Figure 4
Figure 4. Notch signaling is required for pre-HSC development. (A) DAPT treatment completely prevents HSC development in E10.5 AGM, but has a milder effect at E11.5. E10.5 (n = 2) and E11.5 (n = 2) explants were cultured for 5 days without cytokines in the presence of dimethyl sulfoxide (DMSO) or 50 μM DAPT. At the end of the culture, the explants were dissociated and injected into irradiated mice (0.3 ee per mouse); **P < .01, Mann-Whitney test. (B) DAPT treatment disrupts pre-HSC type II maturation. E11.5-sorted VC+CD45+ cells (pre-HSC type II) were coaggregated with OP9. After 5 days of culture with cytokines in presence of DMSO or 50 μM DAPT, the reaggregates were dissociated and injected into irradiated mice (0.2 ee per mouse); n = 3, **P < .01, Mann-Whitney test. (C) DAPT treatment does not affect CFU-C development. E10.5 explants were cultured for 5 days without cytokines in presence of DMSO or 50 μM DAPT. After culture, the development of hematopoietic progenitors (CFU-C) was assessed by performing a colony-forming assay. Bars represent the average number of CFU-Cs per ee and standard errors (n = 3). (D) DAPT does not affect pre-HSC type II maturation into HSCs when added after 24 hours of culture. E11.5-sorted Hes1-GFP+ pre-HSCs type II were cultured for 24 hours prior to addition of DMSO or DAPT. After a subsequent 4 days in culture, the cells were injected into irradiated mice (0.2 ee per mouse); n = 2. (E) Conditional deletion of RBP-Jκ in the E10.5 AGM region. E10.5 RBP-JCKO, RBP-JHet, and wild-type (wt) AGM cells were dissociated, treated individually with 4-OHT for 2 hours, and then cultured as reaggregates without OP9 and without cytokines for 5 days before transplantation. The red triangles represent the recipients whose bone marrow contained RBP-JΔ/Δ dHSCs and showed T-cell phenotype; the blue triangle represents the mouse repopulated with RBP-Jflox/Δ dHSCs (normal T-cell development); black triangles represent recipients whose bone marrow was not analyzed further (0.2 ee per mouse). (F) Numbers of CFU-Cs per ee in E10.5 RBP-JCKO (CKO), RBP-JHet (Het), and WT AGM after culture (4, 3, and 4 embryos, respectively; standard errors are shown). (G) Presence of RBP-Jκ protein after induction of the deletion. E10.5 RBP-JCKO AGM was dissociated and divided into 2 parts, 1 was treated with 4-OHT and the other with methanol (4-OHT vehicle) for 2 hours at 37°C. Twenty-four hours after induction, the presence of RBP-Jκ protein was analyzed by flow cytometry. The dot plots are representative of 4 different AGMs, gated on live cells (EMA−). BFU-E, blood-forming unit-erythrocyte; GM, granulocyte-macrophage; M, macrophage; Mast, mast colonies.

Notch signaling is required for pre-HSC development. (A) DAPT treatment completely prevents HSC development in E10.5 AGM, but has a milder effect at E11.5. E10.5 (n = 2) and E11.5 (n = 2) explants were cultured for 5 days without cytokines in the presence of dimethyl sulfoxide (DMSO) or 50 μM DAPT. At the end of the culture, the explants were dissociated and injected into irradiated mice (0.3 ee per mouse); **P < .01, Mann-Whitney test. (B) DAPT treatment disrupts pre-HSC type II maturation. E11.5-sorted VC+CD45+ cells (pre-HSC type II) were coaggregated with OP9. After 5 days of culture with cytokines in presence of DMSO or 50 μM DAPT, the reaggregates were dissociated and injected into irradiated mice (0.2 ee per mouse); n = 3, **P < .01, Mann-Whitney test. (C) DAPT treatment does not affect CFU-C development. E10.5 explants were cultured for 5 days without cytokines in presence of DMSO or 50 μM DAPT. After culture, the development of hematopoietic progenitors (CFU-C) was assessed by performing a colony-forming assay. Bars represent the average number of CFU-Cs per ee and standard errors (n = 3). (D) DAPT does not affect pre-HSC type II maturation into HSCs when added after 24 hours of culture. E11.5-sorted Hes1-GFP+ pre-HSCs type II were cultured for 24 hours prior to addition of DMSO or DAPT. After a subsequent 4 days in culture, the cells were injected into irradiated mice (0.2 ee per mouse); n = 2. (E) Conditional deletion of RBP-Jκ in the E10.5 AGM region. E10.5 RBP-JCKO, RBP-JHet, and wild-type (wt) AGM cells were dissociated, treated individually with 4-OHT for 2 hours, and then cultured as reaggregates without OP9 and without cytokines for 5 days before transplantation. The red triangles represent the recipients whose bone marrow contained RBP-JΔ/Δ dHSCs and showed T-cell phenotype; the blue triangle represents the mouse repopulated with RBP-Jflox/Δ dHSCs (normal T-cell development); black triangles represent recipients whose bone marrow was not analyzed further (0.2 ee per mouse). (F) Numbers of CFU-Cs per ee in E10.5 RBP-JCKO (CKO), RBP-JHet (Het), and WT AGM after culture (4, 3, and 4 embryos, respectively; standard errors are shown). (G) Presence of RBP-Jκ protein after induction of the deletion. E10.5 RBP-JCKO AGM was dissociated and divided into 2 parts, 1 was treated with 4-OHT and the other with methanol (4-OHT vehicle) for 2 hours at 37°C. Twenty-four hours after induction, the presence of RBP-Jκ protein was analyzed by flow cytometry. The dot plots are representative of 4 different AGMs, gated on live cells (EMA−). BFU-E, blood-forming unit-erythrocyte; GM, granulocyte-macrophage; M, macrophage; Mast, mast colonies.

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