We would like to thank Anne-Laure Duchemin, Denise Duchemin, Nathalie Faggianelli and Basile Gurchenkov for helping to design and make the mold described in this protocol. This work was supported by FRM (DEQ20140329553), the ANR (ANR-15-CE13-0015-01, ANR-12-ISV2-0001-01), the EMBO Young Investigator Program, the European Community, ERC CoG N&#176;682938 Evalve and by the grant ANR-10-LABX-0030-INRT, a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d'Avenir labeled ANR-10-IDEX-0002-02.

1. Preparing molds and mounting agarose
<ol>
	<li>Create a mold with the dimensions shown in Figure 1 using either a 3D printer or traditional mechanical workshop tools.</li>
	<li>Pipette about 1.5 mL of melted 1 % agarose into a 35-mm plastic mounting dish. Place the plastic mold in the liquid agarose, taking care to avoid trapping air between the mold and the agarose. Place the dish at 4 &#176;C, wait till the agarose hardens (this takes about 5 min), then remove the plastic mold.</li>
	<li>To store the mounting dish for later use, cover the dish with its lid, then wrap both the dish and the lid tightly with parafilm. The mounting dish can then be stored lid side facing down for up to 5 days at 4 &#176;C.</li>
	<li>Prepare in advance 1 mL aliquots of 0.7 % low melting point agarose in 1.5 mL Eppendorf tubes to use on the day of mounting. 
	NOTE: The agarose wells of the mounting dish will deform over time as water evaporates if parafilm is not wrapped around the dishes tightly.</li>
</ol>
2. Obtaining Embryos for the Photoconversion
<ol>
	<li>Incross the Tg(fli1a:gal4FFubs, UAS:kaede) line and grow approximately 200 embryos in embryo medium in the dark at 28.5 &#176;C. For more details on how to cross zebrafish lines, please refer to the JoVE Science Education Database11.</li>
	<li>After 5 h but before 24 h, treat embryos with 1-phenyl-2-thiourea (PTU) to prevent pigment formation (0.003 % PTU in embryo medium).</li>
	<li>About 1 h before the start of photoconversion, screen embryos under a fluorescent stereoscope and select 5 healthy embryos that appear to express the brightest green fluorescence. The use of low-intensity light when visualizing embryos is recommended to avoid exposure of the embryos to ambient light in order to avoid photoconverting Kaede in non-specific cells.</li>
</ol>
3. Embedding
<ol>
	<li>Prepare 10 mL of mounting media: 0.02 % tricaine and 50 mM BDM in embryo medium.</li>
	<li>Add 2 mL of mounting medium to the mounting dish and allow 15-30 min for the solution to diffuse into the agarose.</li>
	<li>In the meantime, melt a 1 mL aliquot of 0.7 % low melting point (LMP) agarose by placing the tube in a 70 &#176;C heat block for about 5 min.
	<ol>
		<li>Wait for the LMP agarose to cool slightly, then add 25 &#181;L of 8 mg/mL tricaine solution and 40 &#181;L of 1M BDM solution to the tube of low melting point agarose. Mix by pipetting up and down, then transfer the tube to a 38 &#176;C heat block to keep the LMP agarose in its liquid state.</li>
	</ol>
	</li>
	<li>In a Petri dish with embryo medium, carefully dechorionate the pre-selected embryos under the stereomicroscope using forceps, taking care not to damage the embryos.</li>
	<li>Transfer the embryos to a separate dish containing 2 mL of mounting media.
	<ol>
		<li>When the hearts of the embryos are stopped (takes about 5-10 min), transfer the embryos to the mounting dish and arrange embryos in the wells using forceps so that they lie at a 25 degree angle, tails pointing towards the deeper part of the well, yolk facing up.</li>
		<li>When embryos are roughly in place, remove the media, embed the embryos in ~200 &#181;L of 0.7 % LMP agarose containing tricaine and BDM, and readjust the embryos&#39; positions, tilting the embryos to the left or right as necessary.</li>
	</ol>
	</li>
	<li>Wait until the LMP agarose sets (takes about 5 min), then add mounting medium to the dish. The embryos are now ready to be photoconverted. 
	NOTE: The tricaine and the BDM are used to anesthetize the embryos and to stop the embryos&#39; heart, respectively. Mounting media can be prepared the day before imaging, but make sure to protect the media from light. 
	NOTE: The correct positioning of the embryos is important to allow a clear laser path to the cells one wishes to photoconvert. Position the head of the embryo close to the start of the well so that laser light does not have to travel far to reach the embryo, and so that the embryos can be easily unmounted after photoconversion.</li>
</ol>
4. Photoconversion
<ol>
	<li>On an upright confocal microscope (like Leica SP8) equipped with a 405 nm, 488 nm and 561 nm laser source, locate the embryo&#39;s heart using transmitted white light (brightfield) and an objective lens (like Leica HCX IRAPO L, 25 &#215;, N.A. 0.95 objective). Check for red Kaede fluorescence using the 561 nm laser (i.e., DPSS 561 laser, at ~5 % intensity, detector set at 589-728 nm). Then, visualize the endocardium by using the 488 nm laser (i.e., 30 mW multiline Argon laser at ~5 % intensity, detector set at 502-556 nm).</li>
	<li>Enter &#34;FRAP&#34; mode on the microscope acquisition software.Select ~1 % laser power for both 488 nm and 561 nm lasers.</li>
	<li>Focus on the plane of interest, select a region of interest (ROI), then photoconvert cells using the 405 nm diode laser at 25 % laser power, scanning over the ROI three times. Should insufficient Kaede be photoconverted in the ROI, scan the 405 nm laser over the area three times again. Repeat this step for all ROIs.</li>
	<li>Return to the &#34;TCS SP8&#34; mode on the software and acquire a z-stack, using the 561 nm and 488 nm lasers sequentially. Make sure to select the &#39;bidirectional&#39; option to increase imaging speed. 
	NOTE: How long it takes to photoconvert and image each embryo varies. Heart beat is known to be important for normal heart valve development, so avoid keeping the heart stopped for more than 30 min, even if it means you do not have time to photoconvert all 5 embryos. 
	NOTE: During the photoconversion process, use PMT detectors, while after photoconversion, it is recommended to use hybrid detectors set to &#39;counting&#39; mode. This is because the hybrid detectors are more sensitive and are capable of producing better quality images, but are easily damaged by overexposure.</li>
</ol>
5. Unmounting photoconverted embryos
<ol>
	<li>After photoconversion, use a glass pipette to press down slightly just above the head of the embryo to break the LMP agarose, then gently suck up the embryo.
	<ol>
		<li>Eject the embryo from the glass pipette into a 35-mm Petri dish containing embryo medium with PTU (to wash away BDM containing medium).</li>
		<li>Transfer the embryo to a well in a 6-well plate containing embryo medium with PTU. During this step, make sure to keep note of which embryo goes into which well, as this is essential to correlate the position of photoconverted cells at later developmental stages.</li>
	</ol>
	</li>
	<li>Now that the embryos are removed from the BDM containing medium, their hearts should start beating again in about 5 min. Return embryos to the dark at 28.5 &#176;C to allow embryos to continue to develop normally.</li>
</ol>
6. Imaging photoconverted cells at later embryonic stages
<ol>
	<li>At the desired stage, stop the heart and re-embed the embryos like under Step 3 of this protocol, with the important difference that embryos must be treated with BDM in separate, marked dishes to keep track of embryos.</li>
	<li>For embryos up to 62 hpf, acquire z-stacks of the endocardium using 561 nm and 488 nm for red and green fluorescent signals, respectively. For embryos at stages older than 62 hpf, use a multiphoton laser set to 940 nm to image green Kaede instead of the 488 nm laser.</li>
</ol>
7. Image analysis for embryos under 48 hpf
Note: Tissue movements of the AVC can be difficult to analyze due to its complex three-dimensional structure. To quantify the length of a photoconverted region in the AVC between two non-photoconverted regions or the length of a non-photoconverted region in the AVC between two photoconverted regions, it can be desirable to map the three-dimensional structure onto a two-dimensional map. Images of photoconverted embryos obtained prior to 48 hpf can be analyzed using the following method.
<ol>
	<li>Import the acquired images in Matlab or any similar software.</li>
	<li>Delimitate manually the ROI, i.e. the endocardium, and apply a mask to remove the signal outside this region.</li>
	<li>Apply an intensity threshold on the image to identify the points on the endocardium and plot them in three-dimensions. Isolate the points corresponding to the narrowest and most straight part of the AVC, by erasing manually the points of no interest on the atrium and the ventricle using the erase command in the software.</li>
	<li>Use a cylinder to fit the points representing the AVC so obtained.</li>
	<li>Define a reference system to compare several samples. For example, adopt the center of mass of the AVC points as the origin of the reference system and the axis of the fitted cylinder as the z-axis.
	<ol>
		<li>Make sure that positive z-positions correspond always to the same side of the heart (ventricular or atrial).</li>
		<li>Project the AVC points on the x-y plane and use an ellipse to fit them.</li>
		<li>Rotate the endocardial points around the z-axis so that the major axis of the ellipse and the y-axis of the reference system are parallel and positive y-values correspond to the internal side of the AVC with respect to the embryo.</li>
	</ol>
	</li>
	<li>To segment the AVC, cut the three-dimensional dataset with some planes obtained by rotating the half-plane {y=0, x&#62;0} in the z-direction.
	<ol>
		<li>Project the intensity of the neighboring pixels on these planes and define the endothelium in a consistent manner. For example, draw manually a line from the atrial to the ventricular side of the AVC at half thickness of the endothelium.</li>
		<li>Interpolate the points of the lines representing the endothelium with a three-dimensional spline using the spline toolbox to represent the AVC with a surface. 
		NOTE: The number of the cutting planes determines both the accuracy of the segmentation and the time consumption of this step. Usually, 8 planes are enough to achieve good results.</li>
	</ol>
	</li>
	<li>Define the azimuthal position of each point on the AVC surface using cylindrical coordinates.
	<ol>
		<li>At each azimuthal position define a parametric curve along the surface of the AVC by calculating the arc length on the surface between consecutive points.</li>
		<li>For each parametric curve, define the point on the curve closest to the center of the AVC to zero. The points of the AVC are thus fully described by their azimuthal position and their position on the parametric curve.</li>
	</ol>
	</li>
	<li>Unfold the AVC into a two-dimensional image by using the parametric position of the points.</li>
	<li>Find the edges of the region to quantify as the edges of the binary image thresholding the ratio between the intensities of the photoconverted and the non-photoconverted channels. Manually correct the result if necessary.</li>
	<li>Calculate the arc length between the edges to compute the length of the tissue as a function of the azimuthal position.</li>
</ol>

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No conflict of interest declared.

Timing of photoconversion: Although Kaede remains brightly expressed in EdCs even at 96 hpf, it should be noted that as the embryo grows, laser light diffuses more before it reaches the AVC, making confined photoconversion of Kaede more difficult. At embryonic stages later than 55 hpf, the ballooning of the ventricle and the atrium also means that the violet laser beam used for photoconversion cannot reach AVC cells without first passing through either the atrium or ventricle. This means that beyond 55 hpf, in o...

The zebrafish is currently one of the most important vertebrate models to study cellular and developmental processes in vivo. This is largely due to the zebrafish's optical transparency and amenability to genetics, which makes it a powerful model for applying optical techniques involving genetically encoded photoresponsive protein technologies1. Specific to the study of heart development, zebrafish receive sufficient oxygen via diffusion such that even mutants without heartbeat can survive through the first week of development, permitting analyses on the effects of developmental genes and perturbed blood flow on heart morphogenesis not possible in most vertebrates2.
The zebrafish heart tube is formed by 24 hours post fertilization (hpf). Shortly after its formation, the heart tube starts actively beating. By 36 hpf, a clear constriction separates the atrium from the ventricle. This region of constriction is called the atrioventricular canal (AVC), and cells in this region change from a squamous morphology to a cuboidal morphology3. Zebrafish atrioventricular valve morphogenesis starts around 48 h post fertilization. By 5 days post fertilization, two valve leaflets extend into the AVC and prevent the back flow of blood from the ventricle to the atrium during the cardiac cycle4. Tracking cells during AVC and valve formation is challenging as the rapid beating of the heart makes it difficult to follow cells via traditional time-lapse microscopy5,6. This protocol, adapted from Steed et al., 20167, describes a method that uses the Tg(fli1a:gal4FFubs, UAS:kaede)8 zebrafish transgenic line, in which the photoconvertible protein Kaede is expressed in endothelial cells, including the endocardium. The drug 2,3-butanedione-2-monoxime (BDM) is used to temporarily stop the heart beating, allowing accurate photoconversion of EdCs between 36 and 55 hpf, and high-resolution imaging of photoconverted EdCs at specific developmental time points. It has previously been shown that EdCs photoconverted using this method can remain distinguishable from their unconverted neighbors for five days or more after the time of photoconversion7. This protocol also details a method used for image analysis of photoconverted EdCs in embryos younger than 48 hpf, which has successfully been used to follow tissue movements during AVC development (Boselli et al., in press)9. We hope that readers would find this protocol useful for studying AVC and valve development in normal embryos, and in mutants, morphants, or drug-treated embryos. For a more general protocol relating to cell tracking using photoconvertible proteins during zebrafish development, please view the article by Lombardo et al., 201210.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Necessary equipment for raising fish and collecting eggs (see the Zebrafish Book22 for details).</td><td></td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td></td><td></td><td></td></tr><tr><td>Upright confocal microscope (Equipped with lasers operating at 488 nm, 561 nm, and 405 nm, a tunable multiphoton laser, and a Leica HCX IRAPO L, 25 &amp;times;, N.A. 0.95 objective)</td><td>Leica</td><td>Leica SP8</td><td></td></tr><tr><td>Heat block</td><td>ThermoScientific</td><td>88870001</td><td></td></tr><tr><td>35 mm x 10 mm tissue culture dish</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>6 well plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Petri dish</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Glass pipette</td><td></td><td></td><td></td></tr><tr><td>Mold</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>8 mg/mL Tricaine stock solution&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>1 M BDM stock solution</td><td></td><td></td><td></td></tr><tr><td>UltraPure low melting point agarose</td><td>Invitrogen</td><td>16520-050</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>PTU (1-phenyl-2-thiourea)</td><td>Sigma Aldrich</td><td>P7629</td><td></td></tr><tr><td>Embryo medium: 30x stock solution</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Matlab equipped with the Image Analysis, Curve Fitting, Bio-Formats Toolboxes</td><td>MathWorks</td><td></td><td></td></tr><tr><td>&lt;strong&gt;8 mg/mL Tricaine stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>200 mg of tricaine powder (Ethyl 3-aminobenzoate methanesulfonic acid)</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>25 mL Danieau</td><td></td><td></td><td></td></tr><tr><td>Adjust to pH 7, aliquot and store at -20 &amp;deg;C&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;1 M BDM stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 g BDM</td><td>Sigma-Aldrich</td><td>112135</td><td></td></tr><tr><td>10 mL ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>Aliquot and store at -20 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Embryo medium: 30x stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50.7 g NaCl</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>0.78 g KCl</td><td>Sigma-Aldrich</td><td>7447-40-7</td><td></td></tr><tr><td>1.47 g Magnesium sulfate</td><td>Sigma-Aldrich</td><td>&amp;nbsp;7487-88-9</td><td></td></tr><tr><td>2.1 g Calcium nitrite tetrahydrate</td><td>Sigma-Aldrich</td><td>13477-34-4</td><td></td></tr><tr><td>19.52 g HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td></td></tr><tr><td>Adjust to pH 7.2 and store at room temperature</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Mold&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PlasCLEAR resin</td><td>Asiga</td><td></td><td></td></tr><tr><td>Plus 39&amp;nbsp;Freeform Pico 3D printer</td><td>Asiga</td><td></td><td></td></tr></tbody></table>

following endocardial tissue movements via cell photoconversion in the zebrafish embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

An example of an embryo photoconverted at 48 hpf and imaged again at 80 hpf is shown in Figure 2, Movies 1 and 2. Exposing Kaede to 405 nm light irreversibly converts from the protein from its fluorescent green form to fluorescent red form, enabling the behavior of cells labeled with either the green or their red form to be followed with respect to their differentially colored neighbors during valve formation. It can be seen ...

Watch this Scientific Journal Video about Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo at JoVE.com

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

following-endocardial-tissue-movements-via-cell-photoconversion

Research

JoVE Journal

Developmental Biology

6.4K Views.  Institut de Génétique et de Biologie Moléculaire et Cellulaire. Dit protocol beschrijft een methode voor de photoconversion van Kaede fluorescent proteïne in endocardial cellen van de levende zebrafish embryo waarmee het volgen van endocardial cellen tijdens de atrioventriculaire kanaal en Atrioventriculaire hart ventiel ontwikkeling .

Video: Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Bioengineering

In Vivo Mechanotransduction Study Using Magnetic Beads in the Zebrafish Heart

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction and physical stimuli generated by the beating heart. Intracardiac hemodynamics, driven by blood flow, emerge as fundamental information shaping the development of the embryonic heart. Here, we describe an effective method to manipulate mechanical forces in vivo by grafting a 30 &#181;m to 60 &#181;m diameter magnetic bead in the cardiac lumen. The insertion of the bead is conducted through microsurgery in anesthetized larvae without perturbing heart function and enables artificial alteration of the boundary conditions, thereby modifying flow forces in the system. As a result, the presence of the bead amplifies the mechanical forces experienced by endocardial cells and can directly trigger mechanical stimulus-dependent calcium influx. This approach facilitates the investigation of mechanotransduction pathways that govern heart development and can provide insights into the role of mechanical forces in cardiac valve morphogenesis.

Author Spotlight: Understanding Mechanical Forces Involved in Shaping the Zebrafish Heart

This protocol outlines a method for grafting a magnetic bead into the developing zebrafish heart through microsurgery, enabling the manipulation of mechanical forces in vivo and triggering mechanical stimulus-dependent calcium influx in endocardial cells.

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction ...

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Biology

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Analysis of Zebrafish Cardiac Contraction with Virtual Reality Interaction

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

Author Spotlight: High-Resolution 4D Light-Sheet Imaging and Virtual Reality in Zebrafish for Single-Cell Analysis of Heart Function

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

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