(A) SIM optical sections of a Krt17-GFP[BAC]-expressing zebrafish periderm cell at 48hpf. Cartoon shows the relative location of apical (A) and medial (M) optical sections. (B) Oblique optical section through a cell with endogenously-tagged Krt17. This section shows both the microridge-like pattern at the apical cell surface (arrow) and a filamentous pattern deeper in the cell (arrowhead). (C) Projections and orthogonal views of SIM images of Krt17-GFP[BAC]- and Lifeact-mRuby-expressing cells at the indicated developmental stages. White boxes, regions of magnification in zoom panels. Orthogonal views (XZ) at 19hpf show that Krt17 is not in pegs but at 48hpf localizes alongside F-actin in microridges. Dashed lines outline cell membranes. (D) Section (XY) and orthogonal (XZ) views of SIM images of Krt17-GFP[BAC]- and Lifeact-mRuby-expressing cells at 48hpf. ‘-’: No keratin in protrusion. ‘+’: Keratin partially occupies microridge. ‘++': Keratin fully occupies microridge. Blue lines show the location of XZ optical sections. Dashed lines outline cell membranes. (E) Plot showing keratin localization in actin protrusions of different sizes (area) at 48hpf. The presence of keratin was scored as in D. n = 3 cells from two fish. (F) Dot and box-and-whisker plots of average microridge length per cell in WT cells expressing Lifeact-mRuby, and cells expressing Krt17-GFP[BAC] and Lifeact-mRuby at 48hpf. *p<0.05, the Wilcoxon rank-sum test. n = 24 cells from four fish in Krt17-GFP[BAC]-expressing fish and 69 cells from nine fish in WT. WT data is the same as in Figure 4B. Box-and-whisker plots (F): Middle line shows the median; the upper and lower ends of the box are the 75th and 25th percentiles. Black-and-white images were inverted so that high-intensity fluorescence appears black and low fluorescence is white. Scale bars: 10 µm (A-C, blue line) and 1 µm (C-D white line).
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