© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
LETTERS
A conserved molecular pathway mediates myoblast
fusion in insects and vertebrates
Bhylahalli P Srinivas, Jennifer Woo, Wan Ying Leong & Sudipto Roy
Skeletal muscles arise by fusion of precursor cells, myoblasts,
into multinucleated fibers. In vertebrates, mechanisms
controlling this essential step in myogenesis remain poorly
understood1,2. Here we provide evidence that Kirrel, a
homolog of receptor proteins that organize myoblast fusion in
Drosophila melanogaster3,4, is necessary for muscle precursor
fusion in zebrafish. Within developing somites, Kirrel
expression localized to membranes of fusion-competent
myoblasts of the fast-twitch lineage. Unlike wild-type
myoblasts that form spatially arrayed syncytial (multinucleated)
fast myofibers, those deficient in Kirrel showed a significant
reduction in fusion capacity. Inhibition of Rac, a GTPase and
the most downstream intracellular transducer of the fusion
signal in D. melanogaster1,5,6, also compromised fast-muscle
precursor fusion in zebrafish. However, unlike in
D. melanogaster6, constitutive Rac activation in zebrafish led
to hyperfused giant syncytia, highlighting an entirely new
function for this protein in zebrafish for gating the number and
polarity of fusion events. These findings uncover a substantial
degree of evolutionary conservation in the genetic regulation of
myoblast fusion.
Historically, experimental analysis of vertebrate myoblast fusion has
been confined, almost exclusively, to mammalian muscle precursors in
culture2. Functional studies in vivo are limited, and they have failed to
corroborate a requirement for many of the proteins identified in vitro.
In D. melanogaster, however, systematic genetic screens have led to
more detailed insights into the mechanism of myoblast fusion1,7.
Here, ‘founder’ myoblasts prefigure the position, orientation and
identity of individual muscles, whereas ‘fusion-competent’ myoblasts
(FCMs) fuse with founders and convert them into syncytia. Central to
fusion is a dedicated signaling cascade involving two immunoglobulin
(Ig) domain–containing membrane receptors expressed on founder
cells: Kin-of-IrreC (Kirre) (also known as Dumbfounded (Duf)) and
Roughest (Rst) (also known as Irregular chiasm C-roughest (IrreCRst))3,4. Although myogenic programs of flies and vertebrates have
several points of similarity, they also show fundamental differences in
many aspects of their genetic regulation. With regard to fusion, none
of the molecules identified through in vitro studies of mammalian
myoblast fusion has thus far been implicated in myotube formation in
D. melanogaster. Conversely, there is no evidence as yet to indicate that
the Kirre-Rst pathway is even partially conserved in vertebrates. These
disparities suggest that the mechanism of myoblast fusion may have
significantly diverged during the course of evolution.
In zebrafish embryos, two distinct lineages of muscle precursors can
be distinguished within the developing somites. Slow-twitch myoblasts
are specified in response to Hedgehog signaling and differentiate into a
superficial layer of fibers that express slow isoforms of myosin heavy
chain (MyHC)8–12. However, the vast majority of the myoblasts
develop, by default, into fast-twitch muscles deeper within the
myotome9–12. We have previously shown that slow-twitch myoblasts
are fusion incompetent and mature into mononucleate fibers; by
contrast, precursors of fast muscles fuse with each other to form
syncytial myotubes12. To identify the molecular pathway that regulates
fast-muscle precursor fusion, we explored the possibility that mechanisms that control fusion in D. melanogaster might be functional in
vertebrates, and we searched for homologs of Kirre and Rst in the
zebrafish genome database. Several mammalian Kirre-like proteins,
Kirrels (from ‘Kirre-like’), have already been described13–15; they were
originally discovered based on homology to nephrins, an Ig domaincontaining protein family that functions in renal physiology. We
identified four distinct zebrafish kirrel genes and amplified corresponding cDNA fragments for expression analysis. Consistent with a
role for kirrel genes in muscle development, we observed robust
expression of one of these genes in muscle precursors, beginning at
12 h post fertilization (h.p.f.). At this stage, the embryos have formed
five to six somites; expression was confined to lateral columns of cells
previously identified as fast-muscle precursors10 (Fig. 1a). Expression
was evident in all fast myoblasts but, notably, was excluded from
adaxial cells, non-fusigenic precursors of slow-twitch muscles that are
located medial to the fast myoblasts, immediately adjacent to the
notochord (Fig. 1a,b,d). At 15 h.p.f., the somitic expression intensified
further (Fig. 1b). We first noticed a decline in kirrel mRNA levels in
the anteriormost somites at 18 h.p.f., although newly formed posterior
somites continued to express high levels of the gene (Fig. 1c).
Concomitant with myotome maturation at 24 h.p.f., kirrel expression
disappeared from differentiating muscle fibers (data not shown; see
section on Kirrel protein expression). In addition to fast myoblasts,
kirrel was expressed dynamically in several other cell and tissue types
at specific embryonic stages (Supplementary Fig. 1 online).
Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673. Correspondence should be addressed to S.R. (sudipto@imcb.a-star.edu.sg).
Received 22 November 2006; accepted 27 April 2007; published online 27 May 2007; doi:10.1038/ng2055
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Figure 1 Expression of kirrel mRNA and protein in fast-muscle precursors. (a–c) kirrel mRNA expression (long arrows) in fast-muscle precursors.
(d) Fluorescent RNA in situ hybridization with probes for slow myhc (red) and kirrel (green), showing exclusion of kirrel from adaxial cells. Absence of kirrel
expression from slow myoblasts is also apparent in a and b (short arrows). (e) Kirrel protein expression (red, arrows) in fast myoblasts. (f) High-resolution
image showing Kirrel in discrete puncta (arrows) along cell membranes that were highlighted by b-catenin antibodies (green). (g) Membrane localization of
b-catenin was uniform (arrows). (h–j) Confocal sections through mediolateral width of the myotome (h, most medial; j, most lateral), showing loss of Kirrel
from developing syncytia and elongating fast myocytes (long arrows) and its persistence in unfused cells (small arrows). (k) Kirrel protein was absent from
mature fast fibers. (l) Kirrel expression in unfused myoblasts of the posteriormost somites. Nuclei were labeled with DAPI (blue). a,b,d–g depict dorsal views;
c,h–l represent lateral views, with dorsal to the top. In all panels, anterior is to the left.
We assembled a full-length kirrel cDNA predicted to encode a embryos with monoclonal antibody A4.1025, which recognizes all
protein of 755 amino acid residues (Supplementary Fig. 2 online). MyHC proteins9,17. Unlike wild-type siblings, which formed syncytial
Like Kirre and Rst, zebrafish Kirrel seems to be a type Ia membrane fast fibers by 24 h.p.f., the morphants (that is, the morpholino-injected
protein, with five Ig-like domains in the N-terminal extracellular embryos) showed a notable phenotype, with large clusters of monoregion (approximately 491 residues), a membrane-spanning segment nucleated cells on the surface as well as distributed throughout the
(approximately 25 residues) and a cytoplasmic tail (Fig. 2). The mediolateral width of the myotome (Fig. 3a,b). Subsequently, these
intracellular region at the end of the C terminus contained a PDZ unfused myoblasts differentiated into single-celled mini-muscles
domain–binding motif, and the extreme N terminus contained a (Fig. 3c,d). This phenotypic consequence was confirmed by comparputative signal peptide. Alignment with D. melanogaster Kirre showed ison of wild-type and morphant embryos injected with myogenin:
significant conservation (48% similarity) that encompasses the Ig EGFP that drives EGFP expression in muscle precursors and differdomains and extends to include the transmembrane segment (Sup- entiated muscle fibers. In contrast to multinucleated myotubes of
plementary Fig. 2). We found an equivalent
level of conservation (47%) with Rst. Phylogenetic analysis indicated that this particular
Ig_like
Kirre
Ig
Ig
Ig
Ig
zebrafish Kirrel family member represents a
separate clade in teleosts, with closest similarity to mammalian Kirrel3 (Supplementary
Fig. 2 and ref. 16; see also Ensembl zebrafish
Ig_like
Ig_like
Ig_like
Ig_like
Ig_like
Rst
genome sequence database release Zv6 http://
www.ensembl.org/Danio_rerio/genetreeview?
db¼core;gene¼ENSDARG00000019473).
To establish that kirrel expression correlates
Ig_like
Ig_like
Ig_like
Ig_like
Ig_like
Kirrel
with a requirement for fast myoblast fusion,
we knocked down its activity with an antisense morpholino oligonucleotide directed Figure 2 Structural organization of D. melanogaster Kirre, Rst and zebrafish Kirrel proteins as analyzed
against the 5¢ UTR (kirrel MO1, expected to by the SMART domain annotation tool. Ig or Ig-like domains (green), transmembrane domain (blue),
block mRNA translation) and stained the putative signal peptide (red) and regions of low compositional complexity (pink) are highlighted.
782
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Figure 3 Kirrel is essential for fast-muscle precursor fusion. (a,c) Wild-type embryos stained with monoclonal antibody A4.1025, showing arrays of
multinucleate fast muscle fibers (arrows). (b,d) In kirrel morphants, large numbers of unfused, mononucleate differentiating myocytes were detected (b) that
matured into mini-muscles (d). These mini-muscles elongated and attached to somite borders (long arrows) or remained randomly oriented (short arrows).
(e) Expression of myogenin:EGFP in multinucleated fast fibers of a wild-type embryo (arrows). (f) In a kirrel morphant, myogenin:EGFP labeled
mononucleated, unfused fast myocytes (arrows). (g) Labeling with monoclonal antibody EB165, showing fast muscle fibers in a wild-type embryo (arrows).
(h) Large clusters of unfused fast muscle cells in a kirrel morphant labeled with monoclonal antibody EB165 (arrows). (i) A wild-type embryo stained with
monoclonal antibody F59 that recognizes slow MyHC10,27, showing slow-twitch fibers. (j) The slow muscles appeared to differentiate normally in the
morphant. (k) Somitic expression of Kirrel protein in a wild-type embryo. (l) A kirrel morphant, showing downregulation of Kirrel protein expression.
a–j depict lateral views, with dorsal to the top. k and l show dorsal views. In all panels, anterior is to the left. In a–f, nuclei were labeled with DAPI (blue).
wild-type embryos, in the morphants, EGFP highlighted mononucleate myoblasts that were blocked in the fusion process (Fig. 3e,f; see
Supplementary Fig. 3 online for quantitative analysis of fusion
defects). Expression of the muscle determination gene, myoD18, was
unaffected (Supplementary Fig. 4 online), indicating that the significant lack of fast-muscle precursor fusion was unlikely to have
arisen from a general impairment in their commitment to myogenesis.
The unfused myoblasts also reacted with monoclonal antibody EB165,
which specifically recognizes zebrafish fast MyHC9,19, suggesting that,
notwithstanding the fusion defect, the fast myoblasts were nevertheless
capable of differentiating as mononucleated muscles of the fast-twitch
type (Fig. 3g,h). Development of the slow-twitch lineage appeared
largely unaltered, consistent with the lack of kirrel expression from
their precursors (Fig. 3i,j). However, we noticed that the slow fibers
were no longer located in the characteristic chevron pattern. Moreover, they were often stuck below masses of unfused fast myoblasts
instead of forming an orderly superficial array (data not shown). All of
this is likely to be an indirect consequence of alteration in somite
morphology owing to the lack of properly elongated syncytial fast
myofibers. In addition to a block in fast myoblast fusion, other
obvious consequences of the loss of Kirrel activity were a shortened
anteroposterior axis and aberrant brain morphogenesis (data not
shown), effects that are commensurate with the expression of the
gene in these tissues (Supplementary Fig. 1). These loss-of-function
phenotypes correlated with a marked depletion of the Kirrel
protein product from morphant embryos labeled with an antibody
raised against the C-terminal end of the protein (Fig. 3k,l and
Supplementary Fig. 5 online). Further evidence for the molecular
specificity of the morpholino (including a control mismatch morpholino and a second antisense morpholino, kirrel MO2) are presented
in Supplementary Figures 3, 4 and 5 (see also Supplementary
Methods online).
Like loss of Kirre and Rst, loss of Sticks and stones (Sns), another
Ig-domain containing membrane protein, also precludes myoblast
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fusion in D. melanogaster20. Whereas all myoblasts express Rst,
patterns of Kirre and Sns are mutually exclusive and are restricted
to founders and FCMs, respectively3,4,20. Biochemical evidence indicates that heterophilic interaction of Sns with Kirre and/or Rst
activates the fusion pathway21. Such asymmetry in receptor engagement provides a mechanistic basis for the inherent directionality in
D. melanogaster myoblast fusion, whereby fusion occurs only between
founders and FCMs and not between the two cell types themselves. To
determine whether Kirrel was required cell autonomously for zebrafish
muscle precursor fusion, we generated genetically mosaic embryos by
transplanting kirrel morphant donor blastomeres into wild-type hosts.
Morphant cells fated to form fast myoblasts readily fused with wildtype host cells and made multinucleated chimeric myotubes (Fig. 4a,b
and Supplementary Fig. 3). Notably, in the reciprocal experiment, a
significant proportion of wild-type donor fast-muscle precursors
remained mononucleated and were unable to recruit resident morphant cells into fusion and rescue myotube formation. Instances of
donor host fusions that we noted were almost always confined to
binucleated syncytia (Fig. 4c,d and Supplementary Fig. 3). As
expected, in control transplantations involving morphant donors
into morphant hosts, the majority of donor fast-muscle precursors
remained unfused (Fig. 4e,f, and Supplementary Fig. 3). One interpretation of these results is that Kirrel functions as part of a homophilic adhesion system and that its activity alone is critical for fusion.
kirrel-morphant fast myoblasts that retained threshold levels of protein
activity were able to fuse with neighboring wild-type donor cells and
were able to fuse even more effectively in the presence of large
numbers of wild-type host cells. Alternatively, heterophilic interaction
of Kirrel with an SNS-like receptor could be necessary for zebrafish
fast-muscle precursor fusion. In this view, fast myoblasts are segregated into founder and FCM populations, the latter being the more
abundant cell type and one that expresses the heterophilic partner of
Kirrel. As donor cells would more frequently adopt FCM fate,
extensive fusions of kirrel morphant donor myoblasts in wild-type
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hosts represent morphant FCM fusions with resident Kirrel-positive
founders. Likewise, the substantial numbers of unfused wild-type
donor fast-muscle precursors in morphant hosts denote wild-type
FCMs that failed to fuse with Kirrel-deficient founders of the hosts.
We next used antibodies against Kirrel on wild-type embryos to
follow the distribution of the protein as fast myoblasts progressed
through fusion and formed syncytia. The overall pattern of Kirrel
protein accumulation recapitulated the transcription profile, with high
levels of the protein detectable in all fast-muscle precursors at 15 h.p.f.
(Fig. 1e). High-resolution confocal micrographs of embryos labeled
with antibodies against Kirrel and b-catenin, a plasma membrane
marker, showed localization of Kirrel predominantly to the plasma
membrane (Fig. 1f). At this stage, the myoblasts are very closely
juxtaposed, and it is notable that while b-catenin was uniformly
distributed, Kirrel was enriched in discrete puncta along the opposing
myoblast membranes (Fig. 1f,g). Between 18 and 20 h.p.f., clear
differences emerged in Kirrel distribution along the mediolateral
width of the differentiating myotome. In deeper layers, where elongation and fusion of fast myoblasts was well underway, we noticed a
concomitant loss of Kirrel from nascent myotubes (Fig. 1h). In more
superficial regions, Kirrel expression persisted in unfused myoblasts
but showed obvious signs of decline in those that had begun to
elongate or form syncytia (Fig. 1i,j). At 24 h.p.f., the protein
disappeared from differentiated fast muscle fibers (Fig. 1k). Membranous puncta of D. melanogaster Kirre and Rst are thought to nucleate
the formation of multiprotein ‘fusion complexes’22 that relay the
signal to Rac, a GTPase that triggers reorganization of the actin
cytoskeleton necessary for fusion1. The conspicuous punctate membranous distribution of Kirrel could signify the involvement of
similar protein complexes in mediating fusion among zebrafish
fast myoblasts.
To evaluate this possibility, we examined whether perturbation of
zebrafish rac1, whose expression occurs ubiquitously throughout
embryogenesis (Supplementary Fig. 6 online), also affected myoblast
fusion. Injection of an antisense morpholino designed to block Rac1
translation, but not a control mismatch morpholino, indeed compromised myoblast fusion in wild-type embryos, although we also observed bi- and trinucleated myotubes within their myotomes (Fig. 5d,e;
Supplementary Fig. 3; data not shown). In D. melanogaster,
multiple rac genes act redundantly in myoblast fusion5. As rac1
784
Figure 4 Fusion behavior of kirrel morphant fast-muscle precursors in
genetic mosaics. (a,b) Fast myocytes from a kirrel morphant donor fused
with those of a wild-type host and made chimeric myotubes (green and blue
nuclei in b). Histone2A.F/Z-GFP expressing kirrel morphant donor nuclei are
indicated (long arrows). (c,d) Unfused wild-type histone2A.F/Z-GFPexpressing donor fast-muscle precursors in kirrel morphant hosts (small
arrows). Binucleated syncytia arose from donor-host fusions (long arrows).
(e,f) Unfused histone2A.F/Z-GFP-expressing kirrel morphant donor fast
myocytes in kirrel morphant hosts (small arrows). Binucleated syncytia arose
from donor-donor fusions (long arrows). Myotube and myocyte membranes
were highlighted with antibodies to b-catenin (red). b,d and f show
superimposition of a,b and c with their respective DAPI channels (blue) to
show both donor and host nuclei. All panels depict lateral views of 24-h.p.f.
embryos, with anterior to the left and dorsal to the top. To unambiguously
identify mononucleate donor cells as unfused fast-twitch muscle precursors,
a fourth label, monoclonal antibody F310, was used to visualize expression
of fast muscle–specific myosin light chain (data not shown).
morphants showed significant knockdown of Rac1 protein (Supplementary Figs. 5 and 6), similar redundancy among zebrafish rac genes
must account for the variable expressivity of the fusion defects. In a
converse experiment, we expressed a constitutively active variant of
human Rac1 (caRac), which shares 98% sequence identity with
zebrafish Rac1 (Supplementary Fig. 6), in muscle precursors using
the myogenin promoter. Like loss of function of Rac, constitutive Rac
activity in flies also inhibits myoblast fusion6. Notably, in zebrafish,
caRac led instead to the formation of giant syncytia that contained
supernumerary nuclei, indicative of uncontrolled hyperfusion among
the fast myoblasts (Fig. 5f–i and Supplementary Fig. 3). Serial
confocal sections of the myotome of wild-type embryos showed that
the fast fibers are normally arrayed in multiple layers, with distinct
orientations along the mediolateral width of each hemisomite
(Fig. 5a–c). The number of nuclei within individual fast myotubes
varied (from two to five, typically) in proportion to their lengths
(Fig. 5a–c and Supplementary Fig. 3). Such an arrangement was
clearly disrupted in embryos with hyperfused syncytia (Fig. 5g–i).
Cross-sectional views showed that these giant syncytia often extended
to fill a substantial proportion of the dorsoventral and mediolateral
width of the myotome (Fig. 5j–l). We did not observe any caRacinduced hyperfusion among myoblasts of kirrel morphants or in
embryos with ectopic Hedgehog signaling, which converted all somitic
myoblasts to the slow-twitch lineage (Fig. 5m,n). Thus, the effect of
caRac is dependent on the fusion receptor Kirrel and thus is specific to
fusion-competent fast myoblasts. We conclude that regulated Rac
activation is critical for limiting the number and polarity of fusions
within the myotome. This role of Rac is unique to zebrafish and seems
to be necessary for controlling the size and pattern of the developing
myotubes. How constitutive Rac engenders hyperfusion is presently
unclear. As Rac can remodel the cytoskeleton, and the hyperfusion
effect of caRac is dependent on the availability of Kirrel, we propose
that hyperfusion could ensue from excessive targeting of Kirrelenriched vesicles to the plasma membrane, which use the actin
cytoskeleton for delivery to the cell surface.
Considering limitations of in vitro cell culture models of myoblast
fusion and the technical difficulties in investigating mammalian
myoblast fusion in vivo, our study demonstrates that the zebrafish
myotome is an ideal alternative for analyzing the biology of vertebrate
myoblast fusion. Conservation of the activity of Kirrel and Rac
suggests that other elements of the D. melanogaster fusion pathway
are likely to have parallel functions in zebrafish. However, our data
with caRac imply that varying degrees of diversification in the
deployment of specific molecular components have occurred; this
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Figure 5 Alteration of Rac activity affects fast-muscle precursor fusion. (a–c) Confocal planes from lateral (a) to medial (c) regions of the wild-type myotome,
showing fast fiber layers with three distinct orientations (double-headed arrows). Membrane-localized GFP (green) and b-catenin antibodies (red) were used to
highlight cell membranes. Myofiber nuclei are labeled with DAPI (blue) throughout Figure 5 (arrowheads). (d,e) Unfused myocytes (small arrows) and
binucleated myotubes (long arrows) in a rac1 morphant embryo stained with antibody A4.1025 (d, red) and anti-b-catenin (e, green). (f) Myogenin:EGFP
expression in wild-type multinucleated fast fibers (green, arrows). (g) Single confocal section showing a hyperfused syncytium (arrow) in an embryo that
expressed myogenin:caRac. Supernumerary nuclei are indicated (arrowheads). The embryo was stained with A4.1025 (red) and antibodies against the
hemagglutinin epitope present in caRac (green). (h,i) Single confocal sections of a myogenin:caRac-injected embryo showing hyperfused syncytia visualized
with anti-hemagglutinin (h, green) and anti-b-catenin (i, red). Nuclei (arrowheads) and membranes (arrows) are indicated. (j) y-z section showing single
myotubes (green, arrows) labeled in the embryo shown in f. (k,l) y-z section showing dorsoventral and mediolateral spread of the hyperfused syncytium
(green, arrow) depicted in g and h, respectively. The y-z sections in j–l were taken approximately along the plane of the vertical lines shown in f–h.
(m) Unfused myocytes in a kirrel morphant expressing myogenin:caRac (green, arrows). (n) Mononucleate slow fibers expressing myogenin:caRac (green,
arrows) in an embryo coinjected with dnPKA mRNA to activate Hedgehog signaling. Embryos shown in m and n were labeled with A4.1025 (red) and antihemagglutinin (green). a–i, m and n depict lateral views with anterior to the left and dorsal to the top; j–l represent transverse sections.
could underlie inherent differences in the cellular basis of fusion in
different groups of animals. In mammalian embryos, the first episode
of myogenesis also occurs within the myotomal compartment of the
somites23. It will now be essential to examine whether mammalian
myoblasts use aspects of the Kirrel pathway for fusion into myotubes.
Besides being essential for embryonic muscle development, myoblast
fusion is an obligatory event for postnatal muscle hypertrophy as well
as for the regenerative responses of muscle tissue to injury2. It remains
to be seen whether conservation of the Kirrel pathway will also extend
to cell fusions that occur in these diverse episodes of myogenesis.
METHODS
Zebrafish strains. Wild-type zebrafish and the Tg(H2A.F/Z:GFP) transgenic
strain24 were maintained under standard conditions of fish husbandry. Fertilized eggs were obtained from natural spawning. All experiments with zebrafish
embryos were approved by the Singapore National Advisory Committee on
Laboratory Animal Research.
Cloning of zebrafish kirrel. cDNAs were synthesized from 18- to 20-h.p.f.
embryos using the BD SMART cDNA synthesis kit (Clontech). This cDNA
preparation was used as a template to amplify a fragment of zebrafish kirrel.
Sequences of all primers used in PCR are available in Supplementary Table 1
online. RACE PCRs were performed using the BD SMART RACE PCR kit
(Clontech) followed by cloning of the full-length kirrel gene.
myogenin:EGFP and myogenin:caRac constructs. The zebrafish myogenin
promoter25 was PCR amplified from genomic DNA using primers
having flanking XhoI and HindIII sites. The PCR product (B800 bp) was
cloned into the XhoI-HindIII sites of the pEGFP-1 plasmid (Clontech) to
generate myogenin:EGFP. Human G12V Rac1 (the constitutively active
isoform) cDNA, with three hemagglutinin tags at the N terminus, was
purchased from the University of Missouri-Rolla cDNA Resource Centre. It
was subcloned into the HindIII-XbaI site of the pmyogenin:EGFP vector by
replacing the EGFP fragment.
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RNA in situ hybridizations and antibody staining. Digoxigenin (DIG)-labeled
antisense RNA probes were synthesized using the DIG RNA labeling kit
(Roche). Whole-mount in situ hybridizations were performed following
routine protocols. DIG antisense RNAs, together with those labeled with
fluorescein, were used for the simultaneous detection of kirrel and slow myhc.
For the double-fluorescence in situ hybridization reaction, signals were developed using the Tyramide Signal Amplification (TSA) kit (Molecular Probes).
Whole-mount antibody staining on zebrafish embryos was performed according to published methods. The following antibodies were used: monoclonal
antibody F59 (1:10 dilution), monoclonal antibody A4.1025 (1:20), monoclonal antibody EB165 (1:250), monoclonal antibody F310 (1:20) (Developmental Studies Hybridoma Bank); rabbit anti-hemagglutinin (1:200) (Santa
Cruz Biotech); rabbit and mouse anti b-catenin (1:200) and rabbit anti-GFP
(1:500) (Abcam) and rabbit anti-human Rac1 c-14 (1:200) (Santa Cruz
Biotech). The C-terminal 36 amino acids of Kirrel were fused to the C terminus
of glutathione S-transferase (GST), and the recombinant protein was overexpressed in bacteria, purified and injected into rabbits (iDNA). The resulting
antibodies to Kirrel were affinity purified and used at a dilution of 1:50. For
light microscopy, the Vectastain Elite kit (Vector labs) was used for developing
staining reactions. For confocal microscopy, appropriate AlexaFluor-conjugated
secondary antibodies (1:500; Molecular Probes) were used for signal detection.
The monoclonal antibody F310 was coupled to Alexa 647 using the Zenon
Tricolor labeling kit according to the manufacturer’s instructions (Molecular
Probes). Embryos were counterstained with 4,6-diamidino-2-phenylindole
(DAPI) to visualize cell nuclei when required.
In vitro transcription of capped mRNA. To label cell membranes, we created a
membrane-targeted version of EGFP via farnesyl modification (EGFP-F). The
393.RN3-EGFP-F vector26, containing the EGFP cDNA, was linearized and
transcribed using the Ambion mMessage Machine kit. The pCS2-dnPKA
construct (encoding dominant-negative protein kinase A) was transcribed
using a similar procedure.
Microinjections. Freshly fertilized zebrafish eggs were injected with DNA
(B25 ng/ml), mRNA (B100 ng/ml) or morpholinos (200–300 mM) at the
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one- to two-cell stage. The volume of injected solution per embryo for each of
these reagents was approximately 1 nl. The injected eggs were cultured at 28 1C,
and embryos were fixed at specific developmental stages for further analysis.
Morpholinos were purchased from GeneTools and Open Biosystems and were
dissolved in sterile water at a concentration of 1 mM. Two anti-kirrel morpholinos (MO1 and MO2) and one anti-rac1 morpholino were used in our
experiments. Anti-kirrel MO1 was designed to bind 94 bases 5¢ of the predicted
start codon in the kirrel transcript, whereas the MO2 target sequence resides 57
nucleotides upstream of the MO1 binding site. The anti-rac morpholino was
designed to bind to the translation initiation site of the rac1 mRNA. Oligonucleotides with mismatches relative to target sites recognized by anti-kirrel MO1
and the anti-rac morpholino served as controls. Sequences of all morpholinos are
presented in Supplementary Table 1. Typically, 150 eggs were injected with the
anti-kirrel and the control morpholinos for each experiment; 90% of the embryos
injected with the anti-kirrel morpholinos showed the morphant phenotype, as
judged by morphological criteria. For the fusion defects, at least 20 morphant
embryos were analyzed in detail for each of the markers used. Similar numbers of
eggs were used for injection of the anti-rac1 morpholinos (myogenin:caRac) as
well as for coinjection of myogenin:caRac with kirrel morpholinos and dnPKA
mRNA. We observed inhibition of myoblast fusion in all rac1 morphants
examined (20 embryos), albeit with a more variable expressivity than in the
kirrel morphants. In addition to defects in myoblast fusion, the most readily
apparent phenotype in the rac1 morphants was a shortened embryonic axis,
consistent with a role of Rac1 in regulating convergence-extension movements of
gastrulation. We have not investigated this effect or the possible consequences of
the loss of rac1 in other tissues in any detail. caRac typically produced hyperfused
syncytia with almost 100% expressivity (in 25 embryos examined). However, the
size, shape and number of nuclei within these syncytia were variable. We did not
observe any hyperfusion on coinjection of kirrel morpholino or dnPKA mRNA
with myogenin:caRac (15 embryos examined for each experiment). A detailed
quantification of the fusion defects in morphant and caRac-expressing embryos is
presented in Supplementary Figure 3.
Cell transplantations. Cell transplantations were performed when embryos were
at the high or dome stage. For transplantations involving morphant donors and
wild-type hosts, embryos from the Tg(H2A.F/Z:GFP) transgenic strain, which
expresses nuclear-localized histone2A.F/Z-GFP fusion protein in all cells, were
injected with the anti-kirrel MO1 and used as donors. For transplantation of
wild-type cells into morphant hosts, the Tg(H2A.F/Z:GFP) transgenic strain was
used as a wild-type donor. In the control experiment, which involved transplantation of morphant donor cells into morphant hosts, Tg(H2A.F/Z:GFP)
embryos were injected with kirrel MO1 and used as donors. A quantitative
analysis of donor cell fusions is presented in Supplementary Figure 3.
Microscopy and image preparation. For light microscopy, stained embryos
were cleared and mounted in 70% glycerol and examined using a Zeiss
compound microscope (Axioplan 2) equipped with a Nikon camera
(DMX1200) for digital image capture. Confocal analyses of fluorescent staining
were performed using a Zeiss LSM 510 or an Olympus Fluoview confocal
microscope. All figures were assembled using Adobe Photoshop 6.01.
Accession code. GenBank: kirrel, EF571006.
Note: Supplementary information is available on the Nature Genetics website.
ACKNOWLEDGMENTS
We thank A. Mahadevan for technical assistance; K. Sampath (Temasek Life
Sciences Laboratory) for the 393.RN3-EGFP-plasmid and S.D. Menon, P.W.
Ingham, K. Sampath and members of our laboratory for discussion and
constructive criticism. This work was funded by the Institute of Molecular and
Cell Biology and the Agency for Science, Technology and Research of Singapore.
S.R. is an adjunct faculty member in the Department of Biological Sciences,
National University of Singapore.
AUTHOR CONTRIBUTIONS
B.P.S. and S.R. designed the study; B.P.S., S.R., J.W. and W.Y.L. performed all the
experiments and S.R. wrote the paper with constructive input from B.P.S.
786
COMPETING INTERESTS STATEMENT
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturegenetics
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reprintsandpermissions
1. Chen, E.H. & Olson, E.N. Towards a molecular pathway for myoblast fusion in
Drosophila. Trends Cell Biol. 14, 452–460 (2004).
2. Horsley, V. & Pavlath, G.K. Forming a multinucleated cell: molecules that regulate
myoblast fusion. Cells Tissues Organs 176, 67–78 (2004).
3. Ruiz-Gomez, M., Coutts, N., Price, A., Taylor, M.V. & Bate, M. Drosophila
dumbfounded: a myoblast attractant essential for fusion. Cell 102, 189–198
(2000).
4. Strunkelnberg, M. et al. rst and its paralogue kirre act redundantly during
embryonic muscle development in Drosophila. Development 128, 4229–4239
(2001).
5. Hakeda-Suzuki, S. et al. Rac function and regulation during Drosophila development.
Nature 416, 438–442 (2002).
6. Luo, L., Liao, Y.J., Jan, L.Y. & Jan, Y.N. Distinct morphogenetic functions of similar
small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion.
Genes Dev. 8, 1787–1802 (1994).
7. Taylor, M.V. Muscle differentiation: how two cells become one. Curr. Biol. 12, R224–
R228 (2002).
8. Baxendale, S. et al. The B-cell maturation factor Blimp-1 specifies vertebrate slowtwitch muscle fiber identity in response to Hedgehog signaling. Nat. Genet. 36, 88–93
(2004).
9. Blagden, C.S., Currie, P.D., Ingham, P.W. & Hughes, S.M. Notochord induction of
zebrafish slow muscle mediated by Sonic hedgehog. Genes Dev. 11, 2163–2175
(1997).
10. Devoto, S.H., Melancon, E., Eisen, J.S. & Westerfield, M. Identification of separate
slow and fast muscle precursor cells in vivo, prior to somite formation. Development
122, 3371–3380 (1996).
11. Du, S.J., Devoto, S.H., Westerfield, M. & Moon, R.T. Positive and negative regulation of
muscle cell identity by members of the hedgehog and TGF-beta gene families. J. Cell
Biol. 139, 145–156 (1997).
12. Roy, S., Wolff, C. & Ingham, P.W. The u-boot mutation identifies a Hedgehog-regulated
myogenic switch for fiber-type diversification in the zebrafish embryo. Genes Dev. 15,
1563–1576 (2001).
13. Sellin, L. et al. NEPH1 defines a novel family of podocin interacting proteins. FASEB J.
17, 115–117 (2003).
14. Sun, C. et al. Kirrel2, a novel immunoglobulin superfamily gene expressed primarily in
beta cells of the pancreatic islets. Genomics 82, 130–142 (2003).
15. Ueno, H. et al. A stromal cell-derived membrane protein that supports hematopoietic
stem cells. Nat. Immunol. 4, 457–463 (2003).
16. Mann, C.J., Hinits, Y. & Hughes, S.M. Comparison of neurolin (ALCAM) and neurolinlike cell adhesion molecule (NLCAM) expression in zebrafish. Gene Expr. Patterns 6,
952–963 (2006).
17. Dan-Goor, M., Silberstein, L., Kessel, M. & Muhlrad, A. Localization of epitopes and
functional effects of two novel monoclonal antibodies against skeletal muscle myosin.
J. Muscle Res. Cell Motil. 11, 216–226 (1990).
18. Weinberg, E.S. et al. Developmental regulation of zebrafish MyoD in wild-type, no tail
and spadetail embryos. Development 122, 271–280 (1996).
19. Gardahaut, M.F., Fontaine-Perus, J., Rouaud, T., Bandman, E. & Ferrand, R. Developmental modulation of myosin expression by thyroid hormone in avian skeletal
muscle. Development 115, 1121–1131 (1992).
20. Bour, B.A., Chakravarti, M., West, J.M. & Abmayr, S.M. Drosophila SNS, a member of
the immunoglobulin superfamily that is essential for myoblast fusion. Genes Dev. 14,
1498–1511 (2000).
21. Galletta, B.J., Chakravarti, M., Banerjee, R. & Abmayr, S.M. SNS: Adhesive properties,
localization requirements and ectodomain dependence in S2 cells and embryonic
myoblasts. Mech. Dev. 121, 1455–1468 (2004).
22. Chen, E.H., Pryce, B.A., Tzeng, J.A., Gonzalez, G.A. & Olson, E.N. Control of myoblast
fusion by a guanine nucleotide exchange factor, loner, and its effector ARF6. Cell 114,
751–762 (2003).
23. Buckingham, M. Myogenic progenitor cells and skeletal myogenesis in vertebrates.
Curr. Opin. Genet. Dev. 16, 525–532 (2006).
24. Pauls, S., Geldmacher-Voss, B. & Campos-Ortega, J.A. A zebrafish histone variant
H2A.F/Z and a transgenic H2A.F/Z:GFP fusion protein for in vivo studies of embryonic
development. Dev. Genes Evol. 211, 603–610 (2001).
25. Du, S.J., Gao, J. & Anyangwe, V. Muscle-specific expression of myogenin in zebrafish
embryos is controlled by multiple regulatory elements in the promoter. Comp.
Biochem. Physiol. B Biochem. Mol. Biol. 134, 123–134 (2003).
26. Koprunner, M., Thisse, C., Thisse, B. & Raz, E. A zebrafish nanos-related gene is
essential for the development of primordial germ cells. Genes Dev. 15, 2877–2885
(2001).
27. Crow, M.T. & Stockdale, F.E. Myosin expression and specialization among the
earliest muscle fibers of the developing avian limb. Dev. Biol. 113, 238–254
(1986).
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