Developmental Angiogenesis: Quail Embryonic Vasculature

Scanning Microscopy Volume 2 Number 1 Article 41 8-31-1987 Developmental Angiogenesis: Quail Embryonic Vasculature T. J. Poole S.U.N.Y. Health Science Center at Syracuse J. D. Coffin S.U.N.Y. Health Science Center at Syracuse Follow this and additional works at: https://digitalcommons.usu.edu/microscopy Part of the Biology Commons Recommended Citation Poole, T. J. and Coffin, J.. D (1987) "Developmental Angiogenesis: Quail Embryonic Vasculature," Scanning Microscopy: Vol. 2 : No. 1 , Article 41. Available at: https://digitalcommons.usu.edu/microscopy/vol2/iss1/41 This Article is brought to you for free and open access by the Western Dairy Center at DigitalCommons@USU. It has been accepted for inclusion in Scanning Microscopy by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. Scanning Microscopy, Vol. 2, No. 1, 1988 (Pages 443-448) 0891-7035/88$3.00+.00 Scanning Microscopy International, Chicago (AMF O'Hare), IL 60666 USA DEVELOPMENTALANGIOGENESIS: QUAIL EMBRYONIC VASCULATURE T.J. Poole* and J.D. Coffin Department of Anatomy and Cell Biology, S.U.N.Y. Health Science Center at Syracuse, Syracuse, NY 13210 (Received for publication March 27, 1987 , and in revised form August 31, 1987) Abstract Introduction We have examined the segregation and early Early in this century the or1g1n of embry morphogenesis of the embryonic vasculature by onic vascular endothelium was debated. One side using a monoclonal antibody for immunofluores believed these cells originated in the extra cence and by scanning electron microscopy. This embryonic yolk sac and invaded the embryo and antibody labels the presumptive endothelial the other side thought they arose locally within eel ls (PECs) as they segregate from mesoderm. the embryo. The morphology of the embryonic Similar embryos prepared for SEM revealed finer vasculature was investigated extensively by details of how these segregated cells interact injecting opaque liquids like ink (Evans, 1909). to form the rudiments of the major blood ves The disadvantage of these classic studies was sels. Here we concentrate on the development of that only patent vessels could be visualized. the dorsal aortae and the posterior cardinal This did not reveal much about the early veins. The dorsal aortae form from single PECs segregation and organization of these vessels. which segregate from the lateral mesoderm and By isolating fragments of chick embryos, Reagan aggregate into a loose cord ventral to the (1915) was able to show that blood vessels of somites. These cells become more closely the embryo originate within the body proper, not associated and a lumen forms. The posterior by invasion from the highly vascular embryonic cardinal veins form from a loose plexus of cells yolk sac. segregated from the lateral mesoderm on its Meier (1980) and Hirakow and Hiruma (1981) dorsal surface. These ce lls become intimately were the first to use scanning electron micros associated with the Wolffian ducts. copy to look at early blood vessel formation. The first major blood vessels, the dorsal aortae and posterior cardinal veins, form in situ by the segregation from the mesoderm of mesenchymal cells. They observed single cells segregating from the mesoderm and joining into loose cords or plexuses to form the primordia of the major embryonic blood vessels. They could not identify with certainty, however, which cells were destined to become endothelium in the earliest stages. We have reexamined early embryonic vascul ogenes is taking advantage of a monoclonal antibody, QH-1 (Pardanaud et al., 1987), which labels quail endothelium and other cells of the hematopoietic lineage. This anti body was obtained by immunizing mice with bone marrow cells obtained from 12 day quail embryos. The specificity of this antibody resembles another monoclonal antibody, MB-1 obtained by KEY WORDS: Endothelium, vascular development, immunizing mice with the quail µ chain from quail, blood vessels, antibodies, morphogenesis. plasma (Peault et al., 1983). Some of the labelling studies have been previously presented in abstract form (Coffin and Poole, 1986). *Address for correspondence: Materials and Methods Department of Anatomy and Cell Biology S.U.N.Y. Health Science Center at Syracuse Fertile quail (Coturnix coturnix japonica) 766 Irving Avenue eggs were supplied by the Cornell University Syracuse, NY 13210 Phone No. (315)473-4207 Poultry Science Department. For SEM, embryos 443 T.J . Poole and J.D. Coffin were fixed in 2.5% glutaraldehyde and 2% The posterior cardinal vein develops paraforma l dehyde in O.15M cacodyl ate buffer, pH beneath the ectoderm as a loose plexus whi~h 7 .4. Endoderm or ectoderm was peeled off with eventually migrates medially and becomes closely the aid of fine forceps and sharpened tungsten associated with the Wolffian duct. Figure 8a is wire. The embryos were postfixed in 1% osmium a SEMof a 20 somite embryo from which the ecto tetroxide (in O.lM cacodylate buffer, pH 7.4) derm has been peeled off. The posterior tip of and critical point dried from liquid carbon the Wolffian duct is marked by an arrow. Figure dioxide after passage to absolute ethanol. 9 is an immunofl uorescent view of a similar After sputter coating with gold/palladium, the area. At higher magnifications (Figure 8b), one samples were photographed on a Hitachi S-520 at observes the close association of PECs in this 20kV. plexus as they approach the Wolffian duct. Embryos for antibody labelling as whole Eventually the PECs cluster around the Wolffian mounts were fixed overnight with 10% formalin in duct as can be seen in Figure 10 taken at a more phosphate-buffered saline (PBS). After washing anterior level of a 20 somite embryo. in PBS, embryos were permeabilized in cold absolute methanol in 3 changes with constant Discussion agitation for 3 h. The permeabilized embryos were rehydrated in a graded ethanol series, By utilizing both antibody labelling rinsed in PBS and preincubated overnight in a 3% procedures and scanning electron microscopy, we s•Jlution of bovine serum albumin in PBS. have been able to observe and interpret the Embryos were incubated for 12 h in a 1 :200 earliest stages of vascular development. The dilution of QH-1 ascites fluid, rinsed in PBS dorsal aorta2 arise from eel ls which segregate and incubated with fl uorescei n-conj ugated goat singly frorn the lateral mesoderrn an<l agyregate anti-mouse immunoglobin (Cappel, diluted 1:200), to form longti udinal cords at the lateral edges dehydrated and mounted in Entellan mounting of somites and segmental plates. The posterior medium (VWR). They were photographed on a Leitz cardinal veins form from cells which segregate Orthoplan microscope with epifluorescent from the somatopleure confirming the suggestion illumination. of Hirakow and Hiruma (1981). They form from loose plexuses which move medially and become Results associated with the Wolffian ducts. Vascular development can occur by two Figure la is a scanning electron micrograph distinct morphogenetic processes: 1. in situ (SEM) of a 3 somite quail embryo with the endo aggregation of groups of mesenchymal ceiTsancf derm removed. Scattered single eel ls can be 2. sprouting from pre-existing vessels (Wagner, seen just lateral to the somites and a plexus 1980). We have shown that the posterior further laterally. Figure lb is an immuno cardinal veins and dorsal aortae form by the fl uorescent picture of the same stage embryo first of these processes. The formation of the showing that these eel ls stain with the mono intersegmental arteries occurs by the second clonal antibody, QH-1, and are presumptive process, sprouting off the dorsal aortae. The endothelial cells (PECs). Figure 2 shows a sprouting form of angiogenesis has been studied similar area of a 5 somite embryo revealing that more extensively as it is the mechanism by which in just over 3 h many more PECs have segregated tumors recruit a new vascular supply (Folkman, from the mesoderm. The cells adj a cent to the 1985). We are extending the descriptive analy somites soon become organized into loosely sis in this paper to other embryonic blood associated cords, the rudiments of the dorsal vessel s and utilizing microsurgery and trans aortae. Figure 3a shows the dorsal aorta plantation techniques to reveal the relative rudiment and an extensive plexus which has importance of these two processes in establish formed in more lateral regions of a 6 somite ing embryonic vascular patterns. embryo. Figure 3b is the corresponding antibody image. Shortly thereafter, the dorsal aortae Acknowledgements enlarge and acquire a continuous and sealed off lumen. Figure 4a is a SEMof a 7 somite embryo. We thank Marisa Martini for technical The dorsal aorta on the left side is a broader, assistance, Nancy Snyder for expert typing, and more continuous cord. In Figure 4b is a 7 Paul Kitos and Clayton Buck for the QH-1 anti somite embryo labelled with QH-1 showing the body. This work was supported in part by a organization of the dorsal aortae and their grant from the American Heart Association, relation to the continuous plexus more later Upstate New York Chapter. ally. Figure 5 is the dorsal aorta of another 7 somite embryo at higher magnification. When the References dorsal aorta is fractured and looked at in cross section (Figure 6), one observes not a single Coffin JD, Poole TJ (1986) Embryonic lumen but many small ones with thin processes in vascular development. J. Cell Biol. 103: 195a. between. Soon after it becomes possible to Evans HM (1909) On the development of the fracture open the dorsal aorta to look at its aortae, cardinal and umbilical veins, and the l uminal surface. In this example (figure 7) other blood vessels of vertebrate embryos from from a 9 somite embryo the beginnings of sprouts capillaries.

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