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First published online 31 March 2004
doi: 10.1242/dev.01101

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Apical accumulation of the Drosophila PDGF/VEGF receptor ligands provides a mechanism for triggering localized actin polymerization

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel

View larger version (113K): [in a new window]   Fig. 1. Expression of PVR was detected by antibodies directed against the C-terminal region of the protein, and appeared to follow cell outlines. (A-C) From embryonic stage 14 onwards, prominent expression was detected throughout the embryonic ectoderm. PVR is also expressed in hemocytes (arrowheads) and midline glial cells (arrow). In B, muscles are stained with anti-myosin (green). (D) In the third instar larval eye disc, general cell-membrane association of PVR is observed. (E,F) In the third instar larval wing disc, the receptor is similarly expressed. F is an optical cross-section, showing the uniform distribution of PVR along the apicobasal axis. In all cross-sections, apical is shown towards the top. (G) Expression of PVR persists in the pupal wing.

View larger version (87K): [in a new window]   Fig. 2. Apical localization of PVF1 and PVF3. All panels except insets represent optical cross-sections of third instar larval wing imaginal discs. (I) A section of a pupal wing. Apical is towards the top, and basal towards the bottom. (A) Apical accumulation of endogenous PVF1 in a wild-type wing imaginal disc. (B) Similarly, PVF3 is also apically concentrated. In this panel, live discs were incubated with the PVF3 antibody prior to fixation, thus detecting only the extracellular PVF3. (C) Upon overexpression by MS1096-Gal4, PVF1 maintains its apical localization, whereas PVF3 accumulates within the producing cells (D). (E,F) Overexpression of PVF1 together with PVF3 reduced the portion of apically localized PVF1, which instead accumulated within the cells. In many of the intracellular puncta, colocalization of PVF1 and PVF3 was observed (arrows). The inset shows a section from the basolateral region. (G) Discs expressing sGFP show concentration on the apical side, but after secretion, significant levels were also detected between the cells on the basolateral region. (H) Co-expression of sGFP with PVF3 did not alter its distribution. The inset is a section obtained from the basolateral region. Comparison with the inset in F demonstrates that while PVF1 is trapped within the cells, sGFP is readily detected between the cells. (I) In a wild-type bilayered pupal wing, accumulation of PVF1 on the apical side of both layers is observed. (J) Clones overexpressing PVF1 (marked by GFP) were generated. PVF1 localization was monitored at a laser intensity that detects the overexpressed protein but not the endogenous one. Apical accumulation of PVF1, which was uniform above expressing and non-expressing cells was observed.

View larger version (40K): [in a new window]   Fig. 3. PVF1 binds heparin. The capacity of PVF1 to bind heparin was examined as a possible mechanism for activation of the ligand in the wing imaginal disc. Schneider S2 cells were transfected with a PVF1-expression construct, and medium was collected. Incubation of this medium with heparin beads showed that most of the PVF1 protein was removed by the beads, and could be efficiently eluted by 1.5 M NaCl. PVF1 did not bind to control sepharose beads. By contrast, medium of cells expressing secreted GFP showed that the protein was not bound to the heparin beads. Equal amounts of medium were loaded before and after incubation with the beads. PVF1 was detected by probing the blot with anti-PVF1 serum, and sGFP was followed with anti-GFP.

View larger version (108K): [in a new window]   Fig. 4. Clones lacking endogenous PVR have no apparent wing phenotype. (A,B) Clones homozygous for a Pvr mutation (marked by absence of GFP) show normal tissue organization and F-actin distribution in the larval wing imaginal disc. Note that the size of the mutant clones is similar to that of the wild-type twin clones showing enhanced levels of GFP. As the disc is slightly tilted, this optical section shows the apical domain of the disc on the left part, and the basolateral region on the right. (B) Note that normal F-actin accumulation is observed in the former, and no ectopic accumulation in the latter, regardless of the clone boundaries. (C-E) Similar Pvr mutant clones were analyzed in the pupal wing, marked by the absence of ß-gal staining, or PVR staining. In the case of PVR staining, only a background signal that is not membrane-associated was detected within the clones, implying that the PVR protein expressed by this allele does not contain the C-terminal domain recognized by the antibody. Again, normal tissue organization and F-actin distribution was observed.

View larger version (120K): [in a new window]   Fig. 7. Synergistic interaction between elevated PVR levels and excess apical PVF1. As PVR overexpression leads to pupal lethality, we were able to induce lower levels of PVR by using an unusual insertion of the UAS-Pvr-RNAi construct. Although most Pvr-RNAi constructs lead to a reduced level of PVR (A, arrow), this insertion (termed RNAi-GOF) gave rise to elevated PVR levels (B, arrow). This elevation is also reflected in defects in actin microfilament organization at the basal side of the epithelium (C, arrow). As RNAi-GOF flies were viable, it was possible to examine their pupal wings. We noted that whereas the organization of apical F-actin was unaltered (D,E), a dramatic misorganization was induced at the basal domain (F,G). Expression of adult wing phenotype. Females carrying two copies of MS1096-Gal4 had normal wings (H). Minor defects could be observed with a single dose of MS1096-Gal4 and UAS-RNAi-GOF (I). This phenotype was enhanced when having two copies of the Gal4 driver (J). Overexpression of Pvf1, which accumulates at the extracellular apical side, does not lead to defects in the wing (K). Flies containing a single dose of the driver and UAS-RNAi-GOF and UAS-Pvf1 show a dramatic enhancement of the phenotype (L). This demonstrates that the phenotypes observed following PVR overexpression represent activation of the endogenous pathway by its ligands.

View larger version (133K): [in a new window]   Fig. 5. Uniform PVR activation abolishes cell polarity and generates tumorous wing discs. (A-C) A constitutively activated PVR construct was expressed in the wing imaginal disc, to follow the consequences of uniform PVR activation. Five days after egg lay (AEL), the size of these discs was normal, but aberrant overall tissue organization was observed (compare a wild-type disc in A with B). These larvae failed to pupariate and continued to grow. Fifteen days AEL, these larvae contained wing imaginal discs that were approximately five times larger than normal discs (C). (D,E) Cross-sections show that discs expressing PVR (E) were multilayered, and the cells appeared non-polarized, in contrast to the layered epithelium of a wild-type disc (D). The arrow in E shows a region at the periphery of the disc where MS1096-Gal4 is not expressed, and the simple epithelial structure was retained. (F) Apical section through a wild-type disc shows the ordered organization of F-actin. (G) In MS1096-Gal4/UAS-PVR discs a higher level of F-actin with a highly unorganized distribution is observed. (H) Optical cross-section of this disc shows that within the multilayered structure that is generated, F-actin appears to be distributed in a uniform, non polarized manner within each cell. (I,J) The above observation is corroborated by EM studies. While wild-type disc cells show only a single adherens junction per cell (marked by electron-dense material on both sides of the membrane bilayer, arrows), multiple adherens junctions per cell were identified in the cells expressing PVR. (K) The presence of DLG associated with the membranes of PVR cells suggests that the septate junctions are retained. However, their distribution is no longer polarized. (L) In wild-type epithelial cells, LGL is associated with the plasma membrane. (M,N) In PVR discs, LGL continues to be associated with the membrane (possibly even at higher levels), indicating that the septate junctions that mediate its membrane association are functional. Taken together, these results imply that normal activation of PVR in the wing imaginal disc is polarized, as uniform PVR activation leads to a complete loss of cell polarity. In addition to the disrupted distribution of polarity markers, PVR specifically elevates the levels of F-actin.

View larger version (97K): [in a new window]   Fig. 6. PVR overexpression induces actin polymerization. UAS-Pvr was overexpressed in the wing disc by the dpp-Gal4 driver (A,D,F). Elevation in actin microfilaments was observed in cells overexpressing PVR as monitored by Phalloidin staining (B). Cross-section shows that the excess actin filaments are located in the basolateral area of the cells (C, arrow). Conversely, a reduction in actin monomers was observed in the same region by staining with flourescein-DNaseI (D). Profilin (Chickadee), which binds free actin and affects actin polymerization dynamics, is elevated in the stripe of PVR overexpression (F, arrow). The changes in actin cytoskeleton following PVR overexpression are eventually manifested in a general change in epithelial morphology. An extra and irregular fold is observed and demonstrated with FasIII which marks the outlines of all epithelial cells (H, arrowhead). E and G show the domain overexpressing PVR.

View larger version (85K): [in a new window]   Fig. 8. Polarized activity of PVF1/PVR is specific to the wing imaginal disc. The polarizing activity of PVF1/PVR was followed in other epithelial tissues in which PVR is normally expressed. (A,B) In the eye imaginal disc, induction of Pvf1 by GMR-Gal4 leads to a uniform apicobasal distribution of PVF1, without any apparent defects. (C) Uniform ectodermal expression of Pvr in the embryo (by 69B-Gal4) did not disrupt the polarity of the ectoderm and the secreted cuticle. Arrow shows the position of a hole induced in the dorsal head region. (D,E) Uniform expression of Pvr in the eye disc did not disturb the organization of the epithelium and the distribution of actin. Taken together, these results demonstrate that the apical retention of PVF1, as well as the responses to PVR, are specific to the wing imaginal disc.