ARTICLE |
Correspondence to: M. Luisa IruelaArispe, Molecular Biology Inst., Room 559, UCLA, 611 Charles E. Young Drive, Los Angeles, CA 90095-1570. E-mail: arispe@mbi.ucla.edu
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Summary |
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Chicken embryos are an excellent model system for studies related to vascular morphogenesis. Development in ovo allows manipulations otherwise difficult in mammals, and the use of chickenquail chimeras offers an additional advantage to this experimental system. Furthermore, the chicken chorioallantoic membrane has been extensively used for in vivo assays of angiogenesis. Surprisingly, few markers are available for a comprehensive visualization of the vasculature. Here we report the use of lectins for identification of embryonic chicken blood vessels. Nine lectins were evaluated using intravascular perfusion and directly on sections. Our results indicate that Lens culinaris agglutinin, concanavalin A, and wheat germ agglutinin can be used effectively for visualization of vessels of early chicken embryos (E2.5E4). At later developmental stages, Lens culinaris agglutinin is a better choice because it displays equal affinity for the endothelia of arteries, veins, and capillaries. The findings presented here expand our understanding of lectin specificity in the endothelium of avian species and provide information as to the use of these reagents to obtain comprehensive labeling of the embryonic and chorioallantoic membrane vasculature. (J Histochem Cytochem 51:597604, 2003)
Key Words: angiogenesis, blood vessels, chorioallantoic membrane, assays, endothelial cells
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Introduction |
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CURRENT STUDIES on the morphogenesis of the vascular system in mammals meet with the limitations imposed by intrauterine development. Few species are as well suited to manipulation of the cardiovascular system during development as chickens and quails, with perhaps the exception of zebrafish. Birds are evolutionarily closer to mammals and also offer the advantage of accessibility, because embryos can be cultured ex ovo. However, the lack of reagents for identification of blood vessels in chicken compromises the extension of these studies. Although von Willebrand factor antibodies have been used for identification of large-caliber blood vessels in chicken, this reagent does not consistently label small blood vessels (
Lectins are specific carbohydrate-binding proteins of nonimmune origin that have proven utility for visualization of blood vessels. In addition, lectins have been used to determine sites of leakage/increased permeability (
Interactions of lectins with the endothelium occur through binding to specific glycoprotein moieties on the luminal and/or abluminal surface. Probably because of the great variability of sugar moieties expressed in each species, the interaction of specific lectins with endothelial cells is frequently species-specific (
To investigate the efficacy of lectins as markers for endothelial cells in chick embryos and the chorioallantoic membrane (CAM), we have examined the binding properties of nine lectins, including those frequently used for visualization of murine and human vessels. Our study has focused on lectin binding by intravascular delivery and on tissue sections of early embryos (E2.5E7).
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Materials and Methods |
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Chicken Embryos
Fertile White Leghorn eggs (AA Laboratory Eggs; Westminster, CA) were incubated at 38C in a humidified incubator for 4872 hr. Eggs were opened into 100-mm tissue culture Petri dishes and allowed to develop in a humidified tissue culture incubator at 38C for the times indicated.
Intravital Lectin Perfusion
Fluorescein isothiocyanate (FITC)- and rhodamine-conjugated lectins (Vector Laboratories; Burlingame, CA) were diluted in 1 x PBS and filtered using a 0.22-mm Millipore filter. The solution was warmed to 37C before perfusion. Intracardiac (before E5) or vitelline vein (after E5) injection was performed with the aid of a glass needle and perfusion was achieved by cutting the main yolk artery and allowing solutions to circulate for 35 min. A total of 0.22.5 µg of lectin was delivered in 26 µl depending on the developmental stage. The injection volume was kept to a minimum to avoid rupture of vessels due to volume overload. Pressure was maintained constant by use of a micropump (Harvard Apparatus; Holliston, MA): 50 µl/min before E3 and 100 µl/min thereafter. Lectins used included concanavalin A (ConA), Griffonia simplicifolia isolectin B4 (GSL I-B4), Euonymus europaeus (EE), Lens culinaris agglutinin (LCA), Lycopersicon esculentum (LE), peanut agglutinin (PNA), Ricinius communis agglutinin I (RCA I), Ulex europaeus agglutinin I (UEA I), and wheat germ agglutinin (WGA). When co-injection of two lectins was required, preincubation was performed and monitored under the microscope for possible precipitation. Two controls were performed to ensure lectin specificity: (a) perfusion of unlabeled lectin before injection of fluorophore-conjugated lectin, resulting in complete blockade of binding by fluorophore-conjugated lectin; and (b) tagged lectin together with the cognate hapten sugar moiety (as indicated in Table 1). For these competition experiments, a concentration of 0.2 M of the appropriate sugar was used either on sections (for 2 hr at room temperature) or by intravascular perfusions (1 ml of the particular sugar before lectin injection). In these experiments we saw either a significant decrease in staining or complete inhibition.
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For the purpose of comparison, LM609 antibody (anti-vß3) (Chemicon; Temecula, CA) and anti-VEGFR2 (
Fixation and Sectioning of Embryos
After perfusion, embryos were fixed in 4% paraformaldehyde overnight at 4C and then washed with 1 x PBS three times. Embryos at or younger than E4 were mounted on histological slides using Vectashield (Vector Labs). At E5 and thereafter, embryos were sectioned at 200-µm thickness using a Vibratome (Ted Pella; Redding, CA) and mounted with Vectashield. Evaluation was performed on a MRC1024ES confocal system (Bio-Rad Laboratories; Hercules, CA) equipped with a Nikon E800 microscope and a krypton/argon laser.
Lectin Histochemistry
For evaluation of lectins by direct staining of sections, E7 embryos were fixed in 4% PFA overnight at 4C and sectioned at 200 µm using a vibratome. Sections were incubated with 0.3% Triton in 1 x PBS for 30 min and were then washed to remove excess of detergent. Lectins (5 µg/ml) were incubated for 1 hr at RT and washed extensively for 16 hr.
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Results |
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Nine lectins (ConA, GSL I-B4, EE, LCA, LE, PNA, RCA I, UEA I, and WGA) were selected to evaluate their utility for labeling embryonic chicken vessels. Table 1 includes their known carbohydrate specificity and biological source.
Initial screening was performed by intravascular perfusion of E2.5E3 chicken embryos. In preliminary experiments, we found that LCA and WGA bound well to the luminal surface of vessels, in sharp contrast to UEA I and GSL I-B4, lectins previously known for their ability to label endothelial cells of human and murine vessels, respectively. In subsequent experiments we included either LCA or WGA in combination with other lectins to ensure that perfusion was complete and to compare patterns of binding. The selection of either LCA or WGA was based on compatibility with other lectins. For example, LCA mixed with RCA I resulted in the formation of insoluble precipitates that clogged small vessels during perfusion. Therefore, formation of precipitates was determined by microscopic evaluation and centrifugation before injection into the embryo. Fig 1 shows affinity of each lectin to the endothelium of chickens at E3. For evaluation of uniformity of binding to small- and large-caliber vessels, we decided to focus in the intersomitic area. It should be stressed that although other areas are not shown, binding or lack thereof was uniform and consistent throughout the embryo. In addition to LCA and WGA, ConA (Fig 1B and Fig 1C) and RCA I (Fig 1Q and Fig 1R) bound to the vasculature of early chicken embryos quite well. LE, a lectin routinely used for perfusion of mouse embryos, did not bind as strongly as expected (Fig 1H and Fig 1I). Because it was conceivable that this profile of binding changed over time, we performed similar experiments at later developmental stages (E4, E5, E6, and E7), as discussed below.
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To compare lectin labeling efficacy with a set of known endothelial cell markers, embryos at the same developmental stage were also perfused with anti-vß3 and anti-VEGFR2 (Fig 1V and Fig 1W). Although weak binding was detected, labeling was generally not as complete or as intense as that seen with LCA, ConA, WGA, or RCA I (Fig 1). Additional control experiments included perfusion with (a) unlabeled lectin before injection of fluorophore-conjugated lectin and (b) tagged lectin together with the cognate hapten sugar moiety (as indicated in Table 1). Both these controls resulted in complete and selective blockade of lectin binding (data not shown).
In addition to intravascular delivery, we tested the performance of all nine lectins on tissue sections (Fig 2). Although LE performed poorly when delivered intravascularly, it displayed effective binding of vessels on tissue sections of older embryos (Fig 2A). Surprisingly, WGA did not bind either large- or small-caliber vessels (Fig 2B). We believe that this was the result of delivery (i.e., on sections vs intravascular), as labeling with this lectin was seen on prefixed perfused vessels (Fig 2J). Lack of binding was also seen with UEA I and PNA (Fig 2C and Fig 2D). RCA I, GSL I-B4, and EE bound to capillaries and larger vessels, albeit with lower affinity than LE (Fig 2E2G). LCA and ConA also labeled vessels on sections (Fig 2H and Fig 2I). Nevertheless, in all cases we observed significant background and frequent binding to either epithelium and/or extracellular matrix. Clearly, intravascular injection offered a more selective and intense vascular labeling.
To identify possible variations in lectin affinity that might occur as a consequence of developmental changes, LCA and WGA binding patterns were compared at several stages. Co-injection of these two lectins was performed in embryos at 2.5, 3, 4, 5, and 7 days of development (Fig 3). During early stages of development (Fig 3A3I), both lectins labeled the majority of vessels with equal affinity. However, as a result of vascular expansion, maturation, and remodeling during the developmental stages studied (E5 and E7), WGA showed a progressively more restricted affinity for larger vessels, while LCA continued to display comprehensive binding to the vasculature (Fig 3J3O). Specifically, WGA exhibited higher affinity for some medium- and large-caliber vessels in detriment to small capillaries. Compared to WGA, LCA bound to the luminal surface of large and small vessels more uniformly and revealed the complete vascular network.
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Efficiency of LCA and WGA binding in older embryos was determined on tissue sections after intravascular perfusion. Embryos (E7) were embedded in agarose for generation of 200-µm sections on a vibratome. Sections were mounted on slides and observed by confocal microscopy. Fig 4 shows a cross-section of E7 after co-injection with FITCLCA and rhodamineWGA. The evaluation confirmed previous results and demonstrated that smaller vessels are not as consistently labeled with WGA. In contrast, LCA was able to bind uniformly to endothelial surfaces of different hierarchic vascular networks. Furthermore, tissue sections allowed the clear identification of arteries and veins that appear to interact equally with LCA (Fig 4D). Interaction of venular endothelium with WGA, however, appeared much weaker than its binding to arterial endothelium at this developmental stage (Fig 4E).
Binding of WGA and LCA to CAM vasculature is shown in Fig 5. These findings were similar to those in the intraembryonic vasculature. We found that LCA labeled CAM vessels uniformly (Fig 5A, Fig 5D, and Fig 5G). Binding was detected on vessels of different caliber and variable developmental stage. In contrast, WGA was recruited more selectively to larger vessels (Fig 5H) or to areas in which the vascular plexus appeared to undergo remodeling and fusion (Fig 5B and Fig 5E). Incubation of E7 CAM vasculature with anti-vß3 showed binding to small-caliber vessels and adventitial capillaries of larger vessels (Fig 5J). Interestingly, terminal branches of small-caliber vessels were not recognized by
vß3 as they were with LCA.
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Discussion |
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Visualization of the vasculature in chicken embryos has met with some difficulties owing to the lack of specific reagents. Although PECAM is commonly used for both human and mouse vessels, the antibodies available are species-specific and do not crossreact with chicken vessels. The development of integrin antibodies has been particularly useful for functional analysis. For example, the monoclonal antibody LM 609 directed to the integrin vß3 was injected into developing quail embryos, resulting in significant disruption of capillaries and abnormal vascular patterning (
Lectins are proven tools for comprehensive visualization of the vasculature in several organs and animal species. The most successful approach has been intravascular delivery of the lectin. This method bypasses the problem of cell-type specificity because exposure of the lectin becomes limited to the luminal endothelial surface (
It is unclear which molecule(s) harbors the binding of lectins on the surface of the endothelium. Experiments performed with bovine aortic endothelial cells have indicated that WGA is able to bind to VEGFR2 (
An interesting observation was the gradual change in the affinity of WGA from a more general capillary plexus in early stages of development, to an increasingly restricted binding to larger vessels. It has been recognized that the pattern of lectin binding sites on migrating endothelial cells is both increased and altered compared to quiescent cells (
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Acknowledgments |
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Supported by a grant from the AHA Western affiliates to MLIA.
Received for publication July 10, 2002; accepted November 27, 2002.
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