Fig. 2.

Basal expression of TRα and known T3 target genes in premetamorphic tadpoles is increased in TRβ knockout intestine, accompanied by reduced TR binding to the endogenous TRE region. aTRβ knockout increased TRα and T3 target gene expression in premetamorphic tadpoles. Total RNA of three different tissues, intestine, tail and hindlimb, of stage 54 tadpoles of the three genotypes, was used for qRT-PCR analysis of the expression of TRα and four well-known T3 direct target genes: TRβ, klf9, mmp11 and TH/bzip. The expression levels were normalized against that of rpl8. Asterisks denote statistically significant differences (P < 0.05). Note that in the knockout tadpoles, TRβ mRNA, which had an out-of-frame mutation, was expressed at a higher level, likely due to partial de-repression caused by the lack of TRβ protein expression, just like the other target genes.b ChIP assay reveals reduced TR binding at the TRE region. The intestine sample obtained from at least five tadpoles of both genotypes, wild type and TRβ knockout, at stage 54 and were homogenized together for ChIP assay with antibodies against TR and ID14 as a negative control. The presence of the TRE region or the exon 5, as a negative control, of TRβ gene was determined by PCR. Asterisks (**) indicates statistically significant differences (P < 0.01)