Figure 2.

T3 indirectly induces the mRNA expression of Foxl1 homeologs through Shh signaling. (a) Total RNA was isolated from the intestine of premetamorphic stage 54-tadpoles treated with DMSO (Control, white bars), 50 nM T3+DMSO (T3, gray bars), cycloheximide and anisomycin (Chx, shaded bars) and 50 nM T3+Chx (T3+Chx, black bars) for 6 h following the pretreatment with DMSO or Chx for 1 h. mRNA levels of indicated genes were examined by qRT-PCR and shown relative to those of rpL8.S mRNA, with the value of Control set to 1. Error bars represent the SEM (n = 4). The values were analyzed by ANOVA followed by Scheffe’s post hoc test. Different letters indicate significant differences among the indicated treatment (P < 0.05). When both Control vs T3 and Chx vs T3+Chx were statistically significant, the gene was determined as the direct TH response gene. (b–e) Premetamorphic tadpoles at stage 54 were pretreated with DMSO for 4 days. Then, total RNA was isolated from the intestine at 0 day or 1 day after treatment with 0, 150 or 600 nM SAG. mRNA levels of indicated genes were examined by qRT-PCR and shown relative to those of rpL8.S mRNA, with the value at day 0 set to 1. Error bars represent the SEM (n = 6 for Foxl1.L, n = 5 for Foxl1.S, n = 3 for Gli1.S and IFABP.L). The values were analyzed by ANOVA followed by Scheffe’s post hoc test. Different letters indicate significant differences among the indicated treatment (P < 0.05). (f,g) Premetamorphic tadpoles at stage 54 were treated with 10 nM T3 in the presence of 2.5 mM cyclopamine (CP) or ethanol vehicle for 3 days. Then, total RNA was isolated from the intestine. mRNA levels of indicated genes were examined by qRT-PCR and shown relative to those of rpL8.S mRNA, with the value of T3 plus ethanol vehicle for 3 days set to 1. Error bars represent the SEM (n = 3). The values were analyzed by Student’s t-test. Asterisks indicate that the mRNA levels are significantly different. **P < 0.01.