Summary

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

Published: January 03, 2025
doi:

Summary

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.

Abstract

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 µm to 60 µm 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.

Introduction

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 µm to 60 µm 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.

Protocol

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

  1. Cross the relevant zebrafish line and grow the embryos in Danieau's medium at 28.5 °C. For more detailed instructions on crossing zebrafish lines, refer to the JoVE Science Education Database27.
  2. 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's medium.
  3. 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.

2. Mounting of zebrafish embryos

  1. Prepare 20 mL of Danieau's medium containing 0.02% tricaine.
  2. 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 °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.
  3. Transfer 3-6 dechorionated embryos to a Petri dish containing Danieau's medium supplemented with 0.02% tricaine.
  4. When the embryos are anesthetized, transfer them to a 35 mm x 15 mm glass-bottom dish with minimal medium. Quickly add 300-400 µL 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 µL, 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.

3. Bead grafting

  1. Once the agarose is solidified, deposit approximately 0.5 µL of magnetic beads on top.
    NOTE: Use a 10 µL pipette tip to transfer the magnetic beads. Precise measurement of 0.5 µL 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.
  2. Add a drop of Danieau's medium containing 0.02% tricaine on top of the agarose and the beads.
  3. Select the appropriate bead.
    NOTE: This step is critical. The size of the beads can vary slightly, ranging from 30 µm to 60 µm in diameter, so selecting the appropriate bead by eye, based on the sample and heart size, is essential.
  4. Once the suitable bead is selected, use tweezers to place it on top of the yolk (Figure 2A and Video 1).
  5. Create a yolk lesion or 'hole' 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.
  6. 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.

4. Unmounting of zebrafish embryos

  1. After grafting magnetic beads into all mounted embryos, add 1-2 mL of Danieau's medium containing PTU in the glass-bottom dish.
  2. Using tweezers, gently break the LMP agarose and carefully remove the fish, ensuring that no agarose is left on them.
  3. Using a Pasteur pipette, transfer the embryos to a new Petri dish containing Danieau's medium with PTU and allow them to recover and develop at 28.5 °C.

Representative Results

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 confirming the success of the grafting procedure (Video 4).

These indicators are essential for evaluating the success of bead implantation and determining the suitability of the samples for inclusion in relevant studies. Fish that fail to meet these criteria should be excluded to ensure the accuracy and reliability of the research results.

Figure 1
Figure 1: Mounting of zebrafish embryos. (A) Schematic illustrating how a zebrafish embryo should be mounted in LMP agarose within a glass-bottom dish prior to bead grafting. (B,C) Images depicting the setup for mounting zebrafish embryos before bead grafting. Three anesthetized embryos are positioned in LMP agarose, lying straight with their ventral side facing up. Abbreviations: A = anterior; P = posterior; R = right; L = left; D = dorsal; V = ventral. Scale bar = 1 mm. Please click here to view a larger version of this figure.

Figure 2
Figure 2: The bead grafting process. (A) Widefield images of a zebrafish at 56 hpf, showing the preparation step before bead grafting, where the selected bead is placed on top of the yolk. (B) Widefield images of a zebrafish at 56 hpf, illustrating the sequential steps of grafting a magnetic bead into its beating heart. The bead should be placed into the yolk lesion, created using one arm of the tweezers, and gently pushed anteriorly until it reaches the venous pole. At this point, heart contractions create suction, drawing the bead into the cardiac lumen. Black squares indicate the heart area, and a white asterisk marks the created lesion or 'hole' in the yolk. A white line shows the trajectory that the bead should follow. Abbreviation: hpf = hours post-fertilization. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Representative example of successful bead grafting. (A) Widefield image showing a zebrafish at 56 hpf with a bead successfully grafted into the atrium of its heart. Black lines outline the shape of the heart. (B) A list of criteria for successful bead implantation. Abbreviations: hpf = hours post fertilization; OFT = outflow tract; AVC = atrioventricular canal. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Video 1: Manipulation of the appropriate bead within the agarose. The video demonstrates the process of selecting and positioning the suitable magnetic bead on top of the yolk. It shows the careful handling of the bead with tweezers and illustrates the technique for maneuvering it into the precise location within the agarose. Please click here to download this Video.

Video 2: The bead grafting process. Related to Figure 2. The video demonstrates the technique for creating a lesion or 'hole' in the center of the yolk using one arm of the tweezers. It also shows how to gently maneuver the bead within the yolk, pushing it anteriorly. The video concludes with the successful insertion of the bead into the atrium of the zebrafish heart. Please click here to download this Video.

Video 3: Representative example of successful bead grafting minutes after the procedure. Related to Figure 3. The video shows a zebrafish larva at 56 hpf with a bead successfully implanted into the atrium of its heart. The larva exhibits no hemorrhage 15 min post grafting, blood flow remains unobstructed, and the heart walls maintain their structural integrity. The yolk lesion has already begun to heal. Please click here to download this Video.

Video 4: Representative example of successful bead grafting 24 h post procedure. The video shows a zebrafish larva at 80 hpf with a bead successfully grafted into the atrium of its heart. The larva exhibits no pericardial edema 24 h post grafting, blood flow remains unobstructed, and the heart walls maintain their structural integrity. Please click here to download this Video.

Discussion

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 volume of approximately 350 µL is recommended to adequately cover the embryos while leaving a few millimeters of space above them. If the agarose overflows the glass, it indicates that too much has been added.

The embryos should rest directly on the glass. If an embryo is not fully submerged in the agarose, such as when part of it is floating, draw up approximately 200 µL of LMP agarose. Gently aspirate the embryo and then release it, ensuring the agarose fully surrounds it.

Multiple embryos can be mounted on a single glass-bottom dish. To efficiently mount several embryos at once, perform each step of the mounting process, such as rotating and tilting, for all embryos before proceeding to the next step. In other words, avoid perfectly positioning one embryo before starting on the next. If the agarose sets too quickly, use a larger amount to create a higher dome, which will slow down the cooling process. For beginners, it may be safer to mount fewer embryos at a time.

Selecting the suitable bead based on the sample

This step is crucial. Bead sizes can vary slightly, ranging from 30 µm to 60 µm in diameter. Therefore, selecting the appropriate bead by eye- based on the sample and the heart size, which varies at different developmental time points- is essential. Always thoroughly examine the heart, including the sizes of the atrium, atrioventricular canal (AVC), and ventricle, where the bead will be grafted. Choose a bead that best fits the intended chamber. Larger beads may be used in the atrium to prevent passage through the AVC, however, caution is necessary, as excessively large beads can obstruct blood flow. In the ventricle, smaller beads are appropriate, but if they are too small, they may block the outflow tract.

Manipulation of the magnetic bead within the yolk

Careful manipulation of the magnetic bead within the yolk is essential for successful placement. Avoid pushing the bead too deeply, as it may become lodged. Position the bead at the same level as the cardinal vein by ensuring both are in focus. 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. A small amount of yolk expulsion during this process is expected.

Unmounting of zebrafish embryos

Embryos, particularly those younger than 48 hpf, are vulnerable to damage during unmounting. Therefore, manipulations should be performed with caution. If any LMP agarose remains attached to the embryo, gently remove it using forceps or by carefully aspirating and releasing the embryo with a glass pipette.

Limitations of the method

This protocol describes a microsurgical technique for implanting a magnetic bead into the developing heart of a zebrafish. While this approach provides an effective method for bead grafting, it has certain limitations. Bead grafting can be performed as early as 34 hpf and continues to be feasible up to 6 days post-fertilization (dpf). However, earlier stages can present greater challenges, as the heart chambers have not yet formed, which increases the risk of the bead being extruded from the cardiac lumen back into the yolk or becoming lodged in the outflow tract. Grafting is generally more successful between 48 and 60 hpf, when both heart chambers are formed and the cardinal vein is clearly visible on the yolk's surface.

Additionally, the procedure requires a high degree of precision and skill, especially when manipulating the bead within the yolk. The method is relatively invasive, and there is an inherent risk of damage, which is heightened if the bead size or placement is suboptimal, or if manipulations are not performed with care. Approximately 30% of embryos successfully retain the grafted bead, which can remain implanted in the heart for up to 48 h. However, after 24 h, only ~10% of embryos remain healthy without showing signs of edema. This further highlights the importance of careful monitoring following the procedure.

The significance of the method and potential applications

This method is significant for its ability to precisely manipulate physical forces and study cardiac function and development in zebrafish embryos upon ectopic mechanical stimulation. Unlike genetic10,16,25, pharmacological22, or optogenetic approaches28, bead grafting provides a more direct and mechanical means of influencing heart physiology. Additionally, the presence of the magnetic bead has been shown to induce Ca2+ influx in EdCs13. Therefore, this technique is valuable for examining the role of mechanical forces and Ca2+ influx in cardiac biological processes, investigating cellular responses to shear stress, and studying the mechanotransduction pathways involved in heart function12,13,19. For more precise control over shear forces, this method can be combined with magnetic tweezers, allowing for direct manipulation of the bead within the heart. The applied forces can range from 100 to 2,000 pN, slightly exceeding the endogenous wall shear stresses observed in the AVC, where mechanical forces are most pronounced13. An alternative to using magnetic beads- if the use of magnetic tweezers is not required- would be glass beads, as this approach has been successfully implemented in the past26.

The system represents a complex interplay among the magnetic bead, blood flow, and the cardiac wall. While the presence of the bead increases local shear stress, it may also affect distal shear stress. This interaction can create a cascading phenomenon that influences blood flow dynamics throughout the heart. Consequently, conducting computational fluid dynamics simulations to model these mechanical forces and their variations across different regions can provide critical insights into how the bead impacts overall hemodynamics.

Disclosures

The authors have nothing to disclose.

Acknowledgements

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's Horizon 2020 research and innovation program: GA N°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.

Materials

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

References

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Cite This Article
Vagena-Pantoula, C., Fukui, H., Vermot, J. Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads. J. Vis. Exp. (215), e67604, doi:10.3791/67604 (2025).

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