Abstract
The genetic programs responsible for pulmonary lymphatic maturation prior to birth are not known. To address this gap in knowledge, we developed a novel cell sorting strategy to collect fetal pulmonary lymphatic endothelial cells (PLECs) for global transcriptional profiling. We identified PLECs based on their unique cell surface immunophenotype (CD31+/Vegfr3+/Lyve1+/Pdpn+) and isolated them from murine lungs during late gestation (E16.5, E17.5, E18.5). Gene expression profiling was performed using whole-genome microarrays, and 1,281 genes were significantly differentially expressed with respect to time (FDR q < 0.05) and grouped into six clusters. Two clusters containing a total of 493 genes strongly upregulated at E18.5 were significantly enriched in genes with functional annotations corresponding to innate immune response, positive regulation of angiogenesis, complement & coagulation cascade, ECM/cell-adhesion, and lipid metabolism. Gene Set Enrichment Analysis identified several pathways coordinately upregulated during late gestation, the strongest of which was the type-I IFN-α/β signaling pathway. Upregulation of canonical interferon target genes was confirmed by qRT-PCR and in situ hybridization in E18.5 PLECs. We also identified transcriptional events consistent with a prenatal PLEC maturation program. This PLEC-specific program included individual genes (Ch25h, Itpkc, Pcdhac2 and S1pr3) as well as a set of chemokines and genes containing an NF-κB binding site in their promoter. Overall, this work reveals transcriptional insights into the genes, signaling pathways and biological processes associated with pulmonary lymphatic maturation in the fetal lung.
Highlights
The earliest LEC progenitors express Prox1, a required lymphatic transcription factor, in a distinct subset of anterior cardinal venous endothelial cells at ~E9.5 in the mouse [1,2,3,4] though several groups have suggested additional non-venous origins [5,6,7,8,9,10,11]
Immunophenotypic analysis and isolation of pulmonary lymphatic endothelium To identify genes, gene networks and signaling pathways associated with LEC maturation in the fetal lung, we developed a cell sorting strategy to isolate pulmonary LECs (PLECs) for broad-based transcriptomic profiling
This approach is based on the identification of a unique panel of LEC surface markers that immuno-colocalized with Prox1, the essential transcription factor required for lymphatic development
Summary
The earliest LEC progenitors express Prox, a required lymphatic transcription factor, in a distinct subset of anterior cardinal venous endothelial cells at ~E9.5 in the mouse [1,2,3,4] though several groups have suggested additional non-venous origins [5,6,7,8,9,10,11]. Prenatal pulmonary lymphatic maturation funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call