(A) Morphology of WT mESCs after treatment with CHIR99021 (15 μM for 48 hr; left). Western blots for β-catenin and Oct-4 after control, Oct-4, and β-catenin immunoprecipitates from the indicated cell lines (right).
(B) β-catenin coimmunoprecipitates with Oct-4 from WT mESC lysates after overnight stimulation with Wnt3a-CM.
(C) β-catenin coimmunoprecipitates with Oct-4 from DKO and DKO-TCF4DN mESC lysates, but negligibly from lysates of DKO-TCF4DN mESCs expressing β-catenin-specific shRNAs.
(D) β-catenin coimmunoprecipitates with Oct-4 from lysates derived from WT mESCs expressing stabilized (S33A) β-catenin.
(E) Wnt/β-catenin pathway activation by various means induces the activation of an Oct-4 reporter (PORE).
(F) WT mESCs treated with CHIR99021 for 24 hr displayed a significant increase in the transcript levels for four of five Oct-4 target genes, as assessed by qRT-PCR (p < 0.05, unpaired t test).
(G) Coimmunoprecipitation analysis demonstrates that β-catS33AΔC retains the ability to interact with Oct-4.
(H) Increased activation of an Oct reporter (PORE) in WT mESCs stably expressing both full-length β-catS33A and β-catS33AΔC.
(I) Model of β-catenin action in mESCs. (i) In the absence of Wnt pathway activation, β-catenin is predominantly recruited to cadherin complexes. A small amount of nuclear β-catenin associates with some Oct-4-regulated loci. Under conditions permissive of differentiation, mESCs undergo efficient multilineage differentiation. (ii) Under conditions where the signaling pool of β-catenin is stabilized, nuclear β-catenin coactivates the expression of various genes in concert with either TCF or Oct-4. In this scenario, mESCs exhibit enhanced self-renewal, in part because of β-catenin’s interactions with Oct-4, and mesendoderm-restricted differentiation, resulting from high levels of TCF target gene expression caused by β-catenin-mediated transactivation. (iii) Attenuation of β-catenin/TCF target gene activation, via ectopic expression of a dominant-negative TCF protein (TCFDN), impairs differentiation into all germ layer lineages, including neurectoderm, but enhances retention of pluripotency markers. (iv) mESCs expressing a truncation mutant of stabilized β-catenin (β-ΔC), lacking the C-terminal residues necessary for the efficient transactivation of β-catenin/TCF target genes, exhibit enhanced self-renewal and impeded exit from the pluripotent state. These cells are highly refractory to neurectoderm differentiation.