Este estudio fue financiado por los Institutos Nacionales de Salud (NIH) Core Grant (5P30EY008098) y los siguientes fondos de XW: Subvenciones del NIH R01EY016099 e Investigación para Prevenir la Ceguera Premio al Desarrollo Profesional. Damos las gracias a Kira Lathrop de asistencia en la imagen confocal.

Día 1. Preparación 1. Establecer cruces de pescado Establecer cruces de pares de pez cebra en la noche. Para evitar el apareamiento al azar antes de la hora que desee, utilice separadores para separar los peces de ambos sexos en los cassettes de apareamiento. Que expresan GFP peces transgénicos se utilizan como fuente de blastómeros de los donantes. La línea pt104 transgénicos (Zou et al., 2008), se utiliza aquí, expresa GFP doquier bajo el control del promotor EF1. La expresión de GFP permite la visualización de las células del donante de imágenes en vivo. Otras líneas de peces transgénicos que expresan proteínas fluorescentes adecuados pueden ser utilizados para necesidades específicas. 2. Preparar en forma de cuña de agarosa pozos para la celebración de embriones Vierta derretida 1% de agarosa, preparados de huevo en el agua E3 (Westerfield, 2007), en una placa de Petri. Flotar un molde de plástico que tiene forma de cuña, protuberancias en la solución de agarosa y dejar que la solución de agarosa completamente solidifique. Retire el molde con cuidado y sumerja la cama de agarosa con agua E3. 3. Prepare las agujas del trasplante Utilice un extractor de aguja vertical (David Kopf Instruments) en ajuste de potencia de 11,5 a fabricar agujas tirando pipetas capilares de vidrio (Instrumento Mundial de Precisión). Buenos consejos aguja cónica con ejes largos. Moler las puntas de aguja en un ángulo de 45 grados con una amoladora Micro (Marishige) hasta las aberturas biselado de 40 45μm de diámetro interior se obtiene (Fig. 1A). Para limpiar las puntas de aguja, conectar una manguera a la parte roma de la aguja, conectar la manguera a una jeringa, y luego extraerla, y liberar una solución al 10% de ácido fluorhídrico hasta que toda la suciedad visible desaparezca. Después, lave la punta con agua destilada, seguido de etanol al 100%. Seque al aire las agujas hasta que se evapore el etanol. Es importante asegurarse de que las puntas estén completamente secas: el etanol residual va a arruinar las agujas durante la etapa de pulido de alta temperatura. Para suavizar y pulir las asperezas de las puntas de aguja, cocinar las puntas de aguja con el calor generado por el filamento de un falsificador Micro (Marishige). No permita que las puntas de la aguja toca el filamento durante el pulido. La cantidad de electricidad que pasa a pesar de que el filamento y la distancia entre el filamento y la punta de la aguja puede ser ajustada para alcanzar las condiciones deseadas de calefacción. La temperatura no debe ser demasiado alto: esta se derrite y se deforman las puntas de aguja. Después de las aristas son alisadas, cuidadosamente tocar el filamento se calienta con la punta de la aguja y tire suavemente el filamento de la aguja para generar un extremo en punta (Fig. 1B). Una aguja, suave y limpia es fundamental para obtener blastómeras intactas de los donantes y para no dañar a los embriones de acogida. 4. Prepare una investigación de embriones de posicionamiento Tire de una pipeta de vidrio sobre una llama de gas para obtener una sonda fina de vidrio con un extremo alisado. Esta sonda se utiliza para colocar los embriones en la orientación correcta antes del trasplante. Día 2. Blastómero trasplante 1. Recoger y embriones dechorionate Al día siguiente, quitar los separadores de los cartuchos de cruz para que los peces a su compañero. Colocar los embriones en una placa Petri con agua E3. Luego, los embriones dechorionate manualmente arrancando el corion de los embriones con un par de pinzas finas. Use una pipeta de vidrio con eje doblado cuidadosamente para transferir los embriones dechorionated en agarosa pozos. Evitar cualquier contacto de los embriones con el aire, lo que causa que los embriones a punto de estallar. Deje que los embriones se desarrollan a 28,5 ° C en los pozos de agarosa hasta 3 horas después de la fecundación, o HPF. 2. Establecer un aparato de trasplante Mientras que los embriones se desarrollan, crear un aparato de trasplante mediante la conexión de la aguja del trasplante a la llena de aceite mineral micro-sistema de bombeo. Asegúrese de que el sistema no se escapa y no hay burbujas de aire atrapadas en el sistema. Volver llenar la aguja trasplante completo con aceite mineral, con excepción de los 0,5 a 1 l de espacio en la apertura de la punta. 3. El trasplante de blastómeros Para trasplante de blastómeros, el uso de una sonda de vidrio para orientar el posicionamiento del lado blastómeros de los embriones 3-4-HPF hacia arriba. A continuación, insertar suavemente la aguja del trasplante en un embrión de donante y poco a poco aspirar 05.20 blastómeras en la aguja. Insertar la aguja blastómero lleno en un embrión de acogida y liberación de los blastómeros lentamente. El sitio en el que los blastómeros son liberados influirá en su progenie localizar en las etapas posteriores del desarrollo. Para analizar el desarrollo de la retina y el cerebro, las células liberan en el polo animal. Durante el trasplante, asegúrese de que la punta de la aguja no contacto con el aire. Para reducir al mínimo los daños mecánicos, los embriones de acogida se queda en los pozos de agarosa a 28,5 ° C hasta el día siguiente. Entonces, tTRANSFERENCIA los embriones mosaico de placas de 24 pocillos previamente recubierto con 0,5 ml de agarosa al 1%, con un embrión en cada pozo. 1 ml de agua de huevo E3 se añade a cada pocillo. Para prevenir las infecciones bacterianas, añadir 10 l de 100 veces la penicilina y estreptomicina para el agua de huevo E3. Día 3. Incorporación de imágenes en vivo y de los embriones mosaico 1. Incorporación de embriones Prepare un 1,5% de agarosa de bajo punto de fusión (LMP) en agua de huevo E3 y mantener la solución en un baño de 30 ° C el agua. La transferencia de un embrión de mosaico a un plato de cultivo de tejidos FluoroDish con fondo de cristal de 0,17 mm de espesor (Instrumentos del Mundo de Precisión). Eliminar el agua E3 exceso de salir del embrión apenas cubierta por una cantidad mínima de E3. Añadir 30-50 l LMP fundido 1,5% para el embrión. Rápidamente la posición del ojo o el cerebro lo más cerca del fondo de cristal como sea posible antes de que la solución de agarosa solidifica. Después de que la solución se enfríe, añadir más solución de agarosa para asegurar aún más los embriones en su lugar. Luego, sumerja la agarosa solidificada bloque con 3.2 ml de agua de huevo E3. 2. Microscopía confocal Por microscopía confocal, un objetivo de inmersión en agua invertido (Olympus, PlanApo, 40 X/0.90 WLSM, Japón) con una profundidad de foco grande se utiliza. Una gota de agua se agrega en el objetivo y luego colocar un plato de cultivo de tejidos FluoroDish en el objetivo. Para el desarrollo de la imagen, realizar time-lapse de imágenes de la toma de fotografías de los embriones cada 5 minutos. Dependiendo de la naturaleza de los experimentos, los intervalos de tiempo entre las tomas deben ser ajustados. Los resultados representativos La figura 2 muestra una representación de 24 HPF pez cebra mosaico. La expresión de GFP se destacan las células del donante en verde. Las células neuroepiteliales retina abarcan todo el espesor de la pared de la retina. La película muestra los movimientos de las células del donante en el cerebro de acogida en 24 HPF. Células aviso morfologías cambiar con el tiempo. <img src="/files/ftp_upload/1924/1924fig1.jpg" alt="Figura 1" /> Figura 1. Las formas de las puntas de aguja trasplante. A. La apertura biselada de la punta de una aguja estaba libre de escombros después de lavados con ácido fluorhídrico. B. Una punta se generó en la punta de la aguja. <img src="/files/ftp_upload/1924/1924fig2.jpg" alt="Figura 2" /> Figura 2. Visualización de las buenas prácticas agrarias-que expresan las células del donante en un quimérico embriones de pez cebra en 24 HPF. El panel izquierdo muestra las señales de las buenas prácticas agrarias en el cerebro y la retina. El panel central es una imagen de campo claro del embrión. El panel derecho muestra la imagen fusionada. Movie 1. Imágenes en vivo de las células GFP donante positivo demostrado los grandes cambios en la morfología de las células cerebrales neuroepiteliales durante el desarrollo. Los cuadros individuales de la película fueron recogidos cada 5 minutos entre las 24 y 25 HPF. <a href="http://www.jove.com/files/ftp_upload/1924/1924_Wei_Movie1.wmv">Haga clic aquí para ver la película</a> .

<ol>
	<li>Zou, J., Lathrop, K., Sun, M., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+RPE+maintained+by+Nok+is+essential+for+retinal+epithelial+polarity+and+cellular+patterning+in+zebrafish.">Intact RPE maintained by Nok is essential for retinal epithelial polarity and cellular patterning in zebrafish.</a> Journal of Neuroscience. 28 (50), 13684-13695 (2008).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">A guide for the laboratory use of zebrafish (Danio rerio).</a> THE ZEBRAFISH BOOK. , (2007).</li><li>Ho, R. K., Kane, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+action+of+zebrafish+spt-1+mutation+in+specific+mesodermal+precursors.">Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors.</a> Nature. 348, 728-730 (1990).</li><li>Catalano, A., Raymond, P., Goldman, D., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+dou+yan+Mutation+Causes+Patterning+Defects+and+Extensive+Cell+Death+in+the+Retina.">Zebrafish dou yan Mutation Causes Patterning Defects and Extensive Cell Death in the Retina.</a> Developmental Dynamics. 236 (5), 1295-1236 (2007).</li></ol>

En este vídeo, se demuestra un método para el trasplante de blastómeros para analizar el desarrollo de la retina y el cerebro en el pez cebra. Este protocolo también se puede utilizar para analizar el desarrollo de otros tejidos y órganos, simplemente la liberación de los blastómeros de los donantes a los lugares correspondientes en los embriones de acogida. El etiquetado con las moléculas de las buenas prácticas agrarias se obtiene un fondo más limpio que el etiquetado con tinte fluorescente dextrano conjugad...

E3 agua de huevo: <ul><li> 5 mM NaCl </li><li> 0,17 mM KCl </li><li> 0,33 mM CaCl 2 </li><li> 0,33 mM MgSO 4 </li><li> 0,1% de azul de metileno </li></ul>

transplantation of gfp-expressing blastomeres for live imaging of retinal and brain development in chimeric zebrafish embryos

Las células cambian mucho en su ubicación y la propiedad durante la embriogénesis. Estos cambios están regulados por las interacciones entre las células y su medio ambiente. Embriones quiméricos, que se componen de células de los antecedentes genéticos diferentes, son una gran herramienta para estudiar las interacciones célula-célula mediada por los genes de interés. La transparencia de embriones de pez cebra en las primeras etapas de desarrollo permite la visualización directa de la morfogénesis de los tejidos y órganos a nivel celular. Este sentido, demuestran un protocolo para generar quimérico retinas y cerebros de embriones de pez cebra y para realizar las imágenes en vivo de las células del donante. El protocolo contempla la preparación de las agujas del trasplante, el trasplante de las buenas prácticas agrarias-que expresa blastómeras donantes para GFP-negativos los ejércitos, y el examen de comportamiento de los donantes de células en la microscopía confocal en vivo. Con ligeras modificaciones, este protocolo también se puede utilizar para estudiar el desarrollo embrionario de otros tejidos y órganos en el pez cebra. Las ventajas de utilizar la GFP a las células de los donantes etiqueta también se discuten.

Watch this Scientific Journal Video about Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos at JoVE.com

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Demostramos un protocolo para generar embriones quiméricos de pez cebra para vivir el comportamiento de imagen celular durante la embriogénesis.

El trasplante de las buenas prácticas agrarias-que expresa Blastómeros de imágenes en vivo del desarrollo de la retina y el cerebro en embriones de pez cebra quiméricos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

transplantation-gfp-expressing-blastomeres-for-live-imaging-retinal

Research

JoVE Journal

Biology

10.6K vistas.  University of Pittsburgh. Demostramos un protocolo para generar embriones quiméricos de pez cebra para vivir el comportamiento de imagen celular durante la embriogénesis.

Video: Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

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.

De dos fotones axotomía y de lapso de tiempo de imagen confocal en embriones de pez cebra en vivo

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

Investigación

Biología

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.

Imágenes en vivo del cerebro del embrión de pez cebra mediante microscopía confocal

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 ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

La generación de embriones quiméricos de pez cebra mediante un trasplante

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Imágenes en directo de la motilidad celular y citoesqueleto de actina de las neuronas individuales y las células de la cresta neural en embriones de pez cebra

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

Desarrollo de la imagen y el análisis automatizado de pantallas de química de pez cebra.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

De dos fotones basado en fotoactivación en vivo embriones de pez cebra

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

Dechorionation de embriones de medaka y trasplante de células para la generación de quimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Glicanos imágenes en embriones de pez cebra mediante marcado metabólico y Química Haga clic Bioorthogonal

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imágenes de Apoptotic Liquidación de células vivas durante<em&gt; Drosophila</em&gt; Embriogénesis

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.