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, 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 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 trβ in metamorphosis. trβ-disrupted pre-metamorphic tadpoles exhibited mixed responsiveness to exogenous TH. Specifically, gill resorption and activation of several TH-response genes, including trβ 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 trβ. Surprisingly, trβ-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.

Gephebase, a Database of Genotype-Phenotype Relationships for natural and domesticated variation in Eukaryotes

“…Importantly, the focus of Gephebase is always on genetic variations that emerge naturally -it never includes laboratory variants that were generated by random or directed mutagenesis. Thus the Oca2 CRISPR knockout phenotypes that have been generated in frogs (16) do not have a dedicated Gephebase entry ; the cavefish Oca2 CRISPR/TALEN knockout phenotypes (17,18) do not have a dedicated entry either, but are used as Additional References to support the functionality of the two natural Oca2 null alleles in Gephebase. This makes Gephebase complementary to the Monarch Initiative database, which compiles gene-to-phenotype relationships in humans, as well as in laboratory organisms and mutants generated by reverse genetics, but does not include non-model species such as cavefishes and corn snakes (8,9) .…”

Establishment of knockout adult sea urchins by using a CRISPR‐Cas9 system

“…Therefore, the timing of CRISPR‐Cas9 mediated genome editing is comparable to those of ZFN and TALEN. Ribonucleoprotein (RNP) consisting of the SpCas9 protein in complex with sgRNA has been used in cultured cells and animals (Kim, Kim, Cho, Kim, & Kim, ; Kotani, Taimatsu, Ohga, Ota, & Kawahara, ; Lee et al., ; Sakane et al., ; Shigeta et al., ; Sung et al., ); it shortens the timing of genome editing and overcomes the mosaic property (Kim et al., ; Kotani et al., ). We attempted to accelerate mutagenesis in H. pulcherrimus by using SpCas9 RNP.…”

Overexpression of Lin28a delays Xenopus metamorphosis and down‐regulates albumin independently of its translational regulation domain

“…We cannot rule‐out that Lin28 also regulates the expression or the function of TH receptors during premetamorphosis and prometamorphosis. Loss‐of‐function studies have shown that TH receptors alpha (THRα) and beta (THRβ) are crucial for the proper timing of metamorphosis and the coordination of changes in different organs . Therefore, Lin28 may regulate the function of THRs in specific tissues in a complex network coordinating the timing of metamorphosis with the proliferation and differentiation of progenitor cells in different tissues.…”