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.
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.
