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???displayArticle.abstract??? Thyroid hormone (TH) receptor (TR) expression begins early in development in all vertebrates when circulating TH levels are absent or minimal, yet few developmental roles for unliganded TRs have been established. Unliganded TRs are expected to repress TH-response genes, increase tissue responsivity to TH, and regulate the timing of developmental events. Here we examined the role of unliganded TRα in gene repression and development in Xenopus tropicalis. We used transcription activator-like effector nuclease gene disruption technology to generate founder animals with mutations in the TRα gene and bred them to produce F1 offspring with a normal phenotype and a mutant phenotype, characterized by precocious hind limb development. Offspring with a normal phenotype had zero or one disrupted TRα alleles, and tadpoles with the mutant hind limb phenotype had two truncated TRα alleles with frame shift mutations between the two zinc fingers followed by 40-50 mutant amino acids and then an out-of-frame stop codon. We examined TH-response gene expression and early larval development with and without exogenous TH in F1 offspring. As hypothesized, mutant phenotype tadpoles had increased expression of TH-response genes in the absence of TH and impaired induction of these same genes after exogenous TH treatment, compared with normal phenotype animals. Also, mutant hind limb phenotype animals had reduced hind limb and gill responsivity to exogenous TH. Similar results in methimazole-treated tadpoles showed that increased TH-response gene expression and precocious development were not due to early production of TH. These results indicate that unliganded TRα delays developmental progression by repressing TH-response genes.
Figure 2. TRα TALEN founder developmental phenotypes. A, mCherry mRNA was coinjected with TRα TALEN mRNA into a single blastomere of two-cell stage embryos. Ventral view of the injected tadpole shows mCherry expression only on the left side of its body. B and C, Hind limb and skin phenotype are shown. B, Ventral view of the lower abdomen and proximal part of tail shows different developmental progression in the hind limbs between uninjected and injected sides. White arrows point to side views of the hind limb on each side to show the alteration of not just limb length but stage of limb development. C, Skin on the injected side of a NF stage 57 tadpole shows advanced iridophore development (gold skin color, black triangles). Thicker skin obscures the ultimobranchial body visible only on the uninjected side (white triangle). The dotted line delineates the uninjected and injected sides. D, Histogram of hind limb stage difference in TRα TALEN founders. When the stage of hind limb development on the uninjected side was NF stage 49, the difference in stage compared with the injected side was determined and plotted in the histogram. The hind limb on the injected side was always more advanced, typically by 1 to 1.5 stages (n = 42 injected animals from the same clutch). Tadpoles that lacked a stage difference were not quantified. HL, hind limb.
Figure 3. Hind limb phenotype in F1 offspring. Representative sibling offspring from a pair of TRα TALEN founders were imaged at feeding stage (upper panel). The developmental difference in hind limbs is shown in the lower panels. The hind limbs are bracketed. HL, hind limb.
Figure 4. Derepression of TH-response genes in hind limb phenotype tadpoles. Total RNA from whole bodies of F1 normal or hind limb (HL) phenotype tadpoles at NF stage 48â49 and NF stage 50â51, respectively, was isolated from four clutches [clutches 6, 7, 9, and 11 (Supplemental Table 1)] was isolated to analyze mRNA expression before TH circulation begins at NF stages 54â55. HL phenotype animals had higher expression levels of TRβ (A), ST3 (B), and KLF9 (C) mRNA than normal animals. D, The TALEN-induced mutation in TRα had no effect on its mRNA expression levels, which were not significantly different in normal and hind limb phenotype animals (n = 4â6); bars show expression levels relative to the housekeeping gene rpL8, and error bars represent SD. Significance levels for a one-way ANOVA were: *, P < .05; **, P < 0.01; ***, P < 0.001.
Figure 5. Impaired induction of TH-response genes in hind limb phenotype tadpoles. Total RNA from whole bodies of sibling F1 normal or hind limb (HL) phenotype tadpoles at NF stages 48â49 and NF stages 50â51, respectively, was isolated after treatment with 0, 2, or 10 nM T3 for 24 hours. The mRNA expression levels of TRβ (A), ST3 (B), and KLF9 (C) were significantly higher in normal tadpoles treated with 2 or 10 nM T3. Uninduced levels were increased for TRβ and KLF9 in HL phenotype tadpoles, as in Figure 4. n = 4â6, bars show expression levels relative to the housekeeping gene rpL8, and error bars represent standard deviation. One-way ANOVA showed significant differences for most comparisons, P < .05; *, < 0.01; **, < 0.001; ***.
Figure 6. Reduced hind limb responsivity to TH in hind limb phenotype tadpoles. Normal and hind limb (HL) mutant phenotype tadpoles from the same clutch were treated with 0 or 10 nM T3 for 7 days. Hind limbs in mutant phenotype tadpoles (middle row) were compared with normal tadpoles of the same age (top row, age matched) or at the same stage (bottom row, stage matched). In the absence of T3, hind limbs advanced up to two stages in 7 days in normal and mutant phenotype tadpoles, (compare day 0-T3 and day 7-T3). In the presence of T3 (compare day 0-T3 and day 7+T3), hind limbs in normal tadpoles advanced from NF stage 48 to NF stage 51 (top row) or from NF stage 53 to NF stage 57 (bottom row), whereas hind limbs of mutant phenotype tadpoles advanced from NF stages 53 to NF stage 55. Importantly, the effect of T3 in mutant hind limbs (no difference in stage but slight increase in hind limb length) was greatly reduced compared with the effect in normal tadpoles of the same stage (compare day 7-T3 and day 7+T3). Hind limb images were photographed with the same magnification. This experiment was repeated with a sample size of four to six with similar results.
Figure 7. Reduced gill responsivity to TH in hind limb phenotype tadpoles. Normal and hind limb (HL) mutant phenotype tadpoles were the same individuals as in Figure 6. Gills in mutant phenotype tadpoles (middle row) were compared with normal tadpoles of the same age (top row, age matched) or at the same stage (bottom row, stage matched). In the presence of T3, gill resorption in normal tadpoles (top and bottom rows) occurred to a greater extent than in mutant phenotype tadpoles (middle row) (the clear area seen in mutant phenotype tadpoles is larger than in normal tadpoles, near white brackets). Gill images were photographed with the same magnification. This experiment was repeated with a sample size of four to six with similar results.
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