Primitive Endoderm Differentiates via a Three-Step Mechanism Involving Nanog and RTK Signaling

SummaryDuring 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

Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development

International audienceThe 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

Régulations génétiques contrôlant l'engagement cellulaire au cours du développement murin : différenciation de l'épiblaste versus l'endoderme primitif

A 3.5 jours de développement (J3.5), l'embryon de souris est constitué d'un épithélium externe, le trophectoderme, et d'une masse cellulaire interne (MCI). La MCI est hétérogène, constituée des précurseurs de l'épiblaste (Epi) et de l'endoderme primitif (EPr), représentée par l'expression exclusive de Nanog et de Gata6 respectivement. Lors de l'implantation à E4.5, l'EPr forme un épithélium à la surface de la MCI, en regard de la cavité blastocoelique. L'Epi donnera tous les tissus du nouveau-né. L'EPr permet les premiers échanges nutritionnels entre l'embryon et la mère. Je m'intéresse au rôle de Nanog et de Gata6 dans la détermination et la différenciation de l'Epi et de l'EPr. De plus, je m'intéresse à l'implication de la signalisation RTK dans l'expression de ces deux gènes. Enfin, je cherche à comprendre les interrelations entre Gata6 et Nanog. A l'aide des modèles de souris KO, des modèles in vitro ainsi que des techniques innovantes développées au sein du laboratoire, nous avons mis en évidence que la modulation de l'expression de Nanog, Gata6, Fgf4 et Fgfr2 semble suffisante pour l'engagement des cellules vers un devenir Epi ou EPr. De plus, ces résultats permettent de proposer un nouveau modèle expliquant le rôle de Gata6 et de Nanog dans la spécification des cellules Epi et EPr.At 3.5 days of development (E3.5), the mouse embryo consists of an outer epithelium, the trophectoderm, and an inner cell mass (ICM). The ICM is heterogeneous, composed of the precursors of the epiblast (Epi) and the primitive endoderm (PrE), expressing either Nanog or Gata6 respectively. Upon implantation at E4.5 the EPr forms an epithelium on the surface of the ICM, facing the blastocoelic cavity. The Epi give rise all tissues of the newborn. The PrE allows the first nutritional exchanges between the embryo and the mother. I focus on the role of Nanog and Gata6 in the determination and differentiation of Epi and PrE. In addition I am interested in the involvement of RTK signaling in the expression of both genes. Finally, I seek to understand the relationships between Gata6 and Nanog. Using the transgenic mouse models, in vitro models as well as innovative techniques developed in the laboratory, we have demonstrated that modulating the expression of Nanog, Gata6, FGF4 and FGFR2 seems sufficient for commitment of cells to become an Epi or EPr. Furthermore, these results allow proposing a new model explaining the role of Gata6 and Nanog in the determination and differentiation of Epi and PrE cells

Spécification de l'épiblaste et de l'endoderme primitif lors du développement embryonnaire préimplantatoire chez la souris: Une combinaison entre biologie et modélisation mathématique

Upon its implantation in the uterus of the mother in mammals, the embryo is composed by three morphologically distinct tissues: the Epiblast (Epi), the Trophectoderm (TE) and the Primitive Endoderm (PrE). Both Epi and PrE are formed from the same cell homogeneous population called the Inner Cell Mass (ICM). Based on our studies, we discuss in this review what molecular interactions are necessary for the specification of these two lineages. For this, we have combined a biological approach with mathematical modeling. We have shown the central role of the gene regulation group composed by NANOG, FGF4, GATA6 and FGFR2 for Epi/PrE cell specification.SCOPUS: re.jinfo:eu-repo/semantics/publishe

PC7 and the related proteases Furin and Pace4 regulate E-cadherin function during blastocyst formation

The first cell differentiation in mammalian embryos segregates polarized trophectoderm cells from an apolar inner cell mass (ICM). This lineage decision is specified in compacted morulae by cell polarization and adhesion acting on the Yes-associated protein in the Hippo signaling pathway, but the regulatory mechanisms are unclear. We show that morula compaction and ICM formation depend on PC7 and the related proprotein convertases (PCs) Furin and Pace4 and that these proteases jointly regulate cell-cell adhesion mediated by E-cadherin processing. We also mapped the spatiotemporal activity profiles of these proteases by live imaging of a transgenic reporter substrate in wild-type and PC mutant embryos. Differential inhibition by a common inhibitor revealed that all three PCs are active in inner and outer cells, but in partially nonoverlapping compartments. E-cadherin processing by multiple PCs emerges as a novel mechanism to modulate cell-cell adhesion and fate allocation

PDGF signaling in primitive endoderm cell survival is mediated by PI3K-mTOR through p53-independent mechanism

International audienceReceptor tyrosine kinase signaling (RTK) are key regulators of the formation of the primitive endoderm (PrE) and the epiblast (Epi) from the inner cell mass of the mouse preimplantation embryo. Among them, FGF signaling is critical for PrE cell specification while PDGF signaling is critical for the survival of committed PrE cells. Here, we investigated possible functional redundancies between FGF, PDGF and KIT signaling and showed that only PDGF signaling is involved in PrE cell survival. In addition, we analyzed the effectors downstream of PDGFRα. Our results suggest that the role of PDGF signaling in PrE cell survival is mediated through PI3K-mTOR and independently from p53. Lastly, we uncovered a role for PI3K-mTOR signaling in the survival of Epi cells. Taken together, we propose that survival of ICM cell lineages relies on the regulation of PI3K-mTOR signaling through the regulation of multiple signaling pathways. SIGNIFICANCE STATEMENT: Segregation between embryonic and extra-embryonic lineages occurs at the beginning of mammalian development and requires multiple processes participating in the acquisition and the maintenance of cell identities. In this study, we have characterized the intracellular factors involved in the survival of the epiblast, that will form the embryo proper, and the primitive endoderm, an extraembryonic lineage participating to the formation of the yolk sac, just after their formation. Such better understanding of early embryonic development could benefit to the fields of stem cell biology and assisted reproduction technologies. © AlphaMed Press 2019

A multiscale model of early cell lineage specification including cell division

International audienceEmbryonic 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

Cell Fate Specification Based on Tristability in the Inner Cell Mass of Mouse Blastocysts

International audienceDuring 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

ICM conversion to epiblast by FGF/ERK inhibition is limited in time and requires transcription and protein degradation

Abstract Inner cell Mass (ICM) specification into epiblast (Epi) and primitive endoderm (PrE) is an asynchronous and progressive process taking place between E3.0 to E3.75 under the control of the Fibroblast Growth Factor (FGF)/Extracellular signal-Regulated Kinase (ERK) signaling pathway. Here, we have analyzed in details the kinetics of specification and found that ICM cell responsiveness to the up and down regulation of FGF signaling activity are temporally distinct. We also showed that PrE progenitors are generated later than Epi progenitors. We further demonstrated that, during this late phase of specification, a 4 hours period of FGF/ERK inhibition prior E3.75 is sufficient to convert ICM cells into Epi. Finally, we showed that ICM conversion into Epi in response to inhibition during this short time window requires both transcription and proteasome degradation. Collectively, our data give new insights into the timing and mechanisms involved in the process of ICM specification

Gata6, Nanog and Erk signaling control cell fate in the inner cell mass through a tristable regulatory network

International audienceDuring 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