Fig. 4. Negative regulation of p19^ARF by Tbx3 is required for controlling the proliferation and the hepatobiliary lineage segregation of hepatoblasts. (A) RT-PCR analysis revealed that in Tbx3^-/- mouse livers, expression of p19^ARF and p21^WAF1/CIP1 was upregulated, but p53 expression was unaffected. (B-G) Immunofluorescence staining of p19^ARF was conducted for clonal colonies formed by c-Met⁺ c-Kit^- CD45^- Ter119^- cells isolated from wild-type (B-D) and Tbx3^-/- (E-G) livers after 5 days of culture. Representative data from 18 colonies are shown. (H-J) Cells in clonal colonies formed by Tbx3^-/- liver-derived c-Met⁺ c-Kit^- CD45^- Ter119^- cells were transiently transfected with Myc-Tbx3 and subsequently stained using antibodies against Myc-Tag (H) and p19^ARF (I). Arrowheads indicate Myc-Tbx3-transfected cells. Representative data from 11 colonies are shown. (K-S) Cells in cultures of wild-type liver-derived c-Met⁺ c-Kit^- CD45^- Ter119^- cells were transiently transfected with p19^ARF-IRES-eGFP. Then, the percentage of cells immunoreactive for BrdU (K) or CK7 (L) was analyzed by flow cytometry. Vehicle-transfected cells and non-transfected (eGFP^-) cells were used as controls. Bar charts represent the average of three independent experiments (mean ±s.d.). Immunofluorescence staining of CK7 for vehicle- or p19^ARF-IRES-eGFP-transfected cells confirmed intense CK7 expression in p19^ARF (eGFP)-expressing cells (M-R). Also, CK19 and CK7 transcripts were increased in cells expressing p19^ARF (eGFP), as assessed by RT-PCR following flow-cytometric isolation of eGFP⁺ cells (S). (T) A proposed mechanism underlying the regulation of proliferation and differentiation of hepatoblasts by Tbx3. Scale bars: 50 µm in B-J; 25 µm in M-R.