Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

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 .

1. Induction of ovulation
   1. 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).
   2. 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).
2. 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.
   1. Natural mating
      1. 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).
   2. In vitro fertilization (IVF)
      1. 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.
      2. Make 0.4% Tricaine methanesulfonate (MS222) for euthanasia and inject 300 µL 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.
      3. 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's L-15 Medium (60%) supplemented with 10% fetal bovine serum (FBS); a detailed procedure is described elsewhere¹⁰.
      4. 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 °C in a tightly sealed tube and used for IVF the next day.
      5. 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.
      6. 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'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.
3. (Optional) Injection of antisense morpholino oligonucleotides (MOs) and/or mRNAs
   1. To investigate the effect of a gene of interest on vessel formation, perform the microinjection of antisense MO and/or mRNAs.
   2. 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.
   3. 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.
   4. 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).
   5. 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.
4. Raising
   1. Raise the embryos at 23 °C. Although the speed of development can be slowed down or accelerated by using temperatures below or above 23 °C, respectively (range: 16-26 °C), it is recommended to raise them at 23 °C.
5. (Optional) Treatment with pharmacological reagents
   1. For pharmacological manipulations, administer drugs to the developing embryos by bath application.

2. Preparation of DiI-AcLDL Injection

1. 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.
2. Store DiI-AcLDL stock solution at 4 °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.
3. Fill the prepared glass pipette with a suitable amount of DiI-AcLDL solution (~5 µL) using a microloader.
4. 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.
5. 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 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.

3. Injection Setup

1. Sylgard mold
   1. 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.
2. Anesthesia
   1. 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 µL) and check the pH using pH paper.
   2. Wait until the transferred embryos stop moving. Touch the dorsal fins of the embryos and inspect whether they respond to confirm complete anesthesia.
3. Positioning embryos for injection
   1. 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°) (Figure 2D).
   2. Fill the mold with MS222 solution and place the embryos in the mold.

4. DiI-AcLDL Injection

1. Incision of skin overlying the heart
   1. 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).
   2. 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.
   3. 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' and 2D', arrow). The pipette loaded with DiI-AcLDL can now directly approach the heart (Figure 2D').
2. Injection
   1. 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).
3. Recovery and visual screening
   1. 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).
   2. Discard the tadpoles that have other organs labeled (Figure 3D). Continue until the required number of successfully labeled embryos is obtained.
4. (Optional) Fixation of the embryos
   1. 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 phosphate-buffered saline (PBS) for 1 h at room temperature. Fixed embryos can be stored for several days at 4 °C; protect the injected samples from light to avoid photobleaching of the DiI.

5. Imaging of DiI-AcLDL

1. Stereoscopic imaging
   1. Take photographs under a fluorescence stereoscopic microscope (Figure 3) to assess the overall morphology of the vasculature.
   2. Melt 1% agarose in 1x PBS 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).
2. Fluorescence microscopy (Rhodamine filter set)
   1. 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).
3. Confocal microscopy
   NOTE: For a closer analysis of vascular architecture, use confocal microscopy.
   1. 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.
   2. 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).
      1. Use an acquisition setting for a red emission fluorophore (such as Rhodamine).
      2. 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.
      3. If needed, use the tile scan mode to image the entire vasculature of an embryo. Empirically determine the number of horizontal and vertical tiles.
   3. 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.

6. Quantification of DiI-labeled Vessels

NOTE: DiI-labeled vessels can be traced manually or using software. We use "Simple neurite tracer," 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).

1. ISV lengths
   1. Using Simple neurite tracer, calculate the length of each traced vessel. Calculate the lengths of the 4 rostral-most ISVs.
2. ISV branches
   NOTE: In wildtype embryos, only a few short branches sprout from the rostral-most ISVs at stage 42.
   1. 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.
   2. Total branch number
      1. Once all of the ISVs and the associated branches are traced, calculate the total number of branches (Figure 5B-5D).
   3. Branch order
      1. 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).
   4. Vein complexity index (VCI)
      1. 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).