PMC

T₃ treatment fails to induce dendritic differentiation in early RORa-deficient staggerer PCs in organotypic cultures. (A-D) PCs in organotypic cultures from wild-type (A,C) or RORα-deficient Rora^sg/sg (B,D) P0 mice were revealed by CaBP immunolabeling after 7 DIV without T₃ (A,B) or with 30 nM T₃ (C,D). PCs from wild-type mice responded to T₃ treatment: without T₃, PCs were mostly in stage II (PCs with regressive-atrophic dendrites all around the soma) whereas they were in stages II, III (empty arrows) and IV (empty arrowheads) after 30 nM T₃ treatment (C). PCs from Rora^sg/sg mice were unresponsive to T3 treatment since they remain in stage I (fusiform PCs with long processes of bipolar shape) in the absence (B) or in the presence (D) of 30 nM T₃ treatment. Note the bipolar or fusiform shape of the PCs with long processes but no dendritic arborization in (B,D). Scale bar = 20 μm.

T₃ treatment upregulates Rora promoter activity. HepG2 cells cultured in 12-well plates were co-transfected with 500 ng/well of the pTRα vector, allowing expression of the TR, and 500 ng/well of the p(-487)Rora-Luc reporter vector, which allows expression of the luciferase gene under the control of the human genomic sequences between nucleotides -487 and -45 from the Rora1 translation initiation site, or with 500 ng/well of the promoter-less pGL3-Luc vector. HepG2 cells co-transfected with 500 ng/well of the pTRα vector and of the pDR4-TK-Luc reporter vector were used as a control of the transcriptional effect of T₃ on a TH response element. The luciferase activity of p(-487)Rora-Luc and pDR4-Luc in the absence or the presence of T₃ (30 nM) is expressed relative to that of the promoter-less pGL3-Luc vector (*P < 0.05). Error bars indicate mean ± standard deviation.

Dose-dependent effect of T₃ on PC dendritic differentiation in organotypic cultures. (A-H). Organotypic cultures of P0 cerebella were kept 7 DIV in the absence of T₃ (A,B), or in the presence of 3 nM T₃ (C,D), 30 nM T₃ (E,F) or 100 nM T₃ (G,H). (A,C,E,G) PCs were revealed by CaBP immunolabeling. (B,D,F,H) Quantitative distribution of PCs between stages I to IV. Fusiform PCs with a bipolar shape are defined as stage I (arrow in (A,C)), PCs with regressive atrophic dendrites all around the soma are defined as stage II (white arrowhead in (A,C)), PCs with one or more perisomatic protrusions are defined as stage III (empty arrow in (G)) and PCs with an identified dendritic tree are classified as stage IV (empty arrowhead in (E)). Scale bar = 20 μm. Error bars indicate mean ± standard deviation.

T₃ promotes the early dendritic differentiation of PCs and leads to increased mRNA levels of Rora1 and Rora4 at P0. (A) Quantitative distribution of PCs between stages I and IV. Cultures of P0 cerebella were kept 3 DIV in the absence or the presence of 30 nM T₃. PCs are classified following the same criteria as in Figure 1. (B) P0 organotypic cultures were cultured without T₃ (white bars) or with 30 nM T₃ (black bars). Levels of mRNA were determined by real time RT-PCR and standardized to 18 s rRNA after 0 h, 6 h or 24 h of T₃ treatment. The data are given relative to the mRNA level in untreated slices at the initial time of the culture (0 h). They were obtained from three independent cerebellar slice extracts (**P < 0.005; *** P < 0.0005). Error bars indicate mean ± standard deviation.

T₃ treatment increases the amount of RORα protein and RNA in organotypic cultures. P0 cerebellar slices kept for 7 DIV were cultured in the absence or the presence of 30 nM T₃. (A) Immunoblot analysis and quantification of RORα levels in extracts of untreated or T₃-treated cerebellar slices (*P < 0.05). (B) Left panel: fluorescence density of RORα immunolabeling was measured within each PC nucleus with MetaMorph software. Average values from multiple cells ± SEM are shown (*P < 0.05). Right panel: organotypic cultures after 10 DIV without T₃ (upper row) or with T₃ (30 nM) treatment (lower row). RORα-expressing cells were revealed by RORα immunolabeling (blue), PCs were revealed by CaBP immunolabeling (red) and both PCs and interneurons were revealed by parvalbumin immunolabeling (green). Note the presence of RORα-expressing interneurons (arrow) in the T₃ only treatment. (C) P0 organotypic cultures were cultured without T₃ (white bars) or with 30 nM T₃ (black bars) for 7 days. Levels of mRNA were determined by real time RT-PCR and standardized to 18 s rRNA The data are given relative to the mRNA level in untreated slices at the initial time of the culture (0 DIV). They were obtained from three independent cerebellar slices extracts (*P < 0.05; **P < 0.005). Error bars in (C) indicate mean ± standard deviation.