The heart has fascinated scientists for centuries, from the days of Leonardo da Vinci to modern scientists. Using these three perspectives, we can quantify the motion of the myocardium and endocardium during development while the heart is beating.
Introduction
Introduction
Already in the 15th century, detailed studies of the structure and function of the heart began. The cells that will form the heart begin as the stem cardiac mesoderm on opposite sides of the primitive streak in blastula formation.
Previous Research in Zebrafish
With the advent of new microscopy techniques and transgenic fish, dynamic imaging of the zebrafish heart became possible. By creating a computer model of the zebrafish beating heart, Forouhar and colleagues were able to monitor the blood cells, the contractile wave and the pressure gradient and find that the heart tube behaves more like an impedance pump (Forouhar et al., 2006).
Quail as a Model Organism
Egg incubation time is 21 days, and the chick reaches sexual maturity in four to five months (Grandin and Johnson, 2005). The incubation period of an egg is 16 days, and the quail chick reaches sexual maturity in approximately six weeks (Woodward et al., 1973), resulting in short production.
Quail Heart Morphogenesis
A number of researchers have proposed positional fate maps of heart-forming regions by observing the location of cardiac precursor cells in progressive developmental stages (Dehaan, 1963; Garcia-Martinez & Schoenwolf, 1993; Rawles, 1943; Stalsberg & DeHaan, 1969). These studies have generated a wide range of hypotheses about the spatial and temporal boundaries of the amniote heart-forming region.
Research Goals
Through this research we discover that many of the tools needed to analyze the data have not yet been developed. This study shows the very first images of the early stages of a developing amniotic tube at cellular resolution.
Cardiac looping in the chick embryo: A morphological review with special reference to terminological and biomechanical aspects of the looping process. The role of mechanical forces in dextral rotation during cardiac looping in the chick embryo.
Measuring Contraction and Relaxation within the Developing Amniote
Summary
Introduction
It was then taken in five regions along the length of the heart tube: presumptive atria (A), atria-ventricular border (AV), lower ventricle (LV), upper ventricle (UV), and conus (C). This pattern is observed for the entire length of the heart tube at stages HH10-12.
Results & Discussion
The thickness of the cardiac jelly, defined as the space between the myocardium and the endocardium, was also plotted (see Figure 3). Once the heart tube diameters and heart jelly thickness were known, the fraction of change in diameter and thickness during contraction was calculated (see Materials and Methods and Figure 4). The endocardial inner tube shows a fraction of change compared to the myocardial outer tube, reflecting the nature of the heart tube and the viscoelastic properties of the cardiac jelly.
As the strength of the contraction increases, the amount of time it takes in the heartbeat decreases, allowing the heart to maintain a resting state for longer periods of time.
Materials and Methods
For images where the myocardium could not be imaged using laser light, transmitted light was used to view the myocardium. To analyze the data from each image, Matlab was used to plot both the motion of both the endocardium and the myocardium (see Figure 7B). Diameter of the myocardium = D – A at diastole (red line) Diameter of the endocardium = C – B at diastole (green line).
To measure the contraction, the rates of contractile force and relaxation intensity were calculated by finding the slope of the line (dy/dx) for the myocardial and endocardial layers as observed in Figure 9.
Sato, Y., Poynter, G., Huss, D., Filla, M., Czirok, A., Rongish, B., Little, C., Fraser, S., and Lansford, R., Dynamic analysis of vascular morphogenesis using transgenic quail embryos.
Four-Dimensional Reconstruction of the Quail Heart to Elucidate Pump
Summary
Recent technological advances in dynamic confocal microscopy and four-dimensional reconstruction have shown that the embryonic heart tube in developing zebrafish functions more like a dynamic suction pump than a peristaltic pump, contrary to previous assumptions. All stages of the developing heart between looping and ventricular formation are accessible in ovo and ex ovo, allowing easy manipulation of the embryo for dynamic imaging. Our results using Tie1 transgenic quail embryos show that rapid imaging and four-dimensional reconstruction of the avian heart reveal detailed motions of cardiac contractions in birds that have never been seen before.
From these data, we were able to track individual cell movement in different regions of the heart tube during contraction, which demonstrated the mechanistic purpose of the cardiac jelly as a buffer to remove asynchrony of myocardial cells to create a smooth contraction in the endocardial lining.
Introduction
Loop formation begins when the myofibrils first appear (Manasek et al., 1978), and the dorsal mesocardium is always located at the inner curvature of the loop (Butler, 1952). Over the past decade, much detailed work has been done to model the looping aspects of the early embryonic heart tube. The study and modeling of the looping aspects of cardiac looping is a recent development in the field of cardiac development.
Recent research has been able to correct this problem in cardiac research by creating computer algorithms that can reconstruct a dynamic image of the beating heart in zebrafish (Liebling et al., 2006).
Results & Discussion
At the most caudal end of the heart tube, myocardial cells move in a circular pattern in a clockwise motion. At the cranial end of the heart tube, circular patterns are observed that move in a counterclockwise rotation. Myocardial and endocardial cells in the cross-sectional areas of the heart tube were manually traced during the heartbeat cycle.
Focusing on the myocardial cells in the upper region, the movement of four cells contracts as a group, with some cells lagging behind.
Materials and Methods
Rapid three-dimensional imaging and analysis of the beating embryonic heart reveals functional changes during development. On twisting, turning, lateralization and delivery of the embryonic heart loop: new insights from a simulation model for the heart loop of avian embryos.
Figures
The middle panel shows one frame of the collected data with the two layers of the heart outlined: red is the myocardium, green is the endocardium. The right panel shows a zoomed-in view of the tracks created by Imaris, with the data removed to give a clearer view. Endocardial pathways run approximately parallel to each other, while myocardial pathways become angled to each other along the length of the tube, indicating that a small amount of torsional twisting occurs.
The two layers of the heart are outlined - red is the myocardium, green is the endocardium.
Visualization of Heart Tube Formation and Looping with Cellular
Summary
We found that the left and right regions of the tail portion of the heart tube do not intercalate and move through tissue mechanics. The upper portions of the heart tube are formed by cells already present in the primary heart field region at HH8, and form the heart tube by meeting at the midline. Cells migrating from the sinoatrial regions to the caudal portion of the heart tube complete the elongation of the tube.
The myocardial cells then fuse around the outside of the endocardial cells to form a tube within a tube.
Introduction
The exact process of how the paired primary cardiac fields fuse during heart tube formation and the origin of these cells remains unknown. Dextral looping is the first physical manifestation of the breaking of left-right symmetry in the embryo. After HH12, the chick embryo becomes much more difficult to image due to the increasing complexity of the heart as it continues to rotate and grow in size.
This created a new line of birds where the inner and outer layers of the heart are easily visualized in yellow and red respectively.
Results
In Figures 3B and 3E we see that there is still rapid movement of the endothelial cells in both the sinoatrial regions and the heart tube while looping. This contrasts with the image of the corresponding PGK cells where the sinoatrial regions have essentially stopped moving, while the cells in the heart tube region follow the same movements as the Tie1-labeled cells. The cells that become the endothelial and myocardial layers of the upper ventricle move from the lateral part to the midline to form the heart tube.
In the looping heart, we see that these cells remain tightly packed with their neighbors as they begin to form the lower chamber of the heart tube (see Figure 4D).
Discussion
This is consistent with the conclusion that cells in the heart migrate there from other areas of the embryo and do not divide at accelerated rates to form the heart tube as observed by Stalsberg. The movement patterns of cardiac cells show that myocardial and endocardial cells have predetermined fates by HH8. The secondary heart fields begin their contribution from the conus region at the outflow tract and continue the expansion and development of the heart tube in a caudal direction.
Extending the timelapse imaging to stages HH14 and beyond, while focusing on the cone region, will provide a detailed, dynamic fate map of the contributions of the secondary cardiac fields.
Materials and Methods
Embryos were removed from the egg using a square piece of filter paper with a hole cut through the middle. This was placed over the embryo and the edges of the filter paper were cut to clamp the membranes to the filter paper. After blocking in 5% goat serum in PBST for 1 h, embryos were incubated in a solution of MF20 (1:10 dilution) and QH1 (1:1000 dilution) antibodies overnight at 4°C, both obtained from the Hybridoma Developmental Studies Bank. of the University of Iowa (Moreno-Rodriguez et al., 2006; . Finkelstein and Poole, 2003).
Embryos were rinsed again 3 times for 15 min each in PBST and blocked for 15 min at room temperature.
A study of the localization of organ-forming regions of the chick blastoderm of the main process stage. Regional mitotic activity in the precardiac mesoderm and differentiating heart tube in the chick embryo.
Figures
Panels A-E and K-O are from a time-lapse movie of the developing heart of a double transgenic embryo from HH8 – HH11 [Tg(tie1:H2B::eYFP) X Tg(pgk:H2B::mCherry)]. This allows visualization of the location of the muscle cells during heart tube formation for cell tracking. Pink is the presumed superior ventricular myocardium, purple is the presumed superior ventricular endocardium, yellow is the presumed inferior ventricular myocardium, and white is the presumed inferior ventricular endocardium.
Cells from the different sides do not mix and maintain their neighbor relationships, indicating skin-like behavior during heart tube formation.
Conclusion
The data confirm the material composition of the heart tube as described by Loumes et al. 2008) as a 2-layer fluid-filled elastic tube with a thick jelly-like interior. All these proposed theories about the movement that occurs during heart tube formation and elongation at stages HH9-10 do not tell the whole story. The data presented in this thesis examined all cells on the ventral surface of the embryo.
In addition, individual cell migration in sinoatrial regions contributes cells to the tissue that forms the presumptive left ventricle of the heart tube.
Appendix
Add the saline solution to a sterile 500 ml flask and bring to a boil with a hot plate/stirrer. While the agar is dissolving, collect the thin albumen in a sterile Falcon tube (50 mL) or similar container. Add the egg white to the flask with the dissolved agar and vortex for 30-60 seconds.
After separation is complete, replace the petri dish lids and leave the dishes for several hours or overnight at room temperature to dry.