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Keywords:

  • review;
  • cardiac looping;
  • chick embryo;
  • scanning electron microscopy;
  • morphogenesis

Abstract

  1. Top of page
  2. Abstract
  3. TERMINOLOGICAL PROBLEMS
  4. CONTROVERSIAL INTERPRETATIONS OF MORPHOLOGICAL DATA
  5. PICTORIAL REVIEW OF THE POSITIONAL AND MORPHOLOGICAL CHANGES OF THE LOOPING HEART TUBE
  6. BIOPHYSICAL MECHANISMS INVOLVED IN CARDIAC LOOPING
  7. Acknowledgements
  8. LITERATURE CITED

Understanding early cardiac morphogenesis, especially the process of cardiac looping, is of fundamental interest for diverse biomedical disciplines. During the past few years, remarkable progress has been made in identifying molecular signaling cascades involved in the control of cardiac looping. Given the rapid accumulation of new data on genetic, molecular, and cellular aspects of early cardiac morphogenesis, and given the widespread interest in cardiac looping, it seems worth reviewing those aspects of the looping process that have received less attention during the past few years. These are terminological problems, the “gross” morphological aspects, and the biomechanical concepts of cardiac looping. With respect to terminology, emphasis is given to the unperceived fact that different viewpoints exist as to which part of the normal sequence of morphogenetic events should be called cardiac looping. In a short-term version, which is preferred by developmental biologists, cardiac looping is also called dextral- or rightward-looping. Dextral-looping comprises only those morphogenetic events leading to the transformation of the originally straight heart tube into a c-shaped loop, whose convexity is normally directed toward the right of the body. Cardioembryologists, however, regard cardiac looping merely as a long-term process that may continue until the subdivisions of the heart tube and vessel primordia have approximately reached their definitive topographical relationship to each other. Among cardioembryologists, therefore, three other definitions are used. Taking into account the existence of four different definitions of the term cardiac looping will prevent some confusion in communications on early cardiac morphogenesis. With respect to the gross morphological aspects, emphasis is given to the following points. First, the straight heart tube does not consist of all future regions of the mature heart but only of the primordia of the apical trabeculated regions of the future right and left ventricles, and possibly a part of the primitive conus (outflow tract). The remaining part of the primitive conus and the primordia of the great arteries (truncus arteriosus), the inflow of both ventricles, the primitive atria, and the sinus venosus only appear during looping at the arterial (truncus arteriosus) and venous pole (other primordia). Second, dextral-looping is not simply a bending of the straight heart tube toward the right of the body, as it has frequently been misinterpreted. It results from three different morphogenetic events: (a) bending of the primitive ventricular region of the straight heart tube toward its original ventral side; (b) rotation or torsion of the bending ventricular region around a craniocaudal axis to the right of the body, so that the original ventral side of the heart tube finally forms the right convex curvature and the original dorsal side forms the left concave curvature of the c-shaped heart loop; (c) displacement of the primitive conus to the right of the body by kinking with respect to the arterial pole. Third, dextral-looping does not bring the subdivisions of the heart tube and vessel primordia approximately into their definitive topographical relationship to each other. This is achieved by the morphogenetic events following dextral-looping. This review seeks to bring together data from the diverse disciplines working on the developing heart. Anat Rec 259:248–262, 2000. © 2000 Wiley-Liss, Inc.

During the past few years, an increasing number of original and review articles dealing with cardiac molecular morphogenesis have been published (Moorman and Lamers, 1994; Olson and Srivastava, 1996; Harvey, 1998a, 1999; Srivastava, 1999). These articles document the remarkable progress made in elucidating molecular machineries controlling various aspects of cardiac development. They also demonstrate a renewed interest on the part of developmental biologists in development of the embryonic heart. Particular success has been made in identifying molecular signaling cascades involved in the control of “cardiac looping” (Cooke and Isaac, 1998; Harvey, 1998a,b). Cardiac looping is a term commonly used to characterize a sequence of positional and morphological changes by which the originally straight and symmetric heart tube becomes transformed into an asymmetric heart loop. The renewed interest taken by developmental biologists in cardiac looping arises mainly from the fact that the looping heart is the first organ of vertebrate embryos to develop morphological left-right asymmetry (Icardo, 1996; Cooke and Isaac, 1998; Harvey, 1998b; Levin, 1998; Yost, 1998). Understanding the acquisition of left-right body asymmetry is one of the fundamental topics of developmental biology (for recent reviews, see Brown et al., 1991; Beddington and Robertson, 1999).

Besides developmental biology, there are also other biomedical disciplines interested in cardiac looping and left-right body asymmetry. Based on the postmortem examination of malformed human hearts, for example, pathologists have speculated that certain topographical anomalies of the heart chambers (ventricular inversion) might be explained by an abnormal lateralization of the embryonic heart loop to the left instead of to the right of the body (Lewis and Abbott, 1915, 1916; Van Praagh, 1972). Deviations from the normal left-right asymmetry of the abdominal and thoracic organs can indeed be associated with congenital malformations of the heart (Becker and Anderson, 1989; Anderson et al., 1998). Genetic analyses suggest that some of these human malformation syndromes might develop on specific genetic backgrounds (Belmont, 1998; Kosaki and Casey, 1998). Classical experimental embryology and teratology have produced an immense amount of data supporting the idea that abnormal cardiac looping might be responsible for the development of malformed hearts (Rychter and Lemez, 1959; Okamoto, 1980; Layton and Manasek, 1980; Steding and Seidl, 1984a,b; Tomita et al., 1991; Männer et al., 1993; Yasui et al., 1998). It is, therefore, no wonder that medical disciplines dealing with congenital heart defects have a special interest in understanding cardiac looping from its morphological, biophysical, cellular, molecular, and genetic aspects.

A review of the literature, however, shows that there are a few problems that might hamper the understanding of articles dealing with cardiac looping. Especially readers not completely familiar with this topic might be confused by unperceived problems in terminology and by discrepancies in the morphological description of the looping process. The aim of this article, therefore, is to review these aspects of cardiac looping which, unfortunately, have received less attention during the past few years. Knowledge of the terminological and morphological aspects of cardiac looping might contribute to a better understanding of articles dealing with early cardiac morphogenesis. Such knowledge might also contribute to the elucidation of the mechanisms driving the positional and morphological changes of the embryonic heart. This article, therefore, additionally summarizes the biomechanical concepts of cardiac looping with respect to terminological and morphological aspects.

TERMINOLOGICAL PROBLEMS

  1. Top of page
  2. Abstract
  3. TERMINOLOGICAL PROBLEMS
  4. CONTROVERSIAL INTERPRETATIONS OF MORPHOLOGICAL DATA
  5. PICTORIAL REVIEW OF THE POSITIONAL AND MORPHOLOGICAL CHANGES OF THE LOOPING HEART TUBE
  6. BIOPHYSICAL MECHANISMS INVOLVED IN CARDIAC LOOPING
  7. Acknowledgements
  8. LITERATURE CITED

Our understanding of cardiac looping profits from knowledge on the history of research on the developing heart, which can be traced back for more than 300 years (Malpighi, 1672; von Haller, 1758; Wolff, 1764; Pander, 1817; His, 1881,1885). Albrecht von Haller (1758) seems to have been the first to note that the embryonic heart passes through a developmental phase where it shows the morphology of a loop. Since then, the term “heart loop” has been used in the embryological literature when characterizing the morphology of early embryonic hearts. Although most embryologists of the 18th and 19th centuries were more or less familiar with the morphological and positional changes of the heart loop, this phase of cardiac development did not receive sufficient recognition. The primary interests in cardiac development focused either on the early process of establishment of the heart anlage (for review see His, 1885, p 135–136) or on the processes of formation of the cardiac septa and valves (for reviews, see Lindes, 1865; His, 1885, p 178–184).

Bradley M. Patten (1922) introduced the concept that the formation of the heart loop represents a process that is distinct from the earlier process of establishment of the heart anlage and from the later process of septation of the heart. His description and pictorial review of the formation of the cardiac loop in the chick (Patten, 1922) is a milestone in the history of research on early cardiac morphogenesis. The date of its publication might be regarded as the birth date of the term “cardiac looping.”

Patten's (1922) work formed the foundation of subsequent research on the formation of the heart loop in the chick embryo. It, therefore, might be surprising to note that, at the present time, four different viewpoints exist as to which part of the normal sequence of positional and morphological changes of the developing heart should be called cardiac looping.

In the first or “minimum” version, cardiac looping is also called “dextral-” or “rightward-looping” (DeHaan, 1965; Stalsberg, 1970; Olson and Srivastava, 1996). Dextral-looping comprises only those morphogenetic events leading to the transformation of the originally straight heart tube into a c-shaped bend/loop whose convexity is directed toward the right of the body (DeHaan, 1965; Stalsberg, 1970; Garcia-Peláez and Arteaga, 1993; Icardo, 1996).

In the second version, cardiac looping comprises dextral-looping and subsequent events leading to the transformation of the c-shaped loop into an s-shaped loop (Taber, 1998; de la Cruz, 1998). Thereby, de la Cruz (1998) distinguished three characteristic stages of the looping process in the chick embryo: (1) the c-shaped heart loop at HH-stage 12, (2) the s-shaped heart loop at HH-stage 14, and (3) the terminal stage at HH-stage 16.

In the third and “original” version, the end point of the looping process is set around a stage when the main regional divisions of the mature heart become definitively established (Patten, 1922; Steding and Seidl, 1980; Männer et al., 1993). Cardiac looping comprises dextral-looping, the transformation of the c-shaped loop into the s-shaped loop, and morphogenetic events leading to late positional changes of the conus with respect to the atria (HH-stages 9–24 in the chick).

In the fourth or “maximum” version, cardiac looping comprises the complete sequence of morphogenetic events between the earliest stage of an overt morphological asymmetry of the straight heart tube and the stage when the formation and alignment of the cardiac septa are normally completed (HH-stages 9–34, incubation days 2–8). The period of cardiac looping is divided into an early phase, corresponding to Patten's (1922) phase of cardiac looping (HH-stages 9-24), and a late phase corresponding to the phase of cardiac septation (HH-stages 24–34; Kirby and Waldo, 1995; Bouman et al., 1995, 1997).

Taking into account the existence of four different definitions of the term cardiac looping evidently will prevent some confusion in communications on cardiac looping. Nevertheless, even if readers of contemporary literature on cardiac looping are aware of this terminological problem, they might still be rather unsure. This is because contemporary authors frequently state that cardiac looping brings the subdivisions of the heart tube and the vessel primordia approximately into their definitive topographical relationship to each other (Larsen, 1993; Markwald, 1995; Tsuda et al., 1996; Harvey, 1998a; Taber, 1998; de la Cruz, 1998). Defining cardiac looping in this way obviously conflicts with some of the above-mentioned viewpoints. For example, the regional subdivisions of the c-shaped loop are far away from their definitive topographical relationships to each other. The future ventricles lie cranial and to the right of the future atria, and a morphological subunit representing the anlage of the future great arteries (truncus arteriosus) usually has not appeared at this time point of development (Garcia-Peláez and Arteaga, 1993).

It is clear that the terminological problems of cardiac looping cannot be resolved in a review article such as this one. The presence of striking differences in the terminology of early cardiac morphogenesis, however, points to the need for an open discussion on the following questions: (1) Should we distinguish different subphases within the period of early cardiac morphogenesis? (2) Which subphases should be distinguished? (3) How should these different subphases be termed? Finding a commonly accepted terminology for the normal sequence of positional and morphological changes of the embryonic heart tube might improve communication on the early morphogenesis of the heart.

CONTROVERSIAL INTERPRETATIONS OF MORPHOLOGICAL DATA

  1. Top of page
  2. Abstract
  3. TERMINOLOGICAL PROBLEMS
  4. CONTROVERSIAL INTERPRETATIONS OF MORPHOLOGICAL DATA
  5. PICTORIAL REVIEW OF THE POSITIONAL AND MORPHOLOGICAL CHANGES OF THE LOOPING HEART TUBE
  6. BIOPHYSICAL MECHANISMS INVOLVED IN CARDIAC LOOPING
  7. Acknowledgements
  8. LITERATURE CITED

The understanding of articles dealing with early cardiac morphogenesis does not only profit from knowledge of the terminological problems mentioned earlier. It also profits from knowledge of problems in the interpretation of morphological data. For a long time, the vivid process of early cardiac morphogenesis was studied exclusively on series of postmortem specimens of different embryonic ages. Based on such studies, Davis (1927) claimed that the primordia of all definitive heart chambers and of the great arteries were present as morphologically distinguishable “primitive cavities” at the straight heart tube stage before looping started. He called these cavities, in caudocranial order, the atria, the left ventricle, the bulbus cordis (primordium of the right ventricle), and the aortic bulb (primordium of the great arteries); he stated that these cavities could be distinguished from each other on the base of externally visible furrows (interbulbar sulcus, bulboventricular sulcus, atrioventricular sulcus). Except for some modifications in nomenclature, Davis's (1927) concept has dominated the embryological literature for decades. It can still be found in contemporary textbooks of embryology and developmental biology (Langman, 1981; Larsen, 1993; Gilbert, 1994). Readers not completely familiar with the latest advances in cardiac embryology, therefore, might be surprised when confronted with the fact that Davis's (1927) concept has been disproved by in vivo labeling experiments.

In vivo labeling experiments facilitate the correct identification of the origin and prospective fate of a given portion of the early embryonic heart and the correct description of its positional and morphological changes during cardiac looping. Using in vivo labeling techniques, de la Cruz and coworkers showed that the straight heart tube of chick embryos consisted of the primordia of the apical trabeculated regions of the future right and left ventricles only. The primordia of the inflow and outflow portions of the future ventricles, and of the atria and great arteries, appeared at the venous and arterial poles of the heart during the process of cardiac looping (for reviews, see de la Cruz et al., 1989; de la Cruz and Sanchez-Gomez, 1998).

Divergent interpretations of morphological data were made with respect to the straight (prelooping) heart tube and to the looping heart. For example, the process of transformation of the original straight heart tube into the c-shaped loop (dextral-looping) has frequently been misinterpreted as a bending of the straight heart tube toward the right of the body, so that the original right lateral margin forms its outer convex curvature and the original left lateral margin forms its inner concave curvature (Patten, 1922; Davis, 1927; Stalsberg, 1970). Detailed morphological analyses, however, have shown that the curvature of the heart loop is in fact directed toward its original ventral surface which, in its turn, becomes displaced to the right of the body by torsion around the craniocaudal axis (Schulte, 1916; Butler, 1952; Van Mierop et al., 1963; DeHaan, 1965; Manasek et al., 1984; de la Cruz et al., 1989; Männer et al., 1996; Icardo, 1996; de la Cruz, 1998).

PICTORIAL REVIEW OF THE POSITIONAL AND MORPHOLOGICAL CHANGES OF THE LOOPING HEART TUBE

  1. Top of page
  2. Abstract
  3. TERMINOLOGICAL PROBLEMS
  4. CONTROVERSIAL INTERPRETATIONS OF MORPHOLOGICAL DATA
  5. PICTORIAL REVIEW OF THE POSITIONAL AND MORPHOLOGICAL CHANGES OF THE LOOPING HEART TUBE
  6. BIOPHYSICAL MECHANISMS INVOLVED IN CARDIAC LOOPING
  7. Acknowledgements
  8. LITERATURE CITED

Correct interpretations of the morphological data of early cardiac morphogenesis are only beginning to find their way into current original articles on cardiac looping. A detailed pictorial review, summarizing our current knowledge of the positional and morphological changes of the looping heart tube for a readership not completely familiar with this topic, is still greatly needed. Despite its misinterpretation of dextral-looping, Patten's (1922) article still represents the most detailed publication in this field. The present article, therefore, seeks to update and advance the work of Patten by demonstrating the three-dimensional imaging of the positional and morphological changes of the looping heart tube of chick embryos by using scanning electron microscopy. The chick embryo heart was chosen because the original concept of cardiac looping and the data from in vivo labeling studies both refer to the chick. Furthermore, the chick embryo is still one of the most frequently used model organisms in embryological research.

The heart specimens presented were fixed in ovo in a general dilation (Asami, 1979) to avoid artificial alterations in position and morphology and to obtain a series of specimens of comparable functional states. Specimens were selected from a total number of 120 specimens as demonstrating characteristic steps in the normal sequence of positional and morphological changes of the chick embryo heart between incubation days 2 and 5 (HH-stages 9–24; Hamburger and Hamilton, 1951). This period includes the phase of establishment of the straight heart tube and the phase of cardiac looping as originally understood by Patten (1922). The presentation is subdivided into four sections: (1) the prelooping phase; (2) the phase of dextral-looping; (3) the phase of transformation of the c-shaped heart loop into the s-shaped heart loop; and (4) a phase of late positional changes of the primitive outflow tract (conus) with respect to the atria.

Prelooping Phase: Formation of the Straight, Bilaterally Symmetric Heart Tube

The first morphological manifestation of the straight heart tube is found at HH-stages 8+/9- (six to seven somites; Fig. 1A). This heart tube appears as a short and bilaterally almost symmetric outfolding of the coelomic epithelium covering the ventral wall of the foregut. Besides its broad connection to the foregut, the dorsal mesocardium, the heart tube is also connected ventrally to the wall of the yolk sac via a delicate midsagittal septum called the ventral mesocardium. The insertion line of the ventral mesocardium at the ventral surface of the heart tube marks the ventral midline of the heart. In vivo labeling studies suggest that heart tubes of this stage consist only of the primordium of the apical trabeculated region of the future right ventricle (de la Cruz and Sanchez-Gomez, 1998).

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Figure 1. Ventral views showing the positional and morphological changes of the embryonic heart tube between its first morphological appearance at HH-stage 9- (A) and the end of the phase of dextral-looping at HH-stage 13 (H). A,B: Prelooping stages. Establishment of the straight and bilaterally almost symmetric heart tube concomitantly with the descensus of the anterior intestinal portal (asterisks).The ventral midline of the heart tube is marked by the insertion line of the ventral mesocardium (m); the right (1) and left (2) lateral furrows appear, dividing the heart tube into a cranial and caudal portion. C: Starting point of cardiac looping. Flattening of the right lateral furrow (1) and deepening of its left counterpart (2) are the first signs of morphological left-right asymmetry of the straight heart tube at HH-stages 9+/10-. D: Appearance of the conoventricular sulcus (3) at HH-stages 10/11- is the first sign of the regional division of the heart tube into a primitive ventricular region and a primitive outflow tract (conus). C–F: Bending and lateralization of the primitive ventricular region (v) can be analyzed by using the former insertion line of the ventral mesocardium (shallow furrow marked by the dotted line) as the reference for the original ventral midline of the heart tube. The primitive ventricular region bends toward its original ventral surface and simultaneously flaps toward the right side like a door whose imaginary hinge points are fixed to the craniocaudal axis of the embryo v(c). Compare with Figure 2. Note that the primitive outflow tract does not bend but remains as a straight tube in its original position. G: Morphological appearance of the primitive atria (a) by the end of bending and rightward displacement of the primitive ventricular region at HH-stage 12. H: Rightward kinking of the primitive outflow tract (conus) marks the end point of dextral-looping at HH-stages 12/13. v, primitive ventricular region; c, primitive outflow tract or conus; a, primitive atria. Scale bar = 100 μm.

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Subsequent to its first appearance, the straight heart tube shows a rapid elongation along the craniocaudal axis of the embryo concomitantly with the descensus of the anterior intestinal portal (Figs. 1A,B). A relatively long and bilaterally almost symmetric heart tube is established at HH-stage 9-/9+ (seven to nine somites; Fig. 1B). This heart has a deep midsagittal furrow corresponding to the insertion line of the vanishing ventral mesocardium. Additionally, a bilaterally symmetric pair of lateral furrows appears, dividing the straight heart tube into a long cranial portion and a short caudal portion. In vivo labeling studies (de la Cruz and Sanchez-Gomez, 1998) suggest that these furrows mark the border between the primordia of the apical trabeculated regions of the right (cranial portion) and left ventricles (caudal portion). Hence, they are called the right and left interventricular grooves (de la Cruz and Sanchez-Gomez, 1998). In the following, however, I will call them the right and left lateral furrows.

Dextral-Looping: Transformation of the Straight Heart Tube Into the c-Shaped Heart Loop

Cardiac looping starts at HH-stages 9+/10- (9–10 somites; Fig. 1C). A flattening of the right lateral furrow and a deepening of its left counterpart are the first signs of morphological left-right asymmetry. The ventral mesocardium has almost completely disappeared, but its former insertion line at the cardiac surface is still visible as a shallow midsagittal furrow. Compared to prelooping hearts, there is not only a slight increase in craniocaudal length, but also an increase in diameter that is especially pronounced at the caudal two thirds of the heart tube. This appears to be the first morphological sign of a regional differentiation into a less thickened outflow portion or primitive conus and a more thickened primitive ventricular region. I should note here that what I call the primitive ventricular region does not represent a single ventricular primordium or segment. The term “primitive ventricular region” is used in a purely descriptive way to characterize the region of the heart tube that will form the curvature (then called “primitive ventricular bend,” see below). As stated above, the primitive ventricular region of the straight heart tube is composed of two distinct “segments,” the primordia of the apical trabeculated regions of the future right and left ventricles (de la Cruz and Sanchez-Gomez, 1998). A third segment, called the atrioventricular junction, is added to the primitive ventricular region during dextral-looping (see below).

The regional differentiation of the heart tube into the primitive conus and the primitive ventricular region becomes more prominent at subsequent steps of dextral-looping (Figs. 1D–G). A furrow, the primitive conoventricular sulcus, appears at the junction between the primitive conus and the primitive ventricular region. The formation of the conoventricular sulcus seems to be the consequence of differences in the morphogenetic behavior of the two regions. The primitive conus shows only slight changes in length, form, and position until HH-stage 12. It remains more or less as a straight and vertically oriented tube in the original midsagittal position (Figs. 1D–G). The primitive ventricular region, however, shows a considerable elongation of its caudal portion and adopts the configuration of a c-shaped bend whose convexity is directed toward the right of the body.

The positional and morphological changes of the primitive ventricular region can be followed using the original ventral midline of straight heart tubes as a landmark (Figs. 1C–F and 2A–D). Thereby, it becomes apparent that the primitive ventricular region bends in fact toward its original ventral side. The bending portion of the heart tube simultaneously flaps to the right like a door whose imaginary hinge points are being fixed to the craniocaudal axis of the embryo. Thus, the original ventral and dorsal sides of the straight heart tube become the convex right and concave left sides of the HH-stage 12 ventricular bend, respectively. The original left and right sides of the straight heart tube become the ventral and dorsal sides of the HH-stage 12 ventricular bend, respectively.

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Figure 2. Positional and morphological changes of the embryonic heart tube during the phase of dextral-looping demonstrated by in vivo labeling of the ventral midline of the straight heart tube (A) with four labels (1-4) of india ink. Ventral views of the same heart between HH-stages 9+ (A) and 11+ (D). The heart tube grows in length concomitantly to the descensus of the anterior intestinal portal (asterisks). The primitive ventricular region (labels 2, 3) bends toward its original ventral surface and simultaneously flaps toward the right side like a door whose imaginary hinge points are fixed to the craniocaudal axis of the embryo. Note that label 1 marks the original ventral midline of the primitive outflow tract (conus). Scale bar = 100 μm.

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Rightward flapping of the primitive ventricular region is usually completed at HH-stages 11+ or 12 (Fig. 1G). At this time point, the first morphological manifestation of the primordium of the future atria can be found caudally from its primitive ventricular bend (de la Cruz, 1998). Heart loops of this stage, therefore, are composed of three morphologically distinguishable components. These are in caudocranial sequence: (1) the primitive atrium; (2) the primitive ventricular bend that is subdivided by the original “left” lateral furrow into a proximal and a distal portion; and (3) the primitive conus. In vivo labeling studies have facilitated distinction of three components of the primitive ventricular bend at this stage. The primordia of the apical trabeculated regions of the right and left ventricles and a new component, the atrioventricular segment, forming the connection to the primitive atria (de la Cruz, 1998).

Dextral-looping is commonly said to be finished with completion of the rightward flapping/lateralization of the primitive ventricular bend at HH-stage 12 (Garcia-Peláez and Arteaga, 1993; de la Cruz, 1998). When comparing HH-stage 12 hearts (Fig. 1G) with those of subsequent HH-stages (Figs. 1H and 3A), however, it becomes apparent that the primitive ventricular bend is not the only region of the heart tube that becomes displaced to the right of the body. Soon after completion of the rightward flapping of the ventricular bend, the primitive conus also becomes displaced to the right. This process differs from the rightward displacement of the primitive ventricular bend. The primitive conus does not bend but remains an almost straight tube. During rightward flapping of the primitive ventricular bend, the longitudinal axis of the primitive conus still runs parallel to the craniocaudal axis of the embryo. Flapping of the primitive ventricular bend does not lead to lateralization of the primitive conus but merely to an internal torsion between its caudal and cranial ends (Figs. 1D–G). The displacement of the primitive conus to the right is best described as rightward “kinking” with respect to the arterial pole of the heart loop. Thereby, the longitudinal axis of the primitive conus shifts from its original vertical direction to an almost horizontal direction and an angulation is formed at the border between the primitive conus and the arterial pole of the heart (Figs. 1G,H and 3A). In the past, rightward kinking of the primitive conus did not receive any attention. It is undoubtfully a lateralization process and, therefore, should be regarded as an integral part of dextral-looping.

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Figure 3. Positional and morphological changes of the embryonic heart tube during the transformation from the c-shaped loop into the s-shaped loop. Part 1: HH-stages 14 (A,A'), 15 (B,B'), and 16 (C,C'). Due to the axial torsion of the embryo, ventral views of the hearts were made with respect to the pharyngeal arches (A,B,C) and to the anterior intestinal portal (asterisks) (A',B',C'). The period following dextral-looping is mainly characterized by two morphogenetic events: (1) shortening of the distance between the caudal wall of the primitive conus and the cranial wall of the primitive atria (A–C and A'–C'); and (2) the shift of the primitive ventricular bend from its original position cranial from the primitive atria toward its definitive position caudal to the atria (A–C and A'–C; continued in Fig. 4). These positional changes are accompanied by the morphological appearance of the sinus venosus, by the appearance of lateral expansions of the atrial region, and by general growth of the heart tube. Note the presence of the conoventricular sulcus, demarcating the primitive ventricular bend from the primitive conus, and the presence of the original left lateral furrow, dividing the primitive ventricular bend into a proximal and a distal portion. Note also that the left atrium temporarily lies cranial and to the left of the right atrium and that the proximal portion of the primitive ventricular bend temporarily lies cranial and to the left of the distal portion of the primitive ventricular bend (Figs. 3C,C'; continued in 4A,A',A”). rs, right horn of the sinus venosus; ls, left horn of the sinus venosus; c, primitive conus; v, primitive ventricular bend; a, primitive atria. Scale bar = 100 μm (same magnification as in Fig. 1).

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I should note that some of the data presented above seem to be at variance with those of de la Cruz and coworkers. The present data suggest that the first morphological manifestation of the primitive conus becomes apparent at the beginning of dextral-looping at HH-stage 10 (Figs. 1C–F and 2A–C). Similar data were obtained from studies mapping the fate of the heart-forming area (Rosenquist, 1985; Fig. 10, p 52). Data from the laboratory of de la Cruz, however, suggest that the first morphological manifestation of the primitive conus appears by the end of dextral-looping at HH-stage 12 (for review, see de la Cruz, 1998). This discrepancy is a mystery because it can hardly be explained by the use of different stocks of White Leghorn chicks, or by the fact that the HH-stages usually do not correspond exactly to the developmental stages of the embryonic heart. A solution to this discrepancy could be possible if the conus has a dual origin, partly from the cranial end of the heart tube (as suggested by the present data) and partly from a third heart primordium located just beyond the cranial end of the heart tube (as suggested by the data of de la Cruz). Clarification of the birth date and derivation of the primitive conus surely is a challenge for future research. Thereby, the analysis of mice with a conotruncal segment-specific transgene expression might be promising (Kuo et al., 1999).

Transformation of the c-Shaped Heart Loop Into the s-Shaped Heart Loop

The period following dextral-looping is characterized mainly by two morphogenetic events: (1) shortening of the distance between the caudal wall of the primitive conus and the cranial wall of the primitive atria; and (2) the shift of the primitive ventricular bend from its original position cranial from the primitive atria toward its definitive position caudal to the atria (Figs. 3A–C and 4A,B). These events commence at HH-stages 12/13 (Fig. 1H) and finish at HH-stage 18 (Fig. 4C). They are accompanied by the first morphological manifestation of the sinus venosus at the venous (caudal) pole of the heart (HH-stage 14); by the movement of the sinus venosus from its original position caudal to the atria toward its definitive position dorsal to the atria (HH-stages 14-18); by the rupture and disappearance of the dorsal mesocardium (HH-stages 13–15); by the appearance of lateral expansions of the atrial region (HH-stages 14–18); by the disappearance of the former left lateral furrow (HH-stage 18); and by general growth of the heart tube.

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Figure 4. Positional and morphological changes of the embryonic heart tube during the transformation from the c-shaped loop into the s-shaped loop. Part 2: HH-stages 16 (A,A',A”; same heart as in Figs. 3C,C'), 17 (B,B',B”), and 18 (C,C',C”). Ventral (A,B,C), right lateral (A',B',C'), and left lateral (A”,B”,C”) views. The shift of the primitive ventricular bend from its original position cranial from the primitive atria toward its definitive position caudal to the atria is completed at HH-stage 18 (C,C',C”). Note the changes in the positional relationships of the left and right atrium and of the proximal and distal portions of the primitive ventricular bend. Note also the disappearance of the original left lateral furrow and the general growth of the heart tube. s, sinus venosus; rs, right horn of the sinus venosus; ls, left horn of the sinus venosus; c, primitive conus; v, primitive ventricular bend; ra, atrium; la, left atrium. Scale bar = 100 μm (same magnification as in Fig. 1).

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The shortening of the distance between the caudal wall of the primitive conus and the cranial wall of the primitive atria and the caudal shift of the primitive ventricular bend both correlate with the formation of the cranial and cervical flexures of the embryo (Patten, 1922; Männer et al., 1993). Another conspicuous change in the form of the embryo, namely, the rightward rotation of its body around the craniocaudal axis, also influences the developing heart. This process starts in the embryo head region at HH-stages 12/13 and progresses caudally, reaching the tail region at HH-stages 19/20. In consequence, a torsion reflecting the axial torsion of the embryo is transiently imparted to the heart via its fixed arterial and venous poles. This transiently precludes the possibility of obtaining a single frontal view of the heart. Between HH-stages 12 and 17, standardized frontal views can be made either with respect to the arterial or with respect to the venous pole of the heart (Figs. 3A,A',B,B',C,C').

Another temporary torsion of the heart loop occurs between HH-stages 14 and 17. In consequence, the left atrium temporarily lies cranial and to the left of the right atrium and the proximal portion of the primitive ventricular bend also lies temporarily cranial and to the left of the distal portion of the ventricular bend (Figs. 4A,A',A”,B,B',B”).

The period of formation of the s-shaped heart loop is usually completed at HH-stage 18. At this time point, the primitive ventricular bend occupies its definitive position caudal to the atria. Its convex outer curvature is also directed caudally. HH-stage 18 heart loops are composed of four morphologically distinguishable components (Fig. 4C,C',C”). These are in proximodistal sequence: (1) the sinus venosus, which occupies its definitive position dorsal to the atria; (2) the primitive atria, which have developed two lateral expansions; (3) the primitive ventricular bend consisting of the slender atrioventricular junction and the two expanding primordia of the apical trabeculated region of the left and right ventricle; and (4) the primitive conus, which can be subdivided into a vertical proximal portion and a horizontal distal portion. There still is no morphological manifestation of the tubular anlage of the future great arteries (truncus arteriosus) present.

Late Positional Changes of the Primitive Conus With Respect to the Atria

The period following the establishment of the s-shaped heart loop is characterized mainly by two morphogenetic events (Figs. 5A,A',B,B' and 6A–C) : (1) the morphological appearance of the tubular anlage of the future great arteries (truncus arteriosus) at the arterial pole of the heart; and (2) the shift of the proximal two thirds of the primitive conus from the right lateral position with respect to the atria toward its definitive position ventral to the right atrium. These events are accompanied by general growth of the heart and by considerable expansion of the ventricular bend and primitive atria, which eventually lead to the loss of the original tubular character of the heart by HH-stage 24 (Fig. 5B).

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Figure 5. Morphological appearance of the truncus arteriosus and late positional changes of the outflow tract (primitive conus) with respect to the atria. Ventral (A,B) and right lateral (A',B') views of hearts from HH-stages 21 (A,A') and 24 (B,B'). A,B: Shift of the proximal two thirds of the primitive conus from the right lateral position with respect to the atria toward its definitive position ventral to the right atrium. Note that this event coincides with changes around the inner concave curvature (asterisks) of the heart. Note also that the developing heart has lost its original tubular character at HH-stage 24. A',B': Morphological appearance and elongation of the truncus arteriosus coincides with the positional changes of the primitive conus with respect to the atria. s, sinus venosus; c, primitive conus; v, primitive ventricular bend; ra, atrium; la, left atrium; t, truncus arteriosus. Scale bar = 100 μm (same magnification as in Fig. 1).

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Figure 6. Morphological appearance of the truncus arteriosus and late positional changes of the outflow tract (primitive conus) with respect to the atria. Cranial views of hearts from HH-stages 18 (A), 21 (B), and 24 (C). A: No morphological manifestation of the truncus arteriosus at HH-stage 18. B,C: Morphological appearance and elongation of the truncus arteriosus coincides with the shift of the proximal two thirds of the primitive conus from the right lateral position with respect to the atria toward its definitive position ventral to the right atrium. Arrowheads point to the conotruncal sulcus demarcating the primitive conus from the truncus arteriosus. c, primitive conus; v, primitive ventricular bend; ra, atrium; la, left atrium; t, truncus arteriosus. Scale bar = 100 μm (same magnification as in Fig. 1).

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BIOPHYSICAL MECHANISMS INVOLVED IN CARDIAC LOOPING

  1. Top of page
  2. Abstract
  3. TERMINOLOGICAL PROBLEMS
  4. CONTROVERSIAL INTERPRETATIONS OF MORPHOLOGICAL DATA
  5. PICTORIAL REVIEW OF THE POSITIONAL AND MORPHOLOGICAL CHANGES OF THE LOOPING HEART TUBE
  6. BIOPHYSICAL MECHANISMS INVOLVED IN CARDIAC LOOPING
  7. Acknowledgements
  8. LITERATURE CITED

Various concepts of the biomechanics of cardiac looping have been proposed. Until now, none of them has been proven. The elucidation of the biomechanics of cardiac looping might be hampered, in part, by the terminological problems mentioned above and by misinterpretations of morphological data of the looping process. For example, considering cardiac looping as dextral-looping only (DeHaan, 1965; Stalsberg, 1970; Garcia-Peláez and Arteaga, 1993; Icardo, 1996; Olson and Srivastava, 1996) might have hampered the elucidation of the biophysical mechanisms driving the positional and morphological changes following dextral-looping. The misinterpretation of dextral-looping as bending of the straight heart tube toward the right of the body (Patten, 1922; Davis, 1927; Stalsberg, 1970), on the other hand, obviously has hampered the elucidation of the biomechanics of (rightward) flapping/torsion of the primitive ventricular region. Keeping these problems in mind, it seems meaningful to distinguish among the biomechanics of dextral-looping, the biomechanics of the transformation of the c-shaped loop into the s-shaped loop, and the biomechanics of the late positional changes of the conus.

Biomechanics of Dextral-Looping

In the past, the search for biomechanical factors involved in cardiac looping has focused almost exclusively on dextral-looping. This might be explained by the special interest of developmental biologists in the acquisition of left-right body asymmetry, which might also be responsible for some of the terminological obfuscation mentioned above (e.g., equation of cardiac looping with dextral-looping).

Dextral-looping was originally believed to result from the combined action of forces intrinsic and extrinsic to the heart tube. Patten (1922) claimed that the primitive ventricular region was forced to bend because its growth in craniocaudal length exceeded that of the concomitant increase in distance between the fixed arterial and venous ends of the heart. This bending must be lateral because of further spatial restrictions imparted to the growing heart dorsally by the body of the embryo and ventrally by the yolk. He could not explain, however, why this bending was regularly to the right of the body. Another concept favoring the combined action of extrinsic and intrinsic forces tried to explain the lateralization of the ventricular bend by asymmetric routing of the blood flow to the venous end of the heart (Roux, 1895; Spitzer, 1951). Both ideas, however, seem to have been disproven by experimental embryology. In vitro experiments demonstrated the intrinsic capacity of isolated early heart rudiments to bend (Butler, 1952; for review, see Manning and McLachlan, 1990), and Manasek and Monroe (1972) showed that dextral-looping was not prevented by arresting the circulation of early chick embryos.

Therefore, it has become a generally accepted view that cardiac looping is a process primarily intrinsic to the heart (Manning and McLachlan, 1990). Among the earliest intrinsic mechanisms proposed were differences in the material contribution of the left and right heart “primordia” to cranial and caudal “segments” of the heart tube. These differences were proposed to originate from primary differences of the unfused left and right heart primordia (Van Praagh and DeHaan, 1967) or from processes occurring during the formation of the heart tube, like differential regional cell movement, cell redistribution, or cell proliferation (Lepori, 1967; Stalsberg, 1970). The latter possibility could not be confirmed by studies on the mitotic activity of the early chick embryo heart (Sissman, 1966; Stalsberg, 1969). All these concepts of intrinsic forces, however, were based on the assumption that dextral-looping resulted from bending of the straight heart tube toward the right of the body.

Based on 15 years of experience in research on early cardiac morphogenesis, Manasek et al. (1984) presented the most elaborate model of the biomechanics of dextral-looping. This model took account of the “ventral” bending and the rightward flapping/torsion of the primitive ventricular region. It was proposed that these two components of the looping process both resulted from regulated responses of the myocardial envelope of the heart tube to the deformative forces originating from the internal pressure of the expanding cardiac jelly. The looping heart tube was compared with a cylindrical balloon that becomes inflated with air. Such balloon models have shown that “inflation” of the myocardial envelope of the straight heart tube with cardiac jelly would result in ventral bending if, due to the presence of the dorsal mesocardium, the elastic modulus of its dorsal wall were higher than that of its ventral wall. Expansion of the heart tube would additionally result in a rightward torsion if a handed helical arrangement of myofibrils in the myocardium were to convert the increase in diameter into rotation. Manasek's model, however, was disproved by experimental embryology. Baldwin and Solursh (1989) showed that the degradation of the cardiac jelly with hyaluronidase did not disturb dextral-looping in cultivated rat embryos.

Taber proposed two biomechanical models for the bending of the heart tube (for review, see Taber, 1998). In both models, the dorsal mesocardium is responsible for bending as has been suggested previously by Manasek (see above). The first model assumes that shortening of the remnants of the dorsal mesocardium is the driving force for looping. It is speculated that the dorsal mesocardium is initially under residual tension. When ruptured, its remnants at the dorsal wall of the heart shorten and force the heart tube to bend. The second model assumes that lengthening of the heart tube due to active myocardial cell shape changes is the driving force for looping. The dorsal mesocardium does not elongate and, therefore, passively restricts lengthening of the dorsal wall of the heart tube. Neither model, however, takes account of the rightward flapping/torsion of the primitive ventricular region. Moreover, the fact that the primitive ventricular region bends while the dorsal mesocardium is still intact contrasts with the first model.

At the present time, we have no conclusive concept of the biomechanics of dextral-looping. Some experimental findings point to a role of the myocardial cytoskeleton (Manasek, 1976; Renehan and Kulikowski, 1981; Itasaki et al., 1991). However, after Manasek et al.'s (1984) concept was disproved, the nature of its involvement remained unclear. The only thing that is clear is that myocardial contraction does not play a significant role (Manasek and Monroe, 1972).

There is no doubt that future concepts of the biomechanics of dextral-looping should take account of new genetic, molecular, and cellular data. There is, however, also no doubt that new morphological data should be considered, too. In this respect, account should be taken not only of the ventral bending and rightward flapping/torsion of the primitive ventricular region, but also of the previously neglected process of rightward kinking of the primitive conus. Distinguishing three different morphogenetic events of dextral-looping might facilitate elucidating some of the biophysical mechanisms and molecular machineries driving the looping process. It is conceivable that the bending and lateralization of the primitive ventricular region and the rightward kinking of the primitive conus might be achieved by three different sets of biophysical mechanisms. These mechanisms, in their turn, might be controlled by three different sets of genes. This idea is supported by molecular data. For example, the basic helix-loop-helix transcription factors dHAND and eHAND seem to be involved in the control of growth and bending of the primitive ventricular region but not in the control of its rightward shift. Both genes are preferentially expressed in the myocardium of the outer convex curvature of the heart loop (Thomas et al., 1998). Targeted deletion of dHAND in mice has led to anomalous cardiac looping due to hypoplasia of the future right ventricle, but not due to disturbance of the normal rightward direction of looping (Srivastava, 1999). Moreover, the normal craniocaudal and dorsoventral expression patterns of the HAND genes are not altered in mice with abnormal looping toward the left of the body (Thomas et al., 1998). Other molecules have been identified as candidates providing local control of the lateralization of the primitive ventricular region but not control of its bending. For example, the bicoid-related transcription factor, Pitx2, has been suggested as a candidate mediator for transmitting signals of leftside identity from the lateral plate mesoderm to the developing heart and gut (for review, see Blum et al., 1999). Normal Pitx2 expression in the heart tube is restricted to the primitive myocardium of the left half. Therefore, it has been speculated that Pitx2 might control looping via contractile proteins (Blum et al., 1999). Recent studies, however, have shown that heart tubes loop in the normal direction in Pitx2-deficient mice, suggesting that local control of the lateralization of the primitive ventricular region occurs via a different molecular pathway (Lu et al., 1999; Kitamura et al., 1999). An extracellular matrix molecule called flectin is one canditate to work along this pathway. Flectin shows specific left-right asymmetric expression patterns in the looping heart that correlate with the directionality of the lateralization process (right- or leftward). It has been suggested that randomization of the direction of cardiac looping, after experimental perturbation of the extracellular matrix, might result from perturbing mechanical interactions between flectin and other components of the extracellular matrix (Tsuda et al., 1996, 1998).

Biomechanics of the Transformation of the c-Loop Into the s-Loop

The morphogenetic events directly following dextral-looping have attracted little attention during the past decades. This is astonishing because these events, which are mainly characterized by positional changes along the craniocaudal axis of the embryo, are intimately related to conspicuous changes in the outer morphology of the embryo, namely, the formation of the head flexures (His, 1881; Patten, 1922). The existence of a mechanical interrelationship between the formation of the embryonic head flexures and cardiac looping has been confirmed experimentally. Stiffening the embryonic head and neck regions with a straight hair prevented the formation of the cranial and cervical flexures. It also concomitantly suppressed the caudal shift of the ventricular bend and the shortening of the distance between the caudal wall of the primitive conus and the cranial wall of the primitive atria (Männer et al., 1993). His (1881) and Patten (1922) suggested that the latter two events both result from the compression of the heart loop by the bending head and neck regions. The failure of chick embryos to form the cervical flexure subsequent to experimental removal of the c-shaped heart loop, however, seemed to support the converse view that the cervical region is pulled into an arch by the caudal shift of the ventricular bend (Waddington, 1937; Flynn et al., 1991). Recent experiments have shown that heart-deprived chick embryos will form the cervical flexures when incubated in oxygen-enriched air instead of normal air, indicating that the cervical flexure formation is not caused by the caudal shift of the ventricular bend (Männer et al., 1995). It is, therefore, conceivable that mechanical pressure transmitted from the bending head and neck regions of the embryo to the heart loop might indeed be responsible for the positional changes characterizing the second phase of cardiac looping. Extracardiac forces then might contribute to the looping process to a much greater extent than previously thought (Butler, 1952; Manning and McLachlan, 1990). Thus, understanding of cardiac looping might profit from studies focusing on the biophysical and molecular machineries involved in the formation of the embryonic head flexures. Analyses of pictures published in recent publications suggest that it might be promising to screen the phenotype of mutant or knockout mice for the associated appearance of anomalies in cardiac looping and head flexures (e.g., Fig. 10 in Goh et al., 1997; Fig. 4A in Gory-Fauré et al., 1999; Figs. 6A,E in Constam and Robertson, 2000).

Biomechanics of the Late Positional Changes of the Conus

The positional and morphological changes characterizing the terminal phase of cardiac looping have attracted much more attention than those characterizing the second phase. This is in part because it has been believed that the morphogenesis of certain congenital heart defects could be explained by interferences with these events. Van Praagh et al. (1996) hypothesized that juxtaposition of the right atrial appendage could partially be explained by retainment of the primitive conus in a right-sided position with respect to the atria, possibly caused by growth deficiency of the inflow portion of the developing right ventricle. Others have hypothesized that heart defects with abnormal origin of both great arteries from the morphological right ventricle (double-outlet right ventricle) or abnormal opening of both atria into the morphological left ventricle (double-inlet left ventricle) could be explained by disturbances in the normal changes in positional relationship between the primitive conus and the atrioventricular region, and by disturbances in the subsequent remodeling of the concave inner curvature of the heart (Bouman et al., 1995; Kirby and Waldo, 1995; Markwald et al., 1998). At the present time, however, nothing is known about the biophysical mechanisms involved in the positional changes of the primitive conus. It might be interesting that the morphological appearance and elongation of the tubular anlage of the great arteries (truncus arteriosus) coincides with the ventral shift of the primitive conus, suggesting that the growth dynamics of the truncus arteriosus might have some influence on the late positional changes of the heart loop.

Acknowledgements

  1. Top of page
  2. Abstract
  3. TERMINOLOGICAL PROBLEMS
  4. CONTROVERSIAL INTERPRETATIONS OF MORPHOLOGICAL DATA
  5. PICTORIAL REVIEW OF THE POSITIONAL AND MORPHOLOGICAL CHANGES OF THE LOOPING HEART TUBE
  6. BIOPHYSICAL MECHANISMS INVOLVED IN CARDIAC LOOPING
  7. Acknowledgements
  8. LITERATURE CITED

The author thanks Mrs. Kirsten Falk-Stietenroth, Mrs. Kerstin von Roden, and Mr. Hannes Sydow for technical and photographical assistance; Prof. Gerd Steding for his critical comments on the manuscript; and Mrs. Cyrilla Maelicke for correcting the English manuscript.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. TERMINOLOGICAL PROBLEMS
  4. CONTROVERSIAL INTERPRETATIONS OF MORPHOLOGICAL DATA
  5. PICTORIAL REVIEW OF THE POSITIONAL AND MORPHOLOGICAL CHANGES OF THE LOOPING HEART TUBE
  6. BIOPHYSICAL MECHANISMS INVOLVED IN CARDIAC LOOPING
  7. Acknowledgements
  8. LITERATURE CITED
  • Anderson RH, Webb S, Brown NA. 1998. Defective lateralisation in children with congenitally malformed hearts. Cardiol Young 8: 512531.
  • Asami I. 1979. Development of the outflow tract of the rat embryonic heart. Fifth European Anatomical Congress, Prague. Arg Comm: 14.
  • Baldwin HS, Solursh M. 1989. Degradation of hyaluronic acid does not prevent looping of the mammalian heart in situ. Dev Biol 136: 555559.
  • Becker AE, Anderson RH. 1989. Cardiac pathology. In: BerryCL, editor. Paediatric pathology. 2nd ed. Berlin: Springer-Verlag. p 63124.
  • Beddington RSP, Robertson EJ. 1999. Axis development and early asymmetry in mammals. Cell 96: 195209.
  • Belmont JW. 1998. Recent progress in the molecular genetics of congenital heart defects. Clin Genet 54: 1119.
  • Blum M, Steinbeisser H, Campione M, Schweickert A. 1999. Vertebrate left-right asymmetry: old studies and new insights. Cell Mol Biol 45: 505516.
  • Bouman HGA, Broekhuizen MLA, Baasten AMJ, Gittenberger-de Groot AC, Wenink ACG. 1995. A spectrum of looping disturbances in stage 34 chicken hearts after retinoic acid treatment. Anat Rec 243: 101108.
  • Bouman HGA, Broekhuizen MLA, Baasten AMJ, Gittenberger-de Groot AC, Wenink ACG. 1997. A stereological study of stage 34 chicken hearts with looping disturbances after retinoic acid treatment: disturbed growth of myocardium and atrioventricular cushion tissue. Anat Rec 248: 242250.
  • Brown NA, McCarthy A, Wolpert L. 1991. Development of handed body asymmetry in mammals. Ciba Found Symp 162: 182196.
  • Butler JK. 1952. An experimental analysis of cardiac loop formation in the chick. MA thesis, University of Texas.
  • Constam DB, Robertson EJ. 2000. Tissue-specific requirements for the proprotein convertase Furin/SPC1 during embryonic turning and heart looping. Development 127: 245254.
  • Cooke J, Isaac A. 1998. A cascade of gene action controlling heart asymmetry and torsion in embryonic development. Trends Cardiovasc Med 8: 215220.
  • Davis CL. 1927. Development of the human heart from its first appearance to the stage found in embryos of twenty paired somites. Carnegie Contrib Embryol 19: 245284.
  • DeHaan RL. 1965. Morphogenesis of the vertebrate heart. In: DeHaanRL, UrsprungH, editors. Organogenesis. New York: Holt Rinehart & Winston. p 377419.
  • de la Cruz MV. 1998. Torsion and looping of the cardiac tube and primitive cardiac segments. Anatomical manifestations. In: de la CruzMV, MarkwaldRR, editors. Living morphogenesis of the heart. Boston: Birkhäuser. p 99119.
  • de la Cruz MV, Sanchez-Gomez C, Palomina MA. 1989. The primitive cardiac regions in the straight tube heart (stage 9-) and their anatomical expression in the mature heart: an experimental study in the chick embryo. J Anat 165: 121132.
  • de la Cruz MV, Sanchez-Gomez C. 1998. Straight tube heart. Primitive cardiac cavities vs. primitive cardiac segments. In: de la CruzMV, MarkwaldRR, editors. Living morphogenesis of the heart. Boston: Birkhäuser. p 8598.
  • Flynn ME, Pikalow AS, Kimmelman RS, Searls RL. 1991. The mechanism of cervical flexure formation in the chick. Anat Embryol 184: 411420.
  • Garcia-Peláez I, Arteaga M. 1993. Experimental study of the development of the truncus arteriosus of the chick embryo heart I. Time of appearance. Anat Rec 237: 378384.
  • Gilbert SF. 1994. Developmental biology. 4th ed. Sunderland, MA: Sinauer.
  • Goh KL, Yang JT, Hynes RO. 1997. Mesodermal defects and cranial neural crest apoptosis in α5 integrin-null embryos. Development 124: 43094319.
  • Gory-Fauré S, Prandini MH, Pointu H, Roullot V, Pignot-Paintrand I, Vernet M, Huber P. 1999. Role of vascular endothelial-cadherin in vascular morphogenesis. Development 126: 20932102.
  • Haller A, von. 1758. Sur la formation du coeur. (About the formation of the heart.) Lausanne: Bousquet MM.
  • Hamburger V, Hamilton HL. 1951. A series of normal stages in the development of the chick embryo. J Morphol 88: 4992.
  • Harvey RP. 1998a. Cardiac looping — an uneasy deal with laterality. Cell Dev Biol 9: 101108.
  • Harvey RP. 1998b. Links in the left/right axial pathway. Cell 94: 273276.
  • Harvey RP. 1999. Seeking a regulatory roadmap for heart morphogenesis. Cell Dev Biol 10: 99107.
  • His W. 1881. Mittheilungen zur Embryologie der Säugethiere und des Menschen. (Communications on the embryology of mammals and man.) Arch Anat Physiol 5: 303329.
  • His W. 1885. Anatomie menschlicher Embryonen. Teil III Zur Geschichte der Organe. (Anatomy of human embryos. Part III Development of the organs.) Leipzig: Vogel.
  • Icardo JM. 1996. Developmental biology of the vertebrate heart. J Exp Zool 275: 144161.
  • Itasaki N, Nakamura H, Sumida H, Yasuda M. 1991. Actin bundles on the right side in the caudal part of the heart tube play a role in dextro-looping in the embryonic chick heart. Anat Embryol 183: 2939.
  • Kirby ML, Waldo KL. 1995. Neural crest and cardiovascular patterning. Circ Res 77: 211215.
  • Kitamura K, Miura H, Miyagawa-Tomita S, Yanazawa M, Katoh-Fukui Y, Suzuki R, Ohuchi H, Suehiro A, Motegi Y, Nakahara Y, Kondo S, Yokoyama M. 1999. Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism. Development 126: 57495758.
  • Kosaki K, Casey B. 1998. Genetics of human left-right axis malformations. Cell Dev Biol 9: 8999.
  • Kuo HC, Chen J, Ruiz-Lozano P, Zou Y, Nemer M, Chien KR. 1999. Control of segmental expression of the cardiac-restricted ankyrin repeat protein gene by distinct regulatory pathways in murine cardiogenesis. Development 126: 42234234.
  • Langman J. 1981. Medical embryology, 4th ed. Baltimore: Williams & Wilkins.
  • Larsen W. 1993. Human embryology, 1st ed. New York: Churchill Livingstone.
  • Layton WM, Manasek FJ. 1980. Cardiac looping in early iv/iv mouse embryos. In: Van PraaghR, TakaoR, editors. Etiology and morphogenesis of congenital heart disease. New York: Futura. p 109126.
  • Lepori NG. 1967. Research on heart development in chick embryo under normal and experimental conditions. Monit Zool Ital 1: 159183.
  • Levin M. 1998. Left-right asymmetry and the chick embryo. Cell Dev Biol 9: 6776.
  • Lewis FT, Abbott ME. 1915. Reversed torsion of the human heart. Anat Rec 9: 103105.
  • Lewis FT, Abbott ME. 1916. Reversed torsion of the ventricular bend of the embryonic heart in the explanation of certain forms of cardiac anomaly. Bull Int Assoc Med Mus 6: 111.
  • Lindes G. 1865. Ein Beitrag zur Entwicklungsgeschichte des Herzens. (A study on the development of the heart.) Medical Dissertation Univ Dorpat.
  • Lu MF, Pressman C, Dyer R, Johnson RL, Martin JF. 1999. Function of Rieger syndrome gene in left-right asymmetry and craniofacial development. Nature 401: 276278.
  • Malpighi M. 1672. De ovo incubato. (About the incubated egg.) London: Martyn.
  • Manasek FJ. 1976. Heart development: interactions involved in cardiac morphogenesis. In: PosteG, NicolsonGL, editors. The cell surface in animal embryogenesis and development. Amsterdam: North Holland. p 545598.
  • Manasek FJ, Monroe RG. 1972. Early cardiac morphogenesis is independent of function. Dev Biol 27: 584588.
  • Manasek FJ, Kulikowski RR, Nakamura A, Nguyenphuc Q, Lacktis JW. 1984. Early heart development: a new model of cardiac morphogenesis. In: ZakR, editor. Growth of the heart in health and disease. New York: Raven Press. p 105130.
  • Männer J, Seidl W, Steding G. 1993. Correlation between the embryonic head flexures and cardiac development. An experimental study in chick embryos. Anat Embryol 188: 269285.
  • Männer J, Seidl W, Steding G. 1995. Formation of the cervical flexure: an experimental study on chick embryos. Acta Anat 152: 110.
  • Männer J, Seidl W, Steding G. 1996. Some notes on the description of cardiac looping. Verh Anat Ges 91 (Ann Anat Suppl 178): 9798.
  • Manning A, McLachlan JC. 1990. Looping of chick embryo hearts in vitro. J Anat 168: 257263.
  • Markwald RR. 1995. Overview: formation and early morphogenesis of the primary heart tube. In: ClarkEB, MarkwaldRR, TakaoA, editors. Developmental mechanisms of heart disease. New York: Futura. p 149155.
  • Markwald RR, Trusk T, Moreno-Rodriguez R. 1998. Formation and septation of the tubular heart: integrating the dynamics of morphology with emerging molecular concepts. In: de la CruzMV, MarkwaldRR, editors. Living morphogenesis of the heart. Boston: Birkhäuser. p 4384.
  • Moorman AFM, Lamers WH. 1994. Molecular anatomy of the developing heart. Trends Cardiovasc Med 4: 257264.
  • Okamoto N. 1980. Congenital anomalies of the heart. Embryologic, morphologic and experimental teratology. New York: Igaku-Shoin.
  • Olson EN, Srivastava D. 1996. Molecular pathways controlling heart development. Science 272: 671676.
  • Pander HC. 1817. Beiträge zur Entwickelungsgeschichte des Hühnchens im Eye. (Studies on the development of the chick in the egg.) Würzburg: Brönner.
  • Patten BM. 1922. The formation of the cardiac loop in the chick. Am J Anat 30: 373397.
  • Renehan WE, Kulikowski RR. 1981. Abnormal development in Valium treated embryos. Biophys J 33: 244a.
  • Rosenquist GC. 1985. Migration of precardiac cells from their origin in epiblast until they form the definitive heart in the chick embryo. In: FerransVJ, RosenquistGC, WeinsteinC, editors. Cardiac morphogenesis. Proceeding of the meeting “Selected Topics in Cardiac Morphogenesis,” December 1984. New York: Elsevier. p 4454.
  • Roux W. 1895. Gesammelte Abhandlungen über Entwicklungmechanik der Organismen. (Essays on the mechanics of development of organisms.) 2nd ed. Leipzig: Engelmann.
  • Rychter Z, Lemez I. 1959. Experimenteller Beitrag zur Entstehung der Transposition von Aorta in die rechte Herzkammer der Hühnerembryonen. (Experimental study on the development of transposition of the aorta to the right ventricle.) Verh Anat Ges 55: 310315.
  • Schulte HvW. 1916. The fusion of the cardiac anlages and the formation of the cardiac loop in the cat (Felis domestica). Am J Anat 20: 4572.
    Direct Link:
  • Sissman NJ. 1966. Cell multiplication rates during development of the primitive cardiac tube in the chick embryo. Nature 210: 504507.
  • Spitzer A. 1851. The architecture of normal and malformed hearts. Springfield, IL: Charles C. Thomas.
  • Srivastava D. 1999. HAND proteins: molecular mediators of cardiac development and congenital heart disease. Trends Cardiovasc Med 9: 1118.
  • Stalsberg H. 1969. Regional mitotic activity in the precardiac mesoderm and differentiating heart tube in the chick embryo. Dev Biol 20: 1845.
  • Stalsberg H. 1970. Mechanism of dextral looping of the embryonic heart. Am J Cardiol 25: 265271.
  • Steding G, Seidl W. 1980. Contribution to the development of the heart. Part I: normal development. Thorac Cardiovasc Surg 28: 386409.
  • Steding G, Seidl W. 1984a. Cardiac septation in normal development. In: NoraJJ, TakaoR, editors. Congenital heart disease: causes and processes. New York: Futura. p 481499.
  • Steding G, Seidl W. 1984b. Pathogenesis of congenital heart disease. In: NoraJJ, TakaoR, editors. Congenital heart disease: causes and processes. New York: Futura. p 501512.
  • Taber LA. 1998. Mechanical aspects of cardiac development. Prog Biophys Mol Biol 69: 237255.
  • Thomas T, Yamagishi H, Overbeek PA, Olson EN, Srivastava D. 1998. The bHLH factors, dHAND and eHAND, specify pulmonary and systemic cardiac ventricles independent of left-right sidedness. Dev Biol 196: 228236.
  • Tomita H, Connuck DM, Leatherbury L, Kirby ML. 1991. Relation of early hemodynamic changes to final cardiac phenotype and survival after neural crest ablation in chick embryos. Circulation 84: 12891295.
  • Tsuda T, Philp N, Zile MH, Linask KK. 1996. Left-right asymmetric localization of flectin in the extracellular matrix during heart looping. Dev Biol 173: 3950.
  • Tsuda T, Majumder K, Linask KK. 1998. Differential expression of flectin in the extracellular matrix and left-right asymmetry in mouse embryonic heart during looping stages. Dev Genet 23: 203214.
  • Van Mierop LHS, Alley RD, Kausel HW, Stranahan A. 1963. Pathogenesis of transposition complexes I. Embryology of the ventricles and great arteries. Am J Cardiol 12: 216225.
  • Van Praagh R. 1972. The segmental approach to diagnosis in congenital heart disease. Birth Defects 8: 423.
  • Van Praagh R, DeHaan RL. 1967. Morphogenesis of the heart: mechanism of curvature. Annu Rep Dept Embryol Carnegie Inst 65: 536537.
  • Van Praagh S, O'Sullivan J, Brili S, Van Praagh R. 1996. Juxtaposition of the morphologically right atrial appendage in situs solitus and inversus atria: a study of 35 postmortem cases. Am Heart J 132: 382390.
  • Waddington CH. 1937. The dependence of head curvature on the development of the heart in the chick embryo. J Exp Biol 14: 229231.
  • Wolff CF. 1764. Theorie von der Generation. (Theory of development.) Berlin: Birnstiel.
  • Yasui H, Morishima M, Nakazawa M, Aikawa E. 1998. Anomalous looping, atrioventricular cushion dysplasia, and unilateral ventricular hypoplasia in the mouse embryos with right isomerism induced by retinoic acid. Anat Rec 250: 210219.
  • Yost HJ. 1998. Left-right development from embryos to brain. Dev Genet 23: 159163.