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

Abstract

22 Rationale: Cardiac neural crest cells (CNCCs) contribute greatly to cardiovascular 23 development. A thorough understanding of the cell lineages, transcriptomic states and 24 regulatory networks of CNCC derivatives during normal development is essential for 25 deciphering the pathogenesis of CNCC-associated congenital anomalies. However, the 26 transcriptomic landscape of CNCC derivatives during development has not yet been 27 examined at a single-cell resolution. 28 Objective: We sought to systematically characterize the cell lineages, define the 29 developmental chronology and elucidate the transcriptomic dynamics of CNCC 30 derivatives during embryonic and neonatal development. 31 Methods and Results: We performed single-cell transcriptomic sequencing of 34,131 32 CNCC-derived cells in mouse hearts from eight developmental stages between E10.5 33 and P7. Through single-cell analyses and single-molecule fluorescence in situ 34 hybridization, we confirmed the presence of CNCC-derived mural cells. Furthermore, 35we found the transition from CNCC-derived pericytes to microvascular smooth muscle 36 cells, and identified the genes that were significantly regulated during this transition 37 through pseudo-temporal analysis. CNCC-derived neurons first appeared at E10.5, 38 which was earlier than previously recognized. In addition, the CNCC derivatives 39 switched from a proliferative to a quiescent state with the progression of development. 40Gradual loss of the neural crest molecular signature with development was also 41 observed in the CNCC derivatives. Our data suggested that many CNCC-derivatives 42 had already committed or differentiated to a specific lineage when migrating to the heart. 43Finally, we characterized some previously unknown subpopulations of CNCC 44 derivatives during development. For example, we found that Penk+ cells, which were 45 mainly localized in outflow tract cushions, were all derived from CNCCs. 46 Conclusions:Our study provides novel insights into the cell lineages, molecular 47 signatures, developmental chronology and state change dynamics of CNCC derivatives 48 during embryonic and neonatal development. Our dataset constitutes a valuable 49 resource that will facilitate future efforts in exploring the role of CNCC derivatives in 50 development and disease. 51