1 Philipps-Universität Marburg, Fachbereich Biologie, Entwicklungsbiologie,
Karl-von-Frisch Strasse 8, 35043 Marburg, Germany
2 Philipps-Universität Marburg, Fachbereich Biologie, Spezielle Zoologie,
Karl-von-Frisch Strasse 8, 35043 Marburg, Germany
3 Institut für Neuround Verhaltensbiologie, Westfälische
Wilhelms-Universität Münster, Badestrasse 9, 48149 Münster,
Germany
Author for correspondence (e-mail:
renkawit{at}staff.uni-marburg.de)
Accepted 9 June 2004
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SUMMARY |
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Key words: Myoblast fusion, Attachment, Prefusion complex, Electron dense plaque kette, blow, rols, sns, mbc, crk
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Introduction |
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Drosophila is an appropriate model system with which to study the
basic mechanism of myoblast fusion, because myogenesis is completed within a
few hours during embryogenesis (Bate,
1993) and can be analysed genetically
(Dworak and Sink, 2002
). In
wild-type embryos, myoblast fusion starts at late stage 11 by the formation of
the founder cells, which first fuse with two to three fusion-competent
myoblasts (FCMs) to generate the precursor cells
(Baylies et al., 1998
;
Frasch and Leptin, 2000
;
Paululat et al., 1999a
). Until
stage 14-15, these precursor cells then recruit additional FCMs until the
desired muscle size is reached. At stage 16, muscles are correctly inserted
into their epidermal attachment sites (Fig.
1A,D). Ultrastructural analyses revealed a number of
characteristic steps during later myoblast fusion
(Doberstein et al., 1997
).
Following cell-cell adhesion and alignment, vesicles are transported to the
opposing membranes of precursor and fusion competent myoblasts. Here, they
form the so-called prefusion complex, which is followed by electron-dense
plaques and subsequent membrane breakdown
(Doberstein et al., 1997
).
|
In order to identify further components for myoblast fusion, we screened
EMS-induced mutations and such identified kette mutants displaying a
strong muscle fusion defect. Kette [also called Hem (FlyBase), Nap1 and GEX-3]
is well conserved during evolution
(Baumgartner et al., 1995;
Soto et al., 2002
;
Yamamoto et al., 2001
).
Mutations in kette were first identified based on their embryonic CNS
phenotype, which is based on defects in neurite outgrowth
(Hummel et al., 1999
;
Hummel et al., 2000
).
Subsequently, it was shown that kette affects the formation of the
F-actin cytoskeleton, presumably by regulating the activity of two main
regulators of F-actin nucleation Wasp and Wave (Scar FlyBase)
(Bogdan and Klämbt, 2003
;
Kunda et al., 2003
;
Rogers et al., 2003
). Both
Wasp and Wave are potent activators of the Arp2/3 complex, and their activity
must thus be tightly controlled (Miki and
Takenawa, 2003
). Whereas Wasp is autoinhibited, inhibition of Wave
function requires transacting factors. In vivo, Kette is found in a large
cytosolic protein complex also comprising Sra-1 (also called PIR121, CYFIP),
Abi, HSPC300 and Wave (Eden et al.,
2002
). Upon dissociation of this complex by binding to SH3 domains
or to activated Rac1, Wave is released from the complex and is rendered active
(Eden et al., 2002
). Thus, in
the cytosol Kette keeps Wave in an inactive state. By contrast, genetic data
suggest that at the membrane Kette can activate Wasp function
(Bogdan and Klämbt, 2003
).
In addition to these relatively ubiquitous acting proteins, regulation of
F-actin dynamics is expected to involve cell type specific factors.
We have characterised the muscle phenotype of kette mutants and show that Kette is required for myoblast fusion. Further genetic and phenotypic analyses show that kette interacts with the mesoderm specific expressed gene blow. This interaction is essential for the correct formation of electron-dense plaques and the initiation of membrane breakdown.
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Materials and methods |
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Immunohistological staining and whole-mount in situ hybridisation
For immunohistological staining, the following antibodies were used:
anti-ß3-Tubulin (polyclonal from rabbit)
(Leiss et al., 1988;
Buttgereit et al., 1996
),
anti-ß3-Tubulin (polyclonal from guinea-pig) (D. Buttgereit and R.R.-P.,
unpublished) anti-Kette (97/82) (Bogdan and
Klämbt, 2003
), anti-ß-galactosidase (polyclonal,
Biotrend), anti-Eve (from Developmental Studies Hybridoma Bank), anti-Kr
(Kosman et al., 1998
) and
anti-Alien (Goubeaud et al.,
1996
). Embryos were fixed and stained as described previously
(Stute et al., 2004
). As
detection system we used Vectastain ABC Elite-kit (Vector Laboratories) and
the TSA-kit from Perkin Elmer. The application of the TSA-kit is essential to
visualise Kette. Fluorescent secondary antibodies were obtained from
Dianova.
Whole-mount in situ hybridisation was carried out essentially as described
by Tautz and Pfeifle (Tautz and Pfeifle,
1989). DIG-labelled RNA antisense probes were synthesised by in
vitro transcription using a RNA-DIG-labelling-Kit (Roche) and a sns
cDNA clone in pBSKII kindly provided by Susan Abmayr
(Bour et al., 2000
). Analysis
was performed with a Leica confocal laserscan microscope.
Electron microscopy
Embryos at different developmental stages were dechorionised by bleaching,
prefixed in 18% glutaraldehyde/heptan 1:1 solution and mechanically peeled.
Mutant embryos were selected using an immunohistochemical ß-galactosidase
staining which is restricted to the balancer carrying embryos while homozygous
kette mutants lack lacZ activity
(Stollewerk et al., 1996). The
embryos were fixed by simultaneous glutaraldehyde/osmiumtetroxide-fixation
with postosmication (Franke et al.,
1969
). Afterwards, embryos were stained en bloc with uranylacetate
(Lin et al., 1994
), dehydrated
and embedded in Spurr's resin. After polymerisation semi- and ultra-thin
sections were cut. For analyses in the TEM (Hitachi) ultra-thin sections were
contrasted with lead citrate.
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Results |
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We first analysed the myoblast fusion phenotypes with regard to cell determination and ultrastructure, and then placed Kette within the described fusion cascade in relation to the other known fusion relevant proteins.
kette mutants are characterised by severe distortions in myoblast fusion
In kette mutants, we observe strong disturbances of myogenesis.
Null mutants show many unfused myoblasts until stage 16, which cluster close
to stretched minimuscles, presumably representing founder or precursor cells
(Fig. 1B,E compare with wild type in
1A,D). In addition, the hypomorphic alleles show unfused myoblasts
until stage 15, while at stage 16 hardly any unfused myoblasts are detectable
(Fig. 1C,F). Apparently, in
hypomorphic kette mutants fusion proceeds slower than in the wild
type but muscles are eventually formed. The epidermal attachment, however, is
often missing or incorrect (arrow in Fig.
1F). In order to visualise muscle attachments, we performed
anti-Alien and anti-ß3-Tubulin double labellings, as Alien allows the
visualisation of attachment sites
(Goubeaud et al., 1996). We
could therefore observe that some muscles find their attachment sites, whereas
others do not (Fig. 1L,M). This
suggests that Kette plays a role in both myoblast fusion and muscle
attachment.
We then asked at what level myogenesis was disturbed in kette mutants. As a first point, we excluded migration disturbances, which might affect the mesodermal cells: all kette mutant embryos showed correct dorsal closure that resulted in correct formation of the cardioblasts in the dorsal vessel (Fig. 1B,C,E,F). This indicates that after gastrulation, the mesodermal cells migrate correctly and that signalling from the epidermal cells to the underlying mesodermal cells can take place.
Second, we clarified whether Kette is expressed in the somatic mesoderm
during myoblast fusion. We used the anti-Kette antibody
(Bogdan and Klämbt, 2003)
to determine whether the Kette protein is found in the mesoderm. As shown in
Fig. 1H, Kette is detectable
throughout the entire somatic mesoderm during the myogenic relevant stages.
Double staining for Kette and ß-galactosidase, visualising the founder
cell marker rP298, in wild-type embryos clearly reveals that Kette is
expressed in fusion-competent myoblasts as well as in founder/precursor cells
(Fig. 1K). Thus, we propose
that Kette fulfils an intrinsic function in the somatic mesoderm during
myoblast fusion. Furthermore, in late stage 15 to stage 16, Kette accumulates
at the tips of the mature myotubes that anchor them to the epidermis
(Fig. 1G). This corresponds
well to the incorrect attachment of muscles we observed in the hypomorphic
alleles (Fig. 1M).
To support the idea that Kette fulfils an intrinsic function in the
mesoderm, we employed the UAS-GAL4 system
(Brand and Perrimon, 1993) by
using a twi-GAL4 driver for expression of Kette in the mesoderm of
ketteJ4-48 mutant embryos. The strong fusion phenotype
observed in ketteJ4-48 mutants is almost completely
rescued by mesodermal expression of Kette
(Fig. 1I,J compare with
Fig. 1B,E), which clearly
demonstrates the intrinsic function of Kette in the somatic mesoderm.
Overexpression of Kette in the wild-type background does not disturb muscle
development (data not shown).
In summary, Kette is required for both myoblast fusion and muscle fibre insertion into the epidermis. Expression data and mesoderm-specific rescue experiments both suggest that Kette acts in the mesoderm.
Fusion-competent myoblasts and founder cells are correctly determined in kette mutants
In order to investigate whether the kette mutant phenotype is
caused by abnormal myoblast fate determination or whether it is based on a
specific defect in myoblast fusion, we first tested whether the two myogenic
cell types, the founder cells and the fusion-competent myoblasts, are
determined correctly in kette-null mutants.
Founder cell formation can be traced by analysis of rP298-lacZ,
which is an enhancer trap that expresses ß-galactosidase under the
control of the Duf/Kirre regulatory region and thus allows to visualise all
founder cells (Nose et al.,
1998; Ruiz-Gomez et al.,
2000
). After fusion, each nucleus contains ß-galactosidase
and thus allows to monitor successful fusion. The comparison of rP298
directed ß-galactosidase expression in wild type
(Fig. 2A,C) and
ketteJ4-48 mutant embryos clearly shows that at stage 13,
founder cells are determined properly in time, in space and in number in
kette loss-of-function mutants
(Fig. 2B,D). To monitor the
rP298-positive nuclei relative to the forming muscles we used the
anti ß3-Tubulin antibody for counterstaining. In wild-type embryos the
nuclei of the former FCMs become rP298 positive after fusion with a
founder/precursor cell. Therefore all nuclei of a forming muscle are
rP298 positive (Fig.
2C). kette mutants, however, show less
rP298-positive nuclei, which are surrounded by many FCMs
(Fig. 2D). This is indicative
for fusion defects. The second essential cell population in the myogenic
mesoderm is the pool of FCMs. We analysed sns expression in
kette-null mutants and found that sns mRNA is expressed as
in the wild type (Fig. 2E,F),
suggesting that FCMs are correctly determined in kette mutants.
|
Muscle precursors are established in kette and blow mutants
We previously proposed a two-step fusion model with a first step leading to
precursor cells containing three or four nuclei, followed by a second step
establishing the mature myotubes (Rau et
al., 2001). Therefore, we aimed to clarify whether kette
mutants arrest at the first or second fusion step. The expression of Eve and
Krüppel indicates whether kette mutant embryos are able to
perform the first fusion to precursor cells or are left with mononucleated
founder cells.
Eve is expressed in the nuclei of dorsal muscle 1 (DA1) and in some
pericardial cells. In stage 15-16 wild-type embryos, DA1 contains up to 14
nuclei (Ruiz-Gomez et al.,
1997), while in ketteJ4-48 mutant embryos,
only minimuscles with three or four nuclei are formed
(Fig. 3B).
|
Another gene that is known to regulate myoblast fusion is blown
fuse (blow); blow mutants arrest fusion following the
prefusion complex (Doberstein et al.,
1997). To determine whether blow affects myogenesis at
the first or the second fusion step, we analysed Eve and Krüppel
distribution in blow-null mutants (blow2). As in
kette mutants, blow2 mutants reach the precursor
cell stage and then fail to form mature myotubes and stop myogenesis during
the second fusion step (Fig.
3C,G). The formation of precursor cells in kette mutants
also proves true at the ultrastructural level.
Fig. 4A shows a cluster of two
or three nucleated precursor cells underlying the epidermis. Likewise in
blow2 mutants we could confirm the formation of precursor
cells (Fig. 4B), while in the
mbc mutant embryos, only unfused mononucleated myoblasts are visible
(Fig. 4C).
|
In kette mutants, fusion-competent myoblasts also attach to the precursors as in the wild type, and electron-dense material accumulates into plaques (Fig. 4G). In the wild type, the plaques are 500 nm in length. In kette mutants, however, the plaques are two to three times longer (Fig. 4G). There is no sign of membrane breakdown in the vicinity of the plaques. Instead, residues of the dissolving prefusion complex remain frequently visible until stage 15 (arrow in Fig. 4G).
Because, in contrast to blow, kette mutants still form electron-dense plaques, we postulate that Kette acts downstream of Blow.
kette interacts genetically with blow during myoblast fusion and is able to rescue the blow mutant phenotype
To test the relationship of kette and blow further, we
performed epistasis experiments to detect possible genetic interactions. The
hypomorphic ketteG1-37 allele
(Fig. 5A) and
blow2 loss-of-function mutants
(Fig. 5B) can be distinguished
in the light microscope by ß3-Tubulin expression. We established balanced
Drosophila strains carrying the hypomorphic
ketteG1-37 mutation on the third chromosome and
blow2 on the second chromosome. Loss of one copy of
blow in a ketteG1-37 mutant background results in
an enhanced fusion phenotype of ketteG1-37 mutants
(Fig. 5C). This is even more
prominent in homozygous double mutants
(Fig. 5D). As the blow
phenotype appears dominant, we conclude that Kette is acting later then Blow
during myoblast fusion. This is confirmed by the phenotype of homozygous
blow2 embryos lacking one copy of kette, which
cannot be distinguished from homozygous blow2 mutants
(data not shown). Owing to the fact that removal of one copy of the
blow gene influences the phenotype of ketteG1-37
mutants, we propose that Kette and Blow interact during myoblast fusion.
|
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Discussion |
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Furthermore, we show that founder cells and fusion-competent myoblasts are correctly determined in kette mutants and muscle precursor cells are properly formed during the first myoblast fusion step. Electron microscopic analysis of kette mutants revealed that the second myoblast fusion step is interrupted during formation of the electron-dense plaques and thus kette mutants stop development shortly after blow but before sns15 mutants (Fig. 6).
|
In addition to a role of Kette during myoblast fusion, we noted high
expression of Kette at the growing tips of mature myotubes. These structures
are rich in F-actin and, like growth cones, migrate towards the
muscle-attachment sites (Volk,
1999).
Integration of Kette in a model for myoblast fusion
Within the two-step model of myoblast fusion, we placed kette
relative to other components of the fusion process
(Fig. 6). The initial
recognition between founder cells and fusion-competent myoblasts is mediated
by the Ig-domain proteins Duf/Kirre and Rst in the founder cell. The
extracellular domain of Duf/Kirre interacts with Sns, another member of the
immunoglobulin superfamily, which is expressed in fusion competent myoblasts
(Bour et al., 2000;
Dworak and Sink, 2002
). This
interaction may signal into both cell types and thus initiate the first fusion
step that leads to the formation of precursor cells.
It is possible that Duf/Kirre and Rst, as well as Sns, are also active in
the second series of fusion events leading from the precursor cells to the
mature myotubes. In the precursor cells, the Rols/Ants protein concentrates at
the membrane (Chen et al.,
2003; Menon and Chia,
2001
) and we propose that Rols/Ants is needed to start the second
series of fusion (Rau et al.,
2001
). Chen and Olson (Chen
and Olson, 2001
) have shown that in vitro Rols/Ants binds to the
intracellular domain of Duf/Kirre, and we suggest that this might be the
signal in the precursor cell that recruits further FCMs for fusion
(Fig. 7). In the precursor
cell, this interaction might initiate the formation of the prefusion complex
and subsequently the formation of the electron-dense plaques and finally to
membrane breakdown (Fig.
6).
|
Kette acts together with Blow in the second fusion step
The Blow protein is characterised by a pleckstrin homology (PH) domain that
is often involved in mediation of membrane binding and in regulation of the
cytoskeleton (Lemmon et al.,
2002). As membrane association is important for Kette function
(Bogdan and Klämbt, 2003
),
the observed genetic interaction may reflect a contribution of Blow in
activating Kette. Interestingly, it has been recently reported that Blow binds
to Crk (Giot et al., 2003
),
which in turn is able to associate with the Dock180 homolog Mbc (Galetta et
al., 1999; Nolan et al.,
1998
). This adapter protein is proposed to link to the Duf/Kirre
protein via Rols/Ants in precursor cells
(Chen and Olson, 2001
). We
propose that a similar link between Sns and Mbc in the fusion-competent cells
is mediated by a yet unidentified protein. This scenario would link the
activation of membrane-bound receptors to the regulation of F-actin dynamics
(Fig. 7).
The SH2-SH3 adaptor protein Crk has not yet been studied at the functional
level in Drosophila but it is known from vertebrates that its
orthologue CrkII and Dock180 form a complex after external stimulation
(Hamasaki et al., 1996;
Hasegawa et al., 1996
;
Klinghoffer et al., 1999
;
Lamorte et al., 2003
;
Li et al., 2002
;
Li et al., 2003
;
Ruest at al., 2001
;
Tachibana et al., 1997
;
Thomas et al., 1995
;
Vuori et al., 1996
) and are
able to promote Rac1 activation (Brugnera
et al., 2002
;
Côté and Vuori,
2002
; Guimienny et al.,
2001
; Kiyokawa et al.,
1998
). Rac1, in turn, is acting on the activation of Wave
(Eden et al., 2002
).
The interaction between Blow and Crk is supported by our finding of several
potential binding motives in Blow that are described as potential recognition
sites by both Crk-SH3-domains (Feller,
2001).
Therefore, we postulate that the function of Blow in myoblast fusion is dependent on its binding to Crk for which no mutants exist. We propose that this interaction leads to the activation of kette.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Max-Delbrück-Centrum für Molekulare Medizin,
Robert-Rössle-Strasse 10, 13122 Berlin, Germany
Present address: Institut für Allgemeine und Spezielle Zoologie,
Allgemeine Zoologie und Entwicklungsbiologie, Justus-Liebig-Universität
Giessen, Stephanstrasse 24, 35390 Giessen, Germany
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