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

1 . Electroporation In Ovo
<ol>
	<li>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&#39;s protocol (Table of Materials). Mix pCAGGS-EGFP and pCAGGS-mCherryNuc at a final concentration of 4 &#181;g/&#181;L each. 
	NOTE: Endotoxin-free plasmid DNA purification may be preferred for electroporation.</li>
	<li>Incubate fertile chicken eggs horizontally at 38 &#176;C in 70% relative humidity.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Continue incubation until E5.5.</li>
	<li>Before electroporation, prepare 10 &#181;L of the mentioned plasmid DNA colored with 0.5 &#181;L of Fast Green (25 mg/mL), 10 mL of autoclaved phosphate buffered saline (PBS) containing penicillin (100 units/mL) and streptomycin (100 &#956;g/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.</li>
	<li>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.</li>
	<li>While turning the head with a microspatula, inject 0.1-1 &#956;L 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Flat-Mount Culture on the Cell Insert
<ol>
	<li>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 &#181;g/mL laminin and 80 &#181;g/mL poly-L-lysine to cover the inserts and leave the floated inserts in the cell culture CO2 incubator at 37 &#176;C overnight. 
	NOTE: On the following day, the coated inserts can be stored at 4 &#176;C.</li>
	<li>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 &#956;g/mL streptomycin). Keep the dish in the cell culture CO2 incubator at 37 &#176;C. 
	NOTE: The medium can be used throughout the culture period for three days without change.</li>
	<li>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 &#176;C (temperature controller).</li>
	<li>Cut the head of the embryo with scissors. Pinch the head out with the forceps into the ice-cold Hanks&#39; Balanced Salt solution (HBSS) in a 6-cm cell culture dish.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
</ol>
3. Time-Lapse Imaging
<ol>
	<li>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.</li>
	<li>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 &#181;m interval for the 100 &#181;m 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.</li>
	<li>Set the parameters of the confocal running program described in 3.2 and start imaging.</li>
	<li>After the imaging, combine the confocal images at different z-axis to provide z-stack images with fine focal adjustment at every time point.</li>
	<li>Append the z-stack images at different time-point and construct a time-lapse movie in the AVI format.</li>
</ol>

<ol>
	<li>Mar&#237;n, O., Rubenstein, J. L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+in+the+forebrain.">Cell migration in the forebrain.</a> Annual Reviews of Neuroscience. 26, 441-483 (2003).</li><li>Nadarajah, B., Alifragis, P., Wong, R. O. L., Parnavelas, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+migration+in+the+developing+cerebral+cortex:+Observations+based+on+real-time+imaging.">Neuronal migration in the developing cerebral cortex: Observations based on real-time imaging.</a> Cerebral Cortex. 13, 607-611 (2003).</li><li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipolar+migration:+the+third+mode+of+radial+neuronal+migration+in+the+developing+cerebral+cortex.">Multipolar migration: the third mode of radial neuronal migration in the developing cerebral cortex.</a> Journal of Neuroscience. 23, 9996-10001 (2003).</li><li>Martini, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biased+selection+of+leading+process+branches+mediates+chemotaxis+during+tangential+neuronal+migration.">Biased selection of leading process branches mediates chemotaxis during tangential neuronal migration.</a> Development. 136, 41-50 (2009).</li><li>Polleux, F., Whitford, K. L., Dijkhuizen, P. A., Vitalis, T., Ghosh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cortical+interneuron+migration+by+neurotrophins+and+PI3-kinase+signaling.">Control of cortical interneuron migration by neurotrophins and PI3-kinase signaling.</a> Development. 129, 3147-3160 (2002).</li><li>Watanabe, Y., Yaginuma, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tangential+cell+migration+during+layer+formation+of+chick+optic+tectum.">Tangential cell migration during layer formation of chick optic tectum.</a> Development, Growth and Differentiation. 57, 539-543 (2015).</li><li>Watanabe, Y., Sakuma, C., Yaginuma, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NRP1-mediated+Sema3A+signals+coordinate+laminar+formation+in+the+developing+chick+optic+tectum.">NRP1-mediated Sema3A signals coordinate laminar formation in the developing chick optic tectum.</a> Development. 141, 3572-3582 (2014).</li><li>Watanabe, Y., Sakuma, C., Yaginuma, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersing+movement+of+neuronal+tangential+migration+in+superficial+layers+of+the+developing+chick+optic+tectum.">Dispersing movement of neuronal tangential migration in superficial layers of the developing chick optic tectum.</a> Developmental Biology. 437, 131-139 (2018).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88, 49-92 (1951).</li><li>Cordelie&#768;res, F. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+cell+tracking+and+analysis+in+phase-contrast+videos+(iTrack4U):+development+of+Java+software+based+on+combined+mean-shift+processes.">Automated cell tracking and analysis in phase-contrast videos (iTrack4U): development of Java software based on combined mean-shift processes.</a> PLOS One. 8, e81266 (2013).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li></ol>

The authors declare that they have no competing financial interests.

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 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.

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

visualization of tangential cell migration 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.

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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58506/58506fig1.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58506/58506fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58506/58506fig2.jpg" /> 
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58506/58506fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58506/58506fig3.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58506/58506fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/58506/58506mov1.jpg" /> 
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58506/Movie1.mov" target="_blank">Please click here to view this video. Right-click to download.</a>
<img alt="Movie 2" src="/files/ftp_upload/58506/58506mov2.jpg" /> 
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58506/Movie2.mov" target="_blank">Please click here to view this video. Right-click to download.</a>
<img alt="Movie 3" src="/files/ftp_upload/58506/58506mov3.jpg" /> 
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58506/Movie3.mov" target="_blank">Please click here to view this video. Right-click to download.</a>
<img alt="Movie 4" src="/files/ftp_upload/58506/58506mov4.jpg" /> 
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58506/Movie4.mov" target="_blank">Please click here to view this video. Right-click to download.</a>
<img alt="Movie 5" src="/files/ftp_upload/58506/58506mov5.jpg" /> 
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 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58506/Movie5.mov" target="_blank">Please click here to view this video. Right-click to download.</a>

Watch this Scientific Journal Video about Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum at JoVE.com

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

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 ...

visualization-tangential-cell-migration-developing-chick-optic

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.4K Views.  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.

Video: Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter&#39;s needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis

Here, we present a standardized series of protocols to observe the superior ocular sulcus, a recently-identified, evolutionarily-conserved structure in the vertebrate eye. Using zebrafish larvae, we demonstrate techniques necessary to identify factors that contribute to the formation and closure of the superior ocular sulcus.

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid ...

Visualization and Analysis of Pharyngeal Arch Arteries using Whole-mount Immunohistochemistry and 3D Reconstruction

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart disease. To study the formation of PAAs, we developed a protocol using whole-mount immunofluorescence coupled with benzyl alcohol/benzyl benzoate (BABB) tissue clearing, and confocal microscopy. This allows for the visualization of the pharyngeal arch endothelium at a fine cellular resolution as well as the 3D connectivity of the vasculature. Using software, we have established a protocol to quantify the number of endothelial cells (ECs) in PAAs, as well as the number of ECs within the vascular plexus surrounding the PAAs within pharyngeal arches 3, 4, and 6. When applied to the whole embryo, this methodology provides a comprehensive visualization and quantitative analysis of embryonic vasculature.

Here, we describe a protocol to visualize and analyze the pharyngeal arch arteries 3, 4, and 6 of mouse embryos using whole-mount immunofluorescence, tissue clearing, confocal microscopy, and 3D reconstruction.

Improper formation or remodeling of the pharyngeal arch arteries (PAAs) 3, 4, and 6 contribute to some of the most severe forms of congenital heart ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...