Abstract
Oligodendrocyte differentiation is temporally regulated during development by multiple factors. Here, we investigated whether the timing of oligodendrocyte differentiation might be controlled by neuronal differentiation in cerebellar organotypic cultures. In these cultures, the slices taken from newborn mice show very few oligodendrocytes during the first week of culture (immature slices) whereas their number increases importantly during the second week (mature slices). First, we showed that mature cerebellar slices or their conditioned media stimulated oligodendrocyte differentiation in immature slices thus demonstrating the existence of diffusible factors controlling oligodendrocyte differentiation. Using conditioned media from different models of slice culture in which the number of Purkinje cells varies drastically, we showed that the effects of these differentiating factors were proportional to the number of Purkinje cells. To identify these diffusible factors, we first performed a transcriptome analysis with an Affymetrix array for cerebellar cortex and then real-time quantitative PCR on mRNAs extracted from fluorescent flow cytometry sorted (FACS) Purkinje cells of L7-GFP transgenic mice at different ages. These analyses revealed that during postnatal maturation, Purkinje cells down-regulate Sonic Hedgehog and up-regulate vitronectin. Then, we showed that Sonic Hedgehog stimulates the proliferation of oligodendrocyte precursor cells and inhibits their differentiation. In contrast, vitronectin stimulates oligodendrocyte differentiation, whereas its inhibition with blocking antibodies abolishes the conditioned media effects. Altogether, these results suggest that Purkinje cells participate in controlling the timing of oligodendrocyte differentiation in the cerebellum through the developmentally regulated expression of diffusible molecules such as Sonic Hedgehog and vitronectin.
Highlights
Oligodendrocytes are central nervous system macroglial cells that synthesize myelin, a multilayered membrane ensheathing axons which facilitates rapid nerve conduction [1]
We demonstrated that the maturation of Purkinje cells is one of the key factors controlling the timing of oligodendrocyte differentiation
To analyze the timing of the oligodendrocyte differentiation process in cerebellar slice cultures, we focused on the expression of MBP because this protein is expressed in mature oligodendrocytes
Summary
Oligodendrocytes are central nervous system macroglial cells that synthesize myelin, a multilayered membrane ensheathing axons which facilitates rapid nerve conduction [1]. Oligodendrocyte precursor cells (OPCs) divide and migrate over long distances to reach their final destination where they differentiate into mature oligodendrocytes and produce myelin. Neuron maturation affects oligodendrocyte survival and the timing of myelin formation, OPCs differentiate into mature oligodendrocytes and generate a myelin sheath in the absence of axons in vitro [2,3]. Axonal factors which are directly involved in controlling myelin formation include neuronal electrical activity [10,11] and the downregulation of various molecules in axonal membranes, including Jagged, PSA-NCAM (polysialic acid-neural cell adhesion molecule) and N-cadherin [12,13,14]. Rosenberg and colleagues demonstrated that myelin formation required an axonal microenvironment and a critical density of OPCs [16]
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