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doi: 10.1242/10.1242/dev.00366
1 Howard Hughes Medical Institute, 9 Cambridge Center, Cambridge, MA 02142,
USA
2 Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA
02142, USA
3 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA
02139, USA
4 Institute for Biogenesis Research and Department of Anatomy and Reproductive
Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI
96822, USA
* Author for correspondence (e-mail: jaenisch{at}wi.mit.edu)
Accepted 8 January 2003
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SUMMARY |
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MATERIALS AND METHODS
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Key words: Nuclear cloning, Gene expression, Developmental pluripotency, Oct4, Embryogenesis
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INTRODUCTION |
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;
Briggs and King, 1952), animal
cloning has emerged as a key approach to explore functional changes in the
nuclear genome during differentiation
(Gurdon, 1974;
Gurdon, 1999;
Kikyo and Wolffe, 2000;
Rideout et al., 2001;
Solter, 2000). Studies in
amphibians have revealed that the potential of the nucleus to direct normal
embryonic development decreases progressively with the developmental stage of
the donor cell (Briggs and King,
1960; Gurdon,
1962; Gurdon et al.,
1975). Similarly, in mammals, a direct relationship has been
observed between the state of differentiation of a donor nucleus and its
ability to generate cloned animals upon transfer into the oocyte
(Campbell et al., 1996;
Solter, 2000;
Tsunoda et al., 1987;
Willadsen, 1986). This was
established most clearly in mouse cloning experiments using donor nuclei from
somatic and pluripotent embryonic stem (ES) cells
(Eggan et al., 2001;
Rideout et al., 2000). Somatic
and ES cell clones that develop to the blastocyst stage in vitro develop very
differently in utero after their transfer to recipient animals. As in all
other mammals where nuclear cloning has been successful, only a small fraction
(1-3%) of mouse blastocysts derived from somatic donor nuclei develop to term,
and even fewer reach adulthood (Wakayama
et al., 1998; Wakayama and
Yanagimachi, 1999; Wakayama
and Yanagimachi, 2001). By contrast, up to a third of blastocysts
from mouse clones made by nuclear transfer of ES cell donor nuclei develop to
birth (Eggan et al., 2001;
Rideout et al., 2000). These
observations reinforce the notion that a progressive decrease or loss of
nuclear potency during cell differentiation is a general characteristic of
vertebrate development.
Although as many as 70% of somatic clones can develop to the blastocyst
stage in vitro after nuclear transfer, most arrest soon after implantation
(Wakayama and Yanagimachi,
1999). Homozygous mutant embryos lacking Oct4
(Pou5f1 – Mouse Genome Informatics), a gene that encodes a
POU-domain transcription factor that has an essential role in the control of
developmental pluripotency, form morphologically normal but developmentally
deficient blastocysts (Nichols et al.,
1998; Niwa et al.,
2000). In particular, the cells of Oct4-deficient embryos
fail to form the inner cell mass (ICM) that gives rise to all adult tissues
(Nichols et al., 1998). The
superficially normal development of Oct4-deficient embryos to the
blastocyst stage suggests that the combined activity of maternal, zygotic and
housekeeping genes might be sufficient to produce a phenocopy of normal
preimplantation development. The subsequent failure of embryonic cells in the
blastocyst to form the epiblast leads to the manifestation of developmental
defects observed in Oct4-deficient embryos. The similar developmental
phenotypes observed in Oct4-deficient and cloned blastocysts suggest
that developmental failure in both cases results from the absence of a
pluripotent ICM.
We have examined the hypothesis that the limited developmental potency of cloned embryos is a result of incomplete reactivation of genes that, like Oct4, function specifically in pluripotent embryonic cells. To test this hypothesis, we have identified 10 candidate genes with an expression pattern similar to Oct4 (designated as Oct4-related genes in this paper) and have compared expression of these genes in normal preimplantation embryos to embryos cloned from somatic cumulus cells and pluripotent ES cells.
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). Previously,
electronic analysis of UniGene was used to identify genes whose expression is
enriched in the germ cell lineage
(Rajkovic et al., 2001). The
UniGene mouse dataset can be found at
ftp://ncbi.nlm.nih.gov/repository/UniGene.
Each individual UniGene cluster is a collection of expressed sequence tags
(ESTs) that are grouped together based on sequence similarities. Each EST
record is linked to information about the cDNA library from which it was
sequenced and its biological source (available at
http://www.ncbi.nlm.nih.gov/UniGene/lbrowse.cgi?ORG=Mm).
We examined biological source descriptions of all cDNA libraries that
contribute to the UniGene mouse dataset and identified 105 cDNA libraries of
interest prepared from pre-implantation embryos, gastrulating embryos,
embryonic and adult gonads, posterior regions of early and late gestation
embryos that also contained developing gonads and germ cells, and ES and
embryonic carcinoma (EC) cells. Few cDNA libraries lacking sufficient
biological source descriptions were excluded from our analysis. The UniGene mouse dataset partitions 2.5 million ESTs sequenced from over 500 cDNA libraries into about 88,000 EST clusters. The 105 cDNA libraries of interest to us contributed over 350,000 ESTs to the dataset. Taking these parameters into account, we estimated that a UniGene cluster of six or more ESTs derived from cDNA libraries of interest has a statistically significant chance of identifying a gene specific to tissues containing pluripotent cells. We further restricted electronic selection criteria by requiring that contributing ESTs be derived from at least two distinct stages of development, including one from the preimplantation embryo. This latter criterion was essential to improve selection for genes with a continuous Oct4-like expression pattern and to avoid selecting highly expressed genes specific to a particular cell-type or developmental stage, such as genes specifically involved in spermatogenesis or meiosis.
We then used a custom Perl script to electronically identify 102 UniGene clusters that satisfied the selection criteria. EST contigs were assembled using Sequencher 4.1.2 (Gene Codes Corporation). Subsequent sequence analysis and expression analysis by RT-PCR narrowed the number of clusters of interest to 66. We then performed a more detailed RT-PCR-based expression analysis on embryonic and germ cell samples to identify genes with developmental expression profiles closely matching that of Oct4. Finally, we assessed the reproducibility of detection of candidate genes in single preimplantation embryos and chose 10 Oct4-related genes for further analysis.
Embryonic RNA purification, cDNA synthesis and PCR analysis
Embryos were collected in 100 µl of TRIzol reagent (GibcoBRL) in
non-stick, RNAse-free microtubes (Ambion), extracted twice with 20 µl of
chloroform using Phase Lock Gel Heavy 0.5 ml tubes (Eppendorf) spun at 5000
g. Total RNA was precipitated with an equal volume of
isopropanol in the presence of 1.5 µl of GlycoBlue (Ambion). Residual
genomic DNA was removed using DNA-free reagent (Ambion). RNA was
re-precipitated, washed in 70% ethanol and dissolved in 2 µl of RNAse-free
water (Ambion). First strand cDNA synthesis and universal amplification (four
cycles) were performed using the SMART PCR cDNA synthesis kit (Clontech).
cDNAs was purified using the QIAquick PCR purification kit (Qiagen), eluted in
100 µl of water. PCR products were resolved in 2% agarose gels.
Primordial germ cell and embryonic tissue isolation
PGCs purified from day 10 (E10) of embryonic development of
Oct4-GFP transgenic mouse line (A.B. and D.C.P., unpublished) were
collected directly into TRIzol reagent using a Becton Dickinson cell sorter.
Epiblasts of early gastrulation embryos were dissected cleanly from
extra-embryonic tissues. Embryonic gonads were collected from E12 embryos of
both sexes by carefully dissecting them from mesonephroi. RNA was prepared
from collected tissues using TRIzol reagent and processed for cDNA synthesis
as described above.
Production of cloned embryos from cumulus and ES cell nuclei
Nuclear transfer of ES cell and cumulus cell nuclei into enucleated
metaphase II oocytes was carried out as previously described
(Eggan et al., 2000;
Wakayama et al., 1998;
Wakayama and Yanagimachi,
2001). Three hours after nuclear transfer, oocytes were activated
for 5 hours with 10 mM Sr2+ in Ca2+-free CZB media in
the presence of 5 µg/ml Cytochalasin B. Embryos were cultured in vitro for
96 hours, preimplantation development was assessed by Hoffman contrast
microscopy, and individual embryos were isolated for gene expression
studies.
Embryo culture
All embryo culture was carried out in microdrops on bacterial petri dishes
(Falcon) under mineral oil (Squibb). HEPES-buffered CZB was used for room
temperature operations, while long-term culture was carried out in bicarbonate
buffered KSOM at 37°C under an atmosphere of 5% CO2 in air.
GenBank Accession Numbers
cDNA sequences for examined genes: Pramel4, AF490340;
Pramel5, AF490341; Pramel6, AF490342; Pramel7,
AF490343; Ndp52l1, AF490344; Dppa1, AF490345;
Dppa2, AF490346; Dppa3, AF490347; Dppa4, AF490348;
Dppa5, AF490349.
Primer sequences and RT-PCR conditions for mouse genes: Pramel4, G73527; Pramel5, G73528; Pramel6, G73529; Pramel7, G73530; Ndp52l1, G73531; Dppa1, G73532; Dppa2, G73533; Dppa3, G73534; Dppa4, G73535; Dppa5, G73536; Ptgs2, G73537; Hsh, G73538; Tnfsg6, G73539; Oct4, G73540; Gapd, G73541.
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).
Oct4 encodes a POU-domain transcription factor and is expressed in
pluripotent cells of the early embryo and in the embryonic and adult germ
cells. We were interested in identifying genes whose expression patterns are
like that of Oct4 and that might be involved in establishing or
maintaining developmental pluripotency. We used the following criteria for
electronic selection: (1) expression in cleavage-stage embryos, blastocysts
and in the embryonic germline, and (2) the genes should not be expressed in
somatic cells. An electronic analysis of the UniGene mouse dataset generated a list of 66 candidate genes, ten of which were selected for this study based on their Oct4-like temporal expression pattern and reproducibility of detection in single preimplantation embryos (for details see Materials and Methods). Fig. 1 shows that these 10 candidate genes were expressed during cleavage in early epiblasts and germ cell-containing samples, but they were not expressed in any of the somatic tissues tested. Expression analysis of normal four-cell, morula- and blastocyst-stage embryos established that all but one of these Oct4-related genes were detectable, beginning with the four-cell stage of development (Fig. 2). Strong expression of Dppa1 was readily seen at the morula-to-blastocyst transition.
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). Two
additional mouse Pramel genes, Pramel1 and Pramel3, have
previously been shown to exhibit male germ stem cell-specific expression
(Wang et al., 2001). Products
of three other genes, Dppa2, Dppa3 and Dppa4, share a SAP
putative DNA-binding motif often found in proteins implicated in chromosomal
organization, as well as in various aspects of RNA biology
(Aravind and Koonin, 2000).
Dppa3 was described recently as a primordial germ cell
(PGCs)-specific gene PGC7 (Sato
et al., 2002), as was Stella
(Saitou et al., 2002).
Stella has been implicated in the initiation of the germ cell
lineage, and its expression marks the emerging primordial germ cells in the
epiblast (Saitou et al.,
2002).
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Expression of Oct4-related genes in mouse cumulus cell
blastocysts cloned from cumulus donor nuclei
For expression analyses, total RNA was isolated from individual control and
cloned embryos as well as from cumulus donor cells. Expression of
Oct4 and related genes was not detected in cumulus cells, although
sensitive RT-PCR analysis occasionally revealed small amounts of mRNA
contamination that were probably the result of oocyte lysis during enzymatic
and mechanical disruption of oocyte-cumulus-cell complexes
(Fig. 3). Parthenogenetic and
in vitro fertilized embryos served as controls for the nuclear transfer
procedure and culture conditions. All tested Oct4-related genes were
expressed in all normal control, in vitro fertilized and parthenogenetic
blastocysts (Fig. 2,
Table 2 and data not
shown).
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We also examined the expression of three genes that are active in the
cumulus donor cells but not in normal blastocysts. These genes are
prostaglandin-endoperoxide synthase 2 (Ptgs2)
(Joyce et al., 2001),
hyaluronan synthase homologue (Hsh)
(Fulop et al., 1997b) and
TNF-stimulated gene 6 (Tnfsg6)
(Fulop et al., 1997a). In
contrast to incomplete reactivation of the Oct4-related genes, all
cumulus cell-derived clones uniformly lacked expression of the three `somatic'
genes regardless of the extent of development in vitro
(Fig. 3).
Expression of Oct4-related genes in mouse ES cells and ES
cell-derived cloned embryos
The hypothesis that incomplete reactivation of the Oct4-related
genes may contribute to the early lethality of somatic clones predicts that
these genes should be expressed faithfully in blastocysts derived from nuclei
of donor cells that are expressing these genes. To test this possibility, we
examined expression of the 11 test genes in donor ES cells and in individual
ES cell clones. As expected from the electronic selection criteria, all tested
genes were expressed in undifferentiated ES cells
(Fig. 5). In contrast to
cumulus donor cell-derived clones, all ES cell nuclei-derived clones expressed
all eleven tested genes (Figs 4
and 5,
Table 2 and data not shown). In
addition, unlike somatic clones, ES cell clones correctly recapitulated
temporal regulation of the Dppa1 gene. Like normal embryos in which
Dppa1 expression is upregulated at the morula-to-blastocyst
transition (Fig. 2),
Dppa1 was expressed in blastocyst-stage ES clones but not in
morula-stage clones (Fig. 5).
Thus, the ES cell-derived clones appear to have faithfully recapitulated
expression of the Oct4-related genes with a similar kinetics to the
normal embryos.
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DISCUSSION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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We used an in silico selection method to identify a class of embryonic
Oct4-related genes whose expression is limited to the pluripotent
cells of the early embryo and the germline. Although the biological functions
of these genes have yet to be elucidated, we have found that they serve as
informative markers for pluripotent cell types and thus can be validly used in
the analysis of cloned embryos. The electronic selection that led to the
identification of this set of genes was modeled after the properties and
expression pattern of the Oct4 gene, which has been shown to have an
essential role in early development. Recently, our observations have been
corroborated by the results of an expression profiling study that identified
Dppa2, Dppa4, Dppa5 and Oct4 among genes found at high
levels in mouse ES cells (Ramalho-Santos
et al., 2002). In addition, expression of the
Oct4-related genes is downregulated during ES cells differentiation
(data not shown) in strong agreement with a prior description of
Dppa5 as a cDNA downregulated during EC cell differentiation
(Astigiano et al., 1991).
Finally, one of the Oct4-related genes,
Dppa3/Stella/PGC7, has been implicated in the earliest steps
of germline initiation, further strengthening the argument that these genes
might represent factors with specific roles in early development
(Saitou et al., 2002).
Analysis of expression of the Oct4-related genes in somatic and ES cell-derived clones revealed that successful reactivation of the full set correlates with development of cloned embryos to term. Mouse clones derived from nuclei of ES cells, where the genes are already active, recapitulate their expression pattern with the same kinetics as observed during normal embryogenesis. In contrast to ES clones, almost 40% of the cumulus cell-derived blastocysts, though morphologically normal, failed to reactivate these genes faithfully. Activation of these genes in embryos that were arrested at the pre-blastocyst stage was even less efficient. The significance of this result is difficult to assess as impaired viability and earlier developmental arrest of these embryos might be have been caused by physical damage after nuclear transfer, leading to RNA degradation.
Incomplete reactivation of Dppa genes in cumulus clones strongly agrees
with a recent independent study of Oct4 expression in somatic clones
(Boiani et al., 2002). In that
study, 82% of clones reactivated Oct4. In situ mRNA analysis also
revealed that some of these Oct4-positive clones exhibited aberrant
Oct4 expression in the trophoectoderm. These findings suggest that
somatic clones capable of reactivation of Oct4 may also fail to
reproduce its normal spatial pattern of expression. Our work confirms these
prior findings and attributes incomplete recovery of pluripotency in somatic
clones to a larger set of specific genes.
Our observations could plausibly reflect a more profound transcriptional dysregulation in cumulus cell-derived cloned embryos. Future experiments using single-embryo expression profiling on microarrays will examine the entirety of mouse transcriptome and reveal which genes consistently fail to be expressed in somatic cell-derived cloned embryos. We emphasize, however, that all examined cumulus cell-derived cloned blastocysts expressed Gapd, while the expression of three genes, Ptgs2, Hsh and Tnfsg6, which are normally abundantly expressed in donor cumulus cells, was undetectable in cloned embryos (Fig. 3). These observations indicate that mechanisms controlling gene expression in cumulus cell-derived clones selectively affect whether housekeeping and `somatic' genes are expressed. By contrast, they can only achieve incomplete reactivation of expression of Oct4 and 10 Oct4-related genes.
Why somatic clones fail to reactivate the Oct4-related genes
remains to be determined. It is now generally accepted that epigenetic
mechanisms play important roles in the control of gene expression during
embryogenesis (Reik and Walter,
2001; Renard,
1998; Rideout et al.,
2001; Surani,
2001). However, little is known about the molecular mechanism of
epigenetic reprogramming in the context of normal development and somatic
cloning. Recently, initial studies of bulk genomic DNA methylation in normal
and cloned embryos have been reported
(Barton et al., 2001;
Bourc'his et al., 2001;
Dean et al., 2001;
Fairburn et al., 2002;
Kang et al., 2001;
Mann and Bartolomei, 2002;
Santos et al., 2002). These
studies have revealed significant differences in the pattern and dynamics of
DNA methylation between normal and cloned embryos. However, current approaches
for analysis of epigenetic modifications in single pre-implantation embryos
have significant limitations. Importantly, they lack the resolution to allow
straightforward identification of individual genes whose expression and
epigenetic make-up could be meaningfully correlated with the development of
clones. In this regard, the Oct4-related genes identified in our work
represent a set of loci whose epigenetic control should be examined in the
future. Such analysis might provide specific examples of the role of
epigenetic control of gene expression in the development of cloned and normal
embryos.
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ACKNOWLEDGMENTS |
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DISCUSSION
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