Distinct Wnt-driven primitive streak-like populations reflect in vivo lineage precursors

Anestis Tsakiridis ( 0 Yali Huang 0 Guillaume Blin 0 Stavroula Skylaki 0 Filip Wymeersch 0 Rodrigo Osorno 0 Costas Economou 0 Eleni Karagianni 0 Suling Zhao 0 Sally Lowell 0 Valerie Wilson ) 0 0 MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh , 5 Little France Drive, Edinburgh EH16 4UU, UK During gastrulation, epiblast cells are pluripotent and their fate is thought to be constrained principally by their position. Cell fate is progressively restricted by localised signalling cues from areas including the primitive streak. However, it is unknown whether this restriction accompanies, at the individual cell level, a reduction in potency. Investigation of these early transition events in vitro is possible via the use of epiblast stem cells (EpiSCs), self-renewing pluripotent cell lines equivalent to the postimplantation epiblast. Strikingly, mouse EpiSCs express gastrulation stage regional markers in self-renewing conditions. Here, we examined the differentiation potential of cells expressing such lineage markers. We show that undifferentiated EpiSC cultures contain a major subfraction of cells with reversible early primitive streak characteristics, which is mutually exclusive to a neural-like fraction. Using in vitro differentiation assays and embryo grafting we demonstrate that primitive streak-like EpiSCs are biased towards mesoderm and endoderm fates while retaining pluripotency. The acquisition of primitive streak characteristics by selfrenewing EpiSCs is mediated by endogenous Wnt signalling. Elevation of Wnt activity promotes restriction towards primitive streakassociated lineages with mesendodermal and neuromesodermal characteristics. Collectively, our data suggest that EpiSC pluripotency encompasses a range of reversible lineage-biased states reflecting the birth of pioneer lineage precursors from a pool of uncommitted EpiSCs similar to the earliest cell fate restriction events taking place in the gastrula stage epiblast. - INTRODUCTION Lineage specification in the mouse embryo during gastrulation generates progressively more restricted precursors from initially uncommitted postimplantation epiblast cells (Tzouanacou et al., 2009). Cell fate restriction is spatially organised (Lawson et al., 1991) by localised signalling cues and is reflected by the regionalised expression of lineage-specific markers (Pfister et al., 2007; Arnold and Robertson, 2009; Teo et al., 2011). However, pluripotency is widespread in the epiblast until around the start of somitogenesis (Osorno et al., 2012). A defining feature of gastrulation is the formation of the primitive streak (PS), a posterior structure in which epiblast cells undergo epithelial-to-mesenchymal (EMT) transition, ingressing to give rise initially to the endoderm and mesoderm of the head and heart, and later to progressively more posterior mesoderm types, including somites (Kinder et al., 1999; Kinder et al., 2001). Anterior epiblast that does not encounter the PS instead forms ectoderm-restricted lineages, including the anterior neural ectoderm (Lawson, 1999; Cajal et al., 2012). Clonal analysis showed that some early-ingressing mesodermal derivatives arise from a common mesendodermal (ME) precursor, whereas lateringressing somitic mesoderm is generated by a neuromesodermal (NM) progenitor, which also gives rise to the neurectoderm of the spinal cord (Tzouanacou et al., 2009). One of the earliest markers for the PS is the T-box transcription factor T(Bra), which is expressed both in pre-EMT prospective mesoderm and endoderm in the posterior epiblast, and in post-EMT, nascent mesoderm in the PS and its descendant, the tail bud (Wilkinson et al., 1990; Kispert and Herrmann, 1994; Rivera-Prez and Magnuson, 2005; Burtscher and Lickert, 2009). T(Bra) is also expressed in the node and notochord. Wnt and Nodal signalling are essential for both PS specification (Liu et al., 1999; Ben-Haim et al., 2006) and the initiation of T(Bra) expression (Conlon et al., 1994; Arnold et al., 2000). Despite considerable progress in defining the role of the interactions between signalling pathways and regionalised genetic activity in the induction of PS precursors, the inaccessibility of mouse embryos renders the study of the transition from pluripotency to lineage restriction difficult. Epiblast stem cells (EpiSCs), cell lines derived from the postimplantation epiblast (Brons et al., 2007; Tesar et al., 2007) or from embryonic stem cells (ESCs) in vitro (Guo et al., 2009), represent an attractive model for dissecting early lineage commitment as they comprise the in vitro counterpart of pluripotent cells in the gastrula stage epiblast (Huang et al., 2012). Unlike mouse ESCs but similar to human ES cells (hESCs), self-renewal of EpiSCs, reflected by the simultaneous expression of the key pluripotency factors Oct4 (Pou5f1 Mouse Genome Informatics), Nanog and Sox2, relies on culture in the mesoderm inducers Activin (inhibin Mouse Genome Informatics), a Nodal-like TGF family member, and Fgf2 (Tesar et al., 2007; Vallier et al., 2009; Greber et al., 2010). Like the gastrulating epiblast (Pfister et al., 2007; Arnold and Robertson, 2009), EpiSC lines also express, under self-renewing conditions, lineage-specific markers (Tesar et al., 2007; Bernemann et al., 2011; Teo et al., 2011; Iwafuchi-Doi et al., 2012; Kojima et al., 2014). Variations in their expression at the population level have been shown to result in altered differentiation outcome (Bernemann et al., 2011). However, whether heterogeneous lineage-specific marker expression is an inherent feature of EpiSC pluripotency, reflecting the emergence of pioneer lineage-biased yet uncommitted precursors, or a manifestation of commitment to specific lineages induced by culture conditions is presently unknown. Here we dissect the events underlying the progressive commitment of pluripotent EpiSCs to PS-derived lineages. Using a T(Bra)-based, PS-specific fluorescent reporter, we characterise a major self-renewing fraction of undifferentiated EpiSCs that exhibits early gastrulation, pre-ingression (i.e. epiblast-like) PS characteristics and readily interconverts with reporter-negative cells. PS-like EpiSCs are dependent on endogenous Wnt signalling and are biased towards mesodermal and endodermal differentiation with retention of pluripotency. Further elevation of Wnt activity drives cells out of pluripotency, producing two mutually exclusive cell types resembling ME and NM progenitors present in the PS in vivo. Collectively, our findings suggest that, in vivo, regional gene expression defines cells in the gastrulation stage epiblast that are biased towards, but not committed to, distinct differentiation outcomes. RESULTS Two major subpopulations in EpiSC cultures reflecting PS and neurectoderm Previous studies have reported the expression of lineage-specific markers in self-renewing EpiSC populations (Tesar et (...truncated)