Embryonic heart morphogenesis

Embryonic heart morphogenesis

Literature review

Embryonic heart morphogenesis is a complex and dynamic process that is incompletely understood, but critically important for long-term survival.The heart begins as a linear tube of myocardium lined with endocardial cells that functions like a suction pump (Forouhar et al., 2006), but quickly loops and grows into a four chambered heart complete with valves and septa that pumps in a piston manner. This process involves several key events that happen in three dimensions, including linear heart tube looping, septation of the ventricles, and development of the atrioventricular (AV) valve apparatus, defects in which result in a myriad of clinically relevant congenital heart defects (Collins-Nakai and McLaughlin, 2002; Gittenberger-de Groot et al., 2005). Although the initial heart tube only contains portions of the primitive right ventricle (de la Cruz et al., 2001), eventually all of the chambers are derived from the growing linear heart tube, with smaller contributions from sources such as the anterior heart field and proepicardium. During cardiogenesis, the sizes of these chambers change significantly, but few studies have attempted to quantify them. Two studies by Wenink's group in the 1990s quantified chamber-specific myocardial tissue volume changes over development using a point counting method and found that over two orders of magnitude of heart growth occurs in the embryonic rat and human (Wenink, 1992; Knaapen et al., 1995). It is unclear, however, how the lumen volumes of the different segment/chambers change during development. Studies by Keller et al. have attempted to quantify ventricular volumes at different stages of development and showed that these increase in size as the embryo grows, but how these volumes relate to those of the atria and outflow tract were not assessed (Keller et al., 1996). Many details of cardiac morphogenesis are only now being uncovered in part because of the complexities of the developing geometries. The human heart becomes four chambered by week 8, which is approximately the same time that the embryo can be visualized through ultrasound and, therefore, too late for detailed morphogenic study (Zimmer et al., 1994; Fong et al., 2004). Therefore, embryonic animal models, particularly chick and mouse, have been essential for these investigations. One particular challenge, however, is the extremely small size of these hearts and the rapidity in which they develop. The pumping linear heart tube appears at Hamburger and Hamilton stage (HH) 12 in the chick (approximately?hr of incubation) and generally resembles its mature configuration by HH36 (day 10), or E8.5 to E16.5 in the mouse. Additionally, these hearts are microscopic throughout the developmental window, forcing researchers to use alternate means to visualize these events. Typically, en face microscopic images, scanning electron microscopy (SEM), optical scans, and have been used to describe aspects of morphogenesis, but each has its drawbacks. Photographic images, even with scales, do not have the capacity to measure depth, limiting their utility for three-dimensional (3D) quantification. The 3D serial reconstruction using histological sections has dramatically improved understanding of heart development with the ability to combine geometry and the expressions of cell and/or matrix proteins (Moorman et al., 2000; Groenendijk et al., 2005). However, accuracy of this histological sections and the number of sections processed, its time consuming, but gives our work the possibility acuuratly transform this sections into a 3Dcomputer model.

Optical scanning techniques have also been used to render 3D and 4D volumes of early heart geometries (Liebling et al., 2005; Jenkins et al., 2006; T. Mesud Yelbuz et al.,2002 ), but penetration depth limitations with this technique require imaging early stages and/or using optically clear specimens or preparations. While the exterior walls of the heart are generally smooth with large radii of curvature, the interior “lumens” of the heart, with varying trabecular, septal, and valvular geometries, are far more complex. It is through this lumen that hemodynamic forces (wall shear stress, blood pressure) interact with developing tissue surfaces. Few studies to date have attempted to profile the changing geometry of the interior of the heart. Several studies have used different imaging modalities to explore developing hearts in 3D to identify morphogenic defects (Smith, 2001; Weninger and Mohun, 2002; Schneider et al., 2003; Soufan et al., 2004), but none of these studies focused on quantifying the 3D geometry of the different segments and chambers or as whole heart. Quantification of changing heart chamber volumes would provide insight into the changing function of the early heart, but different imaging technologies may be required to accomplish this in these microscopic tissues.

One technique t hat has been used over the past 15 years to quantify complex geometries at small resolutions is micro-computed tomography (Micro-CT). Micro-CT works by scanning a subject with high-powered Xrays and rendering the dense regions in a volume. The scan head is rotated 360 degrees around the subject, developing a near continuous series of planar slices with no registration defects. Voxel sizes less than 10 _m are readily achievable with Micro-CT (Guldberg et al., 2003), which is superior to those currently achievable with ultrasound (30 _m) and magnetic esonance imaging (100 _m). Micro-CT has mainly been used for visualizing and quantifying bone architecture and development (Guldberg et al., 2004), and several studies have been conducted using this technique to quantify trabecular structures (Chappard et al., 2006). More recent studies using radio-opaque contrast agents have enabled the visualization of microvascular structures, such as those associated , th with bone fracture healing (Duvall et al., 2004) and coronary artery vasa vasorum (Gossl et al., 2004). Micro-CT could be used to visualize and quantify the changing cardiac lumen during embryonic development , and to compare the visualization of the heart lumen using Micro-CT with serial section reconstruction and scanning electron microscopy, measure the volume of each developing segment/ chamber from HH15 through HH36, and determine the 3D structure of the developing atrioventricular valves. Unfortunatly this technique would not provide us with information of full volumetrick measurements of Volume and ratio of chambers of developing chicken heart .( Jonathan T. Butcher et.al.,2007). Another method like video light microscopy has been used to acquire real-time images of the developing beating heart by digitizing the expanding and contracting blood pool in early, transparent hearts. This has been effective for obtaining angiographic-type estimates of ventricular function. However, it is limited to en face views, and, at later, nontransparent stages, only surface morphology is visible (Shottan, D. M. (1988)). Therefore, investigators are confined to using embryos that are transparent during early stages, exposing the heart via surgical intervention or killing the animal at a predetermined stage. High resolution magnetic resonance imaging has been used to produce in vivo cross-sectional images of early development with resolutions as high as 12 mm.( Jacobs, R. E. & Fraser, S. E. 1994) However, the long acquisition times are prohibitive for the in vivo analysis of cardiac dynamics, and structural delineation is plagued by motion artifacts. Furthermore, the technology is expensive and impractical for general use.

High frequency (40-100 MHz) ultrasound backscatter microscopy is capable of 50-mm resolutions to depths of 4-5 mm and has been applied to the analysis of early embryonic development in the mouse.( Turnbull, (1995) Proc. However, imaging requires a transducing medium or direct contact.Also, M-mode echocardiography is problematic in developmental animal models with high heart rates because of the relaxation time of the ultrasound transducer, which limits the rate at which data can be acquired.In some recent works the use of three-dimensional reconstructions of myocardial probes to visualize the atrial components (Alexandre T. Soufan 2006) or quantitative 3D reconstructions of proliferation in the forming heart tube and the mesoderm with its flanking coelomic walls (Gert van den Berg 2009) are only limited to give us partial information(not the look of atria and ventricles) of how chicken heart, during embryonic development, is represented in 3D computer remodeling.

These approaches facilitates understanding of architecture of embryonic heart and gives us the ability to estimate the quantitative amount of a wide spectrum of geometrical parameters of chambers and structure of the wall of the heart. They also serve as new tools for scientific investigation of carcinogenesis and congenital heart disease. But such methods do not yet provide anything like resolution achieved by histology and are therefore of limited use for studies of morphological detail.

In our research, we would like to analyze and compare the heart at diastole and systole. There are several features that can be analyzed: such as intercalated discs, which are connected between two adjacent cells. Sarcomere lengths at the midwall of the left ventricle averaged more at diastole and less at systole. These are changes that are adequate to explain the degree of normal ventricular emptying. They characterize myofiber cross-section, length of cardiomyocytes and papillary muscles and this is our work of the study and understanding of architecture of embryonic heart. Data from imaging will become particularly useful when they can be quantified. Three-dimensional images of chicken embryos in different stages of development must be converted into statistical descriptions with three-dimensional averages and three-dimensional standard deviations. Identification and communication of altered growth and form will become less subjective and more meaningful with statistical data. All this can be used as additional information to estimate the quantitative amount of a wide spectrum of geometrical parameters of chambers and structure of the wall of the heart during cardiac cycle. The present methods are potentially applicable to the study of experimental cardiac disease, and they should permit comparisons between the geometrical and ultrastructural characteristics and the functional states of normal and abnormal hearts.

Recent advances in echocardiography, in particular, the emergence of 3D echocardiography, have encouraged studies of the shape and geometry of non-visualized cardiac structures, such as the annuli. The 3D shape and geometry of the bicaspid valve in the normal population and in cardiac patients had been revealed in detail with 3D echocardiography by several previous studies. Even before the emergence of the 3D echocardiography, the 3D

shape and the function of the bicaspid during cardiac cycle had been explored and uncovered with other 3D imaging techniques. Annular geometry changes during systole. It is generally accepted that the annular size increases

during the latter half of systole after presystolic narrowing of the annulus, and then continues to increase in size from early diastole and reaches a maximum during late diastole. During the process of 3D image acquisition and 3D reconstruction, several limitations were evident. to obtain a 3D reconstruction of a non-visualized cardiac structure, the anatomical reference markers should be defined prior to reconstruction in order to obtain the correct anatomical orientation. Unfortunately this work can only give us a 3D reconstructed images of bicuspid and tricuspid valves but not the relationship to the cavities during the cardiac cycle. Newly developed techniques like laser dissection microscopy in combination with quantitative PCR or mass spectrometry can be used to obtain these estimates. Because of its small size and intricate morphology, it is very laborious to apply these techniques to the developing heart. High-throughput techniques such as serial analysis of gene expression (SAGE) and microarrays provide genetic expression data, but these are devoid of spatial information. The use of staining techniques for mRNA and protein, in situ hybridization (ISH) and immunohistochemistry, respectively, enable the localization of specific mRNA and proteins in tissues with cellular resolution. Combining these staining methods with radioactive probes and autoradiography permits the calibration and subsequent quantification of the staining intensity (Jonker A 1997, Ruijter JM 2001). For organs that are reasonably “amorphous” and composed of isotropic tissue, such as the liver, a limited number of sections can provide a quantitative estimation of gene expression gradients (J. M. Ruijter, J. Hagoort, M. M. Markman, R. G. Gieling, and W. H. Lamers, unpublished observations). However, for organs such as the developing heart, a few random sections would not suffice; the spatial distribution of the specific gene product throughout the whole organ must be mapped. Three-dimensional (3D) reconstructions of embryonic development were originally based on computeraided manual tracing of the organs of interest (Huijsmans DP 1986, Verbeek FJ 1995), and the reconstructed organs were illustrated by medical artists. With the advent of digital cameras, reconstruction methods based on digitized images have become commonplace. A full review of past reconstruction methods would do no justice to those researchers who had to implement their reconstruction protocols with the hardware and software available at that time. Recently, methods were published based on episcopic image capturing (Ewald AJ 2002, WeningerWJ 2002). These episcopic methods, which acquire an image just before sectioning, enable one to accurately obtain aligned high-resolution digital stacks, based on fluorescence.

In a work by (Alexandre T. Soufan,2003)The myocardium and the cardiac lumen of a developmental series of mouse embryos was mapped and reconstructed using their protocol. The myocardium of the heart is defined as the structure that stains with a mix of myocardium-specific mRNA probes using the nonradioactive ISH method. The appearance of the reconstructions shows that hearts from the same embryonic day are virtually identical. Two or three reconstructions of the same embryonic day will probably suffice to obtain a representative reconstruction of that developmental stage. The morphology of the reconstructions closely resembles that seen in whole mount-stained hearts The volume data extracted from the duplicate hearts differ by a maximum of 10%. This “preliminary” biological result indicates that the biological variation may not pose a problem for generalized use of this series. Note that these volumes have been obtained using the Cavalieri principle and are therefore unbiased estimates of the myocardium volumes (Howard CV 1998). Therefore, these volumetric data of the developing heart structures can be used in mathematical and functional models of heart development. One can calculate from these data that the myocardium volume increases 100 times in 6 days (between ED 8.5 and 14.5). Assuming that all cells present in the heart do divide in that period, this volume increase would correspond to 6.6 cell divisions, which is at least one division of each cell every 24 h. However, from literature (Cluzeaut F 1986, Thompson RP 1990) and data obtained from quantitative reconstructions (Soufan AT 2001), we know that myocardial cells differ in cell-cycle duration within hearts and between hearts of different stages. The increase in volume may not only be explained by mitoses, but other mechanisms, such as cellular growth, cell migration, and transdifferentiation (recruitment) have to be taken into account as well. Therefore, an accurate description of the developing heart in dynamics during all stages of development will benefit from data obtained by quantitative and volumetric computer 3D reconstructions.

The past application of the principles of muscle mechanics to the analysis of left ventricular contraction (FRY, D. L., GHIGGS. 1964, Ross, J., JR., COVELL,1966, LEVINE, H. J.,1964) has focused at- tention upon the need for detailed information concerning the anatomy of ventri cular contraction. At present, many gaps exist in our knowledge of the changes in configuration of the muscular walls, the orientation of muscle fibers and the dimensions of the sarcomeres that occur continuously in the ventricle during the cardiac cycle. Such information is essential to the construction of appropriate geometric models for application in echanical analyses of ventricular contraction and also should provide basic insight into correlations between cardiac structure and function in the normal and the abnormal heart. Studies of the external and internal dimensions of the in situ left ventricle by dimension gauges and angiography have provided much important information concerning changes in the shape and volume of the left ventricle during the cardiac cycle, and studies in the isolated papillary muscle and excised left ventricle have investigated relations between sarcomere dimensions, muscle length, and the volume of the passive heart (SPOTNTTZ, H. M.,1966, HORT, W.1960). So far, however, there has been no correlative examination of gross anatomy and ltrastructure in the contracting left ventricle under known hemodynamic conditions. In the work there were a methods described for rapid fixation in systole or diastole have permitted an analysis of the geometry of the ventricular cavity and wall under known hemodynamic conditions. Information gained by the use of these direct measurements and from other, dynamic techniques, should allow the development of appropriate geometrical models for application in analyses of the mechanical properties of ventricular contraction. Changes in ventricular shape, volume, and wall thickness require a continuously variable model which, when correlated with pressure and flow determinations, should eventually permit precise

calculation of stress distribution and fiber shortening throughout the cardiac cycle. Further studies will be undertaken on these ventricles to study the important problem of changes in muscle fiber orientation and

distribution that occur across the ventricular wall during diastole and systole. But fully to be able to analyze geometry the beneficial would be to reconstruct in 3D and to compaire it during all stages not only post natal but prenatal development ,which was difficult to do at that time.

Since the morphogenetic changes of the heart occur three-dimensionally, it is essential to visualize and analyze

heart development in three dimensions. Three-dimensional visualization is also a powerful tool in embryological study and greatly helps us to understand the dynamic morphogenetic movements in the embryo (Yamada et al., 2006). From the early days of human embryology, attempts were made to visualize embryonic structure in three dimensions. Traditionally, 3D reconstruction of embryonic structures used to be made from serial histological sections of embryos, often with the wax plate technique (Born, 1883). However, such reconstruction and drawing methods require an enormous time and special skills and which very volublein a research. Recent advancement in computer science has made computer-assisted reconstruction of biological structures more effectively. Various 3D structures have been reconstructed by this method, and the reconstructed images can be manipulated as desired on the viewing screen. In the area of the developmental study of the heart and great vessels, computer-assisted reconstruction and computer graphics (CG) have been used to visualize the developing heart and vessels of the mouse (Smith, 2001; Schneider et al., 2003), chick (Hiruma and Hirakow, 1995), and human (DeGroff et al., 2003; Abdulla et al., 2004). In mice, the 3D sequential images of the developing heart have been made between E8.5 and E14.5 (Soufan et al., 2003).

In the present study,there were attampes made in reconstruction of the heart and great vessels of staged human embryos with the aid of computer software and compared their luminal structures of embryos. They showed that computer-assisted reconstruction is a useful and powerful tool for analyzing detailed 3D phenotypes in embryos. During embryonic development, dynamic morphogenetic changes occur in a spatially and temporally coordinated

manner. The cardiovascular system is one of the organ systems that undergo drastic morphogenetic movements. The sequential changes of the heart and great vessels in human embryos used to be examined by observing histological sections and wax plate models reconstructed from serial sections (Congdon, 1922; Streeter, 1948), which contributed significantly to human embryology and have been well cited by subsequent embryologists (Cooper and O'Rahilly, 1971; O'Rahilly, 1971). In there study using computer-assisted reconstruction of the heart and great vessels of externally normal embryos largely confirmed the results of those classical reports, although some discrepancies were noted.

In the work (SHIGEHITO YAMADA, 2007) reconstruction of the luminal structure of the hearts and great vessels of staged human embryos from serial histological sections to demonstrate their sequential morphological changes in three dimensions. Anatomical structures were analyzed with a use of 3D images.Not much of information was giving to luminal structure and their volumetric and morphological changes during heart development.

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