Skip to main content
. 2020 Feb 18;9:e52743. doi: 10.7554/eLife.52743

Figure 4. Cell number is regulated by Toll-2 and neuronal activity at the adult critical period.

At adult days 0–2 post-eclosion, within the critical period: (A) Conditional over-expression of Toll-2 increased MyD88 >hisYFP+ cell number in the central brain. Cells were counted automatically in 3D throughout the whole stack with DeadEasy Central Brain, dashed line in all figures indicates ROI quantified. Box-plots, Kruskal-Wallis p<0.0001, post-hoc Dunn test; (B) neither conditional over-expression nor knock-down of Toll-2, altered Toll-2 >hisYFP+ Kenyon cell number, counted automatically with DeadEasy Kenyon Cells, box-plots; (C) conditional over-expression of Toll-2 increased Toll-2 >hisYFP+ cell number in the optic lobe medulla, counted automatically with DeadEasy Optic Lobe. Box-plots, One Way ANOVA p<0.0001, post-hoc Dunnett; (D) pulses of neuronal activation with TrpA1 increased Toll-2 >hisYFP+ cell number in the medulla, and this could be rescued with Toll-2 RNAi knock-down. Box-plots: Left: Un-paired Student t-test, p=0.0058; Right: Un-paired Student t-test, p=0.0225. (E) Knocking-down JNK and cactus and over-expressing activated PI3K (UAS-Dp110CAAX), alone or in combination, in MyD88+ cells with tubGAL80ts, MyD88GAL4 increased cell number in the central brain, consistent with pro-survival signalling downstream of Toll-2. However, over-expressing either wek or MyD88 RNAi knock-down increased cell number in the central brain, and even further in combination, suggesting that Wek also has non-apoptotic functions that antagonise MyD88. Box-plots, Kruskal-Wallis ANOVA p<0.0001, post-hoc Dunn test. (F) By contrast, no statistically significant changes were detected in KCs upon manipulation of any of these downstream effectors, although a mild increase in cell number was observed with UAS-wek, UASMyD88RNAi. Box-plots, One Way ANOVA p=0.0354, post-hoc Dunnett. Dashed lines indicate regions of interest (ROI) for automatic cell counting with DeadEasy. Scale bars: A,C,D,E:50 μm; B,F:25 μm. For genotypes, sample sizes and statistical details, see Supplementary file 2. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. See Figure 4—figure supplement 1.

Figure 4.

Figure 4—figure supplement 1. Toll-signalling downstream factors were found in the brain.

Figure 4—figure supplement 1.

(A,B) sarm was expressed in the adult brain: line sarmGAL4NP760 drives expression of four short isoforms, and sarmGAL4NP0257 of the longest isoform, visualised with UAS-FlyBow. Note prominent expression in (arrows): central brain (CB), some Kenyon cells (KCs) and mushroom body projections along the α,β lobes, subesophageal ganglion (SOG), medulla (me) and the lobula and/or lobula plate (lp). (C,D) JNK signalling visualised with the reporter TRE-RED revealed cells throughout the pupal and adult brain; note the large cells in the optic lobes, reminiscent of the large MyD88+ and Toll-2+ cells (arrowheads). (E,F) Anti-Foxo+ cells were observed both in the pupal and adult CB and optic lobes (OL), insulin producing cells (IPCs) and fan shaped body (fsb) (arrows). Note the large cells in the optic lobes, reminiscent of the MyD88+, Toll-2+ and JNK+ cells (arrowheads). (G–J) NF-κB homologues Dif (G,H) and Dorsal (I,J) were visualised with GFP-fusion to the nuclear isoforms. Dif-GFP was abundant in both pupal (G) and adult brains particularly around antennal lobes (H), arrows). Dorsal-GFP was prominent in the pupal brain (I), and in the adult brain it was found in noduli and optic lobes (J), arrows).