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Keywords:

  • heart development;
  • chick;
  • cardiac progenitors;
  • secondary heart field;
  • cardiac neural crest;
  • cardiac septation;
  • cardiac looping;
  • epicardium;
  • cardiac innervation;
  • cardiac conduction system;
  • coronary vasculature;
  • endocardial cushions;
  • cardiac valve;
  • apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. REFERENCES

Cardiac progenitors of the splanchnic mesoderm (primary and secondary heart field), cardiac neural crest, and the proepicardium are the major embryonic contributors to chick heart development. Their contribution to cardiac development occurs with precise timing and regulation during such processes as primary heart tube fusion, cardiac looping and accretion, cardiac septation, and the development of the coronary vasculature. Heart development is even more complex if one follows the development of the cardiac innervation, cardiac pacemaking and conduction system, endocardial cushions, valves, and even the importance of apoptosis for proper cardiac formation. This review is meant to provide a reference guide (Table 1) on the developmental timing according to the staging of Hamburger and Hamilton (1951) (HH) of these important topics in heart development for those individuals new to a chick heart research laboratory. Even individuals outside of the heart field, who are working on a gene that is also expressed in the heart, will gain information on what to look for during chick heart development. This reference guide provides complete and easy reference to the stages involved in heart development, as well as a global perspective of how these cardiac developmental events overlap temporally and spatially, making it a good bench top companion to the many recently written in-depth cardiac reviews of the molecular aspects of cardiac development. Developmental Dynamics 233:1217–1237, 2005. © 2005 Wiley-Liss, Inc.


HH 1 (FIRST FEW HOURS OF INCUBATION, PRE-STREAK, “EMBRYONIC SHIELD”)

  1. Top of page
  2. Abstract
  3. REFERENCES

Cardiac Progenitors

The earliest identification of cardiac progenitors is before gastrulation in the epiblast cell layer as it separates from the hypoblast (Hatada and Stern, 1994; Yutzey and Kirby, 2002). Early formation of the epiblast and hypoblast, as well as early cardiac lineage analysis has been previously reviewed (Tam and Schoenwolf, 1999).

HH 2 (6–7 HOURS, PRE-GASTRULATION, INITIAL STREAK)

  1. Top of page
  2. Abstract
  3. REFERENCES

Cardiac Progenitors

Precardiac cells are found in the epiblast lateral to the midportion of the primitive streak. Heart precursors are among the first embryonic cells to gastrulate (Tam and Schoenwolf, 1999). Some cardiac lineage diversification may have occurred prior to gastrulation (pre-HH 3) when chick precardiac cells (cardiac progenitors) still reside in the epiblast as described below (Wei and Mikawa, 2000).

HH 3 (12–13 HOURS, ONSET OF GASTRULATION, INTERMEDIATE STREAK)

  1. Top of page
  2. Abstract
  3. REFERENCES

Cardiac Progenitors

Cardiac progenitor cells have been mapped to the anterior two thirds (rostral half) of the primitive streak. The extreme rostral end of the streak (Henson's node), however, contributes cells mainly to prospective head mesenchyme and foregut endoderm, not to cardiac precursors (Schoenwolf et al., 1992). Thus, prospective heart cells extend caudally in the primitive streak 124–750 μm from the anterior end of the streak. These cardiac progenitor cells give rise to endocardium, myocardium, mesocardium, and parietal pericardium (Garcia-Martinez and Schoenwolf, 1993; Cohen-Gould and Mikawa, 1996). Wei and Mikawa (2000) show that the rostral portion of the primitive streak at stage HH 3 consists of at least two distinct subpopulations (cardiomyocyte or endocardial cell lineage), suggesting that these two cell lineages are already segregated within the primitive streak before their migration to the primary heart field. This also suggests that lineage diversification must occur at or before initial primitive streak formation (Wei and Mikawa, 2000). Also, when noncardiogenic cells are transplanted to the primitive streak, they are recruited into the heart, suggesting that signals important for early cardiac specification are present in the primitive streak (Schoenwolf and Garcia-Martinez, 1995; Schultheiss et al., 1995; Yutzey and Kirby, 2002). The very caudal portion of the primitive streak gives rise to lateral plate mesoderm, extraembryonic mesoderm, and endoderm (Schoenwolf et al., 1992). The prospective cardiogenic mesoderm in the primitive streak is arranged in a rostrocaudal sequence (Garcia-Martinez and Schoenwolf, 1993) in a similar order as in the prelooped heart (Fishman and Chien, 1997). The rostro-caudal positioning of cardiogenic cells from HH stages 4–8 has also been shown to affect their fate (Patwardhan et al., 2000). Recently, however, it has been suggested that by stages HH 4–6, the cardiogenic cells are no longer organized in a rostral to caudal pattern (Redkar et al., 2001), which previous studies support (DeHaan, 1963). However, Redkar and colleagues (2001) also suggest that by stages HH 7–8 a definitive rostrocaudal pattern is re-established, relating cell position with their later differentiation, albeit with a considerable overlap of caudal cells into the rostral cells.

HH 4–5 (18–22 HOURS, GASTRULATION, DEFINITIVE STREAK TO HEAD PROCESS, FORMATION OF THE LATERAL PLATE MESODERM)

  1. Top of page
  2. Abstract
  3. REFERENCES

Cardiac Progenitors (Primary Heart Fields)

By stage HH 4, the rostral half of the primitive streak contains no cardiac progenitors, but instead it contains prospective somatic cells (Garcia-Martinez and Schoenwolf, 1993). At stages HH 4–5, cardiac precursor cells are located on both sides of Hensen's node in the forming anterior mesoderm (Rawles, 1936). At HH 4, precardiac mesoderm is specified to some degree. By HH 4–5, mesodermal and endodermal layers become distinct. Thus, precardiac mesoderm can be considered “specified” (Schultheiss and Lassar, 1999). Specified means that cells can differentiate into heart when placed into a neutral environment while determined (higher level of commitment) means that cells can differentiate into heart even when placed into an antagonist environment (non-cardiac regions) (Schultheiss and Lassar, 1999). Embryonic cells become different from one another by a process of specification and as cells become specified, they become fate restricted (Davidson, 1990). However, fate restriction does not imply irreversible commitment. Some embryonic cells are multipotent or totipotent precursors that can give rise to multiple lineages unlike fate-restricted precursors that give rise to a single cell type (Henion and Weston, 1997). These bilateral primary heart fields were first shown by Rawles (1943) to contain cells in the mesoderm that had potential to form myocardium (Abu-Issa et al., 2004). As the cardiac precursor cells move into the lateral plate mesoderm (primary heart fields), they are segregated into the splanchnic mesoderm layer (Linask, 1992; Linask et al., 1997; Abu-Issa et al., 2004). The splanchnic mesoderm layer lies adjacent to the endoderm, which is thought to induce myocardial differentiation (Schultheiss et al., 1995; Lough and Sugi, 2000; Abu-Issa et al., 2004). At HH 5, precardiac cells still lack a complete level of commitment to a cardiac fate. Anterior endoderm in stages HH 4–5 (mid to late gastrula) can induce cardiogenesis from posterior primitive streak (which normally only gives rise to blood and extraembryonic membranes) (Schultheiss and Lassar, 1999). Data suggests that large portions of the primitive streak are competent to become heart, and that the portion of the streak that actually does give rise to heart is determined by which subset of gastrulating mesodermal cells come into contact with the anterior endoderm (Schultheiss and Lassar, 1999). At stage HH 5, both lateral and medial portions of the anterior endoderm have cardiac-inducing properties, whereas only the anterior lateral mesoderm actually differentiates into heart in vivo (Rosenquist and DeHaan, 1966; Schultheiss and Lassar, 1999). Thus, one role of the anterior endoderm is to create a “cardiac field” in the overlying anterior mesoderm, and that other events restrict that field to the region of the embryo that actually becomes heart (Schultheiss and Lassar, 1999). Mouse experiments suggest that the mechanisms of cardiogenesis in chick and mouse are similar (Schultheiss and Lassar, 1999). Redkar and colleagues show that at stage HH 4, the anterior border of the heart-forming region is just above the level of Hensen's node (HN) and extends posteriorly below HN aproximately one quarter the distance of the primitive streak (Redkar et al., 2001). They also show that the medial border is 0.3 mm from the primitive groove (similar to what had previously been described by Rosenquist and DeHaan, 1966), and the lateral border extends almost to the area opeca/area pellucida boundary (Redkar et al., 2001), suggesting that Bmp2 and Nkx2.5 cannot be used to accurately define the heart forming region. This differs from mapping studies by Ehrman and Yutzey (1999), which set the lateral border of the heart-forming region adjacent to the extra embryonic tissue, the medial boundary at the lateral border of the mesoderm overlying the prospective neural plate (more lateral), and the posterior border at the level of Hensen's node (Ehrman and Yutzey, 1999). Fate map data using dye marking and molecular gene expression reveal that there is no exact correspondence of the two types of labeling, providing more evidence to the complexity of defining the primary heart field (Redkar et al., 2001). Thus, clearly more work will be required to determine the exact extent of the primary heart fields (Abu-Issa et al., 2004) (Fig. 1A, caudal pair of red asterisks).

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Figure 1. Cardiac progenitors (primary heart field) and the formation of the bilateral endocardial heart tubes. A: Stage HH 7 chick embryo. The red dots represent mesoderm cells (cardiac progenitors) that have gastrulated through the primitive streak at HH 3–4 and now have formed the primary heart field within the lateral plate splanchnic mesoderm. B: Schematic of a cross-section through the primary heart field at HH 7–8. Note the delaminating splanchnic mesoderm cells that are forming the bilateral endocardial heart tubes. Also note the close approximation of the prospective foregut endoderm with the developing primary heart field splanchnic mesoderm. S, somite; HH, Hamburger and Hamilton staging, 1951. Red asterisks denote areas of debate regarding the extent of the primary heart field, as well as the formation of a cardiac crescent in chick as discussed at stage HH 6–7 in the reference guide.

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HH 6–7 (23–26 HOURS, HEAD FOLD-3 SOMITES, NEURULATION)

  1. Top of page
  2. Abstract
  3. REFERENCES

Cardiac Progenitors (Primary Heart Fields)

By HH 6, the precardiac mesoderm has attained an additional level of commitment (Schultheiss and Lassar, 1999). At HH 7, the first reported markers of terminal myocardial differentiation are detected in the primary heart fields (Bisaha and Bader, 1991; Han et al., 1992). Neural tissue will inhibit the development of cardiac tissue (Schultheiss and Lassar, 1999). Anterior neural tissue has stronger cardiac inhibitory properties than posterior tissue. In addition, the neural crest has a strong inhibitory activity (Schultheiss and Lassar, 1999). Thus, a two-step model of vertebrate cardiac induction may exist (Schultheiss and Lassar, 1999) in which signals from anterior endoderm induce a cardiac field in the overlapping mesoderm and only a subdomain of this field becomes heart in vivo. Signals from surrounding ventral/lateral tissues, which promote cardiogenesis and signals from dorsal/medial structures that inhibit heart formation, may determine the size of the cardiac field, resulting in specification of heart tissue in the anterior ventral mesoderm (Schultheiss and Lassar, 1999). Thus, it is possible that the initial heart-inducing signals may come from the head organizer (Henson's node in chick), whereas the definitive endoderm may provide later signals that reinforce the initial induction of the precardiac mesoderm (Schultheiss and Lassar, 1999). Therefore, it is also important to look for genes that are expressed in the anterior endoderm and neural tissues close to the heart field.

The head fold and the anterior intestinal portal are also forming at this time (Tam and Schoenwolf, 1999). It has long been accepted that heart precursors occupy a crescent in the anterior lateral region of the embryo (Fig. 1A, rostral/medial red asterisk) by stage HH 7 (DeHaan, 1965). Recently, other investigators suggest that the cardiac crescent in chick does not appear until stage HH 8 (Redkar et al., 2001; Abu-Issa et al., 2004), or that the heart arises from paired and separated regions rather than a single crescent (Colas et al., 2000). By stage HH 7–8, the posterior border of the primary heart field is extended to the level of the first somite (Fig. 1A) (DeHaan, 1965; Ehrman and Yutzey, 1999), whereas Redkar and colleagues (2001) show that the posterior border is extended to the fourth somite by stage HH 8.

Primary Heart Tube

Presumptive endocardial cells begin to delaminate from the splanchnic mesoderm via the process of epithelial-to-mesenchymal transformation (EMT) and migrate to start the formation of the bilateral endocardial heart tubes (Fig. 1B, migrating splanchnic mesoderm cells).

HH 8 (26–29 HOURS, 4–6 SOMITES)

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  2. Abstract
  3. REFERENCES

Cardiac Neural Crest Cells

Pre-migratory cardiac neural crest (CNC) cells are located in the region of the neural crest between the mid-otic placode and the caudal limit of somite 3 (Fig. 2A,B) and have been shown to be necessary for proper outflow tract septation, cardiac innervation, aortic arch repatterning, and myocardial function (Kirby et al., 1983, 1985; Kirby and Stewart, 1983; Kirby, 2002; Hutson and Kirby, 2003).

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Figure 2. Origin of cardiac neural crest cells and bilateral endocardial heart tube formation. A: HH 8 chick embryo denoting the origin of the cardiac neural crest cells (green dots) within the cardiac neural folds. Pre-migratory cardiac neural crest cells are located in the region of the neural crest between the mid-otic placode and the caudal limit of somite 3. B: Schematic of a cross-section through the axial level of the premigratory cardiac neural crest (green dots) and the fusing bilateral endocardial heart tubes a HH 8–9. Note that the lateral myocardium is the first to fuse and this condition shown in B persists in the conus until stage 12 (although between HH 10–12 there is a single endothelial tube). C: Schematic of a cross-section through the level of early migrating cardiac neural crest cells (green dots) at HH 10. Tubular heart (the ventricle) has completely fused and has started to beat. Also, note the continued close approximation of the foregut endoderm/pharynx with splanchnic mesoderm (now the secondary heart field) and the dorsal mesocardium of the linear heart tube. Fb, forebrain; Mb, midbrain; HH, Hamburger and Hamilton staging (1951).

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Primary Heart Tube

The process of endocardial tube fusion starts. The first morphological manifestation of the straight heart tube is found at stages HH 8+/9−, which is also considered the prelooping stage (Manner, 2000) (Fig. 2B). The process of heart tube fusion was first verified in the mouse system (DeRuiter et al., 1992).

HH 9 (29–33 HOURS, 7–9 SOMITES)

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  2. Abstract
  3. REFERENCES

Cardiac Neural Crest Cells

CNC cells begin the process of EMT and emigrate from the neural tube at this stage (Boot et al., 2003b) (Fig. 2C). These early-migrating cells (HH 9–10) are eventually located in the condensed mesenchyme and the prongs of the outflow tract aorticopulmonary (AP) septum, as well as the pharyngeal arch arteries (proximal and distal, but not dorsal aorta) (Boot et al., 2003a). Early CNC ablations show a primary role of early-migrating CNC in proper outflow tract septation (Kirby et al., 1985; Boot et al., 2003a). See Hutson and Kirby (2003) or Kirby (2002) for a good review of the molecular pathways that are required for cardiac neural crest cell specification, migration, proliferation, and survival.

HH 10–11 (33–45 HOURS, 10–15 SOMITES)

  1. Top of page
  2. Abstract
  3. REFERENCES

Primary Heart Tube

The tubular heart has completely fused at the level of the presumptive ventricle and begins to beat (Fig. 2C). The straight portion of the outflow tract, however, can still be seen lying open against the ventral pharynx (endoderm) up to stage 12 and the early looping stages (while the ventricles are looping) (Manner, 2000). Thus, at this stage, the straight portion of the outflow tract is not closed to the ventral endoderm, contrary to the common belief that the entire heart tube is fused by stage HH 10–11.

Cardiac Conduction System

At this early stage of heart function, when the primary heart tube is just beginning to beat, the specialized pacemaking and conduction system (central and peripheral) has not yet developed, but the embryonic heart does have the ability to coordinate contractions without a mature pacemaking and conduction system (Pennisi et al., 2002). Interestingly, the tubular heart can spontaneously evoke action potentials even before myocyte cells can contract (Hirota et al., 1979, 1985; Kamino et al., 1981). This occurs through the presence of a primordial pacemaker locus that is present in the farthest posterior segment of the primitive tubular heart around 35 hours of chick development (Kamino et al., 1981; Gourdie et al., 2003). Once the heart tube undergoes looping, the myocardial cells will become contractile (Manasek, 1968). Thus, epithelioid myocytes are electrically active as the primitive heart tube forms, but pacemaking impulses are evoked mainly by myocytes in the posterior inflow tract and the resulting action potentials propagate throughout the heart tube without any local changes in velocity (Pennisi et al., 2002). However, by 42–45 hours, as looping proceeds, a slowly conducting AV canal separating the activation of the faster conduction segments of the atrium and ventricle is forming (Gourdie et al., 2003).

Cardiac Looping

The linear heart tube starts to loop at HH 9+/10− and results in the transformation of the straight heart tube into the c-shaped heart loop (Dextral-looping phase). The process of cardiac looping occurs from stages HH 9–34 and is broken into an early phase (HH 9–24) of cardiac looping and a late phase (HH 24–34) of cardiac septation. The process of looping described here is based on the cardiac looping terminology reviewed by Manner (2000), which is based on previous work (Patten, 1922; DeHaan, 1965; Stalsberg, 1970; Stedin and Seidl, 1980; Manner et al., 1993; Bouman et al., 1995; Kirby and Waldo, 1995; de la Cruz, 1998; Taber, 1998).

Cardiac Neural Crest Cells

Early CNC cell migration continues (Boot et al., 2003a) (Fig. 2C).

Cardiac Progenitors (Primary Heart Field)

The inflow tract, which is believed to form from a caudal extension of the primary heart field, does not lengthen much after stage HH 12 (Waldo et al., 2001). Thus, it is almost completely developed by the time myocardium starts to accrete onto the developing outflow tract from the secondary heart field (Waldo et al., 2001).

Cardiac Progenitors (Secondary Heart Field)

Mjaatvedt and colleagues (2001) suggest that from HH 10–14 there is early formation of the proximal conus (outlet segment of the outflow tract) from a novel “anterior heart field” (AHF) splanchnic mesoderm (Mjaatvedt et al., 2001), also known as the “secondary heart field” (SHF) (Waldo et al., 2001). These results are contrary to the results of Waldo and colleagues (2001) that show a later start (HH 14) to the accretion of SHF cells onto the outflow tract as described below and at stage HH 14 in this review. Also, the results of Waldo et al. (2001) seem to correspond to the morphological description of heart development by Manner (2000). Briefly, the AHF or SHF is located in the splanchnic mesoderm that underlies the floor of the caudal pharynx and is directly continuous with the outflow myocardium (Fig. 2C) (Waldo et al., 2001). The ventral pharyngeal endoderm is capable of inducing heart in non-cardiac mesoderm from stage HH 12 (after body wall closure) until stage HH 16 in chick embryos. The remainder of the conus and the distal portion of the outflow tract (the truncus) forms during later stages (stage HH 16–22) of accretion or recruitment from AHF splanchnic mesoderm (Mjaatvedt et al., 2001). Waldo et al. (2001) also discovered similar accretion between stages HH 14 to HH 22 from anterior splanchnic mesoderm, which they termed the “secondary heart field.” It is important to note here the differences in location of the secondary heart field (anterior heart field) and the extent of contribution to the outflow tract between Mjaatvedt et al. (2001) and Waldo et al. (2001) (Abu-Issa et al., 2004; Eisenberg and Markwald, 2004). Waldo and colleagues believe that the SHF is restricted to the prepharyngeal mesoderm caudal to the outflow tract (see Fig. 4A,B) and gives rise only to the truncus and smooth muscle in the tunica media at the base of the mature aorta and pulmonary trunk (see Fig. 5, yellow dots) (Abu-Issa et al., 2004). Mjaatvedt and colleagues (2001) describe the anterior heart field as being much broader which includes both the prepharyngeal mesenchyme and also the later and more anterior splanchnic mesoderm that extends into middle of the cranial pharyngeal arches (Abu-Issa et al., 2004). They, however, show similar contribution to the distal conus and truncus similar to that described by Waldo et al. (2001). In mouse, the anterior heart field has been described to contribute to an even broader range of myocardium: right ventricle, conus and truncus (Kelly et al., 2001). Abu-Issa et al. (2004) suggest that these differences point to complex patterning in the primary heart field into various subdivisions that converge in a coordinated fashion to form the adult heart. Also, the reported continuous expression of Id2 between the outflow and inflow tract in the splanchnic mesoderm and dorsal mesocardium suggested the need for further investigation on the link between the outflow tract (secondary heart field derived) and the inflow tract (primary heart field derived) (Martinsen et al., 2004). However, it is clear from these studies that the outflow limb of the linear heart tube is accreted onto by splanchnic mesoderm cells during looping, eventually forming the arterial pole of the heart. Thus, for simplicity these splanchnic mesoderm cells are still referred to as the secondary heart field (SHF) in this reference guide.

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Figure 4. Cardiac looping, outflow tract formation, inflow tract formation, and the development of the epicardium. A: Schematic of a looping chick heart at HH 14–17. The outflow tract (yellow) and the inflow tract (blue) represent newly accreted cardiac progenitors during the process of looping. Note the shift of the atrium towards a superior and dorsal position close to the outflow tract. The epicardial cells (blue dots) originate within the splanchnic mesoderm of the sinus venosus and septum transversum (called the proepicardial organ). These cells then migrate out over the developing myocardium of the heart. B: Schematic cross-section of the looping heart at HH 17–18, showing epicardial cells migrating out over the ventricle. Four layers of the heart have now formed: epicardium, myocardium, cardiac jelly, and endocardium. LV, left ventricle; RV, right ventricle.

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Figure 5. A stage HH 34 chick heart showing the derivatives of the major contributors to heart development. The outflow tract (Ao and P) and the ventricles are now completely septated. However, note that atrial septation is not complete until hatching (HH 46). Green dots, cardiac neural crest derivatives; yellow dots, secondary heart field cardiac progenitor derivatives; red dots, primary heart field cardiac progenitor derivatives; blue dots, proepicardial derivatives. Ao, aorta; APP, anterior parasympathetic plexus; Co, coronary vessels; LA, left atrium; LV, left ventricle; P, pulmonary vessel; RA, right atrium; RV, right ventricle.

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HH 12-13- (45–49 HOURS, 16–19 SOMITES)

  1. Top of page
  2. Abstract
  3. REFERENCES

Cardiac Looping

The dextral-looping phase of cardiac looping is completed at stage HH 12 (Manner, 2000).

Cardiac Neural Crest Cells

Late CNC cell migration.

These late-migrating cardiac neural crest cells (HH 12–13-) are restricted to the proximal part of the pharyngeal arch arteries and late CNC ablations show that their primary role is in pharyngeal arch artery remodeling and not AP septum formation (Boot et al., 2003a). However, they are not developmentally restricted and can contribute to the AP septum when transplanted into a younger environment (Boot et al., 2003a). Also note that the outflow attachment to the ventral pharynx shows a dynamic relationship with the pharynx. At stage HH 12, the outflow region joins the pharynx ventral to pharyngeal arch/artery 1, but by stage HH 24 it is located ventral to pharyngeal arches/arteries 4-6 (Waldo et al., 2001).

Endocardial Cushions

Endocardial cushions are first seen as an expansion of the cardiac jelly in the outflow tract and the AV canal. This precedes the actual formation of the cushion mesenchyme (Markwald et al., 1977). Endocardial cushion development has been separated from valve formation in this reference guide to better identify the stages of cushion growth during the septation of the chick outflow tract, but valve development occurs in specific regions of the endocardial cushions as outlined in subsequent stages.

HH 13+ (50–52 HOURS, 20–21 SOMITES)

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  2. Abstract
  3. REFERENCES

Cardiac Looping

Continued cardiac looping from HH 12/13 to HH 18 in which there is a transformation of the c-shaped heart loop into the s-shaped heart loop, results in a shortening of the distance between the caudal wall of the primitive conus and the cranial wall of the primitive atria and a shift of the primitive ventricular bend toward its definitive position caudal to the atria (Manner, 2000). Also from stages HH 13–15, the dorsal mesocardium ruptures and disappears allowing movement of the heart tube (Manner, 2000).

Cardiac Neural Crest Cells

The cardiac neural crest has stopped producing cardiac neural crest cells (Boot et al., 2003b).

HH 14–15 (50–55 HOURS, 22–27 SOMITES)

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  2. Abstract
  3. REFERENCES

Cardiac Conduction System

The AV ring (a portion of the central conduction system) has formed and shows specific expression of the homeobox gene Msx-2 (Chan-Thomas et al., 1993). Lineage tracing studies have suggested that there are different myocyte parental lineages for the central conduction system (AV-node, AV ring, AV bundle, and proximal components of the bundle branches) and the peripheral Purkinje fiber network, which starts to form much later (day 10–20) (Gourdie et al., 1995).

Cardiac Neural Crest Cells

CNC cells have reached aortic arch 3 (Fig. 3A, shown at stage HH 17) (Waldo et al., 1996, 1999).

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Figure 3. Cardiac progenitors (secondary heart field), proepicardium, migrating cardiac neural crest cells, and cardiac looping. A: Lateral view of a HH 17–18 chick embryo denoting the location of the secondary heart field (yellow, based on Gata-4 and Nkx2.5 expression data by Waldo et al., 2001) and a possible extended area of secondary heart field cells in the pharyngeal mesoderm (yellow dashed line) based on research in mouse (Kelly et al., 2001), proepicardium (blue), as well as the cardiac neural crest cells (green dots) that are now migrating through branchial arches 3, 4, and 6. B: Schematic view of a saggital section through a HH 17–18 looping heart as shown in A. Cardiac progenitors from splanchnic mesoderm (SM) cells (yellow, secondary heart field, based on Gata-4 and Nkx2.5 expression data by Waldo et al., 2001) are being accreted onto the looping heart (yellow dots), forming the outflow tract (OFT). DM, dorsal mesocardium; E, eye; Fb, forebrain; H, heart; HH, Hamburger and Hamilton (1951); IFT, inflow tract; Mb, midbrain; Otc, otic vessicle; PC, pericardial cavity; SM, splanchic mesoderm; T, trunk.

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Epicardium (Proepicardial Organ)

From stage HH 14–17, the epicardium originates as villous protrusions of mesothelial cells (coelomic epithelium) on the surface of the sinus venosus and the septum transversum (liver anlage) collectively called the proepicardium or proepicardial organ (Figs. 3A and 4A,B) (Manner, 1992; Mikawa and Fischman, 1992; Poelmann et al., 1993; Mikawa and Gourdie, 1996; Vrancken Peeters et al., 1999; Kirby, 2002). The term proepicardial organ (PEO) was first devised and described in the mouse system (Viragh and Challice, 1981). The proepicardium gives rise to the epicardium, the mesenchymal cells of the subepicardial layer, the endothelium and smooth muscle cells of the coronary vasculature (Fig. 5, blue dots), and the perivascular and intermyocardial fibroblasts (Manner, 1992; Mikawa and Fischman, 1992; Poelmann et al., 1993; Mikawa and Gourdie, 1996; Vrancken Peeters et al., 1999; Kirby, 2002). There are also epicardium-derived cells that contribute a novel population of cells to the myocardial wall of the ventricles and atria (unrelated to the coronary vessels) (at HH stage 25–31), subendocardial region (at HH stage 25–31), the atrioventricular cushions (at HH stage 32–43), and some cells in the atrioventricular valves (at HH stage 32–43) (Gittenberger-de Groot et al., 1998). The myocytes and fibroblasts are important for normal myocardial wall formation and the fibroblasts also produce part of the collagen-rich matrix of the heart (the fibrous heart skeleton) (Gittenberger-de Groot et al., 1998; Vrancken Peeters et al., 1999). It is also important to note here that the endothelium of the coronary vasculature is actually derived from liver cells as shown when pure epicardial primordium transplants (minus the septum transversum/liver anlage) did not lead to endothelial cell formation (Poelmann et al., 1993). Thus, the endothelial cells are not epicardium-derived although the endothelium reaches the heart via the subepicardial layer (Poelmann et al., 1993; Viragh et al., 1993) instead of a direct epithelial-mesenchymal transformation from the epicardium like that of the smooth muscle cells and fibroblasts (Vrancken Peeters et al., 1999). The presence of the epicardium over the myocardium is also very important for proper regulation of cardiomyocyte apoptosis (as described at HH stage 19 and subsequent stages, see Table 1) (Rothenberg et al., 2002; Schaefer et al., 2004) and myocardial proliferation (as described at HH stage 16 and subsequent stages, see Table 1) (Manner, 1993; Gittenberger-de Groot et al., 2000; Perez-Pomares et al., 2002; Pennisi et al., 2003; Stuckmann et al., 2003). Reviews of the molecular markers of the proepicardium can be found in several reports (Perez-Pomares et al., 1998; Kirby, 2002; Morabito et al., 2002).

Table 1. Reference Guide of Cardiac Development and Associated Stagesa
 Stages
  • a

    HH, stages according to Hamburger and Hamilton (1951).

Cell sources that contribute to cardiac development 
 Cardiac Progenitors (CP)HH 1, 2, 3, 4–5, 6–7, 10–11, 21–23
  Primary heart field and primary heart tube formationHH 4–5, 6–7, 8, 10–11
  Secondary heart field and outflow tract accretionHH 10–11, 21–23
 Cardiac neural crest cells (CNC)HH 8, 9, 10–11, 12–13, 13+, 14–15, 21–23
  Outflow tract developmentHH 25–26, 27, 28, 31, 34
  Cardiac innervationHH 27, 35, 36, 40
 Proepicardial organ (PEO)HH 14–15
  EpicardiumHH 17–18, 19–20, 21–23, 27, 28, 32–33
  Coronary vasculature (CV)HH 25–26, 27, 30, 31, 32–33, 34, 35, 36, 38, 40
Cellular events 
 ApoptosisHH 19–20, 21–23, 24, 25–26, 27, 30, 31, 32–33, 35
 Epithelial-mesenchymal transformationSee CP, CNC, PEO, Epicardium, CV, EC
 MigrationSee CP, CNC, PEO, Epicardium, CV, EC
 Proliferation of the myocardiumHH 16, 17–18, 19–20, 27, 29, 34, 46
Cardiac remodeling and development 
 Cardiac loopingHH 10–11, 12–13, 13+, 16, 17–18, 24
 Endocardial cushion (EC) developmentHH 12–13, 16, 19–20, 21–23, 25–26, 29, 34
  Valve developmentHH 21–23, 28, 30, 31, 34, 36
 Cardiac septationSee below
  Outflow tract septationHH 25–26, 27, 28, 31, 34
  Ventricular septationHH 17–18, 19–20, 21–23, 29, 32–33, 34
  Atrial septationHH 16, 21–23, 24, 28, 30, 31, 34, 36, 46
 Pacemaker and conduction systemHH 8, 10–11, 14–15, 28, 29, 34, 36, 46
  Pacemaker (SA-node)HH 28
  Central (AV-node, AV bundles)HH 14–15, 28, 34
  Peripheral (Purkinje fiber network)HH 36, 46

HH 16 (51–56 HOURS, 2.1 DAYS, 26–28 SOMITES)

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Atrial Septation

The interatrial septum is starting to form. A crescent-shaped ridge called the septum primum appears first on the cephalo-dorsal wall of the atrium (Quiring, 1933). In avians, this septum primum eventually is transformed into the mature atrial septum (completely closed only after hatching). Higher vertebrates develop an additional septum called the septum secundium, which eventually fuses with the septum primum to form the adult septum (closed after birth) (Morse et al., 1984).

Cardiac Looping

Continued cardiac looping causes the sinus venosus to move from its original position caudal to the atria towards its definitive position dorsal to the atria during stages HH 14–18 (Fig. 4A) (Manner, 2000). The atria appear during this period (HH 14–18) as lateral expansions in the primitive atrium region (Manner, 2000).

Endocardial Cushions

Both the atrioventricular and outflow tract cushions are emerging as small ridges (Markwald et al., 1977; Ben-Shachar et al., 1985). In the atrioventricular canal and outflow tract, the endocardial cushions form by the initial expansion of extracellular matrix–rich hyaluronan and chondroitin sulfate proteoglycans (Bernanke and Markwald, 1979; Markwald, 1979; Camenisch et al., 2000, 2002b). The myocardium then induces endothelial cells of the endocardium to undergo the process of epithelial-to-mesenchymal transformation (EMT), causing them to disengage cell–cell adhesions, extend filopodia, become migratory, and invade the extracellular matrix (Bolender and Markwald, 1979; Markwald et al., 1979, 1996; Runyan and Markwald, 1983). These mesenchyme cells then contribute to the formation of the mitral and tricuspid valve leaflets, as well as atrial and ventricular septation (Eisenberg and Markwald, 1995) as described in the Valve Development section of this reference guide.

Myocardial Proliferation and Trabeculation

As discussed above, patterning along the dorsoventral and left-right axes is important in determining the right-sided looping (Manner, 2000), but research on the patterning along the transmural axis (across the myocardial wall from the epicardium to the endocardium) is limited and deserves much needed attention (Pennisi et al., 2003). The myocardium has two subcomponents: one consisting of the outermost region, the compact myocardium that contains myocytes that are epithelioid and highly mitotic; and a second consisting of trabeculated myocardium that is less mitotically active (Manasek, 1968; Pennisi et al., 2003). It is at HH stage 16–17 that the luminal appearance of the heart tube begins to change by the formation of trabeculations along the inner myocardial layers at the level of the greater curvature of the looped primitive ventricle (Icardo and Fernandez-Teran, 1987; Sedmera et al., 2000). Ventricular trabeculations coordinate intraventricular conduction (de Jong et al., 1992), increase surface area, enhance contractility, route blood flow, increase diffusion for nourishing the avascular myocardium, and allow the myocardial mass to increase (Rychter and Ostadal, 1971; Rychterova, 1971). Thus, trabeculation is essential for myocardial survival and compact myocardium is essential for myocardial growth as discussed below and at subsequent stages. The compact myocardial layer increases only slightly, from 1–2 cell layers to 3–4 cell layers during the early phase of proliferation (HH stage 12 to 21) prior to major compaction and proliferation (day 8 to 14) (Rychterova, 1971; Sedmera et al., 2000). The increase in myocardial mass at this point, from HH stage 12–21, is mainly due to the forming trabeculations. The initial outer compact layer is a source of new cells (Jeter and Cameron, 1971) and has a higher proliferative/lower differentiation rate as compared to the newly forming trabeculations (Mikawa et al., 1992b). Cardiac myocytes proliferate (albeit at a diminishing rate) until hatching and then the process of hypertrophy (increase in cell size not number/proliferation) of the cardiac myocytes will further increase the size of the heart (Li et al., 1997). The major proliferative period of the compact layer (myocardium adjacent to the epicardium) is from day 8 to day 14, resulting in the expansion of the ventricular wall (Rychterova, 1971, 1978). Delaying the outgrowth of the epicardial layer over the myocardium results in a thin myocardial wall, suggesting that signaling molecules from the epicardium are required to promote proliferation of cardiomyocytes (Manner, 1993; Gittenberger-de Groot et al., 2000; Perez-Pomares et al., 2002; Pennisi et al., 2003; Stuckmann et al., 2003). The precise regulation of cardiomyocyte proliferation and transmural patterning is largely unknown except for the recent work completed on FGF2 and FGFR-1 (Pennisi et al., 2003), as well as erythropoietin and retinoic acid (Stuckmann et al., 2003). However, it is now known that the myocardium is still capable of establishing and maintaining a transmural pattern (not the correct number of myocytes) in an epicardium-independent manner and that the trabecular formation and patterning is regulated by the endocardium rather than the epicardium (Pennisi et al., 2003). Note that the atrial trabeculations appear much later (stage 27) than the ventricular trabeculations (Adelmann and Malpighi, 1966).

HH 17–18 (52–69 HOURS, 2.5 DAYS, 29–36 SOMITES)

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Cardiac Looping

By stage HH 18, the s-shaped heart loop is completed (Fig. 3A) (Manner, 2000) and then from HH 18–24 the proximal two-thirds of the primitive conus shifts toward its definitive position ventral to the right atrium (Manner, 2000).

Epicardium

Villous protrusions of the proepicardium contact the dorsal wall of the heart (Manner, 1992; Perez-Pomares et al., 1998). The epicardial mantle begins to envelop the myocardium (Fig. 4B). It contacts the dorsal tubular heart in the region of the atrioventricular junction.

Myocardial Proliferation and Trabeculation

At stage HH 17–18, trabeculations in the un-septated ventricle run dorsoventrally with no free ends and appear similar in different species (Sedmera et al., 2000).

HH 19-20 (68–72 HOURS, 3.0 DAYS, 37–43 SOMITES)

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Apoptosis

Apoptosis occurs mainly in non-myocardial tissues of the chick heart, which is mostly derived from the endocardium, the epicardium, and the cardiac neural crest (Poelmann et al., 2000) and thus the main locations of apoptosis are in the outflow tract cushions, the atrioventricular cushions, the developing semilunar valves, and the walls of the aorta and the pulmonary trunk (Sumida et al., 1989; Poelmann et al., 1998; Poelmann and Gittenberger-de Groot, 1999). At HH 19, the first substantial number of apoptotic cells (identified by LysoTracker Red) can be detected in the chicken embryo heart at the dorsal inner curvature, the site where a connective tissue bridge has formed between the proepicardial organ and myocardium (Schaefer et al., 2004). This observation suggests this process occurs much earlier than had been previously described (stage HH 24–25) (Pexieder, 1975; Watanabe et al., 1998; Cheng et al., 2002; Rothenberg et al., 2002). Apoptosis in the OFT is detected from HH 19–35 in a very specific pattern as described below, peaking at stages HH 27–32 (Schaefer et al., 2004). It is believed that the epicardium induces apoptosis in cardiomyocytes shortly after making contact with the myocardium (Rothenberg et al., 2002; Schaefer et al., 2004). The pattern of apoptosis (Schaefer et al., 2004) generally follows the pattern reported for epicardial coverage of the OFT (Ho and Shimada, 1978; Hiruma and Hirakow, 1989).

Epicardium

By stage HH 20, the proepicardium has moved over the inner curvature of the heart (AV groove) (Perez-Pomares et al., 1998).

Myocardial Proliferation and Trabeculation

The inner surface of the un-septated ventricle reveals extensive trabeculation, distributed uniformly as far proximally as the atrioventricular canal (Ben-Shachar et al., 1985). In addition, from day 3 to day 7 ventricular myocytes complete almost six cell cycles, showing active proliferation during this period (Pennisi et al., 2003).

Ventricular Septation/ Endocardial Cushions

The dorsal (inferior) and ventral (superior) cushions of the A-V canal have started to form, as well as a primitive interventricular septum (De la Cruz et al., 1983). Thus from stage HH 18–22, a primary interventricular foramin (channel) can be seen (De la Cruz et al., 1983).

HH 21-23 (3.5–4 DAYS, 43–44 SOMITES)

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Apoptosis

A higher incidence of apoptosis can be detected at HH 22–23 on the right dorsal surface of the ventricle near its junction with the proximal OFT (Schaefer et al., 2004). Again, the epicardium has reached this surface of the right ventricle just prior to the apoptosis at stage HH 21, suggesting that the epicardium induces apoptosis within the cardiac myocardium (Manner et al., 2001). Also, proper timing of epicardial outgrowth is important for the ingrowth of coronary arteries into the aorta at later stages (Eralp et al., 2005) (as discussed at HH stage 25–26) and this has been linked to the process of apoptosis (Velkey and Bernanke, 2001). Delayed outgrowth of the epicardium results in defective or absent connections of the coronary system to the systemic circulation (Eralp et al., 2005).

Cardiac Neural Crest Cells

CNC cells have reached the OFT (Waldo et al., 1998; Farrell et al., 1999; Kirby, 2002). CNC cells have two entry points into the heart: the arterial pole (Poelmann et al., 1998) and the venous pole (Poelmann and Gittenberger-de Groot, 1999). CNC arterial pole derivatives contain three different subpopulations: outflow tract septum; smooth muscle cells in the great vessel walls; and ganglionic cells in the outer vessel walls and in the subepicardial space of the heart (Fig. 5, green dots). Additional studies have also revealed that these ganglionic derivatives of CNC form the anterior parasympathetic plexus (APP) of the heart (Fig. 5, green dots labeled APP) (Kirby and Stewart, 1983; Verberne et al., 1998, 2000). CNC cells also migrate through the venous pole of the heart to locations surrounding the prospective conduction system and the atrioventricular (AV) cushions (Poelmann and Gittenberger-de Groot, 1999).

Cardiac Progenitors (Secondary Heart Field)

Secondary myocardium is finished being added to the outflow tract (Mjaatvedt et al., 2001; Waldo et al., 2001).

Endocardial Cushions

Two pairs of endocardial cushions have formed in the outflow tract. One pair is positioned proximally and the other pair is located distally (Qayyum et al., 2001). At stage HH 22, the shape of the AV cushion is changing; the ventral cushion now overlaps the dorsal cushion without fusing (De la Cruz et al., 1983).

Epicardium

The first epicardial cells penetrate into the myocardium at the dorsal wall of the ventricles (Manner, 1999).

Valve Development

Prevalve leaflet formation in the atrioventricular cushion mesenchyme begins (Sugi et al., 2003). The formation of the cardiac valves involves the cellular processes of epithelial-to-mesenchymal transformation (EMT) and cushion cellularization (as discussed in the Endocardial Cushions sections of this reference guide), as well as cell differentiation, proliferation, and apoptosis (discussed at subsequent stages of valve formation). Currently, the genes regulating later aspects of valve and cushion development are poorly understood, but recent studies have expanded our knowledge of these molecules (Camenisch et al., 2002a, b; Dor et al., 2003; Lincoln et al., 2004; Person et al., 2005).

Ventricular Septation

Major trabecular bundles inside the un-septated ventricle at the site of the ventricular groove are seen; however, at this point no solid muscular ventricular septum can be identified (Ben-Shachar et al., 1985).

HH 24 (4.0 DAYS, ALL SOMITES FORMED)

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Apoptosis

A concentration of apoptotic cells is found along a region of the right ventricle near the OFT and on the right dorsal surface at the proximal region of the OFT (Schaefer et al., 2004). Also, at approximately HH stage 24 CNC cells invade the AV cushion mesenchymal tissue and this corresponds to a peak of apoptotic cells in the AV cushions (Poelmann and Gittenberger-de Groot, 1999; Poelmann et al., 2000).

Atrial Septation

The interatrial septum has fused with the dorsal and ventral endocardial cushions as they fuse to divide the atrioventricular canal. As the interatrial septum fuses to the cushions, it obliterates the interatrial communication, foramen primum, but at the same time a secondary interatrial communication in the form of multiple perforations develops in the middle portion of the interatrial septum (similar to the ostium secondium in mammals) allowing continued shunting of blood from the right to left atria, thus bypassing the non-functional pulmonary system (Quiring, 1933; Morse et al., 1984). The new shunt is called the foramina secunda (Morse et al., 1984).

Cardiac Looping

General growth of the heart and considerable expansion of the ventricular bend and the primitive atria have led to the loss of the original tubular character of the heart (Manner, 2000). The late phase of cardiac looping has begun (HH 24–34), which corresponds to the phase of cardiac septation (Bouman et al., 1995, 1997; Kirby and Waldo, 1995; Manner, 2000).

HH 25-26 (4.5–5 DAYS)

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Apoptosis

High levels of apoptosis are evident in both the proximal and distal OFT in contrast to the sparse and evenly distributed apoptotic cells in the ventricles and atria (Schaefer et al., 2004).

Cardiac Neural Crest Cells/Outflow Tract Septation

At stage HH 25, condensed mesenchyme of the aorticopulmonary septum projects into the aortic sac dividing it into pulmonary and aortic channels (Waldo et al., 1998). By stage HH 26, the aorticopulmonary septum has lengthened toward the heart and entered the distal truncus (Waldo et al., 1998).

Coronary Vasculature

Coronary vasculature begins to develop. The first coronary vessels are present within the subepicardial mesenchyme of the dorsal AV groove (Vrancken Peeters et al., 1999). By HH stage 26 onward, the coronary vessels extend further within the subepicardium at the dorsal side of the heart, and also the first endothelium-lined tubes are found in the ventricular myocardium (Vrancken Peeters et al., 1999). It was originally believed that the coronary arteries grow out of the aorta, but researchers determined that the coronary arteries actually grow into the aorta from a capillary ring that encircles the aortic and pulmonary outflow tracts (peritruncal ring of coronary arterial vasculature or bulbar vascular ring) (Bogers et al., 1988, 1989; Waldo et al., 1990, 1994; Hood and Rosenquist, 1992; Poelmann et al., 1993). At this stage, a luminized endothelial plexus appears first in the subepicardial layer of the dorsal wall of the ventricles and then from there it extends into the subepicardial layer and into the myocardium (Manner, 1999). Also, at this stage the subepicardial portion of the endothelial plexus is connected to the sinus venosus.

Endocardial Cushions

A third distal endocardial cushion has developed in the outflow tract (Qayyum et al., 2001). At stage HH 26, the fusion of the AV cushions begins, eventually forming the septum of the AV canal (De la Cruz et al., 1983).

Epicardium

From HH stages 25–31, epicardium-derived cells contribute a novel population of cells to the myocardial wall (unrelated to the coronary vessels) and the subendocardial region. These novel cells migrate to the subendocardial layer from the subepicardial layer by way of transient discontinuities in the thin-walled myocardium (points at which the endocardial lining invaginate through the myocardium, allowing the subepicardial and subendocardial space to contact) (Gittenberger-de Groot et al., 1998). These novel cells, however, are never located within the endocardial lining (Gittenberger-de Groot et al., 1998).

HH 27 (5.0 DAYS)

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Apoptosis

Apoptotic cells from stages HH 27–29 are located in concentrated regions in a proximal site and a distal site within the OFT. The proximal focus of apoptotic cells is a wedge shape with the widest region on the ventral and right side at the base of the OFT (right side under the aorta) (Schaefer et al., 2004). It is important to note that the cardiac neural crest cells embedded in the outflow tract cushions (intracardiac) become apoptotic at about stage HH 27 (Poelmann et al., 1998), whereas the more distally located central mass of cardiac neural crest cells exhibit barely any apoptosis (Bartelings and Gittenberger-de Groot, 1989). The reason for the difference is still poorly understood although it most likely is due to their respective developmental fates. The embedded cardiac neural crest cells are in areas of outflow tract cushions that subsequently myocardialize, while the cardiac neural crest cells in the central mass (at orifice level) remain mesenchymal (Bartelings and Gittenberger-de Groot, 1989; Poelmann et al., 2000). Thus, cardiac neural crest cells in the conus cushions of the outflow tract may induce myocardialization of the septum through activation of latent TGF-beta present in the extracellular matrix (Poelmann and Gittenberger-de Groot, 1999).

Cardiac Innervation

Early cardiac innervation.

Cardiac neural crest cells are now located around the arterial cardiac vagal branches at the arterial pole, the vagal branches along the anterior cardinal veins, and the sinal vagal branch at the venous pole (Verberne et al., 1998).

Cardiac Neural Crest Cells/Outflow Tract Septation

Aorta and pulmonary trunk have lengthened and rotated 20° by stages HH 26–27 (Waldo et al., 1998). In addition, condensed cardiac neural crest cells that surround the aortic arch arteries have begun to move into the walls of the distal aorta and pulmonary trunk (Waldo et al., 1998).

Coronary Vasculature

From HH stage 26 to 31, the endothelium-lined coronary vessels that are connected to the sinus venosus reach the left and right AV groove, as well as the ventral side of the heart (Vrancken Peeters et al., 1999).

Epicardium

The formation of the epicardium is now complete (Manner, 1999). The epicardium derived from the proepicardium covers only the proximal portion of the cardiac outflow tract, whereas the distal portion is covered by an epicardial layer of a different origin, which is continuous with the serosal epithelium at the junction between the outflow tract and the dorsal wall of the pericardial cavity (Manner, 1999).

Myocardial Proliferation and Trabeculation

Trabeculations become transformed into fenestrated trabecular sheets and differences between the trabeculations on the left and right side of the un-septated ventricle become apparent. In chick, the left ventricular trabeculations are thicker and more sheet-like than in the right ventricle, which are finer and shorter (Ben-Shachar et al., 1985; Icardo and Fernandez-Teran, 1987; Sedmera et al., 1997). Also, the progressive thickening (Day 5 onwards) of the compact myocardium results in a multilayered organization of myocardial fibers (three major components: outer longitudinal, middle circular, and inner longitudinal) (Sedmera et al., 2000). Atrial trabeculations first appear approximately at this stage (Adelmann and Malpighi, 1966); however, research on the precise development of the atrial myocardium is lacking probably because of its lesser functional importance (Sedmera et al., 2000). Atrial trabeculations are believed to increase atrial contractility and are actually thought of as pectinate muscles that can traverse the atrial lumen (Sedmera et al., 2000). Their appearance coincides with an increase of the atrioventricular pressure gradient (Hu and Keller, 1995). The venous sinuses and the atrioventricular vestibules, parts of the atrial chambers, remain smooth throughout development (Sedmera et al., 2000).

HH 28 (5.5 DAYS)

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Cardiac Conduction System

Eventually the pacemaking impulses are generated at the sinoatrial node (SA-node), which starts to form at stage HH 27–28 in the right atrium (Lamers et al., 1991; Pennisi et al., 2002). The atrioventricular node (AV-node) and the upper part of the AV-bundle also start to develop at stage HH 28 (Arguello et al., 1988). The AV-node is eventually located at the base of the interatrial septum and close to the endocardium (Arguello et al., 1988). The AV-node slows the pacemaking impulse from the atrium to the ventricular myocardium, providing a deliberate delay that allows the ventricles to fill (after atrial contraction) before the ventricles contract. The impulses are then transmitted to the ventricular conduction network comprising the AV-bundle that originates at the posterior right atrial wall near the atrial septum above the atrioventricular groove. It then extends over the upper margin of the ventricular septal muscle and bifurcates near the aorta, creating the right and left bundle branch. The left branch terminates at the base of the aortic leaflet of the mitral valve. The left and right bundle branches then eventually lead into the Purkinje fibers (Pennisi et al., 2002), which do not start to form until day 10 (Gourdie et al., 1995).

Cardiac Neural Crest/ Outflow Tract Septation

The aorticopulmonary septum has descended deeper and elongated craniocaudally in the truncus, dividing the semilunar valve region into two channels. The prongs of cardiac neural crest cells are now shortened and of equal lengths (Waldo et al., 1998).

Epicardium

Between stages HH 27–29, epicardial cells have now penetrated through all layers of the dorsal wall of the ventricles, atrial wall, outflow tract, and atrioventricular cushions (Manner, 1999).

Valve Development

Atrioventricular (AV) valve formation starts. From stage HH 28–36, the smooth margins of the AV cushions progressively become restructured into valve leaflets (Chin et al., 1992; Wunsch et al., 1994). Thus, cushion cells differentiate into the connective tissue fibroblast cells of the valve leaflets (Wunsch et al., 1994). The valves eventually consist of the fibrous annulus, the leaflets, and the supporting tension apparatus (tendinous cords and papillary muscles) (De la Cruz et al., 1983; Chin et al., 1992). The different material contributions to the valve components have been controversial, but recent lineage studies have shown that the leaflets and chordae tendineae of the mitral and tricuspid valves, the atrioventricular fibrous continuity (connects the septal leaflets of the valves), and the leaflets of the outflow tract semilunar valve arise from endothelial cells of the endocardium (de Lange et al., 2004; Lincoln et al., 2004). De Lange and colleagues (2004) also showed that there is no substantial contribution from myocardial, proepicardial, and neural crest lineages. The mural leaflets of the mitral and tricuspid valves form by protrusion and growth of a sheet of atrioventricular myocardium into the ventricular lumen and the myocardial layer is subsequently removed by apoptosis late in development (de Lange et al., 2004). The aortic leaflet of the mitral valve and the septal leaflet of the tricuspid valve form differently by a direct growth of the endocardial derived mesenchyme of the inferior and superior atrioventricular cushions (de Lange et al., 2004). Cardiac neural crest cells have been shown to greatly contribute to the endocardial cushion mesenchyme in the outflow tract, but it is believed that endocardially derived mesenchyme replaces cardiac neural crest–derived cells during late stages of valve formation via unknown mechanisms (de Lange et al., 2004).

HH 29 (6.0 DAYS)

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Endocardial Cushions

Two additional intercalated endocardial cushions form distally in the outflow tract (Qayyum et al., 2001).

Valve Development

Distinct mitral and tricuspid valve primordia are present by day 6.0 and their distal ends contain highly proliferative cells, supporting an outgrowth mechanism of valve development (Lincoln et al., 2004).

Ventricular Septation

At stages HH 28/29, the interventricular septum has grown towards the atrioventricular cushions and has started to fuse, leaving a small gap called a primary interventricular connection (interventricular tunnel or canal) between the left and right ventricle (De la Cruz et al., 1983; Waldo et al., 1998).

Myocardial Proliferation and Trabeculation

From day 6 to day 8, there is an 80% increase in cell density in the ventricular myocardial compact zone (Pennisi et al., 2003).

HH 30 (6.5 DAYS)

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Apoptosis

A concentration of apoptotic cells is still present just below the aorta (Schaefer et al., 2004) and this region of tissue disappears after stage HH 32.

Coronary Vasculature

Coronary vascularization continues with the formation of a capillary plexus and venous sinusoids derived from epicardial cells (Waldo et al., 1990).

Valve Development

Arterial valvar leaflets start forming at the distal end of the distal cushions (Qayyum et al., 2001).

HH 31 (7.0 DAYS)

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Apoptosis

By stage HH 31–32, the intense focus of apoptotic cells shifts to the epicardial area between the aorta and pulmonary artery and finally to the subpulmonic region (Schaefer et al., 2004). Also, HH stage 31 embryos show elaborate apoptosis of cardiac neural crest cells in the posterior AV- node area and the bundle of His (Poelmann and Gittenberger-de Groot, 1999) and subsequently at stage 32 the conduction system changes its physiological function dramatically. This suggests that cardiac neural crest cells may be instrumental in an as yet poorly understood phase in conduction system differentiation (Poelmann et al., 2000). It has been suggested that cardiac neural crest cells that enter through the dorsal mesocardium induce the final differentiation of specialized myocardial conduction cells in the SA-node, the AV-node area, the penetrating bundle of His, and the left and right bundle branches, extending into the moderator band (Poelmann and Gittenberger-de Groot, 1999).

Cardiac Neural Crest/Outflow Tract Septation

By stages HH 30–31, the prongs in the truncal cushions are very short or have disappeared and septation of the distal two-thirds of the truncus is complete (Waldo et al., 1998).

Coronary Vasculature

The coronary vessels are now found in a regular pattern within the myocardium and are arranged at an equal distance from each other (Vrancken Peeters et al., 1999).

Valve Development

The developing leaflets of the aortic and pulmonary valves no longer lie in the same plane, but have shifted and are now angled relative to each other (Qayyum et al., 2001).

HH 32-33 (7.5–8 DAYS)

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Apoptosis

Apoptosis in the OFT begins to gradually subside (Schaefer et al., 2004).

Coronary Vasculature

The spreading vessel plexus has reached the outflow tract of the heart, forming a peritruncal ring (bulbar vascular ring) around the great arteries (Vrancken Peeters et al., 1999). Endothelial cells lining the vessels of the peritruncal ring have now made contact with the endothelium of the two facing semilunar valve sinuses of the aorta (Vrancken Peeters et al., 1999). Numerous coronary artery orifices can be seen in the left and right aortic sinuses (left coronary artery orifice is usually seen before the right) that are continuous with a penetrating vessel from the peritruncal capillary plexus (Bogers et al., 1989; Waldo et al., 1990; Hood and Rosenquist, 1992; Poelmann et al., 1993; Waldo et al., 1994; Velkey and Bernanke, 2001). Also, multiple vascular channels connect to the left and right aortic sinuses (Waldo et al., 1990; Velkey and Bernanke, 2001) and they disappear as one channel becomes dominant to form the left and right coronary artery stems (Waldo et al., 1990). It is important to note that no coronary artery orifices are seen in the pulmonary artery or the posterior aortic sinus (Waldo et al., 1990; Velkey and Bernanke, 2001), suggesting a controlled invasion of the aorta. There have been observations of transient connections to the posterior aortic sinus, but they do not survive in later stages (Waldo et al., 1990, 1994; Hood and Rosenquist, 1992; Poelmann et al., 1993). The differentiation of smooth muscle cells or fibroblasts around the arterial or venous vessel endothelium has not yet occurred (Vrancken Peeters et al., 1999).

Epicardium

From HH stages 32–43, epicardium-derived cells (EPDCs) contribute a novel population of cells to the atrioventricular cushions by way of the atrioventricular sulcus (Gittenberger-de Groot et al., 1998). In addition, epicardial cells have reached the subendocardial free edges of the developing leaflets of the tricuspid and mitral valves (Gittenberger-de Groot et al., 1998; Manner, 1999). The semilunar valves, however, are negative for novel EPDCs. The novel epicardium-derived cells (EPDCs) are also found in the trabeculae and interestingly scattered in the muscular outflow tract septum (Gittenberger-de Groot et al., 1998). At this point, the myocardial discontinuities are no longer seen. Gittenberger-de Groot and colleages (1998) hypothesize that the specific distribution pattern of EPDCs suggests that this novel population of cells is involved in the induction and organization of purkinje fibers at both subendocardial and periarterial sites in avians.

Ventricular Septation

The primary interventricular connection (interventricular canal) is now a diagonal passageway just below the forming aortic semilunar valve.

HH 34 (8.0 DAYS)

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Cardiac Conduction System

The final mature apex-to-base pattern of ventricular activation coincides with the completion of ventricular septation (Gourdie et al., 2003). Recruitment of cells to the AV-node, His bundle and branches ends after the ventricular septation is complete (Gourdie et al., 1995; Cheng et al., 1999).

Cardiac Neural Crest/ Outflow Tract Septation

OFT septation is complete (Fig. 5). The aortic and pulmonary channels of the distal outflow tract are separated by the aorticopulmonary septum (CNC derived) while the proximal outflow tract is septated by the joining of the two proximal cushions (Kirby et al., 1983; Waldo et al., 1998; Qayyum et al., 2001).

Coronary Vasculature

Coronary arteries have formed (Fig. 5, blue dots). The vessel walls (media and adventitia) of the coronary arteries start to develop (Manner, 1999). The coronary veins, at this stage, however, have not yet acquired a media or adventitia (Vrancken Peeters et al., 1997). After HH stage 32, an area of dense mesenchyme extends from the epicardial lining towards the vessels, which eventually form the future coronary arteries and veins, and mesenchymal condensations appear in the AV groove continuous with the periarterial mesenchyme where arterial media differentiation begins (Vrancken Peeters et al., 1999). Interestingly, prior to HH stage 32 the epicardial epithelium and its underlying myocardial surface are equidistant, but after HH stage 32, the mesothelial lining invaginates into the AV subepicardial mesenchyme, reducing the distance between the epicardial lining and the underlying developing coronary plexus. The mechanisms regulating the movement of this mesothelial sheet are still poorly understood. It is believed that the epithelial-mesenchymal transformation (EMT) of the adjacent surface epicardial epithelium is a major source of the subepicardial mesenchymal cells from which the future smooth muscle cells and fibroblasts of the coronary arteries form (Poelmann et al., 1993; Viragh et al., 1993; Dettman et al., 1998; Vrancken Peeters et al., 1999). As discussed in the endocardial cushion development portion of this reference guide, EMT is a common mechanism in the developing embryo. The myocardium and cardiac ganglia (cardiac neural crest–derived) residing within the subepicardial layer of the AV groove may influence the EMT of the epicardium (Vrancken Peeters et al., 1999). The subepicardial portion of the endothelial plexus is now connected to the aorta (Manner, 1999) and definitive left and right coronary arteries (at this stage only one for each) are continuous with coronary artery orifices in the aortic sinuses (Velkey and Bernanke, 2001).

Endocardial Cushions

There are three endocardial cushions positioned distally and two proximally within the developing outflow tract (Qayyum et al., 2001). The three distal endocardial cushions, and the two intercalated endocardial cushions contribute to the formation of the leaflets and sinuses of the arterial roots. The two proximal cushions give rise to a transient septum that eventually transforms into the muscular component of the subpulmonary infundibulum (Qayyum et al., 2001).

Myocardial Proliferation and Trabeculation

As ventricular septation is completed, the increasing myocardial mass causes the radial arrangement of the trabeculations to change to an apico-basal orientation (Sedmera et al., 2000). The patterns of trabeculation are like fingerprints, strikingly unique for each individual heart (Sedmera et al., 2000). Also, the increasing luminal size compresses the basal portions of the trabeculations into the ventricular wall, significantly increasing the proportion and thickness of the compact myocardium (Rychterova, 1971; Sedmera et al., 1997).

Valve Development

At stage HH 34, the semilunar valves are completely formed and in the orientation of an adult heart. Thus, at this stage the pulmonary valve lies ventral and leftward of the aortic valve (Qayyum et al., 2001).

Ventricular Septation

Ventricular septation is now complete (Waldo et al., 1998). It should be noted that the portion of the interventricular septum that separates the inlet of the right ventricle from the left ventricular infundibulum is not formed by connective tissue as it is in humans, but it still corresponds to the membranous region of the human interventricular septum (De la Cruz et al., 1983). It is also believed that this region is probably formed by different embryological components (Goor et al., 1970).

HH 35 (8–9 DAYS)

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Apoptosis

Apoptotic cells are now restricted to the great vessels with the most intense staining at the branch point of vessels (Schaefer et al., 2004).

Cardiac Innervation

Cardiac neural crest cells are now also located throughout the anterior and posterior plexus (Verberne et al., 1998) (Fig. 5, green dots labeled APP).

HH 36 (10 DAYS)

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Cardiac Conduction System

The critical period of the initial differentiation of the peripheral conduction system (Purkinje fiber network) occurs between days 10–20 (Gourdie et al., 1993). Differentiation of the network of Purkinje fibers begins by the formation of putative conductive cells (Connexin42 positive) surrounding early blood vessels (Gourdie et al., 1995). Interestingly, between days 14–18 there is a dramatic increase in conductive cells, which has been correlated to the growing coronary vascular bed (at day 14, the closed vascular system is functional) (Gourdie et al., 1995). Thus, contractile myocytes can be recruited to form conductive myocytes when exposed to forming coronary vessels, suggesting that the peripheral conduction network of Purkinje fibers is laid down in close temporal and spatial association with the developing coronary vascular bed (Gourdie et al., 1995; Mikawa and Fischman, 1996; Hyer et al., 1999; Mikawa, 1999; Takebayashi-Suzuki et al., 2000). The Purkinje fibers are derived from working myocytes, not from cardiac neural crest cells or primordial epicardial cells (Mikawa et al., 1992a; Gourdie et al., 1995; Cohen-Gould and Mikawa, 1996; Mikawa and Gourdie, 1996; Cheng et al., 1999). However, these extracardiac cells may have an indirect influence on the induction and spatial arrangement of the Purkinje fibers. For example, cardiac neural crest–derived neurons adjacent to blood vessels are critical for the survival of coronary arterial branches (Waldo et al., 1994), which in turn are necessary for proper Purkinje fiber formation. Purkinje fibers are eventually found throughout the subendocardium of the left and right ventricles and they initiate an apex to base contraction of the ventricle. The main function of the Purkinje fiber network is to rapidly transmit impulses to the ventricular muscle (Pennisi et al., 2002). Thus, once the Purkinje fibers have formed, electrical activation initiates at the sinoatrial node and is conducted across the atrial chambers to the AV-node and atrioventricular (AV) ring, then along the AV bundle, and then finally along the network of Purkinje fibers within the ventricular muscle, synchronizing contraction of the ventricles (Lamers et al., 1991; Gourdie et al., 1993, 1995).

Cardiac Innervation

Cardiac neural crest cells differentiate into cardiac ganglion cells in both the anterior and posterior plexus (Verberne et al., 1998).

Valve Development

AV valves are completely formed, although some remodeling continues in later stages (day 10–14) (Chin et al., 1992; Lincoln et al., 2004). The remodeling and maturation of the valve primordia are accompanied by decreased proliferation (Lincoln et al., 2004). Chin and colleagues conclude that the mitral valve leaflets form predominately from the endocardial cushion tissue, and the tricuspid band (in the chick, the mature tricuspid valve is a muscle band instead of leaflets) receives contributions from both the endocardial cushions and surrounding ventricular myocardium (Chin et al., 1992). Thus, only mesenchymal cells and an extracellular matrix (endocardial tissue) compose the mitral valve leaflets while the base of the valve leaflets is composed of a combination of fibroblasts and myocytes. Myocytes, however, also contribute to the tricuspid band, giving it a dual origin of endocardium and ventricular myocardium (Chin et al., 1992).

HH 38 (12 DAYS)

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Coronary Vasculature

Coronary veins have formed. Between days 10–14, the vessel walls (media and adventitia) begin to form in the coronary veins. The vessel walls in the coronary arteries start to form earlier at day 9. Thus, the differentiation of the definitive coronary vessels from the endothelial plexus proceeds in a proximal to distal direction by formation of the medial and adventitia tissue (Manner, 1999).

HH 40 (14 DAYS)

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Cardiac Innervation

Innervation by CNC cells is now complete. CNC cells also contribute to nerve tissue in the adventitia of the large veins at the venous pole and in the adventitia of the coronary arteries (Verberne et al., 1998).

Coronary Vasculature

CNC cells are necessary for survival of the branches of the coronary artery system (Waldo et al., 1994). The coronary vasculature itself may also induce myocytes to differentiate into the conduction system (Purkinje fibers) (Gourdie et al., 1995, 1998; Mikawa and Fischman, 1996; Mikawa, 1999). By stage HH 43, the differentiation of the media and adventitia of the coronary vasculature (arterial and venous) is complete (Vrancken Peeters et al., 1999). The coronary veins, however, contain a very thin media and adventitia, as compared to the coronary arteries (Vrancken Peeters et al., 1997, 1999).

HH 46 (20–21 DAYS): NEWLY-HATCHED CHICK

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Atrial Septation

By two days after hatching, the interatrial septum is completely closed (Quiring, 1933; Morse et al., 1984). De la Cruz (1972) postulates that at the time of hatching there is an increase in peripheral resistance along with a decrease in pulmonary resistance, causing atrial pressure to equalize, allowing the cords separating the secondary foramina to fuse and thus completely close the interatrial septum (De la Cruz, 1972; Morse et al., 1984).

Cardiac Conduction System

Double labeling of Connexin42 and ALD58 confirms that the highly expressing Connexin42 cells adjacent to the blood vessels are Purkinje fibers (Gourdie et al., 1995). Recruitment of cells to the Purkinje network occurs until hatching (Gourdie et al., 1995; Cheng et al., 1999). Several articles review the molecular markers of the developing central and peripheral conduction system (Takebayashi-Suzuki et al., 2000, 2001; Kanzawa et al., 2002; Pennisi et al., 2002; Gourdie et al., 2003; Harris et al., 2004).

Myocardial Proliferation and Trabeculation

The rate of myocyte proliferation diminishes as the chick embryo develops and ceases at hatching, but then increases again during posthatching development (Jeter and Cameron, 1971). Unlike chick, mammalian myocytes seem to completely lose their ability to proliferate shortly after birth. Therefore, the increase in heart mass during avian posthatching development may result from a combination of myocyte proliferation and hypertrophy (Jeter and Cameron, 1971; Li et al., 1997). The process of hypertrophy (increase in cell size, not an increase in cell number/proliferation/hyperplasia), however, causes a dramatic increase in the thickness of the avian compact myocardium especially in the left ventricle, which in birds is five times thicker than that of the right ventricle, allowing efficient pumping at high systemic pressure (King and McLelland, 1984; Sedmera et al., 2000).

SUMMARY

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Our understanding of early heart development has changed rapidly and significantly within the last few years. For example, it was originally thought that the cells comprising the myocardium were derived solely from the paired mesodermal heart fields, but it has been shown that only a fraction of contractile muscle in the mature heart is descended from the primordial myocardial tissue (Eisenberg and Markwald, 2004). The splanchnic mesoderm plays an important role in heart development because it gives rise to three of the major embryonic contributors to heart development, namely the primary heart field, secondary heart field, and proepicardium. Other extracardiac contributors, such as the cardiac neural crest, also contribute greatly to the developing heart. The following is a brief summary of the complex events during chick heart development and the major cardiovascular malformations.

Cardiac progenitor cells in the epiblast at stage HH 3 begin the process of gastrulation via epithelial-to-mesenchymal transformation (EMT) and subsequent migration to form the primary heart fields (splanchnic mesoderm) (Garcia-Martinez and Schoenwolf, 1993; Cohen-Gould and Mikawa, 1996). The transformation of an epithelium into mesenchymal cells (EMT) and migration occurs at many stages during embryonic development (Table 1). Similar to gastrulating epiblast cells, emigrating neural crest cells, proepicardial outgrowth and subsequent coronary vascular development, and cushion formation in the endocardium all use the process of EMT for normal development (Poelmann et al., 2002). At stage HH 7, the presumptive endocardial cells begin to delaminate from the splanchnic mesoderm via the process of EMT and migrate to form the bilateral endocardial heart tubes. Shortly thereafter, at stage HH 8, fusion of the bilateral heart tubes begins and by stage HH 10–11, the single tubular heart begins to beat (Manner, 2000). During the process of looping (stages HH 10–24), additional cardiac progenitor cells are accreted from splanchnic mesoderm onto the linear heart tube forming the outflow tract (aortic and pulmonary vessels, derived from the secondary heart field) (Waldo et al., 2001; Mjaatvedt et al., 2001; Kelly et al., 2001) and inflow tract (sinus venosus and atrium, derived from an extension of the primary heart field) (Waldo et al., 2001).

A complicated process of cardiac septation (stages HH 17–46) then occurs to create a four-chambered heart with a septated outflow tract. Endocardial cushion development (HH 12–34) is a vital part of atrial and ventricular septation. The myocardium induces endothelial cells of the endocardium to undergo the process of EMT and migrate into the extracellular matrix of the developing heart in the outflow tract and atrioventricular canal (Bolender and Markwald, 1979; Markwald et al., 1979, 1996; Runyan and Markwald, 1983). These mesenchyme cells then contribute to the formation of the mitral and tricuspid valve leaflets (HH 21–36), as well as to the atrial (HH 16–46) and ventricular septums (HH 17–34) (Eisenberg and Markwald, 1995). Cardiac neural crest (CNC) cells are an extracardiac population of cells that are also critical for proper septation of the outflow tract and ventricle (HH 25–34) (Kirby et al., 1983, 1985; Kirby and Stewart, 1983; Kirby, 2002; Hutson and Kirby, 2003). CNC cells are also important for cardiac innervation, aortic arch re-patterning, and myocardial function. At stage HH 9, cardiac neural crest (CNC) cells begin EMT and emigrate from the neural tube (Boot et al., 2003b) at the level of rhombomeres 6, 7, and 8. At subsequent stages (HH 10–23), they journey to outflow tract, affecting pharyngeal arch (Waldo et al., 1996) and secondary heart field (Yelbuz et al., 2002) development along the way. Cardiac neural crest cells also give rise to the cardiac innervation (HH 27–40) of the heart and, interestingly, these CNC derived neurons adjacent to blood vessels are critical for the survival of coronary arterial branches (Waldo et al., 1994), which in turn are necessary for proper conduction system formation (Gourdie et al., 1995; Mikawa and Fischman, 1996; Hyer et al., 1999; Mikawa, 1999; Takebayashi-Suzuki et al., 2000). This reflects the interdependence of these populations of cells to form and maintain a functional heart.

Proepicardial cells, another extracardiac population of cells, also undergo EMT and migrate (HH 17–33) out over the surface of the heart, forming the epicardial layer. EMT occurs over the surface of the epicardium, resulting in cells that ingress and migrate into the subepicardial space, eventually differentiating into coronary vessels (HH 25–49) (Poelmann et al., 2002). The presence of the epicardium over the myocardium is also very important for proper regulation of cardiomyocyte apoptosis (HH 19) (Rothenberg et al., 2002; Schaefer et al., 2004) and myocardial proliferation (HH 16–46) (Manner, 1993; Gittenberger-de Groot et al., 2000; Perez-Pomares et al., 2002; Pennisi et al., 2003; Stuckmann et al., 2003). Apoptosis is also an important cellular event that occurs (HH 19–35) mainly in non-myocardial tissues of the chick heart (endocardium, epicardium, and cardiac neural crest) (Poelmann et al., 2000).

Due to the interdependence of these different populations of cells to form a functional heart, defects in one population can cause a cascade of different congenital heart malformations. Congenital heart defects are the most common congenital cause of death in the first year of life and, of those, atrial and ventricular septal defects are the most commonly diagnosed and often have excellent outcomes (Hoffman and Kaplan, 2002; Harris et al., 2003). Abnormal valve formation and morphology are also commonly seen and can be treated (Bartram et al., 2001). In contrast, hypoplastic left heart syndrome is one of the rarest forms of congenital heart disease, but it causes 25% of all congenital heart disease mortalities (Bradley, 1999). Gittenberger-de Groot and colleagues (2005), Kirby (2002), and Hutson and Kirby (2003) have recently reviewed the major types of congenital heart defects and these malformations have been broken into five main categories that include; alignment defects, septation defects, aortic arch anomalies, myocardial and conduction system defects, and coronary vasculature defects (Table 2). Those malformations with a superscript b listed in Table 2 are the resulting cardiovascular defects after cardiac neural crest (CNC) ablation in chick. Persistent truncus arteriosus (complete absence of outflow tract septation) and Tetralogy of Fallot (alignment defect) are most commonly seen after CNC ablation and, therefore, have been studied extensively. Tetralogy of Fallot is a cascade of defects with the primary defect being a malaligned muscular outflow septum that leads to pulmonary stenosis, a VSD, an overriding aorta, and right ventricular hypertrophy. VSDs and ASDs are commonly diagnosed and repaired septation defects. Failure of the ostium primum to fuse with the septum intermedium (region where the superior and inferior atrioventricular cushions fuse) results in ASDs or atrioventricular canal defects (AVCDs). As mentioned in the reference guide, the chick atrial septum develops differently than that of the human. Thus, since chick does not have a septum secundium (a second atrial ridge in humans that septates the atria), ASDs involving the secundium do not occur (Gittenberger-de Groot et al., 2005). Aortic arch anomalies are also a major cardiovascular malformation, but it is important to note the difference between chick and human development of the bilateral pharyngeal arch artery system into a mature aortic arch system. The chick embryo develops a right-sided aortic arch (RAA) system while the human embryo develops a left-sided aortic arch (LAA) system. Thus, aortic arch anomalies in avians can result in an LAA, which would be a normal configuration in humans (Gittenberger-de Groot et al., 2005). Conduction system defects, such as atrial arrhythmias, are also major cardiovascular malformations that are commonly diagnosed. The pacemaking and conduction system (PCS) develops (HH 8–46) through a complex process of cell recruitment that is still poorly understood, resulting in a heart that contracts rhythmically to pump blood in one direction. Coordinated contraction of the atrial and ventricular chamber is through precise initiation and transmission of action potentials through the specialized PCS. The PCS is composed of the sinoatrial (SA) node that generates a pacemaker impulse, the atrioventricular (AV) node that delays an electrical impulse for separating the contraction of the atrial and ventricular chambers of the heart, and the Purkinje system for the fast and coordinated conduction of impulses to, and throughout, the ventricles (Pennisi et al., 2002). Defects can occur anywhere along this complex pathway. The coronary vasculature is also a complex network that is susceptible to genetic and environmental damage. Defects in proepicardial cell migration and EMT can result in partial to complete absence of the main coronary arterial stems. Also, high levels of homocysteine can damage the developing coronary vessels (Boot et al., 2004), as well as the developing cardiac neural crest (Boot et al., 2003c). Thus, the causes of major congenital cardiac malformations (Table 2) are multifactorial and based on genetic and environmental influences. Syndromes with cardiovascular malformations and some teratogens that cause heart defects are listed in Table 2.

Table 2. Major Cardiovascular Malformations and Syndromesa
  • a

    The table was generated from information in recent cardiovascular reviews (Kirby, 2002; Huston and Kirby, 2003; Gittenberger-de Groot et al., 2005).

  • b

    Chick cardiac neural crest ablation phenotypes.

Alignment defects
 Tetralogy of Fallotb
 Pulmonary artery stenosis
 Double-outlet right ventricleb
 Double-inlet left ventricle
 Straddling tricuspid valve
 Tricuspid valve atresia
Septation defects
 AV canal defects
 Subaortic ventricular septal defects
 Ventricular septal defects
 Atrial septal defects
 Persistent truncus arteriosusb
 Transposition of the great arteries
 Aortic stenosis
Aortic arch anomalies
 Interrupted aortic archb
 Double aortic archb
 Left aortic arch (chick)b
 Right aortic arch (mammals)
 Aortic arch hypoplasia
 Coarctation of the aorta
Myocardial and conduction system defects
 Decreased contractilityb
 Decreased excitation-contraction couplingb
 Myocardial hypertrophy
 Hypoplastic left heart syndrome (rare)
 Cardiomyopathy
 Arrhythmias
Coronary vasculature defects
 Partial absence of coronary arterial stems
 Complete loss of coronary arterial (CA) stems with ventricular CA communications
Syndromes
 Alagille
 Carpenter
 Williams
 Holt-Oram
 CHARGE
 DiGeorge
 Velocardiofacial
 Trisomy 21
 Noonans
 Ivemark
Teratogens
 Alcohol
 Retinoic Acid
 Hyperhomocysteine

CONCLUSIONS

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This review is meant to provide a reference guide to the developmental timing of various aspects of chick heart embryology according to the staging of Hamburger and Hamilton (1951) (HH), as well as provide a good bench top companion to the many recently written in depth cardiac reviews. The framework of this reference guide allows a global perspective of how these cardiac developmental events overlap temporally and spatially. Due to constraints of length, many important studies were not referenced and little molecular data was given, but many excellent articles review the molecular biology of the developing heart (Olson and Srivastava, 1996; Perez-Pomares et al., 1998; Baldwin, 1999; Epstein and Buck, 2000; Lohr and Yost, 2000; Maschhoff and Baldwin, 2000; Dees and Baldwin, 2002; Harvey, 2002; Kirby, 2002; McFadden and Olson, 2002; Morabito et al., 2002; Brand, 2003; Hutson and Kirby, 2003; Linask, 2003; Moorman and Christoffels, 2003; Small and Krieg, 2004). Lastly, the combination of the cardiac embryology discussed in this reference guide and the recent genomic analysis of cardiac neural crest and cardiac development (Martinsen and Bronner-Fraser, 1998; Gammill and Bronner-Fraser, 2002; Martinsen et al., 2003; Tabibiazar et al., 2003; Kim et al., 2004) will continue to speed the advancement of our knowledge of heart development.

Acknowledgements

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Thanks to Jamie Lohr and Allison Frasier for critically reading the manuscript and offering helpful suggestions. I also acknowledge the other researchers who have contributed to the field of heart development that were not referenced due to constraints of length. B.J.M. teaches the Human Embryology Course at the University of Minnesota Medical School.

REFERENCES

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