Dataset Information

Functional analysis of thyroid hormone receptor beta in Xenopus tropicalis 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, TR? and TR?, is indispensable for amphibian metamorphosis. However, specific roles for the TR? isoform in metamorphosis are poorly understood. To address this issue, we generated tr?-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 tr? in metamorphosis. tr?-disrupted pre-metamorphic tadpoles exhibited mixed responsiveness to exogenous TH. Specifically, gill resorption and activation of several TH-response genes, including tr? itself and two protease genes, were impaired. However, hind limb outgrowth and induction of the TH-response genes, klf9 and fra-2, were not affected by loss of tr? Surprisingly, tr?-disrupted tadpoles were able to undergo spontaneous metamorphosis normally, except for a slight delay in tail resorption. These results indicate TR? is not required but contributes to the timing of resorptive events of metamorphosis.

SUBMITTER: Sakane Y

PROVIDER: S-EPMC5829506 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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

Sakane Yuto Y Iida Midori M Hasebe Takashi T Fujii Satoshi S Buchholz Daniel R DR Ishizuya-Oka Atsuko A Yamamoto Takashi T Suzuki Ken-Ichi T KT

Biology open 20180122 1

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 trβ-disrupted Xenopus tropicalis tadpoles using the CRISPR-Cas system. We first established a highly efficient and rapid workflow for gene disruption in the ...[more]

PMID: 29358165

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Project description:Tail resorption during anuran metamorphosis is perhaps the most dramatic tissue transformation that occurs during vertebrate development. Earlier studies in highly related anuran species Xenopus laevis and Xenopus tropicalis have shown that thyroid hormone (T3) receptor (TR) plays a necessary and sufficient role to mediate the causative effect of T3 on metamorphosis. Of the two known TR genes in vertebrates, TRα is highly expressed during both premetamorphosis and metamorphosis while TRβ expression is low in premetamorphic tadpoles but highly upregulated as a direct target gene of T3 during metamorphosis, suggesting potentially different functions during metamorphosis. Indeed, gene knockout studies have shown that knocking out TRα and TRβ has different effects on tadpole development. In particularly, homozygous TRβ knockout tadpoles become tailed frogs well after sibling wild type ones complete metamorphosis. Most noticeably, in TRβ-knockout tadpoles, an apparently normal notochord is present when the notochord in wild-type and TRα-knockout tadpoles disappears. Here, we have investigated how tail notochord resorption is regulated by TR. We show that TRβ is selectively very highly expressed in the notochord compared to TRα. We have also discovered differential regulation of several matrix metalloproteinases (MMPs), which are known to be upregulated by T3 and implicated to play a role in tissue resorption by degrading the extracellular matrix (ECM). In particular, MMP9-TH and MMP13 are extremely highly expressed in the notochord compared to the rest of the tail. In situ hybridization analyses show that these MMPs are expressed in the outer sheath cells and/or the connective tissue sheath surrounding the notochord. Our findings suggest that high levels of TRβ expression in the notochord specifically upregulate these MMPs, which in turn degrades the ECM, leading to the collapse of the notochord and its subsequent resorption during metamorphosis.

Dataset Information

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

Publications

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

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