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First published online May 1, 2006
doi: 10.1242/10.1242/dev.02362
Centre Development in Stem Cell Biology, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JQ, UK.
* Author for correspondence (e-mail: josh.brickman{at}ed.ac.uk)
Accepted 3 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Gastrulation, Self-renewal, Embryonic stem cell, Lineage commitment, Pou protein, Pou5f1, Xenopus
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Snape et al., 1987). This
pluripotent feature of early embryonic cells is mirrored in vitro by embryonic
stem (ES) cells (Smith, 2001).
ES cells are self-renewing pluripotent cell lines derived from the ICM of the
mammalian blastocyst that, in mouse, have been shown to be capable of
populating all the lineages of the foetus and adult
(Beddington and Robertson,
1989). Unlike ES cells, early embryonic cells cannot be considered
stem cells as they do not self-renew indefinitely. However, mammalian embryos
still need to maintain a population of non-committed cells through a
significant amount of cell division to generate the number of cells necessary
to complete gastrulation. Although lower vertebrates undergo reductive rather
than proliferative cell division during early cleavage
(Frederick and Andrews, 1994;
Kane et al., 1992), they still
require active mechanisms to ensure against premature cellular
differentiation. Indeed, studies in Xenopus have shown that the
future ectoderm cells and the ventral marginal zone cells are multipotent
until late gastrulation (Domingo and
Keller, 2000; Okabayashi and
Asashima, 2003).
A number of factors have been identified that are necessary for the
maintenance of ES cell pluripotency, including the cytokines LIF and BMP4, and
transcription factors such as Nanog and Oct4 (encoded by Pou5f1,
referred to here as Oct4)
(Chambers, 2004). In addition
to its role in ES cells, Oct4 is also required for the maintenance of the germ
cell lineage (Kehler et al.,
2004) and thus appears to have an in vivo role in maintaining
multipotency. The ICM and then the epiblast express Oct4 until
gastrulation, when Oct4 remains expressed only in the posterior
epiblast and the primitive streak, but not in cells that have undergone
mesendoderm induction (Yeom et al.,
1996; Yoshimizu et al.,
1999). This expression pattern is consistent with Oct4 having a
role in maintaining cells as multipotent until it is the appropriate time for
them to become committed to mesoderm, endoderm or ectoderm.
In the absence of Oct4, ES cells lose the capacity to self-renew and
subsequently differentiate into extra-embryonic trophectoderm
(Niwa et al., 2000).
Similarly, Oct4–/– embryos die at
peri-implantation stages because of the conversion of ICM into trophectoderm
(Nichols et al., 1998). The
involvement of Oct4 in embryonic versus extra-embryonic fate decisions and its
role in the maintenance of primordial germ cells (PGCs) has led to the
proposal that Oct4 arose in mammals to perform these specific functions
(Pesce et al., 1998).
Alternatively, Oct4 and the molecular programmes it regulates might represent
an older evolutionary innovation designed to maintain a non-committed cell
population in the early embryo until the appropriate time for induction of the
germ layers.
Oct4 is a Class V POU domain protein, containing both a POU specific domain
(POUs) and a POU homeodomain (POUh)
(Okamoto et al., 1990;
Rosner et al., 1990;
Scholer et al., 1990). Based
on the degree of conservation within these domains and the linker region, the
POU domain proteins have been divided into five classes. Class V POU domain
proteins (referred to here as PouV proteins) are present in a number of other
species and have been implicated in early embryonic development
(Bachvarova et al., 2004;
Frank and Harland, 1992;
Hinkley et al., 1992;
Takeda et al., 1994;
Whitfield et al., 1993). To
date, a single PouV protein has been identified in teleost fish
(Burgess et al., 2002),
zebrafish Pou2, referred to here as DrPou2. Interestingly, DrPou2 has
recently been shown to have a role in endoderm induction
(Lunde et al., 2004;
Reim et al., 2004).
Although no self-renewing ES cell lines have been generated from lower vertebrates, we considered whether the mechanisms governing self-renewal were conserved. Here, we report that a PouV gene from Xenopus laevis, Xlpou91, has the capacity to maintain murine ES cells in the absence of Oct4. As Xenopus has neither induced PGCs nor extra-embryonic development, we believe that ES cell pluripotency may be based in part on conserved, rather than mammalian-specific, aspects of vertebrate development. Moreover, two other Xenopus genes, Xlpou25 and Xlpou60, and axolotl-Oct4 (AmOct4, also referred to as Axoct4) have some ability to rescue ES cell self-renewal, whereas the zebrafish protein Pou2 has none. Knockdown phenotypes of the three Xenopus XlpouV genes results in elevated expression of endodermal, organizer and neural markers, aspects of which can be rescued by axolotl and mouse Oct4. These data support our hypothesis that PouV proteins represent a class of transcription factor required for the maintenance of multipotent non-committed cell populations in both Xenopus and mouse.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
and cultured according to Slack et al.
(Slack et al., 1984). Embryos
were injected at the two-cell stage in both blastomeres with RNA and/or
morpholino oligonucleotides. Dextran fluorescein (Molecular Probes) was used
as a lineage label (Brickman et al.,
2000).
Animal caps were dissected from stage 8 blastula and cultured in the
presence or absence of partially purified activin (a gift from J. Smith).
Activin units (U) are defined by Cooke et al.
(Cooke et al., 1987). In situ
hybridization was carried out as described in Brickman et al.
(Brickman et al., 2000).
Morpholino oligonucleotides were purchased from GeneTools. Sequences were as
follows: Xlpou25 MO, 5'-ACATGGTGTCCAAGAGCTTGCAGTC-3'; Xlpou60 MO,
5'-GTACAATATGGGCTGGTCCATCTCC-3'; Xlpou91 MO,
5'-GTAGGTCTGTTGGTTATACATGATC-3'. The control morpholino was the
standard provided by GeneTools.
The whole-mount TUNEL staining was carried out as previously described
(Hensey and Gautier,
1997).
In vitro translation
Wild-type XlPouV mRNA or non-complementary (NC) XlpouV mRNA were used as a
template in a Red Nova Lysate Translation kit (Novagen) according to the
manufacturer's instructions in the presence of 35S-methionine.
Translation productions were fractionated by SDS-PAGE and visualized by
autoradiography.
Expression constructs and transcription
Wild-type Xlpou25, Xlpou60, mouse Oct4, DrPou2 and AmOct4, and non
complementary (NC) Xlpou25, Xlpou60 and Xlpou91 expression constructs were
generated by PCR in pCS2+. Plasmids for RNA injection were linearized with
Not1 and mRNA generated according to Smith
(Smith, 1993). cDNAs were
inserted into pCAGIP (Niwa et al.,
2002; Niwa et al.,
1991) for ES cell rescue.
Embryonic stem cell culture and transfection
ES cells were cultured according to Li et al.
(Li et al., 1995). For the
rescue experiments, 2x107 ZHBTc4 ES cells were electroporated
with 100 µg of linearized plasmid DNA followed by culture with or without 2
µg/ml tetracycline (Tc) (Sigma) for 2 days. Cells were then cultured in 2
µg/ml puromycin (Sigma) with or without Tc for 7 days. The resulting
colonies were staining for alkaline phosphatase (AP) activity (Sigma-Aldrich)
or expanded as clonal lines.
Luciferase reporter assays
ZHBTc4 ES cells (1x105) were plated on a 24-well plate
with 2 µg/ml Tc. Twenty-four hours later, 75 ng of reporter plasmids and
150 ng of the test plasmid was transfected according to Brickman et al.
(Brickman et al., 2001). For
Xenopus embryos, 50 pg of reporter plasmid with or without morpholino
was injected into both blastomeres of a two-cell stage embryo.
RNA isolation and real-time RT-PCR
Total RNA was prepared from pools of 15 embryos, 25 animal caps or
106 ES cells using Absolutely RNA RT-PCR (Stratagene). RNA (1
µg) was used as a template for cDNA synthesis. Real-time RT-PCR was
performed using a LightCycler (Roche) and the LightCycler FastStart DNA
MasterPLUS SYBR Green 1 (Roche). Standard curves were generated
either from diluted cDNA derived from control embryos or from plasmid. Samples
were normalized to ornithine decarboxylase (Odc) for Xenopus
embryos and ß-actin for ES cells. PCR primers and conditions can be
provided on request.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), we looked at these regions specifically. Both global and
regionally focused alignments of these proteins failed to reveal any conserved
motifs within these regions indicative of shared functional properties that
might be present within a subset of this family
(Fig. 1A;
Table 1).
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)
(Fig. 1B). Analysis of mRNA expression by in situ hybridization for Xlpou60, Xlpou25 and Xlpou91 was performed (Fig. 1C). Xlpou60 mRNA is maternally expressed and restricted to the animal hemisphere in both unfertilized oocytes and early cleavage stage embryos. Expression remains in the animal and marginal zones of the embryo but is reduced by the late blastula stage and undetectable by the early gastrulation stages. Xlpou25 and Xlpou91 are first transcribed at the onset of zygotic transcription in the animal and marginal zone, similar to Xlpou60. Xlpou25 and Xlpou91 continue to be expressed throughout the process of gastrulation but only in cells that have not undergone involution. Xlpou25 and Xlpou91 expression is then rapidly downregulated as cells begin the process of involution and commitment to their germ layer fate (Fig. 1C). Thus, the composite expression pattern of the Xenopus PouV genes is reminiscent of Oct4.
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Xenopus PouV proteins can rescue self-renewal in Oct4 depleted ES cells
As Oct4 is an absolute requirement for ES cell self-renewal, we could assay
the ability of the different PouV family members to substitute for Oct4
through the use of an inducible Oct4 knockout ES cell system
(Niwa et al., 2000). Briefly,
ZHBTc4 ES cells have both alleles of the endogenous Oct4 gene
inactivated, and expression of Oct4 is maintained within these cells by a
tetracycline (Tc) regulatable Oct4 transgene.
Using this system, we could measure the extent of Oct4 rescue conveyed by the different PouV proteins in three ways: (1) the generation of `ES cell like', alkaline phosphatase (AP) positive colonies (the rescue index); (2) long-term self-renewal (generation of clonal cell lines); and (3) expression of ES cell specific markers.
To ensure that differences in ES cell phenotypes were not due to expression
level or a global defect in transcriptional regulatory activity, we first
tested the ability of all PouV proteins to activate transcription of octamer
binding reporter genes. Co-transfection of the PouV proteins with an Fgf4
enhancer reporter in ZHBTc4 ES cells all resulted in similar levels of induced
transcription (Fig. 2A) with
the exception of mouse Oct4 (V267P), a variant of Oct4 known to be unable to
bind DNA when expressed in ES cells (Niwa
et al., 2002). We also tested the activity of these proteins by
co-transfection with a second reporter gene that contained six reiterated
copies of the octamer binding motif (Niwa
et al., 2002). Interestingly all three Xenopus proteins
were potent activators in this context, whereas mouse Oct4, AmOct4 and DrPou2
were less active to varying degrees (Fig.
2A). The ability of all of these proteins to activate
transcription from the Fgf4 enhancer reporter indicates that any differences
in function they display in ES cells was not due to variability in protein
stability.
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A rigorous test of whether these genes can support long-term self-renewal is to derive and expand ES cell lines in which the introduced genes are stably expressed in the absence of Oct4. The experimental strategy for expanding and analysing the resultant clonal cell lines is shown in Fig. 3A. For each transfection that produced colonies in the absence of Oct4, colonies were picked and expanded to generate clonal lines. All colonies from the mouse Oct4 transfection survived for 10 passages (p10), as did those transfected with Xlpou91. However only 17% and 80% of colonies from the Xlpou25 and Xlpou60 transfection, respectively, survived 10 passages. In cell culture conditions that normally sustain ES cell self-renewal (+LIF) and in the absence of mouse Oct4 expression (Fig. 3B, left panel), both Xlpou91 and mouse Oct4 lines appear undifferentiated, while Xlpou60 and AmOct4 lines contained a significant number of differentiated cells. Xlpou25 lines were almost all differentiated, with clumps of cells that appear to grow as colonies. DrPou2 cell lines, similar to the parental ZHBTc4 cell line, could not be maintained in the absence of mouse Oct4.
ES cell expansion is dependent on the cytokine LIF and, in its absence, ES
cell lines rapidly differentiate (Smith et
al., 1988; Williams et al.,
1988). We observed that all of our cell lines also differentiated
upon LIF withdrawal (Fig. 3B,
centre panel) and thus the ability of these rescued cell lines to
differentiate is not blocked by the presence of the amphibian PouV proteins.
ES cells differentiate in response to increased Oct4 protein levels
(Niwa et al., 2000), an effect
also observed in our PouV cell lines when the Oct4 transgene was reactivated
(Fig. 3B, right panel).
Fig. 3C shows the average expression levels calculated from the individual cell lines derived for each different PouV mRNA transfection. The very low standard deviations of these values illustrate that each clonal cell line, within the set, expressed similar levels of their particular PouV mRNA, indicating that ES cell self-renewal has a specific crucial threshold for each individual PouV protein. Interestingly, the expression level of Xlpou91 transcripts was similar to that of mouse Oct4, whereas the number of transcripts produced in the Xlpou25 and Xlpou60 rescued cell lines proved to be on average 15 fold and 116 fold higher, respectively (Fig. 3C).
Quantitative molecular marker analysis was performed on RNA from the rescued cell lines by real-time RT-PCR (Fig. 3D). We first confirmed that Oct4 expression had been suppressed in these lines. The values presented in Fig. 3D represent the mean level of gene expression calculated from at least two independent cell lines following rescue using the different PouV proteins. Thus, although the real-time PCR technique we employed detected very low residual levels of Oct4 expression on average, we have cell lines supported by each PouV protein that do not express detectable levels of Oct4 (data not shown). Moreover, we have confirmed that this residual level of Oct4 mRNA does not produce detectable levels of Oct4 protein, as determined by immunohistochemistry (data not shown).
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As there is no trophoblast lineage in Xenopus, we considered
whether Oct4 might have a broader function than ES cell self-renewal.
RNAi-mediated knockdown of Oct4 in both mouse and human ES cells suggested
that Oct4 suppressed the endodermal marker Gata4
(Hay et al., 2004), while
expression of both brachyury and Gata4 appeared to be lost following
Oct4 shut down in ZHBTc4 ES cells (Niwa et
al., 2000). Owing to this discrepancy, we thought it important to
examine the response of both mesodermal and endodermal markers to variations
in Oct4 levels. We found that Gata4 expression is maintained in the
absence of Oct4, while brachyury expression was rapidly downregulated in the
ZHBTc4 ES cell line following depletion of Oct4
(Fig. 4E). We also observe that
re-expression of the Oct4 transgene in the mouse Oct4, Xlpou25 and Xlpou91 ES
cell lines results in reduced expression of Gata4 and Mixl1
and an increase in brachyury expression
(Fig. 4F). It has previously
been shown that both an increase or decrease in Oct4 levels leads to ES cell
differentiation (Niwa et al.,
2000). Marker analysis in our cell lines helps to explain this
phenomenon by demonstrating that enhanced Oct4 drives ES cells towards a
brachyury-positive state, whereas reduced levels of Oct4 leads to both
endodermal and trophoblast differentiation.
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). Embryos injected with control MO, Xlpou25 MO or Xlpou60 MO alone developed with no observable phenotype. Protein knockdown with Xlpou91MO alone, Xlpou25 and Xlpou91 MO, or with a combination of all three MOs (XlPouV MO) resulted in a failure in axis elongation, of varying severity (Fig. 4C, Table 2). Although the most prominent phenotype was posterior truncation, we also observed an anterior defect consisting of reduced head and eye size. The depletion of all three XlPouV proteins resulted in the most severe phenotype (Fig. 4C, Table 2).
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).
Depletion of PouV protein in Xenopus embryos also resulted in an
expansion of Xcad3 expression
(Fig. 5D). We also observed an
increase in Xcad3 expression during gastrulation, although at a lower
level (data not shown).
XlPouV protein depletion disrupts the expression of genes associated with early lineage commitment
To investigate whether the loss of posterior tissue was due to increased
cell death of late gastrulating marginal zone cells, whole-mount TUNEL was
performed on XlpouV-depleted embryos and control MO-treated embryos. We
observed no difference between XlpouV depleted embryos and control MO-treated
embryos during late gastrulation (Fig.
5E).
The loss of posterior tissues in the absence of cell death suggests that
Xlpou depletion might have resulted in defects early during cell
specification. Fig. 6A shows
RNA in situ hybridization for a number of gastrulation stage markers in
XlPouV-depleted embryos. Interestingly, we observed a dramatic loss of
Bmp4 and Xom (Xvent2) expression, both genes
associated with the inhibition of differentiation
(Cao et al., 2004;
Constance Lane et al., 2004;
Ying et al., 2003). We also
observed a loss of Fgf8 and brachyury (Xbra) expression,
genes normally expressed in the marginal zone. Expression of marker genes
associated with both endoderm (Mixer, Sox17
and endodermin)
and the organizer region [goosecoid (Gsc), chordin and cerberus] were
all expanded (Fig. 6A,B). These
results were confirmed by real-time RT-PCR analysis of XlPouV-depleted embryos
(Fig. 6B). Depletion of
individual XlPouV proteins revealed some differences in function within the
Xenopus gene family. For example, depletion of Xlpou25 protein alone
did not result in the loss of Xbra and Bmp4 expression or an
increase in Sox17 expression (data not shown). This difference
in the function of Xlpou25, compared with Xlpou60 and Xlpou91, may explain its
reduced capacity to rescue ES cell self-renewal.
Fig. 6C shows the post-gastrulation consequences of XlPouV depletion. All three Xlpou proteins are expressed in the animal hemisphere and following their depletion we found that the neural plate marker Sox2 and a pro-neural gene, Ngnr1, were both upregulated. Marginal zone cells from Xlpou-depleted embryos express high levels of organizer and anterior endoderm markers, consistent with the precocious conversion of uncommitted marginal zone into a committed cell fate. In agreement with the early conversion of an abnormally large region of this tissue to early endoderm, we found an expansion of later Hex expression, which normally marks the earliest endoderm to involute during gastrulation. In normal stage 35 tadpoles, Hex expression is restricted to the descendants of this tissue in the liver, whereas in Xlpou-depleted embryos there is both an expanded liver domain and ectopic Hex expression (Fig. 6C). These embryos also exhibit a dramatic reduction in posterior myosin-light chain 1-3 (MLC1-3) expression. This result is consistent with a premature conversion of uncommitted marginal zone cells to organizer or anterior endoderm early in gastrulation, leaving an insufficient number of cells to form the posterior somites. Thus, the anterior somites appear normal but towards the posterior of the embryo the expression of MLC1-3 is decreased to a point where no segmented somites are visible (Fig. 6C).
Overexpression of XlPouV proteins produced the opposite effects on gene expression to that of XlpouV depletion. At the gastrulation stage, overexpression of all three Xenopus PouV proteins enhanced the expression of Xbra and Bmp4 and reduced the expression of Mixer and goosecoid (Fig. 6D). These results paralleled those obtained from the depletion or overexpression of PouV proteins in ES cells (Fig. 3E,F) and indicate that mammalian and Xenopus PouV proteins have similar downstream targets and may function through similar molecular pathways.
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). The molecular effects of XlpouV knockdown in animal caps was monitored by the expression of specific marker genes. Bmp4 was the only marker analysed that is expressed in un-induced (no activin) animal caps, and we observed a significant decrease in Bmp4 expression in uninduced animal caps following XlPouV protein depletion (Fig. 7A). Although differences could be observed between the control MO and XlpouV MO-treated explants that had not been induced with activin, we do not consider these to be significant because of their very low expression levels (Fig. 7A).
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and endodermin in
XlPouV-depleted explants. Moreover, Gsc and cerberus expression can
only be induced at 16 U/ml activin in control MO-treated animal caps whereas
the lower dose of 8 U/ml activin was sufficient to induce these organizer
markers in the XlPouV depleted animal caps
(Fig. 7B). Thus, depletion of
XlPouV proteins sensitizes animal cap cells to activin treatment, while
reducing expression of marginal zone markers such as Xbra and
Bmp4. We also observed that overexpression of XlPouV proteins in
activin-treated animal caps dramatically reduced the induction of Gsc
(data not shown). |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Reim and Brand,
2002; Reim et al.,
2004).
Although the zebrafish protein DrPou2 was unable to rescue Oct4 null ES
cells or the gastrulation stage XlpouV depletion phenotype, its role in neural
patterning shows some conservation with Xenopus. Xlpou25 and Xlpou91
were found to be required for maintenance of gene expression in the MHB and
the anterior hindbrain. A similar role has been reported for DrPou2 and this
aspect of the XlPouV depletion phenotype is similar to the spiel-ohne-grenzen
(spg) (DrPou2 mutant) phenotype (Belting et
al., 2001; Hauptmann et al.,
2002; Reim and Brand,
2002). Consistent with the conservation of function during neural
development, overexpression of mouse Oct4 mRNA in spg mutant zebrafish can
rescue a defect in Pax2.1 expression in the MHB. However, although
mouse Oct4 rescues this aspect of the zebrafish spg phenotype, there is
currently no evidence for a requirement for Oct4 in neural development. In
addition, despite the rescue of Pax2.1 expression in neural
patterning there is no evidence that mouse Oct4 can rescue the zygotic or
maternal/zygotic defects in endoderm induction. Moreover, DrPou2 induces
endoderm cooperatively with the SRY box protein, Sox32 (also known as
Casanova) (Reim et al., 2004).
As PouV proteins usually bind DNA coordinately with a member of the Sox family
(Dailey and Basilico, 2001)
and no Sox32-like protein has been identified in other vertebrates, the
Sox32-Pou2 endoderm induction mechanism may be unique to teleosts. It is
possible that the genes encoding Sox32 and Pou2 arose from a duplication event
in which an ancient PouV-Sox gene set retained original function and the other
acquired a novel endoderm inducing activity. However, despite the large number
of POU genes present in the sequenced teleost genomes, we have been unable to
find an obvious candidate. This is particularly surprising as sequences
resembling both Pou2 and Oct4 have been identified in sturgeon, a species that
occupies a more basal phylogenetic position than teleosts (Andrew Johnson,
personal communication). The divergent function of DrPou2 in endoderm
induction may explain why it was unable to rescue ES cell self-renewal or the
gastrulation stage XlPouV depletion phenotype.
As Xenopus lacks trophectoderm and the XlpouV depletion does not
affect germ cell development (data not shown), the conserved PouV function in
Xenopus and ES cells argues against Oct4 function being a unique
innovation of mammals for these specific lineages. In support of this, the
relationship between Caudal (Cdx) and PouV proteins does not appear to be
exclusive to trophoblast versus ICM cell fate decisions in mammals.
Xenopus lacks trophectoderm tissue, yet an orthologue of Cdx2,
Xcad3, exists (Pownall et al.,
1996). Loss of PouV proteins in both ES cells and Xenopus
embryos results in ectopic Cdx2
(Nichols et al., 1998;
Niwa et al., 2000) and
Xcad3 expression (Fig.
5D), respectively. Moreover we have found similar sets of genes to
be regulated by PouV proteins in ES cells and during Xenopus
gastrulation. Thus, we conclude that mouse Oct4 is not a mammalian-specific
protein, but rather the function of PouV proteins in suppressing
differentiation and commitment has been adopted by mammals to maintain
pluripotency in ES cells, in part by blocking commitment to the trophoblast
lineage.
A function for PouV proteins during gastrulation
It is likely that the pivotal role of Oct4 in the maintenance of ES cell
pluripotency depends on the ability of this protein to suppress cell fate
commitment in multiple lineages and is therefore derived from an ancient role
in gastrulation. In Xenopus, the future germ layer domains are
specified early by the asymmetrical localization of maternal mRNAs such as
Vg1, VegT, Wnt11 and ectodermin (Dupont et
al., 2005; Joseph and Melton,
1998; Tao et al.,
2005; Xanthos et al.,
2001). Although this process regionalizes the embryo into broad
domains, cells become committed to specific lineages only as they begin
involution in response to these signals. Cells continue to be committed to
their particular cell fate throughout gastrulation, but the embryo requires a
pool of multipotent precursors to enable the formation of more posterior
lineages later during this process. A similar pool of cells is required in
mammalian development and the expression of Oct4, initially throughout the
epiblast and later in the region of the primitive streak, may indicate the
location of these populations (Snape et
al., 1987; Wylie et al.,
1987). ES cell derivation represents the capture of these
epiblast, non-committed populations in vitro. However, in Xenopus,
reductive cell division would make it difficult to capture and sustain these
populations (Frederick and Andrews,
1994). Regardless of the feasibility of creating ES cell lines,
all vertebrates must posses the ability to prevent premature commitment. We
propose here that PouV proteins posses this function, which we have found to
be conserved in a number of vertebrate species.
A model illustrating how precocious differentiation in the absence of PouV
affects cells fate is shown in Fig.
8. In Xenopus, the organizer tissue is the first to
gastrulate and become committed, giving rise to dorsal anterior structures
while cells from the remaining marginal zone gastrulate later and populate
progressively posterior regions of the embryo
(Constance Lane et al., 2004;
Lane and Smith, 1999). In the
PouV-depleted embryos we observe a decrease in the expression of markers
associated with non-committed cell types (Bmp4, Xom and brachyury),
whereas the expression domains of markers associated with early commitment
(organizer and anterior endoderm) were expanded. The concurrent loss and gain
of uncommitted and committed cells, respectively, in the absence of increased
cell death leads us to conclude that there is a premature progression of
uncommitted marginal zone cells to a more committed cell fate early during
gastrulation.
Following PouV depletion in Xenopus, there is a striking
downregulation in the expression of Bmp4. This extrinsic factor has
been associated with maintaining cells in a non-committed state in both ES
cell lines and in Xenopus embryos where expression of a
constitutively active BMP receptor blocks the onset of germ layer commitment
(Constance Lane et al., 2004;
Ying et al., 2003). We found
the expression of both Bmp4 and its downstream target Xom
was dependent on PouV in Xenopus. Interestingly, Xom belongs to the
same class of transcription factors as the ES cell marker, Nanog.
We also observed a reduction in the expression of Xbra in the
PouV-depleted embryos. There is an accumulating body of evidence in mouse that
brachyury is a transient marker of both future mesoderm and endodermal cells
(Kubo et al., 2004;
Wilson et al., 1995). In
addition Xbra interacts directly with components of the BMP
signalling pathway to induce Xom, a direct antagonist of the
organizer associated gene goosecoid
(Messenger et al., 2005).
Thus, PouV proteins may block commitment in bipotent precursors of the
mesoderm and endoderm in part by maintaining brachyury expression.
Loss of XlpouV expression also leads to an expansion of endodermal markers
not associated with the organizer. Interestingly, mouse Oct4 has been shown to
have a role in inhibiting the expression of endodermal associated genes
through an interaction with Foxd3 (Guo et
al., 2002). An increase in endodermal gene expression was also
observed in Oct4-depleted mouse and human ES cells
(Hay et al., 2004).
As the depletion of PouV function in Xenopus results in a premature progression to a committed cell type and XlPouV proteins can sustain undifferentiated ES cells, we propose that there is a conserved role for these proteins in maintaining cell multipotentcy. This would strongly imply that the ability of mouse Oct4 to maintain ES cells as self-renewing and pluripotent is derived from this ancient PouV function.
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ACKNOWLEDGMENTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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