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.
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.
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.
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 .
2. Preparation of DiI-AcLDL Injection
3. Injection Setup
4. DiI-AcLDL Injection
5. Imaging of DiI-AcLDL
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).
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. Alternatively, a tracer mRNA (e.g., mRNA encoding EGFP) can be co-injected with MO. Additionally, drugs can be treated by bath application after hatching (early tailbud stage, around NF stage 26). Dissolve the drug in 0.1x MBS and add it to the embryos. For earlier treatment, embryos can be freed from the membranes with a pair of forceps. DiI-AcLDL can be injected into the heart around stage 33/34 to investigate the dynamics of vessel formation, but typically we inject at stage 37/38 or later (Figure 2, colored regions).
Typical examples of successful and unsuccessful injections (step 4.3) (Figure 3)
Stereoscopic imaging is appropriate for visual screening. If a sufficient amount of DiI-AcLDL is injected into the heart, the PCV should be immediately visible under a fluorescence stereoscope (Figure 3A and 3B). Insufficiently or incorrectly injected embryos are easy to identify (Figure 3C and 3D). Embryos with an insufficient amount of DiI-AcLDL (Figure 3C) can be injected again with the same dye until the vessels are clearly visible. Image successfully injected embryos under a confocal microscope, live or after fixation.
Development of the posterior cardinal vein (PCV) and the intersomitic veins (ISVs)
The PCV extends in the rostal-to-caudal direction, and this extension ends around stage 37/38. ISVs emerge dorsally from the PCV in an anterior-to-posterior wave. This wave of ISV formation begins at stage 36 and ends around stage 40/41; ISVs continue to grow and become lightly branched until stage 43 (Figure 3A and 3B).
Tie2 signaling controls developmental ISV branching (Figure 4)
The PCV and ISVs grow in a stereotypical pattern, and their development is under tight control (Figure 4A). The formation of the ISVs from the PCV can be described as angiogenesis, and therefore it is a useful model to investigate molecular mechanisms underlying angiogenesis. We introduce the result of our recent study showing that developmental signals inhibit Tie2 signaling in ISVs to limit their branching5. The knockdown of Tie2 signaling by antisense MO decreases the length and complexity of ISVs (Figure 4B), and expressing the constitutive active form of Tie2 (caTie2) causes excessive ISV branching (Figure 4C).
Quantification of ISV complexity (Figure 5)
In addition to the lengths and branch numbers of the ISVs, the "vein complexity index" (VCI) can be calculated to assess their complexity (Figure 5A). We adopted the "complexity index" developed by Cohen-Cory and colleagues, which was designed to quantify the complexity of neuronal axonal or dendritic arbors13. In this index, an embryo with more high-order branches receives a higher score than an embryo with the same number of total branches but with more low-order branches. Therefore, a higher VCI indicates that this embryo has a more complex venous network. "Simple neurite tracer" is useful software to keep track of the order and length of individual branches. It is also possible to generate "rendered paths," which is a useful way to visualize the overall architecture of the vasculature (Figures 5B-5D, right panels).
Figure 1: Timeline of the experiments. On the day of fertilization, morpholinos (MOs), DNA, RNA, or protein can be injected into fertilized eggs at NF stage 1 (st1) or 2 (st2). If injected only into the one blastomere at st2, the injected reagent will affect only one lateral side. The uninjected side can be used as a control. Co-inject a tracer (e.g., EGFP mRNA) to identify the injected side. On the second day, early tailbud-stage (~st24) embryos can be treated with pharmacological reagents by bath application. The heart is clearly visible on the third day. DiI-AcLDL can be injected into the heart and blood vessels can be imaged shortly after the injection. Use st33-37 embryos to image the dynamics of vessel growth or st42 embryos to image fully developed vessels. Please click here to view a larger version of this figure.
Figure 2: Location of the DiI-AcLDL injection at st42. Lateral (A-B') and ventral (C-D) views of the stage 42 embryo. The heart is colored in pink in A-C, in images taken from Xenbase (www.xenbase.org). Representative stereoscopic images are shown in A', B', and D. For injection, puncture the skin overlying the heart (C, solid arrow), make a linear incision, and open the skin laterally to expose the heart (red arrows). After making the incision, the heart will be clearly distinguishable for injection (D'). Scale bars = 200 µm. Please click here to view a larger version of this figure.
Figure 3: Expected results (stereoscopic images). Representative fluorescence images from stage 37/38 (A) and stage 42 (B) embryos. The left sides of the embryos are shown. The posterior cardinal vein (PCV) extends caudally, from which intersomitic veins (ISVs) branch dorsally in a rostral-to-caudal wave. Only rostral ISVs are visible at stage 37/38 (A), and most ISVs have formed by stage 42 (B). If an insufficient amount of DiI-AcLDL is injected, the PCV will not be clearly visible, even after 30 min (C). In this case, more DiI-AcLDL can be injected. If DiI-AcLDL is accidentally injected into other organs (D), discard the embryos. Scale bar = 500 µm. Please click here to view a larger version of this figure.
Figure 4: Effects of Tie2 signaling on ISV development. Tie2 gene expression was knocked down by the blastomere injection of translation-blocking antisense morpholino oligonucleotides (Tie2MO). The control morpholino (CoMO) has the same molecular weight but does not target any gene in Xenopus tropicalis. mRNA-encoding constitutively active Tie2 mutant (caTie2) was co-injected to rescue the Tie2 knockdown phenotype. In contrast to the control (A), theTie2MO injection led to a dramatic decrease in the length and number of ISVs (B). This phenotype was over-rescued by caTie2, which showed ISVs with exuberant collateral branches (C). The complexity of ISVs in regions indicated by blue dashed boxes is quantified in Figure 5. Scale bar = 200 µm. Please click here to view a larger version of this figure.
Figure 5: Quantification of vein complexity. (A) The complexity of ISV can be represented by the vein complexity index (VCI). (B-D) VCIs were calculated from the embryos shown in Figure 4. Scale bar = 200 µm. Please click here to view a larger version of this figure.
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 of vessel growth and branching can be imaged in real time4. Using the well-defined venous network, particularly the PCV and rostral-most ISVs, the effects of genetic or environmental perturbation on angiogenesis can be quantitatively assessed.
The most critical step in this protocol is the injection of DiI-AcLDL (step 4). We provide examples of successful and unsuccessful injections (Figure 3). Successfully injected embryos should be screened under a fluorescence microscope, as described in step 4.3. If the PCV is not visible 30 min after injection, not enough DiI-AcLDL was injected (Figure 3C). Unsuccessfully injected embryos may be anesthetized and injected again. In some cases, DiI-AcLDL may be injected into an incorrect location, in which case other organs will be labeled (Figure 3D). Discard incorrectly injected embryos.
The blood vessels of a successfully injected embryo will be visible shortly after injection, and the fluorescence lasts for several hours. Therefore, if the injection is performed before the formation of ISVs, their development can be imaged in a live embryo, providing a powerful tool to investigate development of the cardiovascular system in real time and in vivo4. As Xenopus has been successfully used as a model to screen vascular disrupting agents in mammals14, the experimental approach described here might be combined with pharmacological perturbation experiments and used as a screening platform to find drugs that enhance or inhibit angiogenesis.
Blood vessels can be visualized by genetic tools, as well as by the dye-based labeling methods described here. For example, transgenic Xenopus15 or zebrafish16 that express fluorescence proteins under the control of cell type-specific promoters enable the live imaging of blood and lymphatic vessels, and it is even possible to discriminate arteries from veins16. Although genetic approaches are superior and generate more consistent labeling efficiency, dye-based methods are easily accessible to most laboratories without access to such genetically engineered animals. In Xenopus, the transcardial perfusion of DiI-AcLDL results in the labeling of most endothelial cells4, and arteries and veins are not distinguishable in fluorescence images. Intriguingly, tomato lectin-fluorescein selectively labels arteries in mice, and when co-injected with DiI-AcLDL, arteries and veins can be discriminated17. Although the application was not tested in other tissues or animals, this provides a promising direction to investigate the dynamic development of arteries and veins in Xenopus.
The authors have nothing to disclose.
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 & Medical Technology Development Program of the National Research Foundation (NRF) funded by the ministry of Science, ICT & Future Planning (NRF-2013M3A9D5072551)
35mm Petri dish | SPL | 10035 | Sylgard mold frame |
60mm Petri dish | SPL | 10060 | Embryo raising tray |
Borosilicate Glass | Sutter instrument | B100-50-10 | Needle for injection |
BSA | Sigma | A3059-10G | Coating reagent |
CaCl2 | D.S.P.GR Reagent | 0.1X MBS component | |
Coverslip | Superior | HSU-0111520 | For confocal imaging |
DiI-AcLDL | Thermo Fisher Scientific | L3484 | Vessel staining solution |
FBS | Hyclone | SH.30919.02 | For storage of testis |
Fiber Optical Illuminator | World Precision Instruments | Z-LITE-Z | Light |
Ficoll | Sigma | F4375 | Injection buffer |
Flaming/Brown Micropipette Puller | Sutter instrument | P-97 | Injection needle puller |
Forcep | Fine Science Tool | 11255-20 | For embryo hatching and needle tip cutting |
Glass Bottom dish | SPL | 100350 | For confocal imaging |
hCG | MNS Korea | For priming of frogs | |
HEPES | Sigma | H3375 | Buffering agent |
Incubator | Lab. Companion | ILP-02 | For raising embryos |
KCl | DAEJUNG | 6566-4400 | MBS component |
L15 medium | Gibco | 11415-114 | For storage of testis |
L-cysteine | Sigma | 168149-100G | De-jellying reagent |
MgSO4 | Sigma | M7506 | MBS component |
Microtube | Axygen | MCT-175-C-S | For storage of testis |
MS222 | Sigma | E10521 | Anesthetic powder |
NaCl | DAEJUNG | 7647-14-5 | MBS component |
NaOH | Sigma | S-0899 | pH adjusting reagent |
Paraformaldehyde | Sigma | P6148 | Fixatives |
PBS | BIOSESANG | P2007 | Buffer for imaging |
pH paper | Sigma | P4536-100EA | For confirming pH |
PICO-LITER INJECTOR | Waner instruments | PLI-100A | For injection |
Pin | Pinservice | 26002-10 | For incision |
Pinholder | Scitech Korea | 26016-12 | For incision |
Precision Stereo Zoom Binocular Microscope | World Precision Instruments | PZMIII | For visual screening |
Standard Manual Control Micromanipulator | Waner instruments | W4 64-0056 | For microinjection |
SYLGARD 184 Kit | Dow Corning | For DiI injection | |
Transfer pipette | Korea Ace Scientific Co. | YM.B78-400 | For eggs and embryo collection |