Sterner ZR et al. (2023), Development and metamorphosis in frogs deficien...

XB-ART-59481

Gen Comp Endocrinol 2023 Jan 15;331:114179. doi: 10.1016/j.ygcen.2022.114179.

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Development and metamorphosis in frogs deficient in the thyroid hormone transporter MCT8.

Sterner ZR , Jabrah A , Shaidani NI , Horb ME , Dockery R , Paul B , Buchholz DR .

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Precisely regulated thyroid hormone (TH) signaling within tissues during frog metamorphosis gives rise to the organism-wide coordination of developmental events among organs required for survival. This TH signaling is controlled by multiple cellular mechanisms, including TH transport across the plasma membrane. A highly specific TH transporter has been identified, namely monocarboxylate transporter 8 (MCT8), which facilitates uptake and efflux of TH and is differentially and dynamically expressed among tissues during metamorphosis. We hypothesized that loss of MCT8 would alter tissue sensitivity to TH and affect the timing of tissue transformation. To address this, we used CRISPR/Cas9 to introduce frameshift mutations inslc16a2, the gene encoding MCT8, inXenopus laevis. We produced homozygous mutant tadpoles with a 29-bp mutation in the l-chromosome and a 20-bp mutation in the S-chromosome. We found that MCT8 mutants survive metamorphosis with normal growth and development of external morphology throughout the larval period. Consistent with this result, the expression of the pituitary hormone regulating TH plasma levels (tshb) was similar among genotypes as was TH response gene expression in brain at metamorphic climax. Further, delayed initiation of limb outgrowth during natural metamorphosis and reduced hindlimb and tail TH sensitivity were not observed in MCT8 mutants. In sum, we did not observe an effect on TH-dependent development in MCT8 mutants, suggesting compensatory TH transport occurs in tadpole tissues, as seen in most tissues in all model organisms examined.

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Species referenced: Xenopus laevis
Genes referenced: klf9 slc16a2 slc22a18 thrb tshb
GO keywords: thyroid hormone mediated signaling pathway [+]

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	Fig. 1. CRISPR/Cas9 target site for slc16a2. The gene encoding MCT8, slc16a2, has 6 exons labeled E1-6, with the coding region shown as vertical grey bars and the 5′ and 3′ untranslated regions shown as vertical black bars. The DNA sequences from the L- and S-chromosomes around the CRISPR target site in Exon 1 are shown in the large, boxed area. Gaps in the asterisks below the slc16a2.L and slc16a2.S sequences show sequence divergence between the two slc16a2 loci. The exact CRISPR target site, i.e., the sgRNA sequence, is shaded, and the Cas9 cut site is shown by the black triangle. The 29-base pair deletion in slc16a2.L and the 20-base pair deletion in slc16a2.S are underlined. The forward and reverse primers used in the heteroduplex migration assay (HMA F.P, HMA R.P) and the first transmembrane domain required for MCT8 function are boxed.
	Fig. 2. Expression and tissue distribution of slc16a2.L and slc16a2.S. The expression levels of slc16a2.L and slc16a2.S among adult tissues of X. laevis are represented by shades of gray that indicate log2 of total counts per million (TCM) from RNA-seq data. This figure was obtained using the data visualization tool on XenBase (Karimi et al., 2018).
	Fig. 3. slc16a2 genotyping using the heteroduplex migration assay. All tadpoles used in the growth and development experiments were F3 offspring from the F2 cross Llss × Llss, where both parents were heterozygous for the MCT8 mutation on the L-chromosome and homozygous for the MCT8 mutation on the S-chromosome. The three possible genotypes from this cross were LLss, designated as “wild-type” (WT), Llss, designated as “heterozygous” (HET), and llss, designated as knockout (KO). PCR amplification of a DNA region around the CRISPR target site from each of these genotypes followed by melting and reannealing of the PCR products results in a total of 6 possible allele combinations, namely LL, ll, ss (homoduplexes) and Ll, Ls, ls (heteroduplexes). When run on an 8 % PAGE gel, the reannealed DNA homo- and heteroduplexes can be identified as shown on the gel image based on the allele PCR product sizes. Genotypes were assigned based on their unique set of bands on the gel. n.s. = non-specific.
	Fig. 4. Growth and development in MCT8 mutant tadpoles. Genotyped and individually reared tadpoles were (A) measured for snout-vent length (SVL) and (B) staged according to Nieuwkoop and Faber (NF) every 5 days starting at the beginning of metamorphosis (NF stage 54, limb bud outgrowth) to the end of metamorphosis (NF stage 66, complete tail resorption). In addition, tadpoles were monitored daily to record exact number of days after NF 54 to achieve (C) forelimb emergence (NF 58), (D) metamorphic climax (NF 62), and (E) tail resorption (NF 66). Means and standard error for each genotype are shown, n = 10 per genotype. WT = wild-type (LLss), Het = heterozygous (Llss), MCT8KO = homozygous knockout (llss). No statistical differences were found using one-way ANOVA, p < 0.5.
	Fig. 5. Gene expression of tshb, klf9, and thrb in the brain at metamorphic climax among MCT8 genotypes. Genotyped F3 tadpoles were reared to metamorphic climax (NF 62), then brain including the pituitary was harvested and processed for quantitative PCR to measure RNA expression of the pituitary gene (A) thyroid stimulating hormone beta (tshb) and the TH response genes, (B) Krüppel-like factor 9 (klf9) and (C) TH receptor beta (thrb). Means and standard error for each genotype are shown, n = 7 for wild-type (WT, LLss), 9 for homozygous MCT8 knockout (MCT8KO, llss). No statistical differences were found using one-way ANOVA, p < 0.5.
	Fig. 6. Initiation of metamorphosis among MCT8 genotypes. To detect an effect of MCT8 on the initiation of metamorphosis, we examined start of limb outgrowth (NF 54) with respect to tadpole size. 79 tadpoles were measured for snout-vent length (SVL) and staged according to Nieuwkoop and Faber (NF) at 21 days post fertilization followed by genotyping. We compared (A) hindlimb length normalized to SVL and (B) NF stage among genotypes. Means and standard error for each genotype are shown, n = 14 for wild-type (WT, LLss), 53 for heterozygous (Het, Llss), 12 for homozygous MCT8 knockout (MCT8KO, llss). No statistical differences were found using one-way ANOVA, p < 0.5.
	Fig. 7. Tissue sensitivity to TH in tail and hindlimbs among MCT8 genotypes. Premetamorphic F3 tadpoles (NF 54) were treated for 24 hrs with 0, 0.5, 2, and 5 nM T3 (tri-iodothyronine) added to their rearing water, and then their tails and hindlimbs were harvested and processed for quantitative PCR. The RNA expression of the TH response genes, (A,C) Krüppel-like factor 9 (klf9) and (B, D) TH receptor beta (thrb) in tail (A,B) and hindlimbs (B,C) among MCT8 genotypes. Means and standard error for each genotype are shown, n = 10 for all genotypes and T3 treatments. WT = wild-type (LLss), MCT8KO = homozygous knockout (llss). Letters indicate significant differences among bars determined for each genotype based on non-parametric Kruskal-Wallis test followed by pairwise comparisons using Wilcoxon rank sum exact test (A, B and D) and two-way ANOVA followed by pairwise comparisons using Tukey’s post hoc test (C). p < 0.05.

References [+] :

Baker, Prolactin prevents the autoinduction of thyroid hormone receptor mRNAs during amphibian metamorphosis. 1992, Pubmed, Xenbase