Identification of the protein precursor for thyroid hormone synthesis in the basal chordate ascidian Styela clava

Thyroid hormones (THs) play crucial roles in development, growth, and metabolism across the animal kingdom1,2. Although the mechanism of TH synthesis in vertebrates has been well described, it remains elusive in invertebrates due to the lack of identified orthologs of the thyroglobulin (TG), the protein precursor for TH synthesis3,4. Here, we identified a functional TG protein encompassing multiple TG type 1 (Tg1)-domains, encoded by the ScTg-like gene in proto-vertebrate ascidian Styela clava based on the immunoprecipitation-mass spectrometry combined with phylogenetic and expression pattern analyses. In vitro iodination experiments demonstrated that ScTG-like has the capacity to provide hormonogenic sites for TH synthesis. Furthermore, in vivo gene knockdown of ScTg-like revealed that the larval metamorphosis was significantly inhibited as a consequence of diminished TH levels, indicating that ScTg-like is responsible for the production of endogenous TH. Additionally, an invaginated follicle-like structure was first discovered at the central anterior of the larval trunk where both ScTG-like protein and THs were deposited, suggesting that this structure serves as the location for THs synthesis and storage, functionally similar to vertebrate thyroid follicles. Moreover, we analyzed the structural features of ScTG-like protein and detected the similarly organized Tg1-containing proteins in multiple bilaterian phyla, suggesting that endogenous TH synthesis may be an ancestral, possibly synapomorphic, trait of bilaterians. Our study is the first to identify the TH protein precursor outside vertebrates by providing the direct evidence of endogenous de novo TH synthesis, which offers insights into the evolution of TG protein and the emergence of a major vertebrate endocrine system.