Single-cell transcriptomic landscape of cardiac neural crest cell derivatives during embryonic and neonatal development

ABSTRACT Rationale Cardiac neural crest cells (CNCCs) contribute greatly to cardiovascular development. A thorough understanding of the cell lineages, transcriptomic states and regulatory networks of CNCC derivatives during normal development is essential for deciphering the pathogenesis of CNCC-associated congenital anomalies. However, the transcriptomic landscape of CNCC derivatives during development has not yet been examined at a single-cell resolution. Objective We sought to systematically characterize the cell lineages, define the developmental chronology and elucidate the transcriptomic dynamics of CNCC derivatives during embryonic and neonatal development. Methods and Results We performed single-cell transcriptomic sequencing of 34,131 CNCC-derived cells in mouse hearts from eight developmental stages between E10.5 and P7. Through single-cell analyses and single-molecule fluorescence in situ hybridization, we confirmed the presence of CNCC-derived mural cells. Furthermore, we found the transition from CNCC-derived pericytes to microvascular smooth muscle cells, and identified the genes that were significantly regulated during this transition through pseudo-temporal analysis. CNCC-derived neurons first appeared at E10.5, which was earlier than previously recognized. In addition, the CNCC derivatives switched from a proliferative to a quiescent state with the progression of development. Gradual loss of the neural crest molecular signature with development was also observed in the CNCC derivatives. Our data suggested that many CNCC-derivatives had already committed or differentiated to a specific lineage when migrating to the heart. Finally, we characterized some previously unknown subpopulations of CNCC derivatives during development. For example, we found that Penk + cells, which were mainly localized in outflow tract cushions, were all derived from CNCCs. Conclusions Our study provides novel insights into the cell lineages, molecular signatures, developmental chronology and state change dynamics of CNCC derivatives during embryonic and neonatal development. Our dataset constitutes a valuable resource that will facilitate future efforts in exploring the role of CNCC derivatives in development and disease.