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1.  Noggin Producing, MyoD-Positive Cells are Crucial for Eye Development 
Developmental biology  2009;336(1):30-41.
A subpopulation of cells expresses MyoD mRNA and the cell surface G8 antigen in the epiblast prior to the onset of gastrulation. When an antibody to the G8 antigen was applied to epiblast, labeled cells were later found in the ocular primordial and muscle and non-muscle forming tissues of the eyes. In the lens, retina and periocular mesenchyme, G8-positive cells synthesized MyoD mRNA and the bone morphogenetic protein inhibitor Noggin. MyoD expressing cells were ablated in the epiblast by labeling them with the G8 MAb and lysing them with complement. Their ablation in the epiblast resulted in eye defects, including anopthalmia, micropthalmia, altered pigmentation and malformations of the lens and/or retina. The right eye was more severely affected than the left eye. The asymmetry of the eye defects in ablated embryos correlated with differences in the number of residual Noggin producing, MyoD-positive cells in ocular tissues. Exogenously supplied Noggin compensated for the ablated epiblast cells. This study demonstrates that MyoD expressing cells serve as a Noggin delivery system to regulate the morphogenesis of the lens and optic cup.
PMCID: PMC2783511  PMID: 19778533
epiblast; MyoD; Noggin; eye development
2.  Cells that express MyoD mRNA in the epiblast are stably committed to the skeletal muscle lineage 
The Journal of Cell Biology  2007;178(4):649-660.
The epiblast of the chick embryo contains cells that express MyoD mRNA but not MyoD protein. We investigated whether MyoD-positive (MyoDpos) epiblast cells are stably committed to the skeletal muscle lineage or whether their fate can be altered in different environments. A small number of MyoDpos epiblast cells were tracked into the heart and nervous system. In these locations, they expressed MyoD mRNA and some synthesized MyoD protein. No MyoDpos epiblast cells differentiated into cardiac muscle or neurons. Similar results were obtained when MyoDpos cells were isolated from the epiblast and microinjected into the precardiac mesoderm or neural plate. In contrast, epiblast cells lacking MyoD differentiated according to their environment. These results demonstrate that the epiblast contains both multipotent cells and a subpopulation of cells that are stably committed to the skeletal muscle lineage before the onset of gastrulation. Stable programming in the epiblast may ensure that MyoDpos cells express similar signaling molecules in a variety of environments.
PMCID: PMC2064471  PMID: 17698608
3.  Pluripotency in the Embryo and in Culture 
Specific cells within the early mammalian embryo have the capacity to generate all somatic lineages plus the germline. This property of pluripotency is confined to the epiblast, a transient tissue that persists for only a few days. In vitro, however, pluripotency can be maintained indefinitely through derivation of stem cell lines. Pluripotent stem cells established from the newly formed epiblast are known as embryonic stem cells (ESCs), whereas those generated from later stages are called postimplantation epiblast stem cells (EpiSCs). These different classes of pluripotent stem cell have distinct culture requirements and gene expression programs, likely reflecting the dynamic development of the epiblast in the embryo. In this chapter we review current understanding of how the epiblast forms and relate this to the properties of derivative stem cells. We discuss whether ESCs and EpiSCs are true counterparts of different phases of epiblast development or are culture-generated phenomena. We also consider the proposition that early epiblast cells and ESCs may represent a naïve ground state without any prespecification of lineage choice, whereas later epiblasts and EpiSCs may be primed in favor of particular fates.
Epiblast cells of a blastocyst are pluripotent. Embryonic stem (ES) cells, established from the epiblast, and postimplantation epiblast stem cells (EpiSCs) have distinct properties, culture requirements, and gene expression profiles.
PMCID: PMC3405859  PMID: 22855723
4.  The endogenous retrovirus ENS-1 provides active binding sites for transcription factors in embryonic stem cells that specify extra embryonic tissue 
Retrovirology  2012;9:21.
Long terminal repeats (LTR) from endogenous retroviruses (ERV) are source of binding sites for transcription factors which affect the host regulatory networks in different cell types, including pluripotent cells. The embryonic epiblast is made of pluripotent cells that are subjected to opposite transcriptional regulatory networks to give rise to distinct embryonic and extraembryonic lineages. To assess the transcriptional contribution of ERV to early developmental processes, we have characterized in vitro and in vivo the regulation of ENS-1, a host adopted and developmentally regulated ERV that is expressed in chick embryonic stem cells.
We show that Ens-1 LTR activity is controlled by two transcriptional pathways that drive pluripotent cells to alternative developmental fates. Indeed, both Nanog that maintains pluripotency and Gata4 that induces differentiation toward extraembryonic endoderm independently activate the LTR. Ets coactivators are required to support Gata factors' activity thus preventing inappropriate activation before epigenetic silencing occurs during differentiation. Consistent with their expression patterns during chick embryonic development, Gata4, Nanog and Ets1 are recruited on the LTR in embryonic stem cells; in the epiblast the complementary expression of Nanog and Gata/Ets correlates with the Ens-1 gene expression pattern; and Ens-1 transcripts are also detected in the hypoblast, an extraembryonic tissue expressing Gata4 and Ets2, but not Nanog. Accordingly, over expression of Gata4 in embryos induces an ectopic expression of Ens-1.
Our results show that Ens-1 LTR have co-opted conditions required for the emergence of extraembryonic tissues from pluripotent epiblasts cells. By providing pluripotent cells with intact binding sites for Gata, Nanog, or both, Ens-1 LTR may promote distinct transcriptional networks in embryonic stem cells subpopulations and prime the separation between embryonic and extraembryonic fates.
PMCID: PMC3362752  PMID: 22420414
5.  Human hypoblast formation is not dependent on FGF signalling 
Developmental Biology  2012;361(2-6):358-363.
Mouse embryos segregate three different lineages during preimplantation development: trophoblast, epiblast and hypoblast. These differentiation processes are associated with restricted expression of key transcription factors (Cdx2, Oct4, Nanog and Gata6). The mechanisms of segregation have been extensively studied in the mouse, but are not as well characterised in other species. In the human embryo, hypoblast differentiation has not previously been characterised. Here we demonstrate co-exclusive immunolocalisation of Nanog and Gata4 in human blastocysts, implying segregation of epiblast and hypoblast, as in rodent embryos. However, the formation of hypoblast in the human is apparently not dependent upon FGF signalling, in contrast to rodent embryos. Nonetheless, the persistence of Nanog-positive cells in embryos following treatment with FGF inhibitors is suggestive of a transient naïve pluripotent population in the human blastocyst, which may be similar to rodent epiblast and ES cells but is not sustained during conventional human ES cell derivation protocols.
► Segregation of epiblast and Gata4-positive hypoblast in human blastocysts ► Insensitivity of human hypoblast formation to FGF/Erk inhibition ► Persistence of Nanog-positive cells in human epiblast after FGF/Erk inhibition
PMCID: PMC3368271  PMID: 22079695
Pluripotency; Epiblast; Hypoblast; Fibroblast growth factor; Human ES cell derivation
6.  Myo/Nog Cell Regulation of Bone Morphogenetic Protein Signaling in the Blastocyst is Essential for Normal Morphogenesis and Striated Muscle Lineage Specification 
Developmental biology  2011;359(1):12-25.
Cells that express MyoD mRNA, the G8 antigen and the bone morphogenetic protein (BMP) inhibitor noggin (Nog) are present in the epiblast before gastrulation. Ablation of “Myo/Nog” cells in the blastocyst results in an expansion of canonical BMP signaling and prevents the expression of noggin and follistatin before and after the onset of gastrulation. Once eliminated in the epiblast, they are neither replaced nor compensated for as development progresses. Older embryos lacking Myo/Nog cells exhibit severe axial malformations. Although Wnts and Sonic hedgehog are expressed in ablated embryos, skeletal muscle progenitors expressing Pax3 are missing in the somites. Pax3+ cells do emerge adjacent to Wnt3a+ cells in vitro; however, few undergo skeletal myogenesis. Ablation of Myo/Nog cells also results in ectopically placed cardiac progenitors and cardiomyocytes in the somites. Reintroduction of Myo/Nog cells into the epiblast of ablated embryos restores normal patterns of BMP signaling, morphogenesis and skeletal myogenesis, and inhibits the expression of cardiac markers in the somites. This study demonstrates that Myo/Nog cells are essential regulators of BMP signaling in the early epiblast and are indispensable for normal morphogenesis and striated muscle lineage specification.
PMCID: PMC3192235  PMID: 21884693
MyoD; Noggin; BMP; Blastocyst; Myogenesis
7.  Local cell interactions and self-amplifying individual cell ingression drive amniote gastrulation 
eLife  2014;3:e01817.
Gastrulation generates three layers of cells (ectoderm, mesoderm, endoderm) from a single sheet, while large scale cell movements occur across the entire embryo. In amniote (reptiles, birds, mammals) embryos, the deep layers arise by epithelial-to-mesenchymal transition (EMT) at a morphologically stable midline structure, the primitive streak (PS). We know very little about how these events are controlled or how the PS is maintained despite its continuously changing cellular composition. Using the chick, we show that isolated EMT events and ingression of individual cells start well before gastrulation. A Nodal-dependent ‘community effect’ then concentrates and amplifies EMT by positive feedback to form the PS as a zone of massive cell ingression. Computer simulations show that a combination of local cell interactions (EMT and cell intercalation) is sufficient to explain PS formation and the associated complex movements globally across a large epithelial sheet, without the need to invoke long-range signalling.
eLife digest
A key process during the development of an embryo involves a single layer of cells reorganizing into three ‘germ layers’: the ectoderm, which becomes the skin and nervous system; the mesoderm, which gives rise to the skeleton, muscles and the circulatory and urinogenital systems, and the endoderm, which gives rise to the lining of the gut and associated organs. The process of forming these three layers is known as gastrulation.
To date most experiments on gastrulation in vertebrates have been performed on frog embryos. However, the embryos of amniotes, the group of ‘higher’ vertebrates that comprises reptiles, birds and mammals, differ from those of frogs in a number of ways. Now Voiculescu et al. have used a combination of experimental and computational techniques to shed new light on gastrulation in chick embryos.
Just prior to gastrulation, the cells of the amniote embryo are arranged in a flat disk, one cell thick, called the epiblast. The cells of the epiblast then move to form the mesoderm and endoderm (in a process called epithelial-to-mesenchymal transition). These cell movements also lead to the formation of a structure called the primitive streak that establishes the left-right symmetry of the organism, and also defines the midline of the body.
Now Voiculescu et al. have shown that the epithelial-to-mesenchymal transition starts before the primitive streak appears, and that two main processes drive gastrulation. One involves cells inserting themselves between other cells at the midline of the epiblast, which causes a double whorl-like movement within the plane of the epiblast. At the same time small numbers of cells leave the epiblast, and as these cells accumulate under the epiblast, they initiate a positive feedback effect by which they encourage more cells to leave the epiblast. Voiculescu et al. found that this ‘community effect’ involves signalling by a protein called Nodal. This protein effectively amplifies the epithelial-to-mesenchymal transition and leads to the appearance of the primitive streak at the midline.
Using computational modelling, Voiculescu et al. argue that the movements of gastrulation can be explained entirely based on local interactions between cells, without the need for cells to send signals over long distances to guide cell movements, as had been generally believed.
PMCID: PMC4029171  PMID: 24850665
epithelial–mesenchymal interactions; EMT; cell movements; primitive streak; modelling; computer simulation; chicken
8.  Transcriptional Regulator BPTF/FAC1 Is Essential for Trophoblast Differentiation during Early Mouse Development▿  
Molecular and Cellular Biology  2008;28(22):6819-6827.
The putative transcriptional regulator BPTF/FAC1 is expressed in embryonic and extraembryonic tissues of the early mouse conceptus. The extraembryonic trophoblast lineage in mammals is essential to form the fetal part of the placenta and hence for the growth and viability of the embryo in utero. Here, we describe a loss-of-function allele of the BPTF/FAC1 gene that causes embryonic lethality in the mouse. BPTF/FAC1-deficient embryos form apparently normal blastocysts that implant and develop epiblast, visceral endoderm, and extraembryonic ectoderm including trophoblast stem cells. Subsequent development of mutants, however, is arrested at the early gastrula stage (embryonic day 6.5), and virtually all null embryos die before midgestation. Most notably, the ectoplacental cone is drastically reduced or absent in mutants, which may cause the embryonic lethality. Development of the mutant epiblast is also affected, as the anterior visceral endoderm and the primitive streak do not form correctly, while brachyury-expressing mesodermal cells arise but are delayed. The mutant phenotype suggests that gastrulation is initiated, but no complete anteroposterior axis of the epiblast appears. We conclude that BPTF/FAC1 is essential in the extraembryonic lineage for correct development of the ectoplacental cone and fetomaternal interactions. In addition, BPTF/FAC1 may also play a role either directly or indirectly in anterior-posterior patterning of the epiblast.
PMCID: PMC2573307  PMID: 18794365
9.  The mouse Ovol2 gene is required for cranial neural tube development 
Developmental biology  2006;291(1):38-52.
The Ovo gene family encodes a group of evolutionarily conserved transcription factors and includes members that reside downstream of key developmental signaling pathways such as Wg/Wnt and BMP/TGF-β. In the current study, we explore the function of Ovol2, one of three Ovo paralogues in mice. We report that Ovol2 is expressed during early–mid embryogenesis, particularly in the inner cell mass at E3.5, in epiblast at E6.5, and at later stages in ectodermally derived tissues such as the rostral surface (epidermal) ectoderm. Embryos in which Ovol2 is ablated exhibit lethality by E10.5, prior to which they display severe defects including an open cranial neural tube. The neural defects are associated with improper Shh expression in the underlying rostral axial mesoderm and localized changes of neural marker expression along the dorsoventral axis, as well as with expanded cranial neural tissue and reduced cranial surface ectoderm culminating in a lateral shift of the neuroectoderm/surface ectoderm border. We propose that these defects reflect the involvement of Ovol2 in independent processes such as regionalized gene expression and neural/non-neural ectodermal patterning. Additionally, we present evidence that Ovol2 is required for efficient migration and survival of neural crest cells that arise at the neuroectoderm/surface ectoderm border, but not for their initial formation. Collectively, our studies indicate that Ovol2 is a key regulator of neural development and reveal a previously unexplored role for Ovo genes in mammalian embryogenesis.
PMCID: PMC2891516  PMID: 16423343
Ovo; Ovol2; Neural tube; Brain; Shh signaling; Neuroectoderm; Surface ectoderm (epidermal ectoderm); Neural crest
10.  The Emergence of the Endothelial Cell Lineage in the Chick Embryo Can Be Detected by Uptake of Acetylated Low Density Lipoprotein and the Presence of a von Willebrand-like Factor 
Developmental biology  1989;132(1):230-240.
Vascular endothelial cells from 3- to 10-day-old chicken embryos were identified by the uptake of acetylated low density lipoprotein (Ac-LDL) and the presence of a von Willebrand-like factor. These were determined on cross sections of aortic arches as well as in cell cultures prepared from the arches. To visualize the uptake of Ac-LDL, the fluorescent probe l,r-dioctadecyl-l-3,3,3’,3’-tetramethyl-indo-carbocyanine perchlorate-Ac-LDL (DiI-Ac-LDL) was used. Following injection of the DiI-Ac-LDL probe into the embryonic heart, the endothelium of the aortic arches became specifically labeled. Also, following the administration of the probe to cell cultures, about 5–10% of the cells became DiI-positive. Indirect immunofluorescence with an antibody against von Willebrand (vW) factor also revealed specific staining of the endothelium of the aortic arches as well as of a subset of cells in cultures from aortic arches. These two histochemical markers were further used to identify the emergence of the endothelial cell lineage in the chicken blastodisc. Cultured cells from embryos incubated in ovo for 16 hr exhibited both uptake of DiI-Ac-LDL and expression of a vW-like factor. The proportion of these cells was about 30% of the total cultured cells and increased to over 50% in cultures of embryos incubated in ovo for 20 hr. However, cells positive for uptake of DiI-Ac-LDL and expression of vW-like factor were lacking in cultures of unincubated eggs or eggs incubated for 6–10 hr. We conclude that the very early endothelial cells in the chick blastodisc are already capable of expressing characteristic properties of vascular endothelium.
PMCID: PMC4080922  PMID: 2783918
11.  Cell Lineage Specific Distribution of H3K27 Trimethylation Accumulation in an In Vitro Model for Human Implantation 
PLoS ONE  2012;7(3):e32701.
Female mammals inactivate one of their two X-chromosomes to compensate for the difference in gene-dosage with males that have just one X-chromosome. X-chromosome inactivation is initiated by the expression of the non-coding RNA Xist, which coats the X-chromosome in cis and triggers gene silencing. In early mouse development the paternal X-chromosome is initially inactivated in all cells of cleavage stage embryos (imprinted X-inactivation) followed by reactivation of the inactivated paternal X-chromosome exclusively in the epiblast precursors of blastocysts, resulting temporarily in the presence of two active X-chromosomes in this specific lineage. Shortly thereafter, epiblast cells randomly inactivate either the maternal or the paternal X-chromosome. XCI is accompanied by the accumulation of histone 3 lysine 27 trimethylation (H3K27me3) marks on the condensed X-chromosome. It is still poorly understood how XCI is regulated during early human development. Here we have investigated lineage development and the distribution of H3K27me3 foci in human embryos derived from an in-vitro model for human implantation. In this system, embryos are co-cultured on decidualized endometrial stromal cells up to day 8, which allows the culture period to be extended for an additional two days. We demonstrate that after the co-culture period, the inner cell masses have relatively high cell numbers and that the GATA4-positive hypoblast lineage and OCT4-positive epiblast cell lineage in these embryos have segregated. H3K27me3 foci were observed in ∼25% of the trophectoderm cells and in ∼7.5% of the hypoblast cells, but not in epiblast cells. In contrast with day 8 embryos derived from the co-cultures, foci of H3K27me3 were not observed in embryos at day 5 of development derived from regular IVF-cultures. These findings indicate that the dynamics of H3K27me3 accumulation on the X-chromosome in human development is regulated in a lineage specific fashion.
PMCID: PMC3296731  PMID: 22412909
12.  Identification and Characterization of Side Population Cells in Embryonic Stem Cell Cultures 
Stem Cells and Development  2008;18(8):1155-1166.
Marker and functional heterogeneity has been described for embryonic stem cells (ESCs). This property has been correlated with the presence of ESC subpopulations resembling pluripotent cell lineages of the embryo. The ability to efflux Hoechst (Ho) displayed by side population (SP) cells has proven valuable as a marker to identify multipotent stem cells from a variety of tissues. Here we report that cultures from different ESC lines consistently show an SP population that displays antigens of undifferentiated ESCs, distinct drug efflux properties, and an expression pattern of ABC transporters, inner cell mass (ICM), and epiblast genes, which distinguish it from the non-SP ESC fraction. This SP population contains pluripotent cells that differentiate into ectoderm, mesoderm, and endoderm in embryoid body and teratoma assays. Further, purified SP cells efficiently integrate into developing morulae and contribute to ICM. Under standard ESC culture conditions, SP and non-SP populations display ability to convert into each other; however, an equilibrium establishes between these fractions. Using protocols customized for SP ESCs, we report that cells with similar efflux properties can be identified in the ICM of peri-implanted blastocysts. Our results indicate that ESCs display heterogeneity for the SP marker, and the SP population of these cultures contains cells that phenotypically and functionally resemble efflux-active ICM cells of the peri-implanted embryo. Our observations suggest an involvement of the SP phenotype in ESC maintenance and early embryo development, and support the idea that ESCs are composed of distinct phenotypic and functional pluripotent subpopulations in dynamic equilibrium.
PMCID: PMC3136721  PMID: 19113897
13.  Five Transcription Factors and FGF Pathway Inhibition Efficiently Induce Erythroid Differentiation in the Epiblast 
Stem Cell Reports  2014;2(3):262-270.
Primitive erythropoiesis follows a stereotypic developmental program of mesoderm ventralization and internalization, hemangioblast formation and migration, and erythroid lineage specification. Induction of erythropoiesis is inefficient in either ES/iPS cells in vitro or nonhemangioblast cell populations in vivo. Using the chick model, we report that epiblast cells can be directly and efficiently differentiated into the erythroid lineage by expressing five hematopoietic transcription regulators (SCL+LMO2+GATA2+LDB1+E2A) and inhibiting the FGF pathway. We show that these five genes are expressed with temporal specificity during normal erythropoiesis. Initiation of SCL and LMO2 expression requires FGF activity, whereas erythroid differentiation is enhanced by FGF inhibition. The lag between hematopoiesis and erythropoiesis is attributed to sequential coregulator expression and hemangioblast migration. Globin gene transcription can be ectopically and prematurely induced by manipulating the availability of these factors and the FGF pathway activity. We propose that similar approaches can be taken for efficient erythroid differentiation in vitro.
Graphical Abstract
•Epiblast cells can be directly differentiated into the erythroid lineage in vivo•Direct differentiation bypasses intermediate developmental steps•Five transcription factors promote efficient direct differentiation•Fibroblast growth factor (FGF) inhibition is needed for direct differentiation
Primitive erythropoiesis in development requires step-wise differentiation from a pool of pluripotent epiblast cells. In this work, Weng and Sheng demonstrate that forced expression of five transcription regulators bypasses this requirement and induces erythroid differentiation directly in the epiblast. Inhibition of the FGF pathway activity turns out to be a key ingredient for efficient induction.
PMCID: PMC3964278  PMID: 24672750
14.  MyoD-positive myoblasts are present in mature fetal organs lacking skeletal muscle 
The Journal of Cell Biology  2001;155(3):381-392.
The epiblast of the chick embryo gives rise to the ectoderm, mesoderm, and endoderm during gastrulation. Previous studies revealed that MyoD-positive cells were present throughout the epiblast, suggesting that skeletal muscle precursors would become incorporated into all three germ layers. The focus of the present study was to examine a variety of organs from the chicken fetus for the presence of myogenic cells. RT-PCR and in situ hybridizations demonstrated that MyoD-positive cells were present in the brain, lung, intestine, kidney, spleen, heart, and liver. When these organs were dissociated and placed in culture, a subpopulation of cells differentiated into skeletal muscle. The G8 antibody was used to label those cells that expressed MyoD in vivo and to follow their fate in vitro. Most, if not all, of the muscle that formed in culture arose from cells that expressed MyoD and G8 in vivo. Practically all of the G8-positive cells from the intestine differentiated after purification by FACS®. This population of ectopically located cells appears to be distinct from multipotential stem cells and myofibroblasts. They closely resemble quiescent, stably programmed skeletal myoblasts with the capacity to differentiate when placed in a permissive environment.
PMCID: PMC2150848  PMID: 11684706
MyoD; myoblasts; chicken; fetal; organs
15.  Cardiac progenitor migration and specification 
Cell Adhesion & Migration  2008;2(2):74-76.
The heart is the first organ to function during vertebrate development and cardiac progenitors, are among the first cell lineages to be established from mesoderm cells emerging from the primitive streak during gastrulation. Cardiac progenitors have been mapped in the epiblast of pre-streak embryos. In the early chick gastrula they are located in the mid-primitive streak, from which they enter the mesoderm bilaterally. However, migration routes of cardiac progenitors have never been directly observed within the embryo and the factor(s) controlling their movement are not known. Furthermore, it is not understood how signals controlling cell movement are integrated with those that determine cell fate. Long-term video microscopy combined with GFP labelling and image processing enabled us to observe the movement patterns of prospective cardiac cells in whole embryos in real time. Embryo manipulations and the analysis of explants suggest that Wnt3a plays a crucial role in guiding these cells through a RhoA dependent mechanism involving negative chemotaxis. Wnt3a is expressed at high levels in the amniote primitive streak and ectopic signalling activity caused wider movement trajectories resulting in cardia bifida, which was rescued by dominant-negative Wnt3a. Our studies revealed Wnt3a-RhoA mediated chemo-repulsion as a novel mechanism guiding cardiac progenitors. This activity can act at long-range and does not interfere with cardiac cell fate specification.
PMCID: PMC2634988  PMID: 19262099
cardiac development; cell migration; Wnt signaling; video microscopy; imaging; chicken
16.  Modeling Gastrulation in the Chick Embryo: Formation of the Primitive Streak 
PLoS ONE  2010;5(5):e10571.
The body plan of all higher organisms develops during gastrulation. Gastrulation results from the integration of cell proliferation, differentiation and migration of thousands of cells. In the chick embryo gastrulation starts with the formation of the primitive streak, the site of invagination of mesoderm and endoderm cells, from cells overlaying Koller's Sickle. Streak formation is associated with large-scale cell flows that carry the mesoderm cells overlying Koller's sickle into the central midline region of the embryo. We use multi-cell computer simulations to investigate possible mechanisms underlying the formation of the primitive streak in the chick embryo. Our simulations suggest that the formation of the primitive streak employs chemotactic movement of a subpopulation of streak cells, as well as differential adhesion between the mesoderm cells and the other cells in the epiblast. Both chemo-attraction and chemo-repulsion between various combinations of cell types can create a streak. However, only one combination successfully reproduces experimental observations of the manner in which two streaks in the same embryo interact. This finding supports a mechanism in which streak tip cells produce a diffusible morphogen which repels cells in the surrounding epiblast. On the other hand, chemotactic interaction alone does not reproduce the experimental observation that the large-scale vortical cell flows develop simultaneously with streak initiation. In our model the formation of large scale cell flows requires an additional mechanism that coordinates and aligns the motion of neighboring cells.
PMCID: PMC2868022  PMID: 20485500
17.  Who moves whom during primitive streak formation in the chick embryo 
HFSP Journal  2009;3(2):71-76.
Gastrulation is a critical stage in the development of all vertebrates. During gastrulation mesendoderm cells move inside the embryo to form the gut, muscles, and skeleton. In amniotes the mesendoderm cells move inside the embryo through a structure known as the primitive streak, extending from the posterior pole anterior through the midline of the embryo. Primitive streak formation involves large scale cell flows of a layer of highly polarized epithelial epiblast cells. The epiblast is separated from a lower layer of hypoblast cells through a well developed basal lamina. Recent experiments in which in vivo extracellular matrix dynamics was followed via labeling with fibronectin specific fluorescent antibodies and time-lapse microscopy have suggested that extracellular matrix dynamics essentially coincides with the observed epiblast cell displacements (Zamir et al., 2008, PLoS Biol 6, e247). These observations raise the important question of who moves whom and where do cells derive traction. We discuss these matters and their implications for our understanding of the mechanisms underlying cell flows during primitive streak formation in the chick embryo.
PMCID: PMC2707795  PMID: 19794819
18.  Epigenetic reversion of postimplantation epiblast cells to pluripotent embryonic stem cells 
Nature  2009;461(7268):10.1038/nature08534.
The pluripotent state, which is first established in the primitive ectoderm cells (PE) of blastocysts, is lost progressively and irreversibly during subsequent development1. For example, development of postimplantation epiblast from PE involves significant transcriptional and epigenetic changes, including DNA methylation and X inactivation2, which creates a robust epigenetic barrier and prevents their reversion to a PE-like state. Epiblast cells are refractory to leukaemia inhibitory factor (LIF)-STAT3 signaling, but they respond to Activin/bFGF to form self-renewing epiblast stem cells (EpiSC), which exhibit essential properties of epiblast cells3,4, that differ from embryonic stem cells (ESC) derived from PE5. Here we show reprogramming of advanced epiblast cells from E5.5 - E7.5 embryos with uniform expression of N-cadherin and inactive X chromosome, to ES-like cells (rESC) in response to LIF-STAT3 signaling. Cultured epiblast cells (cEpi) overcome the epigenetic barrier progressively as they proceed with the erasure of key properties of epiblast cells, involving DNA demethylation, X reactivation and expression of E-cadherin. The accompanying changes in the transcriptome result in a loss of phenotypic and epigenetic memory of epiblast cells. Notably, using this new approach, we report reversion of established EpiSC to rESC. Furthermore, unlike epiblast and EpiSC, rESC contribute to somatic tissues and germ cells in chimeras. This is a tractable model to investigate signaling molecule induced epigenetic reprogramming that can promote reacquisition of the fundamental pluripotent state.
PMCID: PMC3863718  PMID: 19816418
19.  Cell movement during chick primitive streak formation 
Developmental biology  2006;296(1):137-149.
Gastrulation in amniotes begins with extensive re-arrangements of cells in the epiblast resulting in the formation of the primitive streak. We have developed a transfection method that enables us to transfect randomly distributed epiblast cells in the Stage XI–XIII chick blastoderms with GFP fusion proteins. This allows us to use time-lapse microscopy for detailed analysis of the movements and proliferation of epiblast cells during streak formation. Cells in the posterior two thirds of the embryo move in two striking counter-rotating flows that meet at the site of streak formation at the posterior end of the embryo. Cells divide during this rotational movement with a cell cycle time of 6–7 h. Daughter cells remain together, forming small clusters and as result of the flow patterns line up in the streak. Expression of the cyclin-dependent kinase inhibitor, P21/Waf inhibits cell division and severely limits embryo growth, but does not inhibit streak formation or associated flows. To investigate the role off cell–cell intercalation in streak formation we have inhibited the Wnt planar-polarity signalling pathway by expression of a dominant negative Wnt11 and a Dishevelled mutant Xdd1. Both treatments do not result in an inhibition of streak formation, but both severely affect extension of the embryo in later development. Likewise inhibition of myosin II which as been shown to drive cell–cell intercalation during Drosophila germ band extension, has no effect on streak formation, but also effectively blocks elongation after regression has started. These experiments make it unlikely that streak formation involves known cell–cell intercalation mechanisms. Expression of a dominant negative FGFR1c receptor construct as well as the soluble extracellular domain of the FGFR1c receptor both effectively block the cell movements associated with streak formation and mesoderm differentiation, showing the importance of FGF signalling in these processes.
PMCID: PMC2556955  PMID: 16725136
Primitive streak; Cell division; Cell intercalation; FGF signalling
20.  Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst 
Development (Cambridge, England)  2014;141(5):1001-1010.
The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established.
PMCID: PMC3929414  PMID: 24504341
Blastocyst; Chimaera; Nanog; Oct4 (Pou5f1); Primitive endoderm; Sox17
21.  Conditional Aurora A deficiency differentially affects early mouse embryo patterning 
Developmental biology  2012;371(1):77-85.
Aurora A is a mitotic kinase involved in centrosome maturation, spindle assembly and chromosome segregation during the cell division cycle. In mice, ablation of Aurora A results in mitotic arrest and pre-implantation lethality, preventing studies at later stages of development. Here we report the effects of Aurora A ablation on embryo patterning at early post-implantation stages. Inactivation of Aurora A in the epiblast or visceral endoderm layers of the conceptus leads to apoptosis and inhibition of embryo growth, causing lethality and resorption at approximately E9.5. The effects on embryo patterning, however, depend on the tissue affected by the mutation. Embryos with an epiblast ablation of Aurora A properly establish the anteroposterior axis but fail to progress through gastrulation. In contrast, mutation of Aurora A in the visceral endoderm, leads to posteriorization of the conceptus or failure to elongate the anteroposterior axis. Injection of ES cells into Aurora A epiblast knockout blastocysts reconstitutes embryonic development to E9.5, indicating that the extra-embryonic tissues in these mutant embryos can sustain development to organogenesis stages. Our results reveal new ways to induce apoptosis and to ablate cells in a tissue-specific manner in vivo. Moreover, they show that epiblast-ablated embryos can be used to test the potency of stem cells.
PMCID: PMC3467101  PMID: 22939930
Mouse; embryo; patterning; Aurora A; gastrulation; epiblast; visceral endoderm
22.  Specific Epiblast Loss and Hypoblast Impairment in Cattle Embryos Sensitized to Survival Signalling by Ubiquitous Overexpression of the Proapoptotic Gene BAD 
PLoS ONE  2014;9(5):e96843.
Early embryonic lethality is common, particularly in dairy cattle. We made cattle embryos more sensitive to environmental stressors by raising the threshold of embryo survival signaling required to overcome the deleterious effects of overexpressing the proapoptotic protein BAD. Two primary fibroblast cell lines expressing BAD and exhibiting increased sensitivity to stress-induced apoptosis were used to generate transgenic Day13/14 BAD embryos. Transgenic embryos were normal in terms of retrieval rates, average embryo length or expression levels of the trophectoderm marker ASCL2. However both lines of BAD-tg embryos lost the embryonic disc and thus the entire epiblast lineage at significantly greater frequencies than either co-transferrred IVP controls or LacZ-tg embryos. Embryos without epiblast still contained the second ICM-derived lineage, the hypopblast, albeit frequently in an impaired state, as shown by reduced expression of the hypoblast markers GATA4 and FIBRONECTIN. This indicates a gradient of sensitivity (epiblast > hypoblast > TE) to BAD overexpression. We postulate that the greater sensitivity of specifically the epiblast lineage that we have seen in our transgenic model, reflects an inherent greater susceptibility of this lineage to environmental stress and may underlie the epiblast-specific death seen in phantom pregnancies.
PMCID: PMC4013130  PMID: 24806443
23.  FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression 
FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.
We find that pharmacological inhibition of FGFR activity blocks migration of cells through the primitive streak of chicken embryos without apparent alterations in the level or intracellular localization of E-cadherin. E-cadherin protein is localized to the periphery of epiblast, primitive streak and some mesodermal cells. FGFR inhibition leads to downregulation of a large number of regulatory genes in the preingression epiblast adjacent to the primitive streak, the primitive streak and the newly formed mesoderm. This includes members of the FGF, NOTCH, EPH, PDGF, and canonical and non-canonical WNT pathways, negative modulators of these pathways, and a large number of transcriptional regulatory genes. SNAI2 expression in the primitive streak and mesoderm is not altered by FGFR inhibition, but is downregulated only in the preingression epiblast region with no significant effect on E-cadherin. Furthermore, over expression of SNAIL has no discernable effect on E-cadherin protein levels or localization in epiblast, primitive streak or mesodermal cells. FGFR activity modulates distinct downstream pathways including RAS/MAPK and PI3K/AKT. Pharmacological inhibition of MEK or AKT indicate that these downstream effectors control discrete and overlapping groups of genes during gastrulation. FGFR activity regulates components of several pathways known to be required for cell migration through the streak or in the mesoderm, including RHOA, the non-canonical WNT pathway, PDGF signalling and the cell adhesion protein N-cadherin.
In chicken embryos, FGF signalling regulates cell movement through the primitive streak by mechanisms that appear to be independent of changes in E-cadherin expression or protein localization. The positive and negative effects on large groups of genes by pharmacological inhibition of FGF signalling, including major signalling pathways and transcription factor families, indicates that the FGF pathway is a focal point of regulation during gastrulation in chicken.
PMCID: PMC3071786  PMID: 21418646
24.  Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions 
Cell Reports  2013;3(6):1945-1957.
Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. The cytokine LIF, necessary for ESC self-renewal, supported the expansion of this population but did not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants.
Graphical Abstract
•Embryos cultured in 2i are blocked before extraembryonic lineage segregation•ESCs cultured in 2i express extraembryonic markers heterogeneously•Single 2i ESCs differentiate into both embryonic and extraembryonic lineages•LIF promotes proliferation of extraembryonically primed ESC populations
Embryonic stem cells (ESCs) are isolated from preimplantation embryos and can contribute to all tissues of the embryo, but not extraembryonic tissues (e.g., placenta). Therefore, they are considered pluripotent, not totipotent. Brickman and colleagues now show that single ESCs cultured in the presence of MEK and GSK3 inhibitors (2i) coexpress embryonic and extraembryonic markers and contribute to both embryonic and extraembryonic tissues (e.g., are totipotent). A role of the cytokine LIF in ESC self-renewal is to support this totipotent population.
PMCID: PMC3701323  PMID: 23746443
25.  Isolation of Oct4-Expressing Extraembryonic Endoderm Precursor Cell Lines 
PLoS ONE  2009;4(9):e7216.
The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4.
Methodology/Principal Findings
Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines (“XEN-P cell lines”), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo.
Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation.
PMCID: PMC2747266  PMID: 19784378

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