Dynamic expression of Lrp2 pathway members reveals progressive epithelial differentiation of primitive endoderm in mouse blastocyst.

International audienceMesenchyme to epithelium transitions are crucial to embryonic development. The early mouse embryo offers an excellent model to study epithelium formation as during the first three days of development two epithelia are formed, the trophectoderm (TE) and the primitive endoderm (PrE). We have previously shown that PrE cells are determined within the blastocyst ICM long before epithelium formation. In this work, we isolated Lrp2 as a novel PrE precursor (pre-PrE) marker by using a microarray strategy that combines a transcriptome analysis of three stem cell lines and early embryos. A detailed expression analysis shows that Lrp2 expression is induced in late E3.5 embryos indicating that pre-PrE cells are progressively maturing prior to polarization into an epithelium. Furthermore, the subcellular location of Lrp2, Disabled-2 (Dab2) and Collagen-IV shows that the epithelial structure is acquired in individual cells through successive steps

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

LIF-Dependent Signaling: New Pieces in the Lego

LIF, a member of the IL6 family of cytokine, displays pleiotropic effects on various cell types and organs. Its critical role in stem cell models (e.g.: murine ES, human mesenchymal cells) and its essential non redundant function during the implantation process of embryos, in eutherian mammals, put this cytokine at the core of many studies aiming to understand its mechanisms of action, which could benefit to medical applications. In addition, its conservation upon evolution raised the challenging question concerning the function of LIF in species in which there is no implantation. We present the recent knowledge about the established and potential functions of LIF in different stem cell models, (embryonic, hematopoietic, mesenchymal, muscle, neural stem cells and iPSC). We will also discuss EVO-DEVO aspects of this multifaceted cytokine

L’embryogenèse précoce des mammifères

Chez les mammifères, les lignages cellulaires embryonnaires et extraembryonnaires qui forment respectivement tout l’embryon et les annexes comme le placenta se séparent dès les premières différenciations cellulaires. En effet, l’embryon de mammifère, n’ayant pas beaucoup de réserves énergétiques, se prépare très tôt à l’implantation pour absorber les nutriments maternels, d’abord par l’intermédiaire du sac vitellin puis du placenta. Ces premières étapes impliquent les processus génétiques et morphogénétiques classiques de la différenciation cellulaire ainsi que des mécanismes propres au début du développement embryonnaire, comme certaines reprogrammations épigénétiques et le maintien de la pluripotence cellulaire. L’analyse des embryons a conduit à l’isolement des cellules souches embryonnaires, découverte couronnée en 2007 par le prix Nobel de médecine (décerné à M. Evans, M. Capecchi et O. Smithies) et qui offre de grands espoirs pour la thérapie cellulaire

[Early embryogenesis in mammals: stem cells and first commitments steps.]

International audienceIn mammals, embryonic and extraembryonic cell lineages segregate during the first steps of cell differentiation in the preimplantation embryo. Indeed, mammal embryos contain very low energy stocks and thus get ready for implantation very early to be able to absorbe nutrients from the mother, first through the yolk sac and then through the placenta. These first steps involve classical genetic and morphogenetic processes as well as specific mechanisms of early embryo development such as epigenetic reprogramming and maintenance of pluripotent cells. Embryo analysis led to the isolation of embryonic stem (ES) cells, granted by the 2007 Nobel prize of Medicine (to M. Evans, M. Capecchi and O. Smithies) and which offer strong hopes for cell therapy. double dagger

Lineage specification in the mouse preimplantation embryo

International audienceDuring 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

Caractérisation de gènes impliqués dans la formation de l'endoderme primitif lors de l'embryogenèse précoce de la souris

De nombreux travaux menés sur l'embryon de souris ont permis de caractériser morphologiquement les 1ères étapes de développement des mammifères. En revanche, les mécanismes moléculaires régissant le déroulement de ces étapes sont encore peu connus à ce jour. Dans l'équipe, notre but est de comprendre ces mécanismes en se focalisant sur la formation de l'endoderme primitif (EPr), le 2ème type cellulaire à se différencier au cours de l'embryogenèse. L'EPr est un tissu extra-embryonnaire qui se forme avant l'implantation dans l'utérus maternel. Bien que ne contribuant pas à la formation de l'embryon lui-même, ce tissu reste néanmoins nécessaire à sa survie et à son développement correct. Afin d'identifier de nouveaux gènes impliqués dans la formation de l'EPr, j'ai analysé un microarray réalisé sur les 1ers stades de l'embryogenèse. Cette analyse a permis de définir de nouveaux gènes précoces, potentiellement spécifiques de l'EPR. En utilisant les techniques de RT-PCR et de localisation in situ, j'ai analysé 2 des gènes sélectionnés : Lrp2 et Dkkl. L'étude approfondie du profil d'expression du récepteur LRP2 entre E2.5 et E4.5 a permis l'élaboration d'un modèle de différenciation épithéliale. Parallèlement à ces travaux, j'ai développé une nouvelle technique pour réaliser des analyses fonctionnelles in vivo. Cette méthode d'électroporation in vivo combine les techniques d'électroporation, d'interférence ARN et de culture d'embryons et ouvre de nombreuses perspectives pour les travaux sur l'embryogenèse précoce.Numerous studies in the mouse embryo have characterised morphological events during preimplantation development. However, molecular mechanisms driving cell fate ad differentiation are not yet well understood. The objective of our group is to analyse the mechanisms of cell differentiation by focusing on primitive endoderm (PrE) formation, the second cell type to differentiate in the embryo. PrE is an extraembryonnic tisssue that appears before implantation. Although PrE does not contribute to the embryo proper, it is necessary for both survival and correct development of the embryo. In order to identify new genes implicated in PrE formation, I analysed microarrays performed on early embryonic stages. This resulted in the identification of new early genes that are potentially specific to PrE. Using RT-PCR and in situ localisation, I analysed two of the selected genes : Lrp2 and Dkkl. A detailed study of LRP2 expression between E2.5 and E4.5 gave rise to a model of epithelial differentiation. In parallel with this work, I developed a new method for in vivo functional analysis. It combines electroporation, RNA interference and embryo culture and open perspectives new for studing early embryogenesis.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF

Primitive endoderm differentiation: from specification to epithelium formation

International audienceIn 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

Facteurs génétiques impliqués dans la différenciation de l'endoderme primitif au cours du développement préimplantatoire chez la souris

Les études menées depuis de nombreuses années sur l'embryogenèse de la souris ont permis sa caractérisation morphologique. Le but de notre équipe est de participer à l'élucidation des facteurs jouant un rôle important dans le développement préimplantatoire murin. Pour cela, nous nous sommes focalisés sur un des 1ers types cellulaires déterminé à ces stades : l'endoderme primitif (EPr). L'EPr est un tissu extra-embryonnaire formant un épithélium indispensable au bon déroulement du développement et à la survie de l'embryon. Afin de mieux caractériser les facteurs ge nétiques et voies de signalisations impliquées dans la différenciation de l'EPr chez la souris, je me suis intéressée à la signalisation rétinoïque ainsi qu'à la voie Wnt canonique. J'ai tout d'abord analysé l'expression des marqueurs de l'EPr et de l'épiblaste (le tissu embryonnaire) par RT-PCR quantitative au cours de la différenciation de cellules F9 en cellules de l'EPr par l'acide rétinoïque. Ainsi, j'ai montré que les marqueurs endodermiques sont induits séquentiellement et dans le même ordre que in vivo. J'ai également adapté ce protocole aux embryons préimplantatoires. Grâce à l'hybridation in situ et l'immunohistochimie j'ai caractérisé les profils d'expression de différents acteurs de la voie des rétinoïdes ainsi que de Dkk1 un inhibiteur spécifique de la voie Wnt canonique.Numerous studies on the mouse embryo have allowed to caracterize the morphological events occuring at early development. Our groups aims to clarify the mechanisms of preimplantation embryogenesis in the mouse, focusing on the differentiation of the primitive endoderm (PrE). The PrE is the second extraembryonic tissue formed before implantation and required for both survival and correct development of the embryo. In order to get a better understanding of how PrE fate is established and the epitelium formed, I focused my study on two signaling pathways : retinoic acid signaling and the Wnt/b-catenin pathway. I analysed the expression of PrE and epiblast markers on a RA-driven F9 cell differentiation by quantitative RT-PCR. I show that PrE-genes are sequencially induced and in the same order as observed in vivo by immunostaining. I also adapted this protocol for preimplantation embryos. By in situ hybridations and immunostainings I characterized the expression profils of different members of the two signaling pathways.CLERMONT FD-BCIU-Santé (631132104) / SudocSudocFranceF