Figure 6.

Figure 6.

(A, B) Lateral micrographs of embryos treated with DMSO (A) or 125 ng/ml PMA (B, C) showing generation of epidermal aggregates (arrowheads). (D, E) Projected confocal images of the trunk of 24hpf WT embryos treated with 0.1% DMSO (D) or 125 ng/ml PMA (E) and immunostained for TP63 (magenta), showing aggregation of TP63-positive cells. (F, G) Projected confocal images superimposed on DIC image of the tail of 48hpf Tg(mpx:eGFP)ⁱ¹¹⁴ embryos treated with 0.1% DMSO (F) or 125 ng/ml PMA (G) showing fin defect and activation of eGFP-positive neutrophils (green, G). (H, I) Single timepoint images at t = 0 (top panels, H) and t = 1800 s (lower panels, H) and kymographs (I) derived from light-sheet movies (Video 7) of the epidermis of 3dpf Tg(krtt1c19e:lyn-tdtomato)^sq16 larvae treated with 0.1% DMSO (left panels) or 37.5 ng/ml PMA (right panels) showing the lack of membrane stability following PMA treatment. (J–K′) Single frames (J, K) and tracks of eGFP- positive neutrophils (J′, K′) from light-sheet (Video 8) showing neutrophils labelled by eGFP and basal keratinocyte cell membranes labelled by lyn-tdTomato in the trunk of a 3dpf Tg(krtt1c19e:lyn-tdtomato)^sq16 larva treated with 0.1% DMSO (J, J′) or 37.5 ng/ml PMA (K, K′) for 18 hr, and imaged every 20 s for 30 min. Track colour in (J′, K′) denotes mean velocity (dark blue 0.0 – red 0.2). (L–O) Projected lateral confocal views of pentafluorobenzenesulfonyl fluorescein (PFBSF) staining of 24hpf WT embryos treated with 0.1% DMSO (L, N) or 125 ng/ml PMA (M, O) showing elevation of H₂O₂ in the trunk (L, M) and tail (N, O). (P, Q) Projected confocal images of eGFP in the tail at 24hpf of WT injected with GCaMP6s RNA, treated with DMSO (P), or with 125 ng/ml PMA (Q). Images are temporal projections of timelapse movies taken at maximum speed intervals (2 min) and projected by time. (R) Plot of PFBSF fluorescent staining intensity of WT embryos treated with 0.1% DMSO or 125 ng/ml PMA in the trunk and tail. n = 6; ANOVA with Bonferroni post-test **p<0.01. (S) Graph comparing eGFP intensities from 24hpf GCaMP6s RNA timelapses in tail following treatment with DMSO and 125 ng/ml PMA. n = 10; t-test. (T–U) Lateral confocal projections of Tg(6xHsa.NFKB:EGFP)^nc1 embryos reporting NfkB signalling levels at 48hpf in WT treated with DMSO (T) and WT treated with 125 ng/ml PMA (U). Scale bars: (A, B, C, F) = 100 µm; (D, J, Q) = 50 µm; (H, I) = 20 µm; (U) = 200 µm.