Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

Summary

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Abstract

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

Introduction

The zebrafish is currently one of the most important vertebrate models to study cellular and developmental processes in vivo. This is largely due to the zebrafish's optical transparency and amenability to genetics, which makes it a powerful model for applying optical techniques involving genetically encoded photoresponsive protein technologies¹. Specific to the study of heart development, zebrafish receive sufficient oxygen via diffusion such that even mutants without heartbeat can survive through the first week of development, permitting analyses on the effects of developmental genes and perturbed blood flow on heart morphogenesis not....

Protocol

1. Preparing molds and mounting agarose

1. Create a mold with the dimensions shown in Figure 1 using either a 3D printer or traditional mechanical workshop tools.
2. Pipette about 1.5 mL of melted 1 % agarose into a 35-mm plastic mounting dish. Place the plastic mold in the liquid agarose, taking care to avoid trapping air between the mold and the agarose. Place the dish at 4 °C, wait till the agarose hardens (this takes about 5 min), then remove the plastic mold.
3. To store the mounting dish for later use, cover the dish with its lid, then wrap both the dish and the lid tightly with parafilm. The mounting dis....

Results

An example of an embryo photoconverted at 48 hpf and imaged again at 80 hpf is shown in Figure 2, Movies 1 and 2. Exposing Kaede to 405 nm light irreversibly converts from the protein from its fluorescent green form to fluorescent red form, enabling the behavior of cells labeled with either the green or their red form to be followed with respect to their differentially colored neighbors during valve formation. It can be seen .......

Discussion

Timing of photoconversion: Although Kaede remains brightly expressed in EdCs even at 96 hpf, it should be noted that as the embryo grows, laser light diffuses more before it reaches the AVC, making confined photoconversion of Kaede more difficult. At embryonic stages later than 55 hpf, the ballooning of the ventricle and the atrium also means that the violet laser beam used for photoconversion cannot reach AVC cells without first passing through either the atrium or ventricle. This means that beyond 55 hpf, in o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Materials
Necessary equipment for raising fish and collecting eggs (see the Zebrafish Book22 for details).
Stereomicroscope
Upright confocal microscope (Equipped with lasers operating at 488 nm, 561 nm, and 405 nm, a tunable multiphoton laser, and a Leica HCX IRAPO L, 25 ×, N.A. 0.95 objective)	Leica	Leica SP8
Heat block	ThermoScientific	88870001
35 mm x 10 mm tissue culture dish	Falcon	353001
6 well plate	Falcon	353046
Petri dish
Forceps
Glass pipette
Mold
Reagents
8 mg/mL Tricaine stock solution
1 M BDM stock solution
UltraPure low melting point agarose	Invitrogen	16520-050
Agarose	Lonza	50004
PTU (1-phenyl-2-thiourea)	Sigma Aldrich	P7629
Embryo medium: 30x stock solution
Software
Matlab equipped with the Image Analysis, Curve Fitting, Bio-Formats Toolboxes	MathWorks
8 mg/mL Tricaine stock solution
200 mg of tricaine powder (Ethyl 3-aminobenzoate methanesulfonic acid)	Sigma-Aldrich	E10521
25 mL Danieau
Adjust to pH 7, aliquot and store at -20 °C
1 M BDM stock solution
1 g BDM	Sigma-Aldrich	112135
10 mL ddH2O
Aliquot and store at -20 °C
Embryo medium: 30x stock solution
50.7 g NaCl	Sigma-Aldrich	7647-14-5
0.78 g KCl	Sigma-Aldrich	7447-40-7
1.47 g Magnesium sulfate	Sigma-Aldrich	7487-88-9
2.1 g Calcium nitrite tetrahydrate	Sigma-Aldrich	13477-34-4
19.52 g HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)	Sigma-Aldrich	7365-45-9
Adjust to pH 7.2 and store at room temperature
Mold
PlasCLEAR resin	Asiga
Plus 39 Freeform Pico 3D printer	Asiga