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???displayArticle.abstract??? Thyroid hormone (T3) receptors (TRs) mediate T3 effects on vertebrate development. We have studied Xenopus tropicalis metamorphosis as a model for postembryonic human development and demonstrated that TRα knockout induces precocious hind limb development. To reveal the molecular pathways regulated by TRα during limb development, we performed chromatin immunoprecipitation- and RNA-sequencing on the hind limb of premetamorphic wild type and TRα knockout tadpoles, and identified over 700 TR-bound genes upregulated by T3 treatment in wild type but not TRα knockout tadpoles. Interestingly, most of these genes were expressed at higher levels in the hind limb of premetamorphic TRα knockout tadpoles than stage-matched wild-type tadpoles, suggesting their derepression upon TRα knockout. Bioinformatic analyses revealed that these genes were highly enriched with cell cycle and Wingless/Integrated (Wnt) signaling-related genes. Furthermore, cell cycle and Wnt signaling pathways were also highly enriched among genes bound by TR in wild type but not TRα knockout hind limb. These findings suggest that direct binding of TRα to target genes related to cell cycle and Wnt pathways is important for limb development: first preventing precocious hind limb formation by repressing these pathways as unliganded TR before metamorphosis and later promoting hind limb development during metamorphosis by mediating T3 activation of these pathways.
Figure S1. Validation of the ChIP samples by analyzing TR binding to TRβ TRE in the hindlimb of wild type (WT) and TRα (-/-) tadpoles without (Control) or with T3 (T3) treatment. The ChIP samples obtained with anti-TR or ID14 antibody was analyzed by qPCR for the presence of the region containing the TRβ TRE (TRE) or TRβ exon 5 (Exon 5), the negative control region lacking a TRE. Note that as expected, only the TRβ TRE region had positive ChIP signals with anti-TR antibody regardless of T3 treatment, while all the other samples had only background ChIP signals. TRα knockout reduced TR binding to the TRβ TRE region.
Figure S2. Independent confirmation of RNA-Seq data by qRT-PCR for selected genes in wild type (WT) and TRα (-/-) hindlimb.
(A) Three known T3 responsible genes (thrb, thibz and klf9) are upregulated by T3 in WT hindlimb, and this upregulation is reduced in TRα (-/-) hindlimb. Hindlimbs were harvested from stage-matched X. tropicalis tadpoles at stage 54 treated with (T3+) or without (T3-) 10 nM T3 for 18 hours and used for RNA-Seq analyses. The expression was normalized to Reads Per Kilobase of exon model per Million mapped reads (RPKM). Each bar represents the mean plus S.D. and the asterisk (*) indicates a significant difference between T3- and T3+ samples, or WT and TRα (-/-) samples (P<0.05).
(B) Relative gene expression of thrb, thibz and klf9 analyzed by qRT-PCR confirms the regulation as found by RNA-Seq analysis. Independent tadpole treatment and RNA isolation were performed as in (A), and qRT- PCR was performed to determine the expression of the genes. Each bar represents the mean plus S.E. and the asterisk (*) indicates a significant difference between the T3- and T3+ samples, or WT and TRα (-/-) samples (P<0.05).
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