Differential requirement for the dual functions of β-catenin in embryonic stem cell self-renewal and germ layer formation

Abstract

Canonical Wnt-signalling has been implicated in mouse and human embryonic stem cell (ESC) maintenance, however its requirement is controversial. β-catenin is the key component in this highly conserved Wnt pathway, acting as a transcriptional transactivator. Yet, β-catenin has additional roles at the plasma membrane regulating cell-cell adhesion, complicating the analyses of cells/tissues lacking β-catenin. We report here the generation of a β-catenin deficient mouse ESC (mESC) line and show that self-renewal is maintained in absence of β-catenin. Cell-adhesion is partially rescued by plakoglobin up-regulation, but fails to be maintained during differentiation. When differentiated as aggregates, wild-type mESCs form descendents of all three germ layers, while mesendodermal germ layer formation and neuronal differentiation are defective in β-catenin deficient mESCs. A Tcf/Lef-signalling defective β-catenin variant, which re-establishes cadherin-mediated cell-adhesion, rescues definitive endoderm and neuroepithelial formation, suggesting that β-catenin cell-adhesion function is more important than its signalling function for these processes.

Wnt Pathway Regulation of Embryonic Stem Cell Self-Renewal

“…Another approach independently taken by two groups (Austin Smith's group and Christine Hartmann's group) found that after conditionally ablating b-catenin from established mESC lines, cells formed abnormally scattered colonies consisting of poorly attached cells (Lyashenko et al 2011;Wray et al 2011), which was similar to the colony morphology of outgrowths from b-catenin-null blastocysts (Haegel et al 1995). Interesting, the abnormal colony morphology returned to normal after several passages despite the continued absence of b-catenin, most likely because plakoglobin, a homolog of b-catenin that also binds to E-cadherin, replaced b-catenin at adherence junctions (Haegel et al 1995;Huelsken et al 2000;Lyashenko et al 2011). Ablating b-catenin failed to block pluripotent cell proliferation in either 2i þ LIF or LIF þ ERK inhibitor conditions (Lyashenko et al 2011;Wray et al 2011).…”

“…Although GSK3 activity is necessary for regulating b-catenin levels, it is also important for cellular effects not normally associated with Wnt signaling, such as glycogen metabolism and translation control (Doble and Woodgett 2003). Moreover, the suggestion that b-catenin was not genetically necessary for mESCs to proliferate in LIF þ serum conditions (Lyashenko et al 2011;Wray et al 2011) presented the possibility that b-catenin-independent effects of GSK3 inhibition and Wnt stimulation may affect pluripotent cell self-renewal. Therefore, it was important to determine if Wnt stimulation/Gsk3 inhibition affects function through b-catenin.…”

“…Interesting, the abnormal colony morphology returned to normal after several passages despite the continued absence of b-catenin, most likely because plakoglobin, a homolog of b-catenin that also binds to E-cadherin, replaced b-catenin at adherence junctions (Haegel et al 1995;Huelsken et al 2000;Lyashenko et al 2011). Ablating b-catenin failed to block pluripotent cell proliferation in either 2i þ LIF or LIF þ ERK inhibitor conditions (Lyashenko et al 2011;Wray et al 2011). b-Catenin-null cells expressed markers of mESCs, including Oct4, Sox2, Nanog, Rex1, and Pecam1 (Lyashenko et al 2011;Wray et al 2011), which was consistent with b-catenin not being absolutely necessary for pluripotency.…”

“…Ablating b-catenin failed to block pluripotent cell proliferation in either 2i þ LIF or LIF þ ERK inhibitor conditions (Lyashenko et al 2011;Wray et al 2011). b-Catenin-null cells expressed markers of mESCs, including Oct4, Sox2, Nanog, Rex1, and Pecam1 (Lyashenko et al 2011;Wray et al 2011), which was consistent with b-catenin not being absolutely necessary for pluripotency. In contrast, EpiSC differentiation following IWP2 and Fz8CRD treatments indicated that Wnt signaling was necessary for self-renewal in a naive state (Berge et al 2011).…”

“…Thus, either a b-catenin-independent effect of Wnt proteins contributes to the naive state and/or the b-catenin-null cells exist in a primed state. Regardless of the state of pluripotency of b-catenin-deficient cells, it is important to note that the b-catenin was necessary for self-renewal under either 2i or LIF þ GSK3-inhibitor conditions (Lyashenko et al 2011;Wray et al 2011), showing that b-catenin is the necessary downstream effector of GSK3 inhibition. Thus, the ability of GSK3 inhibition to support long-term self-renewal functions through b-catenin.…”