Advertisement

Astrocytes pp 49-66 | Cite as

Fluorescence-Activated Cell Sorting-Based Isolation and Characterization of Neural Stem Cells from the Adult Zebrafish Telencephalon

  • Rossella Di Giaimo
  • Sven Aschenbroich
  • Jovica Ninkovic
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1938)

Abstract

Adult mammalian brain, including humans, has rather limited addition of new neurons and poor regenerative capacity. In contrast, neural stem cells (NSC) with glial identity and neurogenesis are highly abundant throughout the adult zebrafish brain. Importantly, the activation of NSC and production of new neurons in response to injuries lead to the brain regeneration in zebrafish brain. Therefore, understanding of the molecular pathways regulating NSC behavior in response to injury is crucial in order to set the basis for experimental modification of these pathways in glial cells after injury in the mammalian brain and to elicit neuronal regeneration. Here, we describe the procedure that we successfully used to prospectively isolate NSCs from adult zebrafish telencephalon, extract RNA, and prepare cDNA libraries for next generation sequencing (NGS) and full transcriptome analysis as the first step toward understanding regulatory mechanisms leading to restorative neurogenesis in zebrafish. Moreover, we describe an alternative approach to analyze antigenic properties of NSC in the adult zebrafish brain using intracellular fluorescence activated cell sorting (FACS). We employ this method to analyze the number of proliferating NSCs positive for proliferating cell nuclear antigen (PCNA) in the prospectively isolated population of stem cells.

Key words

Neural stem cells Zebrafish Neural stem cell purification Intracellular FACS 

References

  1. 1.
    Grupp L, Wolburg H, Mack AF (2010) Astroglial structures in the zebrafish brain. J Comp Neurol 518(21):4277–4287CrossRefGoogle Scholar
  2. 2.
    Adolf B, Chapouton P, Lam CS et al (2006) Conserved and acquired features of adult neurogenesis in the zebrafish telencephalon. Dev Biol 295(1):278–293CrossRefGoogle Scholar
  3. 3.
    Ganz J, Kaslin J, Hochmann S et al (2010) Heterogeneity and Fgf dependence of adult neural progenitors in the zebrafish telencephalon. Glia 58(11):1345–1363CrossRefGoogle Scholar
  4. 4.
    März M, Chapouton P, Diotel N et al (2010) Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon. Glia 58(7):870–888PubMedGoogle Scholar
  5. 5.
    Rothenaigner I, Krecsmarik M, Hayes JA et al (2011) Clonal analysis by distinct viral vectors identifies bona fide neural stem cells in the adult zebrafish telencephalon and characterizes their division properties and fate. Development 138(8):1459–1469CrossRefGoogle Scholar
  6. 6.
    Baumgart EV, Barbosa JS, Bally-Cuif L et al (2012) Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis. Glia 60(3):343–357CrossRefGoogle Scholar
  7. 7.
    Barbosa JS, Sanchez-Gonzalez R, Di Giaimo R et al (2015) Live imaging of adult neural stem cell behaviour in the intact and injured zebrafish brain. Science 348(6236):789–793CrossRefGoogle Scholar
  8. 8.
    Kyritsis N, Kizil C, Zocher S et al (2012) Acute inflammation initiates the regenerative response in the adult zebrafish brain. Science 338(6112):1353–1356CrossRefGoogle Scholar
  9. 9.
    Kizil C, Dudczig S, Kyritsis N et al (2012) The chemokine receptor cxcr5 regulates the regenerative neurogenesis response in the adult zebrafish brain. Neural Dev 7:27CrossRefGoogle Scholar
  10. 10.
    Rodriguez Viales R, Diotel N, Ferg M et al (2015) The helix-loop-helix protein id1 controls stem cell proliferation during regenerative neurogenesis in the adult zebrafish telencephalon. Stem Cells 33(3):892–903CrossRefGoogle Scholar
  11. 11.
    Bernardos R, Raymond P (2006) GFAP transgenic zebrafish. Gene Expr Patterns 6:1007–1013CrossRefGoogle Scholar
  12. 12.
    Kelsh RN, Brand M, Jiang YJ et al (1996) Zebrafish pigmentation mutations and the processes of neural crest development. Development 123:369–389PubMedGoogle Scholar
  13. 13.
    Westerfield M (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th edn. Univ of Oregon Press, EugeneGoogle Scholar
  14. 14.
    Fischer J, Beckervordersandforth R, Tripathi P et al (2011) Prospective isolation of adult neural stem cells from the mouse subependymal zone. Nat Protoc 6(12):1981–1989CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Rossella Di Giaimo
    • 1
  • Sven Aschenbroich
    • 2
    • 3
    • 4
  • Jovica Ninkovic
    • 2
    • 3
    • 4
  1. 1.Department of BiologyUniversity of Naples Federico II, Complesso Universitario Monte S. AngeloNaplesItaly
  2. 2.Institute of Stem Cell Research, Helmholtz Center MunichMunichGermany
  3. 3.Physiological Genomics, Biomedical CenterUniversity of MunichMunichGermany
  4. 4.Institute of Cell Biology, Biomedical CenterUniversity of MunichMunichGermany

Personalised recommendations