High baselines of transcription factor activities represent fundamental obstacles to regulated signaling. Here we show that in Drosophila, quenching of basal activator protein 1 (AP-1) transcription factor activity serves as a prerequisite to its tight spatial and temporal control by the JNK (Jun N-terminal kinase) signaling cascade. Our studies indicate that the novel raw gene product is required to limit AP-1 activity to leading edge epidermal cells during embryonic dorsal closure. In addition, we provide the first evidence that the epidermis has a Basket JNK-independent capacity to activate AP-1 targets and that raw function is required broadly throughout the epidermis to antagonize this activity. Finally, our mechanistic studies of the three dorsal-open group genes [raw, ribbon (rib), and puckered (puc)] indicate that these gene products provide at least two tiers of JNK/AP-1 regulation. In addition to Puckered phosphatase function in leading edge epidermal cells as a negative-feedback regulator of JNK signaling, the three dorsal-open group gene products (Raw, Ribbon, and Puckered) are required more broadly in the dorsolateral epidermis to quench a basal, signaling-independent activity of the AP-1 transcription factor.

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ACTIVITIES of the three signaling subfamilies of the MAP kinase (MAPK) signaling pathway: extracellular signal-related kinase (ERK), p38, and Jun N-terminal kinase ( JNK), are tightly controlled both spatially and temporally in numerous experimental systems and organisms (QI and ELION 2005). It is well-documented that threshold MAPK signaling activities are maintained by a balance of activators (MAPK kinases) and negative-feedback regulators (MAPK phosphatases). Less well considered, however, are the effects of baseline MAPK signaling levels and/or pulses of signaling molecules that are produced in stochastic fashion ("signaling noise") (RAO et al. 2002).

Both signaling baselines and signaling noise represent theoretical obstacles to attaining tightly controlled and/or robust responses to genetically programmed activation of signal cascades such as the MAPK pathway. First, a baseline of signaling activity close to a theoretical activation threshold requires a responding cell to differentially recognize and interpret comparatively small changes in signaling activities. Second, signaling noise, which occurs when various gene products fluctuate about their average values, might drive a process to threshold activation by mere chance. The probability of noise-driven activation is greatest when a baseline of signaling activity is already close to a theoretical activation threshold. Indeed, signaling noise is responsible for many well-characterized stochastic biological events, including, for example, stem cell division and retroviral latency (HASTY et al. 2000; LEMISCHKA 2005). For nonstochastic signaling outcomes, however, theoretical studies dictate genetic programs which ensure minimal signaling baselines and buffered noise. Experimental methods to approach this problem in vivo have been problematic as the gain-of-function phenotypic consequences of ectopic signaling are not always easily predicted.

To genetically dissect the control of MAPK baseline signaling activities, we turned to the Drosophila JNK signaling cascade, a pathway for which the phenotypic consequences of ectopic signaling are understood. In conjunction with its associated transcription factor Jun/Fos (activator protein 1, AP-1), the JNK cascade functions in the fruit fly Drosophila melanogaster as the essential MAPK regulator of several developmental and physiological processes. These include tissue morphogenesis, wound healing, planar cell polarity, programmed cell death, and synaptic plasticity, as well as the immune and oxidative stress responses (for review see STRONACH 2005). Among these processes, it is during tissue morphogenesis, and embryonic dorsal closure in particular, that the role for JNK signaling and the mechanisms of its regulation have been best characterized.

In Drosophila, embryos undergo dorsal closure between 8 and 12 hr after egg lay (AEL). Closure comprises three phases: (1) initiation, (2) spreading, and (3) suturing. At the initiation of closure, leading edge (LE) epidermal cells elongate along their dorsoventral axis. During the spreading (or sweeping) stage of dorsal closure, epidermal cells positioned behind the LE also elongate along their dorsoventral axis, and thus the epidermis spreads dorsally to cover the embryonic dorsal surface. In the final stage—suturing—the epidermal sheets approach one another at the embryonic dorsal midline. Closure is complete when epidermal sheets suture at the dorsal midline. As it is the epidermis that secretes the larval cuticle, mutations in loci required for closure (dorsal-open group loci) are recognized by their embryonic lethal cuticular phenotype, a single large dorsal hole.

Molecular analyses of the dorsal-open group loci have revealed that these code for three classes of gene product. The class I and II dorsal-open group genes, respectively, code for JNK/AP-1 and Dpp signaling components; the class III genes code for structural molecules.

Accumulated data from several labs have led to our understanding that the JNK and Dpp signaling pathways function sequentially to coordinate epidermal spreading during embryonic dorsal closure (for review see KNUST 1997). First, an as yet uncharacterized trigger activates JNK/AP-1 signaling in LE epidermal cells. Next, JNK/AP-1 signaling induces expression of target genes, including dpp and puc, in LE cells. Whereas the puc-encoded phosphatase feeds back to negatively regulate JNK/AP-1 signaling in a cell-autonomous fashion, the dpp-encoded cytokine function is non-cell autonomous. Upon secretion from LE cells, Dpp activates signaling broadly in epidermal cells and thereby induces their elongation. Regulation of the JNK/AP-1 and Dpp signaling pathways is tightly integrated in dorsal closure, but only the JNK/AP-1 pathway is autoregulatory. Although dpp can autoactivate its own expression at some embryonic sites ( JAZWINSKA et al. 1999b), it does not do so in LE epidermal cells (ARORA et al. 1995).

At the cellular level, JNK/AP-1 signaling leads to reorganization of cytoskeletal proteins in LE cells of the epidermal sheets. Whereas actin-, myosin-, and phosphotyrosine-containing proteins accumulate at the LE in wild-type embryos, their accumulation is disrupted in embryos deficient in the AP-1 components Jun ( Jun-related antigen/Jra), or Fos (Kayak/Kay), or in the constituents of the relay system that function to potentiate AP-1 activity via phosphorylation [ JNKKK (Misshapen/Msn), JNKKK (Slipper/Slpr), JNKK (Hemipterous/Hep), JNK (Basket/Bsk)]. That mutations in the genes coding for each of these signaling activators lead to an identical dorsal-open phenotype highlights the nonredundant contribution of each to dorsal closure (for review see STRONACH 2005).

A somewhat surprising discovery, but one that nonetheless underscored the requirement for precise control of JNK/AP-1 signaling during dorsal closure, was the finding that mutations in JNK/AP-1 activators and JNK/AP-1 antagonists both give rise to dorsal-open embryonic lethal phenotypes. As discussed in the current report and elsewhere (BYARS et al. 1999; BRADLEY and ANDREW 2001), mutations in three genetically defined dorsal-open group loci (raw, ribbon, and puckered) lead not to the absence of LE JNK/AP-1 signaling, but rather to its ectopic activity in several rows of the dorsolateral epidermis.

The best characterized of the dorsal-open group subset of JNK/AP-1 antagonists is puckered (puc), which encodes a VH1-like dual-specificity protein tyrosine phosphatase belonging to the MAPK subfamily of mitogen kinase phosphatases (MKPs) (MARTIN-BLANCO et al. 1998). MKPs are characterized by highly conserved protein domains that include the protein tyrosine phosphorylation (PTP) loop and two N-terminal CH2 domains (KEYSE and GINSBURG 1993; MUDA et al. 1996; TANOUE et al. 2000). It is thought, on the basis of these sequence homologies, that the Drosophila MKP Puckered acts by dephosphorylating and thereby inactivating the basket-encoded AP-1 activator JNK (MARTIN-BLANCO et al. 1998). More specifically, puc is expressed in LE cells during dorsal closure and is thought to be part of a feedback loop controlling the magnitude of epidermal JNK signaling. There is not currently a comprehensive explanation for why loss of an intracellular JNK antagonist leads to ectopic AP-1 activity, although it is formally possible that an unidentified secreted JNK-signaling target feeds forward to activate the JNK pathway in paracrine fashion (KNUST 1997). For the studies reported here, we considered an alternative mechanism for ectopic signaling in raw (and puc and rib) mutant embryos—that a second regulatory network directly represses basal JNK and/or AP-1 activities in the embryonic dorsolateral epidermis.

As we report here, our studies of the Drosophila dorsal-open group raw gene reveal an unexpected Basket JNK-independent AP-1 activity in the embryonic dorsolateral epidermis at the time of dorsal closure and identify an essential role for raw in regulating this activity. Our findings provide a compelling explanation for dysregulated signaling in raw (and puc and rib) mutant embryos and have important implications for understanding mechanisms of AP-1 regulation during development in flies and higher eukaryotes. In particular, our studies indicate that the role of raw is primarily to eliminate Basket JNK-independent activity of the AP-1 transcription factor in the embryonic epidermis and thereby establish an AP-1-silenced ground state prior to the onset of dorsal closure and secondarily, to restrict activity of the JNK transcriptional effector AP-1 to the LE subset of epidermal cells during dorsal closure. Furthermore, our studies indicate that raw functions as a general antagonist of AP-1 activity, required for at least three developmental occurrences of JNK/AP-1 signaling in Drosophila. Taken together, our data lead us to suggest that the biological effect of raw-mediated AP-1 antagonism is to establish a sensitized state of competence; this condition then sets the stage for a highly robust, Basket JNK-dependent activation of AP-1 transcriptional activity.

Fly stocks:

raw¹, raw², raw²⁴¹⁸, raw^lex1, raw^lex2, bsk¹, bsk², Jra^IA109, puc^E69, puc^H246, rib¹, and w¹¹¹⁸ mutant lines, balancer lines, and UAS-dpp and 69B-gal4 transgenic lines have been described (BYARS et al. 1999; FLYBASE CONSORTIUM 2003). We obtained UAS-brk transgenics from B. Stronach, Kr-gal4 transgenics from M. Lamka, LE-gal4 transgenics from S. Noselli, and GMR-gal4 transgenics from K. Broadie. Double-mutant lines were generated using standard mating strategies and recombination when necessary. All our lines were tested to ensure that the desired mutations were present. To do this, we assayed for lethality in trans to independently derived alleles of each of the mutants used in our study. All fly lines were maintained on standard cornmeal/molasses/agar medium at 22°, unless otherwise stated.

Phenotypic analyses:

Embryonic lethal cuticular phenotypes were viewed after mounting devitellinized samples in one-step mounting medium [30% CMCP-10 (Masters Chemical Company), 13% lactic acid, 57% glacial acetic acid]. Hybridizations in situ using digoxigenin-labeled RNA probes were performed as described (BYARS et al. 1999). For all our phenotypic studies, we looked at no fewer than 50 mutant samples after development at constant temperature. Embryos and cuticles were analyzed and imaged using dark field, bright field, and DIC optics. In all cases, included figure images are representative of a large experimental set.

Epistasis studies:

Mutants included in our epistasis studies have been defined either molecularly or genetically as null. As genetic definitions of allele strength are sometimes controversial, we note here that it is sometimes appropriate to use hypomorphs in double-mutant epistasis studies (HUANG and STERNBERG 1995). In particular, for epistasis studies of two genes A and B, a gene A hypomorph may be used when B is epistatic to A. In this scenario, the normal function of A would be to negatively regulate B, and AB double-mutant animals would not have functional B activity that could be regulated by residual A activity. We also note that it is appropriate to use either an amorph or a severe loss-of-function hypomorph when a gene has an earlier maternal (and/or zygotic) requirement in development, as long as the chosen allele completely disrupts the process being studied (HUANG and STERNBERG 1995).

Rescue of raw mutant embryonic lethality:

To assess rescue of raw lethality in a tissue-specific manner, lethal phase analyses of embryos derived from matings of w¹¹¹⁸; raw²⁴¹⁸/CyO, Ub-GFP; p[UAS-raw⁺] females to w¹¹¹⁸; raw²⁴¹⁸/CyO, Ub-GFP; gal4 or w¹¹¹⁸; raw²⁴¹⁸ gal4/CyO Ub-GFP males were performed. We employed four independent GAL4 lines to drive expression of the UAS-raw⁺ transgene in raw²⁴¹⁸ homozygotes. Rescued raw²⁴¹⁸ homozygous first instar larvae were identified by their inability to fluoresce. raw²⁴¹⁸/raw²⁴¹⁸ viability was confirmed by single larva PCR using primers directed against GFP.

To assay rescue of raw-dependent lethality, 4-hr collections of embryos derived from matings of w¹¹¹⁸; raw²⁴¹⁸/CyO, Ub-GFP; p[hs-raw⁺] flies were aged as indicated in the text and then subjected to a 1-hr 37^o heat shock. After 36 hr, newly hatched larvae were examined for the presence or absence of the GFP-marked balancer chromosome using fluorescent optics. raw²⁴¹⁸ homozygotes were identified as above.

Plasmid constructions:

P-element rescue constructs pCaSper-raw and pUAST-raw were generated from a full-length (MfeI NotI ) raw cDNA (BYARS et al. 1999). FLAG-tagged raw isoforms were generated by insertion of a 5'FLAG sequence for pUAST-rawFLAG, and by insertion of the MfeI NotI fragment of raw in a pFLAG-CMV-5b (Sigma, St. Louis) derivative in which the ApaLI site was converted to KpnI using the linker TGCATCTCGGTACCC (pFLAG-CMV-raw).

Cell transfection and immunofluorescence:

Anti-FLAG M2 (1:100, Sigma) and anti-c-Myc 9E10 (1:250, Santa Cruz Biotechnology) antibodies were used for immunostains in whole-mount embryos and cell culture. Secondary antibodies and dyes used for this study included anti-mouse FITC (1:200, Jackson Labs), anti-mouse Cy3 (1:500, Jackson Labs), and DAPI ( Jackson Laboratories, West Grove, PA). COS-7 cells, grown in a monolayer at 37° in 5% CO₂, were transiently transfected using the standard calcium method and fixed by acetone/methanol (DEBRY et al. 1997).

raw mutants exhibit ventral cuticular patterning defects that are attributable to ectopic signaling by Dpp:

Our previous phenotypic raw study drew our attention to two highly atypical features (with respect to the dorsal-open group) of the raw mutant phenotype (BYARS et al. 1999). First, we found that in addition to archetypal cuticular defects marking abnormalities in dorsal closure, cuticles derived from raw mutant embryos exhibit ventral cuticular patterning defects, and second, we observed ectopic JNK/AP-1 signaling in the lateral epidermis of dorsal closure stage raw mutant embryos. Deciphering this pleiotropy has been central to our current understanding of Raw function and AP-1 antagonism.

As an initial step toward a better understanding of Raw, we defined an allelic series of mutations that was based upon the severity of the dorsal-closure defect and then we assessed the associated raw-dependent phenotypes. The severity of raw-dependent ventral cuticular patterning defects is positively correlated with the defining defect in dorsal closure. In cuticles derived from embryos homozygous for the null raw¹ allele, ventral denticle belts are nonexistent (Figure 1A). As dorsal closure defects are diminished, ventral denticle belt dysmorphogenesis is similarly diminished; in cuticles derived from embryos homozygous for the hypomorphic raw² and raw²⁴¹⁸ alleles, ventral denticles are evident, however, while properly positioned and shaped, denticles are smaller and less refractile to light, and the belts themselves do not extend as far dorsally in comparison to wild type (Figure 1, C and E). In homozygotes of the weakest allele of raw, raw^lex2, ventral denticles are wild type in position, shape, and form (Figure 1G; see also Figure 3B).

View larger version (111K): In this window In a new window Download PPT slide   FIGURE 1.— raw mutants define an allelic series. (A, C, E, and G) Embryonic cuticles and (B, D, F, and H) dpp mRNA expression in stage 13 whole-mount embryos. (A) Cuticles derived from embryos homozygous for raw1, a null allele, exhibit significant dorsal closure and head involution defects, as well as an absence of ventral denticle belts (*); (B) in whole-mount embryos in situ, leading edge dpp expression extends to a depth of nine cells in the lateral epidermis (). (C) Cuticles derived from embryos homozygous for the raw2 allele, a strong hypomorph, also exhibit significant dorsal closure and head involution defects, as well as severely atrophied ventral denticle belts (*). (D) In whole-mount embryos in situ, dpp expression extends to a depth of seven cells in the lateral epidermis (). (E) Cuticles derived from embryos homozygous for the raw2418 allele, a hypomorph of intermediate strength, exhibit a strong dorsal pucker, in addition to hypertrophied ventral denticle belts (*). (F) In whole-mount embryos in situ, dpp expression extends to a depth of four cells in the lateral epidermis (). (G) Cuticles derived from embryos homozygous for rawlex1, a very weak hypomorph, exhibit a weak dorsal pucker and ventral denticle belts that are indistinguishable from wild type (*). (H) In whole-mount embryos in situ, dpp expression only minimally extends into the lateral epidermis, at most to a depth of two cells laterally (). In this and all subsequent figures, embryos are oriented such that dorsal is up and anterior is to the left.

 

View larger version (71K): In this window In a new window Download PPT slide   FIGURE 3.— raw-group genes exhibit an array of shared loss-of-function phenotypes. (A, E, and I) dpp mRNA transcript expression in whole mount embryos in situ; (B, F, and J) cuticular phenotypes; (C, G, and K) magnification of cuticular denticle phenotypes; and (D, H, and L) trace drawings of denticle magnifications. In wild-type embryos, dpp expression is limited to the single row of leading-edge epidermal cells (A), and the five ventral denticle rows are clearly evident (B–D). pucH246 homozygotes exhibit expanded dpp expression in the lateral epidermis (E), a dorsal cuticular hole (F), and hypertrophied ventral denticle belts that are similar to those in raw2418 hypomorphs (G and H). rib1 homozygotes exhibit expanded dpp expression as well as dorsal closure ( J) and ventral denticle defects (K and L).

 
Next, we assessed the extent of ectopic JNK/AP-1 signaling in raw mutant embryos. In this regard, our demonstration that dpp and puc, two transcriptionally regulated targets of JNK/AP-1 signaling during closure, are ectopically expressed in raw¹ mutant embryos was integral to our characterization of Raw as a JNK/AP-1 signaling antagonist. Whereas dpp and puc expression in wild-type embryos is limited to a single row of cells, corresponding to the LE of the epidermis, dpp and puc expression in the null mutant raw¹ extends from the LE approximately nine cell widths into the lateral epidermis (compare Figures 1B and 3A; see also BYARS et al. 1999).

In an extension of these studies, here we used dpp expression to monitor epidermal JNK/AP-1 activity in several different raw mutants. We found that as the ventral denticle phenotype is correlated with cuticular hole size, so also is the extent of ectopic JNK/AP-1 signaling in the dorsal epidermis. dpp expansion in the epidermis of raw¹ mutant embryos represents the greatest degree of expansion for any of the raw alleles (Figure 1B). In embryos homozygous for raw² and raw²⁴¹⁸, alleles defined via dorsal and ventral cuticular defects to be of intermediate strength, dpp expression expands laterally only four to six cells (Figure 1, D and F). In the weakest allele of raw, raw^lex2, dpp expansion is minimal, extending at most to one additional row of epidermal cells (Figure 1H). Thus, on the basis of the ectopic expression of dpp as a readout for epidermal JNK/AP-1 activity, we conclude that the strength of the raw phenotype correlates with the extent of expanded signaling activity. Notably, it is the dorsal-most epidermis, not just the LE, which is competent for JNK- and/or AP-1-mediated gene expression.

Studies reported by us and others revealed the capacity of pan-epidermal Dpp to disrupt ventral cuticular pattern (STAEHLING-HAMPTON and HOFFMANN 1994; RIESGO-ESCOVAR and HAFEN 1997a; BYARS et al. 1999). In particular, we have shown that although closure occurs normally in UAS-dpp/69B-gal4 transgenics expressing dpp ectopically throughout the embryonic epidermis, ventral cuticular differentiation in these animals is abnormal. Transgenics suffer an embryonic lethality that is associated with the same poorly differentiated ventral cuticle that distinguishes the raw mutant from the dorsal-open group more generally. Notably, the UAS-dpp/69B-gal4-associated cuticle is not dorsalized as (1) dorsal hairs (cuticular markers of dorsal fate) do not extend to ventral cuticular domains, and (2) gastrulation (monitored visually in living embryos) proceeds normally (BYARS et al. 1999).

In a complementary test of dpp effects in the dorsal epidermis of raw mutant embryos, here we employed the UAS-Gal4 system to express the dpp antagonist brinker (brk) pan-epidermally. Our phenotypic characterization of cuticles derived from raw¹/raw¹; UAS-brk/69B-gal4 mutants revealed rescue of raw¹-dependent ventral cuticular defects, but no rescue of the associated dorsal-closure defects (Figure 2, A–C). Comparison of brk expression in wild-type and raw mutant embryos further exposed the extent to which the epidermis is mispatterned in raw mutant embryos. In wild-type embryos undergoing dorsal closure brk is expressed in a wide stripe of lateral epidermis just ventral to the LE, as well as in a more ventrally positioned epidermal stripe (Figure 2D). In contrast, in raw¹ homozygotes both the lateral and the ventral epidermal stripes of brk expression are missing while internal (nonepidermal) domains of expression are unaffected (Figure 2E). Taken together, these data demonstrate that it is Dpp and/or Brinker (but not the JNK/AP-1 signaling cascade) that is responsible for the ventral cuticular defects observed in raw mutant embryos.

View larger version (69K): In this window In a new window Download PPT slide   FIGURE 2.— Ventral denticle abnormalities in raw mutant embryos result from ectopic epidermal Dpp signaling. (A) Cuticles derived from raw1; UAS-brk/69BGAL4 embryos exhibit significant dorsal closure and head involution defects, comparable to those of the null homozygote shown in Figure 1A; the ventral denticle belt phenotype, however, is largely rescued. (B and C) Magnification and trace drawing of ventral denticles in A. brk mRNA transcript expression in situ is shown in stage 13 whole-mount wild-type embryos (D) and raw1 mutant embryos (E). Whereas brk is expressed in lateral and ventral stripes in wild-type embryos, these expression domains are missing in raw1 homozygotes.

 

A subset of dorsal-open group genes (the raw group) exhibits ventral cuticular patterning defects:

Having defined a palette of raw-dependent embryonic phenotypes, we recognized that two other dorsal-open group mutants (puc and rib) exhibit dpp expression patterns and ventral cuticular defects analogous to those that we had observed in raw mutants. Albeit variably expressed, the shared loss-of-function phenotypes are notable, and we reasoned that insights into the function of the novel Raw protein in regulating JNK/AP-1 signaling might be garnered from a molecular and genetic understanding of puc and rib, by themselves and in conjunction with raw. puc codes for a MAPK phosphatase (MARTIN-BLANCO et al. 1998), and rib codes for a putative BTB-POZ-type DNA binding protein (BRADLEY and ANDREW 2001; SHIM et al. 2001).

Whereas most dorsal-open group mutants exhibit striking defects only in dorsal closure, animals homozygous for recessive mutations in raw, puc, and rib die in embryogenesis with defects in dorsal closure and ventral cuticular patterning (Figure 3, B–D, F–H, and J–L). Each of the raw, puc, and rib gene products is required for proper restriction of JNK signaling targets to LE cells during closure, as dpp and/or puc expression are shown in the current report and elsewhere to expand beyond the LE in mutants (BLAKE et al. 1998; BYARS et al. 1999; BRADLEY and ANDREW 2001). Here we show that for all three loci, the extent of ectopic signaling correlates with allele strength, and it is the null alleles that exhibit the broadest signaling domains as measured by the expansion of dpp expression (Figure 3, A, E, and I). Whereas the dpp expression phenotypes in puc and raw mutants are comparable, there is considerably less expansion in the rib mutant in comparison to either raw or puc mutant embryos (compare Figure 1B and Figure 3, A, E, and I; see also BRADLEY and ANDREW 2001). The subset of dorsal-open group genes sharing the raw palette of phenotypes currently includes raw, puc, and rib, and is from hereon referred to as the raw group.

raw and puc function independently to antagonize JNK/AP-1 activity:

Although the raw-group genes share an array of loss-of-function phenotypes, it remained unclear whether these three genes (raw, puc, and rib) act together, in some combination, or independently to restrict JNK/AP-1 signaling to LE cells. The simplest and most common signal attenuating regulatory mechanism in any biochemical network is negative feedback (for review see RAO et al. 2002). Thus, we considered the possibility that the raw and rib gene products function with Puckered in a single negative-feedback loop inactiving JNK via dephosphorylation and consequently inactivating AP-1 indirectly. As an alternative, we reasoned that two or more AP-1 inactivation mechanisms might function in parallel (and thereby combine) to effect a robust signaling response (KERSZBERG 2004).

We employed genetic interaction studies as an initial means to distinguish between linear and parallel roles for raw and puc in antagonizing the JNK/AP-1 pathway. Double-mutant animals harboring hypomorphic mutations can exhibit enhanced phenotypes when two genes function in either common or parallel pathways. Thus, our discovery that hypomorphic mutations in raw and puc enhance one another's cuticular phenotype was not unanticipated; both the dorsal closure and ventral denticle defects are stronger in the raw²⁴¹⁸; puc^E69 double mutants than in either mutant alone (Figure 4, A and B; see also Figure 1E). More notably, animals doubly mutant for null alleles will show evidence of enhancement only when the two gene products function in parallel pathways. Here we show that raw¹; puc^H246 double mutants exhibit a very severe and fully penetrant cuticular defect that is distinct from that of cuticles derived from either raw¹ or puc^H246 single-mutant embryos (Figure 4C; see also Figures 1A and 3F). These null-allele-interaction data indicate that raw and puc function in independent and presumably parallel pathways to antagonize JNK/AP-1 signaling in Drosophila embryos.

View larger version (43K): In this window In a new window Download PPT slide   FIGURE 4.— raw and puc function independently to antagonize JNK/AP-1 signaling. (A) raw2418; pucE69 cuticles exhibit dorsal closure, head involution, and ventral denticle defects that define null alleles of raw (see also Figure 1A). (B) The pucE69 cuticular phenotype is more subtle; it is characterized by a dorsal pucker. (C) raw1; pucH246 cuticles exhibit a novel phenotype, which masks defects in dorsal closure diagnostic of cuticles derived from single-mutant null animals. (D) The rawlex1 rib1 cuticular phenotype is analogous to the rawlex1 (and raw1) null phenotype. Cuticle magnification is identical in all panels.

 
Next, we turned our attention to the relationship between raw and rib. In contrast to our discovery that null alleles of raw and puc enhance one another's phenotype, we found that null alleles of raw mask the weaker rib phenotype. The raw^lex1 rib¹ double-mutant dorsal-closure defect is analogous to that of raw null homozygotes (Figure 4D; see also Figure 1A). Hence, while raw and puc function independently to restrict JNK/AP-1 signaling in epidermal cells during closure, raw and rib (on the basis of their epistatic relationship) likely function together.

Raw could function to antagonize JNK/AP-1 at the level of transcription in the nucleus with Ribbon, or it could antagonize AP-1 activity at the level of signal transduction in the cytoplasm. To begin to distinguish between these possibilities, we examined the subcellular localization of Raw. Two Raw fusion proteins, one harboring an N-terminal Myc tag, the other a C-terminal FLAG tag were expressed transiently in COS cells, and the linked epitopes were detected using standard immunological methods. In both cases, we found the Raw fusion protein to be cytoplasmic (Figure 5). The tagged Raw isoforms are cytoplasmic in embryos as well; however, in contrast to untagged versions of Raw that function in rescue assays in vivo (see Figure 6), the tagged isoforms do not (data not shown).

View larger version (94K): In this window In a new window Download PPT slide   FIGURE 5.— Raw is cytoplasmic. N-terminal Myc-tagged Raw (A–C) is cytoplasmic in COS-7 cells. (D–F) C-terminal FLAG-tagged Raw is cytoplasmic in COS-7 cells. (A) N-terminal Myc-tagged Raw was visualized using an anti-Myc antibody; (D) C-terminal FLAG-tagged Raw was visualized using an anti-FLAG. Nuclei were visualized by staining with DAPI (B and E). Merged images of fluorescent channels are shown (C and F).

 

View larger version (77K): In this window In a new window Download PPT slide   FIGURE 6.— raw-group genes share identical epistatic relationships with genes encoding JNK-signaling molecules. dpp mRNA transcript expression in whole-mount embryos in situ. (A) bsk1, (B) raw1 bsk1, (C) JraIA109; pucH246, (D) JraIA109; rib1, (E) bsk1; pucH246, and (F) bsk1; rib1. In A–F, the position of the leading edge is marked by an arrow (). (G and H) Diagrams showing alternative explanations for the epistatic relationships of JNK-signaling molecules to raw (and other raw-group genes) in leading-edge (LE) and epidermal (EP) cells more generally.

 
Together with the overlapping embryonic patterns of raw and rib expression (BYARS et al. 1999; BRADLEY and ANDREW 2001; SHIM et al. 2001), our genetic interaction studies indicate that raw and rib function together in dorsal closure as they do in several other developmental contexts (BLAKE et al. 1998, 1999). Nevertheless, the gene products' roles in mediating JNK/AP-1 antagonism (at least in dorsal closure) appear to be separable, both in terms of their quantitatively distinct effects on signaling (see Figure 3) and their qualitatively distinct subcellular locales (see Figure 5). Also consistent with this model of separable raw and rib gene functions is our inability to rescue the raw²⁴¹⁸ and rib¹ dorsal-closure phenotypes (n > 100 for each) with functional rib⁺ (BRADLEY and ANDREW 2001) and raw⁺ (this study) transgenes, respectively.

Epidermal JNK/AP-1 regulation is partitioned spatially:

Results from our genetic interaction studies pointed to a function for raw, independent of the Puckered MKP-mediated feedback loop that controls Basket JNK activity cell autonomously. Thus, we considered alternative roles for the raw gene product in modulating JNK and/or AP-1 activity. As an example, Raw might function upstream of the JNK cascade to regulate the activity of an as yet undefined JNK activator (restrictive signaling model); alternatively, Raw might function in parallel to the JNK cascade to repress epidermal JNK and/or AP-1 activity (repression model) (BYARS et al. 1999).

To distinguish between restrictive signaling and repression models of raw function and to establish a regulatory link between raw and the genes encoding members of the JNK signaling cascade, we determined their epistatic relationships. LE dpp expression is a very clear indicator of this relationship since LE dpp transcription is missing in mutants for all JNK/AP-1 signaling components, but is greatly expanded in raw-group mutants (GLISE et al. 1995; RIESGO-ESCOVAR et al. 1996; GLISE and NOSELLI 1997; SU et al. 1998; STRONACH and PERRIMON 2002) (see also Figures 1 and 3). By examining LE dpp expression in embryos doubly mutant for molecularly and genetically defined null alleles of raw and Jra (encoding the Jun component of AP-1), we have shown that Jra is epistatic to raw and thus we identified an upstream role for raw as an antagonist of AP-1 activity (BYARS et al. 1999).

We continued our analysis of raw's epistatic relationships by examining raw's relationship to basket (bsk), the gene encoding the upstream activating JNK ( Jun kinase). For this analysis, we employed two genetically defined bsk null alleles (bsk¹ and bsk²). The bsk¹ and bsk² lesions are distinct, affecting different conserved domains in the Basket JNK protein. bsk¹ corresponds to a missense mutation (G225E) mapping between the Basket kinase subdomains IX and X. This region is required for JNK substrate recognition and is conserved among members of the MAPK family to which the Basket JNK belongs. Molecularly, bsk¹ is classified as a "severe" allele; genetically, however, it is defined as null (SLUSS et al. 1996), and it has been used previously in epistasis studies (STRONACH and PERRIMON 2002). Like bsk¹, bsk² corresponds to an EMS-induced mutation, in this case targeting K316 and creating an in-frame stop. K316 (and its corresponding nonsense mutation) maps within the conserved kinase subdomain XI. Although not demonstrated biochemically, the protein encoded by bsk², like that encoded by bsk¹, is predicted to be enzymatically inactive. Consistent with this prediction are the experimental observations that (1) in bsk² embryos AP-1 activity is undetected in LE dpp expression assays, and (2) the bsk² embryonic lethal cuticular phenotype is analogous to that of the genetically defined bsk¹ and molecularly defined bsk^FLP17E null alleles (RIESGO-ESCOVAR et al. 1996; RIESGO-ESCOVAR and HAFEN 1997a).

Although both bsk¹ and bsk² mutant embryos lack all dpp expression in LE epidermal cells, we found that dpp is broadly expressed in the epidermis of both raw¹ bsk¹ and raw¹ bsk² double mutants (Figure 6, A and B, and data not shown). Thus raw is epistatic to bsk, implying that raw functions either downstream of bsk or independently of bsk.

Together, our observations that (1) Jra is epistatic to raw and (2) raw is epistatic to bsk are consistent with models in which raw, bsk, and Jra function in a single linear pathway. When considered within this linear context, however, our epistasis results place raw squarely between bsk and Jra (Figure 6G), wedged within a well-studied biochemical pathway that has, in all probability, been defined in its entirety. Indeed, Jun ( Jra) is a direct target of Basket-mediated phosphorylation (for review see NOSELLI and AGNES 1999). Genetically defined regulatory pathways are not, however, always linear; sometimes they are branched and/or contain multiple inputs. A more fitting interpretation of our results—that JNK/AP-1 regulation is partitioned in a tissue-specific fashion during closure—is attained using a branched pathway model instead of a linear model. More specifically, our data implicate Raw in epidermal repression of AP-1 activity. While AP-1 activity in LE cells may be dependent upon Basket JNK activity, AP-1 activity in epidermal cells positioned more laterally is (1) Basket independent and (2) repressed by Raw (Figure 6H).

With two additional genetically defined JNK/AP-1 antagonists in hand, we next investigated whether they exhibit similar or different epistatic relationships to components of the JNK/AP-1 cascade. We examined epidermal expression of dpp in Jra puc, Jra rib, bsk puc, and bsk rib double mutants (in all cases using genetically and/or molecularly defined nulls). We found that whereas dpp expression is absent in both Jra puc and Jra rib double mutants (Figure 6, C and D), dpp expression is expanded in both bsk puc and bsk rib double mutants (Figure 6, E and F). Thus, the epistatic relationships of rib and puc to the JNK/AP-1 cascade are identical to that of raw and the JNK/AP-1 cascade. With respect to epidermal AP-1 activity, all three JNK/AP-1 antagonists function upstream of Jra, but independently of the Basket JNK. Overall, these epistasis studies lead us to suggest that (1) the epidermis is broadly competent for Jra/AP-1-mediated transcription, and (2) Basket JNK-mediated phosphorylation of epidermal Jra/AP-1 appears to be dispensable for its activity as a transcription factor.

Raw functions broadly in the embryonic epidermis to establish an AP-1 silenced ground state:

Our studies to this point implicated a role for raw (and the raw-group genes more generally) in a previously uncharacterized epidermal pathway(s) of AP-1 silencing. In a direct test of this model, we assessed the spatial requirements for raw in dorsal closure. To this end, we established a raw transgenic rescue assay and employed targeted expression of a UAS-raw⁺ transgene to determine whether raw expression in any of the three tissues participating in dorsal closure (amnioserosa, leading edge epidermis or epidermis more generally) is sufficient to effect dorsal closure. Normally, raw is expressed in all three tissues (BYARS et al. 1999).

We observed proper epithelial morphogenesis and dorsal closure in raw²⁴¹⁸ homozygotes when we used the 69B-GAL4 driver to express two independently derived UAS-raw⁺ transgenes pan-epidermally. In contrast, we never observed rescue of the raw²⁴¹⁸ phenotypes when we used the Kr-GAL4 or LE-GAL4 drivers that target UAS-raw⁺ gene expression to the amnioserosa or LE epidermis, respectively (Figure 7A). These data indicate that (1) expression of a raw⁺ transgene broadly in the epidermis is sufficient to rescue raw-dependent defects in dorsal closure, and (2) expression of a raw⁺ transgene, when confined to either the amnioserosa or the LE epidermis, is not sufficient to rescue raw-dependent defects in closure. These results, and those from our epistasis studies, indicate that Raw mediates restriction of AP-1 activity in a tissue-autonomous fashion, via the epidermis.

View larger version (17K): In this window In a new window Download PPT slide   FIGURE 7.— Raw functions early in the epidermis to effect dorsal closure. (A) Tissue-specific rescue in raw2418; p[UAS-raw+]/+ transgenics. Rescue was observed only when raw expression was driven by the 69B-GAL4 epidermal driver. In this experimental series, we observed rescue of embryonic dorsal closure in 72% of raw2418 homozygotes. (B) Induction of either of two independently derived hs:raw+ transgenes in raw2418 homozygotes 4–8 hr AEL rescues 68 and 98% of raw– homozygous embryos to the first larval instar stage. Induction of the same transgenes 8–12 hr AEL rescues 39 and 65% of raw– homozygotes. In the absence of heat shock, background rates of rescue range from 9 to 25%.

 
We next tested when expression of a heat-shock inducible raw⁺ transgene can rescue raw-dependent defects in dorsal closure. We found that when hs-raw⁺ (in two independently derived lines) was induced before the start of dorsal closure at 4–8 hr AEL, rescue was very efficient, with rates of 68 and 98%. In contrast, we observed reduced rates of rescue (39 and 65%) when hs-raw⁺ transgenes were induced during dorsal closure, between 8 and 12 hr AEL (Figure 7B). These data indicate that Raw-mediated antagonism of AP-1 baseline activity occurs relatively early in embryogenesis. Moreover, these data are consistent with our hypothesis that raw is required to establish an inactive epidermal AP-1 ground state.

raw functions to antagonize AP-1 activity at multiple developmental time points:

Having established that raw is an essential negative regulator of AP-1 activity in embryogenesis, we next tested whether raw is a dorsal closure-specific antagonist of AP-1 activity or whether raw has a more general role throughout development in the negative regulation of AP-1 prior to its activation by JNK-mediated signaling. Among the best characterized developmental and physiological requirements for JNK/AP-1 signaling in Drosophila are its roles in dorsal closure (GLISE et al. 1995; RIESGO-ESCOVAR and HAFEN 1997a,b), follicle cell morphogenesis (DEQUIER et al. 2001; DOBENS et al. 2001; SUZANNE et al. 2001), and oxidative stress tolerance (WANG et al. 2003). Thus, there are three well-characterized JNK/AP-1-dependent processes in Drosophila, and we show here that each one is affected in raw mutant animals.

During oogenesis, JNK/AP-1 signaling is required for follicle cell (FC) morphogenesis. Reduced or elevated levels of JNK/AP-1 activity, achieved either by mutating or overexpressing components of the JNK cascade in somatic FC clones result in a distinctive oocyte phenotype. Signature defects in oocytes with improper levels of JNK/AP-1 signaling include failures in nurse cell dumping and dorsal appendage morphogenesis, in addition to an overall reduction in oocyte length (DOBENS et al. 2001; SUZANNE et al. 2001). raw might play a role analogous to puc in antagonizing JNK/AP-1 activity during oogenesis as it is expressed in stretch cells (Figure 8A), JNK/AP-1-active somatic FCs that overlie the nurse cells during oogenic stages 10B-11 and one of three migrating cell types in the Drosophila oocyte (DEQUIER et al. 2001).

View larger version (24K): In this window In a new window Download PPT slide   FIGURE 8.— raw antagonizes JNK/AP-1 signaling at multiple Drosophila life stages. (A) raw mRNA transcripts are expressed in stretch cells; for this analysis oocytes were fixed and hybridized with an antisense RNA probe directed against raw. (B–D) raw-dependent gain- and loss-of-function egg phenotypes include size and dorsal appendage defects (indicated by arrows,). (B) +/+, (C) hs-raw+, and (D) raw1 follicle cell clones. (E and F) Quantization of gain- and loss-of-function-size phenotypes. (E) Eggs derived from wild-type females (dotted lines), exposed to either heat shock () or not (), are rarely smaller than 0.63 µm. In similar fashion, eggs derived from females harboring a raw+ transgene that has not been induced by heat shock (solid lines) are also only infrequently smaller than 0.63 µm (). In contrast, a significant fraction of eggs (34.8%) derived from transgenic females after raw+ induction by heat shock (). (F) Eggs derived from females harboring raw1 follicle cell clones exhibit a wider range of sizes than do controls. Twenty-one percent of eggs produced by FLP/FRT raw1 females (solid line) are smaller than 0.63 µm, whereas only 1.1% of eggshells produced by wild-type control females (dotted line) are smaller than 0.63 µm. (G) Reducing JNK/AP-1 signaling extends life span after exposure to oxidative stress. Wild-type adult females have an average lethality of 22.5% 24 hr after exposure to paraquat. Removal of one copy of puc reduces lethality to 2.0%. Removal of one copy of raw, using null (raw1 and rawlex1) or hypomorphic (raw2418) alleles reduces the lethality to 4.5 and 5.0%, respectively.

 
For direct tests of raw function in oocytes, we used a raw¹ FRT40A chromosome in a FLP/FRT-mediated clonal analysis (XU and RUBIN 1993). In this study of raw¹ mutant clones, we found that females with raw mutations in their germ line lay small, unfertilized eggs, many of which are distinguished by short dorsal appendages (Figure 8, B and C). We quantified the size defect and found that in contrast to eggs laid by wild-type females, 71% of which are at least 0.7 µm in length, only 22% of eggs laid by raw¹ FRT40A females grow to this same length. Indeed the majority (57%) is at least 10% shorter (Figure 8E). We also tested whether raw overexpression similarly leads to defects in oogenesis. Five hours after induction of a hs-raw⁺ transgene, females lay unfertilized eggs that are shorter than wild-type controls and exhibit dorsal appendage defects (Figure 8D). Quantization of length defects revealed that 51% of eggs laid by females overexpressing raw exhibit at least a 10% reduction in length in comparison to wild-type control animals (Figure 8F). Overall, the raw-associated loss- and gain-of-function defects mirror those observed in studies of puc, a phosphatase with a molecularly defined role in regulating JNK signaling.

Finally, we tested whether raw functions in oxidative stress tolerance. Animals with elevated levels of JNK signaling and AP-1 activity, achieved either by overexpression of JNK-signaling components or by mutation of the puc-encoded MKP, exhibit an increased resistance to oxidative stress as measured by life span (WANG et al. 2003, 2005). We reasoned that if raw is a general antagonist of the JNK/AP-1 signaling pathway, then elevated levels of AP-1 activity in raw mutants will affect oxidative stress tolerance and aging in a manner analogous to that of puc. To test this hypothesis, we exposed raw and puc adult heterozygotes to oxidative stress in the form of paraquat treatment and measured life span. In comparisons of mortality rates in +/+ homozygotes, puc/+, and raw/+ heterozygotes, we found that as reported previously puc heterozygotes exhibit decreased mortality levels. Notably, we found that a reduction in raw also leads to a mortality decrease (Figure 8G), consistent with raw functioning as an antagonist of JNK signaling during the adult response to oxidative stress. To control for effects of genetic background, we employed two independently derived null alleles of raw. As was the case for puc, the raw heterozygote effect of life span demonstrates a dosage-sensitive requirement for raw in oxidative stress tolerance. Taken together, results from this and the oogenesis study point to a role for raw as a negative regulator of AP-1 activity throughout the Drosophila life cycle.

Our initial molecular and genetic studies of the dorsal-open mutant raw revealed it to encode a widely expressed and novel gene product, required for the restriction of JNK/AP-1 activity to LE epidermal cells (BYARS et al. 1999). The Raw protein sequence yielded no insights into its mechanism of function as the Raw sequence harbors none of the canonical motifs that are associated with nuclear localization, phosphorylation, membrane insertion, or protein secretion. Mechanistic studies of a novel protein can be challenging, but here we report our use of a variety of genetic strategies to probe Raw function and test models of AP-1 silencing. In particular we (1) assessed the epistatic relationship of raw to genes encoding well-characterized JNK-signaling components, (2) identified genes, which we have designated the raw group, that share an array of loss-of-function phenotypes, (3) determined the interaction phenotypes among the raw-group loci, and (4) generated raw transgenics, which we utilized to probe sites of Raw function. Our analyses reveal that raw belongs to a small set of dorsal-open group genes that encode JNK/AP-1 pathway antagonists. Our characterization of raw, and the raw group more generally, has led to a new appreciation of wide-ranging competence for AP-1 activity in early Drosophila embryos. As signal activation is critical for proper development, so also is its silencing.

Regulating AP-1 baselines in Drosophila:

In the current study, we show that although raw functions upstream of Jra as an AP-1 antagonist, its action is independent of the bsk-encoded kinase that is required to activate AP-1 activity in LE cells during closure. In addition, we demonstrate that raw is required broadly in the epidermis to effect normal dorsal closure. Overall, our studies expose the importance of epidermal AP-1 silencing during embryogenesis and lead us to extend existing models for dorsal closure, which have largely confined their focus to mechanisms of JNK/AP-1 activation in LE cells (YOUNG et al. 1993; MARTIN-BLANCO et al. 1998; NOSELLI 1998). In particular, our data indicate that Raw and the other raw-group gene products (Puckered and Ribbon) function to silence Basket JNK-independent AP-1 activity in the embryonic dorsolateral epidermis (Figure 9A). AP-1 silencing, via the combined actions of the raw-group gene products, essentially wipes the epidermal slate clean and primes the system for activation via a still unidentified deterministic signal that acts only in LE cells (Figure 9B).

View larger version (42K): In this window In a new window Download PPT slide   FIGURE 9.— Modeling Raw function. (A) The canonical JNK cascade is active in LE cells and presumably controlled by the negative-feedback regulator Puckered. In the adjacent epidermal (EP) cells, Basket-independent AP-1 activity is silenced by Raw. (B) Baseline/basal levels of AP-1 activity are kept below biologically relevant levels by the actions of Raw, Ribbon, and Puckered. Activation of the JNK-signaling cascade in the leading-edge cells supersedes raw-group gene product-mediated AP-1 silencing. Lastly, biologically appropriate signaling levels are thought to be maintained via the activity of the JNK negative-feedback regulator Puckered.

 
We have yet to molecularly define the AP-1 abnormality in raw-group mutant embryos. Previous studies provide compelling evidence that AP-1 overexpression in Drosophila embryos is not sufficient to disrupt either dorsal closure or development more generally (KOCKEL et al. 1997; ZEITLINGER et al. 1997). It seems unlikely, therefore, that elevated levels of the AP-1 transcription factor in raw-group mutants simply override a requirement for kinase activation in initiating an AP-1-dependent program of gene expression. Instead, we speculate that AP-1 is aberrantly modified in raw-group mutant embryos. It might be that AP-1 escapes inactivation in mutants; either alternatively or additionally, AP-1 in mutants may be inappropriately activated via phosphorylation. In addition to Basket JNK, there are four other Drosophila MAP Kinases (p38a, p38b, Mpk2, and Rolled) that might provide dysregulated kinase activity in mutants. Consistent with this idea is our observation that the oogenesis phenotypes associated with raw (and puc) ectopic expression and mutation have considerable similarity with gain- and loss-of-function phenotypes associated with mutations in the p38 pathway that is required in the germ line for proper oogenesis (SUZANNE et al. 1999). Finally, a kinase-dependent activation model for epidermal Jun provides the most parsimonious explanation for ectopic epidermal signaling observed in puc MPK-deficient embryos. From the perspective of regulated signaling more generally, however, lowering an AP-1 activity baseline in wild-type embryos will (1) provide a means for the clean on/off regulation of JNK/AP-1 that has been predicted in computer simulations and (2) make a less strenuous demand on the input activating signal (SAURO 2004) (Figure 9B).

The different contributions of Raw, Ribbon, and Puckered to JNK/AP-1 regulation:

Our discovery that null alleles of raw and puc interact, with double mutants exhibiting an embryonic lethal phenotype distinct from their shared loss-of-function null phenotypes, revealed the independent contributions of raw and puc to embryogenesis, presumably through their effects on AP-1 antagonism. Drosophila overexpression studies have previously implicated several pathways in the parallel control of AP-1 activity (GRITZAN et al. 2002; KOZLOVA and THUMMEL 2003), but our analysis represents the first direct demonstration of physiologically relevant, parallel regulatory pathways.

The genetic interaction that we documented between null alleles of raw and puc contrasts with the lack of a detectable interaction between null alleles of raw and rib. Moreover, our observation that raw and rib hypomorphs interact genetically during dorsal closure (data not shown) is consistent with previously published data, as well as with findings documenting (1) raw/rib interactions in several other epithelial tissues, including the nervous system, salivary gland, trachea, and gut (BLAKE et al. 1998, 1999) and (2) overlapping raw and rib expression patterns in Drosophila embryos (BYARS et al. 1999; BRADLEY and ANDREW 2001; SHIM et al. 2001). Together, results from these genetic and molecular studies point to roles for raw and rib in a single, previously unrecognized puc-independent AP-1 inactivation system.

Activating JNK signaling in the LE:

In addition to providing evidence for raw-mediated global silencing of AP-1, our study underscores a simultaneous requirement for a biologically appropriate activator of JNK/AP-1 signaling. In this regard, expression of raw in LE cells failed to rescue raw-dependent defects in dorsal closure. Even more notable, however, was our observation that overexpression of raw⁺ in wild-type embryos, and in wild-type LE cells in particular, had no detrimental effects on embryonic development and dorsal closure. From a signaling perspective this result indicates that JNK-dependent AP-1 can be activated despite expression of the wild-type raw gene product, and thus Raw does not function as a binary switch for signaling. Although it is formally possible that LE expression of raw was initiated too late to disrupt JNK/AP-1 signaling and dorsal closure in our LE-gal4/UAS-raw⁺ transgenics, we do not favor this interpretation as the LE-GAL4 driver used here has been shown previously to (1) be an effective driver of at least one gene that is required in LE epidermal cells for closure (LU and SETTLEMAN 1999) and (2) drive expression of a lacZ reporter in LE cells during dorsal closure (SCUDERI and LETSOU 2005).

Our finding that raw expression in LE cells is not sufficient to inactivate AP-1 activity in a cell-autonomous fashion is consistent with models for independent, developmentally regulated triggers of JNK signaling. Indeed, there is abundant experimental support for developmentally regulated activation of JNK signaling in LE cells. JNK/AP-1 activation likely follows an amalgamation of signals, both from the amnioserosa and the epidermis, both in the form of cytoskeletal components and signaling molecules (HARDEN 2002; SCUDERI and LETSOU 2005). Among the best candidates with postulated roles in JNK/AP-1 activation are small GTPases, nonreceptor tyrosine kinases, and integrins (GLISE et al. 1995; STARK et al. 1997; KIYOKAWA et al. 1998; BISHOP and HALL 2000; BROWN et al. 2000; FERNANDEZ et al. 2000). Thus, despite the broad epidermal competence for AP-1 signaling that we have shown in this work, the activation signal is itself limited to only LE cells and functions via an unknown mechanism. Importantly, AP-1 antagonism by raw cannot override its signal-dependent activation in the LE.

Activating brinker in the ventral epidermis:

dpp, when expressed pan-epidermally, leads to a raw-like phenotype: embryonic lethality associated with ventral cuticular defects (RIESGO-ESCOVAR and HAFEN 1997a; BYARS et al. 1999). In a direct assessment of equivalence of raw loss-of-function and dpp gain-of-function ventral cuticular phenotypes, we tested whether pan-epidermal expression of brinker (brk) can rescue raw-dependent defects in the ventral cuticle. The Dpp signaling modifier Brinker functions by negatively regulating dpp target genes (BRAY 1999; CAMPBELL and TOMLINSON 1999; JAZWINSKA et al. 1999a; MINAMI et al. 1999).

In our analysis, we found that although brk is normally expressed in nonoverlapping lateral and ventral domains of the embryonic epidermis, it is undetectable in the epidermis of embryos homozygous for a null allele of raw. We also found that although brk⁺ fails to rescue raw-dependent defects in dorsal closure, it does rescue raw-dependent defects in the ventral cuticle. Together, these data point to an important role for dpp, brk, and/or their target genes in development of the ventral epidermis.

What we cannot discern from our studies is (1) how the nonoverlapping epidermal domains of dpp and brk are established and maintained and (2) if and how epidermal dpp and brk interact during normal embryonic development. In this regard, our previous finding that LE dpp is not autoregulatory makes it unlikely that brk functions in direct fashion to set the LE dpp expression boundary (ARORA et al. 1995). Even more significant is our finding that cuticles derived from Jra raw double mutants exhibit defects in dorsal closure, but not ventral cuticular patterning (BYARS et al. 1999). Indeed, these data highlight the requirement for functional Jun in generating ventral cuticular defects in raw mutant embryos. Taken together then, our data suggest that the effects of JNK/AP-1-activated dpp in the dorsal epidermis of raw mutant embryos are far reaching, extending even to the most ventral regions of the embryo.

Having established a dependence upon Jun for raw-dependent ventral cuticular defects, we postulate that the absence of brk in raw mutant embryos is a direct consequence of ectopic JNK/AP-1 activity in the dorsal epidermis of these mutants. We suspect that ectopic JNK/AP-1 activity leads secondarily to ectopic dpp activity, and that in its turn ectopic dpp activity leads finally to brk repression. An alternative view, that raw might have dual regulatory roles in the epidermis, seems less likely although it is not absolutely excluded by our strictly genetic analysis. In this regard, in addition to its function as a JNK/AP-1 antagonist in the embryonic dorsal epidermis, raw might function independently as a trigger of brk expression in the ventral epidermis. Clearly, the mechanism of raw function and the relationship of dpp to brk in eliciting properly formed ventral cuticle warrant further investigation.

Conclusions and implications for the study of JNK/AP-1 antagonists:

In Drosophila, as in all animals, signaling pathways are finely regulated at several levels. Although there are multiple tiers of regulation operating on the JNK/AP-1 signaling cascade, surprisingly little of the regulation of this pathway is known. Our study of the functions and interactions of a subset of dorsal-open group genes (raw, rib, and puc) has shed some additional light on both old (puc-mediated) and new (raw/rib-mediated) mechanisms of JNK/AP-1 antagonism. Our data indicate that Raw functions to silence Basket JNK-independent AP-1-mediated transcription and to set the stage for JNK-dependent regulation of transcription. Our suggestion that spatial restriction of the JNK/AP-1 signal requires antagonists, as well as activators, is not without precedent in other signaling systems. Many signaling pathways have already been shown to be multilayered and to depend heavily on negative regulation to terminate developmental events, and/or control both the distance and speed that a signal can move (e.g., Nodal; SHEN 2007). In addition, and as we suggest is the case for the Drosophila JNK/AP-1 pathway, reducing basal levels of a signaling pathway can augment the effects of its signaling responses (e.g., Hedgehog and Lef1; LI et al. 2006; FLYNT et al. 2007).

Finally, given the numerous associations of improper JNK/AP-1 activity with human disease, it seems apparent that many cell types have the capacity to signal via the JNK/AP-1 pathway. Presumably, this capacity is diminished (and then tightly regulated) during normal vertebrate development and aging. Viewed from this perspective, our characterization of Raw as an essential AP-1 antagonist establishes a clear basis for future studies of AP-1 regulation.

We thank Cherie Byars and other members of our laboratory for valuable discussions. We also thank Susan Mango and Steve Wasserman for comments on the manuscript, Diana Lim for figure preparation, and Deborah Andrew, Kendal Broadie, Michelle Lamka, Stephan Noselli, and Beth Stronach for fly lines. This work was supported by National Institutes of Health grant R01GM-68083 to A.L.

¹ Present address: California State University, Dominguez Hills, Carson, California 90747.

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Communicating editor: T. SCHÜPBACH

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