A targeted UAS-RNAi screen in Drosophila larvae identifies wound closure genes regulating distinct cellular processes

Abstract

Robust mechanisms for tissue repair are critical for survival of multicellular organisms. Efficient cutaneous wound repair requires the migration of cells at the wound edge and farther back within the epidermal sheet, but the genes that control and coordinate these migrations remain obscure. This is in part because a systematic screening approach for in vivo identification and classification of postembryonic wound closure genes has yet to be developed. Here, we performed a proof-of-principle reporter-based in vivo RNAi screen in the Drosophila melanogaster larval epidermis to identify genes required for normal wound closure. Among the candidate genes tested were kinases and transcriptional mediators of the Jun N-terminal kinase (JNK) signaling pathway shown to be required for epithelial sheet migration during development. Also targeted were genes involved in actin cytoskeletal remodeling. Importantly, RNAi knockdown of both canonical and noncanonical members of the JNK pathway caused open wounds, as did several genes involved in actin cytoskeletal remodeling. Our analysis of JNK pathway components reveals redundancy among the upstream activating kinases and distinct roles for the downstream transcription factors DJun and DFos. Quantitative and qualitative morphological classification of the open wound phenotypes and evaluation of JNK activation suggest that multiple cellular processes are required in the migrating epidermal cells, including functions specific to cells at the wound edge and others specific to cells farther back within the epidermal sheet. Together, our results identify a new set of conserved wound closure genes, determine putative functional roles for these genes within the migrating epidermal sheet, and provide a template for a broader in vivo RNAi screen to discover the full complement of genes required for wound closure during larval epidermal wound healing.

Figure 1.—

Temporal progression of wound closure in control larvae. (A–E) Dissected whole mounts of larvae heterozygous for the e22c-Gal4 driver and UAS-DsRed2-Nuc. Nuclei expressing DsRed2-Nuc are red and membranes immunostained for Fasciclin III are green. (A) Unwounded epidermal sheet. (B–E) Epidermal sheet after wounding: (B) ∼5 min post wounding, (C) 4 hr post wounding, (D) 8 hr post wounding, and (E) 24 hr post wounding. Note that the epidermal sheet is closed. Solid arrowheads, wound-edge cells that retain Fasciclin III staining facing the wound gap; open arrowheads, wound-edge cells that lack Fasciclin III staining facing the wound gap; arrows, elongated epidermal cells; dots, multinucleate epidermal cells. Bar, 100 μm.

Figure 2.—

An epidermal wound reporter allows live scoring of wound closure. (A–C and E–F) Live larvae bearing one copy of the A58 epidermal wound reporter. (A–D) Control (crossed to w¹¹¹⁸). (A) Mock wounded. (B–F) Pinch wounded. (B) Immediately post wounding. (C) Twenty-four hours post wounding. Not all cell membranes are visible, but nuclei are present in the former wound gap. (D) Dissected whole mount of the same larva as in C, immunostained for Fasciclin III (green). The arrowheads mark identical positions in C and D. Faint membranes difficult to see in live larvae are now apparent. (E) Larva expressing a UAS-bsk^DN transgene. (F) Larva expressing a UAS-bsk^RNAi transgene exhibits a wound closure defect similar to UAS-bsk^DN. Arrows, tracheal dorsal trunks. Bar, 100 μm.

Figure 3.—

Quantification of wound closure upon epidermal expression of UAS-RNAi transgenes. (A) Workflow of the UAS-RNAi–based screen for wound closure genes. (B and C) Percentage of open wounds upon expression of UAS-RNAi transgenes targeting indicated genes of the JNK pathway and other SAPK signaling components (B) and genes involved in actin cytoskeletal remodeling (C). Open bars, e22c reporter; shaded bars, A58 reporter; solid bars, Dcr-2;A58 reporter. w¹¹¹⁸ crossed to the respective reporter was used as a negative control and bsk^RNAi as a positive control. Dashed line, arbitrary 15% cutoff for detailed morphological analysis. Daggers indicate insufficient L3 larvae for testing. Lines with two UAS-RNAi inserts targeting the same gene are denoted by ^x2. Genes in boldface type in B indicate members of the canonical JNK pathway. The numbers of scored larvae for each RNAi knockdown using A58 or Dcr-2;A58 in B and C were as follows: n(A58-Gal4) = 82 (w¹¹¹⁸), 56 (msn^x2), 39 (slpr), 39 (Tak1^x2), 31 (Takl1), 35 (Takl2), 32 (Pk92B), 31 (Mekk1), 36 (hep^x2), 38 (Mkk4), 84 (bsk^x2), 42 (DJun/Jra), 33 (DFos/kay), 33 (Ced-12), 41 (mbc), 31 (Rac1), 36 (Cdc42^x2), 36 (SCAR), 37 (Arp14D), 30 (Arp11), 46 (spir), 35 (Pax^x2), and 36 (Gγ1); and n(Dcr-2;A58-Gal4) = 72 (w¹¹¹⁸), 33 (slpr), 35 (Tak1^x2), 44 (Takl1), 31 (Takl2), 33 (Pk92B), 34 (Mekk1), 35 (hep^x2), 36 (Mkk4), 69 (bsk^x2), 65 (DFos/kay), 30 (mbc), 35 (Rac1), 40 (Cdc42^x2), 33 (SCAR), 33 (Arp14D), 37 (Arp11), 46 (spir), 34 (Pax^x2), and 34 (Gγ1); see also Table S1 for n(e22c).

Figure 4.—

UAS-RNAi transgenes affecting wound closure show distinct wound morphologies. (A–I) Dissected whole mounts of larvae heterozygous for e22c-Gal4,UAS-DsRed2-Nuc (red) and the indicated UAS-RNAi transgene were immunostained for Fasciclin III (green) 24 hr post wounding. UAS-RNAi transgenes were grouped into seven classes on the basis of wound morphology. (A) Class I, bsk^x2; (B) class I, DFos/kay; (C) class I, DJun/Jra; (D) class II, hep^x2; (E) class III, SCAR; (F) class IV, Ced-12; (G) class V, mbc; (H) class VI, Rac1; (I) class VII, msn^x2. See results for classification criteria. Stars indicate disorganized epidermal sheet; arrowheads indicate smooth wound edge with pronounced fluorescence; brackets span zones of nuclear crowding. Lines with two UAS-RNAi inserts targeting the same gene are denoted by ^x2. Bar, 100 μm.

Figure 5.—

Quantification of wound closure classes identified by qualitative morphological features. (A) Schematic of parameters quantified for classes of genes showing open wounds at 24 hr post wounding. Hexagons, epidermal cells; solid circles, epidermal nuclei; b, wound area; c, clustering of front line nuclei; d, crowding of nuclei near the wound margin. The lightly shaded area indicates the region where the area occupied by nuclei was measured to assess nuclear crowding. For details of quantification procedures see materials and methods. (B) Quantification of wound area in select classes. n = 35, 10, and 9 for classes I, II, and V, respectively. ***P < 0.001, Mann–Whitney test. (C) Quantification of clusters of front line nuclei in select classes. n = 35, 32, and 10 for classes I, III, and IV, respectively. ***P < 0.001, Mann–Whitney test. For nonsignificant comparison (NS) P = 0.725. (D) Quantification of nuclear area within 50 μm of the wound edge. n = 15, 15, and 10 for classes I, III, and IV, respectively. ***P < 0.001, Mann–Whitney test. For nonsignificant comparison (NS) P = 0.467. (E) Quantification of epidermal syncytium formation in closed wounds of msn^RNAi-expressing larvae. The number of nuclei in each syncytial cell near three wounds of control and msn^{RNAi x2} was measured. The diameter of the bubbles reflects the number of cells with that number of nuclei. Control wounds have on average more syncytial cells with small numbers of nuclei (2–12) while wounds in msn^RNAi-expressing larvae have fewer syncytia with greater numbers of nuclei.

Figure 6.—

JNK activation in larvae expressing UAS-RNAi transgenes affecting wound closure. (A–L) Dissected epidermal whole mounts of unwounded (A and J) or pinch wounded (B–I and K and L) larvae heterozygous for the e22c-Gal4 driver (A–K) or the A58-Gal4 driver (L), the JNK activity reporter msn-lacZ (A–L), and the indicated UAS-RNAi transgene. All whole mounts were stained 6 hr post wounding or post mock wounding with X-Gal to detect β-galactosidase reporter activity (blue). (A) w¹¹¹⁸, unwounded; (B) w¹¹¹⁸, wounded; (C) bsk^x2; (D) DJun/Jra; (E) DFos/kay; (F) hep,Mkk4; (G) SCAR; (H) Ced-12; (I) mbc; (J) Rac1, unwounded; (K) Rac1, wounded; (L) msn^x2. Morphological classes are indicated above the panels. Bar, 100 μm.

Figure 7.—

Model of epidermal cell behaviors in normal and perturbed wound healing. Cell behaviors during wound healing are indicated on a schematic template. On these templates, select cells contacting the wound edge are marked with blue nuclei, whereas select cells within the sheet in close vicinity to the wound are marked with red nuclei. Arrows indicate the direction of cellular migration toward the wound gap. The length of the arrow symbolizes the speed of the migrating cells (longer, fast; shorter, slow). A T-bar indicates that migration is blocked. Wavy arrows show improper directionality of migration. The bright green wound margin of class V indicates pronounced membrane fluorescence. The green central wound area of class VII indicates a large syncytial cell. See text for model details.