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             metamorphosis

	Figure 2. TRα knockout reduces NCoR recruitment and increases target gene expression. (a) ChIP assay reveals reduced TR binding and corepressor recruitment at endogenous TREs in premetamorphic tadpoles. Seven-day-old tadpoles were genotyped, and two days after genotyping, at least five tadpoles of the same genotype were homogenized together for ChIP assays with antibodies against TR, corepressor NCoR, and ID14, an extracellular protein, as a nonspecific control. Note that TRα homozygous knockout dramatically reduced receptor binding or recruitment of the corepressor NCoR at the well-characterized TRE regions of TRβ and TH/bzip genes. Exon 5 of the TRβ gene was also analyzed as a control genomic region lacking TRE. Two asterisks (**) indicate a significant difference between homozygous knockout and the other groups (P < 0.01). (b) TRα knockout increases basal expression of T3 direct target genes in premetamorphic tadpoles. Total RNA from 7-day-old tadpoles was used for real-time PCR analysis of the expression of several well-known T3 direct target genes: TH/bzip (58), Klf9 (60), ST3 (61), and TRβ (57). The expression levels were normalized against that of EF1α. Two asterisks (**) indicate a significant difference between the homozygous knockout and other groups (P < 0.01).
	Figure 3. TRα-knockout tadpoles are resistant to T3-induced precocious metamorphosis. (a) TRα knockout reduces T3 induction of target gene expression in premetamorphic tadpoles. Six-day-old tadpoles were treated with 10 nM T3 for 18 hours. Both T3-treated and control tadpoles were euthanized at the same time. The tail tip was clipped for genotyping and the rest of each animal was homogenized for RNA extraction. At least three animals of the same genotype were mixed together for RNA isolation and subsequent expression analysis by qRT-PCR. The expression levels were normalized against that of EF1α. The fold of T3 induction was obtained by dividing the value for the T3-treated animal by that of the animal of the same genotype without T3 treatment. Two asterisks (**) indicate a significant difference between homozygous knockout and the other groups (P < 0.01). (b) TRα knockout reduced TR binding to TREs in target genes even after T3 treatment. Seven-day-old tadpoles were genotyped. Five tadpoles of each genotype were pooled together and treated with 10 nM T3 for 18 hours at 25°C two days after genotyping. ChIP assay was performed on the whole animals with anti-TR or ID14 antibodies, as described in the legend of Fig. 2. Note that homozygous TRα-knockout animals had dramatically reduced TR binding at well-known target genes. Two asterisks (**) indicate a significant difference between the homozygous knockout and other groups (P < 0.01).
	Figure 4. TRα knockout accelerates premetamorphic tadpole development. (a) TRα-knockout tadpoles are at more advanced stages of development compared with wild-type or heterozygous animals of the same age and similar size. Randomly selected tadpoles were reared separately after tail clipping for genotyping. For each experiment, animals of the same age and similar size were compared after genotyping. One representative animal from one experiment is shown for each genotype. Boxes with dashed borders in the left panel were enlarged and are shown in the right panel. Homozygous TRα-knockout tadpoles always had significantly larger and more advanced hind limb buds. This analysis was repeated seven times from three batches of tadpoles. Altogether, there were 48 homozygous, 118 heterozygous, and 53 wild-type tadpoles. (b) TRα-knockout tadpoles are at more advanced stages of development compared with their age-matched wild-type and heterozygous siblings. Tadpoles in Fig. 4 (a) were staged on the basis of their limb morphology (51), and the stage of the individual animals for each genotype was plotted with the median (darker solid line) for each genotype shown in the figure. Note that homozygous TRα-knockout tadpoles reached a median stage of 51, whereas the wild-type and heterozygous TRα-knockout tadpoles reached a mediate stage of 47.5. The two asterisks (**) indicate a significant difference between homozygous knockout and the other groups (P < 0.01). (c) TRα-knockout tadpoles reach the onset of metamorphosis, i.e., stage 54, at a younger age than do heterozygous and wild-type animals. Tadpoles were genotyped at 7 days of age, and tadpoles of different genotypes were reared under identical conditions. The age in days for each tadpole to reach stage 54 was plotted, with the median (darker solid line) for each genotype shown in the figure. Note that homozygous TRα-knockout tadpoles reached stage 54 significantly sooner than wild-type and heterozygous TRα-knockout tadpoles. There was no statistically significant difference between wild-type and heterozygous TRα-knockout tadpoles. Two asterisks (**) indicate a significant difference between homozygous knockout and the other groups (P < 0.01). (d) The expression of the hind limb–specific gene Tbx4 and forelimb-specific gene Tbx5 is increased in homozygous TRα-knockout tadpoles at 15 days of age. Animals at 7 or 15 days of age [days postfertilization (DPF)] were genotyped by tail clipping, and the rest of the animal for each genotype (three animals for each genotype) were homogenized together for gene expression analysis by qRT-PCR. Two asterisks (**) indicate a significant difference between the homozygous knockout and other groups (P < 0.01).
	Figure 5. TRα knockout increases the growth rate and accelerates development of premetamorphic tadpoles. (a and b) TRα-knockout tadpoles grow larger than their age-matched wild-type (WT) and heterozygous (Het) siblings. Fifty-five randomly selected 11-day-old tadpoles were classified into three categories (small, medium, and large) on the basis of body size. The tadpoles were then photographed and genotyped. (a) One representative photo for each category from each genotype is shown with the number of animals in that category. (b) Bar graph showing the percentage of each category in the three genotypes. Note that most of the homozygous (Hom) knockout animals were in the large category, whereas the Het and WT animals were in the medium or small category. To group the animals, each tadpole was photographed under the same magnification, and the length of the ventral side of the body (minus tail) was measured with ImageJ software (https://imagej.net/). “Large” referred to length >4.7 mm, “small” referred to length <3.2 mm, and the remaining animals, measuring 3.2 mm to 4.7 mm, were categorized as “medium.” (c) The expression of the two GH genes is elevated in the knockout animals. Total RNA was extracted from 11-day-old tadpole samples. Gene expression was analyzed by qRT-PCR. Two asterisks (**) indicate a significant difference between Hom knockout and WT animals (P < 0.01).
	Figure 6. The homozygous TRα-knockout tadpole (Hom) has a smaller body size at the onset of metamorphosis despite a faster growth rate during premetamorphosis. (a) A representative tadpole of each genotype at stage 54, the onset of metamorphosis. Arrows point to the hind limbs. Note that the homozygous TRα-knockout tadpole has a significantly smaller body size but similarly sized hind limbs compared with the heterozygous TRα-knockout (Het) and wild-type (WT) tadpoles at stage 54. (b) TRα-knockout tadpoles have a shorter body length. Animals were allowed to develop to stage 54 regardless of age. The tadpole body length was measured before tail clipping for genotyping. The sample included 15 wild-type, 23 heterozygous TRα-knockout, and 29 homozygous TRα-knockout tadpoles. The body length of individual animals for each genotype was plotted, with the median (darker solid line) for each genotype shown in the figure. Note the median length of the homozygous TRα-knockout tadpoles was about half of that for the heterozygous TRα-knockout and wild-type tadpoles. There was no statistically significant difference between heterozygous TRα-knockout and wild-type tadpoles. Two asterisks (**) indicate a significant difference between the homozygous knockout and other groups (P < 0.01).
	Figure 7. Homozygous TRα-knockout tadpoles (Hom) have a slower rate of metamorphic progression. (a) Homozygous TRα-knockout tadpoles take longer to develop from stage 54 to 58 (prometamorphosis). Stage 54 tadpoles were selected at the same time and reared together. Individual tadpoles were genotyped when they reached stage 58, the beginning of the metamorphic climax. The time required for each animal to reach stage 58 was plotted, with the median (darker solid line) for each genotype shown in the figure. The groups included four wild-type (WT), 10 heterozygous TRα-knockout (Het), and four homozygous TRα-knockout tadpoles. Note that homozygous TRα-knockout tadpoles required over twice as long to reach stage 58 compared with wild-type and heterozygous TRα-knockout tadpoles. Two asterisks (**) indicate a significant difference between the homozygous knockout and other groups (P < 0.05). (b) TRα knockout has no effect at the climax of metamorphosis. The time for each animal to develop from stage 58 to the end of metamorphosis (stage 66) was plotted, with the median (darker solid line) for each genotype shown in the figure. Note that no significant difference was observed among the three genotypes. (c) TRα knockout does not affect the overall development time from fertilization (stage 1) to the end of metamorphosis (stage 66). The time for each animal to proceed from stage 1 to the end of metamorphosis (stage 66) was plotted with the median (darker solid line) for each genotype shown in the figure. Note that no significant difference was observed among the three genotypes.
	Figure 8. Animals lacking TRα complete metamorphosis with lower body weight but longer intestine. (a) The homozygous TRα-knockout animal (Hom) has a lower body weight at stage 66. Tadpoles were euthanized when they reached stage 66, which is considered to be the end of metamorphosis, when the tail is completely resorbed. Intestine was isolated from each animal, and both intestine and animal body were fixed in a 4% solution of MgSO4, ethylene glycol tetraacetic acid, 3-(N-morpholino)propanesulfonic acid, and formaldehyde overnight at 4°C. After washing in 70% ethanol twice, both body weight and intestinal length were measured. The body weight of each animal was plotted, with the median (darker solid line) for each genotype shown in the figure. Note that the homozygous TRα-knockout animal had significantly lower weight. The group included six wild-type (WT), 11 heterozygous TRα-knockout (Het), and 15 homozygous TRα-knockout animals. The asterisk (*) indicates a significant difference between the homozygous knockout and wild-type groups (P < 0.05). (b) homozygous TRα-knockout animals had a longer intestine at stage 66. The length of the intestine of animals described in (a) was measured from the bile duct to the large intestine. After normalization against the body weight for each animal, the relative intestine length was plotted with the median (darker solid line) for each genotype shown in the figure. Given that the length reduction is a hallmark of intestinal metamorphosis (62), the longer intestine length of the homozygous knockout animals suggests incomplete intestinal remodeling. The asterisk (*) indicates a significant difference between the homozygous knockout and wild-type groups (P < 0.05).
	Figure 9. Organ-dependent expression of TRα and TRβ genes during metamorphosis. The expression of TRα and TRβ at premetamorphosis (stage 54) and climax (stage 62) was determined by real-time PCR and is shown relative to the level in the intestine at stage 62 for (a) TRα and (b) TRβ, or (c) as a ratio of the expression level of TRα to that of TRβ, with the ratio in the intestine at stage 62 set to 1. The region in the box with dashed borders in the upper panel (c) is enlarged and shown in the lower panel. Note that the limb has a much higher level of expression of TRα at stage 54 and higher levels of relative TRα/TRβ expression than the intestine, with the tail having the lowest.

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