Name: light-sheet fluorescence microscopy to capture 4-dimensional images of the effects of modulating shear stress on the developing zebrafish heart
Uploaded: 2018-08-10T14:30:00
Description: Watch this Scientific Journal Video about Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart at JoVE.com

The authors would like to express gratitude to William Talbot from Stanford University for providing the Human&#160;Nrg1&#160;cDNA and to Deborah Yelon from UCSD for providing the&#160;wea&#160;mutants. The authors would also like to thank Cynthia Chen for helping with image acquisition. This study was supported by grants NIH HL118650 (to T.K. Hsiai), HL083015 (to T.K. Hsiai), HD069305 (to N.C. Chi and T.K. Hsiai.), HL111437 (to T.K. Hsiai and N.C. Chi), HL129727 (to T.K. Hsiai), T32HL007895 (to R.R. Sevag Packard), HL 134613 (to V. Messerschmidt) and University of Texas System STARS funding (to J. Lee).

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).
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			<td>Model</td>
			<td>Name</td>
	...

<ol>
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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 ...

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 morpholino oligonucleotides (MO) have been shown to reduce erythropoiesis, while erythropoietin mRNA (EPO)6 and Isoproterenol (ISO)7 increase red blood cells and heart rate respectively, and therefore wall shear stress (WSS). Furthermore, ErbB2 signaling, downstream of Notch, promotes cardiomyocyte proliferation and differentiation to generate contractile force, which in turn activates Notch signaling8,9. It is suggested that shear stress governs Notch signaling driven trabeculation for ventricular development. Currently, there are many studies that attempt to further understand the genetic programming events leading to congenital heart defects (CHD)10,11,12, but very little are investigating how mechanical forces influence the forming heart.
In order to investigate the mechanical forces acting on the endocardium, close observation during the developmental period needs to be implemented. However, it is challenging to obtain good quality images of in vivo beating samples due to the inherence of traditional microscopy13. In order to observe the development over time within a sample, physical sectioning and staining, therefore, need to occur13,14,15. Although confocal microscopy is widely used to image the 3-D structure of samples14,16, these imaging systems&#39; acquisition is still limited by slow scanning speeds.
Light-sheet fluorescence microscopy (LSFM) is a unique imaging technique that allows the visualization of in vivo dynamic events with long working distance13. This technique uses a light-sheet fluorescent microscopy to optically section a sample17. Due to illumination of only a thin sheet of light on the sample, there is a reduction in photo-bleaching and photo toxicity13,18. The large field of view and long working distance allows for large samples to stay intact as they are imaged13,14,17. The low magnification allows for a larger area to be imaged, while the long working distance allows for thicker samples to be imaged without compromising the signal-to-noise ratio. Many groups have used LSFM to image entire embryos17, brains14,18, muscles and hearts19 among other tissues, showing the diverse types of samples that can be imaged.
Although previous research demonstrated reduced hemodynamic shear force by occluding the inflow or outflow tracks of the zebrafish heart, the information is solely qualitative. It results in an abnormal third chamber, diminished cardiac looping, and impaired valve formation20. The 4-D LSFM images give a new perspective into the way the hemodynamic shear forces affect the development of the cardiac tissue. These mechanical forces may activate force-sensitive signaling molecules and induce the formation of the trabecular ridges. Because of the added time aspect of 4-D imaging, one is able to track changes in development in real time, which could lead to new revelations that had gone unnoticed previously. The zebrafish is an ideal model for imaging because scientists can observe an entire vertebrate animal versus only cell-cell interactions. Oxygen can also diffuse through the entire embryo, which allows development to occur without depending on the vascular system, unlike in mammalian development. Even though the zebrafish heart lacks the pulmonary organs, which require a four-chambered heart, there is a large number of cardiac genes that are conserved between zebrafish and humans21.
In this manuscript, we describe how to use light-sheet fluorescence microscopy to image the developing trabeculae in zebrafish hearts under various circumstances. First, injection of gata1a4 or tnnt2a5 MOs were used to lower the blood viscosity, and therefore WSS. The morphology of the heart was then recorded. In a separate group of fish, we increased the WSS by administering EPO mRNA6 or isoproterenol7 and observed the results. We also conducted a cell study with different pulsatile or oscillatory flow rates. After imaging each group, we found that WSS sensed by the endocardium via Notch signaling initiates trabeculation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Clontech Hifi PCR pre-mix&#160;</td>
			<td>Takara&#160;</td>
			<td>639298</td>
			<td>PCR mastermix 
			1.1.1.1, 1.1.1.2, 1.1.1.5, 1.1.2.1, 3.1.4</td>
		</tr>
		<tr>
			<td>Human Nrg1 cDNA</td>
			<td>Gift from William Talbot, Stanford University, Stanford, California, USA</td>
			<td>N/A</td>
			<td>Used for trabeculation rescue 
			1.1.1.3, 1.1.1.4, 1.1.2.1</td>
		</tr>
		<tr>
			<td>CFX Connect&#8482; Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855201</td>
			<td>PCR Machine 
			1.1.1.5, 1.1.1.6, 1.1.2.2, 1.1.3.2</td>
		</tr>
		<tr>
			<td>pCS2+</td>
			<td>GE Health</td>
			<td></td>
			<td>Plasmid used to synthesize mRNA 
			1.1.2.1, 1.1.2.4, 1.1.2.5</td>
		</tr>
		<tr>
			<td>Nucleospin purification kit&#160;&#160;</td>
			<td>Clontech</td>
			<td>740609.25</td>
			<td>DNA Purification 
			1.1.2.3, 1.1.2.4, 1.1.6.2</td>
		</tr>
		<tr>
			<td>T4 DNA ligase&#160;</td>
			<td>Clontech</td>
			<td>2011A</td>
			<td>PCR Ligation solution 
			1.1.2.5</td>
		</tr>
		<tr>
			<td>Stellar competent cells</td>
			<td>Clontech</td>
			<td>636763</td>
			<td>E. coli cells used for transformation 
			1.1.2.6, 1.1.3.1, 1.1.3.2</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 transfection reagent</td>
			<td>Life Technologies</td>
			<td>11668027</td>
			<td>Transfection reagent 
			1.1.4</td>
		</tr>
		<tr>
			<td>mMessage SP6 kit</td>
			<td>Invitrogen</td>
			<td>AM1340</td>
			<td>Kit used to synthesize mRNA 
			1.1.6.3</td>
		</tr>
		<tr>
			<td>Aurum Total RNA Mini Kit</td>
			<td>Bio-Rad</td>
			<td>7326820</td>
			<td>Purifies RNA&#160; 
			1.1.6.4, 3.1.2</td>
		</tr>
		<tr>
			<td>GeneTools 4.3.8</td>
			<td>GeneTools</td>
			<td>N/A</td>
			<td>Software for primer design 
			1.2.1, 3.1.3</td>
		</tr>
		<tr>
			<td>EPO cDNA</td>
			<td>Creative Biogene</td>
			<td>CDFH006026</td>
			<td>Increases WSS 
			1.2.2, 1.2.3, 1.1.7</td>
		</tr>
		<tr>
			<td>AG1478</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T4182</td>
			<td>ErbB inhibitor 
			1.3.1</td>
		</tr>
		<tr>
			<td>E3 medium</td>
			<td></td>
			<td></td>
			<td>To grow embryos 
			1.3.1, 1.3.2, 5.1.5</td>
		</tr>
		<tr>
			<td>DAPT</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5942</td>
			<td>&#947;-secretase inhibitor 
			1.3.2</td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9539</td>
			<td>Used for mounting embryos 
			2.1.1.1</td>
		</tr>
		<tr>
			<td>ORCA-Flash4.0 LT&#160;Digital CMOS camera</td>
			<td>Hamamatsu Photonics</td>
			<td>C11440-42U</td>
			<td>Used to capture Images 
			2.1.1.2, 2.1.1.3</td>
		</tr>
		<tr>
			<td>Amira Software</td>
			<td>FEI Software</td>
			<td>N/A</td>
			<td>Visualized and Analysed images into 3D, and 4D 
			2.1.5.1.1-2.1.5.2.8</td>
		</tr>
		<tr>
			<td>Tricaone mesylate&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>886-86-2</td>
			<td>Used to humanely sedated or sacrifice embryos 
			3.1.1</td>
		</tr>
		<tr>
			<td>iScript cDNA Synthesis Kit</td>
			<td>Bio-Rad</td>
			<td>1708890</td>
			<td>Synthesizes cDNA 
			3.1.2</td>
		</tr>
		<tr>
			<td>Eppendorf 5424 microcentrifuge</td>
			<td>Eppendorf</td>
			<td>05-400-005</td>
			<td>Microcentrifuge 
			4.1.1.3</td>
		</tr>
		<tr>
			<td>GI254023X</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>260264-93-5</td>
			<td>ADAM10 inhibitor 
			4.1.2, 4.1.3&#160;</td>
		</tr>
		<tr>
			<td>Isoprenaline hydrochloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>I5627</td>
			<td>Isoproterenol increases WSS 
			5.1.5</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>N/A</td>
			<td>Cardiac mechanics analysis</td>
		</tr>
	</tbody>
</table>

light-sheet fluorescence microscopy to capture 4-dimensional images of the effects of modulating shear stress on the developing zebrafish heart

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.

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...

Watch this Scientific Journal Video about Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart at JoVE.com

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

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.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

This method can help answer key questions in the developmental cardiac mechanobiology field, such as the modulation of shear stress on cardiac trabeculation via notch signaling using a 4D light sheet microscope. The main advantage of this technique is that it illuminates a thin section of sample with a five micron plane, leading to less photo bleaching and photo damage. In addition, our computational algorithm allows us to reconstruct a 4D beating zebrafish heart.The implications of this technique extend toward the diagnosis of congenital cardiac diseases as it allows for the invivo sequential visualization of the cardiac trabeculation in an early developmental stage on through to the later phenotype. Generally, people who are new to this technique will find that thinking in three dimensions over time is difficult to imagine. In addition, it takes time to create the system and accurately align it.To begin, separate the male and female zebrafish in the the breeding tanks by using a divider. Then, lift the divider and allow the male and female zebrafish to breed. Collect the zebrafish embryos and inject morpholino oligo into the embryo to inhibit trabeculation.Next, prepare a one percent agarose gel by adding one gram of agarose to 100 milliliters of distilled water and heat it until all of the agarose is dissolved. Allow the agarose to cool and then use a 20 microliter pipet to load a small plastic tube with one of the prepared embryos and agarose. Secure the tube onto the microscope stage and use the ten x objective.Adjust the objective using the knob so that the top of the embryo is in focus. Take images of the entire fish embryo over multiple cardiac beating cycles using a light sheet thickness of five microns. Collect 500 xy frames with an exposure time of ten milliseconds.Then, move the stage one micron in the z access to a new layer and repeat the imaging procedure. Continue imagine 500 frames per z plane until the entire heart is fully imaged. Next, open the image processing software and begin to stack the images into 3D.To accomplish this, first click on open data. Here, select all of the images that are to be stacked into 3D and then select load. Then add in the voxel size of 0.65 by 0.65 by one micron and click on okay.Inputting the correct voxel size is critical for accurate analysis of the 3D images. Now, click on the multiplaner viewbox and visualize the 3D image. Right-click the blue slice one dot tiff box, select display, and then click on volran.In the edit menu, select options, and then edit color map. Adjust the color using the boxes and then click on okay. While still in the image processing software, click on file, and open the time series data.Select the 3D tiffs that were just made by clicking on load, and enter the same voxel size as before. Right-click the blue slice one dot tiff box, and then select display followed by volran. Now, click on edit.Select options. And then select edit color map. Adjust the color using the boxes and then click on okay.Next, right-click on the time series control, click movie maker, and press the play button in order to watch the movie. To finish up, add a file name, frame size, frame rate, quality equal to one, and enter monoscopic. Then click apply.Finally, export the video. Biomechanical forces, such as hemodynamic shear stress, are intimately involved in cardiac morphogenesis. Here, light sheet fluorescence microscopy was used to visualize the trabecular ridges protruding into the ventricular lumen at 75 hours post fertilization.At 100 hours post fertilization a trabecular network can clearly be seen. In an embryo treated with the gata1aMO microinjection, hematopoiesis and viscosity are reduced by 90 percent. This decreased viscosity attenuated hemodynamic shear stress, resulting in a delayed initiation and a less dense trabecular network.This delay and density of the trabecular network was able to be recovered by up regulating notch related gene expression, rescuing trabecular formation at 75 hours post fertilization and at 100 hours post fertilization. Thus, gata1a morpholino oligonucleotides reduced hemodynamic forces leading to down regulation of notch signaling, whereas Nrg1-mRNA rescue up regulated notch related genes and reinitiated trabeculation. Once mastered, this imaging technique can be completed in approximately two hours if performed properly.After its development, this technique paved the way for researchers in the field of cardiac development to study the factors in the notch gene, which are responsible for the proper formation of trabeculation in zebrafish. After watching this video, you should have a good understanding of how to acquire images using selective plane illumination microscopy and create 4D image files. Don't forget that working with lasers can be extremely hazardous.And precautions, such as wearing appropriate eyewear, should always be taken during this procedure.

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