Early Lineage Segregation Research Articles

Deriving Human Naïve Embryonic Stem Cell Lines from Donated Supernumerary Embryos Using Physical Distancing and Signal Inhibition.

Until recently, naïve pluripotent stem cell lines were not captured from human embryos because protocols were based upon those devised for murine embryonic stem cells. In contrast with early lineage segregation in mouse embryos, human hypoblast specification is not solely dependent upon FGF signaling; consequently, its maturation during embryo explant culture may provide inductive signals to drive differentiation of the epiblast. To overcome this potential risk, here we describe how cells of the immature inner cell mass of human embryos can be physically separated during derivation, achieved via "immunosurgery", to eliminate the trophectoderm, followed by disaggregation of the remaining inner cell mass cells. A modification of a culture regime developed for propagation of human pluripotent stem cells reset to the naïve state is used, which comprises serum-free medium supplemented with various inhibitors of signaling pathways, polarization, and differentiation. Colonies arising from the first plating of an inner cell mass may be pooled for ease of handling, or propagated separately to allow establishment of clonal human naïve embryonic stem cell lines.

Chromatin Regulation in Development: Current Understanding and Approaches.

The regulation of mammalian stem cell fate during differentiation is complex and can be delineated across many levels. At the chromatin level, the replacement of histone variants by chromatin-modifying proteins, enrichment of specific active and repressive histone modifications, long-range gene interactions, and topological changes all play crucial roles in the determination of cell fate. These processes control regulatory elements of critical transcriptional factors, thereby establishing the networks unique to different cell fates and initiate waves of distinctive transcription events. Due to the technical challenges posed by previous methods, it was difficult to decipher the mechanism of cell fate determination at early embryogenesis through chromatin regulation. Recently, single-cell approaches have revolutionised the field of developmental biology, allowing unprecedented insights into chromatin structure and interactions in early lineage segregation events during differentiation. Here, we review the recent technological advancements and how they have furthered our understanding of chromatin regulation during early differentiation events.

Early lineage segregation of the retinal basal glia in the Drosophila eye disc

The retinal basal glia (RBG) is a group of glia that migrates from the optic stalk into the third instar larval eye disc while the photoreceptor cells (PR) are differentiating. The RBGs are grouped into three major classes based on molecular and morphological characteristics: surface glia (SG), wrapping glia (WG) and carpet glia (CG). The SGs migrate and divide. The WGs are postmitotic and wraps PR axons. The CGs have giant nucleus and extensive membrane extension that each covers half of the eye disc. In this study, we used lineage tracing methods to determine the lineage relationships among these glia subtypes and the temporal profile of the lineage decisions for RBG development. We found that the CG lineage segregated from the other RBG very early in the embryonic stage. It has been proposed that the SGs migrate under the CG membrane, which prevented SGs from contacting with the PR axons lying above the CG membrane. Upon passing the front of the CG membrane, which is slightly behind the morphogenetic furrow that marks the front of PR differentiation, the migrating SG contact the nascent PR axon, which in turn release FGF to induce SGs’ differentiation into WG. Interestingly, we found that SGs are equally distributed apical and basal to the CG membrane, so that the apical SGs are not prevented from contacting PR axons by CG membrane. Clonal analysis reveals that the apical and basal RBG are derived from distinct lineages determined before they enter the eye disc. Moreover, the basal SG lack the competence to respond to FGFR signaling, preventing its differentiation into WG. Our findings suggest that this novel glia-to-glia differentiation is both dependent on early lineage decision and on a yet unidentified regulatory mechanism, which can provide spatiotemporal coordination of WG differentiation with the progressive differentiation of photoreceptor neurons.

Epigenetics of human embryonic development

Molecular Biology Substantial epigenetic reprogramming occurs in mammalian early development. Xia et al. investigated the reprogramming dynamics of three key histone modifications in human early embryonic development. The reprogramming in humans is highly species-specific and different than that in mice. A globally permissive chromatin state connects parental and zygotic epigenomes during maternal-to-zygotic transition. Inner-cell mass-specific, but not trophectoderm-specific, genes are asymmetrically patterned by a histone mark during early lineage segregation. Science , this issue p. [353][1] [1]: /lookup/doi/10.1126/science.aaw5118

Early lineage segregation of multipotent embryonic mammary gland progenitors.

SummaryThe mammary gland (MG) is composed of basal cells (BCs) and luminal cells (LCs). While it is generally believed that MG arises from embryonic multipotent progenitors (EMPs), it remains unclear when lineage restriction occurs and what are the mechanisms responsible for the switch from multipotency to unipotency during MG morphogenesis. Here, we performed multicolor lineage tracing and assessed the fate of single progenitors and demonstrated the existence of a developmental switch from multipotency to unipotency during embryonic MG development. Molecular profiling and single cell RNA-seq revealed that EMPs express a unique hybrid basal and luminal signature and the factors associated with the different lineages. Sustained p63 expression in EMPs promotes unipotent BC fate and was sufficient to reprogram adult LCs into BCs by promoting an intermediate hybrid multipotent like state. Altogether, this study identifies the timing and the mechanisms mediating the early lineage segregation of multipotent progenitors during MG development.

O-GlcNAc on PKCζ Inhibits the FGF4-PKCζ-MEK-ERK1/2 Pathway via Inhibition of PKCζ Phosphorylation in Mouse Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ.

The chromatin modifier Satb1 regulates cell fate through Fgf signalling in the early mouse embryo.

The separation of embryonic from extra-embryonic tissues within the inner cell mass to generate the epiblast (EPI), which will form the new organism, from the primitive endoderm (PE), which will form the yolk sac, is a crucial developmental decision. Here, we identify a chromatin modifier, Satb1, with a distinct role in this decision. Satb1 is differentially expressed within 16-cell-stage embryos, with higher expression levels in the inner cell mass progenitor cells. Depleting Satb1 increases the number of EPI cells at the expense of PE. This phenotype can be rescued by simultaneous depletion of both Satb1 and Satb2, owing to their antagonistic effect on the pluripotency regulator Nanog. Consequently, increasing Satb1 expression leads to differentiation into PE and a decrease in EPI, as a result of the modulation of expression of several pluripotency- and differentiation-related genes by Satb1. Finally, we show that Satb1 is a downstream target of the Fgf signalling pathway, linking chromatin modification and Fgf signalling. Together, these results identify a role for Satb1 in the lineage choice between pluripotency and differentiation and further our understanding of early embryonic lineage segregation.

Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis

SummaryNaive pluripotency is manifest in the preimplantation mammalian embryo. Here we determine transcriptome dynamics of mouse development from the eight-cell stage to postimplantation using lineage-specific RNA sequencing. This method combines high sensitivity and reporter-based fate assignment to acquire the full spectrum of gene expression from discrete embryonic cell types. We define expression modules indicative of developmental state and temporal regulatory patterns marking the establishment and dissolution of naive pluripotency in vivo. Analysis of embryonic stem cells and diapaused embryos reveals near-complete conservation of the core transcriptional circuitry operative in the preimplantation epiblast. Comparison to inner cell masses of marmoset primate blastocysts identifies a similar complement of pluripotency factors but use of alternative signaling pathways. Embryo culture experiments further indicate that marmoset embryos utilize WNT signaling during early lineage segregation, unlike rodents. These findings support a conserved transcription factor foundation for naive pluripotency while revealing species-specific regulatory features of lineage segregation.

Heterogeneities in Nanog Expression Drive Stable Commitment to Pluripotency in the Mouse Blastocyst.

The pluripotent epiblast (EPI) is the founder tissue of almost all somatic cells. EPI and primitive endoderm (PrE) progenitors arise from the inner cell mass (ICM) of the blastocyst-stage embryo. The EPI lineage isdistinctly identified by its expression of pluripotency-associated factors. Many of these factors have been reported to exhibit dynamic fluctuations of expression in embryonic stem cell cultures. Whether these fluctuations correlating with ICM fate choice occur invivo remains an open question. Using single-cell resolution quantitative imaging of a Nanog transcriptional reporter, we noted an irreversible commitment to EPI/PrE lineages invivo. A period of apoptosis occurred concomitantly with ICM cell-fate choice, followed by a burst of EPI-specific cell proliferation. Transitions were occasionally observed from PrE-to-EPI, but not vice versa, suggesting that they might be regulated and not stochastic. We propose that the rapid timescale of early mammalian embryonic development prevents fluctuations in cell fate.

Inhibition of transforming growth factor β signaling promotes epiblast formation in mouse embryos.

Early lineage segregation in preimplantation embryos and maintenance of pluripotency in embryonic stem cells (ESCs) are both regulated by specific signaling pathways. Small molecules have been shown to modulate these signaling pathways. We examined the influence of several small molecules and growth factors on second-lineage segregation of the inner cell mass toward hypoblast and epiblast lineage during mouse embryonic preimplantation development. We found that the second-lineage segregation is influenced by activation or inhibition of the transforming growth factor (TGF)β pathway. Inhibition of the TGFβ pathway from the two-cell, four-cell, and morula stages onward up to the blastocyst stage significantly increased the epiblast cell proliferation. The epiblast formed in the embryos in which TGFβ signaling was inhibited was fully functional as demonstrated by the potential of these epiblast cells to give rise to pluripotent ESCs. Conversely, activating the TGFβ pathway reduced epiblast formation. Inhibition of the glycogen synthase kinase (GSK)3 pathway and activation of bone morphogenetic protein 4 signaling reduced the formation of both epiblast and hypoblast cells. Activation of the protein kinase A pathway and of the Janus kinase/signal transducer and activator of transcription 3 pathway did not influence the second-lineage segregation in mouse embryos. The simultaneous inhibition of three pathways--TGFβ, GSK3β, and the fibroblast growth factor (FGF)/extracellular signal-regulated kinases (Erk)--significantly enhanced the proliferation of epiblast cells than that caused by inhibition of either TGFβ pathway alone or by combined inhibition of the GSK3β and FGF/Erk pathways only.

Day‐1 chick development

The first day of chick development takes place inside the mother hen (in utero), during which the embryo progresses from fertilization to late blastula/early gastrula formation. The salient features of developmental anatomy in this period are conserved among the sauropsids (birds and reptiles). Many of these features are also shared in prototherian (monotreme) embryos, whereas metatherian (marsupial) and eutherian (placental) embryos display significant variations. Important for understanding the evolution of early development in amniotes, the knowledge of cellular and molecular mechanisms regulating in utero chick development may also offer valuable insight into early lineage specification in prototherians and conserved features in mammalian early development. This commentary provides a snapshot of what is currently known about intrauterine chick development and identifies key issues that await further clarification, including the process of cellularization, allocation of maternal determinants, zygotic gene activation, mid-blastula transition, cell layer increase and reduction, radial symmetry breaking, early lineage segregation, and role of yolk syncytium in early patterning.

Regulatory microRNA Network Identification in Bovine Blastocyst Development

Mammalian blastocyst formation is characterized by two lineage segregations resulting in the formation of the trophectoderm, the hypoblast, and the epiblast cell lineages. Cell fate determination during these early lineage segregations is associated with changes in the expression of specific transcription factors. In addition to the transcription factor-based control, it has become clear that also microRNAs (miRNAs) play an important role in the post-transcriptional regulation of pluripotency and differentiation. To elucidate the role of miRNAs in early lineage segregation, we compared the miRNA expression in early bovine blastocysts with the more advanced stage of hatched blastocysts. Reverse transcription-quantitative PCR-based miRNA expression profiling revealed eight upregulated miRNAs (miR-127, miR-130a, miR-155, miR-196a, miR-203, miR-28, miR-29c, and miR-376a) and four downregulated miRNAs (miR-135a, miR-218, miR-335, and miR-449b) in hatched blastocysts. Through an integrative analysis of matching miRNA and mRNA expression data, candidate miRNA-mRNA interaction pairs were prioritized for validation. Using an in vitro luciferase reporter assay, we confirmed a direct interaction between miR-218 and CDH2, miR-218 and NANOG, and miR-449b and NOTCH1. By interfering with the FGF signaling pathway, we found functional evidence that miR-218, mainly expressed in the inner cell mass, regulates the NANOG expression in the bovine blastocyst in response to FGF signaling. The results of this study expand our knowledge about the miRNA signature of the bovine blastocyst and of the interactions between miRNAs and cell fate regulating transcription factors.

The Combination of Inhibitors of FGF/MEK/Erk and GSK3β Signaling Increases the Number of OCT3/4- and NANOG-Positive Cells in the Human Inner Cell Mass, But Does Not Improve Stem Cell Derivation

In embryonic stem cell culture, small molecules can be used to alter key signaling pathways to promote self-renewal and inhibit differentiation. In mice, small-molecule inhibition of both the FGF/MEK/Erk and the GSK3β pathways during preimplantation development suppresses hypoblast formation, and this results in more pluripotent cells of the inner cell mass (ICM). In this study, we evaluated the effects of different small-molecule inhibitors of the FGF/MEK/Erk and GSK3β pathway on embryo preimplantation development, early lineage segregation, and subsequent embryonic stem cell derivation in the humans. We did not observe any effect on blastocyst formation, but small-molecule inhibition did affect the number of OCT3/4- and NANOG-positive cells in the human ICM. We found that combined inhibition of the FGF/MEK/Erk and GSK3β pathways by PD0325901 and CHIR99021, respectively, resulted in ICMs containing significantly more OCT3/4-positive cells. Inhibition of FGF/MEK/Erk alone as well as in combination with inhibition of GSK3β significantly increased the number of NANOG-positive cells in blastocysts possessing good-quality ICMs. Secondly, we verified the influence of this increased pluripotency after 2i culture on the efficiency of stem cell derivation. Similar human embryonic stem cell (hESC) derivation rates were observed after 2i compared to control conditions, resulting in 2 control hESC lines and 1 hESC line from an embryo cultured in 2i conditions. In conclusion, we demonstrated that FGF/MEK/Erk and GSK3β signaling increases the number of OCT3/4- and NANOG-positive cells in the human ICM, but does not improve stem cell derivation.

Fibroblast Growth Factor Receptor 2 Homodimerization Rapidly Reduces Transcription of the Pluripotency Gene Nanog without Dissociation of Activating Transcription Factors

Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.

Spatial Restriction of Bone Morphogenetic Protein Signaling in Mouse Gastrula through the mVam2-Dependent Endocytic Pathway

The embryonic body plan is established through positive and negative control of various signaling cascades. Late endosomes and lysosomes are thought to terminate signal transduction by compartmentalizing the signaling molecules; however, their roles in embryogenesis remain poorly understood. We showed here that the endocytic pathway participates in the developmental program by regulating the signaling activity. We modified the mouse Vam2 (mVam2) locus encoding a regulator of membrane trafficking. mVam2-deficient cells exhibited abnormally fragmented late endosomal compartments. The mutant cells could terminate signaling after theremoval of the growth factors including TGF-β and EGF, except BMP-Smad1/Smad5 signaling. mVam2-deficient embryos exhibited ectopic activation of BMP signaling and disorganization of embryo patterning. We found that mVam2, which interacts with BMP type I receptor, is required for the spatiotemporal modulation of BMP signaling, via sequestration of the receptor complex in the late stages of the endocytic pathway.

Differential microRNA expression analysis in blastocysts by whole mount in situ hybridization and reverse transcription quantitative polymerase chain reaction on laser capture microdissection samples

There is cumulating evidence that microRNAs (miRNAs) are important regulators of pluripotency and differentiation and, hence, of early lineage segregation in embryo development. To unravel the function of specific miRNAs, it is important not only to analyze miRNA expression in the entire blastocyst but also to determine the site and level of expression in the inner cell mass (ICM) versus trophectoderm (TE). A new strategy has been developed for miRNA expression analysis in ICM and TE using two complementary techniques. By whole mount in situ hybridization (WISH), it was visualized that bta-miR-155 is mainly expressed in the ICM. However, WISH does not provide quantitative data on expression differences between the two cell types. By reverse transcription quantitative polymerase chain reaction (RT–qPCR) on ICM and TE isolates taken from single blastocysts with laser capture microdissection (LCM), it was quantified that bta-miR-155 was 50-fold higher expressed in ICM than in TE. The possibility to quantify both miRNAs and messenger RNAs (mRNAs) in LCM samples offers the opportunity to analyze the expression of both miRNAs and potential targets in one sample. This article shows that a combination of WISH with LCM and subsequent RT–qPCR is a robust strategy to qualitatively and quantitatively analyze differential miRNA expression in discrete cell types of a single blastocyst.

Role of FoxD3 in maintenance of pluripotency and early lineage segregation in human embryonic stem cells

Role of FoxD3 in maintenance of pluripotency and early lineage segregation in human embryonic stem cells

Dynamic Changes in Epigenetic Marks and Gene Expression During Porcine Epiblast Specification

Given the difficulties in establishing bona fide porcine embryonic stem cells, we considered it would be interesting to investigate histone modifications, X chromosome inactivation (XCI), deacetylation, DNA methylation, and gene expression around the time of inner cell mass (ICM) and epiblast formation in sexed embryos. We found that the porcine epiblast expressed lower levels of NANOG and C-MYC, of which, we speculate may be one indication for the difficulties in obtaining embryonic stem cells (ESCs) from the porcine embryonic epiblast. Our research revealed distinct expression of lineage-specific-, early gastrulation-, and pluripotency-associated genes between the E10 epiblast and trophectoderm and between sexes. We determined that H3K27me3 was hypermethylated in the E6 embryo and hypomethylated in the E10 epiblast. Interestingly, we also observed exclusive localization of H3K4me3 in the E6 ICM, which may be a key marker for early lineage segregation in the pig. We also observed that the methyltransferases of H3K4me3, H3K27me3 and H3K9me3 and the DNA methyltransferases differed between male and female E10 embryos, and between tissues. We consider that epigenetic mechanisms, which are modified by specific enzymes, may be important for both early lineage segregation events and XCI, and these may further effect the levels of downstream-targeted gene expression in the different sexes.

Calcineurin-NFAT Signaling Critically Regulates Early Lineage Specification in Mouse Embryonic Stem Cells and Embryos

Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, the signaling pathways that trigger their transition from self-renewal to differentiation remain elusive. Here, we report that calcineurin-NFAT signaling is both necessary and sufficient to switch ESCs from an undifferentiated state to lineage-specific cells and that the inhibition of this pathway can maintain long-term ESC self-renewal independent of leukemia inhibitory factor. Mechanistically, this pathway converges with the Erk1/2 pathway to regulate Src expression and promote the epithelial-mesenchymal transition (EMT), a process required for lineage specification in response to differentiation stimuli. Furthermore, calcineurin-NFAT signaling is activated when the earliest differentiation event occurs in mouse embryos, and its inhibition disrupts extraembryonic lineage development. Collectively, our results demonstrate that the NFAT and Erk1/2 cascades form a signaling switch for early lineage segregation in mouse ESCs and provide significant insights into the regulation of the balance between ESC self-renewal and early lineage specification.

Small‐molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells

The balance of bone morphogenic protein (BMP), transforming growth factor-β (TGFβ)/activin/nodal, and Wnt signals regulates the early lineage segregation of human embryonic stem cells (ESCs). Here we demonstrate that a combination of small-molecule inhibitors of BMP (Dorsomorphin) and TGFβ/activin/nodal (SB431542) signals promotes highly efficient neural induction from both human ESCs and induced pluripotent stem cells (iPSCs). The combination of small molecules had effects on both cell survival and purity of neural differentiation, under conditions of stromal (PA6) cell coculture and feeder-free floating aggregation culture, for all seven pluripotent stem cell lines that we studied, including three ESC and four iPSC lines. Small molecule compounds are stable and cost effective, so our findings provide a promising strategy for controlled production of neurons in regenerative medicine.