Fig 8. Epidermal dendrite intercalation tunes C4da neuron calcium levels and nociceptive sensitivity.

(A-F) Epidermal dendrite intercalation regulates baseline calcium levels in C4da neurons. (A) Larval preparation for imaging mechanically-evoked calcium responses in C4da neurons. (B) Control and miR-14 mutant larvae exhibit comparable amplitudes of GCaMP6s responses (ΔF/F₀) in C4da neurons to mechanical stimulus (n = 15 larvae each, p = 0.217, Wilcoxan rank sum test). (C) GCaMP6s fluorescence intensity is significantly elevated in miR-14 mutant C4da axons prior to mechanical stimulus and 5 min after mechanical stimulus (n = 15 larvae each, p = 0.0003 pre-stimulus, p = 0.002 post-stimulus, Wilcoxon rank sum test). (E-F) Ratiometric calcium imaging using a GCaMP6s-Cherry fusion protein expressed selectively in C4da neurons (ppk-GAL4, UAS-Gerry). (E) Representative images depict fluorescence intensity of Cherry and GCaMP6s for wild-type control and miR-14 mutant larvae. (F) miR-14 mutants exhibit elevated GCaMP/mCherry fluorescence ratios in C4da axons. Points represent measurements from individual abdominal segments (A2-A8) from 10 larvae of each genotype (n = 64 data points in control larvae, 56 in miR-14 mutants, 66 in miR-14 + ppk>Integrin larvae). *P<0.05, Kruskal-Wallis test followed by Dunn’s post-hoc test. (G) C4da dendrites are confined to territory between muscle adhesion sites at apodemes. (H-J) Mutation of the PS2 integrin ligand Tig prevents dendrite intercalation between apodemes. Representative maximum intensity projections of larvae expressing ppk-CD4-tdTomato to label C4da dendrites and Nrg^GFP to label epidermal junctions are shown for (H) Tig^A1/+ heterozygote control and (I) Tig^A1/X mutant larvae. C4da dendrites are depicted in magenta and apodemes in green using an apodeme mask to subtract Nrg^GFP signal in other cells. (H’ and I’) C4da dendrites are pseudocolored according to their orientation at apodemes (magenta, aligned along apodeme junctional domains; cyan, invading apodeme territory). (J) Tig^A1/X mutant larvae exhibit a significant reduction in junctional dendrite alignment at apodemes and an increase in dendrite invasion into apodeme territory. *P<0.05, ANOVA with a post-hoc Sidak’s test. (K) Tig^A1/X mutant larvae exhibit no significant alteration in dendrite arbor length outside of apodeme domains. No significant difference was detected between the two groups using an unpaired t-test. (L-M) Dendrite intercalation at apodemes tunes nociceptive sensitivity. (L) Tig^A1/X mutant larvae exhibit a significant reduction in rolling responses to noxious mechanical stimulus. (M) Tig^A1/X mutant larvae exhibit a significant reduction in GCaMP/mCherry fluorescence ratios in C4da axons Points represent measurements from individual neurons. *P<0.05, Wilcoxon rank sum test in (L-M).