Identification of taf1 mutations in thyroid mutants. A: the whole morphology of 031003 sibling vs. mutant larvae screened by N-ethyl-N-nitrosourea (ENU). B: thyroid marked by tg in 5 days postfertilization (dpf) embryos using whole mount RNA in situ hybridization (WISH). Nearly 25% of progeny from intercrossed heterozygotes showed an abnormal phenotype. Sibling contained wild-type and heterozygous embryos. C: the tshbα (thyrotrophin) in situ expression pattern in siblings and mutants. D: the 0310003 mutants had lesions in taf1 caused by V241M amino acid substitutions. Verification by Sanger sequencing. E: embryos injected with translation-blocking taf1 morpholinos (taf1-mo) can reproduce the small thyroid phenotype detected at 5 dpf by in situ hybridization. In the control group, embryos were injected with standard control morpholino (sd). Scale bar: 100 μm. tg, thyroglobulin.

031003 mutant presented thyroid dysgenesis phenotypes. A: the body length of the 031003 sibling vs. mutant at 5 days postfertilization (dpf) (ns: no significant difference). B: eye size of the 031003 sibling vs. mutant at 5 dpf. C: confocal examination of mature thyroid follicles marked by triiodothyronine (T3) immunofluorescence at 5 dpf. Scale bar: 100 μm. D: the areas of mature thyroid follicles per embryo were quantified by Imaris software. Whole body length (E) and weight (F) of 2-mo-old adult 031003 zebrafish. G and H: thyroid hormone levels in 2-mo-old adult zebrafish by ELISA. The sera of three individuals were pooled in a tube, and each group was analyzed in triplicate. **P < 0.01, ***P < 0.001, ****P < 0.0001. T4, thyroxine.

Time lapse of thyroid development in the 031003 sibling vs. mutant. The Tg (tg: mCherry) transgenic line used to analyze the time lapse of thyroid development and the volumes of thyroid follicles per embryo were quantified by Imaris software. 55 hpf (A and B), 72 hpf (C and D), 5 dpf (E and F). G: confocal images presenting the activation of the Notch signaling pathway (red) in the Tg(tg:GFP/tp1:nls-mCherry) background at different time points (55 hpf, 72 hpf, 75 hpf, 5 dpf) in zebrafish larvae. Imaging artifacts was due to the beating heart of the Notch reporter. The activation of the Notch signal in the thyroid is indicated by white arrows. H, heart. Scale bar: 100 μm. ***P < 0.001.

Notch signaling was impaired after TATA-box binding protein associated Factor 1 (TAF1) knockdown. Human Nthy-ori 3-1 cells were stably transfected with either control shRNA (Vector) or TAF1-specific shRNA (TAF1 shRNA). A: EdU proliferation assays were performed to determine the proliferative ability (green fluorescence: EdU, blue fluorescence: DAPI). B: the ratio of EdU-positive cells to DAPI-positive cells. C: the results of cell viability analysis verification by CCK8. A: the results of CCK8. D: the expression of TAF1, Notch intracellular domain (NICD), HES1, SLC5A5, and ACTIN was determined by Western blot (n = 3). E: the relative protein expression (protein/ACTIN) was quantified by ImageJ. Scale bars: 100 μm. **P < 0.01, ***P < 0.001.

TATA-box binding protein associated Factor 1 (TAF1) regulated the Notch signaling pathway in vitro and in vivo. A: the chromatin immunoprecipitation (ChIP) assay was performed to detect the binding ability between TAF1 and the NOTCH1 promoter in HEK293T cell lines. B: the ratios of luciferase activity regulated by the NOTCH1 response element to Renilla luciferase in HEK293T cells transfected with vector alone, wild-type TAF1 (WT), mutant TAF1 (MU), or Notch intracellular domain (NICD) overexpression plasmid (NICD). C: the expression of SLC5A5 and ACTIN was determined by Western blot (n = 3). TAF1-inhibited Nthy-ori 3-1 cells were transfected with vector alone, wild-type TAF1 (WT), mutant TAF1 (MU), or NICD overexpression plasmid (NICD). D: the relative protein expression (SLC5A5/ACTIN) was quantified by ImageJ. E and F: the rescue effects of human NICD (Tol2:tg-NICD-2A-mCherry) on thyroid development are displayed. Vector control, embryos injected with Tol2:tg-mCherry. Whole mount RNA in situ hybridization (WISH) (E) and confocal imaging (F) were detected in 5 days postfertilization (dpf) embryos. G, left: under normal conditions, TAF1 could bind to the Notch1 promoter, promoting the activation of Notch signaling. HES1 transcription was then initiated. HES1 upregulated the expression of SLC5A5 and other genes associated with thyroid hormone biosynthesis. Furthermore, thyroid follicular cells can proliferate and mature. Right: the mutant TAF1 was incapable of binding to the Notch1 promoter and impaired Notch signaling. Inadequacy of Notch signaling led to the abnormal proliferation and maturation of thyroid follicular cells. This figure was created using Figdraw and PowerPoint. Scale bars: 100 μm. ***P < 0.001, ****P < 0.0001.