Development 102, 735-748 (1988) 735 Printed in Great Britain © The Company of Biologists Limited 1988 Embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos J. DOUGLAS COFFIN and THOMAS J. POOLE Department of Anatomy and Cell Biology, SUNY Health Science Center at Syracuse, 766 Irving Avenue, Syracuse, NY 13210, USA Summary The development of the embryonic vasculature is from somatic mesoderm at the 7-somite stage to form a examined here using a monoclonal antibody, QH-1, loose plexus which moves niediad and wraps around capable of labelling the presumptive endothelial cells the developing Wolffian duct in later stages. These of Japanese quail embryos. Antibody labelling is first studies suggest two modes of origin of embryonic seen within the embryo proper at the 1-somite stage. blood vessels. The dorsal aortae and cardinal veins Scattered labelling of single cells appears ventral to the apparently arise in situ by the local segregation of somites and at the lateral edges of the anterior presumptive endothelial cells from the mesoderm. The intestinal portal. The dorsal aorta soon forms a intersomitic arteries, vertebral arteries and cephalic continuous cord at the ventrolateral edge of the vasculature arise by sprouts from these early vessel somites and continues into the head to fuse with the rudiments. There also seems to be some cell migration ventral aorta forming the first aortic arch by the 6- in the morphogenesis of endocardium, ventral aorta somite stage. The rudiments of the endocardium fuse and aortic arches. The extent of presumptive endo- at the midline above the anterior intestinal portal by thelial migration in these cases, however, needs to be the 3-somite stage and the ventral aorta extends clarified by microsurgical intervention. craniad. Intersomitic arteries begin to sprout off of the dorsal aorta at the 7-somite stage. The posterior Key words: endothelium, monoclonal antibody, cardinal vein forms from single cells which segregate vasculogenesis, QH-1, quail embryo. Introduction 1912). Subsequent minor vessels are formed by sprouting from preexisting vessels. The disadvantage The morphogenesis of the embryonic vasculature of these classic studies was that only patent vessels commences with the accumulation of presumptive could be visualized. Others have expressed the need endothelial cells (PECs) into loosely associated cords for an endothelial cell marker which could identify following their segregation from the mesoderm (re- presumptive endothelial cells just as they segregate view, Wagner, 1980). Reagen (1915) showed that from the mesoderm and begin organizing into cords blood vessels of the embryo originate within the body (e.g. Hirakow & Hiruma, 1981). The MB-1 (Peault et proper, not by invasion from the highly vascular al. 1983; Labastie etal. 1986) and QH-1 (Pardanaud et extraembryonic yolk sac. The first major blood ves- al. 1987) monoclonal antibodies fill this need as they sels, the dorsal aortae and posterior cardinal veins, label vascular endothelial cells and cells of the haema- form in situ by the segregation of mesenchymal cells topoietic lineage in embryos of the Japanese quail. from the mesoderm. This has been visualized by The sprouting form of angiogenesis has been much scanning electron microscopy (Hirakow & Hiruma, more extensively studied recently as it is the mechan- 1981; Meier, 1980). Many other vessels form by the ism by which tumours recruit a new vascular supply modification of extensive capillary plexuses as shown (Folkman, 1985). Angiogenic factors have also been by the many ink injection studies of Evans (1909, identified in developing systems. Kidney (Risau & 736 /. D. Coffin and T. J. Poole Ekblom, 1986) and brain (Risau, 1986) produce Residual unbound primary Ab was washed away with three angiogenic factors which resemble tumour angiogenic PBS changes then the secondary Ab was applied for 6-12 h factors (Shing et al. 1984; Klagsbrun & Shing, 1985). at 4°C. Any unbound secondary Ab was removed with PBS There is some controversy surrounding the origin of washes. Finally, the embryos were dehydrated in an EtOH embryonic endothelium. For example, Auerbach has series, changed to toluene and mounted on slides in proposed that the specialized endothelium of the Entellan® (VWR). brain differentiates in situ from mesenchymal stem Sections cells (Auerbach & Joseph, 1984); whereas, studies Sections were made by dissecting the embryos from the with marked cells indicate the brain endothelium yolk sac, rinsing them in PBS, then fixing them for 2h derives from the invasion of proliferating capillary minimum in Bouin's solution. After fixation, the embryos sprouts (Stewart & Wiley, 1981). Transplantations were sequentially rinsed in PBS, dehydrated in an EtOH utilizing the nuclear differences between quail and series, changed to toluene and embedded in paraffin. chick or mouse embryonic cells have revealed that the Sections were cut on a rotary microtome at 7/im and endothelium of limb buds (Jotereau & LeDouarin, mounted on albumin-coated slides. Paraffin was washed 1978; Wilson, 1983) and kidney (Ekblom et al. 1982) from the slides with toluene, then the sections rehydrated in clearly arises by invasive sprout penetration. The an EtOH series, followed by.two changes in PBS and one in segregation and directed migration of presumptive 3 % BSA for 30min. Next, the sections were stained for 2h endothelial cells and the importance of other large- in primary Ab and washed for 30min in PBS, then stained scale embryonic foldings, such as the anterior intesti- for 2h in secondary Ab and again washed in PBS. After soaking overnight at 4°C in PBS, the slides were cover- nal portal and lateral body folds, are here examined slipped with a 2 % A'-propyl gallate/80 % glycerol mixture using the monoclonal antibody QH-1 to stain quail (pH8-5) and sealed for microscopy and photography. embryos of 0-22 somites. This descriptive analysis is expanded in similar recent work (Pardanaud et al. 1987) and is a necessary prelude to an experimental Results analysis using microsurgery of the relative contri- butions of cell migration and in situ differentiation to Whole mounts and sectioned embryos were exam- the observed patterns of vascular morphogenesis. ined for immunofluorescent labelling of quail endo- Some of this work has been previously presented in thelium that highlighted the developing vasculature. abstract form (Coffin & Poole, 1986). Specifically, we studied development of the endocar- dium, the dorsal and ventral aortae, the first aortic arch, the intersomitic arteries and the cardinal veins. Materials and methods Results are summarized in Table 1. Immunocytochemistry (A) Endocardium Whole mounts and sections of Japanese quail (Coturnix Construction of the endocardium from individual coturnix japanica) were examined by using indirect immu- endothelial cells is the first step in heart development. nofluorescence to label endothelial cells. A QH-1 mono- Immunofluorescent labelling is first evident with the clonal antibody (Ab) was used first at 1:400 for whole mounts and 1:1000 for sections. This was followed by a goat appearance of PECs at the periphery of the embryo. anti-mouse FITC-conjugated IgG secondary Ab (Accurate These cells are concentrated in angiogenic sites near Biochemical, Westbury, NY) at the same concentrations as the headfold on each side of a Zacchei stage-4 the primary. Both Ab were diluted in 3 % bovine serum embryo (Fig. 1). At the 1-somite (IS) stage, the PECs albumin (BSA) in phosphate-buffered saline (PBS). begin to aggregate into capillary plexuses at the bilateral angiogenic sites and migrate mediad. Thus Whole mounts by 2S (Fig. 2), the enlarging plexuses are connected Whole mounts were prepared using techniques similar to to the extraembryonic circulation laterad, while those reported by Pardanaud et al. (1987). Embryos were mediad they grow into the pericardial coelom above removed from the yolk sac, rinsed in PBS and fixed in 4 % the anterior intestinal portal (AIP). At 2S these formalin/PBS overnight at 4°C. The fixed embryos were plexuses are considered embryonic heart primordia rinsed in PBS then permeabilized with successive changes because their location on either side of the AIP is the of (i) 30min - absolute methanol (aMeOH), (ii) 60min - aMeOH and (iii) 30min- aMeOH; all at 4 °C with constant same as the future vitelline veins and sinus venosus. agitation. After permeabilization, the embryos were rehy- The investing intraembryonic heart primordia fuse drated in an ethanol (EtOH) series (100%, 90%, 70%, at the midline of a 3S embryo. From this point of 50%, 30%, 3min each) then rinsed in PBS. Nonspecific- fusion, directly above the AIP, the ventral aorta binding sites were occupied by incubation in 3 % BSA at elongates toward the head as in a 4S embryo (Fig. 3). 4°C for 6-12h, followed by labelling of specific sites on At 6S the ventral aorta splits, and each of the two endothelial cells with the primary Ab at 4°C, 6-12 h. branches fuse with the dorsal aortae bilaterally to Quail embryonic vascular development 737 Fig. 1. A Zacchei stage-4 embryo whole mount. A cluster of PECs and individual cells are seen around the periphery of the head fold (HF) in this ventral view of the right side. Bar, 100^m. form the first aortic arches (Fig. 4). Thus by 7S, the embryonic heart lies caudal to the paired ventral aortae as the straight descending portion of a 'Y' Fig. 2. Definitive heart primordia (HP) extend medially connected at its caudal extent to the omphalomesen- through the pericardial coelom (PC) of a 2S embryo. tric or vitelline veins (Fig. 5). Notice the large PECs medially and how the HP form a From 7S to 20S, our results agree with previous diffuse capillary plexus (CP) laterally. The neural tube reports (Evans, 1912). In the area surrounding the (NT) and notochord (NC) appear as grey unlabelled pericardial coelom, large capillary strands are seen background.