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First published online 20 March 2008
doi: 10.1242/dev.016634
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Research Report |
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.
* Author for correspondence (e-mail: suzukicks{at}bioreg.kyushu-u.ac.jp)
Accepted 25 February 2008
SUMMARY
Although the T-box family of transcription factors function in many different tissues, their role in liver development is unknown. Here we show that Tbx3, the T-box gene that is mutated in human ulnar-mammary syndrome, is specifically expressed in multipotent hepatic progenitor cells, `hepatoblasts', isolated from the developing mouse liver. Tbx3-deficient hepatoblasts presented severe defects in proliferation as well as uncontrollable hepatobiliary lineage segregation, including the promotion of cholangiocyte (biliary epithelial cell) differentiation, which thereby caused abnormal liver development. Deletion of Tbx3 resulted in the increased expression of the tumor suppressor p19ARF (Cdkn2a), which in turn induced a growth arrest in hepatoblasts and activated a program of cholangiocyte differentiation. Thus, Tbx3 plays a crucial role in controlling hepatoblast proliferation and cell-fate determination by suppressing p19ARF expression and thereby promoting liver organogenesis.
Key words: Tbx3, Liver, Hepatoblast, p19ARF (Cdkn2a), Differentiation, Mouse
INTRODUCTION
The identification of transcription factors that regulate proliferation and
differentiation of organ progenitor cells is crucial for understanding the
fundamental mechanisms of development, regeneration, and disorders of any
given organ. In the vertebrate developing liver, multipotent hepatic
progenitor cells (also known as hepatoblasts) proliferate and give rise to
both hepatocytes and cholangiocytes as descendants
(Lemaigre and Zaret, 2004
).
This essential event in liver organogenesis requires transcription factors
that act either as central inducers or suppressors for the proliferation and
differentiation of these cells. For example, hepatic nuclear factor 4
(Hnf4), a transcription factor in the nuclear receptor family,
activates many downstream genes responsible for hepatocyte differentiation,
including the homeodomain transcription factor Hnf1
(Tian and Schibler, 1991;
Li et al., 2000). The
homeodomain transcription factor Hnf6 (Onecut1) is believed to attenuate early
biliary commitment of hepatoblasts, but later, Hnf6 also positively regulates
cholangiocyte differentiation and bile duct morphogenesis
(Clotman et al., 2002;
Suzuki et al., 2003a). The
cell-lineage restriction into hepatocytes or cholangiocytes is additionally
controlled by the basic leucine-zipper transcription factor C/EBP
(CCAAT/enhancer binding protein ; Cebp), and deletion or
suppression of C/EBP blocks hepatocyte differentiation and
concomitantly induces biliary development
(Tomizawa et al., 1998;
Suzuki et al., 2003a;
Yamasaki et al., 2006).
Our previous studies enabled the prospective isolation of hepatoblasts from
the developing mouse liver by combining flow cytometry and
fluorescence-conjugated antibodies (Suzuki
et al., 2000; Suzuki et al.,
2002). In particular, cells marked by the hepatocyte growth factor
(Hgf) receptor c-Met displayed distinctive activities in response to Hgf
stimulus, including self-renewing cell divisions and differentiation into both
hepatocytes and cholangiocytes (Suzuki et
al., 2002; Suzuki et al.,
2003a). Isolating c-Met+ c-Kit-
CD45- Ter119- (also known as Met, Kit, Ptprc and Ly76,
respectively - Mouse Genome Informatics) cells achieved a much higher
enrichment of hepatoblasts, and thus this method could facilitate the
identification of a discrete set of transcription factors that are activated
in this specific cell population. Using this strategy, we examine here the
developmental role of the T-box family of transcription factors in the
proliferation and differentiation of hepatoblasts.
MATERIALS AND METHODS
Immunostaining
Hepatic tissue sections and cultured cells were fixed and incubated with
primary antibodies against Hnf4 (Santa Cruz, Santa Cruz, CA), Tbx3
(Santa Cruz), BrdU (Amersham, Little Chalfont, UK), E-cadherin (BD
Biosciences, San Jose, CA), N-cadherin (BD Biosciences), albumin (Bethyl,
Montgomery, TX, for tissue sections; Biogenesis, Poole, UK, for cultured
cells), CK7 (Chemicon, Temecula, CA), cleaved caspase 3 (Cell Signaling,
Danvers, MA), PCNA (Santa Cruz), p19ARF (Abcam, Cambridge, UK) and
Myc-Tag (Cell Signaling). Detailed information on the antibodies is available
upon request. After washing, the sections and the cells were incubated with
Alexa 488- and/or Alexa 555-conjugated secondary antibodies specific to the
appropriate species (1:200; Molecular Probes, Eugene, OR), followed by
incubation with DAPI.
Gene expression analysis
Reverse transcriptase (RT)-PCR and quantitative (q)PCR were conducted as
described (Suzuki et al.,
2003a; Suzuki et al.,
2003b). PCR primers and probes are described in our previous
papers (Suzuki et al., 2000;
Suzuki et al., 2002;
Suzuki et al., 2003a) and are
available upon request. For qPCR analysis of p19ARF
expression, we used SYBR Premix Ex Taq II (Takara, Japan) according to the
manufacturer's instructions.
Flow cytometry and cell culture
Single-cell suspensions were prepared from the livers of wild-type,
Tbx3+/- or Tbx3-/- mouse embryos and
incubated with fluorescence-conjugated antibodies as described
(Suzuki et al., 2002). We used
phycoerythrin (PE)-Cy7-conjugated anti-CD45, Ter119 monoclonal antibodies
(mAbs) (Pharmingen, San Jose, CA), allophycocyanin (APC)-conjugated anti-c-Kit
mAb (Pharmingen), and fluorescein isothiocyanate (FITC)-conjugated anti-c-Met
mAb. The c-Met mAb was produced in cultures of a hybridoma cell line raised by
fusing mouse myeloma cells to rat lymphocytes obtained by inoculating rats
with 293T cells expressing the entire coding sequence of mouse c-Met
fused to a C-terminal Flag-Tag (MBL, Nagoya, Japan). BrdU-incorporating cells
were stained using the APC BrdU Flow Kit (BD Biosciences, San Jose, CA). The
fluorescence-labeled cells were analyzed and separated with FACS Aria (BD
Biosciences). For single-cell culture analysis, cells identified on clone
sorting by FACS Aria were cultured in individual wells of
type-IV-collagen-coated 96-well plates, and clonal colonies formed from each
cell were analyzed as described (Suzuki et
al., 2002).
|
RESULTS AND DISCUSSION
The T-box genes, defined by a common DNA-binding T-box domain, are involved
in many aspects of embryonic and extraembryonic tissue development
(Naiche et al., 2005). Until
now, however, there has been no report regarding the contribution of T-box
genes in the developing or adult liver. As an initial approach, we examined
the hepatic expression of multiple T-box genes in E13.5 mouse embryos and
identified the specific expression of Eomes, Tbx3, Tbx6, Tbx10, Tbx12,
Tbx15 and Tbx20 (Fig.
1A). To determine which cell type(s) expressed these genes, cells
that were first fractionated into either CD45+, Ter119+,
c-Kit+, or c-Kit- CD45- Ter119-
cell populations were isolated separately and analyzed
(Fig. 1B). Significantly, only
Tbx3, the T-box gene that is mutated in human ulnar-mammary syndrome
(Bamshad et al., 1997), was
expressed in c-Kit- CD45- Ter119- hepatic
epithelial cells, and Tbx3 expression was restricted to this cell
population (Fig. 1C). Further
fractionation of c-Kit- CD45- Ter119- cells
into c-Met+ or c-Met- cells revealed that Tbx3
expression was much higher in the c-Met+ c-Kit-
CD45- Ter119- hepatoblast population
(Fig. 1B,D). Immunofluorescence
staining revealed that although Hnf4+ primitive hepatic
cells coexpressed Tbx3 in E9.5 and E10.5 hepatic primordia (see Fig. S1A-L in
the supplementary material), by E13.5 Tbx3 was detected only in a portion of
Hnf4+ cells, including primitive hepatic cells and
differentiating hepatocytes (Fig.
1E-I). The Tbx3+ Hnf4+ cells in E13.5
liver were also marked by the epithelial cell marker E-cadherin and were
categorized into albumin-/low (Alb-/low) primitive
hepatic cells and Alb+ differentiating hepatocytes, but scarcely
into cytokeratin 7+ (CK7; also known as Krt7 - Mouse Genome
Informatics) cholangiocytes (Fig.
1J-L). In the later stages of liver development, however, Tbx3
expression decreased and became faint in Hnf4+ cells during
the advancement of hepatocyte differentiation (see Fig. S1M-V in the
supplementary material). These results suggest that Tbx3 plays a role in an
early phase of hepatogenesis, especially in the regulation of hepatoblast
activities.
|
). The E12.5 Tbx3-/- embryonic liver was
much smaller than those from Tbx3+/+ wild-type or
Tbx3+/- heterozygous embryos
(Fig. 2A-C, and data not
shown). Adhesion of epithelial cells in the Tbx3-/- liver
appeared to be normal, but there were many cavities in which epithelial cells
were largely replaced by hematopoietic cells, suggesting a decrease in and an
abnormality of epithelial cells (Fig.
2B-E). Indeed, when compared with cells from the wild-type liver,
the number of BrdU-incorporating or PCNA+ proliferating cells
decreased substantially in the absence of Tbx3
(Fig. 2F-I and see Fig. S2 in
the supplementary material) without any increase in the number of apoptotic
cells (see Fig. S3 in the supplementary material). Additionally, in the
Tbx3-/- embryonic liver, the number of
E-cadherin+ epithelial cells was also significantly reduced,
whereas the number of N-cadherin+ (E-cadherin-)
mesenchymal cells was not affected (Fig.
2J-M). These data demonstrated an essential role of Tbx3 in
activating the proliferation of immature hepatic epithelial cells. Those
Tbx3-deficient epithelial cells were composed of a few primitive
hepatic cells and differentiating hepatocytes that were marked by the
expression of Hnf4 and Alb, as well as a relatively large number of
CK7+ cholangiocytes (Fig.
2N-Q). The percentages of PCNA+ cells in
Hnf4+ cells were 68.8% and 27.3% in the livers of wild-type
and Tbx3-/- embryos, respectively. Quantitative PCR (qPCR)
analysis also demonstrated that the expression levels of genes encoding
hepatocyte differentiation markers [Alb and -1-antitrypsin (AT;
Serpina1 - Mouse Genome Informatics)], primitive hepatic cell markers
[-fetoprotein (Afp) and c-Met], and transcription factors involved in
the early stage of hepatocyte differentiation (C/EBP, Hnf1 and
Hnf4) were all markedly diminished in the Tbx3-/-
embryonic liver, although the expression of genes encoding cholangiocyte
markers [CK19 (Krt19) and CK7] and transcription factors that control
cholangiocyte differentiation (Hnf1β and Hnf6)
(Clotman et al., 2002;
Coffinier et al., 2002) were
all upregulated (Fig. 2R).
Therefore, in the developing liver, Tbx3 acts not only as an activator for
proliferation, but also as a regulator for hepatobiliary lineage
segregation.
|
;
Suzuki et al., 2002). As a
result of deleting the Tbx3 gene, colony formation by
c-Met+ c-Kit- CD45- Ter119- cells
was significantly impaired, and only small colonies formed, despite cell
attachment onto the bottom of wells being normal
(Fig. 3C,D). At day 18 of
culture, differentiating Alb+ hepatocytes (
25%) and
CK7+ cholangiocytes (60%) appeared in colonies formed by
wild-type cells (Fig. 3E,F,H).
In the case of Tbx3-/- colonies, however, 95% of the cells
became CK7+ cholangiocytes, and only a few Alb+
hepatocytes (2%) emerged (Fig.
3G,H). Thus, Tbx3-deficient hepatoblasts suffered severe
defects in proliferation and differentiated more efficiently into
cholangiocytes. To verify these findings further, we introduced Tbx3 short
hairpin RNA (Tbx3-shRNA) into in vitro propagating progeny from a single
hepatoblast derived from the c-Met+ c-Kit-
CD45- Ter119- cell population (see Fig. S4A in the
supplementary material). In the culture of these cells, many cells (80%)
expressed Tbx3 (see Fig. S4B in the supplementary material). Consistent with
the results from the Tbx3-/- liver study, efficient
suppression of endogenous Tbx3 expression inhibited hepatoblast proliferation,
repressed the expression of several genes activated during hepatocyte
differentiation, and enhanced cholangiocyte marker expression (see Fig.
S4C,E-H in the supplementary material). Taken together, we conclude that Tbx3
plays an essential role in hepatogenesis by controlling the proliferation and
the cell-lineage decision of hepatoblasts.
|
). In the
intestine, the molecular mechanisms governing the homeostatic self-renewal of
stem cells and the aberrant proliferation of colorectal cancer cells are
similar (Radtke and Clevers,
2005). Thus, in hepatoblasts, Tbx3 might function as it does in
malignant cancer cells. Tbx3 is known as a transcriptional repressor for
p19ARF (Cdkn2a - Mouse Genome Informatics) and
p14ARF (the human ortholog of p19ARF),
the tumor suppressor genes that stabilize p53 (Trp53) via inactivation of Mdm2
in response to a variety of oncogenic stresses
(Honda and Yasuda, 1999;
Brummelkamp et al., 2002;
Lingbeek et al., 2002;
Kim and Sharpless, 2006). This
evidence suggests that in cancer cells, Tbx3 negatively regulates
p19ARF expression to induce escape from cellular
senescence and to activate mitosis. To determine whether this regulatory
mechanism is also active in hepatoblasts, we first analyzed hepatic
p19ARF expression in wild-type and
Tbx3-/- embryos. Intriguingly, liver cells lacking Tbx3
upregulated the expression of p19ARF and, to a lesser
extent, the cyclin-dependent kinase inhibitor p21WAF1/CIP1
(Cdkn1a - Mouse Genome Informatics)
(Fig. 4A). This
p21WAF1/CIP1 expression might be activated either by
p19ARF-dependent p53 stabilization
(el-Deiry et al., 1993), or as
a direct result of Tbx3 deficiency
(Prince et al., 2004). Cells
in colonies formed by c-Met+ c-Kit- CD45-
Ter119- cells isolated from either wild-type or
Tbx3-/- liver expressed p19ARF at day 5 of
culture, but its expression level was much higher in the cultures of
Tbx3-/- liver cells
(Fig. 4B-G). When Tbx3
expression was restored by transgenes, a significant decrease of
p19ARF expression was exhibited in Tbx3-/-
colony cells (Fig. 4H-J). In
addition, the defect in proliferation of Tbx3-shRNA-expressing hepatoblast
progeny in culture was partially rescued by introducing
p19ARF-small interfering RNA (p19ARF-siRNA) (see Fig.
S4D,E in the supplementary material). These results associate Tbx3 with the
negative regulation of p19ARF in proliferating hepatoblasts.
Moreover, to characterize the relationship between p19ARF
activation and the promotion of cholangiocyte differentiation in the
Tbx3-/- liver, p19ARF was introduced
into cultures of wild-type c-Met+ c-Kit-
CD45- Ter119- cells. Consistent with the data from the
Tbx3-/- liver, the overexpression of
p19ARF resulted in a significant reduction in the number
of proliferating cells (Fig.
4K). Surprisingly, p19ARF overexpression was
also effective in upregulating the expression of CK19 and
CK7 and increased the number of CK7+ cholangiocytes
(Fig. 4L-S). Thus, the growth
arrest induced by active p19ARF in the absence or suppression of
Tbx3 function is sufficient to promote cholangiocyte differentiation from
hepatoblasts.
These findings uncovered unique and unexpected roles for Tbx3 in
controlling the proliferation and the differentiation of hepatoblasts during
liver development (Fig. 4T).
The phenotypic features of Tbx3-/- embryos, including a
diminished liver size, have also been observed in another Tbx3 mutant
mouse line, but such liver defects were only discussed as an effect of
deficiencies in the yolk sac or in hematovascular development
(Davenport et al., 2003). In
our study, although there was no previous evidence to implicate a role for
Tbx3 in hepatogenesis, searching for T-box genes that are expressed
specifically in hepatoblasts led to the identification of Tbx3 as an essential
regulator for the proliferation and differentation of hepatoblasts. Therefore,
the phenotypic alterations in the Tbx3-/- embryonic liver
arose as a direct consequence of the developmental defects of hepatoblasts,
although the subsequent misinteractions of hepatoblasts with other hepatic
components might also be relevant to that phenotype.
The mechanisms controlling the segregation of hepatobiliary lineages have
been a challenging area to understand, as many important genes, including
those encoding Hnf4, Hnf1, Hnf6, Hnf1β and C/EBP,
have been found to be involved (Tian and
Schibler, 1991; Tomizawa et
al., 1998; Li et al.,
2000; Clotman et al.,
2002; Coffinier et al.,
2002; Suzuki et al.,
2003a; Yamasaki et al.,
2006). Our present data indicate that under the loss of Tbx3
function, the growth arrest induced by p19ARF is important for
activating a program of cholangiocyte differentiation in hepatoblasts,
including the upregulation or downregulation of many hepatic transcription
factors. Conversely, the repression of p19ARF expression
by Tbx3 allows hepatoblasts to proliferate and provides these cells with an
alternative fate, such as the differentiation into hepatocytes. These facts
are further supported by evidence that in the developing liver, both
Alb- and Alb+ cells, but few CK7+ cells, are
found in Tbx3+ cells (Fig.
1K,L). Therefore, although a transcriptional hierarchy involved in
the lineage determination of hepatoblasts should be elucidated in future
analyses, Tbx3 might act as a central regulator for maintaining cells in an
undifferentiated state and for activating their proliferation to create the
basis of liver organogenesis, until such a time that its expression ceases or
is superseded by the subsequent activation of other transcription factors
required for differentiation. Because organ progenitor cells consist of a rare
population in each environment, the identification of crucial genes that
regulate their distinctive potentials is difficult. Our method for
prospectively isolating hepatoblasts could be used efficiently to identify new
genes, such as Tbx3, that are fundamentally required for their activities, and
to improve our understanding of the molecular nature of liver development,
regeneration and carcinogenesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/9/1589/DC1
ACKNOWLEDGMENTS
We thank Keiko Sueyoshi and Setsuko Fujii for excellent technical assistance and Yasuhiko Kawakami for helpful suggestions. This work was supported in part by the Program for Improvement of Research Environment for Young Researchers from Special Coordination Funds for Promoting Science and Technology commissioned by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, Grant-in-Aids for Scientific Research from the MEXT of Japan, and a grant from the Leading Project in Japan. Research in the laboratory of J.C.I.B. was supported by funds from Fundacion Cellex, the G. Harold and Leila Y. Mathers Charitable Foundation, the MEC (BFU2006-12247) and the NIH.
Footnotes
Present address: Cellerix SL, Calle Marconi 1, Parque Tecnológico de
Madrid, Tres Cantos, 28760 Madrid, Spain 
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