|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online February 8, 2008
doi: 10.1242/10.1242/dev.017400
1 Laboratory for Pluripotent Cell Studies, RIKEN Center for Developmental
Biology (CDB), 2-2-3 Minatojima-minamimachi, Chu-o-ku, Kobe, Hyogo 650-0047,
Japan.
2 Sir William Dunn School of Pathology, University of Oxford, South Parks Road,
Oxford OX1 3RE, UK.
3 Laboratory for Development and Regenerative Medicine, Kobe University
Graduate, School of Medicine, 7-5-1 Kusunokicho, Chu-o-ku, Kobe, Hyogo
650-0017, Japan.
* Authors for correspondence (e-mails: yayoi.toyooka{at}path.ox.ac.uk; niwa{at}cdb.riken.jp)
Accepted 11 December 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: ES cell, Reversibility, Subpopulation, Rex1 (Zfp42), Oct3/4 (Pou5f1), Mouse
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), and the surviving cells in the outer layer of the epiblast
form a columnar epithelium, which we define as primitive ectoderm (PrE), and
this gives rise to the embryo proper. ES cells are established from the ICM
and epiblast, and may have gained characteristics intermediate between them.
ES cells show almost the same proliferation rate (8-12 hours) as epiblast
cells (11.5 hours) (Snow,
1977), and strongly express many genes expressed in pluripotent
cells in the early embryo, such as Oct3/4, Rex1 and Gbx2
(Saijoh et al., 1996;
Rogers et al., 1991;
Bulfone et al., 1993).
Oct3/4 (also known as Pou5f1)
(Yeom et al., 1991), a
transcription factor shown to be essential for maintenance of pluripotency
(Nichols et al., 1998;
Niwa et al., 2000), is
expressed continuously in the ICM, epiblast and PrE. Rex1 (also known
as Zfp42) is commonly used as a landmark of pluripotency and is
strongly expressed in the ICM, but downregulated in the epiblast and PrE
(Rogers et al., 1991;
Pelton et al., 2002). Hence,
we can distinguish the epiblast and PrE from the ICM by Rex1
expression. However, there have been no previous investigations to determine
whether these genes are differentially expressed in each ES cell or whether
each ES cell has a different pluripotent character.
It is well known that ES cells can mimic cell differentiation events that
occur during early mouse development. In vitro, mouse ES cells have been
suggested to have the ability to differentiate into primitive endoderm,
trophectoderm and many mature somatic cell lineages originating from the
embryonic endoderm, mesoderm and ectoderm (reviewed by
Keller, 2005;
Niwa et al., 2000). Formation
of embryoid bodies (EB) using ES cells can mimic peri-implantation development
in vitro, and has been mainly exploited for both induction of somatic cells
and analysis of the interaction of primitive endoderm and PrE
(Keller, 2005). Several
studies also demonstrated transient expression of Fgf5
(Haub and Goldfarb, 1991;
Hébert et al., 1991), a
gene generally used as a PrE marker, in ES cells cultured in adherent culture
(Niwa et al., 2000;
Shimozaki et al., 2003). These
observations suggest that ES cells differentiate into PrE before they convert
to mature somatic cells even in culture without EB. We noticed that we could
always detect weak Fgf5 expression in undifferentiated ES cell
culture, even under selection for Oct3/4 expression
(Niwa et al., 2000). As
Rex1, Gbx2 (Chapman et al.,
1997) and Fgf5 genes differentially expressed in
pluripotent tissues in different stages were detected in undifferentiated ES
cell culture, we hypothesized that undifferentiated ES cells constitute a
heterogeneous population containing pluripotent cells in various stages
corresponding from the ICM to PrE. To address this issue, we established
knock-in ES cell lines in which the expression of Oct3/4 and
Rex1 could be visualized using fluorescent proteins. We found that
undifferentiated ES cell culture contained ICM-like
(Rex1+/Oct3/4+) and early PrE-like
(Rex1-/Oct3/4+) cells. These
populations showed different differentiation potency in vitro and in vivo, and
could convert into each other's status when cultured in the presence of
leukemia inhibitory factor (LIF). These observations suggest that the status
of undifferentiated ES cells is in fluctuation between the ICM, epiblast and
early PrE.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A knock-in vector for Oct3/4 was designed to replace the coding region of the mouse Oct3/4 gene with an Oct3/4-ECFP fusion gene and IRES (internal ribosome entry site)-puromycin resistance gene, to express Oct3/4-EGFP (or YFP) fusion protein from the recombinant allele. A 4.4 kb fragment containing the coding region of exon 1 to exon 5, and a 2.1 kb fragment containing the untranslated region of exon 5 were amplified from the E14tg2a ES cell genomic DNA and used as the 5' and 3' homologous regions of the targeting vector, respectively. An Oct3/4-ECFP-IRES-puromycin resistance gene unit was ligated between the two DNA fragments. The resulting vector was linearized by NotI digestion and introduced into E14tg2a ES cells by electroporation. Genomic DNAs from puromycin-resistant colonies were screened for homologous recombination by Southern blotting analyses. For detection of recombination, genomic DNA was digested with EcoRI, separated on 0.8% agarose gels and transferred onto nylon membranes. Hybridization with a 300 bp probe produced an 11.6 kb band from the wild-type locus and a 10.3 kb band from the targeted locus.
A knock-in vector for Rex1 was designed to insert an EGFP [or herpes simplex virus 2 thymidine kinase gene (tk2)] and IRES-blasticidin resistance gene into exon 4 of the mouse Rex1 gene. Exon 4 is the first coding exon in the Rex1 gene. A 4.0 kb fragment containing intron 2 and exon 3 and a 1.0 kb fragment of exon 4 were amplified from the ES cell genomic DNA and used as the 5' and 3' homologous regions of the targeting vector, respectively. An EGFP-IRES-blasticidin unit was ligated between the two DNA fragments. The resulting vector was linearized by NotI digestion and introduced into the Oct3/4 knock-in ES cell line. Genomic DNAs from blasticidin-resistant colonies were screened for homologous recombination by Southern blotting analyses. For detection of recombination, genomic DNA was digested with EcoRI, and hybridization with a 300 bp probe produced a 7.9 kb band from the wild-type locus and a 5.7 kb band (for tk2 version; 5.9 kb) from the targeted locus.
RT-PCR and real-time (quantitative) PCR
Total RNA was extracted using Trizol (Invitrogen) in accordance with the
manufacturer's protocol. Total RNA (1.0 µg) was then subjected to
oligo-dT-primed reverse transcription (RT) with ReverTra Ace Kit (Toyobo,
Osaka, Japan). RT-PCR was performed using DNA polymerase (GeneTaq, NipponGene)
on an iCycler (BioRad). For real-time PCR analysis, total RNA was prepared
using Trizol and cDNAs were synthesized with oligodT primer by ReverTraAce
first strand synthesis kit (Toyobo). Q-PCR reactions were performed using the
ExTaq SYBR Green Supermix (Takara) and an iCycler System (Bio-Rad). The amount
of target RNA was determined from the appropriate standard curve and
normalized relative to the amount of Gapdh mRNA. Gene-specific
primers for RT-PCR and Q-PCR were designed based on published sequences
(Table 1).
|
|
Blastocyst injection and chimeric analysis
Rex1/Oct3/4 double-knock-in ES cells were marked by forced
expression of the DsRedT4 gene driven by the CAG promoter, and
EGFP+ and EGFP- cells were purified by FACSAria after
culture for stem cell selection with puromycin. ES cells were injected into
C57BL/6J blastocysts, followed by transfer to the uterus of pseudopregnant ICR
mice. Embryos were dissected at 10.5 days post-coitum (dpc) and DsRed
fluorescence was determined using a fluorescence stereomicroscope
(Olympus).
Mesodermal and neural induction
Induction of mesodermal cells was performed as described
(Nishikawa et al., 1998).
Briefly, aliquots of 104 ES cells were seeded in each well of
type-IV-collagen-coated six-well cluster dishes (Biocoat, Becton Dickinson)
and incubated in alpha-MEM with 10% FCS and 5x10-5 M 2ME.
After a 1-4 day incubation, cells were harvested with cell dissociation buffer
(GibcoBRL), stained with phycoerythrin (PE)-conjugated anti-E-cadherin mouse
antibody ECCD2 (Shirayoshi et al.,
1986) and apophycocyanin (APC)-conjugated anti-Flk1 (also known as
Kdr - Mouse Genome Informatics) mouse antibody AVAS12
(Kataoka et al., 1997). Neural
induction was performed as reported (Ying
et al., 2003a). We measured the proportion of neural cells by
immunostaining for the pan-neural marker NCAM (Ncam1 - Mouse Genome
Informatics). Goat-anti-NCAM antibody (SC-1507, Santa Cruz) and
Alexa-633-conjugated anti-goat-IgG antibody were used as first and second
antibodies, respectively. Stained samples were measured and analyzed by
FACSCalibur (Becton Dickinson).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
Using anti-PECAM-1 antibody, we found that expression of PECAM-1 was
correlated positively with expression of Rex1
(Fig. 2K); this represents
evidence that Rex1+ ES cells have ICM-like properties.
|
), was
significantly abundant in the Rex1+ fraction, and genes
that were strongly expressed in embryos in preimplantation stages and
downregulated in post-implantation embryos (based on EST data of NCBI
UniGene), such as Dppa3, Klf4, Esrrb and Tcl1, were also
highly expressed in Rex1+ cells
(Fig. 3A). By contrast,
transcripts of Fgf5, brachyury (T) and Eomes, which
were upregulated in the PrE and early germ layers
(Pelton et al., 2002;
Tesar et al., 2007), were
detected at higher levels in the Rex1- population
(Fig. 3A). We could also
observe these biased expression patterns in Nanog, Klf4, Tbx3 and Esrrb in
culture of Rex1-GFP knock-in ES cells by immunostaining with
antibodies for them (Fig. 3B).
Nanog, Klf4, Tbx3 and Esrrb tended to be detected in EGFP+
(Rex1+) cells, whereas Foxd3 did not show such a bias,
coincident with the result of Q-PCR. Expression levels of Oct3/4 and Sox2
associated with pluripotency were not much different between the two
populations (Fig. 3A). These
expression patterns of early genes support our hypothesis that
Rex1+ and Rex1- populations have
features of pluripotent cell population in pre- and post-implantation stages,
respectively.
Reversibility of Rex1+ and Rex1- populations
To examine the characteristics of
Rex1-/Oct3/4+ ES cells, we purified
both populations by flow cytometry and grew the cells in culture.
Oct3/4-CFP/Rex1-GFP double-knock-in ES cells were incubated
under puromycin selection, and then Rex1+ or
Rex1- cells were purified using GFP fluorescence. We
cultured purified Rex1+ and Rex1-
cells at more than 98% purity and found that a purified
Rex1+ population generated a Rex1-
population, and that a Rex1+ population emerged from a
purified Rex1- population in a comparatively short period
(Fig. 4A). To exclude the
possibility that this regeneration of another subpopulation was merely due to
contamination, we also performed single-cell culture of
Rex1+ and Rex1- cells.
Rex1+ cells arose from a single Rex1-
cell within 2-3 days (Fig. 4B),
and finally showed the same proportion as the parental double-knock-in cell
line after culture for 10-14 days (data not shown). These observations
indicated that Rex1+ and Rex1- cells
have the ability to convert into each other's status.
The Rex1+ population predominantly contributes to embryonic tissues
It has already been shown that the ICM and epiblast can contribute to
chimera formation, whereas PrE cannot
(Gardner, 1971;
Rossant, 1977;
Beddington, 1983;
Brook and Gardner, 1997). We
performed blastocyst injection of Rex1+ and
Rex1- populations to examine their capacity for
contributing to embryonic tissue in vivo. Rex1/Oct3/4 double-knock-in
ES cells were marked by forced expression of the DsRedT4 gene driven by the
CAG promoter, and EGFP+ and EGFP- cells were purified
from puromycin-resistant cells. While 5 of 22 embryos from blastocysts
injected with Rex1+ cells clearly exhibited chimerism
(Fig. 5A,
Table 2), we could not obtain
embryos with DsRed fluorescence from blastocysts into which
Rex1- cells were injected
(Fig. 5B,
Table 2). These observations
suggested that whereas Rex1+ ES cells have high potency
for contributing to embryonic tissues in chimeras as ICM and epiblast cells in
vivo, Rex1- cells showed poor ability as PrE. We also
noted that pseudopregnant mice into which blastocysts injected with
Rex1- cells were transferred had more vacant deciduas
(ratio of embryos/deciduas: 41/120) than those with blastocysts injected with
Rex1+ cells (22/42). This indicated that embryos injected
with Rex1- cells tend to degenerate, in contrast to those
injected with Rex1+ cells.
|
|
).
Cells were incubated for 1-4 days on type-IV-collagen-coated dishes for
induction of differentiation, then stained with anti-E-cadherin antibody and
Flk1 antibody, and the frequencies of E-cadherin-negative and Flk1-positive
cells were measured as mesodermal cells
(Fig. 7A). The results of
induction of mesoderm indicated that mesodermal differentiation of
Rex1-/Oct3/4+ PrE-like cells was significantly enhanced
compared with the Oct3/4+ fraction, which was composed mainly of
Rex1+/Oct3/4+ cells
(Fig. 7B). We also performed
neural induction using the neural induction system developed by Ying et al.
(Ying et al., 2003b). Although
they used an ES cell line that reported Sox1 expression as GFP fluorescence,
we used an antibody to the pan-neural marker NCAM to detect neural cells
(Fig. 7C). We obtained similar
results to mesoderm induction, with detection of an increasing number of
NCAM+ cells in the Rex1-/Oct3/4+, PrE-like
fraction (Fig. 7D). These
observations, indicating that the ICM-like (Rex1+) population
predominantly differentiated into extra-embryonic cells and that the PrE-like
(Rex1-) population could differentiate efficiently into somatic
lineages, reinforced our assumption that subpopulations in ES cells had
similar features of pluripotent cell lineages in vivo.
Ratio of Rex1+/Rex1- populations was biased under different culture conditions
In addition, we found that the proportion of the two populations was
significantly different between ES cells cultured under serum-free conditions
reported by Ying et al. (N2B27 medium with Bmp4 and LIF) and under those
reported by Ogawa et al. [GMEM medium with LIF, containing KSR and
adenocorticotropic hormone peptide (ACTH) instead of serum] at clonal density.
In KSR/ACTH medium, ES cells mainly formed packed colonies
(Fig. 8A), and most cells
(
96%) were Rex1+ cells
(Fig. 8B). Cells cultured in
N2B27 mainly formed flat colonies (Fig.
8A) containing cells showing low levels of Rex1
expression (or negative) at a high proportion (25-30%;
Fig. 8B). These observations
suggested that these culture conditions supported maintenance or proliferation
of different subpopulations in ES cells with differential efficiency.
|
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
It has been reported that primitive endoderm is necessary for the
establishment of PrE (Li et al.,
2001; Li et al.,
2002; Fassler and Meyer,
1995; Smyth et al.,
1999). The basement membrane deposited from primitive endoderm is
necessary for survival and polarization of adjacent outer epiblast cells to
form the columnar structure of PrE, and inner cells that fail to attach to the
basement membrane undergo apoptosis
(Coucouvanis and Martin,
1995). In early mouse embryos, extracellular matrices (ECMs), such
as laminin 1 and 10, nidogen 1 and 2, perlecan, agrin and collagen IV, have
been reported to be present as basement membrane components. Of these, laminin
1 is well known to be a crucial factor for early development
(Smyth et al., 1999;
Schéele et al., 2005).
Embryoid bodies that lack expression of laminin 1 failed to form the columnar
structure of PrE (Li et al.,
2001; Li et al.,
2002). The results of targeted disruption of integrin B1, a
receptor of laminin, and integrin-linked kinase (ILK), also demonstrated their
importance in PrE formation (Fassler and
Meyer, 1995; Sakai et al.,
2003). Although we found that a small population of PrE cells
emerged in ES cell culture cell-autonomously without any treatment, cell
differentiation is irreversible in real embryos, and epiblasts and PrE cells
do not regain the character of ICM in vivo. A similar reversible phenotype
observed in vitro was reported by Suzuki et al.
(Suzuki et al., 2006). They
induced mesoderm progenitor cells from ES cells by reduction of LIF
concentration in medium. Even the mesoderm progenitor cells contributed only
to mesodermal tissue when injected into blastocysts, and they could repopulate
undifferentiated ES cells in vitro (Suzuki
et al., 2006). It may be possible that although the nature of
pluripotent or stem cell lineages in the early embryo is potentially
reversible, signals from the ECM in the basement membrane function as a niche.
The ECM secreted from the primitive endoderm may fix the PrE status, and cause
the cells to retain the PrE character.
|
;
Rossant, 1977;
Beddington, 1983;
Brook and Gardner, 1997). The
results of chimera experiments in the present study were consistent with their
in vivo data, suggesting that Rex1+ cells are equivalent
to cells in the ICM and epiblast in vivo. We also noted that the survival rate
of embryos injected with Rex1- cells was much lower than
that of embryos injected with Rex1+ cells. We assumed that
ES cells corresponding to different developmental stages are not only omitted
from embryogenesis, but may impede the normal process of development when
injected into the blastocyst. Additionally, we demonstrated the poor ability
of Rex1-/Oct3/4+ cells to differentiate into
primitive endoderm whereas these cells predominantly differentiated into
somatic lineages using a negative selection system, and also that their
differentiation ability seemed interchangeable because their ability for
extra-embryonic lineage was restored in reverted Rex1-
populations. These observations were also consistent with the behavior of PrE
cells in the context of development. We first performed in vitro experiments
with sorted Rex1+ and Rex1- cells, but
we could not obtain clear results regarding the difference in differentiation
ability between Rex1+ and Rex1-
populations. We supposed that this was caused by the quick-reversible
phenotype of both populations in vitro, and we attempted to take advantage of
a negative selection system. The results of chimeric analysis and in vitro
experiments taken together indicated that
Rex1+/Oct3/4+ cells and
Rex1-/Oct3/4+ cells showed different
differentiation abilities and that they were similar in characteristic to ICM
and PrE, respectively, in vivo.
The generation and characterization of PrE-like cells in vitro have been
reported. Rathjen and colleagues reported that they generated early primitive
ectoderm-like (EPL) cells, which showed the character of PrE-like cells, from
ES cells (Rathjen et al.,
1999; Lake et al.,
2000). They obtained EPL cells by culturing ES cells with MEDII
medium, which contained the conditioned supernatant of the hepatocyte cell
line HepG2. They also demonstrated the lack of ability to differentiate into
primitive endoderm, inability to contribute to chimera formation, and
reversible phenotype in response to withdrawal of MEDII. EPL cells expressed
Oct3/4 and Fgf5, and Rex1 at very low levels.
Although it is difficult to compare our data to those of Rathjen's group as
they did not report data from analysis at the single-cell level nor used a
stem cell selection system, we suppose that their EPL cells would be mainly
composed of the Rex1- population.
Furusawa et al. also reported variation in the expression of platelet
endothelial cell adhesion molecule 1 (PECAM-1) and stage-specific embryonic
antigen (SSEA-1; also known as Fut4 - Mouse Genome Informatics) in ES cell
culture. Three sorted populations in ES cells,
PECAM-1+/SSEA-1+, PECAM-1+/SSEA-1-
and PECAM-1-/SSEA-1- cells, could give rise to two other
populations; PECAM-1+/SSEA-1+ cells predominantly
contribute epiblast cells in chimeras, and could differentiate into primitive
endoderm in vitro much more efficiently than the other two populations
(Furusawa et al., 2004). We
confirmed that PECAM-1 expression is nearly consistent with Rex1
expression (Fig. 2K), and thus
the Rex+ subpopulation was thought to contain
SSEA-1+ and SSEA-1- subpopulations. Furusawa and his
colleagues showed that the PECAM-1-/SSEA-1- population showed less
ability to differentiate into Fgf5+ cells compared with
the PECAM-1+/SSEA-1+ population in vitro, which does not
match the characteristics of the Rex1- population we
observed (Furusawa et al.,
2004). This discrepancy might be due to contamination of
differentiated cells into the PECAM-1-/SSEA-1-
population, as they did not use any selection system to exclude differentiated
cell populations.
Recently two independent groups reported that pluripotent stem cells could
be established from mouse post-implantation embryos after E5.5 (termed EpiSCs)
(Tesar et al., 2007;
Brons et al., 2007). EpiSCs
were derived from late epiblast or PrE, probed to be capable of
differentiation into the three germ layers in vivo and in vitro, and had poor
ability to incorporate in preimplantation embryos when they were aggregated
with morulae. They also showed that EpiSCs had a low expression level of
Rex1, Tbx3 and Dppa3, and a higher expression level of genes
that can be detected in post-implantation embryos, such as Fgf5,
Nodal and Eomes, compared with ES cells derived from ICM
(Tesar et al., 2007;
Brons et al., 2007). This
suggests that they have a very similar character to that of the
Rex1- population we observed (refer to
Fig. 3A). The crucial
difference between EpiSCs and the Rex1- population in ES
cells is that Rex1- cells can keep their status only very
transiently but change into Rex1+ within a short period,
whereas EpiSCs seem be able to keep their status consistently. EpiSCs had
quite a different pattern of histone methylation in the promoter region of
several genes from that of ES cells (Tesar
et al., 2007); it might be possible that such an epigenetic status
of cells is crucial to lock the reversibility and continue self-renewal.
|
|
), and
KSR-based medium with ACTH and LIF (Ogawa
et al., 2004). In N2B27 medium, ES cells tended to retain the
epiblast-PrE state, which was unexpected because serum-free conditions were
thought to suppress the appearance of differentiated cells, and as a
consequence ES cells could generally maintain the ICM-like state. Ying and
colleagues reported that inputs from LIF and Bmp4 contained in their medium
were crucial for the maintenance of pluripotency; LIF inhibited cell
differentiation toward the mesodermal and endodermal lineage, whereas Bmp4
suppressed differentiation toward neuroectoderm
(Ying et al., 2003b). It is
possible that ES cells actively maintained the ICM-like state in KSR medium
supplemented with ACTH and LIF, whereas they remained in the undifferentiated
state passively as a consequence of inhibition of commitment to the
differentiation pathway to any cell lineage in N2B27 medium with Bmp4 and LIF.
This result implies that these subpopulations require different factors and
signal transduction pathways for maintenance of their state, and comparison
and verification of each component of these serum-free media may provide some
insight into the factor(s) essential for the transition between
Rex1+ and Rex1- status. These
observations also indicated that different subpopulations could be dominant
under different culture conditions, and this may considerably influence the
results of the experiment in some situations.
|
|
). Shi
et al. showed that two transcription factors essential for maintenance of
pluripotency, Nanog (Chambers et al.,
2003; Mitsui et al.,
2003) and Sox2 (Avilion et al.,
2003), activate expression of Rex1 directly in F9 cells
(Shi et al., 2006). They also
showed that overexpression of Nanog triggers Rex1 expression in P19
cells, which originally did not express Rex1, indicating that Nanog
plays a crucial role in regulation of expression of Rex1 in ES cells.
It is of interest to examine whether overexpression of Nanog and/or Sox2
prevents the fluctuations of Rex1 expression in ES cells.
Rex1-/- F9 cells were able to differentiate into only
parietal endoderm, whereas wild-type F9 cells could differentiate into
primitive, visceral and parietal endoderm. Rex1 was assumed to
regulate the differentiation of F9 cells along several distinct cell lineages
in the early embryo (Thompson and Gudas,
2002). However, we found that Rex1-/- ES cells
could be established and differentiate normally both in vitro and in vivo,
although induction of some of the visceral marker genes were affected, and we
could produce Rex1-/- mice by a conventional
gene-targeting strategy (S. Masui, S. Ohtsuka, R. Yagi, K.T., M. S. H. Ko and
H.N., unpublished). Thus, Rex1 may not be essential for embryogenesis
and maintenance of pluripotency of ES cells.
Although it has already been reported that differentiation into primitive
endoderm can be induced by upregulation of Oct3/4 or forced
expression of GATA factors (Fujikura et
al., 2002; Shimosato et al.,
2007; Niwa et al.,
2000), whereas differentiation into trophectoderm is induced by
downregulation of Oct3/4 and activation of Cdx2
(Niwa et al., 2000;
Niwa et al., 2005), intrinsic
factors crucial for differentiation into somatic lineages have not yet been
identified, and induction of somatic cell lineages can be achieved at present
by the withdrawal of LIF from culture medium. Using our system to detect the
early PrE-like transient population in ES cell culture, it may be possible to
identify the factor(s) crucial for transition of ICM to PrE in vivo.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L., Vivian,
N. and Lovell-Badge, R. (2003). Multipotent cell lineages in
early mouse development depend on SOX2 function. Genes
Dev. 17,126
-140.
Beddington, R. S. P. (1983). The origin of foetal tissues during gastrulation in the rodent. In Development in Mammals. Vol. 5 (ed. M. H. Johnson), pp.1 -32. Amsterdam: Elsevier.
Ben-Shushan, E., Thompson, J. R., Gudas, L. J. and Bergman,
Y. (1998). Rex1, a gene encoding a transcription
factor expressed in the early embryo, is regulated via Oct3/4 and
Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an
adjacent site. Mol. Cell. Biol.
18,1866
-1878.
Brons, I. G., Smithers, L. E., Trotter, M. W., Rugg-Gunn, P., Sun, B., Chuva de Sousa Lopes, S. M., Howlett, S. K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R. A. et al. (2007). Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448,191 -195.[CrossRef][Medline]
Brook, F. A. and Gardner, R. L. (1997). The
origin and efficient derivation of embryonic stem cells in the mouse.
Proc. Natl. Acad. Sci. USA
94,5709
-5712.
Bulfone, A., Puelles, L., Porteus, M. H., Frohman, M. A., Martin, G. R. and Rubenstein, J. L. R. (1993). Spatially restricted expression of Dlx-1, Dlx-2 (Tes-1), Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential transverse and longitudinal segmental boundaries. J. Neurosci. 13,3155 -3172.[Abstract]
Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S. and Smith, A. (2003). Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113,643 -655.[CrossRef][Medline]
Chapman, D. L., Garvey, N., Hancock, S., Alexiou, M., Agulnik, S. I., Gibson-Brown, J. J., Cebra-Thomas, J., Bollag, R. J., Silver, L. M. and Papaioannou, V. E. (1996). Expression of the T-box family genes, Tbx1-Tbx5, during early mouse development. Dev. Dyn. 206,379 -390.[CrossRef][Medline]
Chapman, G., Remiszewski, J. L., Webb, G. C., Schulz, T. C., Bottema, C. D. K. and Rathjen, P. D. (1997). The mouse homeobox gene, Gbx2: genomic organization and expression in pluripotent cells in vitro and in vivo. Genomics 46,223 -233.[CrossRef][Medline]
Coucouvanis, E. and Martin, G. R. (1995). Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83,279 -287.[CrossRef][Medline]
Fassler, R. and Meyer, M. (1995). Consequences
of lack of beta 1 integrin gene expression in mice. Genes
Dev. 9,1896
-1908.
Fujikura, J., Yamato, E., Yonemura, S., Hosoda, K., Masui, S.,
Nakao, K., Miyazaki, J.-i. and Niwa, H. (2002).
Differentiation of embryonic stem cells is induced by GATA factors.
Genes Dev. 16,784
-789.
Furusawa, T., Ohkoshi, K., Honda, C., Takahashi, S. and
Tokunaga, T. (2004). Embryonic stem cells expressing both
platelet endothelial cell adhesion molecule-1 and stage-specific embryonic
antigen-1 differentiate predominantly into epiblast cells in a chimeric
embryo. Biol. Reprod.
70,1452
-1457.
Gardner, R. L. (1971). Manipulations on the blastocyst. Adv. Biosci. 6, 279-296.
Haub, O. and Goldfarb, M. (1991). Expression of the fibroblast growth factor-5 gene in the mouse embryo. Development 112,397 -406.[Abstract]
Hébert, J. M., Boyle, M. and Martin, G. M. (1991). mRNA localization studies suggest that murine FGF-5 plays a role in gastrulation. Development 112,407 -415.[Abstract]
Johnson, M. H. and McConnell, J. M. (2004). Lineage allocation and cell polarity during mouse embryogenesis. Semin. Cell Dev. Biol. 15,583 -597.[CrossRef][Medline]
Kataoka, H., Takakura, N., Nishikawa, S., Tsuchida, K., Kodama, H., Kunisada, T., Risau, W., Kita, T. and Nishikawa, S. I. (1997). Expressions of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells. Dev. Growth Differ. 39,729 -740.[CrossRef][Medline]
Keller, G. (2005). Embryonic stem cell
differentiation: emergence of a new era in biology and medicine.
Genes Dev. 19,1129
-1155.
Lake, J., Rathjen, J., Remiszewski, J. and Rathjen, P. D. (2000). Reversible programming of pluripotent cell differentiation. J. Cell Sci. 113,555 -566.[Abstract]
Li, S., Harrison, D., Carbonetto, S., Fassler, R., Smyth, N.,
Edgar, D. and Yurchenco, P. D. (2002). Matrix assembly,
regulation, and survival functions of laminin and its receptors in embryonic
stem cell differentiation. J. Cell Biol.
157,1279
-1290.
Li, X., Chen, Y., Scheele, S., Arman, E., Haffner-Krausz, R.,
Ekblom, P. and Lonai, P. (2001). Fibroblast growth factor
signaling and basement membrane assembly are connected during epithelial
morphogenesis of the embryoid body. J. Cell Biol.
153,811
-822.
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama, M., Maeda, M. and Yamanaka, S. (2003). The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113,631 -642.[CrossRef][Medline]
Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Schöler, H. and Smith, A. (1998). Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95,379 -391.[CrossRef][Medline]
Nishikawa, S.-I., Nishikawa, S., Hirashima, M., Matsuyoshi, N. and Kodama, H. (1998). Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development 125,1747 -1757.[Abstract]
Niwa, H., Miyazaki, J. and Smith, A. G. (2000). Quantitative expression of Oct3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24,372 -376.[CrossRef][Medline]
Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K., Yagi, R. and Rossant, J. (2005). Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 123,917 -929.[CrossRef][Medline]
Ogawa, K., Matsui, H., Ohtsuka, S. and Niwa, H.
(2004). A novel mechanism for regulating clonal propagation of
mouse ES cells. Genes Cells
9, 471-477.
Pelton, T. A., Sharma, S., Schulz, T. C., Rathjen, J. and
Rathjen, P. D. (2002). Transient pluripotent cell populations
during primitive ectoderm formation: correlation of in vivo and in
vitro pluripotent cell development. J. Cell Sci.
115,329
-339.
Rathjen, J., Lake, J. A., Bettess, M. D., Washington, J. M., Chapman, G. and Rathjen, P. D. (1999). Formation of a primitive ectoderm like cell population, EPL cells, from ES cells in response to biologically derived factors. J. Cell Sci. 112,601 -612.[Abstract]
Robson, P., Stein, P., Zhou, B., Schultz, R. M. and Baldwin, H. S. (2001). Inner cell mass-specific expression of a cell adhesion molecule (PECAM-1/CD31) in the mouse blastocyst. Dev. Biol. 234,317 -329.[CrossRef][Medline]
Rogers, M. B., Hosler, B. A. and Gudas, L. J. (1991). Specific expression of a retinoic acid-regulated, zinc-finger gene, Rex1, in preimplantation embryos, trophoblast and spermatocytes. Development 113,815 -824.[Abstract]
Rossant, J. (1977). Cell commitment in early rodent development. In Development in Mammals. Vol.2 (ed. M. H. Johnson), pp.119 -150. Amsterdam: Elsevier Biomedical Press.
Saijoh, Y., Fujii, H., Meno, C., Sato, M., Hirota, Y., Nagamatsu, S., Ikeda, M. and Hamada, H. (1996). Identification of putative downstream genes of Oct-3, a pluripotent cell-specific transcription factor. Genes Cells 1, 239-252.[Abstract]
Sakai, T., Li, S., Docheva, D., Grashoff, C., Sakai, K., Kostka,
G., Braun, A., Pfeifer, A., Yurchenco, P. D. and Fassler, R.
(2003). Integrin-linked kinase (ILK) is required for polarizing
the epiblast, cell adhesion, and controlling actin accumulation.
Genes Dev. 17,926
-940.
Schéele, S., Falk, M., Franzén, A., Ellin F.,
Ferletta, M., Lonai, P., Andersson, B., Timpl, R., Forsberg, E. and Ekblom,
P. (2005). Laminin alpha1 globular domains 4-5 induce fetal
development but are not vital for embryonic basement membrane assembly.
Proc. Natl. Acad. Sci. USA
102,1502
-1506.
Shi, W., Wang, H., Pan, G., Geng, Y., Guo, Y. and Pei, D.
(2006). Regulation of the pluripotency marker Rex1 by
Nanog and Sox2. J. Biol. Chem.
281,23319
-23325.
Shimosato, D., Shiki, M. and Niwa, H. (2007). Extra-embryonic endoderm cells derived from ES cells induced by GATA factors acquire the character of XEN cells. BMC Dev. Biol. 7, 80.[CrossRef][Medline]
Shimozaki, K., Nakashima, K., Niwa, H. and Taga, T.
(2003). Involvement of Oct3/4 in the enhancement of
neuronal differentiation of ES cells in neurogenesis-inducing cultures.
Development 130,2505
-2512.
Shirayoshi, Y., Nose, A., Iwasaki, K. and Takeichi, M. (1986). N-linked oligosaccharides are not involved in the function of a cell-cell binding glycoprotein E-cadherin. Cell Struct. Funct.11,245 -252.[Medline]
Smyth, N., Vatansever, H. S., Murray, P., Meyer, M., Frie, C.,
Paulsson, M. and Edgar, D. (1999). Absence of basement
membranes after targeting the LAMC1 gene results in embryonic lethality due to
failure of endoderm differentiation. J. Cell Biol.
144,151
-160.
Snow, M. H. L. (1977). Gastrulation in the mouse: growth and regionalization of epiblast. J. Embryo. Exp. Morphol. 42,293 -303.
Suzuki, A., Raya, A., Kawakami, Y., Morita, M., Matsui, T., Nakashima, K., Gage, F. H., Rodriguez-Esteban, C. and Belmonte, J. C. (2006). Maintenance of embryonic stem cell pluripotency by Nanog-mediated reversal of mesoderm specification. Nat. Clin. Pract. Cardiovasc. Med. 3Suppl. 1 , S114-S122.[CrossRef][Medline]
Tesar, P. J., Chenoweth, J. G., Brook, F. A., Davies, T. J., Evans, E. P., Mack, D. L., Gardner, R. L. and McKay, R. D. (2007). New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448,196 -199.[CrossRef][Medline]
Thompson, J. R. and Gudas, L. J. (2002). Retinoic acid induces parietal endoderm but not primitive endoderm and visceral endoderm differentiation in F9 teratocarcinoma stem cells with a targeted deletion of the Rex-1 (Zfp-42) gene. Mol. Cell. Endocrinol. 195,119 -133.[CrossRef][Medline]
Yeom, Y. I., Ha, H.-S., Balling, R., Schöler, H. and Artzt, K. (1991). Structure, expression and chromosomal location of the Oct-4 gene. Mech. Dev. 35,171 -179.[CrossRef][Medline]
Ying, Q. L., Stavridis, M., Griffiths, D., Li, M. and Smith, A. (2003a). Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture. Nat. Biotechnol. 21,183 -186.[CrossRef][Medline]
Ying, Q. L., Nichols, J., Chambers, I. and Smith, A. (2003b). BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115,281 -292.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  I. Chambers and S. R. Tomlinson The transcriptional foundation of pluripotency Development, July 15, 2009; 136(14): 2311 - 2322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hochedlinger and K. Plath Epigenetic reprogramming and induced pluripotency Development, February 15, 2009; 136(4): 509 - 523. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Zhongling Feng, Gang Zhao, and Lei Yu Neural Stem Cells and Alzheimer's Disease: Challenges and Hope American Journal of Alzheimer's Disease and Other Dementias, February 1, 2009; 24(1): 52 - 57. [Abstract] [PDF]
|
|
|
|
 |
|
 Y. Toyooka, D. Shimosato, K. Murakami, K. Takahashi, and H. Niwa Identification and characterization of subpopulations in undifferentiated ES cell culture J. Cell Sci., March 1, 2008; 121(5): e1 - e1. [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
