Figure 5: Upregulated Notch signalling in Snail1-deleted ECs.

(a–c) Transcriptional profiling analysis of cultured ECs derived from E10.5 Snail1^fl/fl embryos and transduced in vitro with Adeno-β-gal or Adeno-Cre to induce Snail1 KO recombination. Pie chart depicts the distribution of total transcripts changed in Snail1-deleted ECs as compared with control ECs (a). Gene ontology (GO) analysis of Snail1-deleted versus control ECs (b). Heat map representation of microarray data highlights the expression levels of key transcription factors, arterial–venous specification genes, EnMT-related genes, as well as Notch signalling target genes (c). (d) qRT-PCR analysis of cultured ECs from a. Snail1 KO ECs presented significantly higher levels of Dll4, Jag1, Notch1, Hey1 and ephrin-B2. Data are presented as mean±s.e.m. (n=3). *P<0.05, **P<0.01, Student’s t-test. (e) Western blot analysis of cultured ECs from a. (f) qRT–PCR analysis of ECs freshly isolated from E10.5 WT and Snail1 KO embryos. ECs isolated from Snail1 LOF embryos displayed significantly higher levels of Dll4, Notch1, Hey1 and ephrin-B2 (n=6 each). Data are presented as mean±s.e.m. *P<0.05, **P<0.01, Student’s t-test. (g–l) Cross-sections from E10.5 WT and Snail1 LOF mutant embryos were co-stained with anti-PECAM-1 and anti-Dll4 antibodies. Cell nuclei were stained with DAPI (blue). (i,l) Magnified areas of boxed regions in h and k, respectively. Yellow arrow depicts Dll4-positive ECs in CV region. Scale bars, 50 μm. (m–r) Cross-sections from E10.5 WT and Snail1 LOF mutant embryos were co-stained with anti-PECAM-1 and anti-ephrin B2 antibodies. Cell nuclei were stained with DAPI (blue). (o,r magnified areas of boxed regions in n and q, respectively. Yellow arrows depict ephrin B2-positive ECs in CV region. Scale bars, 50 μm.