Chapter 14 In the early stages of development, the respiratory, excretory and nutritional requirements of the embryo are provided by simple diffusion. As the conceptus increases in size, diffusion is inadequate for its nutritional, respiratory and excretory needs. Consequently, the mammalian embryo requires a system for delivering oxygen and nutrients to its tissues and for removing its waste products. These requirements are supplied by the cardiovascular system. As one of the first functional systems to develop in the embryo, the cardiovascular system consists of a central pumping organ, the heart, linked to a set of arteries which convey blood to the tissues. Complementary vessels, called veins, carry blood from the tissues back to the heart. An associated network, the lymphatic system, assists in the return of extracellular fluids to the vasculature. Blood vessel formation occurs as a consequence of two sequential processes, vasculogenesis and angiogenesis. Vasculogenesis, the formation of blood vessels from blood islands, commences during the third week of gestation in domestic mammals, first in the yolk sac and later in the allantois. A number of factors including fibroblast growth factor 2 (Fgf‐2), vascular endothelial growth factor (VEGF) and the angiopoietin proteins have an initiating role in vasculogenesis. Fibroblast growth factor induces splanchnic mesodermal cells to form haemangioblasts in the yolk sac. Vascular endothelial growth factors are a family of proteins which are key to vasculogenesis and are expressed at high levels in areas proximal to active blood vessel formation. These signalling factors act on tyrosine kinase receptors such as Flk1 present on both haemangioblasts and angioblasts and subsequently promote the differentiation of angioblasts into endothelial vessels. The contribution of haemangioblasts to the formation of blood vessels and to haematopoiesis is outlined in Figure 14.1. Figure 14.1 Outline of the origin and differentiation of angioblasts and haematopoietic stem cells from a common mesodermal precursor, the haemangioblast. The haematopoietic stem cell initially gives rise to a primitive erythroid lineage but as maturation proceeds definitive erythrocytes and myeloid cells are produced, along with cells from which the lymphoid lineage develops. Angiopoietins promote the interaction between endothelial cells and smooth muscle cells which eventually surround some developing blood vessels. Development of blood vessels involves a complex series of events during which endothelial cells differentiate, proliferate, migrate and become organised into an orderly vascular network. Splanchnic mesodermal cells lining the yolk sac form clusters, referred to as blood islands. With the formation of extra‐embryonic vascular channels, a primitive circulatory system becomes established. In contrast to vasculogenesis, angiogenesis comprises several morphogenic events during which pre‐existing endothelial cells sprout, branch and become canalised. Other processes that occur during angiogenesis include the remodelling of existing vessels through anastomosis and branching accompanied by increases in luminal diameter. This process, a fundamental requirement for embryological development, continues postnatally. VEGF‐a is critial to angiogenesis and is produced by mesenchymal cells. This factor acts on the endothelial cells at points where new vessel formation commences, termed ‘tip’ cells. These tip cells express Delta‐like‐4 (DLL4) which is a ligand to Notch receptor, while adjacent cells express Notch. The expression of Notch receptor prevents these latter cells from responding to VEGF‐a, (unlike the DLL4‐expressing tip cells) thereby spacing the development of new blood vessels. Angiopoietin 1 subsequently interacts with receptor Tie‐2 on endothelial cells at sites where sprouting occurs. At these points, endothelial cells can proliferate and form new vessels. In response to the angiopoietin 1–Tie‐2 interactions, which occur during angiogenesis, endothelial cells release the signalling molecule platelet‐derived growth factor (PDGF) which stimulates migration of mesenchymal cells towards the vascular endothelium. In response to the release of other growth factors by endothelial cells, differentiation of mesenchymal cells into vascular smooth muscle cells occurs. When initially formed, the haematopoietic islands are compact structures. As development progresses, cells at the periphery of the blood islands, under the influence of growth factors, become squamous in shape and surround the centrally‐located cells. The squamous cells form the endothelial lining of the emerging vascular system and the round, centrally‐located cells become the haemoblastic cells or embryonic nucleated erythrocytes (Fig 14.2). Figure 14.2 Sequential stages in the formation of blood vessels and blood cells from blood islands in the yolk sac (A to D). Vascular development occurs under the influence of specific growth factors. Basic fibroblast growth factor, which binds to receptors on splanchnic mesodermal cells, induces them to form haemangioblasts. Vascular endothelial growth factor promotes the differentiation of peripheral haemangioblasts in blood islands into angioblasts which, in turn, differentiate into endothelial cells and form blood vessels. Maturation of the capillary network is influenced by PDGF and transforming growth factor‐β (Tgf‐β). Development of individual channels in the network depends on the volume and direction of blood flow. The channels which convey the greatest volume of blood increase in diameter and acquire additional tissue layers from the surrounding mesoderm, becoming thick‐walled vessels referred to as arteries; the other vessels, veins, remain thin walled. Blood vessels which develop in the foetal membranes, referred to as extra‐embryonic vessels, consist of paired vitelline (yolk sac) and umbilical (allantoic) arteries and veins. Intra‐embryonic formation of blood vessels, which proceeds in a similar manner to extra‐embryonic vasculogenesis, commences soon after blood vessel formation begins in the extra‐embryonic membranes. Subsequently, the extra‐embryonic and intra‐embryonic vessels anastomose, completing the rudimentary circulatory system of the conceptus (Fig 14.3). Figure 14.3 Left lateral view of a mammalian embryo showing the rudimentary cardiovascular system. Early in gestation, the embryo has a pear‐shaped outline and consists of three layers, namely a dorsal layer of ectoderm, a ventral endodermal layer and a middle mesodermal layer. Small discrete spaces in the left and right lateral mesoderm enlarge and coalesce, forming a left and a right intra‐embryonic coelom, thereby splitting the lateral mesoderm into somatic and splanchnic layers. Later, the coelom on the right and the coelom on the left fuse cranial to the developing neural plate, forming an enlarged horseshoe‐shaped coelomic cavity (Fig 14.4). Ventral to the coelom, groups of cells in the splanchnic mesoderm form the cardiogenic plate, which is also horseshoe‐shaped. Within the cardiogenic plate, angiogenic cell clusters give rise to a horseshoe‐shaped structure, the endocardial tube. Later, the lateral limbs of the horseshoe‐shaped vessel form the left and right endocardial tubes. Splanchnic mesodermal cells, which migrate towards and surround the endocardial tubes, form the myoepicardial mantle. At first, this mantle does not attach to the endothelium of the tubes. The intervening space contains a loose, gelatinous reticulum referred to as cardiac jelly. Many of the major intra‐embryonic blood vessels are formed contemporaneously with the endocardial tubes and extra‐embryonic vessels. Mesodermal cells proliferate in a position cranial to the cardiogenic plate and form the septum transversum, which subsequently gives rise to the tendinous part of the diaphragm. The developing embryonic disc undergoes cranio‐caudal and lateral folding. As a consequence of folding of the cranial portion of the embryo, the endocardial tubes and coelom and the septum transversum are displaced caudally. Consequently, the endocardial tubes lie dorsal to the coelom, ventral to the foregut and caudal to the oropharyngeal membrane (Fig 14.5). The caudal displacement of the developing heart is accompanied by rapid growth of the brain in a cranial direction so that it extends over the cardiac area. In this position, the convex segment of the fused endocardial tubes anastomoses with the vitelline veins from the yolk sac (Fig 14.6). Before joining the convex segment of the endocardial tube, the vitelline and umbilical veins pass through the septum transversum. The cranial portions of the dorsal aortae, which are drawn ventrally, form dorso‐ventral loops. These loops, the first aortic arch arteries, fuse with the endocardial tubes (Fig 14.6). With lateral folding of the embryo, the left and right endocardial tubes, surrounded by their muscular layers, gradually converge. Fusion of the medial walls of the endocardial tubes first occurs midway along their length. Later, fusion extends cranially and caudally until a single cardiac tube is formed (Figs 14.6 and 14.7). However, as fusion does not extend along the entire length of the endocardial tubes, the cranial and caudal ends remain separated. The endothelial lining of the single cardiac tube becomes the endocardium, the myoepicardial layer forms the myocardium and, from the visceral layer lining the pericardial cavity, the epicardium is formed. Figure 14.4 Development of the cardiac tube and the coelomic cavity at the embryonic disc stage. Figure 14.5 Sequential stages in the cranio‐caudal folding of the embryo showing the changed relationship of the developing heart to other embryonic structures (A to D). Arrows indicate the direction of folding of the embryo. Figure 14.6 Stages in the formation of the heart from the cardiac tube stage to the development of an S‐shaped structure (A to I). Figure 14.7 Ventral views of the developing cardiac tubes and coelom with corresponding cross‐sections (A to D). The cardiac tube, which is located in the pericardial cavity, is initially suspended by a dorsal mesocardium and anchored by a ventral mesocardium (Fig 14.7). This cardiac tube undergoes differential growth along its length, which results in expanded portions separated by non‐expanded portions. Listed in sequential order from the cranial end, the expanded portions are the truncus arteriosus, the bulbus cordis, the ventricle, the atrium and the sinus venosus (Fig 14.8). The caudal end of the sinus venosus remains bifurcated. The ventral mesocardium persists for only a short period, while the dorsal mesocardium gradually breaks down leaving only the truncus arteriosus and ventricle attached to the pericardium. The atrium and sinus venosus are initially located outside the pericardial cavity in the septum transversum. Because the primitive heart increases in size faster than the pericardial cavity, especially in the bulbo‐ventricular region, a U‐shaped bend, the bulbo‐ventricular loop, forms. As a consequence of this development, the atrium and sinus venosus become drawn into the cavity (Fig 14.8B). The loop occupies a ventral position in the pericardial cavity, to the right of the median plane. Further growth of the developing heart causes the atrium to occupy a position dorsal to the bulbus cordis and ventricle, where it expands towards the truncus arteriosus. The sinus venosus is drawn into the pericardial cavity, and at this stage the developing heart becomes S shaped (Fig 14.8). Figure 14.8 Dorso‐ventral and left lateral views of sequential stages in the differentiation of the cardiac tube, from the bulbo‐ventricular loop stage to the expansion of the bulbo‐ventricular loop ventrally, and the common atrium dorsally (A to D). A number of transcription factors have been implicated in the process of bulbo‐ventricular loop formation. These include Hand‐1 and Hand‐2 transcription factors which are regulated by Nkx‐2.5. As heart development proceeds, Hand‐1 expression becomes confined to the developing left ventricle and Hand‐2 to the developing right ventricle. Deletion of genes which encode Hand‐1 or Hand‐2 factors results in hypoplasia of the ventricle in which they are normally expressed. The T box factors, Tbf‐5 and Tbf‐20, together with Bmp‐4, also influence the formation of the bulbo‐ventricular loop. Differential contraction of the actin cytoskeleton has been proposed as a determining factor in the formation of the bulbo‐ventricular loop. During cardiac morphogenesis, blood vessel formation continues within the embryo. Two major blood vessels which form ventral to the neural tube become the left and right dorsal aortae. Cranially, they fuse with the left and right limbs of the endocardial tubes. Associated with the lateral folding of the embryo, the dorsal aortae caudal to the developing heart fuse, forming a common aorta. In the mesenchyme adjacent to the truncus arteriosus, an additional series of paired aortic arch arteries develop which join the dilated end of the truncus arteriosus with the dorsal aortae (Fig 14.8). Branches of the dorsal aortae, the intersegmental arteries, supply the developing somites. Additional branches supply the yolk sac through the vitelline arteries and the umbilical arteries supply the allantois. Satellite veins are formed which drain the yolk sac and allantois, while the cranial cardinal veins and the caudal cardinal veins convey venous blood from the head and body wall respectively. The venous blood is returned to the caudal end of the primitive heart, the sinus venosus (Fig 14.3). On each side of the developing embryo, the cranial and caudal cardinal veins fuse, forming the common cardinal veins which enter the sinus venosus. At this stage of development, the mammalian cardiovascular system bears a strong resemblance, both morphologically and functionally, to that of the fully formed circulatory system of fish. Bilateral fields of cardiac precursor cells form two populations, referred to as the first and second heart fields. The first heart field forms the heart tube and contributes to the development of the left ventricle and all other parts of the heart except the cardiac outflow tract (pulmonary trunk and aortic regions). Cells derived from the second heart field contribute to the development of the cardiac outflow tract, right ventricle and most of the atria. Both of these fields display distinct expression of marker genes. For example, HCN4 marks the first heart field, while Isl1 is expressed only in the second heart field. Precursors within the two fields give rise to distinct lineages and differentiate according to divergent transcriptional programs. The transcription factor Nkx‐2.5 is central to the initial induction of splanchnic mesodermal cells which ultimately contribute to cardiogenic mesoderm formation. This transcription factor is up‐regulated under the influence of Bmp and Fgf factors. Nkx‐2.5 activates the synthesis of other transcription factors such as members of the GATA family, Mef‐2 and Hand. Nkx‐2.5 and GATAs mutually reinforce each others expression in the developing cardiac tissue. These transcription factors in turn upregulate the expression of cardiac‐specific proteins, including cardiac actin and α‐myosin. In addition, other transcription factors including Nodal and Lefty‐2 influence the pattern of asymmetry, a feature of heart formation. The transcription factor Pitx‐2, which is upregulated by Nodal, is critical for normal heart morphogenesis. Partitions which form in the primordial mammalian heart gradually convert the single pulsating cardiac tube into a complex four‐chambered organ. Although formation of cardiac septa takes place at approximately the same time, for descriptive purposes their formation is described as if they were separate events. The foetal heart continues to function effectively as these ongoing major structural changes occur. In the equatorial region of the atrio‐ventricular canal, two masses of cardiac mesenchymal tissue known as endocardial cushions, which are located between the endocardium and the myocardium, extend towards each other and fuse. The fused endocardial cushions form the septum intermedium, which divides the common atrio‐ventricular canal into left and right atrio‐ventricular openings (Fig 14.9). Figure 14.9 Stages in the division of the common atrio‐ventricular canal into left and right atrio‐ventricular openings, resulting from the fusion of the endocardial cushions and the formation of the septum intermedium at the level of the endocardial cushions. Arrows in A and B indicate direction of growth of endocardial cushions; arrows in C indicate direction of blood flow. During proliferation of the endocardial cushions, a crescent‐shaped fold, the septum primum, arises from the dorsal wall of the common foetal atrium and extends towards the endocardial cushions. The septum primum gradually divides the common atrium into a left and a right atrium (Fig 14.10). As the septum primum grows towards the endocardial cushions, an opening, the foramen primum, persists between the left and right foetal atria. This foramen gradually decreases in size and, when the septum primum reaches the cushions, it eventually closes. Before closure of the foramen primum, however, programmed cell death in the central part of the septum primum results in the formation of a new communication channel between the left and right atria, the foramen secundum (Fig 14.10D). A second membrane, the septum secundum, arises from the dorsal wall of the right atrium, to the right of the septum primum, and extends towards the septum intermedium. The central portion of the septum secundum overlaps the foramen secundum, but does not extend as far as the septum intermedium. The opening which persists between the free edge of the septum secundum and the foramen secundum is known as the foramen ovale. The upper part of the septum primum fuses with the septum secundum while the remaining portion becomes a valve‐like structure for the foramen ovale. The lower margin of the septum secundum divides the blood entering the heart via the caudal vena cava into two streams. The greater amount is directed through the foramen ovale into the left atrium, while a lesser amount is directed through the right atrio‐ventricular opening into the right ventricle. Due to its functional role, the lower margin of the septum secundum is appropriately named the crista dividens. At birth, the foramen ovale closes, completing the separation of the left and right atria. Figure 14.10 Stages in the partitioning of the developing atrium and ventricle, leading to the formation of left and right atria and ventricles (A to F). The arrow in F indicates the direction of blood flow through the foramen ovale. In the early stages of cardiac morphogenesis, blood returning from the left side of the embryo enters the left horn of the sinus venosus. Blood from the right side of the embryo enters the right horn of the sinus. The venous blood entering the sinus venosus enters the embryonic atrium through the sino‐atrial opening, which is regulated by the sino‐atrial valve composed of left and right components. Development of venous shunts between the left and right systemic venous systems leads to the preferential direction of flow to the right side, resulting in enlargement of the right horn of the sinus venosus while the left horn decreases in size. As partitioning of the atrium proceeds, the sino‐atrial opening occupies a position in the right half of the foetal atrium. Gradually, the right horn of the sinus venosus becomes incorporated into the right foetal atrium. In its final form, the right atrium consists of the right foetal atrium which becomes the muscular right auricle, while the right horn of the sinus venosus becomes the thin‐walled sinus venarum into which the venous return from the body enters the heart (Fig 14.11). During morphological adaptation, the left portion of the sino‐atrial valve fuses with the septum secundum, while part of the right portion forms an internal ridge, the demarcation between the auricle and the sinus venarum, termed the crista terminalis. On the external surface a depression, the sulcus terminalis, marks this division. The remainder of the right portion of the sino‐atrial valve contributes to the formation of the valves of the caudal vena cava and coronary sinus. The regressing left horn of the sinus venosus forms part of the coronary venous sinus, which opens into the right atrium.
Cardiovascular system
Development of the cardiac tubes
Molecular aspects of cardiac development
Formation of the cardiac chambers
Partitioning of the atrio‐ventricular canal
Partitioning of the common foetal atrium
Final form of the right atrium
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