Figure 4. Evpl and Ppl are required for microridge morphogenesis and determine their length.

(A) Periderm cells expressing Lifeact-mRuby in WT, evpl^−/−, ppl^−/−, and evpl^−/−;ppl^−/− mutants at 48hpf. Images were inverted so that high-intensity fluorescence appears black and low-intensity fluorescence is white. (B) Dot and box-and-whisker plot of average microridge length per cell at 48hpf from animals of the indicated genotypes. All comparisons between each genotype were significantly different from one another, except where indicated as n.s. *p<0.05, **p<0.01; for all other comparisons ***p<0.0001, the Wilcoxon rank-sum test. The exact P values for all comparisons are shown in Supplementary file 1. n = 27–69 cells from 3 to 9 fish per genotype. Another version of this graph color-coding cells from each animal is provided in Figure 4—figure supplement 2A, as well as a violin plot showing the length distributions of all microridges pooled (Figure 4—figure supplement 2B, Supplementary file 1). To control for animal-to-animal variation, we have also analyzed these data by averaging the microridge length averages for each cell in each animal (i.e. each animal is represented by one number averaging all cells). This approach yielded similar results and are reported in Supplementary file 1. (C) Cells overexpressing Evpl-mRuby[BAC], Ppl-GFP[BAC], and Lifeact-mRuby at 48hpf. (D) Dot and box-and-whisker plot of average microridge length per cell in cells over-expressing Evpl-mRuby[BAC] and Ppl-GFP[BAC] at 48hpf. See Figure 4—figure supplement 7 for categorization into ‘low’ and ‘high’ overexpression groups. ***p<0.0001, the Wilcoxon rank-sum test. n = 41 cells from five fish. Dotted blue line shows the median average microridge length per cell in WT animals (from B). Box-and-whisker plots (B and D): Middle line shows the median; the upper and lower ends of the box are the 75th and 25th percentiles. Scale bars: 10 µm.

Figure 4—figure supplement 1. evpl and ppl mutants.

(A) The evpl gene targeting strategy. Sequence shows the deletion site. Bottom-left panel shows PCR analysis of genomic DNA isolated from adult fish fins with the indicated genotypes. Primers Fw1 and Rev1 were used to detect WT alleles (743 bp) and primers Fw1 and Rev2 were used to detect mutant alleles (576 bp). Bottom-right shows RT-PCR of RNA isolated from adult fish scales with the indicated genotypes. Primers Fw3 and Rev3 were used to detect WT (638 bp) and mutant (167 bp) alleles. (B) The ppl gene targeting strategy. Sequence shows the deletion site. Bottom-left panel shows PCR analysis of genomic DNA isolated from adult fish fins with the indicated genotypes. Primers Fw1 and Rev1 were used to detect the WT (404 bp) allele and primers Fw1 and Rev2 were used to detect the mutant allele (468 bp). Bottom-right shows RT-PCR of RNA isolated from adult fish scales with the indicated genotypes. Primers Fw3 and Rev3 were used to detect WT (571 bp) and mutant (107 bp) alleles.

Figure 4—figure supplement 2. Additional quantification of microridge length and cell area in evpl and ppl mutants.

(A) Dot and box-and-whisker plot of average microridge length per cell of the indicated genotypes, at 48hpf. Same data as in Figure 4B but with colors representing cells from different animals. *p<0.05, **p<0.01, and ***p<0.0001, the Wilcoxon rank-sum test. (B) Box-and-whisker and violin plot of microridge length, pooled from all measured cells, of the indicated genotypes. ***p<0.0001, the Wilcoxon rank-sum test. (C) Dot and box-and-whisker plot of apical cell areas at 48hpf from animals of indicated genotypes. Colors represent cells from different animals. n = 27–69 cells from 3 to 9 fish per genotype. ***p<0.0001, the Wilcoxon rank-sum test. Box-and-whisker plots: Middle line shows the median; the upper and lower ends of the box are the 75th and 25th percentiles. Exact P values for all comparisons are shown in Supplementary file 1.

Figure 4—figure supplement 3. Plakin BAC reporters rescue microridge development in plakin mutants.

(A) Lifeact-GFP-expressing cells mosaically expressing Evpl-mRuby[BAC] in periderm cells. Microridges were rescued by BAC expression (compare to neighboring control cells lacking mRuby). These and subsequent black-and-white images were inverted so that high-intensity fluorescence appears black and low-intensity fluorescence is white. (B) Lifeact-mRuby-expressing cells mosaically expressing Ppl-GFP[BAC] in periderm cells. Microridges were rescued by BAC expression (compare to neighboring control cells lacking GFP). Scale bar: 10 µm.

Figure 4—figure supplement 4. evpl- and ppl-targeting morpholinos disrupt microridge development.

(A) Actin (Lifeact-mRuby) in control morpholino (MO)-, evpl MO-, and ppl MO-injected embryos at 48hpf. These and subsequent black-and-white images were inverted so that high-intensity fluorescence appears black and low-intensity fluorescence is white. (B) Lifeact-GFP-expressing periderm cells in evpl MO-injected embryos, also mosaically expressing Krt5:Evpl-tdTomato. Microridges were rescued by cDNA expression (compare to neighboring control cells lacking tdTomato). (C) Lifeact-mRuby-expressing periderm cells in ppl MO-injected embryos, also mosaically expressing Krt5:Ppl-GFP. Microridges were rescued by cDNA expression (compare to neighboring control cells lacking GFP). Scale bars: 10 µm.

Figure 4—figure supplement 5. Microridges in adult fish were disrupted in evpl and ppl mutants.

Actin (phalloidin staining) of skin covering adult scales in WT, evpl^−/−, ppl^−/−, and evpl^−/−;ppl^−/− mutants. Images were inverted so that high-intensity fluorescence appears black and low-intensity fluorescence is white. Scale bar: 10 µm.

Figure 4—figure supplement 6. evpl and ppl mutant morphologies.

Zebrafish of the indicated genotypes at larval (48hpf) and adult (>3 months) stages. Scale bar: 1 mm for larvae and 5 mm for adults.

Figure 4—figure supplement 7. Evpl and Ppl dictate microridge length.

(A) Images of periderm cells expressing Evpl-mRuby[BAC], Ppl-GFP[BAC], and Lifeact-mRuby at 48hpf. Image is a zoomed out and rotated version of the image used in Figure 4C. Black-and-white images were inverted so that high-intensity fluorescence appears black and low-intensity fluorescence is white. (B) Scatter plot of average microridge length versus mean GFP fluorescence intensity in periderm cells overexpressing Evpl-mRuby[BAC] and Ppl-GFP[BAC]. Cells were divided into high and low fluorescence intensity groups, as indicated, and used in analysis shown in Figure 4D. n = 41 cells from five fish. Scale bar: 10 µm.