Fig. 6

Early inhibition of ceramide synthesis in wild-type embryos recapitulates ACE and apoptosis phenotype.

a Loss of Matriptase inhibition leads to parallel pro-oncogenic and tumour-suppressive pathways. Sphingolipid rheostat-mediated tumour-suppressive methods include S1P-dependent apical cell extrusion, which preserves epithelial integrity, and C₁₆ ceramide-dependent apoptotic cell death, which may lead to loss of epithelial barrier function. When levels of C₁₆ ceramide are above a certain threshold, a negative feedback loop prevents further de novo ceramide synthesis. In hai1a^fr26 mutants by 2 dpf, ceramide levels drop due to sustained SphK activity catalysing S1P production, repression of de novo synthesis is lost, and resultant C₁₆ ceramide levels trigger apoptosis. b-c Epidermal aggregates and apoptotic peridermal cells in the caudal fin fold of 4 dpf WT fish, transiently treated with inhibitor of de novo ceramide synthesis myriocin at 2 dpf, with orthogonal views (b’, c’) showing extruding live (yellow arrowhead) or apoptotic (yellow arrow) peridermal cells. Apoptotic cells are labelled with aCasp3 (red), peridermal cells with krt4:GFP (green), basal keratinocytes with p63 (white), and nuclei using DAPI (blue). Scale bar = 50 μm. d Quantification of the numbers of extruded cells collected per fish at 4 dpf upon inhibition of de novo sphingolipid synthesis by myriocin treatment at 2 dpf. 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. e Quantification of aCasp3-positive cells in the tail fins of 4 dpf embryos upon myriocin treatment, normalised to fin area. f Quantification of proliferating cells in the tail fins of 4 dpf embryos upon myriocin treatment, normalised to fin area. For all graphs, means were compared via two-tailed, unpaired Student’s t-test. N = number of biological replicates, n = number of fish per condition.