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. 2020 Feb 18;9:e52743. doi: 10.7554/eLife.52743

Figure 2. Toll-2 knock-down caused neuronal loss in the pupal and adult brain.

(A,B) Overlapping but distinct expression of Toll-2 and the adaptor MyD88, visualised with MyD88 >tdTomato, Toll-2pTVGAL4 > UASFlyBow, MyD88 >histone YFP and Toll-2pTVGAL4 > UAShistone YFP. Toll-2 is expressed in more Kenyon cells (arrowheads) than MyD88. Note the large MyD88+ cells in the optic lobes (B, arrowhead). (C) Diagram of signalling pathways downstream of Toll receptors regulating cell death and cell survival (adapted from Foldi et al., 2017). (D,E) The pan-neuronal marker anti-Elav co-localises with His-YFP in both MyD88+ and Toll-2+ cells, whereas the pan-glial marker anti-Repo only co-localises with MyD88 >His-YFP+ cells (arrowheads). (F) Drawings showing in green the central brain region of interest (ROI), and Kenyon cells (KCs), used for automatic cell counting with DeadEasy. (G) Toll-2 RNAi knock-down increased the number of anti-Dcp1+ apoptotic cells, in day one pupal central brains (dashed line indicates ROI); cells quantified automatically in the ROI in 3D throughout the stack of images, with DeadEasy software. Left: full projection; right: projection of five optical sections only (5 μm). Quantification in box-plot graph: Student t-test p=0.0295. (H) Toll-2 RNAi knock-down decreased MyD88+ cell number in pupal and adult brains, latter using two independent RNAi lines; MyD88 RNAi knock-down also decreased cell number in the pupal brain (left), but not in the adult brain (right). Dashed lines in (H) indicate central brain ROI used for automatic counting of MyD88 >hisYFP+ cells with DeadEasy Central Brain software. Box-plots: Left: One Way ANOVA p<0.001, and right p<0.0001, post-hoc Dunnett tests. (I) Neither Toll-2 over-expression nor RNAi knock-down altered KC number, in pupal nor adult brains, but Toll-2pTV/Tollp2Δ7-35 mutants had more KCs. Dashed lines in (I) indicate ROI counted automatically with DeadEasy Kenyon Cells software, box-plot graphs on right: Kruskal Wallis ANOVA, both p>0.1. Scale bars: A,B,G left,H: 50 μm; D,E: 10 μm; G right, I: 25 μm. For genotypes, sample sizes and statistical details, see Supplementary file 2. *p<0.05, **p<0.01, ***p<0.001. See Figure 2—figure supplement 1.

Figure 2.

Figure 2—figure supplement 1. Over-expression of Toll-2 does not affect Kenyon cell number.

Figure 2—figure supplement 1.

(A) The MBGAL4 line drives expression virtually only in Kenyon cells (MB-GAL4 >UASFlyBow). (B) The ROI indicates MBGAL4 >UAS HisYFP cells counted automatically with DeadEasy Kenyon cells software in (C). (C) Over-expression of Toll-2 with MBGAL4 does not alter KC number. For sample sizes, genotypes and statistical analysis details see Supplementary file 2.