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...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+tropicalis+as+a+model+organism+for+genetics+and+genomics:+past,+present,+and+future.">Xenopus tropicalis as a model organism for genetics and genomics: past, present, and future.</a> Methods Mol Biol. 917, 3-15 (2012).</li><li>Weisgraber, K. H., Innerarity, T. L., Mahley, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lysine+residues+of+plasma+lipoproteins+in+high+affinity+binding+to+cell+surface+receptors+on+human+fibroblasts.">Role of lysine residues of plasma lipoproteins in high affinity binding to cell surface receptors on human fibroblasts.</a> J Biol Chem. 253 (24), 9053-9062 (1978).</li><li>Showell, C., Conlon, F. 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The authors have nothing to disclose.

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

The overall goal of this procedure is to visualize the vasculature of Xenopus tropicalis tadpoles. This method can help answer key questions in the field of cardiovascular biology such as how angiogenesis is regulated. The main advantage of this technique is that blood vessels in an intact well type embryo can be visualized by a simple fluorescent dye injection.Begin by using a micro loader to fill a tapered glass micro pipette with approximately five microliters of clear diI acetylated LDL solution taking care that no precipitated particles are loaded. Under a dissecting microscope use a fine pair of number 55 forceps to clip the tip of the pipette to adjust the volume of the uploaded complex and set the pressure controlled injection system to eject approximately five nano liters of solution per injection. Then submerge the pipette tip in 0.04%MS-222 in 0.1X modified Barth's saline or MBS in a custom made Sylgard mold.Next transfer Xenopus tropicalis embryo to the Sylgard mold and confirm complete sedation with a lack of response to dorsal fin stimulation. Under the dissecting microscope load the embryo ventral side up into the V shaped indentation of the mold in a slightly oblique position. Tightly fit two 0.1 millimeter minutian pins to a pin holder and use one pin to puncture the skin over the heart which should be visibly pulsing.Insert the pin through the hole in the space between the heart and the skin without puncturing the heart and position the inserted tip within the region where the incision will be made. Then rub the second pin against the inserted pin to make a linear incision and use both pins together to gently widen the incision, exposing the heart. To inject the DiI acetylated LDL solution, insert the glass pipette tip into the heart and inject 50 to 60 nano liters of the DiI acetylated LDL in approximately 10 ejection pulses.When all of the dye solution has been delivered confirm that the heart is still beating and transfer the embryo into a new Petri dish containing 0.1X MBS. After five to 10 minutes the tadpole should recover from anesthesia and begin to move. After an appropriate number of embryos have been injected, visually screen the correctly labeled anesthetized embryos under a fluorescence microscope discarding any animals that have other organs labeled.Embryos that have been insufficiently labeled can be injected again. Then capture images of the correctly labeled embryos. For a more thorough imaging of the blood vessels, fix re anesthetized embryos in 4%paraformaldehyde for one hour at room temperature protected from light for visualization by confocal microscopy.If a sufficient amount of diI acetylated LDL is injected into the heart, the posterior cardinal vein, or PCV, should be immediately visible under a fluorescence microscope. The intersomitic veins, or ISV's, emerge dorsally from the PCV in an anterior to posterior wave beginning at stage 36 and ending around stage 40 to 41 becoming lightly branched around stage 43. The PCV and ISV's grow in a stereotypical angiogenic pattern that is under tight developmental control.Interestingly, the knockdown of TIE-2 signaling by antisense morphalinos decreases the length and the complexity of the ISV's whereas expression of the constitutively active form of TIE-2 induces excessive ISV branching. A Vein Complexity Index can be calculated to assess the complexity of the ISV's and rendered paths can be generated to visualize the overall architecture of the embryonic Xenopus tropicalis vasculature. The original protocol was described for using Xenopus laevis by Levin and Colics and we adapted it for Xenopus tropicalis.Once mastered, this technique can be completed in one hour if it is performed properly. Before this procedure, genetic and pharmacological manipulations can be performed to answer additional questions such as which genes and signaling pathways regulate vascular development. Using this procedure the labeled vasculature can be imaged in an intact animal in real time providing a powerful tool for investigating the dynamics of vascular development.After watching this video you should have a good understanding of how to inject diI acetylated LDL into the heart of a Xenopus tropicalis tadpole to visualize it's vasculature.

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Research

JoVE Journal

Developmental Biology

8.3K Views.  Yonsei University College of Medicine. The overall goal of this procedure is to visualize the vasculature of Xenopus tropicalis tadpoles. This method can help answer key questions in the field of cardiovascular biology such as how angiogenesis is regulated. The main advantage of this technique is that blood vessels in an intact well type embryo can be visualized by a simple fluorescent dye injection.Begin by using a micro loader to fill a tapered glass micro pipette with approximately five microliters of clear diI acetylate...