Centro de Biología Molecular `Severo Ochoa', CSIC, Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain
* Author for correspondence (e-mail: iguerrero{at}cbm.uam.es)
Accepted 13 January 2004
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SUMMARY |
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Key words: Hedgehog signaling, Patched, Morphogenetic gradients, Dynamin, Deep Orange
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Introduction |
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The wing imaginal disc consists of a single-layered sac of polarized
epithelial cells with their apical surfaces orientated towards the disc lumen.
In this epithelium, two populations of cells with different adhesion
affinities divide the field into posterior (P) and anterior (A) cells
(García-Bellido et al.,
1973). The Hh protein synthesized by the P cells is released and
reaches the cells in the A compartment
(Ingham and McMahon, 2001
).
However, just how Hh reaches cells distant from its site of synthesis, and how
its concentration gradient is regulated, is not known. Hh signaling requires
the activity of two transmembrane proteins: Patched (Ptc), the Hh-receptor and
Smoothened (Smo), the activator of the Hh pathway. In the absence of Hh, Ptc
downregulates the activity of Smo. It has been suggested that Ptc increases
Smo turnover when they meet in the same subcellular compartment
(Denef et al., 2000
;
Incardona et al., 2002
). In
addition, it has been proposed that internalization of Hh by Ptc changes the
subcellular localization of Ptc, preventing Smo degradation and activating Hh
signaling (Denef et al.,
2000
). This Hh pathway activation leads to further expression of
ptc, which in turn restricts the range of Hh transport. So, Ptc has
two roles: to activate Hh signaling and to sequester Hh as genetic studies in
Drosophila have shown (Chen and
Struhl, 1996
). Ptc is a membrane protein with 12 transmembrane
domains homologous to sterol-sensing-domain (SSD)-containing proteins
(reviewed by Kuwabara and Labouesse,
2002
). The sterol-sensing domain of Ptc is involved in Hh
signaling but not in Hh sequestration, and it has been proposed to modulate
Smo activity through vesicular trafficking
(Martín et al., 2001
;
Strutt et al., 2001
). The
second function of Ptc, Hh sequestration, appears to regulate the Hh
morphogenetic gradient (Tabata and
Kornberg, 1994
; Chen and
Struhl, 1996
; Martín et
al., 2001
; Strutt et al.,
2001
). Hitherto, it was believed that Hh effectively promoted its
own sequestration (reviewed by Ingham and
McMahon, 2001
) by upregulating ptc transcription. This
negative feedback mechanism restrains the range of Hh signaling. However, the
cellular mechanisms by which Ptc controls the Hh gradient remain unclear.
We investigated the role of Ptc-mediated endocytosis in the control of Hh gradient formation and signaling in the A compartment of the wing imaginal disc. Our data show that Ptc limits the Hh gradient by internalizing Hh through endosomes in a dynamin-dependent manner and that this Hh-Ptc complex is targeted to degradation by lysosomes. We have genetically uncoupled the two proposed functions of Ptc, through the analysis of the ptc14 mutant that is unable to sequester Hh and yet retains a normal capacity to mediate Hh signaling. Our analysis leads to the suggestion that Hh signaling can occur in the absence of Ptc-mediated Hh internalization.
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Materials and methods |
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Chromosomes used were as follows.
FRT42D
P {ry[+t7.2]=neoFRT}42D arm-lacZ
P {ry[+t7.2]=neoFRT}42D P{Ubi-GFP(S65T)nls}
FRT18A
w[1118] P{Ubi-GFP(S65T)nls} P{neoFRT}18A
w[1118] arm-lacZ P{neoFRT}18A
Flipase
Hsp70-flipase chromosomes were provided by G.Struhl.
The amorphic ptc16 (also known as ptcIIW109) allele, the embryonic lethal alleles ptcS2 and ptc14 (also known as ptcIIR87) were from Tübingen (Nüslein-Volhard and Wieschaus, 1980).
In addition, the null dor8 allele
(Shestopal et al., 1997) and
the temperature-sensitive, shits1
(Grigliatti et al., 1973
) and
hhts2 (Ma et
al., 1993
) alleles were used. The restrictive temperatures for the
shits1 and hhts2
alleles are 32°C and 29°C, respectively.
The reporter genes used were dpp-LacZBS 3.0, expressed
as the endogenous RNA in the imaginal disc
(Blackman et al., 1991);
dpp10638 (a dpp mutant with a
lacZ insertion) (Zecca et al.,
1995
); and ptc-lacZ (a lacZ insertion in the
ptc gene), which was a gift from C. Goodman.
The Gal4 drivers for ectopic expression experiments using the
Gal4/UAS system (Brand and
Perrimon, 1993) were the following: c765-Gal4
[ubiquitously expressed in the wing disc
(Nellen et al., 1996
)],
ptc-Gal4 (a gift from J. Campos-Ortega), hh-Gal4 (a
gift from T. Tabata) and AB1-Gal4
(Munro and Freeman, 2000
).
Genotypes of larvae for generating mosaic clones
Mutant clones
Clones were generated by FLP-mediated mitotic recombination.
Larvae of the corresponding genotypes were incubated at 37°C for 1 hour at
24-48 hours after egg laying (AEL), or for 45 minutes at 48-72 hours AEL. The
genotypes of the flies for clone induction were:
y, w, FLP/+; en-Gal4 / UAS-HhGFP; FRT82 arm-lacZ / FRT82B dispS037707
shits1 FRT18A / arm-lacZ FRT18A; FLP/+
shits1 FRT18A / arm-lacZ FRT18A; FLP; hh-Gal4 / HhGFP
shits1; FRT42D ptc16 / FRT42D ubi-GFP; FLP/+
shits1; FRT42D ptc14 / FRT42D ubi-GFP; FLP/+
dor8 FRT18A / arm-lacZ FRT18A; FLP/+
dor8 FRT18A / ubi-GFP FRT18A; dpp-lacZ/+; FLP/+
dor8 FRT18A / ubi-GFP FRT18A; ptc-lacZ/+; FLP/+
FLP122/+; FRT42D ptc14 /FRT42D arm-Lac-Z
FLP122/+; FRT42D ptc14 / dpp10638 FRT42D ubi-GFP
FLP122/+; FRT42D ptc14 / FRT42D arm-lacZ; hhts2
FLP122/+; FRT42D ptc14 / FRT42D arm-lacZ; hh-Gal4 / HhGFP
FLP122/+; FRT42D ptc16 / FRT42D arm-lacZ; hh-Gal4 / HhGFP
Flip-Out clones
To generate random clones of wild-type ptcWTGFP,
ptc14GFP and hhGFP, the actin>CD2>Gal4
(Pignoni and Zipursky, 1997)
or ubx>f+>Gal4, UAS-ßgal
(de Celis and Bray, 1997
)
transgene was used. Larvae of the corresponding genotypes were incubated at
37°C for 15 minutes to induce HS-FLP-mediated recombinant
clones.
Act>CD2>GAL4 / HS-FLP122; ptcWTGFP
Act>CD2>GAL4 / HS-FLP122; ptc14GFP
y, w, HS-FLP122; ubx>f+>Gal4, UAS-ßgal/ UAS-HhGFP
Transgenes
Hh transgene
For the HhGFP fusion protein, the GFP-coding sequence was amplified by PCR
from a pEGFP-N1 vector (Clontech, catalog number 6085-1) and tagged in frame
before cleavage site of Hh by auto-proteolysis (...SH254-GFP-V255HGCF...)
(Fig. 1A).
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UAS-ptcS2
(Martín et al.,
2001).
Functional characterization of HhGFP fusion protein
It was expected that the fusion of the GFP sequence to Hh cDNA at position
between codons 254 (H) and 255 (V) did not interfere neither with the
processing, secretion and diffusion process of Hh nor with the Hh binding to
Ptc (Burke et al., 1999;
Pepinsky et al., 2000
). To
determine whether the auto-proteolysis of HhGFP occurs normally, we analyzed
the presence of the distinct species of Hh by western blot using either
anti-Hh (Tabata and Kornberg,
1994
) or anti-GFP antibodies
(Fig. 1B). In the salivary
gland extracts from AB1-Gal4/UAS-Hh flies, three bands of
45 kDa (U in Fig. 1B, row
1; uncleaved Hh), 25 kDa (C in Fig.
1B, row 1; C-terminal half of Hh) and 20 kDa (N in
Fig. 1B, row 1; N-terminal
active form) were observed using anti-Hh antibody as it was previously
reported (Lee et al., 1994
;
Tabata and Kornberg, 1994
;
Porter et al., 1995
). Three
major bands were also observed in salivary glands extracts from
AB1-Gal4/UAS-Hh GFP flies using either anti-Hh or anti-GFP
antibodies. The molecular weight of these bands corresponded to the ones
expected for the un-cleaved HhGFP chimera (70 kDa, U in
Fig. 1B, rows 2 and 3), for the
HhGFP-Np (46 kDa, N in Fig. 1B,
rows 2 and 3) and for the HhC (25 kDa, C in
Fig. 1B, row 2), indicating
that the Hh-GFP processing occurs normally.
To assay if HhGFP is secreted and spread normally we analyzed the effect of
ectopic HhGFP expressing clones in the wing imaginal disc. In the A
compartment of the wing pouch, these clones were able to induce En expression
autonomously and non-autonomously (Fig.
1C) as does the wild type Hh protein
(Basler and Struhl, 1994;
Zecca et al., 1995
).
Co-localization of Ptc and HhGFP in vesicles was observed in the A cells close
to the AP border in an hh-Gal4/UAS-Hh wing disc
(Fig. 1D,d).
To test if HhGFP was modified by cholesterol we induced dispatched
(disp-) mutant clones in an en-Gal4/UAS-HhGFP wing
disc. It has been reported a membrane accumulation of Hh-Np in the absence of
Disp that is dependent on its cholesterol modification
(Burke et al., 1999). As it was
expected, disp- clones induced in the P compartment showed
an autonomous accumulation of Hh-GFP compared with the wild-type territory
(Fig. 1E). This result
indicates that HhGFP-Np is cholesterol modified.
Finally, to determine if HhGFP can substitute the endogenous function of
Hh, we performed rescue experiments of the hh mutant phenotype.
hhts2 flies raised at the restricted temperature
(29°C) during the larvae period to the end of development are lethal.
However, en-Gal4/UAS-HhGFP; hhts2 mutant flies
raised at the restricted temperature (29°C) for the same developmental
period were rescued to pharates showing normalized notum, legs, abdomen and
genitalia (Fig. 1F). The wings
were present but not extended. In the head, some structures were rescued but
the dorsal head, the ocelli and the eyes were not rescued probably because
en expression begins after the on set of hh expression
(Royet and Finkelstein, 1997).
These results indicate that HhGFP is able to substitute the wild-type Hh
function.
Functional characterization of PtcGFP fusion proteins
To characterize the PtcGFP fusion proteins first, we analyzed the integrity
of the fusion proteins by western blot. We detected the presence of a protein
band of 180 to 200 kDa approximately in extracts of
AB1-Gal4/UAS-PtcWTGFP or UAS-Ptc14GFP
salivary glands using the anti-GFP antibody
(Fig. 2B). This band has a
molecular weight higher than the expected 16,964 kDa based on DNA sequence,
probably because Ptc has six potential glycosylation sites.
Then, we assayed the ability of the PtcWTGFP fusion protein to
sequester Hh when it was expressed in the P compartment. Ectopic expression of
the wild-type Ptc in the P compartment reduces the range of Hh signaling
(Johnson et al., 1995) and
induces the accumulation of Hh
(Martín et al., 2001
).
The expression of Collier, one of the Hh targets, was reduced when
PtcWTGFP was overexpressed in the P compartment
(Fig. 2D). In addition, ectopic
PtcWTGFP clones in the P compartment induced an autonomous
accumulation of Hh (see Fig.
7A). Finally, we observed a downregulation of Smo in
PtcWTGFP ectopic clones in the P compartment
(Fig. 2E) as it has been
reported for the wild type Ptc (Denef et
al., 2000
; Martín et
al., 2001
). Collectively, these data indicate that
PtcWTGFP behaves as the wild-type protein.
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For western blot analysis, protein extracts from salivary glands of
AB1-Gal4/UAS-Hh, UAS-HhGFP, UAS-PtcWTGFP or
UAS-Ptc14GFP flies were prepared in Laemmli buffer and
resolved by SDS-PAGE, immunoblotted and then analyzed using anti-Hh (1/500)
(Tabata and Kornberg, 1994)
and anti-GFP (1/1000) (Molecular Probes, catalog number A-6455) antibodies.
Horseradish peroxidase-conjugated secondary antibodies were used to develop
the signal using the ECL Western Blotting Analysis System (Amersham
Pharmacia).
Labeling the endocytic compartment
Third instar larval discs were incubated in 3.7 mM Red-dextran (lysine
fixable, MW3000; Molecular Probes) in M3 medium at 25°C (pulse) and then
washed five times for 2 minutes in ice-cold M3 medium. The discs were then
incubated for a chase period at 25°C in M3 medium prior to fixation in 4%
paraformaldehyde. To visualize the early endosomes of the endocytic
compartments, the discs were pulsed for 5 minutes without a chase period. For
late endosomes, a 5 minutes pulse and 60 minutes chase were used. This was
followed by fixing in PBS 4% paraformaldehyde (PF) for 40 minutes at 4°C
and in 4% paraformaldehyde in PBS 0.05% Triton X-100 for 20 minutes at room
temperature. The discs were then washed and incubated with antibodies in PBS
0.05% Triton X-100.
Extracellular labeling of HhGFP in shits1 mutant clones
This protocol was modified from that described by Strigini and Cohen
(Strigini and Cohen, 2000).
Larvae containing shi mutant clones where maintained at restrictive
temperature (32°C) for 3 hours, dissected at restrictive temperature and
transferred immediately to ice-cold M3 medium containing anti-GFP antibody.
They were then incubated at 4°C for 30 minutes, washed in ice-cold PBS and
fixed in PBS 4% PF at 4°C. By transferring the discs from 32°C to
ice-cold medium, the cells stop their vesicular trafficking and the proteins
remain at their subcellular locations at the moment of cooling. Incubating
with the anti-GFP antibody under these `in vivo' conditions, without
detergents and prior to fixation, rendered the antibody incapable of
penetrating the cells. Thus, only extracellular antigen was labeled. The
antibody does not label intra-cellular HhGFP. The lack of staining of the
intracellular HhGFP vesicles in the P compartment of the same disc (see
Fig. 3b, arrows) was used as a
control for this specific extracellular anti-GFP antibody staining method.
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Quantification analysis
To quantify the rate of endocytosis of PtcWT and
Ptc14 proteins, ectopic PtcWTGFP and Ptc14GFP
clones in the A compartment were induced and the endocytic compartment was
labeled by incubation with Red-dextran (10 minute pulse). Using Metamorphic
software, the PtcGFP fluorescence in the Red-dextran vesicles was measured and
compared with the total PtcGFP signal in the clone. The resulting ratio was
normalized to the Red-dextran vesicles in the clone cells. For this analysis,
six confocal sections of seven different clones were measured and a total of
1459 vesicles for Ptc14 and 1650 for PtcWT were
analyzed.
The content of HhGFP in the dotted pattern in wild-type and ptc16 mutant clones was also measured with Metamorph analysis software. Each clone analyzed was compared with an equivalent region in the wild type territory of the same wing disc. The result from five different clones from independent experiments shows a ratio between 4 and 5 times more Hh in the wild-type cells.
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Results |
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Here, we asked whether Hh internalization was required for the formation of
the Hh gradient. In other signaling mechanisms, such as that of Dpp, the
internalization of the morphogen is required for gradient formation
(Entchev et al., 2000;
Teleman and Cohen, 2000
). To
test this hypothesis, we used a thermo-sensitive shibire
(shi) mutation. Shibire, the Drosophila Dynamin homolog, is
needed for fission of clathrin-coated vesicles and the internalization of
caveolae (van der Bliek,
1999
). Shi mediates Ptc internalization in Drosophila
embryos. In shits1 mutant embryos, a punctuate
accumulation of Ptc at the plasma membrane was observed
(Capdevila et al., 1994b
),
probably as a consequence of the accumulation of `coated pits' at the plasma
membrane (Kosaka and Ikeda,
1983
). Here, we also observed a punctuate accumulation of both Ptc
and Hh at cell surface in shits1 clones, close to
the AP compartment border of the wing imaginal disc
(Fig. 3A).
To test if the Hh accumulation was extracellular we used HhGFP fusion protein. Hh-Gal4/HhGFP third instar larvae containing shits1 clones were maintained at 32°C for 3 hours, a temperature that blocks Shi-dependent trafficking. Discs were then dissected and incubated with anti-GFP antibody in ice-cold medium prior to fixation. Under these experimental conditions, all trafficking processes were blocked and the antibody only labeled extracellular HhGFP molecules, while GFP fluorescence labeled both the intracellular and extracellular Hh. We noted that in the shi clone, all the green (HhGFP) fluorescence colocalized with the red (antibody-conjugated) fluorescence, indicating that all the retained Hh was extracellular (Fig. 3B,b, arrowheads).
Next, we addressed the question of whether Ptc might be responsible for the
accumulation of Hh at the cell surface when endocytosis was blocked. To
achieve this we made ptc16 clones (an amorphic
allele, which does not produce Ptc protein, also known as
ptcIIW109)
(Capdevila et al., 1994a) in a
shits1 background. We found no build-up of Hh
when Ptc protein was absent (Fig.
3C, red and blue panel, arrow), indicating that Shi-mediated Hh
internalization was severely diminished in the absence of Ptc. We can
therefore conclude that Ptc controls the endocytosis of Hh in a
dynamin-dependent manner.
The accumulation of both Ptc and Hh at cell surface in
shits1 clones, however, did not affect the
activation of the Hh targets inside the clone
(Fig. 3D, asterisk). Moreover,
we did not observe an increase in the distance that Hh moves or signals in the
anterior side of the shits1 clone
(Fig. 3D, arrowhead). These
observations suggest that although internalization of Hh is blocked in the
shits1 mutants, Ptc has already sequestered Hh.
This interpretation is in agreement with the blockage in Dynamin function at a
stage where commitment to endocytic sequestration has already occurred
(Kosaka and Ikeda, 1983;
Ramaswami et al., 1994
;
Guha et al., 2003
).
After internalization, Hh and Ptc are targeted to degradation
We previously reported that Ptc moves Hh to the endocytic compartment in
the wing imaginal disc (Martín et
al., 2001), after which degradation of Hh and Ptc would be
expected. Rab-7 GTPase controls both protein trafficking to late endosomes and
their subsequent fusion with lysosomes (reviewed by
Zerial and McBride, 2001
). A
Rab-7GFP (Entchev et al.,
2000
) was used for labeling late endosomes. Ptc-Hh vesicles were
found to colocalize with Rab-7GFP when this marker was expressed in all wing
imaginal disc cells (Fig. 4A,a,
circles). These results suggest that after internalization, Ptc and Hh go to
late endosomes and lysosomes. Next, we examined whether the targeting of Hh to
the degradative lysosomal pathway had any effect on Hh spreading and
signaling. To block the degradative pathway, we used deep orange
(dor) mutants, one of the mutations that affects eye pigmentation in
Drosophila and is required for normal delivery of proteins to
lysosomes (Sevrioukov et al.,
1999
). dor encodes the homolog of the yeast vacuole
sorting protein Vps18p, a protein that as part of a complex regulates the
function of Ypt7p, the paralog of Rab-7 in yeast
(Rieder and Emr, 1997
;
Price et al., 2000
). Blockage
of the degradative pathway in dor- mutant clones resulted
in the accumulation of both Hh and Ptc
(Fig. 4B).
dor- clones showed an Hh protein gradient in the A
compartment of the wing imaginal disc (Fig.
4B), which was not observed when wild-type discs were stained with
anti-Hh antibodies. As we have observed in
shits1, despite the large accumulation of Hh and
Ptc in dor- clones, the activation of Hh target genes
remains unchanged, as shown by the normal dpp-lacZ (compare mutant
territory in Fig. 4D,
arrowhead, with wild-type territory, arrow) or ptc-lacZ expression
(data not shown). These results indicate that both spreading and signaling are
normal when Hh degradation is blocked in dor- cells.
Furthermore, the intensity of Hh immunofluorescence in the A cells located at
the same distance from the AP border remains the same
(Fig. 4B,C arrowheads),
regardless of whether Hh had crossed wild-type (clone 1) or mutant (clone 2)
territory. These data indicate that Ptc levels control the Hh gradient by
sequestering Hh, and then both Hh and Ptc are targeted to degradation. They
also suggest that Hh signaling is independent of Hh degradation by
lysosomes.
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As PtcS2 is defective only in its signaling function, we used
the ptcS2 allele to test for complementing
alleles of ptc that might be defective for sequestration but not
signaling. In this way, we identified the embryonic lethal
ptc14 allele (also known as
ptcIIR87). Homozygous somatic ptc-null
mutant clones induced in the A compartment produce the described
ptc- mutant phenotype caused by the full activation of Hh
targets (Phillips et al.,
1990; Capdevila et al.,
1994a
; Tabata and Kornberg,
1994
). By contrast, ptc14 mutant
clones did not activate Hh signaling in the A compartment outside the AP
border, as shown by the expression of Hh target genes such as en, col,
dpp, iro or ptc itself (Fig.
5B-F clones 1, 3, 5; data not shown). Large clones (24-48 hours
AEL) abutting the AP border activated En
(Fig. 5B, clone 2), Col
(Fig. 5D, clone 4) and
dpp (Fig. 5F, clone 6)
outside their normal expression domains. In addition to this activation,
short-range, non-autonomous activation of En, Col and dpp extended a
few cells outside the clone boundary (arrowheads in
Fig. 5B,D,F).
ptc14 cells therefore respond to maximum (En),
medium (Col) and low [dpp (Fig.
5F) and Iro (data not shown)] levels of Hh, and Hh spreads
throughout the clone and non-cautiously activates target genes a few cells
outside the clone. To determine whether this effect was due to the presence of
Hh, ptc14 clones were induced in an
hhts2 mutant background. After 30 hours at the
restrictive temperature, ptc14 clones located
neither outside nor close to the AP border activated Hh targets genes
(Fig. 6B,D). These results
indicate that Ptc14 activates the Hh pathway only in the presence
of Hh, similar to wild-type Ptc. Furthermore, the broadened activation of
target genes outside their normal expression domains in clones abutting the AP
border indicates that ptc14 cells do not
sequester Hh efficiently.
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|
Endocytosis defect in the Ptc14 mutant
The extended gradient of Hh across ptc14 mutant cells
could be due to a defect in Ptc-Hh sequestration. The sequence of this allele
confirmed a leucine to glutamic acid change (L83Q) at the first trans-membrane
domain as previously reported (Strutt et
al., 2001). To compare the ability of Ptc14 to
sequester Hh with that of wild-type Ptc, both mutant and wild-type proteins
were C-terminally tagged with GFP. Using these GFP tagged Ptc proteins we
observed that the accumulation of Hh was much lower in Ptc14GFP
ectopic clones than in PtcWTGFP ectopic clones in the P compartment
(compare Fig. 7A,a with
7B,b). This implies that
Ptc14 sequesters Hh quite inefficiently.
Next, we analyzed the internalization of Hh and Ptc in ptc14 clones abutting at the AP compartment border. Fig. 7C shows a wing disc containing a ptc14 clone and its wild-type twin clone. The wild type AP border cells show the normal pattern of Hh and Ptc accumulation in endocytic vesicles (circles in Fig. 7c). In the ptc14 mutant clone, Ptc staining showed the same protein levels as in the wild-type territory (Fig. 7C, green panel). However, Ptc14 was not confined to punctuate structures, and Hh did not co-localize with Ptc14 (compare Fig. 7c and 7c'). This observation is in agreement with the lack of Hh sequestration observed in the ectopic Ptc14 mutant clones induced in the P compartment (Fig. 7B). To analyze this sequestration defect further, we made clones of ptc14 in a shits1 background and found that Hh was not accumulated at the plasma membrane inside the clone (Fig. 7D, asterisk) while in the Ptc wild-type cells anterior to the clone Hh was accumulated (Fig. 7D, arrowhead). This accumulation indicates that Hh can spread further through a ptc14 mutant territory than a wild-type territory. The behavior of Hh in these shits1; ptc14 double mutant cells was similar to that observed in ptc16 clones (without Ptc protein) in a shits1 background (Fig. 3C). This result indicates that Ptc14 does not sequester Hh properly.
To explore a most likely failure in Ptc14-mediated endocytosis
of Hh, we examined the presence of Ptc14 compared with wild-type
Ptc protein in endosomes. In wild-type cells, Ptc accumulates in early
endocytic vesicles (Fig. 8A,a,
circles), while in ptc14 cells Ptc14 does not
accumulate in these structures (Fig.
8B,b, arrowheads). This lack of colocalization of Ptc14
with the endosomal marker could be due to the absence of Ptc14 at
the plasma membrane. To test this possibility, discs expressing
Ptc14GFP ectopic clones were incubated `in vivo' with an anti-Ptc
antibody raised against the first extracellular loop of the Ptc protein
(Capdevila et al., 1994b), in
conditions of blocked trafficking processes (see Materials and methods). The
extracellular labeling of Ptc in both PtcWTGFP
(Fig. 8C) and
Ptc14GFP (Fig. 8D)
ectopic clones indicates the presence of the mutant protein at the plasma
membrane.
|
The two functions of Ptc take place in different subcellular compartments
The complementation of ptc14and
ptcS2 alleles clearly indicates that Ptc has separate
functions (one involved in signaling through an interaction with Smo and the
other in Hh sequestration). But how Ptc performs both functions remains
controversial. One can envision that if both proteins are localized in
different subcellular compartments, indirect complementation might occur.
Thus, while PtcS2 is sequestering Hh in endosomes, Ptc14
could be interacting with Smo to control Hh signaling. Alternatively, both
proteins PtcS2 and Ptc14 might form a complex as part of
the Ptc receptor, as previous studies have suggested
(Johnson et al., 2000;
Martín et al., 2001
).
To discriminate between these two possibilities, we used tagged versions of
PtcWTGFP, PtcS2GFP and Ptc14GFP to analyze
the subcellular distribution of these mutant proteins in imaginal disc (data
not shown) and salivary glands cells. Salivary glands cells are convenient for
this experiments because are large and express endogenous Ptc protein at very
low levels when Hh is not present (Zhu et
al., 2003
). In both systems, both PtcWTGFP and
PtcS2GFP proteins were localized in large vesicles
(Fig. 9A,B). However,
Ptc14GFP was found at the plasma membrane, consistent with the
observations made in ptc14 mutant clones, and also in the
membrane of the secretion vacuoles (Fig.
9C). Next, we examined the sub-cellular distribution of
Ptc14GFP protein when co-expressed with PtcS2 (without
GFP). Under these conditions, Ptc14GFP was localized in vesicles
resembling to PtcWTGFP localization
(Fig. 9D). This change in the
subcellular distribution of Ptc14GFP strongly suggests that
PtcS2 and Ptc14 form a complex that re-establishes the
wild-type function. Furthermore, these observations indicate that Hh signaling
and sequestration take place in different subcellular compartments.
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Discussion |
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Role of Ptc in Hh gradient formation
We have shown here that Hh and Ptc sorting to the endocytic membrane-bound
compartment plays a crucial role in modulating Hh levels during development. A
strong support of the conclusions in this work comes from the analysis of the
ptc14 allele. Although ptc14 mutants
are lethal with a strong ptc- embryonic phenotype,
ptc14 mutant cells in the imaginal discs showed an effect
only when the clone touched the AP compartment border but not in any other
part of the disc. This result indicates that the presence of Hh is required to
reveal a defect in Ptc14 function. This Hh requirement has been
probed by the no activation of the Hh targets in ptc14
cells in the absence of Hh either in the embryos or in the imaginal discs. The
complementation of ptc14 with ptcS2
allele, which is considered as null for blocking Hh signal transduction and
acts as dominant negative (Martín
et al., 2001; Strutt et al.,
2001
), indicates that Ptc14 does not have a greater
sensibility to Hh than the Ptc wild-type protein. Conversely, it is shown here
that there is a decrease of internalization of Hh in ptc14
mutant clones compared with wild-type Ptc territory and an extension of the
range of Hh gradient. Therefore, we can conclude that Ptc14 is
unable to sequester Hh efficiently in either the embryo or imaginal discs and
that the ptc14 embryonic phenotype would be the result of
greater spreading of Hh and not to the constitutive activation of the Hh
pathway.
Ptc14 responds to Hh as the wild-type Ptc protein and activates
the signaling pathway indicating that the interaction of Ptc14 and
Hh is probably normal. However, this Hh-Ptc interaction does not necessarily
imply sequestration. Although Ptc14 occurs at the plasma membrane,
we do not observe internalization of Hh or extracellular Hh accumulation in
ptc14 mutant clones. These results, therefore, suggest
that Hh-Ptc interaction is not sufficient to sequester Hh and that an active
internalization process of Hh mediated by Ptc to control Hh gradient is
required. This Hh internalization mediated by Ptc is Dynamin-dependent, based
on the membrane accumulation of Hh and Ptc in shi mutant clones and
the lack of accumulation of Hh in shits1; ptc16
double mutant clones. However, the initiation of the internalization process
is not blocked in shi mutants because Dynamin is required for fission
of clathrin-coated vesicles after the internalization process has already
started (Kosaka and Ikeda,
1983; Ramaswami et al.,
1994
; Guha et al.,
2003
). This fact would explain why Hh gradient and signaling is
not extended when endocytosis is blocked in shi mutant cells. As
Ptc14 seems to have a problem in entering the endocytic compartment
and we have not found Hh accumulation in shits1;
ptc14 double mutant clones we conclude that the initiation of
the internalization process does not occur in Ptc14. Taken
together, these data indicate that only when Ptc forces Hh to the endocytic
pathway Hh is sequestered in the receiving cells.
The behavior of Hh and Ptc in dor- cells indicates that
after sequestration, Ptc internalizes Hh, and both Hh and Ptc are degraded.
Thus, controlling both endocytosis and degradation of Hh modulates its
gradient. Similar mechanisms have been described for controlling the
asymmetric gradient of Wg in embryonic segments
(Dubois et al., 2001). It is
possible that additional factors may contribute to shaping the Hh gradient,
because in large ptc- clones close to the AP border, which
lack Ptc protein to sequester Hh, an Hh gradient in endocytic vesicles was
also observed, although the range of this gradient was more extended than in
wild-type cells (data not shown). This is consistent with two mechanisms of Hh
internalization in Hh receiving cells as we have observed, one mediated by Ptc
and another not mediated by Ptc.
Role of Ptc in Hh signal transduction
From studies in both vertebrates and Drosophila, it was thought
that Hh protein binds to Ptc (Stone et
al., 1996; Fuse et al.,
1999
). Ptc is then internalized and traffics Hh to endosomal
compartments where both are degraded
(Denef et al., 2000
;
Incardona et al., 2002
), the
entire process triggering activation of the Hh pathway. It is shown here that
Ptc14 responds to Hh as would the wild-type Ptc protein in
activating the pathway. However, Ptc14 does not internalize Hh to
the endocytic compartment because is defective in endocytosis. We therefore
suggest that the massive Hh internalization by Ptc to control the gradient is
not a requirement for Hh pathway signal transduction.
In Hh signal transduction, the cellular mechanisms that regulate Smo
function remain unclear, although the distribution of Ptc/Smo suggests that
Ptc destabilizes Smo levels (Alcedo et al.,
2000; Denef et al.,
2000
; Ingham et al.,
2000
). It has also been proposed that Ptc-mediated Hh
internalization changes the subcellular localization of Ptc preventing Smo
downregulation (Denef et al.,
2000
). Furthermore, in cultured cells, Shh induces the segregation
of Ptc and Smo in endosomes, allowing Smo signaling, independently of Ptc
(Incardona et al., 2002
). It
is known, however, that binding of Shh to Ptc is not sufficient to relieve the
repression of the Hh pathway (Williams et
al., 1999
).
We show that as in wild-type cells, in the absence of Hh, Ptc14
downregulates both Smo levels and Smo activity, while in the presence of Hh,
the normal upregulation of Smo occurs. Consequently, Ptc14 levels
are high at the AP border because upregulation of Ptc by Hh occurs in the
absence of internalization of Hh to the degradative pathway. It might then be
expected that the high levels of Ptc14 not targeted to the
degradative pathway would block Smo activity. However, against all
predictions, the presence of Hh is still able to release Smo activity in
mutant ptc14 cells. Thus, there must be a positive
mediator of Smo activity to overcome the repressive effect of Ptc14
and allow Hh pathway activation in response to Hh. Alternatively, if
Ptc14 is located at the plasma membrane, it could control Smo
activity without entering the endocytic compartment by regulating the entrance
of small molecules, as has been recently proposed
(Taipale et al., 2002). In
fact, Ptc is similar to a family of bacterial proton-gradient-driven
transmembrane molecule transporters known as RND proteins
(Tseng et al., 1999
).
Accordingly, as a membrane transporter, Ptc could indirectly inhibit Smo
through translocation of a small molecule that conformationally regulates the
active state of Smo (Tseng et al.,
1999
). The inter-allelic complementation of Ptc suggests that Ptc
has the oligomeric structure needed for this type of transporter.
Although one of the normal functions of Ptc is to mediate Hh
internalization, the data demonstrate the presence of internalized Hh vesicles
in the absence of Ptc protein. It is therefore suggested that another receptor
mediates Hh internalization in Hh-receiving cells. This molecule could act as
a positive mediator of Hh signaling. Several observations have been published
that cannot easily be reconciled with the idea of Ptc acting as the only
receptor for Hh. For example, it was found that Hh activates signal
transduction in both A and P compartment cells of wing imaginal discs, despite
the absence of Ptc in P cells
(Ramirez-Weber et al., 2000;
Amanai and Jiang, 2001
).
Furthermore, it has been reported that some neuroblasts in Drosophila
embryos, the maturation of which is dependent on Hh, do not express or require
Ptc (Bhat and Schedl, 1997
).
This suggests that a receptor other than Ptc mediates Hh signaling. Recently,
the glypican protein Dally-like, which belongs to the heparan sulfate
proteoglycan protein family, was found to be required for Hh signal
transduction and probably for the reception of the Hh signal in
Drosophila tissue culture cells
(Desbordes and Sanson, 2003
;
Lum et al., 2003
). Dally-like
could act as co-receptor for Hh and it would be interesting to know if
Dally-like is required for Hh endocytosis. In addition, the large glycoprotein
`Megalin' has recently been identified as Shh-binding protein
(McCarthy et al., 2002
).
Megalin is a multi-ligand-binding protein of the low-density lipoprotein (LDL)
receptor family whose function is to mediate the endocytosis of ligands
(Argraves, 2001
) (reviewed by
Christensen and Birn, 2002
).
The finding that megalin-mediated endocytosed N-Shh was not efficiently
targeted to lysosomes for degradation suggests that N-Shh may also traffic in
complexes with Megalin and thus be recycled and/or transcytosed
(Argraves, 2001
). In the Wg
pathway, specific LDL receptor-related proteins are essential co-receptors for
Wnt ligands (Wehrli et al.,
2000
). Further investigation will determine whether LDL
receptor-related proteins could function as co-receptors that internalize Hh
in the absence of Ptc. Alternatively, these proteins could be required for
endocytosis and further delivery of Hh to Ptc in intracellular vesicles,
perhaps facilitating the transcytosis of Hh. A future challenge will be to
find other molecules that internalize Hh and to understand how Hh interacts
with Smo to activate the Hh pathway.
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ACKNOWLEDGMENTS |
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