Fig. 5.

Gata2, Fli1 and Scl bind Gata2-3, Fli1+12 and Scl+19 in vivo. (A) (i and ii) In situ hybridization for Scl expression. (i) Transverse cryosection through a E11.5 embryo at the level of the AGM showing expression in FL, DA (boxed) and neural tissue (arrow). (ii) Magnified view of the boxed area showing expression in the endothelium and in a blood cluster (arrow). (iii and iv) In situ hybridization for Gata2 expression. (iii) Transverse cryosection through the AGM region of a E11.5 embryo showing Gata2 expression in the FL and DA (boxed). Prominent Gata2 expression is also seen in neural tissue (arrows). (iv) Magnified view of the boxed area in iii showing Gata2 expression in the endothelium and in a blood cluster (arrow). (v and vi) In situ hybridization for Fli1 expression. (v) Transverse cryosection through the AGM region of a E11.5 embryo showing Fli1 expression in the FL. (vi) Transverse paraffin section through the AGM region showing expression in the endothelium and blood clusters (arrow). (B) (i–iii) ChIP assays of blood progenitor (416B) and endothelial (MS1) cell lines and primary hematopoietic tissues (abdominal aorta and FL) from E11.5 embryos. Levels of enrichment were normalized to that obtained with a control rabbit antibody and are plotted as a fold-increase over that measured at a control region (Scl+21). (C) Variation in Gata2, Fli1, and Scl gene expression during in vitro differentiation of GFP-Bry ES cells to EBs. (D) Quantitative ChIP assay on in vitro differentiated, unsorted GFP-Bry EBs, of which ≈49% of cells were GFP⁺Flk1⁺ (DP) at day 3 (SI Fig 7). (E) A fully connected triad of haematopoietic TFs. The Gata2/Fli1/Scl triad with putative initiators. Direct binding interactions are represented by solid lines.