Les auteurs remercient Robert Kelsh pour avoir bien voulu partager le poisson zèbre réactif promoteur SOX10.

Une. Collection d&#39;embryons pour Photoconversion <ol><li> Mettre en place au moins dix paires de SOX10: poisson zèbre transgénique Kaede entre 5 et 18 heures dans la soirée. </li><li> Le lendemain matin, tirez diviseurs et ramasser les œufs vers midi. Les transférer à des boîtes de Pétri et les mettre dans 28,5 ° C incubateur. </li><li> Aux environs de 24 heures après la fécondation, nettoyer les boîtes de Pétri en supprimant des embryons morts. Vérifiez le stade de développement des embryons 13 en utilisant la microscopie champ de lumière et tout microscope à fluorescence avec la protéine fluorescente verte (EGFP) de filtre. Kaede sera visible à travers ce filtre. </li><li> Lorsque embryons atteignent 60 transfert de HPF cinq embryons fluorescentes vives dans une boîte de Petri séparée et dechorionate si nécessaire. </li></ol> 2. Montage des embryons pour Photoconversion <ol><li> Montez l&#39;embryon en utilisant un microscope à fluorescence normale. Montage des embryons sur microscope confocal est extrêmement difficile. </li><li> Employez le stand deeau ard E3 ou préparer E3 frais en utilisant la recette suivante: Ajouter le texte suivant pour un litre d&#39;eau distillée dH de l&#39;eau 2 O ajouter ce qui suit à faire 1x E3L: 0,292 g NaCl 5.0 0,013 g KCl 0.17mm 0,044 g 0.33mm CaCl 2 (déshydratant, 96%, réactif ACS) 0,081 g 0,33 mM MgSO 4 (anhydrated) Facultatif: ajouter 200 ul de 0,05% de bleu de méthylène à 1X E3 comme fongicide et remuer. Moyen maintient à la température ambiante pendant 1 semaine. </li><li> Utilisez tricaine standard ou préparer tricaine utilisant recette suivante: Pour 5 L de 0,2% tricaïne mélanger les ingrédients suivants: 45 ml de tris-Cl (1M pH 9,5) 5 LH 2 O 10 g tricaine </li><li> Préparer «jus de film&quot; (50 ml E3, solution de tricaine 0,015%), en ajoutant 3,75 ml de 0,2% à 46,25 ml tricaine E3. </li><li> Préparer agarose en mélangeant 46,25 ml d&#39;eau avec 46,35 mg E3 bas point de fusion (LMP) d&#39;agarose dans un flacon en verre de 50 ml et dissoudre les particules solides par les micro-ondes. Puis ajouter3,73 ml de 0,2% d&#39;agarose à tricaïne et mettre ballon en verre avec de l&#39;agarose à 50 ° C bain d&#39;eau jusqu&#39;à l&#39;utilisation. </li></ol> 3. Préparation et montage de l&#39;embryon <ol><li> Anesthésier embryons avec une solution de tricaine E3/0.015% rendue à l&#39;étape 1.1.2 et le transfert d&#39;embryons à la chambre diapositive. </li><li> Imprégnez-vous de tout excès E3 avec du papier et la vente d&#39;agarose sur l&#39;embryon. </li><li> Position embryon parfaitement dorsale avec des conseils de microloader. Assurez-vous que l&#39;ensemble de l&#39;embryon ne flotte pas au-dessus de la gélose, mais pousser l&#39;ensemble du corps vers le bas. Puis verser la solution de tricaine E3/0.015% par rapport à l&#39;embryon agarose embarqué jusqu&#39;à la chambre est entièrement rempli de liquide. </li></ol> 4. Réglage des paramètres du logiciel sur microscope confocal <ol><li> Utilisation air 20X objectif immersible (ouverture numérique de 0,75, la taille de trou d&#39;épingle 20) pour focaliser l&#39;embryon. Ensuite, appuyez sur le bouton d&#39;éclairage L100 sur le microscope et sélectionner les canaux dans le logiciel. Controls/A1settings ouvrir les icônes voir / d&#39;acquisition et click le bouton de configuration confocale. Ensuite, cliquez sur &quot;auto&quot; et sélectionner les canaux suivants avant de fermer la fenêtre de nouveau: Ch1: DAPI canal 403.4 nm (longueur d&#39;onde de 350 à 410 nm entre peuvent être utilisés) Ch2: 488,0 nm Ch3: 561,9 nm </li></ol> 5. Photoconversion <ol><li> Désactiver Ch1 et 3 et tourner sur Ch2 avant de cliquer sur le bouton supprimer de verrouillage et de numérisation. </li><li> Focus sur les cellules d&#39;intérêt pour photoconversion, qui est le mieux fait en commençant par la tension 1frame/sec et haute (HT) 200, puis aller dans le cadres / sec et l&#39;abaissement de la HV en conséquence jusqu&#39;à ce qu&#39;il atteigne 1/32 images / s et HV autour 100 à 120, en fonction du bruit de fond. </li><li> Lorsque l&#39;embryon est au point, regarder sur le bord droit de l&#39;écran, saisissez le rebord vert et le rendre plus petit de sorte qu&#39;il s&#39;adapte à la zone d&#39;intérêt pour photoconversion et clic droit avec la souris. Plus petit est mieux parce que la photoconversion est irréversible. </li><li> Tournez images / sec à 1 et HV 200 et frappernumériser. Une image zoomée de la zone de photoconversion est maintenant visible. </li><li> cellules Photoconvert en tournant sur Ch1 (403,4 nm). Exposer au laser photoconverting partout 5-60 secondes, selon la taille de la région jusqu&#39;à ce que Kaede vert comme on le voit par la voie de la GFP est presque absent. </li><li> Lorsque le signal de Kaede vert est presque absent, tourner Ch1 off. Tournez Ch2 et 3 sur. Puis cliquez droit sur la fenêtre sur A1 champ de la zone de numérisation et cliquez sur «retour à la taille originale&quot; pour vérifier si les cellules désirées ont été photoconverted. </li></ol> 6. Faire un Z-stack <ol><li> Trouvez l&#39;icône «capture série Z» dans la barre de menus et fixer des limites supérieures et inférieures de Z-pile avant de le démarrer. Réglez LUT. </li></ol> 7. Logiciels-Paramètres Time-lapse <ol><li> Aller à la boîte A1Simple GUI et cochez les paramètres suivants: 1/32 images / s taille 1024 moyenne de 4 ou 8 fois, selon le nombre de Z-piles qui doit être prise dans une boucle </li><li> Aller à:contrôles vue / acquisition / acquisition de séquence ND et mis délai (8-30min), cliquez sur continue et cliquez Z-stack. Définir les frontières comme testé avant et afficher la vidéo. </li><li> Continuez à ajouter E3/0.015% tricaine solution chambre glissière tout au long de la journée. La nuit, remplir la chambre complètement. Cela durera jusqu&#39;à ce que le lendemain matin. </li><li> Le lendemain matin, remplir solution tricaine E3/0.015%, et vérifier si l&#39;embryon est encore en vie. Perte de fluorescence indique la mort. Continuer recharges et contrôles tout au long de la journée. </li><li> Lorsque la structure a terminé la formation, ou l&#39;embryon est mort, terminer le film. </li></ol> 8. Traitement post-production <ol><li> Cliquez sur &quot;projection d&#39;intensité maximale de spectacle» sur la barre d&#39;outils, puis faites un clic droit sur l&#39;image et créer un nouveau document. La vidéo est maintenant visible dans la meilleure qualité possible. Passez à supprimer des images qui ne sont pas nécessaires et ajuster LUT. Enfin enregistrer sous. Avi. Ce format sera parfait pour Windows. Pour Mac logiciel de téléchargement to convertir. mov ou. wmv. </li></ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+at+Birth+of+Cleft+Lip+With+or+Without+Cleft+Palate:+Data+From+the+International+Perinatal+Database+of+Typical+Oral+Clefts+(IPDTOC).">Prevalence at Birth of Cleft Lip With or Without Cleft Palate: Data From the International Perinatal Database of Typical Oral Clefts (IPDTOC).</a> Cleft Palate Craniofac. J. 48 (1), 66-81 (2011).</li><li>Dixon, M. 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=Cleft+lip+and+palate:+understanding+genetic+and+environmental+influences.">Cleft lip and palate: understanding genetic and environmental influences.</a> Nat. Rev. Genet. 12 (3), 167-178 (2011).</li><li>Mangold, E., Ludwig, K. U., Nothen, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakthroughs+in+the+genetics+of+orofacial+clefting.">Breakthroughs in the genetics of orofacial clefting.</a> Trends Mol. Med. 17 (12), 725-733 (2011).</li><li>Kimmel, C. B., Miller, C. T., Moens, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+and+morphogenesis+of+the+zebrafish+larval+head+skeleton.">Specification and morphogenesis of the zebrafish larval head skeleton.</a> Dev. Biol. 233 (2), 239-257 (2001).</li><li>Dougherty, 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=Embryonic+Fate+Map+of+First+Pharyngeal+Arch+Structures+in+the+sox10:+kaede+Zebrafish+Transgenic+Model.">Embryonic Fate Map of First Pharyngeal Arch Structures in the sox10: kaede Zebrafish Transgenic Model.</a> J. Craniofac. Surg. 23 (5), 1333-1337 (2012).</li><li>Trainor, P. A., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+genes,+neural+crest+cells+and+branchial+arch+patterning.">Hox genes, neural crest cells and branchial arch patterning.</a> Curr. Opin. Cell Biol. 13 (6), 698-705 (2001).</li><li>Schilling, T. F., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segment+and+cell+type+lineage+restrictions+during+pharyngeal+arch+development+in+the+zebrafish+embryo.">Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.</a> Development. 120 (3), 483-494 (1994).</li><li>Swartz, M. E., 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=Examination+of+a+palatogenic+gene+program+in+zebrafish.">Examination of a palatogenic gene program in zebrafish.</a> Dev. Dyn. 240 (9), 2204-2220 (2011).</li><li>McCollum, C. W., 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=Developmental+toxicity+screening+in+zebrafish.+Birth+Defects+Res.">Developmental toxicity screening in zebrafish. Birth Defects Res.</a> C. Embryo Today. 93 (2), 67-114 (2011).</li><li>Wada, 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=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132 (17), 3977-3988 (2005).</li><li>Eberhart, 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=Early+Hedgehog+signaling+from+neural+to+oral+epithelium+organizes+anterior+craniofacial+development.">Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development.</a> Development. 133 (6), 1069-1077 (2006).</li><li>Ando, R., 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=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U S A. 99 (20), 12651-12656 (2002).</li><li>Kimmel, C. B., 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=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1993).</li><li>Kawakami, 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=The+zebrafish-secreted+matrix+protein+you/scube2+is+implicated+in+long-range+regulation+of+hedgehog+signaling.">The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling.</a> Curr. Biol. 15 (5), 480-488 (2005).</li><li>Lombardo, V. A., Sporbert, A., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+using+photoconvertible+proteins+during+zebrafish+development.">Cell tracking using photoconvertible proteins during zebrafish development.</a> J. Vis. Exp. (67), e4350 (2012).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Voici une nouvelle méthode pour la visualisation de développement cranio-facial dans le modèle de poisson zèbre est affiché. Les SOX10: Kaede ligne de poisson zèbre transgénique a été utilisé avec succès pour décrire le modèle migratoire de la CNCC en détail est utilisé comme organisme modèle 5. Des études antérieures ont utilisé des repères bruts tels que l&#39;œil aux cellules cibles et se sont appuyés sur l&#39;injection Kaede de l&#39;ARNm, des essais de ...

Les fentes faciales représentent la déformation craniofaciale la plus répandue, avec 1/700-1, 000 livraisons affectées 1. Perturbation du développement cranio-facial embryonnaire précoce peut conduire à la formation de fente labio-palatine (FL / P). Bien que les causes de fissure syndromique ont été largement représenté, les bases génétiques et épigénétiques de formes non syndromiques de clefting buccofaciale doivent encore être mis au jour 2-4. Afin de comprendre l&#39;étiologie et la pathogenèse de ces malformations, il est nécessaire d&#39;élucider le développement des structures craniofaciales sur une base cellulaire. Dans toutes les espèces de vertébrés cellules de la crête neurale crânienne (CNCC) migrent du tube neural dorsal pour remplir les arcs pharyngiens, ce qui contribuera à la formation de structures oro-faciales. Perturbation du développement précoce de la crête neurale embryonnaire peut conduire à la formation de malformations cranio-faciales, y compris CL / P 5-7. Dans annoncecondition des similitudes structurelles entre le poisson zèbre et le développement cranio-facial chez les mammifères (CNCCs résident dans des régions homologues), le réseau de régulation génique est hautement conservée. Il a également été montré que CNCCs développer de la même manière entre les espèces de poisson-zèbre amniote et 8, ce qui rend le poisson zèbre d&#39;un organisme efficace pour l&#39;étude de la génétique et développementale base de CL / P. Il présente de nombreux avantages, y compris de petite taille, rapide et ex-utero le développement embryonnaire, et les taux de reproduction élevé. De plus, l&#39;embryon est optiquement transparent, ce qui prête à l&#39;observation d&#39;événements complexes de développement sous la loupe 9. Il s&#39;agit d&#39;un modèle animal idéal pour l&#39;étude de la migration et la différenciation des cellules de la crête neurale crâniens. Étendre sur le travail déjà publié 8, 10, 11, le modèle migratoire de la CNCC a été décrite en détail à l&#39;aide des SOX10: Kaede modèle transgénique 5. Kaede est une protéine de photo-convertible que turns du vert au rouge après l&#39;activation de photo et permet de retracer CNCCs précisément. Au cours de cette transformation dans le squelette peptidique est clivée, ce qui suggère que la conversion est stable, ce qui signifie les cellules peuvent être suivis à leur destination finale 12. Les lignées transgéniques étiquetés avec kaede sous contrôle transcriptionnel d&#39;SOX10 ont montré que la bouche de amniote et la plaque ethmoïde de poisson-zèbre sont formées de manière homologue par fusion de saillies maxillaires bilatérales (MXP) avec la proéminence frontonasal (FNP), et que la couture de fusion en forme de Y est similaire entre espèces. Parmi d&#39;autres applications, les SOX10: Kaede transgénique modèle de poisson zèbre a été utilisé pour générer des vidéos de embryons de poisson zèbre à différents stades de développement de montrer la formation de structures cranio-faciales normales et anormales. Photoconversion de groupes spécifiques de cellules, il est possible de suivre leur développement. Avec cette méthode, une approche pour créer l&#39;imagerie en direct de développer craniofacstructures ial chez le poisson zèbre est introduit, le rendant facile à démontrer visuellement ce processus complexe du développement. Ce protocole vise à partager l&#39;expérience de la production de ces vidéos à l&#39;aide au développement normal de la plaque ethmoïde dans SOX10: poisson zèbre transgénique Kaede, par exemple. Ce protocole peut en outre être appliquée à faire des vidéos time-lapse de toute structure dérivée de cellules de la crête neurale crânienne chez le poisson zèbre.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>sox10: kaede transgenic zebrafish line</td>
			<td>MGH</td>
			<td></td>
			<td>Available via the Liao lab</td>
		</tr>
		<tr>
			<td>Petri dishes 100x15 mm</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 mmx10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Low melting point (LMP) Agarose</td>
			<td>Invitrogen</td>
			<td>15517022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tek 2 Chamber SlideSystem</td>
			<td>LabTek</td>
			<td>154453</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloaders 200/pk</td>
			<td>Fisher</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R Si Confocal Ti series</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements Software AR3.2 64-bit</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy

Vertébré palatogenesis est un processus de développement très chorégraphiée et complexe, qui implique la migration de la crête neurale crânienne (CNC) des cellules, la convergence et l&#39;extension des protubérances du visage, et la maturation du squelette cranio-facial. Pour étudier la contribution de la crête neurale crânienne à des régions spécifiques du palais de poisson zèbre un de SOX10: Kaede ligne de poisson zèbre transgénique a été généré. Sox10 fournit restriction de la lignée de la protéine Kaede rapporteur de la crête neurale, ce qui rend la cellule étiquetage d&#39;un processus plus précis que colorant traditionnel ou injection d&#39;ARNm rapporteur. Kaede est une protéine de photo-convertible qui passe du vert au rouge après l&#39;activation de photo et permet de suivre précisément les cellules. Les SOX10: Kaede ligne transgénique a été utilisé pour effectuer une analyse de la lignée de délimiter les populations de cellules CNC qui donnent lieu à maxillaire par rapport à des éléments mandibulaires et illustrent homologie des protubérances du visage pour amniotes. Ce protocole décrit l&#39;étape consistant às pour générer une vidéo en time-lapse en direct d&#39;un SOX10: embryon de poisson zèbre Kaede. Développement de la plaque ethmoïde servira un exemple pratique. Ce protocole peut être appliqué à la réalisation d&#39;un time-lapse enregistrement confocale de toute Kaede ou protéine rapporteur photoconvertible similaire chez le poisson zèbre transgénique. En outre, il peut être utilisé pour capturer non seulement normal, mais aussi anormale développement des structures craniofaciales dans les mutants de poisson zèbre.

Dans le SOX10: CNCCs Kaede lignée transgénique, migratoires et post migratoires sont marqués par fluorescence verte. Les cellules marquées par la CNCC vert fluorescent Kaede récapitulent endogène expression de l&#39;ARNm SOX10 5. Parmi d&#39;autres applications, ce modèle animal a été utilisé pour mieux visualiser le développement de structures cranio-faciales charge CNCC. Le développement normal des structures spécifiques et aussi le développement pathologique des m...

Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Visualisation des données expérimentales est devenu un élément clé dans la présentation des résultats à la communauté scientifique. Génération de direct enregistrement time-lapse d&#39;embryons croissance contribue à une meilleure présentation et la compréhension des processus de développement complexes. Ce protocole est un guide pour le marquage des cellules par l&#39;intermédiaire de photoconversion de la protéine chez le poisson zèbre kaede étape par étape.

Visualisation de développement cranio-facial dans les SOX10: Kaede poisson zèbre transgénique ligne à l&#39;aide Time-lapse de microscopie confocale

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

visualization-craniofacial-development-sox10-kaede-transgenic

Research

JoVE Journal

Biology

12.7K vues.  Massachusetts General Hospital.  Visualisation des données expérimentales est devenu un élément clé dans la présentation des résultats à la communauté scientifique. Génération de direct enregistrement time-lapse d'embryons croissance contribue à une meilleure présentation et la compréhension des processus de développement complexes. Ce protocole est un guide pour le marquage des cellules par l'intermédiaire de photoconversion de la protéine chez le poisson zèbre kaede étape pa...

Vidéo: Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy

Surveillance actine démontage Time-lapse microscopie

Recherche

Biologie

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Deux photons axotomie et time-lapse imagerie confocale dans embryons de poissons zèbres vivent

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

En direct l&#39;imagerie du cerveau de poisson zèbre embryonnaires par microscopie confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Imagerie time-lapse de la mitose après transfection de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

Multicolor imagerie time-lapse de poisson zèbre transgénique: Visualisation des cellules souches rétiniennes Activé par ablation cellulaire ciblée neuronale

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Time-lapse de microscopie du début de l&#39;embryogenèse dans les Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Electroporation de Mesenchyme craniofaciale

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Analyse quantitative des migrations au hasard des cellules en utilisant la microscopie vidéo time-lapse

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

À long terme, à haute résolution d&#39;imagerie confocal Time Lapse d&#39;<em&gt; Cotylédon Arabidopsis</emEpiderme&gt; cours de germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analyser craniofaciale morphogenèse chez le poisson zèbre Utilisation de 4D microscopie confocale

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...