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Generation of novel genetic models to dissect resistance to thyroid hormone receptor alpha in zebrafish.

BACKGROUND
Patients with mutations of the thyroid hormone receptor  gene (THRA) show resistance to thyroid hormone (RTH). No amendable mouse models are currently available to elucidate deleterious effects of TR1 mutants during early development. Zebrafish with transient suppressed expression by morpholino knockdown and ectopic expression of TR1 mutants in the embryos have been reported. However, zebrafish with germline transmittable mutations have not been reported. The stable expression of thra mutants from embryos to adulthood facilitated the study of molecular actions of TR1 mutants during development.


METHODS
In contrast to human and mice, the thra gene is duplicated, thraa and thrab. Using CRISPR/Cas9-mediated targeted mutagenesis, we created dominant negative mutations in the two duplicated thra genes in zebrafish. We comprehensively analyzed the molecular and phenotypic characteristics of mutant fish during development.


RESULTS
Adult and juvenile homozygous thrab 1-bp ins (m/m) mutants exhibited severe growth retardation, but adult homozygous thraa 8-bp ins (m/m) mutants had very mild growth impairment. Expression of the growth hormone (gh1) and insulin-like growth factor 1 was markedly suppressed in homozygous thrab 1-bp ins (m/m) mutants. Decreased mRNA and protein levels of T3-regulated keratin genes and inhibited keratinocyte proliferation resulted in epidermis hypoplasia of adult and juvenile homozygous thrab 1-bp ins (m/m) mutants, but not homozygous thraa 8-bp ins (m/m) mutants. RNA-seq analysis showed that homozygous thrab 1-bp ins (m/m) mutation had global impact on the functions of the adult pituitary. However, no morphological defects, nor any changes in the expression of gh1 and keratin genes were observed in the embryos and early larvae. Thus, mutations of either the thraa or thrab gene did not affect initiation of embryogenesis. But the mutation of the thrab gene, but not the thraa gene, is detrimental in post-larva growth and skin development.


CONCLUSIONS
The thra duplicated genes are essential to control temporal coordination in post-larva growth and development in a tissue-specific manner. We uncovered novel functions of the duplicated thra genes in zebrafish in development. These mutant zebrafish could be used as a model for further analysis of TR1 mutant actions and for rapid screening of therapeutics for RTH.