Single-cell RNA-seq of the Developing Cardiac Outflow Tract Reveals Convergent Development of the Vascular Smooth Muscle Cells at the Base of the Great Arteries

Abstract

RationaleCardiac outflow tract (OFT) is a major hotspot for congenital heart diseases (CHDs). A thorough understanding of the cellular diversity, transitions and regulatory networks of normal OFT development is essential to decipher the etiology of OFT malformations.\n\nObjectiveWe sought to explore the cellular diversity and transitions between cell lineages during OFT development.\n\nMethods and ResultsWe performed single-cell transcriptomic sequencing of 55,611 mouse OFT cells from three developmental stages that generally correspond to the early, middle and late stages of OFT remodeling and septation. We identified 17 cell clusters that could be assigned to six cell lineages. Among these lineages, the macrophage and VSMC lineages of the developing OFT have seldom been previously described. Known cellular transitions, such as endothelial to mesenchymal transition, have been recapitulated. In particular, we identified convergent development of the VSMC lineage, where intermediate cell subpopulations were found to be involved in either myocardial to VSMC trans-differentiation or mesenchymal to VSMC transition. Through single-molecule in situ hybridization, we observed that cells expressing the myocardial marker Myh7 co-expressed the VSMC marker gene Cxcl12 in OFT walls, thus confirming the existence of myocardial to VSMC trans-differentiation. Moreover, we found that the Penk+ cluster c8, a relatively small mesenchymal subpopulation that was undergoing mesenchymal to VSMC transition, was associated with the fusion of OFT cushions. We also uncovered the expression dynamics and critical transcriptional regulators potentially governing cell state transitions. Finally, we developed web-based interactive interfaces to facilitate further data exploration.\n\nConclusionsWe provide a single-cell reference map of cell states for normal OFT development, which will be a valuable resource for the CHD community. Our data support the existence of myocardial to VSMC trans-differentiation and convergent development of the VSMC lineage at the base of the great arteries.