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 us...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

<ol>
	<li>Kriegstein, A., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glial+nature+of+embryonic+and+adult+neural+stem+cells.">The glial nature of embryonic and adult neural stem cells.</a> Annu Rev Neurosci. 32 (1), 149-184 (2009).</li><li>Noctor, S. C., Mart&#237;nez-Cerde&#241;o, V., Ivic, L., Kriegstein, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+neurons+arise+in+symmetric+and+asymmetric+division+zones+and+migrate+through+specific+phases.">Cortical neurons arise in symmetric and asymmetric division zones and migrate through specific phases.</a> Nat Neurosci. 7 (2), 136-144 (2004).</li><li>Haubensak, W., Attardo, A., Denk, W., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurons+arise+in+the+basal+neuroepithelium+of+the+early+mammalian+telencephalon:+A+major+site+of+neurogenesis.">Neurons arise in the basal neuroepithelium of the early mammalian telencephalon: A major site of neurogenesis.</a> P Natl Acad Sci USA. 101 (9), 3196-3201 (2004).</li><li>Yoshida, A., Yamaguchi, Y., Nonomura, K., Kawakami, K., Takahashi, Y., Miura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+expression+of+different+transgenes+in+neurons+and+glia+by+combining+in+utero+electroporation+with+the+Tol2+transposon-mediated+gene+transfer+system.">Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system.</a> Genes Cells. 15 (5), 501-512 (2010).</li><li>Tabata, H., 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=Erratic+and+blood+vessel-guided+migration+of+astrocyte+progenitors+in+the+cerebral+cortex.">Erratic and blood vessel-guided migration of astrocyte progenitors in the cerebral cortex.</a> Nat Commun. 13 (1), 6571 (2022).</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> J Neurosci. 23 (31), 9996-10001 (2003).</li><li>Tabata, H., Kanatani, S., 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=Differences+of+migratory+behavior+between+direct+progeny+of+apical+progenitors+and+basal+progenitors+in+the+developing+cerebral+cortex.">Differences of migratory behavior between direct progeny of apical progenitors and basal progenitors in the developing cerebral cortex.</a> Cereb Cortex. 19 (9), 2092-2105 (2009).</li><li>Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mode+of+cell+migration+to+the+superficial+layers+of+fetal+monkey+neocortex.">Mode of cell migration to the superficial layers of fetal monkey neocortex.</a> J Comp Neurol. 145 (1), 61-83 (1972).</li><li>Sekine, K., Honda, T., Kawauchi, T., Kubo, K., 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+outermost+region+of+the+developing+cortical+plate+is+crucial+for+both+the+switch+of+the+radial+migration+mode+and+the+Dab1-dependent+&amp;#34;inside-out&amp;#34;+lamination+in+the+neocortex.">The outermost region of the developing cortical plate is crucial for both the switch of the radial migration mode and the Dab1-dependent &amp;#34;inside-out&amp;#34; lamination in the neocortex.</a> J Neurosci. 31 (25), 9426-9439 (2011).</li><li>Shin, 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=Both+excitatory+and+inhibitory+neurons+transiently+form+clusters+at+the+outermost+region+of+the+developing+mammalian+cerebral+neocortex.">Both excitatory and inhibitory neurons transiently form clusters at the outermost region of the developing mammalian cerebral neocortex.</a> J Comp Neurol. 527 (10), 1577-1597 (2019).</li><li>Sekine, K., 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=Reelin+controls+neuronal+positioning+by+promoting+cell-matrix+adhesion+via+inside-out+activation+of+integrin+&#945;5&#946;1.">Reelin controls neuronal positioning by promoting cell-matrix adhesion via inside-out activation of integrin &#945;5&#946;1.</a> Neuron. 76 (2), 353-369 (2012).</li><li>Morimoto, K., Tabata, H., Takahashi, R., 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=Interactions+between+neural+cells+and+blood+vessels+in+central+nervous+system+development.">Interactions between neural cells and blood vessels in central nervous system development.</a> BioEssays. 230091, (2023).</li><li>Tabata, H., Nagata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoding+the+molecular+mechanisms+of+neuronal+migration+using+in+utero+electroporation.">Decoding the molecular mechanisms of neuronal migration using in utero electroporation.</a> Med Mol Morphol. 49 (2), 63-75 (2016).</li><li>Ishii, K., Kubo, K., 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=Reelin+and+neuropsychiatric+disorders.">Reelin and neuropsychiatric disorders.</a> Front Cell Neurosci. 10, 229 (2016).</li><li>Bosworth, A. P., Allen, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diverse+actions+of+astrocytes+during+synaptic+development.">The diverse actions of astrocytes during synaptic development.</a> Curr Opin Neurobiol. 47, 38-43 (2017).</li><li>Tabata, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosstalk+between+blood+vessels+and+glia+during+the+central+nervous+system+development.">Crosstalk between blood vessels and glia during the central nervous system development.</a> Life. 12 (11), 1761 (2022).</li><li>Wiegreffe, C., Feldmann, S., Gaessler, S., Britsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+confocal+imaging+of+migrating+neurons+in+organotypic+slice+culture+of+embryonic+mouse+brain+using+in+utero+electroporation.">Time-lapse confocal imaging of migrating neurons in organotypic slice culture of embryonic mouse brain using in utero electroporation.</a> J Vis Exp. (125), e55886 (2017).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Gene+Transfer+into+the+Embryonic+Mouse+Brain+Using+in+Vivo+Electroporation.">Efficient Gene Transfer into the Embryonic Mouse Brain Using in Vivo Electroporation.</a> Dev Biol. 240 (1), 237-246 (2001).</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=Labeling+embryonic+mouse+central+nervous+system+cells+by+in+utero+electroporation.">Labeling embryonic mouse central nervous system cells by in utero electroporation.</a> Dev Growth Differ. 50 (6), 507-511 (2008).</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=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103 (4), 865-872 (2001).</li><li>Fukuchi-Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294 (5544), 1071-1074 (2001).</li><li>Matsumoto, K., 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=Study+of+normal+and+pathological+blood+vessel+morphogenesis+in+Flt1-tdsRed+BAC+Tg+mice.">Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice.</a> Genesis. 50 (7), 561-571 (2012).</li><li>Kawai, S., Takagi, Y., Kaneko, S., Kurosawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+three+types+of+mixed+anesthetic+agents+alternate+to+ketamine+in+mice.">Effect of three types of mixed anesthetic agents alternate to ketamine in mice.</a> Exp Anim. 60 (5), 481-487 (2011).</li><li>Meijering, E., Dzyubachyk, O., Smal, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+cell+and+particle+tracking.">Methods for cell and particle tracking.</a> Methods Enzymol. 504, 183-200 (2012).</li><li>Ershov, D., 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=TrackMate+7:+Integrating+state-of-the-art+segmentation+algorithms+into+tracking+pipelines.">TrackMate 7: Integrating state-of-the-art segmentation algorithms into tracking pipelines.</a> Nat Methods. 19 (7), 829-832 (2022).</li><li>Tinevez, J. -. 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A., Bradley, A., Craig, N. 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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160; 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&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>T&#945;1-EGFP</td>
			<td></td>
			<td></td>
			<td>EGFP cDNA from pEGFP-N1 (Clontech, U55762) was inserted into the downstream of the T&#945;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.

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

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

During the development of the cerebral cortex, neurons and gray cells are derived from the ventricular zone and migrate to other part of the brain surface. Many genes are involved in this process, including those responsible for neurodevelopmental and psychiatric disorders. We are addressing their functions on the three behaviors in this process.Recently, we have reported that astrocyte progenitors assume two distinct migration modes:Erratic and blood-vessel guided migration. These observations were made using a combination of serotype-specific labeling and time observation methods introduced in this video. To level cells, we utilize in utero electroporation system, which we developed to visualize individual cells with a high signal to noise ratio.This in vivo gene transfer system also enable us to easily perform gain of function or loss of function experiments on the given genes by electroporation of their expression or neutron vectors. Using this experimental system, we aim to observe the cell behaviors of neurons, glial, and blood vessels, and elucidates the crosstalk between them. The findings from these studies will contribute to understanding the pathogenesis of neurodevelopmental disorders.

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

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.

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

466 vues.  Keio University School of Medicine. Au cours du développement du cortex cérébral, les neurones et les cellules grises sont dérivés de la zone ventriculaire et migrent vers d’autres parties de la surface du cerveau. De nombreux gènes sont impliqués dans ce processus, y compris ceux responsables des troubles neurodéveloppementaux et psychiatriques. Nous abordons leurs fonctions sur les trois comportements dans ce processus.Récemment, nous avons rapporté que les progéniteurs des astrocytes adoptent deux modes d...

Vidéo: Pleins feux sur l’auteur : Explorer la migration cellulaire et les rôles des gènes dans le cerveau en développement

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...

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visualization of tangential cell migration in the developing chick optic tectum

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...

exploring the pharmacological action and molecular mechanism of salidroside in inhibiting mcf-7 cell proliferation and migration

Author Spotlight: Exploring Salidroside&#039;s Molecular Mechanisms in Breast Cancer Treatment 

The present protocol describesa comprehensive strategy for evaluating the pharmacological action and mechanism of salidroside in inhibiting MCF-7 cell proliferation and...

Exploring the Pharmacological Action and Molecular Mechanism of Salidroside in Inhibiting MCF-7 Cell Proliferation and Migration

single cell electroporation in vivo within the intact developing brain

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis...

live-cell imaging assays to study glioblastoma brain tumor stem cell migration and invasion

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment...

live imaging and characterization of microglia dynamics in the zebrafish embryo

Author Spotlight: Using Zebrafish to Explore Microglia Migration During Brain Development

We demonstrate a method that takes advantage of fast scanning confocal microscopy to perform live imaging of microglia cells in the developing zebrafish optic tectum, allowing for the analysis of the dynamics of these cells in...

Elucidating Microglia Behavior in Zebrafish Optic Tectum Development

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biomarker identification for gender specificity of alzheimer's disease based on the glial transcriptome profiles

Author Spotlight: Exploring Sex-Specific Glial Signatures and Therapeutic Leads for Alzheimer&amp;#39;s Disease

This study analyzed single-nuclei transcriptomes of thirty-three individuals with Alzheimer's disease (AD), revealing sex-specific DEGs in glial cells. Functional enrichment analysis highlighted synaptic, neural, and hormone-related pathways. Key genes, namely NLGN4Y and its regulators, were identified, and potential therapeutic candidates for gender-specific AD were...

Sex-Specific Glial Biomarkers in Alzheimer&amp;#39;s Disease: A Transcriptome Analysis

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visualizing the developing brain in living zebrafish using brainbow and time-lapse confocal imaging

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous...

assessing cell cycle progression of neural stem and progenitor cells in the mouse developing brain after genotoxic stress

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain...

a seamless cloning approach for porcine reproductive and respiratory syndrome virus expression vector construction

Author Spotlight: Exploring Cloning Techniques for Full-Length DNA Fragments

Here, we present a protocol to obtain the pVAX1-PRRSV expression vector by introducing suitable restriction sites at the 3' end of the inserts. We can linearize the vector and join DNA fragments to the vector one by one through homologous recombination technology.

Cloning the Full-Length PRRSV Gene in the pVAX1 Expression Vector

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developing a triple cell culture model of the human blood-brain barrier

This video demonstrates the co-culturing of human brain pericyte, astrocyte, and endothelial cells. Each cell layer is formed separately and then cultured together, creating a three-layered system that mimics the cellular arrangement of the blood-brain...

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

This study demonstrates the utility and ease of quantitative cell membrane extension measurement and its correlation to adhesive capacity of cells. As a representative example, we show here that Dickkopf-related protein 3 (DKK3) promotes increased lobopodia formation and cell adhesiveness in adrenocortical carcinoma cells in...

in vitro cell migration and invasion assays

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of...

In vitro Cell Migration and Invasion Assays

a murine model of hyperlipidemia&#45;induced heart failure with preserved ejection fraction

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol presents a detailed approach to replicating a murine model of hyperlipidemia-induced heart failure with preserved ejection fraction (HFpEF). The design combines the administration of adeno-associated virus 9-cardiac troponin T-low-density lipoprotein receptor (AAV9-cTnT-LDLR) and poloxamer-407...

Investigating HFpEF Pathophysiology in a Hyperlipidemia&amp;#45;Induced Mouse Model

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bringing the clinic home: an at-home multi-modal data collection ecosystem to support adaptive deep brain stimulation

Author Spotlight: Automated Deep Brain Stimulation for Parkinson&#039;s Disease - Exploring the Possibilities and Challenges of Home Monitoring 

The protocol shows a prototype of the at-home multi-modal data collection platform that supports research optimizing adaptive deep brain stimulation (aDBS) for people with neurological movement disorders. We also present key findings from deploying the platform for over a year to the home of an individual with Parkinson's...

Optimizing Deep Brain Stimulation for Patients with Neurological Disorders

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gene transfer to the developing mouse inner ear by in vivo electroporation

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

Incubate the flask for 5 minutes at 37 degrees Celsius in a carbon dioxide incubator. View under a microscope to confirm whether the cells are detached from the flask. Add 5 milliliters of warm parasite medium, containing 2% FBS to the flask, and transfer the detached cells to a 50-milliliter centrifuge tube. Centrifuge the cell suspension for three minutes at 200 g, allowing the cells to form a pellet in the bottom of the...

non-invasive modulation and robotic mapping of motor cortex in the developing brain

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in...

preparation of a single-cell suspension from mouse carotid arteries for single-cell sequencing

Author Spotlight: Vascular Tissue Dissociation and Exploring Single-Cell Subclusters for Targeted Therapy 

Here we describe a two-step cell digestion protocol for preparing a single-cell suspension of mouse carotid...

Two-Step Cell Digestion of Mouse Carotid Artery

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brain tumor stem cell (btsc) migration assay for drug treatment studies: an in vitro live-cell imaging technique to analyze the effect of drug treatment on migration of btscs

In this video, we perform brain tumor stem cell migration assay to analyze the effect of the drug of interest on the migration of live brain tumor stem cells using chemotaxis migration plate. Live-cell imaging facilitates the visualization and quantification of cell migration during the...

Brain Tumor Stem Cell (BTSC) Migration Assay for Drug Treatment Studies: An In Vitro Live-cell Imaging Technique to Analyze the Effect of Drug Treatment on Migration of BTSCs