The present work was funded by American Heart Association grants 16SDG30910007 and 18CDA34110338, and by National Institutes of Health grants HL083015, HL111437, HL118650, and HL129727.

All methods described here have been approved by the UCLA Institutional Animal Care and Use Committee (IACUC), and experiments were performed in compliance with protocols approved by the UCLA Office of Animal Research.
1. Breeding Tg(cmlc2:mCherry) zebrafish and collection of embryos
<ol>
	<li>Follow the housing, breeding, and embryo collection procedures as described in previously established husbandry and breeding practices. For details, see Messerschmidt et al.9.</li>
	<li>Treat the collected embryos with 0.003% 1-phenyl-2-thiourea (PTU) in E3 medium 18 h postfertilization to maintain the transparency of the embryos for LSFM imaging.</li>
</ol>
2. Doxorubicin treatment to induce cardiac injury
<ol>
	<li>At 3 days postfertilization (dpf), treat the embryos with doxorubicin at a concentration of 10 &#956;M in E3 fish water medium. After a 24 h treatment to 4 dpf, replace the doxorubicin medium with fresh E3 medium. 
	CAUTION: Doxorubicin is a chemotherapy medication. Appropriate personal protective equipment (PPE) is required and the waste should be disposed of in biohazard waste containers.</li>
</ol>
3. Notch pathway modulation
<ol>
	<li>Treat zebrafish embryos with the Notch pathway inhibitor (2S)-N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine 1,1-dimethylethyl ester (DAPT) at a concentration of 10 &#956;M in E3 fish water medium from 3&#8211;6 dpf.</li>
	<li>Microinject the Notch downstream effectors Notch intracellular domain (NICD) and Neuregulin-1 (Nrg-1) mRNA at concentrations of 10 pg/nL and 5 pg/nL, respectively, into the 1-cell stage zebrafish embryos8,10. 
	NOTE: The microinjection is performed under a microscope with the support of an air pump to accurately control the volume injected. The mRNA microinjection into the cell is done when the fertilized egg is at the first cell stage. For details on the preparation and sequence of the mRNAs, see Chen et al.8. For details on the microinjection and preparation of injection needles, see Rosen et al.10.</li>
</ol>
4. LSFM imaging and post-imaging synchronization
<ol>
	<li>For the LSFM imaging techniques and post-imaging synchronization algorithm, see details in previous publications9,11. 
	NOTE: Briefly, our system utilizes a continuous-wave laser as the illumination source to image all transgenic zebrafish lines. The detection module is composed of two scientific complementary metal-oxide semiconductor (sCMOS) cameras and two sets of filters for dual-channel imaging. The detection module is perpendicularly installed to the illumination plane. Each LSFM frame is acquired within a 20 msec exposure time, while the resolving power in cross section is ~0.65 &#956;m and the step size between consecutive frames is ~2 &#956;m. A 589 nm laser was used to excite mCherry fluorescent signals.</li>
</ol>
5. Reconstruction of the 3D systolic and diastolic heart
<ol>
	<li>Open the folder created by the post synchronization algorithm, then open the &#34;Output&#34; folder. Select the middle plane of the heart and load the entire folder into ImageJ. Find the first diastolic and systolic phase and record the frame number.</li>
	<li>Open the &#34;Output/By State&#34; folder and find the folders that have the same numbers as the frame numbers just recorded. Convert the images in the folder into 3D TIFF (tagged image file format) files and name them &#34;diastole.tif&#34; and &#34;systole.tif&#34;.</li>
</ol>
6. Segmentation of the ventricle
<ol>
	<li>Open the image analyzing software (see Table of Materials). Click File | Open data, and load &#34;diastole.tif&#34; and &#34;systole.tif&#34;. Enter the voxel size according to the imaging settings. 
	NOTE: For the LSFM system used, the typical voxel size is 0.65 &#181;m x 0.65 &#181;m x 2 &#181;m.</li>
	<li>Click the &#34;SEGMENTATION&#34; panel and manually segment out the ventricle part of the heart. The built-in &#34;Threshold&#34; tool that can select all the regions above a certain intensity can facilitate this process. The ventricle is the thicker chamber with a stronger fluorescence. 
	NOTE: Make sure to remove the atrioventricular canal and the outflow tract in the segmented ventricle, because this affects the displacement analysis.</li>
	<li>After the segmentation is done, click the &#34;Project&#34; panel. Right click the &#34;diastole.Labels.tif&#34; and &#34;systole.Labels.tif&#34; tabs in the console and click &#34;Export Data as&#34; to save the data as 3D TIFF files.</li>
</ol>
7. Creation of rectangular parallelepipeds for image registration
<ol>
	<li>Run &#34;prepImage_1.m&#34; in the programming environment (see Table of Materials). Open &#34;prepImage_1.m&#34;, &#34;ImPath&#34; in line 5 so the folder contains the original and segmented TIFF files, and change &#34;slice&#34; in line 4 to the number of slices of the 3D tif files.</li>
	<li>After running the code, it will generate five new 3D TIFF files (&#34;test.tif&#34;, &#34;diastole_200.tif&#34;, &#34;systole_200.tif&#34;, &#34;diaLabel.tif&#34;, and &#34;sysLabel200.tif&#34;) as well as two new folders (&#34;resample_dia&#34; and &#34;resample_sys&#34;).</li>
</ol>
8. Resample systolic and diastolic 3D hearts along the short axis plane
<ol>
	<li>Import all five 3D TIFF files into the image analyzing software (see Table of Materials). 
	NOTE: The voxel size is unchanged.</li>
	<li>Go to the MULTIPLANAR panel. Choose &#34;diastole_200.tif&#34; as the primary data. Align the X-axis (the green line in the XY plane) with the vertical long axis of the ventricle, and align the Z-axis (the red line in the YZ plane) with the horizontal long axis of the ventricle. 
	NOTE: The vertical long axis is determined by finding the longest axis connecting the apex and the outflow tract in the XY plane, and the horizontal long axis is determined by finding the longest axis connecting the apex and the outflow tract in the YZ plane. Rotate the axis by placing the cursor at the end of the axis.</li>
	<li>Choose three random points from the oblique YZ plane (the short axis plane) in a counterclockwise manner and record their 3D position coordinates. 
	NOTE: Make sure the points are chosen in a counterclockwise manner.</li>
	<li>Repeat steps 8.2 and 8.3 for &#34;systole_200.tif&#34;.</li>
	<li>Click the &#34;PROJECT&#34; panel. Create a &#34;Slice&#34; object for &#34;diastole_200.tif&#34; by right clicking on &#34;diastole_200.tif&#34; and searching for &#34;Slice&#34; object. Left click the Slice object just created, and in the Properties panel | Options, check &#34;Set Plane&#34; and choose three points in &#34;Plane Definition&#34;. Enter the coordinates of the three points from step 7.3.</li>
	<li>Repeat step 8.5 for &#34;systole_200.tif&#34;. 
	NOTE: The slice object created should have the name &#34;Slice 2&#34;.</li>
	<li>Right click &#34;diastole_200.tif&#34; and search for &#34;Resample Transformed Image&#34; and create the object. In the Properties panel, choose &#34;Slice&#34; as the &#34;Reference&#34; and click Apply. This should generate an object named &#34;diastole_200.transformed&#34;.</li>
	<li>Right click &#34;diastole_200.transformed&#34; and search for &#34;Resample&#34; and create the object. Choose &#34;Voxel Size&#34; as the &#34;Mode&#34; and change &#34;Voxel Size&#34; to be x = 1, y = 1, and z = 1 in the Properties panel.</li>
	<li>Click &#34;Apply&#34;. This should generate an object named &#34;diastole_200.resampled&#34;. Right click &#34;diastole_200.resampled&#34; and save it as a 3D TIFF file.</li>
	<li>Repeat the same step for &#34;diaLabel.tif&#34; and &#34;test.tif&#34;. Save &#34;diaLabel.resampled&#34; and &#34;test.resampled&#34; as 3D TIFF files. Repeat the same step for &#34;systole_200.tif&#34;, &#34;sysLabel.tif&#34;, and &#34;test.tif&#34; using &#34;Slice 2&#34; as a reference, and save &#34;systole_200.resampled&#34;, &#34;sysLable.resampled&#34;, and &#34;test2.resampled&#34; as 3D TIFF files. 
	NOTE: Make sure there are a total of six TIFF files saved in this step.</li>
</ol>
9. Division of the resampled heart
<ol>
	<li>Import all six resampled files from step 8 to ImageJ. Select a slice of &#34;systole_200.resampled&#34; in which the atrioventricular canal is clearly visualized. Record the number of the slice.
	<ol>
		<li>Use the &#34;Image | Transform | Rotate&#34; function of ImageJ so that the atrioventricular canal is vertical. Apply the same rotation to all files. Close all windows and save all changes.</li>
		<li>Move &#34;diastole_200.resampled&#34;, &#34;diaLabel.resampled&#34;, and &#34;test.resampled&#34; to the &#34;resample_dia&#34; folder, and move &#34;systole_200.resampled&#34;, &#34;sysLable.resampled&#34;, and &#34;test2.resampled&#34; to the &#34;resample_sys&#34; folder.</li>
	</ol>
	</li>
	<li>Open &#34;divider_2_8_pieces.m&#34;. Change &#34;ImPath&#34; in line 5 and &#34;ImPath&#34; in line 395 to the image directory. Change the variable &#34;Middle&#34; in line 22 and line 411 to the slice numbers where the atrioventricular canal is clearly visualized in &#34;systole_200.resampled&#34; and &#34;diastole_200.resampled&#34;.</li>
	<li>Run the code, and in the prompted windows click once at the center of the ventricle and click once at the center of the atrioventricular canal. This needs to be done twice for both systole and diastole images.</li>
</ol>
10. Registration of systolic and diastolic image matrices
<ol>
	<li>Open &#34;register_3.m&#34; and change &#34;ImPath&#34; in line 4 to the image folder path. It might take 5&#8211;20 min to run this code depending on the computation power of the system. 
	NOTE: The artificially created rectangular parallelepipeds in step 7 are used for 3D rigid registration that preserves the distance between two points and angles subscribed by three points. When the end-diastole rectangular parallelepiped (red) is registered to the end-systole rectangular parallelepiped (green), the ensuing discrepant 3D location permits the derivation of a unique matrix of rigid transformation consisting of rotation and translation from the end-diastole matrix to the end-systole matrix (Figure 1H). We perform the registration and regularized energy minimization to denoise the matrix after the transformation using an image processing toolbox (see Table of Materials). For a detailed mathematical description, please see Chen et al.8.</li>
</ol>
11. Output of the displacement vectors
<ol>
	<li>Open &#34;displacement_4.m&#34; and change &#34;ImPath&#34; in line 4 to the image folder path.</li>
	<li>Run &#34;displacement_4.m&#34;, which generates a &#34;vector8.txt&#34; file in the &#34;vectors&#34; folder. Once the &#34;vector8.txt&#34; file is open, there will be an 8 x 4 matrix. Each row of the matrix has four numbers, which are the magnitudes of the X component, Y component, Z component, and the SUM magnitude of the displacement vector of a specific segment of the ventricle. 
	NOTE: The displacement vector is obtained by calculating the displacement of the mass centroid of each segment in 3D space. We calculate the 3D mass centroid (PS and PD) coordinates <img alt="Equation CK" src="/files/ftp_upload/60547/60547eqck.jpg" /> (where k indicates the X, Y, or Z coordinate, respectively) of each segment (I-VI) in the segmentation dataset from systole to diastole (Figure 1J). We define the mass centroid <img alt="Equation CK" src="/files/ftp_upload/60547/60547eqck.jpg" /> in 3D space as follows: 
	<img alt="Equation 1" src="/files/ftp_upload/60547/60547eq1.jpg" /> 
	where Cx = X, Cy = Y, and Cz = Z, Mi = the mass of each segment (I &#8804; i &#8804; VI), m = the number of voxels of each segment, and &#961; = the density function as the segmented region is 1 whereas the rest is 0. The L2-norm of the sub-displacement vectors along the X-, Y-, and Z-axes and the sum displacement vector are calculated during the cardiac cycle. There are a total of eight rows in the matrix. The first row and the eighth row contain the atrioventricular canal and are thus ignored in our analysis. Segments I to VI are represented by the second row to the seventh row.</li>
</ol>

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The authors have declared that no conflict of interest exists.

A rigorous strategy for quantification of segmental myocardial function is critical to assess cardiac mechanics beyond traditional EF, known to be an insensitive and delayed indicator of myocardial injury1,4,12. Hence, there has been a growing interest in markers of early myocardial changes, and a growing body of literature supports myocardial deformation parameters as an early indicator to forecast ventricular dysfunction<sup c...

Chemotherapy-induced cardiac toxicity and ensuing heart failure are one of the main reasons for chemotherapy discontinuation1. Therefore, cardiac functional assessment plays a crucial role in the identification of cardiac toxicity and, more importantly, in the prediction of early cardiac injury following chemotherapy2. However, current approaches for cardiac functional assessment encounter limitations. Methods such as left ventricular ejection fraction (LVEF) provide only global and often delayed cardiac mechanics after injury3,4. Tissue Doppler imaging provides segmental myocardial deformation information but suffers from significant intraobserver and interobserver variability, in part due to ultrasound beam angle dependency5. Two-dimensional (2D) speckle tracking utilizes the B-mode of echocardiography, which theoretically eliminates the angle dependency, but its accuracy is limited by out-of-plane motion6. Therefore, a rigorous approach for quantifying segmental cardiac function is lacking in both research and clinical settings.
In this context, we developed a 4D quantification method for the analysis of segmental cardiac function that we named displacement analysis of myocardial mechanical deformation (DIAMOND), to determine the displacement vectors of myocardial mass centroids in 3D space. We applied DIAMOND for the in vivo assessment of cardiac function and doxorubicin-induced cardiac toxicity with zebrafish (Danio rerio) as the animal model, chosen due to their regenerating myocardium and highly conserved developmental genes7. We further compared segmental DIAMOND displacement with global ejection fraction (EF) determination and 2D strain following doxorubicin treatment. By integrating DIAMOND displacement with 4D light-sheet fluorescent microscopy (LSFM) acquired rendering of embryonic zebrafish hearts, DIAMOND shows that the basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to acute doxorubicin cardiac injury8.

&#160; &#160;

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amira6</td>
			<td>FEI</td>
			<td></td>
			<td>Image analyzing software</td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Millipore Sigma</td>
			<td>D5942-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Millipore Sigma</td>
			<td>D1515-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Millipore Sigma</td>
			<td>E10521-10G</td>
			<td>Tricaine</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td></td>
			<td>Programming environment</td>
		</tr>
		<tr>
			<td>MATLAB Image Processing Toolbox</td>
			<td>MathWorks</td>
			<td></td>
			<td>Image processing toolbox</td>
		</tr>
	</tbody>
</table>

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.

The process by which DIAMOND was developed to assess 3D segmental cardiac function is presented in Figure 1. Following LSFM image acquisition and reconstruction in 3D of the embryonic zebrafish heart (Figure 1A), the true short axis plane was determined as the plane perpendicular to the vertical and horizontal long axes, both of which are determined in a multiplane viewer (Figure 1B). The heart was ...

Watch this Scientific Journal Video about Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish at JoVE.com

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.

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 ...

displacement-analysis-myocardial-mechanical-deformation-diamond

Research

JoVE Journal

Developmental Biology

4.9K Views.  David Geffen School of Medicine at University of California, Los Angeles. 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.

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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained even in embryos that are larger or smaller, within certain limits. Although this phenomenon of scaling has attracted attention for over a century, understanding the underlying mechanisms has been limited, owing in part to a lack of quantitative description of developmental dynamics in embryos of varied sizes. To overcome this limitation, we developed a new technique to surgically reduce the size of zebrafish embryos, which have great advantages for in vivo live imaging. We demonstrate that after balanced removal of cells and yolk at the blastula stage in separate steps, embryos can quickly recover under the right conditions and develop into smaller but otherwise normal embryos. Since this technique does not require special equipment, it is easily adaptable, and can be used to study a wide range of scaling problems, including robustness of morphogen mediated patterning.&#160;

Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change.

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained ...