Hedgehog signalling is required for cell survival in Drosophila wing pouch cells

An appropriate balance between cell survival and cell death is essential for correct pattern formation in the animal tissues and organs. Previous studies have shown that the short-range signalling molecule Hedgehog (Hh) is required for cell proliferation and pattern formation in the Drosophila central wing discs. Signal transduction by one of the Hh targets, the morphogen Decapentaplegic (Dpp), is required for not only cell proliferation, but also cell survival in the pouch cells. However, Hh function in cell survival and cell death has not been revealed. Here, we found that loss of Hh signal activity induces considerable Caspase-dependent cell death in the wing pouch cells, and this process was independent of both Dpp signalling and Jun-N-terminal kinase (JNK) signalling. Loss of Hh induced activation of the pro-apoptotic gene hid and inhibition of diap1. Therefore, we identified an important role of Hh signalling in cell survival during Drosophila wing development.

receptor Patched (Ptc) to derepress the activity of a transmembrane protein Smoothened (Smo) 44,46,47 . The activated Smo maintains Cubitus interruptus (Ci) in an active form 48 . The Ci[act] enters the nucleus and induces target genes expression, including engrailed (en), ptc, Collier (col), and dpp. These target genes are activated in a Hh-concentration dependent manner: The cells close to the AP compartment boundary receive the highest level of Hh and induce the ptc and en; the cells away from the AP compartment boundary will receive the lowest level of Hh and induce the expression of col; the cells between these two type of cells receive the moderate level of Hh and induce the expression of dpp [49][50][51][52] . ptc acting as the target gene of Hh signaling also inhibits Smo expression in the absence of Hh 46 .
Previous studies have demonstrated that Hh plays an important role in the proliferation [38][39][40] and patterning 41,[53][54][55] . Hh also controls cell survival in germ cells 56,57 , neural crest cells 58,59 as well as tumor cells 60,61 in vertebrate. A recent study has shown that in Drosophila eye disc, deregulated Hh signalling promotes cell survival in a non-autonomous manner 62 . However, it is not clear whether Hh signalling is also involved in the control of cell survival in wing disc. Here, we found that Hh signaling plays an important role in the cell survival in the Drosophila wing pouch. Lacking Hh signaling induced cell death is independent of Dpp and JNK signaling pathways.

Results and Discussion
Down-regulation of Hh signalling results in apoptosis in Drosophila wing disc. The wild-type wing disc undergoes rapid proliferation with little apoptosis (Fig. 1A). When down-regulating Hh expression using a temperature-sensitive allele, hh ts46 , apoptosis, indicated by anti-Caspase-3 staining, occurred in the wing pouch (Fig. 1B). Then, the Hh transportation from the posterior to the anterior was blocked by expressing ttv-RNAi in the dpp-Gal4 domain, obvious apoptosis was consistently observed in the central wing discs (Fig. 1C). Then, we assessed whether Smo mediates the role of Hh in regulating apoptosis. Apparent apoptosis was also induced in the central wing discs when smo was inhibited by the expression of smo-RNAi in the dpp-Gal4 domain (Fig. 1D). To further confirm the above results, Hh signalling activity was suppressed by expressing smo PKA12 (a mutation at the PKA site) 35 , smo-RNAi, and ptc in all the wing disc cells (driven by c765-Gal4), wing pouch cells (driven by ms1096-Gal4 and nub-Gal4), and posterior cells (driven by hh-Gal4). All these manipulations caused driver results in small wing discs with massive cell death (red). Note that panel H is also from a 3 rd instar larvae and is shown at the same magnification with other pannels. When ptc is expressed in the whole wing disc, the wing disc size is reduced apparently due to a proliferation defect. obvious apoptosis in the medial wing discs (Fig. 1E-H). These data suggests that suppression of Hh singling, at the levels of transcription, transportation, or signal transduction, induces cell death in the medial Drosophila wing disc, thereby revealing a new role for Hh signalling in cell survival.
Apoptosis induced by the lack of Hh signalling is Dpp-independent. dpp, one target gene of Hh signalling, has been demonstrated to be an important survival factor [24][25][26] . To test whether the apoptosis caused by suppression of Hh signalling is due to the reduction of Dpp signalling, we examined the dpp expression using a dpp-lacZ reporter. In the wild-type background, dpp is expressed in a stripe of cells along the AP boundary ( Fig. 2A). When Hh signalling was suppressed by smo-RNAi, the dpp transcription level was mildly reduced compared with that in wild type ( Fig. 2A and B). Ptc, which is only expressed in a narrow stripe of cells just anterior to the AP compartment boundary by sensing the highest level of Hh, is a direct readout of Hh signalling. To obtain an internal control, we used a dorsal-specific driver, ap-Gal4, to express smo-RNAi (Fig. 2C). Ptc was abolished completely in the ap-Gal4 region (Fig. 2C), while Omb, one of the targets of Dpp signalling, was still detectable. The apoptosis was consistently observed in the ap > smoRNAi wing disc (Fig. S1). These data implied that the cell death might be a direct consequence of the suppression of Hh signalling and not a side effect of the reduction in Dpp signalling. To test this possibility, we co-expressed dpp with smo-RNAi to see whether the apoptosis can be rescued. In the control, dpp was solely expressed in either the dpp-Gal4 or the nub-Gal4 region, and there was no cell death in the pouch region except in the notum region ( Fig. 2D and F). When dpp was co-expressed with smo-RNAi in the dpp-Gal4 domain, the apoptosis was still present in the wing pouch (Fig. 2E). The failure of dpp in the rescue experiment was confirmed in the nub-Gal4 domain (Fig. 2G). Taken together, the cell death caused The apoptosis (red) is still induced even when dpp is co-expressed with smo-RNAi in the dpp-Gal4 (E) and nub-Gal4 (G) domains. A specific marker for the anterior compartment, Ci, is revealed by anti-Ci staining (green), to show the midline of overgrown wing discs. by the suppression of Hh signalling is a direct consequence of the Hh pathway and not a side effect of disturbance in Dpp signalling.

Cell death induced by the lack of Hh signalling is JNK independent. Previous studies have shown
that JNK signalling plays a vital role in cell morphology, cell invasion, and apoptosis 26,31,63,64 . JNK, monitored by puc-lacZ 65 , was activated when Dpp signalling was inhibited by expressing its inhibitor dad (Fig. 3A). However, there was no ectopic puc-lacZ expression when Hh signalling was inhibited ( Fig. 3B and C). Furthermore, the apoptosis was not reduced when JNK signalling was inhibited by co-expressing a dominant negative form of bsk (bsk DN ) (Fig. 3D,E,F,G and J) or by co-expressing hep-RNAi (Fig. 3H,I and J). These data suggested that the apoptosis caused by the suppression of Hh signalling is independent of JNK signalling.
Cell death induced by the lack of Hh signalling is mediated by hid and diap1. Apoptosis is a highly conserved pathway in both invertebrate and vertebrate systems. The key mediators, including Hid, Drice, Dronc, and Diap1, were mentioned in the introduction section. Subsequently, we tested whether there was a link between the cell death pathway and the Hh pathway. When smo was suppressed in the wing pouch, the transcription of hid-lacZ was markedly increased (Fig. 4A and B), while the transcription of diap1-lacZ was reduced ( Fig. 4C and  D). Co-express hid-RNAi and smo-RNAi in the nub-Gal4 domain supppressed the cell death ( Fig. 4E and J). The cell death was also suppressed completely when diap1 was co-expressed with smo-RNAi in the nub-Gal4 domain ( Fig. 4F and J). Next, we examined the roles of an initiator caspase (Dronc) and an effector caspase (Drice). Use of either dronc-RNAi or drice-RNAi partially rescued the apoptosis (Fig. 4G,H and J) compared with the control (Fig. 1F). This cell death was suppressed completely when P35 was co-expressed with smo-RNAi in the nub-Gal4 domain ( Fig. 4I and J). Taken together, we demonstrated that the cell death caused by the suppression of Hh signalling is at least partially mediated by the activation of the proapoptotic gene hid and by inhibition of diap1.

Cell death induced by the lack of Hh signalling led to small adult wings.
To assay the apoptosis effect on adult wing, we measured the size of the medial wing where apoptosis always occur in the manipulations of Hh signalling. Compared with the wild-type adult wing (Fig. 5A), reduction of Hh signalling by hh ts (Fig. 5B and J) and smo-RNAi ( Fig. 5C and J) in the whole wing blade resulted in an obvious reduction in wing size. There was no significant difference in adult wing size between nub > smo-RNAi, GFP and nub > smo-RNAi, hep-RNAi (Fig. 5C, D and K). Suppression of the pro-apoptotic genes hid by hid-RNAi showed rescue effect in adult wing size (Fig. 5C,E and K). Co-expressing diap1 with smo-RNAi in the nub-Gal4 domain had an obvious rescue effect of adult wing size compared with smo-RNAi alone (Fig. 5C,F and K). However, the adult wing size of nub > smo-RNAi, hid-RNAi and nub > smo-RNAi, diap1 did not restore to the wild type size, which may be due to the proliferation effect of Hh signalling. Suppression of the initiator caspase and the effector caspase showed a slight rescue effect in adult wing size (Fig. 5C,G,H and K). Co-expressing the pan-caspase inhibitor P35 could largely rescue the adult wing size (Fig. 5I and K). These data suggest that the wing size is regulated not only by proliferation control, but also by cell survival control of Hh signalling.
The wing veins' pattern was also altered. Consistent with previous reports, lacking Hh signalling lead to loss of L3 and L4 veins (Fig. 5C) 41,55,66 . When the cell death was suppressed by hid-RNAi or dronc-RNAi, the L4 vein was rescued ( Fig. 5E and G). When the cell death was suppressed by diap1 or drice-RNAi, the L3 and L4 veins were only partially rescued up to the proximal region with fusion effect (Fig. 5F and H). The L3 and L4 veins could be completely rescued only when the cell death is suppressed by P35 (Fig. 5I). Therefore, Hh signalling regulates the medial wing pattern formation, at least in part, by control of cell survival.
Various signalling pathways are involved in cell survival. The Hippo/Warts/Yorkie (Hpo/Wts/Yki) pathway is known to control apoptosis. Hpo negatively regulates the transcription factor Yki by phosphorylating it. The dephosphorylation of Yki activates the target gene diap1 to inhibit apoptosis [67][68][69] . Notch and Wingless (Wg) promote cell survival by inhibiting Caspase [70][71][72] . Epidermal Growth Factor Receptor (EGFR) is required for cell survival in the Drosophila eye disc, where it inhibits the pro-apoptotic gene hid [73][74][75] . Dpp is involved in cell survival by activating the downstream target genes omb 24 and sal [25][26][27] . Here, we found that Hh is also involved in cell survival in the Drosophila wing disc through hid and diap1, and we present a model to explain the possible genetic regulation (Fig. 5L). Although hid-RNAi and diap1 can efficiently suppress the cell death induced by smo-RNAi expression (Fig. 4E,F and J), the adult wings are not restored to wild type size (Fig. 5E,F and K). We can not rule out a possibility of compensational mechanism between the Hh-regulated cell survival and proliferation. However, the disruption of any of the above signalling pathways can induce apoptosis. There must be a mechanism by which a cell integrates all of these signals to determine its survival status. Our results suggest that Smo is the most downstream component of Hh signalling that is related to cell survival. To better understand how Hh-Smo signalling promotes cell survival, the potential mediators between Smo and apoptosis pathway need to be identified. In the Drosophila eye, diap1 is up-regulated by deregulated Hh signalling 62 . In the Drosophila wing, diap1 is apparently suppressed under the condition of Hh loss-of-function ( Fig. 4D and D'). Hh has been reported essential for the cell survival in vertebrate [56][57][58][59][60][61] . A recent study has shown that the requirement of Hh in cell survival in pancreatic cancer cells is dependent on the up-regulation of baculoviral IAP repeat-containing 3 (BIRC3) gene which belongs to IAP family 76 . Therefore, Hh is functionally conserved in cell survival control in both vertebrate and invertebrate.

Dissection of larvae.
Wing imaginal discs were dissected from 3 rd instar Drosophila larvae according to a standard protocol and were fixed for 30 min in 4% paraformaldehyde in PBT (PBS with 0.3% Triton X-100). Adult wing imaging. Adult wing images were collected using an inverted microscope (AMG EVOS, America).
Adult wing measurement. The area of the adult wing was measured using Image-J software, and the calculation and measurement were carried out using GraphPad Prism 5 Project.