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 ovulation
	<ol>
		<li>To pre-prime, inject human chorionic gonadotropin (hCG) into the dorsal lymph sac of a pair of frogs (one male and one female) on the day prior to the day of mating. Inject 20 units per female frog and 10 units per male. House a male and a female in the same tank overnight (for more than 18 h).</li>
		<li>To prime, on the following day, inject 200 units of hCG to the pre-primed female and 100 units of hCG to the pre-primed male; the primed female will start to lay eggs in approximately 3 h and will continue to do so throughout the day. Keep the male and female pair together for natural mating or in separate tanks for in vitro fertilization (step 1.2).</li>
	</ol>
	</li>
	<li>Fertilization 
	NOTE: Eggs can be fertilized by natural mating or in vitro fertilization. In natural mating, eggs are fertilized as they are laid, and therefore, the timing of fertilization cannot be controlled. Alternatively, a primed female frog can be housed in a separate tank and unfertilized eggs can be collected for in vitro fertilization, which generates hundreds of embryos fertilized at the same time. The latter approach is useful when blastomere-targeted microinjection will be performed prior to vessel labeling. In this approach, however, a male is sacrificed.
	<ol>
		<li>Natural mating
		<ol>
			<li>Leave primed female and male pair in the same tank. The male will clasp the female, leading to immediate fertilization of the laid eggs. Collect the eggs with a glass or plastic pipette and transfer them to a Petri dish. To prevent the eggs from sticking to the pipette, coat the inner surface of the pipette with 0.5% bovine serum albumin (BSA).</li>
		</ol>
		</li>
		<li>In vitro fertilization (IVF)
		<ol>
			<li>Separate the primed female from the male after priming. The female will start to lay eggs spontaneously in approximately 3 h. Transfer a few laid eggs using a BSA-coated pipette to a Petri dish and check their quality under a microscope. If healthy eggs (clear pigment, elastic ball-shaped) were laid, prepare to dissect out the testes from the primed male.</li>
			<li>Make 0.4% Tricaine methanesulfonate (MS222) for euthanasia and inject 300 &#181;L into the dorsal lymph sac of the primed male. In approximately 10 min, the male will lose his sense of balance and will stay afloat; confirm that the male is euthanized by pinching a hind leg with a pair of blunt forceps and observing no response.</li>
			<li>Dissect out the two testes, clean them by quickly tapping them on lint-free paper, and transfer them into a microtube containing 1 mL of 60% Leibovitz&#39;s L-15 Medium (60%) supplemented with 10% fetal bovine serum (FBS); a detailed procedure is described elsewhere10.</li>
			<li>Gently mince the testes with a pestle to extract the sperm. At this concentration (i.e., 2 testes in 1 mL of 10% FBS in 60% L-15), a few drops of the testis solution (0.1 mL) can fertilize approximately three hundred eggs. The remaining sperm can be stored at 4 &#176;C in a tightly sealed tube and used for IVF the next day.</li>
			<li>Squeeze the eggs from the female into a small Petri dish. Hold the upper part of the female upside down on the palm and put the forefinger between its hind legs. Spread its hind legs with the forefinger and the other hand to expose the cloaca. Touch the cloaca to the Petri dish. Coat the Petri dish with 0.5% BSA to evenly distribute the eggs as a monolayer during IVF.</li>
			<li>Add a few drops (0.1 mL) of sperm-containing L-15 medium to the eggs. For synchronous fertilization, gently stir the sperm solution across the dish. After 4 min at room temperature, fill the Petri dish with 0.01x Modified Barth&#39;s Saline (MBS), incubate for 10 min, and replace the solution with 0.1x MBS. The fertilized eggs will undergo the first cleavage in approximately 1 h at room temperature. 1x MBS is 88 mM NaCl, 1 mM KCl, 2.5 mM NaHCO3, 5 mM HEPES, 1 mM MgSO4, and 0.7 mM CaCl2, pH 7.5.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>(Optional) Injection of antisense morpholino oligonucleotides (MOs) and/or mRNAs
	<ol>
		<li>To investigate the effect of a gene of interest on vessel formation, perform the microinjection of antisense MO and/or mRNAs.</li>
		<li>For such experiments, de-jelly the eggs with 2% L-cysteine in 1x MBS (pH 7.5-7.8). Replace 0.1x MBS in the dish containing the fertilized embryos with 2% L-cysteine in 1x MBS. Gently swirl the dish to mix the solution. 
		NOTE: It usually takes about 5 min for the embryos to be de-jellied. During this step, monitor the eggs frequently to ensure that they are not over-exposed to the solution. If the eggs are over-exposed, they will lose elasticity and become flattened, which will cause aberrant development and lethality.</li>
		<li>Wash the de-jellied eggs five times with 0.1x MBS. Perform the microinjection in 4% Ficoll dissolved in 0.1x MBS. Typically, inject 4 ng of MO and 600 pg of mRNA per blastomere at the two-cell stage. Inject into one cell to manipulate gene expression on only one side of the embryo. Use the uninjected side of the same embryo as a control.</li>
		<li>As another control for the specificity of gene manipulation, inject the same amount of a control MO (CoMO) or control mRNA (e.g., beta-galactosidase mRNA). CoMO must have the same molecular weight as the gene-specific MO and must not target any gene in the Xenopus genome. For easy visualization of the injected side, use fluorescein-tagged MO or co-inject a tracer (e.g., EGFP mRNA).</li>
		<li>Leave the injected embryos in 4% Ficoll for 2 h and then transfer them to a new 60 mm Petri dish filled with 0.1x MBS.</li>
	</ol>
	</li>
	<li>Raising
	<ol>
		<li>Raise the embryos at 23 &#176;C. Although the speed of development can be slowed down or accelerated by using temperatures below or above 23 &#176;C, respectively (range: 16-26 &#176;C), it is recommended to raise them at 23 &#176;C.</li>
	</ol>
	</li>
	<li>(Optional) Treatment with pharmacological reagents
	<ol>
		<li>For pharmacological manipulations, administer drugs to the developing embryos by bath application.</li>
	</ol>
	</li>
</ol>
2. Preparation of DiI-AcLDL Injection
<ol>
	<li>Make a tapering glass pipette using a standard micropipette puller. Place a borosilicate glass tube in the micropipette puller and use the following settings: pressure, 200; heat, 30; pull, 30; velocity, 120; time, 200.</li>
	<li>Store DiI-AcLDL stock solution at 4 &#176;C. Take the upper clear part of the solution for injection, but not the precipitated particles on the bottom of tube. Any debris in the glass pipette will disturb the proper ejection of the solution.</li>
	<li>Fill the prepared glass pipette with a suitable amount of DiI-AcLDL solution (~5 &#181;L) using a microloader.</li>
	<li>Clip off the tip of the glass pipette little by little using a fine pair of forceps (number 55). Set up the pressure-controlled injection system so that approximately 5 nL of solution is ejected at a time; 50-60 nL of DiI-AcLDL solution is enough to stain the vessels of one embryo.</li>
	<li>Do not leave the DiI-AcLDL-loaded pipette in air for an extended period of time in order to avoid drying. Keep the tip of the pipette submerged in 0.04% MS222 in&#160;0.1X MBS solution. Just before injecting it into the embryo (step 4.2.1), check to see whether the same amount of dye ejects.</li>
</ol>
3. Injection Setup
<ol>
	<li>Sylgard mold
	<ol>
		<li>To prepare a small Sylgard mold, mix Sylgard base and curing agent with a weight ratio of 10 to 1. Fill approximately half of a small Petri dish (35 mm or 60 mm dish) with this mixed solution and then leave it to solidify for approximately 48 h at room temperature.</li>
	</ol>
	</li>
	<li>Anesthesia
	<ol>
		<li>Use 0.04% MS222 in 1x MBS (pH 7.5-8.0) for anesthesia. When long anesthesia is needed (e.g., during live imaging), use 0.04% MS222 in 0.1x MBS to reduce the osmotic imbalance. To adjust the pH, add a few drops of NaOH (~20 &#181;L) and check the pH using pH paper.</li>
		<li>Wait until the transferred embryos stop moving. Touch the dorsal fins of the embryos and inspect whether they respond to confirm complete anesthesia.</li>
	</ol>
	</li>
	<li>Positioning embryos for injection
	<ol>
		<li>Indent the surface of the hardened Sylgard mold with a thin and sharp blade. Make a V-shaped concave indentation in which to place the embryos (see Figure 2D). Place an anesthetized embryo, ventral-side up, in a slightly oblique fashion (approximately 30&#176;) (Figure 2D).</li>
		<li>Fill the mold with MS222 solution and place the embryos in the mold.</li>
	</ol>
	</li>
</ol>
4. DiI-AcLDL Injection
<ol>
	<li>Incision of skin overlying the heart
	<ol>
		<li>Prepare two pins (Minutiens, 0.10 mm) to cut the skin overlying the heart. Tightly fix each pin to a pin-holder; the heart should be readily identifiable as it pulsates (Figure 2A-2C, pink).</li>
		<li>Puncture the skin of an embryo with one pin. Insert the pin through the hole in the space between the heart and the skin (Figure 2C, solid arrow). Do not puncture the heart. Position the inserted part of this pin in the region where the incision will be made.</li>
		<li>Make a linear incision by gently rubbing the other pin held outside the skin against the inserted pin. Gently widen this incision with two pins,exposing the heart (Figure 2B&#39; and 2D&#39;, arrow). The pipette loaded with DiI-AcLDL can now directly approach the heart (Figure 2D&#39;).</li>
	</ol>
	</li>
	<li>Injection
	<ol>
		<li>Insert the tip of the loaded glass pipette into the heart and inject 50-60 nL of DiI-AcLDL solution in approximately 10 ejection pulses. Do not inject an excessive amount of solution, as it causes the heart to rupture. Check whether the heart continues to beat after the injection. After every injection, check whether the same amount of DiI-AcLDL is ejected; otherwise, the tip of the pipette may be blocked (see step 2.5).</li>
	</ol>
	</li>
	<li>Recovery and visual screening
	<ol>
		<li>Transfer the injected embryos to a new Petri dish filled with 0.1x MBS for recovery. After 5-10 min, the tadpoles should regain consciousness and start to move. At this time, the labeled vessels can be imaged. It is recommended to visually screen successfully labeled embryos under a fluorescence microscope (Figure 3A and 3B). If an insufficient amount of DiI-AcLDL is injected into an embryo, it can be injected again (Figure 3C).</li>
		<li>Discard the tadpoles that have other organs labeled (Figure 3D). Continue until the required number of successfully labeled embryos is obtained.</li>
	</ol>
	</li>
	<li>(Optional) Fixation of the embryos
	<ol>
		<li>For more thorough imaging of blood vessels, use confocal microscopy; it might be easier to fix the labeled embryos for confocal microscopy. First anesthetize the labeled embryos in 0.04% MS222 in 1x MBS (pH 7.5-8.0) and then fix them in 4% paraformaldehyde in 1X&#160;phosphate-buffered saline (PBS) for 1 h at room temperature. Fixed embryos can be stored for several days at 4 &#176;C; protect the injected samples from light to avoid photobleaching of the DiI.</li>
	</ol>
	</li>
</ol>
5. Imaging of DiI-AcLDL
<ol>
	<li>Stereoscopic imaging
	<ol>
		<li>Take photographs under a fluorescence stereoscopic microscope (Figure 3) to assess the overall morphology of the vasculature.</li>
		<li>Melt 1% agarose in 1x PBS&#160;for fixed embryos or in 1x MBS for live imaging. Pour melted agarose into a Petri dish and wait until it solidifies. Make a shallow indentation on the surface of agarose gel that will fit the embryo to be imaged when lying on its lateral side. Fill the dish with buffer (MS222 solution for anesthetized embryos or 1x PBS for fixed embryos).</li>
	</ol>
	</li>
	<li>Fluorescence microscopy (Rhodamine filter set)
	<ol>
		<li>As DiI is a green-excited and red-emitting dye, image using a standard filter set for rhodamine or spectrally related fluorophores (maximum excitation at 554 nm and emission at 571 nm). Place the injected embryos in the indentations on the agarose mold and take photographs (Figure 3).</li>
	</ol>
	</li>
	<li>Confocal microscopy 
	NOTE: For a closer analysis of vascular architecture, use confocal microscopy.
	<ol>
		<li>If using an inverted microscope, position the embryos on a glass-bottom dish. Add a few drops of 1x PBS and immobilize them by placing a cover slip on top. Remove excess PBS. If live embryos are to be imaged, use anesthetized embryos and use 0.015% MS222 in 0.1x MBS instead of PBS.</li>
		<li>For low-magnification imaging, use a 4X to 10X objective to find the area of interest; the rostral part of the posterior cardinal vein (PCV) is a useful region to investigate developmental angiogenesis (Figure 4, dashed boxes).
		<ol>
			<li>Use an acquisition setting for a red emission fluorophore (such as Rhodamine).</li>
			<li>Make z-stacked images by imaging the lateral half of an embryo. First, focus on the PCV on the lateral side near the objective and then set the lower limit of the focus at the position where the vessels nearest to the objective are in focus. Set the upper limit at the position where the medial part of the PCV being imaged can be focused. Image this entire lateral half of the embryo. Use software that stores metadata on the acquisition settings, such as x, y, and z scales, as they are required to calculate the vessel lengths.</li>
			<li>If needed, use the tile scan mode to image the entire vasculature of an embryo. Empirically determine the number of horizontal and vertical tiles.</li>
		</ol>
		</li>
		<li>For high-magnification imaging, follow the procedures above to image the rostral part of the PCV and 4 rostral-most intersomitic veins (ISVs) (Figure 4, dashed box). Adjust the number of stacks (z-stack) and tiles (tile scan) appropriately.</li>
	</ol>
	</li>
</ol>
6. Quantification of DiI-labeled Vessels
NOTE: DiI-labeled vessels can be traced manually or using software. We use &#34;Simple neurite tracer,&#34; a free ImageJ (NIH) plugin, which allows semi-automatic tracing of tube-like structures such as blood vessels and neurites12. Using this free software, the following parameters can be calculated. A detailed procedure for the use of this software is described elsewhere (https://imagej.net/Simple_Neurite_Tracer)12. Below, we use examples of the vasculature of embryos in which Tie2 signaling is inhibited or enhanced (Figure 4A-4C)5. Antisense Tie2MO-injected embryos exhibit reduces angiogenesis and shortens ISVs (Figure 4B), whereas the co-injection of constitutively active Tie2 mutant mRNA (caTie2 mRNA) over-rescues this phenotype, resulting in exuberant ISV branches (Figure 4C).
<ol>
	<li>ISV lengths
	<ol>
		<li>Using Simple neurite tracer, calculate the length of each traced vessel. Calculate the lengths of the 4 rostral-most ISVs.</li>
	</ol>
	</li>
	<li>ISV branches 
	NOTE: In wildtype embryos, only a few short branches sprout from the rostral-most ISVs at stage 42.
	<ol>
		<li>In Simple neurite tracer, trace these small vessels after making the ISV from which they originate the primary branch. Calculate the following parameters of these branches originating from the ISVs.</li>
		<li>Total branch number
		<ol>
			<li>Once all of the ISVs and the associated branches are traced, calculate the total number of branches (Figure 5B-5D).</li>
		</ol>
		</li>
		<li>Branch order
		<ol>
			<li>As Simple neurite tracer records the order of each branch (Figure 5A), calculate the number of branches for each order. The majority of vessels in wildtype embryos should be primary branches (i.e., ISVs).</li>
		</ol>
		</li>
		<li>Vein complexity index (VCI)
		<ol>
			<li>As VCI is a useful parameter for vessel complexity, which gives more weight to higher-order branches, calculate the VCI for each embryo using the formula in Figure 5A. For example, Tie2MO-injected embryos have a lower VCI compared to control (Figure 5B and 5C), and caTie2 mRNA-injected embryos have a higher VCI (Figure 5D).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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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.

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		<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 ...

visualization-quantitative-analysis-embryonic-angiogenesis-xenopus

Research

JoVE Journal

Developmental Biology

8.3K Views.  Yonsei University College of Medicine. 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.

Video: Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

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 microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

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

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

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

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

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 and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

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