Localized JNK signaling regulates organ size during development

1) To verify the specificity of antibody, the authors used rn-Gal4 and ptc-Gal4 to knock down bsk (Figure 1I, Figure 1—figure supplement 1K-L). Actually ap-Gal4 should be used in this situation, as it is expressed only in the dorsal compartment, and the ventral part could serve as an internal control. Similarly, ap-Gal4 should be used to knock down hep, since some p-JNK staining still presents in the hep mutant clone (Figure 1F').

We thank the reviewers for this suggestion. We now use ap-Gal4 to inhibit JNK signaling in the dorsal compartment and see a specific reduction in pJNK in those cells (ap>puc). This data is now included in the text and figures (paragraph one, subheading “JNK is active in the developing Drosophila wing pouch”; Figure 1—figure supplement 1I). hep^r75 (used in the clonal analysis) is likely not a null allele since hemizygous mutant embryos complete dorsal closure, which likely explains the minor residual pJNK staining in the mutant clones. Unfortunately none of the available UAS-hep^RNAi lines are strong enough to induce the known hep mutant phenotype of a split thorax, nor are they strong enough to abolish pJNK staining (data not shown). So unfortunately we cannot perform this experiment precisely as requested. However, we note that we have done 10 independent experiments to validate the specificity of the antibody and are confident in its fidelity.

2) A control UAS line (e.g. UAS-LacZ or UAS -GFP) should be included to exclude the possibility that expression of any protein by ptc- or rn-Gal4 may disturb wing development and affect wing size.

We now show that expression of UAS-GFP by ptc-GAL4 does not affect wing size (ptc>GFP). We have now included the new data in the text and in the figures (paragraph one, subsection “Localized JNK activity regulates global wing size”; Figure 2—figure supplement 1B–C).

3) Is there any effect on wing disc size by blocking JNK activity?

We now show that blocking JNK causes a reduction in wing disc size (ap>bsk^DN). We have included the new data in the text and in the figures (paragraph one, aforementioned subsection; Figure 2—figure supplement 1H–I).

4) Does ptc>egr-induced wing disc enlargement depends on Bsk?

We now show that ptc>egr-induced disc enlargement depends on bsk (ptc>egr, bsk^DN). In fact, these discs are significantly smaller than even control discs (p= 0.0078). We have now included the new data in the text and in the figures (paragraph two, aforementioned subsection; Figure 2L, disc image in Figure 2—figure supplement 2G).

5) Does ptc>egr increases the adult wing size?

We tried to assess whether ptc>egr causes an increase in adult wing size, but these animals were larval lethal. The overgrowth of the disc likely precludes proper pupation. We have now added this point to the text (paragraph two, aforementioned subsection).

6) To show the increased ptc>egr wing disc size is not a consequence of apoptosis, authors should block apoptosis by expressing p35.

We now show that expression of UAS-p35 with UAS-egr does not abolish the size effect of UAS-egr (ptc>egr, p35). We have now included the new data in the text and in the figures (paragraph two, aforementioned subsection; Figure 2L, disc image in Figure 2—figure supplement 2I).

7) A positive control should be included to validate CCP3 staining (Figure 2—figure supplement 1H, I).

We now show that expression of UAS-bsk^AY, a constitutively active JNK allele, induces CCP3 staining in the wing. We have now included the new data in the figures (Figure 2—figure supplement 1N).

8) There is no evidence that the endogenous JNK signaling regulates cell proliferation. The authors should check cell proliferation (Edu staining) in rn- or ptc>bsk^DN discs. The non-cell autonomous increase of cell proliferation in ptc>egr discs could be triggered by caspase activation, rather than a direct outcome of JNK activation. To discriminate the two possibilities, diap1 should be added to block caspase activation.

We now show that inhibiting JNK signaling causes a reduction in proliferation by phosphorylated histone 3 staining (ap>bsk^DN). We have included this new data in the text and in the figures (paragraph four, aforementioned subsection; Figure 2—figure supplement 1J-K). We now also show that expression of UAS-diap1 does not block the growth effect of UAS-egr (ptc>egr, diap1). We have included this new data in the text and in the figures (paragraph two, same subsection; Figure 2L, disc image in Figure 2—figure supplement 2H).

9) Though Jun is not required by JNK to regulate wing size, what about Fos?

We now show that inhibiting Fos does not alter wing size (rn>kay^RNAi#1,2). We have now included the new data in the text and in the figures (paragraph one, subsection “Non-canonical JNK signaling regulates size”; Figure 3—figure supplement 1G-H).

10) The effect of UAS-Yki on UAS-bsk^DN could be additive effect, but not rescue. The authors should check whether ptc> or rn>bsk^DN-induced small wing phenotype could be suppressed in heterozygous lats mutants.

We now show that the rn>bsk^DN wing phenotype can be partially suppressed in a heterozygous lats mutant background (rn>bsk^DN; lats^e2b-1/+). We have now included the new data in the text and in the figures (paragraph two, same subsection; Figure 3—figure supplement 2C–D).

11) UAS-bsk^DN is probably not strong enough to enhance the ptc>yki^RNAi phenotype, what about UAS-puc?

We now show that UAS-puc does not enhance the ptc>yki^RNAi phenotype (ptc>yki^RNAi, puc). We have now included the new data in the text and in the figures (paragraph three, same subsection; Figure 3—figure supplement 2E–F).

12) Though fj appears to be involved in ectopic Yki-triggered wing growth, is it required by endogenous bsk and yki to regulate wing growth? How do the authors explain this? Is fj acting via control of Ft or Ds in this process? To prove this point more clearly, the authors should overexpress fj with PtcGal4. Also, does the fj-lacZ reporter show a stripe pattern similar to pJNK in the 3rd instar larval wing disc?

We have now overexpressed fj and found it also reduces wing size. Therefore, we cannot simply conclude that fj is required by endogenous Bsk and/or Yki to regulate growth. We have now made this clear in the text and figures (paragraph four, same subsection; Figure 3—figure supplement 2I–J).

fj-lacZ is known to be present in a gradient in the wing disc, highest at the A/P and D/V boundaries, emanating distally (Villano and Katz, 1995). Overall, signaling downstream of Yki is intricate and has not been worked out.

13) Does dTRAF1^RNAi block CiACT-triggered wing growth?

We now show that UAS-dTRAF^RNAi can modulate Ci^ACT-triggered wing growth (ptc>Ci^ACT, dTRAF^RNAi), further strengthening our finding. We have now included the new data in the text and in the figures (paragraph three, subsection “Hh sets up pJNK by elevating dTRAF1 expression”; Figure 5—figure supplement 2).

14) Does expression of dTRAF1 increase wing size in a Bsk-dependent manner?

We tried to determine whether expression of UAS-dTRAF1 in the ptc domain increases wing size in a Bsk-dependent manner, but unfortunately expressing UAS-dTRAF1 is lethal. Nevertheless, it has been shown that dTRAF1 function in the eye is Bsk-dependent (Cha et al., 2003). We have now included the new data and discussed this in the text (p. 11-2, para. 2, line 257-259).

15) Surprisingly, knockdown of Jun did not affect wing size-the authors invoke a non-canonical pathway, but could the knockdown have been incomplete? Also, is there redundancy with Kayak? Similarly, the argument that this signaling is non-canonical is based on the puc-lacZ reporter- could this instead be due to delayed reporter activity, or reduced sensitivity?

Null mutant clones of jun do not show a phenotype in the wing (Kockel et al., 1997). Furthermore, puc-lacZ is both a sensitive and quick JNK signaling reporter, as indicated by its fast and robust response to JNK activation (McEwen and Peifer, 2005). We note that UAS-jun^RNAi is strong enough to show an effect on puc-lacZ expression in the stalk region of the wing (Figure 3—figure supplement 1A–B). We now show that inhibition of kayak does not have an effect on wing size (rn>kay^RNAi), and is not redundant with Jun (rn>jun^RNAi, kay^RNAi). These data are consistent with previous reports that jun/fos do not control wing growth (Kockel et al., 1997). We have now included the new data and discussed this in the text (Paragraph one, subsection “Non-canonical JNK signaling regulates size”; Figure 3—figure supplement 1G–H).

[Editors' note: further revisions were requested prior to acceptance, as described below.] 1) Does expression of dTRAF1 increase wing size in a Bsk-dependent manner?

The authors responded that ptc>dTRAF1 is lethal. What about other Gal4 drivers, e.g. rn-Gal4, ap-Gal4? What Cha et al. showed is that dTRAF1-induced cell death depends on JNK. It remains unknown whether dTRAF1 regulates JNK-dependent cell proliferation and growth. The question is quite crucial for this manuscript.

The other Gal4 drivers mentioned (rn-Gal4 and ap-Gal4) express Gal4 in many more cells than ptc-Gal4, so over-expression of dTRAF1 will certainly be lethal. We show that dTRAF1 is required for growth and cell proliferation, as inhibition of dTRAF1 in the wing leads to a small wing phenotype and a loss of pJNK staining (Figure 5D–I). Indeed, dTRAF1 null mutants fail to grow during the larval stages and have very small imaginal discs (Cha et al., Figure 5). This loss of function experiments show that in addition to cell death, dTRAF1 is involved in regulation of growth.

2) Redundancy of Kayak. Can the authors provide evidence of the efficacy of their Kayak knockdown. The provided experiment showing that double knockdown of Kayak and Jun has no effect on growth is only worthwhile if the knockdowns are effective.

These kayak RNAi lines induced the typical JNK-phenotype, thorax closure defect, when driven by ap-Gal4, confirming their efficacy. We have added this in the legend of Figure 3—figure supplement 1.