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First published online 14 January 2009
doi: 10.1242/dev.031104
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1 Institute of Developmental Biology and Cancer, CNRS UMR 6543, Centre de
Biochimie, Université de Nice, Parc Valrose, 06108 Nice Cedex 02,
France.
2 The Cell Microscopy Centre, Department of Cell Biology, Institute of
Biomembranes, University Medical Centre Utrecht, AZU Room H02.313,
Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
3 Temasek Life Sciences Laboratory, 1 Research Link, Singapore 117604, Republic
of Singapore.
* Author for correspondence (e-mail: pizette{at}unice.fr)
Accepted 2 December 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-like ligand Gurken,
but not in EGFR-expressing cells. Strikingly, we find that GSLs are not
essential for Gurken trafficking and secretion. However, we characterize for
the first time the extracellular Gurken gradient and show that GSLs affect its
formation by controlling Gurken planar transport in the extracellular space.
This work presents the first in vivo evidence that GSLs act in trans to
regulate the EGFR pathway and shows that extracellular EGFR ligand
distribution is tightly controlled by GSLs. Our study assigns a novel role for
GSLs in morphogen diffusion, possibly through regulation of their
conformation.
Key words: Glycosphingolipids, Glycosyltransferases, Egghead, Brainiac, Signaling, Gradient, EGFR, Gurken, Oogenesis, Drosophila
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Sillence and Platt, 2004).
These studies indicate that cell-surface GSLs participate in adhesion through
the binding in trans of lectins or other GSLs. GSLs can also bind in cis to
directly modulate the activity of receptor tyrosine kinases (RTKs) at the
plasma membrane. GSLs are also thought to be involved in vesicular transport
along the exocytic and endocytic pathways, sorting proteins into different
compartments. Lastly, the presence of GSLs in membrane microdomains, which are
considered as signaling platforms, may underlie many of their functions.
There is, however, little in vivo evidence to support any of these presumed
functions. In S. cerevisiae, mutants abolishing all GSL synthesis
fail to show defects in intracellular trafficking
(Lisman et al., 2004). In
C. elegans, GSLs appear to be dispensable throughout life
(Griffitts et al., 2005). In
mammals, the vast majority of GSLs are built on glucosylceramide (GlcCer), and
synthesis branches at the level of the third glycosyl residue to yield three
classes (lacto-, globo- and ganglioseries,
Fig. 1A). Knockout of the mouse
GlcCer synthase gene (Ugcg) leads to early embryonic lethality, for
unclear reasons; assessing the effects of knocking out downstream
glycosyltransferases is complicated by redundancy in these genes and between
different GSLs (reviewed by Sabourdy et
al., 2008). Nonetheless, disrupting the ganglioseries pathway
produces mice that display neurological abnormalities after birth.
Interestingly, in humans, mutations affecting GSL synthesis and degradation
trigger severe neuropathologies (reviewed by
Kolter and Sandhoff, 2006).
Therefore, GSLs are at least required for proper function of the adult nervous
system, but no firm link has yet been established between this requirement and
their proposed cellular roles.
Drosophila melanogaster GSLs are simpler in structure than their
vertebrate counterparts, with a single biosynthetic pathway described to date,
giving rise to a family of differentially elongated molecules
(Seppo et al., 2000). We
previously identified Egghead (Egh) and Brainiac (Brn) as glycosyltransferases
responsible for GSL biosynthesis in the fly, catalyzing the addition of the
second and third glycosyl residues of the GSL oligosaccharide chain
(Fig. 1B)
(Schwientek et al., 2002;
Wandall et al., 2003;
Wandall et al., 2005). We also
showed that there is no redundancy in these enzyme functions and no alternate
biosynthetic pathway. Hence, egh and brn mutants are devoid
of elongated GSLs and provide a useful model system for studies of GSL
functions in vivo. Importantly, mutations in each gene are lethal and cause
identical phenotypes during oogenesis and embryogenesis that are reminiscent
of loss-of-function in the Notch receptor and EGF RTK (EGFR) pathways
(Goode et al., 1996a;
Goode et al., 1996b). Since
the expression of a GSL-dedicated human galactosyltransferase in
Drosophila egh mutants rescues their viability and fertility in a
brn-dependent fashion (Wandall et
al., 2005), these data indicate that Drosophila GSLs are
essential for development, perhaps by modulating signaling.
During Drosophila oogenesis, activation of the EGFR pathway
primarily depends on Gurken (Grk), an EGFR ligand similar to vertebrate
TGF, that is secreted by the oocyte (reviewed by
Nilson and Schupbach, 1999).
The EGFR-Grk couple acts twice to polarize the follicular epithelium as well
as the future embryo along both anteroposterior (AP) and dorsoventral (DV)
axes (Gonzalez-Reyes et al.,
1995; Roth et al.,
1995). Despite ubiquitous expression of EGFR in follicle cells,
its activation is spatially restricted by asymmetric Grk localization. In
early oogenesis, grk mRNA and protein are enriched at the posterior
pole of the oocyte (Fig. 1C),
and Grk activates EGFR in neighboring follicle cells, inducing them to adopt a
posterior fate. At mid-oogenesis, these cells signal back to the oocyte,
resulting in a reorganization of its cytoskeleton, a redistribution of oocyte
maternal determinants along the AP axis, and the movement of the nucleus
towards the anterior oocyte cortex. As grk RNA remains associated
with the oocyte nucleus, a new restricted source of Grk is created to limit
the highest activation of EGFR to the adjacent follicle cells, instructing
them to assume a dorsal identity (Fig.
1C) (Schupbach,
1987; Neuman-Silberberg and
Schupbach, 1993). Respiratory appendages
(Fig. 1D) are eggshell
structures derived from dorsolateral follicular cells and their examination is
an excellent means to monitor EGFR signaling. Indeed, mild Grk or EGFR
loss-of-function causes a fusion of the respiratory appendages owing to the
absence of the dorsal-most cells (weak ventralization,
Fig. 1E). By contrast, a more
severe reduction in EGFR signaling abrogates the formation of these structures
(complete ventralization, Fig.
1F).
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Other
mutant alleles were: brn1.6P6
(Goode et al., 1996b),
egh62d18, molecularly characterized by Wandall et al.
(Wandall et al., 2005), the
top1 allele of Egfr
(Clifford and Schupbach, 1994),
cniAA12 and cniAR55
(Roth et al., 1995) and
grkDC (Queenan et al.,
1999). GAL4 and UAS lines used: nos-GAL4
(Rorth, 1998),
e22c-GAL4 (Duffy et al.,
1998), UASP-brn (Chen
et al., 2007) and UASP-mbgrkmyc
(Ghiglione et al., 2002),
which we remobilized to the third chromosome. brn1.6P6 and
egh62d18 were recombined to FRT101, germ line
clones generated with the hsFLP ovoD1 system
(Chou and Perrimon, 1992), and
follicle cell clones obtained using the e22c-GAL4,UAS-FLP stock
(Bloomington Stock Center).
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In situ hybridization, electron microscopy and immunostaining
Whole-mount in situ hybridization for grk on ovaries was carried
out as described (Vanzo and Ephrussi,
2002). For rho1 and aos mRNAs, amplification was
skipped and Fast Red (Roche) was used as a fluorescent substrate for the
alkaline phosphatase. Probes were prepared according to the manufacturer's
instructions (DIG RNA Labeling Kit, Roche).
Localization of Grk by immunoelectron microscopy was performed as described
(Herpers and Rabouille, 2004).
Conventional immunostaining was as described
(Wandall et al., 2005).
Primary antibodies used: mouse monoclonal anti-MacCer
(Wandall et al., 2005)
undiluted, mouse monoclonal anti-Grk (1D12, DSHB) 1:200, rat polyclonal
anti-EGFR (a kind gift from B. Shilo, Weizmann Institute of Science, Rehovot,
Israel) 1:100. Fluorescently conjugated secondary antibodies were from Jackson
ImmunoResearch. DAPI (Sigma) and fluorescently conjugated phalloidin
(Molecular Probes) were always included in the staining procedure. Note that
for MacCer staining, we used, as a detergent, Tween 20 at 0.05% instead of
Triton X-100 at 0.1%. Extracellular immunostaining was as reported
(Strigini and Cohen,
2000).
Confocal data were acquired with a Leica TCS microscope as single images collected from the focal plane in which the oocyte nucleus is visible, or as image stacks for dorsal views.
Characterization of the extracellular Grk gradient
To compare specimens of different genotypes, egg chambers were stained in
parallel and imaged with identical acquisition settings. The extent of
extracellular Grk diffusion towards the posterior pole was expressed as a
ratio of the length of Grk staining in the extracellular space (delimited by
F-actin staining) over the total length of the extracellular space along the
AP axis (anterior- and posterior-most points were at the boundary with nurse
cells and posterior polar follicle cells, respectively). These distances were
measured using the Freehand tool from ImageJ, on single images of the longest
longitudinal section taken at the level of the dorsal midline.
For dorsal views of the extracellular Grk gradient, quantification of pixel
intensity at a defined level was performed with ImageJ on projections of 1
µm-spaced confocal sections encompassing the whole domain of detectable Grk
expression (typically 
35 sections, roughly corresponding to the dorsal
half of the egg chamber circumference). Average plots from the number of
samples indicated in the text were generated using Microsoft Excel.
Western blot
Ovarian extracts were prepared as described
(Ghiglione et al., 2002). The
rabbit anti-Myc (A-14, Santa Cruz) antibody was used at 1:1000, the mouse
monoclonal anti--Tubulin (DM 1A, Sigma) at 1:10,000 and HRP-conjugated
secondary antibodies (Jackson ImmunoResearch) at 1:5000. Western blots were
developed with ECL reagents (Amersham).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Goode et al., 1996b). As a
result, very few eggs are laid, and those that are exhibit weakly ventralized
eggshells, suggesting a defect in EGFR activity during DV patterning of the
follicular epithelium.
To examine the requirement for GSLs in EGFR signaling, we used the viable
and cold-sensitive brnfs107 allele, in addition to the
lethal egh and brn alleles. We molecularly characterized
brnfs107 and found a single nucleotide substitution
changing the conserved tyrosine residue at position 147 to an asparagine. Even
when raised at 18°C, brnfs107 mutant females are
fertile and rarely display early oogenesis defects, but produce eggs with
fused respiratory appendages (RAs) (Fig.
1G, arrow). At this temperature, the brnfs107
eggshell phenotype is highly penetrant and this allele behaves genetically as
a null for this phenotype (Goode et al.,
1992). Because of its viability and robust eggshell defect, the
brnfs107 allele is useful to study the role of GSLs in
late oogenesis events such as DV patterning.
To assess the level of EGFR activity in the follicular epithelium of the
brnfs107 mutant during DV patterning, we looked at the
expression of argos (aos), which requires amplification of
EGFR signaling, and of rhomboid1 (rho1; rho -
FlyBase), which is induced prior to the amplification phase
(Wasserman and Freeman, 1998).
In wild-type stage 11 egg chambers, aos mRNA is expressed in follicle
cells in a triangle above the oocyte nucleus, being more abundant at the
anterior margin of the epithelium (Fig.
2A,A', arrow). rho1 transcripts are present as two
L-shaped stripes, in a pattern complementary to that of aos
(Fig. 2C,C'). In stage 11
brn mutants, aos and rho1 were still expressed, but
at lower levels. aos mRNA was only detected in the anterior-most
follicle cells (Fig.
2B,B', arrow), whereas rho1 transcripts were
distributed in a single broad domain covering the anterior third of the dorsal
follicular epithelium (Fig.
2D,D', bracket). Strikingly, these expression domains
resemble the wild-type aos and rho1 patterns from stage 10a
egg chambers (see Peri et al.,
1999; Queenan et al.,
1997; Ruohola-Baker et al.,
1993). This suggests that the initial activation of EGFR by Grk
proceeded normally, but that the amplification process itself did not take
place. These observations fit nicely with the fusion of the RA in the
brn mutant because the amplification step and aos expression
are necessary for the splitting of the RA
(Wasserman and Freeman, 1998).
These results provide evidence that GSLs are required during DV patterning of
the follicle cells to achieve high levels of EGFR activity.
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;
Yoon et al., 2006). Given that
the GSL biosynthetic pathway is active in the follicular epithelium
(Wandall et al., 2005), we
might expect GSLs to be required in follicle cells. Nonetheless, clonal
analysis has suggested that egh and brn are required in the
germ line (Goode et al.,
1996a). It is thus possible that GSLs act in both tissues.
To investigate this, we extended our previous analysis of the activity of
the GSL biosynthetic pathway during oogenesis. Since antibodies against
elongated GSLs or Egh and Brn are not available, we used an antibody that
specifically recognizes the non-elongated biosynthetic intermediate produced
by Egh, mactosylceramide (MacCer, Fig.
3A) (Wandall et al.,
2005). Theoretically, MacCer detection indicates that Egh is
active. However, MacCer staining was never seen in wild-type ovaries
(Wandall et al., 2005) (data
not shown), suggesting (1) that Egh is not expressed in this tissue or (2)
that Egh is expressed but that Brn immediately elongates MacCer. In the second
case, abrogating brn function should lead to MacCer accumulation.
When we examined MacCer distribution in brnfs107 ovaries
(Fig. 3B and data not shown),
we found MacCer staining in the germarium (the structure producing the egg
chambers) and early egg chambers. MacCer was again detected at stage 8, when
DV patterning of the follicular epithelium is initiated, and persisted at
least until stage 11. MacCer was present in follicle cells, confirming our
previous observations, but was also strongly detected in both nurse cells and
oocyte. This pattern is not allele-specific, as brn1.6P6
follicle cell and germ line clones generated at different time points gave the
same result (data not shown). Therefore, the GSL pathway is active in both
germ line and somatic follicle cells at the time when DV patterning is being
established.
Next, we tested whether GSLs from follicle cells or the germ line
participate in EGFR signaling. We produced egg chambers with large clones of
follicle cells mutant for the lethal alleles brn1.6P6 or
egh62d18, without affecting the germ line. In these
experiments, the percentage of mutant follicular epithelia was high (65.8% for
brn1.6P6 as determined by MacCer staining; n=38
stage 10 and 11 egg chambers; data not shown), yet none of the eggs derived
from such chambers exhibited defects in the DV polarity of their eggshells
(Table 1A). In agreement with
Goode and colleagues (Goode et al.,
1996a), germ line clones of these alleles led to the formation of
eggs with fused RAs (Table 1B).
Furthermore, restricted expression of a UAS-brn transgene to the
oocyte of brnfs107 mutants completely rescued the
ventralized eggshell phenotype (Table
1C). Altogether, these results indicate that GSLs are required in
the germ line, but not in follicle cells, to support EGFR activity in follicle
cells. This suggests a role in the ligand-producing cell, arguing against a
direct modulation of EGFR activity in cis in this context.
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By contrast, we detected stage-specific changes in Grk protein
distribution. In wild-type egg chambers at stage 8, Grk was concentrated
within the oocyte at its DA corner (Fig.
4C,C'). It was also present at the interface between the
oocyte and the follicular epithelium, just above the oocyte nucleus, as a
result of directional transport (Herpers
and Rabouille, 2004). A small fraction of Grk is seen in adjacent
follicle cells, reflecting uptake of secreted Grk
(Ghiglione et al., 2002;
Peri et al., 1999;
Queenan et al., 1999). At
stages 10a and 10b, the same pattern as previously was observed
(Fig. 4E-G'), but in
addition Grk was prominently localized in the extracellular space between the
DA corner of the oocyte and the follicular epithelium, in dot-like structures.
It was detected there at a considerable distance from the oocyte nucleus,
indicative of Grk diffusion from the site of its secretion.
In the brn mutant, there was no difference to the wild type at stage 8 (Fig. 4D,D'), but from stage 10a, all brn mutant egg chambers displayed abnormal Grk localization (Fig. 4H-I'). Although Grk was still present near the oocyte nucleus, it was noticeably absent from the extracellular space. Moreover, we saw very little evidence of internalized Grk (Fig. 4I', arrow and arrowheads; data not shown). These findings indicate that GSLs are dispensable for initial Grk secretion at the DA corner at stage 8, but that they are implicated either in sustained Grk secretion or in Grk stabilization once released at stage 10a.
GSLs are not necessary for Grk trafficking to the plasma membrane
To discriminate between sustained secretion or stabilization of Grk, we
first sought to determine whether Grk secretion was blocked in stage 10a
brn mutant egg chambers. Grk is synthesized as a transmembrane
molecule that is cleaved in the endoplasmic reticulum (ER) by a member of the
Rho family, possibly Rho2 (Stet - FlyBase)
(Bokel et al., 2006;
Ghiglione et al., 2002;
Guichard et al., 2000;
Urban et al., 2002). Although
this processing is not necessary for Grk trafficking to the plasma membrane,
it is a prerequisite for its secretion and activity
(Bokel et al., 2006;
Ghiglione et al., 2002;
Peri et al., 1999;
Queenan et al., 1999). The
cleaved lumenal portion of Grk is then exported to the Golgi apparatus with
the help of its cargo receptor Cornichon (Cni)
(Bokel et al., 2006;
Roth et al., 1995).
Importantly, when either step is compromised, Grk mislocalizes in the oocyte
cytoplasm. For instance, an uncleaved Grk accumulates in, and is dispersed
throughout, the oocyte cytoplasm (Fig.
5D,D'). In a cni mutant, Grk abnormally diffuses
into the ER lumen (Fig.
5C,C').
In brn mutant oocytes, however, Grk did not appear to accumulate
or mislocalize in the cytoplasm (Fig.
5B,B'), suggesting that its cleavage and export towards the
plasma membrane were normal. Nevertheless, Grk could remain unprocessed in
brn mutants and be rapidly targeted for degradation, preventing its
accumulation and mislocalization. To investigate this, we performed a western
blot on wild-type and brn ovarian extracts. Since endogenous Grk is
notoriously difficult to detect by this technique, we overexpressed a
construct encoding a Myc-tagged Grk protein in the germ line
(Ghiglione et al., 2002). As
in the wild type, Grk was still cleaved in the brn mutant
(Fig. 5E).
To further explore whether Grk follows its normal exocytic route in the
brn mutant, we compared Grk distribution in stage 10a wild-type and
mutant oocytes by immunoelectron microscopy. As previously reported
(Herpers and Rabouille, 2004),
the bulk of intracellular Grk in wild-type samples was found in tER-Golgi
units close to the oocyte nucleus (Fig.
5F). In brn mutants, Grk was also detected in these
organelles (the morphology of which was normal), where its labeling density
was not very different from that in wild-type ovaries
(Fig. 5G and data not shown).
Moreover, we observed neither an accumulation nor a loss of Grk pre- or
post-Golgi (including at the plasma membrane) (data not shown). Altogether,
these data argue against an essential role for GSLs in Grk transport to the
plasma membrane.
GSLs are not required for Grk to be secreted and functional
These last results suggest that Grk is secreted in the brn mutant.
To confirm this, we compared the phenotypes resulting from Grk overexpression
in wild-type versus brn mutant backgrounds
(Table 2). Grk overexpression
in wild-type oocytes induces a range of gain-of-function phenotypes seen as
increasingly dorsalized eggshells
(Neuman-Silberberg and Schupbach,
1994). In the brn mutant, not only did the eggshells lose
their ventralization defect upon Grk overexpression, but they were also
dorsalized. This implies that GSLs are not crucial for the secretion of a
ligand that is, in addition, functional.
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). The authors suggested that in response to EGFR signaling,
Egfr transcription is downregulated. We therefore used the decrease
in EGFR levels as a measure of its activation by Grk. First, we re-examined EGFR distribution in a wild-type background, as well as in a cni mutant as a negative control. As expected, at stage 10a, wild-type egg chambers displayed faint EGFR staining in most follicle cells of the dorsal midline (Fig. 6A,A', arrowheads in A). In cni mutants, in which Grk is not secreted, EGFR staining was uniform (Fig. 6B-B''), establishing that its asymmetrical expression is a consequence of its own activation.
We then looked at EGFR distribution in brn mutant egg chambers. We found that throughout oogenesis, the EGFR pattern was identical to that observed in wild-type samples (Fig. 6C-C'', arrowheads; data not shown). Our data thus show that in the brn mutant, Grk is as active in the maintenance of the spatial regulation of EGFR in DA follicle cells as in wild-type egg chambers. It is however possible that the threshold level of Grk needed to maintain this regulation is lower than that required for proper rho1 and aos expression, and that the role of GSLs is in the regulation of the spatial distribution of secreted Grk.
GSLs shape the extracellular Grk gradient
To examine whether GSLs regulate the spatial distribution of secreted Grk,
we visualized, for the first time, the extracellular Grk gradient in stage 10a
wild-type and brn mutant egg chambers, using conditions that are more
sensitive for the detection of extracellular proteins than conventional
immunostaining (Strigini and Cohen,
2000). We found that this method is suitable to monitor Grk
distribution over the dorsal half of wild-type egg chambers, but the predicted
ventral pool (Chang et al.,
2008; Goentoro et al.,
2006) was below detection.
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Along the DV axis at the level of the source (above the oocyte nucleus,
Fig. 7C), there was a greater
amount of Grk in the brn mutant: the maximal intensity was almost
double that of the wild type, and intensity values of 80 and above (dashed
line) covered a distance approximately twice as long as in the wild type. It
should be noted that follicle cells in this area are not fated to become part
of the RA (Dorman et al.,
2004). This Grk accumulation was restricted to the region
overlying the nucleus in the brn mutant. Just posterior to this
region (Fig. 7D), the maximal
intensity was equal to that in the wild type, although it was still maintained
over a greater area.
Along the dorsal midline, Grk levels in wild-type egg chambers were steeply elevated posterior to the source (Fig. 7E, asterisk) and reached a plateau, before declining. In brn mutants, a plateau of equivalent intensity was also formed, but its length was strongly reduced. In terms of spreading, however, there was still a low amount of Grk (intensity values above 50) that extended as far posteriorly as in the wild type.
The analysis of the extracellular Grk gradient therefore highlighted two major differences in the mutant versus wild-type background: the bulk of Grk diffusion was more efficient along the DV axis at the source and less efficient along the dorsal midline. Since the process that is compromised in the brn mutant is the splitting of the RA that requires high-level EGFR signaling to induce aos expression at the dorsal midline, these data indicate that GSLs play a crucial role in Grk signaling by preventing its diffusion away from the dorsal midline, thus maintaining high levels of extracellular Grk in this region.
Last, we asked whether EGFR, although seemingly normally distributed
(Fig. 6), is involved in the
altered diffusion of extracellular Grk in brn mutant chambers. To
determine how Grk binding to its receptor controls its extracellular movement,
we used the top1 allele of Egfr. This is a
hypomorphic viable allele that bears a point mutation in the ligand-binding
domain (Clifford and Schupbach,
1994), causing homozygous females to lay eggs with fused RAs
(Fig. 1E). In our hands, this
mutant was subviable and we obtained very few top1 egg
chambers of the right stage, all of them oriented in lateral views. This
precluded analysis of the Grk gradient in dorsal views, but allowed us to
measure extracellular Grk spreading at the dorsal midline in longitudinal
confocal sections (Fig.
7F-K'). High and low levels combined, Grk spanned, on
average, 69.4 ± 4.2% of the AP length of the extracellular space in
top1 samples (n=7), versus 54.7 ± 3.4% for
wild-type samples (n=17). This result demonstrates that EGFR limits
the range of extracellular Grk diffusion. By contrast, this value, although
slightly reduced, was not significantly different in the brn mutant
(48 ± 4.6%, n=14), indicating that Grk still efficiently binds
EGFR.
In conclusion, our experiments show that GSLs shape the extracellular Grk gradient and strongly suggest that this is achieved through the direct modulation of Grk diffusion.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Regulation of Grk signaling by GSLs: temporal or spatial?
Surprisingly, we find that GSLs are only involved in the final step of Grk
signaling during the establishment of the DV axis. Prior to DV patterning, Grk
activates EGFR to set the AP axis of the egg (see Introduction). We have
observed, however, that AP polarity is not compromised in egh and
brn alleles (S.P., unpublished). Our analysis of MacCer distribution
during oogenesis also supports the idea that the GSL biosynthetic pathway is
not active at the time AP patterning is established
(Fig. 3). Grk signaling
therefore seems to be more sensitive to GSL function for the determination of
DV fates.
Even during this process, there appear to be differential requirements for
GSL activity. DV patterning proceeds in two distinct temporal phases of EGFR
signaling, but only the second is under the control of GSLs. In a first phase
(between stages 8 and 10a), the EGFR pathway establishes embryonic DV polarity
and dorsal follicle cell fates. This phase culminates in the induction of
rho1 transcription in DA follicle cells and depends on paracrine
signaling mediated by Grk. This initial phase is not overtly affected in
brn mutants as their embryos have a normal DV axis
(Goode et al., 1992) and we
found that rho1 expression was still induced
(Fig. 2). By contrast, the
second phase of EGFR signaling is triggered by rho1 expression and
corresponds to an amplification of EGFR activity needed to split the RA
(Wasserman and Freeman, 1998).
This phase is disrupted in the brn mutant because the expression of
rho1 and aos is not upregulated
(Fig. 2), as exemplified by the
fusion of the RA.
According to Wasserman and Freeman, the amplification phase is independent
of Grk and relies on autocrine EGFR signaling
(Wasserman and Freeman, 1998).
However, we show here that GSLs, unlike the other molecules implicated in this
process, act in the germ line to regulate the distribution of extracellular
Grk (Table 1 and
Fig. 7A-E). This indicates, as
previously suggested by others (Peri et
al., 1999), that this phase is not solely autocrine and that there
is still a need for Grk-mediated paracrine signaling.
At stage 10a, the vitelline membrane is already being deposited as
vitelline bodies in the extracellular space between the oocyte and the
follicular epithelium. Morphological data show that these bodies have not yet
fused, leaving space for a number of interdigitating microvilli emanating from
the oocyte membrane and the apical side of the follicle cells
(King, 1970). Since the
brn mutation specifically affects Grk signaling at this stage, we
propose that GSLs play a role in Grk accessibility to follicle cells and that
this is likely to be mediated through the microvilli. In support of this, at
stages at which elongated GSLs are not essential (AP patterning and the onset
of DV patterning), the oocyte plasma membrane is closely apposed to the apical
side of the follicle cells. This, therefore, supports our hypothesis that GSLs
are only required when Grk is not easily accessible to its receptor.
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). However, from a strong and constant level of Grk over half
the dorsal midline, we expect the expression domains of the Grk primary target
genes to have an identical width along most of the AP axis. This is precisely
what is observed for kekkon-1 and pipe, which are clearly
EGFR primary target genes (Chang et al.,
2008; Peri et al.,
2002; Sapir et al.,
1998; Sen et al.,
1998). We thus suggest that a stripe-shaped source of
extracellular Grk along the dorsal midline, rather than a point-like source of
Grk above the oocyte nucleus, is more efficient in accommodating patterning
across the entire epithelium. Besides contributing to the shape of the Grk gradient, high Grk levels along the dorsal midline might serve to upregulate rho1 expression, leading to higher EGFR activity, aos expression and splitting of the RA primordium. Indeed, we have shown that in the absence of elongated GSLs, weak rho1 expression is retained but it is not upregulated or refined. As mentioned above, Grk signaling is necessary for this step. Since, in the brn mutant, there is a reduction in the high levels of extracellular Grk along the dorsal midline, we propose that a low Grk threshold is sufficient to initiate and maintain rho1 transcription (as well as the spatial regulation of EGFR, Fig. 6C-C''), whereas a higher Grk threshold increases rho1 expression levels.
What could be the basis for the discrepancy between our results and the
mathematical modeling of the Grk gradient? In the latter
(Goentoro et al., 2006), EGFR
expression was assumed to be uniform throughout the follicular epithelium.
However, we showed that at stage 10a, EGFR levels are lower along part of the
dorsal midline (Fig. 7) in a
region coincident with that of high Grk levels. Furthermore, we found that
decreasing Grk binding to EGFR increased Grk spreading
(Fig. 7F-K'). Therefore,
at the dorsal midline, the reduction in EGFR levels might saturate receptor
occupancy. This could allow a large quantity of Grk to remain unbound,
facilitating its movement toward the posterior pole.
Role of GSLs in determining the shape of the extracellular Grk gradient
Our most striking result is that GSLs shape the extracellular Grk gradient
and play a role in Grk diffusion without apparently interfering with the
regulation of Grk diffusion by EGFR (Fig.
6, Fig. 7F-K). But
what could that role be? Grk movement in the extracellular space between the
oocyte and the follicular epithelium is complicated by the formation of the
vitelline membrane (see above). Grk could either be released into the
extracellular space or it could remain associated with the oocyte plasma
membrane and localize to its microvilli. We could not distinguish between
these alternatives, as immunofluorescent staining is of insufficient
resolution and the extracellular space was not well preserved in our
immunoelectron microscopy experiments. Others have nevertheless reported the
presence of Grk on microvilli (Bokel et
al., 2006). GSLs could therefore be important for Grk targeting to
microvilli versus flat portions of the membrane. This, however, is unlikely
because Grk still activates EGFR in the brn mutant
(Table 2 and
Fig. 6), indicating that it can
encounter its receptor. By contrast, what Grk fails to do in the mutant
context is to concentrate along the dorsal midline at a distance from its
point of secretion. This suggests that GSLs function in the planar transport
of Grk along the AP axis, from one oocyte microvillus to the next, a
hypothesis supported by the fact that we found the oocyte microvilli to be
oriented parallel to the AP axis (S.P., unpublished).
An intriguing property of secreted Grk in the brn mutant context
is that it is detected by extracellular staining and not conventional
immunostaining. In an effort to understand the basis for this, we found that
secreted Grk is sensitive to the presence of detergent and to temperature,
suggesting that its conformation relies on the presence of GSLs once it
reaches the cell surface (see Fig. S1 in the supplementary material).
Interestingly, GSLs induce a conformational change in the amyloid
β-protein upon its release from the plasma membrane (reviewed by
Ariga et al., 2008). It is thus
possible that under our experimental conditions, Grk conformation is not fully
restored, modifying its ability to diffuse.
In this case, how could the two processes be linked? There is increasing
evidence that the spreading of secreted molecules depends on elaborate events
involving their multimerization and/or incorporation into higher-order
structures such as lipoprotein particles
(Gallet et al., 2006;
Panakova et al., 2005;
Zeng et al., 2001). It is
therefore tempting to speculate that a change in secreted Grk conformation
that depends on plasma membrane GSLs reflects its packaging into special
structures that are required for its efficient transport along microvilli.
Since mammalian GSLs can be shed from the plasma membrane and are found
circulating with secreted lipoprotein particles (see
Clarke, 1981;
Lauc and Heffer-Lauc, 2006),
GSLs could enhance Grk spreading by delivering it to these particles.
|
), and
palmitoylation is one of the signals that target proteins to membrane
microdomains (reviewed by Pike,
2004). Because Grk recruitment to these domains has not been
addressed and because the detection of extracellular Grk by biochemical means
in ovaries has so far eluded our attempts (S.P., unpublished), understanding
how GSLs regulate Grk diffusion will have to await the generation of better
tools.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/551/DC1
|
Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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