Neurochemical Research

, Volume 41, Issue 1–2, pp 450–462 | Cite as

Keratan Sulfate Regulates the Switch from Motor Neuron to Oligodendrocyte Generation During Development of the Mouse Spinal Cord

  • Hirokazu Hashimoto
  • Yugo Ishino
  • Wen Jiang
  • Takeshi Yoshimura
  • Yoshiko Takeda-Uchimura
  • Kenji Uchimura
  • Kenji Kadomatsu
  • Kazuhiro IkenakaEmail author
Original Paper


Keratan sulfate (KS) is a sulfated glycosaminoglycan and has been shown to bind to sonic hedgehog (Shh), which act as a morphogen to regulate the embryonic spinal cord development. We found highly sulfated KS was present in the floor plate (including lateral floor plate) and the notochord . This expression colocalized with Shh expression. To understand the roles of KS, we analyzed the embryonic spinal cord of GlcNAc6ST-1, KS chain synthesizing enzyme, knock-out (KO) mice. At E12.5, the pMN domain, whose formation is controlled by Shh signaling, became shifted ventrally in GlcNAc6ST-1 KO mice. In addition, the expression patterns of Patched1 and Gli1, two Shh signaling reporter genes, differed between wild type (WT) and GlcNAc6ST-1 KO mice at E12.5. Next, we focused on cell types generated from the pMN domain; namely, motor neurons and subsequently oligodendrocytes. The number of PDGFRα+ [a marker for oligodendrocyte precursor cells (OPCs)] cells was low in the E12.5 mutant spinal cord, while motor neuron production was increased. Thus the switch from motor neuron generation to OPC generation was delayed in the pMN domain. Furthermore, we investigated the cause for this delayed switch in the pMN domain. The number of Olig2, Nkx2.2 double-positive cells was less in GlcNAc6ST-1 KO mice than in WT mice. In contrast, the number of Olig2, Neurogenin2 (Ngn2) double-positive cells related to the motor neuron specification was significantly greater in the KO mice. These results indicate that KS is important for the late phase Shh signaling and contributes to motor neuron to OPC generation switch.


Keratan sulfate Sonic hedgehog signaling Spinal cord Oligodendrocyte Motor neuron 



Autonomic motor neuron


Bone morphogenetic protein


Central nervous system






Keratan sulfate


Myelin basic protein




Oligodendrocyte precursor cell


Platelet derived growth factor receptor alpha


Sonic hedgehog


Somatic motor neuron


Heparan sulfate 6-O-endosulfatase 1


Ventricular zone



This work was supported by a research grant from the Japan Society for the Promotion of Science (JSPS) KAKENHI (26290027) and by a Grant-in-Aid for Scientific Research on Innovative Areas entitled “Neural Diversity and Neocortical Organization” from MEXT (25123721). We thank Spectrography and Bioimaging Facility, NIBB Core Research Facilities for technical support.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11064_2016_1861_MOESM1_ESM.tif (3 mb)
Supplemental Fig. 1 The scheme of the developing spinal cord; Wnt and BMP are secreted from the roof plate and Shh is secreted from the floor plate. They are involved in the patterning of the spinal cord. These twelve domain structures are regulated by the transcription factors expression, such as Math1, Olig3, Pax7, Nkx6.1, Olig2 and Nkx2.2 (TIFF 3021 kb)
11064_2016_1861_MOESM2_ESM.tif (6.5 mb)
Supplemental Fig. 2 The domain structure of the spinal cord appeared normal in the GlcNAc6ST-1 KO mouse at E10.5. At E10.5, WT (a, c, e, g, i, k) and the GlcNAc6ST-1 KO (b, d, f, h, j, l) embryos were analyzed by in situ hybridization with RNA probes for Math1 (a, b), Olig3 (c, d), Pax7 (e, f), Nkx6.1 (g, h), Olig2 (i, j), and Nkx2.2 (k, l). Scale bar = 200 μm (TIFF 6681 kb)
11064_2016_1861_MOESM3_ESM.tif (6.5 mb)
Supplemental Fig. 3 The domain structure of the spinal cord shifted ventrally in the GlcNAc6ST-1 KO mouse at E12.5. At E12.5, WT (a, c, e, g, i, k) and the GlcNAc6ST-1 KO (b, d, f, h, j, l) embryos were analyzed by in situ hybridization with RNA probes for Math1 (a, b), Olig3 (c, d), Pax7 (e, f), Nkx6.1 (g, h), Olig2 (i, j), and Nkx2.2 (k, l). Scale bar = 200 μm (TIFF 6695 kb)


  1. 1.
    Ulloa F, Martí E (2010) Wnt won the war: antagonistic role of Wnt over Shh controls dorso-ventral patterning of the vertebrate neural tube. Dev Dyn 239:69–76. doi: 10.1002/dvdy.22058 PubMedGoogle Scholar
  2. 2.
    Briscoe J, Novitch BG (2008) Regulatory pathways linking progenitor patterning, cell fates and neurogenesis in the ventral neural tube. Philos Trans R Soc Lond B Biol Sci 363:57–70. doi: 10.1098/rstb.2006.2012 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Dessaud E, Yang LL, Hill K, Cox B, Ulloa F, Ribeiro A, Mynett A, Novitch BG, Briscoe J (2007) Interpretation of the sonic hedgehog morphogen gradient by a temporal adaptation mechanism. Nature 450:717–720. doi: 10.1038/nature06347 CrossRefPubMedGoogle Scholar
  4. 4.
    Saha K, Schaffer DV (2006) Signal dynamics in Sonic hedgehog tissue patterning. Development 133:889–900. doi: 10.1242/dev.02337 CrossRefPubMedGoogle Scholar
  5. 5.
    Farshi P, Ohlig S, Pickhinke U, Höing S, Jochmann K, Lawrence R, Dreier R, Dierker T, Grobe K (2011) Dual roles of the Cardin-Weintraub motif in multimeric Sonic hedgehog. J Biol Chem 286:23608–23619. doi: 10.1074/jbc.M110.206474 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Chang SC, Mulloy B, Magee AI, Couchman JR (2011) Two distinct sites in sonic Hedgehog combine for heparan sulfate interactions and cell signaling functions. J Biol Chem 286:44391–44402. doi: 10.1074/jbc.M111.285361 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Nadanaka S, Ishida M, Ikegami M, Kitagawa H (2008) Chondroitin 4-O-sulfotransferase-1 modulates Wnt-3a signaling through control of E disaccharide expression of chondroitin sulfate. J Biol Chem 283:27333–27343. doi: 10.1074/jbc.M802997200 CrossRefPubMedGoogle Scholar
  8. 8.
    Cortes M, Baria AT, Schwartz NB (2009) Sulfation of chondroitin sulfate proteoglycans is necessary for proper Indian hedgehog signaling in the developing growth plate. Development 136:1697–1706. doi: 10.1242/dev.030742 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Alvarez-Medina R, Cayuso J, Okubo T, Takada S, Martí E (2008) Wnt canonical pathway restricts graded Shh/Gli patterning activity through the regulation of Gli3 expression. Development 135:237–247. doi: 10.1242/dev.012054 CrossRefPubMedGoogle Scholar
  10. 10.
    Yu K, McGlynn S, Matise MP (2013) Floor plate-derived sonic hedgehog regulates glial and ependymal cell fates in the developing spinal cord. Development 140:1594–1604. doi: 10.1242/dev.090845 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Shimizu T, Kagawa T, Wada T, Muroyama Y, Takada S, Ikenaka K (2005) Wnt signaling controls the timing of oligodendrocyte development in the spinal cord. Dev Biol 282:397–410. doi: 10.1016/j.ydbio.2005.03.020 CrossRefPubMedGoogle Scholar
  12. 12.
    Fancy SP, Baranzini SE, Zhao C, Yuk DI, Irvine KA, Kaing S, Sanai N, Franklin RJ, Rowitch DH (2009) Dysregulation of the Wnt pathway inhibits timely myelination and remyelination in the mammalian CNS. Genes Dev 23:1571–1585. doi: 10.1101/gad.1806309 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Langseth AJ, Munji RN, Choe Y, Huynh T, Pozniak CD, Pleasure SJ (2010) Wnts influence the timing and efficiency of oligodendrocyte precursor cell generation in the telencephalon. J Neurosci 30:13367–13372. doi: 10.1523/JNEUROSCI.1934-10.2010 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Touahri Y, Escalas N, Benazeraf B, Cochard P, Danesin C, Soula C (2012) Sulfatase 1 promotes the motor neuron-to-oligodendrocyte fate switch by activating Shh signaling in Olig2 progenitors of the embryonic ventral spinal cord. J Neurosci 32:18018–18034. doi: 10.1523/JNEUROSCI.3553-12.2012 CrossRefPubMedGoogle Scholar
  15. 15.
    Nadanaka S, Kinouchi H, Taniguchi-Morita K, Tamura J, Kitagawa H (2011) Down-regulation of chondroitin 4-O-sulfotransferase-1 by Wnt signaling triggers diffusion of Wnt-3a. J Biol Chem 286:4199–4208. doi: 10.1074/jbc.M110.155093 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Danesin C, Agius E, Escalas N, Ai X, Emerson C, Cochard P, Soula C (2006) Ventral neural progenitors switch toward an oligodendroglial fate in response to increased Sonic hedgehog (Shh) activity: involvement of Sulfatase 1 in modulating Shh signaling in the ventral spinal cord. J Neurosci 26:5037–5048. doi: 10.1523/JNEUROSCI.0715-06.2006 CrossRefPubMedGoogle Scholar
  17. 17.
    Funderburgh JL (2002) Keratan sulfate biosynthesis. IUBMB Life 54:187–194. doi: 10.1080/15216540214932 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Weyers A, Yang B, Solakyildirim K, Yee V, Li L, Zhang F, Linhardt RJ (2013) Isolation of bovine corneal KS and its growth factor and morphogen binding. FEBS J 280:2285–2293. doi: 10.1111/febs.12165 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Jacob J, Briscoe J (2003) Gli proteins and the control of spinal-cord patterning. EMBO Rep 4:761–765. doi: 10.1038/sj.embor.embor896 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Lu DC, Niu T, Alaynick WA (2015) Molecular and cellular development of spinal cord locomotor circuitry. Front Mol Neurosci 8:25. doi: 10.3389/fnmol.2015.00025 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Helms AW, Johnson JE (2003) Specification of dorsal spinal cord interneurons. Curr Opin Neurobiol 13:42–49. doi: 10.1016/S0959-4388(03)00010-2 CrossRefPubMedGoogle Scholar
  22. 22.
    Tanabe Y, Jessell TM (1996) Diversity and pattern in the developing spinal cord. Science 274:1115–1123. doi: 10.1126/science.274.5290.1115 CrossRefPubMedGoogle Scholar
  23. 23.
    Zhou Q, Anderson DJ (2002) The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification. Cell 109:61–73. doi: 10.1016/S0092-8674(02)00677-3 CrossRefPubMedGoogle Scholar
  24. 24.
    Ribes V, Balaskas N, Sasai N, Cruz C, Dessaud E, Cayuso J, Tozer S, Yang LL, Novitch B, Marti E, Briscoe J (2010) Distinct Sonic Hedgehog signaling dynamics specify floor plate and ventral neuronal progenitors in the vertebrate neural tube. Genes Dev 24:1186–1200. doi: 10.1101/gad.559910 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Mizuguchi R, Sugimori M, Takebayashi H, Kosako H, Nagao M, Yoshida S, Nabeshima Y, Shimamura K, Nakafuku M (2001) Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Neuron 31:757–771. doi: 10.1016/S0896-6273(01)00413-5 CrossRefPubMedGoogle Scholar
  26. 26.
    Novitch BG, Chen AI, Jessell TM (2001) Coordinate regulation of motor neuron subtype identity and pan-neuronal properties by the bHLH repressor Olig2. Neuron 31:773–789. doi: 10.1016/S0896-6273(01)00407-X CrossRefPubMedGoogle Scholar
  27. 27.
    Fu H, Qi Y, Tan M, Cai J, Takebayashi H, Nakafuku M, Richardson W, Qiu M (2002) Dual origin of spinal oligodendrocyte progenitors and evidence for the cooperative role of Olig2 and Nkx2.2 in the control of oligodendrocyte differentiation. Development 129:681–693PubMedGoogle Scholar
  28. 28.
    Lu QR, Sun T, Zhu Z, Ma N, Garcia M, Stiles CD, Rowitch DH (2002) Common developmental requirement for Olig function indicates a motor neuron/oligodendrocyte connection. Cell 109:75–86. doi: 10.1016/S0092-8674(02)00678-5 CrossRefPubMedGoogle Scholar
  29. 29.
    Soula C, Danesin C, Kan P, Grob M, Poncet C, Cochard P (2001) Distinct sites of origin of oligodendrocytes and somatic motoneurons in the chick spinal cord: oligodendrocytes arise from Nkx2.2-expressing progenitors by a Shh-dependent mechanism. Development 128:1369–1379PubMedGoogle Scholar
  30. 30.
    Takebayashi H, Nabeshima Y, Yoshida S, Chisaka O, Ikenaka K, Nabeshima Y (2002) The basic helix-loop-helix factor olig2 is essential for the development of motoneuron and oligodendrocyte lineages. Curr Biol 12:1157–1163. doi: 10.1016/S0960-9822(02)00926-0 CrossRefPubMedGoogle Scholar
  31. 31.
    Sugimori M, Nagao M, Bertrand N, Parras CM, Guillemot F, Nakafuku M (2007) Combinatorial actions of patterning and HLH transcription factors in the spatiotemporal control of neurogenesis and gliogenesis in the developing spinal cord. Development 134:1617–1629. doi: 10.1242/dev.001255 CrossRefPubMedGoogle Scholar
  32. 32.
    Guillemot F (2007) Spatial and temporal specification of neural fates by transcription factor codes. Development 134:3771–3780. doi: 10.1242/dev.006379 CrossRefPubMedGoogle Scholar
  33. 33.
    Küspert M, Hammer A, Bösl MR, Wegner M (2011) Olig2 regulates Sox10 expression in oligodendrocyte precursors through an evolutionary conserved distal enhancer. Nucleic Acids Res 39:1280–1293. doi: 10.1093/nar/gkq951 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Zhou Q, Choi G, Anderson DJ (2001) The bHLH transcription factor Olig2 promotes oligodendrocyte differentiation in collaboration with Nkx2.2. Neuron 31:791–807. doi: 10.1016/S0896-6273(01)00414-7 CrossRefPubMedGoogle Scholar
  35. 35.
    Pringle NP, Yu WP, Guthrie S, Roelink H, Lumsden A, Peterson AC, Richardson WD (1996) Determination of neuroepithelial cell fate: induction of the oligodendrocyte lineage by ventral midline cells and sonic hedgehog. Dev Biol 177:30–42. doi: 10.1006/dbio.1996.0142 CrossRefPubMedGoogle Scholar
  36. 36.
    Orentas DM, Hayes JE, Dyer KL, Miller RH (1999) Sonic hedgehog signaling is required during the appearance of spinal cord oligodendrocyte precursors. Development 126:2419–2429PubMedGoogle Scholar
  37. 37.
    Uchimura K, Kadomatsu K, El-Fasakhany FM, Singer MS, Izawa M, Kannagi R, Takeda N, Rosen SD, Muramatsu T (2004) N-acetylglucosamine 6-O-sulfotransferase-1 regulates expression of L-selectin ligands and lymphocyte homing. J Biol Chem 279:35001–35008. doi: 10.1074/jbc.M404456200 CrossRefPubMedGoogle Scholar
  38. 38.
    Ding L, Takebayashi H, Watanabe K, Ohtsuki T, Tanaka KF, Nabeshima Y, Chisaka O, Ikenaka K, Ono K (2005) Short-term lineage analysis of dorsally derived Olig3 cells in the developing spinal cord. Dev Dyn 234:622–632. doi: 10.1002/dvdy.20545 CrossRefPubMedGoogle Scholar
  39. 39.
    Caterson B, Christner JE, Baker JR (1983) Identification of a monoclonal antibody that specifically recognaizes corneal and skeletal keratan sulfate. Monoclonal antibodies to cartilage proteoglycan. J Biol Chem 258:8848–8854PubMedGoogle Scholar
  40. 40.
    Mehmet H, Scudder P, Tang PW, Hounsell EF, Caterson B, Feizi T (1986) The antigenic determinants recognized by three monoclonal antibodies to keratan sulphate involve sulphated hepta- or larger-oligosaccharides of the poly(N-acetyllactosamine) series. Eur J Biochem 157:385–391CrossRefPubMedGoogle Scholar
  41. 41.
    Kuhlbrodt K, Herbarth B, Sock E, Hermans-Borgmeyer I, Wegner M (1998) Sox10, a novel transcriptional modulator in glial cells. J Neurosci 18:237–250PubMedGoogle Scholar
  42. 42.
    Zhou Q, Wang S, Anderson DJ (2000) Identification of a novel family of oligodendrocyte lineage-specific basic helix-loop-helix transcription factors. Neuron 25:331–343. doi: 10.1016/S0896-6273(00)80898-3 CrossRefPubMedGoogle Scholar
  43. 43.
    Stolt CC, Rehberg S, Ader M, Lommes P, Riethmacher D, Schachner M, Bartsch U, Wegner M (2002) Terminal differentiation of myelin-forming oligodendrocytes depends on the transcription factor Sox10. Genes Dev 16:165–170. doi: 10.1101/gad.215802 CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Briscoe J, Sussel L, Serup P, Hartigan-O’Connor D, Jessell TM, Rubenstein JL, Ericson J (1999) Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling. Nature 398:622–627. doi: 10.1038/19315 CrossRefPubMedGoogle Scholar
  45. 45.
    Liu Y, Wu Y, Lee JC, Xue H, Pevny LH, Kaprielian Z, Rao MS (2002) Oligodendrocyte and astrocyte development in rodents: an in situ and immunohistological analysis during embryonic development. Glia 40:25–43. doi: 10.1002/glia.10111 CrossRefPubMedGoogle Scholar
  46. 46.
    Scardigli R, Schuurmans C, Gradwohl G, Guillemot F (2001) Crossregulation between Neurogenin2 and pathways specifying neuronal identity in the spinal cord. Neuron 31:203–217. doi: 10.1016/S0896-6273(01)00358-0 CrossRefPubMedGoogle Scholar
  47. 47.
    Sun Y, Nadal-Vicens M, Misono S, Lin MZ, Zubiaga A, Hua X, Fan G, Greenberg ME (2001) Neurogenin promotes neurogenesis and inhibits glial differentiation by independent mechanisms. Cell 104:365–376. doi: 10.1016/S0092-8674(01)00224-0 CrossRefPubMedGoogle Scholar
  48. 48.
    Kicheva A, Bollenbach T, Ribeiro A, Valle HP, Lovell-Badge R, Episkopou V, Briscoe J (2014) Coordination of progenitor specification and growth in mouse and chick spinal cord. Science 345:1254927. doi: 10.1126/science.1254927 CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Kicheva A, Briscoe J (2015) Developmental pattern formation in phases. Trends Cell Biol 25:579–591. doi: 10.1016/j.tcb.2015.07.006 CrossRefPubMedGoogle Scholar
  50. 50.
    Dessaud E, McMahon AP, Briscoe J (2008) Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network. Development 135:2489–2503. doi: 10.1242/dev.009324 CrossRefPubMedGoogle Scholar
  51. 51.
    Cohen M, Kicheva A, Ribeiro A, Blassberg R, Page KM, Barnes CP, Briscoe J (2015) Ptch1 and Gli regulate Shh signalling dynamics via multiple mechanisms. Nat Commun 6:6709. doi: 10.1038/ncomms7709 CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Qi Y, Tan M, Hui CC, Qiu M (2003) Gli2 is required for normal Shh signaling and oligodendrocyte development in the spinal cord. Mol Cell Neurosci 23:440–450. doi: 10.1016/S1044-7431(03)00067-8 CrossRefPubMedGoogle Scholar
  53. 53.
    Al Oustah A, Danesin C, Khouri-Farah N, Farreny MA, Escalas N, Cochard P, Glise B, Soula C (2014) Dynamics of sonic hedgehog signaling in the ventral spinal cord are controlled by intrinsic changes in source cells requiring sulfatase 1. Development 141:1392–1403. doi: 10.1242/dev.101717 CrossRefPubMedGoogle Scholar
  54. 54.
    Barres BA, Hart IK, Coles HS, Burne JF, Voyvodic JT, Richardson WD, Raff MC (1992) Cell death and control of cell survival in the oligodendrocyte lineage. Cell 70:31–46. doi: 10.1016/0092-8674(92)90531-G CrossRefPubMedGoogle Scholar
  55. 55.
    Barres BA, Jacobson MD, Schmid R, Sendtner M, Raff MC (1993) Does oligodendrocyte survival depend on axons? Curr Biol 3:489–497. doi: 10.1016/0960-9822(93)90039-Q CrossRefPubMedGoogle Scholar
  56. 56.
    Hirano K, Ohgomori T, Kobayashi K, Tanaka F, Matsumoto T, Natori T, Matsuyama Y, Uchimura K, Sakamoto K, Takeuchi H, Hirakawa A, Suzumura A, Sobue G, Ishiguro N, Imagama S, Kadomatsu K (2013) Ablation of KS accelerates early phase pathogenesis of ALS. PLoS One 8:e66969. doi: 10.1371/journal.pone.0066969 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Hirokazu Hashimoto
    • 1
  • Yugo Ishino
    • 1
  • Wen Jiang
    • 1
    • 2
  • Takeshi Yoshimura
    • 1
    • 2
  • Yoshiko Takeda-Uchimura
    • 3
  • Kenji Uchimura
    • 3
  • Kenji Kadomatsu
    • 3
  • Kazuhiro Ikenaka
    • 1
    • 2
    Email author
  1. 1.Division of Neurobiology and BioinformaticsNational Institute for Physiological SciencesOkazakiJapan
  2. 2.Department of Physiological Sciences, School of Life SciencesSOKENDAI (The Graduate University for Advanced Studies)HayamaJapan
  3. 3.Department of BiochemistryNagoya University Graduate School of MedicineNagoyaJapan

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