|
Fig. 3 BCAS2 promotes primitive hematopoiesis via activating Wnt signaling. (A, B) Overexpression of BCAS2 increases Wnt3a-induced TOPflash activity in HEK293T cells (A) and mouse embryonic fibroblasts (MEFs) (B). Different amounts of plasmid expressing BCAS2 (0, 80, 160, or 320 ng/well) were transfected into cells, together with the super-TOPflash luciferase and Renilla luciferase vectors. After 36 h of transfection, cells were treated with or without Wnt3a CM for 12 h and harvested for luciferase assays (n=3). *p<0.05; **p<0.01 (Student’s t-test). (C) The Wnt3a-induced TOPflash activity is decreased in BCAS2-deficient cells. HEK293T cells were transfected with shRNA plasmids, along with indicated plasmids, and harvested for luciferase reporter assay (n=3). *p<0.05 (Student’s t-test) (D) Bcas2-cKO MEFs prepared from Bcas2F/F mouse embryos were incubated in medium containing 100 μM tamoxifen for 72 h and then subjected to western blotting and luciferase reporter assay (n=3). **p<0.01 (Student’s t-test). (E, F) Expression analysis of gata1 (E) and hbbe3 (F) in Tg(hsp70l:dkk1b-GFP) embryos after heat shock at 16 hpf. (G, H) Immunofluorescence staining of β-catenin in Tg(gata1:GFP) embryos at 16 hpf. The embryos were injected with 8 ng of the indicated MO at the one-cell stage. The dotted lines show the GFP-positive hematopoietic progenitor cells. The relative fluorescence intensity of nuclear β-catenin was quantified in (H) (n=6). **p<0.01 (Student’s t-test). (I, J) Expression of hbbe3 in bcas2 morphants (I) and bcas2+/Δ14 mutants (J) overexpressing ΔN-β-catenin. Embryos were injected with the indicated MO together with ΔN-β-catenin mRNA at the 1-cell stage and harvested at the 10-somite stage for in situ hybridization. Scale bars, 100 μm (E, F, I, J), 5 μm (G).