Fig. 1

Rasip1 expression in IAHCs in the E10.5 AGM region. A Upper panel: The expression profile of CD45, c-Kit, CD31, and VE-Cad in the E10.5 AGM region. The numbers from 1 to 4 indicate cell populations separated by the expression profile: (1) CD45⁻c-Kit⁻CD31⁺ VE-cad⁻ cells (endothelial cells); (2) CD45⁻c-Kit⁻CD31⁺ VE-cad⁺ cells (endothelial cells); (3) CD45^lowc-Kit^high cells (containing HSPCs); (4) CD45⁺c-Kit⁻ cells (hematopoietic cells). Lower panel: RT-PCR shows the relative expression levels of Rasip1 in these 4 populations. B Whole-mount immunofluorescence staining of Rasip1 (green), CD31 (red), and c-Kit (cyan) in IAHCs of the E10.5 AGM region. The Rasip1 expression on the membrane of IAHCs. The nuclei were stained by Hoechst 33,258 (blue). Scale bar, 20 µm. C Whole-mount immunofluorescence staining of Sox17 (green), Rasip1 (red), and c-Kit (cyan) in IAHCs of the E10.5 AGM region. The nuclei were stained by Hoechst 33,258 (blue). Rasip1 is expressed in most part of c-Kit⁺ Sox17⁺ cells in IAHCs. Scale bar, 20 µm. D A genetic scheme showing the location of the Rasip1 gene and upstream genes in mouse chromosome 7.5 putative Sox17-binding sites were indicated by squares with numbers 1 through 5. Upper panel: Requirement of the proximal putative Sox17-binding site (5) for induction of the Rasip1 gene by Sox17. NIH3T3 cells (1 × 10⁶) were transfected with a pGL3 vector containing the putative Rasip1 enhancer sequences. pRL-CMV was co-transfected as an internal control. The region Rasip1 [1-3] (979 bp) has three putative Sox17- binding sites (1, 2, and 3), and the region Rasip1 [2-3] (602 bp) has two putative Sox17-binding sites (2 and 3). The region Rasip1 [4-5] (720 bp) has putative Sox17-binding sites 4 and 5. Two types of Sox17-binding sites are shown in the enhancer regions: (1) AATGGCG (Sox17-binding sites 2, 4) and (2) ATTGT (Sox17-binding sites 1, 3, 5). Lower panel: Inhibition of the Sox17-induced Rasip1 gene activation by a mutation in the putative Sox17-binding site 5. NIH3T3 cells (1 × 10⁶) were transfected with a pGL3 vector containing the putative Rasip1 enhancer sequences with point mutations in the putative Sox17-binding sites 4 or 5, or both together with plasmids encoding wild-type (WT) Sox17 or Sox17G103R which lost the DNA binding ability. In this experiment, pRL-TK was co-transfected as an internal control. A solid black line indicates the Rasip1 [4-5] region, solid grey lines show the pGL3 vector and Rasip1 enhancer region [1-3] as controls. E. Enzyme-digested chromatin-derived DNA. DNA from the enzyme-digested chromatin was analyzed by electrophoresis. Two bars on the right show the presence of optimally digested DNA F. ChIP assay. The digested chromatin sample was immunoprecipitated with either the anti-Sox17 antibody or normal goat IgG. DNA samples from the immunoprecipitates were subjected to PCR to amplify the region containing the Sox17-binding site 5 (Rasip1 [5]; see Fig. 1D top diagram). The expected PCR fragment length is 223 bp. Similar results were obtained by 2 independent sets of PCR amplifications of the samples from 2 independent preparations of cells from separately obtained E10.5 embryos purchased on different days