Spatiotemporal Gene Coexpression and Regulation in Mouse Cardiomyocytes of Early Cardiac Morphogenesis

Background: Heart tube looping to form a 4-chambered heart is a critical stage of embryonic heart development, but the gene drivers and their regulatory targets have not been extensively characterized at the cell-type level. Methods and Results: To study the interaction of signaling pathways, transcription factors (TFs), and genetic networks in the process, we constructed gene co-expression networks and identified gene modules highly activated in individual cardiomyocytes at multiple anatomical regions and developmental stages using previously published single-cell RNA-seq data. Function analyses of the modules uncovered major pathways important for spatiotemporal cardiomyocyte differentiation. Interestingly, about half of the pathways were highly active in cardiomyocytes at the outflow tract (OFT) and atrioventricular canal, including well-known pathways for cardiac development and many newly identified ones. We predicted that these OFT-atrioventricular canal pathways were regulated by a large number of TFs actively expressed at the OFT–atrioventricular canal cardiomyocytes, with the prediction supported by motif enrichment analysis, including 10 TFs that have not been previously associated with cardiac development (eg, Etv5, Rbpms, and Baz2b). Furthermore, we found that TF targets in the OFT–atrioventricular canal modules were most significantly enriched with genes associated with mouse heart developmental abnormalities and human congenital heart defects, in comparison with TF targets in other modules, consistent with the critical developmental roles of OFT. Conclusions: By analyzing gene co-expression at single cardiomyocytes, our systematic study has uncovered many known and additional new important TFs and their regulated molecular signaling pathways that are spatiotemporally active during heart looping.