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. 2015 Jul 23;6:7739. doi: 10.1038/ncomms8739

Figure 4. The role of the Notch pathway in endothelial to haematopoietic fate decisions.

Figure 4

(a) SOX17 (ChIP) qRT–PCR of E11.0 sorted endothelial cells. Letters denote regions upstream of Notch1, Dll4, and Coup-TFII promoters, and Lef1 as a positive control. Error bars indicate s.e.m.. IgG control set to one for comparisons of fold change, n=3 litters, embryos pooled, P values as indicated. (b) EMSA of putative SOX17-binding sites within ChIP sequences (designated by letters in a). Each lane represents biotin-labelled duplexed oligonucleotides spanning the Lef1 promoter SOX17-binding site (Lef1_Biot). Addition of rSOX17-Flag produces a specific shift, indicating protein–DNA complex (lane 2), which is competed away by unlabelled Lef1 (Lef1_s1), while mutant probe does not compete (Lef1_Δ_s1). Similar designations are used for putative binding sites (and mutants) in Notch1, Dll4 and Coup-TFII sequences. Asterisks denote competition. (c) Schematic of AGM explant analysis depicts in vitro Cre lineage tracing and calculation of hemogenic output (HE ratio); the ratio between per cent labelled (Td+) haematopoietic cells (CD45+CD31−) to per cent labelled (Td+) endothelial cells (CD31+CD45−). (d–g) Each data point represents a separate embryo/AGM explant, littermates are depicted by the same data point colour and shape. Bar indicates group mean. P values calculated on Student's t-test between groups, significance also validated by two-way analysis of variance (Supplementary Table 5) (d) The HE ratio of Notch1 homozygous (f/f) and heterozygous (f/+) mutant explants. f/+ n=18, f/f n=21, 6 litters. (e) Percentage of traced Td+ hemogenic endothelial cluster cells, designated as CD31+CD41+. f/+ n=12, f/f n=13, 4 litters. (f) Percentage of traced (Td+) maturing HSPCs (identified as CD31+CD117+Sca1+CD45+) f/+ n=10, f/f n=6, 3 litters. (g) BrdU+ cells measured after 2-h incubation with BrdU in traced ECs (left) and traced HCs (right) demonstrates a significant increase in HC proliferation, f/+ n=6, f/f n=10, 3 litters. (h) Schema and bar graph of qRT–PCR analyses of sorted endothelial cells from E11 embryos after in vivo Notch1 ablation at E9.5. Error bars indicate s.e.m. (n=3 litters, embryos pooled by genotype). LOF, loss of function.