Cell fate decisions require the integration of various signalling inputs at the level of signal transduction and transcription. Wnt and Notch signalling are two important signalling systems that operate in concert in a variety of systems in vertebrates and invertebrates. My interest lies in understanding how cells integrate signalling inputs to make cell fate decisions, with a focus on the roles of Notch and Wnt signalling in these processes.
The Notch receptor can modulate Wnt signalling by regulating the activity and levels of Armadillo/β-catenin. Further characterization of this role of Notch has shown that whilst Notch is able to suppress ectopic Wnt signalling induced by loss of GSK-3 function, the relationship between Notch and Axin (another member of the Armadillo/β-catenin destruction complex) is more complicated. We observe that there is synergy between Axin and Notch in the regulation of Armadillo/β-catenin. These results combined with other observations collected within the lab over the years have led to the suggestion that Notch and Wnt signalling form an integrated module that plays a particular role during cell fate assignations [4,9]. While programmed interactions between signals and transcription factors determine developmental pathways and generate cell fates, Wnt/Notch signalling determines the efficiency of these processes i.e. they regulate the tempo and probability of a fate decision.
Using mouse embryonic stem cell (mES) as model systems, in collaboration with Fernando Faunes I am currently exploring the inputs of Wnt signalling into cell fate decisions and the mechanism by which this signal transduction pathway exerts its influence. This work, exploring the influence of b-catenin and Wnt signalling on mES cell pluripotency and differentiation has established that b-catenin is a significant potentiator of the pluripotent state in mES cells (also see FFQ page). Further I find that during self-renewal there is negligible transcriptional activity of ß-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-Cadherin. Differentiation triggers a burst of Wnt/ß-catenin transcriptional activity that coincides with the disassembly of the complex. Our results (Also see page of FFQ) establish that ß-catenin, but not its transcriptional activity, is central to pluripotency acting through a ß-catenin: Oct4 complex.
(A) TopFlash assay to assess Wnt/ß-catenin transcriptional activity in mES cells (E14Tg2A) in self renewing (Serum+LIF, blue bars) and differentiating conditions (SRA, red bars) or HEK293T cells (green bars). Results are representative of 2 experiments and the average of 3 independent replicates. (B) E14Tg2A cells were grown in Serum+LIF or Serum+RA for 4 days. 4 hours prior to lysis, cells were exposed to the indicated conditions. Cell lysates were fractionated with ConA and protein expression was assessed by Western blot. (C) Oct4 and ß-catenin containing complexes were immunoprecipitated from membrane associated protein extracts of Tcf3 null (TCF-/-) and their wild-type parental (TCF+/+) mES cells, cells were maintained in 2i media for at least 2 passaged prior to lysis.