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First published online 14 December 2005
doi: 10.1242/dev.02197
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Max-Planck Institute for Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany.
* Author for correspondence (e-mail: eaton{at}mpi-cbg.de)
Accepted 3 November 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Rab5, Rab7, Rab11, Imaginal disc, Polarized endocytosis, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
This concentration gradient activates different genes as a function of
distance from the source, specifying cell identities in developing tissues.
Gradient formation requires both a source of morphogen and a sink. Possible
sinks include degradation in lysosomes of receiving tissue, degradation by
extracellular proteases or release from tissue.
The endocytic pathway in receiving tissue is an important regulator of
morphogen gradients, although it can have opposing effects on the spread of
different morphogens. The TGFß homolog Decapentaplegic (Dpp) depends on
the functions of Dynamin (Shibire - FlyBase) to move over long distances
through the wing disc epithelium of Drosophila
(Entchev et al., 2000) (but
see also Belenkaya et al.,
2004). Dynamin is a GTPase mediating scission of endocytic
vesicles (Sever, 2002); thus,
Dpp molecules are proposed to be actively passed through cells by planar
transcytosis (i.e. successive rounds of internalization and recycling to the
surface). By contrast, internalization restricts the range of FGF-8 movement
in zebrafish - a process called restrictive clearance
(Scholpp and Brand, 2004).
Morphogens of the Wingless (Wg) and Hedgehog (Hh) families harbor
covalently attached lipid moieties
(Pepinsky et al., 1998;
Porter et al., 1996;
Willert et al., 2003;
Zhai et al., 2004). Although
lipid confers high membrane affinity on these proteins, they can escape cell
membranes by binding to Lipoproteins. In the Drosophila wing disc, Wg
and Hh require Lipoproteins to signal over long distances
(Panáková et al.,
2005). Neither Wg nor Hh require Dynamin activity to spread
through receiving disc tissue (Strigini
and Cohen, 2000; Torroja et
al., 2004); therefore, they probably move extracellularly rather
than by planar transcytosis in imaginal discs. Loss of Dynamin function does
perturb the range of Wg signaling in the embryonic ectoderm, however
(Bejsovec and Wieschaus, 1995),
suggesting that different transport mechanisms operate in different
tissues.
Several observations suggest that the endocytic pathway helps shape the Wg
gradient. In imaginal discs, Wg accumulates on the cell surface of Dynamin
mutant clones (Strigini and Cohen,
2000), suggesting that normal cells internalize and degrade Wg. In
embryos, increased Wg lysosomal degradation appears to shorten its signaling
range posteriorly (Dubois et al.,
2001). Furthermore, recycling Wg back to the extracellular space
was proposed to replenish the pool available for movement
(Pfeiffer et al., 2002).
Nevertheless, the respective contributions of internalization, degradation and
recycling in controlling Wg spreading have not been systematically
examined.
The receptors that restrict the spread of Wg remain unidentified. Removing
proteins that mediate Wg internalization and degradation should elevate Wg
concentration on cell surfaces. Conversely, overexpressing such proteins might
cause excess internalization and deplete extracellular Wg. However, ectopic
Frizzled 2 (Fz2) overexpression actually stabilizes extracellular Wg
(Cadigan et al., 1998;
Baeg et al., 2004), suggesting
that this receptor facilitates the spread of Wg. While Wg accumulates on the
surface of cells missing both Frizzled receptors Fz1 and Fz2, it is
nevertheless still internalized; thus, the role of Fz receptors appears
complex (Baeg et al., 2004).
The LDL receptor family protein Arrow is thought to act as a Wg co-receptor;
its role in Wg trafficking is also unclear. Wg accumulates extracellularly on
arrow mutant clones, but this may be an indirect effect; loss of Wg
signaling increases the transcription of dally-like
(Han et al., 2005), of which
the protein product stabilizes Wg at the cell surface (see below). It is not
yet known whether Wg internalization depends on Arrow.
Heparan sulfate proteoglycans (HSPGs) play a crucial, if little understood,
role in promoting Wg spreading. Wg does not accumulate in tissue that cannot
synthesize heparan sulfate (Baeg et al.,
2004), and heparinase treatment in vitro causes Wg to disappear
from both producing and receiving tissue
(Greco et al., 2001). This
mainly reflects the activity of the HSPG Dally-like (Dlp), which is required
for long-range Wg movement and signaling
(Franch-Marro et al., 2005;
Han et al., 2005;
Kirkpatrick et al., 2004;
Kreuger et al., 2004). Dlp
overexpression in imaginal discs causes massive accumulation of extracellular
Wg (Baeg et al., 2001;
Han et al., 2005). Thus, Dlp
is thought to promote the interaction of Wg with the cell surface
(Baeg et al., 2001;
Baeg et al., 2004;
Franch-Marro et al., 2005).
Cleavage of the Dlp gpi anchor by Notum has also been proposed as a mechanism
to promote long-range Wg movement (Kreuger
et al., 2004). The possibility that Dlp might influence the
endocytic trafficking of Wg has not yet been examined.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
When the cassette is present, Gal4 transcribes HcRed and any gene
cloned in the MCS is silent. Heat-shock-mediated induction of Flipase excises
the FRT cassette and triggers expression of the gene in the MCS.
We generated pUHR derivatives containing the following cDNAs:
GFP-Dlp (GFP inserted at a.a. 221); rab5SN subcloned from
pUAST-Rab5SN (Entchev et al.,
2000); rab7TN; rab11SN; rab4SN (donated
by M. González-Gaitán).
Larvae containing pUHR constructs, a GAL4 driver and hs-Flipase were heated for 1 hour 30 minutes to 37.2°C (causing cassette excision in virtually 100% of the cells) and dissected at indicated times.
Inducible tissue-specific RNAi
A 406-bp PCR product generated from the dlp coding sequence was
cloned as an inverted repeat separated by an intron into pUAST. Transgenic
flies were crossed with apterous-Gal4 also expressing ubiquitous
Gal80ts, which represses GAL4 at the permissive temperature of
18° (McGuire et al.,
2003). Dlp dropped to undetectable levels within 36 hours of shift
to 29°. Experiments were performed 40 hours after temperature shift. To
induce Dlp RNAi and Rab5SN sequentially, we first inactivated Gal80 (resulting
in dlp RNAi) and 2 days later heat-shocked larvae to excise the FRT
cassette in pUHR-Rab5SN.
For rab7 RNAi, inverted repeats separated by an intron were cloned into pUHR. For rab11RNAi, inverted repeats were cloned without spacer intron into pUHR. For arrow RNAi, we inserted a 575-bp arrow cDNA fragment into pFRIPE. pFRIPE is a pUAST derivative in which inverted repeats can be inserted in one step by Gateway-based recombination (Invitrogen) on either side of an FRT-HcRed-FRT cassette containing a transcription terminator. The FRT cassette separating the two repeats provides inverted repeat stabilization during Escherichia coli growth, and RNAi inducibility in flies by heat-shock dependent cassette excision, which generates an inverted repeat without spacer.
FP-Rab constructs
To label Rab5, Rab11, Rab4 and Rab7 endosomes, we fused CFP and YFP(Venus)
to the N-terminus of Rabs, cloned them into pCasper4 under control of the
ubiquitous tubulin promoter and generated transgenic flies.
Fluorescence intensity indicates that these constructs drive much weaker
expression than the GAL4/UAS system.
Dextran uptake
Wing imaginal discs were dissected in M3 medium, transferred to medium
containing 5 mg/ml Alexa 488-labeled Dextran 10,000 (Molecular Probes) and
incubated for 10-15 minutes at 29°C. Discs were washed for 10 minutes in
ice-cold PBS and fixed in PBS + 4% formaldehyde.
Immunofluorescence
Antibodies used were mouse anti-Wg
(Strigini and Cohen, 2000),
mouse anti-Dlp (Lum et al.,
2003), rabbit anti-Dfrizzled2
(Strigini and Cohen, 2000),
rat anti-DE-Cadherin2 (Oda et al.,
1994), rabbit anti-Caspase (Cell Signalling Technology),
recognizing Drosophila Drice and Dcp1 (Yu
et al., 2002), rat anti-EGF Receptor
(Jékely and Rørth,
2003). To generate Arrow antibodies, guinea pigs were immunized
with a fragment of the protein corresponding to amino acids 1222-1450.
Antibody specificity was confirmed by staining imaginal discs containing
arrow- clones. Antibody stainings were performed as
described (Strigini and Cohen,
2000), except that for extracellular protein staining, discs were
incubated <3 minutes at room temperature with the Wg antibody before
incubation on ice. Confocal images were acquired at 100x magnification
on a Zeiss Axiovert 200 microscope.
PIPLC treatment
To release gpi-linked proteins, wing imaginal discs dissected from third
instar larvae were transferred to Grace's insect medium containing 10 U/ml of
PIPLC enzyme (Molecular Probes) and incubated at 25°C with mild agitation
for indicated times.
Image analysis
To quantify Wg staining intensity in Fig. S3 (see supplementary material) and Dlp staining intensity in Fig. S6 (see supplementary material), pixel
intensity along different lines centered on the dorsal-ventral boundary were
quantified using the Plot Profile function of ImageJ and values at each point
were averaged.
To quantify co-localization between Wg, CFP-Rab5 and YFP-Rab7 in receiving tissue, we used threshold and despeckle functions of Image J to eliminate cytoplasmic signals from FP-Rab proteins. For Wg, this treatment virtually eliminated the dispersed staining outlining cell boundaries. Wg-expressing cells were excluded. We defined a particle as a signal of at least three contiguous pixels and counted particles automatically using the Analyze particles function of Image J. Co-localization was assessed for single confocal sections using the RGB-co-localization plugin and defined as an overlap of at least three pixels. Similar methods were used to quantify co-localization between membrane proteins and Rab5 or 7 endosomes, but the threshold was set higher to reduce signal from the plasma membrane.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Zerial and McBride, 2001). As
Rab5 is replaced by Rab7, these early endosomes acquire degradative capacity
and mature into lysosomes (Bucci et al.,
2000; Rink et al.,
2005). We used transgenic flies ubiquitously expressing moderate
levels of CFP-Rab5 and YFP-Rab7 to localize these endosomes in discs.
Endosomes positive for Rab5 only were most abundant in the apical- and
basal-most regions of disc cells. The fraction of endosomes containing only
Rab7 increased in central regions (see Fig. S1A in the supplementary
material), suggesting endosomes move centrally as they mature. Consistent with
the model that Rab5 is gradually replaced by Rab7, we observed significant
co-localization between these two markers (see Fig. S1 in the supplementary
material). To test whether Wg was present in these compartments, we examined
co-localization between Wg, CFP-Rab5 and YFP-Rab7 in receiving tissue. Between
43 and 58% of Wg spots co-localized with Rab5 and/or Rab7 in the apical half
of disc cells (Fig. 1).
Basally, between 25 and 29% was found in these compartments (see Fig. S1B in
the supplementary material). Thus a significant fraction of Wg was found in
Rab5- and Rab7-positive endosomes. Non-co-localizing Wg may be extracellular,
or in a different endocytic compartment.
Rab5-dependent endocytosis restricts Wingless spread
Previous studies using a temperature-sensitive dynamin mutant to
block endocytosis indicated that Wg spreads extracellularly in the wing disc
(Strigini and Cohen, 2000). As
Dynamin also functions at the Golgi apparatus and Rab11 endosomes
(Jones et al., 1998;
Pelissier et al., 2003;
Sever, 2002;
van Dam and Stoorvogel, 2002),
we specifically perturbed endocytosis by expressing the dominant negative
Rab5SN (Entchev et al., 2000;
Stenmark et al., 1994) in
different subsets of the wing disc. When Rab5SN expression was induced during
the third larval instar (see Materials and methods; see also experimental
scheme in Fig. S2A in the supplementary material), endocytosis was inhibited
within 2 hours, and cells in the wing pouch began to undergo apoptosis after
6-8 hours (see Fig. S2 in the supplementary material). We therefore limited
our observations to discs that had expressed Rab5SN for 2-6 hours.
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Although Dynamin is needed for Wg secretion from producing cells
(Strigini and Cohen, 2000),
Rab5-dependent endocytosis is dispensable; targeted Rab5SN expression in the
entire wg expression domain had no impact on Wg release even after
long-term Rab5SN expression (up to 23 hours; data not shown).
To identify sites of Wg internalization, we examined the subcellular localization of Wg in Rab5SN-expressing cells. Wg was strongly enriched apical to adherens junctions, and at the very basal side of the epithelium (Fig. 2A-D; quantified in Fig. S3 in the supplementary material), especially when discs were stained specifically for extracellular Wg (Fig. 2C). Polarized accumulation is not an artifact of reduced antibody access to lateral cell membranes, because lateral extracellular Wg accumulation was detected upon Dlp overexpression (Fig. 2E). This suggests that Wg is internalized from apical and basal (but not lateral) surfaces and that internalization normally limits its spread.
Rab7-dependent degradation does not modulate the extracellular spread of Wingless
To investigate whether the balance of Wg recycling versus degradation
affected levels of extracellular Wingless, we perturbed these pathways. In
vertebrate cells, expression of dominant negative Rab7 disperses the lysosomal
compartment and inhibits degradation of endocytosed proteins
(Bucci et al., 2000). In
Drosophila disc cells, Rab7TN expression dispersed the punctate
distribution of YFP-Rab7, leaving CFP-Rab5-labeled endosomes undisturbed (see
Fig. S4A,B in the supplementary material) and caused accumulation of Hedgehog
and Patched (Eugster and Eaton, unpublished), suggesting that lysosomal
degradation is inhibited. Furthermore, YFP-Rab11-labeled recycling endosomes
were more abundant in Rab7TN-expressing cells, suggesting that inhibiting the
degradative pathway increased trafficking to recycling endosomes (see Fig. S4E
in the supplementary material).
Wg-positive endosomes in Rab7TN-expressing tissue were increased in size and number, and were present at a greater distance from the source than in non-expressing control tissue (Fig. 3A). The range of extracellular Wg distribution was unchanged, however (Fig. 3A'). Thus, the increased range of detection of Wg in Rab7TN-expressing tissue is not due to an increase in the distance over which extracellular Wg spreads, but rather to enhanced perdurance of internalized Wg protein. Slowing degradation does not increase the pool of Wg available for extracellular movement.
Wings of adult flies that expressed Rab7TN were slightly upcurved and showed frequent vein deltas near the margin, but no notching or bristle defects indicative of gain or loss of Wg signaling (not shown). RNAi directed against Rab7 reduced RNA levels and produced identical results (see Fig. S4F in the supplementary material). The surprisingly moderate effects of both Rab7 dominant negative expression and RNAi may reflect residual activity of Rab7; alternatively, non-Rab7-dependent degradative pathways may exist in Drosophila.
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;
Lock and Stow, 2005;
Maxfield and McGraw, 2004;
Satoh et al., 2005).
Vertebrate Rab4 directs rapid recycling from early endosomes to the plasma
membrane and is essential for recycling certain receptors
(de Renzis et al., 2002;
Lazzarino et al., 1998;
Seachrist et al., 2000;
van der Sluijs et al.,
1992).
Inducing Rab11SN expression in imaginal discs dispersed wild-type YFP-Rab11
within 4 hours (see Fig. S5A in the supplementary material) and inhibited
recycling of Delta (Emery et al.,
2005), Cadherin (Shotgun - FlyBase)
(Classen et al., 2005) and the
EGF receptor (see Fig. S5C in the supplementary material). Furthermore, Rab7
endosomes are enlarged by Rab11SN expression, suggesting that inhibiting
recycling may increase trafficking to Rab7 endosomes (see Fig. S5B in the
supplementary material). Rab4SN expression prevented endosomal localization of
wild-type YFP-Rab4 (see Fig. S5D in the supplementary material), but did not
affect the morphology of other endosomes (not shown). Expression of Rab11SN,
but not Rab4SN, activated apoptosis within 5 hours
(Fig. 3B inset and not
shown).
Expression of either dominant negative alone, or both Rab11SN and Rab4SN together, did not affect Wg distribution (intra- or extracellular) within 5 hours (Fig. 3B,B' and not shown). Similarly, RNAi directed against Rab11 reduced Rab11 RNA levels (see Fig.S5E in the supplementary material), but did not alter Wg distribution (see Fig. S5F,F' in the supplementary material). Thus, it seems unlikely that either Rab11- or Rab4-dependent recycling pathways contributes to the spread of Wg in imaginal discs, although as yet unknown recycling pathways might exist. These data show that Wg gradient formation is controlled by apical and basal internalization, with little contribution from canonical degradative or recycling pathways.
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). Whether
Arrow (a Lipoprotein receptor family member required for Wg signaling)
internalizes Wg is unknown. To investigate its role in Wg trafficking, we
reduced Arrow protein levels by RNAi in the dorsal compartment. Arrow protein
became undetectable 2 days after RNAi induction
(Fig. 4A), and emerging adults
had phenotypes suggesting loss of Wg signaling
(Fig. 4B). Wg-positive Rab5 and
Rab7 endosomes were more abundant (2.9±0.6-fold, n=3) over a
longer range in Arrow RNAi tissue (Fig.
4C,D). These endosomes were also located more apically than those
of wild-type cells (see Fig. S6A in the supplementary material). Thus Arrow is
not required for Wg internalization, but appears to modulate its trafficking.
As Wg is also detected in endosomes over a longer range when Rab7 activity is
reduced (Fig. 3A), loss of
Arrow may reduce the rate of Wg degradation after endocytosis.
Like arrow mutant clones (Han
et al., 2005), tissue undergoing arrow RNAi accumulated
some extracellular Wg (data not shown); however, this may reflect increased
stabilization caused by elevated Dlp protein levels near the dorsal-ventral
boundary (see Fig. S6B in the supplementary material), consistent with
previous observations (Han et al.,
2005).
Fz2 and Arrow are internalized apically by Rab5-dependent endocytosis
Our results suggest that Wg is internalized from both the apical and basal
disc surfaces. We expected that receptors mediating Wg internalization should
co-accumulate with Wg on these surfaces when endocytosis was blocked. We
therefore examined the subcellular localization of Fz1, Fz2 and Arrow after
Rab5SN induction. In the steady state, Fz2 is found predominantly on the
basal-lateral side of wing epithelial cells
(Wu et al., 2004)
(Fig. 5C, non-Rab5SN-expressing
region). However, after 5 hours of Rab5SN expression, Fz2 accumulated
dramatically on the apical side of the epithelium
(Fig. 5C,D and Fig. S6C in the
supplementary material) and was only slightly elevated on basal and lateral
membranes (Fig. 5C). These data
suggest that, despite its steady state basal-lateral localization, Fz2 is
delivered to the apical side of the cell but does not accumulate there because
of rapid internalization. By contrast, Fz1-GFP localization did not change
obviously under these conditions (not shown). As Fz1 and 2 function
redundantly, it is possible that Fz1 internalization might be more apparent in
the absence of Fz2. Unexpectedly, some Fz2 accumulation may reflect an
increase in synthesis, rather than a decrease in endocytosis, because
Fz2 mRNA levels rose in response to Rab5SN expression
(Fig. 5A). However, the apical
shift in Fz2 subcellular distribution when endocytosis was blocked indicates
that it is normally internalized from the apical surface. As Fz2 is
internalized slowly, if at all, from the basal surface, it is unlikely to
mediate Wg endocytosis there.
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), is lowered by Wg signaling
(Fig. 5B, middle). Arrow
protein is mainly basal-lateral in the steady state
(Fig. 5C, non-Rab5SN-expressing
cells) and is also present in both Rab5 and Rab7 endosomes
(Fig. 5E). In response to
Rab5SN expression, punctate Arrow staining disappeared and Arrow accumulated
at the apical membrane, co-localizing with Fz2
(Fig. 5C,D and Fig. S6C). Thus
Arrow, like Fz2, is internalized apically and is unlikely to mediate basal Wg
endocytosis. While these proteins may internalize Wg from the apical surface,
an Arrow and Fz2-independent Wg internalization pathway must operate at least
on the basal surface. We wondered whether arrow mRNA levels, like those of Fz2, might rise in response to Rab5SN expression. Surprisingly, the opposite was true. Arrow mRNA plunged to undetectable levels by 4 hours after Rab5SN induction (Fig. 5B, right). Arrow protein, however, was not reduced within this time.
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;
Franch-Marro et al., 2005;
Giraldez et al., 2002;
Han et al., 2005) and
localizes predominantly to the basal-lateral membrane in the steady state
(Baeg et al., 2004)
(Fig. 6A,B). As early as 4
hours after inducing Rab5SN expression in the dorsal compartment, endogenous
Dlp dramatically accumulated around the apical-lateral region of expressing
cells and more modestly on the basal-lateral membrane
(Fig. 6A,B and Fig. S6D).
dlp mRNA levels rose within 4 hours 30 minutes of initiating Rab5SN
expression (Fig. 6C),
suggesting that Dlp protein accumulates in part because of increased
synthesis. However, the strong apical shift in Dlp subcellular localization
suggests that Rab5-dependent internalization normally prevents apical-lateral
accumulation of Dlp.
We were surprised that Rab5SN expression trapped Dlp at the cell membrane,
because gpi-linked proteins are thought to be internalized by a
Rab5-independent mechanism (Sabharanjak et
al., 2002). To investigate whether Dlp, like Wg and Arrow, was
internalized into Rab5 endosomes, we examined co-localization between
endogenous Dlp, YFP-Rab7 and CFP-Rab5. Unlike Wg or Arrow, the Dlp signal only
rarely overlaps with that of CFP-Rab5 or YFP-Rab7
(Fig. 6D-G). Therefore,
apical-lateral enrichment of Dlp in Rab5SN-expressing tissue may occur by an
indirect mechanism. For example, Dlp may be recruited there by other proteins
accumulating when Rab5 endocytosis is blocked. Alternatively, Dlp may move
swiftly through these compartments, failing to accumulate to easily detectable
levels.
Wingless is not recruited by elevated Dlp during the endocytosis block
Because Dlp overexpression elevates Wg levels at the plasma membrane
(Baeg et al., 2001;
Franch-Marro et al., 2005;
Giraldez et al., 2002;
Han et al., 2005), we wondered
whether Wg accumulation on Rab5SN-expressing cells was an indirect effect of
increased Dlp. To see whether Wg was bound to these cells via Dlp, we treated
discs that had expressed Rab5SN for 5 hours 30 minutes with
Phosphatidylinositol-Phospholipase C (PI-PLC) to release gpi-linked proteins.
One hour of PI-PLC treatment entirely removed even the high levels of Dlp
present on Rab5SN-expressing cells (Fig.
7C). Wg, however, was not released by this treatment
(Fig. 7D). Therefore, Wg is not
trapped at the surface of Rab5SN-expressing cells by binding to Dlp or any
other gpi-anchored protein. As long as endocytosis is prevented, Wg does not
require glypicans to accumulate extracellularly on disc epithelial cells.
Wingless spreads independently of Dlp during the endocytosis block
Although Dlp is not needed to maintain Wg on the surface of
Rab5SN-expressing cells, it might be required earlier to promote Wg spread
across Rab5SN-expressing tissue. To address this possibility, we used RNAi to
deplete Dlp from the dorsal compartment before Rab5-dependent endocytosis was
blocked (see Materials and methods). In discs that did not express Rab5SN,
inducing dlp-specific RNAi rendered Dlp protein undetectable within 2
days, and reduced the range of extracellular Wg
(Fig. 7E,F), consistent with
the effect of dlp null clones
(Franch-Marro et al., 2005;
Han et al., 2005).
dlp RNAi even abolished Dlp detection in Rab5SN-expressing cells
(Fig. 7H'), which
normally have much higher levels of Dlp on their surface
(Fig. 6A). Despite the absence
of detectable Dlp, blocking Rab5-dependent internalization still caused
accumulation of extracellular Wg throughout the wing pouch within 5 hours 30
minutes (Fig.7G,H). These data
show that Dlp is not needed for the spread of Wg if Rab5-dependent
internalization is prevented. Other HSPGs might then ensure the cell-to-cell
transfer of Wg.
We wondered whether Dlp might normally promote the spreading of extracellular Wg by antagonizing Wg internalization. Dlp overexpression strongly elevates the level of extracellular Wg, especially on lateral membranes (Fig. 2E). To ask whether accumulated Wg was accessible to the endocytic pathway, we compared co-localization of Wg with endosomal markers in Dlp-overexpressing versus normal tissue (Fig. 7I-L). Although Dlp overexpression increased the range over which Wg-positive endosomes were found, their frequency was not significantly higher than wild type in cells near the source (1.3±0.3-fold within the first four cell rows, n=3). This suggests that much of the laterally accumulating Wg on Dlp-overexpressing cells is inaccessible to internalization.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) has been proposed to explain Wg spread
(Bejsovec and Wieschaus, 1995),
other data suggest that, at least in imaginal discs, Wg travels
extracellularly. Loss of Dynamin activity in cell clones within receiving
tissue causes extracellular Wg accumulation
(Strigini and Cohen, 2000),
suggesting that its range of movement is restricted by a Dynamin-dependent
mechanism. Other studies have suggested roles for both degradation and
recycling in controlling the spread of Wg
(Dubois et al., 2001;
Pfeiffer et al., 2002). In
this study, we have used dominant negative Rab GTPases to perturb specific
branches of the endocytic pathway and address these questions.
Our data suggest that the spread of Wg is controlled, like that of
zebrafish FGF-8, by restrictive clearance
(Scholpp and Brand, 2004).
Preventing Rab5-dependent internalization increases the spread of Wg through
disc tissue. While this may also elevate extracellular Wg levels by indirect
mechanisms (for example by modulating extracellular Wg proteolysis or release
from disc tissue), we favor the possibility that the gradient is shaped by
internalization of Wg itself, for several reasons. First, Wg is actually found
in Rab5- and Rab7-positive endosomes. Second, treating living discs with
protease inhibitors does not cause Wg to accumulate (E.M and S.E,
unpublished). Finally, differential centrifugation experiments suggest that
only about 6% of Wg is not membrane-associated
(Panáková et al.,
2005).
Some ligands signal from endocytic compartments after internalization
(Bivona and Philips, 2003;
González-Gaitán,
2003; Miaczynska et al.,
2004). Therefore, we initially wondered whether the high levels of
Wg accumulating after Rab5SN expression could increase signal transduction.
While Rab5SN-expressing cells both accumulate Armadillo and reduce Senseless
expression (not shown), we did not further investigate whether internalization
of Wg is required for signaling because of the striking and unexpected
transcriptional changes caused by blocking Rab5 activity. For example,
transcription of both Fz2 andDlp increases and that of
Arrow plummets within a few hours of initiating Rab5SN expression -
any of these changes by themselves could alter Wg signaling. Although we do
not yet understand this phenomenon, one might imagine that coupling
transcriptional regulation of endocytic receptors to their actual endocytosis
and/or degradation would be an effective homeostatic mechanism.
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). In imaginal discs, we find that Rab7TN expression increases
the abundance of Rab11 recycling endosomes, and that inhibiting recycling via
Rab11SN enlarges the Rab7-positive degradative compartment. Thus, the
recycling and degradative pathways may compete for some cargo in discs as they
do in cultured cells. This raises the possibility that changing the balance of
degradation and recycling might affect the pool of extracellular Wingless
available for spreading. Indeed, Wg appears to be recycled in embryos
(Pfeiffer et al., 2002), and
inhibiting lysosomal degradation in the embryonic ectoderm extends its range
(Dubois et al., 2001).
However, our data suggest that the increase in the range of Wg caused by
inhibiting degradation (at least in imaginal discs) is not the result of
increased recycling and extracellular spread. Although Wg protein is detected
in endosomes over a broader range in imaginal disc tissue expressing Rab7TN,
there is no increase in the range of extracellular Wg. Furthermore, neither
extracellular nor intracellular Wg distribution is affected by inhibiting the
Rab4- or Rab11-dependent recycling pathways. Inhibiting degradation probably
extends the apparent range of Wg by stabilizing internalized protein, raising
its levels above the threshold of detectability in more distant cells.
The idea that apical-basal polarity of epithelial cells might play a role
in regulating morphogen trafficking has been suggested by the observation that
wg mRNA is enriched apically in the embryonic ectoderm. Changing mRNA
localization alters the distribution of Wg protein in receiving tissue
(Simmonds et al., 2001),
raising the intriguing possibility that Wg might be trafficked differently
depending on whether it is secreted apically or basal-laterally. In support of
this idea, our data show that Wingless is internalized specifically from the
apical and basal (but not lateral) surfaces of the disc epithelium. Indeed,
the distribution of Rab5- and Rab7-positive endosomes in general suggests that
the apical and basal surfaces are more endocytically active than other
regions. The apical and basal internalization mechanisms may be distinct; the
known receptors for Wingless, Fz2 and Arrow are internalized mainly from the
apical surface (despite their steady-state basal-lateral localization),
suggesting that basal Wingless endocytosis must be independent of these
proteins. One possibility is that membrane association of Wg via Palmitate is
sufficient to allow its endocytosis - perhaps by mechanisms similar to those
used by gpi-linked proteins (Sabharanjak
et al., 2002). Alternatively, Wg bound to Lipoprotein particles
(Panáková et al.,
2005) might be internalized via Lipoprotein receptors.
We were surprised to observe that the Wg accumulating on the basal side of
disc epithelial cells after Rab5SN expression did not spread onto the lateral
membrane, as no barrier to diffusion has been identified between these
domains. Three possible explanations occur to us. (1) There may indeed be a
`fence' separating the lateral and basal sides of disc epithelial cells.
Neurons have a fence that prevents diffusion of lipid and lipid-linked
proteins between the axon and the cell body, although it does not resemble a
classical intercellular junction
(Kobayashi et al., 1992;
Nakada et al., 2003). (2) It
may be that the receptor(s) that normally internalize Wg basally are linked to
cytoskeletal components, or interact with extracellular matrix (ECM), and are
not free to diffuse. If these Wg receptors were of sufficiently high affinity,
they might trap Wg before it could move laterally. (3) Perhaps Wg itself
interacts efficiently with basal ECM components.
While it seems that endocytosis restricts the spread of Wg on the apical
and basal surfaces, it is not yet clear which receptors might be responsible.
A simple model would predict that removing such a receptor should produce
phenotypes similar to Rab5SN expression, i.e. increased and more extensive
extracellular Wg, and less Wg in endosomes. Conversely, overexpression might
be expected to compress the range of Wg distribution and decrease
extracellular Wg. None of the known receptors behaves in this way. Previous
studies showed that at least a fraction of Wg is still internalized in the
absence of both Fz1 and Fz2 (Baeg et al.,
2004). Furthermore, overexpression of Fz2 causes extracellular Wg
accumulation over longer distances (Cadigan
et al., 1998; Baeg et al.,
2004). We have shown here that loss of Arrow actually increases
the amount of Wg present in Rab5- and Rab7-positive endosomes - more
consistent with a role in Wg degradation after endocytosis. The complexity of
these observations may reflect different mechanisms of Wg endocytosis on the
apical and basal sides of the cell - understanding both these pathways and
their interplay will be necessary to understanding how the Wg gradient
forms.
While internalization limits the range of Wg accumulation, the glypican Dlp
extends it. It has been proposed that Dlp allows Wg to interact with disc
cells, increasing local Wg concentration and restricting its diffusion to the
epithelium (Han et al., 2005).
Our data, however, show that disc cells can accumulate high levels of Wg on
their surface in the absence of Dlp as long as Rab5-dependent internalization
is blocked. This observation is not consistent with a model in which Dlp traps
Wg on the cell surface or helps it transfer from cell to cell. Instead, it
suggests that Dlp normally stabilizes Wg at the cell surface by antagonizing
the effects of Rab5-dependent internalization (see model in
Fig. 8). While Wg is normally
internalized from the apical- and basal-most surfaces of disc cells, Dlp
overexpression recruits Wg to the lateral cell surface. This raises the
possibility that Dlp stabilizes Wg and increases its range by changing its
subcellular localization to protect it from endocytosis. Polarizing the
distribution of morphogens within an epithelium may have a key regulatory role
in the trafficking events leading to gradient formation.
Note added in proof
A recent paper (Piddini et al.,
2005) from the Vincent Laboratory has demonstrated that Arrow
promotes Wingless degradation after internalization by Frizzled 2.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/2/307/DC1
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ACKNOWLEDGMENTS |
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