Supplementary Figure 6: Reduced levels of Ago2 in mz ago2 -/- zebrafish model.

(a) Left, Confocal lateral view of whole mount zebrafish embryos at 48 hpf. Wild-Type and ago2 maternal zygotic homozygous mutant (mz ago2-/-) were stained with the Ab-panAGO-2A8 and secondary only as a control of the total staining background (scale bar = 100 µm). Bar plots indicate average of Ago2 fluorescence intensity normalized for the DAPI staining for each genotype (mean ± SEM, n = 3 replica with 10 embryos / group, * p = 0.0261, unpaired two-sided t-test). Right, RT-qPCR representing miRNAs level in wild-type vs mz ago2 -/- at 72 hpf (mean ± SEM, n = 3 embryos per group, value normalized for one wild-type embro). (b) Left panel, schematics representing the experimental procedure to test defective miRNA-mRNA interaction in the mz ago2-/- fin fold model. In vitro transcribed RNAs encoding for mCherry control and GFP coding sequence upstream of three perfect MREs for miR-24, a miRNA expressed in epidermis²⁹. 25 picograms (pg) of each mRNA was co-injected into wild-type and ago2-/- zebrafish embryos at the one cell stage post fertilization. Right panel, confocal whole mount image of 48 hpf embryos expressing GFP and mCherry (head is to the left, scale bar = 60 µm). GFP and mCherry pixel intensities were quantified and the GFP/mCherry ratio was plotted for each genotype (mean ± SEM, n = 4 embryos / group, *p = 0.0137, unpaired two-sided t-test). (c) and (d) Top, Confocal lateral view of whole mount zebrafish embryos treated as indicated after zebrafish find fold assay (scale bar = 120 µm). White dotted line shows the edge of the wound. Bottom, box plot with minimum, maximum, median and quartiles representing the quantification of each whole mount stained embryos as indicated. Average intensity was calculated for 4 individual fish for PCNA and 4 to 6 fish for TUNEL assay. (Single dots represent single embryos, n.s.= non-significant). Source data can be found in Supplementary Table 8.