T&#945;1 promoter is a gift from P. Barker and F.D. Miller. Dcx promoter is a gift from Q. Lu. hGFAP-Cre was a gift from Albee Messing. The PiggyBac transposon vector system was provided by the Sanger Institute. Flt1-DsRed mice were provided by M. Ema (Shiga University). This work was supported by JSPS KAKENHI (Grant Number JP21K07309 to H. Tabata, JP20H05688 and JP22K19365 to K. Nakajima) and Takeda Science Foundation, Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research, Keio Gijuku Academic Development Funds to K. Nakajima.

The present study was performed with the approval of and following the guidelines of the Animal Care and Use Committee of the Institute for Developmental Research, Aichi Developmental Disability Center (#2019-013), and Keio University (A2021-030).&#160;Timed pregnant ICR (wild-type) mice were obtained commercially (see Table of Materials). To observe the relationship between migrating cells and blood vessels, Flt1-DsRed mice, in which the endothelial cells express DsRed22, were used. Male Flt1-DsRed mice were crossed with female ICR mice (both mice were at the age of 8-24 weeks). All the animals were housed and manipulated under specific pathogen-free (SPF) conditions until sampling. The reagents and equipment used in this study are listed in the Table of Materials.
1. In utero electroporation
<ol>
	<li>Prepare HEPES-buffered saline (HBS) solution by adding an equal volume of autoclaved water to commercially available 2x HBS.</li>
	<li>There are two options of anesthetics; isoflurane (inhalation) and MMB (injection). In the case of using MMB, prepare it as follows. Add 0.75 mL of medetomidine (1 mg/mL), 2 mL of midazolam (5 mg/mL), and 2.5 mL of butorphanol (5 mg/mL) in 19.75 mL of autoclaved water23. 
	NOTE: This anesthetic solution becomes less active within 2 months. Store at 4 &#176;C with light-shield.</li>
	<li>Prepare atipamezole solution. Dilute 0.75 mL of atipamezole (5 mg/mL) with 24.25 mL of autoclaved water.</li>
	<li>Prepare 0.2% (w/v) Fast Green solution in autoclaved water. Sterilize with a filter (pore size 0.22 &#181;m).</li>
	<li>Prepare plasmid using a conventional plasmid purification kit based on alkaline lysis and column purification23 according to the manufacturer&#39;s instructions (see Table of Materials). Dissolve the purified plasmid from 100 mL bacteria culture in 30-50 &#181;L of HBS to prepare the plasmid stock (3-5 &#181;g/&#181;L) (Supplementary File 1).
	<ol>
		<li>Mix and dilute the plasmid stocks with HBS, and add 1/10 volume of 0.2% Fast Green to color the injectant. The components and final concentrations of the plasmids are determined according to their purpose (Table 1).</li>
	</ol>
	</li>
	<li>Make the micropipettes by pulling the glass capillaries using a pipette puller. 
	NOTE: The tip of the pipette is cut obliquely using fine forceps. The outer diameter of the tip is approximately 70 &#181;m. Mark the micropipettes at 2 &#181;L intervals by sucking 2 &#181;L water.</li>
	<li>Inject MMB anesthetic at a dose of 10 &#956;L/g body weight intraperitoneally into a pregnant mouse at an appropriate stage (following institutionally approved protocols). 
	NOTE: The embryonic stages to be manipulated should be determined according to the purpose. To label future II/III pyramidal neurons, E14.5 is suitable. To label glial progenitors, electroporation must be performed on E15-16. Alternatively, the mice can be anesthetized by inhalation of isoflurane (1%-2%).</li>
	<li>After the mouse is deeply anesthetized, place it on a warm plate at 38 &#176;C. 
	NOTE: Ensure that the mouse does not respond to pinching the tail.</li>
	<li>After removing fur around the surgical site using depilatory cream, disinfect the surgical area several times in a circular motion with both an iodine-based scrub and 70% alcohol.&#160;Make a 2 cm long skin incision along the midline from the level of the 2nd most posterior pair of nipples anteriorly, followed by a 2 cm midline incision in the abdominal wall along the linea alba (Figure 1A). 
	NOTE: In the process of incision in the abdominal wall, lift it slightly with fine forceps so as not to cut the internal organs.</li>
	<li>Place a sterile plastic sheet on the skin and a piece of sterile tissue paper on it (Figure 1A). Create a hole in the center to expose the surgical area. 
	NOTE: The sterile plastic sheet and the sterile tissue paper are placed after making the incision because a wide view field is required to position the incision referring to the position of the nipples.</li>
	<li>Wet the paper with sterile PBS, carefully observe the uterine horns from the incision, and draw out the uterine horn through the holes of the plastic sheet and paper.</li>
	<li>Using a micropipette attached to an aspirator tube assembly, inject 1 &#181;L of the plasmid mix into the left or right lateral ventricle through the uterine wall by expiratory pressure (Figure 1A). 
	NOTE: Alternatively, plasmid can be injected by using a microinjector.</li>
	<li>Pinch the head of the embryo with a tweezer-type electrode and apply electronic pulses using an electroporator (35 V for E14-16, 50 V for E17, 50 ms square pulses with 450 ms intervals, 5 times) (Figure 1A). 
	NOTE: Do not pinch the head of the embryos strongly, which will damage the embryos. The actual current should be 40-70 mA.</li>
	<li>Place the uterine horn back into the abdominal cavity.</li>
	<li>Suture the abdominal wall using a 5-0 silk thread.</li>
	<li>Apply atipamezole solution at a dose of 10 &#181;L/g body weight intraperitoneally.</li>
	<li>Suture the skin using a 5-0 silk thread. 
	NOTE: The skin can be closed by using an autoclip.</li>
	<li>Keep the manipulated mice on a warm plate for at least 30 min. The mice will start moving in the cage. 
	NOTE: The procedure of in utero electroporation (from steps 1.7 to 1.16) should be done within 30 min. The typical operation time is 20 min.</li>
</ol>
2. Preparation of slices
NOTE: The freshness of the slices is the key to the success of this experiment. The slices must be prepared (from steps 2.5-2.12) within 2 h.
<ol>
	<li>Prepare the Hanks&#39; balanced salt solution with Ca2+ and Mg2+; HBSS(+). Add 5 mL of CaCl2 (14 g/L, autoclaved) and 5 mL of MgSO4.7H2O (20 g/L, autoclaved) to 500 mL of HBSS(-). Store at 4 &#176;C. Bubble the solution with 95% O2 + 5% CO2 gas for 5 min on ice before use.</li>
	<li>Prepare the culture medium. At least 2.5 mL/dish is needed. Add 2 mL of fetal bovine serum, 25 &#181;L of L-glutamine (200 mM), 200 &#181;L B27 (50x), 50 &#181;L of penicillin + streptomycin (10 K units/mL and 10 mg/mL) to 8 mL of Neurobasal medium.</li>
	<li>Prepare 3% low melting temperature agarose gel. Pore 50 mL of HBSS(+), then add 1.5 mg of low melting temperature agarose, and autoclave it. Melt the agarose in a microwave oven and keep it at 50 &#176;C in a water bath incubator prior to use.</li>
	<li>Add 1.9 mL of culture medium to a glass base dish. Put a cell culture insert on it carefully to not trap air bulbs under the filter, then add 500 &#181;L of culture medium onto the filter (Figure 1B). Place it on ice.</li>
	<li>After anesthetizing the manipulated mice by inhalation of 5% isoflurane, sacrifice them by cervical dislocation (following institutionally approved protocols) at appropriate stages to be observed. 
	NOTE: Two days (48 h) and 3 days after electroporation at E14.5 are suitable for observing the multipolar-bipolar transition and locomotion mode, respectively.</li>
	<li>Take out the embryos and place them in a Petri dish with cold PBS.</li>
	<li>Select the embryos by the expression of GFP or other fluorescent proteins under a microscope.</li>
	<li>After decapitation of the embryos, dissect out the brains6 under a dissecting microscope and keep them in cold HBSS(+).</li>
	<li>Pour the melted 3% low melting temperature agarose gel into a cryomold (see Table of Materials), and then put the brains in it and roll them several times. Place it at room temperature until the agarose completely solidifies, and then place it on ice.</li>
	<li>Slice the brains coronally at the thickness of 350 &#181;m using a vibrating microtome (see Table of Materials).</li>
	<li>Arrange the slices on the filter (maximum 6 slices per filter).</li>
	<li>Withdraw the culture medium from the inside and outside of the filter cup (600 &#181;L in total). In this step, the slices are pressed and fixed onto a filter. After 5 min, add 100 &#181;L of culture medium to the inside of the cell culture insert.</li>
</ol>
3. Time-lapse imaging
<ol>
	<li>Keep the culture chamber mounted on the stage of an inverted laser scan microscope at 37 &#176;C using the heater of the chamber or a hood enclosing the microscope at least 30 min before imaging to prevent Z drift. Supply humidified 5% CO2 + 40% O2 gas to the culture chamber.</li>
	<li>Set the culture dish with slices prepared above in the culture chamber.</li>
	<li>Using a long working distance 20x lens (NA = 0.45), acquire around 10 Z-stack images with 5 &#181;m steps every 15-30 min for 24 h. 
	NOTE: The use of a motorized XY stage is highly recommended. This enables us to obtain images from multiple objective fields. Typically, we obtain 10-20 images from different objective fields in one time-lapse experiment.</li>
</ol>
4. Imaging analysis
<ol>
	<li>Convert XYZT images to XYT by the maximum projection of the Z axis using ImageJ or other software. 
	NOTE: If the slices slid during the observation, adjust the position of the cells within the images using StackRed, an ImageJ plugin, with its function of Rigid Body transformation (see Table of Materials).</li>
	<li>Trace the trajectory of GFP- or other fluorescent protein-positive cells either manually or automatically. Manual and automatic tracing can be performed using MTrackJ24 and TrackMate25,26 (ImageJ plugins), respectively. Automatic tracing using TrackMate is efficient for analyzing many cells in an unbiased manner. However, manual tracing using MTrackJ remains useful for the accurate tracking of individual cells.</li>
</ol>

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

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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+hyperactive+piggyBac+transposase+for+mammalian+applications.">A hyperactive piggyBac transposase for mammalian applications.</a> P Natl Acad Sci USA. 108 (4), 1531-1536 (2011).</li><li>Chen, F., LoTurco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+stable+transgenesis+of+radial+glia+lineage+in+rat+neocortex+by+piggyBac+mediated+transposition.">A method for stable transgenesis of radial glia lineage in rat neocortex by piggyBac mediated transposition.</a> J Neurosci Meth. 207 (2), 172-180 (2012).</li><li>Wang, X., Qiu, R., Tsark, W., Lu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+promoter+analysis+in+developing+mouse+brain+and+genetic+labeling+of+young+neurons+by+doublecortin-DsRed-express.">Rapid promoter analysis in developing mouse brain and genetic labeling of young neurons by doublecortin-DsRed-express.</a> J Neurosci Res. 85 (16), 3567-3573 (2007).</li><li>Sauer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+recombination+with+a+heterospecific+Cre+homolog+identified+from+comparison+of+the+pac-c1+regions+of+P1-related+phages.">DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages.</a> Nucleic Acids Res. 32 (20), 6086-6095 (2004).</li><li>Hermann, M., 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=Binary+recombinase+systems+for+high-resolution+conditional+mutagenesis.">Binary recombinase systems for high-resolution conditional mutagenesis.</a> Nucleic Acids Res. 42 (6), 3894-3907 (2014).</li><li>Kanatani, S., 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=The+COUP-TFII/Neuropilin-2+is+a+molecular+switch+steering+diencephalon-derived+GABAergic+neurons+in+the+developing+mouse+brain.">The COUP-TFII/Neuropilin-2 is a molecular switch steering diencephalon-derived GABAergic neurons in the developing mouse brain.</a> P Natl Acad Sci USA. 112 (36), E4985-E4994 (2015).</li><li>Yozu, M., 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=The+caudal+migratory+stream:+A+novel+migratory+stream+of+interneurons+derived+from+the+caudal+ganglionic+eminence+in+the+developing+mouse+forebrain.">The caudal migratory stream: A novel migratory stream of interneurons derived from the caudal ganglionic eminence in the developing mouse forebrain.</a> J Neurosci. 25 (31), 7268-7277 (2005).</li><li>Kanatani, S., Yozu, M., 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=COUP-TFII+is+preferentially+expressed+in+the+caudal+ganglionic+eminence+and+is+involved+in+the+caudal+migratory+stream.">COUP-TFII is preferentially expressed in the caudal ganglionic eminence and is involved in the caudal migratory stream.</a> J Neurosci. 28 (50), 13582-13591 (2008).</li><li>Kitazawa, A., 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=Hippocampal+pyramidal+neurons+switch+from+a+multipolar+migration+mode+to+a+novel+&amp;#34;climbing&amp;#34;+migration+mode+during+development.">Hippocampal pyramidal neurons switch from a multipolar migration mode to a novel &amp;#34;climbing&amp;#34; migration mode during development.</a> J Neurosci. 34 (4), 1115-1126 (2014).</li></ol>

The authors have no conflicts of interest.

This protocol introduced a method for the time-lapse observation of cells derived from the pallial (cortical) VZ. To label the migrating cells from the VZ, we used in utero electroporation, in which individual cells were clearly labeled with a higher signal-to-noise ratio than in viral vector-mediated labeling. Using in utero electroporation, any type of vector in any combination can be easily introduced into the radial glial cells (neural stem cells) in living embryos. Neurons and glial progenitors can...

During the development of the cerebral cortex, (apical) radial glia in the pallial ventricular zone (VZ) lining the lateral ventricle produce first neurons and then glial progenitors with some overlapping period1. Neurons are also generated from intermediate progenitors or basal radial glia in the subventricular zone (SVZ) adjacent to the VZ, both of which originate from the (apical) radial glia2,3. In mice, radial glial cells produce only neurons on embryonic day (E) 12-14, both neurons and glial progenitors on E15-16, and glial progenitors from E17 onward4. The major population of glial progenitors generated during these embryonic stages preferentially differentiates into astrocytes, although some cells also differentiate into oligodendrocytes5. Neurons and astrocyte progenitors generated at these stages migrate toward the brain surface and enter the cortical plate (future cortical gray matter). Neuronal migration from the VZ to the cortical plate occurs in multiple phases. Neurons first adopt a multipolar morphology just above the multipolar cell accumulation zone (MAZ), overlapping the SVZ or intermediate zone, where they vigorously extend and retract multiple thin processes and slowly migrate (multipolar migration)6,7. After approximately 24 h, neurons transform into a bipolar morphology, extending a thick leading process toward the brain surface and a thin trailing process backward, and migrate linearly toward the brain surface using a radial process extending from the radial glia to the pial surface as a scaffold, which is called locomotion mode2,8. Because neurons in locomotion mode always reach the outermost surface of the cortical plate, passing through their predecessors just under the marginal zone, neurons are aligned in a birthdate-dependent inside-out manner in the cortical plate9,10,11.
In contrast, astrocyte progenitors migrate rapidly to the intermediate zone and cortical plate, with frequent directional changes. This migratory behavior is completely different from neuronal migration and is called erratic migration5. Astrocyte progenitors also migrate along blood vessels in a process called blood vessel-guided migration. Astrocyte progenitors switch between these migration modes and reach the cortical plate5,12. Although the positioning of astrocytes is not strictly determined by their date of production, a mild tendency for early-born astrocytes to settle in the superficial part of the cortical plate has been observed5. Interestingly, astrocytes that settle in the cortical plate are generated in embryonic stages and eventually differentiate into protoplasmic astrocytes, whereas postnatally generated astrocytes do not migrate actively, remain in the white matter, and differentiate into fibrous astrocytes5. How this stage-dependent specification of astrocytic subtypes occurs remains unclear.
A growing number of genes involved in neuronal migration have been identified, including those involved in neurodevelopmental and psychiatric disorders13,14. Therefore, it is crucial to elucidate the effects of mutations in these genes on the behavior of migrating neurons. As previously mentioned, neuronal migration occurs in multiple phases. Time-lapse observations can directly determine the phase that is mainly affected (cell cycle exit, multipolar-bipolar transition, migration speed of locomotion, etc.). However, the molecular mechanisms underlying the specification, migration, and positioning of astrocytes remain largely unknown. Given that astrocytes play crucial roles in synaptogenesis15 and blood-brain barrier formation during brain development16, developmental defects in astrocytes may result in neurodevelopmental disorders. Time-lapse studies on astrocyte progenitors may clarify these molecular mechanisms and their relationship with mental illness.
This protocol provides a method for time-lapse observation of cortical VZ-derived cells. A similar video protocol for the observation of neuronal migration has already been published17. Here, we describe the method for both migrating neurons and astrocyte progenitors. To label these cells with fluorescent proteins, such as green and red fluorescent proteins (GFP and RFP), plasmid mixtures containing appropriate components are introduced into the cortical VZ by in utero electroporation at appropriate stages18,19,20,21. The manipulated embryos are removed at the desired stages, and the brains are sliced and used for time-lapse observations using a laser scanning microscope. The migration speed, direction, and other behaviors, which are never addressed using fixed brain samples, can be examined using this method. Using in utero electroporation, expression, and knockdown/knockout vectors can be easily transferred concomitantly with fluorescent protein vectors, enabling us to conduct gain-of-function and loss-of-function studies of specific genes.

<table><tbody><tr><td>Aspirator tube assembly</td><td>Drummond</td><td>2-040-000</td><td></td></tr><tr><td>Atipamezole (5 mg/mL)</td><td>Meiji</td><td>Mepatia</td><td></td></tr><tr><td>Autoclip</td><td>Becton Dickinson</td><td>427630</td><td>9 mm</td></tr><tr><td>B27 supplement</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>Butorphanol (5 mg/mL)</td><td>Meiji</td><td>Vetorphale</td><td></td></tr><tr><td>Cell culture insert</td><td>Millipore</td><td>PICM ORG 50</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>A1RHD25</td><td>Equipped with a long working distance lens (S Plan Fluor ELWD 20XC)</td></tr><tr><td>Cryomold</td><td>Tissue-Tek</td><td>4566</td><td></td></tr><tr><td>Culture chamber</td><td>Tokken</td><td>TK-NBCMP</td><td>Custom-made</td></tr><tr><td>Electroporator</td><td>NEPA Gene</td><td>NEPA21</td><td></td></tr><tr><td>Fast Green</td><td>Sigma-Aldrich</td><td>F7258</td><td></td></tr><tr><td>Gas mixer</td><td>Tokken</td><td>TK-MIGM01-02</td><td></td></tr><tr><td>Glass base dish</td><td>Iwaki</td><td>3910-035</td><td>Diameter of glass base is 27 mm</td></tr><tr><td>Glass capillaries</td><td>Narishige</td><td>GD-1</td><td></td></tr><tr><td>HBS (2x)</td><td>Sigma-Aldrich</td><td>51558</td><td></td></tr><tr><td>HBSS(-)</td><td>Wako</td><td>084-08345</td><td></td></tr><tr><td>Heater Unit</td><td>Tokken</td><td>TK-0003HU20</td><td>Custom-made, including hood and heater</td></tr><tr><td>hGFAP-Cre</td><td>Addgene</td><td>#40591</td><td>A gift from Albee Messing</td></tr><tr><td>ImageJ</td><td></td><td></td><td>https://imagej.net/ij/</td></tr><tr><td>L-glutamine (200 mM)</td><td>Gibco</td><td>25030</td><td></td></tr><tr><td>Low melting temperature agarose</td><td>Lonza</td><td>50100</td><td></td></tr><tr><td>Medetomidine (1 mg/mL)</td><td>Meiji</td><td>Medetomin</td><td></td></tr><tr><td>Microinjector</td><td>Narishige</td><td>IM-300</td><td></td></tr><tr><td>Midazolam (5 mg/mL)</td><td>Sandoz</td><td>Midazolam</td><td></td></tr><tr><td>MTrackJ</td><td></td><td></td><td>https://imagescience.org/meijering/software/mtrackj/</td></tr><tr><td>Neurobasal medium</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>pCAG-hyPBase</td><td></td><td></td><td>The hyPBase cDNA from pCMV-hyPBase (a gift from Sanger Institute) was inserted into the downstream of the CAG promoter of pCAGGS (a gift from J. Miyazaki).</td></tr><tr><td>pDcx-Dre</td><td></td><td></td><td>The Dcx promoter from Dcx4kbEGFP70 (a gift from Q. Lu) was exchanged with CAG promoter of pCAG-NLS-HA-Dre34 (a gift from Pawel Pelczar, Addgene #51272).</td></tr><tr><td>Penicillin + Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Plasmid purification kit</td><td>Invitrogen</td><td>PureLink HiPure plasmid midiprep kit (K210005)</td><td></td></tr><tr><td>pPB-CAG-LNL-RFP</td><td></td><td></td><td>CAG-LNL cassette from pCALNL-DsRed (a gift from Connie Cepko, Addgene #13769), and TurboRFP cDNA (Evrogen, FP232) were inserted into the cloning site of pPB-CAG.EBNXN (a gift from Sanger Institute).</td></tr><tr><td>pPB-CAG-rDIO-EGFP</td><td></td><td></td><td>The sequence containning synthetic rox sites, synthetic DIO cassette, and EGFP cDNA from pEGFP-N1 (Clontech, U55762) in reverse direction&amp;nbsp; were inserted into the cloning site of pPB-CAG.EBNXN (a gift from Sanger Institute). The sequence is provided in the Supplementary File.</td></tr><tr><td>Puller</td><td>Narishige</td><td>PN-31</td><td></td></tr><tr><td>StackRed</td><td></td><td>a plugin for ImageJ</td><td>http://bigwww.epfl.ch/thevenaz/stackreg/</td></tr><tr><td>Suture needle</td><td>Nazme</td><td>C-24-521-R No.1</td><td>1/2 circle, length 14 mm</td></tr><tr><td>Suture thread</td><td>Nazme</td><td>C-23-B2</td><td>Silk, size 5-0</td></tr><tr><td>Timed pregnant ICR (wild-type) mice</td><td>Japan SLC</td><td>ICR mouse</td><td></td></tr><tr><td>TrackMate</td><td></td><td></td><td>https://imagej.net/plugins/trackmate/index</td></tr><tr><td>Tweezer-type electrode</td><td>BEX or NEPA Gene</td><td>CUY650P5&amp;nbsp;</td><td></td></tr><tr><td>T&amp;alpha;1-EGFP</td><td></td><td></td><td>EGFP cDNA from pEGFP-N1 (Clontech, U55762) was inserted into the downstream of the T&amp;alpha;1 promoter in plasmid 253 (a gift from P. Barker and F.D.Miller)</td></tr><tr><td>Vibrating microtome</td><td>Leica or Zeiss</td><td>Vibrating blade microtome VT1000S or Hyrax V50.</td><td></td></tr></tbody></table>

time-lapse imaging of migrating neurons and glial progenitors in embryonic mouse brain slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Radial glial cells in the pallial VZ produce only neurons until E14, and both neurons and glial cells at E15 and E16. To observe the migratory behaviors of neurons and glial cells simultaneously, we labeled them with enhanced GFP (EGFP) and RFP, respectively, by using a neuron-specific promoter, T&#945;1 promoter27, and human glial fibrillary acidic protein (hGFAP) promoter28, which is preferentially activated in astrocytes. Astrocyte progenitors repeat cell divisi...

Watch this Scientific Journal Video about Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain at JoVE.com

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

time-lapse-imaging-migrating-neurons-glial-progenitors-embryonic

Research

JoVE Journal

Neuroscience

795 Views.  Keio University School of Medicine. Tijdens de ontwikkeling van de hersenschors worden neuronen en grijze cellen afgeleid van de ventriculaire zone en migreren ze naar een ander deel van het hersenoppervlak. Veel genen zijn betrokken bij dit proces, waaronder de genen die verantwoordelijk zijn voor neurologische ontwikkelingsstoornissen en psychiatrische stoornissen. We richten ons op hun functies op de drie gedragingen in dit proces.Onlangs hebben we gerapporteerd dat voorlopers van astrocyten twee verschillende migrati...

Video: Auteur in de schijnwerpers: onderzoek naar celmigratie en genrollen in de zich ontwikkelende hersenen

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

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