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Developmental Biology

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Published: October 24th, 2018

DOI:

10.3791/58506

1Department of Neuroanatomy and Embryology, School of Medicine, Fukushima Medical University

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

The study of cell migration has been progressing with the advancing technique of live imaging. After fluorescent cell labeling, the temporal movement of labeled cells in a culture dish or in vivo can be recorded under video microscopy. In the study of neural development, the morphological changes of migrating cells or elongating axons have been analyzed using time-lapse imaging. For effective imaging, it is essential to apply a suitable method for fluorescent cell labeling and tissue preparation, based on the purpose of the experiment and analysis. For analyzing cell migration in the developing brain, slice culture has been commonly used to observe cell migra....

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1 . Electroporation In Ovo

  1. Prepare the expression plasmid DNA for fluorescent labeling in high concentration. Isolate DNA from 200 mL of bacterial culture by the alkaline lysis method using anion-exchange columns according to the manufacturer's protocol (Table of Materials). Mix pCAGGS-EGFP and pCAGGS-mCherryNuc at a final concentration of 4 µg/µL each.
    NOTE: Endotoxin-free plasmid DNA purification may be preferred for electroporation.
  2. Incubate fertile .......

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Figure 2 shows the visualized superficial tangential migration in a flat-mount culture at an elapsed time (0, 9, 18, 27 h) after onset of recording. Movie 1 is a time-lapse movie of 10 min-intervals over a period of 28 h and 50 min. The frame is selected for focusing on the migrating cells from the labeled lower-left corner of the frame to the unlabeled space (Figure 3A). The mass movement of the.......

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The protocol described above is optimized for detecting cell migration in superficial layers6,8. It is applicable for detecting middle layer migration streams (Movie 5)6,7, just by shifting the timing of the electroporation (E5.5 to E4.5) and the onset of culture and imaging (E7.0 to E6.0).

The presented procedure is composed of cell labeling b.......

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This work was supported by JSPS KAKENHI Grant Number 15K06740 to Y.W.

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Name Company Catalog Number Comments
Materials
NucleoBond Xtra Midi Plus EF MACHEREY-NAGEL 740422.5 endotoxin-free plasmid DNA purification kit
20 ml syringe TERUMO SS-20ESZ
18 gauge needle TERUMO NN-1838R
Fast Green Wako 061-00031
100x penicillin and streptomycin Gibco 15140-122
glass capillary tube Narishige G-1
cell culture insert Millipore Millicell CM-ORG
Laminin SIGMA L2020 coating of culture insert
poly-L-Lysine Peptide Institute 3075 coating of culture insert
glass bottom dish Matsunami D11130H
Opti-MEM Gibco 31985-070 culture medium
F12 Gibco 11765-054 culture medium
fetal bovine serum Gibco 12483 culture medium
chick serum Gibco 16110082 culture medium
10xHBSS Gibco 14065-056
microsurgical knife Surgical specialties cooperation 72-1501
Name Company Catalog Number Comments
Equipment
curved scissors AS ONE No.11
micropipette processor SUTTER INSTRUMENT P97/IVF
forceps-type electrode BEX LF646P3x3
pulse generator BEX CUY21EX electroporator
fluorescence stereoscopic microscope Leica MZ16F
inverted fluorescence microscope Olympus IX81
gas controller Tokken MIGM/OL-2
temperature controller Tokai Hit MI-IBC
laser confocal unit Olympus FV300

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  8. Watanabe, Y., Sakuma, C., Yaginuma, H. Dispersing movement of neuronal tangential migration in superficial layers of the developing chick optic tectum. Developmental Biology. 437, 131-139 (2018).
  9. Hamburger, V., Hamilton, H. L. A series of normal stages in the development of the chick embryo. Journal of Morphology. 88, 49-92 (1951).
  10. Cordelières, F. P., et al. Automated cell tracking and analysis in phase-contrast videos (iTrack4U): development of Java software based on combined mean-shift processes. PLOS One. 8, e81266 (2013).
  11. Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9, 676-682 (2012).

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