These are three videos from our recent publication in Development (“A membrane-associated β-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells.” Development. 2013 Mar;140(6):1171-83. doi: 10.1242/dev.085654) which highlight the behaviour of wildtype and ß-catenin mutant cells in 2i conditions. This study tries to resolve the running controversy about the requirement for ß-catenin signalling. Our results are in keeping with those of Wray et al. (Inhibition of glycogen synthase kinase-3 alleviates Tcf3 repression of the pluripotency network and increases embryonic stem cell resistance to differentiation. Nat Cell Biol. 2011 Jun 19;13(7):838-45) and extend them to reveal an important interaction between ß-catenin and Oct4 associated with pluripotency.
For these movies, cells were plated at low density in 2i (N2B27 supplemented with the MEK inhibitor PD03 and the GSK3 inhibitor Chiron). The wild type cells (E14) rapidly stick together to form the aggregates that are characteristic of these culture conditions. You will see this stickiness in the way individual cells get sucked up into the colonies. On the other hand, notice that the ß-catenin mutant cells have difficulty sticking together and forming the compact structures observed in E14 cells. In ß-catenin mutant cultures, it is possible to observe constant attempts to differentiate which is consistent with the fragile pluripotency of these cells (remember that they are in 2i!). The third movie shows cells whose only ß-catenin lacks the C terminal domain and therefore cannot mediate transcriptional functions. In fact, the biology of ß-catenin suggests that this protein is mostly stable at the membrane and this is what we observe. These cells exhibit an intermediate behaviour between the E14s and the ß-catenin mutants. They adhere to each other though they do not form the tight aggregates that E14 cells do. As these cells are pluripotent this was used by Wray et al to show that the transcriptional function of ß-catenin is not required for the maintenance of pluripotency (and see also Lyashenko et al. Nat Cell Biol. 2011 Jun 19;13(7):753-61).
Of course there is a lot more in these movies and if you notice something, let us know.