Dynamic Three-Dimensional Imaging of Cellular Shape Changes and Protein Expression in the Developing Zebrafish Heart

Abstract

We present our results in dynamic three-dimensional (3D) imaging and quantification of the cellular shape changes and gene expressions of the developing zebrafish heart, in the effort to understand the mechanisms of the embryonic construction of this critical organ. The vertebrate heart is built up through a series of steps taking two flat layers of cells to a hollow heart tube to a multi-layered, multi-chambered, chirally twisted structure of the mature organ. Additionally, the heart is the first organ in the developing embryo to function, through its beating and pumping of the blood, shortly after the formation of the heart tube. Despite this intrinsic dynamic 3D nature of the developing heart, previous works documenting its development consist of largely 2D and/or static imaging (utilizing pharmacological means to stop the beating of the heart), due to the challenges in achieving fast, high 3D-resolution with conventional imaging modalities. To overcome these challenges, we employ 2-photon light sheet microscopy and a wavelet-based synchronization and registration method to achieve the required spatial and temporal resolution to capture the 3D motion of the heart. The high speed 3D imaging and analysis is carried out on several transgenic zebrafish lines that have been recently generated in our lab where proteins important for heart development are fluorescently tagged at their endogenous loci. We thus document not only cellular morphology but also critical genes' expression, with sub-cellular resolution, of the developing heart, over its beating cycle and at different development times. These results provide the necessary groundwork to start deciphering the process where the dynamic changes in cellular shapes, gene expressions, and cellular physical properties participate, in concert with the genetic program, in the development of the vertebrate heart.