Functional analysis of thyroid hormone receptor beta in <i>Xenopus tropicalis</i> founders using CRISPR-Cas

Abstract

Amphibians provide an ideal model to study the actions of thyroid hormone (TH) in animal development because TH signaling via two TH receptors, TRα and TRβ, is indispensable for amphibian metamorphosis. However, specific roles for the TRβ isoform in metamorphosis are poorly understood. To address this issue, we generated <i>trβ-</i>disrupted <i>Xenopus tropicalis</i> tadpoles using the CRISPR-Cas system<i>.</i> We first established a highly efficient and rapid workflow for gene disruption in the founder generation (F0) by injecting sgRNA and Cas9 ribonucleoprotein. Most embryos showed severe mutant phenotypes carrying high somatic mutation rates. Utilizing this founder analysis system, we examined the role of <i>trβ</i> in metamorphosis. <i>trβ</i>-disrupted pre-metamorphic tadpoles exhibited mixed responsiveness to exogenous TH. Specifically, gill resorption and activation of several TH-response genes, including <i>trβ</i> itself and two protease genes, were impaired. However, hind limb outgrowth and induction of the TH-response genes, <i>klf9</i> and <i>fra-2</i>, were not affected by loss of <i>trβ</i> Surprisingly, <i>trβ-</i>disrupted tadpoles were able to undergo spontaneous metamorphosis normally, except for a slight delay in tail resorption. These results indicate TRβ is not required but contributes to the timing of resorptive events of metamorphosis.