Showing papers on "Epiblast published in 2009"
TL;DR: It is proposed that two phases of pluripotency can be defined: naive and primed, and this distinction extends to pluripotent stem cells derived from embryos or by molecular reprogramming ex vivo.
1,640 citations
TL;DR: Evidence is presented that the homeodomain protein Nanog mediates acquisition of both embryonic and induced pluripotency, and that this function is recapitulated in the culmination of somatic cell reprogramming.
1,047 citations
TL;DR: In this article, a single reprogramming factor, Klf4, was introduced into EpiSCs, which activated expression of EScell-specific transcripts including endogenous klf4, and downstream markers of lineage specification.
Abstract: Mouse embryonic stem (ES) cells derived from pluripotent early epiblast
contribute functionally differentiated progeny to all foetal lineages of
chimaeras. By contrast, epistem cell (EpiSC) lines from post-implantation
epithelialised epiblast are unable to colonise the embryo even though they
express the core pluripotency genes Oct4, Sox2 and Nanog . We
examined interconversion between these two cell types. ES cells can readily
become EpiSCs in response to growth factor cues. By contrast, EpiSCs do not
change into ES cells. We exploited PiggyBac transposition to
introduce a single reprogramming factor, Klf4, into EpiSCs. No effect was
apparent in EpiSC culture conditions, but in ground state ES cell conditions a
fraction of cells formed undifferentiated colonies. These EpiSC-derived
induced pluripotent stem (Epi-iPS) cells activated expression of ES
cell-specific transcripts including endogenous Klf4 , and
downregulated markers of lineage specification. X chromosome silencing in
female cells, a feature of the EpiSC state, was erased in Epi-iPS cells. They
produced high-contribution chimaeras that yielded germline transmission. These
properties were maintained after Cre-mediated deletion of the Klf4
transgene, formally demonstrating complete and stable reprogramming of
developmental phenotype. Thus, re-expression of Klf4 in an appropriate
environment can regenerate the naive ground state from EpiSCs.
Reprogramming is dependent on suppression of extrinsic growth factor stimuli
and proceeds to completion in less than 1% of cells. This substantiates the
argument that EpiSCs are developmentally, epigenetically and functionally
differentiated from ES cells. However, because a single transgene is the
minimum requirement to attain the ground state, EpiSCs offer an attractive
opportunity for screening for unknown components of the reprogramming
process.
759 citations
TL;DR: It is proposed that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells, and that in the absence of this signal, the entire ICM can acquire pluripotency.
Abstract: Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. We investigate the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. We find that blockade of Erk signalling from the 8-cell stage does not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naive epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. We probed directly the relationship between naive epiblast and ES cells. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. We propose that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells.
576 citations
TL;DR: This study establishes a robust strategy for reconstituting the mammalian germ cell lineage in vitro via Bmp4 signaling from the extraembryonic ectoderm, which is antagonized by the anterior visceral endoderm (AVE).
489 citations
TL;DR: Reprogramming of advanced epiblast cells from embryonic day 5.5–7.5 mouse embryos with uniform expression of N-cadherin and inactive X chromosome to ES-cell-like cells (rESCs) in response to LIF–STAT3 signalling is shown and reversion of established EpiSCs to rESCs is reported.
Abstract: The pluripotent state, which is first established in the primitive ectoderm cells of blastocysts, is lost progressively and irreversibly during subsequent development. For example, development of post-implantation epiblast cells from primitive ectoderm involves significant transcriptional and epigenetic changes, including DNA methylation and X chromosome inactivation, which create a robust epigenetic barrier and prevent their reversion to a primitive-ectoderm-like state. Epiblast cells are refractory to leukaemia inhibitory factor (LIF)-STAT3 signalling, but they respond to activin/basic fibroblast growth factor to form self-renewing epiblast stem cells (EpiSCs), which exhibit essential properties of epiblast cells and that differ from embryonic stem (ES) cells derived from primitive ectoderm. Here we show reprogramming of advanced epiblast cells from embryonic day 5.5-7.5 mouse embryos with uniform expression of N-cadherin and inactive X chromosome to ES-cell-like cells (rESCs) in response to LIF-STAT3 signalling. Cultured epiblast cells overcome the epigenetic barrier progressively as they proceed with the erasure of key properties of epiblast cells, resulting in 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. Using this approach, we report reversion of established EpiSCs to rESCs. Moreover, unlike epiblast and EpiSCs, rESCs contribute to somatic tissues and germ cells in chimaeras. Further studies may reveal how signalling-induced epigenetic reprogramming may promote reacquisition of pluripotency.
389 citations
TL;DR: These data challenge the paradigm that the three germ layers, formed by gastrulation, constitute the primary branchpoints in differentiation of the pluripotent epiblast toward tissue-specific precursors and may represent the cellular substrate coupling sustained axial elongation and coordinated differentiation of these tissues.
365 citations
TL;DR: It is found that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming, which suggests that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro.
359 citations
TL;DR: It is proposed that Foxa2 regulates a molecular program that induces an epithelial cellular phenotype, supported by the fact thatFoxa2 mutant endodermal cells fail to maintain polarity and do not establish proper cellular junctions, and are thus unable to functionally integrate into the endoderm epithelium.
Abstract: In the mouse, one of the earliest events in the determination of cell fate is the segregation of cells into germ layers during gastrulation; however, the cellular and molecular details are not well defined due to intrauterine development. We were able to visualize a clear sequence of events occurring in the process of germ-layer formation, using immunohistochemistry and time-lapse confocal imaging. The T-box transcription factor brachyury (T) and the Forkhead transcription factor Foxa2 specify mesoderm and endoderm in the posterior epiblast. Fate-specified epiblast cells lose their polarity and undergo epithelial-mesenchymal transition to invade into the primitive streak region, where these cell populations quickly separate and differentiate into morphologically and molecularly distinct Foxa2-positive endoderm and T-positive mesoderm populations. The endoderm cells flatten and acquire apical-basal polarity during intercalation into the outside epithelium in order to establish proper intracellular junctions with pre-existing cells. By contrast, the mesodermal cells become spherical during migration and acquire a mesenchymal fate. Interestingly, axial mesodermal cells are descended from Foxa2-positive epiblast cells that upregulate T protein in the anterior primitive streak region. These cells, as well as Foxa2-positive endoderm cells, are highly polarized and epithelialized, suggesting that Foxa2 promotes an epithelial fate and suppresses a mesenchymal fate. This observation is supported by the fact that Foxa2 mutant endodermal cells fail to maintain polarity and do not establish proper cellular junctions, and are thus unable to functionally integrate into the endoderm epithelium. We propose that Foxa2 regulates a molecular program that induces an epithelial cellular phenotype.
189 citations
TL;DR: The recent clarification of the mechanism by which signaling activities confer the germ cell fate to the epiblast cells underscores the feasibility of generating the mammalian germ cell lineage in vitro.
183 citations
TL;DR: Dynamics of ICM cells from the early to late blastocyst stage are tracked and characterised, and computer modelling suggests that PE formation involves both cell sorting movements and position-dependent induction.
TL;DR: It is shown that EpiSCs have an infinite capacity for generating PGCs, under conditions that sustain their pluripotency and self-renewal, and an in vitro model system is introduced that will be useful for investigations on PGC specification and on mechanisms regulating epigenetic reprogramming in germ cells.
Abstract: Pluripotent epiblast stem cells (EpiSCs) derived from postimplantation embryos exhibit properties that are characteristically different when compared with pluripotent embryonic stem cells (ESCs) derived from mouse blastocysts. However, EpiSCs are relatively less well characterised compared with ESCs. In particular, the relationship between EpiSCs and primordial germ cells (PGCs) is unknown, and is worthy of investigation because PGCs originate from postimplantation epiblast cells in vivo. We show that EpiSCs have an infinite capacity for generating PGCs, under conditions that sustain their pluripotency and self-renewal. These PGCs generated in vitro show appropriate transcriptional and epigenetic reprogramming events and are able to develop further into late germ cells. Notably, the PGCs can, in turn, be induced to undergo dedifferentiation into pluripotent embryonic germ cells (EGCs), which resemble ESCs and not the EpiSC from which they are derived. Our observations demonstrate intrinsic reprogramming during specification of PGCs that results in the erasure of epigenetic memory of EpiSCs following reactivation of the X-chromosome, DNA demethylation and re-expression of key pluripotency genes. This study provides novel insights into the nature and properties of EpiSCs, and introduces an in vitro model system that will be useful for investigations on PGC specification and on mechanisms regulating epigenetic reprogramming in germ cells.
TL;DR: A role for mpk1 in AB polarity formation rather than expected role as a PCP gene is demonstrated, which was further supported by the finding that overexpression of ΔPET/LIM (ΔP/L), a dominant-negative Pk construct, in Xenopus embryo disrupted uniform localization of an apical marker PKCζ, and expanded the apical domain of ectoderm cells.
Abstract: Planar cell polarity (PCP) genes are essential for establishing planar cell polarity in both invertebrate and vertebrate tissues and are known to regulate cellular morphogenesis and cell movements during development. We focused on Prickle, one of the core components of the PCP pathway, and deleted one of two mouse prickle homologous genes, mpk1. We found that the deletion of mpk1 gene resulted in early embryonic lethality, between embryonic day (E)5.5 and E6.5, associated with failure of distal visceral endoderm migration and primitive streak formation. The mpk1−/− epiblast tissue was disorganized, and analyses at the cellular level revealed abnormal cell shapes, mislocalized extracellular matrix (ECM) proteins, and disrupted orientation of mitotic spindles, from which loss of apico-basal (AB) polarity of epiblast cells are suspected. Furthermore, we show mpk1 genetically interacts with another core PCP gene Vangl2/stbm in the epiblast formation, suggesting that PCP components are commonly required for the establishment and/or the maintenance of epiblast AB polarity. This was further supported by our finding that overexpression of ΔPET/LIM (ΔP/L), a dominant-negative Pk construct, in Xenopus embryo disrupted uniform localization of an apical marker PKCζ, and expanded the apical domain of ectoderm cells. Our results demonstrate a role for mpk1 in AB polarity formation rather than expected role as a PCP gene.
TL;DR: It is demonstrated that RA signalling is required at late gastrulation stages for mesodermal and neural progenitors to respond to the Shh signal, and that an interplay between RA, Fgf and Shh signalling is likely to be an important mechanism underpinning the tight regulation of caudal embryonic development.
Abstract: The progressive generation of embryonic trunk structures relies on the
proper patterning of the caudal epiblast, which involves the integration of
several signalling pathways. We have investigated the function of retinoic
acid (RA) signalling during this process. We show that, in addition to
posterior mesendoderm, primitive streak and node cells transiently express the
RA-synthesizing enzyme Raldh2 prior to the headfold stage.
RA-responsive cells (detected by the RA-activated RARE- lacZ
transgene) are additionally found in the epiblast layer. Analysis of
RA-deficient Raldh2 -/- mutants reveals early caudal
patterning defects, with an expansion of primitive streak and mesodermal
markers at the expense of markers of the prospective neuroepithelium. As a
result, many genes involved in neurogenesis and/or patterning of the embryonic
spinal cord are affected in their expression. We demonstrate that RA
signalling is required at late gastrulation stages for mesodermal and neural
progenitors to respond to the Shh signal. Whole-embryo culture experiments
indicate that the proper response of cells to Shh requires two RA-dependent
mechanisms: (1) a balanced antagonism between Fgf and RA signals, and (2) a
RA-mediated repression of Gli2 expression. Thus, an interplay between
RA, Fgf and Shh signalling is likely to be an important mechanism underpinning
the tight regulation of caudal embryonic development.
TL;DR: In the pig, the paternal genome is actively demethylated in the zygote, whereas the maternal genome remains methylated, allowing for trophectoderm and inner cell mass differentiation and this particular and protracted ICM/epiblast biology may contribute to the lack of success in culturing porcine embryonic stem cells.
Abstract: The increasing focus on the pig as a biomedical model calls for studies which investigate morphological and molecular mechanisms during initial embryonic development in this species. In the pig, the paternal genome is actively demethylated in the zygote, whereas the maternal genome remains methylated. The major genome activation occurs at the four-cell stage, when prominent ribosome-synthesizing nucleoli develop in the blastomeres, allowing for trophectoderm and inner cell mass (ICM) differentiation. Unlike in mice, the pluripotency gene OCT4 is initially expressed in both compartments. The ICM differentiates into epiblast and hypoblast approximately at the time of hatching from the zona pellucida, and subsequently the loss of the Rauber's layer results in an uncovered epiblast establishing the embryonic disc again in contrast to mice. This particular and protracted ICM/epiblast biology may contribute to the lack of success in culturing porcine embryonic stem cells. The embryonic disc subsequently becomes polarized by a posterior thickening, which includes ingression of the first extra-embryonic mesoderm. Thereafter, the primitive streak forms and gastrulation results in formation of the somatic germ layers and germline, i.e. the primordial germ cells. The latter remain pluripotent for a period and may be isolated and cultured as embryonic germ cells in vitro.
TL;DR: Cell signaling associated with maintaining pluripotency in human embryonic stem cells is detectable in the porcine epiblast, but not in the inner cell mass, as revealed in this study.
Abstract: The signaling mechanisms regulating pluripotency in porcine embryonic stem cells and embryos are unknown In this study, we characterize cell signaling in the in-vivo porcine inner cell mass and later-stage epiblast We evaluate expression of OCT4, NANOG, SOX2, genes within the JAK/STAT pathway (LIF, LIFR, GP130), FGF pathway (bFGF, FGFR1, FGFR2), BMP pathway (BMP4), and downstream-activated genes (STAT3, c-Myc, c-Fos, and SMAD4) We discovered two different expression profiles exist in the developing porcine embryo The D6 porcine blastocyst (inner cell mass stage) is devoid in the expression of most genes analyzed, with the exception of OCT4 In contrast, the D11 epiblast expressed 10 of the 12 genes investigated Immunocytochemistry confirmed LIFR and bFGF was not expressed in the epiblast, but within the trophectoderm These findings reveal cell signaling associated with maintaining pluripotency in human embryonic stem cells is detectable in the porcine epiblast, but not in the inner cell mass
TL;DR: It is proposed that P19 cells can be used as a simple and efficient screening tool to assess the potential functions of specific molecules in mesoderm formation and axial elongation morphogenesis.
Abstract: Because of their capacity to give rise to various types of cells in vitro, embryonic stem and embryonal carcinoma (EC) cells have been used as convenient models to study the mechanisms of cell differentiation in mammalian embryos. In this study, we explored the mouse P19 EC cell line as an effective tool to investigate the factors that may play essential roles in mesoderm formation and axial elongation morphogenesis. We first demonstrated that aggregated P19 cells not only exhibited gene expression patterns characteristic of mesoderm formation but also displayed elongation morphogenesis with a distinct anterior–posterior body axis as in the embryo. We then showed by RNA interference that these processes were controlled by various regulators of Wnt signaling pathways, namely β-catenin, Wnt3, Wnt3a, and Wnt5a, in a manner similar to normal embryo development. We further showed by inhibitor treatments that the axial elongation morphogenesis was dependent on the activity of Rho-associated kinase. Because of the convenience of these experimental manipulations, we propose that P19 cells can be used as a simple and efficient screening tool to assess the potential functions of specific molecules in mesoderm formation and axial elongation morphogenesis.
TL;DR: Results suggest that abnormalities in vasculature throughout the extraembryonic tissue and the epiblast may contribute to the death of the Pak4-null embryos, and this work suggests that these abnormalities are secondary to defects that occur in response to epiblasts abnormalities.
TL;DR: It is shown that mice lacking all three CYP26 genes manifest duplication of the body axis as a result of expansion of the Nodal expression domain throughout the epiblast of pregastrulation embryos.
Abstract: The abundance of retinoic acid (RA) is determined by the balance between its synthesis by retinaldehyde dehydrogenase (RALDH) and its degradation by CYP26. In particular, the dynamic expression of three CYP26 genes controls the regional level of RA within the body. Pregastrulation mouse embryos express CYP26 but not RALDH. We now show that mice lacking all three CYP26 genes manifest duplication of the body axis as a result of expansion of the Nodal expression domain throughout the epiblast. Mouse Nodal was found to contain an RA-responsive element in intron 1 that is highly conserved among mammals. In the absence of CYP26, maternally derived RA activates Nodal expression in the entire epiblast of pregastrulation embryos via this element. These observations suggest that maternal RA must be removed by embryonic CYP26 for correct Nodal expression during embryonic patterning.
TL;DR: It is demonstrated that Dnmt3b shows a dynamic expression change during pre- and early post-implantation development, consistent with the role for DnMT3b in cell-lineage specification and the creation of lineage-specific DNA methylation patterns.
TL;DR: The round shape and gradual posterior displacement of the APD in the pig appear to be species-specific but correlate with ensuing specific primitive streak and extraembryonic mesoderm development, and the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraEmbryonic membranes and, possibly, the specific adult body form.
TL;DR: It is suggested strongly that the ExEn precursor is a self-renewable entity, indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and provide an in vitro model of early ExEn differentiation.
Abstract: Background: 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 Oct4expressing 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. Conclusions/Significance: 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.
TL;DR: A model of 2-step oligoclonal development of male germ cells in mice is suggested, the second step distinguishing the heritable germ line from cells selected not to participate in forming the next generation.
Abstract: During mouse development, primordial germ cells (PGCs) that give rise to the entire germ line are first identified within the proximal epiblast. However, long-term tracing of the fate of the cells has not been done wherein all cells in and around the germ-cell lineage are identified. Also, quantitative estimates of the number of founder PGCs using different models have come up with various numbers. Here, we use tetrachimeric mice to show that the progenitor numbers for the entire germ line in adult testis, and for the initiating embryonic PGCs, are both 4 cells. Although they proliferate to form polyclonal germ-cell populations in fetal and neonatal testes, germ cells that actually contribute to adult spermatogenesis originate from a small number of secondary founder cells that originate in the fetal period. The rest of the “deciduous” germ cells are lost, most likely by apoptosis, before the reproductive period. The second “actual” founder germ cells generally form small numbers of large monoclonal areas in testes by the reproductive period. Our results also demonstrate that there is no contribution of somatic cells to the male germ cell pool during development or in adulthood. These results suggest a model of 2-step oligoclonal development of male germ cells in mice, the second step distinguishing the heritable germ line from cells selected not to participate in forming the next generation.
TL;DR: Conditional rescue experiments demonstrate that bypassing the primary requirement for Smad4 in the extra-embryonic endoderm allows the epiblast to gastrulate, andSmad4-independent TGF-β signals are thus sufficient to promote mesoderm formation and patterning.
Abstract: Background
Smad4 mutant embryos arrest shortly after implantation and display a characteristic shortened proximodistal axis, a significantly reduced epiblast, as well as a thickened visceral endoderm layer. Conditional rescue experiments demonstrate that bypassing the primary requirement for Smad4 in the extra-embryonic endoderm allows the epiblast to gastrulate. Smad4-independent TGF-β signals are thus sufficient to promote mesoderm formation and patterning. To further analyse essential Smad4 activities contributed by the extra-embryonic tissues, and characterise Smad4 dependent pathways in the early embryo, here we performed transcriptional profiling of Smad4 null embryonic stem (ES) cells and day 4 embryoid bodies (EBs).
TL;DR: It is proposed that the animal cap assay in Xenopus and explant assays in the chick are unsuitable for studying instructive signals in neural induction.
TL;DR: It is hypothesize that very small embryonic/epiblast-like stem cells could be a missing link that support the more than 100-year-old concepts of the embryonic rest or germ line origin hypotheses of cancer development; however, further experimental evidence is needed to support this hypothesis.
Abstract: The morphology of several tumors mimics developmentally early tissues, and tumors often express early developmental markers characteristic of the germ line lineage. The presence of these markers in neoplastic cells could reflect the dedifferentiation of somatic cells in which cancer develops or cancer origination in primitive stem cells closely related to the epiblast/germ line. The identification of primitive germ line-derived very small embryonic/epiblast-like stem cells, which are deposited early in embryogenesis in developing organs and persist in several organs into adulthood, raised the possibility that cancer may originate in these cells. In this review, we hypothesize that very small embryonic/epiblast-like stem cells could be a missing link that support the more than 100-year-old concepts of the embryonic rest or germ line origin hypotheses of cancer development; however, further experimental evidence is needed to support this hypothesis.
TL;DR: The testis performs two basic functions, sperm production and testosterone secretion, and is genetically controlled; expression of the SRY gene directs the embryonic gonads into the pathway leading to the development of testes.
Abstract: The testis performs two basic functions, sperm production and testosterone secretion. Formation of the testis is genetically controlled; expression of the SRY gene directs the embryonic gonads into the pathway leading to the development of testes. By the fourth week of gestation in humans, the primordial germ cells derived from pluripotent cells of the embryonic epiblast proliferate and migrate from the endoderm of the yolk sac into the undifferentiated gonad, which becomes morphologically distinct during the seventh week of gestation in humans. Histological development of the testis is largely completed by the end of the third month of gestation.
TL;DR: It is proposed that the primitive endoderm cells reach the surface by random movement, and subsequently the cells generate an apical/basal polarity that prevents reentry, and the ability to generate epithelial polarity, rather than adhesive affinity, determines the surface positioning of the primitive ENDODerm cells.
Abstract: The classical cell sorting experiments undertaken by Townes and Holtfreter described the intrinsic propensity of dissociated embryonic cells to self-organize and reconcile into their original embryonic germ layers with characteristic histotypic positioning. Steinberg presented the differential adhesion hypothesis to explain these patterning phenomena. Here, we have reappraised these issues by implementing embryoid bodies to model the patterning of epiblast and primitive endoderm layers. We have used combinations of embryonic stem (ES) cells and their derivatives differentiated by retinoic acid treatment to model epiblast and endoderm cells, and wild-type or E-cadherin null cells to represent strongly or weakly adherent cells, respectively. One cell type was fluorescently labeled and reconstituted with another heterotypically to generate chimeric embryoid bodies, and cell sorting was tracked by time-lapse video microscopy and confirmed by immunostaining. When undifferentiated wild-type and E-cadherin null ES cells were mixed, the resulting cell aggregates consisted of a core of wild-type cells surrounded by loosely associated E-cadherin null cells, consistent with the differential adhesion hypothesis. However, when mixed with undifferentiated ES cells, the differentiated primitive endoderm-like cells sorted to the surface to form a primitive endoderm layer irrespective of cell-adhesive strength, contradicting the differential adhesion hypothesis. We propose that the primitive endoderm cells reach the surface by random movement, and subsequently the cells generate an apical/basal polarity that prevents reentry. Thus, the ability to generate epithelial polarity, rather than adhesive affinity, determines the surface positioning of the primitive endoderm cells.
TL;DR: The timing and order of ingression of epiblast cells appears to be controlled by temporal and spatial colinearity of Hox gene expression in the epiblasts, and the collective migration of the mesendoderm away from the streak appears to been controlled by gradients of growth factors of the FGF and VEGF and Wnt families and requires N-cadherin expression.
TL;DR: The evidence suggests that germ cell specification is an integration of the repression of the somatic program, re-acquisition of potential pluripotency, and ensuing genome-wide epigenetic reprogramming.
Abstract: Specification of the germ cell lineage is fundamental in development and heredity. In mice, and presumably in all mammals, germ cell fate is not an inherited trait from the egg, but is induced in pluripotent epiblast cells by signaling molecules. Recent studies are beginning to uncover the signaling requirements and key transcriptional regulators for the specification of the germ cell lineage in mice, as well as the distinct properties that the specified germ cells acquire uniquely. Accordingly, the evidence suggests that germ cell specification is an integration of the repression of the somatic program, re-acquisition of potential pluripotency, and ensuing genome-wide epigenetic reprogramming. The accumulated knowledge will be critical for the reconstitution of this key lineage in vitro, which may provide a useful foundation for reproductive and regenerative medicine.