Arterialization requires the timely suppression of cell growth

Arteries are thought to be formed through the induction of a highly conserved arterial genetic programme in a subset of vessels that will later experience an increase in oxygenated blood flow. The initial steps of arterial specification require both VEGF and Notch signalling. Here, we combined inducible genetic mosaics and transcriptomics to modulate and define the function of these signalling pathways in cell proliferation, arteriovenous (AV) differentiation and mobilization. We observed that endothelial cells (ECs) with high VEGF or Notch signalling are intrinsically biased to mobilize and form arteries; nevertheless, they are not genetically pre-determined, and can also form veins. Mechanistically, we found that higher VEGF and Notch signalling in pre-arterial capillaries suppresses Myc-dependent metabolic and cell-cycle activities, promoting the incorporation of ECs in arteries. Mosaic lineage tracing studies revealed that ECs completely lacking the Notch-Rbpj transcriptional activator complex rarely form arteries; however, these cells regained the ability to form arteries when Myc function was suppressed. Thus, arterial development does not require the direct induction of a Notch-dependent arterial differentiation programme, but rather the timely suppression of endothelial cell-cycle progression and metabolism, a process preceding arterial mobilization and complete differentiation. Overall design: To isolate and profile coronary ECs with normal, half or full loss of Dll4 signalling, we intercrossed Pdgfb-iCreERT2-IRES-EGFP mice with wildtype females (abbreviated here as Wt samples) or Dll4flox/flox Pdgfb-iCre-ERT2-EGFP mice with wildtype (abreviated here as Het samples) or Dll4 flox/flox females (abreviated here as KO samples). Tamoxifen was administered at E13.5 and several litters of independent females were collected at E14.5. Different groups of hearts carrying EGFP expression were obtained: controls (wt), Dll4 heterozygous mutants (het), and Dll4 homozygous mutants (KO). Embryonic hearts were dissected and then filtered through a 70-µm cell strainer, incubated for 30min at 4C with APC rat-anti-mouse CD31, and CD31+GFP+ ECs sorted by FACS. RNA was isolated from the sorted endothelial cells of embryonic hearts with RNAeasy Micro kit – Qiagen. cDNA amplification (SMART-Seq v4 Ultra Low Input RNA Kit – Clontech-Takara), library preparation (Nextera XT DNA library preparation kit - Illumina) and single read Next generation sequencing (Illumina HiSeq 2500) were performed at the Genomics Unit at Centro Nacional de Investigaciones Cardiovasculares (CNIC).