Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

Summary

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Abstract

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Introduction

Biomechanical forces, such as hemodynamic shear stress, are intimately involved in cardiac morphogenesis. In response to hemodynamic shear forces, myocardial ridges and grooves develop in a wave-like trabecular network in alignment with the direction of the shear stress across the atrioventricular (AV) valve¹. Cardiac trabeculation is necessary to increase contractile function and myocardial mass². Mutations in Notch signaling pathways result in congenital heart defects in humans and other vertebrates³. For example, gata1a⁴ and tnnt2a⁵

Protocol

The following methods were performed in compliance with UTA and UCLA IACUC protocols. These experimental groups were used with transgenic Tg(cmlc2:gfp), the wea (weak atrium) or clo (cloche) mutants: (a) wild-type (WT) control, (b) gata1a MO, and (c) tnnt2a MO injections (Table 1).

Model	Name	Modified Genes	Phenotype	Reference
Control....

Results

LSFM was used in this manuscript in order to acquire high-resolution 2-D and 3-D pictures. As seen in Figure 1A and 1B, the illumination lens directs the light sheet at the sample. Because of the thinness of the light sheet, only a single plane is illuminated. The detection lens is positioned perpendicular to the illumination lens and is focused on the illuminated plane (Figure 1B). The light sheet from the illum.......

Discussion

In this protocol, we have shown that 4-D imaging can be used to track the development of a trabecular network in response to changes in biomechanical forces. In particular, the shear stress experienced by endothelial cells initiates the Notch signaling cascade, which in turn promotes trabeculation. In this manuscript, we have shown that (1) gata1a MO injection decreased hematopoiesis and therefore it reduced wall shear stress, (2) tnnt2a MO injection inhibited ventricular contractile function to reduce .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Clontech Hifi PCR pre-mix	Takara	639298	PCR mastermix 1.1.1.1, 1.1.1.2, 1.1.1.5, 1.1.2.1, 3.1.4
Human Nrg1 cDNA	Gift from William Talbot, Stanford University, Stanford, California, USA	N/A	Used for trabeculation rescue 1.1.1.3, 1.1.1.4, 1.1.2.1
CFX Connect™ Real-Time PCR Detection System	Bio-Rad	1855201	PCR Machine 1.1.1.5, 1.1.1.6, 1.1.2.2, 1.1.3.2
pCS2+	GE Health		Plasmid used to synthesize mRNA 1.1.2.1, 1.1.2.4, 1.1.2.5
Nucleospin purification kit	Clontech	740609.25	DNA Purification 1.1.2.3, 1.1.2.4, 1.1.6.2
T4 DNA ligase	Clontech	2011A	PCR Ligation solution 1.1.2.5
Stellar competent cells	Clontech	636763	E. coli cells used for transformation 1.1.2.6, 1.1.3.1, 1.1.3.2
Lipofectamine 2000 transfection reagent	Life Technologies	11668027	Transfection reagent 1.1.4
mMessage SP6 kit	Invitrogen	AM1340	Kit used to synthesize mRNA 1.1.6.3
Aurum Total RNA Mini Kit	Bio-Rad	7326820	Purifies RNA  1.1.6.4, 3.1.2
GeneTools 4.3.8	GeneTools	N/A	Software for primer design 1.2.1, 3.1.3
EPO cDNA	Creative Biogene	CDFH006026	Increases WSS 1.2.2, 1.2.3, 1.1.7
AG1478	Sigma-Aldrich	T4182	ErbB inhibitor 1.3.1
E3 medium			To grow embryos 1.3.1, 1.3.2, 5.1.5
DAPT	Sigma-Aldrich	D5942	γ-secretase inhibitor 1.3.2
Agarose	Sigma-Aldrich	A9539	Used for mounting embryos 2.1.1.1
ORCA-Flash4.0 LT Digital CMOS camera	Hamamatsu Photonics	C11440-42U	Used to capture Images 2.1.1.2, 2.1.1.3
Amira Software	FEI Software	N/A	Visualized and Analysed images into 3D, and 4D 2.1.5.1.1-2.1.5.2.8
Tricaone mesylate	Sigma-Aldrich	886-86-2	Used to humanely sedated or sacrifice embryos 3.1.1
iScript cDNA Synthesis Kit	Bio-Rad	1708890	Synthesizes cDNA 3.1.2
Eppendorf 5424 microcentrifuge	Eppendorf	05-400-005	Microcentrifuge 4.1.1.3
GI254023X	Sigma-Aldrich	260264-93-5	ADAM10 inhibitor 4.1.2, 4.1.3
Isoprenaline hydrochloride	Sigma-Aldrich	I5627	Isoproterenol increases WSS 5.1.5
MATLAB	Mathworks	N/A	Cardiac mechanics analysis