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. 2021 Jan 12;31(1):128–142. doi: 10.1089/thy.2020.0022

FIG. 3.

FIG. 3.

T3-induced larval epithelial cell death is reduced in TRα−/− tadpoles. (A) Apoptotic cells in the TRα−/− intestine are reduced during T3-induced metamorphosis. The cross sections of the intestine from stage 54 tadpoles treated with 10 nM T3 for 0–3 days were double-stained with TUNEL for apoptotic cells and DAPI for DNA. The dotted white lines depict the epithelium–mesenchyme boundary. Scale bar indicates 20 μm. (B) The boxed regions in (A) are shown at a higher magnification. White arrowheads denote TUNEL-positive cells. Scale bar indicates 10 μm. (C) Quantification of apoptosis as detected in (A, B). The TUNEL-positive cells (green) in the epithelium were counted with the ImageJ software and normalized against the total cells in the epithelium as determined by DAPI staining. Note that apoptotic cell number reached the peak after two days of T3 treatment in wild-type tadpoles, while it was still low in the TRα−/− intestine even after T3 treatment for three days. The asterisk (*) indicates a significant difference between the TRα−/− tadpoles and wild-type tadpoles (p < 0.05). Experiment was repeated three times, and each bar represents the mean + S.E. (D) The mRNA levels of apoptosis-regulatory genes in the intestine are significantly induced by T3 treatment of wild-type but not TRα−/− premetamorphic tadpoles. Total intestinal RNA was isolated from wild-type and knockout stage 54 tadpoles treated with 10 nM T3 for up to 0–3 days. The mRNA levels of caspase3 (casp3), caspase9 (casp9), and Bcl-2-associated X protein (bax) were determined by real-time PCR and shown as relative levels to those in the intestine of wild-type animals at stage 54. Note that the wild-type intestine had much higher levels of casp3, casp9, and bax after one and two days of T3 treatment compared with the knockout one. The asterisk (*) indicates a significant difference between TRα−/− and wild-type tadpoles (p < 0.05). Experiment was repeated twice, and each bar represents the mean + S.E. TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.