Ddc, ple, msn, and kkv are transcriptionally activated in epidermal cells around wounds. (A) Image of wounded embryo, visualized with DIC optics, fixed 30 min after wounding. Arrow shows entry wound; dotted box shows the region imaged with fluorescence optics in frames B–E. (B–E) Ddc (B), msn (C), kkv (D), and ple (E) transcripts were simultaneously detected in the embryo around the aseptic wound using hapten-labeled probes (35). No signals were detected around wounds in embryos fixed immediately after wounding and hybridized with probes (data not shown).

ple epidermal wound enhancer binding site requirements. (A) Diagram of WT and mutant ple-WE1 enhancers. White boxes indicate regions of sequence conservation in drosophilids; red blocks denote mutant sequences. Wound responses of the elements are indicated at right. (B–G) Dashed lines outline the embryos; arrows indicate wound sites. (B) ple-WE1 was activated at epidermal wounds. (C) Mutation of two AP-1-like consensus sites abolished ple-WE1 wound-enhancer activity. (D) Mutation of a GRH consensus site strongly reduced ple-WE1 wound-enhancer activity. (E–G) Mutation of 2 CREB-like sites (E), or 2 EXD-like sites (F), or 14 HOX-like binding site had no effect on ple-WE1 enhancer function (G).

Sequence requirements of the Ddc epidermal wound enhancer. (A) Diagram of WT and mutant Ddc .47 epidermal wound–response enhancers. White blocks indicate conserved regions with other drosophilids; red blocks denote mutant sequences. Functional wound enhancers are indicated by “+,” nonfunctional enhancers by “−.” (B–E) Dashed lines outline embryos, arrows indicate wound sites, and fluorescent nuclei surrounding wounds show GFP or DsRed reporter gene activation provided by wound enhancers. (B) Ddc .47 activated GFP-reporter expression around wounds in late-stage embryonic epidermis. (C) Ddc .47 activated GFP-reporter expression around wounds in early adult abdominal epidermis. (D) A mutation of AP-1-like consensus sites (APm) in Ddc .47 abolished wound-enhancer function. (E) Ddc Gap had reduced wound-enhancer function. Previous results showed that a GRH site mutant (GRHm) in Ddc .47 abolished wound-enhancer function (11).

Genetic requirements of epidermal wound–response enhancers. DsRed fluorescent protein reporter genes attached to the ple-WE1, kkv-WE1, and msn-WE1 epidermal wound enhancers were introduced into grh^IM and fos/kay^sro1 mutant backgrounds balanced with Kruppel-GFP chromosomes carrying positive WT grh or fos/kay genes. DsRed signals around wound sites from Kruppel-GFP negative embryos (homozygous mutants) were compared with the signals observed in wounded Kruppel-GFP positive embryos (heterozygous for the mutant allele). Dashed lines show embryo boundaries; white arrows indicate puncture-wound sites. (A and B) msn-WE1 wound-enhancer function in a grh^IM/+ compared to a grh^IM/grh^IM embryo. (C and D) ple-WE1 wound-enhancer function in a grh^IM/+ compared to a grh^IM/grh^IM embryo. (E and F) kkv-WE1 wound-enhancer function in a grh^IM/+ compared to a grh^IM/grh^IM embryo. (G and H) msn-WE1 wound-enhancer function in a kay^sro1/+ compared to a kay^sro1/kay^sro1 embryo. (I and J) ple-WE1 wound-enhancer function in a kay^sro1/+ compared to a kay^sro1/kay^sro1 embryo. (K and L) kkv-WE1 wound-enhancer function in a kay^sro1/+ compared to a kay^sro1/kay^sro1 embryo.

Conserved AP-1-like and GRH consensus site clusters identify kkv and msn epidermal wound enhancers. (A and B) Diagrams of 5′ regions of the msn (A) and kkv (B) genes, with conserved AP-1-like and GRH consensus sites indicated by A and G, respectively, and nonconserved matches indicated in small case. Sites were found using GenePalette (60). The potential wound-enhancer regions that were tested overlie the diagrams. (C) Diagrams of the WT and mutant kkv-WE1 enhancers. In the 5′–3′ orientation, they were tested in reporter gene constructs. AP-1-like consensus binding site mutants (APm); GRH consensus binding site mutants (GRHm). White boxes indicate regions of conservation in drosophilids. Mutant sites are indicated by red boxes. (D and E) msn-WE1 (D) and kkv-WE1 (E) DNA epidermal wound enhancers driving DsRed fluorescent reporter protein expression around wound sites. Dashed lines indicate embryo boundaries; arrows indicate entry-wound sites. (F and G) kkv-WE1 enhancers with mutations in either AP-1-like or GRH consensus sites. See SI Appendix for all DNA sequences.

GRH and FOS bind crucial sequences in the Ddc and ple wound enhancers. (A) Oligonucleotide DNA probes including the GRH consensus sites from Ddc and ple epidermal wound–response enhancers were tested in EMSA assays for binding to full-length Drosophila GRH protein (see Materials and Methods). Probes with mutant GRH sites were used to test binding-site specificity. Lanes 1 and 2 show Ddc or ple DNA probes with no GRH protein. Lanes 3 and 4 show GRH protein with WT and mutant Ddc probes, respectively. Lanes 5 and 6 show GRH protein with WT and mutant ple probe, respectively. Arrow indicates GRH–DNA complexes. The bands at the bottom of the frame are unbound probe. (B) Oligonucleotide probes including the AP-1-like site from the Ddc wound–response enhancer were tested in EMSA assays for binding to full-length Drosophila JUN, FOS-B, and FOS-D proteins (see Materials and Methods). Probes with mutant AP-1-like sites were used to test the specificity of binding. Lanes 1 and 2 show WT or mutant probes, no protein; NS denotes nonspecific shifted probe complexes that result from reticulocyte lysate alone; unbound probe is not shown. Lanes 3 and 4 show JUN protein with WT and mutant probes, respectively. Lanes 5 and 6 show FOS-B protein with WT and mutant probes, respectively. Lanes 7 and 8 show FOS-B and JUN proteins with WT and mutant probes, respectively. Lanes 9 and 10 show FOS-D protein with WT and mutant probes, respectively. Lanes 11 and12 show FOS-D and JUN with WT and mutant probes, respectively. The positions of the various shifted protein/DNA complexes are noted on the side.