Cardiac Embryology

Precardiac to Cardiac Tissues: Commitment and Formation of Primary Axes

The tissue regions giving rise to the heart have been mapped by the application of dyes, particles, and radiolabels at stages when the vertebrate embryo comprises two layers: the epiblast (or primitive ectoderm) and the hypoblast (or primitive endoderm). The process of gastrulation, the movement of precardiac epiblast cells through the primitive streak, appears to be important in specification ( Fig. 71.2 A ). These cells migrate laterally to form the left and right heart fields (see Fig. 71.2 B ), which then create the horseshoe-shaped first and second heart fields around the end of the primitive streak, the primitive node. The second heart fields are adjacent to and influenced by cardiac neural crest cells, a process critical to understanding how the later steps in cardiogenesis are affected by neural crest–associated genetic syndromes.

Transcription factors are proteins that bind as part of complexes to specific DNA sequences and influence the expression of genes ( Fig. 71.3 ). Such factors influence many aspects of cardiogenesis and include members of the following families: the helix-loop-helix proteins (dHand and eHand), zinc finger proteins (GATAs), homeobox gene proteins (NKX2.5), and MADS box proteins related to serum response factor. Of the zinc finger proteins, GATA4, 5, and 6 are much-studied members of the evolutionarily conserved transcription factor subfamily that recognizes a “GATA” DNA sequence motif. These have been implicated in the early specification of embryonic tissue destined to become heart.

An example from the homeobox gene family demonstrates how the study of fruit flies has advanced our understanding of human heart development. The dorsal vessel pumps hemolymph (insect blood) and is considered the insect heart. It is derived from mesoderm under the influence of homeobox genes. The mutation of one of these genes, tinman , results in absence of the dorsal vessel. Frog and mouse homologues of these homeobox genes were identified and localized in expression to the developing heart. Transgenic mice lacking a tinman homologue ( NKX2.5 ) do not lack hearts but die at 9 days of development with severely abnormal hearts. Humans in whom the tinman homologue NKX2.5 is mutated suffer atrial septal defects with associated AV conduction delay and other complications. The fruit fly findings led to the study of a set of genes critically important to many aspects of human heart development.

The Tubular and Looping Heart

Within each side of the first heart fields primitive endocardial tubes form, come together, and fuse as the lateral body portions fold to create the primitive heart tube. The most caudal cells of the right and left heart fields will form the most ventral portion of the primitive heart tube (asterisks in Fig. 71.2 ). The inflow portion of the heart quickly gains pacemaker activity and initiates slow, unidirectional contractions along the tube. Concurrent with this process, endocardial and myocardial cells—which form the layers of the developing heart—emerge from a common precursor. The myocardium secretes a thick layer of acellular extracellular matrix, also known as cardiac jelly, which creates a distinct layer between the myocardium and the endocardium ( Fig. 71.4 ). This tissue progression requires cell differentiation, sorting, and polarization. Evidence supports both negative and positive influences from the surrounding neural tissue and endoderm on these processes.

The primitive heart tube undergoes three connected steps, looping, convergence, and wedging, all of which are crucial to normal cardiac septation. Looping is the first obvious structural left–right lateralization in the developing embryo ( Fig. 71.5 ). The heart tube elongates and folds into an “S” shape to the right. The direction of looping will determine the ultimate ventricular positions. Looping to the right will give the normal Dextro, or “D,” looping. If the primitive tube loops to the left, this will result in Levo, or “L,” looping and result in the left ventricle on the right side and the right ventricle on the left side. The left–right handedness of looping is directed by monocilia in the primitive node many stages before looping starts. These cilia are angled 40 degrees posterior and rotate in a clockwise direction, resulting in a leftward laminar flow of fluid containing key signaling molecules. Mutations in genes responsible for ciliary ultrastructure result in syndromes such as Kartagener syndrome. Importantly, the primitive atrium is not involved in this portion of looping, so atrial position is not severely disturbed by abnormal looping. Atrial positions are affected by situs abnormalities as seen in heterotaxy syndromes. Left–right embryologic abnormalities have a wide range of known genetic causes in which mutations interfere with early breaking of left–right symmetry, abnormal signal transduction or gene expression in the lateral plate mesoderm, and abnormal morphogenesis of internal organs.

Convergence is the process of proper orientation of the inflow and outflow tracts ( Fig. 71.6 ). The inflow tract rises cranially behind the outflow tract such that the inflow and outflow tracts are aligned, with the outflow tract ventral to the inflow tract. Abnormalities can lead to irregularities in atrial and ventricular septa and outflow tracts. Convergence and looping are dependent on lengthening of the polar ends of the heart tube, which is achieved by the addition of cells from the second heart field. The original primitive heart tube will grow to become the left ventricle and portions of the atrioventricular canal, and additions from the second heat field will direct formation of the outflow tracts, right ventricle, and atria. As cardiac neural crest cells influence the second heart field, it becomes easier to understand how neural crest cell–associated disease such as DiGeorge syndrome, 22q11 deletion syndromes, and velocardiofacial syndromes result in abnormalities of these cardiovascular structures.

Occurring in parallel with convergence and septation (see Figs. 71.5 and 71.6 ) is wedging ( Fig. 71.7 ). Here, the outflow tract rotates counterclockwise approximately 45 degrees, resulting in the future aorta residing behind the future pulmonary artery (see Fig. 71.7 ). The aortic portion shortens, and the future site of the aortic valve “wedges” between the future tricuspid and mitral valves. This separates the mitral valve from the septum, which is why the mitral valve has no connections to the septal wall. Irregularities in wedging result in conotruncal defects such as tetralogy of Fallot and double outlet right ventricle.

How is the program for cardiac muscle differentiation turned on and regulated? This question has been approached by studying the transcription factors that bind to promoter regions of the cardiac-specific cytoskeletal genes coding for molecules that appear in the early tubular heart. The discovery of MyoD, a helix-loop-helix DNA-binding protein that controls skeletal muscle expression, led investigators to search for similar factors in cardiac muscle. Current findings support the idea that both positive and negative regulators of cardiac genes exist and that they work in complexes that define the spatiotemporal pattern of expression of cardiac genes (see Fig. 71.3 ).

Closely related to the question of cardiac-specific gene regulation is the question of regional specification of the heart tube. Segmentation, although not as obvious in the vertebrate heart as it is in the insect body plan, is important in cardiogenesis. Chamber-specific expression of a number of endogenous genes and promoter-driven reporter genes suggests that the outflow tract, the left and right ventricles, the atria, and the sinus venosus can in some cases be considered separate cardiac segments. However, many of the endogenous cardiac genes do not strictly follow chamber-specific expression but rather appear to be modularly controlled based on their position along the anteroposterior axis (see Fig. 71.2 ).

An understanding of segment specification has been greatly advanced by the study of the homeobox genes in the fruit fly. The identity and differentiation of segments are determined by interactions among homeobox and other genes. These interactions occur in the mesoderm, which gives rise to the heart tissues and the tissues adjacent to the mesoderm.

The vertebrate heart tube normally loops to the right side of the embryo. Sidedness appears to be influenced by factors acting at a time when the heart is not recognizable as a distinct organ structure. Transplantation studies using the early chicken embryo suggest that left and right sidedness are already determined in the precardiac mesoderm stages. Sidedness can also be regulated in part by retinoids. Retinoic acid–soaked bead implants in chicken embryos or application of all- trans retinoic acid in mouse embryos cause transposition of the great arteries. Abnormalities in the direction of cardiac looping also occur in humans. These abnormalities are linked to an autosomal recessive gene defect that has been detected in the Amish population and another gene defect in the X chromosome (Xq24-q27.1) in the general population. Interestingly, abnormalities in genes associated with left–right asymmetry may also be responsible for apparently isolated cases of congenital heart defects. Mutations in the laterality CFC1 gene have manifested as transposition of the great arteries in humans without other obvious laterality defects.

Two mouse mutants have abnormalities in sidedness. The iv/iv mutant mouse develops L-looping and dextrocardia 50% of the time. A large percentage of these mice have a combination of cardiac defects, including persistent sinus venosus, atrial and ventricular septal defects, common AV canal, double-outlet right ventricle, tetralogy of Fallot, and transposition of the great arteries. This gene has been mapped to mouse chromosome 12, which is homologous to human chromosome 14. The inv/inv mutant mouse develops situs inversus close to 100% of the time. Mutation screens using the zebrafish have uncovered a number of genes involved in laterality specification and indicate that there are organ-specific laterality regulators. Identification of the genes involved in these human, mouse, and zebrafish mutations could provide valuable insight into the mechanisms of normal dextral looping of the heart.