During preimplantation mouse development, the inner cell mass (ICM) differentiates into two cell lineages--the epiblast and the primitive endoderm (PrE)--whose precursors are identifiable by reciprocal expression of Nanog and Gata6, respectively. PrE formation depends on Nanog by a non-cell-autonomous mechanism. To decipher early cell- and non-cell-autonomous effects, we performed a mosaic knockdown of Nanog and found that this is sufficient to induce a PrE fate cell autonomously. Strikingly, in Nanog null embryos, Gata6 expression is maintained, showing that initiation of the PrE program is Nanog independent. Treatment of Nanog null embryos with pharmacological inhibitors revealed that RTK dependency of Gata6 expression is initially direct but later indirect via Nanog repression. Moreover, we found that subsequent expression of Sox17 and Gata4--later markers of the PrE--depends on the presence of Fgf4 produced by Nanog-expressing cells. Thus, our results reveal three distinct phases in the PrE differentiation program.

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.

Pluripotent mouse embryonic stem cells maintain their identity throughout virtually infinite cell divisions. This phenomenon, referred to as self-renewal, depends on a network of sequence-specific transcription factors (TFs) and requires daughter cells to accurately reproduce the gene expression pattern of the mother. However, dramatic chromosomal changes take place in mitosis, generally leading to the eviction of TFs from chromatin. Here, we report that Esrrb, a major pluripotency TF, remains bound to key regulatory regions during mitosis. We show that mitotic Esrrb binding is highly dynamic, driven by specific recognition of its DNA-binding motif and is associated with early transcriptional activation of target genes after completion of mitosis. These results indicate that Esrrb may act as a mitotic bookmarking factor, opening another perspective to molecularly understand the role of sequence-specific TFs in the epigenetic control of self-renewal, pluripotency and genome reprogramming.

Embryonic development is a self-organised process during which cells divide, interact, change fate according to a complex gene regulatory network and organise themselves in a three-dimensional space. Here, we model this complex dynamic phenomenon in the context of the acquisition of epiblast and primitive endoderm identities within the inner cell mass of the preimplantation embryo in the mouse. The multiscale model describes cell division and interactions between cells, as well as biochemical reactions inside each individual cell and in the extracellular matrix. The computational results first confirm that the previously proposed mechanism by which extra-cellular signalling allows cells to select the appropriate fate in a tristable regulatory network is robust when considering a realistic framework involving cell division and three-dimensional interactions. The simulations recapitulate a variety of in vivo observations on wild-type and mutant embryos and suggest that the gene regulatory network confers differential plasticity to the different cell fates. A detailed analysis of the specification process emphasizes that developmental transitions and the salt-and-pepper patterning of epiblast and primitive endoderm cells from a homogenous population of inner cell mass cells arise from the interplay between the internal gene regulatory network and extracellular signalling by Fgf4. Importantly, noise is necessary to create some initial heterogeneity in the specification process. The simulations suggest that initial cell-to-cell differences originating from slight inhomogeneities in extracellular Fgf4 signalling, in possible combination with slightly different concentrations of the key transcription factors between daughter cells, are able to break the original symmetry and are amplified in a flexible and self-regulated manner until the blastocyst stage.

During development, interactions between transcription factors control the specification of different cell fates. The regulatory networks of genetic interactions often exhibit multiple stable steady states; such multistability provides a common dynamical basis for differentiation. During early murine embryogenesis, cells from the inner cell mass (ICM) can be specified in epiblast (Epi) or primitive endoderm (PrE). Besides the intracellular gene regulatory network, specification is also controlled by intercellular interactions involving Erk signaling through extracellular Fgf4. We previously proposed a model that describes the gene regulatory network and its interaction with Erk signaling in ICM cells. The model displays tristability in a range of Fgf4 concentrations and accounts for the self-organized specification process observed in vivo. Here, we further investigate the origin of tristability in the model and analyze in more detail the specification process by resorting to a simplified two-cell model. We also carry out simulations of a population of 25 cells under various experimental conditions to compare their outcome with that of mutant embryos or of embryos submitted to exogenous treatments that interfere with Fgf signaling. The results are analyzed by means of bifurcation diagrams. Finally, the model predicts that heterogeneities in extracellular Fgf4 concentration play a primary role in the spatial arrangement of the Epi/PrE cells in a salt-and-pepper pattern. If, instead of heterogeneities in extracellular Fgf4 concentration, internal fluctuations in the levels of expression of the transcription factors are considered as a source of randomness, simulations predict the occurrence of unrealistic switches between the Epi and the PrE cell fates, as well as the evolution of some cells toward one of these states without passing through the previous ICM state, in contrast to what is observed in vivo.

Targeted deletion of PC7 and the related proprotein convertases Furin and Pace4, combined with live imaging of their activities, unmasks their overlapping and complementary functions in morula compaction and ICM formation in mouse blastocysts and in E-cadherin precursor processing.

The transcription factors Nanog and Gata6 are critical to specify the epiblast versus primitive endoderm (PrE) lineages. However, little is known about the mechanisms that regulate the protein stability and activity of these factors in the developing embryo. Here we uncover an early developmental function for the Polycomb group member Bmi1 in supporting PrE lineage formation through Gata6 protein stabilization. We show that Bmi1 is enriched in the extraembryonic (endoderm [XEN] and trophectodermal stem [TS]) compartment and repressed by Nanog in pluripotent embryonic stem (ES) cells. In vivo, Bmi1 overlaps with the nascent Gata6 and Nanog protein from the eight-cell stage onward before it preferentially cosegregates with Gata6 in PrE progenitors. Mechanistically, we demonstrate that Bmi1 interacts with Gata6 in a Ring finger-dependent manner to confer protection against Gata6 ubiquitination and proteasomal degradation. A direct role for Bmi1 in cell fate allocation is established by loss-of-function experiments in chimeric embryoid bodies. We thus propose a novel regulatory pathway by which Bmi1 action on Gata6 stability could alter the balance between Gata6 and Nanog protein levels to introduce a bias toward a PrE identity in a cell-autonomous manner.

Furin trafficking, and that of related proprotein convertases (PCs), may regulate which substrates are accessible for endoproteolysis, but tools to directly test this hypothesis have been lacking. Here, we develop targeted biosensors that indicate Furin activity in endosomes is 10-fold less inhibited by decanoyl-RVKR-chloromethylketone and enriched >3-fold in endosomes compared to the trans-Golgi network (TGN). Endogenous PC7, which resists this inhibitor, was active in distinct vesicles. Only overexpressed PC7 activity reached the cell surface, endosomes, and the TGN. A PLC motif in the cytosolic tail of PC7 was dispensable for endosomal activity, but it was specifically required for TGN recycling and to rescue proActivin-A cleavage in Furin-depleted B16F1 melanoma cells. In sharp contrast, PC7 complemented Furin in cleaving Notch1 independently of PLC-mediated TGN access. Our study provides a proof in principle that compartment-specific biosensors can be used to gain insight into the regulation of PC trafficking and to map the tropism of PC-specific inhibitors.