In humans, congenital heart diseases occur in approximately twelve out of 1000 live births (Hoffman et al. 2004
). Although heart development in all vertebrates, from fishes to humans, follows the same general pattern, much of the pioneering research on the developing heart has been conducted in zebra fish and chicks. Since the rapid progression of transgenic technologies, the mouse has become the major animal model in which to study normal and abnormal cardiovascular development. Because cardiogenesis is a very complicated process and vital to the survival of the embryo, one of the main and relatively common causes of in utero lethality of any post-implantational mutant mouse embryo/early fetus is defective cardiovascular development (Conway et al. 2003
; Copp 1995
; Papaionnou and Behringer 2005
). Currently, phenotyping the embryonal mouse heart is an arduous task due to limited resources. Current resources include a detailed, descriptive anatomical histology atlas of mouse development (Kaufman 1992
; Theiler 1972
), an online tutorial of normal mammalian development using scanning electron micrographs (Sulik and Bream 1999
), a high-resolution magnetic resonance histology atlas of the embryonic and neonatal mouse (Petiet et al. 2008
) and the Edinburgh 3D mouse embryo anatomy atlas (http://genex.hgu.mrc.ac.uk/
). However, there is a need for a more detailed, stage-by-stage, descriptive anatomic and histological reference on the developing mouse heart. Because of the potential for strain differences when evaluating normal development or developmental defects, it is crucial to always compare to concurrent controls, with littermates considered the ideal source of comparison.
Early Mouse Heart Development
The heart is the first organ to develop and function in the embryo. Cardiomyocytes differentiate from precursor cells in the primitive streak and move anterior-laterally to form bilateral paired cardiogenic plates (myocardial primordial) in the mouse embryo at E7.5. Subjacent to these plates, endothelial cells differentiate and form right and left endocardial heart tubes (Kaufman and Bard 1999
). These endothelial-lined vessels align in a parallel fashion with each other and then fuse across the ventral midline to form a single beating heart tube by E8.0 with rostral arterial (aortic sac) and caudal venous poles (right and left sinus horns). At first, the contractions are irregular, but by E9.0 a regular heartbeat is established. The venous pole acts as the initial pacemaker, and the wave of muscle contraction is then propagated along the tubular heart (Kaufman and Navaratnam 1981
The heart tube consists of the outer myocardial and the inner endothelial cell layers. Between these two layers is a space filled with an extracellular matrix, the cardiac jelly (DeHaan 1965
; Kaufman and Navaratnam 1981
; Wessels and Markwald 2000
). The heart tube subsequently loops to the right during E8.5–10.5, with the venous pole moving cranially and dorsally (). At E8.5, three regions can be distinguished by the bulging morphology: bulbus cordis (future right ventricle), primitive left ventricle, and common atrial chamber behind the primitive left ventricle () (Kaufman and Bard 1999
). Concomitantly with the rightward looping, lengthening of the tube and further ballooning of the future chambers takes place. Myocardium from the pharyngeal arch region (second or anterior heart field) adds to the lengthening outflow tract after the formation of the initial heart tube (first heart field) (Kirby 2002
; Kirby 2007
; Yelbuz et al. 2002
). From E9.5, several segments can be distinguished (). The right and left sinus horns empty all of the systemic blood into the common atrial chamber. The common atrial chamber is connected to the primitive left ventricle by an atrioventricular canal. The primitive left ventricle communicates with the future right ventricle via the bulboventricular canal. The outflow tract, conus and truncus (arteriosus), connects the future right ventricle to the aortic sac. Further development of the atrial and ventricular parts is dependent on the expansion or ballooning of the chambers. The atrial chambers expand on both sides of the developing arterial pole, whereas ventricular chambers form in the ventricular loop along the outer curvature (), with the ballooning producing the interventricular septum between the pouches (Anderson et al. 2006
Figure 1 Early cardiovascular development of the mouse. The schematic illustrations of the mouse heart from E8.5 (A), E9.5 (B) and E10.5 (C) illustrate the early developmental events of the cardiogenesis. The aortic sac (AS) contributes to the aortic arch arteries (more ...)
In the atrioventricular canal and in the common outflow tract, localized swellings of the inner heart wall arise at E9.5 (Kisanuki et al. 2001
; Mjaatvedt and Markwald 1989
). These swellings are termed atrioventricular and outflow tract cushions or ridges, and they will contribute to all septal and valvular structures that are needed to produce the four chambers of the heart as well as the two separate outflow channels, the aorta and the pulmonary trunk, respectively (Fananapazir and Kaufman 1988
Three populations of extracardiac cells are incorporated into the heart through waves of cell migration: pro-epicardium from the mesenchyme of the septum transversum; dorsal mesocardium, which is incorporated into the prospective atria from the body wall; and cardiac neural crest cells, which migrate through pharyngeal arches to the outflow tract. The pro-epicardium invests the external surface of the entire heart, including the outflow tract during and right after the looping E9.0-11.0 (Komiyama et al. 1987
). In addition, endothelial and smooth muscle cells of the coronary vasculature, as well as connective tissue, are formed by the epithelial-mesenchymal transformation of the epicardium (Perez-Pomares et al. 1997
). In addition to forming a primary atrial septum (septum primum), dorsal mesocardium is important in connecting the primitive atrial chamber to the midline of the body of the embryo and to the pulmonary pit, the entrance point of the pulmonary vein (Webb et al. 1999
). The cardiac neural crest cells contribute fundamentally to the developing aortic arch arteries and to the septation of the outflow tract and the formation of outflow tract valves (Gitler et al. 2003
; Kirby 2007
Principal Features of Embryonal/Fetal Circulation
By E8.5, the primitive circulation has been established. The inflow of blood to the venous pole of the primitive heart comes from three sources: oxygenated blood from the placenta via the umbilical veins, and deoxygenated blood from the body (via the right and left anterior and posterior cardinal veins, common cardinal veins and horns of sinus venosus) and from the yolk sac (via the vitelline veins) (Kaufman and Bard 1999
). The collection point of blood from the two sinus horns is called the sinus venosus, and the junction of the sinus venosus with the primitive atrium is called the sinoatrial junction. As this junction gradually shifts to the right, two craniocaudally oriented valvelike structures, the right and left venous valves, are produced. These valve leaflets will meet cranially and form a septum called the septum spurium, which does not contribute to atrial partitioning (Anderson et al. 2006
; Kirby 2007
At E11, concomitantly with the development of the lungs in the body wall behind the heart, a primary pulmonary vein starts to canalize within the dorsal wall of the common atrial chamber, at the site of the mesocardial pulmonary pit and between the left and right pulmonary ridges (Anderson et al. 2006
; Kirby 2007
). These ridges demarcate the site of the persisting dorsal mesocardium within the atrial wall. The right pulmonary ridge becomes especially prominent and is the structure that has long been known as the vestibular spine (His 1880
From the heart, the largely oxygenated blood goes through the outflow tract to the branchial (pharyngeal) arches and then around the primitive pharynx to the dorsal aortae, most of it supplying the rostral regions of the embryo. In the mid-abdominal region, the paired dorsal aortae fuse to form a single midline vessel, which gives off the vitelline artery supplying the primitive yolk sac. More caudally, the single dorsal aorta bifurcates again and gives rise to the common iliac arteries and then the paired umbilical arteries, which carry the deoxygenated blood to the placenta and the various arterial branches to the pelvic viscera.
By the time looping has finished, the aortic sac at the arterial end has given rise to six bilaterally symmetric vessels known as pharyngeal or aortic arch arteries. The pharyngeal arch arteries arise sequentially along the anterior-posterior axis, each traversing a pharyngeal arch before joining to the paired dorsal aortae (Hiruma et al. 2002
; Kaufman 1992
). The first and second pharyngeal arch arteries develop around E8.5–9.5 but are disrupted by E10.5. However, their distal parts persist in mice and are transformed into the mandibular and stapedial and hyoid arteries, respectively (Hiruma et al. 2002
). The third pharyngeal arch arteries are evident at E9.5, and fourth and sixth pharyngeal arch arteries at E10.0 and E10.5, respectively. The fifth pharyngeal arch arteries never fully form in mammals. The arch arteries undergo extensive remodeling to ultimately form a mature aortic arch and proximal pulmonary arteries.
Apoptosis during Heart Development
Programmed cell death, or apoptosis, occurs in the normally developing heart at specific times and regions, and it is involved in the developmental remodeling of tissues by targeting transient cells and allowing for further tissue differentiation (Jacobson et al. 1997
). The primary sites of apoptosis in the normal developing heart are the outflow tract and atrioventricular cushions, the walls and developing valves of the aorta and pulmonary trunk, and the upper part of the interventricular septum (Icardo 1996
; Pexieder 1975
; Poelmann et al. 2000
). Apoptosis also plays an essential role in the ventricular morphogenesis (E11.0–16.0) (Abdelwahid et al. 1999
). Outside the heart, remodeling of the originally symmetric pharyngeal arch arteries toward the unilateral left-sided aortic arch coincides with a highly spatiotemporal apoptosis pattern (Molin et al. 2002
). Though apoptosis is a normal phenomenon in embryonic heart development, aberrant patterns of apoptosis may cause cardiac malformation, including septation and coronary anomalies and interrupted aortic arch segments (Poelmann and Gittenberger-de Groot 2005
Defective Mouse Heart Development
To date, over a thousand mutant mice with abnormal heart morphology have been reported in the literature (). A majority of these defects cause lethality during embryonic/early fetal development. Early lethality (E8.5–11.0) may be due to the inadequate establishment of embryonic-maternal circulation, linear heart tube formation defects, and/or poor cardiac function (Conway et al. 2003
). The classic sign of poor cardiac function is edema. The progressive fluid build-up within the pericardial cavity may by itself result in further functional defects, and finally lethality. Failure to undergo correct looping and chamber formation of the primitive heart tube is rarely fatal per se, but it may lead to subsequent lethal misalignment defects.
The most common abnormal cardiovascular phenotypes found in mutant pre- or perinatal mice.
At later stages (E11.0 onward), improper septation of the primitive ventricles and atria, failure to establish the divided outflow tract, or inadequate establishment of the cardiac conduction system may result in embryonic lethality. At birth, failure of the in utero cardiac system to adapt to adult life and close the inter-atrial and aorta-pulmonary trunk shunts may lead to death (Conway et al. 2003
). Defects in embryonal/fetal heart development may cause lethality even later in postnatal life. It is also worth noting that a single developmental defect typically results in subsequent additional defects. However, especially in transgenic mice, several unrelated primary defects may arise.
Herein, the further development of the murine heart from E11.5 to E18.5 is described and demonstrated in detail with labeled representative histological images of different stages and orientations.