Fig. 4.

Scl induction rescues hematopoiesis from Hh inhibition in EBs. (A) Gene expression time course measured by quantitative PCR for Scl, Gata2, and Runx1 during the course of cyc treatment during days 2–5 of differentiation (n = 2). P values were derived from one-way ANOVA for correlated samples. (B) CFUs from day 6 whole EBs that were Scl-overexpressed and/or cyc-treated during days 2–5 or 3–5 of differentiation (n = 4). (C) Flow cytometric quantification of CD41⁺c-Kit⁺ hematopoietic precursors from day 6 whole EBs after Scl induction and/or cyc treatment. Horizontal bars indicate mean values (n = 4). (D) Blast and core colony formation during Scl overexpression and/or cyc treatment as assessed by BL-CFC assay (n = 2). (E) Gene expression profile by quantitative PCR in day 6 whole EBs that were Scl-overexpressed and/or cyc-treated during days 2–5 of differentiation (n = 2). (F) Flow cytometric quantification of VE-cadherin⁺CD41⁺ double-positive and VE-cadherin⁺CD41⁻ single-positive cells over the course of EB differentiation (n = 2). (G) CFU potential of sorted VE-cadherin⁺ cells from day 6 EBs that were Scl-overexpressed and/or cyc-treated during days 2–5 of differentiation (n = 4). (H) Images of cells from hemato-endothelial culture at 10× objective (Upper) and flow cytometry for CD45⁺ cells from hemato-endothelial culture (Lower). VE-cadherin⁺CD41⁻CD45⁻ cells were sorted from day 6 EBs and grown in hemato-endothelial culture in conjunction with Scl induction for 3–4 d. (I) Effect of Scl overexpression via dox treatment and/or cyc treatment on sorted VE-cadherin⁺ cells from E9 to E10 yolk sacs that were infected with lentiviruses for dox-inducible Scl. (n = 5).