Thyroid hormone (T3) mediates concentration-dependent downregulation of lws2 and upregulation of trβ2 in both wildtype and trβ2 gain-of-function transgenics. (A) Confocal images of whole-mount retinas from 4dpf wildtype and trβ2 GOF transgenic larvae treated for 48 h with increasing concentrations of T3 or DMSO (control), followed by HCR in situ hybridization. lws1 (green) and lws2 (magenta) mRNA expressions are shown. N = 6 larvae. In wildtype retinas (top row), lws2 mRNA levels progressively decrease with increasing T3 concentration. In the transgenic line (bottom row), this reduction is more subtle. Insets display lws2 expression only. (B) RT-qPCR analysis of wildtype and transgenic larvae showing differential regulation of lws1 and lws2 following 48 h of T3 treatment. (C) Whole-mount retinas from wildtype and transgenic larvae showing the pattern of trβ2 mRNA expression relative to lws + cones under DMSO and T3 treatment. Insets provide higher magnification of lws and trβ2 co-expression. T3 upregulates trβ2 expression in a concentration-dependent manner, while lws2 downregulation is less pronounced in transgenic larvae compared to wildtype. Scatter and box-and-whisker plots represent fold change in transcript expression (2-ddCT). Each point represents a biological replicate (n ≥ 6). P-values were calculated using Kruskal-Wallis one-way ANOVA with post hoc testing adjusted by Dunn’s correction. Statistical significance is denoted as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. hpt: hours post treatment. D = Dorsal N = Nasal. Scale bars in A and B = 50 μm.
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