In Vitro Analysis of Mouse Mesencephalic Neural Crest Development

Kazuo Ito1, Kyohei Fujita1

1 Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka, Japan
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.23
DOI:  10.1002/0471142301.ns0323s56
Online Posting Date:  July, 2011
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Abstract

The neural crest is a unique structure in vertebrates. Neural crest cells play important roles in the formation of organs that characterize the vertebrate body plan. In this unit, we describe a primary culture method for mouse mesencephalic neural crest cells. The neural crest cells cultured by this method actively proliferate and differentiate into various cell types that originate from cranial neural crest cells, such as chondrocytes, neurons, and glia. Therefore, this primary culture method is useful for analyzing the development of mouse mesencephalic neural crest cells. Curr. Protoc. Neurosci. 56:3.23.1‐3.23.8. © 2011 by John Wiley & Sons, Inc.

Keywords: mesencephalon; neural crest cells; primary culture

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Primary Culture of Mouse Mesencephalic Neural Crest Cells
  • Support Protocol 1: Preparation of Chick Embryo Extract (CEE)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Primary Culture of Mouse Mesencephalic Neural Crest Cells

  Materials
  • Distilled water (DDW) sterilized by autoclave
  • Culture medium sterilized by filtration (see recipe)
  • E8.0‐8.5 mouse embryos (6 to 12 somite stages)
  • Hanks' balanced salt solution sterilized by filtration (HBSS; see recipe)
  • Collagen‐coated plastic 35‐mm tissue culture dishes, sterile
  • 37°C humidified incubator containing 5% CO 2
  • 60‐mm glass dishes, sterile
  • Stereomicroscope
  • Fine scissors sterilized by autoclave
  • Fine forceps sterilized by autoclave
  • Three‐well blood agglutination plate sterilized by autoclave
  • 1‐ml disposable plastic measuring pipets, sterile
  • Electrically sharpened tungsten needles sterilized by flame
  • Disposable glass Pasteur pipets sterilized by autoclave
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.NOTE: Steps 2 to 10, 12, 15, and 16 are performed under a stereomicroscope. All steps should be carried out on a clean bench.

Support Protocol 1: Preparation of Chick Embryo Extract (CEE)

  Materials
  • 11‐day‐old chick embryos
  • 70% ethanol
  • HBSS sterilized by filtration (see recipe)
  • α‐modified minimum essential medium with ribonucleotides and deoxyribonucleotides (α‐MEM; Sigma, cat. no. M0644) sterilized by filtration
  • Ice
  • 38°C incubator
  • Curved fine scissors sterilized by autoclave
  • Straight fine scissors sterilized by autoclave
  • Large forceps sterilized by autoclave
  • 500‐, 300, and 100‐ml beakers sterilized by autoclave
  • 100‐mm plastic petri dishes, sterile
  • Fine forceps sterilized by autoclave
  • 1‐liter beaker covered with double‐layer gauze sterilized by autoclave
  • Large scissors sterilized by autoclave
  • 50‐ml disposable syringe, sterile
  • 1‐liter conical flask with a cap containing a stirring rod sterilized by autoclave
  • Scale
  • Centrifuge tubes for refrigerated centrifugation sterilized by autoclave
  • Centrifuge
  • Ultracentrifuge tubes sterilized by autoclave
  • Ultracentrifuge
  • 10‐ml disposable plastic measuring pipets, sterile
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.NOTE: Steps 3 to 11, 13, and 14 should be performed on a clean bench.
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Figures

Videos

Literature Cited

Literature Cited
   Baroffio, A., Dupin, E., and Le Douarin, N.M. 1988. Clone‐forming ability and differentiation potential of migratory neural crest cells. Proc. Natl. Acad. Sci. U.S.A. 85:5325‐5329.
   Chan, W.Y. and Tam, P.P.L. 1988. A morphological and experimental study of the mesencephalic neural crest cells in the mouse embryo using wheat germ agglutinin‐gold conjugate as the cell marker. Development 102:427‐442.
   Cohen, A.M. and Konigsberg, I.R. 1975. A clonal approach to the problem of neural crest determination. Dev. Biol. 46:262‐280.
   Hall, B.K. 1999. The Neural Crest in Development and Evolution. Springer, New York.
   Ijuin, K., Nakanishi, K., and Ito, K. 2008. Different downstream pathways for Notch signaling are required for gliogenic and chondrogenic specification of mouse mesencephalic neural crest cells. Mech. Dev. 125:462‐474.
   Ito, K. and Takeuchi, T. 1984. The differentiation in vitro of the neural crest cells of the mouse embryo. J. Embryol. Exp. Morph. 84:49‐62.
   Ito, K. and Morita, T. 1995. Role of retinoic acid in mouse neural crest cell development in vitro. Dev. Dyn. 204:211‐218.
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   Nakanishi, K., Chan, Y.S., and Ito, K. 2007. Notch signaling is required for the chondrogenic specification of mouse mesencephalic neural crest cells. Mech. Dev. 124:190‐203.
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   Ota, M. and Ito, K. 2006. BMP and FGF‐2 regulate neurogenin‐2 expression and the differentiation of sensory neurons and glia. Dev. Dyn. 235:646‐655.
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   Sieber‐Blum, M. and Cohen, A. M. 1980. Clonal analysis of quail neural crest cells: They are pluripotent and differentiate in vitro in the absence of noncrest cells. Dev. Biol. 80:96‐106.
   Sugiura, K. and Ito, K. 2010. Roles of Ets‐1 and p70S6 kinase in chondrogenic and gliogenic specification of mouse mesencephalic neural crest cells. Mech. Dev. 127:169‐182.
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