Endocrine Abstracts

Thyroid hormone receptors (TRs) modulate various physiological functions in many organs. The conservation of TRα and TRβ isoforms during evolution of vertebrates suggests different roles for these TRs in thyroid hormone-dependent regulation of gene expression. TRα and TRβ are widely distributed and overlapping in several tissues, and their functional divergence is mainly attributed to their variable tissue expression patterns and intrinsic properties of specific TR isoforms have not been extensively investigated. Here, we want to verify whether the two TRs can be interchangeable in their tissue actions by comparing the phenotypical alterations in zebrafish embryos ubiquitously expressing human TR (hTR) mRNAs, injected in zygotes at 1-2 cells stage at a concentration of 50 pg/embryo. We previously showed that zebrafish TRs (zTR) can efficiently interact with their human homologues and the microinjection of wild-type hTRs does not affect the normal embryonic development. Conversely, the dominant-negative hTRα1-E403X or hTRβ2-E464X mRNAs results in distinct phenotypes despite their ubiquitous expression in zebrafish tissues. Embryos injected with hTRα1-E403X present aberrant angiogenesis, reduced heart looping and bradycardia, whereas only the embryos expressing hTRβ2-E464X show the typical RTHβ biochemical signature (high T4/T3 with unsuppressed tshba levels) confirming a dominant-negative effect on the pituitary negative feedback. Then, we generated mutant chimeras by switching the α1 and β2 functional domains: N-terminus A/B domain (A/B), DNA binding domain (DBD), hinge region (HR) and ligand binding domain (LBD). Multi-step PCR was used to generate wild-type and mutant chimeric fragments where the hTRα1 domain has been replaced with that of hTRβ2, and vice versa. Using tshba expression as marker of TRβ2-dependent activity, we attempted to discriminate which domain dictates the specific function. Several experimental replicates indicate the chimeric E403X mutant containing the A/B of hTRβ2 linked to DBD-HR-LBD of hTRα1 as functionally similar to the hTRβ2-E464X on tshba expression, suggesting that the A/B domain of TRβ2 is both required and sufficient to mediate this specific transcriptional effect, and that the variable structural properties of TRs represent another relevant factor dictating the isoform-specific actions in vivo.