Name: multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos
Uploaded: 2017-08-09T15:00:00
Description: Watch this Scientific Journal Video about Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos at JoVE.com

Los autores agradecen a Thomas Schilling para amablemente regalar el pescado Tg (sox10:eGFP) y Mary Halloran para amablemente regalar pescado Tg(foxd3:GFP) .

The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA).
1. Embryo Collection for Time-lapse Imaging
<ol>
	<li>Between 3 and 9 pm, set up male and female adult Tg(sox10:EGFP) or Tg(foxd3:GFP) transgenic zebrafish in a divided breeding tank for pairwise mating. 
	NOTE: The Tg(sox10:EGFP) and Tg(foxd3:G...

<ol>
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La proyección de imagen multi-photon Time-lapse permite el seguimiento en vivo de poblaciones celulares transitorias y migratorias. Esta poderosa técnica se puede utilizar para el estudio de los procesos embrionarios en tiempo real, y en el presente estudio, los resultados de este método mejorado el conocimiento actual de la migración de células de cresta neural y el desarrollo. Anteriores estudios Time-lapse de imágenes típicamente utilizan la microscopía de láser confocal. 29 <...

Enfermedades congénitas oculares pueden causar ceguera de la infancia y a menudo debido a las anormalidades de la cresta neural craneal. Las células de la cresta neural son células transitorias que surgen a partir del tubo neural y forman numerosos tejidos en todo el cuerpo. 1 , 2 , 3 , 4 , 5 las células de la cresta Neural, derivadas del prosencéfalo y mesencéfalo, dan lugar a los huesos y el cartílago de la cara y regiones frontales, iris, córnea, red trabecular y esclera en el segmento anterior del ojo. 4 , 6 , 7 , 8 células de la cresta Neural desde el rhombencephalon forma que arcos de la faringe, mandíbula y tracto de salida cardíaco. 1 , 3 , 4 , 9 , 10 estudios han puesto de relieve las aportaciones de la cresta neural para ocular y periocular desarrollo, haciendo hincapié en la importancia de estas células en el desarrollo del ojo vertebrado. De hecho, alteración de la diferenciación y migración de células de cresta neural conducen a anomalías craneofaciales y oculares como se observa en el síndrome de Axenfeld-Rieger y síndrome de Peters Plus. 11 , 12 , 13 , 14 , 15 , 16 , 17 por lo tanto, una comprensión global de la migración, proliferación y diferenciación de estas células de la cresta neural se proporciona la penetración en la complejidad subyacente a enfermedades oculares congénitas.
El pez cebra es un organismo modelo de gran alcance para estudiar desarrollo ocular, como las estructuras del ojo de pez cebra son similares a sus contrapartes mamíferas, y muchos genes son conservados evolutivamente entre pez cebra y mamíferos. 18 , 19 , 20 además, embriones de pez cebra son transparentes y ovípara, facilitando la visualización del desarrollo del ojo en tiempo real.
Ampliando el trabajo previamente publicado,6,7,20 el patrón migratorio de las células de la cresta neural fue descrito usando múltiples fotones fluorescencia Time-lapse de imagen en líneas de pez cebra transgénico con proteína verde fluorescente (GFP) bajo el control transcripcional de SRY (sex-determining región Y)-caja de 10 (sox10) o caja del Forkhead D3 regiones reguladoras del gen de (foxd3). 21 , 22 , 23 , 24. proyección de imagen de múltiples fotones fluorescencia Time-lapse es una técnica poderosa que combina las técnicas ópticas avanzadas de láser microscopía con larga excitación de fluorescencia de varios fotones de longitud de onda para capturar imágenes de alta resolución, tridimensionales de las muestras etiquetadas con fluoróforos. 25 , 26 , 27 el uso del multi-photon laser tiene distintas ventajas sobre el estándar microscopia confocal, incluyendo penetración creciente del tejido y disminución fluoróforo blanqueo.
Usando este método, dos poblaciones distintas de las células de la cresta neural varía en el momento de la migración y vías migratorias fueron las células de la cresta neural es decir positivo de foxd3, discriminados en el mesenchyme periocular y el ojo en desarrollo y sox10-positivo de la cresta neural células de mesénquima craneofacial. Con este método, se introduce un enfoque para visualizar la migración de migración de la cresta de los nervios oculares y craneofacial en el pez cebra, lo que es fácil observar la migración de la cresta neural regulado en tiempo real durante el desarrollo.
Este protocolo proporciona información para la generación de vídeos Time-lapse durante el desarrollo temprano del ojo en Tg (sox10:EGFP) y Tg (foxd3:GFP) de pez cebra transgénico, por ejemplo. Este protocolo se puede aplicar aún más para la visualización de alta resolución, tridimensional en tiempo real del desarrollo temprano de cualquier estructura ocular y craneofacial derivado de las células de la cresta neural en el pez cebra. Además, este método puede ser aplicado además para la visualización del desarrollo de otros tejidos y órganos en el pez cebra y otros modelos animales.

<table border="0" cellpadding="0" cellspacing="0" width="919">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Breeding Tanks with Dividers</td>
			<td style="width:175px;">Aquaneering</td>
			<td style="width:104px;">ZHCT100</td>
			<td style="width:391px;">Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">M205 FA Combi-Scope</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;">Stereofluorescence Microscope - FusionOptics and TripleBeam</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">7760-5KG</td>
			<td style="width:391px;">Double PE sack. CAS No. 7647-14-5, EC Number 231-598-3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Potassium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">1049380500</td>
			<td style="width:391px;">Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Calcium Chloride Dihydrate</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">C79-500</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 10035-04-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Magnesium Sulfate (Anhydrous)</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">MX0075-1</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Methylene Blue</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">284-12</td>
			<td style="width:391px;">Glass bottle; 25 g. Powder, Certified Biological Stain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Bicarbonate</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">SX0320-1</td>
			<td style="width:391px;">Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">N-Phenylthiourea</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">P7629-25G</td>
			<td style="width:391px;">&#62;98%. CAS Number 103-85-5, EC Number 203-151-2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Dimethylsulfoxide</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">D8418-500ML</td>
			<td style="width:391px;">Molecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tricaine Methanesulfonate</td>
			<td style="width:175px;">Western Chemical Inc.</td>
			<td style="width:104px;">MS222</td>
			<td style="width:391px;">Tricaine-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Low-Melt Agarose</td>
			<td style="width:175px;">ISC Bioexpress</td>
			<td style="width:104px;">E-3112-25</td>
			<td style="width:391px;">GeneMate Sieve GQA Low Melt Agarose, 25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Open Bath Chamber</td>
			<td style="width:175px;">Warner Instruments</td>
			<td style="width:104px;">RC-40HP</td>
			<td style="width:391px;">High Profile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Glass Coverslips</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">12-545-102</td>
			<td style="width:391px;">Circle cover glass. 25 mm diameter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">High Vacuum Grease</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">14-635-5C</td>
			<td style="width:391px;">2.0-lb. tube. DOW CORNING CORPORATION 
			1658832</td>
		</tr>
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multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos

Ojo congénita y anomalías craneofaciales reflejan alteraciones en los nervios de la cresta, una población transitoria de células migratorias que dan lugar a numerosos tipos de células en todo el cuerpo. Entender la biología de la cresta neural ha sido limitada, lo que refleja una falta de modelos genéticamente manejables que pueden ser estudiado en vivo y en tiempo real. Pez cebra es un modelo de desarrollo particularmente importante para el estudio de las poblaciones de células migratorias, como la cresta neural. Para examinar la migración de la cresta neural en el ojo en desarrollo, se implementó una combinación de las técnicas ópticas avanzadas de láser, microscopía de excitación de fluorescencia de varios fotones de onda larga para capturar videos de alta resolución, tridimensionales en tiempo real del ojo en desarrollo en embriones de pez cebra transgénico, es decir, Tg (sox10:EGFP) y Tg (foxd3:GFP), como sox10 y foxd3 han demostrado en numerosos modelos animales para regular la diferenciación temprana de la cresta neural y probablemente representan los marcadores para células de la cresta neural. La proyección de imagen multi-photon Time-lapse fue utilizada para discernir el comportamiento y los patrones migratorios de dos poblaciones de células de cresta neural que contribuye al desarrollo temprano del ojo. Este protocolo proporciona información para la generación de vídeos Time-lapse durante la migración de la cresta neural del pez cebra, como un ejemplo y puede aplicarse más para visualizar el desarrollo temprano de muchas estructuras en el pez cebra y otros organismos modelo.

Imágenes de Time-lapse de fluorescencia de varios fotones generan una serie de videos que revelaron los patrones de migración de las células de la cresta de los nervios craneales que dan lugar a las estructuras craneofaciales y segmento anterior del ojo en la Tg (sox10:EGFP) y Tg (foxd3:GFP) líneas de pez cebra. Por ejemplo, sox10 -células cresta neural positiva entre 12 y 30 hpf migran desde el borde del tubo neural en la región craneofacial (Vide...

Watch this Scientific Journal Video about Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos at JoVE.com

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

La proyección de imagen de lapso tiempo multi-Photon para visualizar el desarrollo en tiempo real: visualización de la migración de las células de cresta Neural en embriones de pez cebra

Se implementó una combinación de las técnicas de microscopía con largo excitación de fluorescencia de varios fotones de longitud de onda del láser ópticas avanzadas para capturar imágenes de alta resolución, tridimensionales en tiempo real de la migración de la cresta neural en Tg (sox10:EGFP) y los embriones de pez cebra de Tg (foxd3:GFP).

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

The overall goal of multi-photon time lapse imaging is to capture a high-resolution three dimensional real time images of migratory cell populations. This method can help answer key questions in developmental biology, such as mapping migration pathways of different cell populations. The main advantage of this technique over traditional confocal microscopy is that multi-photon lasers have deeper tissue penetration and decreased phototoxicity.This increases the usability in thicker tissues and allows for longer periods of image acquisition. To set up for embryo collection between 3:00 and 9:00 p.m. assemble breeding tanks and fill them with reverse osmosis, or RO water.Transfer up to three females and three males into opposing sides of the divided tank. The next morning, remove the divider shortly after the lights turn on in the vivarium. Allow undisturbed mating for 20 minutes or until sufficient numbers of embryos are produced.The embryos will be at the bottom of the breeding tank. When eggs are observed, lift the slotted inner tank out along with the fish, and quickly place them into a clean, solid outer tank filled with RO water. Use an egg collecting screen to collect eggs from the outer tank and transfer the eggs to Petri dishes containing 30 milliliters of 1X embryo medium.Incubate the collected eggs at 28.5 degrees Celsius. For transgenic embryos, at 24 hours post-fertilization or HPF, remove the dead eggs and assess the developmental stage of the embryos. Using forceps, remove the chorions from three to four embryos that express GFP.Under a dissecting fluorescence stereo microscope with a standard 460 to 490 nanometer band pass excitation filter, screen for GFP positive embryos. Transfer these embryos to a separate Petri dish containing 30 milliliters of fresh 1X embryo medium. After 20 HPF, place the screened and dechorionated GFP positive embryos in 0.003%PTU solution to inhibit pigmentation.Avoid initiating treatment earlier as it can have adverse effects on neural crest and neuroepithelial development. Add 0.4 grams of LowMelt Agarose powder to 20mL of 1x embryo medium. Heat the mixture for one to two minutes or until the solution is clear and all particles are dissolved.Aliquot approximately 1mL of the Agarose solution into fresh 1.5mL centrifuge tubes for storage. To set up the open bath chamber, place a small amount of high vacuum grease on the base of the open bath chamber. Place a circular glass cover slip onto the base and screw the top of the open bath chamber onto the base until it is tight.Pipette approximately 500-700 microliters of 2%Agarose solution into the open bath chamber until the base is approximately 3/4s filled. Wait 30 seconds to allow the Agarose to cool slightly without completely polymerizing and subsequently transfer a single embryo to the center of the base. Under a fluorescent stereo microscope, use a 10 microliter micro pipette tip to orient the embryo and position it near the bottom of the Agarose so that it doesn't float away when the embryo medium is added.A critical step is to make sure that the embryo is positioned correctly in the Agarose. In these experiments, we orient the embryo laterally but the embryo can also be positioned to focus on dorsal and ventral areas. Monitor and reposition the embryo until the Agarose has set.Then use a transfer pipette to entirely fill the assembled open chamber with time-lapse embryo medium. After placing the entire set up onto the stage of the microscope, according to the text protocol, use the five times objective to locate the embryo. Then manually raise the stage to the highest position and use the fine focus to position the embryo in the middle of the microscope range.Manually lower the stage and change the five time objective to the 25 time water immersion objective. Carefully raise the stage to bring the embryo back into focus. In the software, click on the XYTZ mode for obtaining multiple images at time or T intervals in the XY plane over a depth of Z.Use the epifluorescence or bright field view to find the depth of focus in the area of interest which will demarcate the z-stack.For these experiments, the lateral edge of the eye is the beginning of the z-stack. When focused here, in the software click the begin button. After focusing down to the mid-line of the embryo, which is the end of the z-stack, click the end button.A critical step is to make sure that the Z step encompasses the area of interest. In our case, we want to capture the width of the eye from the lateral to the medial edges. Click on the menu for adjusting the acquisition time and the imaging frequency.For adequate recovery of the fluor4 and survival of the embryo, allow for a ratio of at least one to three between z-stack acquisition when laser power is on and recovery time when laser power is off. With appropriate time for embryo recovery, set the time between z-stacks in the designated window. Then set the total length of time for the experiment in the appropriate window.Now, turn on the live image setting to make final adjustments to the laser settings. Adjust laser transmission, gain and offset slider bars within the software to optimize the fluorescence image. Also, adjust the orientation of the embryo as needed depending on the length of the experiment, anticipated growth of the embryo, et cetera.Make sure that the area of interest remains within the frame through the duration of the experiment. During time-lapse acquisition, turn off the epifluorescence light source, cover the stage with the laser safety box which is adequate for protection against background light, then press start. Access the open bath chamber through the sliding doors on the laser safety box to refill it with time lapse embryo medium every 8-12 hours.Following imaging acquisition, open the files in the image processing software, highlight the correct image series. In the software, choose the process menu, click on 3D deconvolution and apply to deconvolve each z-stack. Import individual tif files into video processing software, then select all tif files and drag them into the video editor.Adjust the length of each image within the video to 0.1 seconds. Finally, export the video as a mov or mp4 file. Seen here by multi-photon fluorescence imaging are the migration patterns of cranial neural crest cells that give rise to the cranial facial structures and anterior segment of the eye in transgenic sox10:EGFP and foxd3:GFP zebrafish lines.Sox 10 positive neural crest cells between 12 and 30 hpf migrate from the edge of the neural tube into the cranial facial region. The cells from the prosencephalon and mesencephalon migrate dorsally and ventrally to the developing eye to populate the periocular mesencine. As indicated here, the sox10 positive cells form the frontal nasal process.Neural crest cells from the rhombencephalon migrate ventrally and give rise to the pharyngeal arches. Time lapse imaging of foxd3 positive neural crest cells between 30 and 60 hpf show that these cells migrated between the optic cup and surface ectoderm and though the ocular fissure marked by the arrow and coalesced around the lens marked by the asterisk forming the iris stroma. Once mastered, this technique from start to finish can be done in four days if it is performed properly.When attempting this procedure, it is important to remember to choose embryos with a high amount of fluorescence. Setting up the software initially can be challenging but once the parameters are determined, additional changes are typically minimal. After watching this video, you should have a good understanding of how to use multi-photon time-lapse microscopy to image migrating cells in the embryo.

Research

JoVE Journal

Developmental Biology

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

analizando<em&gt; En Vivo</em&gt; Migración de células usando trasplantes de células y time-lapse en embriones de pez cebra

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Investigación

Biología del Desarrollo

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Análisis para el Desarrollo de riñón de pez cebra con time-lapse de uso de un microscopio de disección equipado para seccionamiento óptico

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Cinética Medición y visualización en tiempo real de la reprogramación somática

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Inducción de la hipoxia en los sapos vivos y los embriones de pez cebra

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Desarrollo de un ensayo In Vitro para cuantificar Progenitor y vástago hematopoyética células (HSPCs) en el desarrollo de embriones de pez cebra

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Citometría de flujo en tiempo real de células vivas para investigar la afluencia del calcio, formación de poro y fagocitosis por los receptores P2X7 en células progenitoras neurales adultas

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Un método de montaje en capas para la microscopía confocal de lapso de tiempo extendido de embriones enteros de pez cebra

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Visualización del cerebro en desarrollo en el pez cebra vivo mediante imágenes confocales de barco iris y lapso de tiempo

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

Aislamiento, cultivo e inducción adipogénica de células madre originales adiposas de raíz de la cresta neuronal del tejido adiposorio

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...