1 Institut de Génétique et de Biologie Moléculaire et
Cellulaire, CNRS/INSERM/ULP, BP 10142, 67404 Illkirch Cedex, C.U. de
Strasbourg, France
2 Institut für Genetik, Universität Mainz, D-55099 Mainz,
Germany
3 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot
76100, Israel
* Author for correspondence (e-mail: angela{at}titus.u-strasbg.fr)
Accepted 7 June 2004
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SUMMARY |
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Key words: Drosophila melanogaster, glide/gcm, glide2/gcm2, Tendon cell differentiation, Muscle attachment, Locomotion
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Introduction |
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Our knowledge on the formation of junctions between muscles and tendon
cells (called attachment sites) derives from fly studies
(Volk, 1999). Initially,
subsets of epidermal cells within each embryonic segment acquire the
competence to form tendon cells. This early muscle-independent step is under
the control of the Stripe transcription factor
(Frommer et al., 1996
;
Lee et al., 1995
). Competent
tendon cells provide guidance cues that direct myopodia to their attachment
sites. Once muscles and target tendon cells get in contact with each other,
myopodia stop growing (Bate,
1990
; Swan et al.,
2004
) and only competent tendon cells that are in contact with
muscles maintain tendon cell marker expression
(Becker et al., 1997
). This
late muscle-dependent differentiation step is characterised by the expression
of delilah (Armand et al.,
1994
) and ß1tubulin
(Buttgereit, 1993
;
Buttgereit et al., 1991
;
Yarnitzky et al., 1997
) and
leads to the formation of hemiadherens junctions (HAJs), cell-specific
junctions associated with extracellular matrix components deposited between
muscles and tendon cells (Tepass and
Hartenstein, 1994
). Mutations in HAJ components, as for example in
genes encoding the ßPS integrin ortholog
(Brown, 1994
;
Leptin et al., 1989
;
MacKrell et al., 1988
;
Newman and Wright, 1981
) or
its ligand Tiggrin (Bunch et al.,
1998
), lead to complete lack of adhesion at attachment sites,
which results in muscle detachment and rounding
(Bokel and Brown, 2002
;
Prokop et al., 1998
).
Interestingly, although ßPS integrin is expressed in muscles and tendon
cells, most accumulation occurs in muscles and only the muscular component
seems required in the process
(Martin-Bermudo and Brown,
1996
). Thus, establishment of HAJs triggers muscletendon
cell interactions that are necessary for the formation of functional muscle
attachment sites (Clark et al.,
2003
; Martin-Bermudo,
2000
; Martin-Bermudo and
Brown, 2000
). This terminal step of tendon cell differentiation
that allows muscle contraction to generate locomotion is still poorly
understood.
glial cell deficient/glial cell missing (glide/gcm) and
glide2/gcm2, which we will refer to as glide and
glide2 throughout the text for the sake of simplicity, form a gene
complex on the second chromosome and encode homologous transcription factors
(Akiyama et al., 1996;
Alfonso and Jones, 2002
;
Hosoya et al., 1995
;
Jones et al., 1995
;
Kammerer and Giangrande, 2001
;
Miller et al., 1998
;
Ragone et al., 2001
;
Schreiber et al., 1997
;
Van De Bor and Giangrande,
2002
; Van De Bor et al.,
2000
; Vincent et al.,
1996
). When both genes are missing, glial cells transform into
neurons, whereas overexpression of either glide or glide2 is
sufficient to induce gliogenesis, indicating that the glide complex triggers
the glial fate (Akiyama-Oda et al.,
1998
; Alfonso and Jones,
2002
; Bernardoni et al.,
1999
; Bernardoni et al.,
1998
; Hosoya et al.,
1995
; Jones et al.,
1995
; Kammerer and Giangrande,
2001
; Miller et al.,
1999
; Vincent et al.,
1996
).
Here, we reveal a novel role of the glide complex during Drosophila development. We show that both glide and glide2 are expressed in a subpopulation of embryonic tendon cells. In the absence of both genes, muscles are disorganised and HAJs are abnormal, even though all known tendon cell-specific markers, including stripe itself, are still expressed. Our data also show that the glide complex controls a new molecular pathway required in terminal tendon cell differentiation and tendon cellmuscle interactions. Finally, we show that the same transcription factor is used for different purposes during development (fate choice in neurogenic region, terminal differentiation in tendon cells) in response to positional cues that control its expression and its specificity.
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Materials and methods |
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Immunohistochemistry
Embryos were immunolabelled as in Vincent et al.
(1996). The following primary
antibodies were used: guinea-pig anti-Stripe (1:300)
(Becker et al., 1997
), mouse
anti-Repo (1:100) (DSHB), rabbit anti-ß gal (1:500) (Cappel), rabbit
anti-Alien (1:1000) (gift of A. Paululat), rabbit anti-Myosin heavy chain
(MHC) (1:1000) (gift of D. P. Kiehart), rabbit anti-GFP (1:500) (Molecular
Probes). Secondary antibodies conjugated with FITC, Cy3 (Jackson) were used at
1:400. Phalloidin-TRITC was used (1:100) (Molecular Probes) to label muscle
F-actin in late embryos. Embryos were mounted in Vectashield medium (Vector)
and analysed using conventional (Axiophot, Zeiss) or confocal (DMRE, Leica)
microscopes.
RNA analysis
In-situ hybridisation was as in Bernardoni et al.
(Bernardoni et al., 1999) and
Kammerer and Giangrande (Kammerer and
Giangrande, 2001
). stripe a and Ent2 mRNAs were
detected using specific probes corresponding to nucleotide 665 to 1583
(accession number NM 079671) and 681 to 1851 (accession number NM 135205),
respectively. stripe a/b mRNAs were detected using a common probe
corresponding to nucleotide 422 to 1140 (accession number NM 169786).
Transmission electron microscopy
Thirty-minute egg lays were kept at 25°C until stage 16. After manual
devitellinization, embryos were fixed overnight at 4°C in 0.1 M
Na-phosphate buffer (pH 7.4) containing 4% paraformaldehyde and 3%
glutaraldehyde, postfixed for 1 hour in 1% osmium tetroxide in 0.1 M
Na-phosphate buffer (pH 7.4) and dehydrated in graduated ethanol series ending
with propylene oxide treatment. Embedding used an araldite-epon (48-hour
polymerization at 60°C). Semi-thin sections were stained with toluidine
blue and visualised under light microscope to determine the region of
interest. Ultra-thin sections (60-70 nm) were contrasted with uranyl acetate
and lead citrate, and examined using a Philips EM208 electron microscope.
glide-glide2 embryos were genotyped by X-Gal staining.
Larval behaviour and histology
Locomotion was measured on 3.5 cm 2.5% agarose plates marked with a 0.5 cm
grid by counting the total number of squares crossed in 10 minutes by each
larva [modified from Naimi et al. (Naimi
et al., 2001)]. For muscle visualisation, larvae were
progressively dehydrated in ethanol series ending with methyl salicylate
treatment and examined under polarised light (Axiophot, Zeiss).
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Results |
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Somatic muscles of glide-glide2 embryos did not show any
abnormality until stage 16 [11.50-14.50 hours after egg-laying (AEL) stages,
according to Campos-Ortega and Hartenstein
(Campos-Ortega and Hartenstein,
1997)] (data not shown). Moreover, late markers (delilah
and ß1tubulin) were still expressed (data not shown), indicating
that muscles had established contacts with tendon cells
(Yarnitzky et al., 1997
), a
process normally achieved by stage 15 (11.20-11.50 hours AEL)
(Bate, 1990
;
Swan et al., 2004
).
By contrast, defects became obvious by early stage 17 (14.50-16.00 hours
AEL): ventral longitudinal muscles bypassed their target tendon cells, reached
the midline and attached to other muscles
(Fig. 2B,D). This highly
penetrant phenotype (93% of embryos, n=14), present in one or more
segments, affected a muscle that attached to the ventral
glide-expressing tendon cell. Midline crossing muscles seemed to be
still attached since they did not display the compact round shape of detached
muscles observed in ßPS integrin embryos
(Brown, 1994;
Leptin et al., 1989
;
MacKrell et al., 1988
;
Newman and Wright, 1981
). This
was also confirmed by the presence of ßPS integrin, a typical marker of
attachment sites (Brown,
2000
), along the ends of crossing muscles (arrow,
Fig. 4G). Altogether, our
results suggest that terminal tendon cell differentiation is affected,
resulting in defective muscle attachment sites. Interestingly, glide
embryos carrying one dose of glide2 displayed similar but less severe
and less penetrant muscle defects (compare
Fig. 2E with
Fig. 2D) (one midline crossing
per embryo, 44% penetrance, n=16), whereas embryos only lacking
glide2 or glide2 embryos carrying one dose of glide
showed no defects (data not shown). Finally, glide embryos, which
lack most glial cells, did not show any defect either (data not shown),
indicating that muscle phenotypes are not due to nervous system
alteration.
|
|
Tissue-specific repression of glide complex activity affects muscle organisation and locomotion
Muscle attachment defects were mostly observed ventrally
(Fig. 2), whereas the glide
complex is expressed in all segment border tendon cells
(Fig. 1). Two lines of evidence
suggested that such a confined phenotype was due to the pleiotropic effect of
the glide-glide2 deficiency. First, lethality occurred
before all attachment sites were fully differentiated and functional. Second,
neuromuscular defects indirectly caused by lack of glia probably affected the
contractions that normally occur at the end of embryogenesis. As a
consequence, mutant tendon cells were not as solicited as wild-type ones.
To assess the role of the glide complex at the attachment site in the
absence of nervous system phenotypes, we produced a tissue-specific
glide-glide2 loss of function using a dominant negative (DN)
approach. For this purpose, we generated a fusion protein between the Glide
DNA binding domain and the Engrailed repressor domain. Chimeric genes with
this repressor domain have already been successfully used to produce DN
mutations (Jaynes and O'Farrell,
1991; Vickers and Sharrocks,
2002
). Since Glide and Glide2 DNA-binding domains are highly
homologous and bind to the same site
(Kammerer and Giangrande,
2001
; Miller et al.,
1998
), the glideDN construct probably inhibits
targets of both proteins and, indeed, when expressed in the central nervous
system (CNS), it blocks glial differentiation (B.A. and G.M.T.,
unpublished).
To specifically repress the glide complex dependent programme in tendon
cells, we used two drivers. No muscle phenotype was detected in
stripe-gal4; UAS-glideDN flies (data not shown),
probably because the driver is expressed too late to efficiently repress glide
complex activity. By contrast, the earlier driver ptc-gal4 induced
muscle defects similar to those observed in embryos lacking
glide-glide2 (see ptc::glideDN in
Fig. 2G). To provide further
evidence that the muscle defects are specifically due to
glideDN, we also generated a point mutation in the DNA
binding domain (glideN7-4DN). The
glideN7-4 allele corresponds to a null, due to the fact
that the mutant protein is not able to bind DNA
(Miller et al., 1998;
Vincent et al., 1996
).
Therefore, the glideN7-4DN construct corresponds to an
inactive form of glideDN, providing a negative control.
The findings that ptc::glideN7-4DN embryos did not show
muscle defects (Fig. 2H) and
were perfectly viable until the adult stage indeed validate the DN approach.
Finally, possible mesodermal contribution to the observed phenotype was
excluded in two ways: the ptc-gal4 driver was not expressed in
muscles, and mesodermal GlideDN expression using the
twi-gal4 driver did not induce attachment defects (data available
upon request).
Most ptc::glideDN embryos died just prior to eclosion, later than glide-glide2 embryos, few escapers reaching the second instar larval stage. Such escapers displayed major locomotion defects, such as uncoordinated movements and abnormal muscle contractions (see Movie 1 at http://dev.biologists.org/cgi/content/full/131/18/4521/DC1). Moreover, ptc::glideDN animals were sluggish (Fig. 3A and Movie 1) and tended to make turns instead of following the same direction, the stereotyped behaviour shown by wild-type animals (data not shown). Larval muscle organisation was severely altered, indicating that muscle attachment is impaired (Fig. 3C,E) and that the glide pathway is required in tendon cells during larval life as well. Finally, when analysed at the end of stage 17 (17-19 hours AEL), ptc::glideDN embryos displayed severe muscle attachment defects in longitudinal and ventral longitudinal muscles (Fig. 3I-K) that probably account for the late embryonic lethality. By contrast, ptc::glideN7-4DN animals displayed no locomotion defects (see Movie 1 and Fig. 3A) and showed no embryonic and larval muscle defects (Fig. 3B,D,F-H).
|
Attachment sites are defective in glide-glide2 embryos
glide-glide2 attachment sites were analysed by electron
microscopy. Hemiadherens junctions (HAJs), which are normally found at contact
sites between muscles and tendon cells
(Fig. 4A), contain
cell-specific integrins that link the extracellular matrix components to the
cytoskeleton of each cell (Prokop et al.,
1998; Tepass and Hartenstein,
1994
). Adhesion of HAJs to the extracellular matrix formed an
electron-dense material (arrowhead in Fig.
4B) that was absent in glide-glide2 embryos
(Fig. 4C), indicating that the
junction was defective. Moreover, the tight interdigitation observed between
wild-type muscle and tendon cell was not present in mutant animals, strongly
suggesting adhesion defects (Fig.
4C). Finally, ßPS integrin accumulation at the position of
attachment sites was decreased in glide-glide2 embryos (arrowheads in
Fig. 4D,G). The HAJ phenotypes
were probably due to the loss of both tendon cell and muscular ßPS, since
HAJs and muscles are normal in animals lacking only the tendon cell-specific
integrin PS1 or its ligand Laminin
(Gotwals et al., 1994
;
Prokop et al., 1998
). In
conclusion, muscle attachment sites were defective in glide-glide2
embryos.
Epistatic interactions between the glide complex and the stripe-induced pathway
stripe is the earliest known gene to be expressed in presumptive
tendon cells (Frommer et al.,
1996; Lee et al.,
1995
). It encodes two isoforms (a and b) that differ by their
transcription start site but have the same DNA-binding domain
(Frommer et al., 1996
;
Lee et al., 1995
). While the
role of stripe a is unknown, stripe b ectopic expression
induces the expression of tendon cell markers such as shortstop (also
called kakapo) (Gregory and
Brown, 1998
; Strumpf and Volk,
1998
; Subramanian et al.,
2003
), alien
(Goubeaud et al., 1996
),
delilah (Armand et al.,
1994
), ß1tubulin
(Buttgereit, 1993
;
Buttgereit et al., 1991
) and
stripe a (Becker et al.,
1997
; Vorbruggen and Jackle,
1997
). In stripe embryos, tendon cell differentiation
does not occur and most markers are less, or not at all, expressed
(Frommer et al., 1996
).
We asked whether stripe exerts its function through glide and found no effect on glide transcription in stripe b gain- and loss-of-function embryos (Fig. 5E,F and Fig. 5G,H, respectively). Thus, glide represents the first identified gene that is expressed independently of stripe. To further clarify the role of the glide complex, we analysed the expression of stripe a and b in embryos either lacking or ectopically expressing glide or glide2. Loss of glide-glide2 had no effect on stripe a or b expression (data not shown). By contrast, ectopic glide expression (en::glide) specifically induced stripe b transcription (Fig. 5A-D and Fig. 6B). Interestingly, while stripe b ectopic expression (en::stripe b) induced tendon cell marker expression, ectopic glide (en::glide) induced only stripe b and alien (compare Fig. 6D with Fig. 6E). This may depend on the delayed stripe b ectopic expression in glide gain-of-function experiments. These results suggest that glide and stripe act on common targets such as alien.
|
|
All stripe-induced tendon cell markers (shortstop,
delilah, ß1tubulin, how) were still expressed in
glide-glide2 embryos (data not shown), implying that the glide
complex controls the expression of yet unknown targets. Functional microarray
experiments based on glide overexpression in the nervous system, as
well as computational analyses based on the presence of Glide binding sites
(GBSs), have identified potential glide targets
(Egger et al., 2002;
Freeman et al., 2003
). We
analysed several of them for tendon cell expression and identified
Ent2 (Equilibrative nucleoside transporter 2)
(Fig. 6G), which is also
expressed in glial cells and contains eight GBSs
(Freeman et al., 2003
).
glide was necessary (data not shown) and sufficient
(Fig. 6H) to induce
Ent2 expression. Also glide2, but not stripe b,
ectopic expression induced Ent2 expression (data not shown and
Fig. 6I, respectively). Thus
Ent2 represents a target specific to the glide complex. To summarise,
the glide complex did not depend on stripe and controlled a new
molecular pathway independently of stripe.
Regulation of glide expression in tendon cells
The glide complex set-up of expression precedes muscle interaction with
competent tendon cells and is probably muscle-independent, as confirmed by the
observation that glide was still expressed in mesoderm-lacking
embryos (data not shown). Segment polarity genes such as hedgehog
(hh) and wingless (wg) are involved in the
determination of segment border cells
(Piepenburg et al., 2000),
some of which later become tendon cells
(Volk and VijayRaghavan,
1994
). We therefore tested whether patterning genes control
glide expression. patched (ptc) encodes a
transmembrane receptor involved in the repression of Hh signal transduction
(Alcedo and Noll, 1997
). In
ptc embryos, the Hh signalling pathway is constitutively activated
(Forbes et al., 1993
;
Ingham and Hidalgo, 1993
),
leading to ectopic cells with segment border identity (compare
Fig. 7A with
Fig. 7F)
(Nusslein-Volhard and Wieschaus,
1980
; Piepenburg et al.,
2000
) that expressed stripe and glide
(Fig. 7G-J). In wg
embryos, neither glide (compare
Fig. 7B,C with
Fig. 7L,M) nor stripe
(compare Fig. 7D,E with
Fig. 7N,O) was expressed in
epidermis, in agreement with the loss of segment border cell identity (compare
Fig. 7A with
Fig. 7K)
(Nusslein-Volhard and Wieschaus,
1980
; Nusslein-Volhard et al.,
1984
). Consistent results were obtained in other segment polarity
mutants (lines, naked) (data not shown). Thus, stripe and
glide expression were independently controlled by positional
cues.
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Discussion |
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Terminal tendon cell differentiation requires the glide complex to establish functional muscle attachment sites
glide and glide2 are required in segment border tendon
cells. By contrast to the nervous system, where these genes are transiently
expressed and play a role in fate choice, their transcripts are present in
tendon cells until the end of embryogenesis. Moreover, the glide complex is
not controlled by the stripe tendon cell fate determinant and is not
necessary for the expression of stripe targets. Finally, the glide
complex does not trigger tendon cell differentiation. Thus, glide and
glide2 do not play a role in fate choice in the epidermis.
Several observations indicate that the glide complex controls terminal
tendon cell differentiation and thereby affects attachment site integrity.
First, glide-glide2 embryos do express genes (delilah and
ß1tubulin) that are activated by the establishment of
muscle-tendon cell contact (Yarnitzky et
al., 1997) and only show muscle defects after stage 16, once these
contacts have been established. Second, glide-glide2 tendon cells and
muscles are unable to form a functional attachment site, as shown by muscle
midline crossing and defective HAJs. Third, tendon cell-specific inactivation
of glide complex activity results in massive muscle disorganisation and
defective locomotion. Interestingly, locomotion defects have also been
observed in larvae mutant for flapwing, a gene encoding a phosphatase
1ß, known for its role in muscle attachment maintenance
(Raghavan et al., 2000
). It
will be interesting to determine whether glide complex targets represent
Flapwing substrates, even though it is clear that Flapwing also affects a
glide-independent pathway since it is expressed and required both in
tendon cells and muscles (Raghavan et al.,
2000
).
Although Stripe represents the tendon cell fate determinant
(Frommer et al., 1996;
Lee et al., 1995
), it is
expressed until the end of embryogenesis, suggesting an additional, late,
role. Indeed, while early inactivation of the stripe pathway (stage
11) by the dominant negative approach and null mutations induce the same
phenotype of detached muscles, late inactivation (stage 15) induces a muscle
midline crossing phenotype similar to the one we observed in
glide-glide2 embryos (Vorbruggen
and Jackle, 1997
). Thus, the glide complex and stripe are
necessary for terminal tendon cell differentiation and probably act on common
targets such as alien. Interestingly, the glide complex also controls
its own pathway, independently of stripe
(Fig. 9). Understanding the
relative contribution of the two pathways will require the identification of
the stripe and glide targets. Finally, the observation that
tendon cell-specific mutations alter muscle organisation highlights the
importance of cellcell interactions throughout muscle and tendon cell
development. Identifying mutations affecting either cell type will help us
elucidate the bases of such interactions.
|
While dorso/ventral (D/V) patterning contribution to stripe
expression and tendon cell differentiation is not yet elucidated, a microarray
study has identified glide as a target of Dorsal
(Stathopoulos and Levine,
2002), the maternally provided D/V patterning factor
(Belvin and Anderson, 1996
).
However, we found no Dorsal binding sites in the glide promoter,
suggesting that this regulation is also indirect. Altogether, our results show
that stripe and glide expression is mutually independent and
associated with segment border cell identity. Therefore, positional cues
trigger the expression of Glide and Stripe, which in turn control several
aspects of tendon cell differentiation. Detailed analyses of the
glide promoter will allow us to identify the transcription factors
upstream from glide in tendon cells.
glide is able to induce glial- (ventral and anterior) or tendon
cell- (dorsal and posterior) specific markers depending on D/V and A/P cues,
indicating that the two fates (glial versus tendon cell) are mutually
exclusive and that cell-specific factors depending on patterning genes dictate
Glide specificity. In line with this are the data on Abrupt, a BTB-zinc finger
transcription factor that is thought to act as a repressor
(Hu et al., 1995). Indeed, the
loco Glide target is expressed only in glial cells in wild-type
embryos (Granderath et al.,
1999
), but is also expressed in tendon cells in abrupt
embryos (Granderath et al.,
2000
). Abrupt seems to be expressed throughout the epidermis
(Hu et al., 1995
) and does not
regulate glide expression
(Granderath et al., 2000
),
indicating that it normally represses the Glide glial pathway in tendon cells.
Thus, expression of tendon cell-versus glial-specific markers depends on the
balance of positive and negative factors. Whether such factors interact
directly with Glide and/or act on glide targets remains to be
elucidated.
Vertebrate tendons
Like fly attachment sites, the vertebrate musculoskeletal system requires
the coordinated formation of distinct types of tissues. The cellular and
molecular bases of vertebrate tendon development are not well understood,
mostly due to lack of markers and mutations that specifically affect this
process. The identification of Scleraxis, a bHLH transcription factor
(Cserjesi et al., 1995)
expressed in tendons and their progenitors
(Brown et al., 1999
;
Schweitzer et al., 2001
) has
opened the way to study vertebrate tendon development
(Brent et al., 2003
;
Dubrulle and Pourquie, 2003
;
Schweitzer et al., 2001
). It
will be interesting to determine whether glide orthologs
(Akiyama et al., 1996
;
Altshuller et al., 1996
;
Kammerer et al., 1999
;
Kanemura et al., 1999
) are
expressed and required in vertebrate tendons as well.
Fly HAJs resemble vertebrate myotendinous junctions (MTJs), as, in both
cases, muscle attachment to tendon cells requires integrins and extracellular
matrix components (Benjamin and Ralphs,
1997; Brown,
2000
). Alterations of vertebrate MTJs result in muscular
dystrophy, as shown by the phenotype of mice double mutant for
dystrophin and utrophin, two redundant genes encoding
proteins that link muscle intracellular cytoskeleton to extracellular matrix
at the MTJ (Deconinck et al.,
1997
; Deconinck et al.,
1998
; Grady et al.,
1997
; Tinsley et al.,
1998
). Moreover, the dystrophin zebrafish ortholog is
necessary for attachment site integrity
(Bassett et al., 2003
).
A single Drosophila gene (dmDLP) displays homology with
both dystrophin and utrophin and is characterised by
transcripts regulated by different promoters
(Greener and Roberts, 2000;
Neuman et al., 2001
).
Interestingly, one transcript is expressed in segment border cells during
embryonic development. It will be interesting to analyse the contribution of
this protein to fly attachment site integrity and its interaction with the
glide pathway.
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ACKNOWLEDGMENTS |
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Footnotes |
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