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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.
doi:10.1016/j.ydbio.2009.09.022
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.
doi:10.1083/jcb.200703060
PMCID: PMC2064471  PMID: 17698608
3.  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.
doi:10.1083/jcb.200105139
PMCID: PMC2150848  PMID: 11684706
MyoD; myoblasts; chicken; fetal; organs
4.  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.
Highlights
► 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
doi:10.1016/j.ydbio.2011.10.030
PMCID: PMC3368271  PMID: 22079695
Pluripotency; Epiblast; Hypoblast; Fibroblast growth factor; Human ES cell derivation
5.  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.
doi:10.1016/j.ydbio.2006.04.451
PMCID: PMC2556955  PMID: 16725136
Primitive streak; Cell division; Cell intercalation; FGF signalling
6.  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.
doi:10.1128/MCB.01058-08
PMCID: PMC2573307  PMID: 18794365
7.  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
8.  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.
doi:10.1016/j.ydbio.2005.12.003
PMCID: PMC2891516  PMID: 16423343
Ovo; Ovol2; Neural tube; Brain; Shh signaling; Neuroectoderm; Surface ectoderm (epidermal ectoderm); Neural crest
9.  Live Imaging of Whole Mouse Embryos during Gastrulation: Migration Analyses of Epiblast and Mesodermal Cells 
PLoS ONE  2013;8(7):e64506.
During gastrulation in the mouse embryo, dynamic cell movements including epiblast invagination and mesodermal layer expansion lead to the establishment of the three-layered body plan. The precise details of these movements, however, are sometimes elusive, because of the limitations in live imaging. To overcome this problem, we developed techniques to enable observation of living mouse embryos with digital scanned light sheet microscope (DSLM). The achieved deep and high time-resolution images of GFP-expressing nuclei and following 3D tracking analysis revealed the following findings: (i) Interkinetic nuclear migration (INM) occurs in the epiblast at embryonic day (E)6 and 6.5. (ii) INM-like migration occurs in the E5.5 embryo, when the epiblast is a monolayer and not yet pseudostratified. (iii) Primary driving force for INM at E6.5 is not pressure from neighboring nuclei. (iv) Mesodermal cells migrate not as a sheet but as individual cells without coordination.
doi:10.1371/journal.pone.0064506
PMCID: PMC3704669  PMID: 23861733
10.  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.
doi:10.1371/journal.pone.0010571
PMCID: PMC2868022  PMID: 20485500
11.  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.
doi:10.1016/j.ydbio.2011.08.007
PMCID: PMC3192235  PMID: 21884693
MyoD; Noggin; BMP; Blastocyst; Myogenesis
12.  Method for Whole Mount Antibody Staining in Chick 
The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.
doi:10.3791/956
PMCID: PMC2783013  PMID: 19488030
13.  Genesis of embryonic stem cells. 
Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre-implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality, however, continuous expansion of pluripotent cells does not occur in vivo, and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency, the transcription factor Oct-4, in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct-4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct-4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059, which suppresses activation of the mitogen-activated protein kinases Erk1 and Erk2, results in increased persistence of Oct-4-expressing cells. Oct-4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline-competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time-window. Procedures that extend this time-window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation.
doi:10.1098/rstb.2003.1327
PMCID: PMC1693233  PMID: 14511487
14.  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.
doi:10.1016/j.ydbio.2012.08.010
PMCID: PMC3467101  PMID: 22939930
Mouse; embryo; patterning; Aurora A; gastrulation; epiblast; visceral endoderm
15.  FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression 
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-213X-11-20
PMCID: PMC3071786  PMID: 21418646
16.  New Methods for Chicken Embryo Manipulations 
The capacity to image a growing embryo while simultaneously studying the developmental function of specific molecules provides invaluable information on embryogenesis. However, until recently, this approach was accomplished with difficulty both because of the advanced technology needed and because an easy method of minimizing damage to the embryo was unavailable. Here we present a novel way of adapting the well-known EC culture of whole chick embryos to time-lapse imaging and to functional molecular studies using blocking agents. The novelty of our method stems from the ability to apply blocking agents ex ovo as well as in ovo. We were able to study the function of a set of molecules by culturing developing embryos ex ovo in tissue culture media containing these molecules or by injecting them underneath the live embryo in ovo. The in ovo preparation is particularly valuable since it extends the period of time during which the developmental function of the molecule can be studied and it provides an easy, reproducible method for screening a batch of molecules. These new techniques will prove very helpful in visualizing and understanding the role of specific molecules during embryonic morphogenesis, including blood vessel formation.
doi:10.1002/jemt.20753
PMCID: PMC2797828  PMID: 19582831
in ovo; chicken culture; imaging; embryo manipulation
17.  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.
doi:10.1089/scd.2008.0391
PMCID: PMC3136721  PMID: 19113897
18.  Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions 
Cell Reports  2013;3(6):1945-1957.
Summary
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
Highlights
•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.
doi:10.1016/j.celrep.2013.04.034
PMCID: PMC3701323  PMID: 23746443
19.  Reciprocal Repression between Sox3 and Snail Transcription Factors Defines Embryonic Territories at Gastrulation 
Developmental Cell  2011;21(3):546-558.
Summary
In developing amniote embryos, the first epithelial-to-mesenchymal transition (EMT) occurs at gastrulation, when a subset of epiblast cells moves to the primitive streak and undergoes EMT to internalize and generate the mesoderm and the endoderm. We show that in the chick embryo this decision to internalize is mediated by reciprocal transcriptional repression of Snail2 and Sox3 factors. We also show that the relationship between Sox3 and Snail is conserved in the mouse embryo and in human cancer cells. In the embryo, Snail-expressing cells ingress at the primitive streak, whereas Sox3-positive cells, which are unable to ingress, ensure the formation of ectodermal derivatives. Thus, the subdivision of the early embryo into the two main territories, ectodermal and mesendodermal, is regulated by changes in cell behavior mediated by the antagonistic relationship between Sox3 and Snail transcription factors.
Graphical Abstract
Highlights
► Snail and Sox3 are reciprocal direct transcriptional repressors ► Snail/Sox3 reciprocal repression defines ectodermal versus mesendodermal territories ► Snail2 induces cell delamination without inducing mesodermal or endodermal fates ► The Snail/Sox3 relationship is conserved in mouse embryos and human cancer cells
doi:10.1016/j.devcel.2011.07.005
PMCID: PMC3256632  PMID: 21920318
20.  MyoD-positive epiblast cells regulate skeletal muscle differentiation in the embryo 
The Journal of Cell Biology  2006;175(2):283-292.
MyoD mRNA is expressed in a subpopulation of cells within the embryonic epiblast. Most of these cells are incorporated into somites and synthesize Noggin. Ablation of MyoD-positive cells in the epiblast subsequently results in the herniation of organs through the ventral body wall, a decrease in the expression of Noggin, MyoD, Myf5, and myosin in the somites and limbs, and an increase in Pax-3–positive myogenic precursors. The addition of Noggin lateral to the somites compensates for the loss of MyoD-positive epiblast cells. Skeletal muscle stem cells that arise in the epiblast are utilized in the somites to promote muscle differentiation by serving as a source of Noggin.
doi:10.1083/jcb.200605037
PMCID: PMC2064569  PMID: 17060497
21.  DNA Dendrimers Localize Myod mRNA in Presomitic Tissues of the Chick Embryo 
The Journal of Cell Biology  2000;149(4):825-834.
MyoD expression is thought to be induced in somites in response to factors released by surrounding tissues; however, reverse transcription-PCR and cell culture analyses indicate that myogenic cells are present in the embryo before somite formation. Fluorescently labeled DNA dendrimers were used to identify MyoD expressing cells in presomitic tissues in vivo. Subpopulations of MyoD positive cells were found in the segmental plate, epiblast, mesoderm, and hypoblast. Directly after laying, the epiblast of the two layered embryo contained ∼20 MyoD positive cells. These results demonstrate that dendrimers are precise and sensitive reagents for localizing low levels of mRNA in tissue sections and whole embryos, and that cells with myogenic potential are present in the embryo before the initiation of gastrulation.
PMCID: PMC2174576  PMID: 10811824
myogenesis; epiblast; segmental plate; in situ hybridization; muscle transcription factor
22.  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.
doi:10.2976/1.3103933
PMCID: PMC2707795  PMID: 19794819
23.  Transcriptional control of GABAergic neuronal subtype identity in the thalamus 
Neural Development  2014;9:14.
Background
The thalamus is often defined as the ‘gateway to consciousness’, a feature that is supported by the specific connectivity and electrophysiological properties of its neurons. Inhibitory GABAergic neurons are required for the dynamic gating of information passing through the thalamus. The high degree of heterogeneity among thalamic GABA neurons suggests that, during embryonic development, alternative differentiation programmes exist to guide the acquisition of inhibitory neuron subtype identity.
Results
Taking advantage of the accessibility of the developing chick embryo, we have used in ovo manipulations of gene expression to test the role of candidate transcription factors in controlling GABAergic neuronal subtype identity in the developing thalamus.
Conclusions
In this study, we describe two alternative differentiation programmes for GABAergic neurogenesis in the thalamus and identify Helt and Dlx2 as key transcription factors that are sufficient to direct neuronal progenitors along a specific differentiation pathway at the expense of alternative lineage choices. Furthermore, we identify Calb2, a gene encoding for the GABA subtype marker calretinin as a target of the transcription factor Sox14. This work is a step forward in our understanding of how GABA neuron diversity in the thalamus is achieved during development and will help future investigation of the molecular mechanisms that lead up to the acquisition of different synaptic targets and electrophysiological features of mature thalamic inhibitory neurons.
doi:10.1186/1749-8104-9-14
PMCID: PMC4065548  PMID: 24929424
Helt; Dlx2; Sox14; GABAergic thalamus; Neurogenesis; Diencephalon
24.  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.
doi:10.1371/journal.pone.0096843
PMCID: PMC4013130  PMID: 24806443
25.  E-Cadherin Promotes Incorporation of Mouse Epiblast Stem Cells into Normal Development 
PLoS ONE  2012;7(9):e45220.
Mouse epiblast stem cells (mEpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs) in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.
doi:10.1371/journal.pone.0045220
PMCID: PMC3445497  PMID: 23028858

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