Ce projet de recherche a été soutenue par un supplément ARRA, 5 R01 NS056380. Un soutien supplémentaire a été fourni par le vecteur viral de base et le noyau de microscopie de l&#39;Emory Neuroscience NINDS les principales installations de subvention, P30NS055077. Nous remercions la souris transgénique Emory et Gene Targeting base pour dériver la lignée de souris à partir de GENSAT; Greg Pazour pour la lignée cellulaire SSTR3-GFP IMCD3 stable et Bradley Yoder pour le SSTR3-GFP lentiviral construisons. Les anticorps monoclonaux ont été obtenus à partir des études du développement Hybridoma Bank, élaboré sous les auspices du NICHD, et entretenus par l&#39;Université de l&#39;Iowa, Département des sciences biologiques, Iowa City, IA 52242. Toutes les procédures d&#39;animaux ont été approuvés par le IACUC et le Comité de biosécurité de l&#39;Université Emory.

souris adultes sont euthanasiés par dislocation cervicale mécanique. Toutes les procédures d&#39;animaux ont été approuvés par le IACUC et le Comité de biosécurité de l&#39;Université Emory. 1. Embryon Generation <ol><li> Cross tamoxifène Cre inductible ligne, CAGGCreER, et la ligne de journaliste dsRedCre (Tg (CAG-Bgeo,-DsRed * MST) 1Nagy) (Figure 1) 5,6. Pour suivre la chronologie relative de la réponse Shh dans les cellules filles, croix CAGGCreER et DsRed ligne avec le Olig2-eGFP BAC souris transgéniques (Tg (Olig2-EGFP) EK23Gsat) (Figure 1) 7,8. </li><li> Dissoudre le tamoxifène dans l&#39;éthanol à 100%, et d&#39;effectuer une injection intrapéritonéale de 2,5 mg de tamoxifène par 40 g de poids corporel des femelles gravides à 6,5 embryonnaire (E6.5) pour induire l&#39;expression Cre et l&#39;étiquetage DsRed dans un petit sous-ensemble de cellules. </li><li> 48 heures plus tard disséquer embryons, d&#39;identifier les embryons positifs DsRed utilisant le microscope à fluorescence, les transférer à la boîte de culture et observateurse divisions cellulaires simples lors de l&#39;imagerie ex vivo (voir ci-dessous). </li></ol> 2. Whole embryon de souris Culture <ol><li> Disséquer E8.5 embryons en milieu de lavage préchauffé contenant DMEM/F12 (1:1) complété avec 10% de sérum de veau nouveau-né et 1% de pénicilline / streptomycine (P / S) 9. </li><li> Directement après dissection, place embryons sur ° étage de chauffage 37 ° C sous le microscope fluorescent et d&#39;identifier que la GFP et / ou DsRed positive (voir ci-dessous). </li><li> Transfert jusqu&#39;à 2 embryons dans une goutte 500 ul de milieux de culture pré-équilibrée contenant 50% sérum de rat d&#39;un mâle Sprague-Dawley et 50% DMEM/F12 (1:1) sans rouge de phénol additionné de L-glutamine et 1% des 1 M HEPES dans NaCl 0,85% et P / S 9. </li><li> Appliquer une couche mince (0,1 cm) d&#39;huile minérale légère équilibrée sur le moyen pour éviter l&#39;évaporation et le transfert de la boîte de culture contenant les embryons à la 37 ° C, 5% de CO 2 incubateur. </li></ol> 3.Infection virale <ol><li> Afin de cils de l&#39;étiquette dans le neuroépithélium, ajouter 5-10 pi de SSTR3-GFP lentivirus, environ 2 millions de virions, à une baisse de 500 ul équilibrée de milieu de culture contenant un embryon E8.5. </li><li> Après 18 heures de culture, de transférer l&#39;embryon infecté dans quelques gouttes de milieu de lavage pour laver le virus et préparer tranche du tube neural. </li></ol> 4. Neural Tube Slice Préparation pour l&#39;imagerie en direct <ol><li> Disséquer tube neural de l&#39;embryon E8.5 en milieu de lavage préchauffé en utilisant une taille de micro-couteau 0.025 mm, sur un 1% d&#39;agar plat enduit. </li><li> Placez le neural face ventrale du tube isolé dans une goutte 150 ul de milieu de culture équilibrée sans rouge de phénol sur le mm enduit plat à fond de verre poly-L-lysine 35. </li><li> Mettez de petites quantités d&#39;un mélange 1:1 en 100% pur de la vaseline et la cire de bougie fondue (bougie d&#39;IKEA) autour du tube neural monté, et appuyez doucement par une étroite bande de lamelle de verre afin d&#39;immobiliser l&#39;échantillon. </li><li> Couvrir le plat avec une couche mince (~ 0,1 cm) d&#39;huile minérale légère équilibrée (Figure 2). </li></ol> 5. Vivre Imaging and Time-lapse microscopie confocale <ol><li> Placez le plat sous le Nikon A1R Laser Microscope inversé confocal équipé d&#39;une chambre environnementale qui régule la température, réglé à 37 ° C et 5% de CO 2. </li><li> Utilisez 60x objectif à immersion d&#39;huile d&#39;enregistrer GFP cils étiquetés et cellules positives DsRed, tandis que l&#39;utilisation de l&#39;objectif à immersion d&#39;huile 40x pour surveiller Olig2-GFP et des cellules positives DsRed. </li><li> Ouvrez le logiciel NIS Elements pour mettre en place les conditions d&#39;imagerie time lapse. Chaque 10 min acquérir z piles allant jusqu&#39;à 25 um avec un espacement de 1,5 um (40x) et jusqu&#39;à 8 pm avec un espacement de 0,4 um (60x objectif). Utilisez 488 et 561 nm les longueurs d&#39;onde d&#39;excitation et le canal transmis si nécessaire. Acquérir des images à 512 x 512 taille. Mettre en place plusieurs régions définies par l&#39;utilisateur de intEREST pour effectuer un enregistrement en même temps. Exposition optimale à la puissance du laser et de la luminosité de l&#39;image a été ajusté par l&#39;utilisation intensité du laser bas (jusqu&#39;à 11% pour mCherry 561; jusqu&#39;à 4,5% EGFP 405/488), vitesse de balayage 1, ligne moyenne 2 et la taille du trou d&#39;épingle (61 um pour dsRED; 28,1 um pour Olig2; 72 um pour SSTR3GFP; 61,3 um pour dsRED et SSTR3GFP; 58 um pour dsRED et Olig2). L&#39;utilisation de reporters fluorescentes codées génétiquement nous a permis de détecter la luminosité avec une puissance laser de faible. </li><li> Analyser les données enregistrées en utilisant un logiciel 3D Imaris de reconstruction. </li></ol> 6. Immunofluorescence <ol><li> Fixer des embryons ou des tubes neuraux isolés dans du paraformaldéhyde 4% / 0,1 M de tampon phosphate sur la glace (4 ° C) pendant 1 heure dans un plat en verre. </li><li> Lavez échantillons 2 h dans du PBS sur la glace (4 ° C) (variation PBS quelques fois) et de mettre en saccharose à 30% / 0,1 M de tampon phosphate au cours de la nuit ou jusqu&#39;à ce que les embryons évier. </li><li> Intégrer dans l&#39;OCT, gel sur la glace sèche et le ranger dans -80 ° C. Perform sectionner le cryostat (10 mm). </li><li> Laver les lames pendant 10 min dans une solution de lavage contenant 1% de sérum de mouton inactivés par la chaleur et de 0,1% de Triton X-100 dans du PBS. </li><li> Diluer anticorps primaires dans la solution de lavage à la suite de concentrations: Rat monoclonal anti-DP (5F8), 1:200; lapin anti-Arl13b 1:1500 sérum et anticorps monoclonal de souris anti-Arl13b 01h05 (295B/54); lapin anti-Olig2 1:300; anticorps monoclonaux de souris Pax6, chut et Nkx2.2-all 01h10 et de lapin polyclonaux Ki67 1:500. Ajouter environ 150 pi par diapo dans chambre humidifiée plat, couvrir avec du parafilm pour éviter le dessèchement et laisser sur la nuit à 4 ° C. </li><li> Laver les lames dans une solution de lavage à trois reprises, à 20 min à chaque fois à la température ambiante. </li><li> Diluer anticorps secondaire Alexa Fluor 488, 568, 350 dans les solutions de lavage à 1:200 concentration. Utilisez Hoechst 1:3,000 ou TO-PRO-3 1:500 pour colorer les noyaux. Ajouter 150 ul par diapositive, laisser 1 heure à température ambiante en chambre humide abri de la lumière. </li><li> Laver les lames dans la solution de lavage two fois, 30 min à chaque fois à la température ambiante. </li><li> Mont glisse dans 80% ProLong réactif et vue anti-fade or dans les 24 heures. </li></ol>

<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>

Aucun conflit d&#39;intérêt déclaré.

Notre système ex vivo nous permet d&#39;observer directement divisions cellulaires uniques dans le neuroépithélium développement en temps réel. À titre d&#39;exemple, nous avons examiné les divisions cellulaires dans le tube neural embryonnaire de la souris et surveillés soit la formation cil ou réponse Shh. Nous avons confirmé nos résultats d&#39;imagerie (n = 24) ont été cohérents avec les résultats des sections fixes (n = 178) indiquant notre technique fournit des données ph...

<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

Nous avons développé un système qui intègre l&#39;imagerie en direct de marqueurs fluorescents et des tranches de culture du neuro-épithélium embryonnaire de la souris. Nous avons profité de lignées de souris existants pour la cellule génétique lignée de traçage: une ligne Cre inductible par le tamoxifène et une ligne de journaliste exprimant Cre DsRed lors de la recombinaison Cre-médiation. En utilisant un niveau relativement bas du tamoxifène, nous étions capables d&#39;induire la recombinaison dans un petit nombre de cellules, ce qui permet de suivre les divisions cellulaires individuels. En outre, nous avons observé la réponse transcriptionnelle à Sonic Hedgehog (Shh) de signalisation utilisant un Olig2-eGFP transgénique ligne 1-3 et nous avons suivi la formation des cils en infectant la tranche cultivé avec le virus exprimant le marqueur de cils, SSTR3-GFP 4. Afin d&#39;image à l&#39;neuroépithélium, nous avons récolté embryons à E8.5, isolé du tube neural, fixé la tranche de neurones dans des conditions de culture appropriées dans la chambre de formation d&#39;image et réalisée en accéléré imagerie confocale. Notre exvivo méthode imagerie en temps réel permet de tracer des divisions cellulaires simples pour évaluer la chronologie relative de la formation des cils primaires et la réponse Shh d&#39;une manière physiologiquement pertinents. Cette méthode peut être facilement adapté en utilisant des marqueurs fluorescents distinctes et fournit le terrain, les outils permettant de surveiller le comportement des cellules in situ et en temps réel.

Ici, nous avons effectué ex vivo l&#39;imagerie en direct de divisions cellulaires simples dans le neuroépithélium souris E8.5. Pour marquer les cellules individuelles, nous avons provoqué la recombinase Cre dans un sous-ensemble de cellules contenant une ligne de journaliste Cre celle exprimée DsRed lors de la recombinaison 5,6 (figure 3A). Ainsi, 48 heures plus tard, nous avons pu observer des divisions cellulaires simples pendant ex imagerie in vivo ...

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

Ici, nous développons les outils nécessaires pour<em&gt; Ex vivo</em&gt; Imagerie en temps réel à tracer des divisions cellulaires simples dans la souris E8.5 neuroépithélium

Ex vivo d&#39;imagerie en temps réel de simples divisions cellulaires dans neuroépithélium de souris

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

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Neuroscience

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

10.3K vues.  Emory University School of Medicine. Ici, nous développons les outils nécessaires pour Ex vivo Imagerie en temps réel à tracer des divisions cellulaires simples dans la souris E8.5 neuroépithélium

Vidéo: Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Multiple-Mouse Imaging neuroanatomiques par résonance magnétique

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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Neurosciences

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Imagerie à haute résolution en direct du comportement des cellules dans le développement neuro-épithélium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

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.

Imagerie en direct de la mitose dans les développement Cortex souris embryonnaires

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

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex Vivo Imaging postnatal du cervelet granules migration cellulaire utilisant confocale Macroscopie

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Simultané<em&gt; Ex vivo</em&gt; Tests fonctionnels de deux rétines par<em&gt; In vivo</em&gt; Système Électrorétinogramme

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

Fluorescence de cellule unique résolution vivre Imaging of Drosophila horloges circadiennes dans cerveau larvaire Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of individual cells. Since an increase of intracellular calcium (Ca2+) in the cytosol often occurs because of the electrical activity, or in response to a myriad of other stimuli, this process can be monitored by the imaging of cells loaded with fluorescent calcium-sensitive dyes.&#160; However, it is difficult to image this response in an individual cell type within whole tissue because these dyes are taken up by all cell types within the tissue. In contrast, genetically encoded calcium indicators (GECIs) can be expressed by an individual cell type and fluoresce in response to an increase of intracellular Ca2+, thus permitting the imaging of Ca2+ signaling in entire populations of individual cell types. Here, we apply the use of the GECIs GCaMP3/6 to the mouse neuromuscular junction, a tripartite synapse between motor neurons, skeletal muscle, and terminal/perisynaptic Schwann cells. We demonstrate the utility of this technique in classic ex vivo tissue preparations. Using an optical splitter, we perform dual-wavelength imaging of dynamic Ca2+ signals and a static label of the neuromuscular junction (NMJ) in an approach that could be easily adapted to monitor two cell-specific GECI or genetically encoded voltage indicators (GEVI) simultaneously. Finally, we discuss the routines used to capture spatial maps of fluorescence intensity. Together, these optical, transgenic, and analytic techniques can be employed to study the biological activity of distinct cell subpopulations at the NMJ in a wide variety of contexts.

Ex Vivo Imaging of Cell-specific Calcium Signaling at the Tripartite Synapse of the Mouse Diaphragm

Here we present a protocol to image calcium signaling in populations of individual cell types at the murine neuromuscular junction.

Ex Vivo Imagerie du Calcium cellulaire spécifique de signalisation à la Synapse Tripartite du diaphragme souris

The electrical activity of cells in tissues can be monitored by electrophysiological techniques, but these are usually limited to the analysis of ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Transpupillaire Imagerie in vivo à deux photons de la rétine de souris

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imagerie du calcium chez la souris Olive inférieure

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Alignement du fibergramme OCT de la rétine de la souris avec des images confocales

Alignment of Visible-Light Optical Coherence Tomography Fibergrams with Confocal Images of the Same Mouse Retina

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has been increasingly applied to obtain an objective assessment of neural damage in eye diseases. Ex vivo confocal imaging of the same retina is often necessary to validate the in vivo findings especially in animal research. In this study, we demonstrated a method for aligning an ex vivo confocal image of the mouse retina with its in vivo images. A new clinical-ready imaging technology called visible light optical coherence tomography fibergraphy (vis-OCTF) was applied to acquire in vivo images of the mouse retina. We then performed the confocal imaging of the same retina as the &#34;gold standard&#34; to validate the in vivo vis-OCTF images. This study not only enables further investigation of the molecular and cellular mechanisms but also establishes a foundation for a sensitive and objective evaluation of neural damage in vivo.

Pleins feux sur l’auteur : Progrès de l’imagerie rétinienne in vivo et ex vivo pour améliorer le diagnostic et le traitement du glaucome 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

Alignement de fibrogrammes de tomographie par cohérence optique en lumière visible avec des images confocales de la même rétine de souris

In recent years, in vivo retinal imaging, which provides non-invasive, real-time, and longitudinal information about biological systems and processes, has ...