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. 2002 Dec;3(12):1195-200.
doi: 10.1093/embo-reports/kvf242. Epub 2002 Nov 21.

PVF2, a PDGF/VEGF-like growth factor, induces hemocyte proliferation in Drosophila larvae

Anne-Isabelle Munier  1 , Daniel Doucet, Emmanuel Perrodou, Daniel Zachary, Marie Meister, Jules A Hoffmann, Charles A Janeway Jr, Marie Lagueux
Affiliations

PVF2, a PDGF/VEGF-like growth factor, induces hemocyte proliferation in Drosophila larvae

Anne-Isabelle Munier et al. EMBO Rep. 2002 Dec.

Abstract

Blood cells play a crucial role in both morphogenetic and immunological processes in Drosophila, yet the factors regulating their proliferation remain largely unknown. In order to address this question, we raised antibodies against a tumorous blood cell line and identified an antigenic determinant that marks the surface of prohemocytes and also circulating plasmatocytes in larvae. This antigen was identified as a Drosophila homolog of the mammalian receptor for platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF). The Drosophila receptor controls cell proliferation in vitro. By overexpressing in vivo one of its putative ligands, PVF2, we induced a dramatic increase in circulating hemocytes. These results identify the PDGF/VEGF receptor homolog and one of its ligands as important players in Drosophila hematopoiesis.

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Figures

Figure 1
Figure 1
Generation of monoclonal antibodies against cell surface antigens. (A) Flow cytometric analysis: live mbn-2 cells are gated according to their forward and side scatter profiles (FSC- and SSC-Height). (B) Flow cytometric detection of mbn-2 cells labeled with antibody. A secondary FITC-labeled antibody allows the detection of stained cells (black curve). Cells incubated only with the secondary FITC antibodies served as control to measure fluorescence background (plain gray curve). (C) Mitotic rate of mbn-2 cells measured by [3H]thymidine incorporation incubated with different antibodies (1–15; 18G is number 6). C, control: cells without antibody. E, cells incubated with 20-hydroxyecdysone; this ecdysteroid was used as a control because it has been shown to impair proliferation of mbn-2 cells (Gateff et al., 1980; Dimarcq et al., 1997). (D) The effect of 18G on cell proliferation increases in a dose-dependent manner. Black bars, 1 × 105 cells/well; gray bars, 5 × 104 cells/well. (E) Western blot analysis (under non-reducing conditions) of mbn-2 cell and larval blood cells (B.C.) extracts using 18G antibody. (F) Western blot analysis (under non-reducing conditions) using 18G antibody of S2 cell extracts after mock transfection (control) or transfection with Gfp dsRNA and Pvr dsRNA (experiments were performed as in Duchek et al., 2001). Molecular weight markers are indicated (in kDa).
Figure 2
Figure 2
The 18G antibody stains prohemocytes and circulating plasmatocytes. Blood smears from Oregon third instar larvae (A and B) or from hopTum-l third instar larvae (C and D). Dissected and dilacerated lymph glands of Oregon third instar larvae (E and F) or from hopTum-l third instar larvae (G and H). Staining were performed using 18G antibody followed by a peroxidase-conjugated goat anti-mouse IgG (A, C, E and G) or for controls an irrelevant IgG (human CD45) mouse antibody followed by a peroxidase-conjugated goat anti-mouse IgG (B, D, F and H). The arrowhead points to plasmatocytes, the thin arrows to prohemocytes and the thick arrows to lamellocytes.
Figure 3
Figure 3
Ectopic expression of PVF2 promotes the proliferation of hemocytes. (A) Blood drop of a third instar larva overexpressing PVF2 (w; UAS-Pvf2/+; e33C-GAL4/+). (B) Blood drop of control larva (w; UAS-Pvf2/+; TM6B/+). (C) Blood drop of a third instar larva overexpressing PVF1 (w; UAS-Pvf1/+; e33C-GAL4/+). (D) Blood drop of a control larva (w; UAS-Pvf1/+; TM6B/+). Nuclei of blood cells are stained with DAPI.
Figure 4
Figure 4
Blood cell types present in larvae overexpressing PVF2. (A and B) Plasmatocytes are evidenced by their phagocytotic activity revealed by the presence of India ink (arrowheads; Lanot et al., 2001). (A) Blood smears of larvae overexpressing PVF2 (w; UAS-Pvf2/+; e33c-GAL4/+). (B) Blood smears of control larva (w; UAS-Pvf2/+; TM6B/+). (C and D) Dividing cells, prohemocytes, are evidenced by immunostaining with an anti-phosphohistone H3 antibody. (C) Blood smears of larvae overexpressing PVF2 (w; UAS-Pvf2/+; e33c-GAL4/+). (D) Blood smears of control larva (w; UAS-Pvf2/+; TM6B/+). (E and F) The presence of crystal cells is revealed by a 10 min heating at 60°C; with this treatment, they turn black (Rizki et al., 1980). (E) No crystal cells are observed in larvae overexpressing PVF2 (w; UAS-Pvf2/+; e33c-GAL4/+), as opposed to (F) control larvae (w; UAS-Pvf2/+; TM6B/+).
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