Figure 5: Inactivation of Hedgehog signalling induces autophagy in the Drosophila larval fat body.
(a,b) Control (a) or hh[ts2] (b) larvae were incubated at permissive temperature (18 °C) to the third instar stage, then cultured at non-permissive temperature (29 °C) in well-fed conditions for 24 h. Dissected fat body tissue was incubated in Lysotracker Red and DAPI and imaged unfixed. (c) Lysotracker Red-labelled fat body containing single-cell flipout clones (GFP positive) overexpressing patched. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed. (d) Confocal image of a GFP-positive patched-expressing clone, with mCherry-Atg8a expressed in all cells. Fat body was dissected from well-fed larvae and imaged after formaldehyde fixation. (e) GFP-marked flipout clones of CiCell-expressing cells. Fat body was dissected from well-fed larvae, incubated in Lysotracker Red/DAPI and imaged unfixed. (f) Confocal image of fixed mCherry-Atg8a expressing fat body containing a GFP-marked clone of CiCell-expressing cells. (g) Cells homozygous for the null costal2[2] allele (marked by lack of GFP) were generated by flp/FRT-mediated recombination in mCh-Atg8a expressing fat body. Larvae were starved 4 h before dissection and fixation. Scale bar represents 25 μm in a, b, c and e, and 10 μm in d and f. Genotypes: (a) y,w; (b) hh[ts2/ts2]; (c) hs-flp; UAS-ptc/+; Act>CD2>GAL4 UAS-GFPnls/+; (d) hs-flp; UAS-ptc/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/+; (e ) hs-flp; UAS-CiCell/+; Act>CD2>GAL4 UAS-GFPnls/+;(f ) hs-flp; UAS-CiCell/+; r4:mCherry-Atg8a Act>CD2>GAL4 UAS-GFPnls/+; (g ) hs-flp; FRT42D cos2[2]/Cg-GAL4 UAS-mChAtg8a[2L] FRT42D UAS-GFPnls.
