In vivo time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during zebrafish development Brandon B Kirby 1,3 , Norio Takada 1,3 , Andrew J Latimer 1 , Jimann Shin 1 , Thomas J Carney 2,4 , Robert N Kelsh 2 & Bruce Appel 1 Myelinating oligodendrocytes arise from migratory and proliferative oligodendrocyte progenitor cells (OPCs). Complete myelination requires that oligodendrocytes be uniformly distributed and form numerous, periodically spaced membrane sheaths along the entire length of target axons. Mechanisms that determine spacing of oligodendrocytes and their myelinating processes are not known. Using in vivo time-lapse confocal microscopy, we show that zebrafish OPCs continuously extend and retract numerous filopodium-like processes as they migrate and settle into their final positions. Process remodeling and migration paths are highly variable and seem to be influenced by contact with neighboring OPCs. After laser ablation of oligodendrocyte-lineage cells, nearby OPCs divide more frequently, orient processes toward the ablated cells and migrate to fill the unoccupied space. Thus, process activity before axon wrapping might serve as a surveillance mechanism by which OPCs determine the presence or absence of nearby oligodendrocyte-lineage cells, facilitating uniform spacing of oligodendrocytes and complete myelination. Rapid conduction of nerve impulses in vertebrates requires that most large-diameter axons be wrapped by myelin. In the CNS, myelin sheaths are formed by oligodendrocytes, one of the major classes of glial cells 1,2 . During development, discrete subpopulations of neural precursors give rise to OPCs. OPCs proliferate and migrate from their origins, eventually becoming uniformly distributed throughout the gray and white matter of the brain and spinal cord 3 . During late embryogenesis and early postnatal life, many OPCs stop dividing and differentiate as oligodendrocytes, which extend fine membrane pro- cesses that wrap axons. Each oligodendrocyte can wrap numerous axons, and each axon is ensheathed by periodically spaced processes extending from multiple oligodendrocytes. Uniform and periodic spacing of myelin sheaths on axons is a critical feature of saltatory conduction, but whether spacing is determined by OPCs or positional cues within axons is unknown. To investigate behavior of OPCs as they migrate and make contact with axons in vivo, we created transgenic zebrafish that express fluorescent proteins in oligodendrocyte-lineage cells and performed time-lapse imaging during normal development and after laser micro- surgical removal of nearby cells. Our data establish three key points. First, OPCs constantly remodel numerous filopodium-like processes as they migrate and for many hours before they wrap axons. Second, OPCs often retract processes and change direction of migration after making contact with a neighboring OPC. Third, OPCs can divide and migrate to replace nearby oligodendrocyte-lineage cells removed by laser microsurgery. These results raise the possibility that OPCs survey their environments by filopodial activity and adjust their migration and division according to the density and distribution of nearby OPCs and oligodendrocytes. Density-dependent regulation of migration and division ensures a uniform distribution of oligodendrocytes, facilitating uniform myelination. In mice, genetically ablated subpopulations of OPCs are rapidly replaced by nearby cells, suggesting that OPCs compete for space 4 . In humans, OPCs seemingly divide and migrate to replace oligo- dendrocytes lost from disease or injury 5 . Our data point toward filopodium-based surveillance a mechanism that regulates myelination and, possibly, remyelination. RESULTS Migrating OPCs rapidly remodel filopodium-like processes The transgenic line Tg(nkx2.2a:megfp) expresses a membrane-tethered enhanced green fluorescent protein (mEGFP) under control of nkx2.2a regulatory sequences carried on a bacterial artificial chromosome (BAC) 6 . The transgene expresses EGFP in a pattern similar to nkx2.2a RNA, marking a subset of oligodendrocyte-lineage cells (data not shown). To examine spinal cord OPC behaviors, we mounted Tg(nkx2.2a:megfp) embryos on their sides and performed time-lapse imaging using spinning disc confocal microscopy from 36 h post- fertilization (hpf), when OPCs are first specified in ventral spinal cord of zebrafish, to 72 hpf, when axon wrapping is initiated (Fig. 1 and Received 14 September; accepted 24 October; published online 12 November 2006; doi:10.1038/nn1803 1 Department of Biological Sciences, Vanderbilt University, 465 21 st Avenue South, Nashville, Tennessee 37232, USA. 2 Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK. 3 These authors contributed equally to this work. 4 Present address: Spemann Laboratories, Max-Planck- Institut fur Immunobiologie, Stuebeweg 51, Freiburg D-79108, Germany. Correspondence should be addressed to B.A. (b.appel@vanderbilt.edu). 1506 VOLUME 9 [ NUMBER 12 [ DECEMBER 2006 NATURE NEUROSCIENCE ARTICLES © 2006 Nature Publishing Group http://www.nature.com/natureneuroscience