Oncogenic BRAF disrupts thyroid morphogenesis and function via Twist expression

Thyroid cancer is common, yet the sequence of alterations that promote tumor formation are incompletely understood. Here we describe a novel model of thyroid carcinoma in zebrafish that reveals temporal changes due to BRAFV600E. Through the use of real-time in vivo imaging we observe disruption in thyroid follicle structure that occurs early in thyroid development. Combinatorial treatment using BRAF and MEK inhibitors reversed the developmental effects induced by BRAFV600E. Adult zebrafish expressing BRAFV600E in thyrocytes developed invasive carcinoma. We identified a gene expression signature from zebrafish thyroid cancer that is predictive of disease free survival in patients with papillary thyroid cancer. Gene expression studies nominated TWIST2 as a key effector downstream of BRAF. Using CRISPR/Cas9 to genetically inactivate a TWIST2 orthologue, we suppressed the effects of BRAFV600E and restored thyroid morphology and hormone synthesis. These data suggest that expression of TWIST2 plays a role in an early step of BRAFV600E-mediated transformation. Overall design: 3 embryo tg-TOM (tg:TdTomato), 3 embryo tg-BRAFV600E-TOM, 3 adult tg-TOM and 5 adult tg-BRAFV600E-TOM biological replicates were sequenced. Strains with tg:TdTomato express the TdTomato fluorophore under control of the zebrafish thyroglobulin promoter (tg).