THE ANATOMICAL RECORD 235:261-274 (1993) Early Formation of the Vascular System in Quail Embryos M.C. DERUITER, R.E. POELMANN, M.M.T. MENTINK, L. VANIPEREN, AND A.C. GITTENBERGER-DE GROOT Department of Anatomy and Embryology, University of Leiden, Leiden, The Netherlands ABSTRACT The relation between vascular development and translocation of the splanchnic mesodermal layers was studied in presomite to 20-somite quail embryos by scanning electron microscopy. In addition, serially sectioned embryos were stained immunohistochemically with monoclonal antibodies (aQH1 or aMB1) specific for endothelial and hemopoietic cells. By the formation of the foregut the anterior borders of the two splanchnic mesodermal layers of a presomite embryo are translocated to the lateral and ventral sides of the foregut and fuse in the ventral midline of a 4-somite embryo. Meanwhile the splanchnic mesoderm differentiatesinto a splanchnic mesothelial layer and a plexus of endothelial cells, facing the endoderm. From 4 somites onward the foregut is covered by a single endothelial plexus. At first the endothelial precursors bordering the anterior intestinal portal and those in the area of the ventral mesocardium lumenize, subsequently giving rise to the endocardium of the heart tube. Hereafter, the pharyngeal arch arteries and the dorsal aortae develop from the remaining precursors. During formation of the pharyngeal arches, the pharyngeal arch arteries maintain their connections with the splanchnic plexus through the developing ventral pharyngeal veins. After disappearance of the dorsal mesocardium, the midpharyngeal endothelial strand, which is a longitudinal strand of proendocardial cells, remains connected to the foregut. This strand will contribute to the formation of the pulmonary venous drainage into the left atrium. A bilateral accumulation of cardiac jelly developing between the promyocardium and proendocardial plexus only suggests that the heart develops from two tubes. The proendocardial layer, however, is not divided by the ventral mesocardium but initially forms just one endocardia1heart tube. C 1993 Wiley-Liss, Inc. Key words: Angiogenesis, Coelom, Embryo, Endocardium, Mesocardium, Myocardium, Pharyngeal arch arteries, Quail, Scanning Electron Microscopy, Somatic mesoderm, Splanchnic mesoderm, Vasculogenesis In current descriptions on the development of the embryonic vascular system, two possible mechanisms are distinguished: vasculogenesis and angiogenesis. Vasculogenesis is defined as the differentiation of mesodermal cells into endothelial precursors, which assemble to form endothelial-lined blood vessels (Poole and Coffin, 1989, 1991; Risau, 1991). Poole and Coffin (1991) subdivide vasculogenesis into two types. On the one hand, the dorsal aortae and the yolk sac vessels develop from endothelial precursors, which differentiate from the mesoderm close to where they assemble into endothelial-lined vessels (vasculogenesis type I). On the other hand, the vitelline veins and the endocardium, for instance, derive from endothelial precursors, which migrate as single cells from the yolk sac to the site of vessel formation before they assemble (vasculogenesis type 11). 0 1993 WILEY-LISS. INC. Angiogenesis is the other mechanism involved in vessel formation. Blood vessels, e.g., the dorsal intersegmental arteries (Poole and Coffin 1991), the second to the sixth pair of pharyngeal arch arteries (Bockman et al., 1990), and vessels in the neural epithelium (Noden, 1991), grow by sprouting and branching from existing vessels. A new sprout extends into the surrounding tissue as a coherent structure by mitosis of migratory endothelial cells at its tip (Ausprunk and Folkman, 1977; Noden, 1991; Poole and Coffin, 1989). Angiogenesis seems to be more prevalent in the em- Received January 30, 1992; accepted June 2, 1992. Address reprint requests to Prof. Dr. A.C. Gittenberger-de Groot, Department of Anatomy and Embryology, University of Leiden, P.O. Box 9602, 2300 RC Leiden, The Netherlands. 262 M.C. DERUITER ET AL. bryo than vasculogenesis and will occur during the entire lifespan (Risau, 1991). In addition, angiogenesis, in contrast to vasculogenesis, is not restricted or closely related to the association of mesoderm with endoderm, as in vessel formation in liver, lungs, stomach, intestine, and pancreas (Pardanaud et al., 1989, Sherer, 1991). Quail-chick transplantation experiments of Noden (1989, 1991) and Wilms and coworkers (1991) indicate that most embryonic mesodermal populations, except for the prechordal mesoderm and ectodermal-derived mesenchyme, contain endogenous endothelial precursor cells. This means that the mechanisms of angiogenesis and vasculogenesis are probably not explicitly separable in the embryo. Another recent study, concerning the development of the coronary vasculature (Poelmann et al., 19901, also shows that both angiogenesis and vasculogenesis contribute (simultaneously) to the same vasculature. In a previous study (DeRuiter e t al., 1992) we described in presomite to 6-somite mouse embryos the remodelling of a n initially two-dimensional, in situ differentiated horseshoe-shape endothelial plexus between the splanchnic mesothelium and the endoderm. It seemed that during remodelling caused by the outgrowth of the head folds and the development of the foregut, this plate of endothelial cells and precursors does not only contribute to the endocardium of the heart, a s is generally assumed, but also to the vitelline veins, dorsal aortae, and the pharyngeal arch arteries. It is interesting whether this finding is also pertinent for the avian embryo. In avian embryos the development of the endocardium and dorsal aortae is generally described as different echelons of in situ differentiation (vasculogenesis). The endocardial cells arise from the two lateral mesodermal compartments, bilateral to the anterior intestinal portal in the 1-somite embryo, which have to migrate medially to establish the two endocardial heart tubes in the 3-somite embryo (Manasek, 1968; Poole and Coffin, 1991). Both separated endocardial tubes interconnect in the 6-somite embryo with the dorsal aortae, which have already developed in the 3-somite embryo at the dorsal side of the foregut subjacent to the head mesenchyme (Coffin and Poole, 1988). In the 8somite embryo, the left and right endocardial tube will fuse to establish the single heart tube (Patten, 1971; Romanoff, 1960; Viragh et al., 1989). Moreover, Viragh and coworkers (1989) mentioned that endocardial cells can probably also arise by vasculogenesis from the intermesocardial space, that is, the area between the two somatic layers of the ventral mesocardium, and from the splanchnic mesoderm covering the endoderm of the foregut. This last finding seems to contradict the descriptions of Bockman and coworkers (19901, who stated that the pharyngeal arch arteries develop in the same area of the foregut by angiogenesis. A great number of transplantation experiments (Noden, 1989,1991; Pardanaud et al., 1989; Poole and Coffin, 1989; Risau et al., 1988), blocking experiments (Poole and Coffin, 19911, and even descriptive studies (Coffin and Poole, 1988; Pardanaud, 1987) mainly concentrate on the mechanisms of vessel formation, but these important studies pay no or only little attention to the establishment of vascular patterns in relation to other morphological events in organogenesis. To understand the possible mechanisms of vessel formation in the avian embryo, we studied the formation of the endocardium, pharyngeal arch arteries, dorsal aortae, and vitelline veins in relation to spatially very complicated changes, as the formation of the foregut and translocations of splanchnic and somatic mesodermal layers. Therefore the developing vasculature of presomite to 20-somite quail embryos was investigated with a combination of scanning electron microscopy and serially sectioned embryos incubated with a Q H l (Pardanaud et al., 1987) or aMBl (Peault et al., 1983). These antibodies are specific for endothelial and hemopoietic cells of quail embryos. MATERIALS AND METHODS Japanese quail embryos (Coturnix coturnix japonica) of 20 to 50 h r incubation were used for this study. The quail embryos were staged according to the age-determination criteria of Hamilton and Hamburger (1951). This classification was used to describe the quail embryos rather than the criteria of Zacchei (1961), which are less detailed. Scanning Electron Microscopy Quail embryos, stage HH5 to HH13 (presomite to 20 somites), were removed from the yolk in phosphatebuffered saline (PBS), pH 7.2, and fixed in half strength Karnovsky’s fixative (1965) for a t least 1 hr. To expose the endocardium and myocardium, various covering layers as the yolk sac and foregut endoderm, splanchnic and somatic mesothelium or ectoderm were dissected away from the embryo. Moreover, part of these embryos were sectioned frontally, sagittaly, or transversely with iridectomy scissors. Hereafter they were rinsed in 0.1 M sodium-cacodylate buffer (pH 7.2) and postfixed for 2 h r a t 4°C in 1%OsO, in the same buffer, followed by dehydration in graded ethanol. The preparations were critical point dried over CO, by conventional methods, sputter-coated with gold for 3 min (Balzers MED 0101, and studied in the scanning electron microscope (Philips SEM 525M). After a first evaluation remnants of dissected layers or a next layer were peeled off with very small pieces of double-sided Scotch tape, tweezers, or a bird feather, whereafter the specimens were coated again with gold. lmmunohistochernistry Quail embryos, stage HH8 to HH13 (4 to 20 somites), were fixed for 24 h r in periodate-lysin-paraformaldehyde fixative (McLean and Nakane, 1974) a t 4°C. After embedding in paraplast, the embryos were serially sectioned transversely a t 3 pm. Deparaffinated and rehydrated sections were incubated with QH1 (Pardanaud et al., 1987) or MB1 (Peault et al., 1983) monoclonal antibodies, which detect the quail endothelial and hemopoietic cells, diluted in PBS with 0.05% Tween-20 and 0.1% BSA. Overnight incubation was followed by washing in PBS with 0.05% Tween-20. The second incubation, for 2 hr, with rabbit antimouse conjugated to horse radish peroxidase was 11300 diluted in the same buffer as the primary antibody. After washing in PBS buffer, the staining reaction was performed with 0.04% diamino benzidine tetrahydrochloride in 0.05M tris-maleic acid (pH 7.6) with 0.07% imidazole 263 FORMATION OF THE QUAIL EMBRYONIC VASCULATURE and 0.06% hydrogen peroxide for 10 min, followed by washing in the buffer. Last, the sections were briefly (10 sec) counterstained with Mayer’s hematoxylin. RESULTS At first, the vascular system in bird embryos develops as a two-dimensional plexus of endothelial cells and precursors, located in the mesoderm facing the endoderm. The formation of the head folds and the foregut from the blastodisc remodels this vasculature into a three-dimensional structure. To understand the development of the endothelial lining of the heart, arteries, and veins, we relate this to the topographical and morphological changes of the mesoderm, endoderm, and coelomic cavities, occurring between stages HH5 to HH13 (presomite to 20 somites). A PP General Morphology In embryos of stage HH5, two flat primitive streakderived mesodermal compartments are present bilaterally to the notochord and prechordal plate. The mesoderm (Fig. 1A) is anteriorly bounded by the proamnion, which initially only consists of ectoderm and endoderm (Fig. 2). The left and right mesodermal compartments (Figs. lA, 2) continue laterally as the lateral plate mesoderm extending toward the (extra-embryonic) area pellucida and area opaca. In presomite embryos of stage HH6, two coelomic cavities have arisen in the anterior parts of the lateral mesoderm (Fig. 1B). These future pericardial regions divide the lateral mesoderm into a dorsal somatic layer facing the ectoderm and a ventral splanchnic layer that faces the yolk sac endoderm. From this splanchnic mesoderm, the heart will form. The splanchnic mesoderm starts to differentiate into both a splanchnic mesothelium and a n endothelial plexus adjacent to the endoderm. Furthermore, a part of the splanchnic mesoderm will differentiate into promyocardium. In the scanning microscope the hinge-point of the splanchnic and so- B so sp]Lnl C Abbreviations A acv AIP cc CJ D Dao E Ec En F H Lm mes N nt P Pm PP Pr so SP ss V Vm YS anterior anterior cardinal vein anterior intestinal portal coelomic cavity cardiac ,jelly dorsal dorsal aorta endocardia1 tube endocardium ectoderm endoderm foregut head mesenchyme or paraxial mesoderm or right head fold lateral mesoderm midpharyngeal endothelial strand notochord neural tube posterior promyocardial prechordal plate proamnion somatic mesoderm splanchnic mesoderm subcephalic space ventral ventral mesocardium yolk sac Fig. 1 .A-C. A schematic representation of the left and right mesodermal compartments a t stages HH5 (A), HH6 (B), and HH8 (C) in a ventral view. The ventrally situated endoderm and dorsally positioned ectoderm are not drawn. The hinge-point of the splanchnic mesoderm (Sp) and somatic mesoderm (So) is marked by a row of diverticula, which is used as a landmark to indicate the translocations of the various mesodermal layers. At stage HH8 the left and right lateral mesoderm (Lm) start to fuse ventral to the foregut. matic mesoderm is marked by a row of diverticula (Fig. 1; see also Fig. 5C) that extends toward the paraxial mesoderm. To understand the transformations described below, this row of diverticula is taken as a landmark. Transformation of the Cardiac Mesoderm The neural plate grows rapidly from stage HH5 onward, forming the two head folds (Fig. 2). The head folds in stage HH6- are elevated from the yolk sac and grow dorsally over the proamnion (Fig. 3), which is gradually incorporated into the ventral side of the 264 M.C. DERUITER ET AL. Fig. 2. A midsagittally sectioned presomite embryo (HH5). 'I'he right mesodermal compartment is bounded by the proamnion (Pr). The anterior endodermal fold indicates the developing foregut (F). Bar = 100 pm. Fig. 3. A midsagittally sectioned presomite embryo (HH6-). The buccopharyngeal membrane (*) is part ofthe proamnion (Pr), which is incorporated into the ventral side of the head. The yolk sac iYS) and the foregut (F) are lined by the endoderm (En). Bar = 50 pm. head. By the same process invagination of the yolk sac endoderm, causing a crescent-like fold, indicates the development of the foregut (Figs. 2, 3). Dorsally the foregut is lined by endoderm adjacent to the head mesenchyme and ventrally by endoderm belonging to the proamnion. At stage HH6- the anterior part of the proamnion can be referred to as the buccopharyngeal membrane (Fig. 3). At this stage the anterior border of the mesoderm with the proamnion is translocated ventrally toward the ventrolateral side of the foregut in a 1-somite embryo (compare A and B, Fig. 1). The incorporated proamnion is bordered anterolaterally by the head mesenchyme and posterolaterally by the lateral mesodermal compartment (Figs. lB, 4B). In a disk-like HH5-staged embryo, the transition zone of the head mesenchyme and lateral mesoderm is parallel to the notochord (Fig. 1A). From stage HH6 onward the anterior part of the transition zone is translocated into a plane more perpendicular to the notochord (Figs. lB, C; see also Figs. 4B, 5C). The left and right lateral mesoderm are brought together at the ventral side of the foregut to fuse in the 4-somite embryo (HH8) (Fig. 5B). The mesoderm grows into the crescent-like fold, marking the anterior intestinal portal (Figs. 4A, 5A). During translocation the coelomic cavities enlarge greatly. The number of diverticula (Fig. 5C) decreases dramatically in embryos with 10-12 somites (HH10 to HH11). In a 1-somite embryo two conical coelomic cavities are present in the lateral mesodermal compartments at the ventral side of the foregut (Fig. 4A). Only a small anterior part of both coelomic cavities is situated subjacent to the ectoderm of the head folds (Fig. 4B). In the midline the coelomic cavities are separated by a zone of -80 pm wide containing only a few mesodermal cells (Fig. 4A,B), which are referred to as the ventral mesocardium (seebelow).Subsequently,the coelomic cavities in the 4-somite embryo enlarge in an anterior direction (compare Figs. 4B, 5B). The medial borders of the lateral mesodermal compartments are marked by an indentation in the ventral ectodermal layer of the head (Fig. 5A). Together with the enlargement of the coelomic cavities, the fusion of the lateral mesoderm proceeds anteriorly in embryos with 5-6 somites. In a 6-somite embryo (HH9-) the distance between the endoderm of the anterior intestinal portal and the ectoderm of the proamnion is enlarged compared t o the 4-somite embryo (Figs. 5A, 6A). It is not clear t o what extent the relative translocation by ventral bending and the growth of the lateral mesoderm contributes to the fusion of the left and right coelomic cavities at the ventral side of the foregut. Together with the translocation of the lateral mesoderm, the relative position of the splanchnic mesoderm and the somatic mesoderm is changed too. In the coelomic lining of a 3-4-somite embryo (HH8), a large part of the splanchnic mesoderm, initially adjacent to the yolk sac endoderm, is translocated to the dorsal aspect of the coelomic cavity (Figs. l C , 5B). Consequently, part of the somatic mesoderm moves t o the ventral aspect of the coelomic cavity, which is thus anteriorly lined by somatic mesoderm and posteriorly by splanchnic mesoderm (Figs. 4B, 5A,B). The extraembryonic lateral mesoderm retains its original position with respect to the coelomic cavity (see Fig. 11A). Ventral to the foregut in the 4-somite embryo, the coelomic cavities are still separated medially by the ventral mesocardium (Fig. 5B). It now consists of a bilayer of squamous cells that connects the dorsal splanchnic layers with the ventral splanchnic and somatic lining of the coelomic cavity. The ventral mesocardium starts to disrupt in the 5-somite embryo (HH8) at the level of the ectodermal fold of the proamnion (Fig. 6A). Injection of Indian ink (DeRuiter, unpublished results) into one of the coelomic cavities of a 4-5-somite embryo filled the opposite cavity, confirming the presence of the first small connections through the ventral mesocardium. Although in a 5-somite embryo, the left and right dorsal splanchnic layers are separated by the presence of the ventral mesocardium (Fig. 6A,B), they have joined in the midline of a 6somite embryo. After fusion of the splanchnic layers, the ventral mesocardium may partly persist. The last remnant of the ventral mesocardium is visible until the stage HHlO (10 somites) as a row of irregularly oriented promyocardial cells in the midline of the heart. FORMATION OF THE QUAIL EMBRYONIC VASCULATURE 265 Flg. 4. A Ventral view of a 1-somite embryo after dissection of the endodem (En) of the yolk sac. The lateral mesodermal compartments are situated in the fold of the anterior intestinal portal (AIP). The coelomic cavity (cc) divides the lateral mesoderm into a splanchnic (Sp) and a somatic mesodermal layer. Squares indicate the areas of C and D. B: The same 1-somite embryo after partial dissection of the ectoderm (Ec) of the proamnion (F’r) and the head fold. Arrows indicate the transition zone of the lateral mesoderm and the head mes- enchyme (H). Dots indicate the ectodermal fold of the proamnion. The area of the proamnion between the lateral mesodermal compartments is the area of the ventral mesocardium. C:The endothelial precursors (arrows) are clearly distinguishable in the lateral extraembryonic areas. D: The proendocardial cells (arrows) are embedded in the splanchnic mesoderm of the heart forming regions. Bars = A and B, 100 pm; C, 20 pm; D, 200 p,m. * = area of the buccopharyngeal membrane. Promyocardium border the anterior intestinal portal. Before the 8somite stage, the cuboidal promyocardial cells that were situated at the ventral side of the coelomic cavity are translocated to its dorsal side. From a 4-somite stage onward, cardiac jelly is found between the promyocardial layer and the proendocardium (Fig. 6A,B). The first indication of cardiac jelly is seen within the limbs of the heart primordium, whereafter it can also be detected in the medial part. The presence of cardiac jelly is restricted to the area of cuboidal splanchnic mesothelial cells (Figs. 6A,B, 10A,B,C). Therefore, the promyocardium is sharply outlined against the squamous splanchnic mesothelium. The splanchnic mesothelia in 1to 4-somite embryos, which line the intraembryonic coelomic cavities (Fig. 5C), mainly consist of cuboidal cells or promyocardial cells. These promyocardial cells do not extend to the lateral rows of diverticula, but blend into a n area of squamous splanchnic mesothelial cells (Fig. 5C). Through the fusion of the coelomic cavities, which starts in the 5-somite embryo, a crescent-like promyocardial layer is formed, bordering the anterior intestinal portal in the 6-somite embryo. The crescent-shape heart primordium can be divided into a medial part in front of the foregut and two caudo-lateral limbs, which 266 M.C. DERUITER ET AL. Fig. 5. A Ventral view of a 4-somite embryo (HH8) after dissection of large parts of the endoderm of the yolk sac. The lateral mesodermal compartments have fused in the endodermal fold (En) of the anterior intestinal portal (AIP). An indentation (large arrow) in the ectoderm (Ec) of the head indicates a medial border of the lateral mesoderm. An extended plexus of proendocardial cells (small arrows) borders the anterior intestinal portal. B: The same 4-somite embryo, but after dissection of ectoderm of the head and the proamnion. Dots indicate the ectodermal fold of the proamnion, which is the borderline between ventrally situated somatic (So) and splanchnic (Sp) mesoderm. The coelomic cavities are separated by the ventral mesocardium (Vm). C: Lateral view of the left coelomic cavity of a 4-somite embryo (HH8). The row of diverticula marks the hinge-point of the splanchnic and somatic (So) mesoderm. Note the curving of this line. The splanchnic mesothelium consists of bulging promyocardial cells (pm) and flat mesothelial cells (p). Bars = A and B, 100 km; C, 50 pm. * = area of the buccopharyngeal membrane. It is obvious that the volume of the cardiac jelly in the medial part (midportion) of the heart primordium does not increase evenly. Only a very small amount of cardiac jelly is present in the midline area of the ventral mesocardium, through which the promyocardium, proendocardium (see below), and endoderm remain in close contact, seen as a deep furrow in the medial part of the heart tube (Fig. 6A,B). After disappearance of the ventral mesocardium, the endocardium remains connected to the promyocardium in the midline of the medial part (Fig. 7; see also Fig. 10A,C).As a result the endocardial tube in a 9-somite embryo is bilaterally flanked by two compartments of cardiac jelly (see Fig. 10A,C). The promyocardium shows a n indentation (see Fig. 10B) a t the site of the original ventral mesocardium just after its disappearance. Finally, the formation and disappearance of the dorsal mesocardium must be described here to understand the formation and translocation of the endocardium. In a 5-somite embryo the dorsal side of the heart is completely connected to the endoderm of the foregut (Fig. 6B). In a n 11-somite embryo the lateral borders of the promyocardial layer come closely together forming the characteristic thin dorsal mesocardium in the medial part of the heart (Fig. 7). This thin part of the dorsal mesocardium disappears in the 15-somite embryo (HH12) resulting in a heart tube completely surrounded by the coelomic cavity. Endocardial Plexus As already described, part of the splanchnic mesoderm differentiates into a splanchnic mesothelium, lining the coelomic cavity, and a n endothelial vascular plexus facing the endoderm. To describe the formation of a lumenized vascular system from stem cells, it is necessary to define the different subsequent components. Squamous cells, which line the lumen of a blood vessel and are recognized by the aMBl and a Q H l antibodies, are defined as endothelial cells. Positively stained cells, which do not line a lumen, are considered to be endothelial precursors. In the scanning electron microscope they are recognizable as isolated, globular cells, which are often connected to similar cells in appearance. The part of the endothelial vascular plexus related to the promyocardial area of the splanchnic mesoderm is considered to be the endocardium. It consists of endocardial and proendocardial cells, from which only the first one line a lumen. In a 1-somite embryo, a n extensive vascular plexus is present between the splanchnic mesothelium and the endoderm of the yolk sac. In the lateral extraembryonic areas, the endothelial precursors can be distinguished within the splanchnic mesoderm as seen by scanning microscopy (Fig. 4C). The vascular plexus extends medially to the heart forming regions, where the proendocardial cells are embedded in the splanchnic meso- FORMATION OF THE QUAIL EMBRYONIC VASCULATURE 267 Fig. 6. A: Ventral view of a 5-somite embryo after dissection of the endoderm and the ventrally situated somatic and splanchnic mesoderm of the coelomic cavity. Dots indicate the ectodermal fold of the proamnion. With regard to the 4-somite embryos, the coelomic cavities have enlarged in a n anterior direction during the formation of the medial part of the heart. The promyocardial layers are separated by the ventral mesocardiurn (Vm). The promyocardial (pm) limbs protrude into the coelomic cavity by the presence of cardiac jelly. Note the diverticula (arrows) between the splanchnic (Sp) and somatic (So) mesothelial layers. B: Transversely sectioned 5-somite embryo through the medial part of the heart primordium. The cardiac jelly (CJ) is restricted to the area of the promyocardium. The dorsal meso- cardium between the dots is a wide area. The proendocardial plexus (arrows) covers the endoderm (En) of the foregut (F), but is not separated by the ventral mesocardium (Vm). Some of the proendocardial cells have lumenized (star). C: Medial view of the left limb of the heart primordium of a 5-somite embryo. Arrows indicate the proendocardial cells. Bars = A, 100 pm; B, 30 pm; C, 50 pm. derm (Fig. 4D).Endothelial precursors in the areas of the future dorsal aortae were not detectable in any of the 1-somite embryos as seen by scanning microsCOPY. In both limbs and the medial part of the heart of 3 to 4-somite embryos (HHK), many proendocardial cells are seen (Fig. 5A; see also Fig. 9B). Most of these cells are connected to similar cells giving the appearance of a plexus. In the medial part this plexus of proendocardial cells extends bilaterally to the head mesenchyme, flanks the foregut (Fig. 8A), and is called the splanchnic plexus (see below). In a 4-somite embryo (HH8) proendocardial cells are also situated in the area of the now translocated proamnion (the future ventral mesocardium) (Fig. 9A), by which a crescent-shape plexus of proendocardial cells arises. Fig. 7. A transversely sectioned 11-somite embryo through the medial part of the heart, showing the characteristic dorsal mesocardium (between the dots). The endocardial tube (E) loses its contact with the endoderm (En) of the foregut (F), but some of the endocardial cells remain connected to the endoderm (arrow). Bar = 50 km. 268 M.C. DERUITER ET AL. Fig. 8. A Dorsal view of the splanchnic mesoderm of a 3-somite embryo after dissection of the endoderm of the foregut and the complete head mesenchyme. The transition zone of the lateral mesoderm and the head mesenchyme is indicated with dots. The plexus of proendocardial cells (arrows), subjacent to this medial part of the heart, extends laterally to the head mesenchyme. B: Ventral view of the head mesenchyme in a 4-somite embryo after dissection of the endo- derm of the foregut. The longitudinal strand indicates the right dorsal aorta (arrows). C : The same area (as in B) of a 6-somite embryo. The longitudinal strand, indicating the dorsal aortae (arrows), is more pronounced than in the 4-somite embryo. Note the endothelial precursors (arrows) in the transition zone of the head mesenchyme and the lateral mesoderm (white and black dots). Bars = A, B, C, 50 pm. Formation of the Endocardial Tube phasized that the ventral mesocardium, lacking cardiac jelly, does not separate the endothelial plexus into a right and a left part (Fig. 6B). After disappearance of the ventral mesocardium (about 8 somites), the developing endocardial tube remains connected to both the ventrally situated promyocardium and the dorsally situated endoderm. It still shows the largest lumen bilaterally to the connection with the promyocardium (Fig. 10B). This is usually explained as two fusing heart tubes interconnected by small canals. A dorsal view on the endocardial plexus shows that it still consists of one By the production of cardiac jelly from stage HH8 (4 to 5 somites) onward, the endocardial plexus and the promyocardial layer both in the limbs and in the medial part are separated. Meanwhile, the proendocardial cells lumenize and are defined, therefore, as endocardium. In the medial part of the heart, the endocardium starts to lumenize bilaterally to the ventral mesocardium in the 5-somite embryo (Fig. 6B). It must be em- FORMATION OF THE QUAIL EMBRYONIC VASCULATURE 269 enchyme (Fig. 8A). Two longitudinal, irregular strands of endothelial precursors can be recognized in the head mesenchyme, giving rise to the dorsal aortae (Fig. 8B). In the 6-somite embryo they are more pronounced (Fig. 8C), but they will not become lumenized before the 8-somite stage. The development of the dorsal aortae from the endothelial plexus flanking the foregut (splanchnic plexus) is comparable to the formation of the dorsal aortae more caudally in the area of the midgut. In a 7-somite embryo the yolk sac vasculature extends to the head mesenchyme where the dorsal aortae are formed (Fig. 11A). First Pair of Pharyngeal Arch Arteries The splanchnic plexus between the endocardium and the dorsal aortae indicates the location of the future pharyngeal arch arteries. From stage HH8 (4 somites) onward the number of endothelial precursors increases, predominantly in the anterior aspect of the splanchnic mesoderm. In 6-8-somite embryos, these endothelial precursors are oriented in characteristic, parallel V-shaped strands converging in the midline (Fig. 10D,E). In the 8-somite embryo the anteriorly situated strands of endothelial precursors, subjacent to the squamous splanchnic mesothelium, lumenize and form the first pair of pharyngeal arch arteries (Fig. 10E). The endothelial cells and precursors, situated caudally to the first pair of pharyngeal arch arteries, will contribute in later stages to the next pharyngeal arch arteries. Second and Third Pair of Pharyngeal Arch Arteries Flg. 9. Transverse sections of a 4-somite quail embryo incubated with QHl. A Proendocardial cells (arrows) are present in the translocated proamnion between the left and right lateral mesodermal compartments (Lm). B: Some proendocardial cells in the limbs of the heart already line a lumen and are defined as endocardial cells (arrows). Bars = A and B, 5 km. uninterrupted plate (Fig. 10D). Until the 12-somite stage, the medial part of the heart is reorganized into an endocardial tube, whereas remnants of the former endocardial plexus can be recognized as cell strands within the endocardial tube (Fig. lOC). Midpharyngeal Endothelial Strand During the formation of the dorsal mesocardium, the endocardial tube becomes enveloped within the myocardium and thus loses contact with the foregut endoderm (Fig. 7). However, over the complete length of the heart tube a strand of endocardial cells, formerly part of the endocardial tube, remains connected to the foregut endoderm. After the disappearance of the dorsal mesocardium in the 15-somite stage, this midpharyngeal endothelial strand runs from the arterial to the venous pole (Fig. 11B) and will persist until stage HH20. During this period the strand lumenizes and contributes to part of the venous system of the oesophagus, larynx, trachea, and bronchi as well as the pulmonary veins draining into the left atrium. Dorsal Aortae From the 3-somite stage onward, the endocardium extends bilaterally around the foregut to the head mes- After disappearance of the dorsal mesocardium, the splanchnic plexus situated caudally to the first pair of pharyngeal arch arteries expands and aligns. The splanchnic plexus in the surroundings of the aortic sac and the dorsal aortae mainly consists of endothelial cells, whereas in the transition zone of the lateral mesoderm to the head mesenchyme only endothelial precursors are present. In the 20-somite embryo an irregularly lumenized pair of second pharyngeal arch arteries has arisen from the cranial aspect of the splanchnic plexus. Caudal to these arteries the splanchnic plexus has the same features as in the 15somite embryo and will contribute to the third pharyngeal arch arteries (Fig. 11D). By the increase in number of mesenchymal cells around the first pair of pharyngeal arch arteries, these endothelial tubes lose their contact with the splanchnic mesothelium. In the 20-somite embryo only the proximal parts of the arteries, close to the aortic sac, remain connected to the splanchnic plexus (Fig. 1lC). In later stages these parts of the splanchnic plexus will lumenize and contribute to the ventral pharyngeal veins, which provide part of the ventral venous drainage of the facial region. DlSCUSSiON In a combination of scanning electron microscopy and immunohistochemically stained serial sections, we studied the development of the vascular system in the quail embryo between stage HH5 and HH13. By partial dissection of the endoderm, splanchnic and somatic mesothelia, or ectoderm, the subjacent developing vascular system, consisting of characteristic globular en- 270 M.C. DERUITER ET AL. Fig. 10. A Anterior view of a transversely sectioned 9-somite embryo, posteriorly through the medial part of the heart. The endocardial tube (E) is bilaterally flanked by two compartments of cardiac jelly (CJ). The cardiac jelly is restricted to the promyocardium (pm); see also B and C. B: Transversely sectioned 7-somite embryo, anteriorly through the medial part of the heart. Subjacent to the longitudinal indention (arrowhead)of the promyocardium the endocardial tube is narrowed. Note the proendocardial cells (small arrows). C : Transverse section anteriorly through the medial part of the heart tube of a llil2-somite embryo. Within the endocardial tube many strands of rounded endocardial cells, covered by a lot of villi, are present. These are remnants of the endocardial plexus. D: Dorsal view of the endocar- dium and the laterally situated splanchnic endothelial plexus of an 8-somite embryo after dissection of the foregut endoderm and the head mesenchyme. The endocardium (E) is not divided by the ventral mesocardium. Note the parallel V-shape strands of endothelial precursors (arrows).The transition zone of the lateral mesoderm and the head mesenchyme is at the right side indicated by dots. E: Ventral view of the endocardium and splanchnic plexus of a 8-somite embryo after dissection of the promyocardium and the lumenized parts of the endocardium. The first pair of pharyngeal arch arteries (I) has developed from the splanchnic plexus (white arrows). Bars = A and B, 50 pm; C , 20 km; D and E, 100 wm. dothelial precursors without a lumen andior squamous endothelial cells lining a lumen, could be visualized in the electron microscope. The quail embryos, incubated with the monoclonal antibodies QH1 or MB1, show the endothelial precursors even before they have segregated from the splanchnic mesoderm. The splanchnic mesothelium consists of areas of squamous and cuboidal cells. The squamous splanchnic mesothelia give rise to part of the pericardial lining (Manasek et al., 1984). The cuboidal cells will differentiate into myocardium as can be deduced from DeJong and coworkers (1990) who showed that these cells already contain atrial and ventricular isomyosins in the 4-somite chick embryo. In our study we defined them a s promyocardial cells, although they do not contract before stage HHlO (DeJong et al., 1990; Johnson et al., 1974; Patten, 1971). Translocation of the Splanchnic Mesoderm To understand the establishment of the vascular system, which initially differentiates from the lateral and the paraxial mesoderm (e.g., Drake et al., 1990; Manasek, 1968; Noden, 1989,1991; Poole and Coffin, 1988; Wilms et al., 1991), it was necessary to investigate the translocations of these mesodermal layers. It appears that the hinge-point of splanchnic and somatic meso- FORMATION OF THE QUAIL EMBRYONIC VASCULATURE 271 Fig. 1 1. Light micrographs of transverse sections incubated with MB1. A: Section just caudal to the anterior intestinal portal of a 7-somite embryo. The yolk sac vasculature extends medially to the paraxial mesoderm (p), where the dorsal aortae (arrow) are formed. B: Section through the medial part of the heart tube of a 15-somite embryo. By the formation of the dorsal mesocardium (between the dots) a strand of endocardia1 cells becomes situated extracardially: the mid- pharyngeal endothelial strand (mes). C: Twenty-somite embryo. The proximal parts of the first and second pair of pharyngeal arch arteries (I and 11)are connected with the splanchnic vascular plexus (arrows), the future ventral pharyngeal veins. D: Section caudal to that in C through the same 20-somite embryo. This part of the endothelial plexus (arrows) will contribute to the third pair of pharyngeal arch arteries (arrows). Bars = A, B, D, 5 pm; C, 10 pm. derm, which is marked by a characteristic row of diverticula, aids in understanding the translocations of the mesodermal layers. To our knowledge only Manasek and coworkers (1984) mentioned this demarcation zone in the scanning electron microscope. This hinge-point is also important as a central feature between the lateral plate mesoderm and the head mesenchyme. According to Drake and coworkers (1990), part of the paraxial mesoderm (primary mesoderm) separates into the splanchnic and somatic mesothelium. During the enlargement of the coelomic cavity and growth of the promyocardium, many diverticula are visible in our quail embryos, but their number is reduced after the disappearance of the dorsal mesocardium in stages older than 15 somites. As already suggested for the mouse embryo (DeRuiter et al., 1992), the mesodermal transition zone may very well contribute to the promyocardium and pericardium, during the period in which the dorsal mesocardium is present. Interestingly, cardiac-specific myosin is demonstrated in voluntary head muscles (Bredman et al., 1990, 1991). Although the striated muscles are generally described to be derived from the paraxial mesoderm and/or prechordal plate (Jacob et al., 1984; Noden, 19831, we speculate that the transition zone marked by the diverticula may also contribute to the formation of the head muscles in very early stages. Formation of the Splanchnic Plexus Our results show that from the 1-somite stage onward, the extraembryonic vasculature is continuous with that of the heart forming regions. Part of the extraembryonic vasculature lines a lumen, whereas the intraembryonic endothelial precursors are situated between the mesothelial cells. It looks as if we are dealing with a gradient of locally segregating endothelial cells from the splanchnic mesoderm. Drake and Jacobson (1988) also described in the chick embryo that vascularization progresses from the lateral and cranial regions to more medial and caudal levels. On the contrary, Poole and Coffin (1991) suggested that endothelial precursors, which derive from the lateral regions of the splanchnic mesoderm (Manasek, 19681, migrate to 272 M.C. DERUITER ET AL. the foregut and head (vasculogenesis type 11, as defined by Poole and Coffin, 1991). Additional experiments are necessary to clarify the correlation between long-distance migration of endothelial cells and the possibility of in situ differentiation. In literature the ventral mesocardium is seen as a septum between the left and the right side of the plexus (Stalsberg and DeHaan, 1969; Viragh et al., 1989). Nevertheless, Viragh and coworkers (1989) described mesenchymal cells, which are indistinguishable from the proendocardial cells between the mesothelial layers of the ventral mesocardium (intermesocardial space) of a 5-somite chick embryo. With both scanning electron microscopy and immunohistochemistry, we have not found a discontinuity in the midline of the endothelial plexus related to the ventral mesocardium. It is understandable that figures depicting the ventricular segments, presenting with two lumens of the endocardial plexus next to the midline, could have been misinterpreted in literature (see below). Superficially, it seems as if the various major vessels as the endocardial tube, pharyngeal arch arteries, and dorsal aortae develop independently and not as parts of the same plexus. Local expansion and lumenization of the splanchnic plexus occurring at a considerable speed is in contrast with the sparse endothelial precursors flanking the foregut. This local expansion can be caused by recruitment of endothelial precursors from adjacent areas and/or by local cell division. Although the first pair of pharyngeal arch arteries is not lumenized before stage HH9, the pattern of the pharyngeal arch system is seen as early as stage HH7 (3 somites). Coffin and Poole (19881, demonstrating the splanchnic plexus in in toto incubations of 12-somite embryos, mentioned capillary strands in the region of the developing pharyngeal arch arteries. Viragh and coworkers (1989) described the plexus consisting of “proendocardial cells and capillaries,” attached to fine filamentous extracellular material that migrate toward the ventral mesocardium in the 4-8-somite stage. We have found them by immunohistochemistry to be present already during the formation of the proendocardial tube and the dorsal aortae (3-7 somites), but also later on during the formation of the pharyngeal arch arteries (6 to 20 somites). splanchnic plexus in the lung buds and to the central pulmonary veins. Chang (1931)described that the dorsal side of the proendocardial tube in a 5-somite chick embryo is firmly attached to the foregut endoderm. From that side, “angioblasts” may spread along the foregut to give rise to the splanchnic plexus. After disappearance of the dorsal mesocardium only the lumenized pulmonary veins are connected to the splanchnic plexus (Chang, 1931). A study of embryos with 20 somites (HH13) and older (DeRuiter et al., in preparation) will show that the midpharyngeal endothelial strand lumenizes and contributes to the venous drainage of the oesophagus, larynx, trachea, bronchi, and the central pulmonary veins into the left atrium. This implies that these developing veins, a s is the case with the ventral pharyngeal veins, do not sprout from the atrium to establish a venous system, but rather develop from pre-existing endothelial precursors. Our studies have shown the formation of a strand of endothelial cells (midpharyngeal endothelial strand). These cells are initially part of the endocardial heart tube, but with the disappearance of the dorsal mesocardium they become separated from the endocardium. Visualization of this midpharyngeal endothelial strand was only possible with the use of the aMBl and a Q H l monoclonal antibodies. A high concentration of electron-dense material consisting of laminin, fibronectin, and crossbanded collagen (Johnson et al., 1974; Manasek et al., 1984; Waterman and Balian, 1980) attaches the endocardial cells to the foregut endoderm. We assume that the cells of the midpharyngeal endothelial strand refer to the same angioblasts as described by Buell (1922) and Chang (1931). Angiogenesis Versus Vasculogenesis Indications of invasive outgrowth of newly forming vessels into the mesenchyme (angiogenesis) were not seen in any of the embryos. Therefore, our observations support the mechanism of vasculogenesis (differentiation of mesodermal cells into endothelial cells) in the stages studied. This corresponds with the quail-chick transplantation experiments (Noden, 1989, 1991; Wilms et al., 1991), which indicate that the paraxial and lateral mesoderm and the prechordal plate have angiogenic potentials, respectively. Angiogenesis as a suggested mechanism for the formation of the pharyngeal arch arteries (Bockman e t al., 1990) could not be Formation of the Veins demonstrated, corresponding also with a n earlier study Literature on the development of the pharyngeal on the formation of the vascular system in the mouse arch system pays hardly any attention to the formation embryo (DeRuiter et al., 1992). An initially two-dimenof the veins in the pharyngeal arches (Congdon, 1921; sional, in situ differentiated, horseshoe-shape endotheMoffat, 1956; Padget, 1948). Our results show that both lial plexus (vasculogenesis) does not contribute to the the pharyngeal arch artery and the ventral pharyngeal endocardium of the heart, but also to the vitelline vein develop from the same splanchnic plexus. During veins, the dorsal aortae, and the pharyngeal arch arthe growth of the pharyngeal arches the arterial and teries. the venous systems remain connected to each other. Fusion of Two Heart Tubes? Outgrowth from existing veins into a precursor-free After joining of the two mesodermal compartments pharyngeal arch to be followed by connection to the in the 4-6 somite stage no additional fusion of any part artery was not observed. of the promyocardial and proendocardial layers is obMidpharyngeal Endothelial Strand served. The foregut of a 4 somite embryo is covered by According to Buell (1922), the first “angioblasts” in one single endothelial plexus, which is not divided by the dorsal mesocardium are not present before 20 the ventral mesocardium. According to Manasek somites. These cells sprout in the mesenchyme around (19681, Patten (19711, Romanoff (19601, and Viragh the foregut, contributing to the formation of the and coworkers (1989), the embryonic chick heart arises FORMATION OF THE QUAIL EMBRYONIC VASCULATURE by fusion of two heart primordia developed in both splanchnic mesodermal layers. They describe that each heart primordium differentiates into a proendocardial tube as well as a myocardial envelope. With the formation of the foregut, the two heart tubes comprising both the proendocardium and promyocardium approach each other and fuse at their full length in the 9-somite embryo by the disappearance of the ventral mesocardium (Viragh et al., 1989). The fusion should begin at the anterior end progressing caudally (Manasek et al., 1984). Part of the data presented here showing a single endocardial plexus is described earlier by Steding and coworkers (1980). They stated that one endothelial strand in the midline of the medial part extends and lumenizes to form a single endocardial tube. Manasek and coworkers (1984) showed scanning pictures of stage-HH10-chick embryonic hearts containing a longitudinal furrow along the midline of the myocardial tube. They stated that although the ventral mesocardium has disappeared, this furrow indicates the place of fusion between the left and right splanchnic mesodermal layers. In our view this furrow does not indicate the site of fusion of the ventricular and atrial segments. Although the ventral mesocardium was indeed located at this place, this furrow is caused by the attachment of the endocardium and myocardium flanked by two compartments of cardiac jelly. If the suggested caudo-cranial fusion of two heart tubes takes place, the foregut endoderm has to be entrapped between the two heart tubes (Patten, 1971). No signs of such a reconstruction of the foregut endoderm is observed in our material, nor satisfactorily described in the above mentioned studies. After the formation of the crescent-like promyocardial layer in the 4-5-somite embryo, the medial part of the promyocardial layer enlarges in anterior direction. The medial part of the promyocardial layer, giving rise to the ventricular segments (Stalsberg and DeHaan, 19691, shows only later production of cardiac jelly and formation of an actual endocardial tube compared to the caudal parts of the heart. This indicates that the ventricular part develops through an anterior expansion rather than by fusion of the advanced caudal parts. Moreover, the sinus venoms and atrial segments become more and more distinct in the caudal parts, but continue to border the complete anterior intestinal portal. This is confirmed by the labelling experiments of DeLaCruz and coworkers (1977), who stated that the inflow tract (the trabeculated part of the ventricle) arises by fusion of the lateral splanchnic mesodermal compartments a t stage HH9-. At this stage the outflow segment is not yet present but will be constituted at the distal end of the heart tube at stage HH12. ACKNOWLEDGMENTS This study was supported by a grant of the Netherlands Heart Foundation (Grant 87.062). The added skillful technical assistance of Mrs. I. VanderPlas was greatly appreciated. We also express thanks to Dr. M Coltey (Nogent-sur-Marne, France) for providing the MB1 and QH1 antibodies. The authors are indebted to Mr. J. Lens and Mr. L.D.C. Verschragen for their excellent photographic work, and to Mrs. H.E. DeVries and Mrs. J.T. Wossels for preparing the manuscript. 273 LITERATURE CITED Ausprunk, D.H., and J . 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