The Effects of Small Molecule Inhibitors of Wnt/β-Catenin Signaling Pathway on Murine Embryonic Stem Cells.

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

The earliest lineage decisions during mouse embryonic development occur as early as the eight-cell stage when individual blastomeres begin to express molecular markers of the inner cell mass (i.e. embryo) and trophectoderm (i.e. placenta). Immediately preceeding implantation, asymmetric cell divisions in the embryo help maintain a population of stem cells while simultaneously providing progenitors with different developmental potential. Previous gene expression analyses have implicated the involvement of the Wnt/β-catenin signaling pathway during preimplantation embryonic development. A library of specific, small molecule inhibitors of the Wnt/β-catenin signaling pathway have been developed in our laboratory and tested in a variety of chemogenomic studies. One such small molecule, ICG-001, has provided clear evidence that the highly homologous transcriptional coactivators, CBP and p300, have distinct functions in regulating the expression of Wnt/β-catenin genes. Consequently, we have developed an innovative model proposing that TCF/β-catenin/CBP-mediated transcription is critical for proliferation without differentiation (e.g. in stem cells). The switch of coactivators by β-catenin is the essential first step in the initiation of differentiation with the TCF/β-catenin/p300 complex driving transcription of genes associated with differentiation. Another small molecule, IQ-1, prevents β-catenin from switching coactivator usage from CBP to p300, thereby allowing proliferation of stem cell populations without differentiation. In this study, we propose that the differential usage of these coactivators is involved during lineage decisions in preimplantation mouse embryos. Embryonic stem (mES) cells have been employed in this investigation as a cellular model for early murine development and to investigate the effects of small molecule inhibitors on cell fate. Real-time RT-PCR has been used to examine gene expression of both pluripotency- and differentiation-associated markers. Three-day treatment of mES with ICG-001 resulted in a time- and dose-dependent increase in the expression of markers associated with early trophectoderm development, including Cdx2, Hand1 and Fgfr2. Interestingly, the steady state levels of Oct4 mRNA appear to be unaffected by ICG-001 at this time point. On the other hand, three day treatment of mES with IQ-1 had no effect on the expression of Cdx2, Hand1 or Fgfr2; the expression of Oct4 mRNA was also unaffected by IQ-1 treatment. Intracellular localization of markers associated with either pluripotency (e.g. Oct4) or differentiation (e.g. Cdx2) has also been investigated in mES using immunocytochemistry in conjunction with confocal microscopy. All untreated mES express Oct4 protein; however, treatment of mES for 24 hours with ICG-001 results in homogeneous populations containing both Oct4-positive and Oct4-negative cells. Therefore, these data provide the first evidence that the Wnt/β-catenin signaling pathway could be involved during early lineage decisions in mouse preimplantation development. Results also suggest the involvement of CBP during mES self-renewal and that the switch from CBP- to p300-mediated transcription leads to differentiation. Future investigations should attempt to clarify the molecular mechanisms involved as they may have important ramifications in the field of regenerative medicine.