The authors acknowledge funding from NINDS/NIH, R00-NS064197 and NINDS/NIH, R01NS083897 (both to D.L.S.).

1. Preparation of Media (Figure 1, Step 1)
<ol>
	<li>Slice Culture Medium
	<ol>
		<li>25 ml of slice culture medium is sufficient to prepare 5 glass bottom dishes with 2 slices per well.</li>
		<li>In a 50 ml conical tube, add 250 &#181;l of a 100x N2 solution and 500 &#181;l of a 50x B27 solution without vitamin A. Add DMEM/F12 to a volume of 22.5 ml.</li>
		<li>Filter sterilize solution and subsequently add 1.25 ml of heat inactivated horse serum and 1.25 ml of fetal bovine serum.</li>
		<li>Incubate solution in a wa...

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+ventricular+zone+neural+precursors+contributes+to+neuronal+fate+diversity+in+the+postnatal+neocortex.">Heterogeneity in ventricular zone neural precursors contributes to neuronal fate diversity in the postnatal neocortex.</a> Journal of Neuroscience. 30, 7028-7036 (2010).</li><li>Konno, 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=Neuroepithelial+progenitors+undergo+LGN-dependent+planar+divisions+to+maintain+self-renewability+during+mammalian+neurogenesis.">Neuroepithelial progenitors undergo LGN-dependent planar divisions to maintain self-renewability during mammalian neurogenesis.</a> Nat Cell Biol. 10, 93-101 (2008).</li><li>LoTurco, J. J., Manent, J. -. 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F., Ang, E., 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=Mitotic+spindle+rotation+and+mode+of+cell+division+in+the+developing+telencephalon.">Mitotic spindle rotation and mode of cell division in the developing telencephalon.</a> Proc Natl Acad Sci USA. 100, 2890-2895 (2003).</li><li>Takahashi, T., Nowakowski, R. S., Caviness, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+cycle+of+the+pseudostratified+ventricular+epithelium+of+the+embryonic+murine+cerebral+wall.">The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall.</a> J Neurosci. 15, 6046-6057 (1995).</li><li>Pilaz, L. -. 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V., 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=Mitotic+spindle+orientation+predicts+outer+radial+glial+cell+generation+in+human+neocortex.">Mitotic spindle orientation predicts outer radial glial cell generation in human neocortex.</a> Nature Communications. 4, 1665 (2013).</li><li>Postiglione, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Inscuteable+Induces+Apical-Basal+Spindle+Orientation+to+Facilitate+Intermediate+Progenitor+Generation+in+the+Developing+Neocortex.">Mouse Inscuteable Induces Apical-Basal Spindle Orientation to Facilitate Intermediate Progenitor Generation in the Developing Neocortex.</a> Neuron. 72, 269-284 (2011).</li><li>Silver, D. L., 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+exon+junction+complex+component+Magoh+controls+brain+size+by+regulating+neural+stem+cell+division.">The exon junction complex component Magoh controls brain size by regulating neural stem cell division.</a> Nat Neurosci. 13, 551-558 (2010).</li><li>Pulvers, J. N., 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=Mutations+in+mouse+Aspm+(abnormal+spindle-like+microcephaly+associated)+cause+not+only+microcephaly+but+also+major+defects+in+the+germline.">Mutations in mouse Aspm (abnormal spindle-like microcephaly associated) cause not only microcephaly but also major defects in the germline.</a> Proc Natl Acad Sci USA. , 107-16595 (2010).</li><li>Feng, Y., Walsh, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+regulation+by+Nde1+controls+cerebral+cortical+size.">Mitotic spindle regulation by Nde1 controls cerebral cortical size.</a> Neuron. 44, 279-293 (2004).</li><li>Megraw, T. L., Sharkey, J. T., Nowakowski, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdk5rap2+exposes+the+centrosomal+root+of+microcephaly+syndromes.">Cdk5rap2 exposes the centrosomal root of microcephaly syndromes.</a> Trends Cell Biol. 21, 1-11 (2011).</li><li>Bond, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+centrosomal+mechanism+involving+CDK5RAP2+and+CENPJ+controls+brain+size.">A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size.</a> Nat Genet. 37, 353-355 (2005).</li><li>Shu, T., 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=Doublecortin-like+kinase+controls+neurogenesis+by+regulating+mitotic+spindles+and+M+phase+progression.">Doublecortin-like kinase controls neurogenesis by regulating mitotic spindles and M phase progression.</a> Neuron. 49, 25-39 (2006).</li><li>Nelson, B. R., Hodge, R. D., Bedogni, F., Hevner, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Interactions+between+Intermediate+Neurogenic+Progenitors+and+Radial+Glia+in+Embryonic+Mouse+Neocortex:+Potential+Role+in+Dll1-Notch+Signaling.">Dynamic Interactions between Intermediate Neurogenic Progenitors and Radial Glia in Embryonic Mouse Neocortex: Potential Role in Dll1-Notch Signaling.</a> Journal of Neuroscience. 33, 9122-9139 (2013).</li><li>Hu, D. J. -. 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=Dynein+recruitment+to+nuclear+pores+activates+apical+nuclear+migration+and+mitotic+entry+in+brain+progenitor+cells.">Dynein recruitment to nuclear pores activates apical nuclear migration and mitotic entry in brain progenitor cells.</a> Cell. 154, 1300-1313 (2013).</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> Nature Publishing Group. 7, 136-144 (2004).</li><li>Tyler, W. A., Haydar, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Genetic+Fate+Mapping+Reveals+a+Novel+Route+of+Neocortical+Neurogenesis,+Which+Is+Altered+in+the+Ts65Dn+Mouse+Model+of+Down+Syndrome.">Multiplex Genetic Fate Mapping Reveals a Novel Route of Neocortical Neurogenesis, Which Is Altered in the Ts65Dn Mouse Model of Down Syndrome.</a> Journal of Neuroscience. 33, 5106-5119 (2013).</li><li>Noctor, S. 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The major advantage of the protocol we have described is that it provides a dynamic temporal resolution of mitosis of neural progenitors. Typically, assays to visualize mitosis in the developing brain are performed using immunofluorescence of fixed tissue sections. But this approach only provides a snapshot of mitosis at one time point.
There are several steps that are most critical for imaging mitosis in brain slices: 1) The brain slices should remain attached to the agarose, to preserve the ...

The overall goal of this protocol is to describe how to perform live imaging of neural progenitor mitosis in embryonic brain slices. Using live imaging of brain slices in culture, this protocol provides a simple method to assay multiple aspects of mitosis in neural progenitors in an environment highly similar to an in vivo setting. It can be applied to brains of mutant animals and/or brains that have been manipulated with in utero electroporation)1-5. This technique is also ideal to test the effect of pharmacological agents on neural precursors&#39; mitosis, by simply adding an agent to the culture medium. In sum, this article will make a technically challenging protocol accessible to those studying neurogenesis.
During neurogenesis, distinct neural progenitor populations undergo precise divisions to generate neurons that eventually contribute to the six cortical layers of the adult neocortex6-8. Early in cortical development, the neural precursor pool expands as neuroepithelial (NE) cells divide symmetrically to self-renew. NE cells then convert into radial glial cells (RGCs). Initially RGCs divide symmetrically to produce two new RGCs, however during the bulk of neurogenesis, RGCs&#39; main mode of division is asymmetric. In asymmetric division, 1 RGC gives rise to a new RGC and either a post-mitotic neuron, or a more specialized progenitor (either a short neural precursor (SNP), an outer radial glia (ORG), or an intermediate progenitor (INP)2,3,7,9. INPs, SNPs, and ORGs can then generate neurons at the sub-ventricular, ventricular, and basal regions of the cortex, respectively. Hence, cell division of progenitors is a fundamental process for generating neurons of the neocortex.
Numerous studies point to a correlation between specific mitotic traits of RGCs and the fate of daughter cells. Haydar et al. and Takahashi et al. have shown that RGC mitotic duration and cell cycle length increase as neurogenesis proceeds, a finding echoed in follow up studies10-13. A number of studies have suggested that mitotic spindle orientation relative to the ventricle influences aspects of neurogenesis and corticogenesis, including types of neurons generated and location of progeny in the brain, respectively3,10,14-16. Whether cleavage plane orientation directly influences cell fate is controversial, but the conclusion remains that this mitotic parameter impacts neurogenesis. Further underscoring the importance of mitosis is the observation that many genes involved in the mechanics of mitosis are crucial for neurogenesis and for proper brain development17-20.
Mitosis is a dynamic process, yet to date most studies detailing neural progenitor mitosis utilize analysis of fixed tissue sections or imaging of neural progenitors via in vitro cell culture. Thus, the mainstream methods to evaluate mitosis only provide a snapshot of this process and fail to uncover how cells behave in a tissue. Live imaging of neural progenitor mitosis has increasingly become a critical tool for understanding neural progenitor function. For examples please see these references4,8,10,21-25. Several outstanding protocols have been published for preparation and imaging of brain slices26,27. However to date, a comprehensive protocol for imaging and analysis of mitosis has not been described nor demonstrated in video.
This technique offers several significant advantages over fixed analysis of brain sections. Time-lapse analysis of brain slices enables generation of significantly more data points that can be analyzed in a flexible fashion. First, data is gathered at individual time points over the course of several minutes or several hours. One can analyze individual time points (to create a static montage) or can combine different time points into movies. Second, confocal imaging of slices enables generation of data at different Z sections in brain slices. As a result, individual sections can be analyzed. Alternatively, stacks of individual sections can be combined into a maximum intensity projection. Third, analysis is done in the context of a tissue, revealing how cells divide relative to neighboring cells and structures. Fourth, it is ideally suited to analysis of mutants that show some evidence of mitotic defects. Together this protocol will help clarify critical steps to aid investigators who wish to carry out live imaging of neural progenitor mitosis in their own laboratories.

<table border="0" cellpadding="0" cellspacing="0" style="width:739px;" width="738">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:143px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:261px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100X N2</td>
			<td>Life technologies</td>
			<td>17502048</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50X B27 without vitamin A</td>
			<td>Life technologies</td>
			<td>12587010</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12</td>
			<td>Life technologies</td>
			<td>11320033</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat-inactivated horse serum</td>
			<td>Sigma Aldrich</td>
			<td>H1138-500ml</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat-inactivated calf serum</td>
			<td>Sigma Aldrich</td>
			<td>F4135-500ML</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FGF</td>
			<td>R&#38;D Sytems</td>
			<td>3139-FB-025</td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGF</td>
			<td></td>
			<td></td>
			<td style="width:261px;">for culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X HBSS</td>
			<td>Life technologies</td>
			<td>14065-056</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hepes Free Acid</td>
			<td>Sigma Aldrich</td>
			<td>&#160;H4034</td>
			<td style="width:261px;">dilute to 1M (pH7.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.5M D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8769</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9M NaHC03</td>
			<td>Life technologies</td>
			<td>25080-094</td>
			<td style="width:261px;">for the dissection of the embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">low-melting agarose</td>
			<td>Fisher&#160;</td>
			<td>BP165-25</td>
			<td style="width:261px;">for generating slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Loctite 404 glue</td>
			<td>Loctite 404</td>
			<td>46551</td>
			<td style="width:261px;">keep at 4&#730;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">syto11</td>
			<td>Life technologies</td>
			<td>S7573</td>
			<td style="width:261px;">Make 5&#181;l aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mg/ml collagen type I&#160;</td>
			<td>Life technologies</td>
			<td>A1048301</td>
			<td style="width:261px;">for culturing slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass bottom dish</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td style="width:261px;">for culturing slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">petri dishes</td>
			<td></td>
			<td></td>
			<td style="width:261px;">for dissection of the embryos</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">digital thermometer</td>
			<td></td>
			<td></td>
			<td style="width:261px;">to measure the temperature of the agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spatula</td>
			<td></td>
			<td></td>
			<td style="width:261px;">to transfer brains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paintbrush</td>
			<td></td>
			<td></td>
			<td style="width:261px;">alternative to transfer brains</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vibratome</td>
			<td>Leica</td>
			<td>VT1000s</td>
			<td style="width:261px;">for generating slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting microscope</td>
			<td></td>
			<td></td>
			<td style="width:261px;">dissecting out embryos</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">imaging microscope</td>
			<td></td>
			<td></td>
			<td style="width:261px;">A confocal microscope is required, it needs to be equipped with an incubation chamber</td>
		</tr>
	</tbody>
</table>

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.

The success of this assay and the observation of multiple mitotic cells during one live imaging session largely depend upon both the integrity and anatomical level of the slice where acquisitions are made. As discussed below, the anatomical level of a slice is an important factor. Figure 3A shows rostro-caudal and medial-lateral locations where we have been most successful. For additional discussion of this subject please see Noctor26. The integrity of the slice can vary in different regions o...

Watch this Scientific Journal Video about Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex at JoVE.com

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

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 ...

live-imaging-of-mitosis-in-the-developing-mouse-embryonic-cortex

Research

JoVE Journal

Neuroscience

15.1K visualizações.  Duke University Medical Center.  Neural mitose de células progenitoras é um parâmetro crítico da neurogénese. Muito do nosso entendimento da mitose progenitor neural é baseado na análise de tecido fixo. Imagens ao vivo em fatias de cérebro de embriões é uma técnica versátil para avaliar a mitose com resolução temporal e espacial de alta em um ambiente controlado. 