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Home , Angiogenesis, Anterior cardinal vein, Aortic arches, Common cardinal veins, Dorsal aorta, Heart development

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 of the major vessel primordia in quail

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 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 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 soon forms a intersomitic , 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 , ventral aorta somite stage. The rudiments of the endocardium fuse and . 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 at the 7-somite stage. The posterior Key words: , monoclonal antibody, cardinal vein forms from single cells which segregate , 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 . 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 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 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 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 . 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 (NC) appear as grey unlabelled pericardial coelom, large capillary strands are seen background. Bar, 150/im. extending from the sinus venosus to the extraembry- onic circulation (Fig. 6). As the head and heart move farther apart the strands appear to break away and PECs and islets lying over the segmental plate on degenerate, each side of the notochord (Fig. 7). As development proceeds, more PECs are seen (B) Dorsal aortae migrating mediad and the dorsal aortae become Dorsal aorta development begins concomitant with bilateral longitudinal lines of PECs and islets. At 4S heart formation. Just below the bilateral primitive the aortae appear as vascular cords extending from angiogenic sites, PECs migrate mediad indepen- the head to just beyond the last pair of somites dently of the forming heart. These cells are destined (Fig. 3). This in situ coalescence of PECs continues as to form the paired dorsal aortae. Therefore, by the IS the aortae form their lumens and extend further into stage the PECs at each lateral angiogenic site have the head, eventually joining the first arch (Fig. 4). already been segregated for two different fates, one Caudally, the dorsal aortae elongate with the body. population directed toward and From the 7S to 12S stages, the dorsal aortae appear as another toward the dorsal aortae. The latter aggre- two large vessels bilaterally (Figs 5,8). They become gate into angiogenic islets that appear as isolated attached to the extraembryonic circulation by a capillary strands. At IS these vessels consist of a few capillary plexus at 12S, which becomes the vitelline 738 J. D. Coffin and T. J. Poole at later stages (Figs 8, 9A). At 15S the dorsal (C) Aortic arches aortae move closer together as the lateral body folds First arch development occurs at 4S-6S by fusion of adhere to close the AIP (Fig. 9B). Later, a fusion the ventral and dorsal aortae in the head. As men- occurs between the two vessels just caudal to the tioned above, the ventral aorta bifurcates at 6S and heart, forming the . the paired dorsal aortae extend well into the head of this stage embryo (Fig. 3). These two events lead to the fusion of the two ventral aortic branches with the paired dorsal aortae to form the first aortic arch. It is poorly defined at 6S (Fig. 4), but quite obvious at 7S (Fig. 5). Other nonspecific capillary strands can be seen between the ventral and dorsal aortae at 12S (Fig. 8) that might be primordia of any of the other five aortic arches, but this could not be confirmed. The internal carotid arteries are seen at 10S as sprouts from the cranial portion of the first arch (Fig. 6). These sprouts later extend rostrally into the head to become the internal carotid arteries proper (Fig. 10). Thus, the first arch is attached by the to a large capillary plexus that forms over the developing brain. The anterior cardi- nal vein and the vertebral arteries also join this plexus (Fig. 9C, D). Scattered PECs are seen over the neural tube at 15S (Fig. 11), when the cephalic plexus is first obvious (Fig. 12). By 19S the cephalic plexus is well developed, extending over the dorsal surface of

Fig. 4. First arch formation at 6S. The ventral aorta (VA) bifurcates and the branches fuse with the dorsal aortae (DA) to form the first aortic arch (AA) on each side of the neural tube (NT) in the embryonic head. Bar, 50/mi.

Fig. 3. A composite of a 4S embryo. The heart primordia (HP) have fused at the midline above the AIP and are joined laterally to the extraembryonic circulation. The ventral aorta (VA) has grown cranially from the HP fusion point. The dorsal aortae (DA) appear as paired longitudinal broken lines of islets and PECs. The DA extend through the AIP well into the head where they are slightly out of focus in this photo. Caudally the segmental plate (SP) shows some labelling. The neural tube (NT), notochord (NC), and somites (S) are in the background. Bar, 150ftm. Quail embryonic vascular development 739

Fig. 6. The cranial part of a 10S embryo. Broken vascular strands (VS) that extended from the vitelline veins (VV) and sinus venosus (SV) to the extraembryonic circulation (EC) are seen. The well-developed first aortic arches (AA) are obvious as are the embryonic heart (EH), the ventral aortae (VA), and the dorsal aortae (DA). The EH has begun to bend slightly to the right forming the bulbus chordus (BC). Note the first small sprouts of the internal carotid arteries (1CA) from the apices of the aortic arches (AA). Bar, 75j

the brain and forming a vascular ring around the avascular optic cup (Fig. 13). Fig. 5. A composite of a 7S embryo. The embryonic heart (EH) is now developing between the ventral aortae (D) lntersomitic and vertebral arteries (VA) and the vitelline veins (VV, formerly the HP). The lntersomitic arteries begin to sprout from the dorsal dorsal aortae (DA) are large longitudinal vessels growing aortae at about 7S. Once the intersomitic arteries caudally while maintaining a diffuse connection with the have reached the medial surface of the somite they extraembryonic circulation. The aortic arches (AA) are obvious in the head but the cranial portions of the DA turn right angles to grow over that surface, fuse with are out of the focal plane. Bar, 200 jim. other sprouts and form the vertebral arteries adjacent to the neural tube. Thus the vertebral arteries grow caudad as longitudinal links between the intersomitic arteries at their most medial extent. There were some 740 J. D. Coffin and T. J. Poole ICA

Fig. 7. A IS embryo showing PECs that segregated from the primitive angiogenic sites laterally to migrate medially, form angiogenic islets (ISL), and develop into the dorsal aortae. Bar, 75 fim.

indications that vertebral artery rudiments already exist cranially before the intersomitic arteries start to form, but this could not be confirmed. The first few intersomitic and interlocking ver- tebral arteries form between the second and third somites of an 8S embryo (Fig. 14). In the head, at 10S the vertebral arteries extend farther rostrally than the Fig. 8. This composite of a 12S embryo shows the large dorsal aortae (DA) running from the aortic arches (AA) most cranial somite. Other presumptive intersomitic cranially to a level caudal to the last somite where they arteries are seen as incomplete sprouts from the aorta show a firm attachment to the extraembryonic circulation between more caudal somites (Fig. 15). As develop- by capillary plexuses (CP) that will become the vitelline ment continues to the 15S stage, vertebral arteries arteries. Note the avascular area lateral to each DA that grow rapidly past the more caudal somites as their contained many diffuse earlier (Figs 3, 5). The embryonic heart has a distinct bulbus chordus (BC). corresponding intersomitic arteries reach the medial Internal carotid artery (ICA) sprouts are seen off the tops edge of the somites (Figs 9B, 16). Meanwhile, at their of the aortic arches (AA). Also note the intersomitic cranial extent, the vertebral arteries grow well into arteries (ISA) running between the DA and the vertebral the head and join the plexus there (Fig. 11). arteries (VTA). Bar, 250^m. Quail embryonic vascular development 741

PCV

VVLA

Fig. 9. Transverse sections of a 15S embryo. Bars, 150^m. A is from a caudal area through the (VLA) in the splanchnopleure. In the somatopleure, labelled capillary strands (CS) and plexuses are seen that will contribute to the . B is a plane just caudal to the heart. The large vitelline veins (W) are shown as are the AIP, the posterior cardinal veins (PCV), the vertebral arteries (VTA), and the dorsal aortae (DA). In 9C, the section is through the (TA) of the heart. The dorsal aortae (DA) and cephalic plexuses (CeP) are also shown. Further cranially, D shows the ventral aortae (VA) after they exit from the heart. Also labelled are the DA that will join the VA by the first aortic arch; and the cephalic plexus (CeP) into which the , vertebral artery and the internal carotid artery flow.

(E) Cardinal veins the posterior cardinal vein grows caudad from the Cardinal vein PECs can be seen early, at 5S in the common cardinal vein, these islets contribute to the somatopleure, between the mesoderm and the ecto- extension of it. At 20S the posterior vein is a fine derm (Fig. 17). These cells slowly migrate mediad elongate capillary plexus closely associated with the from each side of the embryo toward the sites of the Wolffian duct (Fig. 19B). future common cardinal vein or duct of Cuvier. Soon after the first segment of the vertebral artery forms at 8S, processes are seen running from these PECs Discussion between the somites (Fig. 14). These processes join the vertebral artery mediad, presumably to become Use of the monoclonal antibody QH-1 has begun the intersomitic veins dorsal to the intersomitic arter- reconsideration of classical models of embryonic ies (Fig. 18). Laterally, PECs continue to aggregate vascular development. Pardanaud et al. (1987) and as the common cardinal vein that forms in situ Poole & Coffin (1988) have recently reported on the (Fig. 16). By 20S the common cardinal vein is well early formation of the heart, dorsal aorta and pos- developed, as are the intersomitic veins and arteries terior cardinal veins. We have carried these studies that connect the aorta, the vertebral artery and the considerably farther to describe the origins of the cardinal veins (Fig. 18). vitelline arteries and veins, ventral aorta, first aortic Farther caudad at the level of the segmental plate, arch, internal carotid arteries, cephalic plexus, ver- nonspecific angiogenic islets form capillary strands on tebral arteries, intersomitic arteries and veins, and the lateral plate beneath the ectoderm (Fig. 19A). As the cardinal veins. 742 /. D. Coffin and T. J. Poole

Fig. 10. Internal carotid artery (ICA) sprouts from the Fig. 11. Some individual angiogenic islets (ISL) are first aortic arches (AA) that have begun to stretch into labelled sitting on the dorsal surface of the embryonic the head of a 15S embryo. Notice the conspicuous ventral brain (EB) at 15S. These may contribute to a developing aortae (VA). Bars, 50/im. cephalic plexus that forms in the head. Notice the vertebral arteries (VTA) adjacent to the neural tube (NT), the anterior cardinal vein (ACV) more laterally However, of equal import to this new descriptive and the optic cups (OC) destined to form the eyes. Bar, anatomy of the embryonic vasculature are the ques- 75 /zm. tions that arise about its morphogenesis. We envision embryonic vascularization as a programmed process in development where the principal blood vessels are then the epicardium (Manasek, 1968). It appears that formed by differentiated PECs that undergo directed the endothelium establishes the pattern for the deve- cell migration to form definitive structures in situ. loping heart and blood vessels. The QH-1 mono- Thus it is essential to understand how the PECs clonal antibody proved quite useful as an endothelial undergo cell-cell recognition and adhesion, use cell- marker. The QH-1 epitope is expressed soon after the matrix interactions and differentially express certain PECs segregate from the lateral plate. Our results genes during angiogenesis to form vascular patterns. show that blood vessels, individual cells and angio- We must, therefore, use modern techniqes to re- genic islets, are conspicuously labelled. The definitive examine the origin and behaviour of PECs and the vasculature then results from the growth and modifi- interaction between other developmental processes cation (i.e. regression, selective cell death, etc.) of and . the early vessels described here. Hence the basic The definitive embryonic heart consists of three morphological data, illustrated in Fig. 20, that are layers: an inner endocardium made of endothelial necessary for further studies on embryonic vascular cells, a muscular myocardium and an outer epicar- development have been established. dium. In the chick, the endocardium has been shown Ink injection studies had shown the embryonic to develop first, followed by the myocardium and heart forming at the 6S-8S stages by the fusion of Quail embryonic vascular development 743 large patent blood vessels at the midline (Evans, 1912). The dorsal and ventral aortae and other embryonic blood vessels were thought to form shortly thereafter, originating from these initial vessels or by the modification of existing capillary plexuses (Evans, 1909). The data presented here and by Pardanaud et al. (1987) and by Poole & Coffin (1988) indicate that the heart and dorsal aorta form early. Heart primor- dia are undergoing directed growth toward the mid- line of the pericardial coelom at IS. Other PECs destined for the dorsal aorta segregated from the initial cluster of individual PECs as early as Zacchei stage 4. We are not certain, however, as to what caused the separation of PECs for the early heart and for the

Fig. 14. A high magnification dorsal view of an 8S whole Fig. 12. In this 15S whole mount, the anterior cardinal mount near the site of the forming common cardinal vein. veins (ACV) are seen growing up over the dorsal surface PECs migrate in the somatopleure toward this focal point of the brain to contribute to the cephalic plexus that will and send processes between the somites that join the cover the entire surface at later stages. At the bottom of vertebral artery (VA). We presume that these processes the photo, a flexure (F) between the metencephalon and form the intersomitic veins dorsal to the intersomitic the rhombencephalon is noticeable. Bar, 15 (im. arteries. Bar,

dorsal aorta from each other. It could be that the two populations differentially express an epitope that leads to altered behaviour between them. Alterna- tively, differences in the surrounding may direct the PECs onto separate migratory pathways. The extent of migration of PECs is unclear and we are currently examining this question by using blockages and transplants. The physical force of the forming body folds could also present barriers that favour migration in certain directions. The mechan- ism for the directed migration of PECs in embryonic vasculogenesis is unknown, but it probably involves a combination of some or all or the forementioned Fig. 13. This lateral view of the head of a 19S embryo events. shows the well-developed cephalic plexus (CeP) that Another area where populations of PECs separate surrounds the optic cup (OC) and extends up over the is in the development of the dorsal aortae and dorsal surface of the brain. Bar, 50^m. posterior cardinal veins. The cells of the dorsal aortae 744 /. D. Coffin and T. J. Poole

Fig. 15. A ventral view of a 10S embryo. Lateral PECs Fig. 16. Ventral view of a 15S embryo showing the are migrating toward an area where the common cardinal developing (CCV), the vertebral veins are forming. Intersomitic arteries (ISA) are slightly arteries (VTA), some intersomitic arteries (ISA) and ISA out of focus as are some forming intersomitic veins (ISV). sprouts. Bar, 60 ^m. Notice the vertebral arteries (VTA) medially and the ISA sprouts out of the focal plane caudally between the somites (S). Bar, 100nm. addition, we often noticed labelling in the segmental plate, which could have some significance in aorta, migrate between the endoderm and mesoderm in the intersomitic artery or cardinal vein development. splanchnopleure, while cardinal vein PECs migrate Vascular morphogenesis in the embryo seems to between the ectoderm and mesoderm in the somato- occur via two different mechanisms, by sprouting pleure. On a whole mount of 4S-10S embryos, the from existing blood vessels or capillary plexuses, or two populations are seen in the same field, but in by the in situ aggregation of migrating PECs. These different focal planes. The question remains as to two types of formation are evident in when these populations separate. Before the forma- early stage embryos (1S-3S). The heart develops by tion of the first somite, the lateral plate mesoderm is sprouting of the lateral heart primordia and the dorsal divided by the intraembryonic coelom, creating a aorta by the in situ aggregation of migrating PECs. space between the two layers with medial and lateral We report here that the patterns for the embryonic points of communication. We do not know whether heart, the ventral aortae, the first aortic arch and the PECs are trapped in their respective layers when the dorsal aortae are all established by the 6S stage of mesoderm splits, or if they differentiate from the development. Other embryonic vessels will sub- mesodermal layers after the coelom forms. The PECs sequently develop from these vessels. Intersomitic could also segregate at the periphery and differen- arteries sprout from the dorsal aortae, beginning at tially migrate into the splanchnopleure for the aorta, the 7S stage. Internal carotid arteries sprout from the and into the somatopleure for the cardinal vein. In apices of the first aortic arch at about 10S. However, Quail embryonic vascular development 745

the cardinal veins may or may not depend on these Table 1. Sequence of embryonic vascular vessels for their development. development The above discussion alluded to the segregation of Stage cardinal vein and aortic PECs. But cardinal vein (somites, S) Description formation proceeds much more slowly than the devel- 0 PECs appear at bilateral angiogenic sites near opment of the dorsal aortae. The cardinal vein headfolds. pnmordia appear as large islets over the lateral plate IS PECs aggregate into angiogenic islets then into mesoderm until the 7S stage. They then form the plexuses at each site as heart primordia; other common cardinal vein near the AIP as described, but PECs from site migrate caudomedially for dorsal aortae. 2S Heart primordia begin medial sprouting from each side through pericardial coelom above AIP; caudally migrating PECs begin to form islets that align as bilateral craniocaudal dorsal aortae primordia. 3S Heart primordia reach midline to fuse; dorsal aortae as broken lines of islets extend into headfold. 4S Ventral aorta sprouts from point of fusion of heart primordia and grows craniad; dorsal aortae appear as solid lines from head to segmental plate. 5S Ventral aorta has bifurcated at its cranial extent and grows toward dorsal aortae; cardinal vein PECs are apparent and begin to migrate medially. 6S Ventral and dorsal aortae fuse in head to form first arches; cardinal vein PECs migrate to primitive angiogenic sites. 7S Intersomitic arteries begin to sprout from dorsal aorta between cranial somites; cardinal vein PECs reach area dorsal to intersomitic artery sprouts. 8S First and second intersomitic arteries link medially to vertebral arteries; cardinal vein PECs send processes between somites to link with vertebral artery segments. 9S Heart begins to bend to the right to form bulbus Fig. 17. A dorsal view of a 5S embryo showing cardinal chordus; more intersomitic arteries form and vein PECs and angiogenic islets (ISL) in the vertebral artery lengthens; intersomitic veins begin to form from cardinal vein PEC processes; somatopleure. In the background are the developing common cardinal vein plexuses form. dorsal aortae and the extraembryonic circulation. Bar, 10S Heart bends farther to right; dorsal aortae 175 fim. lengthen; vitelline artery formation begins; internal carotid arteries sprout from first arch. 12S Heart has complete right bend and begins left bend more craniad; dorsal aorta attached to extraembryonic circulation by vitelline artery plexus; common cardinal veins are capillary plexuses. 15S Heart is 'S' shaped with right and left bends; dorsal aortae begin to fuse midline under AIP; common cardinal veins as large plexuses that anterior and posterior cardinal veins are extending from; several nonspecific islets have moved medially toward forming posterior cardinal veins; vertebral arteries extend well into head to attach to cephalic plexus. 20S Heart is convoluted and compact in pericardial coelom; dorsal aortae are fused in abdomen; large plexus over brain connected to internal carotid Fig. 18. Lateral view of a 20S embryo at the level of the arteries; posterior cardinal veins are thin plexuses common cardinal vein (CCV) that is well developed at near pronephros that extend caudad from common this stage. The head would be to the right of this photo. cardinal veins; caudal islets attaching to cardinal Attached to the CCV are some intersomitic veins (ISV) veins. that extend to the vertebral artery (VTA) above. Out of the focal plane are some intersomitic arteries (ISA) in the background. Bar, 50 ^m. 746 J. D. Coffin and T. J. Poole

Fig. 19. Dorsal views of a 15S (A, bar, 100^m) and of a 20S (B, bar, 50/.im) embryo, at the level of the caudal extent of the developing posterior cardinal vein, near the segmental plate (SP). As shown in A, the posterior cardinal vein elongates caudally from the common cardinal vein and is joined by PECs and islets (ISL) that migrate medially from the lateral plate in the somatopleure. A fine cardinal vein plexus (CVP) is thus formed, shown in B, that surrounds the pronephros and sends strands between the somites to contribute to the vasculature there. the islets remain unincorporated farther caudad. PEC recruits other mesenchymal cells as it migrates. However, when the pronephric duct begins to move But the PECs seem capable of forming islets either caudad, these islets form plexuses near the duct, and before or after they reach their definitive position. then merge with the developing posterior cardinal Whole mounts and sectioned tissues of early-stage vein. There are probably morphogenetic interactions embryos stained with QH-1 proved useful for describ- between the cardinal vein and pronephros, but their ing the early events of embryonic vascular develop- role in the formation of either structure is uncertain. ment. This process involves two mechanisms for Studies using scanning electron microscopy in con- neovascularization. One method is by in situ localiz- junction with immunofluorescence have yielded some ation of migrating PECs to a vascular cord that then preliminary data on PEC behaviour (Poole & Coffin, 1988). It seems that the PECs migrate over the1 enlarges and forms a lumen as a definitive blood surface of the mesoderm until they recognize a vessel, e.g. the dorsal aortae. The second method is particular stimulus that causes them to become seden- by sprouting of existing vessels, e.g. the intersomitic tary and contribute to a blood vessel. They may arteries. These methods employ directed cell mi- migrate as individual cells, or as angiogenic islets. gration and selective cell-cell and cell-matrix These islets appear as small clusters of PECs that are phenomena to guide the migration of PECs and capable of migration as a group. It is unknown determine the location of the vessels. However, many whether the islet cells are clonal, the result of PEC questions remain for future studies. Use of microsur- mitosis during migration or whether the migrating gery is proving useful for understanding how the Quail embryonic vascular development 141

B

Fig. 20. Diagrams summarizing the morphogenesis of the major vessel primordia: (A) Zacchei stage 4, (B) one pair of somites, (C) two pairs of somites, (D) four pairs of somites, (E) six pairs of somites, (F) twelve pairs of somites. patterns are formed and what types of interactions Biology of Endothelial Cells (ed. E. A. Jaffe), pp. are involved in embryonic angiogenesis. 393-400. Boston: Martinus Nijhoff. COFFIN, J. D. & POOLE, T. J. (1986). Embryonic vascular development. /. Cell Biol. 103, 195a. We thank Paul Kitos (Kansas University) and Clayton A. EKBLOM, P., SARIOLA, H., KARKIKNEN, M. & SAXEN, L. Buck (Wistar Institute) for the QH-1 monoclonal antibody, (1982). The origin of the glomerular endothelium. Cell and Marisa Martini for technical assistance. This work was Differ. 11, 35-39. supported in part by a Grant-in-Aid from the American EVANS, H. M. (1909). On the development of the aortae, Heart Association, Upstate New York Chapter. cardinal and umbilical veins, and the other blood vessels of vertebrate embryos from capillaries. Anal. Rec. 3, 498-518. References EVANS, H. M. (1912). The development of the vascular system. In Human , vol. II (ed. F. Keibel AUERBACH, R. & JOSEPH, J. (1984). Cell surface markers & F. P. Mall), pp. 570-709. Philadelphia: J. P. on endothelial cells: a developmental perspective. In Lippincott Co. 748 /. D. Coffin and T. J. Poole

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