Following Motion of Early Heart Development at the Cellular Level Using Confocal Microscopy in Transgenic Quail Embryos

Citation

Yang, Jennifer Chuen-Hsien (2011) Following Motion of Early Heart Development at the Cellular Level Using Confocal Microscopy in Transgenic Quail Embryos. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Y0YB-2312. https://resolver.caltech.edu/CaltechTHESIS:08312010-135100976

Abstract

The heart has fascinated scientists for centuries, from the days of Leonardo da Vinci to the modern scientist. As tools have developed over the years, we have begun to create a different perspective of how the heart develops and functions from original ideas. Genetics, molecular biology, and microscopes have allowed us to examine the heart with a level of detail that could not have been imagined by da Vinci.

The heart is a complex organ to study due to its innate nature of contracting and relaxing for the entire life of an organism. The motions of systole and diastole complicate any measurements and analysis that can be done. In general, most research has been done in either fixed tissue or dead organisms, neither being ideal for studying a live heart. But current research aims to change our knowledge of cardiogenesis through dynamic imaging of live tissue. With the progress made in the last few years, we have had to revise many of our assumptions about heart development and pumping mechanics. Now, we are at a new stage of research that requires the quantification of cardiogenesis to understand the requirements of heart development to prevent future diseases from occurring.

The following work divides the heart into three areas: 1-dimensional dynamic imaging and analysis of contractions and relaxation to achieve temporal resolution, 4-dimensional modeling of a beating heart during tube formation and early looping to observe individual cellular motion, and 4-dimensional fate mapping of the developing heart tube to visualize development with cellular resolution. With these three perspectives, we are able to quantify the movements of the myocardium and endocardium during development while the heart is beating. This novel approach will give us a wealth of information never seen before.

We use transgenic Japanese quail to highlight the nuclei of individual endothelial cells in the vasculature to analyze the inner tube of the heart, and another transgenic with a ubiquitous marker that fluoresces in every cell of the embryo. The quail heart is comparable to the human heart in that both have 4-chambered hearts, yet the quail is a much easier organism to study. The embryo is transparent and develops outside the mother. The quail embryo can be imaged ex ovo on inverted microscopes, allowing for ease of setup in a controlled environment.

With the use of dynamic imaging and innovative computer software, we were able to reconstruct the live heart while it is beating and developing. By dynamically imaging the endocardium and myocardium, we were able to study the interactions between the two layers by offering unsurpassed visual access to the morphogenetic events of cardiogenesis. Questions that have remained elusive are now being answered with extensive analysis from the data collected. Quantification of cardiogenesis is possible and a detailed fate-map has been produced from this research.

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