Identification of Thyroid Hormones and Functional Characterization of Thyroid Hormone Receptor in the Pacific Oyster <i>Crassostrea gigas</i> Provide Insight into Evolution of the Thyroid Hormone System

<div><p>Thyroid hormones (THs) play important roles in development, metamorphosis, and metabolism in vertebrates. During the past century, TH functions were regarded as a synapomorphy of vertebrates. More recently, accumulating evidence has gradually convinced us that TH functions also occur in invertebrate chordates. To date, however, TH-related studies in non-chordate invertebrates have been limited. In this study, THs were qualitatively detected by two reliable methods (HPLC and LC/MS) in a well-studied molluscan species, the Pacific oyster <i>Crassostrea gigas</i>. Quantitative measurement of THs during the development of <i>C</i>. <i>gigas</i> showed high TH contents during embryogenesis and that oyster embryos may synthesize THs endogenously. As a first step in elucidating the TH signaling cascade, an ortholog of vertebrate TH receptor (TR), the most critical gene mediating TH effects, was cloned in <i>C</i>. <i>gigas</i>. The sequence of <i>CgTR</i> has conserved DNA-binding and ligand-binding domains that normally characterize these receptors. Experimental results demonstrated that <i>CgTR</i> can repress gene expression through binding to promoters of target genes and can interact with oyster retinoid X receptor. Moreover, <i>CgTR</i> mRNA expression was activated by T4 and the transcriptional activity of <i>CgTR</i> promoter was repressed by unliganded <i>CgTR</i> protein. An atypical thyroid hormone response element (CgDR5) was found in the promoter of <i>CgTR</i>, which was verified by electrophoretic mobility shift assay (EMSA). These results indicated that some of the <i>CgTR</i> function is conserved. However, the EMSA assay showed that DNA binding specificity of <i>CgTR</i> was different from that of the vertebrate TR and experiments with two dual-luciferase reporter systems indicated that l-thyroxine, 3,3′,5-triiodothyronine, and triiodothyroacetic acid failed to activate the transcriptional activity of <i>CgTR</i>. This is the first study to functionally characterize TR in mollusks. The presence of THs and the functions of <i>CgTR</i> in mollusks contribute to better understanding of the evolution of the TH system.</p>