Fig. 3

Sphingosine kinase activity modulates cell death in hai1a^fr26 mutant epidermis.

a–d S1P levels are elevated in hai1a^fr26 mutants at 4 dpf. a–c Whole mount immunostaining in the caudal fin fold, anti-S1P (white), nuclei are labelled with DAPI (blue). c shows an individual extruding cell, with the orthogonal view shown in c’. d Quantification of S1P fluorescence in the caudal fins of embryos, normalised to fin area. For full dataset including SphK inhibition, see Fig. S3g. Scale bars: (a, b) 200 µm, (c) 20 µm. e Quantification of the numbers of extruded cells collected per fish at 4 dpf upon inhibition of sphingosine kinase by MPA08 treatment. White bars show the numbers of live cells, blue bars show the numbers of dead cells, with the proportion of dead cells indicated with blue text. f Survival curves showing the effect of sphingosine kinase inhibition on embryo viability. g–j Apoptotic cells in the caudal fin fold of 4 dpf hai1a^hi2217 mutant embryos, with and without MPA08 treatment. Apoptotic cells are labelled with aCasp3 (red), basal keratinocytes with p63 (green), and nuclei using DAPI (blue). Scale bar = 50 μm. l–o Apoptotic cells in the caudal fin fold of 4 dpf hai1a^fr26 mutant embryos, with and without MPA08 treatment. Apoptotic cells are labelled with aCasp3 (red), basal keratinocytes with p63 (green), and nuclei using DAPI (blue). Scale bar = 50 μm. q, r Apoptotic cells in the caudal fin fold of 4 dpf hai1a^fr26 mutant embryos upon S1P treatment. Apoptotic cells are labelled with aCasp3 (red), basal keratinocytes with p63 (green), and nuclei using DAPI (blue). Scale bar = 50 μm. k, p, s Quantification of numbers of aCasp3-positive cells in the tail fins of embryos, normalised to fin area. t–v Apoptotic cells in the caudal fin fold of 4 dpf wild-type embryos, treated with ceramide to induce apoptosis (u), or concomitant treatment with ceramide and S1P (v). Apoptotic cells are labelled with aCasp3 (red), basal keratinocytes with p63 (green), and nuclei using DAPI (blue). Scale bar = 50 μm. w Quantification of numbers of aCasp3-positive cells in the tail fins of embryos, normalised to fin area. For (d), means of controls and mutants were compared using an unpaired, two-tailed Student’s t-test. For all other graphs, means were compared using one-way ANOVA with post-hoc Tukey’s multiple comparison test. N = number of biological replicates, n = number of fish per condition.