RESEARCH ARTICLE STEM CELLS AND REGENERATION Gata6, Nanog and Erk signaling control cell fate in the inner cell mass through a tristable regulatory network Sylvain Bessonnard 1,2,3, * , ‡ , Laurane De Mot 4, ‡ , Didier Gonze 4 , Manon Barriol 1,2,3 , Cynthia Dennis 1,2,3 , Albert Goldbeter 4,5 , Geneviè ve Dupont 4,§ and Claire Chazaud 1,2,3,§ ABSTRACT During blastocyst formation, inner cell mass (ICM) cells differentiate into either epiblast (Epi) or primitive endoderm (PrE) cells, labeled by Nanog and Gata6, respectively, and organized in a salt-and-pepper pattern. Previous work in the mouse has shown that, in absence of Nanog, all ICM cells adopt a PrE identity. Moreover, the activation or the blockade of the Fgf/RTK pathway biases cell fate specification towards either PrE or Epi, respectively. We show that, in absence of Gata6, all ICM cells adopt an Epi identity. Furthermore, the analysis of Gata6 +/- embryos reveals a dose-sensitive phenotype, with fewer PrE-specified cells. These results and previous findings have enabled the development of a mathematical model for the dynamics of the regulatory network that controls ICM differentiation into Epi or PrE cells. The model describes the temporal dynamics of Erk signaling and of the concentrations of Nanog, Gata6, secreted Fgf4 and Fgf receptor 2. The model is able to recapitulate most of the cell behaviors observed in different experimental conditions and provides a unifying mechanism for the dynamics of these developmental transitions. The mechanism relies on the co-existence between three stable steady states (tristability), which correspond to ICM, Epi and PrE cells, respectively. Altogether, modeling and experimental results uncover novel features of ICM cell fate specification such as the role of the initial induction of a subset of cells into Epi in the initiation of the salt-and-pepper pattern, or the precocious Epi specification in Gata6 +/- embryos. KEY WORDS: Epiblast, Primitive endoderm, Cell lineage specification, Gata6 mutants, Mathematical model, Multistability, Preimplantation, Bifurcation, Mouse INTRODUCTION In the mouse, two differentiation processes take place before the implantation of the egg in the uterus. The first one gives rise to the inner cell mass (ICM) and the trophoblast (TE). The second one is the differentiation of the ICM into primitive endoderm (PrE) and epiblast (Epi). Two antagonistic transcription factors control the differentiation of the ICM into Epi and PrE: Nanog is required for the differentiation into Epi cells (Mitsui et al., 2003; Silva et al., 2009; Messerschmidt and Kemler, 2010; Frankenberg et al., 2011), whereas Gata6 is necessary to produce the PrE epithelium in vitro and in vivo (Morrisey et al., 1998; Koutsourakis et al., 1999; Capo- Chichi et al., 2005; Cai et al., 2008; Morris et al., 2010). The zygotic expression of these genes starts around the 2/4-cell stage (Guo et al., 2010; Miyanari and Torres-Padilla, 2012), and from the 8-cell [embryonic day (E) 2.5] to the 32-cell (E3.0), stage, Gata6 and Nanog proteins accumulate in almost all the cells (Dietrich and Hiiragi, 2007; Plusa et al., 2008). From E3.0-E3.25, their expression becomes mutually exclusive asynchronously within the ICM cells. Hence, at E3.75, the ICM contains two distinct cell populations that have a salt-and-pepper pattern: Gata6-expressing PrE progenitors and Nanog-expressing Epi progenitors (Rossant et al., 2003; Chazaud et al., 2006; Kurimoto et al., 2006; Plusa et al., 2008; Guo et al., 2010). These two populations are then sorted, so that the PrE forms a layer of cells separating the Epi from the blastocoel (Rula et al., 2007; Plusa et al., 2008; Meilhac et al., 2009). After specification, PrE progenitors activate several tissue-specific genes, such as Pdgfra, Sox17, Gata4, Dab2 and Lrp2, which are required for their maturation (Stephenson et al., 2012; Artus and Chazaud, 2014). Experimental findings indicate that Nanog and Gata6 inhibit each other’s expression. First, the invalidation of Nanog induces the expression of Gata6 in the whole ICM (Frankenberg et al., 2011), while forced expression of Gata6 in ES cells downregulates Nanog and pluripotency markers (Fujikura et al., 2002; Shimosato et al., 2007). Moreover, Nanog can bind to Gata6 promoter and directly decreases its activity in vitro (Singh et al., 2007). Besides the Nanog and Gata6 network of interactions, the Fgf/RTK signaling pathway also plays a crucial role in the balance between Epi and PrE cell fate specification. Embryos mutant for Grb2 – an adaptor of the Erk signaling pathway – do not produce any PrE cells, whereas all ICM cells express Nanog (Chazaud et al., 2006). Likewise, culturing wild-type embryos with a Mek inhibitor abolishes the expression of Gata6 and induces Nanog expression (Nichols et al., 2009; Yamanaka et al., 2010). Conversely, if these embryos are cultured with recombinant Fgf4, they present a larger proportion of cells differentiating into PrE (Yamanaka et al., 2010). Interestingly, there is a window of plasticity between E2.5 and E4.0 where ICM cells can change their identity through the influence of their Fgf/RTK environment (Yamanaka et al., 2010; Grabarek et al., 2012; Arias et al., 2013). Inhibiting the Erk signaling pathway also prevents ES cell differentiation into PrE and maintains them in a pluripotent state (Cheng et al., 1998; Burdon et al., 1999; Hamazaki et al., 2006; Ying et al., 2008). Experiments modulating the Fgfr/Erk pathway in Nanog mutants revealed that, in a first phase around E2.5, Gata6 expression is induced by the Erk pathway. Afterwards, Erk signaling progressively becomes dispensable for the maintenance of Gata6 expression in the absence of Nanog, but remains necessary to counteract the Nanog-induced Gata6 Received 4 March 2014; Accepted 29 July 2014 1 Clermont Université , Université d’Auvergne, Laboratoire GReD, Clermont-Ferrand F-63000, France. 2 Inserm, UMR1103, Clermont-Ferrand F-63001, France. 3 CNRS, UMR6293, Clermont-Ferrand F-63001, France. 4 Unité de Chronobiologie thé orique, Faculté des Sciences, Université Libre de Bruxelles (ULB), Campus Plaine, CP 231, Brussels B-1050, Belgium. 5 Stellenbosch Institute for Advanced Study (STIAS), Wallenberg Research Center at Stellenbosch University, Stellenbosch 7600, South Africa. *Present address: Ecole Polytechnique Fé dé rale de Lausanne (EPFL) SV ISREC, Station 19, Lausanne CH-1015, Switzerland. ‡ These authors contributed equally to this work § Authors for correspondence (genevieve.dupont@ulb.ac.be; claire.chazaud@udamail.fr) 3637 © 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 3637-3648 doi:10.1242/dev.109678 DEVELOPMENT