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>&lt;strong&gt;Materials&lt;/strong&gt;</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>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</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. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de ontwikkelingsbiologie, zoals de mechanismen van neuronale celmigratie. Het belangrijkste voordeel van deze techniek is dat u individuele en collectieve celmigratie op lange termijn detecteren. Na het isoleren van 200 milliliter bacteriële cultuur door de alkalische lyse methode, meng de twee plasmiden, zodanig dat de uiteindelijke concentratie van elk is vier microgram per microliter.Broed de vruchtbare kippenei...

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

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Biology

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, proliferation, and differentiation. Time-lapse imagining offers unique insight to the behavior of cells during eye development because it allows us to visualize oculogenesis in vivo. Zebrafish are an excellent model to visualize this process due to their highly conserved vertebrate eye and their ability to develop rapidly and externally while remaining optically transparent. Time-lapse imaging studies of zebrafish eye development are greatly facilitated by use of the transgenic zebrafish line Tg(rx3:GFP). In the developing forebrain, rx3:GFP expression marks the cells of the single eye field, and GFP continues to be expressed as the eye field evaginates to form an optic vesicle, which then invaginates to form an optic cup. High resolution time lapse imaging of rx3:GFP expression, therefore, allows us to track the eye primordium through time as it develops into the retina. Lightsheet microscopy is an ideal method to image ocular morphogenesis over time due to its ability to penetrate thicker samples for fluorescent imaging, minimize photobleaching and phototoxicity, and image at a high speed. Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. The resulting dataset can provide valuable insights into the process of ocular morphogenesis, as well as perturbations to this process as a result of genetic mutation, exposure to pharmacological agents, or other experimental manipulations.

Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software.&#160;

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo 

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video