January 22, 2018;
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, 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.
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Fig. 1. trβ sgRNA/Cas9 RNP leads to trβ gene disruption in X. tropicalis founders. (A) Schematic illustration of the sgRNA targeting sequences on the 2nd exon and 2nd intron of the trβ gene in X. tropicalis. One of the sgRNAs was designed in the coding region (trβ sgRNA-1) and the other was designed across the exon-intron junction (trβ sgRNA-2). Highlights in red and gray denote the protospacer adjacent motif (PAM) and the 20 bp target sequences of sgRNA, respectively. Exon and intron sequences are indicated by capital and small letters, respectively. (B) Genotyping by HMA in uninjected and injected embryos. PCR products encompassing sgRNA target sites were analyzed using a microchip electrophoresis system. Heteroduplex bands and multiple short bands are shown in the sgRNA/Cas9 RNP-injected embryos (crispants). Arrowheads, wild-type original bands; curly brackets, up-shifted heteroduplex bands; square brackets, deleted bands. (C) RT-PCR was performed using total RNA from stage 61/62 tadpoles with primers designed upstream and downstream of the 2nd exon. In addition to the original band (arrowhead), about 101 bp deleted bands were detected in crispants caused by skipping of the 2nd exon (asterisk) in trβ crispants. Heteroduplex bands were also detected in crispants (brackets). Each lane represents an individual animal. Ui, uninjected control.
Fig. 2. Amplicon sequencing analysis of on-target sites in trβ crispants. (A) Image of trβ crispants at stage 66. Genomic DNA was extracted from whole bodies of eight trβ crispants and the sgRNA target region was PCR-amplified using barcoded primers. PCR amplicons were subjected to amplicon sequencing according to Materials and Methods. Numbers indicate each crispant that was analyzed by amplicon sequencing in B. (B) The results of wild-type and mutant allele reads in each trβ crispant. All sequenced reads were classified into three groups; wild-type reads, frameshift and/or splice donor site mutation reads, in-frame mutation reads.
Fig. 3. Morphological changes induced by T3 treatment in trβ crispants. (A) Uninjected control tadpoles and trβ crispants at pre-metamorphic stage (stage 52–54) were treated with or without 10 nM T3 for 3 days and morphological changes observed. After 3 days of T3 treatment, regression of gills was observed in all uninjected control tadpoles (n=44/44), whereas these morphological changes were not induced in most of the trβ crispants (n=51/54). In contrast, morphological change did not occur in the gills of both uninjected control tadpoles and trβ crispants without T3 treatment. (B) Hind limb development was induced by T3 treatment in all uninjected control tadpoles (n=44/44) and trβ crispants (n=54/54). N values represent the total numbers of tadpoles in three independent experiments.
Fig. 4. Impaired induction of TH-response genes in trβ crispants. mRNA expression of five TH response genes, thyroid hormone receptor β (trβ), krüppel-like factor 9 (klf9), fos-related antigen-2 (fra-2), matrix metallopeptidase 13 (mmp13), and fibroblast activation protein alpha (fapa), was analyzed by qPCR. Uninjected control tadpoles and trβ crispants at pre-metamorphic stage (stage 52–54) were treated with or without 10 nM T3 for 3 days and at least four tadpoles were pooled per sample (n=3) in three independent experiments. The expression levels were normalized by rpl8. Expression changes by T3 induction were calculated relative to those of control animals without T3 treatment. Error bars represent ±s.d. (n=9). The three independent experiments exhibited similar trends. Asterisks indicate significant differences between the uninjected control and trβ crispants: *P<0.005; Welch's t-test.
Fig. 5. Delay of tail resorption in trβ crispants during natural metamorphosis. (A) The days from fertilization to stage 61 are indicated in a box plot for uninjected control tadpoles (n=46) and trβ crispants (n=39). There were no significant differences. (B) The days from stage 61 to stage 66 are indicated in a box plot for uninjected control tadpoles (n=45) and trβ crispants (n=38). trβ crispants required significantly more days to reach stage 66 (Welch's t-test, *P<0.05). N values represent the merged total numbers of tadpoles from two independent experiments. Box and whiskers show the interquartile range and maximum and minimum data points, respectively. (C) Time course of tail resorption in uninjected control tadpoles and trβ crispants. Uninjected control tadpoles and trβ crispants of the same age were selected from the same clutch at stage 61 and gill resorption observed at 1 days, 3 days, and 5 days after stage 61 (day 0). Uninjected control tadpoles completed tail resorption by day 5. In contrast, the tail remained in trβ crispants on day 5 (an arrowhead in the high magnification image) and a longer time was required to reach the end of metamorphosis.
Fig. 6. F1 trβ mutants accomplish natural metamorphosis. (A) Image of F1 offspring produced by mating of the F0 crispants. (B) Mutation sequences from each F1 offspring. PCR products of trβ target site from six F1 froglets were subcloned and sequenced. Sequences highlighted in red and gray denote protospacer adjacent motif (PAM) and protospacer sequence, respectively. Splice donor site is underlined. The deleted and inserted nucleotides are shown by dashes and blue letters, respectively. Capital and small letters indicate exon and intron, respectively.
A role for basic transcription element-binding protein 1 (BTEB1) in the autoinduction of thyroid hormone receptor beta.