ASC speck formation in keratinocytes leads to cell death. (A) Time-lapse imaging of speck formation in keratinocyte (top) and muscle cell (bottom) in 3 dpf Tg(HSE:asc-mKate2) larva at 3 hphs. (B) Drastic, morphological changes occur only in keratinocytes. Tg(HSE:asc-mKate2) larvae, and negative siblings were stained with acridine orange and imaged at 2.5 and 15 hphs. 3D rendering of individual larvae manually segmented to exclude the head, heart and yolk regions. (B′) Acridine orange spots in segmented region were quantified using 3D image analysis software (white spots); spots positive in the red channel were excluded (magenta spots). (C) Histogram of acridine orange spots in each group shows only transgenic larvae at 15 hphs have significantly higher cell death (one-way ANOVA; ****, P < 0.0001). (D) Time-lapse imaging of Tg(HSE:asc-mKate2, krt4:GFP) larvae at 3 hphs showing morphological changes in EVL keratinocyte upon speck formation (white arrowhead). Enlarged view of EVL keratinocyte (dashed white outline) of single plane with the brightfield (D′). (E) Time-lapse imaging of Tg(HSE:asc-mKate2) injected with lynGFP mRNA for membrane visualization at 8 hphs. Epidermal layer shows extrusion and gap closure after speck formation. (E′) Single plane showing extruded keratinocyte. (F) Time-lapse imaging of 3-dpf Tg(krtt1c19e:Tomato) larva transiently expressing HSE:asc-tGFP, showing plasma membrane collapse and cell extrusion after speck formation in keratinocytes. All time-lapses are included in Video 5. Bars, 30 µm.