Inhibition of the thyroid hormonogenic H2O2 production by Duox/DuoxA in zebrafish reveals VAS2870 as a new goitrogenic compound.

Thyroid hormone (TH) synthesis requires extracellular hydrogen peroxide generated by the NADPH oxidases, DUOX1 and DUOX2, with maturation factors, DUOXA1 and DUOXA2. In zebrafish, only one duox and one duoxa gene are present. Using a thyroid-specific reporter line, we investigated the role of Duox and Duoxa for TH biosynthesis in zebrafish larvae. Analysis of several zebrafish duox and duoxa mutant models consistently recovered hypothyroid phenotypes with hyperplastic goiter caused by impaired TH synthesis. Mutant larvae developed enlarged thyroids and showed increased expression of the EGFP reporter and thyroid functional markers including wild-type and mutated duox and duoxa transcripts. Treatment of zebrafish larvae with the NADPH oxidase inhibitor VAS2870 phenocopied the thyroid effects observed in duox or duoxa mutants. Additional functional in vitro assays corroborated the pharmacological inhibition of Duox activity by VAS2870. These data support the utility of this new experimental model to characterize endocrine disruptors of the thyroid function.

Small-Molecule Screening in Zebrafish Embryos Identifies Signaling Pathways Regulating Early Thyroid Development.

Background: Defects in embryonic development of the thyroid gland are a major cause for congenital hypothyroidism in human newborns, but the underlying molecular mechanisms are still poorly understood. Organ development relies on a tightly regulated interplay between extrinsic signaling cues and cell intrinsic factors. At present, however, there is limited knowledge about the specific extrinsic signaling cues that regulate foregut endoderm patterning, thyroid cell specification, and subsequent morphogenetic processes in thyroid development. Methods: To begin to address this problem in a systematic way, we used zebrafish embryos to perform a series of in vivo phenotype-driven chemical genetic screens to identify signaling cues regulating early thyroid development. For this purpose, we treated zebrafish embryos during different developmental periods with a panel of small-molecule compounds known to manipulate the activity of major signaling pathways and scored phenotypic deviations in thyroid, endoderm, and cardiovascular development using whole-mount in situ hybridization and transgenic fluorescent reporter models. Results: Systematic assessment of drugged embryos recovered a range of thyroid phenotypes including expansion, reduction or lack of the early thyroid anlage, defective thyroid budding, as well as hypoplastic, enlarged, or overtly disorganized presentation of the thyroid primordium after budding. Our pharmacological screening identified bone morphogenetic protein and fibroblast growth factor signaling as key factors for thyroid specification and early thyroid organogenesis, highlighted the importance of low Wnt activities during early development for thyroid specification, and implicated drug-induced cardiac and vascular anomalies as likely indirect mechanisms causing various forms of thyroid dysgenesis. Conclusions: By integrating the outcome of our screening efforts with previously available information from other model organisms including Xenopus, chicken, and mouse, we conclude that signaling cues regulating thyroid development appear broadly conserved across vertebrates. We therefore expect that observations made in zebrafish can inform mammalian models of thyroid organogenesis to further our understanding of the molecular mechanisms of congenital thyroid diseases.