Running Head: miR-7a in oligodendrogenesis Specification and Maintenance of Oligodendrocyte Precursor Cells from Neural Progenitor Cells: Involvement of MicroRNA-7a

The generation of myelinating cells from multipotential neural stem cells in the central nervous system (CNS) requires the initiation of specific gene expression programs in oligodendrocytes (OLs). We reasoned that microRNAs (miRNAs) could play an important role in this process by regulating genes crucial for OL development. Here we identified miR-7a as one of the highly enriched miRNAs in oligodendrocyte precursor cells (OPCs), overexpression of which in either neural progenitor cells (NPCs) or embryonic mouse cortex promoted the generation of OL lineage cells. Blocking the function of miR-7a in differentiating NPCs led to a reduction in OL number and an expansion of neuronal populations simultaneously. We also found that overexpression of this miRNA in purified OPC cultures promoted cell proliferation and inhibited further maturation. In addition, miR-7a might exert above effects partially by directly repressing proneuronal differentiation factors including Pax6 and NeuroD4, or pro-OL genes involved in oligodendrocyte maturation. These results suggest that miRNA pathway is essential in determining cell fate commitment for oligodendrocyte and thus provide a new strategy for modulating this process in OL loss diseases. Introduction Oligodendrocytes (OLs) play a critical role in the central nervous system (CNS) by producing insulating protein membranes that ensheath axons. Significant damages to OLs result in demyelination and hinder effective communication among neurons. Correspondingly, CNS demyelinating conditions, such as spinal cord injury and multiple sclerosis will result in severe http://www.molbiolcell.org/content/suppl/2012/06/11/mbc.E12-04-0270.DC1.html Supplemental Material can be found at: 2 motor, sensory and cognitive impairment. To obtain proper remyelination, the knowledge regarding the regulators of OL development has been a major focus in understanding the mechanisms promoting differentiation of oligodendrocyte precursor cells (OPCs). Myelinating oligodendrocytes are derived from multipotential neural progenitor cells (NPCs). The process of creating a properly functional OL, including cell fate specification, OPCs migration, maturation, and myelination, is regulated by the dynamic interplay between transcription factors, epigenetic factors, microRNA (miRNA) regulators, and other cell-extrinsic signals (Emery, 2010; Yu et al., 2010). miRNAs are small, noncoding RNAs that regulate gene expression by post-transcriptional targeting RNA-induced silencing complex (RISC) to cognate messenger RNA (Bartel, 2004). Studies using Nestin-Cre line to delete the miRNA-processing enzyme Dicer in the entire CNS show that Dicer function or the maturation of miRNA is required for the proper specification and development of both neurons and oligodendrocytes during embryonic stages (Kawase-Koga et al., 2009). Moreover, disruption of Dicer function in specified OPCs and OLs by expressing Cre from the Olig1 or Olig2 promoter induces the failure of normal OL differentiation, as well as myelin formation (Dugas et al., 2010; Zhao et al., 2010; Zheng et al., 2010). Therefore, these studies indicate that mature miRNA activity is required at various stages of OL development, including the initial production of fate-specified OPCs, the differentiation of mature OLs, the generation of compact CNS myelin during development, and the maintenance of functional myelin sheaths in adult animals (Dugas and Notterpek, 2011). Several groups have investigated the roles of individual miRNA in promoting functional CNS myelination. miR-219 and miR-338 are two of the most highly induced miRNAs in differentiating OLs and they are individually necessary and sufficient to promote normal OPC differentiation into OLs in vitro and in vivo (Dugas et al., 2010; Zhao et al., 2010); miR-23a and miR-23b have been identified as miRNAs that are induced approximately 5-fold during OL maturation, and overexpression of either one can enhance OL differentiation, probably by reducing the expression of a gene that inhibits normal OL maturation (Lin and Fu, 2009); miR138 is also induced in differentiating OLs, and it specifically promotes the early stages of OL differentiation, while simultaneously suppressing the later stage of OL differentiation (Dugas et al., 2010; Zhao et al., 2010). In contrast to the identification of miRNAs in promoting OL differentiation, a set of OPC-enriched miRNAs has also been identified (Lau et al., 2008; Shin et al., 2009; Letzen et al., 2010), among which members of the miR-17-92 cluster were found to be