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

  • ️Sun Jan 01 2017

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

Juan Lu et al. Sci Rep. 2017.

Abstract

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.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1

Hh signalling activity is required for cell survival in Drosophila wing disc. In this and subsequent figures, wing discs are oriented with dorsal up and anterior left. (A) In the wild-type wing disc, there is no obvious apoptosis indicated by anti-Caspase-3 staining (red). The smo expression pattern is revealed by anti-Smo staining (green). (B) Heterozygote of a hh temperature-sensitive mutant allele showing the induction of apparent cell death (red). (C) Suppression of Hh transportation from posterior to anterior by expressing ttv-RNAi in the dpp-Gal4 domain (inset panel, green) results in apparent cell death (red). (D) Smo (green) is suppressed by expressing smo-RNAi in the dpp-Gal4 domain, and that induces massive cell death (red). (E) Suppressing smo by expressing a mutant smo PKA in all the wing pouch cells induces massive cell death (red). ms1096-Gal4 is expressed in all the wing pouch cells with a higher activity within the dorsal compartment (See inset panel, green). (F and G) Suppressing smo by expressing smo-RNAi in large regions induces massive cell death (red). The nub-Gal4 domain covers the pouch region (See inset panel in F, green). hh-Gal4 is expressed only within the posterior compartment (See inset panel in G, green). (H) Suppressing Smo activity by expressing the inhibitor gene ptc in all the wing cells under the c765-Gal4 driver results in small wing discs with massive cell death (red). Note that panel H is also from a 3rd 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.

Figure 2
Figure 2

Cell death caused by Hh signalling reduction is dpp-independent. (A) The dpp expression pattern in wild-type wing discs is revealed by the dpp-lacZ enhancer trap line (red). (B) dpp-lacZ is still present (red) when Smo (green) is suppressed within the dpp-Gal4 domain. (C) Omb (red) is still detectable when smo is inhibited in the dorsal compartment by the dorsal-specific driver ap-Gal4. The Hh target Ptc is apparently inhibited within the dorsal compartment (green). The dotted line indicates the boundary between the dorsal and ventral compartments. (D and F) In control experiments, dpp is expressed within the dpp-Gal4 domain (D) and the nub-Gal4 domain (F). No cell death occurs within the wing pouch regions (dotted regions) except for a patch of dead cells (red) in the presumptive hinge domains, which might be a side effect of overgrowth induced by excess Dpp. (E and G) 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.

Figure 3
Figure 3

The cell death induced by suppressing Hh signalling is JNK-independent. (A) Ectopic JNK signalling activity can be revealed by using a puc-lacZ. In a positive control experiment, puc-lacZ reporter (red) is activated when Dpp signalling is suppressed by expressing dad within the nub-Gal4 domain. (B,C) Suppression of Hh signalling by expressing smo-RNAi does not induce ectopic puc-lacZ (red). (D–I) Inhibition of JNK signalling by expressing a dominant negative form of bsk DN (E and G) or by suppressing an upstream effector (by expressing hep-RNAi) (I) can not rescue the apoptosis induced by expressing smo-RNAi. (D,F and H) The control experiments show no apoptosis when bsk DN or hep-RNAi is expressed alone. (J) Statistics for the apoptotic cell number per wing disc of each genotype mentioned above. ns stands for no significant difference.

Figure 4
Figure 4

Cell death induced by the lack of Hh signalling activity requires the activation of the pro-apoptotic gene hid and the reduction of diap1. (A) In the control nub > GFP wing disc, there is no apparent hid-lacZ expression (red). (B) The pro-apoptotic gene hid (red) is activated in the wing pouch when smo-RNAi is expressed within the nub-Gal4 domain. (C,D) diap1-lacZ (red) is apparently reduced in the medial region of the nub > smo-RNAi wing discs (D) compared with the control (C). (E) Suppressing pro-apoptotic gene hid by expressing hid-RNAi efficiently suppresses the cell death compared with the control (Fig. 1F). (F) Co-expression of diap1 with smo-RNAi efficiently suppresses the cell death. (G) Suppressing initiator caspase activity by expressing dronc-RNAi largely suppresses the cell death induced by smo-RNAi expression. (H) Suppressing effector caspase activity by expressing drice-RNAi reduces the cell death induced by smo-RNAi expression. (I) Co-expression of P35 with smo-RNAi suppresses the cell death completely. (J) Statistical analysis for the apoptotic cell number per wing disc of each genotype mentioned above. Means ± SEM indicated *** are significantly different (pairwise comparisons performed using t-tests, p < 0.0001).

Figure 5
Figure 5

The phenotypes of adult wings. (A) Wild-type adult wing. The area between L2 and L5 veins is measured. The region between L2 and L5 veins of each control wing (A and C) is marked in green and compared in each manipulation (B,D–H) because most of the cell death induced by the suppression of Hh signalling occurs in the presumptive region between the L2 and L5 veins. (B) The hh ts adult wing is smaller than the wild-type wing (A). (D) Co-expressing hep-RNAi does not rescue the small size between L2 and L5 compared with the expression of smo-RNAi (C). (E) Co-expressing hid-RNAi increases the indicated area compared with the control wing in C. (F) Co-expressing diap1 increases the indicated area compared with the control wing in C. (G) Co-expressing dronc-RNAi increases the indicated area compared with the control wing in C. (H) Co-expressing drice-RNAi increases the indicated area compared with the control wing in C. (I) The wing area is rescued by co-overexpressing P35. (J and K) Statistics for the green-marked wing regions in each genotype mentioned above. Means ± SEM indicated by ** or *** are significantly different (pairwise comparisons performed using t-tests, p < 0.001 or p < 0.0001). (L) A model of the genetic pathway regulating cell survival by Hh signalling.

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