Fig. 3. Tbx3 regulates the proliferation and the cell-lineage determination of hepatoblasts. (A) CD45⁺, Ter119⁺, and c-Kit⁺ cells were gated and removed from the initial wild-type and Tbx3^-/- mouse liver tissue specimens (E12.5). The c-Kit^- CD45^- Ter119^- hepatic epithelial cells were then fractionated based on c-Met expression. For the in vitro colony assay, the sorting gate was set for the c-Met⁺ c-Kit^- CD45^- Ter119^- cell population. The ratios of c-Met⁺ cells in c-Kit^- CD45^- Ter119^- cells and in unfractionated total cells are shown by the percentage values outside and within the parenthesis, respectively. Wild-type cells stained with an isotype control antibody were used as a control. (B,C) Clonal colony formation upon 5-days single-cell culture of c-Met⁺ c-Kit^- CD45^- Ter119^- cells isolated from wild-type (B) or Tbx3^-/- (C) livers. (D) Numbers of large, medium and small colonies per 150 wells, as formed by wild-type or Tbx3^-/- liver-derived c-Met⁺ c-Kit^- CD45^- Ter119^- cells after 5 days of single-cell culture. The average of three independent experiments (mean ±s.d.). (E-G) Co-immunofluorescence staining of Alb and CK7 was conducted for clonal colonies formed by c-Met⁺ c-Kit^- CD45^- Ter119^- cells isolated from wild-type (E,F) and Tbx3^-/- (G) livers after 18 days of culture. Insets denote individual cells labeled with DAPI. (H) Following co-immunofluorescence staining of Alb and CK7, the percentage of Alb⁺, CK7⁺, Alb/CK7⁺, and Alb/CK7^- cells in each colony was determined. The average of 24 colonies (mean ±s.d.). Scale bars: 500 µm in B,C; 100 µm in E-G.