Download Mutations in a novel gene, myoblast city, provide evidence

Development 121, 1979-1988 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
1979
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
2Department
of Zoology, University of Cambridge, Downing St, Cambridge CB2 3EJ, UK
of Biochemistry and Molecular Biology, The Pennsylvania State University, 459 N. Frear Laboratory, University Park,
PA 16802-4500, USA
3Howard 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
SUMMARY
We have used mutations in the newly identified gene
myoblast city to investigate the founder cell hypothesis of
muscle development in Drosophila melanogaster. In
embryos mutant for myoblast city the fusion of myoblasts
into multinucleate muscles is virtually abolished. Nevertheless, a subset of the myoblasts develop specific musclelike characteristics, including gene expression appropriate
to particular muscles, migration to the appropriate part of
the segment, correct position and orientation, and contact
by motor neurons. We suggest that this subset of myoblasts
represents the proposed muscle founder cells and we draw
an analogy between these founder cells and the muscle
pioneers described for grasshopper muscle development.
INTRODUCTION
bouring cells, which then begin to express S59, ap or vg themselves, and the resulting fused groups go on to form specific
muscles (Dohrmann et al., 1990; Bourgouin et al., 1992; Bate,
1993; Bate et al., 1993). S59 and ap contain homeobox
sequences and so are implicated in DNA binding and transcriptional control (Dohrmann et al., 1990; Cohen et al., 1992).
vg is related to a family of proteins involved in protein-protein
interactions (Williams et al., 1991). All three proteins are
localised to the nucleus. The function of such genes in muscle
development is not known, but it is possible that they are
involved in specifying the identity of the muscle precursors in
which they are expressed. The best evidence for such a
function is provided by ap. In the absence of ap function there
is variable loss of one or more of the ap expressing muscles
and strikingly, when ap is expressed ubiquitously under heatshock control, extra muscles develop in a similar location and
position to some of the wild-type ap-expressing muscles, while
the rest of the muscle pattern is apparently normal (Bourgouin
et al., 1992).
The idea of the muscle founder cell in Drosophila was
preceded by the observation of pioneering muscle cells in the
grasshopper embryo. Muscle pioneers in the grasshopper
stretch out and span the future territory of the muscle they
prefigure. The pioneers form a scaffold with which mesodermal cells fuse to form the mature musculature (Ho et al.,
1983). Superficially, the muscle pioneers of the grasshopper
are different from the muscle founder cells of Drosophila,
because the pioneers extend processes to span their territory
The larval muscles of Drosophila melanogaster form a
complex but highly regular array on the body wall of the larva.
The muscles are formed during embryogenesis by the fusion
of myoblasts, cells of the somatic mesoderm. Midway through
development, small syncytia containing two or three nuclei
appear at defined points in the somatic mesoderm (Bate, 1990).
These syncytia are termed ‘muscle precursors’, as each one
gives rise, by a continuing process of fusion and
extension/migration, to a larval muscle, whose identity is
defined by its size, position, orientation on the body wall of the
larva and pattern of innervation.
Interestingly, fusion does not take place randomly among
myoblasts, but always begins at particular points in the
mesoderm in close contact with the ectoderm (Bate, 1990). A
model for muscle development has been proposed in which
muscle differentiation begins with the segregation of founder
cells – single cells in the somatic mesoderm – which seed the
fusion process and which may in some way confer identity on
the developing muscle (Bate, 1990; Dohrmann et al., 1990;
Bate et al., 1993).
This view is supported by the observation that genes such
as S59, apterous (ap) and vestigial (vg) are each expressed in
a small number of larval muscles, and that earlier, myoblasts
expressing these genes behave like the predicted founder cells.
Expression of these genes begins in the mesoderm in a small
number of cells before fusion. These cells fuse with neigh-
Key words: Drosophila, embryo, muscle, myogenesis, founder cell,
cell fusion, myoblast city
1980 E. Rushton and others
before fusion takes place, while the proposed founder cells
first initiate fusion and form multinucleate muscle precursors.
It is these precursors in Drosophila that resemble the muscle
pioneers of the grasshopper in that they start to explore their
territory and form attachments to the epidermis. This difference in timing may simply reflect the more rapid process of
embryogenesis in Drosophila as compared with the grasshopper. While the existence of founder cells in Drosophila
has yet to be demonstrated, they have been proposed as a
model to explain early events in the formation of the muscle
pattern.
Here we describe the embryonic phenotype of lethal
mutations in a gene called myoblast city (mbc). The principal
characteristic of these mutations is lack of myoblast fusion,
and we use this phenotype to examine the founder cell hypothesis in Drosophila. We present evidence in support of the
founder cell hypothesis and show that, in the absence of
fusion, two classes of myoblasts exist. The first class
resembles muscle pioneers in that these cells stretch out and
span muscle territories, while the second class appears to constitute a pool of myoblasts with which the pioneers would
normally fuse.
MATERIALS AND METHODS
Genetic methods
All marker mutations and balancer chromosomes used are described
in FlyBase (1994).
(a) Deficiencies in the 95 region were created as follows: The
P{ry+, lacZ}A189.2F3 line (Bellen et al., 1989) was obtained from
the Bloomington Stock Center. It carries an enhancer trap element at
95A, and homozygotes are phenotypically wild type. Males homozygous for P{ry+, lacZ}A189.2F3 were irradiated with 4000 rads of X
rays. Approximately 26,000 chromosomes were screened for the loss
of the ry+ marker. A single deficiency, Df(3R)mbc-R1, with breakpoints in 95A5-7 and 95D6-11 was recovered in this screen.
A parallel mutagenesis utilised an insertion of the plasmid
H{ry+har1} at 95A (Blackman et al., 1989). This hobo-associated ry+
insertion (referred to as TnM2) is homozygous lethal, with no obvious
muscle 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 chromosomes
were examined for loss of the ry+ insertion. A single deficiency,
Df(3R) mbc-30, with breakpoints in 95A5-7 and 95C10-11 was
recovered.
(b) Four alleles of mbc were identified in several screens, on the
basis of failing to complement mbc deletions for embryonic viability:
Homozygous red e males were fed 25 mM EMS in 5% sucrose (Lewis
and Bacher, 1968). Mutagenized third chromosomes were recovered
over the TM3 Sb balancer chromosome and test crossed against the
Df(3R)mbc-R1 chromosome. Three independent lethal alleles of mbc
(mbcC1, mbcC2 and mbcC3) were recovered from a total of 1,440 chromosomes tested.
In a parallel analysis, isogenic ru st e males were fed 30 mM EMS
in 10% sucrose. Mutagenized third chromosomes were recovered over
a TM3 (Sb, ry) balancer and tested for complementation of Df(3R)
mbc-30. One lethal allele of mbc (mbcS4) was recovered from a total
of approximately 2400 chromosomes tested. Independent recombination mapping localised this allele to cytological region 95.
Immunohistochemical methods
Eggs were laid and allowed to develop on yeasted apple-juice agar
plates at 25°C. Embryos were fixed for 20 minutes in heptane and 4%
paraformaldehyde in phosphate buffer pH7.0, and devitellinized in a
1:1 mixture of heptane and methanol with vigorous shaking.
Embryos were blocked prior to incubation with antibody, in
phosphate-buffered saline, 0.3% Triton-X (PBS-TX) + 0.5% bovine
serum albumin (Sigma). Anti-myosin, used at a dilution of 1:1000 was
a gift from Dan Kiehart (Kiehart and Feghali, 1986). Anti-S59 was
produced in collaboration with Fernando Jimenez and Mary Baylies,
using an S59 cDNA clone kindly sent by Manfred Frasch (Dohrmann
et al., 1990). Rabbit anti-S59 was used at a dilution of 1:5000. AntiVG was provided by Sean Carroll and used at a dilution of 1:100
(Williams et al., 1991). Anti-Connectin was provided by Rob White
and used at a dilution of 1:10 (Meadows et al., 1994). Anti-Groovin
was provided by Talila Volk and used at a dilution of 1:3. Anti-Peroxidasin was provided by J. Fessler and L. Fessler and used at a
dilution of 1:1000.
Incubation with antibody took place overnight at 4°C. All washes
were 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, antiS59 or anti-VG and developed in the presence of nickel chloride
(giving a blue-black stain), then incubated in anti-myosin and
developed in the absence of nickel chloride (for a pale brown stain).
The anti-myosin-stained embryos shown in Fig. 1 were aged by
making one-hour egg-lays and allowing the embryos to develop for
the appropriate length of time at 25°C. All other embryos were staged
using morphological criteria.
Embryos were examined and photographed using a Zeiss Axiophot
microscope.
RESULTS
Mutations in a new gene, mbc, severely affect
muscle development
Alleles of mbc were recovered in a screen designed to make
mutations in the 95 region of the third chromosome. This
region includes the gene nautilus (nau), a Drosophila
homologue of vertebrate MyoD. nau is expressed from an early
stage in developing muscles (Michelson et al., 1990). Results
for nau will be published elsewhere; here we concentrate on
the phenotype of mbc.
A total of 4 alleles of the mbc locus were isolated: mbcC1,
mbcC2, mbcC3 and mbcS4. Deletion mapping using deficiency
chromosomes (described in Materials and Methods) maps the
mbc locus between 95A5-7 and 95C10-11 on the right arm of
the third chromosome. Deficiencies that remove the nau coding
sequence complement the mbc mutations (Erickson and
Abmayr, unpublished results), thus mbc is not nautilus.
All known mbc alleles are recessive and cause embryonic
lethality. Epidermal development and differentiation appear
normal: cuticle preparations of mbc mutant embryos reveal no
abnormalities in segmentation or patterning (data not shown).
The embryos lie motionless in the vitelline membrane and fail
to hatch. Examination of the mbc mutant embryos with a
polarised light microscope (Drysdale et al., 1993) shows a
striking lack of differentiated muscle.
The same muscle phenotype was consistently found in
embryos homozygous for Df(3R)mbc-R.1, Df(3R)mbc-30 and
all four mbc alleles, and also in embryos carrying any combination of deficiency and point mutation. This suggests that all
mutations generated so far have a null phenotype.
Founder cells and Drosophila myogenesis 1981
Fig. 1. Wild-type and mbcC1 mutant embryos stained using an antibody to muscle myosin. Staged embryos were obtained by making hour-long
egg collections and allowing them to develop to the appropriate age at 25°C. (A) 13- to 14-hour old wild-type embryo showing mature muscle
pattern. White arrow points to muscle 25, black arrow points to muscle 29. v marks ventral oblique muscle group. (B) 13- to 14-hour old mbc
mutant embryo showing unfused myoblasts lying in a segmented array. (C) 16- to 17-hour old mbc mutant embryo showing the two
populations of myoblasts: stretched and rounded. (D) Enlarged view of myoblasts in a 14- to 15-hour old embryo. Black arrowhead points to a
possible founder cell for a ventral oblique muscle. White arrowheads point to possible founders for muscle 25. (E) Enlarged view of ventral
oblique 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 the
characteristic 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 fusion
In order to make a detailed analysis of the muscle phenotype
of embryos mutant for mbc, we used an antibody against
Drosophila muscle myosin to reveal the muscles during development (Kiehart and Feghali, 1986). In wild-type embryos,
muscle myosin is first expressed approximately 9 hours after
egg laying (AEL). At this time, myoblast fusion is under way
and every muscle is represented by a precursor (Bate, 1990).
Myosin expression begins in a small number of precursors
lying ventrally and laterally and rapidly extends to all the pre-
1982 E. Rushton and others
cursors and some of the cells that are about to fuse with them.
express myosin until at least 17 hours AEL, when cuticle
By 13 hours AEL, the muscle pattern is complete and muscle
formation prevents further antibody staining. Initially these
attachments are forming on the epidermis (Fig. 1A).
cells are only slightly elongated, but from about 15 hours AEL
In embryos mutant for mbc, myosin expression begins on
they stretch and send out long processes (Fig. 1C). These two
schedule at 9 hours AEL, in cells that appear by their position
myoblast populations and their behaviours are reminiscent of
to belong to the somatic mesoderm. Because of their position
the two myoblast types postulated to exist during wild-type
and muscle myosin expression, we identify these cells as
myogenesis. The rounded myoblasts in mbc mutants might
myoblasts. As in the wild-type, expression begins in a few cells
constitute the pool of myoblasts available for fusion, whereas
ventrally and laterally, and by 10 hours, apparently extends to
the elongated cells might represent the founder cells, whose
most of the cells of the somatic mesoderm. The myosinspecial properties are revealed in this mutant because fusion
expressing cells are clearly organised into an array which
fails to occur.
resembles the pattern of wild-type muscle precursors, with a
To explore this idea, we stained mbc mutant embryos using
segmentally repeated arrangement of ventral, lateral and dorsal
antibodies against S59 and VG, the products of two genes that
cell clusters (Fig. 1B). Strikingly, with only a few exceptions,
are expressed in putative founder cells (Fig. 4).
these myoblasts fail to fuse. Initially, all the cells are rounded,
vg and S59 expression in mbc mutants
but from 11 hours AEL, a subset of mononucleate cells become
slightly elongated, lying in positions and orientations similar
The early expression of S59 is identical in wild-type and mbc
to the multinucleate muscle precursors in wild-type embryos
mutant embryos. S59 expression begins in a consistent pattern
(Fig. 1C,D,F).
of a small number of cells in the somatic mesoderm. Here we
The elongated myoblasts first appear at about 11 hours AEL
focus on the sequence of events in a single abdominal hemisegand increase in number until about 13-15 hours AEL. After this
ment. In both wild-type and mbc mutant embryos, expression
time some myoblasts become much longer, some now
begins in a single ventral cell between 6 and 7 hours AEL,
which divides to give rise to two cells, known collectively as
spanning distances two or three times the length of normal
muscles. These myoblasts occasionally have more
than one nucleus, indicating that a small number
of cell fusions occur. At 13 hours AEL, there is
still a large number of rounded myosin-expressing
myoblasts, but from 14 hours onward, this population diminishes. Some cells may simply lose
myosin expression, as many faintly stained and
unstained rounded cells can be seen using
Nomarski optics. However, cell death is also
involved, as myosin-positive cells engulfed by
macrophages can be seen in preparations which
have been doubly stained for myosin and peroxidasin (Nelson et al., 1994; Fig. 2).
Groovin is expressed in the epidermis at muscle
attachment sites (Volk and VijayRaghavan, 1994).
To investigate whether the elongated myoblasts
were indeed finding their correct attachment sites,
embryos were double-labelled using anti-myosin
and anti-Groovin. Fig. 3 shows myosin-positive
cells stretching out and making contact with
Groovin-positive epidermal cells, which appear
normal in mbc mutant embryos. We conclude that
at least some of the elongated myoblasts succeed
in forming attachments with appropriate cells in
the epidermis, and that myoblast fusion is not
required for the normal patterning of muscle
attachment sites on the epidermis.
In summary, there appear to be two populations
of myoblasts within embryos mutant for mbc.
Both populations are first visible by myosin
expression at 9 hours AEL, as rounded cells, and
one population remains rounded as long as they
are detectable, decreasing in number from about
Fig. 2. 17-hour old embryos, double stained with anti-Peroxidasin (blue-black) and
13 hours AEL. The other type of cell, in contrast,
anti-myosin (brown). Peroxidasin is expressed by macrophages (Nelson et al.,
can be distinguished as elongated cells from about
1994). (A) Wild type. Arrow points to macrophage. (B) mbcC2/mbcC3 mutant
11 hours AEL, often situated in positions and
embryo. Arrows in B point to macrophages that have engulfed many cells,
orientations recognisably like those of wild-type
including some expressing myosin. Anterior is to the right, dorsal is up. Scale bar,
10 µm.
muscle precursors, and these cells continue to
Founder cells and Drosophila myogenesis 1983
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 I
begin expression at about 7 hours AEL; these are known as
Group II. Group III is the last to appear, at about 8 hours AEL,
consisting of two cells lying dorsally and at the same level in
the anteroposterior axis as the cells of Group II.
Subsequently, S59-expressing cells undergo movements and
pattern refinements and this rearrangement is identical in mbc
mutants and wild-type embryos, as follows. After Group I has
divided into two cells, cell Ib remains in the same place, while
cell Ia migrates across the segment border in both mbc mutants
and the wild-type. The behaviour of Group II in mbc mutants
appears at first sight to be different from that of the wild-type,
in that only one of the original four cells maintains S59
expression after germ band retraction (Fig. 4B,D). The original
observations of Dohrmann et al. (1990) suggested that in the
wild-type, all the cells of Group II maintain expression, contributing eventually to muscle 27 (muscle nomenclature
according to Crossley (1978). However, more recent observations (Carmena, Bate and Jimenez, unpublished data) show that
in fact, only one of the cells of Group II does so. The other
cells gradually lose S59 expression and each contribute to
separate muscles. The loss of S59 expression in three of the
four cells of Group II in mbc mutants, therefore, exactly
follows the sequence of S59-expression in wild-type embryos.
Group III also maintains S59 expression in only one cell in
embryos mutant for mbc (Fig. 4B,D). Once again it is probable
that this corresponds to events in the wild-type as it is likely
that the two cells of Group III give rise to two separate muscles,
only one of which (muscle 18) expresses S59 (Carmena, Bate
and Jimenez, unpublished data). Oddly, cell Ib does behave
abnormally in mbc. In wild-type embryos, cell Ib continues to
express S59 and gives rise to muscle 25. In mbc mutants,
however, cell Ib loses expression (Fig. 4B,D). This is the more
curious, in that we do see a putative founder cell for muscle
25 in mbc mutant embryos which have been stained for
myosin.
In embryos mutant for mbc, therefore, the initial pattern of
expression of S59 is almost identical to wild-type, suggesting
that the segregation and movement of founder cells is normal
in these mutants. There is, however, a dramatic difference in
S59 expression between wild-type and mbc mutant embryos,
in that muscle fusion fails to occur in mbc mutants and there
is no concomitant increase in the number of nuclei positive for
S59 (Fig. 4B,D). Thus, fusion is required for the recruitment
of cells to express S59, as predicted by Dohrmann et al. (1990).
The S59-positive cells remain as single cells, lying in approximately the same position as the muscle they would normally
have 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 to
the S59-expressing muscles of one hemi-segment; Roman numerals refer to the group from which the muscles arose. Group Ia is muscle 5, Ib is
muscle 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) S59
expression in a 10-hour old mbc mutant embryo. Arrows point to the S59-expressing cells; Roman numerals refer to the group, as in A. Note
that 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, and
group 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-type
embryo showing mature pattern of S59 expression. Note that group Ia (muscle 5) is no longer expressing S59. (D) 16-hour old mbc mutant
embryo, showing mature pattern of S59 expression. Here, as in C, cell Ia no longer expresses S59. All groups are now reduced to one S59expressing cell only. (E) vg expression in a 13-hour old wild-type embryo. The four ventral muscles of one hemisegment are bracketed and
marked 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 mutant
embryo. 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 fused
muscles 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 by
antibody staining. In 3% of cases (n=200), two cells can be
seen instead of one and we assume that this is caused by the
rare fusion events that occasionally take place in mbc mutant
embryos.
Like S59, vg is expressed in a small number of mesodermal
cells (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 dorsal
muscles is difficult to examine because vg expression in the
epidermis overlies and partly conceals the dorsal mesodermal
vg expression. Here we concentrate on ventral mesodermal vg
expression. Ventrally, vg expression in wild-type and in mbc
mutants begins in one cell per abdominal hemisegment during
the extended germ band stage of development and soon
increases to three or four cells. By 10 hours AEL, the cells lie
Founder cells and Drosophila myogenesis 1985
in a small cluster in the posterior of the segment. So far, the
pattern of vg expression is identical in wild-type and mbc
mutant embryos. In wild-type, the vg-expressing cells further
increase in number and resolve into four ventral longitudinal
muscles (Fig. 4E). In mbc mutants, however, the vg-expressing cells do not increase in number, but otherwise behave in a
similar fashion to the wild-type ones. The four cells separate
and align themselves in a dorsoventral pattern in the positions
normally taken by the ventral longitudinal muscles (Fig. 4F).
Thus we conclude, firstly, that as for S59 expression, increase
in the number of vg-expressing cells is due to fusion and
recruitment. Secondly, the vg-expressing cells contain the
necessary information to migrate to their correct positions in
the segment.
Position and orientation of the founder cells
The above results suggest that founder cells are segregated
normally in mbc mutants and behave normally in every respect
save that of fusion. To confirm that these cells correspond to
the stretched myoblasts seen in the myosin-stained preparations, mbc mutants were double stained using antibodies to
myosin and VG or S59 (Fig. 5). In mbc mutants vg or S59
positive nuclei are clearly seen in stretched myosin-expressing
cells which span the territory that in wild-type is occupied by
an S59 or vg-expressing muscle. We have never seen a vg or
S59-expressing nucleus with a rounded cytoplasm. It is not
always possible to see the cytoplasm of these cells owing to
the many rounded myoblasts which surround them. However
where the cytoplasm can be distinguished, the orientation of
the cell is consistent with the orientation of the wild-type
muscle which it represents. For example, S59-expressing
muscle 27 was examined in 192 segments, and in 155 of these
segments it was possible to distinguish a myosin-stained
process with an S59-expressing nucleus. Of these, 138 (89%)
were correctly oriented, running from ventral-anterior to
dorsal-posterior. S59-expressing muscle 18 was harder to distinguish from surrounding myoblasts, with clear processes
visible in only 121 out of 220 segments examined. However,
of these 121 processes, 115 (95%) were correctly oriented. We
conclude, therefore that myoblasts which express S59 or vg
contain information which enables them to find their correct
position and orientation.
Innervation in mbc mutants
A feature of normal Drosophila development is specific
innervation of particular muscles by particular motor
neurons. The Connectin protein is expressed on the surface
of a subset of developing motor neurons and muscles and may
be involved in mediating homophilic adhesion between them,
prior to synapse formation (Nose et al., 1992; Meadows et
al., 1994). We stained mbc mutant embryos with an antibody
to the Connectin gene product and showed that a subset of
myoblasts in the appropriate parts of the segment express
Connectin on their surface, while the surrounding myoblasts
do not. Moreover, Connectin-expressing myoblasts are
contacted specifically by Connectin-expressing nerves (Fig.
6). This strongly suggests that these Connectin-expressing
myoblasts have an identity which is recognised by motor
neurons and which is not shared by the surrounding pool of
myoblasts.
DISCUSSION
The phenotype of mbc mutants supports the
founder cell hypothesis
In this paper we describe the phenotype of mutants of a newly
identified gene, mbc, which is required for normal myogenesis
in Drosophila. In normal Drosophila embryos, muscles form
by fusion of adjacent myoblasts. Each muscle is a unique
element in a distinctive pattern and each has its own position,
size, orientation, attachment sites and innervation. In mbc
mutant embryos, myoblasts fail to fuse and no multinucleate
muscles are formed, yet a subset of myoblasts retains the characteristics of position, orientation and specific innervation.
Two possible models could explain how fusion of myoblasts
generates individual muscles each with its own identity and
characteristics. In the first model, myoblasts are specified as a
group in which all the cells contain the information as to which
muscle they are about to form. According to this model, each
myoblast is specified to form a particular muscle and no
myoblast in a group is unique. If fusion were to fail in this
system, one would expect to see all the unfused myoblasts
behaving in the same way, perhaps all sending out processes
to span the territory of the muscle they would normally form.
In the second model, a single cell is specified to become a particular muscle and this cell is capable of seeding the process
of fusion in the surrounding cells, which then take on the
identity of the cell with which they have fused. In this second
model, we might expect to see a mass of myoblasts without
identity or distinguishing characteristics and a small population of myoblasts that have some of the characteristics of the
muscles they would normally form. These characteristics
might include the expression of certain genes, and exploration
of the territory normally covered by that muscle.
In support of the second model, there are indeed, in mbc
mutants, two different populations of myoblasts, one that
remains rounded throughout embryogenesis and one that
becomes elongated. This apparent subdivision could be a result
of random behaviour of the myoblasts, as a consequence of
failure of fusion or some other aspect of the mbc mutant
phenotype. However, in embryos mutant for mbc we can
identify S59 and vg-expressing cells with the stretched myosinexpressing cells, in preparations that have been double-stained.
For example, some vg-expressing cells have processes that
span the region which in wild-type would be spanned by vgexpressing muscles. The orientation of these single-cell
‘muscles’ is not always accurate, but this is most likely because
these myoblasts explore their surroundings later than wild-type
muscle precursors, and the surfaces over which they migrate
may be expressing different proteins. Moreover, the unfused
myoblasts themselves may make the terrain confusing to
exploring cells. It is perhaps the more surprising therefore that
so many of the founder cells we see are in the correct orientation.
In mbc mutants, S59 or vg-expressing cells appear at the
correct time and place, and migrate correctly, but fail to recruit
surrounding cells to S59 or vg expression, showing that the S59
and vg-expressing cells are a distinct population of myoblasts
with their own identity. Clearly, as predicted by the founder
cell hypothesis, neighbouring myoblasts cannot acquire this
identity 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 mutant
embryo showing S59-positive nuclei with the correct position and orientation for muscle 18 (arrowheads). Dots as in A. (C) Wild-type embryo
showing 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. Arrows
point 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 and
on nerves making contact with these myoblasts. We argue
therefore that these particular myoblasts have an identity which
can be recognised by the outgrowing motor axons and which
is not shared by the surrounding myoblasts. This observation
is also consistent with the founder cell hypothesis, as it shows
that only a subset of myoblasts are able to specify a characteristic pattern of innervation.
Many genes may be required for fusion to occur successfully. There are already several reports of genetic loci, which
have not so far been completely characterised, but whose
mutant phenotypes show partial or complete loss of fusion.
These include two P-element induced mutations, rolling stone
and P-20 (Paululat et al., 1994a,b), several first-chromosome
deficiency lines, and runt (Drysdale et al., 1993). Furthermore,
myoblast fusion fails when a constitutively active form of
Drac1, the Drosophila homologue of Rac, is expressed in the
mesoderm. The function of Drac1 is not known, but it is
expressed in the mesoderm from about 6 hours AEL. Embryos
that express the mutated Drac1 have a number of features in
common with embryos mutant for mbc, including stretched
myosin-expressing cells and a Connectin expression pattern
which appears identical to that of mbc mutants (Luo et al.,
1994). This suggests that the myoblast morphology described
for mbc mutants is indeed a secondary effect due to lack of
fusion and not a phenomenon restricted to mbc mutants.
To summarise, therefore, we suggest that the stretched cells
in mbc are founder-cell like. They constitute a special population of cells which in wild-type cannot be distinguished morphologically because by the time muscles are stretching and
extending processes, the cells are part of a syncytium (Bate,
1990). In mbc mutants however, the founder cells are revealed
because of the lack of myoblast fusion.
There is a clear analogy between the cells described here and
the muscle pioneer cells of the grasshopper (Ho et al., 1983).
Both are large, distinctive cells which extend processes to
explore their surroundings and both stretch out to span the territories of the future muscle. A prediction from the founder cell
hypothesis is that if a founder cell is removed, the muscle it
should have formed would be missing. This prediction was
tested in the grasshopper. When individual muscle pioneers
were ablated, no muscle subsequently formed, though the
myoblasts that normally contributed to it were still present
(Ball et al., 1985).
We therefore believe that myogenesis is essentially identical
in grasshopper and Drosophila. Both require a special class of
cells that initiate fusion. Drosophila’s more rapid development
may explain the different sequence of events, in that founder
cells form a syncytium before spanning their territories, while
the grasshopper pioneers are visible first as single, stretching
cells. This rapid development obscures the essential similarity
of the founder cells to the pioneer cells, and this similarity is
Founder cells and Drosophila myogenesis 1987
Fig. 6. 16-hour old embryos stained with anti-Connectin. Muscles 21-24 are bracketed. Arrows point to the nerve that makes contact on these
muscles. (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 is
revealed, which resemble grasshopper muscle pioneer cells,
and we identify these cells with the founder cells that have been
proposed to explain the process of muscle development in
Drosophila.
The phenotype of mbc mutants gives us insight into
other aspects of myogenesis
There are some puzzling aspects of the phenotype of mbc
mutants, some of which reveal unexpected features of normal
myogenesis. Firstly, the observation, as revealed by myosin
staining, that the population of stretched cells in mbc mutants
does not emerge until some time after muscles have spanned
their territories in wild-type embryos. It certainly is not the case
that muscle development in general is delayed in mbc mutants,
since muscle-specific proteins such as S59, VG and myosin are
all expressed on schedule, and the S59 and vg-expressing
myoblasts variously migrate, divide and lose expression at the
correct times. It may be that normal muscle growth and
extension occurs in two phases. At first growth may be passive,
as a result of fusion of myoblasts into the syncytium. Once
fusion is complete, further extension must take place actively
by the process of sending out muscle growth cones. Possibly
it is this late stage of active extension that is revealed in
embryos mutant for mbc.
Secondly, in mbc mutants, rounded myoblasts decline in
number from about 14 hours AEL onwards, as detected by antimyosin staining. It appears that this is due in part to cells losing
myosin expression, and in part to cell death, as detected by the
presence of macrophages that have engulfed myosin-express-
ing cells. In either case, it seems that fusion is a requirement
in non-founder cell myoblasts to maintain viability and/or a
muscle fate. Founder cells, on the other hand, appear to contain
the information necessary to sustain myosin expression and
other aspects of muscle differentiation. It could be that the distinctive characteristics of founder cells depend at least in part
on contact with the epidermis. Three observations support this
idea: firstly, that S59 and vg-expressing cells arise in close
contact with the ectoderm and remain in contact (our own
observations). Secondly, that the stretched cells in mbc mutants
appear to contact the epidermis, and thirdly, that in neurogenic
mutants, lack of epidermis leads to premature loss of
expression of such genes as S59 and vg (Bate et al., 1993).
We cannot explain the curious observation that in mbc
mutants, S59 expression is lost from cell 1B. As described
above, Group I of the S59-expressing cells is first seen as one
cell, which divides into two. Cell Ia migrates across the
segment border, loses S59 expression in both wild-type
embryos and mbc mutants and in wild-type embryos becomes
incorporated into muscle 5. Cell Ib migrates a couple of celldiameters posteriorly and fuses with its neighbours to form
S59-expressing muscle 25. The loss of S59 expression from
cell Ib in mbc mutants is therefore a mystery and the only light
we can shed on the puzzle comes from the observation that in
mbc all the cells that retain S59 expression are lying in contact
with the anterior margin of the engrailed- (en) expressing
stripe in the ectoderm (our unpublished observations). It may
be that this is in some sense a ground state for mesodermal S59
expression. All the mesodermal S59-expressing cells arise in
contact with the en stripe, and cell Ib is the only S59-expressing 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 Ib
with en-expressing cells.
Conclusion
To conclude, we here present evidence in support of the
founder cell hypothesis of larval muscle development in
Drosophila. This model suggests that single cells of the
somatic mesoderm are selected and set aside to initiate the
process of muscle fusion. In addition, these founder cells
contain the information that gives the future muscle its
identity. This information is required from a very early stage,
as shown by the patterns of gene expression and cell migrations of the single-cell muscle founders. It is also required
later in development to determine the position and orientation of the muscle and its innervation pattern. We have shown
here that single cells of the somatic mesoderm are capable of
displaying the above characteristics even in the absence of
fusion and we therefore identify these cells as muscle founder
cells.
We thank Jacob Harrison for technical assistance, and Mary
Erickson, Ana Carmena and Fernando Jimenez for communicating
results prior to publication. We also thank Mary Baylies, Kendal
Broadie, Andreas Prokop, Helen Skaer and Mike Taylor for critically
reading earlier versions of this manuscript and making many helpful
suggestions. 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 Medical
Institute.
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(Accepted 20 April 1995)