Regionally specific levels and patterns of keratin 8 expression in the mouse embryo visceral endoderm emerge upon anterior-posterior axis determination.

The mechanical properties of the different germ layers of the early mammalian embryo are likely to be critical for morphogenesis. Cytoskeleton components (actin and myosin, microtubules, intermediate filaments) are major determinants of epithelial plasticity and resilience to stress. Here, we take advantage of a mouse reporter for Keratin 8 to record the pattern of the keratin intermediate filaments network in the first epithelia of the developing mouse embryo. At the blastocyst stage, Keratin 8 is strongly expressed in the trophectoderm, and undetectable in the inner cell mass and its derivatives, the epiblast and primitive endoderm. Visceral endoderm cells that differentiate from the primitive endoderm at the egg cylinder stage display apical Keratin 8 filaments. Upon migration of the Anterior Visceral Endoderm and determination of the anterior-posterior axis, Keratin 8 becomes regionally distributed, with a stronger expression in embryonic, compared to extra-embryonic, visceral endoderm. This pattern emerges concomitantly to a modification of the distribution of Filamentous (F)-actin, from a cortical ring to a dense apical shroud, in extra-embryonic visceral endoderm only. Those regional characteristics are maintained across gastrulation. Interestingly, for each stage and region of the embryo, adjacent germ layers display contrasted levels of keratin filaments, which may play a role in their adaptation to growth and morphological changes.

NANOG initiates epiblast fate through the coordination of pluripotency genes expression.

The epiblast is the source of all mammalian embryonic tissues and of pluripotent embryonic stem cells. It differentiates alongside the primitive endoderm in a "salt and pepper" pattern from inner cell mass (ICM) progenitors during the preimplantation stages through the activity of NANOG, GATA6 and the FGF pathway. When and how epiblast lineage specification is initiated is still unclear. Here, we show that the coordinated expression of pluripotency markers defines epiblast identity. Conversely, ICM progenitor cells display random cell-to-cell variability in expression of various pluripotency markers, remarkably dissimilar from the epiblast signature and independently from NANOG, GATA6 and FGF activities. Coordination of pluripotency markers expression fails in Nanog and Gata6 double KO (DKO) embryos. Collectively, our data suggest that NANOG triggers epiblast specification by ensuring the coordinated expression of pluripotency markers in a subset of cells, implying a stochastic mechanism. These features are likely conserved, as suggested by analysis of human embryos.

Integrated pseudotime analysis of human pre-implantation embryo single-cell transcriptomes reveals the dynamics of lineage specification.

Understanding lineage specification during human pre-implantation development is a gateway to improving assisted reproductive technologies and stem cell research. Here we employ pseudotime analysis of single-cell RNA sequencing (scRNA-seq) data to reconstruct early mouse and human embryo development. Using time-lapse imaging of annotated embryos, we provide an integrated, ordered, and continuous analysis of transcriptomics changes throughout human development. We reveal that human trophectoderm/inner cell mass transcriptomes diverge at the transition from the B2 to the B3 blastocyst stage, just before blastocyst expansion. We explore the dynamics of the fate markers IFI16 and GATA4 and show that they gradually become mutually exclusive upon establishment of epiblast and primitive endoderm fates, respectively. We also provide evidence that NR2F2 marks trophectoderm maturation, initiating from the polar side, and subsequently spreads to all cells after implantation. Our study pinpoints the precise timing of lineage specification events in the human embryo and identifies transcriptomics hallmarks and cell fate markers.

Ex Vivo Culture for Preimplantation Mouse Embryo to Analyze Pluripotency.

A couple of days after fertilization of a mouse oocyte by a sperm, two sequential cell differentiation events segregate pluripotent cells that can be identified by the presence of specific markers. Early mammalian embryos are relatively easy to recover as they are not yet implanted in the uterus matrix. Several decades of experimentation have enabled to find appropriate media to culture them, and therefore provide an excellent way to test different experimental setups such as the use of signaling inhibitors. We provide here a commonly used protocol to culture preimplantation embryos as well as a method to detect pluripotent cells in blastocysts.

Hair Growth Activity of Three Plants of the Polynesian Cosmetopoeia and Their Regulatory Effect on Dermal Papilla Cells.

Hair loss is becoming increasingly prevalent as dietary and living habits change. The search for natural products to limit hair loss has led to tapping into traditional cosmetic knowledge. We studied three plants of the Polynesian cosmetopoeia, Bidens pilosa, Calophyllum inophyllum and Fagraea berteroana, to determine their ability to promote hair growth. Their chemical content was characterized by liquid chromatography coupled to mass spectrometry (LC-MS). Their proliferative activity on dermal papilla cells (DPCs) was assessed via MTT assay and molecular targets were evaluated by RT-qPCR analysis of seven factors involved in the modulation of the hair cycle, CCND1, LEF1, DKK1, WNT5A PPARD, TGFΒ1, PPARD and RSPO2. Our results show that our extracts significantly increased proliferation of dermal papilla cells. Furthermore, LC-MS/MS analysis revealed a diversity of molecules, flavonoids, iridoids and organic acids, some known for hair-inducing properties. Finally, specific extracts and fractions of all three plants either upregulated CCND1, LEF1 and PPARD involved in stimulating hair follicle proliferation and/or lowered the gene expression levels of hair growth inhibiting factors, DKK1 and TGFB1. Our findings suggest that extracts from B. pilosa, C. inophyllum and F. berteroana are interesting candidates to stimulate hair growth.

Netrin-1 promotes naive pluripotency through Neo1 and Unc5b co-regulation of Wnt and MAPK signalling.

In mouse embryonic stem cells (mESCs), chemical blockade of Gsk3α/ß and Mek1/2 (2i) instructs a self-renewing ground state whose endogenous inducers are unknown. Here we show that the axon guidance cue Netrin-1 promotes naive pluripotency by triggering profound signalling, transcriptomic and epigenetic changes in mESCs. Furthermore, we demonstrate that Netrin-1 can substitute for blockade of Gsk3α/ß and Mek1/2 to sustain self-renewal of mESCs in combination with leukaemia inhibitory factor and regulates the formation of the mouse pluripotent blastocyst. Mechanistically, we reveal how Netrin-1 and the balance of its receptors Neo1 and Unc5B co-regulate Wnt and MAPK pathways in both mouse and human ESCs. Netrin-1 induces Fak kinase to inactivate Gsk3α/ß and stabilize ß-catenin while increasing the phosphatase activity of a Ppp2r2c-containing Pp2a complex to reduce Erk1/2 activity. Collectively, this work identifies Netrin-1 as a regulator of pluripotency and reveals that it mediates different effects in mESCs depending on its receptor dosage, opening perspectives for balancing self-renewal and lineage commitment.

Regulation of the ERK signalling pathway in the developing mouse blastocyst.

Activation of the ERK signalling pathway is essential for the differentiation of the inner cell mass (ICM) during mouse preimplantation development. We show here that ERK phosphorylation occurs in ICM precursor cells, in differentiated primitive endoderm (PrE) cells as well as in the mature, formative state epiblast (Epi). We further show that DUSP4 and ETV5, factors often involved in negative-feedback loops of the FGF pathway, are differently regulated. Whereas DUSP4 presence clearly depends on ERK phosphorylation in PrE cells, ETV5 localises mainly to Epi cells. Unexpectedly, ETV5 accumulation does not depend on direct activation by ERK but requires NANOG activity. Indeed ETV5, like Fgf4 expression, is not present in Nanog mutant embryos. Our results lead us to propose that in pluripotent early Epi cells, NANOG induces the expression of both Fgf4 and Etv5 to enable the differentiation of neighbouring cells into the PrE while protecting the Epi identity from autocrine signalling.

Computational models for the dynamics of early mouse embryogenesis.

Early embryonic development, from the zygote to the blastocyst, is a paradigm of a dynamic, self-organised process. It involves gene expression, mechanical interactions between cells, cell division and inter- and intracellular signalling. Imaging and transcriptomic data have significantly improved our understanding of early embryogenesis in mammals. However, they also reveal a great level of complexity. How the genetic, mechanical, and regulatory processes interact to ensure reproducible development is thus much investigated by computational modelling, which allows a dissection of the mechanisms controlling cell fate decisions. In this review, we discuss the main types of modelling approaches that have been used to investigate the dynamics of preimplantation mammalian development. We also discuss the insights provided by modelling into our understanding of the specification processes leading to the three types of cells in the embryo 4.5 days after fertilization: the trophectoderm, the epiblast and the primitive endoderm.

Primitive Endoderm Differentiation: From Specification to Epithelialization.

At the time of implantation, the mouse blastocyst has developed three cell lineages: the epiblast (Epi), the primitive endoderm (PrE), and the trophectoderm (TE). The PrE and TE are extraembryonic tissues but their interactions with the Epi are critical to sustain embryonic growth, as well as to pattern the embryo. We review here the cellular and molecular events that lead to the production of PrE and Epi lineages and discuss the different hypotheses that are proposed for the induction of these cell types. In the second part, we report the current knowledge about the epithelialization of the PrE.

A multiscale model of early cell lineage specification including cell division.

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.

Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/ß-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo.

Lineage specification in the mouse preimplantation embryo.

During mouse preimplantation embryo development, totipotent blastomeres generate the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm. In recent years, studies have shown that this process appears to be regulated by differences in cell-cell interactions, gene expression and the microenvironment of individual cells, rather than the active partitioning of maternal determinants. Precisely how these differences first emerge and how they dictate subsequent molecular and cellular behaviours are key questions in the field. As we review here, recent advances in live imaging, computational modelling and single-cell transcriptome analyses are providing new insights into these questions.

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

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.

Assessment of cell lineages and cell death in blastocysts by immunostaining.

During the last decade it has been shown that most mammalian blastocysts consisted of three cell lineages. Immunofluorescence with multiple antibodies enables to identify each cell type allowing an easy detection of eventual defects. It is complementary to RT-PCR experiments as this technique allows to look at cell position and to analyze and count the proportions between the different cell types. Thus after any kind of embryo manipulation such as nuclear transfer (NT), the analysis of the three cell lineages by immunofluorescence will provide criteria for good or poor development.

Primitive endoderm differentiation: from specification to epithelium formation.

In amniotes, primitive endoderm (PrE) plays important roles not only for nutrient support but also as an inductive tissue required for embryo patterning. PrE is an epithelial monolayer that is visible shortly before embryo implantation and is one of the first three cell lineages produced by the embryo. We review here the molecular mechanisms that have been uncovered during the past 10 years on PrE and epiblast cell lineage specification within the inner cell mass of the blastocyst and on their subsequent steps of differentiation.

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

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.

A close look at the mammalian blastocyst: epiblast and primitive endoderm formation.

During early development, the mammalian embryo undergoes a series of profound changes that lead to the formation of two extraembryonic tissues--the trophectoderm and the primitive endoderm. These tissues encapsulate the pluripotent epiblast at the time of implantation. The current model proposes that the formation of these lineages results from two consecutive binary cell fate decisions. The first controls the formation of the trophectoderm and the inner cell mass, and the second controls the formation of the primitive endoderm and the epiblast within the inner cell mass. While early mammalian embryos develop with extensive plasticity, the embryonic pattern prior to implantation is remarkably reproducible. Here, we review the molecular mechanisms driving the cell fate decision between primitive endoderm and epiblast in the mouse embryo and integrate data from recent studies into the current model of the molecular network regulating the segregation between these lineages and their subsequent differentiation.

Fluorescent mRNA labeling through cytoplasmic FISH.

RNA in situ hybridization (ISH) has been widely used in cell and developmental biology research to study gene expression. Classical ISH protocols use colorimetric staining approaches, such as the assay with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP), which do not allow the implementation of multiple probe analyses and do not enable investigators to achieve cellular resolution. Here we describe a protocol to determine the presence of target cytoplasmic RNA via cytoplasmic fluorescence ISH (cFISH), an approach that renders possible the visualization of specific RNA strands from the whole tissue down to the cell. This fluorescence technique, adapted here for use in mouse embryos, enables researchers to implement multiple labeling by combining several RNA probes and/or antibodies in immuno-cFISH. Depending on the options chosen, the protocol can be completed within 2 or 3 d.

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

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.

Primitive endoderm differentiates via a three-step mechanism involving Nanog and RTK signaling.

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.