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Figure 4
Somatosensory neurons are necessary for formation and maintenance of epidermal sheaths.
( A–G) Timecourse of sheath formation. Maximum intensity projections show dual labeling of sheaths by epidermal PLCδ-PH-GFP and the c4da-specific marker ppk-CD4-tdTomato ( A, D) or PLCδ-PH-GFP signal alone ( B, E) at 48 and 120 h AEL. ( C, F) Composites show portions of c4da dendrite arbors (shaded purple) wrapped by sheaths labeled by PLCδ-PH-GFP. ( G) Plot shows mean and standard deviation values for the proportion of c4da dendrite arbors wrapped by PLCδ-PH-GFP or cora-positive sheaths at the indicated time points. See also Figure 4—figure supplement 2 for images of cora labeling of sheaths at 72 and 120 h AEL. ( H) Once formed, sheaths persist. Plot shows sheath dynamics; the proportion of sheaths from eight neurons that grew, retracted, or were stable over a 24 h time-lapse is shown. See also Figure 4—figure supplement 1 for time-lapse images. ( I–K) Epidermal sheath formation following genetic ablation of c4da neurons. Maximum intensity projections show dual labeling of anti-cora staining to label sheaths and anti-HRP staining to label PNS neurons ( I) and the individual markers alone ( J, K) at 120 h AEL for a larva expressing the pro-apoptotic gene reaper ( rpr) specifically in c4da neurons under control of ppk-Gal4. ( L–N) Epidermal sheath formation following laser ablation of larval c2da, c3da, and c4da neurons. Images show dual labeling of epidermal sheaths with anti-cora staining and sensory neurons with anti-HRP staining ( L) and the individual markers alone ( M, N) at 120 h AEL in a hemisegment in which c2da, c3da, and c4da were ablated with a focused laser beam at 72 h AEL. Red arrowheads in ( M) mark cora-positive structures (also visible in Figure 1R and Figure 4—figure supplement 2) located in the dorsal-medial portion of the hemisegment, corresponding to the position of the dorsal pharyngeal sense organ. These structures are not co-labeled by other epidermal sheath markers and cora immunoreactivity persists following epidermal cora(RNAi) (Figure 5—figure supplement 2), suggesting that these structures are distinct from the epidermal sheaths that wrap sensory neurites. ( O–T) Somatosensory dendrites are required for sheath maintenance. Maximum projections of confocal stacks show time-lapse images of da neurons labeled with membrane-targeted mRFP ( O) and epidermal sheaths ( P) immediately prior to c4da dendrite severing at 108 h AEL and 12 h post-severing at 120 h AEL ( R, S). White dashed lines outline the anterior-dorsal portions of the c4da arbor that are ensheathed prior to severing and the location those sheaths would occupy if they persisted post-severing. ( Q, T) Traces depict unensheathed c4da dendrites in black and ensheathed c4da dendrites in green, the arrow marks the site of dendrite severing, and the gray box marks the quadrant in which c4da dendrites and associated epidermal sheaths are lost post-severing. ( U–Z) Epidermal sheath formation in zebrafish injected with a morpholino targeting neurog1 to prevent somatosensory neuron development. Maximum intensity projections of confocal z-stacks showing lateral views through the zebrafish epidermis at 72 hpf. Note the lack of ensheathment channels (green arrowheads) in neurog1(MO)-injected embryos. Yellow and cyan arrows indicate the lateral cell membranes of periderm and basal cells, respectively. ( AA) Somatosensory axons are required for sheath maintenance in zebrafish. Arrowhead and arrow indicate sheaths associated with a severed and intact axon, respectively.