This research project was supported by an ARRA Supplement, 5 R01 NS056380. Additional support was provided through the Viral Vector Core and the Microscopy Core of the Emory Neuroscience NINDS Core Facilities grant, P30NS055077. We thank the Emory Transgenic Mouse and Gene Targeting Core for deriving the mouse line from GENSAT; Greg Pazour for the stable SSTR3-GFP IMCD3 cell line; and Bradley Yoder for the Sstr3-GFP lentiviral construct. Monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD, and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. All animal procedures were approved by the IACUC and the Biosafety Committee at Emory University.

Adult mice are euthanized by mechanical cervical dislocation. All animal procedures were approved by the IACUC and the Biosafety Committee at Emory University.
1. Embryo Generation
<ol>
	<li>Cross tamoxifen- inducible Cre line, CAGGCreER, and dsRedCre reporter line (Tg(CAG-Bgeo,-DsRed*MST)1Nagy) (Figure 1) 5,6. To monitor the relative timing of Shh response in the daughter cells, cross CAGGCreER and dsRed line with the Olig2-eGFP BAC transgenic mice (Tg(Olig2-EGFP...

<ol>
	<li>Yamada, T., Pfaff, S. L., Edlund, T., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+pattern+in+the+neural+tube:+motor+neuron+induction+by+diffusible+factors+from+notochord+and+floor+plate.">Control of cell pattern in the neural tube: motor neuron induction by diffusible factors from notochord and floor plate.</a> Cell. 73, 673-686 (1993).</li><li>Ericson, J., Muhr, J., Placzek, M., Lints, T., Jessell, T. M., Edlund, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+induces+the+differentiation+of+ventral+forebrain+neurons:+A+common+signal+for+ventral+patterning+within+the+neural+tube.">Sonic hedgehog induces the differentiation of ventral forebrain neurons: A common signal for ventral patterning within the neural tube.</a> Cell. 81, 747-756 (1995).</li><li>Briscoe, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeodomain+Protein+Code+Specifies+Progenitor+Cell+Identity+and+Neuronal+Fate+in+the+Ventral+Neural+Tube.">Homeodomain Protein Code Specifies Progenitor Cell Identity and Neuronal Fate in the Ventral Neural Tube.</a> Cell. 101, 435-445 (2000).</li><li>Berbari, N. F., Johnson, A. D., Lewis, J. S., Askwith, C. C., Mykytyn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+ciliary+localization+sequences+within+the+third+intracellular+loop+of+G+protein-coupled+receptors.">Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors.</a> Mol. Biol. Cell. 19 (4), 1540-1547 (2008).</li><li>Vintersten, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+red:+red+fluorescent+protein+expression+in+mouse+ES+cells,+embryos,+and+adult+animals.">Mouse in red: red fluorescent protein expression in mouse ES cells, embryos, and adult animals.</a> Genesis. 40 (4), 241-246 (2004).</li><li>Hayashi, S., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+recombination+in+diverse+tissue+by+a+tamoxifen-inducible+form+of+Cre:+a+tool+for+temporally+regulated+gene+activation/inactivation+in+the+mouse.">Efficient recombination in diverse tissue by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse.</a> Dev. Biol. 244 (2), 305-318 (2002).</li><li>Gong, S., Zheng, C., Doughty, M. L., Losos, K., Didkovsky, N., Schambra, U. B., Nowak, N. J., Joyner, A., Leblanc, G., Hatten, M. E., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+expression+atlas+of+the+central+nervous+system+based+on+bacterial+artificial+chromosomes.">A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.</a> Nature. 425 (6961), 917-925 (2003).</li><li>Mukouyama, Y. S., Deneen, B., Lukaszewicz, A., Novitch, B. G., Wichterle, H., Jessell, T. M., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olig2++neuroepithelial+motoneuron+progenitors+are+not+multipotent+stem+cells+in+vivo.">Olig2+ neuroepithelial motoneuron progenitors are not multipotent stem cells in vivo.</a> Proc. Natl. Acad. Sci. U.S.A. 103 (5), 1551-1556 (2006).</li><li>Jones, E. A. V., Crotty, D., Kulesa, P. M., Waters, C. W., Baron, M. H., Fraser, S. E., Dickinson, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+in+vivo+imaging+of+postimplantation+mammalian+embryos+using+whole+embryo+culture.">Dynamic in vivo imaging of postimplantation mammalian embryos using whole embryo culture.</a> Genesis. 34, 228-235 (2002).</li><li>Piotrowska-Nitsche, K., Caspary, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+individual+cell+divisions+in+mouse+neuroepithelium+shows+asymmetry+in+cilium+formation+and+Sonic+hedgehog+response.">Live imaging of individual cell divisions in mouse neuroepithelium shows asymmetry in cilium formation and Sonic hedgehog response.</a> Cilia. 1 (6), (2012).</li><li>Nowotschin, S., Ferrer-Vaquer, A., Hadjantonakis, A. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mouse+development+with+confocal+time-lapse+microscopy.">Imaging mouse development with confocal time-lapse microscopy.</a> Methods in Enzymology. 476, 351-377 (2010).</li></ol>

No conflicts of interest declared.

Our ex vivo system enables us to directly observe single cell divisions within the developing neuroepithelium in real time. As an example we examined cell divisions within the mouse embryonic neural tube and monitored either cilium formation or Shh response. We confirmed our imaging results (n = 24) were consistent with results from fixed sections (n = 178) indicating our technique provides physiologically relevant data.
Our technique relies on Cre induction being l...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Z/RED line (STOCK Tg(CAG-Bgeo,-DsRed*MST)1Nagy/J</td>
			<td>Jackson Laboratory</td>
			<td>005438</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Olig2-eGFP line (STOCK Tg(Olig2-EGFP)EK23Gsat/Mmcd</td>
			<td>MMRRC,</td>
			<td>010555-UCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CAGGCreER</td>
			<td>Jackson Laboratory</td>
			<td>003724</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1)</td>
			<td>GIBCO</td>
			<td>21041-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Newborn calf serum</td>
			<td>Lonza</td>
			<td>14-416F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rat Serum SD male</td>
			<td>Harlan Bioproducts</td>
			<td>4520</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1M Hepes</td>
			<td>BioWhittaker</td>
			<td>17-737E</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>GIBCO</td>
			<td>21041-025</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Light mineral oil</td>
			<td>Sigma</td>
			<td>M8410</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sstr3-GFP lentivirus</td>
			<td>Emory Viral Core</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micro-knife, size 0.025 mm</td>
			<td>Electron Microscopy Sciences</td>
			<td>62091</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-L-lysine coated glass bottom dish</td>
			<td>MatTek</td>
			<td>P35GC-0-10-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>100% petroleum jelly</td>
			<td>Kroger</td>
			<td>FL9958c</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A1R Laser Scanning Confocal Inverted Microscope</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NIS Elements software</td>
			<td>Nikon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Imaris 3D software</td>
			<td>Bitplane AG</td>
			<td>Imaris 7.2.3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1850</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat-inactivated sheep serum</td>
			<td>Invitrogen</td>
			<td>16210-072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Parafolmaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffer</td>
			<td>Lab made</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rat monoclonal anti-RFP (5F8)</td>
			<td>Chromotek</td>
			<td>110411</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti-Arl13b serum</td>
			<td>NeuroMab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-Arl13b 1:5</td>
			<td>NeuroMab</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit anti-Olig2</td>
			<td>Chemicon</td>
			<td>AB9610</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonals Pax6</td>
			<td>Developmental Hybridoma Bank</td>
			<td>Pax6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonalsShh</td>
			<td>Developmental Hybridoma Bank</td>
			<td>5E1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mouse monoclonals Nkx2.2</td>
			<td>Developmental Hybridoma Bank</td>
			<td>74.5A5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal Ki67</td>
			<td>Abcam</td>
			<td>AB15580</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568</td>
			<td>Molecular Probes</td>
			<td>A11031</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Alexa Fluor 350</td>
			<td>Molecular Probes</td>
			<td>A11046</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Fisher</td>
			<td>AC22989</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TO-PRO-3</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ProLong Gold anti-fade reagent</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

ex vivo live imaging of single cell divisions in mouse neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Here we performed ex vivo live imaging of single cell divisions within the E8.5 mouse neuroepithelium. To label individual cells, we induced Cre recombinase in a subset of cells containing a Cre reporter line that expressed dsRed upon recombination 5,6(Figure 3A). Thus, 48 hr later we were able to observe single cell divisions during ex vivo imaging (Figures 4A-D). Concomitantly, we monitored when the labeled cells became Shh-responsive by including a Shh tra...

Watch this Scientific Journal Video about Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium at JoVE.com

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

ex-vivo-live-imaging-of-single-cell-divisions-in-mouse-neuroepithelium

Research

JoVE Journal

Neuroscience

10.3K Görüntüleme.  Emory University School of Medicine. Burada için gerekli araçları geliştirmek Ex vivo Canlı görüntüleme fare E8.5 neuroepithelium tek hücre bölünmeleri izlemek için