Differential plasticity of epiblast and primitive endoderm precursors within the ICM of the early mouse embryo

Cell differentiation during pre-implantation mammalian development involves the formation of two extra-embryonic lineages: trophoblast and primitive endoderm (PrE). A subset of cells within the inner cell mass (ICM) of the blastocyst does not respond to differentiation signals and forms the pluripotent epiblast, which gives rise to all of the tissues in the adult body. How this group of cells is set aside remains unknown. Recent studies documented distinct sequential phases of marker expression during the segregation of epiblast and PrE within the ICM. However, the connection between marker expression and lineage commitment remains unclear. Using a fluorescent reporter for PrE, we investigated the plasticity of epiblast and PrE precursors. Our observations reveal that loss of plasticity does not coincide directly with lineage restriction of epiblast and PrE markers, but rather with exclusion of the pluripotency marker Oct4 from the PrE. We note that individual ICM cells can contribute to all three lineages of the blastocyst until peri-implantation. However, epiblast precursors exhibit less plasticity than precursors of PrE, probably owing to differences in responsiveness to extracellular signalling. We therefore propose that the early embryo environment restricts the fate choice of epiblast but not PrE precursors, thus ensuring the formation and preservation of the pluripotent foetal lineage.     

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.     

DDX21 is a p38-MAPK-sensitive nucleolar protein necessary for mouse preimplantation embryo development and cell-fate specification

Successful navigation of the mouse preimplantation stages of development, during which three distinct blastocyst lineages are derived, represents a prerequisite for continued development. We previously identified a role for p38-mitogen-activated kinases (p38-MAPK) regulating blastocyst inner cell mass (ICM) cell fate, specifically primitive endoderm (PrE) differentiation, that is intimately linked to rRNA precursor processing, polysome formation and protein translation regulation. Here, we develop this work by assaying the role of DEAD-box RNA helicase 21 (DDX21), a known regulator of rRNA processing, in the context of p38-MAPK regulation of preimplantation mouse embryo development. We show nuclear DDX21 protein is robustly expressed from the 16-cell stage, becoming exclusively nucleolar during blastocyst maturation, a localization dependent on active p38-MAPK. siRNA-mediated clonal Ddx21 knockdown within developing embryos is associated with profound cell-autonomous and non-autonomous proliferation defects and reduced blastocyst volume, by the equivalent peri-implantation blastocyst stage. Moreover, ICM residing Ddx21 knockdown clones express the EPI marker NANOG but rarely express the PrE differentiation marker GATA4. These data contribute further significance to the emerging importance of lineage-specific translation regulation, as identified for p38-MAPK, during mouse preimplantation development.     

Cell lineage determination in the mouse

During the peri-implantation development of the mouse embryo from the blastocyst through gastrulation, Pou5f1 (OCT-4) down-regulation is closely linked to the initial step of lineage allocation to extraembryonic and embryonic somatic tissues. Subsequently, differentiation of the lineage precursors is subject to inductive tissue interactions and intercellular signalling that regulate cell proliferation and the acquisition of lineage-specific morphological and molecular characteristics. A notable variation of this process of lineage specification is the persistence of Pou5f1 activity throughout the differentiation of the primordial germ cells, which may underpin their ability to produce pluripotent progeny either as stem cells (embryonic germ cells) in vitro or as gametes in vivo. Nevertheless, intercellular signalling still plays a critical role in the specification of the primordial germ cells. The findings that primordial germ cells can be induced from any epiblast cells and that they share common progenitors with other somatic cells provide compelling evidence for the absence of a pre-determined germ line in the mouse embryo.     

Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency

Embryonic stem cells (ESCs) generated from the in-vitro culture of blastocyst stage embryos are known as equivalent to blastocyst inner cell mass (ICM) in-vivo. Though several reports have shown the expression of germ cell/pre-meiotic (GC/PrM) markers in ESCs, their functional relevance for the pluripotency and germ line commitment are largely unknown. In the present study, we used mouse as a model system and systematically analyzed the RNA and protein expression of GC/PrM markers in ESCs and found them to be comparable to the expression of cultured pluripotent cells originated from the germ line. Further, siRNA knockdown experiments have demonstrated the parallel maintenance and independence of pluripotent and GC/PrM networks in ESCs. Through chromatin immunoprecipitation experiments, we observed that pluripotent cells exhibit active chromatin states at GC marker genes and a bivalent chromatin structure at PrM marker genes. Moreover, gene expression analysis during the time course of iPS cells generation revealed that the expression of GC markers precedes pluripotency markers. Collectively, through our observations we hypothesize that the chromatin state and the expression of GC/PrM markers might indicate molecular parallels between in-vivo germ cell specification and pluripotent stem cell generation.     

Staufen1 is expressed in preimplantation mouse embryos and is required for embryonic stem cell differentiation

Pluripotent mouse embryonic stem (mES) cells derived from the blastocyst of the preimplantation embryo can be induced to differentiate in vitro along different cell lineages. However the molecular and cellular factors that signal and/or determine the expression of key genes, and the localisation of the encoded proteins, during the differentiation events are poorly understood. One common mechanism by which proteins can be targeted to specific regions of the cell is through the asymmetric localisation of mRNAs and Staufen, a double-stranded RNA binding protein, is known to play a direct role in mRNA transport and localisation. The aims of the present study were to describe the expression of Staufen in preimplantation embryos and mES cells and to use RNA interference (RNAi) to investigate the roles of Staufen1 in mES cell lineage differentiation. Western blotting and immunocytochemistry demonstrated that Staufen is present in the preimplantation mouse embryo, pluripotent mES cells and mES cells stimulated to differentiate into embryoid bodies, but the Staufen staining patterns did not support asymmetric distribution of the protein. Knockdown of Staufen1 gene expression in differentiating mES cells reduced the synthesis of lineage-specific markers including Brachyury, alpha-fetoprotein (AFP), PAX-6, and Vasa. There was however no significant change in either the gene expression of Nanog and Oct4, or in the synthesis of SSEA-1, all of which are key markers of pluripotency. These data indicate that inhibition of Staufen1 gene expression by RNAi affects an early step in mES cell differentiation and suggest a key role for Staufen in the cell lineage differentiation of mES cells.     

Enhancing myogenic differentiation of pluripotent stem cells with small molecule inducers

Pluripotent stem cells are able to differentiate into many types of cell lineages in response to differentiation cues. However, a pure population of lineage-specific cells is desirable for any potential clinical application. Therefore, induction of the pluripotent stem cells with lineage-specific regulatory signals, or small molecule inducers, is a prerequisite for effectively directing lineage specification for cell-based therapeutics. In this article, we provide in-depth analysis of recent research findings on small molecule inducers of the skeletal muscle lineage. We also provide perspectives on how different signaling pathways and chromatin dynamics converge to direct the differentiation of skeletal myocytes.     

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity

The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.