We thank Y. A. Pan, J. Livet and Z. Tobias for technical and intellectual contributions. This work was supported by the National Science Foundation (Award 1553764) and the M.J. Murdock Charitable Trust.

Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at Lewis &#38; Clark College.
1. Microinjection of Zebrafish Embryos
<ol>
	<li>Set up wild type, adult zebrafish in sex-segregated mating tanks the afternoon prior to performing microinjections39,40.</li>
	<li>Prepare the DNA solution in the morning of the microinjections. Dilute hsp:Zebrabow11...

The authors have nothing to disclose.

This protocol describes a method to visualize clones of progenitor cells and neurons in the developing zebrafish hindbrain and follow them in vivo using Brainbow and time-lapse confocal microscopy11. The major advantage of this protocol in comparison to in vitro or ex vivo studies is the ability to directly observe the proliferative zone of the vertebrate brain in its natural milieu over time. This technique builds on previous studies&#160;that labeled a single clone using retroviral vectors. In c...

In the early phases of development, pools of specialized progenitor cells divide repeatedly in proliferative zones, producing diverse arrays of daughter cells. The cells born during this developmental period will then differentiate and travel to form the nascent organs. In the nervous system, progenitors such as radial glia give rise to immature neurons in ventricular zones. As neurons migrate away from ventricles and mature, the expanding tissue eventually forms the highly complex structures of the brain1,2,3,4,5,6. The coordination between division of progenitors and differentiation and migration of neurons will determine the eventual size, shape, and thus function of the brain, directly impacting behavior7,8,9,10. While tight control over these processes is clearly crucial for normal brain development, the global mechanisms that regulate these dynamics are not well understood. Here we describe a tool to study nervous system development at a cellular resolution, allowing researchers to visualize progenitor cells and neurons in vivo in the developing zebrafish brain with Brainbow and track their behavior over time via time-lapse confocal microscopy11. The approach can also be adapted to visualize other parts of the developing embryo.
To observe and distinguish among cells in the developing zebrafish brain, we have adapted the Brainbow cell-labeling technique11. Brainbow utilizes the randomly determined, combinatorial expression of three distinct fluorescent proteins (FPs) to label a population of cells. While the default expression for Brainbow expression is the red FP dTomato, recombination by the enzyme Cre recombinase results in expression of mCerulean (cyan fluorescent protein, CFP) or yellow fluorescent protein (YFP)12,13. The combined amount of each FP expressed in a cell gives it a unique hue, allowing clear visual distinction from neighboring cells. Additionally, when a progenitor cell divides, each daughter cell will inherit the color from its mother cell, producing color-coded clones and allowing researchers to trace cell lineage11,14. While originally used to analyze neuronal circuitry in mice12, Brainbow has since been expressed in a wide variety of model organisms, including zebrafish15.
Our technique builds on previous multicolor labeling and imaging methods to directly image multiple color-coded clones over time in living zebrafish. Due to their optical transparency as embryos, zebrafish are well suited to imaging experiments16, and previous studies have utilized Brainbow in zebrafish to study a variety of tissues, including the nervous system11,15,17,18,19,20,21,22,23,24,25,26,27. The ability to directly image into the living organism, along with their rapid ex utero development, make zebrafish a valuable model of vertebrate development. In contrast to the mammalian brain, the entire proliferative zone of the zebrafish hindbrain is readily available for imaging without disruption to its endogenous environment6. This allows experiments to be conducted in the living organism, rather than in in vitro or fixed tissue preparations. In contrast to fixed imaging experiments, in vivo studies allow for a longitudinal design, producing hours of data that can be analyzed for patterns, thus increasing the likelihood of observing relatively rare events. Depending upon the speed and length of the events of interest, researchers may choose to perform short (1&#8211;2 h) or long (up to ~16 h) time-lapse imaging experiments. By using the zebrafish heat shock promoter 70 (hsp70, hsp), Brainbow expression can be temporally controlled28,29. Additionally, the mosaic expression induced by this promoter is well suited for labeling and tracking many clones11.
The ability to visually identify multiple clones within the living brain is an advantage of this method. Important previous studies that investigated the role of clones within development of the nervous system utilized retroviral vectors to label a single progenitor cell and its progeny using a single FP or other readily visualized protein. Such labeling allows researchers to observe a single clone over time, either in vitro or in vivo2,30,31,32,33,34,35,36,37,38. In contrast to methods to track the behavior of cells within one clone, the distinct colors of Brainbow allow researchers to observe dynamics among clones. Additionally, by using Brainbow to label many clones within the brain, additional data on clonal behavior is collected relative to techniques that label a single clone11. Importantly, the approaches described here can be expanded to generate developmental comparisons between fish that have undergone different genetic or pharmacological manipulations18. Overall, these advantages make time-lapse in vivo confocal imaging of Brainbow-expressing zebrafish ideal for researchers exploring development of the vertebrate nervous system, particularly those interested in the role of clones.

<table>
	<tbody>
		<tr>
			<td>1.5mL transfer pipet</td>
			<td>Globe Scientific, Inc.</td>
			<td>134020</td>
			<td></td>
		</tr>
		<tr>
			<td>1-phenyl-2-thiourea (PTU)</td>
			<td>Alfa Aesar</td>
			<td>L06690</td>
			<td>Diluted to 0.2 mM in E3 to prevent embryo pigmentation</td>
		</tr>
		<tr>
			<td>50ml conical tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td>For heat shocking embryos</td>
		</tr>
		<tr>
			<td>6 lb nylon fishing line</td>
			<td>SecureLine</td>
			<td>NMT250</td>
			<td>For making embryo manipulators</td>
		</tr>
		<tr>
			<td>7.5mL transfer pipet</td>
			<td>Globe Scientific, Inc.</td>
			<td>135010</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C3881</td>
			<td>For E3</td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Puritan</td>
			<td>867-WC NO GLUE</td>
			<td>For making embryo manipulators</td>
		</tr>
		<tr>
			<td>Cre recombinase</td>
			<td>New England Biolabs</td>
			<td>M0298M</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital dry bath</td>
			<td>Genemate</td>
			<td>490016-616</td>
			<td>Used to store LMA at 40&#176;C</td>
		</tr>
		<tr>
			<td>Epifluorescence dissection scope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary tubes</td>
			<td>World Precision Instruments</td>
			<td>TW100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Forma Scientific</td>
			<td>3158</td>
			<td>To maintain embryos at 28&#176;C</td>
		</tr>
		<tr>
			<td>Injection plate molds</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp water bath</td>
			<td>Fisher Scientific</td>
			<td>2320</td>
			<td>For heat shocking embryos</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>AMRESCO</td>
			<td>0395</td>
			<td>For E3 and for DNA solution for injections</td>
		</tr>
		<tr>
			<td>Laser-scanning confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM710</td>
			<td></td>
		</tr>
		<tr>
			<td>LE agarose</td>
			<td>Genemate</td>
			<td>E3120</td>
			<td>To create agarose injection plates</td>
		</tr>
		<tr>
			<td>Low-melt agarose (LMA)</td>
			<td>AMRESCO</td>
			<td>J234</td>
			<td></td>
		</tr>
		<tr>
			<td>Mating tanks</td>
			<td>Aquaneering, Inc.</td>
			<td>ZHCT100</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>Sigma</td>
			<td>M9140</td>
			<td>For E3</td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma</td>
			<td>9397</td>
			<td>For E3</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>M3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>MS-222 Tricaine-S</td>
			<td>Western Chemical, Inc.</td>
			<td></td>
			<td>Stock made at 4 mg/mL in reverse osmosis (RO) water, then added dropwise to E3 to final concentration of 0.2 mM to anesthetize embryos</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>J.T. Baker</td>
			<td>4058-01</td>
			<td>For E3</td>
		</tr>
		<tr>
			<td>Petri dishes (90 mm, 60 mm)</td>
			<td>Genesee Scientific</td>
			<td>32-107G</td>
			<td>To house embryos and create imaging chamber (60 mm)</td>
		</tr>
		<tr>
			<td>Phenol red</td>
			<td>Sigma</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft stitch ring markers</td>
			<td>Clover Needlecraft, Inc.</td>
			<td>354</td>
			<td>For creating imaging chamber with Petri dish</td>
		</tr>
		<tr>
			<td>Super glue (Ultra gel control)</td>
			<td>Loctite</td>
			<td>1363589</td>
			<td>For making embryo manipulators</td>
		</tr>
		<tr>
			<td>Syringe needles</td>
			<td>Beckton Dickinson</td>
			<td>BD329412</td>
			<td>For dechorionating embryos</td>
		</tr>
	</tbody>
</table>

visualizing the developing brain in living zebrafish using brainbow and time-lapse confocal imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

This section illustrates examples of results that can be obtained using the in vivo multicolor time-lapse imaging approach described here. We show that Brainbow color-coded clones of cells in the proliferative ventricular zone of the developing zebrafish hindbrain14 (Figure 1).
Typically, when Brainbow-labeled cells were arranged along a particular radial fiber, they shared the same color (Figure 1D), which cou...

Watch this Scientific Journal Video about Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging at JoVE.com

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

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

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

This method can help answer fundamental questions about the dynamics of neural progenitor cells and their progeny which underlie vertebrate brain development. The main advantage of this technique is the ability to directly visualize multiple clusters of clonally related cells within the living brain over many hours of development. The protocol can also be applied to studying clonal and progenitor dynamics in other developing systems of the zebrafish embryo.Visualization using this method is particularly useful for directly observing fundamental processes that occur during neural development. On the afternoon prior to performing micro injections, set up wild type adult zebrafish in sex segregated mating tanks. On the next morning prepare the DNA solution according to manuscript directions and inject approximately 4.2 nano liters of this solution into one cell zebrafish embryos within 45 minutes of fertilization.Maintain the injected embryos in E three medium and a 28 degrees Celsius incubator for 24 hours, then call the dead and deformed embryos from the group. Transfer up to 20 healthy embryos into a 50 milliliter tube, fill it with 10 milliliters of E three and place a cap on top of each tube. Place a rack with the 50 milliliter tubes upright in a 37 degrees Celsius water bath for 80 to 90 minutes, making sure that the water level in the bath is higher than the E three in the tube.Remove the tube rack from the water bath and place it in the 28 degrees Celsius incubator. Allow up to one hour for the E three to cool and the embryos to reacclimate to the temperature. Then transfer them to Petri dishes with 0.2 millimolar PTU and E three kept warm in the 28 degrees Celsius incubator.At two to four hours after the heat shock, examine the embryos under a standard fluorescence dissection microscope for expression of CFP or YFP which indicates successful Brainbow recombination. Select embryos with a robust FP expression throughout and transfer them to a separate dish with PTU. Image them one, two, or three days post fertilization.Expression which appears dim under a fluorescence microscope may actually be well visualized in confocal time-lapse imaging. Prior to the day of the experiment, prepare the imaging chamber and embryo manipulation tool. To prepare the imaging chamber, carefully superglue a plastic ring to the center of a 60 millimeter Petri dish.Construct the manipulator by super gluing a small length of nylon fishing line to the end of a four inch wooden swab stick. If necessary, dechorionate the embryos under a dissection microscope prior to mounting. To mount the embryos, transfer the fish to the center of the plastic ring in the imaging chamber and remove as much excess E three as possible with a fine tipped transfer pipette.Use a clean transfer pipette to cover the fish with one percent low melt agarose and E three, filling the entire plastic ring with a layer of agarose. Then gently pull the embryo up into the pipette tip and back into the agarose without introducing any air bubbles. Use an embryo manipulator to quickly orient the fish and the agarose before it hardens.If imaging with an upright microscope, position the embryo as close to the upper surface of the agarose as possible, making sure that they are parallel to the bottom of the imaging chamber with their tail straight. Wait for the agarose to harden, then fill the imaging chamber with E three, adding as much as possible to account for evaporation over the course of imaging. Place the imaging chamber with the fish and E three on the confocal microscope.Then select the objective with a high numerical aperture and a long working distance. Find a region with appropriately dense and bright labeling of cells and set up the acquisition parameters. This is an example using Zen software, but settings will vary depending on microscope and laser lines available.Prepare three tracks to image each FP channel sequentially. Use an argon laser to excite CFP at 458 nanometers and YFP at 514 nanometers, and use a DPSS 561 nanometer laser to excite dTomato. Collect the missions for the three at the appropriate wavelengths.Select the Z stack range to image and a time interval between 10 and 30 minutes to track mitotic and apoptotic events. Finally, select the length the imaging session and run the experiment. After imaging is complete, save the raw data in CZI format if using Zen or another format compatible with Fiji, then import the images into Fiji using the bio formats importer.In vivo multicolor time-lapse imaging was used to show Brainbow color coded clones of cells in the proliferative ventricular zone of the developing zebrafish hindbrain. Brainbow labeled cells arranged along a particular radial fiber shared the same color. Which can be quantified as the relative RGB channel weights.This suggests that these radio groups were clones of dividing cells and that their similar color could be used to identify them as being clonally related. A quantitative color analysis showed that daughter cells express the same color as their mother's cell. But that neighboring radio clusters of cells can be distinguished from one another.Numerous clones of related cells can be followed simultaneously over hours in vivo, allowing for a multiplex lineage analysis and comparison. Quantification of color expression in clones at two and then again at three days post fertilization showed that Brainbow expression remained relatively constant from two to three days. Time-lapse imaging revealed numerous cells undergoing interkinetic nuclear migration and cell division from one to two days post fertilization, making it possible to study the cell cycle.An average cell cycle of 8.4 hours was calculated which is comparable to previous measurements in zebrafish. Furthermore, this imaging technique was used to observe individual cells undergoing the stereotypical morphological changes associated with apoptosis, such as membrane blebbing and cell fragmentation. Because this method is performed in vivo, zebrafish can be rescued from the imaging chamber and maintained for imaging or other experiments at later time points.The use of this technique allows us to explore new questions about the roles of clonally related cells during neural development.

visualizing-developing-brain-living-zebrafish-using-brainbow-time

Research

JoVE Journal

Developmental Biology

8.3K vistas.  Lewis & Clark College. Este método puede ayudar a responder preguntas fundamentales sobre la dinámica de las células progenitoras neuronales y su progenie que subyacen al desarrollo cerebral de los vertebrados. La principal ventaja de esta técnica es la capacidad de visualizar directamente múltiples grupos de células relacionadas clonalmente dentro del cerebro vivo durante muchas horas de desarrollo. El protocolo también se puede aplicar al estudio de la dinámica clonal y progenitora en otros sistemas en de...