Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Summary

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.

Abstract

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.

Introduction

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 migration^1,2,3. The slice culture system is also used for detecting tangential cell migration^4,5, 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 tectum^7,8. 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.

Protocol

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 chicken eggs horizontally at 38 °C in 70% relative humidity.
3. After 2.5 days, eliminate 5 mL of albumen (egg white) from the pointed side of the egg using 20 mL syringe with an 18 gauge needle and seal the needle hole with tape.
4. Cut the top of the egg shell with curved scissors to open a hole 2 cm in diameter and check the developmental stage of the embryo under the stereomicroscope (stage 17 of Hamburger and Hamilton⁹). Seal the hole again with tape.
   NOTE: This step can be performed at any time during E2.5-E3.0. Steps 1.3 and 1.4 lower the level of the embryo in the egg to avoid a tight attachment of the growing embryo and the blood vessels to the top shell.
5. Continue incubation until E5.5.
6. Before electroporation, prepare 10 µL of the mentioned plasmid DNA colored with 0.5 µL of Fast Green (25 mg/mL), 10 mL of autoclaved phosphate buffered saline (PBS) containing penicillin (100 units/mL) and streptomycin (100 μg/mL), and micropipettes made from glass capillary tubes using a micropipette processor. Cut the distal tip of the micropipette to make an appropriate size of pore depending on the amount of DNA for injection.
7. Cut the sealed top shell with scissors to reopen a hole 2.5 cm in diameter and set the egg stably under the stereo microscope. Add a few drops of PBS into the egg. Peel off the allantoic membrane covering the embryo without hemorrhage using two fine forceps. Remove the amnion from the head of the embryo.
8. While turning the head with a microspatula, inject 0.1-1 μL of the colored DNA into the ventricular cavity of the left optic tectum using a micropipette connected to a suction tube.
   NOTE: The amount of DNA injected depends on the purpose of the experiment. Concentrated DNA solution should sink and attach along the ventricular wall.
9. Place a pair of forcep-type electrodes (3 mm square electrodes with 7 mm distance) so that the target area of the optic tectumis placed in between (Figure 1, Step 1).
   NOTE: The DNA is electroporated to the anode side.
10. Charge a pre-pulse of 30 V, 1 ms with a 5 ms interval and four subsequent pulses of 6 V, 5 ms with a 10 ms interval using the pulse generator.
    NOTE: The electronic condition depends on the size and distance of the electrodes. It should be strong enough to achieve efficient transfection, but it must be modest enough to assure the normal development of the target tissue.
11. Seal the hole with tape and continue incubation. Soak the electrodes in PBS to remove albumen with an interdental brush in a plastic dish. Repeat 1.7-1.11 for each egg.

2. Flat-Mount Culture on the Cell Insert

1. One day before the flat-mount culture, prepare the coated cell culture insert. Float some cell culture inserts on the autoclaved distilled water in a 10-cm cell culture dish. Load a solution of 8 µg/mL laminin and 80 µg/mL poly-L-lysine to cover the inserts and leave the floated inserts in the cell culture CO₂ incubator at 37 °C overnight.
   NOTE: On the following day, the coated inserts can be stored at 4 °C.
2. On day of the culture at E7.0, remove the laminin-poly-L-lysine solution from the insert and place the insert in a glass bottom dish (Figure 1, Step 2) filled with 1.1 mL of culture medium (60% reduced serum medium, 20% F12, 10% fetal bovine serum, 10% chick serum, 50 units/mL penicillin, 50 μg/mL streptomycin). Keep the dish in the cell culture CO₂ incubator at 37 °C.
   NOTE: The medium can be used throughout the culture period for three days without change.
3. Prepare the culture setup. Set a humid chamber unit on an inverted confocal microscope with a gas flow of 40% O₂ and 5% CO₂ (gas controller) at 38 °C (temperature controller).
4. Cut the head of the embryo with scissors. Pinch the head out with the forceps into the ice-cold Hanks' Balanced Salt solution (HBSS) in a 6-cm cell culture dish.
5. Isolate the electroporated optic tectum using two fine forceps. Transfer the tectum with a plastic dropper into another dish filled with ice-cold HBSS. Use the concaved glass dish based with black silicon as a saucer for cutting.
6. Check the position of the labeling under the fluorescence stereoscopic microscope. Cut out the tectal tissue surrounding the labeling area with a microsurgical knife. Make sure the direction of the tissue in the tectum (anterior-posterior, dorsal-ventral).
7. Transfer the labeled tissue with a plastic dropper to the insert so that the pia side is attached to the insert. Lay the tectal tissue in the desired direction and remove excess HBSS (Figure 1, Step 2).
8. Repeat 2.4-2.7 in order to prepare other tissues on the same insert. Place the dish in the prewarmed chamber in the inverted confocal microscope (Figure 1, Step 3).

3. Time-Lapse Imaging

1. Check the fluorescent labelling and focus the microscopic field in the inverted confocal microscope. Start the laser confocal units and try sampling the confocal scan to adjust the direction and position of the tissue along the X and Y-axis in the field. Use the 10X or 20X objective lens without immersion oil.
2. Select the scanning size (e.g., 512x512 dpi). Decide the interval and total range of the confocal scan along the z-axis. Take a 5 or 10 µm interval for the 100 µm range. Decide the time interval of the confocal imaging and total imaging duration (e.g., 10 min intervals over 48 h).
   NOTE: To avoid laser phototoxicity, prohibit scanning in the high scanning size with short z-intervals for the long z-range, or with short time intervals during the long imaging duration. For example, when applying a high scanning size (e.g., 1024x1024 dpi), decrease the number of scans along the z-axis.
3. Set the parameters of the confocal running program described in 3.2 and start imaging.
4. After the imaging, combine the confocal images at different z-axis to provide z-stack images with fine focal adjustment at every time point.
5. Append the z-stack images at different time-point and construct a time-lapse movie in the AVI format.

Results

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...

Discussion

The protocol described above is optimized for detecting cell migration in superficial layers^6,8. 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...

Materials

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