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. 2020 Feb 14;30(2):300–313. doi: 10.1089/thy.2019.0366

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Copyright 2020, Mary Ann Liebert, Inc., publishers

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FIG. 6. — TR is required for larval tissue resorption. (A) Representative photos of the heads of TRα ^(+/+)TRβ ^(+/+) and TRα ^(−/−)TRβ ^(−/−) tadpoles at stage 61, the climax of metamorphosis. The developmental stage was determined based on the length of the NO and BO with NO = BO at stage 61 (note that the boundaries of NO and BO could be easily determined based on morphological differences by microscopy). (a) TRα ^(+/+)TRβ ^(+/+) and (b) TRα ^(−/−)TRβ ^(−/−⁾. The black line in the top panel spans the width from the outer edge of the gills, and the white line spans the width from the outer edge of the eyes. The yellow box encloses the head region. Higher magnification of areas boxed with white lines in (a, b) is shown in (a′, b′). An area of the dorsal skin used for gene expression studies in D is shown in the dashed box. Scale bars: 5 mm. (B) TRDKO inhibits gill repression at the metamorphic climax. The ratio of the width at the gill position [black line in the top panel of (A)] to the width at the eye position [white line in the top panel of (A)] was calculated as a measure of the relative gill size. Data are expressed as mean plus SE (TRα ^(+/+)TRβ ^(+/+) [n = 9] and TRα ^(−/−)TRβ ^(−/−) [n = 6]). The asterisk (*) indicates a significant difference when compared with that for TRα ^(+/+)TRβ ^(+/+) (p < 0.01). (C) TRDKO inhibits the reduction in head size associated with jaw remodeling and gill resorption. The head area, enclosed by yellow box in the top panel of (A), was measured by the ImageJ software and is presented as mean plus SE (TRα ^(+/+)TRβ ^(+/+) [n = 9] and TRα ^(−/−)TRβ ^(−/−) [n = 6]). The asterisk (*) indicates a significant difference when compared with that for TRα ^(+/+)TRβ ^(+/+) (p < 0.01). (D) TRDKO blocks the downregulation of larval keratin genes but allows the expression of adult keratin genes at the metamorphic climax. The dorsal skin, as shown in the area enclosed by dashed boxes in A, of the WT (TRα ^(+/+)TRβ ^(+/+)) at stages 54, 61, and 66, and TRDKO (TRα ^(−/−)TRβ ^(−/−⁾) tadpoles stages 54 and 61 were dissected for total RNA extraction and qRT-PCR analysis of the expression of a larval skin marker, larval keratin (xlk), and two adult skin markers, adult keratins b and c (xak-b and xak-c). The qRT-PCR for all genes and on all samples was performed simultaneously. The expression levels were normalized against that of rpl8. The groups included 5, 3, 3 WT (TRα ^(+/+)TRβ ^(+/+)) tadpoles at stage 54, 61, and 66; and 5 TRβ homozygous (TRα ^(−/−)TRβ ^(−/−)) tadpoles each at stages 54 and 61, respectively. Note that, as expected, the larval keratin gene was repressed by stage 61, while adult keratin genes were upregulated by stage 61 in WT animals. Interestingly, in the double knockout animals at stage 61, no repression of the larval keratin gene was observed, although the adult keratin genes were activated as in the WT animals. Different lowercase letters denote statistically significant differences (p < 0.05).