Fig. 2. Defects in proliferation and differentiation of hepatic epithelial cells in Tbx3^-/- embryos. (A) Morphology of Tbx3^+/+ wild-type and Tbx3^-/- mouse embryos (E12.5). Besides the abnormal development of Tbx3^-/- hind limbs (upper and middle panels), the Tbx3^-/- liver was much smaller than that of wild type (bottom panel). (B-E) Hematoxylin and Eosin (HE) staining of liver from wild-type (B,D) and Tbx3^-/- (C,E) embryos. (F-I) BrdU immunofluorescent images of wild-type (F,H) and Tbx3^-/- (G,I) liver cells. (J-Q) When compared with the wild-type liver (J,L,N,P), the number of E-cadherin⁺ epithelial cells, but not of N-cadherin⁺ (E-cadherin^-) mesenchymal cells, was significantly smaller in the absence of Tbx3 (M). Within Tbx3-deficient liver epithelial cells, primitive hepatic cells and differentiating hepatocytes that expressed Hnf4 and Alb constituted only a small population (O,Q), whereas the number of CK7⁺ cholangiocytes was relatively large (Q). Insets denote individual cells as labeled with DAPI. (R) Gene expression analysis by qPCR for E12.5 liver from wild-type, Tbx3^+/- and Tbx3^-/- embryos. Two mice of each genotype were analyzed separately. All data were normalized to the values of a wild-type liver and fold differences are shown. Bar represents mean ±s.d. (n=3). Scale bars: 200 µm in B,C; 100 µm in F-I; 50 µm in D,E,J-Q.