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. 2021 Oct 4;10:e74047. doi: 10.7554/eLife.74047

Figure 3. Perk is necessary and sufficient to induce gstD-GFP expression.

(A–D) Representative eye imaginal discs with gstD-GFP (green) alone channels (A–D), and gstD-GFP (green) channels merged with anti-Rh1 (red) (A’-D’). The genotypes of the discs are as follows: (A) GMR-Gal4; gstD-GFP/+, (B) GMR-Gal4; gstD-GFP/UAS-Rh1G69D, (C) GMR-Gal4; gstD-GFP/UAS-Rh1G69D; Perke01744 (D) GMR-Gal4; gstD-GFP/UAS-Perk. (E) Quantification of gstD-GFP pixel intensity fold change from posterior eye discs of the indicated genotypes from A-D. (F) qRT-PCR of gstD2 normalized to RpL15 in S2 cells treated with 10 ug/mL tunicamycin for 0, 4, or 8 hr. Cells were either pre-treated with control dsRNA (GFP) or those targeting Perk. (G–I) gstD-GFP (green) containing GMR > Rh1G69D eye discs with either control mosaic clones (G), or those with Perk loss-of-function clones (H, I). Homozygous Perk e01744 clones in H’ and I are marked by the absence of β-galactosidase (red) expression. Note that gstD-GFP expression occurs broadly in the background of control mosaic clones, but is largely absent in Perk homozygous mutant clones. (I, I’) A magnified view of the dotted inset in (H’). (I’) β-galactosidase only channel (marking Perk-positive cells; in white). Scale bar = 50 μm. * = p < 0.05, ** = p < 0.005, *** = p < 0.001.

Figure 3.

Figure 3—figure supplement 1. PERK’s kinase domain and eIF2α phosphorylation mediate gstD-GFP induction.

Figure 3—figure supplement 1.

(A) A schematic diagram of eIF2α phosphorylation regulation. (B–G) Late third instar larval imaginal discs expressing gstD-GFP (green). Posterior is to the right. (B–E) Eye imaginal discs. Expression of a Perk transgene with a kinase domain mutation (PerkKD) through the GMR-Gal4 driver does not induce gstD-GFP expression (B), whereas the wild type Perk transgene strongly induces this reporter (C). Expression of gadd34 RNAi through the GMR-Gal4, ey-Gal4 drivers induces gstD-GFP at the posterior edge of the eye disc (E), whereas gstD-GFP is not detected in the no RNAi control (D). (F, G) Wing discs. No RNAi control shows low basal levels of gstD-GFP expression (F), whereas gadd34 RNAi expression through the hh-Gal4 driver induces gstD-GFP throughout the domain of Gal4 expression (G). Scale bars in E, G = 50 μm.