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First published online September 7, 2007
doi: 10.1242/10.1242/dev.006569
1 Institut de Génomique Fonctionnelle de Lyon, Université de Lyon,
Université Lyon 1, CNRS, INRA, Ecole Normale Supérieure de Lyon,
France.
2 Department of Anatomy and Developmental Biology, University College London,
London, UK.
3 Roslin Institute, Roslin, Midlothian EH25 9PS, UK.
4 Inserm, U384, Faculté de Médecine, 28, place Henri Dunant,
Clermont-Ferrand, F-63001, France.
5 Département de Physiologie Animale, INRA, France.
Author for correspondence (e-mail:
bepain{at}u-clermont1.fr)
Accepted 16 July 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Nanog, Oct4, Avian homologue, cPouV, Stem cells
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), Nanog
(Mitsui et al., 2003;
Chambers et al., 2003), Sox2
(Avilion et al., 2003) and
FoxD3 (Hanna et al., 2002).
Oct4 is found in oocytes and is expressed in cleavage stage cells up to the
morula stage (Kirchhof et al.,
2000), and subsequently in the epiblast of the pre-primitive
streak stage embryos. Oct4 expression is downregulated in
trophectodermal cells but maintained in the inner cell mass, becoming
restricted to primordial germ cells and oocytes
(Kehler et al., 2004;
Boiani et al., 2002). In vitro,
Oct4 is expressed in proliferating murine and primate (including
human) ESC, as well as in tumourigenic cells such as embryonal carcinoma (EC)
(Ben-Shushan et al., 1995) and
germ cell tumour (GCT) cells (Looijenga et
al., 2003).
Oct4 appears to control cell-fate decisions of ESC in vitro.
Inhibition of Oct4 expression in mouse ESC (mESC) causes a loss of
proliferation and the induction of trophectodermal and endodermal markers
(Velkey and O'Shea, 2003;
Hay et al., 2004). By
contrast, overexpression of Oct4 leads to primitive endoderm
differentiation (Niwa et al.,
2000) and it appears that a fine balance between Oct4 and
Cdx2 expression controls the choice between embryonic and
trophectoderm cell fates (Niwa et al.,
2005; Tolkunova et al.,
2006).
Oct4 contains a POU-specific domain and a POU homeodomain and belongs to
the class V POU homeodomain family of transcription factors. A complex of
proteins including Oct4 and Sox2 has been found to regulate expression of the
growth factor Fgf4 (Dailey et al.,
1994; Ambrosetti et al.,
1997) and of the transcription factors Utf1
(Nishimoto et al., 1999),
Zfp42 (Rex1) (Ben-Shushan et al.,
1998), Fbx15 (also known as Fbxo15 - Mouse Genome Informatics)
(Tokuzawa et al., 2003), Nanog
(Kuroda et al., 2005;
Rodda et al., 2005) and Sox2
itself (Tomioka et al., 2002).
Different nuclear receptors participate in the regulation of Oct4
expression including Sf1 (Barnea and
Bergman, 2000), Lrh-1 (Nr5a2)
(Gu et al., 2005a), Gcnf
(Nr6a1) (Fuhrmann et al.,
2001; Gu et al.,
2005b), CoupTF (Nr2f2)
(Ben-Shushan et al., 1995) and
Rar/Rxr heterodimers, the latter being responsible for the downregulation of
Oct4 expression by retinoic acid
(Schoorlemmer et al., 1994;
Pikarsky et al., 1994).
Oct4 expression is under the control of its own protein
(Okumura-Nakanishi et al.,
2005; Chew et al.,
2005) through specific response elements located in its own
promoter (Yeom et al., 1996;
Nordhoff et al., 2001;
Gu et al., 2005a;
Gu et al., 2005b).
Nanog expression is also confined to pluripotent tissues and cell
lines and its overexpression is able to maintain mESC in an undifferentiated
state, even in the absence of Lifr/gp130 stimulation. Inhibition of
Nanog expression in mESC results in their differentiation into
primitive endoderm (Chambers et al.,
2003; Mitsui et al.,
2003).
To date, this relationship between Oct4 and/or Nanog and
stem cell pluripotency has only been demonstrated in mammals. Indeed, in
zebrafish, it was reported that the pou2 gene (also known as
spg and pou5f1 - ZFIN), initially identified by a mutation
that caused neural and endoderm defects, is the fish homologue of the
mammalian Oct4 gene based on protein similarities, chromosomal
syntenic relationship and developmental expression pattern, but not in terms
of function (Burgess et al.,
2002). No evaluation of a putative role in fish ESC pluripotency
was described in an assessment of murine Oct4 activity in medaka ESC
(Hong et al., 1998). The
X. laevis Pou91 (XlPou91) gene product, encoded by one of
three X. laevis PouV genes, has been demonstrated to have a similar
activity to the mouse Oct4 gene in mESC and to participate in the
maintenance of putative stem cell populations during early development
(Morrison and Brickman,
2006).
Given that Oct4 appears to be so important in the maintenance of
pluripotency, a report suggesting that the chicken genome lacks a homologue of
Oct4 (Soodeen-Karamath and
Gibbins, 2001) was very surprising. Indeed, no corresponding
sequence was identified in the chicken genome annotation, even in the latest
release (Ensembl 42, December 2006).
Here we report the isolation of chicken PouV (cPouV) and
Nanog (cNanog), homologues of mammalian Oct4 and
Nanog. Both genes are expressed in early embryos before gastrulation
and thereafter in germ cells. Taking advantage of chick ESC (cESC)
(Pain et al., 1996;
Petitte et al., 2004), we
demonstrate that chicken PouV and Nanog are required for the
maintenance of cESC pluripotency and for continued proliferation. Together,
these findings show that the mechanisms by which these two genes regulate
pluripotency and self-renewal are not exclusive to mammals.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) were reverse transcribed. The cDNAs were subject to a
subtractive liquid hybridisation procedure
(http://www.genome-express.com/).
The transcripts enriched in cESC were subcloned, sequenced and filtered
sequences assembled using PHRAP software. Target sequences delivered by the
assembly process were subject to BLAST analysis.
Library screening and cloning of cPouV
The cDNA library from chicken embryonic stem cell mRNA
(Acloque et al., 2001) was
screened using T7 or T3 vector primers and internal sequences P06(381)S
(5'-GTTGTCCGGGTCTGGTTCT-3') or P06(382)AS
(5'-GTGGAAAGGTGGCATGTAGAC-3') derived from the 1P06g01 initial
clone. A 5'-RACE strategy was developed with the P06RAAS2
(5'-TGAGTGAAGCCCAGCATGAT-3') primer followed by a second
amplification with P06RAAS1 (5'-AACATCTTCCCATAGAGCGTGC-3') and
AnchPS (5'-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTT-3') primers. A
second round of amplification using P06(pL7-2)AS
(5'-TGCTTGAGGTCCTTGGCAAA-3') and PCRprimseq primers led to the
isolation of 300 bp upstream of the 1P06g01 clone, including an in-frame ATG.
A full-length cDNA was cloned into pGEM-T-easy (Promega) using primers
P06EcoRIS (5'-ATGAATTCATGCATGTAAAAGCCAAA-3') and P06EcoRIAS
(5'-ATGAATTCTCAGTGGCTGCTGTTGTT-3').
RNA extraction and RT-PCR
Real-time RT-PCR was performed using the MXP-3000P PCR-system (Stratagene)
using Mix-Quantitect SYBR Green (Qiagen). Samples were run in duplicate and
gene expression levels were calculated using the 
Ct method
(http://www.gene-quantification.info)
with the chicken ribosomal gene RS17 (X07257) as reference. The
number of independent experiments performed is indicated in each figure
legend.
In situ hybridisation
Hens' eggs were incubated for 0-36 hours and embryos staged according to
Eyal-Giladi and Kochav (Eyal-Giladi and
Kochav, 1976) for pre-primitive streak stages and according to
Hamburger and Hamilton (Hamburger and
Hamilton, 1951) for later stages. Embryos were subjected to
whole-mount in situ hybridisation (Streit
and Stern, 2001). Fluorescent Vasa and cPouV
probes were labelled with digoxigenin and fluorescein, respectively, and
successively revealed using an HRP-coupled anti-digoxigenin and an HRP-coupled
anti-fluorescein antibody and the TSA-Plus Cyanine3/Fluorescein system (Perkin
Elmer). SSEA-1 labelling (DSHB, Iowa) was performed on frozen sections (15
µm) and revealed with an anti-mouse IgM conjugated to Texas Red (Abcam).
Adjacent sections were processed for in situ hybridisation as previously
described (Strähle et al.,
1994).
Expression constructs
Reverse-transcribed chicken embryonic stem cell mRNA was used with
P06GFPEcoRIS (5'-GGGAATTCGCATGTAAAAGCCAAA-3') and P06GFPKpnIAS
(5'-ATGGTACCTCAGTGGCTGCTGTTGT-3') primers to amplify the
cPouV coding region. The product was subcloned into pEGFP-C1
(Clontech) to produce the pGFP-cPouV expression vector. cPouV cDNA
was amplified with P06EcoRIS (5'-ATGAATTCATGCATGTAAAAGCCAAA-3')
and P06EcoRIAS (5'-ATGAATTCTCAGTGGCTGCTGTTGTT-3') primers and
cloned into pCAGIP (Niwa et al.,
2000). The 1.8 kb pou2 zebrafish coding sequence was
amplified from a pCSL2-Pou2 template using Pou2EcoRIS
(5'-ATAGAATTCTATGACGGAGAGAGCGCAG-3') and Pou2EcoRIAS
(5'-GTAGAATTCTTAGCTGGTGAGATGACCC-3') primers and cloned into
pCAGIP. Murine Oct4 and Nanog coding sequences were reverse
transcribed from mESC total RNA with primer pairs mOct4EcoRIS
(5'-ATGAATTCTGCTGGACACCTGGCTTC-3') with mOct4EcorIAS
(5'-ATGAATTCTTAACCCCAAAGCTCCAG-3') and mNanogXhoIS
(5'-GTCTCGAGATGAGTGTGGGTCTTCC-3') with mNanogNotIAS
(5'-ATGCGGCCGCTCATATTTCACCTGGT-3'), respectively, then inserted
into pCAGIP. The cNanog coding sequence was obtained from
reverse-transcribed cESC total RNA using cNanogEcoRIS
(5'-ATGAATTCATGAGCGCTCACCTGGCC-3') and cNanogEcoRIAS
(5'-ATGAATTCCTAAGTCTCATAACCATT-3') primers and cloned into
pCAGIP.
Transactivation test
The p(ATGCAAAT)x3-luc reporter gene was constructed by inserting
double-stranded oligonucleotides Oct4BS
(5'-CTAGCATGCAAATAACAGCGCGCATGCAAATAACAGCGCATGCAAATAACAGCGCCCC-3')
and Oct4BAS
(5'-GGGGCGCTGTTATTTGCATGCGCTGTTATTTGCATGCGCGCTGTTATTTGCATG-3')
into the pGL3 vector (Promega). To construct the pPE-luc reporter gene,
a 1.4 kb fragment from the mOct4 distal enhancer was amplified from
pGOF18PE-GFP using ODES
(5'-GTACGCGTGAATTCAGACAGGACTGCTGGGC-3') and SVAS
(5'-AGCATCACAAATTTCACAAATAAAGAATTCACGGCTTT-3') primers
(Hong et al., 2004) and
subcloned into pGL3. For luciferase assays, ZHBTc4 cells were plated at
1x105 cells per well with 2 µg/ml tetracyclin. Twenty-four
hours later, 75 ng of reporter plasmid, 150 ng of the test plasmid and 10 ng
of the Renilla reporter plasmid were co-transfected using 600 ng
FuGENE 6 (Roche) and incubated overnight before fresh medium was added with 2
µg/ml tetracyclin. Cell lysates were analysed 48 hours after transfection
as described by the manufacturer (Promega).
RNA interference (RNAi) vector construction
pFLNeo was obtained by inserting into pBSK the 2 kb PCR-amplified
product mU6Neo, derived from the mU6Neo-ApaIDXhoI
template (Coumoul et al.,
2004) using mU6SmaIS
(5'-ATCCCGGGGTATATCCGACGCCGCCAT-3') and mU6HindIIIAS
(5'-ATAAGCTTAACAAGGCTTTTCTCC-3') primers. Double-stranded short
hairpin (sh) RNA was cloned into pFLNeo, generating
pFLNeo-XshRNA vectors for each gene to be targeted. The
oligonucleotides containing the HindIII and XhoI sites used
for generating the 21 bp shRNA sequence were: cPouV-shRNA-2S
(5'-AGCTTAAGATGTTCAGCCAGACCACCTTCAAGAGAGGTGGTCTGGCTGAACATCTTTTTTTTC-3')
and cPouV-shRNA-2AS
(5'-TCGAGAAAAAAAAGATGTTCAGCCAGACCACCTCTCTTGAAGGTGGTCTGGCTGAACATCTTA-3')
against cPouV; cNanog-shRNA-1S
(5'-AGCTTAACAGAAACCTTCAGGCTGTGTTCAAGAGACACAGCCTGAAGGTTTCTGTTTTTTTTC-3')
and cNanog-shRNA-1AS
(5'-TCGAGAAAAAAAACAGAAACCTTCAGGCTGTGTCTCTTGAACACAGCCTGAAGGTTTCTGTTA-3')
against cNanog; as well as cNanog-shRNA-3S
(5'-AGCTTAAGGCCAAGAGCCGCACAGCTTTCAAGAGAAGCTGTGCGCTCTTGGCCTTTTTTTTC-3')
and cNanog-shRNA-3AS
(5'-TCGAGAAAAAAAGGCCAAGAGCCGCACAGCTTCTCTTGAAAGCTGTGCGGCTCTTGGCCTTA-3')
and cOct6-shRNA-3S
(5'-AGCTTAAGCAGCGGCGGATCAAGCTGTTCAAGAGACAGCTTGATCCGCCGCTGCTTTTTTTTC-3')
and cOct6-shRNA-3AS
(5'-TCGAGAAAAAAAAGCAGCGGCGGATCAAGCTGTCTCTTGAACAGCTTGATCCGCCGCTGCTTA-3')
against cOct6. The Cre-ERT2 coding sequence
(Feil et al., 1997) was cloned
into the pCIFL-Hygro vector, derived from pCINeo (Promega), by replacing the
neomycin cassette with a hygromycin cassette to produce pCre-ERT2-Hygro.
Cell maintenance and transfection
cESC were maintained and transfected as previously described
(Pain et al., 1996;
Pain et al., 1999). For
kinetic experiments, formation of cEB was achieved by allowing dissociated
proliferating cESC to float in bacterial dishes. When used, retinoic acid was
added at 10-7 M 24 hours after plating and considered as T=0.
Cycloheximide and actinomycin D were added to the culture medium at 10
µg/ml for various times as indicated.
ZHBTc4 cells were maintained as described
(Niwa et al., 2000).
Expression of the endogenous murine Oct4 can be downregulated by
addition of 1 µM doxycyclin (Sigma). For transfection,
5x106 cells were electroporated (BioPulser, BioRad) at 575
µF with 25 µg of the various linearised vectors. From twenty-four hours
after electroporation, doxycyclin was added at daily intervals, and puromycin
was added 72 hours after electroporation at 1 µg/ml and administered daily
for 6 days.
RNAi induction and proliferation assay
Once transfected and selected with 200 µg/ml neomycin for 7 days,
resistant clones of cESC were pooled, transfected with the pCre-ERT2-Hygro
vector and selected for 7 days with 0.75 µg/ml hygromycin. Clones were
numbered, picked and individually observed during the induction of shRNA
expression by adding 1 µM 4-hydroxytamoxifen to the medium. Morphology was
assessed by direct microscopic observation and Wright Giemsa staining. For
proliferation kinetic assays, clones were picked individually, the cells
dispersed and plated in six wells in 250 µl medium. After 24 hours,
4-hydroxytamoxifen was added and proliferation was assessed at different times
using two wells per time point using Cell Proliferation Kit II (XTT)
(Roche).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Saitou and Nei, 1987). This
novel predicted protein is part of the PouV protein subfamily, which contains
XlPou91, the D. rerio Pou2 and mammalian Pou5f1 proteins and exhibits
high similarity with the other members of the family
(Fig. 1B,C). At the genome level, the novel gene was mapped to chicken chromosome GGA17, specifically between primers SEQ0256 and SEQ0257 described in the ChickRH6 whole-genome radiation hybrid (WGRH) panel (http://chickrh.toulouse.inra.fr/). Syntenic comparison identified a relationship between this chicken gene, XlPou91 and zebrafish pou2. This relationship appears to be absent, either deleted or displaced, in mammalian species, despite the presence of adjacent syntenic loci on mouse chromosome 2 (data not shown).
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Cloning of chicken Nanog cDNA
A chicken Nanog gene was predicted in the chicken genome
annotation at reference ID ENSGALG00000014319 on chicken chromosome 1 (GGA1).
Primers designed using this sequence were used to isolate a clone from the
chicken embryonic stem cell library with a 930 bp ORF (GenBank accession
DQ867025). Comparative analysis and phylogenetic tree construction revealed
that this sequence is closely related to mammalian Nanog genes and
that the predicted protein exhibits high similarity with the other Nanog
proteins (Fig. 1D). This
sequence contains a homeodomain of 57 aa, located between aa 98 and 155, but
does not have the WWW repeat in the C-terminus that is characteristic of the
mammalian Nanog subfamily (Pan and Pei,
2005). In contrast to the recently reported chicken Nanog sequence
identified in silico (Canon et al.,
2006), our cloned protein does not indicate the existence of a 112
aa segment after aa 50 that could correspond to a putative alternatively
spliced form.
cPouV and cNanog are highly expressed in proliferating cESC and downregulated during differentiation of cESC
To determine the expression profiles of cNanog and cPouV,
we performed real-time RT-PCR experiments showing that proliferating cESC
express high levels of cPouV and cNanog
(Fig. 2A,D, time 0). cESC can
be induced to differentiate either as cEB, by preventing cell attachment, or
following treatment with chemical inducers such as DMSO or retinoic acid (RA)
(Pain et al., 1996). During a
5-day RA treatment, cPouV expression was almost completely abolished
in parallel to similar reductions in expression of the markers alkaline
phosphatase (AP) and telomerase reverse transcriptase (Tert)
(Fig. 2A). Chicken
Gcnf expression was also strongly downregulated, as was expression of
Sox2 and Nanog, although with a more complex profile. By
contrast, Rar
expression was upregulated following RA
treatment (Fig. 2A). Treatment
with cycloheximide, known to block de novo protein synthesis, did not affect
the downregulation of cPouV and Gcnf transcription, whereas
Nanog transcription was no longer responsive to RA, suggesting that
downregulation of cPouV and Gcnf transcription are direct
transcriptional events following RA treatment
(Fig. 2B). Moreover, following
actinomycin D treatment, which blocks transcription, a 50% decrease in the
expression of cPouV and Gcnf was observed 8 to 12 hours
after addition of the drug. A decrease of greater than 50% in Nanog
mRNA levels was seen as early as 30 minutes after treatment, suggesting that
it has a very short half-life (Fig.
2C).
Expression of cPouV and Nanog was also strongly downregulated during formation of cEB (Fig. 2D), as was expression of Sox2 and AP. By contrast, Sox3, Cdx2 and Gata6 were induced, suggesting a complex differentiation process during formation of these three-dimensional bodies (Fig. 2E).
cPouV and cNanog are expressed dynamically in early chick embryos
To determine the likely sites at which cPouV and cNanog
function during normal development, transcripts were identified in embryos
during progressive stages of development using whole-mount in situ
hybridisation. cPouV mRNA was found to be ubiquitously expressed in
the epiblast of pre-primitive streak stage embryos and in a salt-and-pepper
fashion in the forming hypoblast (Fig.
3A,B,B'). As the primitive streak started to form,
transcripts were strongly localised in the epiblast of the streak itself
(Fig. 3C,C') and in the
mesoderm emerging from it, whereas expression in the lower layer tended to
decrease (Fig.
3D-F,D',F'). Expression in the area opaca was lost by
stage 3+ (Fig. 3F).
At later stages, cPouV continued to be expressed in the mesoderm, but
was undetectable in the endoderm (Fig.
3G-I). At stage 8 and subsequently, cPouV was strongly
expressed in the neural plate and neural tube with particularly strong
expression in the anterior hindbrain/posterior midbrain
(Fig. 3I). Later, at stage 9
and subsequently, cPouV was still expressed in neural tissue and
expression appeared in primordial germ cells
(Fig. 3J).
cNanog showed a different pattern. In pre-streak embryos, transcripts were detected in the whole epiblast but not in the forming hypoblast (Fig. 4A,B,B'). As the primitive streak started to form, transcripts disappeared from the primitive streak epiblast but were still expressed throughout the area pellucida epiblast (Fig. 4C-E,C',D'). At the end of gastrulation (stage 4-4+), cNanog mRNA was quickly downregulated in the epiblast and persisted in a crescent anterior to the emerging head process (Fig. 4F-H). As the neural plate formed (stages 6-8), expression in the epiblast was restricted to the anterior neural plate (Fig. 4H-J,H').
In conclusion, following initial, high levels of expression in early pluripotent epiblast cells, cPouV and cNanog present a very restricted in vivo pattern of expression during early embryonic development.
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cNanog expression was also detected by QRT-PCR in heart, brain, kidney and gonads, but at levels 20-, 25-, 90- and 100-fold lower, respectively, than in cESC (data not shown). In early embryos, Nanog was also expressed in scattered cells in the germinal crescent: these cells are likely to correspond to the future germ cells (Fig. 4I and arrowhead in H'). Later in development, cNanog was still expressed in germ cells at stage 33 (Fig. 5I-K) identified as SSEA-1-positive, in a similar manner to the cPouV-expressing cells (Fig. 5L,M). However, cNanog expression became weaker at this stage compared to the previous stages. This expression profile was observed in both male and female embryos (data not shown).
In conclusion, expression of cPouV and Nanog becomes restricted to germ cells at later stages of embryonic development.
Overexpression of Oct4-related genes in cESC and mESC
In order to compare cPouV function with its orthologues, coding
sequences of cPouV, murine Oct4 (mOct4),
XlPou91 and zebrafish pou2 were transfected into cESC and
mESC. In cESC, overexpression of cPouV using the pCAGIP vector
impaired the isolation of proliferating clones. Using pCMV-based vectors, gene
expression analysis revealed a 4-fold induction of cPouV, but a
strong decrease in expression of Nanog and Tert. By
contrast, strong upregulation of Gata4, Gata6 and Cdx2,
associated with differentiation, was observed
(Fig. 6A). Ectopic expression
of XlPou91 induced a similar expression profile, with an increase in
endogenous cPouV and of differentiation markers Gata4, Gata6
and Cdx2, and a strong decrease in Nanog and Tert
expression (Fig. 6B). By
contrast, overexpression of mOct4 did not modify cPouV or
Nanog endogenous expression levels and induced only a slight increase
in endogenous chicken Cdx2 gene expression
(Fig. 6B).
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), it was not possible to isolate clones of cells
overexpressing mOct4 after transfection of mESC with the neomycin
resistance overexpression plasmid. The same pCAGIP vector was used to
overexpress cPouV and XlPou91 in mESC, but clones could only
be isolated of mESC expressing XlPou91. Endogenous expression of
Oct4 was maintained and expression of Gata4, Gata6 and
Cdx2, as well as of mesendodermal markers including Hnf1,
brachyury, Sox17 and laminin B1, was observed
(Fig. 6C). Using a pCMV-based
expression vector, cPouV expression enabled identification of clones
presenting a similar expression profile, i.e. with a maintenance of endogenous
Oct4 expression but only a slight increase in Gata4, Gata6, Cdx2,
Hnf1, brachyury, Sox17 and laminin B1 expression
(Fig. 6D). In conclusion, high ectopic expression of cPouV impairs the proliferation of both cESC and mESC, but a moderate level of expression of exogenous cPouV is tolerated by cESC and mESC with an associated modification of the observed gene expression profile.
cPouV is able to rescue partially Oct4-deficient ZHBTc4 mESC
A good test of whether cPouV is functionally equivalent to its
murine counterpart Oct4, is to assay the ability of the chick gene to
rescue the ZHBTc4-inducible cells in which endogenous Oct4 expression
is downregulated by addition of doxycyclin. Transfection of ZHBTc4 cells with
expression vectors for mOct4 or XlPou91 allowed isolation of
proliferating clones in the presence or absence of doxycyclin,
(Fig. 7A,B) as predicted
(Niwa et al., 2000;
Morrison and Brickman, 2006).
In the presence of doxycyclin, expression of cPouV was able to
support the growth of slowly proliferating AP-positive colonies
(Fig. 7C) with a rescue index
(the ratio between the number of clones in the presence versus the absence of
doxycyclin) of 0.5 (Fig. 7D),
as compared with 1.0 for expression of mOct4 and 3.5 for
XlPou91. However, the colonies recovered after cPouV
expression were limited in their capacity to be passaged or amplified and
exhibited a differentiated morphology. No clones were obtained after
expression of zebrafish pou2 in the presence of doxycyclin.
Real-time RT-PCR analysis performed on RNA from the clones generated by cPouV complementation revealed a complete loss of endogenous mOct4 mRNA, but high expression of the exogenous cPouV mRNA (data not shown). Expression of pluripotency-associated markers such as Nanog, Sox2, Utf1 and Zfp42 (Rex1) was maintained at the same level in cells complemented by XlPou91 as in cells complemented by mOct4 (and expression was even higher for Tert and Fgf4). Expression of these markers was reduced, but detectable, in cells complemented by cPouV, with the exception of Sox2 and Fgf4 for which no expression could be detected in the presence of cPouV (Fig. 7E).
To test the ability of this gene to transactivate specific
Oct4-responsive elements, promoters containing either the
mOct4 consensus binding site (ATGCAAAT), or the 1.4 kb PE
fragment from the mOct4 promoter
(Yeom et al., 1996;
Hong et al., 2004), linked to
a luciferase reporter, were transfected into ZHBTc4 cells. These promoters
were activated in ZHBTc4 cells treated with doxycyclin in the presence of the
expression vectors coding for mOct4, XlPou91 or cPouV, as
measured by luciferase activity (Fig.
7F). This interesting result suggests that the cPouV protein is
able to recognise mOct4-response elements and activate
transcription.
In conclusion, these experiments suggest that the cPouV gene is able to partially rescue the loss of mOct4 function in mESC, and does interact with and activate mOct4-dependent regulatory elements.
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In contrast to mESC, cESC are not dependent on a single cytokine for their
proliferation and survival (Pain et al.,
1996) (our unpublished results). Overexpression of cNanog
conferred the ability of the cESC to grow in a low-serum medium in the absence
of growth factors and cytokines that are usually required for proliferation
(Fig. 8I). The clones obtained
proliferated actively and were easily passaged and amplified (data not shown).
It was particularly surprising to obtain proliferative avian primary stem
cells in the presence of only 1% foetal bovine serum. Interestingly, under
these drastic conditions, mNanog expression did not have any
pronounced effect on the chicken cells. Real-time RT-PCR analysis confirmed
overexpression of cNanog, the maintenance of Tert and
reduced, but detectable, expression of cPouV and AP
(Fig. 8J-K).
In conclusion, we have shown that cNanog functions in a very similar way to mNanog in mESC and has a more dramatic effect in cESC, where overexpression permits maintenance of the stem cell phenotype in the absence of growth factors and in low serum.
Inactivation of cPouV or cNanog inhibits ES cell proliferation and induces differentiation
To assess cPouV and cNanog function, constructs
expressing shRNAs were designed to knockdown transcripts of these genes, using
a tamoxifen-inducible Cre system to activate the expression of the shRNAs.
Following induction of Cre recombinase activity by tamoxifen addition, a rapid
and dramatic morphological change was observed, involving changes associated
with differentiation (Fig.
9A-D). These changes were seen in 
60% of the clones when
specific shRNAs were used against cPouV and cNanog
(Fig. 9G) This morphological
change was observed even in the presence of growth factors and was accompanied
by a loss of AP activity and of SSEA-1 antibody staining
(Fig. 9H), plus a growth rate
alteration 48 and 96 hours after Cre induction
(Fig. 9I). Comparison of the
gene expression profiles between differentiated clones and clones that
continued to proliferate revealed strong inhibition of endogenous
cPouV expression as well as of cNanog and Gata4 and
strong induction of Gata6 (Fig.
9J). No upregulation of Cdx2 was detected, nor of other
mesendodermal markers such brachyury or Hnf3ß. Similar
experiments involving inhibition of another POU-domain gene, Oct6
(Levavasseur et al., 1998),
did not change the endogenous level of cPouV and proliferating clones
were obtained (Fig. 9G,J).
|
In conclusion, inhibition of either cPouV or cNanog leads to a loss of proliferation of cESC and to the induction of differentiation.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
Both overexpression and disruption of Oct4 in mESC leads to a loss of
pluripotency and induces the cells to differentiate into primitive endoderm,
characterised by high Gata6 expression
(Li et al., 2004), and into
trophectoderm expressing Cdx2
(Niwa et al., 2000;
Strumpf et al., 2005;
Niwa et al., 2005;
Tolkunova et al., 2006). In
vivo, it is now thought that complex regulatory mechanisms lead to restricted
expression in early pregastrulation embryos
(Gu et al., 2005a;
Boiani et al., 2002) and in the
germ line (Kehler et al.,
2004; Yeom et al.,
1996).
Nanog, a homeodomain transcription factor, was identified as another key
factor maintaining the pluripotency of mammalian ESC
(Chambers et al., 2003;
Mitsui et al., 2003;
Hart et al., 2004). In mESC,
Nanog overexpression has been shown to substitute for the requirement
for growth factors in the maintenance of self-renewal. Disruption of
Nanog leads to a loss of pluripotency and to induction of
differentiation towards an endoderm-like state
(Mitsui et al., 2003).
The existence and equivalent functions of homologues of these genes in
non-mammalian vertebrates are still debated. Functional assays were used to
identify the zebrafish pou2 gene as the Oct4 homologue
(Burgess et al., 2002), but
this gene appears to be mainly involved in the endoderm-specification cascade
(Reim et al., 2004;
Lunde et al., 2004). In
Xenopus, XlPou91, a PouV gene, plays a significant role in
the maintenance of pluripotent cells during early development and was shown to
rescue Oct4 depletion in mESC. XlPou91 knockdown in vivo
using morpholinos induces expression of Xcad3, which is considered to
be the Xenopus homologue of Cdx2
(Morrison and Brickman, 2006).
These data are consistent with the idea that PouV family members,
including murine Oct4, could act to prevent premature commitment of
pluripotent cells present in vertebrate embryos prior to and during
gastrulation.
cESC have been isolated and maintained in culture for long periods
(Pain et al., 1996;
Petitte et al., 2004;
Van de Lavoir et al., 2006).
These cells were derived from the culture of pre-primitive streak blastodermal
cells and are characterised by the presence of typical ESC markers such as AP,
Tert activity and reactivity with particular antibodies including ECMA-7,
SSEA-1, SSEA-3 and EMA-1 (Pain et al.,
1996; Petitte et al.,
2004).
|
Third, cPouV is expressed in a complex pattern in the embryo,
being expressed widely in the early epiblast and later becoming restricted to
specific regions, including the mesoderm and nervous system. This initial
expression in multipotent epiblast cells, which then becomes restricted once
the cells start to be committed, is also shared by the zebrafish,
Xenopus and mouse homologues, which have been implicated in
regulation of early neural development and patterning
(Ramos-Mejia et al., 2005;
Burgess et al., 2002;
Reim and Brand, 2002;
Morrison and Brickman, 2006).
During late development, cPouV expression becomes more restricted to
migrating and proliferating germ cells, as demonstrated by co-localisation
with Cvh-positive cells in the developing gonads. This
germ-line-restricted expression is a feature shared with its murine
counterpart, in contrast to the zebrafish and Xenopus homologues. We
conclude that cPouV plays a similar role to its mammalian and
non-mammalian homologues in pregastrulating embryos, but functions more like
the mammalian homologue in germ cells.
Another feature of this chicken gene is its ability to induce
differentiation when overexpressed in cESC. Expression of cPouV in
cESC and mESC alters the morphology and reduces the growth rate of the ESC,
and inhibits isolation of clones from cells in which cPouV is
expressed from a very strong promoter. Expression using a moderate CMV
promoter induces expression of differentiation markers such as Gata4,
Gata6 and Cdx2. In mouse, these markers are associated with
endodermal and trophectodermal lineages, but their function during early
chicken development is still unknown. A similar profile of gene expression is
obtained in parallel experiments with XlPou91, suggesting common
target genes. Overexpression of cPouV in mESC led to a more moderate
phenotype, with a slight induction of differentiation markers such as
Gata6 and Cdx2, and also of Hnf1, brachyury and
Sox17, which are known in mouse to be strongly expressed in
mesendoderm structures (Tada et al.,
2005; Yasunaga et al.,
2006). cPouV is able to trigger a differentiation
programme when overexpressed in both mESC and cESC.
|
).
In the presence of cPouV, endogenous mouse Nanog expression
in mESC is maintained at a low but detectable level. Expression of Utf1,
Zfp42 (Rex1) and Tert is maintained, but expression of
Sox2 and Fgf4 is completely abolished. These observations
could explain the limited ability of these clones to be passaged and
amplified. However, under these same conditions, the various factors are able
to transactivate specific promoters containing either the Oct4
consensus binding site or the mouse endogenous Oct4 promoter,
suggesting that cPouV is able to substitute functionally for mouse
Oct4. The N-terminal domains of the mouse and chicken genes are
highly diverged. It is probable that stringent interprotein interactions are
required for full activity of the chicken protein. A first attempt to test
this hypothesis, by constructing molecular chimaeras between the mouse
Oct4 N-terminus and the cPouV homeodomain and C-terminus,
proved unable to restore complete ZHBTc4 cell proliferation (data not shown),
suggesting that other mechanisms and/or partners are likely to be
required.
We also report the isolation of the chicken functional orthologue of
Nanog (cNanog) from proliferating cESC and demonstrate that
cNanog also plays a role in the maintenance of self-renewal and
pluripotency of cESC. The sequence we cloned is shorter than the one recently
reported by Canon et al. (Canon et al.,
2006). The cNanog expression profile differs both in
vitro and in vivo from that of cPouV. Specifically, cNanog
expression is downregulated by differentiation but with a different
time-course than cPouV in both RA-induced differentiation and cEB
formation. In contrast to cPouV, cNanog transcription is maintained
and possibly increased in the presence of cycloheximide and the mRNA half-life
appears to be reduced in the presence of actinomycin, suggesting a short
half-life of the mRNA. In vivo, the expression pattern of cNanog is
also different from that of cPouV, with a more rapid disappearance
from the epiblast and a subsequent restriction to the anterior neural plate.
cNanog expression is detected in migrating germ cells, as was also
observed for Nanog in the mouse
(Yamaguchi et al., 2005), but
also in germ cells of developing gonads.
|
Finally, inhibition of expression of cPouV and cNanog, using an inducible knockdown approach, promotes rapid growth arrest within 48 hours of shRNA induction. This inhibition of proliferation is accompanied by an induction of differentiation as detected by altered morphology, loss of SSEA-1 labelling and expression of Gata6 and Cdx2. This suggests that these two genes play a key role in the maintenance of the pluripotent character of cESC.
|
|
ACKNOWLEDGMENTS |
|---|
PE-GFP plasmid, Dr Deng
for the mU6 promoter, Dr Brandt for the Spg plasmid, Dr Vignal for
the cPouV localisation and Dr Brunet for his help in phylogenetic
analysis. F.L. was supported by an INRA fellowship. This work was supported by
Region Rhône-Alpes, GenAnimal program and by the Vivalis Company. The
work in the laboratory of C.D.S. was supported by the MRC, BBSRC, National
Institutes of Health (NIMH) and the European Union Network of Excellence
`Cells into Organs' and in the laboratory of H.M.S. by the award of a BBSRC
studentship to D.J.M. |
Footnotes |
|---|
|
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