Skip to main content
. 2024 Dec 20;13:RP102222. doi: 10.7554/eLife.102222

Figure 3. DNT-2 and Toll-6 maintain PAM neuron survival in the developing and adult brain.

Figure 3.

(A) Illustration of PAM neuronal cell bodies and experimental temporal profile. DANs are shown in green, DNT-2A neurons in magenta, and MB in dark grey. The left hemisphere shows the anterior brain with PAL and PAM DAN neurons (green) and DNT-2 neurons (magenta); the right shows the posterior brain, with the calyx and other DAN neurons (PPM1, PPM2, PPM3, PPL1, PPL2, green). (B–D) Fruit flies were kept constantly at 25°C, from development to adult. Analyses done in adult brains. (B) DNT-237/DNT-218 mutants had fewer histone-YFP+-labelled PAM neurons (THGAL4, R58E02-GAL4>hisYFP). Unpaired Student’s t-test. (C) Toll-6 RNAi knock-down in all DANs (THGAL4, R58E02-GAL4>hisYFP, Toll-6 RNAi) reduced Histone-YFP+-labelled PAM cell number. Unpaired Student’s t-test. (D) DNT-237/DNT-218 mutants had fewer PAMs stained with anti-TH antibodies. Over-expressing Toll-6CY in DANs (THGAL4, R58E02 GAL4>hisYFP, Toll-6CY) rescued TH+ PAM neurons in DNT-237/DNT-218 mutants, demonstrating that DNT-2 functions via Toll-6 to maintain PAM cell survival. Welch ANOVA p<0.0001, post hoc Dunnett test. (E–H) Adult-specific restricted over-expression or knock-down at 30°C using the temperature-sensitive GAL4 repressor tubGAL80ts. (E) Adult-specific DNT-2 RNAi knock-down in DNT-2 neurons decreased Tomato+ PAM cell number (tubGAL80ts, R58E02-LexA, DNT-2 GAL4>LexAOP-Tomato, UAS DNT-2-RNAi). Unpaired Student’s t-test, p=0.005. (F) Adult-specific Toll-6 RNAi knock-down in Toll-631 heterozygous mutant flies in DANs, reduced Histone-YFP+ PAM cell number (tubGAL80ts; THGAL4, R58E02-GAL4>hisYFP, Toll-6 RNAi/Toll631). Unpaired Student’s t-test. (G) PAMs were visualised with anti-TH. Left: tubGAL80ts, Toll-6>Toll-6-RNAi knock-down decreased TH+ PAM cell number. Unpaired Student’s t-test. Right: tubGAL80ts, DNT-2>DNT-2-RNAi knock-down decreased TH+ PAM cell number, whereas DNT-2FL over-expression increased PAM cell number. Kruskal–Wallis ANOVA, p=0.0001, post hoc Dunn’s test. (H) Adult-specific tubGAL80ts, DNT-2>DNT-2RNAi knock-down increased the number of apoptotic cells in the brain labelled with anti-DCP-1. Dcp-1+ cells co-localise with anti-TH at least in PAM clusters. One-way ANOVA, p<0.0001, post hoc Bonferroni’s multiple comparisons test. DANs>histone-YFP: all dopaminergic neurons expressing histone-YFP, genotype: THGal4 R58E02Gal4>UAS-histoneYFP. PAMsLexA>tomato: restricted to PAM DANs: R58E02LexA >LexAop-nlstdTomato. Controls: GAL4 drivers crossed to wild-type Canton-S. Scale bars: (B–G) 30 µm; (H) 20 µm. Graphs show boxplots around the median. p-values over graphs in (D, G right, H) refer to group analyses; stars indicate multiple comparisons tests. *p<0.05, **p<0.01, ***p<0.001. For further genotypes, sample sizes, and statistical details, see Supplementary file 2.

Figure 3—source code 1. DeadEasy Central Brain was used to automatically count the number of PAM neuron nuclei labelled with His-YFP in adult brains.
Figure 3—source code 2. DeadEasy Dopaminergic-3D was developed and used to automatically or semi-automatically count the number of PAM neurons stained with anti-TH antibodies in adult brains.
Figure 3—source code 3. Cells filter, to function together with DeadEasy Dopaminergic-3D.
Figure 3—source data 1. Quantitative results used to generate graphs in Figure 3B.
Figure 3—source data 2. Quantitative results used to generate graphs in Figure 3C.
Figure 3—source data 3. Quantitative results used to generate graphs in Figure 3D.
Figure 3—source data 4. Quantitative results used to generate graphs in Figure 3E.
Figure 3—source data 5. Quantitative results used to generate graphs in Figure 3F.
Figure 3—source data 6. Quantitative results used to generate graphs in Figure 3G.
Figure 3—source data 7. Quantitative results used to generate graph in Figure 3H.