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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2009 Jan 23;106(7):2224–2229. doi: 10.1073/pnas.0810219106

Multiple transcription factor codes activate epidermal wound–response genes in Drosophila

Joseph C Pearson 1,1, Michelle T Juarez 1, Myungjin Kim 1, Øyvind Drivenes 1,2, William McGinnis 1,3
PMCID: PMC2630199  PMID: 19168633

Abstract

Wounds in Drosophila and mouse embryos induce similar genetic pathways to repair epidermal barriers. However, the transcription factors that transduce wound signals to repair epidermal barriers are largely unknown. We characterize the transcriptional regulatory enhancers of 4 genes—Ddc, ple, msn, and kkv—that are rapidly activated in epidermal cells surrounding wounds in late Drosophila embryos and early larvae. These epidermal wound enhancers all contain evolutionarily conserved sequences matching binding sites for JUN/FOS and GRH transcription factors, but vary widely in trans- and cis-requirements for these inputs and their binding sites. We propose that the combination of GRH and FOS is part of an ancient wound–response pathway still used in vertebrates and invertebrates, but that other mechanisms have evolved that result in similar transcriptional output. A common, but largely untested assumption of bioinformatic analyses of gene regulatory networks is that transcription units activated in the same spatial and temporal patterns will require the same cis-regulatory codes. Our results indicate that this is an overly simplistic view.

Keywords: cuticle, grainy head, fos, wound repair

Animals have evolved systems to sense and repair epidermal wounds and to fight microbial infections that follow wounding. Many wound responses are carried out by homologous genetic pathways in arthropods and vertebrates and thus apparently had evolved in a common ancestor of bilateral animals. These responses include pathways mediating reepithelialization (1–9), responses to microbial invasion (10), and regeneration of epidermal barrier layers (11, 12). The outermost epidermal barrier in mammals is the stratum corneum, a constantly regenerated layer of crosslinked keratinocytes, proteins, and lipids (13). The analogous protective barrier for arthropods is the cuticle, comprised of crosslinked chitin, proteins, and lipids (14). Although mammalian and arthropodal epidermal barriers are constructed in largely different ways, they share a homologous genetic pathway controlling barrier repair that involves transcription factors of the Grainy head (GRH) family (11, 12).

One central question in the control of epidermal wound repair is how signals are integrated at the level of transcription to activate the large battery of effector genes that mediate wound responses. Although many genes are known to be activated in epidermal cells after wounding (15–17), little is known about the cis-regulatory enhancers that mediate wound-induced gene activation. In Drosophila, there are only a few known genes activated at epidermal wound sites (11, 14, 17). Two of these genes encode the enzymes Dopa decarboxylase (Ddc) and Tyrosine hydroxylase (encoded by ple) (11). These 2 enzymes are limiting steps in the pathway to produce the highly reactive quinones that crosslink chitin and cuticle proteins during the construction and repair of cuticular barriers (18).

To better characterize the transcriptional wound response in Drosophila embryos, we have mutagenized minimal Ddc and ple epidermal wound enhancers, showing that both require AP-1-like and GRH consensus sites. We then searched for AP-1 and GRH consensus binding sites in the regulatory DNA for other epidermal wound–response genes, which led to the identification of epidermal wound enhancers for the genes krotzkopf verkehrt (kkv) and misshapen (msn). Ddc, ple, kkv, and msn are all transcriptionally activated within minutes after epidermal wounding. Three of the genes use overlapping transcriptional codes involving GRH and FOS to activate their epidermal wound enhancers, but kkv uses a fundamentally different code for wound activation. Whereas the common wound–response cis-regulatory codes we describe will be useful in the identification of new epidermal wound enhancers in both vertebrates and invertebrates, the evidence for alternative wound–response codes provides insight into how genetic control of wound healing has evolved in metazoans.

Results

Minimal Ddc Epidermal Wound Enhancers.

By using reporter gene constructs in Drosophila embryos, we previously identified a 0.47-kb DNA fragment just upstream of the Ddc gene that functioned as an epidermal wound enhancer in late embryos (Fig. 1A) (11). This enhancer can activate GFP reporter expression in a zone around aseptic epidermal punctures in late embryos (Fig. 1B), in larvae just before molts (data not shown), and in very young adults (Fig. 1C). The Ddc .47 wound-enhancer DNA contains 2 regions upstream of the basal promoter that are highly conserved among all sequenced drosophilid species: CR1 (44 nt) and CR2 (13 nt) (Fig. 1A and SI Appendix). The CR2 sequence consists largely of a high-affinity GRH binding site (ACCGGTT) (12, 19, 20), which is required for wound-enhancer function (11). Ddc .47 CR1 contains a sequence matching the consensus binding site (TGANTCA) for AP-1, a transcription factor consisting of a JUN-FOS dimer (21).

Fig. 1.

Fig. 1.

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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).

To test for AP-1-like site function in the Ddc .47 wound enhancer, we mutated the conserved AP-1-like site in CR1 and a second AP-1-like site that lies between CR1 and CR2 (Fig. 1A). This construct is unable to activate reporter expression after wounding (Fig. 1D). We also deleted 355 bp between the conserved regions (Ddc Gap) (Fig. 1A), to test whether any required DNA elements are located within the region between CR1 and CR2. This deletion mutant functions as an epidermal wound enhancer, but the number of cells that activate reporter gene expression is reduced compared with WT Ddc .47 (Fig. 1E). In summary, both AP-1-like and GRH consensus sequences are absolutely required for Ddc .47 wound activation, and a minimal enhancer of 117 bp containing these sites is sufficient for modest activation in cells around wound sites. However, additional sequences within .47 Ddc contribute to the strength of this epidermal wound enhancer.

An Epidermal Wound Enhancer from the ple Gene Requires GRH and AP-1-Like Sequences.

We previously identified 2 epidermal wound enhancers upstream of ple by searching for conserved clusters of AP-1-like and GRH consensus sites (11). We have refined the boundaries of the distal 3-kb ple fragment that contains a wound enhancer, delimiting the enhancer to a 687-bp DNA fragment that resides 3.45 kb upstream of ple (Wound Enhancer 1 or WE1) (Fig. 2 A and B). In addition to conserved AP-1-like and GRH consensus sites, analysis of this ple wound enhancer revealed conserved regions with consensus binding sites for other transcription factors. Of these sites, we noted putative binding sites for CREB homodimers (TGACGTMA) (22), EXD/PBX homodimers (WGATTGAW) (23–25), and Hox monomers (YMATTA) (26, 27). To test the importance of these sites, we mutated them in the context of ple-WE1 (Fig. 2A). Mutating the consensus AP-1-like sites abolished ple wound-enhancer function (Fig. 2C), whereas mutation of the GRH consensus site resulted in a consistent reduction of wound-enhancer function (Fig. 2D). In contrast, mutations in the CREB, EXD, or HOX consensus sites had no consistent effect on ple WE1 wound-dependent activation (Fig. 2 E–G). Therefore, activation of Ddc and ple wound enhancers depends on AP-1-like and GRH consensus binding sites.

Fig. 2.

Fig. 2.

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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).

To test whether sequences matching the GRH and AP-1-like consensus sites in the Ddc and ple wound enhancers bound GRH and AP-1 family proteins in vitro, we used EMSAs (SI Text) (28). An oligonucleotide including a GRH consensus site (ACCGGTT) from the Ddc wound enhancer binds full-length GRH protein with high affinity and specificity (Fig. 3A) (19). The GRH-like site in the ple WE1 element (ACTCGTTT) is a weaker match to an optimal site (AACCGGTTT) (20), and oligonucleotides with this site bind GRH protein with low affinity and specificity (Fig. 3A).

Fig. 3.

Fig. 3.

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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.

EMSA experiments using full-length Drosophila JUN and FOS-B proteins (the fly kay gene produces 4 isoforms of FOS, A–D), indicate that they bind as heterodimers with high specificity and affinity to AP-1-like sites from CR1 of the Ddc wound enhancer, whereas JUN or FOS-B homodimers had lower affinities (Fig. 3B). Because the AP-1-like site in the ple enhancer has the same consensus sequence as the one in the Ddc enhancer, we did not test JUN/FOS binding to ple WE1 AP-1-like sites. In contrast to mammalian FOS, Drosophila FOS-B protein can bind as a homodimer to AP-1-like consensus sites, albeit with lower affinity than JUN/FOS heterodimers (Fig. 3B) (29). To explore the possibility that other Drosophila FOS isoforms might bind wound-enhancer AP-1-like sites, we also tested FOS-D binding to an oligonucleotide containing the Ddc AP-1-like site. As seen in Fig. 3B, FOS-D homodimers apparently have higher affinity with Ddc AP-1-like sites than FOS-B or JUN homodimers, albeit less than JUN/FOS-B or JUN/FOS-D heterodimers. The relevance of FOS-D homodimer binding will be seen later in this report. From the above, we concluded that the mutations we introduced into the GRH and AP-1-like consensus sites in the Ddc and ple wound enhancers would eliminate binding of GRH, JUN, or FOS transcription factors in vivo.

Identification of Epidermal Wound–Response Enhancers.

We next wished to determine whether clusters of AP-1-like and GRH consensus binding sites were a common feature of epidermal wound enhancers. Two other candidate genes for activation in embryonic epidermal cells after wounding were kkv, which encodes chitin synthase (30, 31), and msn, which encodes an upstream activating kinase in the JNK pathway (32). Chitin synthase is required for the final step in the production of chitin, a major component of Drosophila exo- and endocuticle (33), and we reasoned that kkv transcription was likely to be activated at epidermal wound sites to promote cuticle regeneration. msn transcription was likely to be activated around embryonic wounds, because previous results have shown a lacZ reporter gene inserted into the promoter region of msn is activated in larval and adult epidermal cells around wound sites (14, 34).

Using multiplex in situ hybridization (35), we tested for the transcriptional activation of kkv, msn, Ddc, and ple around epidermal wounds in late-stage embryos (Fig. 4). All 4 genes were transcriptionally induced within 30 min in a zone of cells surrounding wounds. Thus, it is possible that all 4 genes are regulated by the same wound-signaling pathways and combinatorial transcriptional codes. The accumulation of fluorescently labeled probes at wound sites was not an artifact of enhanced accessibility, as no localized probe signals were observed in embryos that were wounded, immediately fixed, and then hybridized (data not shown).

Fig. 4.

Fig. 4.

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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).

To identify candidate epidermal wound enhancers regulating kkv and msn, we surveyed these loci for evolutionarily conserved clusters of AP-1-like and GRH consensus binding sites. The 5′ region of the third intron of msn has a cluster of 2 conserved GRH sites and 1 conserved AP-1-like site. A 1.2-kb DNA fragment containing these sites functioned as an epidermal wound enhancer (msn-WE2) (Fig. 5 A and D).

Fig. 5.

Fig. 5.

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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.

The kkv first intron has 5 GRH consensus sites and 4 AP-1-like sites. We tested the wound enhancer function of 2 overlapping DNA fragments that each contained 4 GRH sites (2 conserved) and 3 AP-1-like sites (1 conserved). One of these fragments functioned as an epidermal wound enhancer (kkv-WE1) (Fig. 5 B and E), whereas the other did not (kkv2) (Fig. 5B and data not shown). To test whether AP-1-like and GRH consensus sites were required for the function of the kkv wound enhancer, we mutated the sites in the context of the 2.2-kb kkv-WE1 element (Fig. 5C). To our surprise, kkv-WE1 reporters with either AP-1-like (Fig. 5F) or GRH consensus sites (Fig. 5G) mutated were still induced in epidermal cells around wound sites. This evidence strongly suggests that the kkv wound enhancer is activated by a different transcription factor code than the Ddc and ple wound enhancers. A table of the quantitative responses of different mutant wound-enhancer lines is provided in Table S1.

Genetic Requirements for the Induction of ple, kkv, and msn Wound Enhancers.

To test trans requirements for activation of the ple, kkv, and msn wound enhancers, we first tested their function in grh mutants (Ddc wound-enhancer function was previously shown to be dependent on grh genetic function) (11). In grh mutants, the msn-WE1 enhancer was not activated around embryonic wounds (Fig. 6 A and B), but the ple WE1 enhancer was activated to approximately normal levels (Fig. 6 C and D). This finding is seemingly at odds with the requirement of a GRH consensus binding site in ple-WE1 and suggests that another required factor acts through the low-affinity ple“GRH” consensus site in ple-WE1. The kkv-WE1 enhancer showed no significant reduction in function in grh homozygotes (Fig. 6 E and F), consistent with the kkv-WE1 enhancer's lack of dependence on GRH consensus binding sites.

Fig. 6.

Fig. 6.

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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 grhIM and fos/kaysro1 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 grhIM/+ compared to a grhIM/grhIM embryo. (C and D) ple-WE1 wound-enhancer function in a grhIM/+ compared to a grhIM/grhIM embryo. (E and F) kkv-WE1 wound-enhancer function in a grhIM/+ compared to a grhIM/grhIM embryo. (G and H) msn-WE1 wound-enhancer function in a kaysro1/+ compared to a kaysro1/kaysro1 embryo. (I and J) ple-WE1 wound-enhancer function in a kaysro1/+ compared to a kaysro1/kaysro1 embryo. (K and L) kkv-WE1 wound-enhancer function in a kaysro1/+ compared to a kaysro1/kaysro1 embryo.

We had previously found that a Ddc wound enhancer was activated normally in mutants for jun (JraIA109) and fos (kay1) (11). At first glance, this finding seems paradoxical, because AP-1 consensus sites (binding sites for JUN/FOS dimers), are required for the activity of Ddc and ple epidermal wound enhancers. The jun/Jra mutation we tested is a null allele for the locus (36), and, thus, excludes JUN as an activator of Ddc wound enhancers. However, the fos/kay1 allele does not eliminate the function of all the Drosophila FOS isoforms (37–39).

We thus considered the possibility that other FOS isoforms might act through the required AP-1-like binding sites in the Ddc and ple wound enhancers. One intriguing candidate is the FOS-D protein isoform, encoded in transcripts that are abundantly expressed in late embryonic epidermis (39). In addition, a mutation in the fos/kay gene called shroudP54 (or fos/kaysroP54) is caused by a transposable element insertion in the promoter for FOS-D, and fos/kaysroP54 and other fos/kaysro mutants have fragile, poorly differentiated cuticles that resemble those of grh mutants (39).

We therefore tested whether Ddc, ple, kkv, and msn wound enhancers could be induced in the epidermis of fos/kaysro1 mutants. The epidermal wound enhancers msn-WE1 (Fig. 6 G and H), ple-WE1 (Fig. 6 I and J), and Ddc .47 (data not shown) were not active in fos/kaysro1 mutants, whereas kkv-WE1 showed normal wound activation in fos/kaysro1 mutants (Fig. 6 K and L). The absence of wound-enhancer activation for ple, Ddc, and msn in fos/kaysro1mutants was not due to a failure of epidermal development or function, as shown by the normal induction of kkv-WE1 in the mutants. The activation of kkv-WE1 in wounded fos/kaysro1 mutants is consistent with the finding that kkv-WE1 is also unaffected by mutation of AP-1-like consensus binding sites. Given that Drosophila FOS-D homodimers can bind with high affinity and specificity to wound-enhancer AP-1-like sites (Fig. 3B), it is possible that FOS-D does not require a heterodimeric binding partner on wound enhancers, but it is also possible that FOS-D heterodimerizes with a Drosophila basic-leucine zipper protein other than JUN on wound enhancers (40).

Discussion

One principal conclusion of our findings is that the activation of Ddc and msn epidermal wound enhancers requires the genetic function of both grh and fos/kaysro. The current evidence indicates that fos/kaysro mutants reduce or abolish the function of the Drosophila FOS-D isoform (39). The grh and fos/kaysro gene functions are required not only for the activation of epidermal barrier-repair genes in Drosophila embryos but also for the generation of a normal epidermal barrier during embryonic development (11, 30, 39, 41, 42). The current evidence suggests that the combinatorial roles of Drosophila grh and fos/kaysro in activating epidermal barrier-repair genes might be conserved by their mammalian homologs. In mice, one of the grh-like genes is required for embryonic wound repair and development of a normal epidermal barrier, and this is accomplished at least in part via activation of downstream target genes that are required for skin barrier formation (12, 43, 44). Some mammalian fos and jun genes have been implicated in epidermal barrier development and control of epidermal wound repair, although their genetic roles in the epidermis are still being explored. It is known that mouse fos and jun paralogs are expressed in differentiating epidermis and up-regulated in wounded epidermal cells (45). Also, in the cells surrounding epidermal wounds, there is some genetic evidence that certain jun and fos paralogs regulate wound healing, although whether they act to accelerate or retard (or both) the process of wound healing is still unclear (46–49).

On the basis of the evidence just described, we propose that GRH and FOS family proteins are part of an ancient, evolutionarily conserved code that serves to activate an immediate transcriptional response in epidermal cells near wound sites. A recent report found that the Drosophila GADD45 gene is strongly activated around epidermal wounds (17) and that it may be controlled by the same activation code because a cluster of GRH and FOS (AP-1) consensus binding sites exist ≈2 kb upstream of the GADD45 transcription start site. However, there must be other epidermal wound transcriptional codes, because the ple wound enhancer required fos/kaysro function (but little or no input from grh), and the kkv wound enhancer required neither of these inputs. A common assumption is that similar combinations of transcription factors will control transcription units that are activated in the same temporal and spatial patterns. Our results indicate that, at least for Drosophila wound enhancers, this assumption is incorrect.

The signals that are sensed by Drosophila cells surrounding wounds are as yet unknown. The functional activation of either or both of the GRH or FOS-D proteins may depend on a receptor tyrosine kinase pathway, because both phospho-tyrosine and diphospho-ERK levels increase rapidly around wound sites and because ERK inhibition reduces the function of a Ddc epidermal wound enhancer (11). It is known that Drosophila GRH and FOS proteins can be phosphorylated by ERK in vitro (50–52), but at present it is unknown whether GRH and FOS are phosphorylated in a wound-dependent manner in the epidermis.

Although JUN kinase function is apparently not required for activation of the Ddc wound enhancer (11), the immediate transcriptional activation of msn, which encodes a JUN kinase kinase kinase kinase (32), is consistent with previous findings that the JNK pathway is required for wound-dependent reepithelialization (14, 34, 52, 53). The immediate activation of msn transcription after wounding (Fig. 4) suggests that robust JNK activity after epidermal wounding might be dependent on an immediate response involving GRH, FOS-D, and ERK (11). Genetic linkages between the reepithelialization response and the epidermal barrier-repair response are still poorly understood, but in a biological sense the two responses must be coordinated, with reepithelialization occurring in concert with barrier regeneration. The activation of the msn epidermal wound enhancer in a manner that depends on grh and fos/kaysro suggests that msn may mediate crosstalk between the reepithelialization and cuticle barrier-repair pathways during the complex process of wound repair. Ongoing genetic screens in Drosophila are likely to clarify the relationships between the reepithelialization and epidermal barrier–repair pathways, as well as identify diffusible signals and receptors that mediate the immediate response to epidermal wounding.

Materials and Methods

Drosophila Stocks and Genomic DNA.

D. melanogaster strain w1118 was used for germline transformation (54, 55), for in situ hybridizations, and as a source for genomic DNA. Fly stocks for Drosophila pseudoobscura, Drosophila virilis, Drosophila immigrans, and Drosophila hydei were supplied by the Tucson Drosophila Stock Center. Genomic DNA was prepared by using standard procedures. kaysro1 mutants were obtained from the Bloomington Stock Center. Transgenic lines containing epidermal wound-enhancer test constructs were obtained by using injected w1118 embryos. The reporter constructs were tested in pH-Stinger vectors that contain GFP (56) or DsRed (57) reporter genes.

Wounding Procedure.

Embryos were collected on apple juice agar plates and aged to 15–17 h at 25 °C. Embryos were washed into mesh baskets, dechorionated in bleach for 1 min, then washed copiously with water. Embryos were then transferred to a clean slab of apple juice agar and aligned for 30–60 min at 18 °C, transferred to slides with double-sided tape, then covered in a 1:1 ratio of 700:27 weight halocarbon oil or PBS. Embryos were then wounded laterally with fresh microinjection needles made from an automated puller mounted on a micromanipulator, allowed to recover for 3–8 h at room temperature, and visualized under fluorescent light in either a compound microscope or a Leica SP2 confocal microscope to determine wound response. At least 2 independent experiments with at least 20 successfully wounded embryos were performed, testing at least 2 independent transformant lines. Assays involving altered enhancers were performed in parallel to unaltered enhancers, impaling all embryos using a micromanipulator so that the needle protruded 1 embryo-width from the exit wound, and responses rated on a scale of “no response, weak, moderate, strong.” Images were obtained by wounding embryos with microinjection needles by hand and imaged on a Leica SP2 confocal microscope, selecting representative embryos to image. Pixel-intensity levels of images were adjusted for clarity; Adobe Photoshop despeckle, blur, and sharpen functions were used occasionally to enhance clarity. Original images are available on request.

In Vitro Synthesis of Proteins.

The following plasmid clones were used to produce Drosophila GRH, JUN, FOS-D, and FOS-B proteins in reticulocyte lysates; pcDNAMyc/His-GRH, pcDNAMyc/His-DJUN, pcDNAMyc/His-DFOS-B, and pcDNAMyc/His-DFOS-D. PCR synthesized ORFs for these proteins were inserted into pcDNA3.1(−)/Myc-His A plasmids (pcDNAmyc; Invitrogen). The primers used to synthesize the ORFs are reported in SI Text.

Synthesis of proteins was performed in a quick-coupled transcription/translation system (TNT kit, Promega) in vitro, programmed with 0.05–0.3 μg of pcDNAMyc/His-GRH, pcDNAMyc/His-DJUN, pcDNAMyc/His-DFOS-B, or pcDNAMyc/His-DFOS-D plasmids. For assaying expression level of TNT products, proteins were subjected to 10% SDS/PAGE and transferred to PVDF membranes. The blots were incubated for 1 h with a 1/1,000 dilution of anti-myc antibody 9E10 (Developmental Studies Hybridoma Bank). The blots were washed, incubated with goat anti-mouse antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch), and visualized by chemiluminescence as described by the supplier (SuperSignal West Pico chemiluminescent substrate; Pierce). EMSAs were performed as described in the SI Text.

Multiplex Fluorescent in Situ Hybridization.

Probes were generated from partial or full cDNA clones from the Drosophila Gene Collection (58, 59). Probe labeling and hybridization protocol was as described in Dave Kosman's multiplex FISH protocol (35).

Supplementary Material

Supporting Information
supp_106_7_2224__index.html (748B, html)

Acknowledgments.

We thank Kimberly Mace for fly stocks, constructs, and experimental advice; Oyvind Drivenes for cloning kkv fragments into DsRed H-Stinger; Israel Lidsky for several pilot experiments; Scott Wheeler for useful comments on the manuscript; the Tucson and Bloomington Stock Centers for supplying fly stocks; and the Drosophila Genomics Resource Center and Biogem for supplying cDNA clones. This research was supported by National Institutes of Health Grant R01GM077197 (to W.M.), Korea Research Foundation Grant KRF-2008-357-C00126 (to M.K.), National Institutes of Health Training Grant T32GM007240 (to J.C.P.), and National Institutes of Health Institutional Research and Academic Career Development Award GM68524 (to M.T.J.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0810219106/DCSupplemental.

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