Digital and molecular reconstruction of zebrafish heart morphogenesis by single-cell transcriptomics and light-sheet microscopy

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Vertebrate cardiac development depends on molecular pathways that control the generation and allocation of progenitor cell populations and guide morphogenesis. Mechanical forces exerted by blood flow and cardiac contractility are important triggers of these processes by impacting the proliferation of endocardial progenitor cells and by strongly affecting the morphogenesis of heart valves. However, a comprehensive understanding of the mechanisms by which endocardial cells are rendered sensitive to fluid flow shear forces and how these biomechanical stimuli trigger endocardial growth and morphogenesis is lacking. Even less is known about the modes by which the mechanical impact of fluid flow shear forces on endocardial cells is integrated and transmitted to neighboring myocardial cells during cardiac morphogenesis. Here, we propose to combine cardiac tissue RNA-seq analyses with in vivo imaging of cardiac development at a cellular resolution in zebrafish. We have recently worked out conditions for in vivo imaging of heart valve morphogenesis in zebrafish embryos and established a protocol for Tomo-seq, which provides high spatial resolution of whole-transcriptome data. Deciphering cardiac gene expression in combination with light-sheet based imaging of zebrafish embryonic hearts will help to identify lineage relationships of cardiac progenitor cells and unravel the molecular orchestration of single cell behaviors during valvulogenesis. One hypothesis that we aim to address is the question whether valvulogenesis is based on conserved endoMT processes of cardiac progenitor cells and, if so, to elucidate how biomechanical stimuli impact the regulation of these processes. Since biomechanical forces vary in different regions of the heart and endocardial cells show different responsiveness to these cues, we also propose the existence of distinct endocardial populations with varying thresholds of mechanosensitivity. We aim to test this hypothesis by dissecting the molecular signature of the endocardium at a single cell level and in dependence of biomechanical forces. Finally, single cell data will be utilized to probe for distinct cell populations in vivo and their behaviors will be studied using live imaging under conditions that are restrictive or permissive for the formation of cardiac valves.