![Figure 2. Both the Runx1 P1 and P2 promoters are active in the emerging hematopoietic system when combined with the Runx1 +23 enhancer. (A) Summary of P1 and P2 promoter activity in P1 LacZ +23 and P2 LacZ +23 F0 transgenic mouse embryos. The activity of the hsp68LacZ +23 reporter construct9 is shown for comparison. +23-specific staining indicates staining in the E8 YS blood islands, in hematopoietic clusters and few endothelial/mesenchymal cells of the E10 dorsal aorta and vitelline and umbilical arteries, and in E12 FL hematopoietic cells, as previously observed in hsp68LacZ +23 F0 transgenic embryos.9 Nonspecific staining, or no staining, is presumably the result of random integration of the constructs at or near endogenous enhancers, or in heterochromatin, respectively. (B) Representative photographs of the +23-specific Xgal staining in P1 LacZ +23 (Bi-iii) and P2 LacZ +23 (Biv-vi) F0 transgenic embryos. (Bi,iv) Staining in E8 YS blood islands. (Bii,v) Staining in emerging clusters and few endothelial cells in the E10 dorsal aorta. (Biii,vi) Staining in E12 FL hematopoietic cells. ▶ points at examples of Xgal-stained cells. Figure S3 shows negative control. (C) Schematic of the Runx1 P1 promoter region showing the location and sequence of the deeply conserved pair of Runx motifs. Basepairs critical for DNA binding were mutated as shown. Pink denotes the area of noncoding sequence conservation spanning the promoter; light blue, the UTR; dark blue, the coding sequence of exon 1. (D) Summary of mutated P1 promoter activity in P1mut LacZ +23 F0 transgenic mouse embryos. The lack of expression in the E8 P1mut LacZ +23 F0 transgenic embryos is probably a frequency issue (only 2 of 16 P1 LacZ +23 embryos [Figure 2A] were Xgal+). (E) Mutation of the deeply conserved P1 Runx motifs does not alter the activity of the P1mutLacZ +23 construct in vivo. Representative Xgal staining in P1mutLacZ +23 F0 transgenic mouse embryos in E10 YS (Ei), in emerging hematopoietic clusters of the E10 vitelline artery (Eii) and dorsal aorta (Eiii), and in E12 FL cells (Eiv). ▶ points at examples of Xgal-stained cells. Photographs were taken using a Nikon Eclipse E600 microscope equipped with a 20× Nomarski objective and a Nikon DXM 1200c Digital Camera (Nikon, Tokyo, Japan) and processed using Adobe Photoshop (Adobe Systems Europe, Uxbridge, United Kingdom). Scale bar represents 100 μm.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/113/21/10.1182_blood-2008-12-193003/6/zh89990936270002.jpeg?Expires=1724235214&Signature=slLz8eYJbfz1Y6Swu0cCwcY0nyrDR09NObbUc~RefV1-Qrmf0NvUT431K2YbgGaB4JiYjuTELvasD7--cdq46sVG3-BUyLY7yjGe0g3LxV-DvKMXgDjADy9uy7AU1QzKUtwPY18hDqWhAXrVJOXEcPJ75tGysXxRcWRcC29dCt3ngQENc~EI1JYLVXEHg0NU2c093ZeYMNp4llQh5-eYdXu98pCoxSOwNHHVkmxE1pmZXa3icWU9t0pf8M07NvYQ0dKBvXe88bRJkVt6S6nntgAjiNUgWxu4dRkS5RiG-9PwXU53lP8N10~WEMdCodIGBy7D68vZPEqC2AbNzhZGSA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Both the Runx1 P1 and P2 promoters are active in the emerging hematopoietic system when combined with the Runx1 +23 enhancer. (A) Summary of P1 and P2 promoter activity in P1 LacZ +23 and P2 LacZ +23 F0 transgenic mouse embryos. The activity of the hsp68LacZ +23 reporter construct9 is shown for comparison. +23-specific staining indicates staining in the E8 YS blood islands, in hematopoietic clusters and few endothelial/mesenchymal cells of the E10 dorsal aorta and vitelline and umbilical arteries, and in E12 FL hematopoietic cells, as previously observed in hsp68LacZ +23 F0 transgenic embryos.9 Nonspecific staining, or no staining, is presumably the result of random integration of the constructs at or near endogenous enhancers, or in heterochromatin, respectively. (B) Representative photographs of the +23-specific Xgal staining in P1 LacZ +23 (Bi-iii) and P2 LacZ +23 (Biv-vi) F0 transgenic embryos. (Bi,iv) Staining in E8 YS blood islands. (Bii,v) Staining in emerging clusters and few endothelial cells in the E10 dorsal aorta. (Biii,vi) Staining in E12 FL hematopoietic cells. ▶ points at examples of Xgal-stained cells. Figure S3 shows negative control. (C) Schematic of the Runx1 P1 promoter region showing the location and sequence of the deeply conserved pair of Runx motifs. Basepairs critical for DNA binding were mutated as shown. Pink denotes the area of noncoding sequence conservation spanning the promoter; light blue, the UTR; dark blue, the coding sequence of exon 1. (D) Summary of mutated P1 promoter activity in P1mut LacZ +23 F0 transgenic mouse embryos. The lack of expression in the E8 P1mut LacZ +23 F0 transgenic embryos is probably a frequency issue (only 2 of 16 P1 LacZ +23 embryos [Figure 2A] were Xgal+). (E) Mutation of the deeply conserved P1 Runx motifs does not alter the activity of the P1mutLacZ +23 construct in vivo. Representative Xgal staining in P1mutLacZ +23 F0 transgenic mouse embryos in E10 YS (Ei), in emerging hematopoietic clusters of the E10 vitelline artery (Eii) and dorsal aorta (Eiii), and in E12 FL cells (Eiv). ▶ points at examples of Xgal-stained cells. Photographs were taken using a Nikon Eclipse E600 microscope equipped with a 20× Nomarski objective and a Nikon DXM 1200c Digital Camera (Nikon, Tokyo, Japan) and processed using Adobe Photoshop (Adobe Systems Europe, Uxbridge, United Kingdom). Scale bar represents 100 μm.
