THE ANATOMICAL RECORD PART A 288A:989–999 (2006) Vascular Patterning of the Quail Coronary System During Development ROBERT J. TOMANEK,* HEIDI K. HANSEN, AND EDUARD I. DEDKOV Department of Anatomy and Cell Biology and Cardiovascular Center, University of Iowa Carver College of Medicine, Iowa City, Iowa ABSTRACT Recent studies have provided insights into speciﬁc events that contribute to vasculogenesis and angiogenesis in the developing coronary vasculature. This study focused on the developmental progression of coronary vascularization beginning with tube formation and ending with the establishment of a coronary arterial tree. We used electron microscopy, histology of serial sections, and immunohistochemistry in order to provide a comprehensive view of coronary vessel formation during the embryonic and fetal periods of the quail heart, a species that has been used in a number of studies addressing myocardial vascularization. Our data reveal features of progenitor cells and blood islands, tubular formation, and the anatomical relationship of a transformed periarterial tubular network and sympathetic ganglia to the emergence and branching of the right and left coronary arteries. We have traced the pattern of coronary artery branching and documented its innervation. Finally, our data include the relationship of ﬁbronectin, laminin, and apoptosis to coronary artery growth. Our ﬁndings bring together morphological events that occur over the embryonic and fetal periods and provide a baseline for studies into the mechanisms that regulate the various events that occur during these time periods. Anat Rec Part A, 288A:989–999, 2006. Ó 2006 Wiley-Liss, Inc. Key words: vasculogenesis; angiogenesis; arteriogenesis; epicardium The coronary vasculature develops via closely regulated events that involve vasculogenesis, angiogenesis, and arteriogenesis. The cells that contribute to the various vessel types of the coronary system arise from the epicardium, which in turn is derived from the proepicardium. Development of the coronary vasculature has recently been reviewed (Majesky, 2004; Tomanek, 2005). Numerous studies have provided insights into signaling events and the process of epicardium development and cell differentiation of coronary precursor cells (reviewed in Olivey et al., 2004). The epicardial-derived cells are a source of endothelial, smooth muscle, and pericytic cells. Formation of a capillary network occurs as the compact region of the left ventricle expands and subsequently vascular tubes penetrate the aorta at the left and right coronary cusps, thus establishing the two coronary ostia. This pattern has been documented in mammalian and avian species. Quail and chicken have been used in studies that have addressed various aspects of myocardial vascularization, Ó 2006 WILEY-LISS, INC. including the roles of VEGF and FGF2 (Tomanek et al., 1998). Several key ﬁndings, published between 1989 and 1996, are based on these species. The in-growth of a capillary plexus into the aorta was ﬁrst documented in 1989 (Bogers et al., 1989). Mikawa and Fischman (1992) used retroviral tagging to establish independent precursors for cardiac myocytes, endothelial cells, smooth muscle cells, and perivascular ﬁbroblasts. Poelmann et al. Grant sponsor: The National Institutes of Health; Grant number: R01 HL075446. *Correspondence to: Dr. Robert J. Tomanek, Department of Anatomy and Cell Biology, 1-402 BSB, University of Iowa, Iowa City, IA 52242. Fax: 319-335-7198. E-mail: firstname.lastname@example.org Received 4 May 2006; Accepted 24 May 2006 DOI 10.1002/ar.a.20365 Published online 4 August 2006 in Wiley InterScience (www.interscience.wiley.com). 990 TOMANEK ET AL. (1993) veriﬁed that an extracardiac source provided these precursors and described the path of the subepicardial precursors in forming vessels at the sinus venosus and myocardium. Subsequent to the ﬁnding that the proepicardium provides cells for the coronary vasculature (Viragh et al., 1993), proof that the proepicardium is a source of endothelial and vascular smooth muscle cells was provided by Mikawa and Gourdie (1996). Recent studies have used whole mounts to describe arterialization of the myocardium (Kattan et al., 2004) and endothelial cell penetration of the aorta to form the proximal coronary arteries (Ando et al., 2004). Most recently, we have documented the role of key VEGF family members as regulators of coronary artery formation (Tomanek et al., 2006). This study also found that red blood cells of cardiac blood islands are derivatives of the proepicardium. Because of the existence of an endothelium-speciﬁc marker for quail (QH1 antibody), this species is a good model of coronary vessel formation, as the vasculature is easily and reliably identiﬁable. We have utilized quail heart explants in order to document the regulation of vasculogenesis and angiogenesis by speciﬁc growth factors and their interactions (Yue and Tomanek, 1999, 2001; Tomanek et al., 2001, 2002; Tomanek and Zheng, 2002; Holiﬁeld et al., 2004). Other studies have used chicken-quail chimeras to answer questions regarding the progenitor cells that contribute to the coronary circulation (Vrancken Peeters et al., 1997, 1999; Bergwerff et al., 1998; Gittenberger-de Groot et al., 1998, 2004; Perez-Pomares et al., 1998; Manner, 1999; Eralp et al., 2005; Van Den Akker et al., 2005) or have examined speciﬁc aspects of coronary vessel (Bogers et al., 1989; Dettman et al., 1998; Perez-Pomares et al., 2002; Ando et al., 2004; Kattan et al., 2004; Pennisi and Mikawa, 2005). These studies, together with our in vitro studies, have precipitated a number of questions regarding the sequence of events encompassing vasculogenesis, angiogenesis, and arteriogenesis. Accordingly, the current study addressed the temporal and spatial aspects of myocardial vascularization. The data, based on serial microscopic sections, immunohistochemistry, and electron microscopy, were obtained in order to deﬁne vascular patterning in the coronary system during development in this species. ter paraformaldehyde ﬁxation, then frozen in isopentane (precooled with liquid nitrogen). Sections were then cut with a cryostat and used for double immunostaining for smooth muscle a-actin and either neuroﬁlaments, ﬁbronectin, laminin, or the TUNEL technique. A few hearts were injected with India ink (Higgins, Bellwood, IL) and then ﬁxed by immersion in glutaraldehyde. These hearts were processed, embedded in Spurrs, and ultrathin sections were prepared for electron microscopy. The sections were viewed with a Hitachi 7000 electron microscope. Immunohistochemistry The following antibodies were purchased from the Developmental Studies Hybridoma Bank, University of Iowa (Iowa City, IA): ﬁbronectin (B3/d6) and quail endothelial cell surface marker (QH1). Cy-3-conjugated monoclonal smooth muscle a-actin (Clone 1A4) and polyclonal laminin antibodies were purchased from Sigma (St. Louis, MO). Neuroﬁlament polyclonal antibody (150 KD) was from Chemicon (Temecula, CA). Antibodies for VEGF and FGF-2 (bFGF) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Both parafﬁn-embedded and frozen hearts were used for immunohistochemical staining procedures. Nonspeciﬁc binding was blocked with 5% normal goat serum, 1% BSA in PBS. Incubation with primary and secondary antibodies was in the dark at room temperature. For ﬁbronectin and QH1, we used Alexa Fluor 488 antimouse-conjugated secondary antibodies (Molecular Probes, Eugene, OR). For neuroﬁlaments and laminin, we used Alexa Fluor 488 antirabbit secondary antibodies. Staining for VEGF and FGF-2 utilized the alkaline phosphatase-conjugated to the secondary antibody (goat, antirabbit). TUNEL Technology for Apoptosis Paraformaldehyde-ﬁxed frozen sections were used to detect in situ apoptosis. We employed an ApopTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon) according to the manufacturer’s instructions. The method, TUNEL, is based on the modiﬁcation of genomic DNA utilizing deoxynucleotidyl transferase for detection of positive cells. All nuclei were revealed by nuclear counterstain with propridium iodide (DAPI). MATERIALS AND METHODS Fertile quail embryos (Northwest Game Girds, Kennewick, WA) were incubated under humiﬁed conditions at 388C. We staged embryos either by days of incubation or by the system of Hamburger and Hamilton (1951). Tissue Preparation Hearts from embryos incubated (in ovo) for 5–18 days were ﬁxed by perfusing 4% paraformaldehyde buffered with PBS via the apex of the left ventricle. The basal half of the heart was processed, embedded in parafﬁn, and serial sections (6 mm thick) were placed on glass slides. Slides were then deparafﬁnized, rehydrated, and stained with hematoxylin and eosin, or the sections were viewed prior to deparafﬁnization under dark-ﬁeld microscopy and slides representing select areas of the heart and great vessels processed for immunostaining. A subset of hearts was cryoprotected in 5% sucrose/PBS af- RESULTS Coronary Cells and Precursors: Electron Microscopy Coronary vascular cells are derivatives of the epicardium/subepicardium. As documented by electron microscopy, delamination of epicardial cells (Fig. 1A) necessitates the breaking of tight junctions (Fig. 1B). The subepicardial population includes a variety of cells, some of which migrate to the myocardium and are active in protein synthesis, as indicated by a rich endoplasmic reticulum (Fig. 1B). Phagocytosis occurs in this region and less commonly in the myocardium (Fig. 1D and E) and frequently cellular remnants are enclosed in a cytoplasmic ring. In some instances, glycogen is present, indicating that the degenerated cells were cardiomyocytes. Some areas of the subepicardium, which vary greatly in thickness depending on the region, also include erythrocytes (Fig. 1C). These cells DEVELOPMENT OF CORONARY VASCULATURE 991 Fig. 1. Electron micrographs of epithelial-derived coronary vascular cells. A: Two mesothelial cells (arrows) delaminating from the epicardium (E8 heart). B: Tight junction (arrow) between two mesothelial cells (asterisk) in E10 heart. Note the large subepicardial cells with rough endoplasmic reticulum indicative of protein synthesis. C: Mesothelial cells (Mes) of the epicardium with underlying (subepicardial) cells (E7 heart). The latter often include erythrocytes (asterisk). D: Subepicardial region with cellular remnants surrounded by a cell ring with thin cytoplasm (between arrows) in an E7 heart. E: A similar degenerative proﬁle (between arrows) consisting of cell elements including contractile ﬁlaments (E9 heart). The debris lies within a tubular structure just under the mesothelium (Mes) and is another example of phagocytosis in this region. F: Subepicardial network of cells with multiple thin processes. A cell remnant with glycogen is seen (arrow). Note the capillaries (asterisk) in this E6 specimen. G and H: Blood islands (demarcated by arrowheads) in an E10 heart. They are seen in the subepicardium (G) and in the myocardium (E10 heart) with adjacent mature capillaries (asterisk). Scale bars ¼ 5 mm. are present prior to establishment of the coronary circulation and are also found in blood islands before and after coronary arteries are formed (Fig. 1G and H). Blood islands occur most frequently in the subepicardium and at the base of the aorta, but may also be seen within the myocardium (Fig. 1H). The expanded subepicardium, typically seen at the aortic root and A-V groove, contains a network of cells with long multiple cellular processes abutting one another (Fig. 1F). The earliest signs of tubulogenesis, i.e., formation of capillaries, is observed in this network of cells (Fig. 1F). Vascular tubes (capillaries) vary in their morphology, which may be in part accounted for by their stage of development. Cells may be relatively thick or very narrow (Fig. 2). A tube proﬁle may consist of two or more cells or a single cell with a large vacuole (not shown). Mature forms have a scant cytoplasm and usually an adjacent pericyte (Fig. 2C, E, and F). The development of a myocardial tubular (capillary) network develops between E6 and E8, prior to the establishment of the coronary ostia and a functional coronary circulation at E8–E9 (Tomanek et al., 2006). We injected India ink via the apex of beating E7 or E9 hearts to determine the extent of perfusion of the compact region of the left ventricle. As seen in Figure 2, we failed to ﬁnd ink carbon particles in vascular tubes at E7, even when erythrocytes were present (Fig. 2C). In contrast, nearly all vessels in E9 hearts contained the tracer (Fig. 2D–F). These data support the conclusion that the tubular network of the ventricles is not, to a signiﬁcant extent, open to the sinusoidal system of the trabecular spongy portion of the developing ventricles. Temporal Aspects of Myocardial Vascularization We double-immunostained tissue sections with antibodies against QH1 and smooth muscle a-actin to reveal endothelial lined channels and arteries/arterio- 992 TOMANEK ET AL. Fig. 2. Election micrographs of capillaries from E7 (A–C) and E9 (D–F) beating hearts perfused via the ventricular apex with India ink. Capillaries are in various stages of development. Arrows indicate ink carbon particles in E9 capillaries, but not in those of E7. Note that even when erythrocytes are present in E7 capillaries, they are devoid of carbon particles. Intraluminal erythrocytes are likely derived from blood islands at this stage of development. Scale bar ¼ 5 mm. les and veins (Fig. 3). These micrographs show the early stage of tubulogenesis in the compact region of the myocardium (E6.5) and its progression through E18, which corresponds to the end of the fetal period. Tubular density increases during the next few days (E8, E9, E10). Although both coronary stems are formed by E9, the coronary arteries develop their tunica media gradually in a base-apex direction and accordingly smooth muscle is not seen at the base-apex midpoint in the E9 specimen. After this time, coronary vessels increase in diameter by remodeling and more branches are formed, as evidenced by muscle cell recruitment to endothelial-lined channels (E10–E18). The growth of the vasculature parallels the growth of the ventricular wall, as previously documented by us in chicken embryos (Tomanek et al., 1999). In the quail, wall thickness increases sharply from 60 mm at E7 to over 200 mm at E10 (Fig. 3). During the next 6 days, the increase is more gradual as wall thickness reaches about 340 mm at E16. The diameter of the left coronary artery in general increases in proportion to the expansion of the ventricle’s compact region. Coronary Ostia and Coronary Artery Growth As previously described (Ando et al., 2004), an endothelial network develops around the aortic root at E6– E7, as seen in Figure 4A. Penetration of the aortic wall by a tubular network to form the two coronary ostia occurs between E8 and E9 (Fig. 4B) as previously documented (Ando et al., 2004; Tomanek et al., 2006). At the site of developing coronary ostia, we have consistently noted the presence of a blood island. The formation of the two coronary ostia is not simultaneous as indicated by the observation that E8 hearts frequently have only one coronary artery. The onset of coronary artery formation in E8 embryos (Fig. 4B) is characterized by a narrow channel through the aortic wall. The aortic smooth muscle cells realign themselves at this site from a circumferential alignment to one more in an outward direction, i.e., parallel to the coronary artery. This pattern is maintained through the embryonic/fetal period as seen in an E16 specimen (Fig. 4C). Micrographs of coronary arteries at their site of origin during development in E14 and E16 hearts are seen in Figure 4D–H. We consistently noted three features at this site. First, multiple branches occur close to the root of the coronary artery. Second, a vascular plexus consisting of capillaries and venules surrounds or is in close proximity of the coronary artery. This appears to be an expansion of the tubular plexus noted in the stages prior to the development of the coronary ostia. Thus, the tubular plexus, seen prior to coronary artery formation and contributing to the artery’s formation, does not regress in its entirety. Rather, the portion of the plexus at the coronary artery stems enlarges and surrounds these vessels. We noted this as a consistent feature in all hearts (E9–18). Third, parasympathetic ganglia are adjacent to the root of the coronary artery and present along its sites of branching. The patterns of coronary artery growth and spatial distribution were examined by following their course in serial sections, and a summary is illustrated in Figure 4I. The left coronary artery develops numerous branches, as is seen at the interventricular groove. These course toward the apex and posteriorly. The right coronary artery divides at its root into a large septal artery and a smaller right coronary artery division. The septal courses through the middle of the septum and provides branches anteriorly and posteriorly. Subsequently, with further development and an increase in LV wall thickness, the large branches of the left coronary artery are positioned somewhat closer to the epicardium. Vascular remodeling of major branches occurs simultaneously with muscularization of the endothelium-lined channels. Thus, both the two main coronary arteries and their major branches increase in diameter. For example, as the lumen of the left coronary artery is formed at E8, it is only 5–15 mm in diameter, but enlarges to about 30–40 mm at E9. We immunostained selective serial sections for VEGF and FGF-2 to determine preferential staining of various cells and structures (Fig. 5). Although both angiogenic growth factors are expressed throughout the myocardium, we noted selective staining of some subepicardial cells. VEGF immunoreactivity was intense in the cells of the epicardium and subepicardium, and in a select population of cells near the root of coronary arteries, and in blood islands (Fig. 5A–C). Most were spindle-shaped; they appear to be specialized components of the subepicardial population. At the root of the aorta, we noted that some smooth muscle and endothelial cells of small vessels stained distinctly for FGF-2, while larger vessels displayed a very weak reactivity (Fig. 5D). DEVELOPMENT OF CORONARY VASCULATURE 993 Fig. 3. Micrographs of left ventricular (LV) cross-sections immunoreacted for QH1 to label endothelium and smooth muscle a-actin to label arterioles and arteries. The epicardium is on the left and endocardium on the right of all micrographs. Tubulogenesis progresses as LV wall thickness increases from early onset at E6.5 through subsequent stages. Mean wall thickness 6 SE (n ¼ 4–7 per gestation day) of the compact myocardium is shown in the graph (lower right corner). Formation of the coronary ostia occurs at E8–E9, after which time coronary artery branches develop progressively from base to apex (stained red in these micrographs). Most of these vessels are situated intramurally in the ventricular wall and increase in size over time. Note major arteries and veins at E18 (seen in the bottom of the ﬁgure). V, vein; S, sinusoid. Scale bars ¼ 100 mm in E6.5 through E16 and 200 mm in E18. Development of the Arterial Wall: Innervation, Extracellular Matrix, and Apoptosis seen in later stages (E12 and E15), the nerve ﬁbers can be seen in contact with the tunica media. The numbers of nerve ﬁbers associated with vessels increased notably between E10 and E15. Thus, nerve ﬁber growth appears to follow arteries, with innervation becoming more evident in the later stages of development. Our data indicate a close association between vascular smooth muscle cells and two key extracellular components: ﬁbronectin and laminin (Fig. 5H–M). Developing arterioles/arteries are characterized by an intermingling Having deﬁned the temporal/spatial patterning of the coronary arterial system, we focused on development of the arterial wall. Based on immunohistochemical reactivity of neuroﬁlaments, we show that innervation of the coronary arteries and arterioles is progressive (Fig. 5E–G). In smaller vessels, as seen in E10, the nerve ﬁbers tend to lie near the vessel, while with further development, 994 TOMANEK ET AL. Fig. 4. Micrographs of coronary artery development. A: Immunostained with QH1. B: H&E-stained. C: 1 mm section of Spur’s embedded heart stained with Richardson’s solution. D–H: Immunostained with QH1 and smooth muscle a-actin. A: Base of aorta (ao), in an E7 specimen surrounded by capillary plexus (arrows) immunostained for endothelium. This capillary plexus will penetrate the aorta at E8–E9 to form the coronary artery stems. B: Early formation (E8) of a coronary artery (arrows) whose lumen is conﬂuent with the lumen of the aorta (20). C: Aortic wall (E16) near coronary artery ostia (not seen in this ﬁeld). Coronary artery branch on surface of aorta (arrow). The aortic smooth muscle cells are disorganized around the site of coronary ostia (within broken lines) in contrast to their characteristic circumferential arrangement (asterisk). D–G: Coronary artery roots and branches in E14 (D and E) and E16 (F and G) embryos. D: Coronary artery (ca) at the level of the aortic valve (av) in close proximity to a plexus of capillaries and venules. pa, pulmonary artery. E: Coronary artery (ca) stem emerging through the wall of the aorta. Again, note the capillary/venous plexus closely opposed to the artery (arrow heads). Some of these vascular channels are associated with cells positive for smooth muscle a-actin. F: A coronary artery stem at the aorta. Note that immunostaining for smooth muscle a-actin is more intense in the coronary artery than in the aorta. Branching of the main coronary artery (as indicated by the arrow) occurs close to the aorta. G: Branching of the main coronary artery at its root. Note the capillary/venous plexus (arrow heads) surrounding the coronary artery; most of its components are in the venular diameter range (> 10 mm). H: Major coronary artery branches (E16). Several branches of the left coronary artery are seen in the left ventricular free wall at the lower left coroner of this micrograft. The three arteries seen in the septum are divisions of the large septal artery, which is the main stem of the right coronary artery. rv, right ventricle; lv, left ventricle; sep, septum. I: Diagram illustrating the branching and distribution of the right and left coronary arteries. Note the periaortal cell and tubular network and its presence as a venous-capillary plexus at the coronary stems; the parasympathetic ganglia are consistently seen at the coronary artery stems and their branches. Scale bars ¼ 25 mm. of smooth muscle cells with a ﬁbronectin network. The latter is generously distributed at sites of vessel formation. As seen in Figure 5H, smooth muscle cells align themselves with ﬁbronectin, which appears to form a tract for them as they are recruited to vascular tubes. Regions containing early vessel formation are characterized by a generous ﬁbronectin network. We previously showed that ﬁbronectin in the developing rat heart pre- cedes capillary formation (Rongish et al., 1996). Thus, the origin of the extracellular component is likely from ﬁbroblasts. With further development, ﬁbronectin, as seen in E12 and E15 hearts, forms a ﬁne network within the tunica adventitia and extends into the tunica media. Laminin is somewhat similarly distributed, i.e., closely associated with smooth muscle cells, but additionally forms a ring somewhat removed from the media. This DEVELOPMENT OF CORONARY VASCULATURE 995 Fig. 5. Immunostaining for VEGF (A–C), FGF-2 (D), neuroﬁlaments (E–G), ﬁbronectin (H–J), laminin (K–M), and apoptosis (N–P). A: VEGF immunoreactivity is evident throughout the myocardium, but intense in the epicardial and subepicardial cells (E9 heart). B: VEGF expression is seen in a select population of cells (arrows) at the root of the coronary artery (ca), as well as the myocardium (left bottom of micrograph) in this E15 heart. C: Some cells (arrows) in a blood island stain intensely for anti-VEGF antibody (E11 specimen). D: FGF-2 expression is selectively intense in small vessels (arrows) at the aortic root (E14 specimen). Note that 3 embryonic days are represented in the micrographs as follows: E, H, K, N (E10), F, I, L, O (E12), and G, J, M, P (E15). All of these specimens were immunoreacted for smooth muscle actin (shown in red). Neuroﬁlament reactivity (green proﬁles), a marker for nerve ﬁbers, follows arterioles and arteries (E–G). Nerve ﬁbers are seen at the junction with smooth muscle (arrows) in E10 specimen; as vessels mature, nerve ﬁbers extend into vessels’ tunica media. Arterial/arteriolar development is closely associated with ﬁbronectin (H–J). Smooth muscle cells (arrows) are intermingled with a relatively dense ﬁbronectin network during early formation of the media in arterioles (H). In more mature vessels (I and J), ﬁbronectin ﬁbers surround smooth muscle cells in the media and also continue into the adventitia (arrows). Laminin also exists as a network within the media (K–M), but in addition encircles the vessels (arrows). N–P: Apoptotic nuclei (green) are found scattered throughout the myocardium. In arterioles/arteries, they can be seen in both the adventitia and media in various sized vessels. In O, several apoptotic nuclei are seen at a branching point. characteristic circle of laminin was noted in both developing and more mature vessels (Fig. 5K–M). Finally, we used TUNEL to localize apoptotic cells in the arterial wall. Apoptotic nuclei were encountered within the myocardium, subepicardium, and in arterioles/arteries. Since remodeling of vessels is necessary for both increases in their diameters and pruning of the tubular network, it is not surprising to ﬁnd 996 TOMANEK ET AL. apoptotic ﬁgures in the media and adventitia. They were seen in small as well as large arterioles/arteries (Fig. 5N–P). tubulogenesis. The magnitude of growth of the compact component of the left ventricle during the embryonic period, as noted by wall thickness, is very similar in quail (the current study) and chicken (Sedmera et al., 2000). DISCUSSION Since formation of the coronary vasculature is strikingly similar in avian and mammalian species (Tomanek, 2005), data obtained from quail and chicken reﬂect developmental patterns and mechanisms in man and other mammals. Experiments on avian hearts provide two distinct advantages over those using mammalian species, namely, accessibility and cost. The current study provides the ﬁrst temporal-spatial documentation of coronary vascularization throughout the entire embryonic/fetal period. Tubulogenesis Hirakow (1983) noted that the ﬁrst deﬁnitive blood vessels in the developing human heart resembled blood islands, with ‘‘primitive erythroblasts.’’ Perez-Pomares et al. (1998) found that 20% of the cells within the expanding subepicardium in the embryonic hamster heart were VEGFR-2-positive, suggesting that they were endothelial or erythrocyte progenitors. Subsequently, CD45-positive hematopoietic precursors were shown to precede blood vessel formation in the quail heart (Kattan et al., 2004). Most recently, using retroviral tagging, we documented that erythrocytes in blood islands are derivatives of the proepicardium (Tomanek et al., 2006). Thus, the proepicardium is a source of not only all vascular components (Mikawa et al., 1992, 1996), but also erythrocytes that associate with endothelial cells that form vascular channels. Our current study and previous work (Tomanek et al., 2002) also document a very strong expression of VEGF protein in epicardial and subepicardial cells. Thus, this ligand is available in high quantity at the site of cells that delaminate from the mesothelium of the epicardium and that express the VEGFR-2 receptor. The earliest tube formation (capillaries) occurs in the subepicardium, where there is the highest level of VEGF expression. Vrancken Peeters et al. (1997) noted that the myocardial tubular network has nonluminized strands that connect to the sinusoids that are continuous with the ventricular lumen. They suggested that ﬂow through the capillary bed prior to connections to the aorta is possible. Subsequently, Manner (2000) grafted quail proepicardium to chick hearts at E3 and incubated the chimeras until E10. He found small patches of quail-derived endocardium in communication with the coronary vasculature, which he described as ‘‘ventriculo-coronary communications.’’ Although the question of ﬂow between the ventricular lumen and the coronary system is not entirely resolved, our electron micrographs of ink-injected beating hearts show that capillary proﬁles lack carbon particles prior to the establishment of the coronary arteries. Even when erythrocytes were present in the vascular tubes of E7 hearts, carbon particles were lacking. Thus, blood islands lacked connections with the ventricular lumen. As the compact region of the ventricle expands, tubulogenesis keeps pace, a ﬁnding consistent with our previous work on embryonic chicken hearts (Tomanek et al., 1999). In that study, we showed that experimentally accelerated myocardial growth is matched by enhanced Coronary Arterial System: Arteriogenesis Since the ﬁrst evidence that the coronary arteries are formed by ingrowth, rather than outgrowth, into the aorta (Bogers et al., 1989), this concept has been corroborated by a variety of approaches in several species (Waldo et al., 1990; Poelmann et al., 1993; Tomanek et al., 1996). Ando et al.(2004) showed that endothelial strands in the quail penetrated the aorta at E6 and E7 and fused by E8 to form the coronary stems. Our own ﬁndings agree with this temporal sequence, but we also provide evidence that the tubular network at the coronary ostia expands into a venous-capillary plexus, surrounds the coronary artery stems, and persists through venous-capillary plexus, and the coronary arteries and the parasympathetic ganglia remain in close proximity throughout this period (Fig. 4). It is not surprising, as documented in this study, that parasympathetic ganglia are closely associated with the roots of the two coronary arteries and also with their immediate branches. The presence of these ganglia has been documented to be necessary for normal development of coronary artery vascular smooth muscle (Hood and Rosenquist, 1992). Their study used neural crest ablation to demonstrate a spatial disordering of smooth muscle, the presence of only one coronary ostium and anomalous coronaries from the subclavian artery. Moreover, cardiac ganglion cells in the chick have been shown to originate from the cardiac neural crest (Verberne et al., 1998). The latter are not coronary artery smooth muscle precursors, but rather are positioned at the base of coronary arteries (Waldo et al., 1994), a ﬁnding that suggests their signaling may contribute to formation of the coronary ostia. The locations of the intracardiac ganglia in human neonate (Smith, 1971) and adult (Pauza et al., 2000) have been described in detail. The formation of the coronary ostia at E8–E9 is dependent on a variety of factors (reviewed by Tomanek, 2005). Foremost is a functional epicardium, including the timing of its outgrowth (Eralp et al., 2005). Our recent work has shown that VEGF family members, especially VEGF-B, play a critical role in the formation of coronary ostia (Tomanek et al., 2006). Further growth and remodeling of the coronary arteries have not received much attention. That arteriolar growth is dependent on FGF-2 has been demonstrated in neonatal rats (Tomanek et al., 2001). In that study, we showed that arteriolar growth was attenuated in response to anti-FGF-2 neutralizing antibodies. When both FGF-2 and VEGF are experimentally decreased, there occurs a shift in the arteriolar hierarchy, i.e., length density of the smallest arterioles is decreased and increased in the largest arterioles. Moreover, precocious expression of FGF-2 induces abnormal coronary artery branching (Mikawa, 1995). Current studies in our laboratory on embryonic quail indicate that PDGF-B, which is expressed in coronary endothelial cells (Van Den Akker et al., 2005), in combination with FGF-2 are critical for coronary artery formation (data not shown). DEVELOPMENT OF CORONARY VASCULATURE As noted in the current study, coronary artery growth is rapid. Ratajska et al. (2000) noted that coronary artery diameter in the rat increases fourfold in a 3-day period prior to birth. Our ﬁndings in the quail show a progressive increase in coronary artery diameter that follows the increase in ventricular thickness. By analysis of serial sections, we have also noted a consistent pattern of branching and distribution of the coronary arterial tree, including a dominant septal artery. The substantial increases in artery diameter must involve a rapid remodeling process, which occurs with increased ﬂow, as shown in adult models of remodeling (Gibbons and Dzau, 1994; Sho et al., 2003). It has been reported that coronary artery formation predates the periarterial Purkinje ﬁbers (Harris et al., 2002). The periarterial Purkinje ﬁbers thus follow the course of coronary arteries. Interestingly, the large main septal artery lies between the two main bundle branches of the conduction system. A recent review of apoptosis in the chick heart notes that the greatest numbers of apoptotic ﬁgures occur in the outﬂow tract in HH 19–26 hearts, with a concentration noted just below the aorta at HH 31 (Martinsen, 2005). This concentration, also noted by Schaefer et al. (2004), at this developmental stage corresponds to the site of coronary ostia formation. Velkey and Bernanke (2001) documented apoptosis within the aorta at the site of penetration by the peritruncal capillary plexus, as well as pruning of the capillary plexus around the aorta, a ﬁnding that is consistent with observations on quail (Ando et al., 2004). Although it is well known that apoptosis functions in vascular pruning and reduction in vessel diameter (reviewed by Fisher et al., 2000), less attention has been paid to its role in angiogenesis and arteriogenesis. In this regard, it has been shown that inhibition of apoptosis attenuates capillary formation in vivo (Segura et al., 2002). It has been noted that apoptosis occurs mainly in the ‘‘nonmyocardial compartment’’ (Poelmann et al., 2000), with a high rate in neural crest cells (Poelmann et al., 2005). Apoptotic ﬁgures were noted in coronary arteries of HH 40 (E14) chick hearts (Cheng et al., 2002). Our study documents apoptosis in various-sized arterial vessels at E10 and later in both the medial and adventitial tunics. This suggests a role for apoptosis in coronary vessel remodeling. It is important to understand apoptosis in the developing heart because it plays a role in cardiac malformations (Fisher et al., 2000). The importance of the extracellular matrix in endothelial morphogenesis and stabilization has been reviewed (Davis and Senger, 2005). Evidence is provided to support the idea that ﬁbronectin and collagen matrices stimulate tubulogenesis, whereas laminin-rich matrices affect stabilization. This supports our previous ﬁnding on rat embryonic development of the coronary vasculature (Rongish et al., 1996). In that study, we documented a role for ﬁbronectin as scaffolding for vascular cells and suggested that laminin deposition coincides with tube formation. The current study, which focused on the arterial system, also indicates the importance of ﬁbronectin for cell migration as evidenced by smooth muscle cell contact with ﬁbronectin strands. We show that in addition to the presence of ﬁbronectin and laminin in the artery’s media, the latter forms a distinct ring outside of the tunica adventitia. Both of these noncollagenous gly- 997 coproteins are cell adhesion molecules that are highly expressed in the embryonic human coronary arteries (Kim et al., 1999). However, our ﬁndings should not be interpreted to mean that ﬁbronectin and/or laminin are the only extracellular matrix components regulating vessel development. For example, ﬁbulin-1 and ﬁbulin-2 are expressed in tunica adventitia and basement membrane of the endothelium in human embryonic blood vessels (Miosge et al., 1996). Perspective Myocardial vascularization includes many phenomena that are spatially and temporally regulated. The current study illustrates the progression of myocardial vascularization in the quail during the embryonic and fetal periods, i.e., E6–E18. Most of our data concern the development of the arterial hierarchy, i.e., arteriogenesis, a component of vascular development that has received less attention than the earlier stages of vascularization, i.e., vasculogenesis and angiogenesis. Our ﬁndings, some of which conﬁrm and complement data from other studies, provide new insights into several developmental events. Using electron microscopy, we show some features of coronary precursors, including blood islands, and demonstrate that the tubular network is almost entirely without connections to the endocardium. The hierarchy of the arterial system, based on serial sections, has been revealed and its development noted with respect to innervation, extracellular matrix, and apoptosis. Thus, the ﬁndings presented here provide an overview of a relatively broad period of vascular development. Our goal in providing these data was to chart the components of vascular development and provide a foundation for future studies on the mechanism underlying formation of the coronary vasculature. LITERATURE CITED Ando K, Nakajima Y, Yamagishi T, Yamamoto S, Nakamura H. 2004. Development of proximal coronary arteries in quail embryonic heart: multiple capillaries penetrating the aortic sinus fuse to form main coronary trunk. Circ Res 94:346–352. Bergwerff M, Verberne ME, DeRuiter MC, Poelmann RE, Gittenberger-de Groot AC. 1998. Neural crest cell contribution to the developing circulatory system: implications for vascular morphology? Circ Res 82:221–231. 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