Introduction Oligodendrocytes are post-mitotic cells that myelinate axons in the vertebrate central nervous system (CNS). Like the majority of other cells in the CNS, oligodendrocytes are generated from pluripotent neuroepithelial cells of the neural tube. Oligodendrocyte development begins in the embryo, when oligodendrocyte precursor cells (OPCs) arise in very restricted regions of the ventral ventricular zone of the developing brain and spinal cord (Noll and Miller, 1993; Ono et al., 1997; Pringle et al., 1998; Timsit et al., 1995; Yu et al., 1994). The restriction of OPC development to these regions depends on localised positive and negative signals (Wada et al., 2000; Woodruff et al., 2001). The initial appearance of OPCs in the developing spinal cord, for example, depends on positive signals from the adjacent notochord: removal of the notochord in Xenopus, mouse or chick embryos inhibits the formation of these cells (Maier and Miller, 1997; Pringle et al., 1996), whereas transplantation of an additional notochord adjacent to the dorsal spinal cord induces their ectopic formation (Maier and Miller, 1997; Orentas and Miller, 1996). One such positive signal is Sonic hedgehog (Shh), which is secreted by both the floor plate and the notochord along the rostro-caudal axis (Echelard et al., 1993; Placzek et al., 1993; Roelink et al., 1994). Shh can induce the development of oligodendrocytes in dorsal spinal cord explants (Pringle et al., 1996; Roelink et al., 1995), and neutralising anti-Shh antibodies can block oligodendrocyte development in ventral regions of CNS explants (Orentas et al., 1999; Tekki-Kessaris et al., 2001). Furthermore, mice with a targeted deletion of the Nkx2.1 gene lack Shh expression in the most anterior domain of the ventral hypothalamus, and OPCs fail to appear in this region at the appropriate stage of development (Sussel et al., 1999; Tekki- Kessaris et al., 2001). The development of OPCs from neuroepithelial cells has mostly been studied in the rodent neural tube. In the mouse spinal cord, for example, the first OPCs expressing the platelet- derived growth factor receptor α (PDGFRα29 are detected as a narrow band in the ventral neuroepithelium around E12.5-13, well after neuronal development begins at E9 (Hardy, 1997; Pringle et al., 1998). Some proteins that are characteristic of oligodendrocytes and their precursors are expressed earlier than PDGFRα, however, and may identify the earliest stages of oligodendrocyte lineage specification. These include the Olig1 and Olig2 basic helix-loop-helix gene regulatory proteins, which are expressed in the same region of the spinal cord as PDGFRα at E13, but are expressed as early as E9 (Lu 3657 Oligodendrocytes are post-mitotic cells that myelinate axons in the vertebrate central nervous system (CNS). They develop from proliferating oligodendrocyte precursor cells (OPCs), which arise in germinal zones, migrate throughout the developing white matter and divide a limited number of times before they terminally differentiate. Thus far, it has been possible to purify OPCs only from the rat optic nerve, but the purified cells cannot be obtained in large enough numbers for conventional biochemical analyses. Moreover, the CNS stem cells that give rise to OPCs have not been purified, limiting one’s ability to study the earliest stages of commitment to the oligodendrocyte lineage. Pluripotent, mouse embryonic stem (ES) cells can be propagated indefinitely in culture and induced to differentiate into various cell types. We have genetically engineered ES cells both to positively select neuroepithelial stem cells and to eliminate undifferentiated ES cells. We have then used combinations of known signal molecules to promote the development of OPCs from selected, ES-cell-derived, neuroepithelial cells. We show that the earliest stages of oligodendrocyte development follow an ordered sequence that is remarkably similar to that observed in vivo, suggesting that the ES-cell-derived neuroepithelial cells follow a normal developmental pathway to produce oligodendrocytes. These engineered ES cells thus provide a powerful system to study both the mechanisms that direct CNS stem cells down the oligodendrocyte pathway and those that influence subsequent oligodendrocyte differentiation. This strategy may also be useful for producing human cells for therapy and drug screening. Key words: Oligodendrocyte, Development, ES cells, Genetic selection Summary Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells Nathalie Billon 1, *, Christine Jolicoeur 1 , Qi Long Ying 2 , Austin Smith 2 and Martin Raff 1 1 MRC Laboratory for Molecular Cell Biology and Cell Biology Unit and the Biology Department, University College London, London WC1E 6BT, UK 2 Centre for Genome Research, University of Edinburgh, King’s Buildings, West Mains Road, Edinburgh EH9 3JQ, UK *Author for correspondence (e-mail: n.billon@ucl.ac.uk) Accepted 7 July 2002 Journal of Cell Science 115, 3657-3665 © 2002 The Company of Biologists Ltd doi:10.1242/jcs.00049 Research Article