Convergence of cMyc and β-catenin on Tcf7l1 enables endoderm specification.

Gillian Morrison,Austin Smith,Roberta Scognamiglio,Andreas Trumpp

doi:10.15252/embj.201592116

Abstract

The molecular machinery that directs formation of definitive endoderm from pluripotent stem cells is not well understood. Wnt/β-catenin and Nodal signalling have been implicated, but the requirements for lineage specification remain incompletely defined. Here, we demonstrate a potent effect of inhibiting glycogen synthase kinase 3 (GSK3) on definitive endoderm production. We find that downstream of GSK3 inhibition, elevated cMyc and β-catenin act in parallel to reduce transcription and DNA binding, respectively, of the transcriptional repressor Tcf7l1. Tcf7l1 represses FoxA2, a pioneer factor for endoderm specification. Deletion of Tcf7l1 is sufficient to allow upregulation of FoxA2 in the presence of Activin. In wild-type cells, cMyc contributes by reducing Tcf7l1 mRNA, while β-catenin acts on Tcf7l1 protein. GSK3 inhibition is further required for consolidation of endodermal fate via upregulation of Sox17, highlighting sequential roles for Wnt signalling. The identification of a cMyc/β-catenin-Tcf7l1-FoxA2 axis reveals a de-repression mechanism underlying endoderm induction that may be recapitulated in other developmental and patho-logical contexts.

Highlights

Nodal/Activin signalling is required for endoderm specification in the embryo (Brennan et al, 2001; Vincent et al, 2003) and steering differentiation of pluripotent stem cells towards definitive endoderm (Kubo et al, 2004; Gadue et al, 2006; Morrison et al, 2008)
We first examined the response of naıve embryonic stem (ES) cells to a wellestablished cocktail of endoderm differentiation inducers (Kubo et al, 2004; Gadue et al, 2006; McLean et al, 2007; Morrison et al, 1 Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK 2 Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany 3 Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany 4 Department of Biochemistry, University of Cambridge, Cambridge, UK
Gillian Morrison et al Tcf7l1 is a roadblock to endoderm specification

Summary

Introduction

Nodal/Activin signalling is required for endoderm specification in the embryo (Brennan et al, 2001; Vincent et al, 2003) and steering differentiation of pluripotent stem cells towards definitive endoderm (Kubo et al, 2004; Gadue et al, 2006; Morrison et al, 2008). We have a limited understanding of the signalling processes that activate FoxA2 expression and initiate molecular specification of definitive endoderm. The heterogeneity of ES cells maintained in the presence of serum (Hayashi et al, 2008; Silva & Smith, 2008) can be a confounding factor when attempting to optimise lineage specification and delineate initial molecular and cellular transitions. Upon release from the naıve ground state conditions, ES cells readily undergo developmental progression and lineage specification (Hayashi et al, 2011; Marks et al, 2012; Betschinger et al, 2013; Buecker et al, 2014; Kalkan & Smith, 2014; Yang et al, 2014)

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