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First published online March 6, 2009
doi: 10.1242/10.1242/dev.026302
1 Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
2 The Huck Institutes of Life Sciences, The Pennsylvania State University,
University Park, Pennsylvania, PA 16802, USA.
3 Department of Anatomy and Cell Biology, University of Kansas Medical Center,
3901 Rainbow Boulevard, MS 3038, Kansas City, KS 66160, USA.
Author for correspondence (e-mail:
sma{at}stowers-institute.org)
Accepted 23 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: Myoblast fusion, Sns, Hbs, Cell adhesion, Muscle development, Drosophila
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Founder myoblasts are specialized cells that dictate muscle identity, and
confer on each muscle fiber unique features that include size, shape, pattern
of innervation and attachment. FCMs represent a larger naïve group of
cells that are lacking the complex attributes characteristic of mature muscle
(Abmayr and Kocherlakota,
2005). These cells come under the influence of
founder-cell-specific muscle-identity genes, becoming entrained to the
myogenic program of the founder cell with which they fuse. The initial fusion
event occurs between a founder cell and one or two FCMs to form a muscle
precursor, whereas subsequent fusions occur between the developing syncitium
and additional FCMs.
In Drosophila, cell adhesion molecules of the immunoglobulin
superfamily (IgSF) direct the above events, and include kin of irre
(kirre; also called dumbfounded, or duf),
roughest (rst; IrreC), sticks and stones
(sns) and hibris (hbs)
(Artero et al., 2001;
Bour et al., 2000;
Dworak et al., 2001;
Ruiz-Gomez et al., 2000;
Strunkelnberg et al., 2001).
The kirre and rst loci result from gene duplication
(Strunkelnberg et al., 2003)
and are orthologs of syg-1 in Caenorhabditis elegans
(Shen and Bargmann, 2003) and
neph1-4 in mammals (Sellin et
al., 2003). Kirre is exclusive to the founder cells
(Ruiz-Gomez et al., 2000),
whereas Rst is present in founder cells and at least some FCMs
(Strunkelnberg et al., 2001).
Although no role has been identified for Rst in the FCMs, Kirre and Rst
function redundantly in the founder cell
(Strunkelnberg et al., 2001).
Embryos lacking both kirre and rst exhibit no myoblast
fusion, a defect that is rescued by mesodermal expression of either gene
(Ruiz-Gomez et al., 2000;
Strunkelnberg et al., 2001).
The FCM-specific IgSF proteins Sns and Hbs share 48% identity
(Artero et al., 2001;
Bour et al., 2000;
Dworak et al., 2001). Like
their orthologs C. elegans syg-2
(Shen et al., 2004) and
vertebrate nephrin (Kestila et
al., 1998), Sns and Hbs are predicted to include nine Ig domains
and one fibronectin type-III domain in their extracellular regions. Their
cytoplasmic domains differ in length, corresponding to 374 amino acids and 165
amino acids, respectively. Sns is restricted to the FCMs, appears on their
surface just before fusion, and is often coincident with Kirre or Rst at
points of cell-cell contact (Bour et al.,
2000; Galletta et al.,
2004). Hbs is also restricted to the FCMs, where it declines
slightly before Sns. In cells that express both proteins, Sns and Hbs
co-localize at discrete points on the cell surface
(Artero et al., 2001). Despite
these similarities, Sns and Hbs have distinct roles from each other in the
FCMs. Whereas embryos lacking sns exhibit a dramatic absence of
multinucleate syncitia, embryos lacking hbs exhibit only a modest
perturbation of myoblast fusion, which does not impair their survival.
Moreover, although some studies have suggested that Hbs acts antagonistically
to limit Sns activity (Artero et al.,
2001), others suggest that Hbs acts positively to direct limited
myoblast fusion in the absence of Sns
(Menon et al., 2005).
Sns appears to act as a receptor for Kirre and Rst, mediating the ability
of FCMs to recognize and adhere to founder cells. Intracellular pathways
downstream of these proteins then direct myoblast fusion. Downstream of Kirre
is the guanine nucleotide exchange factor Schizo (Loner), which probably
activates Rac1 via the GTPase Arf51F (Arf6)
(Chen et al., 2003). The
cytoplasmic domain of Kirre is also linked to the non-conventional guanine
nucleotide exchange factor Mbc (Erickson
et al., 1997) through interaction with Rolling pebbles (Rols;
Antisocial, or Ants) (Chen and Olson,
2001). Whereas Kirre and Rols are exclusive to the founder cells,
some of this machinery is present and may be required in both founder cells
and FCMs. For example, expression of Mbc exclusively in the founder cells is
insufficient to rescue the mutant phenotype
(Balagopalan et al., 2006). Mbc
functions in concert with Ced-12 (Elmo) to activate the small GTPases Rac1 and
Rac2, which are essential for myoblast fusion
(Geisbrecht et al., 2008;
Hakeda-Suzuki et al., 2002).
Activated Rac1 then modulates polymerization of Actin 5C (F-actin) through the
Arp14D/66B (Arp2/3) complex via SCAR
(Berger et al., 2008;
Richardson et al., 2007) and
the regulatory factor Hem (Kette) (Hummel
et al., 2000; Schroter et al.,
2004). Arp14D/66B-directed actin polymerization is also regulated
in the FCMs through the action of WASp and the FCM-specific WASp-interacting
protein Verprolin 1 (Vrp1; also termed D-wip and solitary,
or sltr) (Berger et al.,
2008; Kim et al.,
2007; Massarwa et al.,
2007; Schafer et al.,
2007). Consistent with the involvement of actin remodeling
proteins, dynamic foci of Actin 5C are present at sites of myoblast fusion and
modulated by these proteins (Kesper et
al., 2007; Richardson et al.,
2007). Although Kim et al.
(Kim et al., 2007) suggest
that the WASp/Vrp1 complex is connected to the Sns cytodomain via the SH2-SH3
adaptor protein Crk (Galletta et al.,
1999), the biochemical interactions necessary for activation of
the pathway are not well understood. Indeed, multiple redundant functional
domains in the Sns cytodomain have the potential to mediate a spectrum of
interactions (Kocherlakota et al.,
2008).
To better understand the role of Sns in myoblast fusion, identify mechanisms through which fusion can occur in its absence, and resolve the relative contribution of Hbs, we undertook a detailed examination of Sns and Hbs. We report herein that Hbs acts positively to direct myoblast fusion, is capable of driving significant fusion even in the absence of Sns and functions interchangeably with Sns in the first fusion events between founder cells and FCMs. Re-examination of the genetic interaction between sns and hbs with new methods of visualization and quantitation also support a model in which Hbs functions positively to direct myoblast fusion. The ability of chimeric proteins of Sns and Hbs to rescue fusion in sns mutant embryos supports a model in which Hbs functions less efficiently than Sns, a limitation that rests primarily within its cytoplasmic domain. Finally, our data establish conclusively that all fusion requires signaling pathways that are downstream of Sns and Hbs.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), mbcD11.2
(Erickson et al., 1997),
lmd1 (Duan et al.,
2001), Df(1)w67k30
(Ruiz-Gomez et al., 2000),
rstirreC1
(Strunkelnberg et al., 2001),
hbs459 and hbs2593
(Artero et al., 2001). Stocks
described elsewhere include mef2Gal4
(Ranganayakulu et al., 1998),
UAS-sns-HA, snslacZ and snsGal4
(Kocherlakota et al., 2008)
and UAS-hbs (Artero et al.,
2001). The snss660 allele was a gift from
Elizabeth Chen. The snsD1, hbs459 double mutant
stock was generated by EMS mutagenesis of an isogenized
hbs459 chromosome to generate mutations in sns
(see Fig. S1 in the supplementary material). The molecular lesions in
snss660 and snsD1 correspond to Y333X
at nucleotide position 1547 and W215R at nucleotide position 1191,
respectively. Transgenic stocks were generated by Genetic Services (Cambridge,
MA). The transgenes were genetically recombined into
snsXB3 or snsZf1.4 and balanced with
CyO, P{ry[+t7.2]=en1}wgen11 from the Bloomington
Stock Center.
Cloning and constructs
The following constructs have been described: pUAST-sns-HA
(Kocherlakota et al., 2008),
pUAST-hbs (Artero et al.,
2001), pUAST-sns20-5HA
(Kocherlakota et al., 2008).
To generate pUAST-kirre-HA, an EcoRV-SalI fragment
that includes the entire kirre-HA coding sequence from
pRmHa3-kirre-HA (Galletta et al.,
2004) was sub-cloned into pBSK, recovered as a
NotI-KpnI fragment and cloned into pUAST
(Brand and Perrimon, 1993). To
generate pUAST-hbs
ICD-HA an HA epitope tag followed
by a stop codon and an XbaI restriction site were introduced after AA
1114 of Hbs. The pUAST-sns-V5, pUAST-sns-Flag,
pUAST-hbs-HA, pRmHa3-sns-V5, pRmHa3-hbs-HA and
pRmHa3-kirre-Flag constructs were each engineered using PCR. In each,
a single repeat of the epitope tag was inserted after the last codon of the
corresponding open reading frame (ORF). Domain swap constructs of sns
and hbs were generated by PCR using the following chimeric
oligonucleotides: (5'-CAGCGCCGCAAGAAAGTGTCTCAGAGCGAAGCGGA-3') for
pUAST-SETHC-HA,
(5'-CATGGCTGCTGAGGTGATTCCAATTATGACCAAATTCGGTGTGTTGC-3') for
pUAST-SEHTC-HA and
(5'-GAATGCCGCCAGAGAGATGCCAATGATCATCACATTGGGCAGTTCGTC-3') for
pUAST-HESTC-HA. Chimeric sequences for SETHC-HA and
SEHTC-HA were substituted for Sns in pUAST-sns
(Galletta et al., 2004) using
AflII and XbaI. The chimeric sequence for HESTC-HA
was substituted for Hbs in pUAST-hbs
(Artero et al., 2001) using
BglII and XbaI. An HA epitope tag, stop codon and
XbaI site were engineered to follow the last amino acid in each
chimeric ORF. The entire cDNA region of all constructs was sequenced before
injection. Of note, this analysis found the cytoplasmic sequence of
hbs to be consistent with that published by Dworak and colleagues
(Dworak et al., 2001).
Immunohistochemistry
Embryos were collected and processed as described
(Erickson et al., 1997).
Homozygous mutant embryos were identified by absence of β-galactosidase
(β-gal) activity. Primary antibodies to myosin heavy chain (MHC) (1:1000,
D. Kiehart), Even skipped (Eve, 1:1000, M. Frasch), Pericardin (Prc, 1;10,
Developmental Society Hybridoma Bank), Kruppel (Kr, 1:300, East Asian
Distribution Center for Segmentation Antibodies at National Institute of
Genetics, Division of Developmental Genetics, Mishima, Japan) and rabbit
polyclonal to Nautilus (Nau, AA 29-143, 1:100) were used in this study.
Colorimetric detection was performed using biotinylated anti-mouse and
anti-rabbit IgG (1:200) and the Vectastain ABC Elite Kit (Vector Laboratories,
Burlingame, CA) according to the manufacturer's instructions. Embryos were
imaged using a Zeiss Axioplan2. Fluorescent detection used
Alexa-Fluor-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA).
Stained embryos were imaged using a Zeiss LSM-510 confocal microscope and
analyzed using AIM (Zeiss) and Imaris (Bitplane) software.
Statistical analysis
Nuclei were visualized in late stage 15 embryos and were manually
quantitated from confocal z-series. For mutant alleles, three (Eve
and Kr) or four (Nau) abdominal hemisegments were analyzed per embryo. For
rescue of snsXB3, hbs2593 double mutants, six
(Eve and Kr) abdominal hemisegments were analyzed. Data are represented as the
mean number of nuclei per hemisegment ± s.e.m., where n equals
the number of hemisegments. Unfused myoblasts were visualized by anti-Sns
antisera or antibodies to β-gal (Cappel, MP Biomedicals) in conjunction
with snslacZ (Kocherlakota et
al., 2008). The number of unfused myoblasts in late stage 15
embryos was quantitated using Imaris software (Bitplane), with manual editing
of confocal z-series. Quantitation of unfused myoblasts assayed three
abdominal hemisegments in rescues using Hbs and Sns-Hbs chimeras and four
abdominal hemisegments in Sns overexpression and sns, hbs genetic
interaction. The mean number of unfused myoblasts is indicated ±
s.e.m., where n equals the total number of embryos.
S2 cell culture, transfection and aggregation
S2 cells were grown and transiently transfected as described
(Cherbas and Cherbas, 1998).
Plasmids pUAST-sns-V5, pUAST-sns-HA, pUAST-sns-Flag
and pUAST-hbs were co-transfected with pWAGal4
(Ishimaru et al., 2004) as
described in Results. Cells transfected with pRmHa3-sns-V5,
pRmHa3-hbs-HA or pRmHa3-kirre-Flag were induced for 16 hours
with 0.7 mM CuSO4. Immunoprecipitations used cells transfected with
pUAST constructs grown for 24 hours after DNA removal. Aggregations were
carried out as described (Beiber,
1994; Galletta et al.,
2004), with the following modifications: cells were transfected
separately with pRmHa3-sns-V5, pRmHa3-hbs-HA or
pRmHa3-kirre-Flag and then mixed at a 1:1:1 ratio at a final
concentration of 4.5x106 cells/ml. Aggregation results from
three independent experiments are presented as the percentage of Sns- or
Hbs-positive cells not in contact with Kirre-positive cells ±
s.e.m.
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) using anti-V5 resin (Invitrogen, Carlsbad, CA), anti-HA
clone3F10-affinity matrix (Roche Applied Science, Indianapolis, IN) or
anti-FLAG-M1-agarose (Sigma, St Louis, MO), as indicated. Proteins were eluted
either by boiling in Laemmli buffer, for immunoblots, or by incubation with an
HA peptide for PNGaseF digestion. PNGaseF (Elizabethkingia
meningosepticum, EMD chemicals, Gibbstown, NJ) digestion was as described
(Hanford et al., 2004).
Immunoblots used rabbit anti-Hbs (1:1000; gift of Mary Baylies), mouse
anti-phosphotyrosine (1:1000; Upstate Biotechnology),
horseradish-peroxidase-conjugated, anti-mouse, anti-rabbit or anti-rat
(1:5000; GE Healthcare Lifesciences), anti-HA (1:3000; Roche, Indianapolis,
IN), anti-V5 (1:5000; Invitrogen, Carlsbad, CA), anti-HA HRP conjugate
(1:5000; Roche, Indianapolis, IN) anti-V5 HRP conjugate (1:5000; Invitrogen,
Carlsbad, CA), and anti-FLAG (1:5000; Sigma, St Louis, MO). |
RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). By the
time that Eve declines at Stage 15, the DA1 muscle of wild-type embryos
contains an average of 11 nuclei (Menon et
al., 2005) (Fig.
1Q). As a control, we confirmed that the DA1 founder cell
undergoes no fusion in mbc mutant embryos
(Fig. 1D-F,P)
(Beckett and Baylies, 2007;
Menon et al., 2005;
Schroter et al., 2004). By
comparison, bi-and tri-nucleate muscle precursors are reproducibly observed in
embryos lacking sns (Fig.
1G-I,P). Although delayed compared with the initial fusion event
observed in wild-type embryos, this Sns-independent fusion was observed as
early as stage 12. It was observed consistently across abdominal hemisegments
by stage 15 (Fig. 1Q-R). Like
mbc, however, embryos lacking both kirre and rst
exhibit no fusion by late stage 15 (Fig.
1J-L,P) (Menon et al.,
2005). Thus, precursor formation can occur through a
Kirre/Rst-mediated process that is independent of Sns. This fusion is not the
result of Kirre- or Rst-mediated homotypic interaction between founder cells,
as it is not observed in embryos lacking lmd
(Fig. 1M-O,P)
(Menon et al., 2005) in which
FCMs are not specified (Duan et al.,
2001; Furlong et al.,
2001; Ruiz-Gomez et al.,
2002). Thus, the fusion events that occur in sns mutant
embryos are dependent on the presence of FCMs.
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;
Strunkelnberg et al., 2001)
and is capable of interacting homotypically
(Dworak et al., 2001;
Galletta et al., 2004). To
address whether it is sufficient to direct interaction between founder cells
and FCMs in the absence of sns, the Eve-positive nuclei in the DA1
founder cell were quantitated in embryos mutant for
snss660 alone and rstirreC1;
snss660 double mutants. At stages 15 and 16, embryos of both
genotypes exhibited multiple Eve-positive nuclei
(Fig. 2A,B), indicating that
Rst does not substitute for Sns in the FCMs.
Like Sns (Bour et al., 2000)
and Rst (Strunkelnberg et al.,
2001), the IgSF protein Hibris is detected in all or a large
subset of FCMs (Artero et al.,
2001). Studies have reported that Hbs acts antagonistically to Sns
(Artero et al., 2001). By
contrast, an analysis of DA1 similar to that described above revealed a lower
number of Eve-positive nuclei in sns, hbs double mutant embryos
(Menon et al., 2005). To
resolve this apparent difference, we examined the Eve-expressing DA1 founder
cell, the Kr-expressing DA1 and dorsal oblique muscle 1 (DO1) founder cells
and the Nau-expressing ventral acute muscle 1 (VA1) founder cell. Multiple DA1
nuclei were present in sns mutant embryos, as revealed by expression
of Eve (Fig. 2C,E) and Kr
(Fig. 2E). Multiple Kr-positive
nuclei were also observed in DO1 (Fig.
2E). Finally multiple Nau-positive nuclei were apparent in VA1
(Fig. 2G). Thus, fusion occurs
in the absence of sns in the DO1, DA1 and VA1 founder cells. By
comparison, representative embryos revealed that DA1, DO1 and VA1 all remain
mononucleate in most hemisegments of snsXB3,
hbs2593 mutant embryos
(Fig. 2D,F,H). We therefore
conclude that Hbs substitutes for Sns in formation of the bi- and tri-nucleate
precursors for muscles DA1, DO1 and VA1.
Hbs has features and behaviors in common with Sns
The above results indicate that Hbs functions redundantly with Sns in
formation of muscle precursors. Consistent with this result, Hbs has several
features in common with Sns. Hbs directs adhesion with S2 cells expressing
Kirre protein at a rate comparable to that of Sns
(Fig. 3A). Sns and Hbs also
share similar modes of protein modification. In particular, Sns and Hbs are
typical of many cell adhesion molecules in that their extracellular domains
are modified by N-linked glycosylation
(Fig. 3B). We previously
demonstrated that Sns was phosphorylated on tyrosines when expressed
pan-mesodermally in the embryo using mef2Gal4 and that these
tyrosines impact the ability of Sns to drive myoblast fusion
(Fig. 3C)
(Kocherlakota et al., 2008).
Analogous studies reveal that Hbs is similarly phosphorylated on tyrosines
(Fig. 3C), and many of these
tyrosines align with those in Sns (Fig.
3D). Lastly, the Sns and Hbs cytodomains both include two PxxP
motifs, which have the potential to interact with SH2-domain-containing
proteins and have been shown to play a role in Sns, one of which is closely
aligned and highlighted (Fig.
3D) (Kocherlakota et al.,
2008).
Hbs sequences can drive some myoblast fusion in Sns mutant embryos
Although endogenous Hbs acts positively to direct formation of bi and
tri-nucleate muscle precursors, and has many features in common with Sns, it
is unable to replace Sns in formation of mature myofibers. Possible
explanations are that Hbs is spatially restricted, that it is present at lower
levels and/or that it functions less efficiently than Sns. Alternatively, Hbs
may lack sequences that mediate interactions crucial for progression beyond
precursor formation, the `second step' in myoblast fusion. To address these
possibilities, we examined the ability of full-length Hbs and Hbs/Sns domain
swaps to rescue fusion in sns mutant embryos when expressed either in
the FCMs or pan-mesodermally under the control of snsGal4
(Kocherlakota et al., 2008) or
mef2Gal4 (Fig. 4; see
Fig. S3 in the supplementary material). These studies demonstrate that Hbs can
act positively to direct myoblast fusion beyond the precursor stage, but
inefficiently and only when overexpressed
(Fig. 4D-F; see Fig. S3D-F,P in
the supplementary material). Moreover, we infer from these results that the
Hbs cytodomain must contain sequences capable of directing the necessary
downstream events.
|
We did not observe significant or consistent pattern defects in these rescued embryos to suggest that the presence of Hbs or a balance between Sns and Hbs favors the formation of some muscles to the detriment of others. Rather, we observed a fairly consistent gradient in which fusion in all muscles was improved by swapping Sns sequences for those of Hbs. Furthermore, significant muscle formation was observed at stage 14 using UAS-hbs, compared with the sns loss-of-function mutation alone, suggesting that higher expression is a significant driving factor behind increased muscle formation in these rescued embryos (Fig. 4S-U). We conclude from these results that several domains within Hbs function inefficiently in directing myoblast fusion compared with the analogous sequences in Sns, but that these domains are nonetheless capable of driving significant fusion. We hypothesize that weak protein-protein interactions between these Hbs domains and their downstream effectors limit the ability of endogenous Hbs to function, but that these interactions occur under Gal4-driven conditions in which higher levels of Hbs are present.
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),
contributing to the conclusion that Hbs functions to limit the action of Sns,
and implying that excess Sns is deleterious in some way. To first determine
the consequences of misregulated or excess Sns, we overexpressed a functional
UAS-sns transgene in the musculature of wild-type embryos using
mef2Gal4. An sns-promoter reporter transgene
(snslacZ) was incorporated to facilitate quantitation of unfused
myoblasts (Kocherlakota et al.,
2008). As shown in Fig.
5A, excess Sns has little impact on muscle pattern or on the
number of unfused myoblasts compared to controls. We then compared the number
of unfused myoblasts in hbs2593/hbs459
trans-heterozygous embryos, which are hypomorphic and null alleles of
hbs, respectively (Artero et al.,
2001), and hbs2593/hbs459 mutant
embryos that were heterozygous for snsXB3
(Fig. 5B). This analysis used
an snsXB3, hbs2593 double mutant that has been
described (Artero et al., 2001;
Menon et al., 2005). To
eliminate a potential contribution to this mutant phenotype by mutations
elsewhere on the snsXB3, hbs2593 recombinant
chromosome, we also generated snsD1, hbs459
(see Fig. S1 in the supplementary material). Unfused myoblasts were
preferentially detected by staining for Sns and quantitated. In short, the
limited number of unfused myoblasts in embryos mutant for hbs
actually increased rather than decreased upon removal of one copy of
sns. Thus the loss of hbs is deleterious to myoblast fusion
and removal of one copy of sns enhances this effect, supporting our
model that sns and hbs both act positively to drive myoblast
fusion.
Mechanistically, Hbs can substitute for Sns in early myoblast fusion and
rescue extensive myoblast fusion when overexpressed in sns mutant
embryos, yet it interferes with fusion when overexpressed in whole or in part
in a wild-type embryo (Artero et al.,
2001; Dworak et al.,
2001). One explanation for this behavior was provided by proteomic
studies that revealed an interaction between Sns and Hbs in the somatic
musculature (data not shown). To validate this potential interaction, S2 cells
were transiently transfected with combinations of pUAST-hbs,
pUAST-sns-Flag and pWAGal4, the interacting proteins identified by
immunoprecipitation and immunoblot (Fig.
5C). Hbs co-precipitated with Sns in extracts from cells
expressing Sns and Hbs but not from cells expressing Hbs alone. This
interaction occurs in cis, as Hbs was not detected by interaction with Sns
upon mixing of independently transfected cells before lysate preparation (data
not shown). As observed for the interaction between the IgSF proteins
CAM-related/downregulated by oncogenes (Cdo) and Brother of Cdo (Boc)
(Kang et al., 2002), both the
extracellular and intracellular domains of Sns were capable of mediating
interaction with Hbs (see Fig. S4 in the supplementary material). To confirm
this association in the embryonic mesoderm, recombinant flies containing both
UAS-hbs and UAS-sns-V5 transgenes were mated to flies
expressing mef2Gal4. Lysates were prepared from Stage 9-15 embryos
and analyzed by immunoprecipitation. Again, Hbs efficiently co-precipitated
with Sns in lysates from embryos expressing Hbs and Sns-V5 but not from
embryos expressing only Hbs (Fig.
5C). Thus, Sns and Hbs are present in a hetero-oligomer in S2
cells and in the developing embryonic musculature. Similar studies using V5
and HA-tagged Sns (Materials and methods) revealed that Sns was also present
in homo-oligomers in S2 cells and in embryos
(Fig. 5D). In combination with
the observation that Hbs functions less efficiently than Sns in driving
myoblast fusion (Fig. 4), this
finding supports a scenario in which excess Hbs could sequester Sns in a less
functional or nonfunctional complex, leading to the observed defects in
myoblast fusion upon Hbs overexpression
(Artero et al., 2001;
Dworak et al., 2001).
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), which
lacks a functional cytoplasmic domain and is unable to drive myoblast fusion
in sns mutant embryos
(Kocherlakota et al., 2008),
and a hbs transgene in which the cytoplasmic domain is truncated in a
region homologous to that of the sns20-5 lesion
(UAS-hbsICD-HA). These transgenes under control of
mef2Gal4 were then assayed for their ability to rescue formation of
Eve-positive (DA1) or Kr-positive (DO1) bi- and tri-nucleate muscle precursors
in snsXB3, hbs2593 double mutant embryos. Even
by stage 15, past the time at which multinucleate precursors are first seen in
sns mutant embryos (Fig.
1; Fig. 6E), no
fusion was observed upon expression of either transgene
(Fig. 6A,C,F). In fact, we were
unable to detect fusion in the DA1 or DO1 muscles as late as mid-stage 16 upon
expression of truncated Sns or Hbs (Fig.
6B,D), after which the reporters were no longer detectable. We
interpret these data to indicate that the first step in myoblast fusion, in
which a muscle precursor is formed from fusion between a founder cell and one
or two FCMs, requires interactions that occur downstream of Sns and Hbs and,
in conjunction with the data of Fig.
3, are mediated by their cytoplasmic domains.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), but
these studies addressed hbs function in only one aspect of muscle
development and did not resolve conflicts with the previously reported
function of hbs. Our finding that Hbs is inefficient in directing
fusion beyond precursor formation may account for the previously observed
consequences of UAS-hbs overexpression
(Artero et al., 2001).
Moreover, genetic interactions between hbs and sns observed
at later stages of muscle development clearly indicate that Hbs acts
positively to direct myoblast fusion in a manner similar to that of Sns.
Finally, the cytodomain of either Hbs or Sns must be present for precursor
formation, suggesting that intracellular events downstream of these cell
surface proteins are important during or before fusion for generation of
muscle precursors.
A revised role for Hbs
Sns and Hbs function redundantly in the initial fusion event between
founder cells and FCMs. As observed in other mutants
(Beckett and Baylies, 2007),
precursor formation in sns mutant embryos is delayed over that
occurring in wild-type embryos, but is readily observed in stage 13 embryos in
at least some segments. By contrast, no fusion was observed by late stage 15
in sns, hbs double mutant embryos. Although we cannot eliminate the
possibility of a temporal delay of fusion in sns, hbs double mutants
because reporter expression declines after this stage, we favor a model in
which a crucial first step is not occurring in the absence of both Sns and
Hbs. Using new FCM reporters that facilitate quantitation of unfused
myoblasts, re-examination of the hbs loss-of-function phenotype
reveals that the loss of one copy of sns actually worsens the
hbs mutant phenotype, as expected if these proteins have some
functional redundancy. Finally, both snsGal4 and mef2Gal4
directed Hbs can drive a significant amount of fusion in sns mutants,
arguing that Hbs is capable of directing fusion beyond precursor
formation.
Although Hbs can rescue the sns mutant phenotype beyond precursor
formation, replacing any domain of Hbs with the comparable domain of Sns
improves the ability of the chimeric protein to rescue fusion over that
achieved by Hbs alone. The activity of the Hbs cytodomain is most dramatically
different from that of Sns, providing an explanation for the observation that
intact Hbs or a membrane-anchored Hbs cytoplasmic domain both interfere with
myoblast fusion in wild-type embryos
(Artero et al., 2001;
Dworak et al., 2001). Rather
than acting as an antagonist of Sns, these high levels of Hbs probably
interfere competitively with endogenous Sns. First, an excess of Hbs may drive
its interaction with a limiting component that is normally used more
efficiently by Sns. Alternatively, given their ability to form hetero- and
homodimers in vivo, excess Hbs may sequester Sns in a less functional form.
Although our data do not fully resolve this issue, the co-localization of Hbs
and Sns is consistent with the latter model
(Artero et al., 2001). Of note,
dimer formation between the related IgSF proteins Boc and Cdo can be directed
by sequences in both the extracellular and intracellular domains
(Kang et al., 2002), and both
the extracellular and intracellular domains of Sns are capable of mediating
its interaction with Hbs, raising the possibility that either full-length Hbs
or a membrane-anchored cytodomain may sequester Sns under conditions of
overexpression.
Implications for the regulation of Sns
The finding that Hbs functions positively but much less efficiently than
Sns in directing later rounds of myoblast fusion provides an explanation for
the previously observed behavior of Hbs in overexpression assays
(Artero et al., 2001;
Dworak et al., 2001).
Additionally, our data appear to be inconsistent with a model in which excess
Sns is deleterious, as inferred if a decrease in sns copy number
compensates for the loss of hbs
(Artero et al., 2001). We
cannot eliminate the possibility that Sns activity is negatively regulated.
Possible mechanisms could include limitations in the machinery for tyrosine
phosphorylation, such that unphosphorylated Sns even in excessive amounts
would be unable to transduce a signal to downstream events. Downstream targets
of Sns may also be limiting, such that no further activation of the pathway
can be accomplished by Sns. We also note that Sns protein is transient,
appearing just before fusion and being eliminated shortly thereafter. Despite
the issue of whether Sns activity is regulated in some fashion, our data are
not consistent with a model in which its activity is negatively regulated by
endogenous Hbs.
Hbs does not replace Sns: implications for signal transduction mediated by IgSF proteins
Current models for myoblast fusion suggest that it occurs in two steps that
differ genetically and/or temporally. Consistent with the two genetically
distinct steps, fusion does not occur in embryos mutant for genes encoding the
guanine nucleotide exchange factors Schizo
(Chen et al., 2003), Mbc
(Beckett and Baylies, 2007;
Menon et al., 2005;
Schroter et al., 2004) or, as
discussed herein, Duf and Rst (Menon et
al., 2005). By contrast, precursor formation is observed in
embryos lacking the Hem-2/Nap1 homolog Kette
(Schroter et al., 2004), the
Kirre-associated protein Rols (Chen and
Olson, 2001; Rau et al.,
2001), the Arp14D/66B regulators WASp and Vrp1
(Berger et al., 2008;
Kim et al., 2007;
Massarwa et al., 2007;
Schafer et al., 2007) or, as
described herein, Sns. These data support a model in which the molecular
requirements for precursor formation differ from those for subsequent myotube
formation (Berger et al., 2008;
Doberstein et al., 1997;
Kesper et al., 2007). An
alternative model, using three dimensional analyses and quantitating fusing
myoblasts over time, revealed that fusion occurs in two temporal phases,
comprising an initial phase of limited fusion between cells that are in close
proximity and a second phase when most myoblast fusion occurs
(Beckett et al., 2007).
Moreover, precursor formation is temporally delayed in embryos lacking
molecules such as Rols and Kette, suggesting that these molecules do influence
the first step in fusion (Beckett et al.,
2007; Richardson et al.,
2008).
The present study does not address whether the genetic requirements for
precursor formation differ from those for subsequent rounds of fusion, or
whether these steps utilize the same set of proteins. Our data do not
eliminate the possibility of two distinct genetic steps, with Sns and Hbs
acting redundantly in precursor formation but not in later events. Hbs is
capable of directing precursor formation in the absence of Sns. However, the
ability of Hbs to drive fusion beyond precursor formation when in excess, and
the observation that removal of one copy of sns enhances fusion
defects in hbs mutants, suggests that Hbs can assist in later rounds
of myoblast fusion. These data are consistent with models in which molecular
interactions in precursor formation and subsequent fusion differ kinetically
but not genetically (Beckett et al.,
2007; Richardson et al.,
2008). One possibility, independent of the process of fusion
itself, is that Sns and Hbs differ in their ability to drive FCM cell
migration. Although the role of cell migratory behavior in myoblast fusion is
unclear, the ability to migrate may contribute to the rate of fusion
(Beckett et al., 2007;
Richardson et al., 2008).
While these questions remain to be addressed, the present study advances our
understanding of fusion by resolving the interaction of two proteins that
function early in the process, thereby providing additional perspectives for
sorting out the different mechanisms of myoblast fusion.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1159/DC1
|
Footnotes |
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* These authors contributed equally to this work 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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