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. 2022 Feb 18;11:e71705. doi: 10.7554/eLife.71705

Figure 5. eIF2α phosphorylation can induce Xrp1 expression and cell competition.

All panels show single confocal planes from third instar wing imaginal discs, mosaic for the genotypes indicated. All the sections pass through the central region of the disc proper containing nuclei in all genotypes, as indicated by the DNA stain in blue in some panels. (A) Clones expressing white RNAi (green). Clones induced by 7 min heat shock. (B) Clones expressing PPP1R15 RNAi (green)were fewer and smaller than the control (compare panel A). Clones induced by 7 min heat shock. (C) Clones expressing PPP1R15 RNAi (white) contain phosphorylated eIF2α (see C’). (D) Clones induced by 25 ± 5 min heat shock, which results in larger clone areas (white). Labelled clones expressing PPP1R15 RNAi reduced translation rate (see D’). (E) Labelled clones expressing PPP1R15 RNAi (green) underwent competitive apoptosis at interfaces with wild type cells (activated caspase Dcp1 in magenta; see also E’). (F) Nub-Gal4 drives gene expression in the wing pouch, shown in green for RFP, with little expression of Xrp1-HA (magenta; see also F’). (G) PPP1R15 RNAi induces Xrp1-HA expression in the wing pouch (magenta; see also G’). (H) Clones co-expressing PPP1R15 RNAi and Xrp1 RNAi (green) lacked competitive apoptosis (activated caspase Dcp1 in magenta; see also H’). (I) Clones expressing PPP1R15 RNAi (green). Experiment performed in parallel to panel H. Note competitive apoptosis at interfaces with wild type cells (activated caspase Dcp1 in magenta; see also I’), and smaller clone size. Cell death at the basal surface of the same disc shown in Figure 5—figure supplement 1F. (J) Clones co-expressing PPP1R15 RNAi and Xrp1 RNAi (white) showed less eIF2α phosphorylation than for PPP1R15 RNAi alone (compare panel C). Sample prepared in parallel to panel C (in the same tube from fixation to staining). (K) Xrp1 knock-down restored normal translation rate to cell clones expressing PPP1R15 RNAi (green; see also K’). Sample prepared in parallel to panel D (in the same tube from fixation to staining). Additional data relevant to this Figure is shown in Figure 5—figure supplement 1. Genotypes: A: {hs:FLP}/+; act> CD2> Gal4, UAS-GFP / UAS – RNAiw, B: {hs:FLP}/+; act> CD2> Gal4, UAS-GFP / UAS – RNAiPPP1R15 (line: BL 33011) (samples were processed on the same day, not on the same tube), C: {hs:FLP}/+; UAS – RNAiPPP1R15 /TRE-dsRed; act> CD2> Gal4, UAS-GFP /+(line: v107545) (processed in parallel with 5 J), D: {hs:FLP}/+; act> CD2> Gal4, UAS-GFP / UAS – RNAiPPP1R15 (line: BL 33011), E: {hs:FLP}/+; UAS – RNAiPPP1R15 /+; act> CD2> Gal4, UAS-GFP /+ (line: v107545),F: nubGal4, UAS-RFP/+; Xrp1-HA/RNAiw, G: nubGal4, UAS-RFP/ UAS – RNAiPPP1R15; Xrp1-HA/+ (line: v107545), H, J, K: {hs:FLP}/+; UAS – RNAiPPP1R15 / UAS-RNAiXrp1; act> CD2> Gal4, UAS-GFP /+ (line: v107545) (5 H processed in parallel with 5I. Also, 5 K processed in parallel with Figure 5—figure supplement 1B) (line RNAiPPP1R15: v107545 and line RNAiXrp1: v107860), I: {hs:FLP}/+; UAS – RNAiPPP1R15 /TRE-dsRed; act> CD2> Gal4, UAS-GFP /+ (line RNAiPPP1R15: v107545).

Figure 5.

Figure 5—figure supplement 1. eIF2α phosphorylation induces Xrp1 expression and cell competition.

Figure 5—figure supplement 1.

Single confocal planes from third instar wing imaginal discs. (A) Clones expressing PPP1R15-RNAi had increased p-eIF2α levels (A’). (B) Clones expressing PPP1R15-RNAi had reduced translation (OPP) (B’). (C) Basal section of the same disc shown in Figure 5E. More dying PPP1R15-RNAi cells labeled for active caspase (magenta, see also C’) accumulate basally at the boundaries with the wild-type cells. (D) nub-Gal4 driving PPP1R15 RNAi in the wing pouch (green) led to Xrp1-HA expression (magenta; see also D’). (E) Basal confocal section of the wing disc also shown in Figure 5H, mosaic for cells expressing PPP1R15 RNAi and Xrp1 RNAi (green). Even at these basal levels, apoptosis was almost completely rescued by Xrp1 knockdown (magenta, see also E’). (F) Basal confocal section of wing disc mosaic for wild-type cells and cells expressing PPP1R15 RNAi also shown in Figure 5I and a parallel control to panel E. Note extensive cell death basally (magenta, see also F’), as well as smaller clone size (green). Genotypes: A: {hs:FLP}/+; act> CD2> Gal4, UAS-GFP / UAS – RNAiPPP1R15 (line: BL 33011), B: {hs:FLP}/+; UAS – RNAiPPP1R15 /+; act> CD2> Gal4, UAS-GFP /+ (line: v107545) (processed in parallel with Figure 5K), C: {hs:FLP}/+; UAS – RNAiPPP1R15 /+; act> CD2> Gal4, UAS-GFP /+ (line: v107545) (basal side of the same disc in Figure 5E), D: nubGal4, UAS-RFP/ UAS – RNAiPPP1R15; Xrp1-HA/+ (line: Bl 33011), E: {hs:FLP}/+; UAS – RNAiPPP1R15 /+; act> CD2> Gal4, UAS-GFP /+ (line: v107545) (basal side of the same disc in Figure 5H), F: {hs:FLP}/+; UAS – RNAiPPP1R15 /UAS-RNAiXrp1; act> CD2> Gal4, UAS-GFP /+ (line RNAiPPP1R15: v107545 and line RNAiXrp1: v107860) (basal side of the same disc in Figure 5I).
Figure 5—figure supplement 2. eIF2α phosphorylation reduces bristle size.

Figure 5—figure supplement 2.

Minute-like short, thin bristles occur on adults containing clones depleted for PPP1R15, for example compare highlighted posterior scutellar bristle (black arrow) with normal contralateral bristle (white arrow). Genotype: p{hs:FLP}/+; UAS- RNAi PPP1R15/+;act> CD2> Gal4, UAS- GFP /+ (line: Bl 33011).
Figure 5—figure supplement 3. eIF2α phosphorylation is not required for cell competition.

Figure 5—figure supplement 3.

All panels show single confocal planes from third instar wing imaginal discs, mosaic for the genotypes indicated. (A) When mitotic recombination generated twin clones of RpL19+/+/+ cells (brightly labeled) and RpL19+/- ± (black) in the RpL19+/+ background (gray), the RpL19+/- ± did not survive. This was also shown by the absence of mCherry expression, which would have been labelled white in panel A’. (B) Clones of perk-/- RpL19+/- ± (black) were also not seen after recombination in the perk+/- RpL19+/+ background (gray) led to twin clones of perk+/+ RpL19+/+/+ cells (brightly labeled). Any clones of perk-/- RpL19+/- ± would also have been detected through mCherry expression, which would have been labelled white in panel B’. (C) Clones of both RpL36+/+/+/+ cells (brightly labeled) and RpL36+/+ cells (black) survived in the RpL36+/+/+ background (gray). (D) No clones of RpL36+/- ± (black) were seen after recombination in the RpL36+/+ background (gray) generated twin clones of RpL36+/+/+ cells (brightly labeled). (E) No clones of perk-/- RpL36+/- ± (black) were seen after recombination in the perk+-/- RpL36+/+ background (gray) generated twin clones of perk+/+ RpL36+/+/+ cells (brightly labeled). (F) Clones of perk-/- RpL36+/+ cells (black) were readily obtained after recombination in the perk+-/- RpL36+/+/+ background (gray) generated twin clones of perk+/+ RpL36+/+/+/+ cells (brightly labeled). Genotypes: A,A’: y w, hs-FLP; Act5C > GAL4 w, UAS-mCherry-CAAX, Df(2 R)M60E; FRT82B, ubi-GFP-nls, tubP-GAL80, M{RpL19 genomic}ZH-86Fb/FRT82B. B,B’: y w, hs-FLP; Act5C > GAL4 w, UAS-mCherry-CAAX, Df(2 R)M60E; FRT82B, ubi-GFP-nls, tubP-GAL80, M{RpL19 genomic}ZH-86Fb/FRT82B Perknull C: FM7/hs-FLP; FRT82B p{RpL36+} p{arm:LacZ}/FRT82B. D: Df(1)R194/hs-FLP; FRT82B p{RpL36+} p{arm:LacZ}/FRT82B. E: Df(1)R194/hs-FLP; FRT82B p{RpL36+} p{arm:LacZ}/FRT82B PERKnull F: FM7/hs-FLP; FRT82B p{RpL36+} p{arm:LacZ}/FRT82B PERKnull The FM7 and Df(1)R194 genotypes used in panels C-F were unmarked in wing discs. The experiment with FRT82B led to the two classes of discs with and without clones shown in panels C and D, and the experiment with FRT82B perk- led to the two classes of discs with and without clones shown in panels E and F. A parallel experiment with FRT82B Xrp1-, which rescues RpL36+/- ± (Lee et al., 2018) led only to wing discs with clones equal in size and frequency to reciprocal twin clones.