bcas2 is expressed in the ICM and required for primitive hematopoiesis.

(A) WISH assay showing bcas2 expression in the ICM at the 18-somite stage and 22 hpf. The dotted lines represent the section position and the black arrowheads indicate the ICM region. n, notochord. (B) Double FISH assay showing the expression pattern of bcas2 and gata1 in the ICM at 22 hpf. Scale bar, 50 μm. (C-D) Comparison of bcas2 expression in cloche mutants (C) or bcas2 heterozygous mutants (D) along with their corresponding siblings. (E-F) Expression analysis of gata1 and hbbe3 in bcas2Δ7+/- and bcas2Δ14+/- embryos. (G) Hemoglobin detection using o-dianisidine staining in bcas2 homozygous mutant at 36 and 48 hpf. (H) Representative images of yolk sac from the hemangioblast-specific Bcas2 knockout mice and their siblings. Bcas2F/F females were crossed with Bcas2F/+;Flk1-Cre males to induce the deletion of Bcas2 in hemangioblasts. Scale bars, 1 mm.

bcas2 is required for hematopoietic progenitor differentiation.

(A-C) Expression analysis of hemangioblast markers npas4l, scl, gata2 (A), erythroid progenitor markers gata1, hbbe3 (B), and myeloid marker pu.1 (C) in bcas2Δ14+/- embryos and their wild-type siblings at indicated stages. (D) Expression changes of gata1 in bcas2Δ14+/-embryos overexpressing BCAS2 at the 10-somite stage. The indicated embryos were injected with or without 300 pg of human BCAS2 mRNA at the one-cell stage. (E) Expression of endothelial marker fli1a in bcas2Δ14+/- and sibling embryos at the 10-somite stage. (G) Confocal imaging of bcas2Δ14+/-and control sibling Tg(kdrl:GFP) embryos at 54 hpf. Scale bars, 500 μm.

BCAS2 promotes primitive hematopoiesis via activating Wnt signaling.

(A-B) Overexpression of BCAS2 increases Wnt3a-induced TOPflash activity in HEK293T cells (A) and 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. *P < 0.05; **P < 0.01 (Student’s t-test). (C-E) The Wnt3a-induced TOPflash activity is decreased in BCAS2-deficient cells. HEK293T Cells were co-transfected with shRNA plasmids, along with indicated plasmids, and harvested for western blot analysis (C) or luciferase reporter assay (D). Bcas2-cKO MEFs prepared from Bcas2F/Fmouse embryos were incubated in medium containing 100 μM tamoxifen for 72 h and then subjected to western blotting and luciferase reporter assay (E). *P < 0.05; **P < 0.01 (Student’s t-test). (F-G) Expression analysis of gata1 (F) and hbbe3 (G) in Tg(hsp70l:dkk1b-GFP) embryos after heat shock at 16 hpf. (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 and collected at the 10-somite stage. The dotted lines show the GFP-positive hematopoietic progenitor cells. Scale bars, 5 μm. (I-J) Expression of hbbe3 in bcas2 morphants (I) and bcas2Δ14+/-mutants (J) overexpressing ΔN-β-catenin. Embryos were injected with the indicated MO together with ΔN-β-catenin mRNA at the one-cell stage and harvested at the 10-somite stage for in situ hybridization.

BCAS2 is essential for β-catenin nuclear accumulation.

(A-C) BCAS2 enhances LiCl-induced TOPflash activity in HEK293T cells. Cells were transfected with BCAS2 expression plasmids (A), shRNA plasmids (B), or S37A-β-catenin expression plasmids (C), together with the TOPflash luciferase and Renilla luciferase vectors. After transfection, cells were subsequently treated with or without 100 ng/ml LiCl for 12 h and assayed for luciferase activity. *P < 0.05; **P < 0.01 (Student’s t- test). (D-E) Bcas2-cKO MEFs were incubated with tamoxifen for 24 h and then treated with or without 100 ng/ml LiCl. The nuclear accumulation of β-catenin was analyzed using immunofluorescence (D) and western blotting (n=3) (E) Scale bars, 10 μm. (F) SW480 cells were transfected with the indicated shRNA constructs, and the endogenous β-catenin protein was detected using immunofluorescence 48 h after transfection. The expression of GFP served as a transfection control. The arrowheads indicate the cells transfected with indicated shRNA constructs. Scale bars, 10 μm. (G) Bcas2-cKO MEFs were cultured in the presence of tamoxifen for 24 h and then treated with 20 μM MG132 for 6 h. The expression of BCAS2 and β-catenin was measured by immunofluorescence. Scale bars, 10 μm.

BCAS2 functions in CRM1-mediated nuclear export of β-catenin.

(A) Tamoxifen-treated Bcas2-cKO MEFs were incubated with 20 nM LMB for 3 h. The expression of Bcas2 and β-catenin was analyzed using immunofluorescence. The arrowheads show the cells with nuclear β-catenin accumulation. Scale bars, 10 μm. (B) SW480 cells were transfected with the indicated shRNA constructs and then treated with LMB for 3 h before immunostaining. GFP was regarded as a transfection control. The arrowheads indicate the transfected cells. Scale bars, 10 μm. (C) Immunofluorescence staining of β-catenin in bcas2 morphants with Tg(gata1:GFP) background. Embryos were exposed to 20 nM LMB from the bud stage. The dotted lines indicate the GFP-positive hematopoietic progenitor cells. Scale bars, 5 μm. (D) bcas2Δ14+/- embryos were treated with 20 nM LMB for 6 h and then subjected to WISH assay to analyze the expression of gata1 at the indicated stages.

BCAS2 interacts with β-catenin.

(A-C) Flag-tagged β-catenin was co-transfected with or without HA-tagged BCAS2 into HEK293T cells. Cell lysates were immunoprecipitated using anti-Flag antibody. Eluted proteins were analyzed by western blotting using indicated antibodies. In panel C, for Wnt signaling activation, cells were treated with Wnt3a CM for 5 h before harvest. (D-E) YN-BCAS2 and YC-β-catenin were either individually or collectively transfected into HeLa cells. The expression of YN-BCAS2 and YC-β-catenin was analyzed with anti-GFP antibody (D). The reconstituted YFP fluorescence in living cells was detected by confocal laser scanning microscopy with excitation at 488 nm (E). Scale bars, 10 μm. (F) Schematics of full-length and deletion mutants of β-catenin. (G) HEK293T cells were transfected with HA-tagged BCAS2 and Flag-tagged deletion mutants of β-catenin. Cell lysates were then immunoprecipitated using anti-Flag antibody followed by western blot analysis. (H) GST pull-down assays were performed using bacterially expressed GST, GST-ARM1-12, and His-BCAS2.

BCAS2 sequesters β-catenin in the nucleus via its CC domains.

(A) Schematics of full length and deletion mutants of BCAS2. (B-C) HEK293T cells were transfected with Flag-β-catenin and indicated deletion mutants of BCAS2. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Eluted proteins were immunoblotted using anti-HA (B) or anti-GFP antibodies (C) for BCAS2 detection. (D) HEK293T cells transfected with the indicated plasmids were treated with 100 ng/ml LiCl for 12 h, and then subjected to luciferase assay. Note that overexpression of BCAS2 without the CC domains failed to increase LiCl-induced TOPflash activity. Data represent the mean ± SD of three independent experiments. ns, not significant; **P < 0.01 (Student’s t-test). (E) Immunofluorescence staining of β-catenin in Tg(gata1:GFP) embryos injected with 8 ng bcas2 MO and 300 pg of full length BCAS2 mRNA or ΔCC1-2 mRNA at the one-cell stage. Scale bars, 5 μm. (F) Transcripts of gata1 were evaluated by WISH in bcas2Δ14+/- embryos injected with 300 pg of BCAS2 mRNA or ΔCC1-2 mRNA.