Fig. 7

Twist expression is required for BRAF^V600E mediated thyroid follicle disruption.

(A) Results from an in silico screen of transcription factor (n = 1665) expression in human PTC correlated with BRAF^V600E gene signature performed by GSEA. Top hits represented in red. (B) CrisprVariants analysis of twist3 gene editing in zebrafish embryos. tg-BRAF^V600E-TOM embryos were microinjected with rCas9 and either a non-targeting sgRNA or a twist3-specific sgRNA. Individual embryos were isolated and a clonal analysis of the twist3 target locus was performed. Non-targeting embryos (n = 5, grouped) had no evidence of deletions at the twist3 locus, 21/21 clones were wild-type. Twist3 targeted embryos (n = 5, one per column) had a range of small indels at the targeting site. (C) Representative confocal images of 5 dpf fixed tg-BRAF^V600E-TOM embryo injected with either non-targeting or twist3 targeting sgRNA. Anti-T4 immunostaining performed to identify T4-positive (green) follicles. (D) Quantification of follicle rescue in embryos injected with a non-targeting versus twist3 targeting sgRNA in BRAF^V600E-TOM embryos. Follicle rescue was observed in 0/142 non-targeting sgRNA injected embryos vs. 57/173 (32.9%) of twist3 sgRNA injected embryos, p<1.0 × 10⁻³, two-tailed Fisher exact test. (E) FACS plot of TdTomato-positive cells isolated from twist3 sgRNA injected tg-BRAF^V600E-TOM embryos. (F) Alignment of DNA chromatogram from a clone isolated from twist3 sgRNA injected TOM+ cells harboring a three nucleotide deletion in the twist3 gene. The twist3 sgRNA sequence is underlined in red.