This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Development/Plasticity/Repair Age-dependent decline in fate switch from NG2 cells to astrocytes after Olig2 deletion Hao Zuo1, William M. Wood1, Amin Sherafat1, Robert A. Hill1, Q. Richard Lu2 and Akiko Nishiyama1,3 1University of Connecticut, Department of Physiology and Neurobiology, 75 North Eagleville Road, Storrs, CT 06269-3156, USA 2Cincinnati Children's Hospital Medical Center, Department of Pediatrics, Cincinnati, OH 3University of Connecticut Institute of System Genomics and University of Connecticut Institute of Brain and Cognitive Sciences DOI: 10.1523/JNEUROSCI.0712-17.2018 Received: 14 March 2017 Revised: 31 December 2017 Accepted: 23 January 2018 Published: 30 January 2018 Author contributions: HZ, WMW, AS, and RAH performed experiments and contributed to figures, HZ, WMW, AS, and AN wrote the manuscript, QL contributed mouse line as well as design and discussion of the study. Conflict of Interest: The authors declare no competing financial interests. This study was funded by NIH R01NS049267, NIH R01 NS074870, and NIH R01 NS073425 to AN, NIH R01NS072427 and R01NS075243 (QRL), and Connecticut Stem Cell Program 06SCB03 (AN). The Leica SP8 confocal microscope was purchased using NIH Shared Instrumentation Grant S10OD016435 (PI, AN) and is maintained by Dr. Chris O'Connell, Director of Advanced Microscopy Facility. We thank Youfen Sun for maintaining the transgenic mouse colony. We thank Dr. Ben Deneen (Baylor College of Medicine) for the rabbit anti-NFIA antibody and Dr. Michael Wegner (Erlangen University) for the guinea pig anti-Sox10 antibody. The authors declare no competing financial interests. Correspondence should be addressed [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0712-17.2018 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 Age-dependent decline in fate switch from NG2 cells to astrocytes after Olig2 deletion 2 3 Hao Zuo1*, William M. Wood1*, Amin Sherafat1, Robert A. Hill1,4, Q. Richard Lu2, and Akiko Nishiyama1,3 4 5 1University of Connecticut 6 Department of Physiology and Neurobiology 7 75 North Eagleville Road 8 Storrs, CT 06269-3156 9 USA 10 [email protected] 11 *co-first authors 12 2Cincinnati Children’s Hospital Medical Center 13 Department of Pediatrics 14 Cincinnati, OH 15 3University of Connecticut Institute of System Genomics and University of Connecticut Institute of Brain 16 and Cognitive Sciences 17 4Present address: Yale University School of Medicine, Department of Neurology 18 19 Running title: Age-dependent decline of OPC fate plasticity 20 21 Author contributions: HZ, WMW, AS, and RAH performed experiments and contributed to figures, HZ, 22 WMW, AS, and AN wrote the manuscript, QL contributed mouse line as well as design and discussion of the 23 study. 24 25 Journal section: Development/Plasticity/Repair 26 27 Word count: 28 Abstract 236 29 Significance statement 118 30 Introduction 466 31 Materials and Methods 961 32 Results 3,046 33 Discussion 1,500 34 Figure legends 1,301 35 Bibliography 1,810 36 Total 9,438 37 38 Number of figures: 5 39 Number of tables: 1 40 1 41 ABSTRACT 42 NG2 cells are a resident glial progenitor cell population that is uniformly distributed throughout 43 the developing and mature mammalian central nervous system (CNS). Those in the postnatal CNS 44 generate exclusively myelinating and non-myelinating oligodendrocytes and are hence equated with 45 oligodendrocyte precursor cells (OPCs). Prenatally, NG2 cells in the ventral gray matter of the 46 forebrain generate protoplasmic astrocytes as well as oligodendrocytes. The fate conversion from 47 NG2 cells into protoplasmic astrocytes is dependent on downregulation of the key oligodendrocyte 48 transcription factor Olig2. We previously showed that constitutive deletion of Olig2 in NG2 cells 49 converts NG2 cells in the neocortex into protoplasmic astrocytes at the expense of oligodendrocytes. 50 In this study, we show that postnatal deletion of Olig2 in both male and female mice caused NG2 51 cells in the neocortex but not in other gray matter regions to become protoplasmic astrocytes. 52 However, NG2 cells in the neocortex became more resistant to astrocyte fate switch over the first 53 three postnatal weeks. Fewer NG2 cells differentiated into astrocytes and did so with longer latency 54 after Olig2 deletion at postnatal day 18 (P18) compared to deletion at P2. The high-mobility group 55 transcription factor Sox10 was not downregulated for at least one month after Olig2 deletion at P18 56 despite an early transient upregulation of the astrocyte transcription factor NFIA. Furthermore, 57 inhibiting cell proliferation in slice culture reduced astrocyte differentiation from Olig2-deleted 58 perinatal NG2 cells, suggesting that cell division might facilitate nuclear reorganization needed for 59 astrocyte transformation. 60 61 2 62 SIGNIFICANCE STATEMENT 63 NG2 cells are glial progenitor cells that retain a certain degree of lineage plasticity. In the 64 normal postnatal neocortex, they generate mostly oligodendrocyte lineage cells. When the 65 oligodendrocyte transcription factor Olig2 is deleted in NG2 cells in the neocortex, they switch their 66 fate to protoplasmic astrocytes. However, the efficiency of the fate switch decreases with age over 67 the first postnatal three weeks and is reduced when cell proliferation is inhibited. As the neocortex 68 matures, sustained expression of oligodendrocyte lineage-specific key transcription factor Sox10 69 becomes less dependent on Olig2. Taken together, our findings suggest a gradual stabilization of the 70 oligodendrocyte lineage genes and loss of lineage plasticity during the first three weeks after birth, 71 possibly due to nuclear reorganization. 72 73 74 3 75 INTRODUCTION 76 NG2 cells, also called NG2-glia or polydendrocytes, are widely distributed throughout the 77 developing and mature mammalian central nervous system (CNS) (Nishiyama et al., 2009, 2016). 78 Genetic fate mapping studies have confirmed that in the postnatal CNS, NG2 cells either self-renew 79 or differentiate into myelinating and non-myelinating oligodendrocytes, and thus they are 80 commonly equated with oligodendrocyte precursor cells (OPCs). They express two OPC signature 81 proteins NG2 (Cspg4 gene product) and platelet-derived growth factor receptor alpha (Pdgfra), 82 which are downregulated upon their terminal differentiation into postmitotic oligodendrocytes. NG2 83 cells remain proliferative throughout life, but the rate of their proliferation declines with age 84 (Dawson et al., 2003; Psachoulia et al., 2009; Young et al., 2013). 85 Olig2 is a basic helix-loop-helix (bHLH) transcription factor that is broadly expressed in the 86 germinal zones and is required for oligodendrocyte specification and maturation (Zhou and 87 Anderson, 2002; Lu et al., 2002; Takebayashi et al., 2002; Maire et al., 2010; Wegener et al., 2015). 88 During oligodendrocyte specification, Olig2 positively regulates transcription of Sox10, which 89 belongs to the SoxE family of high-mobility group (HMG)-domain transcription factors and is 90 expressed and functionally important throughout all stages of the oligodendrocyte lineage 91 (Kuhlbrodt et al., 1998; Stolt and Wegner, 2016; Küspert et al., 2011). The onset of NG2 and Pdgfra 92 expression immediately follows Sox10 induction in committed oligodendrocyte lineage cells as they 93 emigrate from the germinal zone (reviewed in Nishiyama et al., 2016). 94 A subpopulation of NG2 cells in the prenatal CNS also generate protoplasmic astrocytes in a 95 region-specific manner (Zhu et al., 2008a, b; Ge et al., 2012; Huang et al., 2014). NG2 cells emerge 96 in the telencephalon around embryonic day 14.5 (E14.5, Zhu et al., 2008a). Subsequently a 97 subpopulation of them in the ventral forebrain downregulate Olig2 and become protoplasmic 98 astrocytes, while other NG2 cells in the same region develop along the oligodendrocyte lineage. 99 When Olig2 is constitutively deleted in NG2 cells, those in the neocortex switch their fate to 100 become protoplasmic astrocytes at the expense of oligodendrocytes (Zhu et al., 2012), underscoring 101 the critical role for Olig2 in maintaining NG2 cells in the oligodendrocyte lineage. In the normal 102 adult CNS, NG2 cells do not produce astrocytes (Rivers et al., 2008; Dimou et al., 2008; Zhu et al., 4 103 2011; Kang et al., 2010), and deletion of Olig2 in the injured adult brain has yielded variable results 104 (Dimou et al., 2008; Tatsumi et al., 2008; Komitova et al., 2011). To investigate the temporal 105 changes in Olig2-dependent lineage plasticity of NG2 cells, we examined the fate of NG2 cells in 106 the neocortex after deleting Olig2 at different postnatal ages. We show that there was an 107 age-dependent delay and decline astrocyte differentiation from NG2 cells after Olig2 deletion, 108 which was correlated with delayed downregulation of Sox10. Furthermore, inhibiting cell division 109 in slice cultures attenuated astrocyte differentiation from NG2 cells. 110 111 5 112 MATERIALS AND METHODS 113 Generation of tamoxifen-inducible NG2 cell-specific Olig2 conditional knockout mice 114 Triple transgenic mouse line NG2creERTM:YFP:Olig2fl/fl