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Fold formation at the compartment boundary of Drosophila wing requires Yki signaling to suppress JNK dependent apoptosis

Compartment boundaries prevent cell populations of different lineage from intermingling. In many cases, compartment boundaries are associated with morphological folds. However, in the Drosophila wing imaginal disc, fold formation at the anterior/posterior (A/P) compartment boundary is suppressed, probably as a prerequisite for the formation of a flat wing surface. Fold suppression depends on optomotor-blind (omb). Omb mutant animals develop a deep apical fold at the A/P boundary of the larval wing disc and an A/P cleft in the adult wing. A/P fold formation is controlled by different signaling pathways. Jun N-terminal kinase (JNK) and Yorkie (Yki) signaling are activated in cells along the fold and are necessary for the A/P fold to develop. While JNK promotes cell shape changes and cell death, Yki target genes are required to antagonize apoptosis, explaining why both pathways need to be active for the formation of a stable fold.

requirements even within one epithelium. For example, in Drosophila gastrulation, the ventral furrow forms by apical constriction whereas the dorsal folds arise by a basal shift of the adherens junctions 26 .
In the larval wing disc pouch (the future wing blade), the normal, graded expression of Dpp and Wg does not instruct folding but rather is involved in maintaining the appropriate position-specific cell shape 27 . In the columnar main epithelium, loss of Dpp signaling causes extrusion of cells correlated with loss of the apical microtubule web 28,29 . Similarly, loss of Dpp targets Optomotor-blind (Omb) or Spalt lead to retraction of cells toward basal membrane 30,31 . Dpp signaling cell-autonomously promotes and maintains the elongated columnar shape of wing disc cells by regulating Rho1 and the regulatory light chain of non-muscle myosin II 32 . Wg signaling cell-autonomously promotes and maintains the columnar shape of wing disc cells through maintaining Vestigial (Vg) expression 33 . The Wg gradient, centered on the D/V boundary, instructs similarly shaped gradients of DE-cadherin concentration and apical cell circumference (high and constricted, respectively, close to the D/V boundary) 34 . The loss of Adenomatous polyposis coli (APC), a negative regulator of Wg signaling, leads to apical constriction and invagination independent of its effect on the DE-cadherin level. Wg, too, acts by activation of Rho1 and Myosin II 35 .
The folds which separate parasegments in the Drosophila embryonic ectoderm separate fields of cells that are related by lineage (compartments) 36 . But even in the absence of lineage restriction, groups of epithelial cells differing in gene expression and fated to develop into different structures tend to be separated by a fold. For instance, in the wing imaginal disc, which gives rise to adult notum, hinge, and wing blade, several folds orthogonal to the proximo-distal axis separate gene expression domains without being lineage restriction boundaries 37,38 . The most distal of these, the blade/hinge fold develops under control of the Omb-related T-box transcription factors Dorsocross (Doc) 39 . The proximal notum/hinge fold requires the complementary expression of Omb in hinge and Iroquois complex (Iro-C) in notum 40 . In contrast, the A/P compartment boundary is not associated with a fold and remains morphologically inconspicuous throughout development 41 , even though it derives from the corresponding infolded parasegmental boundary in the embryonic ectoderm 42 . It is conceivable that fold formation was selected against because of the structural requirement for the adult wing as a flight appendage. Indeed, fold formation is actively suppressed by a genetic program. Omb which is expressed in most of the pouch 43 is required to maintain the normal epithelial structure at the A/P boundary. Reduction of Omb level in the pouch causes an apical morphogenetic defect at the A/P boundary due to contraction of cells along their apical-basal axis 44 . We here investigate the mechanisms of boundary fold formation elicited by Omb loss. We found that A/P fold formation is dependent on activation of JNK signaling induced by loss of omb. Loss of omb also induced Yki activity which promoted cell survival and attenuated the pro-apoptotic activity of JNK. Our results reveal a network of signaling pathways induced by loss of omb that controls cell shape and ensures cell survival of folded cells at the A/P boundary.

Results and Discussion
We and others have shown before that Omb prevents aberrant apical fold formation at the A/P boundary 44,45 . When Omb is directly or indirectly repressed in the P compartment, the A/P boundary of the wing develops a deep apical fold in the larval wing disc and a cleft in the adult wing 44 . We here use this model system to investigate the mechanisms of boundary fold formation under three genetic manupulations, ptc-Gal4 UAS-tkv, en-Gal4 UAS-omb-RNAi, and nub-Gal4 UAS-omb-RNAi (Fig. S1).

JNK is ectopically activated to initiate A/P fold formation.
To investigate potential effectors downstream of Omb, we monitored the expression or activity of candidate targets. JNK signaling is an important pathway in the regulation of wing disc morphogenesis 46,47 . We monitored JNK pathway activity by monitoring transcription of the JNK target gene puckered (puc) 48 . In both ptc> tkv and en> omb-RNAi wing discs, puc was activated along the A/P fold (Fig. 1A,B). puc was initially activated in cells adjacent to the fold in early-mid L3, then its expression extended further into the A and P compartments (Fig. S2). These data indicate that JNK signaling is activated in the process of A/P fold formation.
We next asked whether JNK activation is required for A/P fold generation. Co-expressing omb-RNAi and a dominant negative form of JNK, bsk DN 49 , was sufficient to suppress A/P fold formation (Fig. 1C).
This suggests that activation of JNK signaling is required in this process. To test for sufficiency of JNK activation for A/P fold formation, we activated JNK by expressing hep CA (encoding a constitutively active form of JNKK 50 ) for a short duration controlled by dpp-Gal4 and tub-Gal80 ts (continuously activation of hep CA induced severe apoptosis thereby disturbing observation of cell morphology). Under these conditions folds occurred throughout the dpp-Gal4 expression domain (Fig. 1D). The broad anterior activation of JNK signaling did not lead, however, to a discrete A/P fold.
The matrix metalloproteinase 1 (Mmp1) is induced by ectopic activation of JNK during morphological reorganization of epithelia [51][52][53][54][55] . When the JNK pathway was activated for 24 h in the dpp-Gal4 expression domain, Mmp1 was broadly induced anterior to the A/P boundary, similar to the plexus of epithelial folds observed under these conditions (Fig. 1E). However, in en> omb-RNAi wing discs, Mmp1 accumulated in a discrete stripe of A/P fold cells on both sides of the fold (Fig. 1F and F'). Co-expression of omb-RNAi and Mmp1-RNAi with nub-Gal4 rescued the A/P fold with full penetrance (Fig. 1G). Uniform reduction of omb expression on both sides of the A/P boundary, like posterior omb reduction, leads to A/P fold formation (Fig. S1E and F).
However, expression of Mmp1 with dpp-Gal4 along the A/P boundary did not generate a fold (Fig. S3). These data suggests that either additional gene expression changes, induced by the loss of omb, are necessary for A/P fold formation or that Mmp1 must be induced on both sides of the A/P boundary.
Yki-Diap1 signaling is activated parallel to the JNK pathway. It  JNK-dependent apoptosis in the wing pouch 50 . However, omb knock-down did not induce apparent apoptosis at the A/P boundary 44 , although JNK signaling was activated ( Fig. 1A and B). We assume that the apoptosis pathway is repressed in this case. Yki signaling can be induced by the JNK pathway 56 . Yki targets such as Death-associated inhibitor of apoptosis 1 (Diap1) and the microRNA bantam (ban) can repress apoptosis [57][58][59] . We analyzed transcription of the Yki target expanded (ex 60 and observed that ex-lacZ was up-regulated at the A/P fold generated by en> omb-RNAi (Fig. 2B), suggesting an activation of Yki signaling during A/P fold formation. This was confirmed in nub> omb-RNAi wing discs (Fig. 2C). Upregulation at the A/P fold was also observed for Diap1 (Fig. 2E). The Yki target ban is suppressed by Omb in the medial wing discs 61 . Consistently, ban was up-regulated in the medial wing disc of nub> omb-RNAi larvae, with the strongest enhancement along the A/P boundary (Fig. 2G). Therefore, during A/P fold generation, ban and Diap1 were both activated and could suppress potential apoptosis induced by changes in cell shape and JNK activation.
In order to determine whether the Yki targets were induced as a consequence of JNK signaling, we co-expressed bsk DN and omb-RNAi in the nub-Gal4 domain. When JNK pathway and A/P fold were suppressed, ex-lacZ expression was still activated at the A/P boundary (Fig. S4A). This also held for Diap1 and ban expression ( Fig. S4B and C). This suggests that in A/P fold formation Yki can be activated even when the JNK pathway is blocked. Yki activation, thus, appears to occur parallel to JNK signaling and is not sufficient for fold formation.
To test whether the suppression of cell death by Yki signaling is required for omb-loss induced A/P fold formation, yki-RNAi was co-expressed with omb-RNAi in the nub-Gal4 domain. As shown in Fig. 3A-A", co-expressing yki-RNAi was sufficient to suppress the formation of A/P fold. Severe cell death occurred in this double knock-down. When p35 was co-expressed with omb-RNAi and yki-RNAi in the nub-Gal4 domain to inhibit apoptosis, cell death was effectively suppressed (Fig. 3B and B') and the A/P fold appeared again ( Fig. 3B and C). These data indicate that Yki signaling is required for A/P fold by ensuring cell survival.
Generally, abnormal activation of the JNK pathway induces apoptosis. For instance, expression of activated tumor genes or mutation in tumor suppressor genes lead to JNK-induced cell invasion and apoptosis 55,62-64 . However, in omb-knocked-down wing discs, the activation of JNK pathway did not cause cell death along the A/P fold 44 . We suggest that cell death is suppressed by the simultaneous induction of a cell survival pathway. Yki has an important role in promoting cell survival by driving the expression of downstream genes such as Diap1 and ban 65 . We found these genes upregulated along the A/P fold (Fig. 2). This suggests that Yki antagonizes apoptosis along the fold. Previous studies identified JNK as a promoter of Yki activity in the wing disc 56,63,66 . But this regulatory relationship is not absolute 62,63,67 . We found that co-expression of omb-RNAi and bsk DN had no effect on ex, Diap1, and ban expression (Fig. S4). This indicates that, at the A/B boundary, Yki is activated parallel to JNK signaling.  Larvae were raised at 25 °C. For efficient expression of RNAi transgenes, larvae were raised at 29 °C. Larvae containing Gal80 ts -Gal4 combinations were raised at 18 °C and then were shifted to 29 °C that allows GAL4 to function and activate transcription of UAS controlled transgenes.