Dendrite intercalation between epidermal cells tunes nociceptor sensitivity to mechanical stimuli in Drosophila larvae
Fig 7
miR-14 regulation of epidermal gap junction assembly controls dendrite position and nociceptive sensitivity.
(A-B) miR-14 mutation affects epidermal barrier function. Maximum intensity projections show C4da arbors (ppk-CD4-tdGFP) and rhodamine-conjugated dextran labeling in cross-section of wild-type control and miR-14 mutant larvae. Dashed lines indicate the position of the orthogonal xz sections (middle), and bottom images show xz maximum intensity projections. Arrows indicate apically-shifted dendrite branches and carets mark apical dextran infiltration at cell-cell junctions. (C) Schematic depicting position of epidermal junctional complexes (left), alleles used for genetic interaction studies (center), and markers used for analysis of junctional assembly (right). (D-F) ogre and Inx2 genetically interact with miR-14 to regulate dendrite position. Representative images show 120 h AEL C4da neurons from (D) miR-14Δ1/+ heterozygous mutant and (E) miR-14Δ1/+, Inx2G0118/+ double heterozygous mutant larvae. (F) Morphometric analysis of C4da dendrites in larvae heterozygous for miR-14 and the indicated epidermal junction genes showing the mean number of dendrite-dendrite crossing events per neuron normalized to dendrite length. *P<0.05, ANOVA with post-hoc Dunnett’s test. (G) ogre and Inx2 genetically interact with miR-14 to regulate mechanical nociceptive sensitivity. Plots depict the rolling probability and frequency of multiple roll responses evoked by 25 mN von Frey fiber stimulation in larvae of the indicated genotypes. *P<0.05 compared to miR-14 heterozygous controls, Fisher’s exact test with a post-hoc BH correction (G). (H-O) miR-14 regulates GJ assembly. Maximum projection images show C4da dendrites (green) and ogre immunoreactivity in the epidermis of a wild-type control larva (H). (I) Zoomed images corresponding to hatched box in (H) show the relative position of C4da dendrites and ogre at epidermal cell-cell junctions. Arrows mark sites of disconiuties in junctional ogre immunoreactivity, which most frequenly occurs at tricellular junctions (I’).Orthogonal sections show ogre distribution at a representative bicellular junction (outlined with hatched lines) in xy (J) or xz projections (K). C4da dendrites are confined to the basal face of a continuous belt of ogre immunoreactivity in control larvae. (L-O) miR-14 mutation disrupts organization of ogre immunoreactivity at epidermal cell-cell junctions. (L-M) The belt of ogre immunoreactivity exhibits irregularity in width, signal intensity, and frequent discontinuities (arrows). (N, O). C4da dendrites intercalate into gaps in ogre and immunoreactivity and penetrate apically into the GJ domain. (P-R) Selective epidermal overexpression of Inx genes suppresses miR-14 mutant dendrite alignment and mechanonociception defects. (P) ogre immunoreactivity at GJs is reduced in miR-14 mutants. Plot depicts intensity of ogre immunoreactivity signal normalized to arm immunoreactivity at epidermal cell-cell interfaces in control and miR-14 mutant larvae. P<0.05, unpaired t-test with Welch’s correction. (Q) GJ belt width is reduced in miR-14 mutants. Violin plot depicts the distribution of GJ belt widths at epidermal cell-cell interfaces in control and miR-14 mutant larvae Each data point represents the average GJ belt width measured across the full length of a cell-cell interface. *P<0.05, Kolmogorov-Smirnov test. (R) Composite images show C4da dendrites pseudocolored green to label epidermal junctional alignment in a miR-14 mutant (left) and a mir-14 mutant expressing UAS-ogre selectively in epidermal cells (right). (R) Plot depicts the fraction of C4da dendrite arbors aligned along epidermal junctions at 120 h AEL for the indicated genotypes. NS, not significant, *P<0.05, Kruskal-Wallis test followed by Wilcoxon rank sum test with BH correction. (S) Fraction of larvae of the indicated genotypes that exhibit nocifensive rolling responses to 25 mN von Frey stimulation. NS, not significant, *P<0.05, Fisher’s exact test with a BH correction. (T) Epidermis-specific Inx gene knockdown increases epidermal junctional alignment of C4da dendrites. The plot depicts the proportion of C4da dendrite arbors aligned along epidermal junctions in larvae expressing the indicated RNAi transgenes. Quantitative analysis of Inx protein knockdown and representative images of dendrite phenotypes are shown in S9 Fig. *P<0.05, Kruskal-Wallis test followed Dunn’s multiple comparisons test.