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First published online November 26, 2007
doi: 10.1242/10.1242/dev.014027
1 Division of Genetics, Department of Medicine, Brigham and Women's Hospital and
Harvard Medical School, Boston, MA 02115, USA.
2 Centro Andaluz de Biología del Desarrollo (CABD), Universidad Pablo de
Olavide-CSIC, Carretera de Utrera Km. 1, 41013 Sevilla, Spain.
3 National Heart, Lung and Blood Institute, National Institutes of Health,
Bethesda, MD 20892, USA.
Authors for correspondence (e-mails:
bestmar{at}upo.es;
michelsonam{at}nhlbi.nih.gov)
Accepted 21 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PS1-βPS integrin
expressed in tendon cells. These experiments uncovered a previously
unrecognized role for the PS1-βPS integrin in the formation of
muscle projections during early stages of myotendinous junction development.
We propose that Perd regulates projection of myotube processes toward and
subsequent differentiation of the myotendinous junction by priming formation
of a protein complex through its intracellular interaction with Grip and its
transient engagement with the tendon cell-expressed laminin-binding
PS1-βPS integrin.
Key words: Muscle attachment, Myotendinous junction, Myogenesis, NG2, MCSP, Integrins, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The embryonic body wall muscles of Drosophila have a stereotypic
pattern that is readily investigated. Here, myofiber development starts with
the specification of two types of myoblasts, founder cells and
fusion-competent myoblasts, which fuse with each other to form nascent
multinucleated myotubes. Founder cells contain all of the information each
muscle needs for its development, including its size, position, orientation,
innervation and specific attachment to the epidermis
(Bate, 1990
;
Baylies and Michelson, 2001).
These growing myotubes then migrate underneath the ectoderm, and eventually
they stably adhere to specific epidermis-derived tendon cells to form apodemes
or muscle attachment sites (Baylies et al.,
1998; Schnorrer and Dickson,
2004). During the migration phase, myotubes elongate and multiple
radiating projections or filopodia - similar to neuronal growth cones - are
observed at the leading edge of the muscle. By stages 15-16, most muscles have
a smooth surface with both poles stably associated with separate tendon cells
(Bate, 1990;
Bate, 1993;
Schnorrer and Dickson, 2004).
In both Drosophila and vertebrates, the correct development of the
myotendinous junction involves cross-talk between these two cell types
(Volk, 1999). Where tendon
cells not only serve as attachment sites but also provide guiding cues for the
migrating myotube, the muscle is essential for tendon-specific gene expression
and terminal differentiation (Bate,
1990; Frommer et al.,
1996; Volk, 1999;
Vorbruggen and Jackle,
1997).
Despite this detailed morphological view of myogenesis, the molecular cues
that guide myotube migration, muscle-epidermal recognition and apodeme
formation remain largely unknown. Interestingly, all of the proteins that are
currently known to be involved in muscle migration have been shown to
participate in axon guidance and/or synaptic junction signaling, raising the
possibility that both neurons and muscles utilize similar molecular mechanisms
for target recognition (Schnorrer and
Dickson, 2004). One of the few known non autonomous signals
provided by tendon cells is the secreted protein Slit - the ligand for the
Roundabout (Robo) receptors - which is necessary for migration of the
embryonic ventral longitudinal muscles (VLMs)
(Kidd et al., 1998;
Kramer et al., 2001).
Similarly, the receptor tyrosine kinase, Derailed, controls lateral transverse
muscle (LTM) guidance (Callahan et al.,
1996).
In addition to the slit-Robo signaling pathway, the multi-PDZ
domain-containing protein, Glutamate receptor interacting protein (Grip), is
required for VLM guidance through its involvement in the formation of cellular
extensions (Swan et al.,
2004). PDZ domains facilitate protein-protein interactions and are
capable of recruiting multiple proteins to form a molecular complex that
mediates specific and localized signaling
(Bilder, 2001;
Sheng and Sala, 2001). Indeed,
PDZ domain 7 of Grip is essential for its function by binding to the cell
adhesion protein, Echinoid (Ed). This interaction suggests that these two
proteins form a signaling complex during muscle morphogenesis
(Swan et al., 2006).
Once a myotube has reached its attachment site, cell surface adhesion
molecules become localized toward the tendon cell to form a stable connection
that is capable of withstanding muscle contraction
(Brown et al., 2000).
Integrins, which are major cellular and extracellular matrix transmembrane
receptors, are localized at the myotendinous junction where they play an
important role in stabilizing this intercellular connection. Thus, integrin
mutant embryos contain spherical myotubes secondary to detachment following
muscle contraction (Bokel and Brown,
2002; Brown et al.,
2000). Each integrin consists of a heterodimer composed of one
and one β subunit. In Drosophila, there are two
integrins relevant to somatic muscle development, PS1-βPS, which
is expressed in tendon cells, and PS2-βPS, which is expressed in
the muscle. The integrin subunits are encoded by the genes, multiple
edematous wing (mew, PS1), lethal myospheroid
(mys, βPS), and inflated (if, PS2). It
is thought that the attachment of muscles to the epidermis is a sequential
process, in which the muscle first transiently comes in close contact with the
epidermal cell, thereby initiating assembly of a hemiadherens-type junction.
Integrin activation follows, which, in turn, triggers strong attachment of the
integrins to the extracellular matrix and stabilization of the attachment
prior to muscle contraction (Brown et al.,
2000; Prokop et al.,
1998; Tepass and Hartenstein,
1994).
Here, we report the identification of a novel gene that is essential for
development of a subset of muscle attachments in the Drosophila
embryo, a gene which we named perdido (perd; `lost' in
Spanish). perd encodes a single-pass transmembrane cell adhesion
protein that contains two extracellular globular-laminin domains and a small
intracellular domain with a PDZ-binding consensus sequence. In vivo
visualization of muscle development both in perd ethane methyl
sulphonate (EMS)-induced mutants and in wild-type embryos injected with
perd double-stranded RNA (dsRNA), revealed that specific ventral
muscles fail to form proper guidance projections and do not attach stably to
their tendon cells. Consequently, affected muscles detach upon contraction,
and rounded myotubes appear in place of elongated myofibers. This phenotype
resembles that observed with loss-of-function of the PDZ domain-containing
Grip protein and each of the three integrin subunits. Using a newly developed,
sensitive, RNA interference (RNAi)-based method to rapidly and efficiently
test genetic interactions, we demonstrate that perd interacts with
Grip, mys and mew. In addition, we have found that Perd
protein binds directly to PDZ domain 7 of Grip, and that this interaction is
required to localize Grip to the muscle plasma membrane. We hypothesize that
perd primes formation of a protein complex at the myotendinous
junction by transient binding of its extracellular domain to Mew - the
integrin subunit expressed by the tendon cell - and by localizing Grip to the
internal surface of the muscle membrane. Together, this Perd-dependent
mechanism activates a signaling pathway within the muscle that is essential
for myotube guidance, recognition and attachment. Of note, while this
manuscript was in preparation, a related paper was published in which
equivalent findings were reported for Grip and perd
(referred to there as kon-tiki). However, an additional connection
between perd (kon-tiki) and integrin function was not
established by that work (Schnorrer et
al., 2007).
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Flies carrying the 5053Gal4 line
(Swan et al., 2004) (J. Lopez,
personal communication to FlyBase 1998) and UAS-lacZ were used to
visualize the morphology of the VL1 muscle in perd
(perdF1-3UAS-lacZ/perdF1-3;
5053Gal4/+), and Gripex36
(Swan et al., 2004) mutants
(Gripex36/Y; UAS-lacZ/+; 5053Gal4/+). Myosin
heavy chain (MHC)-tau-GFP (Chen and Olson,
2001) and UAS-srcEGFP; 5053Gal4/+ (encoding a membrane-bound form
of GFP) embryos were used to visualize the muscle pattern upon dsRNA
injections.
EMS mutagenesis
yw flies isogenic for chromosome II were starved for 12 hours and
fed for 18 hours with 25 mM EMS in 5% sucrose. Crosses between 3090 single
male progeny of mutagenized males and Df(2L)M36-S5/CTG females were tested for
lethality. Lethal chromosomes were collected over the CTG balancer and fly
lines were established for subsequent phenotypic analysis.
In situ hybridizations and immunohistochemistry
Histochemical in situ hybridization was done as described by Estrada et al.
(Estrada et al., 2006).
RNA-labeled probes for perd and Grip were obtained from
Drosophila Gene Collection (DGC) clones LD31354 and RE14068,
respectively. Fluorescent in situ hybridizations followed standard protocols
(Kosman et al., 2004).
Antibody stainings were carried out as described previously
(Carmena et al., 1998). The
following primary antibodies were used: rabbit and mouse anti-MHC (D. Kiehart,
Duke University, Durham, NC), rabbit anti-Grip (S. Sigrist,
Max-Planck-Society, Göttingen, Germany), guinea pig anti-Kruppel (D.
Kosman, UCSD, San Diego, CA), rabbit and mouse anti-β-galactosidase
(Cappel, Promega), rabbit and mouse anti-GFP (Invitrogen, Clontech), and
anti-digoxigenin-alkaline phosphatase (Roche).
RNA interference assays and in vivo microscopy
RNAi assays were carried out as described by Estrada et al.
(Estrada et al., 2006). Any
gene segment matching any other predicted gene at 18 consecutive nucleotides
was excluded to avoid off-target effects
(Kulkarni et al., 2006). To
test for synergistic interactions, each individual dsRNA to be tested was
first injected at high concentration (2 mg/ml) and the embryos assessed for a
robust and penetrant phenotype. Dilutions were then tested until a critical
concentration was ascertained at which a majority of embryos appeared
completely wild-type and the remainder were only mildly affected. Images of
all GFP-expressing embryos were scored into four phenotypic classes: wild
type, marginally affected (effects were equivocal or limited to a single
hemisegment), clearly affected (improperly attached muscles visible in
multiple hemisegments), or unscorable (embryos exhibiting the severely
aberrant morphology observed at low frequency as a result of all injections,
including lacZ and buffer controls). For examples of each class, see
Fig. S1 in the supplementary material. For the quantification of various
titration experiments, see Fig. S2 in the supplementary material. The critical
concentration varied widely for different dsRNAs - compare, for example, Fig.
S2A,D and G in the supplementary material. Titrations were performed in the
presence of a constant dsRNA concentration, achieved by inclusion of an
appropriate concentration of inactive control (lacZ) dsRNA. This
proved to be crucial, as the concentration of inactive dsRNA could profoundly
influence the effect of an identical dose of test dsRNA; 20 µg/ml of
perd dsRNA has a very strong effect in the absence of any inactive
dsRNA (Fig. S2H in the supplementary material; total dsRNA concentration in
this experiment was 20 µg/ml), but little effect when lacZ dsRNA
was added to a final concentration of 2 mg/ml (Fig. S2G in the supplementary
material).
For each interaction experiment, tubes containing negative control (500 µg/ml lacZ dsRNA), dsRNA 1 at its critical concentration, dsRNA 2 at its critical concentration, or both tested dsRNAs together, each at its critical concentration (plus lacZ dsRNA to 500 µg/ml in all cases) were coded by a different individual so that the identity of the dsRNAs was not known to the operator at the time of either the injection or the scoring of the embryos. dsRNAs were injected, alone and in combination, into at least 30 MHC-tau-GFP embryos per sample. We estimate the injected volume to be 10-100 pl per embryo. Late stage 17 embryos were photographed for GFP fluorescence, and the resulting image files were computationally scrambled using a simple Perl program (see Fig. S3 in the supplementary material).
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2 test. The resulting P value can be interpreted as
the probability of obtaining the observed distribution of phenotypes if the
effects of each dsRNA were independent and additive. Any interaction that we
considered reliable evidence for synergy was characterized by a clear shift
toward more severely affected embryos and a P value less than 0.001.
A more detailed version of this protocol is available from the authors by
request.
Biochemical interaction assays
The intracellular domain of Perd was cloned by PCR, inserted into pET30a(+)
(Novagen), and expressed as a fusion protein with an amino-terminal 6xHis tag
and an S-Tag, then purified by nickel affinity chromatography and eluted in
binding buffer (Kumar and Shieh,
2001) supplemented with 0.5 M imidazole. Isolated PDZ domains from
Grip [PDZ1: aa71-171, PDZ2 (not shown): 172-274, PDZ3: 267-408, PDZ4: 468-559,
PDZ5: 559-669, PDZ6: 809-964, PDZ7: 972-1058] were cloned by PCR, inserted
into pGEX-5X-1, and expressed as GST fusion proteins. Bacteria expressing
soluble protein (including GST only) were resuspended in binding buffer, lysed
by sonication, cleared by centrifugation, applied to GST-Bind resin (Novagen)
at saturating concentrations, and incubated on ice for 1 hour. Bound resin was
washed with excess binding buffer, then purified Perd fusion protein was
applied and incubated on ice for 1 hour. Resin was washed twice with binding
buffer at room temperature, and bound proteins were eluted in reducing gel
sample buffer and western blotted in duplicate according to standard
protocols. GST fusion proteins were detected with rabbit anti-GST and
anti-rabbit HRP conjugate; Perd fusion protein was detected with S-protein HRP
conjugate (Novagen) and visualized by chemiluminescence (Pierce).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We identified hundreds of genes specifically expressed in
founder cells or fusion-competent myoblasts, one of which is the gene CG10275.
Embryonic in situ hybridization experiments in wild-type and informative
mutant backgrounds showed that CG10275 is specifically expressed in founder
cells at stage 11, when cell fate specification occurs
(Estrada et al., 2006).
Detailed analysis of CG10275 RNA expression revealed that it is present in
only a subset of Kruppel-positive founder cells
(Fig. 1A). Later in embryonic
development, CG10275 RNA is restricted to myotubes at the site of attachment
with the epidermal tendon cells (arrows in
Fig. 1B). Other tissues also
express this gene, including the visceral mesoderm, the heart, the gonadal
mesoderm, the endoderm and the central nervous system. This pattern of
expression suggests that CG10275 could be playing important roles in the
development of the Drosophila embryo.
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;
Estrada et al., 2006). In such
an RNAi experiment, the band of ventral longitudinal muscles was abnormal,
presenting globular-shaped muscles instead of elongated myofibers that span
the entire intersegmental space (Fig.
1E,F, arrows; compare control lacZ dsRNA in
Fig. 1C,D). This phenotype
suggests that loss of CG10275 function causes defects in muscle attachment,
but does not reveal when in development the abnormality initially occurs.
CG10275 encodes a single pass type I transmembrane protein of 2355 amino
acids and molecular weight of 267 kDa, with two predicted laminin G domains at
the extracellular amino terminus. The intracellular domain is small and ends
in a class II PDZ binding motif (Fig.
1G), suggesting that it may form part of a protein complex with
one or more PDZ domain-containing proteins. It appears (based on the
bidirectional best BLAST hit) to be orthologous to the mammalian NG2/AN2/MCSP
proteoglycan; however, the predicted open reading frame lacks the signal
sequence for that gene. As the translation initiation site in the predicted
open reading frame is not supported by available experimental evidence, we
performed 5' RACE to determine the true N terminus of the protein and
discovered an additional exon 
5 kb upstream of the predicted initiation
site, with a canonical TATA box and high-quality splice donor site (data not
shown). The predicted protein product of this transcript begins with a classic
signal sequence.
The expression pattern of CG10275, its RNAi phenotype and the molecular nature of the encoded protein motivated us to undertake an EMS mutagenesis screen to isolate mutant alleles for this gene. We screened 3090 mutagenized second chromosomes for embryonic lethality over a deficiency that uncovered the gene CG10275 (Df(2L)M36-S5), and found nine chromosomes that failed to complement the deficiency and each other. These alleles showed abnormalities in the ventral band of muscles, as ascertained by MHC inmunostainings. After sequencing all 12 annotated exons of CG10275 from five independent mutant alleles, we found that four of them created early stop codons in the protein (H2-5=Q404*, F1-3=K868*, F2-5=Q1095* and 187(C2)=L1462*; asterisks in Fig. 1G), and one of them contained a four-nucleotide deletion, resulting in a frameshift and early termination of the protein (after T1216, with an additional 50 amino acids translated out of frame; Fig. 1G). Phenotypic analysis of these mutants showed that ventral longitudinal muscles were rounded and teardrop-shaped, whereas dorsal and lateral transverse muscles were normal (Fig. 1H,I). The fact that homozygous mutant embryos for each of these alleles shows a phenotype identical to that of the same allele placed over the Df(2L)M36-S5 deficiency suggests that these are null alleles. The molecular lesions associated with these mutations (translational terminations prior to the transmembrane and intracellular domains) are consistent with this conclusion. Since the morphology of the mutant muscles indicates that they are not attached to the epidermal tendon cells, we named the gene CG10275, perdido (perd), which means `lost' in Spanish. Of note, the previously identified RNAi phenotype for CG10275 is the same - albeit less severe - as that exhibited by the EMS alleles of this same gene.
To better understand the phenotype of perd mutant embryos, we focused on the development of a single ventral longitudinal muscle (VL1). This muscle can be visualized using the VL1-specific 5053Gal4 line (Swan 04; J. Lopez, personal communication to FlyBase 1998). At stage 13, wild-type VL1 muscles have an elongated, rectangular shape, and both ends of the myotube are already attached to the segment border tendon cells (Fig. 2A,D). Later in development, this muscle maintains its elongated shape, even at stages 16 and 17 when muscle contraction has started and the myotendinous junction is under tension (Fig. 2C,F). By contrast, perd mutant VL1 muscles begin, at stage 13, to exhibit an abnormal teardrop shape, ending in thin projections oriented toward the appropriate tendon cells, instead of having a wide rectangular surface of attachment (Fig. 2G). As development proceeds, these muscles become more and more dysmorphic, assuming rounded and irregular shapes (Fig. 2H,I). This phenotype suggests abnormal differentiation of the attachment site. Occasionally, in later stages, we observe random projections originating from the mutant myotubes, as if they were searching for a new attachment after failing to find one in the normal location (arrow in Fig. 2I).
These results indicate that the adhesion transmembrane protein Perd selectively affects the development of a subset of muscles by regulating myotube projections and subsequent muscle attachment during formation of the myotendinous junction.
A synergistic genetic interaction between perdido and the Glutamate receptor interacting protein is revealed by a sensitized RNAi interaction assay
Having analyzed the aberrant muscle phenotype of perd mutant
embryos, we noticed a striking similarity to the abnormalities associated with
mutations in Grip (Swan et al.,
2004). Grip is required for proper formation of cellular
extensions during myotube guidance (Swan
et al., 2004). At the end of embryogenesis, both Grip and
perd mutants contain rounded, detached muscles that span half a
hemisegment (Swan et al.,
2004) (Fig. 3A-C).
Also, in both mutants the ventral longitudinal and segment border muscles are
affected, but the lateral transverse muscles are spared
(Fig. 3A-C). Early in
myogenesis, the morphology of affected muscles is similar in perd and
Grip mutants, where myotubes start extending thin projections to the
proper tendon cell, but the apodeme does not differentiate properly and the
muscles detach and round up as development proceeds
(Fig. 3D-F). These observations
led us to conclude that perd and Grip affect myogenesis in a
qualitatively similar manner. However, a quantitative analysis of null alleles
of both genes indicates that the expressivity of the phenotype is lower in
Grip mutants (Fig. 3J,K). These
phenotypes, together with the fact that perd and Grip
transcripts are coexpressed in the same myoblasts
(Fig. 3G-I), suggest that these
two genes could be functioning in a common developmental pathway within the
same cells. To test this hypothesis, we developed a method to rapidly detect
genetic interactions using RNAi in living embryos.
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2 test) than either dsRNA
alone or than the distribution predicted to result from an additive effect of
the individual dsRNAs (see Materials and methods for details of the additive
prediction and statistical approach; Fig.
4E). We verified the specificity of the strategy by testing for a
genetic interaction between perd or Grip and the gene
mind bomb 2 (mib2), mutations of which produce a similar
muscle morphology phenotype and which affects a similar and overlapping group
of muscles (Estrada et al.,
2006). In the experiments involving mib2, no
statistically significant interaction occurred
(Fig. 4F,G). This RNAi-based
method suggests that perd and Grip interact specifically in
the formation of muscle attachments. It also represents a new, rapid and
generalizable assay to test for genetic interactions in living embryos, one
that is potentially more sensitive than classical genetic methods which are
limited by the strengths of the alleles that are available to be combined.
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)
(Fig. 5C-H). Since
perd encodes a muscle transmembrane protein, and it interacts
genetically and biochemically with Grip, we hypothesized that it might be
responsible for the translocation of Grip to the plasma membrane. Indeed, in
perd mutant myotubes, Grip protein is distributed throughout the
cytoplasm and fails to localize to the muscle membrane
(Fig. 5J,K,M,N). This finding
suggests that Perd is the protein responsible for establishing the normal
subcellular distribution of Grip at muscle attachment sites where the two
proteins may form a signaling complex.
The integrin genes mys and mew are required to form proper muscle guidance projections and interact genetically with perdido
Phenotypic analysis of perd loss-of-function embryos indicates
that this gene encodes an important muscle transmembrane protein regulating
muscle projections during the early phases of myotendinous junction formation.
The spherical muscles observed in perd mutant embryos are reminiscent
of the phenotypes of the integrin mutants, mys and if
(Fig. 6B-D)
(Brabant and Brower, 1993;
Brown, 1994;
Leptin et al., 1989;
Prokop et al., 1998;
Wright, 1960). This phenotype
- together with the fact that Perd contains laminin domains, to which
integrins are known to bind (Gotwals et
al., 1994) - led us to investigate whether there is a genetic
interaction between integrin genes and perd.
To address this question, we first studied the phenotypes of embryos
separately injected with dsRNA specific for the genes mys, mew and
if. RNAi for each gene resulted in embryos with many rounded muscles.
This result is consistent with the phenotype reported for mys and
if null zygotic alleles. However, the mew RNAi result
contrasts with the minor effects that have been described for mew
null alleles (Roote and Zusman,
1995). Although our dsRNAs are routinely designed to minimize the
likelihood of off-target effects (Estrada
et al., 2006) (also, see Materials and methods), we tested an
additional dsRNA corresponding to a non-overlapping region of the mew
gene and obtained identical results (data not shown). We therefore proceeded
with titration and co-injection experiments to assay potential interaction of
each integrin subunit with perd and with Grip, as
well as with each other. mys was not included in this analysis
because its requirement for both PS1 and PS2 function would make the result
uninformative. The genetic interaction between mew and if -
previously observed at the level of muscle ultrastructure
(Prokop et al., 1998) - is
clearly detected in this assay (Fig.
6E). As predicted, both perd and Grip interact
significantly with mew (Fig.
6F,G). Strikingly, the distribution of muscle phenotypes observed
when perd and if dsRNA are combined is indistinguishable
from the additive effects of each individual dsRNA
(Fig. 6H), whereas
Grip and if interact strongly
(Fig. 6I). This result suggests
that the PS2 integrin (comprising the if and mys gene
products) may participate in muscle attachment in a manner different from the
inferred PS1-Perd complex, possibly by stabilizing myotendinous junctions
after they have formed.
To test this possibility, we visualized the development of muscle VL1 in
vivo with RNAi directed against each of the integrin subunit genes. These
results, along with those for perd, Grip, and lacZ dsRNAs,
are shown in Fig. 7. Each row
shows four time points after injection of the same embryo; t=0
represents the time at which we were first able to visualize the muscle by GFP
fluorescence (that is, during stage 14), and the last panel shows stage 17
embryos, at which point the muscles are clearly contracting when directly
visualized by fluorescence microscopy. In the lacZ dsRNA-injected
embryos, the muscles are clearly elongating at the earliest time at which they
are visible (Fig. 7A,B).
Embryos injected with mew or mys dsRNA exhibit an aberrant
phenotype, appearing more rounded and with less robust projections than in
age-matched control embryos, as early as stage 14-15
(Fig. 7N,V). In this regard
they resemble embryos injected with perd dsRNA, where the muscles
have a rounded shape early in development, suggesting that PS1 integrin is
necessary for the formation of proper projections and muscle attachment. By
contrast, if dsRNA-injected embryos have numerous muscles that extend
normally and achieve an apparently mature morphology (arrowheads in
Fig. 7R,S), but which then
round up late in development, after muscle contraction starts (arrowheads in
Fig. 7T). This finding is
consistent with the established role of integrins in the stabilization of the
attachment between muscle and tendon cells
(Brown et al., 2000).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Very little is known about how individual muscles acquire their specific
morphologies. It is thought that the restricted expression of certain
transcription factor genes, known as `muscle identity genes', and their
regulation of downstream targets play an important role in this process
(Baylies et al., 1998;
Frasch, 1999). perd
is both expressed and required in a subset of muscle founder cells, although
there are more cells expressing perd than muscles with a detectable
mutant phenotype. These observations suggest that perd encodes some
of the specific information that founder cells provide to a given muscle so
that it can acquire its particular characteristics, in this case related to
the formation of the attachment to the tendon cell. It remains unkown if there
are similar transmembrane proteins with an equivalent role in the muscles that
do not express perd.
Inactivation of perd function, either by RNAi or by chemically
induced null alleles, causes embryonic muscle projections to appear dysmorphic
at early stages of embryogenesis and prevents them from forming stable
attachments to the appropriate tendon cells, resulting in rounded, unattached
muscles. There could be several reasons for such a phenotype, including
failure of muscle guidance, of migration, and/or of attachment to the tendon
cell. To better understand the function of perd, we carried out a
series of genetic interaction experiments with genes having similar
phenotypes. To this end, we developed a new method to assess genetic
interactions by testing for phenotypic synergy between low doses of different
gene-specific dsRNAs injected into live embryos. Our results suggest that
RNAi-based interaction experiments are more sensitive than allelic genetic
interaction because we can achieve greater reductions in mRNA levels than is
possible in a heterozygous condition (even with null alleles), and we can
simultaneously reduce both maternal and zygotic mRNA contributions in the
embryo (Kennerdell and Carthew,
1998). This experimental protocol is readily adapted to explore
any pairwise genetic interaction when a suitable phenotype can be scored.
One gene with a similar phenotype to perd is Grip, which
is required in muscles for proper formation of cellular extensions during
guidance (Swan et al., 2004);
thus, mutations in this gene result in rounded muscles. Using our RNAi assay,
we have found a genetic interaction between these two genes, supporting the
idea that they could be playing related roles during the formation of the
myotendinous junction. In addition to this similarity, we noticed that the C
terminus of Perd resembles a PDZ binding consensus, leading us to test a
possible biochemical interaction between the intracellular domain of Perd and
isolated PDZ domains from Grip. The finding of a biochemical interaction
between Perd and a functionally essential Grip PDZ domain, PDZ7
(Swan et al., 2006), together
with the observation that Grip fails to localize to the attachment site in
perd mutants, suggests that Perd is physically recruiting Grip to the
muscle membrane. We hypothesize that Perd may be priming a molecular complex
at the muscle membrane attachment site by localizing Grip and other proteins
through its multiple PDZ protein interaction domains.
Quantification of the affected muscles in Grip and perd
null mutant embryos demonstrates that the Grip phenotype is of lower
expressivity, suggesting that perd has additional functions
independent of Grip. In order to investigate other possible
perd interactors, we focused on the study of Drosophila
integrins, for three reasons. First, loss-of-function mutations in
mys and if result in spherical unattached embryonic muscles
(Brown et al., 2000), similar
to the terminal phenotype of perd mutants. Second, Perd contains two
laminin domains, which are known integrin ligands
(Gotwals et al., 1994). Third,
integrins can bind both to extracellular matrix molecules and to transmembrane
receptors (Brown et al., 2000).
We found that interfering with the function of the mys, mew and
if genes by injection of gene-specific dsRNAs into live embryos
generates unattached, rounded muscles. Interestingly, these results are
comparable with loss-of-function mutations in mys and if,
but are more severe than previously described for mew; only 10% of
mew mutant embryos were reported to have muscle abnormalities
(Roote and Zusman, 1995).
Inactivating both the maternal and zygotic mRNAs by RNAi might account for the
higher number of affected muscles in embryos injected with mew dsRNA.
In addition, double mutants for mew and if show muscle
detachments at an earlier stage than if single mutants, providing
another line of evidence that mew has a role in muscle attachment
(Roote and Zusman, 1995). We
carried out a detailed analysis of these phenotypes using single myotube
imaging in live embryos for which integrin function was blocked by RNAi, where
we indeed uncovered an early role for both mys and mew in
myogenesis, that is, in the formation of proper projections and attachments to
the tendon cells, similar to perd and Grip dsRNA-injected
embryos. By contrast, embryos injected with if dsRNA exhibited normal
muscle projections at early stages.
There are several possible molecular functions for perd. Perd
could be a receptor for guidance cues, although the fact that muscles
initially migrate toward and make contact with the correct tendon cell in
perd mutants suggests that it is not involved in directed migration.
Alternatively, Perd could serve as a muscle receptor for an anchoring molecule
in the tendon cell; the rounding up of muscles and the rare misdirected
projections in later stages suggest that it is the differentiation of the cell
contacts between the muscle and the tendon cell that is impaired in
perd mutants. In fact, it has been described that, as development
progresses, the cellular interactions mediated by integrins change from small
transient contacts, involved in cell recognition and migration, to the larger,
more stable adhesive contacts known as hemiadherens junctions
(Brown et al., 2000;
Prokop et al., 1998). Indeed,
in the careful ultrastructural analysis of Prokop et al. the authors point out
that although the distance between cell membranes at mature myotendinous
junctions is too great for direct contact between integral membrane proteins
of the muscle and tendon cells, "At early muscle attachments, closer
cell contacts with an extracellular gap of only about 15 nm are also
seen." Thus, we hypothesize that Perd serves as a receptor for the
tendon-expressed PS1 integrin, encoded by mew and mys, and
that Perd also recruits the PDZ protein, Grip, to regulate muscle projection
and differentiation of the myotendinous junction
(Fig. 8A). A series of results
supports this hypothesis: the biochemical and genetic interaction between
Grip and perd; the fact that localization of Grip to the
membrane is dependent on perd; the fact that Perd contains laminin
domains and that PS1 is an integrin of the laminin-binding class
(Gotwals et al., 1994); and
the occurrence of rounded muscles before muscle contraction in embryos
injected with mys or mew dsRNA, phenotypes similar to those
observed in perd mutants and in embryos injected with perd
dsRNA.
During preparation of this manuscript, Schnorrer and colleagues
(Schnorrer et al., 2007),
described the phenotypic characterization of loss-of-function mutations in
CG10275 (which they named kon-tiki), and physical and functional
interaction of the corresponding protein with Grip. The present work not only
corroborates but also extends their findings by demonstrating a requirement
for Perd in localizing Grip protein to developing myotendinous junctions, and
by proposing a mechanism by which this localization occurs: recognition of
tendon cell-expressed PS1 integrin heterodimers by Perd.
Interestingly, the vertebrate orthologues of Perd (NG2 in rats, AN2 in mice
and MCSP in humans) are chondroitin sulphate proteoglycans required for cell
migration and communication during the normal development of certain cell
types, such as glia and mesenchyme progenitors. MCSP has also been implicated
in tumor cell metastasis in which cellular adhesion and migration are
abnormally regulated (Stallcup,
2002). Similar to Perd, these mammalian proteins also interact
with integrins or Grip proteins in different cellular contexts that promote
cell migration and signaling, although the underlying molecular mechanisms are
largely unkown (Eisenmann et al.,
1999; Fukushi et al.,
2004; Iida et al.,
1998; Iida et al.,
1995; Stegmuller et al.,
2003) (Fig. 8B-D).
Our findings for the function of Perd provide a new model in another cellular
and in vivo system, the myotendinous junction, in which it may be possible to
better understand the function of the vertebrate orthologues in the regulation
of cell adhesion and metastasis. In addition, our novel dsRNA interaction
method offers a fast, generalizable and directed way to test hypothetical
interactions in an experimentally tractable model system.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/24/4469/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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indicates the position of a four-nucleotide deletion in
H1-4, resulting in a frameshift and early termination. (H,I)
Immunostaining for myosin heavy chain shows that genetic loss of perd
function causes the same muscle phenotype as RNAi, in which ventral
longitudinal and segment border muscles have a rounded or teardrop shape
(arrows), whereas lateral transverse muscles are normal (arrowhead).
