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

<ol>
	<li>Streisinger, G., Walker, C., Dower, N., Knauber, D., Singer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-296 (1981).</li><li>Tu, S., Chi, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+in+cardiac+development+and+congenital+heart+birth+defects.">Zebrafish models in cardiac development and congenital heart birth defects.</a> Differentiation. 84 (1), 4-16 (2012).</li><li>Stainier, D. 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. L., Vignes, H., 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=Mechanically+activated+piezo+channels+modulate+outflow+tract+valve+development+through+the+Yap1+and+Klf2-Notch+signaling+axis.">Mechanically activated piezo channels modulate outflow tract valve development through the Yap1 and Klf2-Notch signaling axis.</a> Elife. 8, e44706 (2019).</li><li>Vignes, 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=Extracellular+mechanical+forces+drive+endocardial+cell+volume+decrease+during+zebrafish+cardiac+valve+morphogenesis.">Extracellular mechanical forces drive endocardial cell volume decrease during zebrafish cardiac valve morphogenesis.</a> Dev Cell. 57 (5), 598-609.e595 (2022).</li><li>Steed, E., 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=klf2a+couples+mechanotransduction+and+zebrafish+valve+morphogenesis+through+fibronectin+synthesis.">klf2a couples mechanotransduction and zebrafish valve morphogenesis through fibronectin synthesis.</a> Nat Commun. 7, 11646 (2016).</li><li>Chow, R. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</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&#160;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>Reagents</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&#39;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>Danieau&#39;s medium (60x stock solution)</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&#183;2H2O</td>
			<td>Sigma-Aldrich</td>
			<td>C3306</td>
			<td></td>
		</tr>
		<tr>
			<td>9.78 g MgCl2&#183;6H2O&#160;</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>4 mg/mL tricaine stock solution</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 &#176;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.

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

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

We focus on understanding how mechanical forces shape the zebrafish heart using advanced imaging techniques. By combining our knowledge of biology, physics, microscopy, and computing, we develop tools for optical imaging and image analysis to study how mechanical stimuli influence the way the cardiovascular system develops. A beating heart generates several type of forces, such as pressure for the shear and contractor force.Although each of these forces has a function in vitro culture systems, it is difficult to separate these parameters in the in vivo heart. By developing our approach, we aim to tackle this challenging task. We have established a novel approach to assess, adapt biological output caused by external force stimulation, and we verified our force spin and constant signal in crosscutting the cardiac cells.As a result this approach enables us to impetrate other aspects of tissue neurogenesis in the world of mechanical biology. This method enables the study of heart function in zebrafish embryo via targeted mechanical stimulation. Unlike genetic, pharmacological, or optogenetic approaches, pit grafting offers a more direct way to influence heart physiology through mechanical means.Pit grafting is viable for examining the roles of mechanical forces and calcium influx in cardiac biological processes. It provides means to deepen our understanding of the mechanical transduction pathways involved in heart development and function.

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...

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 ...

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Research

JoVE Journal

Developmental Biology

Magnetic Bead Grafting in the Zebrafish Cardiac Lumen for Controlled Force Amplification: Unraveling Mechanotransduction in Heart Valve Development

To begin, place three to six dechorionated embryos in a Petri dish containing Danios medium supplemented with 0.02%tricaine. Once the embryos are anesthetized, transfer them to a 35 by 15 millimeter glass bottom dish with minimal medium. Quickly add 300 to 400 microliters of 1%low melting point 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. Next, deposit approximately 0.5 microliters of magnetic beads on top of the agarose. Then add a drop of Danios medium containing 0.02%tricaine on top of the agarose and the beads.Based on the sample and heart size, select the appropriate bead and use tweezers to place it on top of the embryo yolk. Create a yolk lesion or hole in the center of the yolk using one arm of the tweezers. Place the bead into the lesion and gently push it anteriorly until it reaches the venous pole.If suction alone is insufficient to move the bead, gently push it toward the atrium, applying enough pressure to guide it into the pericardial cavity. After grafting the magnetic beads into the embryos, add one to two milliliters of Danios medium containing PTU to the glass bottom dish. Then using tweezers, gently break the low melting point agarose and carefully remove the embryos.Finally, using a Pasteur pipette, transfer the embryos to a new Petri dish containing Danios medium with PTU and allow them to recover and develop at 28.5 degrees Celsius. The magnetic bead was correctly positioned within the atrium of the zebrafish heart, allowing for unobstructed blood flow and no observed hemorrhage with the heart walls maintained.

999 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.

In Vivo Mechanotransduction Study Using Magnetic Beads in the Zebrafish Heart

Summary