Molecular pathogenesis of congenital heart disease mediated by neural crest and second heart field cells

ABSTRACT The cardiac outflow tract (OFT) with adjoining major arteries is susceptible to developmental insults during embryogenesis that leads to congenital heart disease. Part of the reason for their vulnerability is because the morphogenesis of the OFT requires the interaction of both neural crest cells (NCCs) and adjacent second heart field (SHF) mesoderm cells. This takes place during dynamic expansion of the embryonic pharyngeal apparatus. To understand these two juxtaposed populations, we performed single cell RNA-sequencing (scRNA-seq) after lineage purification, focusing mainly on NCCs. Cardiac NCCs (CNCCs) have traditionally been defined positionally rather than molecularly by specific gene markers of cell fate progression because of their multipotency and changing genetic profiles. Using scRNA-seq, we were able to identify putative CNCCs in the pharyngeal apparatus. This was achieved by using expression of early vascular smooth muscle genes, such as Acta2, as a guide. From this, we identified three CNCC populations at mouse embryonic day, E10.5. We refer to them as the Tbx2 and Tbx3 (Tbx2/3), Isl1 and Acta2 populations, based upon distinct expression of these genes. We suggest that the Tbx2/3 and Isl1 lineage cells may independently evolve and contribute to smooth muscle cells of the pharyngeal arch arteries and OFT, respectively. This proposal is set to explore the origin and fate trajectories of these three populations by dual lineage tracing, by determining the function of genes within, and to build gene regulatory networks controlling their development. Not only will we examine changes in NCCs but also will evaluate surrounding SHF mesoderm cells because altering CNCCs might affect these progenitor cells as well. We also have scRNA-seq data on the SHF mesoderm cells purified from mouse embryos. On the other hand, it is possible that alteration of genes required in the SHF can disrupt CNCC development. As one specific example, Tbx1, the gene for 22q11.2 deletion syndrome is expressed in the SHF but not CNCCs, but it greatly affects their function. We will test the idea that in Tbx1 null mutant embryos, CNCCs fail to progress from Sox10 expressing progenitors and are not able to enter the OFT. Many of the genes to be investigated in this program are associated with congenital heart disease in human patients. Overall, this program will help identify molecular aspects of CNCCs and the orchestration of CNCC-SHF cell fates in embryogenesis.