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	Fig. 1. Most TR-bound genes found by ChIP-Seq are bound by TR constitutively in the wild-type intestine and many are upregulated by T3.a Venn diagram of the TR target genes identified by ChIP-Seq in wild-type intestine with or without T3 treatment. The 3308 and 4319 TR-bound genes were identified from the 11,469 and 17,930 ChIP-seq peaks present in all three replicates of the −T3 and +T3 samples, respectively, based on the presence of TR-binding peak(s) in the coding region (including the introns) and/or 5 kb upstream or downstream of the cording region. Those peaks that could not be assigned to any known genes are indicated as “NA or Not Annotated” in Supplementary Data 1. Note that most genes were bound by TR constitutively. b Venn diagram showing overlap between the TR-bound genes identified by ChIP-Seq and T3-upregulated genes in the intestine based on previous RNA-seq study except using Xenbase for gene annotation and a cutoff of 1.5 fold regulation (cut-off threshold, FDR < 0.05)19. Of the 4605 TR-bound genes identified by ChIP-Seq, 19% were shown as the T3-upregulated genes. Conversely, 36% of the T3-upreguated genes were found to be TR-bound. c Venn diagram showing overlap between the TR-bound genes identified by ChIP-Seq and T3-downregulated genes in the intestine based on previous RNA-seq study except using Xenbase for gene annotation and a cutoff of 1.5 fold regulation (cut-off threshold, FDR < 0.05)19. Of the 4605 TR-bound genes identified by ChIP-Seq, 9% were shown as the T3-downregulated genes. Conversely, 26% of the T3-downreguated genes were found to be TR-bound.
	Fig. 2. GO terms and biological pathways related to cell cycle or hedgehog signaling that are enriched among TR-bound genes in intestine.a Top 10 enriched GO terms related to cell cycle. The GO terms that were enriched among genes bound by TR in the wild-type intestine were ranked based on FDR as shown in Supplementary Data 5. The most significant GO terms related to cell cycle were plotted here (see Supplementary Data 18 for raw data). b Top 10 enriched pathways related to cell cycle or hedgehog signaling. The biological pathways that were enriched among genes bound by TR in the wild-type intestine were ranked based on FDR as shown in Supplementary Data 6. The most significant pathways related to cell cycle or hedgehog signaling were plotted here (see Supplementary Data 19 for raw data).
	Fig. 3. TR-binding and regulation of genes in the hedgehog signaling pathway in intestine based on ChIP-Seq and RNA-Seq data.The hedgehog signaling pathway was visualized with regard to genes regulated by T3 based on RNA-Seq and/or bound by TR based on ChIP-Seq. The arrows show functional interaction: green for activation; red for inhibition. Letters in red indicate upregulated genes in wild-type intestine after T3 treatment. Letters in black indicate genes with no change in wild-type intestine after T3 treatment. Red boxes indicate ChIP-Seq detected genes both with and without T3 treatment, and purple boxes indicate genes identified by ChIP-Seq only in wild type with T3 treatment. Note that many genes including Sonic hedgehog (Hh) were upregulated by T3 and also bound by TR. No downregulated genes were found by RNA-Seq in this pathway.
	Fig. 4. Most of TR-bound genes are common between wild-type and TRα−/− intestine with or without T3 treatment.a Venn diagram of the TR target genes identified by ChIP-Seq in TRα−/− intestine with or without T3 treatment. Note that like in the wild-type intestine (Fig. 1a), most genes were bound by TR constitutively. b Venn diagram showing overlap between the TR-bound genes identified by ChIP-Seq in TRα−/− intestine and T3-upregulated genes in the intestine based on previous RNA-seq study19. Of the 3327 TR target genes identified by ChIP-Seq, 7% were shown as the T3-upregulated genes in TRα−/− intestine. Conversely, 27% of the T3-upreguated genes in TRα−/− intestine were found to be TR-bound. Both ratios were lower than those for the wild-type animals (Fig. 1b). c Venn diagram of comparison of genes detected by ChIP-Seq between wild-type (WT) and TRα−/− intestine. Of the 4605 TR target genes in wild-type intestine, nearly 70% or 3129 genes were bound by TR in TRα−/− intestine, presumably by TRβ, indicating that most of TR target genes in the intestine are commonly recognized by both TRα and TRβ. d Venn diagram comparison was made between 896 genes upregulated by T3 among all TR-bound detected in the wild-type intestine (Fig. 1b) and 1476 genes bound by TR only in the wild-type intestine (c). Of the 896 T3-upregulated genes that are bound by TR in wild-type intestine, 32% genes were genes bound by TR only in wild-type intestine, similar to the percentage of the genes bound by TR only in the wild-type intestine within all genes bound by TR in the wild-type intestine (1476 out of 4605). Similarly, of the genes bound by TR only in the wild-type intestine, 19% were upregulated by T3, just like the fraction of all TR-bound genes upregulated by T3 (Fig. 1b). e Venn diagram comparison was made between 896 genes upregulated by T3 among all TR-bound detected in the wild-type intestine (Fig. 1b) and 3129 genes bound by TR in both wild-type and TRα−/− intestine (c). Of the 896 T3-upregulated genes that are bound by TR in wild-type intestine, 68% genes were genes bound by TR in both wild-type and TRα−/− intestine, similar to the percentage of the genes bound by TR in both wild-type and TRα−/− intestine within all genes bound by TR in the wild-type intestine (3129 out of 4605). Similarly, of the genes bound by TR in both wild-type and TRα−/− intestine, 19% were upregulated by T3, again just like the fraction of all TR-bound genes upregulated by T3 (Fig. 1b). The results in D/E suggest that the genes bound by TR only in the wild-type intestine or both wild-type and TRα−/− intestine respond to T3 similarly in the wild-type animals.
	Fig. 5. T3 regulation profile of ChIP-Seq detected genes.Heatmap showing the fold change of gene expression in wild-type or TRα−/− intestine after 18 h T3 treatment as measured by RNA-Seq19, for TR-bound genes detected by ChIP-Seq in only the wild-type intestine (left), both wild-type and TRα−/− intestine (middle) or only TRα−/− intestine (right). Note that higher fractions of the TR-bound genes were upregulated (red) or downregulated (blue) by T3 were present in the wild-type-specific or common TR-bound genes than TRα−/−-specific TR-bound genes and that TRα knockout reduced T3-regulation, i.e., leading to lighter red or blue, for individual genes, suggesting that TRα−/− is important for gene regulation during the treatment. Note that the blank region between the red and blue areas are genes whose expression has no or little change after T3 treatment of wild-type or TRα knockout animals. The color range shows fold changes in log2 scale.
	Fig. 6. TRα-dependent enrichment of cell cycle pathways among genes bound by TR in the wild-type intestine.a Top 10 cell cycle pathways differentially enriched among genes bound by TR in wild-type intestine. The pathways that were enriched among genes bound by TR in the wild-type intestine (Supplementary Data 6) were subtracted by the pathways that were enriched among genes bound by TR in TRα−/− intestine (Supplementary Data 12) to obtain the pathways only enriched in the wild-type intestine (Supplementary Data 14). The most significant 10 cell cycle-related pathways were plotted here (see Supplementary Data 20 for raw data). b TR-binding and regulation of genes in the pathway for cell cycle based on ChIP-Seq and RNA-Seq data. The pathway for cell cycle was visualized with regard to genes regulated by T3 based on RNA-Seq and/or bound by TR based on ChIP-Seq. The arrows show functional interaction: green for activation. Letters in red and blue indicate upregulated and downregulated genes, respectively, in wild-type intestine after T3 treatment. Letters in black indicate genes with no change in wild-type intestine after T3 treatment. Red boxes indicate ChIP-Seq detected genes both with and without T3 treatment, and purple boxes indicate genes identified by ChIP-Seq only in wild type with T3 treatment. Note that Cyclin A and CDK1 were among the genes directly bound by TR and upregulated by T3.
	Fig. 7. Inhibiting CDK1 signaling blocks intestinal remodeling during T3-induced metamorphosis.a Experimental design for treatment with CDK1 and Foxo1 inhibitors. Wild-type tadpoles at stage 54 were treated with 2 μg/ml of CDK1 inhibitor JNJ-7706621 or 1 μg/ml of phosphorylation inhibitor of Foxo1 as1842856 for 2 days in the presence and absence of T3. Most of tadpoles treated with the inhibitors in the presence of T3 died at 3 days. Thus, intestinal remodeling was analyzed after 2 days of T3 treatment. b JNJ-7706621 and as1842856 inhibits T3-induced shortening of the intestine. Tadpoles at stage 54 were treated with or without 10 nM T3 for 2 days in the presence or absence of indicated inhibitor. Intestinal length was measured from bile duct junction to colon and normalized against body length. Each group included more than 4 tadpoles. Each bar represents the mean plus S.E. The asterisk (*) indicates a significant difference between the T3 treated tadpoles and control animals (P < 0.05) (see Supplementary Data 21 for raw data). c T3-induced intestinal morphological changes (epithelial folding) and apoptosis in the intestinal epithelium were inhibited by JNJ-7706621 and as1842856. Tadpoles were treated as above. Cross-sections of the intestine were stained with MGPY, which stains DNA blue and RNA red (top), and apoptotic cells were detected by TUNEL staining (bottom). TUNEL positive cells were stained green and nuclei were stained by Hoechst 33342 (blue). Bar indicates 20 μm (see Supplementary Data 21 for raw data). d Quantification of the apoptotic cells in c. TUNEL positive epithelium region was measured by using ImageJ software and normalized against the total epithelium region as determined by DAPI staining (see Supplementary Data 22 for raw data).

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