By examining the ontogenetic changes in stemness and pluripotency genes during embryogenesis and the transitions from ICM to early outgrowth, an established ESC line and early differentiating ESCs, we have gained insight into how these genes relate to these developmental transitions in the rhesus monkey. First it is clear that some of these genes are not highly expressed in all totipotent or pluripotent cell types, contrary to their previous assignment as “stemness” or “pluripotency” genes. Some are predominantly maternal mRNAs present in oocytes and embryos before transcriptional activation but diminish before the blastocyst stage. Others are well expressed in morulae or early blastocysts but are poorly expressed in later blastocysts or ICMs. We failed to detect LIN28
mRNA in rhesus monkey oocytes and embryos. A study by Assou et al. (Assou et al., 2009
) reported LIN28
mRNA expression in human oocytes and ESCs. Additionally, Lin28
mRNA was reported on a mouse array for oocytes (Zeng et al., 2004
), and it can be detected on rhesus monkey MII oocyte arrays at a comparatively low level (Lee et al., 2008
). These results indicate that, although these genes are increasingly expressed and may maintain pluripotency or stemness at later stages or in cultured cell lines, their expression in the oocyte and early embryo may be limited and they may not be required for early totipotency. Thus, their role as stemness genes in these stages requires further testing.
Second, we find that over a third of the mRNAs detected were downregulated during the initial transition to culture from ICM to early outgrowth. Many of the genes examined are upregulated as ESC lines are established. A frequent pattern observed was elevated expression in the ICM, downregulation in early outgrowths, and then elevated expression once again as ESCs are established (e.g., POU5F1, CDH1, SOX17, EOMES, FZD7, ASCL2
). This pattern of expression may reflect either an adaptive response to the culture environment or an initial loss of pluripotency by some of the cells as outgrowths, followed by ongoing selection for a sub-population of cells that have entered a highly proliferative state. The increased expression of GSC
mRNA in EOs is consistent with some portion of cellular differentiation during this initial culture period. Our results coincide with those of Reijo Pera et al (Reijo Pera et al., 2009
) in human ESC, which compared global gene expression between individual ICM clusters and human embryonic stem cells and found that these two cell types are significantly different in regards to gene expression, with fewer than one half of all genes expressed in both cell types.
As expected, early differentiation in ESCs was marked by downregulation of mRNAs associated with cell proliferation and self-renewal, including CDH1, CTNNB1, FZD7, SALL4 and SOX17. This was accompanied by upregulation of some mRNAs encoding lineage or differentiation markers, including CDX2, KRT8.
The divergence of ESC and TSC lineages was marked by downregulation in TSC of mRNAs more characteristic of somatic lineages (GRB2, LEFTY1, LIN28, POU5F1
), and a divergence in keratin expression, with KRT7
expressed more highly in TSCs. Similarly, the SOX2
mRNA, which is expressed in a variety of specific progenitor cells and tumors (Gangemi et al., 2009
; Graham et al., 2003
; Phi et al., 2008
; Taranova et al., 2006
) was expressed highly in TSCs, again correlating with a more restricted cell fate. These results thus confirm that some genes associated with pluripotent stem cells (e.g., LIN28, SOX2
) correlate more with proliferative state than with pluripotency.
Some of the genes employed to induce pluripotent stem cells from somatic cells (iPS genes) appear unlikely to play a role as stemness or pluripotency genes in normal embryos. For example, the LIN28
mRNA was not detected in oocytes or embryos by this method (a comparatively low, positive signal was obtained on microarrays; (VandeVoort et al., 2009
; Zheng et al., 2009
), the KLF4
mRNA was expressed transiently at the morula stage and not detected in stem cell samples, the KLF2
mRNA was expressed predominantly as a maternal mRNA, and the MYC
mRNA expression was very low in oocytes and embryos but upregulated in established ESC lines. POU5F1
, and to a lesser degree SOX2
mRNAs displayed the most consistent expression across developmental stages, however the SOX2
mRNA was upregulated in TSCs as compared to pluripotent ESCs. Thus, these mRNAs do not follow a simple expression pattern of consistent high expression in totipotent or pluripotent cells. Their expression even in stem cells is clearly developmentally regulated, and their requirement for establishing or maintaining pluripotency may be stage- and cell type-dependent. We propose that when introduced into differentiated cells to create iPS cells, some of these genes may act to induce pluripotency through mechanisms that are outside of their normal functions and outside of normal ontogenetic processes, by activating downstream target genes that promote cell proliferation. The forced re-entry into the cell cycle may provide an opportunity for genes like NANOG
to reactivate other genes leading finally to a pluripotent state.
The expression of mRNAs that regulate pluripotency and self-renewal (POU5F1, NANOG, SOX2
has not been described for nonhuman primate oocytes and early cleavage stage embryos. The expression of POU5F1
mRNA has been reported in oocytes and preimplantation embryos in several species, including rabbit, (Kobolak et al., 2009
) mouse (Monti and Redi, 2009
) and human (Monk et al., 2008
). Interestingly, the pattern of POU5F1
expression found in human oocytes and embryos (Monk et al., 2008
) is similar to our results in the rhesus monkey. Patterns of increasing gene expression as human preimplantation embryos developing in vitro for NANOG, CDX2
(Kimber et al., 2008
) were also similar to those found in this study; however, earlier expression was sometimes noted for the rhesus monkey. The interactions among these factors, especially during the transition from maternal to embryonic gene expression is not well understood and may be critical for later processes that guide cell fate decisions morulae and blastocysts.
The relative quantities of the mRNA for these critical transcription factors are described here for the first time throughout primate embryo development, stem cell maintenance and early ESC differentiation. Stem cell pluripotency and initiation of differentiation are regulated by the relative levels of many interacting transcription factors (Cauffman et al., 2005
; Loh et al., 2006
; Pan et al., 2006
(a.k.a. OCT4) has been reported in human oocytes and embryos (Cauffman et al., 2005
) and the later stages of rhesus embryo development (Harvey et al., 2009
). However, only demonstrating the presence of a protein through immunostaining does not provide information on the subtle changes that appear to control the determination of which lineage, ICM or TE, that is selected during early embryo development. Although it has been speculated that NANOG
expression precedes POU5F1
in the inner cell mass of rhesus embryos (Harvey et al., 2009
), our data clearly show expression of these two genes throughout oocyte maturation and early embryo development and that NANOG
is expressed at a much higher level that POU5F1
; similarly, SOX2
are expressed throughout this time period. CDX2
interactions with POU5F1
appear essential for trophectoderm differentiation in mouse embryos (Niwa et al., 2005
is observed in rhesus blastocysts (Douglas et al., 2009
), but our study is the first to quantify the mRNA level of CDX2
in morulae and later blastocysts, embryonic stages in which differentiation is occurring, as well as during early differentiation of ESCs. Our data support the hypothesis that CDX2
is involved in differentiation in primate as well as murine early embyo development. The relative levels of POU5F1
may be critical in controlling early ESC differentiation and the size of the ICM, a factor that is associated with improved implantation rates and embryo survival in human infertility clinics.
Trophoblasts are a group of ectodermal epithelial tissues, so the expression of KRT7
, 8 and 18 in the TSC is not surprising. Trophoblast cells express keratins, and differences in keratin expression patterns are seen for different types of trophoblasts (Muhlhauser et al., 1995
). Increases in keratins KRT7, 8 and 18 were associated with the extravillous trophoblast compared to the villous trophoblast (Ahenkorah et al., 2009
). Because most studies on human and macaque trohpoblast cells are performed on cells isolated from term placentae (Douglas and King, 1989
) it is difficult to determine the relative significance of the expression of these particular keratins in TSC that are derived directly from the trophectoderm of blastocysts. It is possible that TSC express KRT7
, 8 and 18 until differentiation into specific types of trophoblasts, some of which may require downregulation.
The expression in TSC of the trophoblast markers CDX2
IS in agreement with the previous study on these cells and also confirm the finding that POU5F1
(OCT4) is expressed at very low levels (Vandevoort et al., 2007b
). The expression of POU5F1, SOX2
are associated with pluripotency and self-renewal (Table S2
). High expression levels of SOX2
in TSC have not been reported, but are exhibited in this study at levels similar to all undifferentiated ESC cell lines; this seems to indicate that these genes are also important in maintaining the stemness of TSC.