- Title
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A conserved morphogenetic mechanism for epidermal ensheathment of nociceptive sensory neurites
- Authors
- Jiang, N., Rasmussen, J.P., Clanton, J.A., Rosenberg, M.F., Luedke, K.P., Cronan, M.R., Parker, E.D., Kim, H.J., Vaughan, J.C., Sagasti, A., Parrish, J.Z.
- Source
- Full text @ Elife
Epidermal PIP2 accumulation marks sites of dendrite ensheathment. |
Screen for epithelial markers that accumulate at sites of c4da dendrite contact. |
c4da neurons are enclosed by epidermal sheaths. |
Molecular markers of epidermal sheaths in larval zebrafish. |
Epithelial sheaths form adjacent to somatosensensory neurons in a modality-specific manner. |
( |
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Somatosensory neurons are necessary for formation and maintenance of epidermal sheaths. |
Representative ddaC c4da neuron imaged at 84 h ( |
Dual-labeling of cora and c4da neurons at 72 h AEL ( |
Maximum projections of confocal stacks show ( |
( A–D) Time of arrival of PIP2 and other sheath markers. Images show dual labeling of sheaths by PLCδ-PH-Cerulean and dArf6-GFP ( A) or GMA-GFP to label F-actin ( C) in larvae additionally expressing the c4da-specific marker ppk-CD4-tdTomato. ( B, D) Plots show mean and standard deviation values for the proportion of c4da dendrite arbors ensheathed by structures labeled by the indicated markers at the indicated times. All sheath structures labeled by dArf6-GFP and GMA-GFP were labeled by PLCδ-PH-Cerulean. ( E) Time-lapse images show dual labeling of sheaths by PLCδ-PH-Cerulean and GFP-cora1-383 in larvae additionally expressing the c4da-specific marker ppk-CD4-tdTomato. Cyan arrows mark single-positive (Cerulean-positive) sheaths that convert to double-positive (Cerulean-positive and GFP-positive), magenta arrows mark double-positive sheaths that persist. See also Figure 5—figure supplement 1 for time-lapse images depicting dArf6-GFP and GMA-GFP recruitment to PLCδ-PH-Cerulean sheaths. ( F) Quantification of marker recruitment. Bars depict the proportion of PLCδ-PH-Cerulean-positive GFP-negative sheaths at 104 h AEL that are labeled by the indicated markers at 120 h AEL. More than 100 sheaths (from six independent time-lapse series) were scored for each marker combination. ( G) Timing of accumulation of ensheathment channel markers in the zebrafish epidermis. ( H) Epistatic relationship between markers. The indicated RNAi transgenes were expressed in the epidermis and effects on ensheathment were assessed (see Figure 5—figure supplement 2 for accompanying images). Plots show mean and standard deviation values for the proportion of c4da dendrite arbors wrapped by PLCδ-PH-GFP or cora-positive sheaths. n = 8 neurons each; ***p<0.001 relative to control; one way ANOVA with post-hoc Dunnett’s test. ( I) Model depicting the timing of arrival of sheath components. |
Time-lapse images show dual labeling of epidermal sheaths by PLCδ-PH-Cerulean and GMA-GFP ( |
Live-imaging of PIP2 in basal cells [ |
( |
Representative images of 120 h AEL c4da neurons from ( A) control larvae and larvae expressing ( B) PI4K(RNAi), ( C) dominant-negative shibire (shiDN), ( D) temperature-sensitive shibire ( shits), ( E) epidermal cora(RNAi), and ( F) epidermal shg(RNAi) larvae are shown. Larvae were reared at 25° C, with the exception of larvae in ( D) which were reared at 25° C for 4 days and then shifted to the non-permissive temperature 29° C for 1 day prior to imaging. ( G–H) Morphometric analysis of dendrites from c4da neurons of the indicated genotypes. Plots show mean and standard deviation for ( G) the number of terminal branches and ( H) terminal branch length. Data points, measurements from an individual neuron; ***p<0.001 relative to control; one way ANOVA with post-hoc Dunnett’s test. ( I–L) Time-lapse analysis of epidermal sheath control of terminal dendrite dynamics. C4da neurons were imaged over an 18 h time-lapse (96–114 h AEL) and growth (green) and retraction (magenta) were pseudocolored in a composite of the two time-points. Representative composite images are shown for c4da neurons from ( I) Gal4-only control, ( J) epidermal PI4K(RNAi), and ( K) epidermal cora(RNAi) larvae. ( L–P) Quantification of terminal dendrite dynamics. ( L) The fraction of terminal dendrites that were growing, stable, or retracting over the time-lapse is shown. ***p<0.001 compared to controls, Chi-square analysis. ( M) Epidermal ensheathment regulates the extent of terminal dendrite dynamics. Box plots depict mean values and 1st/3rd quartile, whiskers mark minimum/maximum values. ***p<0.001 compared to Epi-Gal4 control; one way ANOVA with post-hoc Dunnett’s test. ( N) Epidermal ensheathment regulates dendrite turnover. C4da neurons were imaged over a 12 h time-lapse (72–84 or 108–120 h AEL) and all terminal dendrites were scored as persistent (present at both time points) or transient. Each bar represents measurements from a single neuron. Terminal dendrites at the later time-point, when c4da neurons are extensively ensheathed, were significantly more likely to persist. ( O) Quantification of terminal dynamics in ensheathed and unensheathed terminal dendrites from 108 to 120 h AEL. ***p<0.001, Chi-square analysis with post-hoc Bonferroni adjustment for multiple comparisons. Pairwise comparisons are indicated. ( P) Dynamic portions of dendrite arbors are less extensively ensheathed. Mean and standard deviation values for the proportion of c4da dendrite arbors, terminal dendrites, and new terminal dendrite growth (12 h time-lapse) wrapped by PLCδ-PH-GFP sheaths at 120 h AEL. ***p<0.001, Chi-square analysis with post-hoc Bonferroni adjustment for multiple comparisons. Pairwise comparisons are indicated. ( Q) Distribution of branching events during 12 h time-lapse imaging. Each bar represents a single neuron. ( R–U) Epidermal ensheathment regulates dendrite structural plasticity. Class IV neurons in newly eclosed 2nd instar control ( R), epidermis PI4k(RNAi) ( S), and epidermis cora(RNAi) ( T) larvae were ablated with a focused laser beam and imaged 48 h post-ablation. Images depict dendrite growth of spared neurons into unoccupied territory following laser ablation and hatched boxes demarcate the territory occupied by the ablated neuron. ( U) Scatter plot depicting mean and standard deviation for dendrite invasion of the indicated mutants. The number of samples analyzed for each treatment is indicated. ***p<0.001 relative to control; one way ANOVA with post-hoc Dunnett’s test. |
Representative images of 120 h AEL c4da neurons from ( |
C4da neurons were imaged over a 12 h time-lapse in larvae additionally expressing the epidermal sheath marker PLCδ-PH-GFP. Maximum projections show a representative neuron at 108 h ( |
Larvae were transferred at 72 h AEL to 35 mm dishes containing unmodified cornmeal-molasses agar (mock) or cornmeal-molasses agar supplemented with 20 µM PBP10 and assayed for behavior responses ( |