Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
* Author for correspondence (e-mail: benny.shilo{at}weizmann.ac.il)
Accepted 14 January 2004
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SUMMARY |
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Key words: Drosophila, PVR, PVF, VEGF, PDGF, Epithelial polarity, Wing epithelium, Actin organization
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Introduction |
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Among the molecular components of adherens junctions, cadherins and
catenins comprise the core of an adhesive interaction that connects subapical
actin belts of adjacent cells with each other
(Tepass, 2002). This
association with the cytoskeletal network is necessary for stable cell-cell
adhesion and for the integration of these contacts with the morphology of
epithelial cells. Several signaling pathways that are activated by cell
adhesion have been identified, most notably those regulated by small Rho-like
GTPases (Braga, 2002
). However,
the molecular mechanisms governing the interactions between adherens junction
components and the actin cytoskeleton remain largely unresolved issues.
Septate junctions are maintained by localization of intracellular PDZ
domain proteins such as Discs large (DLG) and Scribble. In combination with
their ability to multimerize, PDZ proteins have the potential to assemble
large multiprotein complexes at the cell membrane
(Bilder, 2001).
The generation and maintenance of polarity in epithelial cells is
intimately linked to the polarized distribution of transmembrane and secreted
proteins. The junctions between cells form a barrier not only to diffusion of
extracellular molecules between the cells, but also to movement of molecules
within the plasma membrane. Thus, in order to localize a transmembrane protein
to a particular cellular compartment (apical or basolateral), it must be
targeted to secretory vesicles that will specifically fuse with that membrane
compartment (Mostov and Cardone,
1995; Simons and Ikonen,
1997
; Wandinger-Ness et al.,
1990
).
In this study, we identify broad expression of the Drosophila PDGF/VEGF receptor (PVR) in epithelial tissues. We specifically examined possible roles of the PVR pathway in the wing disc epithelium. Two of the PVR ligands, PVF1 and PVF3, are deposited within the apical extracellular space, suggesting that polarized apical activation of the receptor may take place. Clones for null alleles of Pvr showed no phenotypes in the wing disc or pupal wing. However, when uniform activation of PVR was induced by a constitutively dimerizing receptor, loss of epithelial polarity, uniform appearance of adherens junctions and tumorous growth were observed in the wing disc. Elevation of full-length PVR levels also gave rise to prominent phenotypes, characterized by significantly higher levels of F-actin in the basolateral area of the epithelium. Taken together these results point to the importance of polarized apical PVR activation in maintenance of the wing disc monolayered epithelium, via regulation of localized actin microfilament polymerization.
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Materials and methods |
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Fly strains
The following Gal4 driver lines were used to express the various
transgenic constructs: 69B-Gal4 (expressed uniformly in the embryonic
ectoderm), actin-Gal4 (expressed ubiquitously), MS1096-Gal4
(expressed in the pouch of the wing imaginal disc), patched-Gal4 and
dpp-Gal4 (expressed along the anterior-posterior border of the wing
disc), GMR-Gal4 (expressed in differentiating photoreceptor cells),
and eyeless-Gal4 (expressed in all eye precursor cells). For
generation of Pvf1 `flip out' clones the pUAST-Pvf1 line was
crossed to a line containing hs-flp, UAS-GFP and
actin>CD2>Gal4. To generate larval clones, a 30-minute heat
shock at 37°C was applied at 72-96 hours AEL. Flies containing
Pvr were obtained from P. Rorth.
The Pvr mutant VegfrC2195 [obtained from
Exelixis (Cho et al., 2002)]
was recombined with FRT 40A. Clones were generated following a cross
to hs-flp; FRT 40A, ubi-GFP (Bloomington #5629) or hs-flp; FRT
40A, arm-lacZ (Bloomington #6579). Three heat shocks of 1.5 hours each at
37°C were applied, starting at 24-48 hours AEL, at 24 hour intervals.
Antibodies
EST SD02385 was used to insert the region encoding PVR C-terminal residues
1291-1509 into pRSETB. Recombinant protein was purified on a NINTA column, and
injected into rats. Typically, the antibody is diluted 1/200 for
immunohistochemical staining. EST LD28763 was used to insert the region
encoding PVF1 residues 27-303 into pRSETA. Recombinant protein was injected
into rats. The antibody is diluted 1/100 for staining. To generate antibodies
recognizing PVF3, PCR amplification of the region encoding residues 22-359 was
carried out. The template used was Vegf 27Ca cDNA. The fragment was cloned
into pRSETA. The purified recombinant protein was injected into guinea pigs,
and the antibody used at a dilution of 1/100 for staining.
Rabbit anti-DLG was obtained from P. Bryant, mouse anti-LGL antibodies were obtained from F. Matsuzaki, Profilin antibodies (chi1J) from the Developmental Studies Hybridoma bank, TRITC-labeled phalloidin was purchased from Sigma, and Alexa 488 phalloidin and fluorescein conjugate deoxyribonuclease I from Molecular Probes. Monoclonal anti-GFP antibodies were obtained from Roche, and diluted 1/10 for staining, and rabbit anti-beta galactosidase from Cappel (diluted 1/1000). Secondary antibodies were obtained from Jackson ImmunoResearch, and diluted at 1/300 for staining.
Staining and microscopy
Antibody staining of wing discs was according to Neumann and Cohen
(Neumann and Cohen, 1997).
Staining of extracellular distribution of PVF1 and PVF3 was as described
previously (Strigini and Cohen,
2000
). For extracellular staining, antibodies were used at a
dilution of 1/30 for PVF1 or PVF3, and 1/3 for anti-GFP. Incubation with these
antibodies was carried out on ice, to block endocytosis. Staining of pupal
wings was as described previously (Fristrom
et al., 1994
) with the following modifications: Pupal discs were
fixed at 4°C for 48 hours, and mounted in Aquamount after staining.
For sections, wing discs were fixed in 4% glutaraldehyde and 4% formaldehyde, embedded in JB4 (Polysciences), and stained in Hematoxylin and Eosin after sectioning.
For EM, discs were fixed by immersion in freshly prepared 3% paraformaldehyde, 2% glutaraldehyde in cacodylate buffer containing 5 mM CaCl2 (pH 7.4) for 2 hours at room temperature and for 12 hours at 4°C. After washing, the tissue was post fixed in osmium tetroxide, 0.5% potassium dichromate and 0.5% potassium hexacyanoferrate in cacodylate buffer for 1 hour. The tissue was stained and blocked with 2% aqueous uranyl acetate followed by ethanol dehydration. The samples were embedded in EMBED 812. Thin sections were cut using a diamond knife, stained with 2% uranyl acetate and Reynolds's lead citrate, and examined with a transmission electron microscope (Philips, CM12) at an accelerating voltage of 120 kV.
Heparin binding assay
Schneider S2 cells were transiently transfected with actin-Gal4 and
pUAST-PVF1 or pUAST-sGFP plasmids, and grown in serum-free medium for 3 days.
Medium was collected and incubated for 3 hours with Heparin-Sepharose cl-6B
beads (Pharmacia). Beads were washed with PBS, and bound material eluted with
1.5 M NaCl. The presence of PVF1 or sGFP in the medium was monitored by
western blotting with anti-PVF1 or anti-GFP, respectively.
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Results |
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Expression of the ligands PVF1 and PVF3
Three putative PVR ligands have been identified following
Drosophila genome sequencing. One of them, termed PVF1, was also
identified in a functional ovary misexpression screen and shown to activate
the receptor in cell culture assays. It represents the major PVR ligand in
border cells, a migratory subset of ovarian follicle cells
(Duchek et al., 2001). PVF2 was
shown to be required for viability and proliferation of larval hemocytes
(Munier et al., 2002
). The
third ligand, PVF3, has a redundant role with the other two ligands, in
promoting embryonic hemocyte migration (Cho
et al., 2002
). We generated antibodies against PVF1 and
demonstrated their capacity to recognize this protein in embryos, in a pattern
similar to the one observed for the Pvf1 transcript by RNA in situ
hybridization (not shown). These antibodies do not cross react with the other
two ligands, as demonstrated by their failure to detect expression of PVF2 and
PVF3 in the embryonic midline. In larval wing discs, PVF1 is specifically
localized to the apical side of the disc epithelium
(Fig. 2A). In this domain the
disc forms a sac-like structure, with the layer of squamous peripodial cells
on the opposite side. The pupal wing is a bilayered structure, with the basal
side of each layer facing the other, and the apical sides facing outward. PVF1
localization in the pupal wing remained apical
(Fig. 2I). Antibodies were also
raised against PVF3, and shown to detect the expected embryonic midline
expression (not shown). In the wing disc, they detect a distinct apical
distribution, similar to PVF1.
|
Overexpression of PVF1, using MS1096-Gal4, a strong wing-specific driver, retained the apical localization of PVF1 (Fig. 2C). Despite the high levels of apical PVF1 (one to two orders of magnitude above endogenous levels), we did not detect any spread of the ligand to the basolateral side, indicating that the cell junctions form an efficient barrier to PVF1 diffusion towards the basolateral domain. Incubation of live discs overexpressing PVF1 with the antibody demonstrated that the majority of apical PVF1 is extracellular (not shown). The restricted apical localization of overexpressed PVF1 was maintained at the pupal stage (not shown).
Both endogenous PVF1 and PVF3 accumulate on the apical side of the disc
epithelium. This is unusual, as other ligands used for patterning the wing
disc (e.g. WG) were shown to be localized at the basolateral side
(Strigini and Cohen, 2000). To
ask if PVF1 and PVF3 use a common machinery for their secretion which results
in apical accumulation, we overexpressed both ligands simultaneously.
Overexpression of PVF1 alone did not perturb its apical accumulation. By
contrast, overexpressed PVF3 was detected in a punctate pattern within the
wing disc cells (Fig. 2D). The
ligand failed to be secreted, probably owing to saturation of specific
components of the secretory pathway, and no staining was detected when the
extracellular PVF3 was probed by staining live discs (not shown).
Co-expression of PVF1 with PVF3 led to a significant reduction in the level of
apical PVF1, and elevated levels in punctate structures within the cells
(compare Fig. 2C with 2E). In
many of these puncta, colocalization of PVF1 and PVF3 was observed
(Fig. 2F, inset).
The capacity of PVF3 overexpression to inhibit secretion of PVF1 is specific. When expressed alone, secreted GFP (sGFP) could be detected in both apical and basolateral domains (Fig. 2G). No alterations in this pattern were observed following coexpression with PVF3 (Fig. 2H). Optical sections show that sGFP was located between the cells in the basolateral region, while PVF3 was trapped within the cells (Fig. 2H, inset). These results indicate that PVF1 and PVF3 use a discrete secretory mechanism, leading to exclusive apical deposition, which is distinct from the bulk flow, represented by sGFP.
We next examined whether PVF1 is sequestered following secretion, or free to diffuse within the apical domain. As overexpressed PVF1 maintained its restricted apical localization, we expressed the ligand in clones of cells marked by GFP (`flipout' clones), and monitored its distribution over adjacent cells. Detection was performed under conditions of low laser illumination intensity, which identify only overexpressed PVF1 but not the endogenous ligand. Excess PVF1 could be uniformly detected within the entire apical space of the disc epithelium (Fig. 2J). This result indicates that following apical secretion of PVF1, the ligand is not trapped on the apical extracellular surface of the producing cells, but is capable of extensive lateral diffusion.
Heparin binding properties of PVF1
One of the hallmarks of vertebrate VEGF molecules is their capacity to bind
heparin. This property is mapped to a distinct domain located C-terminal to
the receptor-binding domain. Removal of this domain in one of the mammalian
VEGF splice isoforms (VEGF121), generates a molecule that is
capable of binding the receptor, while failing to bind heparin
(Park et al., 1993a). Although
PVF1 shows no distinct homology to the heparin-binding domain of VEGF, it may
still have the capacity to bind heparin. To test for heparin-binding
properties of PVF1, medium derived from Schneider S2 cells overexpressing PVF1
was collected. Incubation with heparin-Sepharose beads showed a specific and
efficient binding of PVF1. Most of the protein was removed from the medium.
The bound PVF1 was eluted from the beads at 1.5 M NaCl. No such heparin
binding was detected for the control secreted GFP protein
(Fig. 3).
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Overexpression of PVR induces actin microfilament polymerization
The dramatic effect of -PVR expression prompted us to study the
phenotypic consequences resulting from overexpression of PVR. Ubiquitous
expression of a full-length construct of PVR in the wing disc results in pupal
lethality. When we analyzed the wing disc upon overexpression of PVR in a
specific domain along the anteroposterior border using the dpp-Gal4
driver, we noticed a significant elevation in the level of F-actin along the
basolateral area of the epithelium (Fig.
6B,C). Conversely, the levels of G-actin were slightly lower than
in the surrounding cells (Fig.
6D) indicating that the elevation in PVR levels enhanced actin
polymerization, rather than induced actin transcription. The elevation in
actin polymerization was accompanied by elevation in the Chickadee (Profilin)
protein that binds actin monomers (Fig.
6F). Consequently, an irregular fold was formed within the domain
expressing PVR (Fig. 6H).
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We analyzed wing discs that expressed RNAi-GOF under the control of ptc-Gal4. In accordance with the phenotypes caused by UAS-Pvr expression, F-actin organization was altered specifically at the basal side of the epithelium (Fig. 7C). Elevation in Chickadee levels was also observed (not shown). Examination of components of the adherens junction (phospho-tyrosine) as well as septate junctions (DLG protein) revealed no change in the localization or the levels of these proteins (not shown).
We also examined the effects of expressing RNAi-GOF in the pupal wing, to determine the basis for the adult blistering phenotype. In the pupal wing, F-actin in each layer is normally enriched not only at the apical side as in larval discs, but also in the basal region (Fig. 7D,F). Although the organization of apical F-actin was unaffected, an ectopic and highly irregular accumulation of F-actin at the basolateral side of MS1096Gal4/RNAi-GOF pupal wings was observed (Fig. 7E,G).
The specificity of the gain-of-function effect was strengthened by genetic interaction experiments. Females heterozygous for the driver MS1096-Gal4 and RNAi-GOF show a mild wing phenotype (Fig. 7I). When the ligand PVF1 was overexpressed, accumulation of excess ligand at the apical side was observed (Fig. 2C), but no apparent phenotype ensued (Fig. 7K). If expression of the RNAi-GOF construct leads to a gain-of-function phenotype, excess apical PVF1 should result in a more severe phenotype. Indeed, co-expression of RNAi-GOF with Pvf1 gave rise to an enhanced wing phenotype that was fully penetrant. Females containing a single copy each of MS1096-Gal4, UAS-RNAi-GOF and UAS-Pvf1 showed a dramatic enhancement. The wings were not only blistered, but also reduced in size and highly folded (compare Fig. 7L with 7I,K). Thus, the excess apical PVF1 enhanced the effect of RNAi-GOF.
PVF localization and PVR activation phenotypes are tissue specific
In view of the retention of PVF1 and PVF3 on the apical extracellular
surface of the wing imaginal disc, we examined whether a similar localization
also takes place in other epithelial tissues. PVF1 was overexpressed in the
embryonic ectoderm and the eye imaginal disc. In both cases, no polarized
localization of the ligand could be observed either within or outside the
cells (Fig. 8A,B). Furthermore,
no deleterious phenotypes resulted from this manipulation
(Fig. 8A). As the uniform
distribution of ectopic PVF1 in the embryonic ectoderm and eye disc is
expected to trigger the receptor uniformly in the responding cells we
concluded that apical localization of PVF1 in these tissues is not
necessary.
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Discussion |
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Expression of PVR and its ligands
Examination of PVR protein revealed a broad expression in epithelial
tissues in the embryo from stage 14, and in the imaginal discs. PVR expression
is not confined along the apicobasal axis of the cells. In contrast to the
uniform distribution of the receptor, there is restricted apical localization
of the ligands PVF1 and PVF3 within the wing disc epithelium.
The mechanism responsible for the apical accumulation of PVF1 and PVF3 in
the wing disc is intriguing. As cell junctions are likely to form barriers
that can not be bypassed by exogenous ligand, apical accumulation may imply
preferential secretion of PVR ligands at the apical compartment. It is
possible that PVF1 and PVF3 are targeted to vesicles that are specifically
marked for secretion at the apical surface. The observation that PVF3
overexpression compromises the secretion of PVF1 but not that of sGFP,
supports such a possibility. The presence of distinct secretory vesicles which
are targeted to apical versus basolateral compartments has been previously
observed (Jacob and Naim,
2001).
What further interactions do PVF1 and PVF3 undergo, once secreted to the
apical extracellular compartment? One possible interaction involves binding to
heparan-sulfate proteoglycans on the cell surface. The vertebrate VEGF
proteins have a defined heparin-binding domain at the C terminus that is
distinct from the receptor-binding moiety
(Park et al., 1993b). The
equivalent C-terminal domain of PVF1 does not show a distinct homology to the
heparin-binding domain of VEGF. We have shown, however, that PVF1 secreted by
S2 cells can bind heparin beads. To examine if PVF1 is trapped on the cell
surface following secretion, we created marked clones of cells overexpressing
PVF1. We find that the ligand is uniformly redistributed along the entire
apical surface, including the surface of cells not secreting the ligand.
Although the ligand is capable of spreading readily within the apical plane,
it is incapable of crossing the cell junctions, and is thus excluded from the
basolateral extracellular compartment.
What is the role of PVR in the wing disc?
Accumulation of PVF1 and PVF3 at the extracellular apical compartment
implies that the PVR receptor is activated in a polarized fashion. Does such
an apically polarized pattern of activation play a role in shaping the wing
disc epithelium? The most direct way to examine PVR function in the wing disc
is to generate clones for null Pvr alleles, and follow their
phenotype. The Pvr-mutant clones were similar in size to their
wild-type twins, and within the clones no aberrant morphology or
misorganization of actin was detected. We thus conclude that PVR has a
redundant role in the wing. Nevertheless, a series of dramatic wing phenotypes
is induced following expression of various PVR constructs. Our analysis leads
us to propose that these phenotypes represent gain-of-function circumstances
following inappropriate activation of PVR on the basolateral side of the wing
disc epithelium.
We were first drawn to this interpretation by the dramatic effects of
non-restricted and constitutive receptor activation, achieved by expression of
PVR in the wing disc epithelium. The epithelium lost its polarity,
multiple cell layers were generated, and giant tumorous discs were formed.
Ectopic accumulation of F-actin around the circumference of the cells was
observed, and corroborated by the identification of multiple adherens
junctions in EM images.
The phenotype created by expressing PVR in the wing disc is
reminiscent of the phenotype described for loss of the septate junction
proteins DLG and Scribbled, as well the LGL protein. We believe that the
alterations in cell polarity following
PVR expression are less severe
than the dlg, scribble or lgl mutant phenotypes. Although
excess adherens junctions are established and the septate junctions are
mislocalized, the LGL protein, which requires intact septate junctions for its
insertion into the membrane, is found associated with the membrane in wing
discs expressing
PVR. The tumorous growth of the cells is believed to
be a secondary consequence of the loss of polarity, which may lead to
impairment of cell-cell communication.
We examined also the consequences of misexpressing full-length PVR. We
noticed significantly higher levels of F-actin in the basolateral area of the
cells expressing PVR, while the level of actin monomers was lower. This
indicates that PVR has a localized effect on actin polymerization, rather than
a general role in actin monomer synthesis. We also noticed elevation in
Profilin (chickadee) protein levels. Profilin binds actin monomers in a way
that inhibits nucleation and elongation of pointed ends but promotes rapid
elongation of uncapped barbed ends, leading to depletion of the actin monomer
pool (Amann and Pollard,
2000).
Overexpression of the ligands alone did not lead to any phenotype, while even mild overexpression of the receptor resulted in pronounced phenotypes. Moreover, these phenotypes were strongly enhanced by elevating the levels of PVF1 in the apical domain. There are two possible explanations for the overexpression phenotype. The overall levels of receptor activation may be important. The receptor could be present in limited amounts, so that increasing its levels allows more ligand at the apical side to bind and activate receptors. Alternatively, polarized activation of the receptor that normally takes place is disturbed, because of redistribution of the ligand. This may happen by recycling of the ligand-bound receptor inside the cells. The fact that elevation in PVR levels resulted in basolateral polymerization of actin, while the ligand is normally found on the apical side supports this possibility. Mislocalized activation of the receptor may also take place because of spontaneous dimerization caused by the higher levels of the receptor.
It is interesting to note that while PVR gave rise to a dramatic
phenotype when expressed in the wing disc or the follicular epithelium, no
apparent phenotypes were observed following expression in the embryonic
ectoderm or the eye disc. Some of the intracellular elements that may be
essential for relaying the signals resulting from PVR activation could thus be
expressed or active only in a restricted set of tissues.
In the embryonic ectoderm and eye disc where PVR was inactive, we
also failed to see apical accumulation of PVF1. The correlation between the
capacity of the wing epithelium to localize the ligands apically, on the one
hand, and to respond to uniform PVR activation, on the other, strengthens the
notion that apical activation of PVR is instructive in this tissue.
What can the ectopic phenotypes teach us with regards to the normal
downstream responses to PVR activation in the wing epithelium? The primary
defect upon overexpression of PVR is misorganization of the actin cytoskeleton
at the basolateral side. In addition, expression of the constitutively active
receptor results in multiple adherens junctions. We thus suggest that apically
restricted PVR activation provides signals that facilitate the formation of
F-actin at the adherens junctions. This role is reminiscent of the activity of
PVR in the border cells of the ovary, where polarized activation by PVF1,
expressed in the oocyte, participates in guiding the migration of the border
cells (Duchek et al., 2001). It
is tempting to suggest that polarized PVR activation regulates migration or
cell polarity, using a common set of intracellular responses leading to
localized actin polymerization.
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ACKNOWLEDGMENTS |
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