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 migrating cells (GFP; upper left panel, Movie 1) and their nuclei (mCherry-Nuc; upper right panel) can be observed with the merged movie (lower panel, Movie 1). Directionality of the cell migration can be examined by focusing on the dispersing cells from the labeled center to all directions (Movie 2, Figure 3B).
The clear images of the nuclear movement (Movie 2; mCherry-Nuc, right panel) allows us to trace the cell nuclear migration by automatic tracking using a Particle Tracker plugin10 of a Fiji image processing application of ImageJ11 (Movie 3, Figure 3B). Temporal changes of the trajectories of the tangential migration can be visualized to prove the dispersing migration in omni-directions.
Individual cell behavior with the sequential morphological change of the leading process, trailing process and nuclei can be manifested with higher magnification images of 5 min-intervals (Movie 4, Figure 3C).
Another type of tangential migration in the middle layers7 can be visualized using a similar protocol (Movie 5, Figure 3D). Bidirectional linear migration along the axon fasciculus running dorsal to ventral (top to down) is evident7.
Figure 1. Protocol flow chart. Step 1: Electroporation in ovo. Step 2: Laying tectal tissue so that the pia side is attached to the insert. Step 3: Inverted fluorescent microscope with laser confocal unit. Please click here to view a larger version of this figure.
Figure 2. Visualization of superficial tangential migration in flat-mount culture. The tangentially migrating cells (GFP; upper panel) and the nucleus (mCherry-Nuc; lower panel) in the superficial layers of the optic tectum are shown at 0, 9, 18, 27 h after onset of the culture from E7.0. The cells at the lower-left corner of the frame are labeled at 0 h (see Figure 3A). Scale bar: 100 µm. Please click here to view a larger version of this figure.
Figure 3. Schematic figure illustrating the mount conditions for time-lapse imaging. The shape of the fluorescent labeling (green), initial direction of cell migration (magenta), and video frame (black square) were illustrated with magnification of the objective lens (obj) and digital zoom (zoom) in the upper field, with day of electroporation (EP) and onset of culture (culture) in the bottom field. (A) Movie 1, (B) Movie 2 and 3, (C) Movie 4, (D) Movie 5. Please click here to view a larger version of this figure.
Movie 1. Visualization of superficial tangential migration in a flat-mount culture. Movement of the tangentially migrating cells (GFP; upper left panel) and their nuclei (mCherry-Nuc; upper right panel) in the superficial layers of the optic tectum after onset of the culture from E7.0. The merged image is shown in the lower panel. The cells at the lower-left corner of the frame are labeled at 0 h (see Figure 3A), and the time-lapse images were captured over 28 h and 50 min. Scale bar: 100 µm. Please click here to view this video. Right-click to download.
Movie 2. Dispersing movement of the tangentially migrating cells (GFP; left panel) and their nuclei (mCherry-Nuc; right panel) from the center of both panels are shown over 48 h (see Figure 3B). Scale bar: 100 µm. Please click here to view this video. Right-click to download.
Movie 3. Trajectories of tangential migration. Displacement of the cell nucleus (the right panel of Movie 2) was tracked to visualize the trajectories of the tangential migration. Scale bar; 100 µm. Please click here to view this video. Right-click to download.
Movie 4. Individual cell behavior in higher magnification. Movement of the individual cells (GFP; left panel) and their nuclei (mCherry-Nuc; right panel) are shown in higher magnification over 24 h (see Figure 3C). Branching process of the leading process can be recognized. Scale bar: 100 µm. Please click here to view this video. Right-click to download.
Movie 5. Middle layer migration. Movement of the tangentially migrating cells (GFP; left panel) and their nucleus (mCherry-Nuc; right panel) in the tectal middle layers are shown over 24 h after onset of the culture from E6.0 (see Figure 3D). Linear migration along dorso-ventral axis (top to down) is remarkable. Scale bar: 100 µm. Please click here to view this video. Right-click to download.
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 by electroporation in ovo, flat-mount culture and time-lapse confocal imaging (Figure 1). First of all, it is a prerequisite that the culture conditions should be optimized to keep the tissue healthy and growing normally as in vivo. It is also crucial to ensure that the orientation of the tissue is suitable for detecting cell migration. For such purposes, we apply a flat-mount culture on cell insert, which facilitates observation of horizontal cell dispersion and supplies rich medium with high oxygen. After ensuring the culture condition and orientation, it is critical for visualization to adjust the conflicting conditions of better fluorescent labelling with the least electronic and photo damages. For better labeling, we can choose an expression vector with an efficient promoter and electroporate concentrated DNA. For achieving least damage, it is important to moderate the electric condition of the electroporation, lower the DNA concentration, and minimize the total time of laser irradiation.
An advantage of this protocol using the flat-mount culture on the cell insert is that we can observe tangential cell migration in the long term. Generally, it is difficult to determine how long the tissue in culture maintains the physiological conditions in comparison to that in ovo. At the very least, superficial tangential migration continues over 72 h after onset of culture at E7.0, which shows normal cellular dispersion similar to that in ovo8. In addition, fresh tissue is prepared at the start of the culture and imaging to observe migration at later stages. It is also advantageous that cell displacement can be followed over a long period because the cells remain moving horizontally in the superficial flat sheets of the tectum, which is close to the objective lens. Using the confocal system also facilitates tracking cell movement along the z-axis. On the other hand, a disadvantage of this method is that the flat-mount culture may not always be relevant to recapitulate other types of migration such as radial migration. When the method is applied to observe radial migration in the tectal slice on the insert, the thickness of the tectal tissue does not increase as rapidly as that in ovo. The slice culture method in the collagen gel may be better to recapitulate such layer development.
Since our method enables the observation of horizontal movement parallel to the culture insert, it can potentially be applied for detecting sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue. For example, after labeling commissural axons in the developing neural tube by electroporation, movements of pre- and post-crossing axons over the floor plate can be visualized in the open-book culture incising the roof plate. Provided that the appropriate culture condition on the cell insert is available for reproducing in vivo conditions, our method provides an effective technique to visualize horizontal movements of various cell types and structures.
The authors have nothing to disclose.
This work was supported by JSPS KAKENHI Grant Number 15K06740 to Y.W.
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 |