First published online 20 March 2008
doi: 10.1242/dev.016634
Development 135, 1589-1595 (2008)
Published by The Company of Biologists 2008
Tbx3 controls the fate of hepatic progenitor cells in liver development by suppressing p19ARF expression
Atsushi Suzuki1,2,*,
Sayaka Sekiya1,
Dirk Büscher3,
,
Juan Carlos Izpisúa Belmonte3,4 and
Hideki Taniguchi2,5
1 Division of Organogenesis and Regeneration, Post-Genome Science Center,
Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi,
Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.
2 Research Unit for Organ Regeneration, Center for Developmental Biology, RIKEN,
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
3 Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N.
Torrey Pines Rd., La Jolla, CA 92037, USA.
4 Center of Regenerative Medicine in Barcelona, Doctor Aiguader 88, 08003
Barcelona, Spain.
5 Department of Regenerative Medicine, Graduate School of Medicine, Yokohama
City University, 3-9 Fuku-ura, Kanazawa-ku, Yokohama, Kanagawa 236-0004,
Japan.
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Fig. 1. Specific expression of Tbx3 in hepatoblasts isolated from the developing
mouse liver. (A) Of the 17 mouse T-box genes, Eomes, Tbx3,
Tbx6, Tbx10, Tbx12, Tbx15 and Tbx20 were expressed in the E13.5
mouse liver, as assessed by RT-PCR. The upper band in the Tbx3 lane
represents a transcript variant of this gene
(Bamshad et al., 1999 ).
(B) Flow-cytometric fractionation of E13.5 liver cells into
CD45+, Ter119+, c-Kit+, c-Kit-
CD45- Ter119-, c-Met- c-Kit-
CD45- Ter119-, and c-Met+ c-Kit-
CD45- Ter119- cell populations. Cells in each fraction
were sorted and analyzed. (C,D) RT-PCR analyses of T-box gene
expression in cells sorted from each fraction shown in B. Erythroid
cell-specific glycophorin A was recognized by anti-Ter119 antibody.
(E,F) Hnf4 (E) and Tbx3 (F) immunofluorescent images of
E13.5 liver sections. Insets denote individual cells as labeled with DAPI
(blue). (G-I) Co-immunofluorescence staining of Hnf4 and Tbx3
showed that a subpopulation of Hnf4+ cells was marked by
either strong or weak staining of Tbx3. (J) All Tbx3+ cells
expressed E-cadherin. (K,L) Tbx3 was expressed in
Alb-/low primitive hepatic cells (arrowheads) and in a
subpopulation of Alb+ differentiating hepatocytes (K), and in a few
CK7+ cholangiocytes (L). Scale bars: 100 µm in E,F; 50 µm in
I; 25 µm in J-L.
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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.
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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.
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Fig. 4. Negative regulation of p19ARF 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
p19ARF and p21WAF1/CIP1 was
upregulated, but p53 expression was unaffected. (B-G)
Immunofluorescence staining of p19ARF 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 p19ARF (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 p19ARF-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 p19ARF-IRES-eGFP-transfected cells confirmed intense
CK7 expression in p19ARF (eGFP)-expressing cells (M-R). Also,
CK19 and CK7 transcripts were increased in cells expressing
p19ARF (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.
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