Initiation of trophectoderm lineage specification in mouse embryos is independent of Cdx2.

The separation of the first two lineages - trophectoderm (TE) and inner cell mass (ICM) - is a crucial event in the development of the early embryo. The ICM, which constitutes the pluripotent founder cell population, develops into the embryo proper, whereas the TE, which comprises the surrounding outer layer, supports the development of the ICM before and after implantation. Cdx2, the first transcription factor expressed specifically in the developing TE, is crucial for the differentiation of cells into the TE, as lack of zygotic Cdx2 expression leads to a failure of embryos to hatch and implant into the uterus. However, speculation exists as to whether maternal Cdx2 is required for initiation of TE lineage separation. Here, we show that effective elimination of both maternal and zygotic Cdx2 transcripts by an RNA interference approach resulted in failure of embryo hatching and implantation, but the developing blastocysts exhibited normal gross morphology, indicating that TE differentiation had been initiated. Expression of keratin 8, a marker for differentiated TE, further confirmed the identity of the TE lineage in Cdx2-deficient embryos. However, these embryos exhibited low mitochondrial activity and abnormal ultrastructure, indicating that Cdx2 plays a key role in the regulation of TE function. Furthermore, we found that embryonic compaction does not act as a 'switch' regulator to turn on Cdx2 expression. Our results clearly demonstrate that neither maternal nor zygotic Cdx2 transcripts direct the initiation of ICM/TE lineage separation.

Oct1 regulates trophoblast development during early mouse embryogenesis.

Oct1 (Pou2f1) is a transcription factor of the POU-homeodomain family that is unique in being ubiquitously expressed in both embryonic and adult mouse tissues. Although its expression profile suggests a crucial role in multiple regions of the developing organism, the only essential function demonstrated so far has been the regulation of cellular response to oxidative and metabolic stress. Here, we describe a loss-of-function mouse model for Oct1 that causes early embryonic lethality, with Oct1-null embryos failing to develop beyond the early streak stage. Molecular and morphological analyses of Oct1 mutant embryos revealed a failure in the establishment of a normal maternal-embryonic interface due to reduced extra-embryonic ectoderm formation and lack of the ectoplacental cone. Oct1(-/-) blastocysts display proper segregation of trophectoderm and inner cell mass lineages. However, Oct1 loss is not compatible with trophoblast stem cell derivation. Importantly, the early gastrulation defect caused by Oct1 disruption can be rescued in a tetraploid complementation assay. Oct1 is therefore primarily required for the maintenance and differentiation of the trophoblast stem cell compartment during early post-implantation development. We present evidence that Cdx2, which is expressed at high levels in trophoblast stem cells, is a direct transcriptional target of Oct1. Our data also suggest that Oct1 is required in the embryo proper from late gastrulation stages onwards.

Efficient derivation of pluripotent stem cells from siRNA-mediated Cdx2-deficient mouse embryos.

In the early mammalian embryo, lineage separation of and subsequent crosstalk between the trophectoderm (TE) and inner cell mass (ICM) are required to support further development. Previous studies have shown that the homeobox transcription factor Cdx2 is required for TE differentiation and that lack of Cdx2 expression causes death of embryos at the peri-implantation stage. In this study, we effectively eliminated Cdx2 transcripts by microinjection of siRNA into embryos and evaluated the effect on efficiency of deriving embryonic stem cells (ESCs). By this approach, we successfully created nonviable embryos similar to reported knockout embryos. Accordingly, the efficiency of ESC derivation dropped from 19.1% in control blastocysts to 2% in Cdx2-deficient blastocysts, indicating loss of pluripotency in the ICM. Strikingly, when 8-cell stage embryos were cultured under ESC culture conditions before lineage separation, fully functional pluripotent stem cell lines were obtained, with efficiency even greater than that for control embryos. These results demonstrate that Cdx2 plays an essential role within the microenvironment created by the TE to support ICM pluripotency but that the ESC culture system, with mouse embryonic fibroblasts, could rescue the pluripotent cell population for efficient ESC derivation.