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><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 ...

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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 ...

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Research

JoVE Journal

Developmental Biology

5.0K Views.  David Geffen School of Medicine at University of California, Los Angeles. Diamond is de eerste open source methode voor de kwantitatieve beoordeling van de vierdimensionale segmentale cardiale functie in zebravis embryo's. Onze methode heeft een micrometerresolutie die lokale hartmechanica in 3D-ruimte kan kwantificeren. Geen enkele andere huidige aanpak staat dat toe.Diamond ontrafelt nieuwe verwondingspatronen in doxorubicine-geïnduceerde cardiale toxiciteit. Daarom geloven we dat onze methode kan worden gebruikt als een platform voor een hoge doorvoer in...

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

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 ...

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 ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its ...

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

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Biology

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 ...

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 ...

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

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