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First published online 27 February 2008
doi: 10.1242/dev.015982
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1 Riken Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku
Kobe 650-0047, Japan.
2 National Institute of Genetics and the Graduate School for Advanced Studies,
1111 Yata, Mishima, Shizuoka-ken 411-8540, Japan.
3 Department of Biology, Tokyo Metropolitan University, Minami-Ohsawa 1-1,
Hachioji, Tokyo 192-0397, Japan.
4 Department of Life Science, Kobe University Graduate School of Sciences and
Technology, Kobe 657-8501, Japan.
Author for correspondence (e-mail:
shayashi{at}cdb.riken.jp)
Accepted 24 January 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Src, E-cadherin, Armadillo, Drosophila, Trachea, Cancer
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ribeiro et al., 2004;
Zallen and Wieschaus, 2004).
The cell adhesion molecule E-cadherin is concentrated at the adherens
junctions (AJs) and plays a key organizing role in epithelial morphogenesis
(Takeichi, 1991) through its
interaction with β-catenin and 
-catenin, which forms a dynamically
regulated molecular link between cadherins and the actin cytoskeleton
(Drees et al., 2005;
Yamada et al., 2005). In
addition, β-catenin transduces Wnt/Wg signaling
(Peifer et al., 1991) by
entering the nucleus and stimulating transcription through an association with
TCF (Molenaar et al., 1996).
Whether cell junctions act as an alternate trigger for signaling through
Armadillo (Arm) is not understood.
Members of the Src family of non-receptor protein tyrosine kinases (SFK)
represent good candidates for regulators of AJ function
(Thomas and Brugge, 1997). The
activation of Src in cultured epithelial cells downregulates E-cadherin and
causes cell dissociation (Behrens et al.,
1993), and is implicated in the promotion of the
epithelial-mesenchymal transition (EMT)
(Boyer et al., 1997). By
contrast, a recent study suggests that Src might also act positively on cell
adhesion (McLachlan et al.,
2007). Src elicits effects on a number of signaling pathways and
multiple mammalian SFKs have overlapping functions, making genetic analyses
via gene disruption difficult (Thomas and
Brugge, 1997). Thus, functional studies of mammalian SFKs in the
context of epithelial morphogenesis have progressed slowly.
Here, we used the Drosophila tracheal system to study the role of
Src in epithelial morphogenesis. This respiratory system is formed by a series
of dynamic remodeling steps of the tubular ectodermal epithelia
(Samakovlis et al., 1996) to
form a complex three-dimensional tubular network that infiltrates the entire
body cavity of the larva. All of these processes occur in the absence of cell
division and proceed seamlessly without the loss of epithelial integrity
because of efficient cell rearrangement and the remodeling of cell junctions.
Tracheal cell adhesion is defective in E-cadherin mutants
(Tanaka-Matakatsu et al.,
1996; Uemura et al.,
1996), and the inhibition of E-cadherin turnover via elevated Rac
GTPase activity causes disintegration of the tracheal epithelium
(Chihara et al., 2003),
suggesting that E-cadherin is a key modulator of cell adhesion in the
trachea.
This prompted us to study how Drosophila homologs of Src
(Simon et al., 1985;
Takahashi et al., 2005) might
regulate adherens junction turnover. We provide evidence that Src42A is
preferentially activated in the AJs of epithelia undergoing morphogenesis.
Src42A activation influences E-cadherin in two distinct, and disparate, ways,
in that it antagonizes E-cadherin-mediated cell adhesion while simultaneously
stimulating the transcription of E-cadherin. These two opposing outcomes are
accompanied by an increased mobilization of AJs. We propose that the
activation of Src42A in AJs promotes adherens junction turnover, thereby
promoting cell rearrangement in rapidly remodeling epithelial tissues.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Toba et al., 1999), to screen
for gene activities causing EMT-like transformation in tracheal cells. In an
initial screen, 5858 independent insertion lines of UASG-containing
GS elements were crossed with an eye-specific GMR-Gal4 driver to
select lines causing dominant phenotypes in the eye. Next, 380 lines having
the rough eye phenotype were crossed to a strain carrying the trachea-specific
btl-Gal4 driver and the UAS-GFP-moesin marker. An additional
142 lines lacking the rough eye phenotype were also tested. GS11022
(Src42A) and GS9618 (Src64B) were analyzed for further
study.
Fly stocks and genetics
We used strong loss-of-function alleles of Src genes:
Src42A26-1 (Takahashi
et al., 2005), Src42Amyri
(Tateno et al., 2000) and
Src64BP1 (Dodson et
al., 1998). The following strains were used in this study:
trachealess enhancer trap line 1-eve-1
(Perrimon et al., 1991),
UAS-wg (Lawrence et al.,
1995), UAS-armS10
(Pai et al., 1997),
UAS-TCF
N (van de
Wetering et al., 1997), UAS-E-cadherin-GFP
(Oda and Tsukita, 1999b),
UAS-D-catenin-GFP
(Oda and Tsukita, 1999a),
shg-lacZ (Uemura et al.,
1996), UAS-GFP-moesin
(Chihara et al., 2003),
UAS-Src42AYF (Tateno
et al., 2000), UAS-Src42A-RNAi (NIG Stock Center) and
btl-Gal4 (Shiga et al.,
1996). Src42ADN was constructed by introducing
the K295M mutation at the catalytic center of the kinase domain and was cloned
into the pUAST vector (Brand and Perrimon,
1993).
Immunostaining and imaging
The following primary antibodies were used: rat anti-Esg
(Fuse et al., 1994); mouse
anti-tracheal luminal antigen 2A12, mouse anti-Armadillo N27A1 and mouse
anti-septin 4C9H4 (Developmental Studies Hybridoma Bank); rabbit
anti-β-galactosidase (Cappel); mouse anti-GFP B-2 and rabbit anti-GFP
(MBL); rabbit anti-Src PY418 (Biosource International); rat anti-E-cadherin
(DCAD2) (Oda et al., 1994) and
rabbit anti-Src42A (Takahashi et al.,
2005). Chicken anti-Src42A antibody was raised against the
full-length GST-Src42A protein. For cell surface staining, S2 cells
transfected with an E-cadherin-GFP expression vector, with or without Src42A
vectors, were fixed with ice-cold 4% paraformaldehyde in PBS for 10 minutes.
After blocking with 1% BSA in PBS, the cells were incubated with DCAD2
antibody in PBS. After washing, the cells were permeabilized by 0.1% Triton
X-100 in BSA+PBS, and processed with the standard procedure to detect GFP and
DCAD2. Immunostaining was detected with secondary antibodies labeled with
Alexa 488 (Molecular Probes), Cy3 or Cy5 (Amersham Biosciences). Stained
specimens were mounted in Vectorshield (with DAPI, Vector Laboratories) and
imaged using confocal microscopes (Olympus FV500 and Olympus FV1000) with a
water immersion objective [60x, numerical aperture (NA) 1.2]. Time-lapse
images were obtained using a confocal microscopes equipped with a motorized
stage (Olympus FV1000) and an oil immersion objective (60x, NA 1.42).
Typically 20-25 1-µm optical sections were taken every 180 seconds for up
to 6 hours at 25°C and projected images were converted to QuickTime movies
as previously described (Kato et al.,
2004). FRAP experiment was performed on the dorsal branch (DB) of
stage 12 embryos (btl>D-catenin-GFP and
btl>E-cadherin-GFP) using a confocal microscope equipped with two
scanners (Olympus FV1000). GFP signal in a part of the AJ was photo bleached
by 405-nm diode laser irradiation and images were taken by continuous
recording (256x192 pixel, 3 z-stacks/frame/2.6 seconds). Image
stacks were processed by iSEMS software
(Wada et al., 2007) in order
to computationally compensate for the vigorous movement of tracheal cells
during recording. A fluorescence recovery curve was drawn according to Rabut
and Ellenberg (Rabut and Ellenberg,
2005). The statistical significance of the difference between
t
and Mf values of control and
experimental data was evaluated by Student's t-test (two-sided). Six
control, 5 Src42Amyri, 11 Src42Amyri;
Src64P1, 10 Src42ARNAi and 10
Src42AACT btl>D-catenin-GFP embryos
were used.
Cell culture, western blotting and immunoprecipitation
For transient transfections of S2 cells, 106 cells were cultured
in 35-mm culture dishes for 24 hours and then transfected with pUAST-based
expression vectors and the actin 5C-Gal4 driver (generous gift of Dr Yash
Hiromi, National Institute of Genetics, Mishima, Shizuoka-ken, Japan) using
Effectene (Qiagen). Src42AACT has a Y511F mutation, and a fly
strain carrying its UAS construct (Tateno
et al., 2000) was used for tracheal experiments. Two days after
transfection, cells were lysed in 150 µl lysis buffer [1.0% (w/v) NP40,
0.5% deoxycholate, 150 mM NaCl, 50 mM Tris-HCl, pH 8.0] containing protease
inhibitor cocktail (Complete Mini, Roche). Cell lysates were cleared by
centrifugation at 20,000 g for 10 minutes, and the
supernatants immunoprecipitated for 12 hours with anti-GFP beads (MBL) or
anti-Arm bound to 10 µl of protein G Sepharose 4B beads at 4°C, and
then subjected to SDS-PAGE and western blotting. For some western blotting
experiments, UAS-lacZ was included as an internal control for
transfection efficiency; this confirmed that the change in E-cadherin level
upon elevation or reduction of Src42A activity was robust (data not shown).
Surface biotinylation was performed with Sulfo-NHS-Biotin (Pierce), as
described by the manufacturer.
mRNA quantification
Poly A+ mRNA, isolated from 30 embryos using an mRNA purification kit
(Amersham Biosciences), was reverse transcribed with SuperScript II
(Invitrogen) reverse transcriptase using oligo-dT as a primer. mRNA was
quantified using a Prism 7000 Sequence Detection System (ABI) with SYBR Green
PCR Master mix (ABI). Data obtained from duplicate mRNA preparations were
standardized using rac1 mRNA as an internal control.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Piwnica-Worms et al.,
1987). pSrc was detected preferentially in AJs of epithelial
tissues (Fig. 1B-D), whereas
the anti-Src42A antibody detected signals outlining many types of cells
(Fig. 1D)
(Takahashi et al., 2005). Both
of these signals were greatly reduced in the deletion mutant
Src42A26-1 (Fig.
1E), and the low-level signal detected in this mutant may reflect
maternal Src42A. Overexpression of Src42A in the trachea greatly
increased the pSrc signal and caused a loss of epithelial integrity
(Fig. 1F). Overexpression of
Src64B caused a similar tracheal defect but did not significantly affect pSrc
staining (Fig. 1G), most likely
because the amino acid sequence in the vicinity of the Src64B
autophosphorylation site (Y416) differs from that of Src42A. We conclude that
the observed pSrc staining mainly represents the activation of Src42A in AJs,
although a minor contribution by other proteins cannot be ruled out. During tracheal development, pSrc expression became elevated in invaginating tracheal primordia (Fig. 1B) and its expression continued in tracheal branches undergoing cell rearrangement (Fig. 1D). pSrc staining was highly enriched in epithelial tissues undergoing various morphogenetic processes, including in the segmental furrow (Fig. 1D, arrowhead), the hindgut, the posterior spiracle and the salivary gland (data not shown). The parasegmental groove is the first morphological sign of segmentation and is formed by a transient inward shift of the apical cell surface and nucleus (Fig. 1H). pSrc was enriched in this groove (Fig. 1H) but was subsequently reduced when the groove reverted to a flat epithelia (data not shown). These observations demonstrated that Src activity is dynamically upregulated in the AJs of tissues undergoing morphogenesis.
Src is required for tracheal cell morphogenesis
To investigate the role of Src epithelial morphogenesis, we assessed
loss-of-function phenotypes of Src in the embryonic tracheal system
(Fig. 2; see also Movies 1-4 in
the supplementary material). Extensive cell rearrangement is involved in
partitioning the proper number of cells to each of the primary branches, and
later in converting multicellular tubules into unicellular ones, as revealed
by single lines of the AJ marker -catenin-GFP
(Fig. 2A)
(Ribeiro et al., 2004). The
dorsal branch (DB) 1 to 9 consists of a pair of cells at the tip (fusion cell
and terminal cell) and 2 to 7 stalk cells (4.3±0.03, n=44),
and is extended in concert with dorsal closure (DC) of the epidermis
(Fig. 2A)
(Kato et al., 2004). In
Src42Amyri mutant embryos, the DB lagged behind DC and
contained fewer stalk cells (2.6±0.02, n=46,
P<3x10-10; two-sided Student's t-test;
Fig. 2B). The dorsal trunk (DT)
became a zigzag shape, indicating that increased pulling forces applied by the
shortened DBs deformed the DT. To test whether the phenotype of
Src42Amyri reflects a trachea-autonomous function of
Src42A, we reduced Src42A expression by expressing a hairpin RNA
construct of Src42A (see Fig. S1 in the supplementary material for
its effect in the wing disc) and removing one zygotic copy of Src42A.
The mutants reproduced the Src42A loss-of-function phenotype in the
trachea. The number of stalk cells was reduced (3.6±0.1, n=9,
P<0.05) and the phenotypes of delayed DB extension and zigzagged
DT were observed (Fig. 2C).
Further reduction of Src function by the Src42Amyri;
Src64BP1 double mutation exacerbated the phenotype. Many DBs
were arrested in a multicellular state despite the progression of DC
(Fig. 2D). The phenotype in the
trachea was much more severe than the relatively minor defects observed in
dorsal closure (Takahashi et al.,
2005). Perhaps the prolific rearrangement of cells in the trachea
makes this tissue highly sensitive to changes in Src activity. The expression
of Spalt, which inhibits cell rearrangement
(Ribeiro et al., 2004),
remained limited to the DT (see Fig. S2 in the supplementary material),
suggesting that the transformation of branch-specific cell fate is unlikely to
account for the phenotype. In summary, the reduction of Src activity caused a
variety of phenotypes in the DBs, including the reduction of cell number,
delayed branch extension and delayed cell intercalation.
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-catenin-GFP,
which has been used as a marker for AJs
(Oda and Tsukita, 1999a;
Ribeiro et al., 2004).
Although E-cadherin is a core component of AJs, whether its production is rate
limiting in junctional remodeling in embryos has not been well clarified
because zygotic shg mutants undergo a substantial degree of
epithelial morphogenesis, including the formation of apparent unicellular
branches (Tepass et al., 1996;
Uemura et al., 1996). However,
in time-lapse images of shg mutants, we noted that AJ signals became
increasingly faint and discontinuous, and that cell attachment in the tracheal
branches was loosened and broken at several places
(Fig. 3C, compare with
Fig. 3B; see also Movie 5 in
the supplementary material). These phenotypes were more severe in the
unicellular DB than in the multicellular DT
(Fig. 3C). We next increased
the dosage of E-cadherin several fold by expressing the E-cadherin-GFP fusion
protein in tracheal cells, and then compared its effect on DB elongation with
that of control GFP-moesin-expressing embryos
(Fig. 3A,D; see also Movies 6,
7 in the supplementary material). E-cadherin-GFP delayed elongation of the DBs
(Fig. 3I) and cell
rearrangement, as evidenced by the fact that a multicellular tubule still
remained after 3 hours of recording (Fig.
3D, inset, arrowhead marking dual lines of the AJ; compare with
Fig. 3A), suggesting that
excess E-cadherin is inhibitory to the morphogenesis of DBs. Despite the
delay, tracheal development was eventually completed
(Oda and Tsukita, 1999b).
These results suggest that the expression of E-cadherin must be maintained at
a proper level to allow the smooth morphogenesis of unicellular DB.
We next studied the role of activated Src in E-cadherin-dependent cell
adhesion. Src42AACT caused disintegration of the tracheal
epithelium in a manner similar to that of Src42A or Src64B overexpression
(Fig. 3E; see also Movie 6 in
the supplementary material). Src42AACT-expressing trachea marked
with AJ markers (GFP-moesin, -catenin-GFP, E-cadherin-GFP;
Fig. 3E,F,H) had rounded cells
and discontinuous luminal cavities with deteriorating apicobasal polarity, and
an occasional detachment of the tip cells of the DBs
(Fig. 3F). Time-lapse
recordings demonstrated that tracheal cells expressing Src42AACT
failed to stabilize cell junctions and to extend branches, although they
retained filopodia activity (see Movies 6, 7 in the supplementary material).
The reduction of one of the zygotic copies of shg enhanced the
phenotype of Src42AACT: DTs were broken at many places, AJ
accumulation of -catenin-GFP was much reduced and cells were more
rounded (compare Fig. 3G with
3F). Furthermore, co-expression
of E-cadherin-GFP significantly restored the branch elongation defects that
were due to Src42AACT (see Fig.
3E,H,J). The tracheal cells maintained apicobasal polarity for at
least 2 hours longer and dorsal branches were extended
(Fig. 3H), indicating that a
high level of E-cadherin partially suppresses the effect of
Src42AACT. The results suggest that E-cadherin is a rate-limiting
component of Src42A-mediated cell junctional destabilization.
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-catenin-GFP recovered to 30.6% (±2.2,
n=6) of the original level, with the half time of recovery
(t) being 16.7±1.6 seconds
(Fig. 4D). These values were in
the same order as those obtained for E-cadherin-GFP [mobile fraction
(Mf)=34.7±9.5 %,
t=23.7±6.8 seconds] and in good agreement
with the values obtained from mammalian tissue culture cells
(Yamada et al., 2005). Because
a change of E-cadherin level altered tracheal branch migration
(Fig. 3), we chose
-catenin-GFP as a neutral marker of AJs for subsequent FRAP
analyses.
As shown in Fig. 4D, the
mild reduction of Src activity in the Src42Amiri mutant
did not significantly alter t and Mf.
However, upon strong reduction of Src activity by a Src42A; Src64B
double mutation, t was increased to 28.1 seconds
(P>0.05), while Mf was not significantly altered.
Furthermore, tracheal-specific RNAi knockdown of Src42A
(btl>Src42ARNAi) increased
t (22.0 seconds, P<0.05), suggesting
that cell-autonomous activity of Src42A contributes to the fast turnover of
AJs. By contrast, Src42AACT increased Mf to 56.2%
(P>0.005). Src42AACT also increased
t, probably to due to the increased Mf.
Unlike Src double mutants, btl>Src42ARNAi slightly
increased Mf. The difference between the two Src loss-of-function
conditions might be due to the persistent presence of tension applied by
wild-type tissues in btl>Src42ARNAi. Taken together,
the results demonstrate that Src is required for mobilization of the AJs.
Src downregulates E-cadherin and Armadillo
To study the regulation of E-cadherin and Arm by Src in a defined system,
we used Drosophila S2 cells. Expressed E-cadherin-GFP stabilized
endogenous Arm (Fig. 5A. The
amounts of both E-cadherin and Arm were reduced by Src42AACT, and
were elevated by a dominant-negative form of Src, Src42ADN
(Fig. 5A,B).
Src42AACT specifically reduced the amount of the slower mobility
form of Arm that was known to be hyperphosphorylated
(Fig. 5A, band labeled H). As
revealed by cell surface antibody staining and cell surface biotinylation
(Fig. 5B,C; see also Materials
and methods), Src42AACT was found to deplete E-cadherin from the
cell surface, and, consequently, resulted in a loss of cell adhesion
(Fig. 5B, data not shown). It
was previously shown that the E-cadherin/Arm complex includes Src42A
(Takahashi et al., 2005). We
now found that immunoprecipitated Arm includes Src42A
(Fig. 5A). Src42ADN
in whole-cell extract reacted with pSrc, possibly as a result of
transphosphorylation by endogenous Src proteins. However, Y400 of Src42A,
which associates with Arm, was under phosphorylated in Src42ADN
cells and hyperphosphorylated in Src42AACT cells
(Fig. 5A), suggesting that the
Y400 phosphorylation status of Src42A bound to Arm inversely correlates with
cell adhesion activity. In embryos lacking most of the zygotic Src activities
(Src42Amyri, Src64BP1 double-mutants), we
observed an 
70% increase of E-cadherin protein
(Fig. 5D). These observations
suggest that Src proteins normally downregulate E-cadherin.
Src upregulates E-cadherin transcription
The results so far demonstrated that Src is a negative regulator of
E-cadherin and AJs. However, the effect Src on the level of E-cadherin protein
was modest: by antibody staining only a small change in the level of
E-cadherin protein in tracheal cells was observed in Src mutants (data not
shown). To explore the additional control of E-cadherin by Src, we studied
Arm. In wild-type embryos, Arm is localized at apical cell-cell adhesion sites
in a pattern similar to that of E-cadherin
(Fig. 6A). In contrast to the
observation in S2 cells (Fig.
5A), Src42AACT increased the Arm signal in tracheal
cell cytoplasm (Fig. 6D).
Furthermore, Src42AACT greatly increased the expression of Escargot
(Esg), a known target of Wg/Arm signaling in the trachea
(Chihara and Hayashi, 2000;
Llimargas, 2000)
(Fig. 6B,E,L), whereas
Src42Amyri mutants reduced expression
(Fig. 6H). Thus, Src42A
stimulates Esg expression in the trachea. The transcription factor TCF acts
downstream of Arm (van de Wetering et al.,
1997). Its dominant-negative form, TCFN, suppressed the
increase in the number of Esg-expressing cells caused by Src42A
hyperactivation (Fig. 6J),
suggesting that Src activates Esg through the Arm-TCF pathway.
|
). shg-lacZ expression was greatly stimulated by the
hyperactivation of Wg signaling (Fig.
6M), and was reduced by TCFN
(Fig. 6I), further suggesting
that shg is regulated by the canonical Wg pathway. shg-lacZ
was also stimulated by Src42AACT in a TCF-dependent manner
(Fig. 6F,K). The positive
effect of Src42A on E-cadherin transcription was confirmed by the direct
measurement of E-cadherin mRNA in embryos ubiquitously expressing
Src42AACT or wild-type Src42A (Src42AGS,
Fig. 6N). Src42AACT
and Src42AGS increased E-cadherin mRNA by about threefold,
comparable to induction by Wg, whereas the level of control mRNA
(rac1) did not change significantly
(Fig. 6N). Additionally, Src42A
increased arm mRNA in these embryos
(Fig. 6N). These results
suggest that Src42A upregulates E-cadherin and Esg transcription via the
transcriptional activation and stabilization of Arm.
Antagonistic activities of the Src42A and Wnt pathway on tracheal cell adhesion
Our analyses identified two opposing effects of Src on AJs: the inhibition
of cell adhesion and the upregulation of E-cadherin by stimulating its
transcription by activating Arm and TCF. Although Src42AACT
strongly destabilized cell adhesion, it did not cause complete cell
dissociation (Fig. 3E,F,H). In
some cases, we observed Src42AACT-expressing tracheal cells that
were detached from one branch and re-attached to the neighboring branch,
suggesting that Src42AACT-expressing tracheal cells retain a
significant level of homophilic cell adhesion
(Fig. 7A; see also Movie 8 in
the supplementary material). We speculated that the elevated synthesis of
E-cadherin would have compensated for its inactivation upon hyperactivation of
Src42A, thereby preventing the complete diminution of cell adhesion.
Consistent with this idea, we found that the shg mutation enhanced
the effect of Src42AACT; tracheal cells were now completely
dissociated (Fig. 7E). To
specifically address the significance of Src-mediated activation of Arm, we
observed the phenotype of TCFN overexpression. TCFN blocked
tracheal branching, but did not disrupt epithelial integrity
(Fig. 7C), suggesting that the
downregulation of TCF-dependent expression of E-cadherin was not rate limiting
at the normal level of Src activity. Strikingly, Src42AACT and
TCFN co-expression synergistically caused tracheal cells to lose cell
polarity and dissociate (Fig.
7F; Movie 9 in the supplementary material). Antibody staining of
these embryos demonstrated that Src42AACT mildly reduced
E-cadherin. The additional presence of TCFN synergistically caused the
near complete removal of E-cadherin (see Fig. S3 in the supplementary
material). These results demonstrated that TCF-dependent transcription helps
to maintain epithelial integrity under conditions of high Src activity.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-catenin-GFP, and a loss of some TCF target gene expression. We thus
suggest that Src is a key coordinator of junctional remodeling in epithelia
undergoing rapid cell rearrangement. Wild-type tracheal cells undergoing rapid cell rearrangement sometimes dissociated from the tracheal system (Fig. 7B, arrowhead). Those cells stayed in the body cavity until engulfed by hemocytes. By contrast, tracheal cells expressing Src42AACT and dissociated from branches acquired the ability to re-associate with other tracheal cells (Fig. 7A). The enhancement of both de-adhesion and re-adhesion by Src42AACT mimics the behavior of tumor cells undergoing metastasis, suggesting that the dual function of Src42A on E-cadherin might provide a molecular clue for understanding tumor metastasis.
The role of Src in Arm-dependent transcription
A negative role of activated Src on E-cadherin-dependent cell adhesion has
been observed in cases of embryonic ectoderm undergoing dorsal closure
(Takahashi et al., 2005). We
noted that the cell dissociation effect of Src42AACT was much
stronger on tracheal epidermis than on dorsal ectoderm (S.H., unpublished). We
suggest that the vigorous cell rearrangement and high level of Src42A activity
in the tracheal epithelium lead to a rapid decline of E-cadherin protein, and
make the de novo synthesis of E-cadherin essential, as demonstrated by the
loss of AJs in shg mutant embryos (this study)
(Tanaka-Matakatsu et al.,
1996).
We have shown here that Src42A increased the accumulation of Arm and
promoted the TCF-dependent stimulation of Esg and E-cadherin expression,
demonstrating that Src42A plays a previously unrecognized role in stimulating
the transcriptional activity of Arm. The increase of arm mRNA due to
Src42A suggests that the activation of Arm involves a positive transcriptional
feedback mechanism. Furthermore, release of Arm from the AJs, and its
stabilization, may also contribute to the activation of Arm. The stimulation
of E-cadherin transcription by Src42A may be a direct consequence of Arm
activation, as stimulation of E-cadherin synthesis by Wg was observed in
cultured Drosophila cells (Wodarz
et al., 2006).
|
N (Fig. 7). We
suggest that the transcriptional stimulation of the E-cadherin gene by Arm
compensates for the elevated degradation rate of E-cadherin due to
Src42AACT.
Wg controls tracheal branching by activating Arm and the transcription of
Esg (Chihara and Hayashi, 2000;
Llimargas, 2000). As Esg
expression also requires Src42A in the trachea, this observation suggests that
the simultaneous activation of Wg and Src is required to increase the level of
Arm sufficiently for the transcriptional activation of Esg. By contrast, the
expression of spalt, another Wg target gene in the trachea, was not
altered by a loss or gain of Src activity. In addition, Src42A;
Src64B double mutant embryos did not show any segmentation defects that
were characteristic of Wg class segment polarity mutations. These results
suggest that the role of Src in Wg signaling is limited, perhaps because the
level of Arm accumulation caused by normal levels of Src is less than the
threshold of activation of most of Wg target genes.
The regulation of Src
Although Src is known to regulate cell-matrix adhesion through the integrin
pathway (Frame et al., 2002),
preferential localization of pSrc in AJs indicates that AJs are the major site
of Src function in Drosophila embryos. We have shown that pSrc is
enriched in epithelia having high morphogenetic activity or undergoing sharp
deformation. During tracheal development, the tip of the DB is tightly
associated with the epidermis, and its elongation is coupled to dorsal
closure, a unidirectional movement of the epidermis
(Kato et al., 2004). This
implies that branch elongation is driven at least in part by an externally
applied force that generates tension in the tracheal epithelium. It was
reported that the mechanical stretching of cultured cells activates Src
through an interaction with actin filaments
(Han et al., 2004;
Wang et al., 2005). We
speculate that mechanical deformation and tension applied to AJs through
morphogenetic movements in tracheal development are good candidates for
triggering Src activation.
The regulation of epithelial morphogenesis by Src
Our results suggest that E-cadherin is under dual control by Src
(Fig. 7G). Introducing a
repressor of E-cadherin to this system would change the balance toward cell
dissociation, as has been observed in Sna/Slug-induced EMT in vertebrate
development (Cano et al.,
2000). Thus, the effect of Src on the synthesis and degradation of
E-cadherin can be modulated depending on the cellular context, and this may
explain the various functions of Src in developmentally programmed EMT or in
metastasis during cancer progression. We suggest that the positive and
negative control of E-cadherin by Src during rapid cell rearrangements in the
tracheal epithelium represent an equilibrium between epithelial and transient
mesenchymal states. The widespread expression of pSrc in epithelial tissues
indicates that the equilibrium state of cell adhesion is common in
development, the advantage being to buffer the various mechanical stresses
arising during morphogenetic movement.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/7/1355/DC1
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ACKNOWLEDGMENTS |
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