Single-nuclear multiome (RNA & ATAC) profiling of Pax3-Cre lineage traced endothelium from E9.5 mouse embryos and bulk ATAC-seq from E13.5 mouse embryos

Background: The formation of the lymphatic system has been a subject of controversy for over a century. Although the accepted view is that lymphatic endothelial cells originate exclusively from veins, there have been reports of non-venous derived lymphatics in multiple tissues. Methods: We performed single-nuclear multiome sequencing analyses on 3,801 individual nuclei isolated from Pax3Cre/+;Rosa26tdTomato mouse embryos at E9.5, the temporal window in which lymphatic endothelium initially emerges. Additionally, we performed bulk ATAC-seq analyses following the OmniATAC protocol on FACS-sorted ECs from Pax3Cre/+;Rosa26tdTomato embryos at E13.5. Results: These analyses revealed the existence of angioblasts which arise from paraxial mesoderm and directly differentiate into lymphatic endothelium. Our findings support the notion that lymphatic endothelial cells also have non-venous origins and explain the discrepancies between previous studies. Single-nuclear multiome (RNA & ATAC) profiling, endothelial progenitors and endothelial cells (ECs) were isolated from Pax3Cre/+;Rosa26tdTomato embryos at ~E9.5, to define the transcriptional and chromatin accessibility landscape of paraxial mesoderm as it differentiates into angioblasts and endothelial cells. Single nuclear multiome-seq was performed using the 10xChromium Next GEM Single Cell Multiome ATAC + Gene Expression kit. For bulk ATAC-seq, ECs were FACS sorted from Pax3Cre/+;Rosa26tdTomato embryos at E13.5 and divided into tdTomato positive (spos) and negative blood ECs (dneg), and tdTomato positive lymphatic ECs (dpos) using podoplanin (PDPN) as a marker.