Molecular characterization of Left-Right symmetry breaking in the mouse embryo

The asymmetric morphology of the mammalian heart is essential to its function as the organ of pulmonary and systemic double circulation. Left-right asymmetry is established by a leftward flow in the node that results in the asymmetric expression of Nodal. This triggers a cascade of asymmetric expression of downstream genes, such as Pitx2c, in the lateral plate mesoderm that gives rise to the first morphologically recognizable primordial heart structure, the cardiac crescent. Relatively little is known about gene expression asymmetries in the cardiac crescent that might underpin asymmetric cardiac morphogenesis. To systematically identify asymmetrically expressed genes, we performed a single-cell transcriptional analysis of manually dissected left and right halves of the cardiac crescent at stages spanning symmetry breaking. This revealed both left and right-sided genes that have not previously been implicated in left-right symmetry breaking. Some of these were expressed in multiple cell types but showed asymmetric expression in only a sub-set of cell types. We validated these findings using multiplexed in situ Hybridization Chain Reaction (HCR) and high-resolution volume imaging to characterize the expression patterns of select genes. Using Dnahiv/iv mutant embryos that show randomized situs, we established that all the genes tested tracked the asymmetric expression of Pitx2c, indicating their asymmetric expression also arose from the early asymmetries at the node. This study provides a high-fidelity molecular characterization of left-right symmetry breaking during cardiac crescent formation, providing a basis for future mechanistic studies on asymmetric cardiac morphogenesis.