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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 migration parallel to the slice section, such as radial cell migration1,2,3. The slice culture system is also used for detecting tangential cell migration4,5, but it is not suitable for directional analysis in cases where the cells disperse perpendicular to the slice section.
The optic tectum is composed of a multilayered structure, formed by radial and tangential cell migration during embryonic development. Tectal layer formation depends primarily on radial migration of postmitotic neuronal precursor cells from the ventricular zone, and their final destination in the layers correlates with their birth date in the ventricular zone6. As for tangential migration, we previously reported two streams of migrations in the middle and superficial layers in a developing chick optic tectum. In the middle layers during E6-E8, the bipolar cells with a long leading process and a thin trailing process migrate dorsally or ventrally along the axon fasciculus of tectal efferent axons that run dorso-ventrally7. After this axophilic migration, the cells differentiate into multipolar neurons located in the deep layers. In the superficial layers during E7-E14, the migrating cells disperse horizontally by reforming a branched leading process and scatter into multiple directions8. After dispersing migration, the latter cells eventually differentiate into superficial neurons of various morphologies. In both cases, a flat-mount culture is efficient to observe cell movement parallel to the pial surface.
Here, we present a protocol for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum7,8. 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 and migration direction. The goal of this method is to facilitate detection of both individual cell behavior in the long term and the collective action of a group of cells in the horizontal plane.
1 . Electroporation In Ovo
2. Flat-Mount Culture on the Cell Insert
3. Time-Lapse Imaging
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...
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...
The authors declare that they have no competing financial interests.
This work was supported by JSPS KAKENHI Grant Number 15K06740 to Y.W.
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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