A blood-borne PDGF/VEGF-like ligand initiates wound-induced epidermal cell migration in Drosophila larvae

Abstract

Epidermal cell migration is critical for restoration of tissue structure and function after damage. However, the mechanisms by which differentiated cells neighboring the wound sense the wound and assume a motile phenotype remain unclear. Here, we show that Pvr, a receptor tyrosine kinase (RTK) related to platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) receptors, and one of its ligands, Pvf1, are required for epidermal wound closure. Morphological comparison of wound-edge cells lacking Pvr or the Jun N-terminal kinase (JNK) signaling pathway previously implicated in larval wound closure suggests that Pvr signaling leads wound-margin epidermal cells to extend actin-based cell processes into the wound gap while JNK mediates transient dedifferentiation of cells at the wound margin. Genetic epistasis experiments reinforce the conclusion that the JNK and Pvr signaling pathways act in parallel. Tissue-specific knockdown and rescue experiments suggest that epidermally derived Pvf1 may be sequestered in the blood and that tissue damage exposes blood-borne Pvf1 to Pvr receptors on wound-edge epidermal cells and initiates the extension of cell processes into the wound gap. These results uncover a novel mechanism of sensing tissue damage and suggest that PDGF/VEGF ligands and receptors may play a conserved autocrine role in epidermal wound closure.

Figure 1

Pvr is an Epidermal Membrane Protein Required for Wound Closure (A) Percentage of larvae of each genotype that showed a defect in epidermal wound closure when knocked down via UAS-RNAi or a UAS-dominant negative transgene. N = 30. (B-D) Dissected epidermal wholemounts immunostained with anti-Fasciclin III (top panels, red) to label membranes and reveal wound architecture 24 hours post-wounding. (B) Larva expressing epidermal UAS-Pvr^RNAi. An open wound persists. (B′-B″) An unwounded larva of the same genotype expresses epidermal Fasciclin III (B′, green) but not Pvr (b″, blue). (C) Larva homozygous for Pvr^c02859. An open wound persists. (C′-C″) An unwounded larva of the same genotype expresses epidermal Fasciclin III (C, green) but not Pvr (C″, blue). (D) Larva homozygous for Pvr^c03211. Wound closure is normal. (D′-D″) An unwounded larva of the same genotype expresses epidermal Fasciclin III (D′, green) and Pvr (D″, blue). Scale bar for (B-D) is 100 μm.

Figure 2

The JNK and Pvr Signaling Pathways Control Distinct Cellular Aspects of Wound Closure (A-E) TEM of transverse sections of epidermal wholemounts of pinch wounded larvae. Control larvae (A-C) contained either w; Pxn-Gal, UAS-nlacZ (A-B) or w;; A58-Gal4, UAS-nlacZ to mark the wound site for sectioning. (A) Control larva, 8 hr post-wounding. Arrowhead, point of detachment from overlying cuticle. Bracket, 50 μm long thin process extending underneath cellular debris and into the wound gap. (B) Control larva, 4 hr post-wounding. Arrowhead, as in a. Note close apposition of process and wound site debris. (C) Control larva, 16 hr post-wounding. A new cuticle traps wound-site debris between itself and the old cuticle. Arrowhead, point of divergence between new and old cuticles. (D) Larva expressing UAS-Pvr^RNAi in the epidermis. Asterisk, cytoplasmic bulge of leading edge cell. Arrowhead, as in a. (E) Larva expressing UAS-bsk^DN in the epidermis. Arrowhead, terminus of cell process that continues to secrete cuticle. In all panels: d, wound site debris; e, epidermis; m, muscle; nc, new cuticle; oc, old cuticle; arrow, direction of original wound gap. All bars 10 μm. Bar in C is for B and C and bar in E is for D and E.

Figure 3

Epidermally-Produced Pvf1 Ligand is Required for Wound Closure (A-C) w, UAS-dsRed2Nuc, A58-Gal4 heterozygous larvae were pinch-wounded, dissected, fixed and immunostained for Fasciclin III (green) 24 hours post-wounding. Red, epidermal nuclei. (A) Female larva heterozygous for Pvf1^null. Wound closure is normal. (B) Male larva hemizygous for Pvf1^null. An open wound persists. (C) Larva in which A58-Gal4 drives pan-epidermal expression of UAS-Pvf1^RNAi. An open wound persists. (D) Percentage of larvae of each genotype that showed a defect in epidermal wound closure. Larvae mutant for Pvf1 or expressing UAS-Pvf1^RNAi within the epidermis show defects in wound closure, while larvae mutant for Pvf2 or expressing UAS-Pvf2^RNAi or larvae mutant for Pvf3 or expressing UAS-Pvf3^RNAi show no defects in wound closure. By contrast, fat body or blood cell expression of UAS-Pvf1^RNAi does not perturb wound closure (middle). N = 30. Scale bar in C for A-C, 100 μm.

Figure 4

The Proper Spatial Presentation of Pvf1 is Required for Wound Closure (A-F) Dissected epidermal wholemounts immunostained with anti-Fasciclin III (red). (A) Pvf1^null/Y male larvae with A58-Gal4. Open wound. (B) Larva in which A58-Gal4 drives pan-epidermal expression of UAS-Pvf1. Open wound. (C) Larva in which A58-Gal4 drives pan-epidermal expression of UAS-Pvf1 in a Pvf1^null mutant background. Ectopic Pvf1 expression in the epidermis fails to rescue the wound closure defect of Pvf1^null. (D) Pvf1^null/Y male larvae with Fat body-Gal4. Open wound. (E) Larva in which Fat body-Gal4 drives expression of UAS-Pvf1. Closed wound. (F) Expression of UAS-Pvf1 via Fat body Gal4 in a Pvf1^null mutant background. Ectopic Pvf1 expression rescues the wound closure defect of Pvf1^null. (G) Percentage of larvae of each genotype that showed a defect in wound closure. Expression of UAS-Pvf1^RNAi via epidermal A58-Gal4 completely blocks wound closure, expression from the weaker e22c-Gal4 driver gives a partial block, and expression from Fatbody-Gal4 rescues the wound closure defect of the Pvf1^null mutant. By contrast, epidermal overexpression of Pvf2 (Pvf2^d02444) or Pvf3 (UAS-Pvf3) does not block wound closure. N = 30. Scale bar in F for A-F, 100 μm.

Figure 5

Model of Pvr and JNK Signaling in Larval Epidermal Wound Closure Pvr signaling (green, left side of wound) controls cell process extension into the debris-filled (brown) wound gap. Hemolymph-borne Pvf1 (green circles) originally produced by the epidermis (blue cells) binds to exposed Pvr receptors (green glyphs) at the wound edge to activate signaling. JNK signaling (red, right side of wound) is activated through an as yet undiscovered ligand (red diamonds) and receptor (red glyphs) or mechanical signal. Once JNK is activated it suppresses secretion of and attachment to cuticle (purple) near the wound gap. Thick black line, basal lamina. At a real wound both pathways would be active at all wound margins. See text for details.