Figure 3.
Loss of Matriptase inhibition leads to apical cell extrusion of peridermal and basal keratinocytes, accompanied by engulfment of basal cells in the epidermis. (A) WT sibling, (B–G) hai1ahi2217mutants at 48 hpf. (A) Transverse cryosection image of regular bilayered epidermis with outer krt4:GFP peridermal layer labeled in green and p63:dsRed basal keratinocytes in red. (B) Cryosection image of extruding peridermal cell (green) separating from the surface of the epithelium. Note the F-actin accumulation at the base of the extruding cell (white). Asterisks in B′′ indicate nuclei of cells within the plane of the rest of the epidermis. (C) Cryosection image of extruding basal cell (red), engulfed by overlying peridermal cells (green); white arrow in C indicates F-actin accumulation (white) at base of engulfed cell (compare with D). Yellow arrows in C′′′ indicate peridermal and basal cell nuclei. (D) En face whole-mount image of an extruding basal cell (red; p63 antibody) in the epidermis, in the center of an F-actin ring (white) formed by surrounding peridermal cells (green). Single plane image; see also Video 1 for the full z-stack. (E) YZ orthogonal view of confocal z-stack depicting single peridermal cell (green) engulfing a basal keratinocyte (red; p63 antibody). The krt4:GFP plasmid was injected into fertilized eggs to label single peridermal cells. (F) Live image of embryo injected at the single-cell stage with krt4:TomatoCAAX and p63:GFPCAAX constructs, showing intact cell membrane of engulfed basal keratinocytes (F′, green), surrounded by cell membrane of host peridermal cell (F, red, arrow). Arrowhead in F points to cell membrane at the surface of host cell. (G) Live image of embryo injected at the single-cell stage with p63:lifeact-Ruby (red) construct to label single basal keratinocytes, showing actin accumulating at rear of invading basal cell (G′; orthogonal view) located within extruding cell cluster (G, en face view). (H) Quantification of rosette-like figures (extrusion events) in median fin folds of untreated and pharmacologically treated hai1ahi2217mutants. Percentages of rosettes containing (engulfed) basal cells distal of the peridermal rosette (compare with D) are indicated in red. n = 5–7 embryos per condition in two independent experiments. For quantifications and statistical significances, see legend of Fig. 1. Scale bars in A and B, 5 µm; and in C–G, 10 µm.

Loss of Matriptase inhibition leads to apical cell extrusion of peridermal and basal keratinocytes, accompanied by engulfment of basal cells in the epidermis. (A) WT sibling, (B–G) hai1ahi2217mutants at 48 hpf. (A) Transverse cryosection image of regular bilayered epidermis with outer krt4:GFP peridermal layer labeled in green and p63:dsRed basal keratinocytes in red. (B) Cryosection image of extruding peridermal cell (green) separating from the surface of the epithelium. Note the F-actin accumulation at the base of the extruding cell (white). Asterisks in B′′ indicate nuclei of cells within the plane of the rest of the epidermis. (C) Cryosection image of extruding basal cell (red), engulfed by overlying peridermal cells (green); white arrow in C indicates F-actin accumulation (white) at base of engulfed cell (compare with D). Yellow arrows in C′′′ indicate peridermal and basal cell nuclei. (D) En face whole-mount image of an extruding basal cell (red; p63 antibody) in the epidermis, in the center of an F-actin ring (white) formed by surrounding peridermal cells (green). Single plane image; see also Video 1 for the full z-stack. (E) YZ orthogonal view of confocal z-stack depicting single peridermal cell (green) engulfing a basal keratinocyte (red; p63 antibody). The krt4:GFP plasmid was injected into fertilized eggs to label single peridermal cells. (F) Live image of embryo injected at the single-cell stage with krt4:TomatoCAAX and p63:GFPCAAX constructs, showing intact cell membrane of engulfed basal keratinocytes (F′, green), surrounded by cell membrane of host peridermal cell (F, red, arrow). Arrowhead in F points to cell membrane at the surface of host cell. (G) Live image of embryo injected at the single-cell stage with p63:lifeact-Ruby (red) construct to label single basal keratinocytes, showing actin accumulating at rear of invading basal cell (G′; orthogonal view) located within extruding cell cluster (G, en face view). (H) Quantification of rosette-like figures (extrusion events) in median fin folds of untreated and pharmacologically treated hai1ahi2217mutants. Percentages of rosettes containing (engulfed) basal cells distal of the peridermal rosette (compare with D) are indicated in red. n = 5–7 embryos per condition in two independent experiments. For quantifications and statistical significances, see legend of Fig. 1. Scale bars in A and B, 5 µm; and in C–G, 10 µm.

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