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.

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

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Developmental Biology

2.6K 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 ...

Mechanical Vessel Injury in Zebrafish Embryos

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

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

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

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

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

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

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. This tissue complexity confounds the identification of causal link between extrinsic cues and cellular dynamics. A synthetically reconstituted multicellular system overcomes this problem by enabling researchers to test for a specific cue while eliminating others. Here, we present a method to reconstitute cell contact patterns with isolated Caenorhabditis elegans blastomere and adhesive polystyrene beads. The procedures involve eggshell removal, blastomere isolation by disrupting cell-cell adhesion, preparation of adhesive polystyrene beads, and reconstitution of cell-cell or cell-bead contact. Finally, we present the application of this method to investigate the orientation of cellular division axes that contributes to the regulation of spatial cellular patterning and cell fate specification in developing embryos. This robust, reproducible, and versatile in vitro method enables the study of direct relationships between spatial cell contact patterns and cellular responses.

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

Tissue complexities of multicellular systems confound the identification of causal relationship between extracellular cues and individual cellular behaviors. Here, we present a method to study the direct link between contact-dependent cues and division axes using C. elegans embryo blastomeres and adhesive polystyrene beads.

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...