Figure 4. Analysis of the Notch pathway during aortic hematopoiesis.

(A, B) Mutual exclusion between cjagged2 and runx1 during aortic hematopoiesis. In situ hybridization, adjacent sections separated by 7μm.

(A) Runx1 expression extends to the whole floor (arrowheads) except to ECs in the middline.

(B) Cjagged2 expression is lost (arrowheads) in cells up-regulating runx1.

(C, D) Q-PCR analysis of the Notch pathway in AGM ECs at the hematopoietic stage in chicken (C) and mouse (D) embryos i.e. respectively E3.5 and E11.5. ECs (pink) were sorted on the basis of AcLDL upake (chicken) and CD144 (mouse) expression. HCs (green) were retreived by including the expression of CD45.

(E) Hes1 expression analysis by Q-PCR in the E11.5 Runx1⁺ hemogenic endothelium compared to increasing stages of hematopoietic maturation characterized by their combinatorial expression of CD41 and CD45. A decrease in Hes1 expression associated to HC maturation from the endothelium is clearly visible.

(F, G) Detection of Active Notch following pTP1-Venus reporter construct electroporation in the aorta.

(F) HC cluster stage. Two clusters are visible (white arrows). The green staining (active Notch) is restricted to ECs and absent from the clusters.

(G) Same section double stained for active Notch (green) and CD45 (red) to visualize the clusters. Bar=40μm

(H–L) Hematopoietic production analysis of E9.5 mouse P-Sps treated or not with DAPT.

(H) Number of cells in vehicle- and DAPT-treated aortas after 7 days of coculture on OP9 layer. Differences in cell number are not significant.

(I, J) Percentages of CD45⁺ cells produced by E9.5 mouse P-Sp after 3 (I) and 6 (J) days of culture with or without DAPT. A significant increase of CD45⁺ cell production is visible at 3 days in the DAPT-treated samples compared to the vehicle-treated samples. The inverse is observed at 6 days of culture. Error bars represent the results from four independent assays.

(K) Percentage of 7AAD⁺ cells at 3 (D3) and 6 (D6) days following mouse P-Sp culture incipience. NS: not significant

(L) Percentage of wells positive for D7-CAFCs. A significant decrease in CAFCs is observed in the DAPT-treated samples in keeping with previously published data (Robert-Moreno et al., 2008).

The error bars represent s.e.m. Three stars indicates p<0.01, two p<0.25, one p<0.5, NS, not significant

See also Figure S3.