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First published online 25 February 2009
doi: 10.1242/dev.031567
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Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA.
* Author for correspondence (e-mail: rcripps{at}unm.edu)
Accepted 28 January 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Drosophila, TGFβ, Adult myogenesis, Crossveinless, Founder cell
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). It
is also apparent that several muscle diseases affect some muscle groups to a
greater extent than other groups; thus, understanding how individual skeletal
muscles arise is a central challenge in the field.
Although there is still much to learn regarding muscle patterning in
mammalian muscle specification, some significant insight has been afforded by
invertebrate systems. In grasshopper embryos, Ho et al.
(Ho et al., 1983) were the
first to identify, among the myoblast pool, individual larger cells that
appeared to seed the formation of individual muscle fibers. These `muscle
pioneers' were subsequently identified in Drosophila
(Bate, 1990), where it was
shown that single skeletal muscle fibers arise from the fusion of a muscle
pioneer or `founder' cell with a small number of fusion-competent
myoblasts.
Each embryonic founder cell is also largely responsible for the acquisition
of fiber-specific phenotypes, such as patterns of gene expression, innervation
and muscle attachment locations. This was concluded based upon myoblast fusion
mutants, where unfused founder cells still attempt to make appropriate
orientations and connections (Rushton et
al., 1995) (reviewed by Baylies
and Michelson, 2001). Furthermore, specific muscle phenotypes
arise from individual patterns of regulatory gene expression within founder
cells (Crozatier and Vincent,
1999; Knirr et al.,
1999; Clark et al.,
2006). Thus, understanding the genetic pathways that contribute to
founder cell specification will impact our understanding of muscle
specification. Along these lines, signaling pathways including the Wingless
pathway (Cox and Baylies,
2005) and the epidermal growth factor pathway
(Buff et al., 1998) contribute
to founder cell selection in the embryo. Nevertheless, there are still several
parts of this specification process that have yet to be uncovered.
During Drosophila metamorphosis, most of the larval skeletal
muscles degenerate and are replaced by new muscles arising from imaginal
myoblasts (Crossley, 1978;
Currie and Bate, 1991;
Fernandes et al., 1991). These
adult myoblasts are specified during embryogenesis and many become associated
with the imaginal discs (Poodry and
Schneiderman, 1970; Bate et
al., 1991). Subsequently, the adult myoblasts migrate from the
discs to the future locations of the muscles, and myoblasts from each disc
give rise to a variety of physiologically distinct muscles
(Lawrence, 1982). However,
mechanisms that control the specification of many of these muscles have yet to
be fully elucidated.
Does the founder cell model of muscle development also hold true for adult
myogenesis in Drosophila? This question is important given the
complex organization of the adult skeletal musculature: individual fibers can
span several segments and multiple fibers are often arranged into larger
muscles that are more characteristic of those found in mammals. In the adult
thorax, there are two major types of muscles (reviewed by
Bernstein et al., 1993): the
fibrillar indirect flight muscles (IFMs) are adapted to contract at high
frequency to provide the power for flight. The IFMs comprise six pairs of
medial fibers termed the dorsal longitudinal muscles (DLMs), and three pairs
of lateral muscles termed the dorsoventral muscles (DVMs). In addition to the
fibrillar flight muscles, physiologically distinct tubular muscles are located
laterally and ventrally in the thorax. Tubular muscles function in walking,
jumping and angling the wings. Tubular muscles each are composed of several
individual fibers grouped together. Most prominent among the tubular muscles
is the tergal depressor of the trochanter (TDT, or `jump') muscle, a large
fiber found in all Diptera studied which attaches the dorsal notum to the
second pair of legs. This muscle is essential for the escape response of the
fly (Nachtigall and Wilson,
1967).
Several groups have demonstrated that the formation of some adult skeletal
muscle fibers are associated with cells showing the characteristics of
founders. These include the abdominal muscles, where each individual muscle is
pre-figured by a cell expressing the canonical founder cell marker,
dumbfounded/kirre, usually detected as an enhancer trap termed
duf-lacZ, or rp298
(Ruiz-Gomez et al., 2000).
Precursors of the adult indirect flight muscles also express duf-lacZ
(Dutta et al., 2004), and the
ablation of these founder cells significantly disturbs the formation of the
DVMs (Atreya and Fernandes,
2008). Interestingly, whereas founders have been observed to
prefigure adult muscle development, relatively little is known of the
mechanisms responsible for their specification at this stage. In fact, the
process of singling out founder myoblasts, which in the embryo requires in
part lateral inhibition via the Notch pathway
(Carmena et al., 1995;
Carmena et al., 1998), appears
to occur in a Notch-independent manner in the adult
(Dutta et al., 2004). Thus,
understanding specification of adult muscles should provide further new
insight into muscle specification mechanisms.
In this work, we have analyzed the basis of a mutation that affects the morphology of the jump muscle of the adult thorax. This mutation, which causes a reduction in TDT fiber number and defects in the morphology of the muscle, arises from mutation of the crossveinless (cv) gene, the established function of which is to modulate TGFβ signaling in the specification of wing crossveins. We demonstrate that cv functions in muscle development as part of the TGFβ pathway, which is activated autonomously in the adult myoblasts in order to control the number of founder cells specified for the TDT. By manipulating the TGFβ pathway, the TDT, which normally comprises 20-30 muscle fibers, can be modified to consist of as little as five fibers or as many as 50 fibers. Overall, these studies define an important function for the TGFβ pathway in adult muscle specification that might also be used in the formation of the more complex muscles found in higher animals.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) at 25°C
unless indicated. Stocks carrying cv1, cv43,
dpp10638, X chromosome deficiencies, the second chromosome
deficiency covering gbb, and Gal4 driver lines (unless
noted) were obtained from the Bloomington Drosophila Stock Center.
UAS-Dad (Tsuneizuni et al.,
1997) and UAS-tkv*
(Adachi-Yamada et al., 1999)
were from Stuart Newfeld (Arizona State University, AZ, USA); UAS-cv
was from Larry Marsh (UC Irvine, CA, USA); 1151-Gal4
(Anant et al., 1998) was from
L. S. Shashidhara (CCMB, Hyderabad, India); and duf-lacZ was from
Upendra Nongthomba (India Institute of Science, Bangalore, India). The
wild-type strain Simms-L was locally captured in Albuquerque (NM, USA) and
determined to be conspecific based upon visual examination and crossing with
established laboratory strains.
Preparation of samples for microscopy
Samples were prepared for paraffin sectioning as described by Lyons et al.
(Lyons et al., 1990) and
modified by Cripps et al. (Cripps et al.,
1998). Sections were cut at 8-12 µm, and stained with
Hematoxylin and Eosin (Sigma) for evaluation of TDT structure. Stained slides
were dehydrated through 100% ethanol, soaked in xylene, and mounted in
Cytoseal-XYL (VWR Scientific Products). TDT fibers were counted from both
sides of the thorax and treated as independent samples. Averages for each
genotype were calculated and comparisons were performed using Student's
t-test at
Graphpad.com.
Cryosections were prepared by embedding adult flies in OCT medium followed by freezing. Sections were cut at 15 µm at -18°C and air dried. Next, samples were fixed for 5 minutes at room temperature with 1.9% v/v formaldehyde in 1xPBS, washed and used for antibody staining as described in the following section.
For pupal dissections, newly pupariated animals were marked, and aged for the appropriate time until harvesting and dissection. All pupal samples were dissected in a Sylgard-coated petri dish (Dow Corning) and pinned open. After dissection, samples were fixed for 30 minutes on ice with 5% formaldehyde in 1xPBS, washed in PBTx [1xPBS, 0.2% v/v Triton-X100, 0.2% w/v Blocking Agent (Roche)], and then subjected to blocking and antibody incubations (see below).
For documentation of adult wings, wings were removed from adult flies and stored in 70% (v/v) ethanol overnight, then transferred twice to 100% ethanol. Wings were next soaked in 100% xylene, and mounted using Cytoseal-XYL (VWR Scientific Products) for photography.
Immunostaining and in situ hybridization
Fixed and washed samples were subjected to immunostaining essentially as
described by Patel (Patel,
1994) and modified by Molina and Cripps
(Molina and Cripps, 2001).
Primary antibodies used were: anti-βPS-integrin 1:10
(Brower et al., 1984)
(University of Iowa Developmental Studies Hybridoma Bank, IA); anti-Z(210)
1:100 (Vigoreaux et al., 1991)
(kindly supplied by Jim Vigoreaux, University of Vermont, VT, USA); anti-MEF2
1:2000 (Lilly et al., 1995)
(kindly supplied by Bruce Paterson, NIH); rabbit anti-β-galactosidase
1:1000 (AbCam); and mouse anti-β-galactosidase (Promega). For
immunofluorescence, secondary antibodies were Alexa conjugated (Molecular
Probes), and mixed with Alexa-488 phalloidin at 1:500 (Molecular Probes) and
DAPI used at 2 µg/ml (Sigma). For immunohistochemistry, secondary
antibodies and detection were carried out using the Vectastain Elite staining
kit and diaminobenzidine (DAB) substrate according to the manufacturer's
recommendations.
In situ hybridization was carried out using digoxigenin-labeled probes
(Roche) according to the method of O'Neill and Bier
(O'Neill and Bier, 1994).
Probes for cv were generated from the plasmid pOT2/SD27025
(Drosophila Genomics Resource Center, Indiana University, IN, USA): sense
probes were synthesized from plasmid cut with XhoI using T7 RNA
polymerase; antisense probes were generated from plasmid cut with
EcoRI using SP6 RNA polymerase.
Images were collected using an Olympus BX-51 stereomicroscope using either DIC or fluorescence optics. Digitally captured images were assembled into figures using Adobe Photoshop.
Jump testing
Jump tests were performed essentially as described by Cripps et al.
(Cripps et al., 1994).
Briefly, flies lacking wings and aged 2-3 days after eclosion were induced to
jump from a platform elevated 10 cm above a piece of white paper. The landing
location was marked and the lateral distance from the edge of the platform to
the landing point was measured in mm. Average distances were calculated for
more than 20 individuals for each genotype and compared using Student's
t-test.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Peckham et al.,
1990). The cells are visualized in paraffin sections cut
horizontal to the axis of the muscle (Fig.
1A), and also are outlined in anti-β-PS integrin stains of
horizontal cryosections (Fig.
1B). Large and small cells can additionally be distinguished based
upon the presence (large cells) or relative absence (small cells) of the
Z(210) Z-disc-associated protein (Fig.
1C) (Vigoreaux et al.,
1991).
Control animals in some of our publications have shown fewer fibers than
the canonical 26-28 (Baker et al.,
2005), prompting us to evaluate TDT fiber number in a range of
strains. The number of small cells only occasionally deviated from four (a
range of three to five); however, there was significant variation in the
number of large cells (summarized in Table
1). Some strains showed the published 26-28 large fibers
(Fig. 1A), other lines showed
as few as 18 large fibers (Fig.
1B). These data include a locally caught strain named Simms-L,
which showed an average of 22 large cells. Nevertheless, in all but one of
these strains, the shape and arrangement of the fibers was maintained. Given
the importance of this muscle to the escape response of the fly, we sought to
investigate the genetic basis of this variation in more detail.
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). Given
the large reduction in fiber number in the y cv1 v f car
strain, we defined the cause of this phenotype.
Identification of cv as the gene responsible for TDT defects
Using standard genetic crosses and analyzing TDT fiber number in
individuals, the reduction in fiber number in the X-chromosome stock arose
from a recessive mutation that mapped to the marked X chromosome (data not
shown).
To localize the mutation, we crossed y cv1 v f car with a wild-type strain to generate y cv1 v f car/+ + + + + females, which were then backcrossed to wild-type males. Male recombinant offspring from this cross were collected, genotyped using the visual markers, and individually assessed for TDT fiber number. The results are shown in Table 2. In all cases the TDT fiber phenotype segregated according to the allele of cv that was present: cv+ recombinants showed a normal fiber number, whereas cv1 recombinants showed defective TDTs. These results placed the TDT mutation in the proximity of cv.
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Given the close linkage of the TDT mutation to the cv1
allele, and given that cv resides at 5A13 of the X chromosome, we
next tested whether mutation of cv might be responsible for the TDT
phenotype by analyzing cv43, an allele which is null for
cv gene function, and which has a genetic background distinct to that
of cv1 (Vilmos et al.,
2005). cv43 homozygotes also showed a severe
TDT phenotype (Fig. 2D), and
cv1/cv43 female heterozygotes had defective TDT
structure (Fig. 2E). These
studies demonstrated that mutation of cv, in addition to affecting
the crossveins of the adult wing (Bridges,
1920), affects the normal development of the adult jump
muscle.
We also determined whether genetic rescue of the cv mutation would
rescue the jump muscle phenotype. We used the Gal4-UAS system
(Brand and Perrimon, 1993) to
determine whether Gal4-driven expression of cv was sufficient to
rescue the mutant phenotype. We also followed the crossveinless phenotype in
the wing to control for our manipulations; wild type and mutant are indicated
in Fig. 3A,B.
When we generated a homozygous stock of the genotype w1118 cv43; UAS-cv, this stock showed some rescue of the crossvein phenotype in the wings (Fig. 3C, left panel). Upon sectioning these adults, the TDT phenotype was also rescued (Fig. 3C, right panel). We attribute this finding to the UAS-cv transgene used being slightly leaky, such that in the homozygous condition it generates sufficient Cv protein to rescue the two phenotypes. By contrast, when we studied w1118 cv43; UAS-cv/+ (i.e. mutants carrying just one copy of the UAS-cv transgene), adults showed the mutant crossvein and TDT phenotypes (Fig. 3D), indicating that two copies of the UAS construct were required for rescue.
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), or the mesodermal driver 24B-Gal4
(Brand and Perrimon, 1993). In
male offspring for both of these cases (offspring of the genotype
w1118 cv43/Y; UAS-cv/ptc-Gal4 or
w1118 cv43/Y; UAS-cv/+; 24B-Gal4/+),
significant rescue was observed for both the crossvein and the TDT
(Fig. 3E,F).
The observation that either a mesodermal or an ectodermal driver could
rescue the wing and muscle phenotypes of cv43 can be
explained by the demonstration that Cv is a secreted protein
(Shimmi et al., 2005;
Vilmos et al., 2005), and
might diffuse from a source to the target tissue. It is interesting to note
that 24B-Gal4 can direct sufficient Cv synthesis that the wing vein
phenotypes can be rescued. In this instance, it is possible either that
24B-Gal4 is expressed at some levels outside of the mesoderm, or that
the production of Cv in this background sufficiently stabilizes the ligand to
which it binds in order to allow signaling over large distances.
To determine whether TDT defects are a common phenotype among mutants showing wing crossvein alterations, we studied the fiber number in mutants for crossveinless-2 (cv-2) and detached (det). In both of these cases, no obvious fiber defect was observed (Table 1), indicating that cv must play either a unique or a more crucial function in muscle development than other members of this mutant class.
Effects of reduced fiber number upon TDT function
We next determined whether reductions in the number of TDT fibers affected
TDT function. This muscle is solely responsible for the jump response in flies
(Nachtigall and Wilson, 1967;
Elliot et al., 2007), thus we carried out jump tests of wild-type, mutant and
rescued flies. For w1118 controls, the jumping distance
was 48 ±2 mm, whereas w1118 cv43 males
jumped 38±3 mm. This difference was significant using the Student's
t-test (P=0.02). When we analyzed the jumping ability of
w1118 cv43; UAS-cv rescued males flies (the
same genotype used in Fig. 3C),
the jumping distance was rescued to 50±3 mm, essentially
indistinguishable from wild type. These experiments revealed that the TDT
defects in cv mutants result in subtle, albeit significant defects in
jump muscle performance. Clearly, reduction in TDT fiber number causes severe
abnormalities in both muscle morphology and muscle performance.
TGFβ signaling controls TDT fiber number
Recently, cv was shown to be a component of the TGFβ
signaling pathway, facilitating Dpp signaling in the formation of crossveins
(Shimmi et al., 2005;
Vilmos et al., 2005). To
determine whether the cv muscle phenotype arises from disruptions in
TGFβ signaling, we tested the effects upon TDT development of activation
and repression of the TGFβ pathway.
To achieve this, we firstly crossed the adult myoblast driver 1151-Gal4 to a line carrying UAS-tkv*, an autonomous constitutive activator of the TGFβ pathway. We analyzed TDT structure in adult offspring (termed 1151>UAS-tkv*) using both paraffin sections and immunostained cryosections. Compared with wild type (Fig. 4A-C), the mutant offspring showed a striking increase in the number of TDT fibers, although each individual fiber was somewhat smaller than in wild-type muscles (Fig. 4D,E). We consistently observed over 50 fibers per TDT, approximately twice the normal number. By immunostaining we observed that most of the fibers showed accumulation of Z(210), a characteristic of the large TDT cells (Fig. 4F). Thus, fiber number, but not fiber fate, was altered in these mutants.
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The opposing effects upon TDT fiber number of activating or repressing the TGFβ pathway provided strong evidence for the involvement of this pathway in TDT development. Moreover, as we used a Gal4 driver line active in the adult myoblasts, we also conclude that the TGFβ pathway is activated in the myoblasts themselves, rather than in an adjacent cell population.
Specification of TDT muscle fibers
To define the cellular basis for the TDT defects, we studied the early
development and specification of the TDT in duf-lacZ at late larval
and early pupal time points. By correlating this expression with markers of
the muscle lineage, we defined crucial events in TDT formation, which could be
compared with cv mutants. First, the TDT is pre-figured by the
specification of founder cells at the proximal region of the T2 leg imaginal
disc. The founders were somewhat difficult to discern during the late larval
stage (Fig. 5A), either because
they were just being specified, or because the duf-lacZ reporter was
only just becoming activated. In the early pupal stage [2 hours after puparium
formation (APF)] 12-15 duf-lacZ-expressing founder cells were
observed in this region of the imaginal disc
(Fig. 5B), and by 8 hours APF
had increased to an average number of 21.5±1.3 founder cells
(Fig. 5C).
As pupal development proceeded, this group of founder cells migrated laterally and dorsally, among a large number of MEF2-positive myoblasts. By 16 hours APF, the TDT founders had increased in number to 25.4±1.5, similar to the final number of large plus small fibers in the mature TDT of duf-lacZ adults (Fig. 5D; Table 1). Later than 16 hours APF there was no significant increase in the number of founder cells for the TDT, and instead the formation of linear fibers began to take place.
These observations first suggested that the fibers of the TDT each develop
according to the founder cell model. Second, the specification of TDT founders
takes place over several hours at the end of the larval stage and early during
pupal development. These findings are consistent with the observations of
Rivlin et al. (Rivlin et al.,
2000) who elegantly followed the appearance and migration of
`muscle pioneers' for the TDT, which are probably coincident with the founder
cells. In addition, similar data are presented by Atreya and Fernandes
(Atreya and Fernandes, 2008),
although their study did not focus specifically upon TDT development.
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Interestingly, we observed that the initial specification of TDT founders
was normal in the cv mutants. By 8 hours APF, the number of founder
cells in the mutants was 20.2±0.8, which was not significantly
different from wild type at this timepoint. By 16 hours APF, however, there
were slightly fewer TDT founder cells observed in the cv43
mutants, averaging 18.3±0.5 founder cells
(Fig. 6C). This number was
significantly less than wild-type at 16 hours APF (P<0.01,
Student's t-test), and identified this timepoint as the first at
which defects became apparent in the cv mutants. This value of
18 founder cells is slightly larger than the number of large plus small
fibers in the cv mutants, and it is possible that there is additional
loss of founder cells prior to the initiation of fiber formation.
At 24 hours APF, the formation of individual fibers could be easily discerned in both wild-type (Fig. 6B) and cv43 mutants (Fig. 6D). However, the number of fibers was clearly reduced in the mutants compared with controls, and the mutant fibers were also disorganized. Taken together, these studies indicated that in the cv mutants the process of founder cell specification was stalled, and that the reduction in founder cell numbers was the direct cause of the fiber number defect.
We next determined whether founder cell number and the formation of initial TDT fibers was altered when we interfered with TGFβ signaling via expression in the myoblasts of activated tkv or of Dad. Consistent with our observations of fiber number in the adults, we saw striking effects upon founder cell and fiber number in young mutant pupae. For expression of activated tkv, the number of founder cells was clearly increased at 16 hours APF. This is apparent in comparing Fig. 6E with 6A. The increase in founders was also reflected by an increase in the total number of fibers that were initially specified (Fig. 6F).
When we expressed Dad in the adult myoblasts, the opposite effect was observed: there were significantly fewer founder cells (only two or three can be discerned in Fig. 6G), although we also noted that there was an overall reduction in the total number of myoblasts, as determined by anti-MEF2 staining. At 24 hours APF, TDT fibers were difficult to identify as they were so few in number, and also showed severe hypoplasty (Fig. 6H).
Taken together, the studies shown in Fig. 6 define for the first time a crucial role for TGFβ signaling in the specification of TDT founder cells. These results will be further evaluated in the Discussion.
TGFβ ligands controlling TDT development
We next sought to identify the TGFβ ligand responsible for controlling
TDT fiber number. Ligand encoded by the dpp gene is expressed at high
levels in the imaginal discs and during pupal development
(Masucci et al., 1990) and
expression of lacZ from a dpp enhancer trap lies close to
the TDT founder cells throughout early pupal development (data not shown).
Furthermore, ligand encoded by glass bottom boat (gbb) is
also expressed broadly in the imaginal discs
(Khalsa et al., 1998). Thus,
we focused upon these genes as potential contributors to TDT founder cell
specification.
To determine whether Dpp or Gbb might be important for TDT founder specification, we tested whether double-heterozygotes for a ligand-encoding gene and for cv mutant alleles showed significantly fewer TDT fibers than did single heterozygotes for either gene mutation. The results of this analysis are presented in Fig. 7A,B. For Dpp, we observed, first, that the number of fibers in the dpp10638/+ heterozygotes was reduced slightly compared with that of cv43/+, suggesting that haploinsufficiency for dpp might be an important factor in muscle specification. More importantly, the combination of both cv and dpp mutant alleles resulted in a fiber count further reduced relative to either single heterozygotes alone, and which was significant based upon Student's t-test (Fig. 7A). These studies identified Dpp as at least one of the TGFβ ligands that impacts TDT fiber number during pupal muscle development.
For Gbb, we also investigated whether haploinsufficiency would exacerbate the TDT phenotype in a cv43/+ background. In this instance, we observed a more striking effect upon fiber number in the double heterozygotes (Fig. 7B). The double mutants displayed a highly significant reduction in fiber number when compared with controls. These data suggest that both Dpp and Gbb ligands might play important roles in specification of TDT muscle fibers.
To complement these data, we studied the pupal expression pattern of
cv by in situ hybridization. We generated cv sense and
antisense probes, and validated their functionality in embryos. As reported by
Vilmos et al. (Vilmos et al.,
2005), we observed specific expression of cv surrounding
the embryonic tracheal pits (Fig.
7C), whereas no signal was obtained from a sense control probe
(Fig. 7D).
In 16 hour APF pupal samples, we also observed expression of cv in the forming cuticle, close to where the TDT myoblasts had migrated (Fig. 7E). Although the signal was relatively weak, it was reproducibly greater than in sense probe control preparations that were stained in parallel (Fig. 7F). We are currently generating promoter-lacZ gene fusions of cv, in order to expand upon these data. These findings will be presented elsewhere.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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The TGFβ pathway has roles in a range of developmental processes, and
the addition of adult muscle development here extends a detailed list
(reviewed by Kollias and McDermott,
2008). In mammals, TGFβ can affect muscle development in
several ways, including inhibition of differentiation (e.g.
Yanagisawa et al., 2001) and
inhibition of muscle regeneration in vivo
(Cohn et al., 2007).
Interestingly, the TGFβ molecule myostatin acts in mammalian muscle
development to modulate the number of muscle fibers: in myostatin mutants in a
variety of mammalian models, there is a profound increase in both muscle fiber
number and muscle mass (reviewed by
Kollias and McDermott, 2008).
Despite these similarities, there is not yet sufficient evidence to suggest
that Cv-mediated modulation of the Dpp pathway plays a similar role in
Drosophila to that played by myostatin in mammals. This is at least
partly because the effects of cv mutants that we observe here are
restricted to a small subset of the adult musculature. A Drosophila
gene named Myoglianin and encoding a molecule with strong sequence
similarity to myostatin has been described, although no mutant alleles have
been characterized (Lo and Frasch,
1999).
Our genetic interaction data suggest roles for both Dpp and Gbb in TDT
fiber specification. Each of these ligands function in wing vein development
(e.g. Khalsa et al., 1998;
Ralston and Blair, 2005);
thus, a combinatorial role for them in founder cell specification would not be
unprecedented. We also note that the Drosophila genome encodes a
number of additional TGFβ-related molecules
(O'Connor et al., 2006), and
such molecules, in addition to Dpp and Gbb, might contribute to TDT founder
cell specification.
Although manipulation of the TGFβ pathway showed clear effects upon the numbers of TDT founder cells, we also note that inhibition of the pathway, via UAS-Dad, caused a decrease in the number of total myoblasts as visualized by MEF2 staining. This observation suggests that, in addition to founder cell specification, the TGFβ pathway in adult myoblasts impacts either myoblast proliferation or myoblast survival. This observation is consistent with our finding that, in cv mutants, the number of founder cells reduces slightly as pupal development proceeds.
Specification of TDT founder cell number appears to be subject to
significant variability in Drosophila. This observation contrasts
sharply with many of the other muscles of the animal, which show relatively
invariant fiber numbers. These include the skeletal body wall muscles of the
larva (Bate, 1993), and the
indirect flight muscles of the adult
(Cripps and Olson, 1998;
Farrell et al., 1996). Perhaps
the variability in TDT fiber number is a reflection of the multi-step
TGFβ pathway responsible for its specification, where variation in one or
a few of the signaling components required for founder cell specification will
ultimately impact the number of founder cells specified. Similar to the
studies presented here, a genetic approach should be able to identify
additional genes whose products function in this founder specification pathway
and are responsible for the strain-specific differences in TDT fiber number
that we have characterized. The identified genes might encode novel new
members of the TGFβ signaling pathway active in myoblasts, or might
identify mild alleles of known pathway members.
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;
Thompson et al., 2001;
Sebastin et al., 2005). This
variability also has a strong heritable component
(Thompson et al., 1921), and
there is also significant variation in the size of this muscle
(Sebastin et al., 2005).
Similarly, the plantaris major also shows variable loss in humans
(Vanderhooft, 1996). Our
studies, which define how muscle fibers in the adult can be specified, point
to potential mechanisms that might contribute to this variability in higher
animals. |
Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Adachi-Yamada, T., Nakamura, M., Irie, K., Tomoyasu, Y., Sano,
Y., Mori, E., Goto, S., Ueno, N., Nishida, Y. and Matsumoto, K.
(1999). p38 mitogen-activated protein kinase can be involved in
transforming growth factor beta superfamily signal transduction in Drosophila
wing morphogenesis. Mol. Cell. Biol.
19,2322
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Anant, S., Roy, S. and VijayRaghavan, K.
(1998). Twist and Notch negatively regulate adult muscle
differentiation in Drosophila. Development
125,1361
-1369.[Abstract]
Atreya, K. B. and Fernandes, J. J. (2008).
Founder cells regulate founder number but not fiber formation during adult
myogenesis in Drosophila. Dev. Biol.
321,123
-140.[CrossRef][Medline]
Baker, P. W., Kelly Tanaka, K. K., Klitgord, N. and Cripps, R.
M. (2005). Adult myogenesis in Drosophila
melanogaster can proceed independently of Myocyte enhancer factor-2.
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