Fig. 4.

Thyroid hormone regulates DISP3 expression. A, Analysis of DISP3 expression by immunohistochemistry in the retina of 16-d-old chicken embryos treated at d 14 either with PBS (as a control) or an initial dose of thyroid hormone (T₃, 3 μg) for 24 h before a second dose was administered. Sections were also stained with the control antibody RA4 to monitor the presence of retinal ganglion and bipolar cell layers at this developmental stage. Arrows mark the retinal ganglion cell layer. Scale bar, 100 μm. GCL, Ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium. B, Quantification of DISP3 immunostaining shown in Fig. 4A. Intensity of staining was expressed as relative OD. The intensity of background staining was measured at the outer plexiform layer where no specific DISP3 staining was observed. The results shown are from three experimental animals. Triplicate spots from three parallel sections in each cell layer were measured and presented as the mean ± sd. Statistical analysis of the data was carried out using the ANOVA method (F = 27,2; P < 0.01). C, RT-PCR analysis of Disp3 expression in various cell lines. RGC5, Rat retinal ganglion cell line; Y79, human retinoblastoma cell line; HW3.5, mouse hippocampal progenitor cell line; C17.2, mouse cerebral neuronal cell line; TM4, mouse testis cell line. For all cell lines analyzed, 31 cycles of PCR were performed except for the TM4 cell line where 33 cycles were required. Gapdh was used as a control. D, Regulation of Disp3 mRNA by T₃ in TM4 and Y79 cells. Cells were grown in standard medium (−) or medium containing the thyroid hormone receptor antagonist KB044146 (Inh) or thyroid hormone (T₃) at the final concentration of 5 × 10⁻⁷ or 10⁻⁷ m, respectively. The qPCR results shown are from three representative experiments assayed in triplicate and are presented as the mean ± sd.