Apical cell extrusion and self-healing in the hai1a^hi2217 mutant are mediated by S1P signaling. (A–F) Localization of transgene encoded, transiently expressed, and EGFP-tagged WT mouse Sphk1 (krt19:EGFP-Sphk1WT; C–F) and, as negative control, a C-terminally truncated version of Sphk1 lacking the PA-binding site (krt19:EGFP-Sphk1NT, A and B) in single basal keratinocytes of zebrafish WTs or hai1a^hi2217 mutants at 48 hpf. (G) Quantification of Sphk1 cortical localization, determined as the ratio of average cortical fluorescence measured in a radius of 2 µm from the cell border, to the average fluorescence in the interior of the cell. n = 3–13 cells per condition. (H) Quantification of cell extrusion numbers at 48 hpf upon blockage of S1P signaling, either by mutation of the s1pr2 gene or by sphingosine kinase inhibition (MPA08), or upon FIPI treatment upstream of the S1P pathway. n = 19–39 embryos. (I–K) Alternative method of quantification of extruded cell numbers, counting the number of actin rosettes per embryo tail in krt4:lifeact-ruby (white in J and K) hai1a morphants upon drug treatment. n = 6–8 embryos. (L–P and L′–P′) Bright field images of embryo tails at 48 hpf and 96 hpf, showing that blockage of cell extrusion in hai1a^hi2217 mutants, either by s1pr2 mutation or by continual FIPI treatment from 10–96 hpf, leads to worsening of the epithelial phenotype over time (compare M′ with O′ and P′). See also Fig. S4. (L′′–P′′ and L′′′–P′′′) BrdU (red) incorporations at 96 hpf demonstrate increased proliferation rates upon blockage of cell extrusion. Double BrdU and p63 (green) labeling reveals proliferating basal keratinocytes (arrowheads in L′′′–P′′′). (Q) Quantification of proliferation rates of basal keratinocytes at 96 hpf after blockage of apical cell extrusion. n = 7–16 embryos. For quantifications and statistical significances, see legend of Fig. 1. Scale bar in A–F, 10 µm; in L–P′, 200 µm; and in J, K, and L′′–P′′′, 50 µm.