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 beca de doctorado de S.B. está financiada por la Región de Bretaña (número de subvención ARED COH20020) y la Universidad de la Sorbona. I.T.is beca de doctorado está financiada por la Región de Bretaña (número de subvención ARED COH18020) y la Universidad Norvegian NMBU. Este proyecto ha recibido apoyo financiero del CNRS a través de los programas interdisciplinarios del MITI. MRic es miembro de la infraestructura nacional France-BioImaging apoyada por la Agencia Nacional de Investigación de Francia (ANR-10-INBS-04).

1. Producción de Saccharina latissima gametophytes
<ol><li>Recolectar esporófitos maduros de S. latissima de la naturaleza como se describió anteriormente20,21. Asegúrese de que los esporófitos seleccionados estén desprovistos de epífitas (pequeños organismos visibles en la superficie de la cuchilla) o parásitos internos (que se encuentran en las áreas blanqueadas o manchas en la cuchilla). 
</li>
	<li>Con u...

<ol>
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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. 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La ablación celular local con láser permite la ablación temporal y espacial con un alto nivel de precisión. Sin embargo, su eficiencia puede verse obstaculizada por la falta de accesibilidad de las células objetivo; por ejemplo, todas las células son de un embrión tridimensional. Este protocolo se desarrolló en el embrión del alga Saccharina latissima, que desarrolla una lámina monocapa en la que todas las células se pueden distinguir y destruir fácilmente individualmente con un rayo láser.
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La ablación con láser se ha utilizado durante décadas para estudiar el desarrollo embrionario. La irradiación de células embrionarias con un rayo láser permite monitorear el potencial regenerativo y la modificación del linaje celular durante la embriogénesis e investigar el impacto de la ablación dirigida en la división celular y el destino celular. Los organismos modelo utilizados en los métodos de ablación con láser son típicamente animales, como insectos 1,2, nematodos 3,4, vertebrados 5,6 y ocasionalmente plantas 7,8. Además, se utilizó un enfoque de microablación láser en el alga marrón Fucus en 1994 y 1998 para demostrar el papel de la pared celular en la fotopolarización del embrión temprano 9,10.
Las algas pardas pertenecen al grupo Stramenopiles, divergieron en la raíz del árbol eucariota hace 1.600 millones de años. Como resultado, son filogenéticamente independientes de otros organismos multicelulares, como animales y plantas11. Saccharina latissima pertenece al orden Laminariales, más comúnmente conocido como algas marinas, y se encuentran entre los organismos más grandes de la tierra, alcanzando tamaños de más de 30 m. Saccharina sp. es un gran alga marina utilizada para muchas aplicaciones como alimentos y piensos, y sus polisacáridos se extraen para su uso en las industrias agrícola, farmacológica y cosmética en todo el mundo12, 13. Su cultivo, principalmente en Asia y más recientemente en Europa, requiere la preparación de embriones en criaderos antes de liberar juveniles en mar abierto. Como todas las algas marinas, tiene un ciclo de vida bifásico compuesto por una fase gametofítica microscópica, durante la cual un gametofito haploide crece y produce gametos para la fertilización, y una fase esporófita macroscópica diploide, donde se desarrolla una gran cuchilla plana desde su sujeción unida al fondo marino o las rocas. El esporófito libera esporas haploides en la madurez, completando así el ciclo de vida 14,15,16.
S. latissima presenta algunos rasgos morfológicos interesantes17. Su embrión se desarrolla como una lámina plana monocapa 15,18,19 antes de adquirir una estructura multicapa coincidiendo con la aparición de diferentes tipos de tejidos. Además, Laminariales es uno de los únicos taxones de algas pardas cuyos embriones permanecen adheridos a su tejido gametofítico materno (Desmarestiales y Sporochnales también lo hacen15). Esta característica ofrece la oportunidad de estudiar el papel del tejido materno en este proceso de desarrollo y comparar los mecanismos de control materno en algas marrones con los de animales y plantas.
Este artículo presenta el primer protocolo completo para la ablación con láser en un embrión temprano de algas marinas. Este protocolo que involucra la técnica UV ns-pulsada da como resultado la destrucción específica de células embrionarias individuales para estudiar sus respectivos roles durante la embriogénesis. El procedimiento ofrece un enfoque confiable para investigar las interacciones celulares y el destino celular durante la embriogénesis en 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

En Saccharina latissima, el embrión se desarrolla como una lámina celular monocapa llamada lámina o cuchilla. Cada célula embrionaria es fácil de observar, fácilmente distinguible de sus vecinas, y puede ser dirigida individualmente. Durante décadas, la ablación con láser se ha utilizado para estudiar el desarrollo embrionario. Aquí, se desarrolló un protocolo para la ablación con láser específica de la célula para embriones tempranos del alga marrón S. latissima. El trabajo presentado incluye: (1) la preparación de embriones de Saccharina , con una descripción de los parámetros críticos, incluidas las condiciones de cultivo, (2) los ajustes de ablación con láser y (3) el monitoreo del crecimiento posterior del embrión irradiado mediante microscopía de lapso de tiempo. Además, se proporcionan detalles sobre las condiciones óptimas para transportar los embriones desde la plataforma de imágenes hasta el laboratorio, lo que puede afectar profundamente el desarrollo embrionario posterior. Las algas pertenecientes al orden Laminariales muestran patrones de embriogénesis similares a Saccharina; por lo tanto, este protocolo puede transferirse fácilmente a otras especies en este taxón.

Se cultivaron gametofitos de S. latissima , y se indujo la gametogénesis para producir cigotos y embriones. Doce días después de la inducción de la gametogénesis, los embriones se sometieron a ablación con láser. Aquí, el experimento tuvo como objetivo evaluar el papel de células específicas en el desarrollo general de embriones de S. latissima . La célula más apical, la célula más basal y las células medianas fueron atacadas. Después del escaneo de baldosas, toda la placa de Petri (<str...

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 destrucción de células específicas en el embrión es una herramienta poderosa para estudiar las interacciones celulares involucradas en el destino celular. El presente protocolo describe técnicas para la ablación con láser de células diana en el embrión temprano del alga marrón Saccharina latissima.

Ablación dirigida con láser en el embrión 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 ...

Este protocolo es el primer protocolo completo de ablación con láser en embriones tempranos de algas marinas. El procedimiento ofrece un enfoque confiable para investigar el destino celular y la interacción durante la embriogénesis en algas pardas. La ablación celular local con láser permite la ablación temporal y especial con un alto nivel de precisión y tejido en las células.Este enfoque es un método prometedor para estudiar los destinos celulares y la interacción en el embrión temprano de algas pardas. Se puede aplicar a otros sistemas de algas con pocos cambios. Comience por obtener imágenes de toda la placa de Petri para localizar los embriones para su selección durante la etapa de ablación y supervise el desarrollo posterior de los embriones seleccionados.Inicie el análisis de mosaicos. Registre la posición de los cuatro puntos cardinales de la placa de Petri. Introduzca los parámetros definidos en el manuscrito de texto y adquiera imágenes transmitidas o fluorescentes de toda la placa de Petri a baja resolución.Guarde la imagen de escaneo de mosaico y manténgala abierta en la ventana del software de adquisición de imágenes. Cambia el objetivo sin quitar la placa de Petri. Encuentre embriones de interés en la exploración.Para calibrar el láser, abra el controlador láser y el paquete de software de adquisición de imágenes. A continuación, inicialice la ruta del láser. Sincronice ambos paquetes de software haciendo clic en iniciar adquisición en el paquete de software del controlador láser.Configure los parámetros para la ablación con láser y haga clic en en vivo. Seleccione un área vacía en el plato y baje el nivel del escenario a 20 micrómetros por debajo de la superficie superior de la placa de Petri para centrarse en el fondo de vidrio. Para encontrar un área de interés haciendo clic en el botón elija AOI y haga clic en los bordes de la imagen en el paquete de software del controlador láser UV.Haga clic en iniciar calibración y seleccione calibración manual para establecer las trayectorias del láser de ablación y del láser de imágenes. Asegúrese de que todas las persianas estén abiertas. Seleccione una potencia láser lo suficientemente alta como para ver un punto negro en el centro de la imagen en vivo correspondiente al orificio en el deslizamiento de la cubierta de vidrio.Haga clic en el punto negro central con el cursor del mouse y seleccione 18 puntos adicionales propuestos por el software para completar el procedimiento de alineación. Haga clic en el modo de clic y disparo. Verifique la calibración en el mismo deslizamiento de la cubierta y haga clic en la capa de vidrio donde el impacto del láser es visible.Seleccione un embrión de interés en el escaneo de mosaico y mueva el escenario haciendo clic en él. Inicie la serie temporal. Pruebe los parámetros de lapso de tiempo en vivo y ajuste si es necesario.Inicie una grabación de lapso de tiempo en el software de adquisición de imágenes a la velocidad máxima. Ajuste el zoom para enfocar el área de interés y visualizar todo el embrión. Para seguir al embrión durante el registro de la ablación, haga clic en el botón, adquiera la última imagen.Ajuste los parámetros al 45% de transmisión láser correspondiente a un máximo de 40 microvatios y una duración de tiempo de pulso de milisegundos. Aplique la irradiación dañina en las células embrionarias de interés utilizando el software del controlador láser click and fire function. Bajo 688 nanómetros, monitoree la eyección de cloroplastos autofluorescentes del citoplasma.Si el contenido de la celda permanece en la celda, utilice la función de clic y disparo una vez más para aumentar el tamaño de la brecha en la celda. Repita este proceso hasta que se libere la mayor parte del contenido de la celda, manteniendo el número de disparos al mínimo. Detenga el registro del lapso de tiempo después de que el embrión se haya estabilizado y no se pueda detectar ningún movimiento intercelular adicional.Actualice la anotación en la imagen de escaneo de mosaico, sobrescriba el escaneo de mosaico existente y guarde la imagen. Determine la tasa de supervivencia monitoreando el número de embriones que se desarrollan después de la ablación con láser y compárelos con los que mueren. Determine el retraso en el crecimiento midiendo la longitud de los embriones inyectados con láser todos los días y comparándola con embriones intactos.Encuentre el daño adyacente monitoreando la reacción de las células adyacentes a las células ablacionadas. Las células objetivo de interés fueron la célula más apical, la célula más basal y las células medianas. Después de escanear toda la placa de Petri, se identificó un embrión de interés como un candidato adecuado para el disparo con láser.La célula liberó su contenido cuando se disparó con un rayo láser UV de pulso a una potencia del 45%, mientras que la célula adyacente a las células irradiadas se expandió al espacio intercelular. La posición de los embriones irradiados se registró cada 24 horas durante 10 días. La mayoría de los embriones irradiados continuaron desarrollándose, pero mostraron alteración del crecimiento.En contraste, los embriones probados con otros parámetros del láser mostraron rápidamente signos de estrés severo, como blanqueamiento celular, desvanecimiento o cambios de forma. Casi todos los embriones disparados de esta manera murieron dentro de los cinco días posteriores al experimento. Algunos parámetros, como el formato de imagen o el zoom, no deben cambiarse después de la calibración.Se requiere monitoreo durante y después de la ablación con láser para tener una idea de lo que sucedió. La ablación con láser está allanando el camino hacia nuevos descubrimientos en el estudio del desarrollo de las algas pardas y una comprensión más profunda de las interacciones de diferentes metabolitos o componentes de sus células.

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 Implantación en el embrión de pez cebra

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 ...

Investigación

Biología del Desarrollo

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.

La formación de imágenes estructuras subcelulares en el embrión vivo de pez cebra

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 microinyección de embriones y la electroporación en el Chordate<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.

La ablación quirúrgica de ensayo para estudio de la regeneración de los ojos en planarias

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 y Microrheology en la celda de yema de embrión de pez cebra

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.

Eficacia de la extracción de aire en dos peces salmónidos, trucha arco iris (Oncorhynchus Mykiss) y trucha (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.

Transferencia de embriones mínimamente invasiva y vitrificación de embriones en la etapa óptima del embrión en el modelo 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.

Sistema basado en la microinyección en Vivo implantación de cardiomiocitos embrionarios en el embrión aviar

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.

Reconstitución in Vitro de patrones de contacto de células espaciales con Caenorhabditis elegans Embryo Blastomeres aislados y perlas de poliestireno adhesivo

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.

Ensayo de regeneración y ablación láser basado en microscopio confocal en células interneuromas de peces cebra

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