Recovery of dyshormonogenesis phenotypes in zebrafish duox crispants. (A) Epifluorescence live imaging of Tg(tg:nlsEGFP) zebrafish. Ventral views of the head region (anterior to the right, scale bar: 100 µm) and magnified views of the thyroid region (GFP channel only, scale bar: 50 µm) are shown for non-injected controls and duox crispants at 55 hpf, 80 hpf, and 6 dpf. No thyroid phenotypes were detectable at 55 hpf and 80 hpf but by 6 dpf, the majority of duox crispants displayed a goitrous thyroid enlargement with severe hyperplasia. (B) Whole-mount immunofluorescence staining of Tg(tg:nlsEGFP) zebrafish (6 dpf) for EGFP (thyroid cells) and thyroxine (colloidal T4). Epifluorescence imaging of the thyroid region in 6 dpf larvae (ventral views, anterior to the right, scale bar: 50 µm) revealed reduced T4 content in duox crispants as the most prevalent phenotypic alterations. Most of these hypothyroid larvae displayed concurrent thyroid hyperplasia. (C) Distribution of allelic variants as determined by Illumina HiSeq analysis of individual duox crispants confirmed high mutagenesis efficiency in larvae affected by thyroid dyshormonogenesis. The percentage of WT alleles (no variant call), in-frame indels, or frameshift indels is shown for N = 4 larvae per phenotypic category (median values with interquartile range).
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