 25, black arrow points to muscle 29. v marks ventral oblique muscle group. (B) 13- to 14-hour old mbcmutant embryo showing unfused myoblasts lying in a segmented array. (C) 16- to 17-hour old mbc mutant embryo showing the twopopulations of myoblasts: stretched and rounded. (D) Enlarged view of myoblasts in a 14- to 15-hour old embryo. Black arrowhead points to apossible founder cell for a ventral oblique muscle. White arrowheads point to possible founders for muscle 25. (E) Enlarged view of ventraloblique muscles in a wild-type embryo. Muscle 29 is marked with arrowheads, and its characteristic process is marked with arrows.(F) Enlarged view of ventral myoblasts in a 14- to 15-hour old mbc mutant embryo. Arrows point to possible precursors of muscle 29, with thecharacteristic process. Anterior is to the left and dorsal is up. Scale bars, 30 µm (A,B,C); 10 µm (D,E,F).

Mutations in mbc cause a failure of fusionIn order to make a detailed analysis of the muscle phenotypeof embryos mutant for mbc, we used an antibody againstDrosophila muscle myosin to reveal the muscles during devel-opment (Kiehart and Feghali, 1986). In wild-type embryos,

muscle myosin is first expressed approximately 9 hours afteregg laying (AEL). At this time, myoblast fusion is under wayand every muscle is represented by a precursor (Bate, 1990).Myosin expression begins in a small number of precursorslying ventrally and laterally and rapidly extends to all the pre-

1982 E. Rushton and others

hour old embryos, double stained with anti-Peroxidasin (blue-black) andn (brown). Peroxidasin is expressed by macrophages (Nelson et al., Wild type. Arrow points to macrophage. (B) mbcC2/mbcC3 mutantrrows in B point to macrophages that have engulfed many cells,ome expressing myosin. Anterior is to the right, dorsal is up. Scale bar,

cursors and some of the cells that are about to fuse with them.By 13 hours AEL, the muscle pattern is complete and muscleattachments are forming on the epidermis (Fig. 1A).

In embryos mutant for mbc, myosin expression begins onschedule at 9 hours AEL, in cells that appear by their positionto belong to the somatic mesoderm. Because of their positionand muscle myosin expression, we identify these cells asmyoblasts. As in the wild-type, expression begins in a few cellsventrally and laterally, and by 10 hours, apparently extends tomost of the cells of the somatic mesoderm. The myosin-expressing cells are clearly organised into an array whichresembles the pattern of wild-type muscle precursors, with asegmentally repeated arrangement of ventral, lateral and dorsalcell clusters (Fig. 1B). Strikingly, with only a few exceptions,these myoblasts fail to fuse. Initially, all the cells are rounded,but from 11 hours AEL, a subset of mononucleate cells becomeslightly elongated, lying in positions and orientations similarto the multinucleate muscle precursors in wild-type embryos(Fig. 1C,D,F).

The elongated myoblasts first appear at about 11 hours AELand increase in number until about 13-15 hours AEL. After thistime some myoblasts become much longer, some nowspanning distances two or three times the length of normalmuscles. These myoblasts occasionally have morethan one nucleus, indicating that a small numberof cell fusions occur. At 13 hours AEL, there isstill a large number of rounded myosin-expressingmyoblasts, but from 14 hours onward, this popu-lation diminishes. Some cells may simply losemyosin expression, as many faintly stained andunstained rounded cells can be seen usingNomarski optics. However, cell death is alsoinvolved, as myosin-positive cells engulfed bymacrophages can be seen in preparations whichhave been doubly stained for myosin and peroxi-dasin (Nelson et al., 1994; Fig. 2).

Groovin is expressed in the epidermis at muscleattachment sites (Volk and VijayRaghavan, 1994).To investigate whether the elongated myoblastswere indeed finding their correct attachment sites,embryos were double-labelled using anti-myosinand anti-Groovin. Fig. 3 shows myosin-positivecells stretching out and making contact withGroovin-positive epidermal cells, which appearnormal in mbc mutant embryos. We conclude thatat least some of the elongated myoblasts succeedin forming attachments with appropriate cells inthe epidermis, and that myoblast fusion is notrequired for the normal patterning of muscleattachment sites on the epidermis.

In summary, there appear to be two populationsof myoblasts within embryos mutant for mbc.Both populations are first visible by myosinexpression at 9 hours AEL, as rounded cells, andone population remains rounded as long as theyare detectable, decreasing in number from about13 hours AEL. The other type of cell, in contrast,can be distinguished as elongated cells from about11 hours AEL, often situated in positions andorientations recognisably like those of wild-typemuscle precursors, and these cells continue to

Fig. 2. 17-anti-myosi1994). (A)embryo. Aincluding s10 µm.

express myosin until at least 17 hours AEL, when cuticleformation prevents further antibody staining. Initially thesecells are only slightly elongated, but from about 15 hours AELthey stretch and send out long processes (Fig. 1C). These twomyoblast populations and their behaviours are reminiscent ofthe two myoblast types postulated to exist during wild-typemyogenesis. The rounded myoblasts in mbc mutants mightconstitute the pool of myoblasts available for fusion, whereasthe elongated cells might represent the founder cells, whosespecial properties are revealed in this mutant because fusionfails to occur.

To explore this idea, we stained mbc mutant embryos usingantibodies against S59 and VG, the products of two genes thatare expressed in putative founder cells (Fig. 4).

vg and S59 expression in mbc mutantsThe early expression of S59 is identical in wild-type and mbcmutant embryos. S59 expression begins in a consistent patternof a small number of cells in the somatic mesoderm. Here wefocus on the sequence of events in a single abdominal hemiseg-ment. In both wild-type and mbc mutant embryos, expressionbegins in a single ventral cell between 6 and 7 hours AEL,which divides to give rise to two cells, known collectively as

1983Founder cells and Drosophila myogenesis

Fig. 3. 17-hour old embryos double stained with anti-myosin (brown) and anti-Groovin (blue-black). (A,C) Wild type, (B,D) mbcC2/mbcC3

mutant embryos. C and D show enlarged views. Arrows in D point to attachment of myoblasts to Groovin-expressing cells in an mbc mutant.Anterior is to the right, dorsal is up. Scale bars, 30 µm (A,B); 10 µm (C,D).

Group I. Four cells posterior and slightly ventral to Group Ibegin expression at about 7 hours AEL; these are known asGroup II. Group III is the last to appear, at about 8 hours AEL,consisting of two cells lying dorsally and at the same level inthe anteroposterior axis as the cells of Group II.

Subsequently, S59-expressing cells undergo movements andpattern refinements and this rearrangement is identical in mbcmutants and wild-type embryos, as follows. After Group I hasdivided into two cells, cell Ib remains in the same place, whilecell Ia migrates across the segment border in both mbc mutantsand the wild-type. The behaviour of Group II in mbc mutantsappears at first sight to be different from that of the wild-type,in that only one of the original four cells maintains S59expression after germ band retraction (Fig. 4B,D). The originalobservations of Dohrmann et al. (1990) suggested that in thewild-type, all the cells of Group II maintain expression, con-tributing eventually to muscle 27 (muscle nomenclatureaccording to Crossley (1978). However, more recent observa-tions (Carmena, Bate and Jimenez, unpublished data) show thatin fact, only one of the cells of Group II does so. The othercells gradually lose S59 expression and each contribute toseparate muscles. The loss of S59 expression in three of thefour cells of Group II in mbc mutants, therefore, exactlyfollows the sequence of S59-expression in wild-type embryos.

Group III also maintains S59 expression in only one cell inembryos mutant for mbc (Fig. 4B,D). Once again it is probablethat this corresponds to events in the wild-type as it is likelythat the two cells of Group III give rise to two separate muscles,only one of which (muscle 18) expresses S59 (Carmena, Bateand Jimenez, unpublished data). Oddly, cell Ib does behaveabnormally in mbc. In wild-type embryos, cell Ib continues toexpress S59 and gives rise to muscle 25. In mbc mutants,however, cell Ib loses expression (Fig. 4B,D). This is the morecurious, in that we do see a putative founder cell for muscle25 in mbc mutant embryos which have been stained formyosin.

In embryos mutant for mbc, therefore, the initial pattern ofexpression of S59 is almost identical to wild-type, suggestingthat the segregation and movement of founder cells is normalin these mutants. There is, however, a dramatic difference inS59 expression between wild-type and mbc mutant embryos,in that muscle fusion fails to occur in mbc mutants and thereis no concomitant increase in the number of nuclei positive forS59 (Fig. 4B,D). Thus, fusion is required for the recruitmentof cells to express S59, as predicted by Dohrmann et al. (1990).The S59-positive cells remain as single cells, lying in approx-imately the same position as the muscle they would normallyhave given rise to, and continue to express S59 at least until

1984 E. Rushton and others

Fig. 4. S59 and vg expression in wild-type and mbcC1 mutant embryos. (A) S59 expression in a 10-hour old wild-type embryo. Arrows point tothe S59-expressing muscles of one hemi-segment; Roman numerals refer to the group from which the muscles arose. Group Ia is muscle 5, Ib ismuscle 25, II is muscle 27 and III is muscle 18 (Dohrmann et al., 1990). Note that group III occurs only in the abdominal segments. (B) S59expression in a 10-hour old mbc mutant embryo. Arrows point to the S59-expressing cells; Roman numerals refer to the group, as in A. Notethat group Ia consists only of a single cell, Ib is absent (arrowhead), group II consists of 3-4 cells at this stage, but will reduce to one cell, andgroup III consists of one cell, though in four segments it is possible to make out a second, very faint cell, which is losing S59 expression(marked with an open arrow in one segment). Note that, as in A, group III occurs only in abdominal segments. (C) 16-hour old wild-typeembryo showing mature pattern of S59 expression. Note that group Ia (muscle 5) is no longer expressing S59. (D) 16-hour old mbc mutantembryo, showing mature pattern of S59 expression. Here, as in C, cell Ia no longer expresses S59. All groups are now reduced to one S59-expressing cell only. (E) vg expression in a 13-hour old wild-type embryo. The four ventral muscles of one hemisegment are bracketed andmarked v. Nuclei are polarised to either end of the muscles. Some of the dorsal muscles can also be seen, out of focus, bracketed and marked d.Arrowhead points to a vg-expressing cell, which is not a muscle, but may be a sense organ. (F) vg expression in a 13-hour old mbc mutantembryo. Labels as in E. Note that the single, unfused vg-expressing muscle cells are lined up along the segment in the same array as the fusedmuscles in wild type. Anterior is to the right and dorsal is up. Scale bar, 30 µm.

17 hours AEL – the limit of our ability to detect protein byantibody staining. In 3% of cases (n=200), two cells can beseen instead of one and we assume that this is caused by therare fusion events that occasionally take place in mbc mutantembryos.

Like S59, vg is expressed in a small number of mesodermalcells (Bate, 1993; Bate et al., 1993). In the wild-type embryo,these cells contribute to ventral muscles 6, 7, 12 and 13 and

dorsal muscles 1, 2, 3 and 4. Early development of the dorsalmuscles is difficult to examine because vg expression in theepidermis overlies and partly conceals the dorsal mesodermalvg expression. Here we concentrate on ventral mesodermal vgexpression. Ventrally, vg expression in wild-type and in mbcmutants begins in one cell per abdominal hemisegment duringthe extended germ band stage of development and soonincreases to three or four cells. By 10 hours AEL, the cells lie

1985Founder cells and Drosophila myogenesis

in a small cluster in the posterior of the segment. So far, thepattern of vg expression is identical in wild-type and mbcmutant embryos. In wild-type, the vg-expressing cells furtherincrease in number and resolve into four ventral longitudinalmuscles (Fig. 4E). In mbc mutants, however, the vg-express-ing cells do not increase in number, but otherwise behave in asimilar fashion to the wild-type ones. The four cells separateand align themselves in a dorsoventral pattern in the positionsnormally taken by the ventral longitudinal muscles (Fig. 4F).Thus we conclude, firstly, that as for S59 expression, increasein the number of vg-expressing cells is due to fusion andrecruitment. Secondly, the vg-expressing cells contain thenecessary information to migrate to their correct positions inthe segment.

Position and orientation of the founder cellsThe above results suggest that founder cells are segregatednormally in mbc mutants and behave normally in every respectsave that of fusion. To confirm that these cells correspond tothe stretched myoblasts seen in the myosin-stained prepara-tions, mbc mutants were double stained using antibodies tomyosin and VG or S59 (Fig. 5). In mbc mutants vg or S59positive nuclei are clearly seen in stretched myosin-expressingcells which span the territory that in wild-type is occupied byan S59 or vg-expressing muscle. We have never seen a vg orS59-expressing nucleus with a rounded cytoplasm. It is notalways possible to see the cytoplasm of these cells owing tothe many rounded myoblasts which surround them. Howeverwhere the cytoplasm can be distinguished, the orientation ofthe cell is consistent with the orientation of the wild-typemuscle which it represents. For example, S59-expressingmuscle 27 was examined in 192 segments, and in 155 of thesesegments it was possible to distinguish a myosin-stainedprocess with an S59-expressing nucleus. Of these, 138 (89%)were correctly oriented, running from ventral-anterior todorsal-posterior. S59-expressing muscle 18 was harder to dis-tinguish from surrounding myoblasts, with clear processesvisible in only 121 out of 220 segments examined. However,of these 121 processes, 115 (95%) were correctly oriented. Weconclude, therefore that myoblasts which express S59 or vgcontain information which enables them to find their correctposition and orientation.

Innervation in mbc mutantsA feature of normal Drosophila development is specificinnervation of particular muscles by particular motorneurons. The Connectin protein is expressed on the surfaceof a subset of developing motor neurons and muscles and maybe involved in mediating homophilic adhesion between them,prior to synapse formation (Nose et al., 1992; Meadows etal., 1994). We stained mbc mutant embryos with an antibodyto the Connectin gene product and showed that a subset ofmyoblasts in the appropriate parts of the segment expressConnectin on their surface, while the surrounding myoblastsdo not. Moreover, Connectin-expressing myoblasts arecontacted specifically by Connectin-expressing nerves (Fig.6). This strongly suggests that these Connectin-expressingmyoblasts have an identity which is recognised by motorneurons and which is not shared by the surrounding pool ofmyoblasts.

DISCUSSION

The phenotype of mbc mutants supports thefounder cell hypothesisIn this paper we describe the phenotype of mutants of a newlyidentified gene, mbc, which is required for normal myogenesisin Drosophila. In normal Drosophila embryos, muscles formby fusion of adjacent myoblasts. Each muscle is a uniqueelement in a distinctive pattern and each has its own position,size, orientation, attachment sites and innervation. In mbcmutant embryos, myoblasts fail to fuse and no multinucleatemuscles are formed, yet a subset of myoblasts retains the char-acteristics of position, orientation and specific innervation.

Two possible models could explain how fusion of myoblastsgenerates individual muscles each with its own identity andcharacteristics. In the first model, myoblasts are specified as agroup in which all the cells contain the information as to whichmuscle they are about to form. According to this model, eachmyoblast is specified to form a particular muscle and nomyoblast in a group is unique. If fusion were to fail in thissystem, one would expect to see all the unfused myoblastsbehaving in the same way, perhaps all sending out processesto span the territory of the muscle they would normally form.In the second model, a single cell is specified to become a par-ticular muscle and this cell is capable of seeding the processof fusion in the surrounding cells, which then take on theidentity of the cell with which they have fused. In this secondmodel, we might expect to see a mass of myoblasts withoutidentity or distinguishing characteristics and a small popula-tion of myoblasts that have some of the characteristics of themuscles they would normally form. These characteristicsmight include the expression of certain genes, and explorationof the territory normally covered by that muscle.

In support of the second model, there are indeed, in mbcmutants, two different populations of myoblasts, one thatremains rounded throughout embryogenesis and one thatbecomes elongated. This apparent subdivision could be a resultof random behaviour of the myoblasts, as a consequence offailure of fusion or some other aspect of the mbc mutantphenotype. However, in embryos mutant for mbc we canidentify S59 and vg-expressing cells with the stretched myosin-expressing cells, in preparations that have been double-stained.For example, some vg-expressing cells have processes thatspan the region which in wild-type would be spanned by vg-expressing muscles. The orientation of these single-cell‘muscles’ is not always accurate, but this is most likely becausethese myoblasts explore their surroundings later than wild-typemuscle precursors, and the surfaces over which they migratemay be expressing different proteins. Moreover, the unfusedmyoblasts themselves may make the terrain confusing toexploring cells. It is perhaps the more surprising therefore thatso many of the founder cells we see are in the correct orienta-tion.

In mbc mutants, S59 or vg-expressing cells appear at thecorrect time and place, and migrate correctly, but fail to recruitsurrounding cells to S59 or vg expression, showing that the S59and vg-expressing cells are a distinct population of myoblastswith their own identity. Clearly, as predicted by the foundercell hypothesis, neighbouring myoblasts cannot acquire thisidentity in the absence of fusion.

We have shown in mbc mutant embryos that Connectin is

1986 E. Rushton and others

Fig. 5. 16- to 17-hour old embryos double-stained with anti-myosin (pale brown stain) and anti-S59 (A,B) or anti-vg (C,D) (blue-black stain).(A) wild-type embryo about 15-hours old, showing muscle 18 (arrowheads). Dots mark position of main tracheal trunk. (B) mbcC1 mutantembryo showing S59-positive nuclei with the correct position and orientation for muscle 18 (arrowheads). Dots as in A. (C) Wild-type embryoshowing vg-expressing ventral longitudinal muscles. Arrows point to muscle attachment sites, arrowheads mark segment borders. (D) mbcC1

mutant embryo showing vg-positive cells spanning the segment in the correct region of the embryo for the ventral longitudinal muscles. Arrowspoint to putative muscle attachment sites, arrowheads mark segment border. Anterior is to the right and dorsal is up. Scale bar, 10 µm.

expressed on the surface of a subset of unfused myoblasts andon nerves making contact with these myoblasts. We arguetherefore that these particular myoblasts have an identity whichcan be recognised by the outgrowing motor axons and whichis not shared by the surrounding myoblasts. This observationis also consistent with the founder cell hypothesis, as it showsthat only a subset of myoblasts are able to specify a charac-teristic pattern of innervation.

Many genes may be required for fusion to occur success-fully. There are already several reports of genetic loci, whichhave not so far been completely characterised, but whosemutant phenotypes show partial or complete loss of fusion.These include two P-element induced mutations, rolling stoneand P-20 (Paululat et al., 1994a,b), several first-chromosomedeficiency lines, and runt (Drysdale et al., 1993). Furthermore,myoblast fusion fails when a constitutively active form ofDrac1, the Drosophila homologue of Rac, is expressed in themesoderm. The function of Drac1 is not known, but it isexpressed in the mesoderm from about 6 hours AEL. Embryosthat express the mutated Drac1 have a number of features incommon with embryos mutant for mbc, including stretchedmyosin-expressing cells and a Connectin expression patternwhich appears identical to that of mbc mutants (Luo et al.,1994). This suggests that the myoblast morphology describedfor mbc mutants is indeed a secondary effect due to lack offusion and not a phenomenon restricted to mbc mutants.

To summarise, therefore, we suggest that the stretched cellsin mbc are founder-cell like. They constitute a special popula-tion of cells which in wild-type cannot be distinguished mor-phologically because by the time muscles are stretching andextending processes, the cells are part of a syncytium (Bate,1990). In mbc mutants however, the founder cells are revealedbecause of the lack of myoblast fusion.

There is a clear analogy between the cells described here andthe muscle pioneer cells of the grasshopper (Ho et al., 1983).Both are large, distinctive cells which extend processes toexplore their surroundings and both stretch out to span the ter-ritories of the future muscle. A prediction from the founder cellhypothesis is that if a founder cell is removed, the muscle itshould have formed would be missing. This prediction wastested in the grasshopper. When individual muscle pioneerswere ablated, no muscle subsequently formed, though themyoblasts that normally contributed to it were still present(Ball et al., 1985).

We therefore believe that myogenesis is essentially identicalin grasshopper and Drosophila. Both require a special class ofcells that initiate fusion. Drosophila’s more rapid developmentmay explain the different sequence of events, in that foundercells form a syncytium before spanning their territories, whilethe grasshopper pioneers are visible first as single, stretchingcells. This rapid development obscures the essential similarityof the founder cells to the pioneer cells, and this similarity is

1987Founder cells and Drosophila myogenesis

Fig. 6. 16-hour old embryos stained with anti-Connectin. Muscles 21-24 are bracketed. Arrows point to the nerve that makes contact on thesemuscles. (A,C) Wild type; (B,D) mbcC1 mutant. Anterior is to the right, dorsal is up. Scale bars, 30 µm (A,B); 10 µm (C,D).

revealed only in a mutant where fusion does not take place.Under these special conditions, a particular subset of cells isrevealed, which resemble grasshopper muscle pioneer cells,and we identify these cells with the founder cells that have beenproposed to explain the process of muscle development inDrosophila.

The phenotype of mbc mutants gives us insight intoother aspects of myogenesisThere are some puzzling aspects of the phenotype of mbcmutants, some of which reveal unexpected features of normalmyogenesis. Firstly, the observation, as revealed by myosinstaining, that the population of stretched cells in mbc mutantsdoes not emerge until some time after muscles have spannedtheir territories in wild-type embryos. It certainly is not the casethat muscle development in general is delayed in mbc mutants,since muscle-specific proteins such as S59, VG and myosin areall expressed on schedule, and the S59 and vg-expressingmyoblasts variously migrate, divide and lose expression at thecorrect times. It may be that normal muscle growth andextension occurs in two phases. At first growth may be passive,as a result of fusion of myoblasts into the syncytium. Oncefusion is complete, further extension must take place activelyby the process of sending out muscle growth cones. Possiblyit is this late stage of active extension that is revealed inembryos mutant for mbc.

Secondly, in mbc mutants, rounded myoblasts decline innumber from about 14 hours AEL onwards, as detected by anti-myosin staining. It appears that this is due in part to cells losingmyosin expression, and in part to cell death, as detected by thepresence of macrophages that have engulfed myosin-express-

ing cells. In either case, it seems that fusion is a requirementin non-founder cell myoblasts to maintain viability and/or amuscle fate. Founder cells, on the other hand, appear to containthe information necessary to sustain myosin expression andother aspects of muscle differentiation. It could be that the dis-tinctive characteristics of founder cells depend at least in parton contact with the epidermis. Three observations support thisidea: firstly, that S59 and vg-expressing cells arise in closecontact with the ectoderm and remain in contact (our ownobservations). Secondly, that the stretched cells in mbc mutantsappear to contact the epidermis, and thirdly, that in neurogenicmutants, lack of epidermis leads to premature loss ofexpression of such genes as S59 and vg (Bate et al., 1993).

We cannot explain the curious observation that in mbcmutants, S59 expression is lost from cell 1B. As describedabove, Group I of the S59-expressing cells is first seen as onecell, which divides into two. Cell Ia migrates across thesegment border, loses S59 expression in both wild-typeembryos and mbc mutants and in wild-type embryos becomesincorporated into muscle 5. Cell Ib migrates a couple of cell-diameters posteriorly and fuses with its neighbours to formS59-expressing muscle 25. The loss of S59 expression fromcell Ib in mbc mutants is therefore a mystery and the only lightwe can shed on the puzzle comes from the observation that inmbc all the cells that retain S59 expression are lying in contactwith the anterior margin of the engrailed- (en) expressingstripe in the ectoderm (our unpublished observations). It maybe that this is in some sense a ground state for mesodermal S59expression. All the mesodermal S59-expressing cells arise incontact with the en stripe, and cell Ib is the only S59-express-ing cell in mbc mutants to lose contact with it (Dohrmann et

1988 E. Rushton and others

al., 1990; our observations). It may be that in the wild-type,some aspect of fusion overrides the need for contact of cell Ibwith en-expressing cells.

ConclusionTo conclude, we here present evidence in support of thefounder cell hypothesis of larval muscle development inDrosophila. This model suggests that single cells of thesomatic mesoderm are selected and set aside to initiate theprocess of muscle fusion. In addition, these founder cellscontain the information that gives the future muscle itsidentity. This information is required from a very early stage,as shown by the patterns of gene expression and cell migra-tions of the single-cell muscle founders. It is also requiredlater in development to determine the position and orienta-tion of the muscle and its innervation pattern. We have shownhere that single cells of the somatic mesoderm are capable ofdisplaying the above characteristics even in the absence offusion and we therefore identify these cells as muscle foundercells.

We thank Jacob Harrison for technical assistance, and MaryErickson, Ana Carmena and Fernando Jimenez for communicatingresults prior to publication. We also thank Mary Baylies, KendalBroadie, Andreas Prokop, Helen Skaer and Mike Taylor for criticallyreading earlier versions of this manuscript and making many helpfulsuggestions. This work is funded by grants from The Wellcome Trust(E. R., R. D. and M. B.) and the National Science Foundation (S. A.).A. M. is an Assistant Investigator of the Howard Hughes MedicalInstitute.

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(Accepted 20 April 1995)