Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

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 circulation^2,3,4. Furthermore, among the many significant features of zebrafish is the optical transparency of their embryos, which enables non-invasive monitoring of the developing heart^5,6,7,8.

Mechanical forces continuously provide feedback to the heart valve morphogenetic programs^{9,10,11,12,13,14,15,16,17,18,19,20,21,22}, and aberrant blood flow is widely acknowledged as a shared factor in various cardiovascular disorders^23,24. 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 defects^{10,13,14,22,26}. 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.

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 con...

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 v...

Materials

Name	Company	Catalog Number	Comments
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.