Kaede‐Centrin1 Labeling of Mother and Daughter Centrosomes in Mammalian Neocortical Neural Progenitors

Janice H. Imai1, Xiaoqun Wang2, Song‐Hai Shi1

1 BCMB Allied Program, Weill Cornell Medical College, New York, New York, 2 Developmental Biology Program, Memorial Sloan‐Kettering Cancer Center, New York, New York
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5A.5
DOI:  10.1002/9780470151808.sc05a05s15
Online Posting Date:  October, 2010
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Abstract

The importance of the centrosome in regulating basic cellular processes and cell fate decisions has become increasingly evident from recent studies tracing the etiology of developmental disorders to mutations in genes encoding centrosomal proteins. This unit details a protocol for a fluorescence‐based pulse labeling of centrioles of neural progenitor cells in the developing neocortex of mice. In utero electroporation of Kaede‐Centrin1 followed by in utero or ex vivo photoconversion allows a direct monitoring of the inheritance of centrosomes containing centrioles of different ages in dividing neocortical neural progenitors (i.e., radial glial cells). This is achieved by combining the irreversible photoconversion capacity of the Kaede protein from green to red fluorescence with the faithful duplication of the centrosome during each cell cycle. After two mitotic divisions following photoconversion, mother centrosomes containing the original labeled centriole appear in both red and green fluorescence, and can be distinguished from daughter centrosomes which appear in green fluorescence only. This facilitates the study of the inheritance and behavior of the mother and daughter centrosomes in asymmetric cell divisions in the developing mammalian neocortex. Curr. Protoc. Stem Cell Biol. 15:5A.5.1‐5A.5.12. © 2010 by John Wiley & Sons, Inc.

Keywords: centrosome; Kaede‐Centrin1; mother and daughter centrosomes; photoconversion; neocortex; radial glia progenitor cell; in utero electroporation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Utero Electroporation of the Kaede‐Centrin1 Plasmid
  • Basic Protocol 2: In Utero Photoconversion of the Kaede Reporter
  • Alternate Protocol 1: Ex Vivo Photoconversion in Brain Slices and Time‐Lapse Imaging
  • Support Protocol 1: Tissue Preservation, Imaging, and Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Utero Electroporation of the Kaede‐Centrin1 Plasmid

  Materials
  • Timed‐pregnant female mouse, E13.5
  • Isofluorane
  • Ethanol and iodine wipes
  • Phosphate‐buffered saline, sterile, 37°C
  • Plasmid DNA (Kaede‐Centrin1, 3.0 µg/µl; contact S.‐H. Shi, shis@mskcc.org) mixed with Fast Green dye (Fisher Biotech) (1% w/v in PBS, 1 µl dye solution per 10 µl DNA solution); the use of endotoxin‐free plasmid DNA (e.g., prepared using Qiagen Endotoxin‐free Maxiprep kit) is recommended
  • Antibiotic‐PBS solution: penicillin (100 IU/ml)/streptomycin (100 mg/ml) in PBS, warmed to 37°C
  • Antibiotic/analgesic solution (Duane Reade Triple Antibiotic Ointment plus Pain Relief; contains Bacitracin, zinc, neomycin sulfate, polymyxin B sulfate, and the analgesic pramoxine)
  • Isoflurane induction chamber (VetEquip, e.g., cat. no. 901807)
  • Isofluorane dispenser (VetEquip) and nose cone
  • Heating pad
  • Disposable underpads
  • Hair clipper
  • Surgical instruments
  • 10‐ml sterile syringe
  • Sterile gauze
  • Sterile spatula
  • Glass capillary injection needles (tip diameter ∼100 µm; beveled; see recipe)
  • Electroporation system (BTX, ECM830, Harvard Apparatus)
  • Silk or nylon sutures
  • Wound clips (7‐mm; CellPoint Scientific)
  • Cotton‐tipped applicators

Basic Protocol 2: In Utero Photoconversion of the Kaede Reporter

  Materials
  • Pregnant mouse with electroporated embryos at E13.5 ( protocol 1)
  • Source of violet light (e.g., a fluorescence dissection microscope with a mercury lamp and a 4′,6‐diamidino‐2‐phenylindole [DAPI] filter)
  • Wound clip remover
  • Additional reagents and equipment for anesthesia of the mouse and surgically removing and replacing embryos ( protocol 1)

Alternate Protocol 1: Ex Vivo Photoconversion in Brain Slices and Time‐Lapse Imaging

  • Pregnant mouse with electroporated embryos at E13.5 ( protocol 1)
  • 4% (w/v) agarose in artificial cerebrospinal fluid (ASCF; see recipe)
  • Brain slice culture medium (see recipe)
  • Vibratome (Leica Microsystems)
  • Slice culture insert (Millicell, Millipore)
  • Glass‐bottom petri dish (MatTek Corporation)
  • Inverted microscope (e.g., Axiovert 200, Zeiss) with a mercury lamp and a DAPI filter
  • Humidified incubator (37°C and 5% CO 2)

Support Protocol 1: Tissue Preservation, Imaging, and Analysis

  Materials
  • 4% Avertin (2,2,2‐tribromoethanol; Sigma) in PBS
  • 4% paraformaldehyde (PFA), freshly prepared (cold)
  • Pregnant mouse with electroporated embryos that have been subjected to photoconversion ( protocol 2)
  • Phosphate‐buffered saline (PBS), pH 7.4 (cold)
  • 4% (w/v) paraformaldehyde in PBS, freshly prepared
  • 3% to 4% (w/v) agarose in PBS
  • PBS with 0.03% (w/v) sodium azide
  • 1‐ml syringe with 30‐G, 1‐in. needle for Avertin injection
  • Dissection dish (60‐mm Pyrex dish lined with 7 to 8 mm of Sylgard 184 Silicone Elastomer)
  • Insect pins (Fine Scientific Tools)
  • Dissection microscope
  • Dissection tools for removing brain (fine forceps, scissors, spatula)
  • Two 10‐ml syringes connected by a stopcock, with 30‐G, 0.5‐in needle attached to tubing (Fig. A)
  • Vibratome (Leica Microsystems)
  • 48‐well culture plate
  • Microscope equipped for confocal laser scanning microscopy (Olympus FV1000)
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Figures

Videos

Literature Cited

Literature Cited
   Anderson, C.T. and Stearns, T. 2009. Centriole age underlies asynchronous primary cilium growth in mammalian cells. Curr. Biol. 19:1498‐1502.
   Daza, R.A.M., Englund, C., and Hevner, R.F. 2007. Organotypic slice culture of embryonic brain tissue. Cold Spring Harbor Protoc. doi:10.1101/pdb.prot4914.
   Delattre, M. and Gonczy, P. 2004. The arithmetic of centrosome biogenesis. J. Cell Sci. 117:1619‐1630.
   Elias, L. and Kriegstein, A. 2007. Organotypic slice culture of E18 rat brains. J. Vis. Exp. 6:235.
   Götz, M. and Huttner, W.B. 2005. The cell biology of neurogenesis. Nat. Rev. Mol. Cell Biol. 6:777‐788.
   Mitsuhashi, T. and Takahashi, T. 2009. Genetic regulation of proliferation/differentiation characteristics of neural progenitor cells in the developing neocortex. Brain Dev. 31:553‐557.
   Molyneaux, B.J., Arlotta, P., Menezes, J.R., and Macklis, J.D. 2007. Neuronal subtype specification in the cerebral cortex. Nat. Rev. Neurosci. 8:427‐437.
   Nigg, E.A. and Raff, J.W. 2009. Centrioles, centrosomes, and cilia in health and disease. Cell 139:663‐678.
   Schatten, H. and Sun, Q.Y. 2010. The role of centrosomes in fertilization, cell division and establishment of asymmetry during embryo development. Semin. Cell Dev. Biol. 21:174‐184.
   Tabata, H. and Nakajima, K. 2008. Labeling embryonic mouse central nervous system cells by in utero electroporation. Dev. Growth Differ. 50:507‐511.
   Wang, X., Tsai, J.W., Imai, J.H., Lian, W.N., Vallee, R.B., and Shi, S.‐H. 2009. Asymmetric centrosome inheritance maintains neural progenitors in the neocortex. Nature 461:947‐955.
   Yamashita, Y.M. and Fuller, M.T. 2008. Asymmetric centrosome behavior and the mechanisms of stem cell division. J. Cell Biol. 180:261‐266.
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