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First published online 23 April 2008
doi: 10.1242/dev.017103
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1 Department of Biochemistry, Chang Gung University, Tao-Yuan, 333,
Taiwan.
2 Department of Anatomy, Chang Gung University, Tao-Yuan, 333, Taiwan.
* Author for correspondence (e-mail: pai{at}mail.cgu.edu.tw)
Accepted 26 March 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Gurken, Egfr, Cbl, Morphogen gradient, Endocytosis, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Tabata
and Takei, 2004; Zhu and
Scott, 2004). Cell surface molecules, such as morphogen receptors
and heparan sulfate proteoglycans (HSPG)
(Hacker et al., 2005), shape
morphogen gradients by influencing their spreading. To understand the
generation of positional information, it is important to assess how far a
morphogen travels and how the morphogen gradient is maintained.
Gurken, a transforming growth factor 
(TGF) homolog, is a
member of the Epidermal growth factor receptor (Egfr) ligand family. In
Drosophila, this family includes a soluble secreted protein, Vein
(Schnepp et al., 1996), and
three transmembrane proteins, Keren (Krn), Spitz (Spi) and Gurken (Grk)
(Neuman-Silberberg and Schupbach,
1993; Reich and Shilo,
2002; Schweitzer et al.,
1995). The activation of Spi requires a proteolytical process
involving Star and Rhomboid 1 (Rho). Star promotes the translocation of Spi
from the endoplasmic reticulum to the Golgi apparatus where Rho, an
intramembrane serine protease, cleaves Spi
(Lee et al., 2001;
Urban et al., 2001).
Similarly, the transmembrane domain of Gurken is essential for its activity in
the oocyte (Queenan et al.,
1999), and Star and brother of rhomboid
(brho; stet-FlyBase) are required for a cleavage process of
Gurken to produce the secreted soluble active form of Gurken
(Ghiglione et al., 2002).
Egfr signaling activated by Gurken during oogenesis is required for axes
determination in the future embryo (Roth,
2003). Gurken exhibits a striking asymmetrical localization at
both RNA and protein level during middle stages of Drosophila
oogenesis. The 5' and 3' untranslated regions, as well as part of
the coding region of gurken mRNA target its localization tightly to
the future dorsal anterior corner (Thio et
al., 2000; Van De Bor et al.,
2005) and by efficient sorting through the transitional
endoplasmic reticulum (tER)-Golgi units, Gurken protein is also directionally
transported to this dorsal anterior region
(Herpers and Rabouille, 2004).
The spatial restriction of Gurken activates the Egfr in a restricted number of
follicle cells, leading to the establishment of dorsal follicle cell fates
(Neuman-Silberberg and Schupbach,
1996). The further patterning of the dorsal egg shell structure is
carried out by positive and negative feedback pathways of Egfr signaling
(Freeman, 1998). Gurken
activates the Egfr pathway to a maximum in the dorsal midline region where Rho
expression is induced. Rho facilitates the activation of Spi that diffuses and
activates Egfr in a border dorsal region. The active form of Spi is
sequestered by the negative regulator Argos, which is expressed in the dorsal
midline (Golembo et al., 1996;
Klein et al., 2004). In
addition, the negative regulator Pointed is also induced by high Egfr
signaling at the dorsal midline (Deng and
Bownes, 1997). As a consequence, the domain of high levels of Egfr
signaling is split into two regions that promote dorsal appendage
formation.
Whether Gurken travels to the ventral side of the egg chamber to influence
the ventral follicle cell fate has never been completely resolved. Our
previous examination of the expression of the Egfr target gene pipe,
which is transcriptionally repressed by Egfr signaling, indicates that some
level of Egfr activity is suppressed by Cbl in ventral follicle cells
(Pai et al., 2000). In the
absence of Cbl, pipe is repressed on the ventral side and this
repression requires Gurken (Pai et al.,
2000). Other studies suggested that Gurken indirectly regulates
pipe expression through another diffusible morphogen induced by Notch
signaling, which would be activated by interaction between mirror
(mirr) and fringe (fng)
(Jordan et al., 2000).
However, further analyses of mirr and fng mutant follicle
cell clones indicated that these two genes have no effects on pipe
expression. Moreover, the role of Spi, which is activated by rho, in
regulating pipe expression has also been ruled out by examination of
large rho mutant clones (Peri et
al., 2002). These data suggest that the Pipe domain is directly
defined by a long-range Gurken gradient. Based on indirect estimation through
measuring the repression of pipe and mathematical modeling, the
Gurken protein level at the ventral side of the egg chamber was estimated to
be 10% of that at the dorsal side
(Goentoro et al., 2006).
However, Gurken was never directly detected at the ventral side of wild-type
egg chambers, possibly owing to low levels and rapid turnover. We reasoned
that if Gurken directly activates the Egfr in ventral follicle cells then,
using better imaging approaches, Gurken should be detected in these follicle
cells in which Cbl is required for Egfr signal attenuation.
In mammals, the Egfr is dimerized and autophosphorylated upon ligand
activation, followed by recruitment of various proteins recognizing
phospho-tyrosine to transduce or terminate Egfr signaling
(Schlessinger, 2002). Cbl
family proteins interact with the C-termini of activated Egfr molecules either
indirectly through the adaptor protein Grb2 or directly through the tyrosine
kinase binding (TKB) domain of Cbl
(Schmidt and Dikic, 2005). The
E3 ligase activity of Cbl promotes the ubiquitylation of activated Egfr while
they are associated, and the Egfr-Cbl complex is then internalized by multiple
routes (Schmidt and Dikic,
2005). The ubiquitylated receptor binds to Epsin family proteins,
and goes through a non-clathrin-dependent, lipid-raft-dependent route
(Sigismund et al., 2005). In
addition, Cbl serves as an adaptor for interaction with the CIN85/endophilin
complex, leading to dynamin-dependent clathrin-mediated receptor endocytosis
(Dikic, 2003;
Petrelli et al., 2002;
Soubeyran et al., 2002).
Previously, we have demonstrated that both Cbl isoforms (CblS and CblL)
downregulate Egfr signaling, and that the efficiency of Egfr signaling
attenuation is controlled by the levels of the CblL isoform
(Pai et al., 2006). The
genetic interaction with shibire, a homologue of dynamin
(Chen et al., 1991;
van der Bliek and Meyerowitz,
1991), further suggested that CblL may promote the internalization
of activated Egfr to reduce signaling (Pai
et al., 2006).
Here, we report the use of a HRP (horseradish peroxidase)-Grk fusion protein that allows us to directly trace the fate of Gurken protein in follicle cells during signaling. We found that the HRP-Grk fusion protein is internalized with the Egfr into follicle cells, a process that is mediated by Shibire. The Grk-Egfr complex trafficks through Rab5/7-associated endocytic pathways to the MVBs (multivesicular bodies) and the lysosome for signal termination. Gurken was visualized not only in the dorsal but also in ventral follicle cells. We document that Cbl facilitates the internalization of the Grk-Egfr complex into follicle cells using clonal analysis of loss of function mutations, as well as over-expression studies. Our results indicate that the distribution of the Gurken morphogen is controlled by the endocytosis of the Grk-Egfr complex through Cbl.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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); topIP02 and topCO
(Clifford and Schupbach, 1994);
UAS-shiTS1 (Kitamoto,
2001); UAS-GFP-Rab5
(Wucherpfennig et al., 2003);
UAS-GFP-Rab7 (Entchev et al.,
2000); UAS-nls-GFP
(Shiga et al., 1996);
EQ1 and GR1 Gal4 lines
(Queenan et al., 1997);
CblF165 (Pai et al.,
2000); UAS CblS-A1; UAS CblL-A18; and
A12 (Pai et al.,
2006). e22c-Gal4
(Duffy et al., 1998) or
GR1-Gal4 was used to drive expression of UAS-Flipase in follicle
cells.
DNA constructs
To generate HRP-grk, DNA encoding HRP
(Connolly et al., 1994) was
inserted into the gurken genomic fragment, which is a 5 kb
EcoRV fragment derived from the original rescuing fragment
(Neuman-Silberberg and Schupbach,
1993) as shown in Fig.
2 of the publication by Thio et al.
(Thio et al., 2000). This 5 kb
fragment was subcloned into pBSKS as two separate plasmids, A5 (5'
EcoRV site at the beginning to EcoRV site in the third
intron) and A6 (EcoRV site in the third intron to the Eag
site at the end). The Grk fragment in A5 was first cloned into pcDNA using the
EcoRV site, and then DNA encoding HRP was inserted into pcDNA grk
A5 using standard PCR-based cloning at the SacII site. This
HRP-Grk A5 fragment was cloned into A6 at the EcoRV site.
DNA encoding the HRP-Grk fusion protein was then cloned into pCaSpeR4 with the
ApaI-EagI fragment and the construct was introduced in
w1118 hosts by P element-mediated transformation using
standard methods.
Antibodies
The antibodies used for immunocytochemistry are the monoclonal anti-Gurken
antibody 1D12 made in mouse (Queenan et
al., 1999); the goat anti-HRP antibody (Jackson ImmunoResearch,
1:250 dilution); the anti-CblL 8C4 ascites at 1:100 dilution
(Pai et al., 2006); the
anti-c-myc antibody 9E10 (Oncogene, at 1:50 dilution); a rabbit anti-Myc
antibody (Santa Cruz Biotechnology); the rabbit anti-Egfr antibody, which was
prepared according to the previously reported method
(Jekely and Rorth, 2003) and
used at 1:50; the Alexa Fluor 488 or 546 goat anti-mouse antibody (Molecular
Probes); the Alexa Fluor 488 or 546 rabbit anti-goat antibody (Molecular
Probes); and the TRITC-conjugated donkey anti-mouse antibody (Jackson
ImmunoResearch). The antibodies used for immunoelectron microscopy were goat
anti-HRP (Sigma; 1:1000 dilution) and rabbit anti-goat IgG (DakoCytomation
Denmark, Glostrup, Denmark). The antibodies were detected with protein A-gold
conjugated with 10 nm gold (Cell Microscopy Center, Utrecht, The
Netherlands).
Immunofluorescent histological staining and in situ hybridization
To induce Myc expression, e22cFLP/grkHFHRP-grk;
CblF165FRT80B/ 
MFRT80B
adult females were subjected to heat shock for 1 hour at 37°C before
dissection (Xu and Rubin,
1993). Ovaries were dissected in PBS and fixed for 20 minutes in
200 µl 4% paraformaldehyde (PF) saturated with 600 µl heptane and 0.25%
NP40. Staining procedures were performed as described
(Neuman-Silberberg and Schupbach,
1996) with minor modifications. To improve the penetration,
ovaries were incubated with 1% TritonX-100 in PBST with 1% BSA before
blocking, in which 10% normal rabbit serum or 1% BSA was used to reduce the
background. To visualize actin, ovaries were incubated with 1 unit of
phalloidin (Molecular Probes) for 30 minutes. For staining DNA, 0.5 µg/ml
DAPI (Sigma) was added to the ovaries for 5 minutes. Egg chambers were further
dissected and mounted in glycerol for examination under fluorescence confocal
microscopy. The RNA hybridization procedure was carried out as described
previously (Tautz and Pfeifle,
1989). A digoxigenin (Dig)-labeled RNA probe was made using the
DIG RNA labeling Kit (Roche). The probe was detected with alkaline phosphatase
conjugated anti-DIG antibody (1:5000 dilution, Roche) and the NBP/BCIP
developing kit (Promega).
Immunoelectron microscopy and quantification
Ovaries were dissected and fixed in a mixture of 0.2% glutaraldehyde and 2%
paraformaldehyde in 0.1 M phosphate buffer for 3-4 hours. The individual egg
chambers were embedded in 12% gelatin. The blocks were trimmed to size,
immersed in PVP/sucrose (15%/1.7M) at 4°C overnight, mounted on aluminum
pins in the proper orientation and stored in liquid nitrogen. Frozen sections
were prepared by using an ultracryomicrotome (Reichert Ultracut S/Reichert
FCS, Leica; Vienna, Austria). To obtain ultrathin sections that included the
oocyte nucleus, 0.5 µm semithin sections were first cut, at-60°C, and
examined with a light microscope after staining with 0.1% Toluidine Blue in 1%
boric acid. When the nucleus was reached, the cutting temperature was lowered
to-100°C and section thickness switched to 55 nm. Ultrathin sections were
picked up with a drop of an equal volume mixture of methylcellulose
(2%)/sucrose (2.3 M). The ultracryomicrotomy and protocol used for
immunolabeling has been described in detail
(Liou et al., 1996). Samples
were viewed under a JOEL JEM-1230 transmission electron microscope. To compare
the endocytic activity of follicle cells at different zones, the volume
density of MVB/lysosomes per follicle cell profile was estimated by the
point-counting method (Griffiths,
1993). Only those cell profiles exhibiting MVB/lysosomes were
graphed. Sampling was from 12 different grids (nine OreR and three
grkHF/grkHF) from seven egg chambers (five
OreR and two grkHF/grkHF). Cell
profiles analyzed (pn) are indicated in the figure. Total areas quantified
were 3227.23 µm2 for OreR and 693.39 µm2
for grkHF/grkHF.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Queenan et al., 1999). These
punctate signals in the posterior follicle cells are very likely to be the
Grk-Egfr complex in endocytic vesicles after the complex is internalized.
To test this possibility, we examined the effect of a dominant-negative
form of Shibire [UAS-shits
(Kitamoto, 2001)] on the
distribution of Gurken. Under a non-permissive temperature, these punctate
signals in follicle cells were dramatically reduced
(Fig. 1C,D), suggesting that
Gurken accumulates in posterior follicle cells through a Dynamin-dependent
process. The localization of Gurken in endocytic vesicles was further
confirmed by the colocalization with a GR1-Gal4 driven expression of
the early endosomal marker (GFP-Rab5)
(Wucherpfennig et al., 2003)
and a late endosomal marker (GFP-Rab7)
(Entchev et al., 2000),
respectively (Fig.
1E-H'). These data indicate that Gurken is internalized into
posterior follicle cells through the Shibire and Rab5/7-associated endocytic
pathway at the stage when the posterior follicle cell fate is defined.
After stage 8, the oocyte nucleus moves to the anterior of the egg chamber, and Gurken is localized around the oocyte nucleus. During this stage, the Gurken signal in follicle cells was only detected in 17% (n=24) of egg chambers, and the expression was restricted to a few follicle cells adjacent to the nucleus of oocyte (Fig. 1I-L). The punctate signals within individual follicle cells possibly correspond to the endocytic compartments, similar to that which was observed in the posterior follicle cells.
An active HRP-Grk fusion protein
Previous studies have successfully used HRP-Boss
(Sunio et al., 1999) and
HRP-Wingless fusion proteins (Dubois et
al., 2001) to study ligand distribution in endocytic trafficking.
To visualize the endocytic trafficking of Gurken during stages 8 to 10, we
made an HRP-Grk fusion to use as a tracer because HRP is relatively stable
within the destructive environment of the endosome and lysosomal compartments.
As 5' and 3' UTR, as well as part of the coding region of
gurken, are important for the correct localization of gurken
RNA in Drosophila oogenesis (Thio
et al., 2000; Van De Bor et
al., 2005), HRP was inserted into exon 3 of the gurken
genomic sequence, between the signal peptide and the EGF repeat
(Fig. 2A). We expected that
this fusion protein, as driven by the endogenous gurken promoter,
could mimic the endogenous expression and trafficking of the Gurken protein,
and would still be detected after processing through endocytosis.
|
|
;
Pai et al., 2000;
Peri et al., 2002), which
indicated that some level of Egfr activity in ventral follicle cells is
activated by Gurken. To test whether the internalization of HRP-Grk in dorsal and ventral follicle cells is Dynamin dependent, HRP signal was examined in the temperature-sensitive and dominant-negative shibire mutant UAS-shits. The punctate HRP signals were dramatically reduced both in dorsal and ventral follicle cells during stage eight to nine at non-permissive temperature (Fig. 2J-M), suggesting that Dynamin has a crucial role in the internalization process of Gurken. To follow the trafficking route of HRP-Grk, we marked early endosomes with UAS-GFP-Rab5 driven by GR1-Gal4, expressed in all follicle cells, starting from the germarium. Some HRP-Grk signals were colocalized with Rab5 both in dorsal and ventral follicle cells (59%, n=44, Fig. 2N-Q). Similarly, colocalization of HRP-Grk and GFP-Rab7 (36%, n=33), a late endosomal marker, was also detected (data not shown).
We then asked whether the internalization of Gurken is dependent on its
interaction with Egfr. We generated an anti-Egfr antibody to monitor
colocalization of HRP-Grk and Egfr. The specificity of this antibody was
tested in mosaic egg chambers with Egfr mutant clones
(Fig. 3A-C). Indeed, HRP-Grk
and Egfr were colocalized (62%, n=39) in dorsal
(Fig. 3D-F) and ventral (data
not shown) follicle cells as revealed by double staining with anti-HRP and
anti-Egfr antibodies. We then generated Egfr mutant clones in the follicular
epithelium to test whether HRP signal in follicle cells is dependent on the
presence of Egfr, and hence corresponds to the HRP-Grk-Egfr complex
internalized into signal receiving cells. Two torpedo
(top)/Egfr null mutant alleles, Egfr[CO] and Egfr[IP02],
were tested. The molecular information of Egfr[IP02] suggests that this
mutated Egfr has no cytoplasmic domain for signaling
(Clifford and Schupbach, 1994).
In the Egfr mutant follicle cells, marked by the absence of GFP signal, the
HRP signal was significantly reduced or entirely absent
(Fig. 3G-I). The fact that the
HRP signal in follicle cells is Egfr-dependent suggests that Gurken binding
triggers the entry of the ligand-receptor complex into follicle cells. We also
found that, once inside the cells, Egfr localizes to Rab5 positive vesicles
near the apical region and to Rab7-positive vesicles in the region between the
apical membrane and the nucleus, respectively
(Fig. 3J-O). Given that
internalized Gurken in both dorsal and ventral follicle cells depends on a
functional Egfr, these data indicate that Egfr is activated by Gurken not only
in dorsal but also in ventral follicle cells. The results also show that the
ligand-receptor complex is mainly internalized through the Dynamin- and
Rab5/7-associated endocytic pathway at the stage when Gurken defines the
dorsoventral cell fate.
The Grk-Egfr complex is degraded in the lysosomal compartment
To visualize the subcellular localization of HRP-Grk, we applied
immuno-electron microscopy using an anti-HRP antibody. Herpers and Rabouille
(Herpers and Rabouille, 2004)
studied the exocytic pathway of Gurken in stage 9 Drosophila oocytes
by immunoelectron microscopy using the Gurken antibody, and demonstrated that
the Gurken protein is directionally transported through the tER-Golgi units at
the dorsal-anterior corner of the oocyte. We first tested whether HRP-Grk
trafficks through the same exocytic pathway. Consistent with their
observation, we found that HRP-Grk positive tER-Golgi units were significantly
concentrated in the D/A corner of the ooplasm (7.7
16.9 gold/units),
whereas the labeling density in tER-Golgi units located in the ventral or
posterior of the ooplasm was much weaker (0.30.8 gold/units)
(Fig. 4C-E and data not show).
Gurken protein gradually reached the highest peak at middle stage 9 in the
tER-Golgi units at the dorsal anterior corner, and declined at late stage 9
(Fig. 4E). This expression
pattern corresponds well with the pattern detected by immunostaining with the
Gurken antibody, in which no Gurken signal can be detected after stage 10B
(Neuman-Silberberg and Schupbach,
1996). We also checked the labeling in the space between the
oocyte and the overlying follicle cells to examine the extracellular gradients
of Gurken. However, a strong labeling even in wild-type egg chambers not
expressing HRP-Grk was detected, which might result from trapped gold
particles within this region (data not shown).
We then examined the fate of the Grk-Egfr complex in the follicle cells at
stage 9. Gold particles in the follicle cells corresponding to Gurken in our
cryosectioned fixed stage 9 egg chambers showed a distinct pattern. Except for
some nonspecific binding to mitochondria, major gold particles were located in
unidentified small vesicles, the MVB, and the lysosome in both dorsal and
ventral follicle cells (Fig.
4F,G). We previously demonstrated that the internalization of the
Grk-Egfr complex plays a major role in terminating Egfr signaling, as
dorsalized egg shell phenotypes resulting from elevated Egfr signaling were
observed when shibire activity was blocked
(Pai et al., 2006). Taken
together, our data indicate that the internalized Gurken travels through the
early and late endosomes, and is targeted to degradation in the MVB/lysosome
compartment. Consistent with our immunostaining result, this signaling
attenuation process occurs both in the dorsal and ventral follicle cells,
again indicating that Gurken has reached the ventral side of the egg chamber
to activate the Egfr at ventral follicle cell plasma membrane. Interestingly,
we noted that after early stage 9, the lateral follicle cells contained a
larger lysosomal area than did the dorsal/ventral follicle cells
(Fig. 4H-J). This difference
was not observed in a gurken mutant background, indicating an early
response to Egfr signaling in these cells
(Fig. 4K).
|
). To further
investigate the precise mechanisms by which Cbl regulates Egfr signaling, we
examined the pattern of Gurken distribution in egg chambers with
overexpression of CblL driven by GR1-Gal4. When analyzing Gurken by
immunofluorescence, it is not possible to distinguish between Gurken protein
that is still at the oocyte membrane versus Gurken protein that is
extracellular or bound to the follicle cell surface. In egg chambers with
over-expressed CblL, at this level of resolution, we found that Gurken was
markedly reduced in late stage 9 egg chambers when Gurken should have reached
its highest expression peak. This reduction was seen in more than 40%
(n=190) of egg chambers with CblL overexpression
(Fig. 5C,D). Some variation of
Gurken immunostaining was seen in about 10% of control wild-type egg chambers
(n=96) (Fig. 5D). The
reduction occurred only at the protein level, while the mRNA was not changed,
as indicated by an in situ hybridization assay (data not shown). By contrast,
overexpression of CblS had much more subtle effects
(Fig. 5B,D). Furthermore, in
contrast to the reduction in the intercellular and membrane region, the
internal punctate HRP signals were dramatically increased in follicle cells
with medium overexpression of CblL, but were not detectable in cells with high
expression levels of CblL (Fig.
5E-H). EQ1 and GR1-Gal4 driven expression in the follicle
cells is somewhat patchy, resulting in follicle cells with both higher and
somewhat lower expression levels, allowing a direct comparison of the effects
of higher versus intermediate levels of CblL (see Fig. S2 in the supplementary
material). This suggests that in cells with high levels of CblL the
receptor-ligand complexes may be undergoing very rapid trafficking and
degradation. In the CblL overexpressing follicle cells, colocalization of
HRP-Grk punctate signals with Rab5 (41%, n=27,
Fig. 5I), Rab7 (67%,
n=21, Fig. 5J), and
Egfr (51%, n=41, Fig.
5K) were detected. Taken together, these results suggest that more
Grk-Egfr complexes are in the follicle cells with the help of CblL than that
in wild-type cells without CblL overexpression. This leads to a reduction of
Gurken outside the follicle cells and an increase of Gurken inside the
follicle cells. As a consequence, the promotion of Grk-Egfr endocytosis by
CblL can change the overall distribution of the Gurken morphogen. In summary,
Cbl negatively regulates Egfr signaling by facilitating the internalization
and degradation of Grk-Egfr complex and thus helps to shape the pattern of
Gurken protein expression.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
However, a direct examination of the Gurken gradient has never been possible,
as it has been very difficult to distinguish the distribution of Gurken in the
oocyte, in the extracellular space between the oocyte and the follicle cells
or on the follicle cell membrane with the resolution of confocal microscopy in
immunostaining assays. We tried to examine Gurken through TEM, but nonspecific
binding of the anti-Gurken antibody in the space between the oocyte and the
follicle cell made this impossible. Nevertheless, it is important to know how
far Gurken can travel in the egg chamber to evaluate whether all ranges of
Egfr activation are determined by one ligand, Gurken, or whether they might
also be affected by other signals. We therefore took an alternate approach and
examined the ligand-receptor complex in the signal-receiving cells.
|
), and
Egfr signaling in the late endosome has been shown to mediate through a
p14/MP1 scaffold (Teis et al.,
2002). However, in Drosophila follicle cells, the
function of internalization of the Grk-Egfr complex is mainly to terminate
signaling, as blocking endocytosis by interfering with Dynamin function
results in an excess Egfr signaling phenotype
(Pai et al., 2006). In other
words, the Egfr signaling mainly occurs on the cytoplasmic membrane in
follicle cells.
Our data showed that a significant amount of HRP-Grk was detected in
ventral follicle cells by immunostaining and immunoelectron microscopy using
an anti-HRP antibody. Direct secretion of Gurken from the ventral surface of
the oocyte to the ventral follicle cells is very unlikely, given results from
tER studies carried out by both Rabouille and colleagues, as well as by us.
Furthermore, the autonomous effect of Cbl clones on the vesicular
signal of HRP-Grk in dorsal and ventral follicle cells suggest that the major
spreading of Gurken in the egg chamber was not achieved through transcytosis
(Fig. 6C-H). Our results are
much more compatible with a mechanism where Gurken diffuses as a free secreted
form or in a complex with some extracellular molecule such as HSPG
(Belenkaya et al., 2004;
Hacker et al., 2005), and
distributes to the dorsal and ventral side in the space between the oocyte and
follicle cells. This interpretation is also consistent with a quantification
model of the Gurken morphogen in which 10% of Gurken was predicted to appear
on the ventral side of egg chambers compared with dorsal levels
(Goentoro et al., 2006). Taken
together, our data demonstrate that Gurken acts as a long-range morphogen,
which directly determines the fates of ventral follicle cells.
|
;
Teleman et al., 2001).
Goentoro and colleagues have shown that overexpression of Egfr in the follicle
cells traps Gurken near the dorsal anterior corner and prevents its diffusion,
leading to an expansion of Pipe expression
(Goentoro et al., 2006). This
finding suggests that in the wild-type situation, most Egfr is bound with
Gurken in dorsal follicle cells, and excess free Gurken diffuses to the
lateral/ventral side of egg chambers to form the gradient.
Here, we report that endocytosis also plays a crucial role in shaping the
gradient of the Gurken protein, which is similar to observations for Wingless
and Fgf8 (Dubois et al., 2001;
Scholpp and Brand, 2004).
Dubois and colleagues had shown that endocytosis regulates the asymmetry of
the Wingless gradient using HRP-Wingless to monitor Wingless degradation. It
has been well established that Cbl acts as an endocytic adaptor to mediate the
internalization of Egfr in mammals (Rubin
et al., 2005; Schmidt and
Dikic, 2005). Our previous data showed that overexpression of CblL
resulted in the reduction of Egfr signaling in eggshell patterning and
repression of its target gene, argos
(Pai et al., 2006). We
demonstrate here that Cbl facilitates the endocytosis of the Grk-Egfr complex
into follicle cells based on our clonal analysis of Cbl mutant
follicle cells (Fig. 6C-H).
Very strikingly, neighboring wild-type cells contained more punctate HRP-Grk
signals (Fig. 6E,F), indicating
that more Gurken was available to these wild-type cells. This result suggests
that larger quantities of free Gurken can travel towards the posterior when
the Grk-Egfr complex cannot efficiently enter the dorsal anterior cells in the
absence of Cbl. However, this subtle change in Gurken distribution
cannot be revealed through examining the overall Gurken gradient by
immunostaining (Fig. 6A).
Conversely, more Grk-Egfr complexes were removed from the membrane to the
Rab5/7-associated endocytic pathway in follicle cells with over-expressed CblL
when compared with those in wild-type cells, and under these conditions,
Gurken diffusion was limited (Fig.
5C,H-K). This observation indicates that CblL serves as a
rate-determining component of the endocytic pathway. Increased levels of CblL
effectively promote the endocytosis of Egfr. Furthermore, the significant
reduction of Gurken protein in egg chambers with CblL overexpression at stage
9 suggests that internalization of the Grk-Egfr complex may lead to a rapid
recycling of Egfr on the cytoplasmic membrane of follicle cells and thus allow
more binding to Gurken. In conclusion, we demonstrate here that increasing the
rate of endocytosis by overexpression of CblL can modify the Gurken
distribution.
Interestingly, we noticed that the area of the lysosome and MVB vesicle in
the lateral follicle cells was bigger than that in the dorsal or ventral
follicle cells in wild-type egg chambers, suggesting that the endocytic
activity was higher in the lateral follicle cells
(Fig. 5H-J). This difference
was observed after early stage 9 and was Gurken dependent. The high endocytic
activity in lateral follicle cells could set up a sharp boundary for the
Gurken morphogen between the dorsal and ventral side of the egg chamber. This
hypothesis is consistent with the quantification model
(Goentoro et al., 2006), which
predicted a steep reduction for the Gurken morphogen, from 63% to 22% at the
lateral region. Thus, in response to early Gurken/Egfr activity, during stage
9 and later, once free Gurken binds to the Egfr in lateral follicle cells, the
ligand-receptor complex may be internalized quickly as these lateral follicle
cells may have a very active endocytic machinery. Because of a high activity
of a degradation mechanism in the lateral follicle cells, only very low levels
of Gurken would then be able to travel to the ventral region, leading to a low
level of Egfr signaling in the ventral follicle cells, which would ensure the
robustness of the Gurken gradient.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/11/1923/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
|---|
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Belenkaya, T. Y., Han, C., Yan, D., Opoka, R. J., Khodoun, M., Liu, H. and Lin, X. (2004). Drosophila Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans. Cell 119,231 -244.[CrossRef][Medline]
Chen, M. S., Obar, R. A., Schroeder, C. C., Austin, T. W., Poodry, C. A., Wadsworth, S. C. and Vallee, R. B. (1991). Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature 351,583 -586.[CrossRef][Medline]
Clifford, R. and Schupbach, T. (1994). Molecular analysis of the Drosophila EGF receptor homolog reveals that several genetically defined classes of alleles cluster in subdomains of the receptor protein. Genetics 137,531 -550.[Abstract]
Connolly, C. N., Futter, C. E., Gibson, A., Hopkins, C. R. and
Cutler, D. F. (1994). Transport into and out of the Golgi
complex studied by transfecting cells with cDNAs encoding horseradish
peroxidase. J. Cell Biol.
127,641
-652.
Deng, W. M. and Bownes, M. (1997). Two signalling pathways specify localised expression of the Broad-Complex in Drosophila eggshell patterning and morphogenesis. Development 124,4639 -4647.[Abstract]
Dikic, I. (2003). Mechanisms controlling EGF receptor endocytosis and degradation. Biochem. Soc. Trans. 31,1178 -1181.[Medline]
Dubois, L., Lecourtois, M., Alexandre, C., Hirst, E. and Vincent, J. P. (2001). Regulated endocytic routing modulates wingless signaling in Drosophila embryos. Cell 105,613 -624.[CrossRef][Medline]
Duffy, J. B., Harrison, D. A. and Perrimon, N. (1998). Identifying loci required for follicular patterning using directed mosaics. Development 125,2263 -2271.[Abstract]
Entchev, E. V. and Gonzalez-Gaitan, M. A. (2002). Morphogen gradient formation and vesicular trafficking. Traffic 3,98 -109.[CrossRef][Medline]
Entchev, E. V., Schwabedissen, A. and Gonzalez-Gaitan, M. (2000). Gradient formation of the TGF-beta homolog Dpp. Cell 103,981 -991.[CrossRef][Medline]
Freeman, M. (1998). Complexity of EGF receptor signalling revealed in Drosophila. Curr. Opin. Genet. Dev. 8,407 -411.[CrossRef][Medline]
Ghiglione, C., Bach, E. A., Paraiso, Y., Carraway, K. L., 3rd,
Noselli, S. and Perrimon, N. (2002). Mechanism of activation
of the Drosophila EGF Receptor by the TGFalpha ligand Gurken during oogenesis.
Development 129,175
-186.
Goentoro, L. A., Reeves, G. T., Kowal, C. P., Martinelli, L., Schupbach, T. and Shvartsman, S. Y. (2006). Quantifying the Gurken morphogen gradient in Drosophila oogenesis. Dev. Cell 11,263 -272.[CrossRef][Medline]
Golembo, M., Raz, E. and Shilo, B. Z. (1996). The Drosophila embryonic midline is the site of Spitz processing, and induces activation of the EGF receptor in the ventral ectoderm. Development 122,3363 -3370.[Abstract]
Griffiths, G. (1993). Fine structure Immunocytochemistry. Berlin, Heidelberg: Springer-Verlag.
Hacker, U., Nybakken, K. and Perrimon, N. (2005). Heparan sulphate proteoglycans: the sweet side of development. Nat. Rev. Mol. Cell Biol. 6, 530-541.[CrossRef][Medline]
Herpers, B. and Rabouille, C. (2004). mRNA
localization and ER-based protein sorting mechanisms dictate the use of
transitional endoplasmic reticulum-golgi units involved in gurken transport in
Drosophila oocytes. Mol. Biol. Cell
15,5306
-5317.
Hoeller, D., Volarevic, S. and Dikic, I. (2005). Compartmentalization of growth factor receptor signalling. Curr. Opin. Cell Biol. 17,107 -111.[CrossRef][Medline]
Jekely, G. and Rorth, P. (2003). Hrs mediates downregulation of multiple signalling receptors in Drosophila. EMBO Rep. 4,1163 -1168.[CrossRef][Medline]
Jordan, K. C., Clegg, N. J., Blasi, J. A., Morimoto, A. M., Sen, J., Stein, D., McNeill, H., Deng, W. M., Tworoger, M. and Ruohola-Baker, H. (2000). The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation. Nat. Genet. 24,429 -433.[CrossRef][Medline]
Kitamoto, T. (2001). Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons. J. Neurobiol. 47, 81-92.[CrossRef][Medline]
Klein, D. E., Nappi, V. M., Reeves, G. T., Shvartsman, S. Y. and Lemmon, M. A. (2004). Argos inhibits epidermal growth factor receptor signalling by ligand sequestration. Nature 430,1040 -1044.[CrossRef][Medline]
Lecuit, T. and Cohen, S. M. (1998). Dpp receptor levels contribute to shaping the Dpp morphogen gradient in the Drosophila wing imaginal disc. Development 125,4901 -4907.[Abstract]
Lee, J. R., Urban, S., Garvey, C. F. and Freeman, M. (2001). Regulated intracellular ligand transport and proteolysis control EGF signal activation in Drosophila. Cell 107,161 -171.[CrossRef][Medline]
Liou, W., Geuze, H. J. and Slot, J. W. (1996). Improving structural integrity of cryosections for immunogold labeling. Histochem. Cell Biol. 106, 41-58.[CrossRef][Medline]
Neuman-Silberberg, F. S. and Schupbach, T. (1993). The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGF alpha-like protein. Cell 75,165 -174.[CrossRef][Medline]
Neuman-Silberberg, F. S. and Schupbach, T. (1996). The Drosophila TGF-alpha-like protein Gurken: expression and cellular localization during Drosophila oogenesis. Mech. Dev. 59,105 -113.[CrossRef][Medline]
Pai, L. M., Barcelo, G. and Schupbach, T. (2000). D-cbl, a negative regulator of the Egfr pathway, is required for dorsoventral patterning in Drosophila oogenesis. Cell 103,51 -61.[CrossRef][Medline]
Pai, L. M., Wang, P. Y., Chen, S. R., Barcelo, G., Chang, W. L., Nilson, L. and Schupbach, T. (2006). Differential effects of Cbl isoforms on Egfr signaling in Drosophila. Mech. Dev. 123,450 -462.[CrossRef][Medline]
Peri, F., Technau, M. and Roth, S. (2002). Mechanisms of Gurken-dependent pipe regulation and the robustness of dorsoventral patterning in Drosophila. Development 129,2965 -2975.[Medline]
Petrelli, A., Gilestro, G. F., Lanzardo, S., Comoglio, P. M., Migone, N. and Giordano, S. (2002). The endophilin-CIN85-Cbl complex mediates ligand-dependent downregulation of c-Met. Nature 416,187 -190.[CrossRef][Medline]
Queenan, A. M., Ghabrial, A. and Schupbach, T. (1997). Ectopic activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase, dorsalizes both the eggshell and the embryo. Development 124,3871 -3880.[Abstract]
Queenan, A. M., Barcelo, G., Van Buskirk, C. and Schupbach, T. (1999). The transmembrane region of Gurken is not required for biological activity, but is necessary for transport to the oocyte membrane in Drosophila. Mech. Dev. 89, 35-42.[CrossRef][Medline]
Reich, A. and Shilo, B. Z. (2002). Keren, a new ligand of the Drosophila epidermal growth factor receptor, undergoes two modes of cleavage. EMBO J. 21,4287 -4296.[CrossRef][Medline]
Roth, S. (2003). The origin of dorsoventral
polarity in Drosophila. Philos. Trans. R. Soc. Lond. B Biol.
Sci. 358,1317
-1329; discussion 1329.
Rubin, C., Gur, G. and Yarden, Y. (2005). Negative regulation of receptor tyrosine kinases: unexpected links to c-Cbl and receptor ubiquitylation. Cell Res. 15, 66-71.[CrossRef][Medline]
Schlessinger, J. (2002). Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110,669 -672.[CrossRef][Medline]
Schmidt, M. H. and Dikic, I. (2005). The Cbl interactome and its functions. Nat. Rev. Mol. Cell Biol. 6,907 -919.[CrossRef][Medline]
Schnepp, B., Grumbling, G., Donaldson, T. and Simcox, A.
(1996). Vein is a novel component in the Drosophila epidermal
growth factor receptor pathway with similarity to the neuregulins.
Genes Dev. 10,2302
-2313.
Scholpp, S. and Brand, M. (2004). Endocytosis controls spreading and effective signaling range of Fgf8 protein. Curr. Biol. 14,1834 -1841.[CrossRef][Medline]
Schupbach, T. (1987). Germ line and soma cooperate during oogenesis to establish the dorsoventral pattern of egg shell and embryo in Drosophila melanogaster. Cell 49,699 -707.[CrossRef][Medline]
Schweitzer, R., Shaharabany, M., Seger, R. and Shilo, B. Z.
(1995). Secreted Spitz triggers the DER signaling pathway and is
a limiting component in embryonic ventral ectoderm determination.
Genes Dev. 9,1518
-1529.
Shiga, Y., Tanaka-Matakatsu, M. and Hayashi, S. (1996). A nuclear GFP/β-galactosidase fusion protein as a marker for morphogenesis in living Drosophila. Dev. Growth Differ. 38,99 -106.[CrossRef]
Sigismund, S., Woelk, T., Puri, C., Maspero, E., Tacchetti, C.,
Transidico, P., Di Fiore, P. P. and Polo, S. (2005).
Clathrin-independent endocytosis of ubiquitinated cargos. Proc.
Natl. Acad. Sci. USA 102,2760
-2765.
Soubeyran, P., Kowanetz, K., Szymkiewicz, I., Langdon, W. Y. and Dikic, I. (2002). Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF receptors. Nature 416,183 -187.[CrossRef][Medline]
Sunio, A., Metcalf, A. B. and Kramer, H.
(1999). Genetic dissection of endocytic trafficking in Drosophila
using a horseradish peroxidase-bride of sevenless chimera: hook is required
for normal maturation of multivesicular endosomes. Mol. Biol.
Cell 10,847
-859.
Tabata, T. and Takei, Y. (2004). Morphogens,
their identification and regulation. Development
131,703
-712.
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[CrossRef][Medline]
Teis, D., Wunderlich, W. and Huber, L. A. (2002). Localization of the MP1-MAPK scaffold complex to endosomes is mediated by p14 and required for signal transduction. Dev. Cell 3,803 -814.[CrossRef][Medline]
Teleman, A. A., Strigini, M. and Cohen, S. M. (2001). Shaping morphogen gradients. Cell 105,559 -562.[CrossRef][Medline]
Thio, G. L., Ray, R. P., Barcelo, G. and Schupbach, T. (2000). Localization of gurken RNA in Drosophila oogenesis requires elements in the 5' and 3' regions of the transcript. Dev. Biol. 221,435 -446.[CrossRef][Medline]
Urban, S., Lee, J. R. and Freeman, M. (2001). Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases. Cell 107,173 -182.[CrossRef][Medline]
Van De Bor, V., Hartswood, E., Jones, C., Finnegan, D. and Davis, I. (2005). gurken and the I factor retrotransposon RNAs share common localization signals and machinery. Dev. Cell 9,51 -62.[CrossRef][Medline]
van der Bliek, A. M. and Meyerowitz, E. M. (1991). Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature 351,411 -414.[CrossRef][Medline]
Wucherpfennig, T., Wilsch-Brauninger, M. and Gonzalez-Gaitan,
M. (2003). Role of Drosophila Rab5 during endosomal
trafficking at the synapse and evoked neurotransmitter release. J.
Cell Biol. 161,609
-624.
Xu, T. and Rubin, G. M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117,1223 -1237.[Abstract]
Zhu, A. J. and Scott, M. P. (2004). Incredible
journey: how do developmental signals travel through tissue? Genes
Dev. 18,2985
-2997.
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