Wnt signaling is sufficient to perturb oligodendrocyte maturation
Introduction
Myelin, the lipid membrane that ensheathes axons, is necessary for the rapid and effective conduction of neural signals. In the central nervous system, it is synthesized by oligodendrocytes (OLs). These specialized glial cells arise in ventricular zones as oligodendrocyte precursors (OPCs) and progress through several stages before myelinating axons (Pringle and Richardson, 1993, Ono et al., 1995). During this period, OPCs are exposed to a variety of environmental signaling factors that can be inductive or inhibitive of developmental processes, including differentiation (Miller, 2002). Although much of OL development is well characterized, the factors that affect OL differentiation and the mechanisms by which they operate are less well understood.
In the spinal cord, the majority of OPCs originate in the ventral ventricular zone beginning at 12.5 dpc. The specification of these precursors is controlled by floor plate signals in the ventral spinal cord, notably sonic hedgehog (Shh), which induces expression of transcription factors necessary for the stages of OL development (Lu et al., 2000, Zhou et al., 2000, Zhou and Anderson, 2002). OPCs then migrate from the subventricular zone to populate the spinal cord, eventually contacting axons and expressing myelin proteins. Recent evidence suggests, however, that there are populations of dorsally generated OLs, although their contribution to the general pool may be small and late (Cai et al., 2005, Vallstedt et al., 2005, Kessaris et al., 2006). The predominantly ventral origin of OPCs is thought to result, in part, because of specific OL inhibitors present in the roof plate and adjacent structures (Wada et al., 2000). These dorsal tissue areas contain several families of signaling factors with overlapping expression patterns that may repress multiple aspects of OL development (Wine-Lee et al., 2004).
One of these families, the bone morphogenic proteins (BMPs), has been extensively studied in this regard. Treatment of OPC cell cultures with BMP4 or BMP4 overexpression in vivo induces astrogliogenesis at the expense of OL differentiation (Grinspan et al., 2000, Mekki-Dauriac et al., 2002, Gomes et al., 2003, Miller et al., 2004, See et al., 2004). BMPs, however, may not be the only family of factors that dorsalize the neural tube to affect OL development. BMPs interact with other signaling molecules, among them the Wnts, to regulate dorsal–ventral patterning and other developmental processes (Soshnikova et al., 2003, Wine-Lee et al., 2004, Ille et al., 2007, Zechner et al., 2007). For example, upregulating the Wnt signaling pathway in neural precursor cells increases the expression of BMPs, which may coordinately regulate OL differentiation (Kasai et al., 2005). The extent of their interaction relating to signaling in OL development, however, is not well understood.
In the developing mammalian system, Wnt signaling is involved in regulating embryonic patterning, cell proliferation, migration, specification, and differentiation, and has stage and context specific effects (Dorsky et al., 1998, Patapoutian and Reichardt, 2000, Coyle-Rink et al., 2002, Braun et al., 2003, Hirabayashi et al., 2004, Karim et al., 2004, Kalani et al., 2008). Wnt signaling is active during central nervous system development, in dorsal spinal cord from E9.5 to E12.5 (Wine-Lee et al., 2004, Shimizu et al., 2005), and in the subventricular zone of the developing CNS at E14.5 (Kalani et al., 2008). In the canonical Wnt pathway, Wnt binds with LRP5/6 to frizzled (Fz) receptors, which, through a signal cascade, results in the dephosphorylation and thereby accumulation of β-catenin in the cytoplasm (Mi and Johnson, 2005). Ordinarily, β-catenin is sequestered in a complex with GSK-3β, APC, and axin, marking it for degradation. Dephosphorylated β-catenin collects in the cytoplasm and translocates to the nucleus, where it activates TCF-Lef transcription factors (Wodarz and Nusse, 1998, Staal et al., 2002). GSK-3β inhibitors, such as LiCl, prevent phosphorylation of β-catenin, allowing nuclear translocation and initiation of the signaling cascade. The role of Wnt signaling during OL development has not been extensively investigated. Recent studies have demonstrated that Wnt signaling has inhibitory effects on OPC differentiation in zebrafish and in mouse spinal cord explants and culture (Shimizu et al., 2005, Kim et al., 2008). We have investigated the role of Wnt/β-catenin signaling on OL maturation in primary cultures of OPCs and in a mutant mouse in which β-catenin is constitutively active.
Using the Cre-Lox system, we created a mutant mouse that expresses constitutively active β-catenin in CNP positive cells. The ubiquitin binding site on the β-catenin gene is flanked by LoxP sites, so upon Cre induced recombination, the resulting transgene expresses a β-catenin protein that is not targeted for degradation and remains perpetually transcriptionally active. CNP is present in cells of the OL lineage and is essential for axonal survival, hence its expression is preserved throughout life (Lappe-Siefke et al., 2003). Using this approach, we can study the effects of constitutively active canonical Wnt signaling in all cells of OL lineage.
In normal rat OPC cultures, the addition of Wnt3a significantly decreases the number of immature and mature OLs generated during differentiation. Similarly, cultures prepared from CNP-Cre mice in which β-catenin was constitutively activated show a marked reduction in numbers of immature and mature OLs relative to cultures taken from control littermates. Spinal cord sections show significant decreases in mature OLs and myelin proteins at postnatal stages, and, although less pronounced, this decrease persists until adulthood. Similarly, semi-thin preparations of myelin show fewer myelinated axons at postnatal stages, although this effect recovers by adulthood. These findings indicate that constitutively activated canonical Wnt signaling delays myelination by inhibiting the differentiation of OPCs, and that this signaling pathway is a regulator of OL development.
Section snippets
Wnt3a inhibits oligodendrocyte differentiation via the canonical Wnt signaling pathway
To determine whether Wnt3a signaling affects the progression of the OL lineage in culture, we generated purified primary OPC cultures from rat and mouse forebrain, grew them on 100 mm dishes or 12 mm coverslips, and then removed growth factors and added differentiation medium (DM) when the cells became confluent. After 3 days in DM, 20% of the cells had differentiated and could be labeled with antibody to galactocerebroside (GalC), an early marker of differentiation (Figs. 1A, B). Eighty-six
Discussion
We have investigated the role of canonical Wnt signaling during OL development both in vitro, using cultures from normal rats and from mice in which a dominant stable form of β-catenin is active, and in vivo, examining spinal cords from these same mutant mice. We showed a significant inhibition of OPC differentiation in cultures with activated canonical Wnt signaling. In spinal cord sections, we observed a decrease in numbers of OLs and myelin protein early in postnatal development of β-Cat-CA
Cell culture generation and treatment
All experiments were performed in accordance with the guidelines set forth by the Children's Hospital of Philadelphia Institutional Animal Care and Use Committee. To generate cultures of purified OPCs from newborn rats, a mixed population of cells was harvested from Sprague–Dawley rats and seeded on 100 mm Petri dishes in serum-containing medium as previously described (See et al., 2004). After 24 h, the cell cultures were switched to a serum free growth medium, containing Neurobasal medium
Acknowledgments
We thank Dr. Steven Scherer (University of Pennsylvania) and the members of his laboratory for assistance with the semi-thin sections. We thank Jennifer Minarcik for assistance with western blots. We also thank Dr. James Garbern (Wayne State University) for the gift of the antibody to aspartoacylase. This work was supported by Nat'l Multiple Sclerosis Society RG 4105-A7 (to JBG).
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