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Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
| Correspondence to: Dr Yasuhisa Matsui, Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi 980-8575, Japan (e-mail: ymatsui{at}idac.tohoku.ac.jp). |
| Received for publication April 21, 2009; accepted for publication October 5, 2009. |
Time-critical extracellular stimuli as well as the intrinsic functions of
transcriptional regulatory molecules play essential roles in fate
determination and differentiation of mouse primordial germ cells (PGCs). We
found that the precursor cells of PGCs require E-cadherin–mediated
cell-cell interaction and the functions of transcription factor Oct3/4 to be
specified to PGCs. In addition, transcriptional factors commonly regulating a
number of PGC-specific genes appear important for PGC development, and we
demonstrated that PGC-specific expression of the mil-1 gene is
controlled by germ cell–conserved regulatory sequences in the 5'
flanking region. Once they have undergone specification and differentiation,
PGCs normally give rise to gametes, but they maintain the potential to be
converted into pluripotential stem cells upon activation of particular
signaling pathways.
Key words: Primordial germ cell, E-cadherin, Oct3/4, mil-1, GFP, EG cell
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After the onset of gastrulation, the PGC precursors in the proximal region of the epiblast move towards the posterior end of the embryo and form a cluster of cells. In the cluster, the precursors interact with each other via the functions of E-cadherin, and this interaction is essential for the precursor cells to finally differentiate into PGCs. Several mechanisms by which E-cadherin regulates PGC determination are possible. The simplest model is that E-cadherin itself transmits instructive signals among the precursor cells for PGC determination. For example, it is known that homophilic interactions of E-cadherin transmit signals by sequestering β-catenin from lymphoid enhancer factor (Hecht and Kemler, 2000), and similar mechanisms might work in the PGC precursors. E-cadherin might also facilitate interaction among surface signaling molecules on adjacent cells to transmit signals (Carmeliet et al, 1999).
Oct3/4 Controls the Final Step of Germ Cell Specification
On the other hand, transcriptional regulators within the PGC precursors may
more directly control their fate, and Blimp1 and Prdm14 play key roles in germ
cell specification and the further differentiation of PGC precursors
(Ohinata et al, 2005;
Yamaji et al, 2008). In
Blimp1-null embryos, PGC-like cells are initially formed. However, those cells
aberrantly express somatic genes such as the Hox genes, cannot undergo proper
differentiation into PGCs, and shortly disappear in the embryo
(Ohinata et al, 2005). PGC
development is also severely affected in Prdm14-deficient embryos, in which a
few PGCs exist but they do not increase in number, and cannot undergo proper
epigenetic reprogramming (Yamaji et al,
2008). Although Blimp1 is necessary to suppress somatic genes in
the PGC precursors, the precursors most likely also require activation of
PGC-specific genes. Because Oct3/4 is specifically expressed in the germ cell
lineage, including the PGC precursors during specification, and is able to
activate gene expression as a transcriptional regulator
(Pesce et al, 1998), it is
likely that Oct3/4 also functions as an activator for PGC specification.
Knockout of Oct3/4 in mice is lethal at the preimplantation stage (Nichols et al, 1998). Using this information, the possible functions of Oct3/4 during PGC specification were investigated using an embryonic stem (ES) cell line in which the Oct3/4 gene was manipulated (Niwa et al, 2000; Okamura et al, 2008). In these ES cells, both alleles of the endogenous Oct3/4 gene were disrupted by gene targeting, and cell pluripotency was maintained by an Oct3/4 transgene under control of the Tet-Off promoter. This ES cell line has another expression vector of Oct3/4 fused to green fluorescent protein (GFP) and the ligand-binding domain of the glucocorticoid receptor, which is activated by dexamethasone. In the absence of the drugs, the ES cells can maintain pluripotency via the Oct3/4 protein from the Tet-Off transgene. In the presence of tetracycline or its derivative, doxycycline, the cells lose the functions of the Oct3/4 protein, whereas Oct3/4 activity is restored by the addition of dexamethasone (Okamura et al, 2008). This ES cell line was used to generate chimeric embryos, and PGC precursors of the chimeric embryos were cultured with doxycycline or with both doxycycline and dexamethasone to examine Oct3/4 function during PGC specification. With doxycycline, the Oct3/4 activity was completely suppressed in the cultured ES-derived cells, and in this case, the specified PGCs were only GFP-negative host-derived cells. By contrast, with both doxycycline and dexamethasone, Oct3/4 activity was restored, and numerous ES-derived cells became PGCs. These findings indicate that PGC specification from precursor cells depends on the activity of Oct3/4 (Okamura et al, 2008).
Oct3/4 may activate the expression of PGC-specific genes and may induce the final step of PGC specification. Although Oct3/4 is continuously expressed in pluripotential cells from earlier developmental stages, previous results indicate that the expression of Sox2, a heterodimeric partner of Oct3/4, is up-regulated at the time of PGC specification (Yabuta et al, 2006), suggesting that Oct3/4 becomes functional with regard to PGC specification after up-regulation of cooperative factors such as Sox2.
Transcriptional and Epigenetic Regulation of PGC-Specific Gene Expression
We previously identified a gene preferentially expressed in PGCs
(Tanaka and Matsui, 2002).
This gene, mil-1, is a homologue of the human interferon-induced gene
family, IFITM, and is identical to fragilis, which was isolated by Dr
Surani's group (Saitou et al,
2002). mil-1 is specifically expressed in the nascent PGC
cluster at the posterior end of 7.25-day embryos. mil-1 expression is
also found in PGCs migrating along the hindgut and colonizing embryonic gonads
(Saitou et al, 2002;
Tanaka and Matsui, 2002).
Because mil-1 is specifically expressed in PGCs from their formation onwards, the transcriptional regulation of mil-1 may correlate with mechanisms of PGC determination and their subsequent development. The transcriptional regulatory elements for PGC-specific expression were identified by generating transgenic mice containing the flanking regions of the mil-1 gene fused to the GFP reporter gene. A 3-kb region of the 5' flanking region was found to be sufficient for PGC-specific expression of mil-1 (Tanaka et al, 2004). In the transgenic embryos, the entire epiblast was GFP positive at 6.75 days, but the PGC precursors at the posterior end of the embryo showed stronger GFP expression. After 7.25 days and onwards, the GFP signals were preferentially observed in PGCs, and were mostly identical to the expression of the endogenous mil-1 gene.
Regulatory elements within the 3-kb flanking region were identified using deletion constructs, and the region between 1.8 and 2.2 kb upstream from the transcriptional start site was found to be particularly important for mil-1 expression in PGCs, as well as for suppression of mil-1 in somatic cells (Tanaka et al, 2004). This region is thought to include critical elements for PGC-specific expression, and was found to have sequence similarity with the flanking regions of other PGC-specific genes. Genes including Stella, alkaline phosphatase, Oct3/4, and nanos are specifically or preferentially expressed in PGCs, and they also have the consensus sequence within their flanking regions. This element, named ICE, contains a sequence that is similar to the Alu family of short interspersed transposable elements that are known to act as transcriptional regulatory elements (Tanaka et al, 2004), suggesting that this conserved element is involved in the expression of some PGC-specific genes. We speculate that putative transcriptional regulators commonly controlling a number of PGC-specific genes might bind to this element.
Some PGC-specific genes are demethylated at the time of their activation (Maatouk et al, 2006). The expressions of mouse vasa homologue, meiosis-specific genes, Scp3, and Dazl are induced at around embryonic day 11, when PGCs settle in the embryonic gonads, and DNA demethylation of the flanking regions of these genes concomitantly occurs. In addition, embryos deficient in a maintenance DNA methyltransferase, Dmnt1, prematurely express these genes in migrating PGCs (Maatouk et al, 2006). All of these results support the idea that DNA demethylation of these genes leads to their expression. Although the mil-1 gene is expressed in PGCs from much earlier stages, we believe that DNA demethylation is also involved in its expression.
Mechanisms of Pluripotential Stem Cell Formation From PGCs
Once the fate of PGCs is determined, they normally differentiate only to
eggs or sperm, and not to any types of somatic cells. However, they are
occasionally converted to pluripotential stem cells in embryonic testis of
particular strains of mice such as 129Sv, and subsequently develop into
teratomas consisting of various differentiated cell types
(Stevens, 1983). PGCs also
develop into pluripotential stem cells known as EG cells in culture in the
presence of particular growth factors, such as Steel factor, LIF, and bFGF
(Matsui et al, 1992;
Resnick et al, 1992),
indicating that the cooperative functions of these molecules are critical for
PGCs to be converted to pluripotential cells. Previous reports have also
indicated that the intracellular signaling molecules linked to the growth
factor receptors are involved in pluripotential cell development from PGCs
(Figure). For example,
inactivation of PTEN, a lipid phosphatase antagonizing PI3K, in PGCs
stimulates EG cell formation in culture and teratoma development in testis
(Kimura et al, 2003). In
addition, conditional activation of Akt in cultured PGCs enhances the
efficiency of EG cell formation and can be substituted for bFGF
(Kimura et al, 2008). Because
PI3K is an upstream regulatory molecule of Akt, the PI3K-Akt signaling
pathway, which is probably stimulated by bFGF, most likely plays a role in
pluripotential cell development from PGCs. Another molecule regulating the
conversion of PGCs to pluripotential cells is an RNA-binding protein, Dnd1.
Deficiency in the Dnd1 gene in the 129Sv genetic background causes
testicular teratoma at high rates
(Youngren et al, 2005). A
recent report indicated that Dnd1 inhibits the interaction of microRNAs with
their target messenger RNAs (Kedde et al,
2007), but the molecular mechanisms of the Dnd1 effects on
teratoma formation are still unknown.
A recent report by Dr Surani's group indicated that expression of Bimp1, a key molecule for fate determination and subsequent development of PGCs as described above, was quickly down-regulated in PGCs after starting culture (Durcova-Hills et al, 2008). In addition, c-myc and klf4, which are involved in reprogramming of somatic cells to pluripotential cells, were up-regulated, and those 2 genes are targets of Blimp1. Blimp1 is also known to recruit histone deacetylase (HDAC) to its targets, and it was demonstrated that trichostatin A, an inhibitor of HDAC, accelerated EG cell formation, which means histone deacetylation prevents EG cell formation (Durcova-Hills et al, 2008). Suppression of particular gene expression by histone deacetylation may also critically affect conversion of PGCs to pluripotential cells.
Summary
In this manuscript, I first briefly summarized molecular mechanisms of PGC
determination in mouse. During formation of PGCs from their precursor cells,
E-cadherin–mediated cell-cell interaction is necessary, and this
interaction should transmit essential signals for fate determination of germ
cells. In addition, the function of Oct3/4 in the precursor cells is required
for the final step of PGC specification. The specified PGCs express a number
of specific genes, including mil-1, whose expression is regulated by
the regulatory sequences within its flanking region, and those sequences are
also found in flanking regions of other PGC-specific genes, suggesting common
regulatory mechanisms shared among PGC-specific genes. I finally described
molecular cascades such as the PI3K-Akt and the histone
deacetylation–mediated reprogramming process, regulating conversion of
PGCs into pluripotential EG cells. Studies concerning PGC development
described here may shed light on molecular mechanisms controlling
establishment of totipotency that only germ cells possess, and on correlation
between germ cells and pluripotential cells.
Footnotes
Supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.
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