AgResearch Crown Research Institute, Ruakura Campus, East Street, Hamilton 2001, New Zealand
Author for correspondence (e-mail:
peter.pfeffer{at}agresearch.co.nz)
Accepted 7 March 2005
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SUMMARY |
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Key words: Elf5, Extraembryonic ectoderm, Trophoblast stem cells, AVE, Mesoderm
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Introduction |
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The proliferative potential of the pTE and ExE is dependent on a population
of trophoblast stem (TS) cells that can be isolated from these tissues at
blastocyst to gastrula stages (Tanaka et
al., 1998; Uy et al.,
2002
). FGF signalling from the ICM, and subsequently from the
epiblast, is required for maintaining these TS cells. Removal of Fgf4 in TS
cell cultures leads to the differentiation of TS cells into giant cells and
the loss of early ExE-specific markers, such as Eomes and
Cdx2 (Tanaka et al.,
1998
). Eomes and Cdx2 appear to be crucial to
the formation and maintenance of TS cells within the pTE as no TS cells can be
derived from null mutants of Eomes
(Russ et al., 2000
) or
Cdx2 (Rossant et al.,
2003
), in accordance with the in vivo trophoblast defects in these
embryos. The EPC, which overlies the ExE, is devoid of TS cells
(Uy et al., 2002
). It contains
differentiated trophectodermal cells that are thought to form the
spongiotrophoblast (Cross et al.,
2003
).
Towards the end of gastrulation, the ExE forms a bilayer with
extraembryonic mesoderm and becomes separated from the epiblast by the
exocoelomic cavity (Kaufman,
1995). This bilayer is termed the chorion and is deflected towards
the proximal pole of the conceptus. At around embryonic day (E) 8.5, the
allantoic mesoderm attaches and fuses with the basal layer of the chorion.
Chorionic trophoblast cells begin to differentiate into syncytiotrophoblast
cells and villi progenitors, and, in conjunction with allantoic cells, will
form the chorioallantoic placenta essential for maternal-foetal nutrient and
gaseous exchange and therefore embryonic survival
(Cross et al., 2003
;
Rossant and Cross, 2001
).
However, the ExE also fulfils an earlier inductive function by signalling
to the subjacent epiblast during germ cell formation
(Yoshimizu et al., 2001) and
embryonic patterning (Beck et al.,
2002
). These roles of the ExE are mediated by at least two
distinct pathways involving the TGFß superfamily members Bmp4 and Nodal.
Bmp4 is expressed at gastrulation stages in the ExE adjacent to the
epiblast, and, in chimeric loss-of-function mutants in which extraembryonic
Bmp4 expression is selectively ablated, neither primordial germ cells
nor extraembryonic mesoderm is formed
(Lawson et al., 1999
).
Secondly, Nodal activity in the epiblast is necessary for both mesoderm and
anterior visceral endoderm (AVE) formation
(Brennan et al., 2001). The AVE
is formed by migration of a group of distal visceral endoderm cells to one
side of the egg cylinder well before gastrulation commences. It secretes
antagonists into the adjacent epiblast thereby restricting Nodal activity and
thus mesoderm formation to the opposite (posterior) side of the egg cylinder
(Lu et al., 2001
). However,
Nodal translation generates Pro-Nodal, which has to be cleaved by
endoproteases to generate the fully active Nodal signalling molecule. This
cleavage is performed by Furin/Spc1 and Spc4/Pace4, which are expressed in,
and secreted from, the ExE (Beck et al.,
2002
). Double loss-of-function mutants for these two proteases
closely resemble Nodal-deficient embryos and do not form AVE or
mesoderm (Beck et al.,
2002
).
We describe here the identification of a novel key gene involved in
maintaining the polar trophectoderm/ExE lineage. This gene is Elf5
(ESE2 in humans), which encodes a transcription factor belonging to
the Ets superfamily. It is characterised by a DNA-binding Ets domain, is able
to bind to a subset of Ets-binding sites and can transactivate constructs
containing Ets-binding sites upstream from a minimal promoter
(Oettgen et al., 1999).
Elf5 has previously been shown to be expressed in foetal and adult
epithelial cells of organs such as the mammary and salivary glands, kidney,
prostate and lung in mice and in humans
(Oettgen et al., 1999
;
Zhou et al., 1998
).
Significantly, placenta of pregnant mice at E9.5 and later exhibited
Elf5 expression, as assayed by northern blots
(Zhou et al., 1998
). Presently
no in vivo role for Elf5 has been demonstrated though Elf5 expression
appears to be increased significantly in mouse mammary tumors relative to in
normal mammary tissue (Galang et al.,
2004
).
We report here on the early expression of Elf5 in the ExE lineage and demonstrate an essential function of Elf5 for the generation of this tissue. We discuss our findings in relation to trophoblast stem cell maintenance and epiblast-ExE interactions.
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Materials and methods |
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Genotyping by PCR
PCR genotyping was performed using the two allele, three primer PCR
strategy. The common primer A (5'-GAGCAATGGGAATAAACAGGG) was located
within the long arm at the 3' end of intron 1 and could give a 410 bp
product only on the targeted allele with primer B
(5'-TGGATGTGGAATGTGTGCGA) located within the puromycin cassette. On the
wild-type allele, primer A gave a 578 bp product in combination with primer C
(5'-GGAGAAAGGTGGGGAGGATAA). The inserted puromycin cassette
prevented the primer A-C combination from yielding a product with the targeted
allele as template. Tail tips were digested in Proteinase K buffer [100 mM
Tris (pH 8.0), 5 mM EDTA, 0.1% SDS, 200 mM NaCl] at 55°C, with shaking,
for 2 hours to overnight, boiled for 5 minutes, centrifuged and then 0.25-1
µl used in a standard 25 µl PCR reaction containing 1 Unit Taq
polymerase, 1.5 mM Mg2+, 10 pmol of primer A, and 5 pmol of primers
B and C. PCR conditions were 94°C for 4 minutes, followed by 35 cycles of
94°C for 30 seconds, 62°C 45 seconds and 72°C for 1 minute.
Whole-mount in situ hybridisation
The whole-mount in situ hybridisation protocol has been described
(Nagy et al., 2003). Staining
reactions were carried out for 2 hours up to 5 days, with littermates always
treated in the same vessels in the same way. Embryos were genotyped after
photography by PCR. Mouse Elf5 cDNA clones were isolated by screening
a mammary gland cDNA library with a bovine Elf5 fragment. The
Elf5 antisense probe used covered nucleotides 9 to 334 of the
reference sequence NM_010125, excluding the conserved ETS domain. A 530 bp
mouse Hex fragment was cloned into pGEM-Teasy (Invitrogen) in the
T7/sense orientation using the primers 5'-CCCTCTGTACCCGTTCCC and
5'-CCGATGACTGTCATCCAGC and a Ta of 50°C.
Trophoblast stem (TS) cell culture
E6.5 embryos from Elf5+/ matings were separated
into proximal and distal halves. The distal half was used for genotyping. The
proximal half was stripped of visceral endoderm
(Nagy et al., 2003) and
treated at 37°C for about 10 minutes with 0.25% pronase in Tyrodes Ringer
saline. Cells were dispersed by brief pipetting and plated on primary feeder
cells (Nagy et al., 2003
;
Tanaka et al., 1998
;
Uy et al., 2002
). We used 70%
conditioned medium (Tanaka et al.,
1998
) from the outset. Several colonies were observed in wild-type
and Elf5+/ cultures by 3 days after dissociation. After the
third passage, TS cultures were grown in the absence of feeder cells. For the
Fgf4/heparin withdrawal experiment, sixth passage TS cultures were grown in
four-well dishes to 30% confluency, whereupon growth was continued for 5 days
in conditioned medium with or without Fgf4 and heparin. Cells were pelleted
and subjected to real-time PCR as described below.
Real-time RT-PCR
RNA was isolated using TRIZOL (Invitrogen) and reverse transcribed with
Superscript3 (Invitrogen) and oligo-dT, according to the manufacturer's
instruction. Real-time PCR was performed using SYBR-Green master mix (Applied
Biosystems) and the following primers (introns spanned; amplicon size in base
pairs):
PCR conditions were 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds, 56°C for 30 seconds, 72°C for 30 seconds, and 78°C for 10 seconds, followed by dissociation curve analysis. RT-minus controls were run routinely and representative PCR products analysed on agarose gels to ensure spcecificity of reactions. Amplification efficiencies were monitored by standard curves using serially diluted samples and ranged from 1.6 to 1.9. Relative copy numbers were calculated and normalised against actin.
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Results |
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The absence of ExE and a visible ExE-EmE constriction suggested that the
ectodermal layer of the Elf5 mutant egg a marker for undifferentiated
embryonic ectoderm (Scholer et al.,
1990). We found that Oct4 expression in mutants extended
to the proximal end of the egg cylinder
(Fig. 4K,L), confirming the
absence of ExE tissue well before the commencement of gastrulation. Similarly
Otx2, normally restricted to the anterior embryonic epiblast
(Perea-Gomez et al., 2001
),
was expressed across the entire mutant ectoderm, reaching the EPC region
(Fig. 4M,N). We conclude from
these marker studies that in the absence of Elf5 activity, the ExE is not
formed, resulting in embryos composed of EPC directly abutting the embryonic
ectoderm.
Elf5 is required for the maintenance of trophoblast stem cells
Why were Elf5-/- embryos depleted of ExE by E5.5? The
ExE is a direct derivative of the polar trophectoderm (pTE) of the blastocyst.
Yet unlike Cdx2 and Eomes deficient embryos, which die
around the implantation stage as a result of defects in trophectoderm and pTE
cells, respectively (Chawengsaksophak et
al., 1997; Russ et al.,
2000
), Elf5-/- embryos implant and develop at
expected Mendelian ratios to E6.5 (Table
1). Furthermore, Cdx2 marking the pTE was still expressed
in E4.5 Elf5-/- mutants
(Fig. 4A). This suggests that
pTE formation is not impaired in Elf5 deficient embryos.
However, both pTE and ExE contain trophoblast stem (TS) cells
(Tanaka et al., 1998;
Uy et al., 2002
), and a
failure to maintain these stem cells after implantation would be expected to
affect ExE formation. Indeed, our observations of the absence of expression of
the undifferentiated TS cell markers Cdx2, Eomes and Fgfr2
in Elf5-/- embryos suggested that TS cells are not
maintained in mutant embryos. We therefore attempted to isolate TS cells from
Elf5-/- embryos by culturing E6.5 dissociated proximal
ectoderm tissue on primary feeder cells in the presence of Fgf4 and heparin
(Tanaka et al., 1998
;
Uy et al., 2002
). Notably,
TS-like colonies were only formed from wild-type and
Elf5+/ proximal ectoderm (n=18/18),
whereas Elf5-/- tissue formed no colonies (n=0/4;
Fig. 5A,B).
|
Extraembryonic ectoderm is required for patterning of the embryo proper
Elf5 deficient embryos not only exhibit defects in the ExE lineage
but also display severe patterning defects in the embryo proper. Although we
observed expression of Elf5 solely in the ExE and chorion using
whole-mount in situ hybridisation, low levels of expression in other regions
might have escaped our detection and be partly or wholly responsible for the
phenotype seen in the embryo proper. We therefore wished to determine whether
patterning defects occurred in embryos lacking Elf5 function only in the
epiblast. To this end, we performed a tetraploid rescue experiment. Wild-type
eight-cell-stage tetraploid cells known to contribute only to extraembryonic
tissue were aggregated with diploid cells derived from four- to
eight-cell-stage embryos of Elf5+/
xElf5+/ matings and allowed to develop to
E10.5, when Elf5-/- embryos are either dead or severely
retarded and malformed. Statistically, one quarter of the chimeric embryos
would be expected to contain epiblast with an Elf5-/-
genotype. Genotyping revealed that four out of 15 embryos were composed solely
of Elf5-/- cells (Fig.
6A). These four embryos were morphologically normal
(Fig. 6B,C), proving that
Elf5 is not required in embryonic tissues for development up to this
developmental stage.
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Discussion |
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We suggest that Elf5 acts at the next step, being required for maintaining trophoblast stem cell potential beyond the implanted blastocyst stage (Fig. 9A). Thus in the absence of Elf5, TS cells within the pTE are no longer maintained, instead differentiating into EPC precursors that will differentiate finally into sphongiotrophoblasts and giant cells. By egg cylinder stages, the ExE is absent whereas the EPC trophoblast is still seen. Several lines of evidence support this scenario. The normal implantation rates of Elf5 deficient embryos implies that Elf5 is only required after the formation of trophectoderm. The presence of an EPC in mutants means that the pTE must have formed correctly, as it gives rise to this lineage. This is supported by the correct pTE-specific expression of Cdx2 at E4.5 in Elf5-/- embryos. That TS cells are no longer maintained in Elf5 mutants past the pTE stage is supported by the absence of the TS-cell containing ExE and the observation that TS cells could not be derived from the proximal half of the egg cylinder of Elf5 mutant embryos. Moreover, TS cell markers such as Cdx2, Eomes and Fgfr2 were no longer expressed in mutant embryos. Lastly, Elf5 is expressed in TS cell lines and lost upon their differentiation after FGF removal. Elf5 can therefore be considered to be a lineage-determining factor, required for the formation of the ExE lineage by virtue of maintaining TS cells.
The role of the extraembyonic ectoderm in patterning the embryo
The absence of ExE from its inception in Elf5 deficient embryos
has provided a novel mouse model allowing an examination of the role of this
tissue in the development of the embryo proper. Patterning of the embryo
involves two distinct steps, the first being the establishment of the AVE
signalling centre thereby preparing the adjacent epiblast for anterior
induction by subsequent mesendodermal signalling. Thereafter, the primitive
streak with its associated organiser activities is formed on the opposite side
of the egg cylinder. Both events require Nodal signalling within the epiblast
(Brennan et al., 2001;
Lu et al., 2001
). In turn,
Nodal activity has been shown to be dependent on the ExE, which secretes the
proteases Furin and Spc4 to cleave and thereby activate the Nodal precursor in
the epiblast. In the absence of both proteases, mesoderm and AVE formation are
abolished (Beck et al., 2002
).
Why do Elf5-/- embryos, which do not have an ExE, still
form the AVE? We propose (Fig.
9B) that in the absence of ExE, Nodal cleavage is mediated by
secretion of Spc4, which is strongly expressed in the EPC located adjacent to
the epiblast in Elf5-/- pregastrula embryos. Once
proteolytically activated, Nodal can then amplify its own expression
(Fig. 7D,
Fig. 9B) via its autoregulatory
intronic enhancer, leading to AVE formation and migration
(Brennan et al., 2001
;
Norris et al., 2002
).
Although the EPC may substitute for the ExE at stages preceding
gastrulation, this is not the case at later stages. The loss of Nodal
transcription at E6.5 in type I Elf5 mutants explains the absence of
expression of Eomes, Fgf8, T and Cripto, all of which are
downstream targets of Nodal activity and required for posterior patterning and
mesoderm formation. Significantly, the absence of transcription from the
Nodal locus at E6.5 in Elf5 mutants differs from
ExE-containing Nodal null mutants and Furin/Spc4 double
mutants, which do exhibit transcripts in the proximal epiblast
(Beck et al., 2002;
Brennan et al., 2001
). These
mutants differ from Elf5 deficient embryos in two fundamental ways
they have no AVE and they do contain ExE. Could the continued presence
of the AVE in Elf5 mutants result in the absence of posterior
markers? We consider this to be unlikely as the Nodal-repressive AVE is
restricted to only one side of the mutant egg cylinders. We would thus favour
the alternate hypothesis (Fig.
9C), that a protease independent signal emanating from the ExE
(absent in Elf5 but present in Nodal mutants) is required
for Nodal transcription at E6.5. Potential candidates for such
signalling are Bmp4 and/or Bmp8b, which are expressed in the ExE, but not the
EPC (Fujiwara et al., 2002
;
Ying et al., 2000
).
ExE-derived Bmp4 has been shown to induce posterior genes in the epiblast and
is required (in a mouse background strain-dependent fashion) for the
generation of a normal primitive streak
(Beck et al., 2002
;
Fujiwara et al., 2002
;
Winnier et al., 1995
). In
Nodal-null mutants, Bmp4 is present in the ExE at E6.5
(Brennan et al., 2001
) and thus
could theoretically contribute to transcription from the Nodal locus,
as it does in other contexts (Fujiwara et
al., 2002
; Piedra and Ros,
2002
; Schlange et al.,
2002
).
Whatever the identity of the signals emanating from the ExE are, our
ExE-deficient mouse model and tetraploid rescue experiments strongly support
the proposed inductive role of this extraembryonic tissue in primitive streak
formation, thus suggesting that this tissue is equivalent to the avian
posterior marginal zone (Bachvarova et al.,
1998).
Conclusion
We have found a novel factor exquisitely restricted to and required for the
formation of the ExE, and have created a mouse model that clearly separates
two temporally distinct requirements for the ExE in instructing the patterning
of the epiblast. Whereas the early ExE function in anterior patterning via AVE
establishment can be replaced presumably by Spc4 secreted from the EPC, there
is an essential requirement for ExE in initiating gastrulation and posterior
patterning of the embryo proper. Furthermore, we now can identify three genes
that consecutively function to determine cell choices in the maintenance of TS
cells. First, Cdx2 for the trophectoderm/ICM choice at the morula
stage; second, Eomes for the polar/mural trophectoderm choice at the
blastocyst stage; and third, Elf5 in the ExE/EPC decision at the
implanted blastocyst stage.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/10/2299/DC1
* These authors contributed equally to this work
Present address: Academic Medical Genetics, The Children's Hospital at
Westmead, Locked Bag 4001, Westmead, NSW 2145, Australia
Present address: Department of Physiology, University of Otago, PO Box 913,
270 Great King Street, Dunedin, New Zealand
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