Functional analysis of thyroid hormone receptor beta in Xenopus tropicalis founders using CRISPR-Cas

Amphibians provide an ideal model to study the actions of thyroid hormone (TH) in animal development because TH signaling via two TH receptors, TRa and TRb, is indispensable for amphibian metamorphosis. However, specific roles for the TRb isoform in metamorphosis are poorly understood. To address this issue, we generated trb-disrupted Xenopus tropicalis tadpoles using the CRISPR-Cas system. 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 trb in metamorphosis. trb-disrupted premetamorphic tadpoles exhibited mixed responsiveness to exogenous TH. Specifically, gill resorption and activation of several TH-response genes, including trb itself and two protease genes, were impaired. On the other hand, hind limb outgrowth and induction of the TH-response genes, klf9 and fra-2, were not affected by loss of trb. Surprisingly, trb-disrupted tadpoles were able to undergo spontaneous metamorphosis normally, except for a slight delay in tail resorption. These results indicate TRb is not required but contributes to the timing of resorptive events of metamorphosis.