Name: visualization and quantitative analysis of embryonic angiogenesis in xenopus tropicalis
Uploaded: 2017-05-25T14:00:00
Description: Watch this Scientific Journal Video about Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis at JoVE.com

This study was inspired by the work of Levine et al., which described this experimental method and provided a comprehensive description of vascular development in Xenopus laevis. We thank the members of our laboratory for their input. This study was supported by the Yonsei University Future-leading Research Initiative of 2015 (2015-22- 0095) and the Bio &#38; Medical Technology Development Program of the National Research Foundation (NRF) funded by the ministry of Science, ICT &#38; Future Planning (NRF-2013M3A9D5072551)

All experiments complied with protocols approved by the Yonsei University College of Medicine Institutional Animal Care and Use Committees.
1. Preparation of Xenopus tropicalis Embryos
NOTE: Xenopus tropicalis embryos were produced as previously described10, with slight modification. Xenopus tropicalis embryos were staged according to the tables of Nieuwkoop and Faber11 .
<ol>
	<li>Induction of ovulatio...

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The protocol presented here was first developed by Ali H. Brivanlou and colleagues to investigate developmental events during vascular formation in Xenopus laevis4, but, as shown in this manuscript, it can be applied to other small animals. Dye injection into the heart is simple to perform, and the entire vascular network can be imaged under a fluorescence dissection microscope, as well as a confocal microscope. If the dye is injected into the heart during vessel development, the dynamics...

Vasculogenesis, the formation of new blood vessels from newly born endothelial cells, and angiogenesis, the formation of new vessels from pre-existing vessels, are two distinct processes that shape embryonic vasculature1. Any dysregulation in these processes results in various heart diseases and structural abnormalities of vessels. Furthermore, tumor growth is associated with uncontrolled vessel growth. As such, molecular mechanisms underlying vasculogenesis and angiogenesis are the subject of intense investigation2.
Xenopus and zebrafish are attractive vertebrate models for vasculogenesis and angiogenesis studies, for several reasons. First, their embryos are small; therefore, it is relatively easy to image the entire vasculature. Second, embryonic development is rapid; it only takes a couple of days for the entire vasculature to develop, during which time the developing vasculature can be imaged. Third, genetic and pharmacological interventions before and during vessel formation are easy to perform, such as through the microinjection of antisense morpholino nucleotides (MOs) into the developing embryo or through the bath application of drugs3,4,5.
The unique advantage of Xenopus over zebrafish is that embryological manipulations can be performed because Xenopus follows stereotypical holoblastic cleavages and the embryonic fate map is well defined6. For example, it is possible to generate an embryo in which only one lateral side is genetically manipulated by injecting an antisense MO to one cell at the two-cell stage. It is also possible to transplant the heart primordium from one embryo to another to determine whether the gene exerts its function by a cell-intrinsic or -extrinsic mechanism7. Although these techniques have mostly been developed in Xenopus laevis, which is allotetraploid and is therefore not ideal for genetic studies, they can be directly applied to Xenopus tropicalis, a closely related diploid species8.
One way to visualize the vasculature in a live Xenopus embryo is to inject a fluorescent dye to label the blood vessels. Acetylated low density lipoprotein (AcLDL) labeled with a fluorescent molecule such as DiI is a very useful probe. Unlike unacetylated LDL, AcLDL does not bind to the LDL receptor9 but is endocytosed by macrophages and endothelial cells. The injection of DiI-AcLDL into the heart of a live animal results in the specific fluorescent labeling of endothelial cells, and the entire vasculature can be imaged by fluorescence microscopy in live or fixed embryos4.
Here, we present detailed protocols for the visualization and quantification of blood vessels using DiI-AcLDL in Xenopus tropicalis (Figure 1). We provide key practical points, with examples of successful and unsuccessful experiments. In addition, we provide a straightforward method for the quantitative analysis of vascular complexity, which might be useful in assessing the effects of genetic and environmental factors on the shaping of the vascular network.

<table border="0" cellpadding="0" cellspacing="0" width="539">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">35mm Petri dish</td>
			<td style="width:113px;">SPL</td>
			<td style="width:93px;">10035</td>
			<td style="width:180px;">Sylgard mold frame</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">60mm Petri dish</td>
			<td style="width:113px;">SPL</td>
			<td style="width:93px;">10060</td>
			<td>Embryo raising tray</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Borosilicate Glass</td>
			<td style="width:113px;">Sutter instrument</td>
			<td style="width:93px;">B100-50-10</td>
			<td>Needle for injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">BSA</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">A3059-10G</td>
			<td>Coating reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">CaCl2</td>
			<td style="width:113px;">D.S.P.GR Reagent</td>
			<td style="width:93px;"></td>
			<td>0.1X MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Coverslip</td>
			<td style="width:113px;">Superior</td>
			<td style="width:93px;">HSU-0111520</td>
			<td>For confocal imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">DiI-AcLDL</td>
			<td style="width:113px;">Thermo Fisher Scientific</td>
			<td style="width:93px;">L3484</td>
			<td>Vessel staining solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">FBS</td>
			<td style="width:113px;">Hyclone</td>
			<td style="width:93px;">SH.30919.02</td>
			<td>For storage of testis</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Fiber Optical Illuminator</td>
			<td style="width:113px;">World Precision Instruments</td>
			<td style="width:93px;">Z-LITE-Z</td>
			<td>Light</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Ficoll</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">F4375</td>
			<td>Injection buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Flaming/Brown Micropipette Puller</td>
			<td style="width:113px;">Sutter instrument</td>
			<td style="width:93px;">P-97</td>
			<td>Injection needle puller</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Forcep</td>
			<td style="width:113px;">Fine Science Tool</td>
			<td style="width:93px;">11255-20</td>
			<td style="width:180px;">For embryo hatching and 
			needle tip cutting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Glass Bottom dish</td>
			<td style="width:113px;">SPL</td>
			<td style="width:93px;">100350</td>
			<td>For confocal imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">hCG</td>
			<td style="width:113px;">MNS Korea</td>
			<td style="width:93px;"></td>
			<td>For priming of frogs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">HEPES</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">H3375</td>
			<td>Buffering agent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Incubator</td>
			<td style="width:113px;">Lab. Companion</td>
			<td style="width:93px;">ILP-02</td>
			<td>For raising embryos</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">KCl</td>
			<td style="width:113px;">DAEJUNG</td>
			<td style="width:93px;">6566-4400</td>
			<td>MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">L15 medium</td>
			<td style="width:113px;">Gibco</td>
			<td style="width:93px;">11415-114</td>
			<td>For storage of testis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">L-cysteine</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">168149-100G</td>
			<td>De-jellying reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">MgSO4</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">M7506</td>
			<td>MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Microtube</td>
			<td style="width:113px;">Axygen</td>
			<td style="width:93px;">MCT-175-C-S</td>
			<td>For storage of testis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">MS222</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">E10521</td>
			<td>Anesthetic powder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">NaCl</td>
			<td style="width:113px;">DAEJUNG</td>
			<td style="width:93px;">7647-14-5</td>
			<td>MBS component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">NaOH</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">S-0899</td>
			<td>pH adjusting reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Paraformaldehyde</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">P6148</td>
			<td>Fixatives</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">PBS</td>
			<td style="width:113px;">BIOSESANG</td>
			<td style="width:93px;">P2007</td>
			<td>Buffer for imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">pH paper</td>
			<td style="width:113px;">Sigma</td>
			<td style="width:93px;">P4536-100EA</td>
			<td>For confirming pH</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">PICO-LITER INJECTOR</td>
			<td style="width:113px;">Waner instruments</td>
			<td style="width:93px;">PLI-100A</td>
			<td>For injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Pin</td>
			<td style="width:113px;">Pinservice</td>
			<td style="width:93px;">26002-10</td>
			<td>For incision</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">Pinholder</td>
			<td style="width:113px;">Scitech Korea</td>
			<td style="width:93px;">26016-12</td>
			<td>For incision</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Precision Stereo Zoom Binocular Microscope</td>
			<td style="width:113px;">World Precision Instruments</td>
			<td style="width:93px;">PZMIII</td>
			<td>For visual screening</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:153px;">Standard Manual Control Micromanipulator&#160;</td>
			<td style="width:113px;">Waner instruments</td>
			<td style="width:93px;">W4 64-0056</td>
			<td>For microinjection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:153px;">SYLGARD 184 Kit</td>
			<td style="width:113px;">Dow Corning</td>
			<td style="width:93px;"></td>
			<td>For DiI injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:153px;">Transfer pipette</td>
			<td style="width:113px;">Korea Ace Scientific Co.</td>
			<td style="width:93px;">YM.B78-400</td>
			<td>For eggs and 
			embryo collection</td>
		</tr>
	</tbody>
</table>

visualization and quantitative analysis of embryonic angiogenesis in xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Timeline of experiments (Figures 1 and 2)
Shortly after fertilization, targeted microinjection can be performed to modulate gene expression. For example, an antisense MO that specifically binds to the initiation codon of the endogenous Tie2 mRNA can be injected, inhibiting the translation of Tie2 target mRNA by steric hindrance. A MO can be conjugated to fluorescein for the easy visual screening of successfully injected embryos. Al...

Watch this Scientific Journal Video about Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis at JoVE.com

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

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Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...