Figure 5
Figure 5. The Lyl1 promoter region and endogenous Lyl1 are expressed early during hematopoietic specification, yet Lyl1 cannot compensate for loss of Scl in hematopoietic differentiation assays using Scl−/− ES cells. (A) Flow cytometric analysis of E11.5 fetal liver demonstrates that enhancer-positive cells are largely c-kit+ and Ter119− consistent with a hematopoietic progenitor phenotype. FDG indicates fluorescein di-β-D-galactopyranoside fluorescent β-galactosidase/lacZ substrate. (B) E11.5 fetal liver cells expressing the Lyl1P transgene are enriched for hematopoietic progenitors; lacZ-positive and lacZ-negative cells were sorted by FACS and assessed for hematopoietic colony-forming activity. BFU indicates erythroid burst-forming units; GM, granulocyte-macrophage colonies; Mix, multipotent colonies. Error bars indicate SD. (C) Expression of the Lyl1P transgene in E7.5 mouse embryos; X-gal staining of a representative E7.5 transgenic embryo. Staining in the extraembryonic mesoderm seen by wholemount analysis (left panel) was confirmed on histologic sections (right panel). (D) Analysis of Scl and Lyl1 expression by real-time PCR at the onset of hematopoietic development. Flk1+ cells were isolated from day 3.3 embryoid bodies generated from E14.1 ES cells and grown in hemangioblast conditions for up to 4 days. RNA and cDNA were prepared for Flk1+ cells and day 1 to day 4 cultures and analyzed by real-time PCR. The results are represented as femtomole template per 50 ng total RNA (see “Materials and methods”) and are representative of 3 independent experiments. (E) Scl−/− ES cells were transfected with MSCV constructs expressing Scl or Lyl1 cDNA or MSCV control vector. Day 5 embryoid bodies derived from transfected ES cells were harvested, and single-cell suspensions were replated in methylcellulose-containing cytokines to induce hematopoietic colony formation. EryP indicates primitive erythroid colonies; Def, definitive hematopoietic colonies including macrophage, macrophage-erythrocyte, mix, and granulocyte-macrophage colonies. (F) Representative pictures of Scl-rescued colonies showing primitive colonies (top panel) and definitive colonies (middle and bottom panels). (G) Colonies from methylcellulose cultures were harvested, stained for CD45 and Mac1 expression, and analyzed by flow cytometry.

The Lyl1 promoter region and endogenous Lyl1 are expressed early during hematopoietic specification, yet Lyl1 cannot compensate for loss of Scl in hematopoietic differentiation assays using Scl−/−ES cells. (A) Flow cytometric analysis of E11.5 fetal liver demonstrates that enhancer-positive cells are largely c-kit+ and Ter119− consistent with a hematopoietic progenitor phenotype. FDG indicates fluorescein di-β-D-galactopyranoside fluorescent β-galactosidase/lacZ substrate. (B) E11.5 fetal liver cells expressing the Lyl1P transgene are enriched for hematopoietic progenitors; lacZ-positive and lacZ-negative cells were sorted by FACS and assessed for hematopoietic colony-forming activity. BFU indicates erythroid burst-forming units; GM, granulocyte-macrophage colonies; Mix, multipotent colonies. Error bars indicate SD. (C) Expression of the Lyl1P transgene in E7.5 mouse embryos; X-gal staining of a representative E7.5 transgenic embryo. Staining in the extraembryonic mesoderm seen by wholemount analysis (left panel) was confirmed on histologic sections (right panel). (D) Analysis of Scl and Lyl1 expression by real-time PCR at the onset of hematopoietic development. Flk1+ cells were isolated from day 3.3 embryoid bodies generated from E14.1 ES cells and grown in hemangioblast conditions for up to 4 days. RNA and cDNA were prepared for Flk1+ cells and day 1 to day 4 cultures and analyzed by real-time PCR. The results are represented as femtomole template per 50 ng total RNA (see “Materials and methods”) and are representative of 3 independent experiments. (E) Scl−/− ES cells were transfected with MSCV constructs expressing Scl or Lyl1 cDNA or MSCV control vector. Day 5 embryoid bodies derived from transfected ES cells were harvested, and single-cell suspensions were replated in methylcellulose-containing cytokines to induce hematopoietic colony formation. EryP indicates primitive erythroid colonies; Def, definitive hematopoietic colonies including macrophage, macrophage-erythrocyte, mix, and granulocyte-macrophage colonies. (F) Representative pictures of Scl-rescued colonies showing primitive colonies (top panel) and definitive colonies (middle and bottom panels). (G) Colonies from methylcellulose cultures were harvested, stained for CD45 and Mac1 expression, and analyzed by flow cytometry.

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