Fig. S4

Effects of the goitrogenic compound VAS2870 on thyroid differentiation marker expression and cell proliferation. Tg(tg:nlsEGFP) larvae were treated with VAS2870 (25, 50 μM) from 52 to 120 hpf. Negative controls included embryo medium (Ctrl) and 0.025% DMSO (vehicle). Treatment with 197 μM PTU served as a positive control. (A) Live imaging of EGFP reporter expression was performed for quantification of thyroidal EGFP reporter signal (epifluorescence analyzes) and total TFC number (confocal analyzes) in 100 hpf larvae. Scatter plots show the median with the interquartile ranges. Each dot represents the global EGFP intensity or the total cell number for individual fish. Each treatment was replicated in three independent experiments with minimum three embryos per condition analyzed. (B) Percentage of thyroid proliferating cells (number of EdU+/EGFP+ cells divided by the TFC number (DAPI+/EFGP+)) in treated or vehicle control (DMSO) 120 hpf Tg(tg:nlsEGFP) larvae. Scatter plots show the median with the interquartile ranges. Each dot represents the % of proliferating cells of individual fish (n=7-9). (C) Pituitary tshb mRNA expression detected by WISH in 120 hpf treated larvae from 52 hpf. Representative coronal sections are shown (dorsal view, anterior to the top, scale bars: 50 μm). (D) At the thyroid level (arrow heads), increased expression of TSH-dependent functional markers (tg, slc5a5/nis, tpo, duox and duoxa) was detected in 120 hpf treated larvae. Representative larvae are shown in ventral views of the head region (anterior to the left). Scale bars: 100 μm. (E) Comparative fold change mRNA expression (mean ± SD, n=3) of tg, slc5a5/nis, tpo and tshb genes using DMSO treated larvae as controls. Mann-Whitney tests were performed to compare DMSO treated larvae with all other conditions.