Understanding Cardiac Tube Formation in Developing Drosophila Embryos using Light Sheet Microscopy and Cardiac Drug Screening

Abstract

Heart development begins with the formation of a primitive tube, which is mediated by coordinated cell movements. In Drosophila, the heart is formed from 52 bi-lateral pairs of cardiac precursors that migrate dorsally and medially to join their counterparts. The cells must undergo distinct morphological changes in order to form a lumen. 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 melanogaster and the ability to perform live imaging of its embryos, we have developed a light-sheet microscopy platform and quantitative image analysis to characterize cell behaviours and molecular rearrangements during heart tube formation in living Drosophila embryos. Automated image analysis allows quantitative comparison of the dynamics of tube formation across embryos. To identify the pathways that regulate collective cell movements during heart development, we are conducting a pharmacological screen for inhibitors of cardiac precursor migration. We are particularly interested in the role of the cytoskeleton as both actin and myosin are important for cell movements in heart development. The kinase Rho-kinase (Rok) phosphorylates and activates the myosin light chain, and thus inhibiting Rho-kinase results in impaired myosin contractility. Preliminary experiments suggest that Rok may be important for the coordinated movement of cardioblasts during Drosophila heart morphogenesis. Embryos injected with water (controls) developed normally but those injected with 10 mM Y-27632, a Rok inhibitor had disrupted coordination of cardioblasts, leading to defects in heart tube formation. Together, our novel tools will allow us to identify pathways critical for cardiac precursor migration, polarization, and cell-cell adhesion.