A bimodal influence of thyroid hormone on cerebellum oligodendrocyte differentiation

Abstract

Thyroid hormone (T(3)) can trigger a massive differentiation of cultured oligodendrocytes precursor cells (OPC) by binding the nuclear T(3) receptor α1 (TRα1). Whether this reflects a physiological function of TRα1 remains uncertain. Using a recently generated mouse model, in which CRE/loxP recombination is used to block its function, we show that TRα1 acts at two levels for the in vivo differentiation of OPC in mouse cerebellum. At the early postnatal stage, it promotes the secretion of several neurotrophic factors by acting in Purkinje neurons and astrocytes, defining an environment suitable for OPC differentiation. At later stages, TRα1 acts in a cell-autonomous manner to ensure the complete arrest of OPC proliferation. These data explain contradictory observations made on various models and outline the importance of T(3) signaling both for synchronizing postnatal neurodevelopment and restraining OPC proliferation in adult brain.

Fig. 1.

Expression pattern of different Cre-mice in the cerebellum, using ROSA26-lox-STOP-lox-EYFP (R26YFP) as reporter. A–F, Olig2 immunostaining in red; YFP immunostaining in green,. Colocalization between YFP and Olig2 (yellow) indicates Cre-mediated recombination in OPC. White lines delineate the Purkinje cell layer. A, PDGFRα-Cre ER^T2 (tamoxifen injection at P1. Immunostaining at P15) indicates cre-mediated recombination in all cerebellar cell types including oligodendrocyte lineage. B, L7-Cre (P15). Recombination is restricted to Purkinje cells. C, Glast-Cre ER^T2 (tamoxifen injection at P1. Immunostaining at P15). Recombination is restricted to the astrocytes and not found in Olig2+ OPC. D, Ptfa1-Cre (P15): Recombination in GABAergic neurons, but not in oligodendrocytes lineage at P15. E and F, Cnp-Cre (P15). Recombination limited to the oligodendrocytes lineage and Purkinje cells. Stars indicate YFP+Olig2+ OPC. G, Cnp-Cre (P8, green; YFP, red; parvalbumin, blue: DAPI) indicates an expression of Cre recombinase from in Purkinje cells at this stage. H, Cnp-Cre (P3 green: YFP, red: parvalbumin). Absence of recombination at P3 in Purkinje cells at this stage. Scale bar, 100 μm. I, Cell lineages in the cerebellum and recombination patterns observed in various Cre and Cre-ER^T2 transgenic mice. X indicates Cre expression. Counting and nomenclature are summarized in Table 1. GFP, Green fluorescent protein.

Fig. 2.

Delayed postnatal differentiation of OPC after ubiquitous TRα1^L400R expression. A, Quantification of mRNA relative abundance in whole cerebellum at different ages (P8, P15, and P21). Comparison between ubiquitous mutants (TRα^AMI/S) mice (n = 6, in black) and control littermates (n = 6, in gray). PDGFRα is specific of OPC, and Mbp is a marker of mature OL. P15 wild-type whole cerebellum RNA was used as a reference. B, Myelin basic protein quantification at P15 by Western blot analysis (n = 5), normalized by β-tubulin (β-Tub) indicates a delay in myelination in mutant expressing the TRα1^L400R ubiquitously (TRα^AMI/S). Data are expressed ± sd. *, Significant difference between mutants and wild types, P < 0.05. MBP, Myelin basic protein.

Fig. 3.

An indirect effect of T₃ for OPC differentiation at P15. A, Olig2 staining at P15 (in green. DAPI Counterstain blue) indicates an increased density of OPC in TRα^AMI/S (ubiquitous expression), TRα^AMI/N (brain specific), and TRα^AMI/P^T2/P5 (pan-cerebellar expression from P5) compared with littermate control. See Table 2 for counts. B, Olig2 staining in the white matter of control (without Cre), oligodendrocyte specific (TRα^AMI/C), GABAergic lineage specific (Purkinje cell and GABAergic interneurons, TRα^AMI/P), and astrocyte specific from P1 (TRα^AMI/G^T2/P1) indicate a not cell-autonomous effect of thyroid hormone in oligodendrocyte differentiation at P15. C, Same staining at P21 indicates a transient phenotype, mainly observable at P15. D, 24 h BrdU labeling of proliferating cells. Double BrdU/Olig2+ (red/green, colocalization in yellow) E, Counting of double BrdU/Olig2+ cells indicates an increased density in proliferating OPC at P15 when TRα1^L400R is expressed in GABAergic neurons (TRα^AMI/P) mutants or in astrocytes (TRα^AMI/G^T2/P1) but not in OPC (TRα^AMI/C). Data are expressed ± sd. *, Significant difference between mutants and wild types, P < 0.05. Scale bar, 50 μm.

Fig. 4.

TRα1^L400R stimulates adult OPC proliferation in a cell-autonomous manner. A–H, Immunostaining at P60 of Olig2+ OPC (red; DAPI counterstain in blue) compared with control (A and E). Density is increased when TRα1^L400R is expressed in the oligodendrocyte lineage (B and F, TRα^AMI/C, 12-fold increase compared with littermate controls) or in all cell types starting from P30 (C and G, TRα^AMI/P^T2/P30, 14-fold increase compared with littermate controls), but not if expression is restricted to GABAergic neurons (D and H, TRα^AMI/P). I, YFP/Olig2 immunostaining confirms that Cre recombination occurs in the oligodendrocyte lineage of TRα^AMI/P^T2/P30/R26YFP mice (green, YFP; red, Olig2; yellow, merge). J and K, Expression of TRα1^L400R in oligodendrocyte lineage increases the number of YFP+/EdU+ cells at P60, 7 d after EdU injection (YFP, red; and EdU, green). J, Control CrexR26YFP K, TRα^AMI/CxR26YFP. L and M, Colocalization of Olig2 and EdU in TRα^AMI/C white matter (blue, DAPI; gree:, EdU; red, Olig2). n = 4 for each genotype. Scale bar, 200 μm (panels A–L), 10 μm (panel M).

Fig. 5.

Hypothetical model proposed for the influence of T₃ signaling on OPC differentiation. During the first postnatal week, T₃ exerts its cell-autonomous function only in Purkinje cells and astrocytes, modifying neurotrophic factor secretion and cell contacts, and indirectly favoring the commitment of proliferating OPC toward terminal differentiation. Although ubiquitous expression of TRα1^L400R affects BDNF, Shh, and PDGF secretion, only secretion of NT3 by granular neurons is presented. By contrast, adult OPC are sensitive to the cell-autonomous signaling by T₃/TRα1, which is required to prevent uncontrolled proliferation (second mode of action).