Name: targeted laser ablation in the embryo of saccharina latissima
Uploaded: 2022-03-11T15:00:00
Description: Watch this Scientific Journal Video about Targeted Laser Ablation in the Embryo of Saccharina latissima at JoVE.com

La bourse de doctorat de S.B. est financée par la Région Bretagne (numéro de subvention ARED COH20020) et la Sorbonne Université. I.T.is bourse de doctorat est financée par la Région Bretagne (numéro de subvention ARED COH18020) et l’Université norvégienne NMBU. Ce projet a reçu le soutien financier du CNRS à travers les programmes interdisciplinaires du MITI. MRic est membre de l’infrastructure nationale France-BioImagerie soutenue par l’Agence nationale de la recherche Français (ANR-10-INBS-04).

1. Production de Saccharina latissima gametophytes
<ol><li>Recueillir des sporophytes matures de S. latissima dans la nature comme décrit précédemment20,21. Assurez-vous que les sporophytes sélectionnés sont dépourvus d’épiphytes (petits organismes visibles à la surface de la lame) ou de parasites internes (présents dans les zones blanchies ou les taches sur la lame). 
</li>
	<li>À l’aide d’un scalpel,...

<ol>
	<li>Montell, D. J., Keshishian, H., Spradling, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+studies+of+the+role+of+the+Drosophila+oocyte+nucleus+in+pattern+formation.">Laser ablation studies of the role of the Drosophila oocyte nucleus in pattern formation.</a> Science. 254 (5029), 290-293 (1991).</li><li>Shivakumar, P. C., Lenne, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+ablation+to+probe+the+epithelial+mechanics+in+drosophila.">Laser ablation to probe the epithelial mechanics in drosophila.</a> Methods In Molecular Biology. 1478, 241-251 (2016).</li><li>Bargmann, C. I., Avery, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+killing+of+cells+in+Caenorhabditis+elegans.">Laser killing of cells in Caenorhabditis elegans.</a> Modern Biological Analysis of an Organism. 48, 225-250 (1995).</li><li>Fouad, A. D., Liu, A., Du, A., Bhirgoo, P. D., Fang-Yen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+laser+ablation+with+tunable+lesion+size+reveals+multiple+origins+of+seizure-like+convulsions+in+Caenorhabditis+elegans.">Thermal laser ablation with tunable lesion size reveals multiple origins of seizure-like convulsions in Caenorhabditis elegans.</a> Scientific Reports. 11 (1), 5084 (2021).</li><li>Johnson, C. S., Holzemer, N. F., Wingert, R. 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Cellular integration of signalling pathways in plant development.</a> Proceedings of the NATO Advanced Study Institute. , 237-250 (1998).</li><li>Berger, F., Taylor, A., Brownlee, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+determination+by+the+cell+wall+in+early+fucus+development.">Cell fate determination by the cell wall in early fucus development.</a> Science. 263 (5152), 1421-1423 (1994).</li><li>Bouget, F. Y., Berger, F., Brownlee, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Position+dependent+control+of+cell+fate+in+the+Fucus+embryo:+role+of+intercellular+communication.">Position dependent control of cell fate in the Fucus embryo: role of intercellular communication.</a> Development. 125 (11), 1999-2008 (1998).</li><li>Bringloe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogeny+and+evolution+of+the+brown+algae.">Phylogeny and evolution of the brown algae.</a> Critical Reviews in Plant Sciences. 39 (4), 281-321 (2020).</li><li>Saifullah, S., Olsen, Y., Surilayani, D., Hand&#229;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+of+the+brown+seaweed,+saccharina+latissima:+a+review.">Carbohydrate of the brown seaweed, saccharina latissima: a review.</a> Joint proceedings of the 2nd and the 3rd International Conference on Food Security Innovation (ICFSI 2018-2019). , 180-182 (2021).</li><li>Zhang, X., Thomsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techno-economic+and+environmental+assessment+of+novel+biorefinery+designs+for+sequential+extraction+of+high-value+biomolecules+from+brown+macroalgae+Laminaria+digitata,+Fucus+vesiculosus,+and+Saccharina+latissima.">Techno-economic and environmental assessment of novel biorefinery designs for sequential extraction of high-value biomolecules from brown macroalgae Laminaria digitata, Fucus vesiculosus, and Saccharina latissima.</a> Algal Research. 60, 102499 (2021).</li><li>Kanda, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+gametophytes+of+some+japanese+species+of+laminariales.">On the gametophytes of some japanese species of laminariales.</a> Scientific papers of the Institute of Algological Research, Faculty of Science. 1 (2), 221-260 (1936).</li><li>Fritsch, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The structure and reproduction of the algae. Volume 2. , (1945).</li><li>Bartsch, I., 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+genus+Laminaria+sensu+lato+:+recent+insights+and+developments.">The genus Laminaria sensu lato : recent insights and developments.</a> European Journal of Phycology. 43 (1), 1-86 (2008).</li><li>Theodorou, I., Charrier, B., Boutet, A., Schierwater, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+2:+Brown+algae:+ectocarpus+and+saccharina+as+experimental+models+for+developmental+biology.">Chapter 2: Brown algae: ectocarpus and saccharina as experimental models for developmental biology.</a> Handbook of Marine Model Organisms in Experimental Biology - Established and Emerging. , 485 (2021).</li><li>Drew, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reproduction+and+early+development+of+laminaria+digitata+and+laminaria+saccharina.">The reproduction and early development of laminaria digitata and laminaria saccharina.</a> Annals of Botany. 24 (1), 177-189 (1910).</li><li>Yendo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+costaria,+undaria,+and+laminaria.">The development of costaria, undaria, and laminaria.</a> Annals of Botany. 25 (99), 691-715 (1911).</li><li>Forbord, S., Steinhovden, K., R&#248;d, K., Hand&#229;, A., Skjermo, J., Charrier, B., Wichard, T., Reddy, C. R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+protocol+for+Saccharina+latissima.">Cultivation protocol for Saccharina latissima.</a> Protocols for Macroalgae Research. , 37-59 (2018).</li><li>Bartsch, I., Charrier, B., Wichard, T., Reddy, C. R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+clonal+stock+cultures+and+hybridization+of+kelps.">Derivation of clonal stock cultures and hybridization of kelps.</a> Protocols for Macroalgae Research. , 61-78 (2018).</li><li>Theodorou, I., Opsahl-Sorteberg, H. -. G., Charrier, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+zygotes+and+embryos+of+the+kelp+saccharina+latissima+for+cell+biology+approaches.">Preparation of zygotes and embryos of the kelp saccharina latissima for cell biology approaches.</a> Bio-protocol. 101, 4132 (2021).</li><li>L&#252;ning, K., Dring, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproduction+induced+by+blue+light+in+female+gametophytes+of+Laminaria+saccharina.">Reproduction induced by blue light in female gametophytes of Laminaria saccharina.</a> Planta. 104 (3), 252-256 (1972).</li><li>de Medeiros, G., 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=Cell+and+tissue+manipulation+with+ultrashort+infrared+laser+pulses+in+light-sheet+microscopy.">Cell and tissue manipulation with ultrashort infrared laser pulses in light-sheet microscopy.</a> Scientific Reports. 10 (1), 1942 (2020).</li><li>Liang, X., Michael, M., Gomez, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mechanical+tension+at+cell-cell+junctions+using+two-photon+laser+ablation.">Measurement of mechanical tension at cell-cell junctions using two-photon laser ablation.</a> Bio-protocol. 6 (24), 2068 (2016).</li><li>Ebbing, A., Pierik, R., Bouma, T., Kromkamp, J. C., Timmermans, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+light+and+biomass+density+influence+the+reproduction+of+delayed+Saccharina+latissima+gametophytes+(Phaeophyceae).">How light and biomass density influence the reproduction of delayed Saccharina latissima gametophytes (Phaeophyceae).</a> Journal of Phycology. 56 (3), 709-718 (2020).</li><li>Rabill&#233;, H., Billoud, B., Tesson, B., Le Panse, S., Rolland, &. #. 2. 0. 1. ;., Charrier, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brown+algal+mode+of+tip+growth:+Keeping+stress+under+control.">The brown algal mode of tip growth: Keeping stress under control.</a> PLoS Biology. 17 (1), 2005258 (2019).</li></ol>

L’ablation laser cellulaire locale permet une ablation temporelle et spatiale avec un haut niveau de précision. Cependant, son efficacité peut être entravée par la non-accessibilité des cellules cibles; par exemple, toutes les cellules sont d’un embryon tridimensionnel. Ce protocole a été développé sur l’embryon de l’algue Saccharina latissima, qui développe une lame monocouche dans laquelle toutes les cellules peuvent être facilement distinguées et détruites individuellement avec un faiscea...

L’ablation au laser est utilisée depuis des décennies pour étudier le développement embryonnaire. L’irradiation des cellules embryonnaires à l’aide d’un faisceau laser permet de surveiller le potentiel de régénération et la modification de la lignée cellulaire au cours de l’embryogenèse et d’étudier l’impact de l’ablation ciblée sur la division cellulaire et le devenir cellulaire. Les organismes modèles utilisés dans les méthodes d’ablation au laser sont généralement des animaux, tels que les insectes 1,2, les nématodes 3,4, les vertébrés 5,6 et parfois les plantes 7,8. De plus, une approche de micro-ablation au laser a été utilisée sur l’algue brune Fucus en 1994 et 1998 pour démontrer le rôle de la paroi cellulaire dans la photopolarisation de l’embryon précoce 9,10.
Les algues brunes appartiennent au groupe des Stramenopiles, qui ont divergé à la racine de l’arbre eucaryote il y a 1,6 milliard d’années. En conséquence, ils sont phylogénétiquement indépendants des autres organismes multicellulaires, tels que les animaux et les plantes11. Saccharina latissima appartient à l’ordre des Laminariales, plus communément appelés varechs, et ils sont parmi les plus grands organismes sur terre, atteignant des tailles de plus de 30 m. Saccharina sp. est une grande algue utilisée pour de nombreuses applications telles que l’alimentation humaine et animale, et ses polysaccharides sont extraits pour être utilisés dans les industries agricoles, pharmacologiques et cosmétiques du monde entier12, 13. Sa culture, principalement en Asie et plus récemment en Europe, nécessite la préparation d’embryons dans des écloseries avant de relâcher des juvéniles en pleine mer. Comme tous les varechs, il a un cycle de vie biphasique composé d’une phase gamétophytique microscopique, au cours de laquelle un gamétophyte haploïde se développe et produit des gamètes pour la fécondation, et d’une phase sporophytique macroscopique diploïde, où une grande lame plane se développe à partir de sa retenue attachée au fond marin ou aux rochers. Le sporophyte libère des spores haploïdes à maturité, complétant ainsi le cycle de vie 14,15,16.
S. latissima présente des caractéristiques morphologiques intéressantes17. Son embryon se développe sous la forme d’une feuille plane monocouche 15,18,19 avant d’acquérir une structure multicouche coïncidant avec l’émergence de différents types de tissus. De plus, Laminariales est l’un des seuls taxons d’algues brunes dont les embryons restent attachés à leur tissu gamétophyte maternel (Desmarestiales et Sporochnales en font trop15). Cette fonctionnalité offre l’occasion d’étudier le rôle du tissu maternel dans ce processus de développement et de comparer les mécanismes de contrôle maternel chez les algues brunes avec ceux des animaux et des plantes.
Cet article présente le premier protocole complet pour l’ablation au laser dans un embryon de varech précoce. Ce protocole impliquant la technique UV ns-pulsed entraîne la destruction spécifique de cellules embryonnaires individuelles pour étudier leurs rôles respectifs au cours de l’embryogenèse. La procédure offre une approche fiable pour étudier les interactions cellulaires et le devenir cellulaire au cours de l’embryogenèse chez les laminariales.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>25 mm glass bottom petri dish</td>
			<td>NEST</td>
			<td>801001</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclaved sea water</td>
			<td>-</td>
			<td></td>
			<td>Collected offshore near the Astan buoy (48&#176;44.934 N 003&#176;57.702 W) close to Roscoff, France, at a depth of 20 m.</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>MED 2</td>
			<td>83.3951</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 40 &#181;m</td>
			<td>Corning / Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture cabinets</td>
			<td>Snijders Scientific Plant Growth Cabinet ECD01</td>
			<td></td>
			<td>Any other brand is suitable provided that the light intensity, the photoperiod and the temperature can be controlled.</td>
		</tr>
		<tr>
			<td>LSM 880 Zeiss confocal microscope</td>
			<td>Carl Zeiss microscopy, Jena, Germany</td>
			<td></td>
			<td>Ablation and imaging were performed using a 40x/1.2 water objective</td>
		</tr>
		<tr>
			<td>Pellet pestles</td>
			<td>Sigma Aldrich</td>
			<td>Z359947</td>
			<td>Blue polypropylene (autoclavable)</td>
		</tr>
		<tr>
			<td>Provasoli supplement</td>
			<td>-</td>
			<td></td>
			<td>Recipe is available here: http://www.sb-roscoff.fr/sites/www.sb-roscoff.fr/files/documents/station-biologique-roscoff-preparation-du-provasoli-2040.pdf</td>
		</tr>
		<tr>
			<td>Pulsed 355 laser (UGA-42 Caliburn 355/25)</td>
			<td>Rapp OptoElectronic, Wedel, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Paramount</td>
			<td>PDSS 11</td>
			<td></td>
		</tr>
		<tr>
			<td>SysCon software</td>
			<td>Rapp OptoElectronic, Wedel, Germany</td>
			<td></td>
			<td>Laser-driver software</td>
		</tr>
		<tr>
			<td>ZEN software</td>
			<td>Carl Zeiss microscopy, Jena, Germany</td>
			<td></td>
			<td>Imaging software, used together with the SysCon software; Black 2.3 version</td>
		</tr>
	</tbody>
</table>

targeted laser ablation in the embryo of saccharina latissima

Chez Saccharina latissima, l’embryon se développe sous la forme d’une feuille cellulaire monocouche appelée la lame ou lame. Chaque cellule embryonnaire est facile à observer, facilement reconnaissable de ses voisines et peut être ciblée individuellement. Pendant des décennies, l’ablation au laser a été utilisée pour étudier le développement embryonnaire. Ici, un protocole d’ablation au laser spécifique aux cellules a été développé pour les embryons précoces de l’algue brune S. latissima. Les travaux présentés comprennent: (1) la préparation d’embryons de Saccharina , avec une description des paramètres critiques, y compris les conditions de culture, (2) les paramètres d’ablation au laser, et (3) la surveillance de la croissance ultérieure de l’embryon irradié à l’aide de la microscopie time-lapse. En outre, des détails sont fournis sur les conditions optimales pour le transport des embryons de la plate-forme d’imagerie vers le laboratoire, ce qui peut affecter profondément le développement ultérieur des embryons. Les algues appartenant à l’ordre des Laminariales présentent des schémas d’embryogenèse similaires à saccharina; ce protocole peut ainsi être facilement transféré à d’autres espèces de ce taxon.

Des gamétophytes de S. latissima ont été cultivés et la gamétogenèse a été induite pour produire des zygotes et des embryons. Douze jours après l’induction de la gamétogenèse, les embryons ont subi une ablation au laser. Ici, l’expérience visait à évaluer le rôle de cellules spécifiques dans le développement global des embryons de S. latissima . Les cellules les plus apicales, les cellules les plus basales et les cellules médianes ont été ciblées. Après le balayage des carreaux...

Watch this Scientific Journal Video about Targeted Laser Ablation in the Embryo of Saccharina latissima at JoVE.com

Targeted Laser Ablation in the Embryo of Saccharina latissima

La destruction de cellules spécifiques dans l’embryon est un outil puissant pour étudier les interactions cellulaires impliquées dans le destin cellulaire. Le présent protocole décrit les techniques d’ablation au laser de cellules ciblées dans l’embryon précoce de l’algue brune Saccharina latissima.

Ablation laser ciblée dans l’embryon de Saccharina latissima

In Saccharina latissima, the embryo&#160;develops as a monolayered cell sheet called the lamina or the blade. Each embryo cell is easy to observe, readily ...

Ce protocole est le premier protocole complet d’ablation au laser sur embryon de varech précoce. La procédure offre une approche fiable pour étudier le devenir cellulaire et l’interaction au cours de l’embryogenèse chez les algues brunes. L’ablation cellulaire locale au laser permet une ablation temporelle et spéciale avec un haut niveau de précision et de tissu sur les cellules.Cette approche est une méthode prometteuse pour étudier le destin cellulaire et l’interaction dans l’embryon précoce d’algues brunes. Il peut être appliqué à d’autres systèmes d’algues avec peu de changements. Commencez par imager l’ensemble de la boîte de Pétri pour localiser les embryons à sélectionner pendant l’étape d’ablation et surveiller le développement ultérieur des embryons sélectionnés.Lancez l’analyse des vignettes. Notez la position des quatre points cardinaux de la boîte de Pétri. Entrez les paramètres définis dans le manuscrit textuel et acquérez des images transmises ou fluorescentes de l’ensemble de la boîte de Pétri à basse résolution.Enregistrez l’image de numérisation par vignette et gardez-la ouverte dans la fenêtre du logiciel d’acquisition d’image. Changez l’objectif sans retirer la boîte de Pétri. Trouvez des embryons d’intérêt sur le scan.Pour calibrer le laser, ouvrez le pilote laser et le progiciel d’acquisition d’images. Ensuite, initialisez le chemin laser. Synchronisez les deux progiciels en cliquant sur Démarrer l’acquisition dans le progiciel du pilote laser.Configurez les paramètres de l’ablation au laser et cliquez sur live. Sélectionnez une zone vide sur la boîte et abaissez le niveau de la scène à 20 micromètres sous la surface supérieure de la boîte de Pétri pour vous concentrer sur le fond en verre. Pour trouver une zone d’intérêt en cliquant sur le bouton choisir AOI et en cliquant sur les bords de l’image dans le progiciel du pilote laser UV.Cliquez sur Démarrer l’étalonnage et sélectionnez l’étalonnage manuel pour définir les trajectoires du laser d’ablation et du laser d’imagerie. Assurez-vous que tous les volets sont ouverts. Sélectionnez une puissance laser suffisamment élevée pour voir un point noir au centre de l’image en direct correspondant au trou dans le glissement du couvercle en verre.Cliquez sur le point noir central avec le curseur de la souris et sélectionnez 18 points supplémentaires proposés par le logiciel pour compléter la procédure d’alignement. Cliquez sur le mode clic et tir. Vérifiez l’étalonnage sur le même couvercle et cliquez sur la couche de verre où l’impact laser est visible.Sélectionnez un embryon d’intérêt sur le scan de tuiles et déplacez la scène en cliquant dessus. Démarrez la série chronologique. Testez les paramètres de laps de temps en direct et ajustez-les si nécessaire.Démarrez un enregistrement en accéléré dans le logiciel d’acquisition d’images à la vitesse maximale. Ajustez le zoom pour vous concentrer sur la zone d’intérêt et visualisez l’embryon entier. Pour suivre l’embryon pendant l’enregistrement de l’ablation, cliquez sur le bouton, acquérez la dernière image.Réglez les paramètres sur une transmission laser de 45%, ce qui correspond à un maximum de 40 microwatts et à une durée d’impulsion d’une milliseconde. Appliquez l’irradiation dommageable sur les cellules embryonnaires d’intérêt à l’aide de la fonction de clic et de tir du logiciel du pilote laser. Sous 688 nanomètres, surveillez l’éjection des chloroplastes autofluorescents du cytoplasme.Si le contenu de la cellule reste dans la cellule, utilisez à nouveau la fonction de clic et de tir pour augmenter la taille de la brèche dans la cellule. Répétez ce processus jusqu’à ce que la plupart du contenu de la cellule soit libéré, en maintenant le nombre de tirs au minimum. Arrêtez l’enregistrement en accéléré une fois que l’embryon s’est stabilisé et qu’aucun autre mouvement intercellulaire ne peut être détecté.Mettez à jour l’annotation sur l’image de numérisation de vignette, remplacez la numérisation de vignette existante et enregistrez l’image. Déterminez le taux de survie en surveillant le nombre d’embryons qui se développent après l’ablation au laser et comparez-les à ceux qui meurent. Déterminez le retard de croissance en mesurant la longueur des embryons injectés au laser chaque jour et en la comparant à des embryons intacts.Trouvez les dommages adjacents en surveillant la réaction des cellules adjacentes aux cellules ablées. Les cellules d’intérêt ciblées étaient les cellules les plus apicales, les cellules les plus basales et les cellules médianes. Après avoir scanné l’ensemble de la boîte de Pétri, un embryon d’intérêt a été identifié comme un candidat approprié pour la prise de vue au laser.La cellule a libéré son contenu lorsqu’elle a été filmée avec un faisceau laser UV à impulsions à 45% de puissance, tandis que la cellule adjacente aux cellules irradiées s’est étendue dans l’espace intercellulaire. La position des embryons irradiés a été enregistrée toutes les 24 heures pendant 10 jours. La plupart des embryons irradiés ont continué à se développer, mais ont montré une altération de la croissance.En revanche, les embryons testés avec d’autres paramètres laser ont rapidement montré des signes de stress sévère tels que le blanchiment cellulaire, la décoloration ou les changements de forme. Presque tous les embryons ainsi abattus sont morts dans les cinq jours suivant l’expérience. Certains paramètres, tels que le format d’image ou le zoom, ne doivent pas être modifiés après l’étalonnage.Une surveillance pendant et après l’ablation au laser est nécessaire pour avoir un aperçu de ce qui s’est passé. L’ablation au laser ouvre la voie à de nouvelles découvertes dans l’étude du développement des algues brunes et à une compréhension plus profonde des interactions de différents métabolites ou composants de leurs cellules.

targeted-laser-ablation-in-the-embryo-of-saccharina-latissima

Research

JoVE Journal

Developmental Biology

Targeted Laser Ablation in the Embryo of Saccharina latissima

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Microbead implantation dans l&#39;embryon de poisson zèbre

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Recherche

Biologie du développement

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Imagerie Structures subcellulaires dans le Embryo Living Zebrafish

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

La micro-injection et l&#39;électroporation d&#39;embryons dans le chordés<em&gt; Ciona intestinalis</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

Chirurgical Ablation Essai pour étude de la régénération des yeux dans planaires

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

AFM et microrhéologie dans la cellule de jaune de œuf embryon de poisson zèbre

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout (Oncorhynchus Mykiss) and Brown Trout (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on fish abdomens to expel the eggs. An alternative method uses the pressure of gas injected into the body cavity, which causes the subsequent release of the eggs. This method is believed to have less negative effects on both the welfare and egg quality of the broodstocks. Herein, we compare the results of air and hand stripping methods with respect to one-year survival and egg quantity and quality in two salmonid fish, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta morpha fario). Our results indicate that air stripping yielded a better quality of eggs and higher one-year survival rate in rainbow trout. In addition, air stripping resulted in lower mortality rate than the group subjected to hand stripping (25% vs. 35%). The pH and hatching rate of the hand stripped group was lower than those of the air stripped group. In the case of brown trout, the quality of eggs obtained by both hand and air-stripping methods was similar; however, the one-year losses in fish were higher in air stripped group (15% compared to 0% in hand stripped fish). Although the advantages of air stripping method over hand stripping in terms of egg quality might not be observed in all salmonid species, the air-stripping procedure might be a promising option to be adopted in hatcheries as it ensures a high level of reproducibility and efficiency.

Effectiveness of the Air Stripping in Two Salmonid Fish, Rainbow Trout Oncorhynchus Mykiss and Brown Trout Salmo Trutta Morpha fario

The principal aim of this study is to standardize and test the pneumatic method (air stripping) of collecting eggs in rainbow trout and brown trout. This method allows effective and simple collection of the eggs without the necessity of fish abdomen massage.

Efficacité du Stripping Air dans deux poissons salmonidés, truite arc-en-ciel (Oncorhynchus Mykiss) et la truite brune (Salmo Trutta Morpha fario)

Egg collection is one of the most crucial procedures during fish reproduction in salmonid hatcheries. Classic methods involve the use of hand massage on ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Transfert d’embryon et vitrification d’embryons au stade optimal de l’embryon dans le modèle Rabbit

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Microinjection-based System for In Vivo l’Implantation des Cardiomyocytes embryonnaires chez l’embryon aviaire

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. This tissue complexity confounds the identification of causal link between extrinsic cues and cellular dynamics. A synthetically reconstituted multicellular system overcomes this problem by enabling researchers to test for a specific cue while eliminating others. Here, we present a method to reconstitute cell contact patterns with isolated Caenorhabditis elegans blastomere and adhesive polystyrene beads. The procedures involve eggshell removal, blastomere isolation by disrupting cell-cell adhesion, preparation of adhesive polystyrene beads, and reconstitution of cell-cell or cell-bead contact. Finally, we present the application of this method to investigate the orientation of cellular division axes that contributes to the regulation of spatial cellular patterning and cell fate specification in developing embryos. This robust, reproducible, and versatile in vitro method enables the study of direct relationships between spatial cell contact patterns and cellular responses.

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

Tissue complexities of multicellular systems confound the identification of causal relationship between extracellular cues and individual cellular behaviors. Here, we present a method to study the direct link between contact-dependent cues and division axes using C. elegans embryo blastomeres and adhesive polystyrene beads.

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Essai confocal d’ablation et de régénération au microscope dans les cellules interuromast de Zebrafish

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...