Understanding Mechanisms of Coordinated Cardiac Tube Formation in Developing Drosophila Embryos using Light Sheet Microscopy and Pharmacological Inhibition

McFaul, Christopher 1, 2, 5 ; Yip, Christopher 2, 3, 5 ; Fernandez-Gonzalez, Rodrigo 2, 4

1. MD/PhD Program, University of Toronto; 2. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 3. Departments of Biochemistry,  Chemical Engineering and Applied Chemistry, University of Toronto; 4. Department of Cell and Systems Biology, University of Toronto; 5. Donnelly Centre for Cellular & Biomedical Research, University of Toronto

Heart development begins with the formation of a primitive tube, both in vertebrates and invertebrates. Improper heart tube formation leads to cardia bifida, a lethal congenital disorder. Heart tube formation is mediated by coordinated cell movements. In the fruit fly Drosophila melanogaster, the heart is formed from 52 contra-lateral pairs of cardiac precursors (cardioblasts) that migrate dorsally and medially to join their counterparts. While the genetic pathways that induce cardiac cell specification have been clearly defined, the cellular and molecular mechanisms that regulate collective cell migration during heart tube formation are not well understood. Leveraging the simplicity and pharmacological tractability of Drosophila, and the ability to perform live imaging of its embryos, we have developed a light-sheet microscopy platform and quantitative image analysis tools to characterize cell behaviours and molecular rearrangements during heart tube formation in living Drosophila embryos. Our system enables identification and tracking of cardiac precursors and the overlying epidermal cells. Semi-automated image analysis allows quantitative comparison of the dynamics of tube formation across embryos to investigate the mechanisms of heart tube closure.

Supracellular networks formed by the cytoskeletal protein actin, and the molecular motor myosin II coordinate cell-cell movements during tissue development (e.g. zebrafish epiboly or mouse eyelid closure) and in embryonic wound repair. Using live imaging, we recently found the presence of an actin cable at the trail end of each of the two rows of cardiac precursors, suggesting that cytoskeletal cables may coordinate cardioblast movements. To investigate the role of actomyosin-based contraction in the  regulation of collective cell movements during heart development, we injected embryos with Y-27632, a pharmacological inhibitor of the kinase Rho-kinase, the main activator of myosin-based contraction. Rho-kinase inhibition resulted in uncoordinated cardioblast movements and disrupted heart tube formation, suggesting that myosin contractility is necessary for heart tube closure. We are currently taking advantage of the genetic tractability of Drosophila to conduct RNAi-based, tissue-specific knock down of Rho-kinase to elucidate the role of myosin-based contraction in each of the three major tissues involved in collective cell movement during Drosophila heart formation (cardiac, epidermal, extraembryonic). Together, our novel tools will allow us to identify pathways critical for collective cell behavior during the earliest steps of heart development.