UvA-DARE (Digital Academic Repository)
Growth of the developing heart
van den Berg, G.
Publication date
2011
Link to publication
Citation for published version (APA):
van den Berg, G. (2011). Growth of the developing heart.
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Scope
Of all life-births, 0.6% is accompanied by moderate to severe congenital heart malformations; 2% if potentially serious cases of bicuspid aortic valves are included. These high incidences still exclude congenital arrhythmias, “trivial” defects (such as self-limiting septal defects), and cardiomyopathies that present later in life [1]. Of this high number of children born with congenital heart disease (CHD), many require expert care and, not infrequently, multiple operations. They experience a life-time hindrance of normal-day activities, and normal personal development is impaired. It is easy to imagine that CHD has a major impact on both patient (the child) and his or her family.
Congenital heart disease originates from errors that occur during embryonic development. Therefore, knowledge of the mechanisms of cardiac development is of great importance for understanding the basis of congenital heart disease. Such understanding is a firm prerequisite for development of eventual treatments of CHD. Even more, the mechanisms of cardiac development are also of interest for adult cardiac disease [2]. Given the inability of adult myocardium to sufficiently divide and thus regenerate new muscle after damage, many researchers are studying the mechanisms of embryonic growth to translate these findings to new regenerative strategies.
The heart is the first organ to become functional during embryonic development. At 3 weeks in human, 7 days in mouse, and 2 days in chicken development, sheets of mesodermal cells differentiate into cardiac muscle. By folding and fusion of the mesoderm a tube-like structure is formed, which starts to pump soon after its formation. This pumping occurs in a peristaltic fashion and is always initialized at the venous inlet of the heart [3,4]. As the vascular system develops in connection with the heart, a unidirectional flow is established, by which the forming blood cells from the blood islands that cover the yolk sac are distributed throughout the embryo. This vascular transport system supplies the embryo with nutrients, which it needs to grow.
The heart starts as a simple sluggishly pumping tube, composed of primary myocardium which is slow conducting and poorly contractile. As the heart grows and lengthens, the ventral aspect of the straight heart tube rotates towards the right and the heart loops [5]. At the outer curvatures of the looped heart, primary myocardium will locally differentiate into the “working” myocardium of the cardiac chambers (Figure). This working myocardium upgrades its contractile apparatus [6], allowing for stronger contraction. The conduction velocity is also increased by local expression of fast-conducting gap-junctions (review: [7]). Remaining poorly differentiated, or “primary”, myocardium is located as zones flanking the forming chambers. These regions of primary myocardium are connected via the inner curvatures of the heart, and are located at the atrioventricular canal, the atrial floor, and the outflow tract. They remain slow conducting and function as sphincter-like valves [8].
As the heart develops further, septation occurs to serve the forming parallel circuits of the pulmonary and systemic circulation. This division occurs by a combination of muscular septation, and division of non-muscular cushion tissue in the atrioventricular canal and the outflow tract. In the end, a four chambered heart is generated with a concordant attachment of the respective left and right atria and ventricles, so enabling the function of the separate systemic and pulmonary
circulatory circuits. Remaining primary myocardium will differentiate in the sinu-atrial and atrioventicular nodes, whereas fast conducting purkinje fibers form along the ventricular septum and trabeculae [9].
The last decades, insight into the molecular mechanisms of normal and abnormal development of the heart has increased exponentially [10,11]. An example of such a recent insight is the finding that certain T-box transcription factors locally inhibit chamber differentiation, thereby retaining primary myocardium in a poorly differentiated state and even forcing it to develop into nodal tissue (review: [9,12]). Another example is the significant shift in paradigm caused by molecular evidence that addition of precursor cells to the myocardial lineage is a mayor parameter of cardiac growth [13-18].
The morphogenesis of the heart is complex and occurs rapid. Therefore, molecular mechanisms that control heart development can only be fully exploited if also placed within this rapidly changing three-dimensional context. Furthermore, absence of a clear spatiotemporal image of cardiac morphogenesis, potentially leading to discussions based on differences in interpretation, rather than observation. Complicating the formation of such an image is that communication of the intricate morphogenesis of the heart mostly relied on the presentation of single sections or on schematic illustrations. Also, prior to the work presented in this thesis, basic knowledge on local proliferation during cardiac development, an important parameter of growth, was scarce and conflicting. For instance, both rapid [19] and slow [20] proliferation was concluded to be the mode of growth in the early heart.
Focus of this thesis
This thesis attempts to clarify the growth of early embryonic heart by using three-dimensional (3D) reconstructions to communicate intricate morphological changes during cardiac development. Furthermore, to gain insight into local cardiac proliferation new methods of combining 3D reconstructions with local information on proliferation rates were developed, and applied to chicken and mouse development. In chapter 1, we review classic morphological studies and combine these, now often forgotten, findings with recent data on cardiac development. This review shows that current concepts of cardiac development are not new, and could have been deduced from classic literature. In chapter 2 a review is given of the proliferation, differentiation and lineage separation of the cell types of the developing heart.
Chapter 3 describes the development of the techniques used for the quantification
and visualization of proliferation rates as they are presented in this thesis. The method uses a 3D matrix to determine local BrdU-labelled nuclear fractions (an established measure of proliferation rate). Such fractions are then mapped onto morphological reconstructions, resulting in a quantitative 3D reconstruction of proliferation rate. In
chapter 4 the methods described in the previous chapter are expanded enabling the
calculation and visualization of actual cell-cycle times. This was accomplished by the use of two different halogenated thymidine analogues (IdU and CldU) with different exposure times. Per location in the embryonic heart, the two different labeling indices can then be converted into a local cell-cycle length. Chapter 5 clarifies, using 3D reconstructions, the complex morphology of the normal formation of the venous
Schematic illustration of formation of the four chambered heart by local differentiation of the early heart tube. Straight tube: oft – outflow tract, v – primary ventricle. Looping tube: oft – outflow tract, rv – right ventricle, lv – left ventricle, ra – right atrium, la – left atrium. Septating heart: ra – right atrium, la – left atrium, av-canal – atrioventricular canal, lv – left ventricle, rv – right ventricle. 4-chambered heart: vcs – vena cava superior, vci – vena cava inferior, san – sinu-atrial node, ra – right atrium, la – left atrium, avn – atrioventricular node, rv – right ventricle, lv – left ventricle, his/purkinje – his bundle / purkinje fibers.
pole of the heart, leading to insights into the highly variable spectrum of abnormal pulmonary venous return. In chapter 6 we apply quantitative 3D reconstructions to a series of chicken embryos and show that the early tubular heart forms its chambers by a local increase in proliferation, which is preceded by a local increase in myocardial cell volume. In chapter 7 we proceed to calculate the proliferation rate of the forming ventricle in chicken, and show that this is a very rapidly proliferating structure with a doubling time of 8.5 hours. In contrast, we show that the early straight heart tube does not proliferate (having a doubling time of 5.5 days), but nevertheless grows rapidly. We then show that the precursors of this early heart originate from a single pool of cells, and that these precursors are added to both the inflow and the outflow of the developing heart. When proliferation is locally inhibited in the proliferative growth zone, cardiac defects occur at both the inflow and the outflow pole of the heart. In chapter 8 we present a comprehensive overview of the growth of the early mouse heart. We clarify the difficult morphology of the mouse heart and show that, unlike chicken development, many development processes occur simultaneously. Nevertheless, proliferation is shown to drop upon initial myocardium formation, and to increase with differentiation into chamber myocardium. Further quantification of general and trabecular cardiac growth, along with an overview of the patterns of proliferation almost spans the entire gestational period of mouse development. Taken together, the work presented in this thesis adds to an understanding of cardiac growth and forms a comprehensive spatiotemporal description of the development of the heart. A mini-disc containing interactive versions of most of the 3D reconstructions presented in the above mentioned chapters is added to this thesis. The reader is encouraged to use these interactive reconstructions to form a comprehensive spatial image of cardiac morphogenesis.
Reference List
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