Fig. 3.

Downregulation of insulin receptor enhances ‘ventral targeting’ and functional restoration in a daf-16-dependent manner. (A) Pathway diagram showing how insulin signaling regulates lifespan. (B,C) RI values (B) and percentage of ‘ventral targeting’ (C) corresponding to the ‘non-fusion’ regrowth events in the mutants affecting either DAF-2 or downstream kinases at 24 h post-axotomy performed at the A3 stage. Similar analysis was also performed in daf-16(lf) and daf-2(e1368ts); daf-16(lf). For B and C, N (independent replicates)=3-9. (D) An example of a ‘ventral targeting’ event in daf-2(e1386ts) at A3 stage. The neuron is co-expressing Pmec-4::mCherry::RAB-3 (tbIs227) and Pmec-7::GFP (muIs32). The depth-coded image of the GFP channel was presented separately to show the regrowth along the VNC (double-headed yellow arrow). The arrowheads indicate mCherry::RAB-3 enrichment along the VNC. (E,F) The RI values (E) and the percentage of ‘ventral targeting’ events (F) in the mutants affecting IIS at 24 h post-axotomy performed at the L4 stage. For E and F, N=3-6. (G) The percentage ‘ventral targeting’ events in wild type (WT) and daf-16(lf) after axotomy performed at the L4 stage. N=5-8. (H) Depth-coded confocal image of the multibranch regrowth pattern in daf-16(lf). (I) RI values in wild type and daf-16(lf) at 24 h and 48 h post-axotomy performed at the L4 stage. N=5-8. Broken y-axes were used in the plots in B and E. Red arrows indicate the site of injury, and arrowheads indicate the mCherry::RAB-3 punctae. Data are mean±s.d. *P<0.05; **P<0.01; ***P<0.001; ns, not significant [ANOVA with Tukey's multiple comparisons test (B,E,I); Fisher's exact test (C,F,G)].