We thank the members of the Vermot lab for discussions and comments on the protocol. We are grateful to all the staff members of the Imperial College London fish facility. This project has received funding from the European Research Council (ERC) under the European Union&#39;s Horizon 2020 research and innovation program: GA N&#176;682939, Additional Ventures (award number 1019496), the MRC (MR/X019837/1) and the BBSRC (BB/Y00566X/1). CV-P was supported by a Bioengineering Departmental Scholarship (Imperial College London). HF was supported by the JSPS KAKENHI (23H04726 and 24K02207), the JST FOREST program (23719210), the Uehara Memorial Foundation, the Cell Science Research Foundation, the Takeda Medical Research Foundation, and the Novartis Research Foundation.

The procedures for working with zebrafish embryos described in this protocol adhere to the European directive 2010/63/EU and Home Office guidelines under the project licence PP6020928.
1. Obtaining zebrafish embryos for bead grafting
<ol>
	<li>Cross the relevant zebrafish line and grow the embryos in Danieau&#39;s medium at 28.5 &#176;C. For more detailed instructions on crossing zebrafish lines, refer to the JoVE Science Education Database27.</li>
	<li>Treat the embryos with 1-phenyl-2-thiourea (PTU) from 24 h post fertilization (hpf) to inhibit pigment formation, using a concentration of 0.003% PTU in Danieau&#39;s medium.</li>
	<li>If using a fluorescently labeled line, examine the embryos under a fluorescence stereoscope before beginning bead grafting, and select 10-20 healthy embryos that exhibit bright fluorescence. Gently dechorionate the preselected embryos under the stereomicroscope using forceps, ensuring that the embryos are not damaged.</li>
</ol>
2. Mounting of zebrafish embryos
<ol>
	<li>Prepare 20 mL of Danieau&#39;s medium containing 0.02% tricaine.</li>
	<li>Prepare 2 mL aliquots of 1% low melting point (LMP) agarose containing 0.02% tricaine in 2 mL microcentrifuge tubes. Place the tubes in a 38 &#176;C heat block to maintain the LMP agarose in a liquid state. 
	NOTE: The 1% LMP agarose can be prepared in advance for use on the day of mounting and bead grafting.</li>
	<li>Transfer 3-6 dechorionated embryos to a Petri dish containing Danieau&#39;s medium supplemented with 0.02% tricaine.</li>
	<li>When the embryos are anesthetized, transfer them to a 35 mm x 15&#160;mm glass-bottom dish with minimal medium. Quickly add 300-400 &#181;L of 1% LMP agarose containing tricaine to create a dome. Using forceps, position the embryos so they lie straight, in contact with the glass, with the ventral side facing up, as shown in Figure 1. Wait ~4 min for the agarose to solidify. 
	NOTE: The quantity of agarose used for mounting embryos is very important. Aim for a dome of approximately 350 &#181;L, which should provide enough coverage while leaving a few millimeters of space above the embryos. For beginners, mounting a smaller number of embryos at a time may be a safer approach.</li>
</ol>
3. Bead grafting
<ol>
	<li>Once the agarose is solidified, deposit approximately 0.5 &#181;L of magnetic beads on top. 
	NOTE: Use a 10 &#181;L pipette tip to transfer the magnetic beads. Precise measurement of 0.5 &#181;L is not necessary, as the beads are highly concentrated in the suspension. Simply inserting the tip into the vial after spinning down will be sufficient to collect an adequate quantity of beads.</li>
	<li>Add a drop of Danieau&#39;s medium containing 0.02% tricaine on top of the agarose and the beads.</li>
	<li>Select the appropriate bead. 
	NOTE: This step is critical. The size of the beads can vary slightly, ranging from 30 &#181;m to 60 &#181;m in diameter, so selecting the appropriate bead by eye, based on the sample and heart size, is essential.</li>
	<li>Once the suitable bead is selected, use tweezers to place it on top of the yolk (Figure 2A and Video 1).</li>
	<li>Create a yolk lesion or &#39;hole&#39; in the center of the yolk using one arm of the tweezers (Figure 2B and Video 2). 
	NOTE: The depth of the lesion should not penetrate too deeply into the yolk; it should be approximately level with the cardinal vein. The diameter of the hole created will correspond to the tip of the tweezer arm. Ensure the lesion is made in the yolk and avoid contacting the cardinal veins by aiming below them. A small amount of yolk will typically come out when the hole is created.</li>
	<li>Place the bead into the lesion created in step 3.5 and gently push it anteriorly until it reaches the venous pole. There, the heart contractions will create suction, drawing the bead into the cardiac lumen (Figure 2 and Video 2). 
	NOTE: The venous wall is breached during bead insertion, and the high density of the yolk helps to minimize extensive bleeding. The yolk lesion typically heals within a few minutes after bead insertion. Some embryos may exhibit pericardial edema 20-24 h after bead grafting.</li>
</ol>
4. Unmounting of zebrafish embryos
<ol>
	<li>After grafting magnetic beads into all mounted embryos, add 1-2 mL of Danieau&#39;s medium containing PTU in the glass-bottom dish.</li>
	<li>Using tweezers, gently break the LMP agarose and carefully remove the fish, ensuring that no agarose is left on them.</li>
	<li>Using a Pasteur pipette, transfer the embryos to a new Petri dish containing Danieau&#39;s medium with PTU and allow them to recover and develop at 28.5 &#176;C.</li>
</ol>

In Vivo Mechanotransduction Study Using Magnetic Beads in the Zebrafish Heart

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+genetics+and+vertebrate+heart+formation.">Zebrafish genetics and vertebrate heart formation.</a> Nat Rev Genet. 2 (1), 39-48 (2001).</li><li>Brown, D. R., Samsa, L. A., Qian, L., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+study+of+heart+development+and+disease+using+zebrafish.">Advances in the study of heart development and disease using zebrafish.</a> J Cardiovasc Dev Dis. 3 (2), 13 (2016).</li><li>Hoo, J. Y., Kumari, Y., Shaikh, M. F., Hue, S. M., Goh, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+A+versatile+animal+model+for+fertility+research.">Zebrafish: A versatile animal model for fertility research.</a> Biomed Res Int. 2016 (2016), 9732780 (2016).</li><li>Bakkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+to+study+cardiac+development+and+human+cardiac+disease.">Zebrafish as a model to study cardiac development and human cardiac disease.</a> Cardiovasc Res. 91 (2), 279-288 (2011).</li><li>Briggs, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish:+a+new+model+organism+for+integrative+physiology.">The zebrafish: a new model organism for integrative physiology.</a> Am J Physiol Regul Integr Comp Physiol. 282 (1), R3-R9 (2002).</li><li>Veldman, M. B., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+developmental+model+organism+for+pediatric+research.">Zebrafish as a developmental model organism for pediatric research.</a> Pediatr Res. 64 (5), 470-476 (2008).</li><li>Duchemin, A. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+forces+regulate+zebrafish+heart+valve+delamination+by+modulating+Nfat+signaling.">Cardiac forces regulate zebrafish heart valve delamination by modulating Nfat signaling.</a> PLoS Biol. 20 (1), e3001505 (2022).</li><li>Fukui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioelectric+signaling+and+the+control+of+cardiac+cell+identity+in+response+to+mechanical+forces.">Bioelectric signaling and the control of cardiac cell identity in response to mechanical forces.</a> Science. 374 (6565), 351-354 (2021).</li><li>Vignes, H., Vagena-Pantoula, C., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+control+of+tissue+shape:+Cell-extrinsic+and+-intrinsic+mechanisms+join+forces+to+regulate+morphogenesis.">Mechanical control of tissue shape: Cell-extrinsic and -intrinsic mechanisms join forces to regulate morphogenesis.</a> Semin Cell Dev Biol. 130, 45-55 (2022).</li><li>Juan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+pkd+and+piezo+gene+family+members+are+required+for+atrioventricular+valve+formation.">Multiple pkd and piezo gene family members are required for atrioventricular valve formation.</a> Nat Commun. 14 (1), 214 (2023).</li><li>Juan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cardiac+contractions+using+Cre-lox+and+degron+strategies+in+zebrafish.">Control of cardiac contractions using Cre-lox and degron strategies in zebrafish.</a> Proc Natl Acad Sci U S A. 121 (3), e2309842121 (2024).</li><li>Duchemin, A. L., Vignes, H., Vermot, J., Chow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+in+cardiovascular+morphogenesis+and+tissue+engineering.">Mechanotransduction in cardiovascular morphogenesis and tissue engineering.</a> Curr Opin Genet Dev. 57, 106-116 (2019).</li><li>Kalogirou, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+flow+dynamics+regulate+atrioventricular+valve+morphogenesis.">Intracardiac flow dynamics regulate atrioventricular valve morphogenesis.</a> Cardiovasc Res. 104 (1), 49-60 (2014).</li><li>da Silva, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=egr3+is+a+mechanosensitive+transcription+factor+gene+required+for+cardiac+valve+morphogenesis.">egr3 is a mechanosensitive transcription factor gene required for cardiac valve morphogenesis.</a> Sci Adv. 10 (20), eadl0633 (2024).</li><li>Bartman, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+myocardial+function+affects+endocardial+cushion+development+in+zebrafish.">Early myocardial function affects endocardial cushion development in zebrafish.</a> PLoS Biol. 2 (5), E129 (2004).</li><li>Steed, E., Boselli, F., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+driven+cardiac+valve+morphogenesis.">Hemodynamics driven cardiac valve morphogenesis.</a> Biochim Biophys Acta. 1863 (7 Pt B), 1760-1766 (2016).</li><li>Vermot, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversing+blood+flows+act+through+klf2a+to+ensure+normal+valvulogenesis+in+the+developing+heart.">Reversing blood flows act through klf2a to ensure normal valvulogenesis in the developing heart.</a> PLoS Biol. 7 (11), e1000246 (2009).</li><li>B&#228;ck, M., Gasser, T. C., Michel, J. B., Caligiuri, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+factors+in+the+biology+of+aortic+wall+and+aortic+valve+diseases.">Biomechanical factors in the biology of aortic wall and aortic valve diseases.</a> Cardiovasc Res. 99 (2), 232-241 (2013).</li><li>Hahn, C., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanotransduction+in+vascular+physiology+and+atherogenesis.">Mechanotransduction in vascular physiology and atherogenesis.</a> Nat Rev Mol Cell Biol. 10 (1), 53-62 (2009).</li><li>Boselli, F., Steed, E., Freund, J. B., Vermot, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+shear+stress+patterns+predict+the+orientation+of+convergent+tissue+movements+in+the+embryonic+heart.">Anisotropic shear stress patterns predict the orientation of convergent tissue movements in the embryonic heart.</a> Development. 144 (23), 4322-4327 (2017).</li><li>Hove, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracardiac+fluid+forces+are+an+essential+epigenetic+factor+for+embryonic+cardiogenesis.">Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis.</a> Nature. 421 (6919), 172-177 (2003).</li><li>JoVE Science Education database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+II:+Mouse,+zebrafish,+and+chick.+Zebrafish+breeding+and+embryo+handling.">Biology II: Mouse, zebrafish, and chick. Zebrafish breeding and embryo handling.</a> JoVE. , (2023).</li><li>Arrenberg, A. B., Stainier, D. Y., Baier, H., Huisken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+cardiac+function.">Optogenetic control of cardiac function.</a> Science. 330 (6006), 971-974 (2010).</li></ol>

The authors have no conflicts of interest to declare.

Critical steps in the protocol and troubleshooting
Mounting of zebrafish embryos
The quantity of agarose used to mount the embryos is important. The dome formed should not be excessively large, as this can hinder the manipulation of the bead from the surface to the embryos. Conversely, it should not be too small; having multiple beads atop the agarose, positioned close to the embryos and their yolk, can cause confusion. A v...

Since its introduction in the late 1970s1, the zebrafish (Danio rerio) has emerged as a powerful model system for studying the intricacies of cardiac development and congenital heart disorders. Unlike most vertebrates, including mouse and chick embryos, which rely on a functional cardiovascular system and cannot survive early heart defects, zebrafish provide a unique advantage by enabling the investigation of severe heart phenotypes. This is due to their small size, which facilitates sufficient oxygen supply through passive diffusion, allowing survival even in the absence of heart contraction and active blood circulation2,3,4. Furthermore, among the many significant features of zebrafish is the optical transparency of their embryos, which enables non-invasive monitoring of the developing heart5,6,7,8.
Mechanical forces continuously provide feedback to the heart valve morphogenetic programs9,10,11,12,13,14,15,16,17,18,19,20,21,22, and aberrant blood flow is widely acknowledged as a shared factor in various cardiovascular disorders23,24. In zebrafish, cardiac valve development relies on heart contraction and mechanical forces generated by the beating heart. Multiple zebrafish mutants have demonstrated the significance of heart-generated mechanical stimuli in valvulogenesis. Remarkably, the complete absence of heart contraction and, consequently, blood flow due to mutations of cardiac troponin T (tnnt2) in silent heart (sih) mutants results in the absence of tissue convergence and endocardial cell (EdC) clustering during early morphogenetic stages25.
Intracardiac hemodynamics and mechanical forces generated by the blood flow emerge as fundamental epigenetic components shaping the development of the zebrafish embryonic heart. Numerous studies suggest that proper cardiac morphogenesis in zebrafish requires distinct flow stimuli, and deviations from these physiological patterns lead to heart valve defects10,13,14,22,26. Here, we describe an effective method, adapted from Fukui et al.13, to manipulate mechanical forces in vivo by grafting a 30 &#181;m to 60 &#181;m diameter magnetic bead within the developing zebrafish beating heart. The technique involves microsurgical insertion of a bead into the cardiac lumen of anesthetized larvae without perturbing heart function. The presence of the bead leads to the amplification of the mechanical forces experienced by EdCs, directly triggering mechanical stimulus-dependent calcium influx13. This approach enables the investigation of mechanotransduction pathways that regulate heart morphogenesis and offers a means to deepen our understanding of the role of mechanical forces in valve formation.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Essential equipment for zebrafish raising, breeding, and embryo collection</td><td></td><td></td><td></td></tr><tr><td>Glass-bottom dish (35 mm x 15&amp;nbsp;mm)</td><td>VWR International</td><td>734-2905</td><td></td></tr><tr><td>Heat block</td><td>Eppendorf</td><td>EP5382000031</td><td>Eppendorf ThermoMixer C</td></tr><tr><td>Jewelers forceps</td><td>Sigma-Aldrich</td><td>F6521-1EA</td><td>Dumont No. 5, L 4 1/4 in., Inox alloy</td></tr><tr><td>Microcentrifuge tubes 2 mL</td><td>Eppendorf</td><td>30120094</td><td></td></tr><tr><td>Pasteur pipette</td><td></td><td></td><td></td></tr><tr><td>Petri dish</td><td></td><td></td><td></td></tr><tr><td>Stereomicroscope</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>4 mg/mL tricaine stock solution</td><td></td><td></td><td></td></tr><tr><td>Danieau&#039;s medium (60x stock solution)</td><td></td><td></td><td></td></tr><tr><td>PureCube Glutathione MagBeads</td><td>Cube Biotech</td><td>32201</td><td></td></tr><tr><td>PTU (1-phenyl-2-thiourea)</td><td>Sigma-Aldrich</td><td>P7629</td><td></td></tr><tr><td>UltraPure low melting point agarose</td><td>Invitrogen</td><td>16520-050</td><td></td></tr><tr><td>&lt;strong&gt;Danieau&#039;s medium (60x stock solution)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>34.8 g NaCl</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>1.6 g KCl</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>5.8 g CaCl2&amp;middot;2H2O</td><td>Sigma-Aldrich</td><td>C3306</td><td></td></tr><tr><td>9.78 g MgCl2&amp;middot;6H2O&amp;nbsp;</td><td>Sigma-Aldrich</td><td>442611-M</td><td></td></tr><tr><td>Dissolve the ingredients in H2O to a final volume of 2 L. Adjust the pH to 7.2 using NaOH, then autoclave.</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;4 mg/mL tricaine stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>400 mg of tricaine powder (Ethyl 3-aminobenzoate methanesulfonate salt)</td><td>Sigma-Aldrich</td><td>A5040</td><td></td></tr><tr><td>97.9 mL double-distilled H2O</td><td></td><td></td><td></td></tr><tr><td>2.1 mL 1 M Tris (pH 9)</td><td></td><td></td><td></td></tr><tr><td>Adjust the pH to 7, then aliquot and store at -20 &amp;deg;C.</td><td></td><td></td><td></td></tr></tbody></table>

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.

Examples of successful bead grafting are shown in Figure 3, Video 3, and Video 4. The magnetic bead was correctly positioned within the atrium of the zebrafish heart, allowing for unobstructed blood flow and no observed hemorrhage. Additionally, the heart walls maintained their structural integrity without collapsing (Figure 3 and Video 3). After 24 h, the embryo showed no signs of pericardial edema, further con...

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.

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 ...

manipulating-mechanical-forces-developing-zebrafish-heart-using

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

2.7K Views.  Imperial College London. 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.

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

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 ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

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.

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 ...

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

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

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 ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

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Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Microbead Implantation in the Zebrafish Embryo

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

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

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

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

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

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos