Differentiation and specification of unspecialized cells into other cell types is a crucial process of development. Thus, understanding the molecular mechanisms governing the downstream lineage determination and commitment is critical to dissect fundamental developmental pathways. As indicated above, the embryonic genome at early cleavage stages is transcriptionally quiescent and development is supported and regulated by maternally inherited factors present at the time of fertilization in the oocyte (Minami et al., 2007
). The transition in developmental regulation occurs gradually with activation of the embryonic genome in a species-specific manner and a complete loss of dependence on oocyte factors takes place by the blastocyst stage. Thus, TE and ICM cells are self sustained and their development is maintained by endogenously expressed factors. However, oocyte-specific transcriptional and epigenetic factors are essential for “natural” reprogramming of highly specialized and terminally differentiated gametic genomes into trophectodermal and pluripotent ICM lineages. Currently, little is known about these maternal factors and their role in induction of totipotency remains elusive.
Studies in the mouse suggested that embryonic Cdx2
functions as a gatekeeper for committing totipotent cells to the TE lineage during early preimplantation development (Niwa et al., 2005
; Ralston and Rossant, 2008
). Here, we present evidence that CDX2
plays a similar role in primates. Our results also indicate that maternal CDX2
transcripts are present in monkey oocytes, with their level increasing dramatically after fertilization. These results were also confirmed by detection of CDX2 protein in the zygotic pronuclei by ICC. This observation was somewhat unexpected and has not been reported for other species including the mouse. The function of CDX2 in primate oocytes soon after fertilization is unclear and remains to be addressed. CDX2
mRNA levels were low in cleavage stage embryos and undetectable at the protein level except in the second polar body. Onset of embryonic expression was evident at the compact morula stage, where the protein was ubiquitously expressed in all nuclei. In contrast, CDX2 protein became confined to the outer TE layer in early cavitating blastocysts and was not detected in the ICM. This pattern of expression in blastocysts was similar to that reported in the mouse suggesting that CDX2 is an important transcription factor during primate TE specification.
On the other hand, embryonic activation of OCT4
in monkey embryos occurs slightly earlier than CDX2
, at the 16-cell stage and in blastocysts it is initially expressed in both the ICM and TE cells (Mitalipov et al., 2003
). OCT4 protein is downregulated in the TE of hatched blastocysts while strong expression is maintained in the ICM. In our study, CDX2 and OCT4 were co-expressed initially in all cells of the compact morula and in TE cells of early and expanded blastocysts. However, their expression becomes mutually exclusive in individual TE and ICM cells as development continues to the more advanced blastocyst stages. Studies in the mouse indicate that overexpression of Cdx2
expression in ESCs (Strumpf et al., 2005
). Modulation of the OCT4
expression ratio in ESCs can determine their fate. A high OCT4:CDX2
ratio supports the maintenance of the pluripotent ICM, whereas a low ratio promotes differentiation into TE (Niwa et al., 2005
). This pattern of CDX2
expression suggests that the interplay and mutual repression between these two transcription factors could be important for the segregation and specification of the first two lineages in primate embryos.
Furthermore, inhibition of CDX2 protein synthesis secondary to anti-sense morpholino injection into unfertilized monkey oocytes suggests translational down-regulated of maternal and embryonic CDX2 protein synthesis throughout preimplantation development. Thus, the inhibition of protein synthesis by morpholinos would appear to be an effective tool for investigating gene function in primate embryos. Despite their normal appearance, CDX2-deficient monkey blastocysts were unable to expand and hatch from the zona pellucida, suggesting that CDX2 is likely responsible for the maintenance of cell junctions in the TE. Our results also indicate that although CDX2 has an important role in the development of a fully functional TE and possibly in the downregulation of the ICM-associated pluripotency genes, initial blastocoel formation does not seem to require either maternal or zygotic CDX2
. These observations are consistent with available evidence that Cdx2
is not essential for certain early aspects of the TE specification during preimplantation mouse embryo development (Meissner and Jaenisch, 2006
; Niwa et al., 2005
; Strumpf et al., 2005
). However, Cdx2
-deficient TE cells were unable to proliferate and form giant trophoblast cell outgrowths in vitro
or produce TE stem cells upon culture on feeder layers (Niwa et al., 2005
). Moreover, mouse Cdx2
knockout embryos fail to implant and grow into a fetus (Ralston and Rossant, 2008
Similar to the mouse (Chawengsaksophak et al., 2004
), depletion of CDX2 did not seem interfere with the formation of the pluripotent ICM lineage in primate embryos based on the observation that OCT4
expression was unaffected. We were able to rescue the pluripotent lineage by plating CDX2-deficient embryos onto feeder layers and subsequently isolating ESC lines. Two ESC lines were derived at a 50% efficiency which is slightly higher than our average for control embryos (30%) (Mitalipov et al., 2006
). Moreover, both cell lines exhibited a normal diploid karyotypes, expressed key ESCs markers and differentiated into various cell and tissue types representing all the three embryonic germ layers.
In summary, we demonstrated the expression pattern of CDX2 during early primate embryo development. Knockdown of CDX2 protein resulted in embryonic arrest due to inability to form the TE. These results provide evidence that CDX2 plays an essential role in formation of the functional TE lineage during primate embryonic development.