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

Abstract

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.

On the involvement of the second heart field in congenital heart defects

“…These include the identification of a population of FHF cells termed the juxtacardiac field, located at the lateral boundary between the embryo and extraembryonic mesoderm, that contributes to growth of the early cardiac primordium along its ventrolateral margin, in contrast to SHF contributions along the dorsomedial margin [25][26][27]. Single-cell transcriptomics has also revealed differences in gene expression between left and right SHF cells, consistent with the recent finding that the SHF is a driver of looping morphogenesis [28,29]. Defects in cardiac looping, or in the prior establishment of embryonic laterality, have been implicated in a spectrum of CHD [29,30].…”

Notch3is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping

“…BMP (20–22), HH (23), NOTCH (24–26) and WNT (27–29) signalling have been shown to be required upstream of Nodal for asymmetry. Transcriptomic screens have been performed to identify asymmetric genes in embryos outside the node or in cardiac precursors (50,51). Yet, asymmetric candidate genes were either not validated or not analysed functionally, leaving open the question of which are genuine asymmetry factors.…”

“…To uncover asymmetric factors in organ specific precursors, rather than the left-right organiser, recent transcriptomic screens were performed on micro-dissected tissues excluding the node (50,51). Whereas NODAL signalling is transient (12,52), these screens were based on pooled embryos, which limits their resolution.…”