Inner Cell Mass - Regulation of Cellular Specification

Regulation of Cellular Specification

Since segregation of pluripotent cells of the inner cell mass from the remainder of the blastocyst is integral to mammalian development, considerable research has been performed to elucidate the corresponding cellular and molecular mechanisms of this process. There is primary interest in which transcription factors and signaling molecules direct blastomere asymmetric divisions leading to what are known as inside and outside cells and thus cell lineage specification. However, due to the variability and regulative nature of mammalian embryos, experimental evidence for establishing these early fates remains incomplete.

At the transcription level, the transcription factors Oct4, Nanog, Cdx2, and Tead4 have all been implicated in establishing and reinforcing the specification of the ICM and the TE in early mouse embryos.

• Oct4: Oct4 is expressed in the ICM and participate in maintaining its pluripotency, a role that has been recapitulated in ICM derived mouse embryonic stem cells. Oct4 genetic knockout cells both in vivo and in culture display TE morphological characteristics. It has been shown that one transcriptional target of Oct4 is the Fgf4 gene. This gene normally encodes a ligand secreted by the ICM, which induces proliferation in the adjacent polar TE.

• Nanog: Nanog is also expressed in the ICM and participates in maintaining its pluripotency. In contrast with Oct4, studies of Nanog-null mice do not show the reversion of the ICM to a TE-like morphology, but demonstrate that loss of Nanog prevents the ICM from generating primitive endoderm.

• Cdx2: Cdx2 is strongly expressed in the TE and is required for maintaining its specification. Knockout mice for the Cdx2 gene undergo compaction, but lose the TE epithelial integrity during the late blastocyst stage. Furthermore, Oct4 expression is subsequently raised in these TE cells, indicating Cdx2 plays a role in suppressing Oct4 in this cell lineage. Moreover, embryonic stem cells can be generated from Cdx2-null mice, demonstrating that Cdx2 is not essential for ICM specification.

• Tead4: Like Cdx2, Tead4 is required for TE function, although the transcription factor is expressed ubiquitously. Tead4-null mice similarly undergo compaction, but fail to generate the blastocoel cavity. Like Cdx2-null embryos, the Tead4-null embryos can yield embryonic stem cells, indicating that Tead4 is dispensable for ICM specification. Recent work has shown that Tead4 may help to upregulate Cdx2 in the TE and its transcriptional activity depends on the coactivator Yap. Yap’s nuclear localization in outside cells allows it to contribute to TE specificity, whereas inside cells sequester Yap in the cytoplasm through a phosphorylation event.

Together these transcription factors function in a positive feedback loop that strengthens the ICM to TE cellular allocation. Initial polarization of blastomeres occurs at the 8-16 cell stage. An apical-basolateral polarity is visible through the visualization of apical markers such as Par3, Par6, and aPKC as well as the basal marker E-Cadherin. The establishment of such a polarity during compaction is thought to generate an environmental identity for inside and outside cells of the embryo. Consequently, stochastic expression of the above transcription factors is amplified into a feedback loop that specifies outside cells to a TE fate and inside cells to an ICM fate. In the model, an apical environment turns on Cdx2, which upregulates its own expression through a downstream transcription factor, Elf5. In concert with a third transcription factor, Eomes, these genes act to suppress pluripotency genes like Oct4 and Nanog in the outside cells. Thus, TE becomes specified and differentiates. Inside cells, however, do not turn on the Cdx2 gene, and express high levels of Oct4, Nanog, and Sox2, These genes suppress Cdx2 and the inside cells maintain pluripotency generate the ICM and eventually the rest of the embryo proper.

Although this dichotomy of genetic interactions is clearly required to divide the blastomeres of the mouse embryo into both the ICM and TE identities, the initiation of these feedback loops remains under debate. Whether they are established stochastically or through an even earlier asymmetry is unclear, and current research seeks to identify earlier markers of asymmetry. For example, some research correlates the first two cleavages during embryogenesis with respect to the prospective animal and vegetal poles with ultimate specification. The asymmetric division of epigenetic information during these first two cleavages, and the orientation and order in which they occur, may contribute to a cell’s position either inside or outside the morula,.