DNA methylation dynamics during epigenetic reprogramming in the germline and preimplantation embryos.

Methylation of DNA is an essential epigenetic control mechanism in mammals. During embryonic development, cells are directed toward their future lineages, and DNA methylation poses a fundamental epigenetic barrier that guides and restricts differentiation and prevents regression into an undifferentiated state. DNA methylation also plays an important role in sex chromosome dosage compensation, the repression of retrotransposons that threaten genome integrity, the maintenance of genome stability, and the coordinated expression of imprinted genes. However, DNA methylation marks must be globally removed to allow for sexual reproduction and the adoption of the specialized, hypomethylated epigenome of the primordial germ cell and the preimplantation embryo. Recent technological advances in genome-wide DNA methylation analysis and the functional description of novel enzymatic DNA demethylation pathways have provided significant insights into the molecular processes that prepare the mammalian embryo for normal development.

Temporal reduction of LATS kinases in the early preimplantation embryo prevents ICM lineage differentiation.

Cellular localization of the Yes-associated protein (YAP) is dependent on large tumor suppressor (LATS) kinase activity and initiates lineage specification in the preimplantation embryo. We temporally reduced LATS activity to disrupt this early event, allowing its reactivation at later stages. This interference resulted in an irreversible lineage misspecification and aberrant polarization of the inner cell mass (ICM). Complementation experiments revealed that neither epiblast nor primitive endoderm can be established from these ICMs. We therefore conclude that precisely timed YAP localization in early morulae is essential to prevent trophectoderm marker expression in, and lineage specification of, the ICM.

A genetic and developmental pathway from STAT3 to the OCT4-NANOG circuit is essential for maintenance of ICM lineages in vivo.

Although it is known that OCT4-NANOG are required for maintenance of pluripotent cells in vitro, the upstream signals that regulate this circuit during early development in vivo have not been identified. Here we demonstrate, for the first time, signal transducers and activators of transcription 3 (STAT3)-dependent regulation of the OCT4-NANOG circuitry necessary to maintain the pluripotent inner cell mass (ICM), the source of in vitro-derived embryonic stem cells (ESCs). We show that STAT3 is highly expressed in mouse oocytes and becomes phosphorylated and translocates to the nucleus in the four-cell and later stage embryos. Using leukemia inhibitory factor (Lif)-null embryos, we found that STAT3 phosphorylation is dependent on LIF in four-cell stage embryos. In blastocysts, interleukin 6 (IL-6) acts in an autocrine fashion to ensure STAT3 phosphorylation, mediated by janus kinase 1 (JAK1), a LIF- and IL-6-dependent kinase. Using genetically engineered mouse strains to eliminate Stat3 in oocytes and embryos, we firmly establish that STAT3 is essential for maintenance of ICM lineages but not for ICM and trophectoderm formation. Indeed, STAT3 directly binds to the Oct4 and Nanog distal enhancers, modulating their expression to maintain pluripotency of mouse embryonic and induced pluripotent stem cells. These results provide a novel genetic model of cell fate determination operating through STAT3 in the preimplantation embryo and pluripotent stem cells in vivo.

ß-catenin-mediated adhesion is required for successful preimplantation mouse embryo development.

ß-catenin (CTNNB1) is integral to cell adhesion and to the canonical Wnt signaling pathway. The effects of maternal and zygotic CTNNB1 on embryogenesis have each been separately assessed, whereas the effect of its total absence has not. As the 'traditional' conditional Ctnnb1 knockout alleles give rise to truncated CTNNB1 fragments, we designed a new knockout allele incapable of CTNNB1 production. Mouse embryos lacking intact maternal/zygotic CTNNB1 from two knockout strains were examined in detail. Preimplantation embryos are formed, yet abnormalities in their size and shape were found throughout pre- and early postimplantation development. In the absence of the zona pellucida, embryos lacking CTNNB1 undergo fission and these separated blastomeres can become small trophoblastic vesicles, which in turn induce decidual reactions. Comparing the severity of this defective adhesion phenotype in embryos bearing the null allele with those carrying the 'traditional' knockout allele suggests a hypomorphic effect of the truncated CTNNB1 protein fragment, an important observation with possible impact on previous and future studies.

Optimal histone H3 to linker histone H1 chromatin ratio is vital for mesodermal competence in Xenopus.

Cellular differentiation during embryogenesis involves complex gene regulation to enable the activation and repression of genes. Here, we show that mesodermal competence is inhibited in Xenopus embryos depleted of histones H3 and H3.3, which fail to respond to Nodal/Activin signaling and exhibit concomitant loss of mesodermal gene expression. We find that transcriptional activation in gastrula embryos does not correlate with promoter deposition of H3.3. Instead, gastrulation defects in H3.3/H3-deficient embryos are partially rescued with concurrent depletion of the linker histone H1A. In addition, we show that linker histone H1-induced premature loss of mesodermal competence in animal cap explants can be abrogated with the overexpression of nucleosomal H3.3/H3. Our findings establish a chromatin-mediated regulatory mechanism in which a threshold level of H3 is required to prevent H1-induced gene repression, and thus facilitate mesodermal differentiation in response to inductive signaling.

The nuage mediates retrotransposon silencing in mouse primordial ovarian follicles.

Mobilization of endogenous retrotransposons can destabilize the genome, an imminent danger during epigenetic reprogramming of cells in the germline. The P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) pathway is known to silence retrotransposons in the mouse testes. Several piRNA pathway components localize to the unique, germline structure known as the nuage. In this study, we surveyed mouse ovaries and found, for the first time, transient appearance of nuage-like structures in oocytes of primordial follicles. Mouse vasa homolog (MVH), Piwi-like 2 (PIWIL2/MILI) and tudor domain-containing 9 (TDRD9) are present in these structures, whereas aggregates of germ cell protein with ankyrin repeats, sterile alpha motif and leucine zipper (GASZ) localize separately in the cytoplasm. Retrotransposons are silenced in primordial ovarian follicles, and de-repressed upon reduction of piRNA expression in Mvh, Mili or Gasz mutants. However, these null-mutant females, unlike their male counterparts, are fertile, uncoupling retrotransposon activation from sterility.

Controlling Endogenous Retroviruses and Their Chimeric Transcripts During Natural Reprogramming in the Oocyte.

Complete genomic reprogramming happens twice during the life of a genome, once during the formation of gametes in their parents and once after their union at fertilization. For that matter complete genomic reprogramming happens twice in the same parental cell, the oocyte, when it is forming and after it matures and receives the paternal gamete. Control of these processes in this unique single cell is epigenetic, and our understanding of it is based on information gleaned from imprinting, X chromosome inactivation, and activation and silencing of endogenous retroviruses (ERV). Activation of ERVs is attributed to demethylation of chromatin and DNA, whereas silencing requires methylation, attributed to the nuage proteins, which engage the Piwi-interacting RNA pathway and other posttranscriptional mechanisms. This reprogramming process has evolved throughout speciation because in mammals, but not fish, flies and worms, nuage-component muations affect male and female gametogenesis differentially. Transcription of ERVs is derepressed in both sexes in nuage-mutant mice, but whereas males are sterile, females are fertile. Using a proteomic approach we now report molecular interactions between nuage proteins and components of the oocyte cytoplasmic lattice and speculate how this interaction could preserve ERV/host chimeric gene products affecting female fertility.

Developmental fate and lineage commitment of singled mouse blastomeres.

The inside-outside model has been invoked to explain cell-fate specification of the pre-implantation mammalian embryo. Here, we investigate whether cell-cell interaction can influence the fate specification of embryonic blastomeres by sequentially separating the blastomeres in two-cell stage mouse embryos and continuing separation after each cell division throughout pre-implantation development. This procedure eliminates information provided by cell-cell interaction and cell positioning. Gene expression profiles, polarity protein localization and functional tests of these separated blastomeres reveal that cell interactions, through cell position, influence the fate of the blastomere. Blastomeres, in the absence of cell contact and inner-outer positional information, have a unique pattern of gene expression that is characteristic of neither inner cell mass nor trophectoderm, but overall they have a tendency towards a 'trophectoderm-like' gene expression pattern and preferentially contribute to the trophectoderm lineage.

myc maintains embryonic stem cell pluripotency and self-renewal.

While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditionally knocked out both c- and N-myc using myc doubly homozygously floxed mESC lines (cDKO). Both lines of myc cDKO mESC exhibited severely disrupted self-renewal, pluripotency, and survival along with enhanced differentiation. Chimeric embryos injected with DKO mESC most often completely failed to develop or in rare cases survived but with severe defects. The essential nature of myc for self-renewal and pluripotency is at least in part mediated through orchestrating pluripotency-related cell cycle and metabolic programs. This study demonstrates that endogenous myc genes are essential for mESC pluripotency and self-renewal as well as providing the first evidence that myc genes are required for early embryogenesis, suggesting potential mechanisms of myc contribution to iPS cell formation.

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.

Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos.

A comprehensive analysis of transposable element (TE) expression in mammalian full-grown oocytes reveals that LTR class III retrotransposons make an unexpectedly high contribution to the maternal mRNA pool, which persists in cleavage stage embryos. The most abundant transcripts in the mouse oocyte are from the mouse transcript (MT) retrotransposon family, and expression of this and other TE families is developmentally regulated. Furthermore, TEs act as alternative promoters and first exons for a subset of host genes, regulating their expression in full-grown oocytes and cleavage stage embryos. To our knowledge, this is the first example of TEs initiating synchronous, developmentally regulated expression of multiple genes in mammals. We propose that differential TE expression triggers sequential reprogramming of the embryonic genome during the oocyte to embryo transition and in preimplantation embryos.

Assessing the Safety of Human Pluripotent Stem Cells and Their Derivatives for Clinical Applications.

Pluripotent stem cells may acquire genetic and epigenetic variants during culture following their derivation. At a conference organized by the International Stem Cell Initiative, and held at The Jackson Laboratory, Bar Harbor, Maine, October 2016, participants discussed how the appearance of such variants can be monitored and minimized and, crucially, how their significance for the safety of therapeutic applications of these cells can be assessed. A strong recommendation from the meeting was that an international advisory group should be set up to review the genetic and epigenetic changes observed in human pluripotent stem cell lines and establish a framework for evaluating the risks that they may pose for clinical use.

ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice.

Preeclampsia (PE) is a gestational hypertensive syndrome affecting between 5 and 8% of all pregnancies. Although PE is the leading cause of fetal and maternal morbidity and mortality, its molecular etiology is still unclear. Here, we show that ELABELA (ELA), an endogenous ligand of the apelin receptor (APLNR, or APJ), is a circulating hormone secreted by the placenta. Elabela but not Apelin knockout pregnant mice exhibit PE-like symptoms, including proteinuria and elevated blood pressure due to defective placental angiogenesis. In mice, infusion of exogenous ELA normalizes hypertension, proteinuria, and birth weight. ELA, which is abundant in human placentas, increases the invasiveness of trophoblast-like cells, suggesting that it enhances placental development to prevent PE. The ELA-APLNR signaling axis may offer a new paradigm for the treatment of common pregnancy-related complications, including PE.

Honoring the work and life of Leroy C. Stevens. A symposium as part of the International Stem Cell Initiative Workshop.

In 2016, a symposium was convened in Leroy C. Stevens' honor, in association with a meeting of the International Stem Cell Initiative (ISCI). ISCI, funded internationally, is composed of a group of ~100 scientists from many countries, under the leadership of Peter Andrews, who have worked together to characterize a significant number of human pluripotent stem cell lines, to monitor their genetic stability and their differentiation into mature cell types and tissues in vitro and in vivo. Those at the ISCI meeting puzzled through one of the thorniest problems in the therapeutic use of the differentiated derivatives of pluripotent stem cells for human therapy; namely, pluripotent stem cells can differentiate into any cell type in the adult organism, but they also have the capacity for unlimited self-renewal, hence if mutated they may have tumorigenic potential. The meeting considered how these cells might become genetically or epigenetically abnormal and how the safety of these cells for human therapeutic uses could be assessed and assured. The symposium was an opportunity to pay tribute to Leroy Stevens and to the basic science origins of this newest aspect of regenerative medicine. It was a time to reflect on the past and on how it can influence the future of our field.

Development of Teratocarcinomas and Teratomas in Severely Immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/Szj (NSG) Mice.

The embryonic portion of 7-day-old mouse embryos transplanted to extrauterine sites of syngeneic adult animals gives rise to teratoid tumors, which may be either benign [teratomas (T)] or malignant [teratocarcinomas (TC)]. The incidence of embryo-derived TC varies from one mouse strain to another, indicating that some strains are TC-permissive whereas others are relatively TC-nonpermissive. Embryos of a TC-permissive mouse strain (DBA/2J) and a TC-nonpermissive one (C57BL/6J) were transplanted into NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NSG) mice to determine their tumorigenic potential in the absence of functional adaptive and innate immune responses in the hosts. C57BL/6J embryos transplanted to NSG mice gave rise to TC in 31% of cases, whereas the incidence of TC produced from DBA/2J transplanted embryos was 71%. The NSG embryos transplanted to syngeneic hosts gave rise to TC in 67% of cases, allowing the classification of NSG as a TC-permissive strain. A previously reported correlation between teratocarcinoma and splenomegaly was also observed in the NSG mice. The capacity of these tumors to differentiate into the cells and tissues of the normal embryo is mapped through a detailed histological analysis. These data suggest that teratocarcinogenesis, in the absence of host innate and adaptive immunity, is largely determined by the genetic background of the embryo.

Erase-Maintain-Establish: Natural Reprogramming of the Mammalian Epigenome.

The genetic information is largely identical across most cell types in a given organism but the epigenome, which controls expression of the genome, is cell type- and context-dependent. Although most mature mammalian cells appear to have a stable, heritable epigenome, a dynamic intricate process reshapes it as these cells transition from soma to germline and back again. During normal embryogenesis, primordial germ cells, of somatic origin, are set aside to become gametes. In doing so their genome is reprogrammed-that is, the epigenome of specific regions is replaced in a sex-specific fashion as they terminally differentiate into oocytes or spermatocytes in the gonads. Upon union of these gametes, reprogramming of the new organism's epigenome is initiated, which eventually leads, through pluripotent cells, to the cell lineages required for proper embryonic development to a sexually mature adult. This never-ending cycle of birth and rebirth is accomplished through methylation and demethylation of specific genomic sites within the gametes and pluripotent cells of an organism. This enigmatic process of natural epigenomic reprogramming is now being dissected in vivo, focusing on specific genomic regions-that is, imprinted genes and retrotransposons, where TRIM28 molecular complexes appear to guide the transition from gamete to embryo.

Proteotypic classification of spontaneous and transgenic mammary neoplasms.

INTRODUCTION: Mammary tumors in mice are categorized by using morphologic and architectural criteria. Immunolabeling for terminal differentiation markers was compared among a variety of mouse mammary neoplasms because expression of terminal differentiation markers, and especially of keratins, provides important information on the origin of neoplastic cells and their degree of differentiation. METHODS: Expression patterns for terminal differentiation markers were used to characterize tumor types and to study tumor progression in transgenic mouse models of mammary neoplasia (mice overexpressing Neu (Erbb2), Hras, Myc, Notch4, SV40-TAg, Tgfa, and Wnt1), in spontaneous mammary carcinomas, and in mammary neoplasms associated with infection by the mouse mammary tumor virus (MMTV). RESULTS: On the basis of the expression of terminal differentiation markers, three types of neoplasm were identified: first, simple carcinomas composed exclusively of cells with a luminal phenotype are characteristic of neoplasms arising in mice transgenic for Neu, Hras, Myc, Notch4, and SV40-TAg; second, 'complex carcinomas' displaying luminal and myoepithelial differentiation are characteristic of type P tumors arising in mice transgenic for Wnt1, neoplasms arising in mice infected by the MMTV, and spontaneous adenosquamous carcinomas; and third, 'carcinomas with epithelial to mesenchymal transition (EMT)' are a characteristic feature of tumor progression in Hras-, Myc-, and SV40-TAg-induced mammary neoplasms and PL/J and SJL/J mouse strains, and display de novo expression of myoepithelial and mesenchymal cell markers. In sharp contrast, EMT was not detected in papillary adenocarcinomas arising in BALB/cJ mice, spontaneous adenoacanthomas, neoplasms associated with MMTV-infection, or in neoplasms arising in mice transgenic for Neu and Wnt1. CONCLUSIONS: Immunohistochemical profiles of complex neoplasms are consistent with a stem cell origin, whereas simple carcinomas might originate from a cell committed to the luminal lineage. In addition, these results suggest that the initiating oncogenic events determine the morphologic features associated with cancer progression because EMT is observed only in certain types of neoplasm.

A tudor domain protein SPINDLIN1 interacts with the mRNA-binding protein SERBP1 and is involved in mouse oocyte meiotic resumption.

Mammalian oocytes are arrested at prophase I of meiosis, and resume meiosis prior to ovulation. Coordination of meiotic arrest and resumption is partly dependent on the post-transcriptional regulation of maternal transcripts. Here, we report that, SPINDLIN1 (SPIN1), a maternal protein containing Tudor-like domains, interacts with a known mRNA-binding protein SERBP1, and is involved in regulating maternal transcripts to control meiotic resumption. Mouse oocytes deficient for Spin1 undergo normal folliculogenesis, but are defective in resuming meiosis. SPIN1, via its Tudor-like domain, forms a ribonucleoprotein complex with SERBP1, and regulating mRNA stability and/or translation. The mRNA for the cAMP-degrading enzyme, PDE3A, is reduced in Spin1 mutant oocytes, possibly contributing to meiotic arrest. Our study demonstrates that Spin1 regulates maternal transcripts post-transcriptionally and is involved in meiotic resumption.

Single-cell DNA-methylation analysis reveals epigenetic chimerism in preimplantation embryos.

Epigenetic alterations are increasingly recognized as causes of human cancers and disease. These aberrations are likely to arise during genomic reprogramming in mammalian preimplantation embryos, when their epigenomes are most vulnerable. However, this process is only partially understood because of the experimental inaccessibility of early-stage embryos. Here, we introduce a methodologic advance, probing single cells for various DNA-methylation errors at multiple loci, to reveal failed maintenance of epigenetic mark results in chimeric mice, which display unpredictable phenotypes leading to developmental arrest. Yet we show that mouse pronuclear transfer can be used to ameliorate such reprogramming defects. This study not only details the epigenetic reprogramming dynamics in early mammalian embryos but also suggests diagnostic and potential future therapeutic applications.

BCL-XL mediates the strong selective advantage of a 20q11.21 amplification commonly found in human embryonic stem cell cultures.

Human embryonic stem cells (hESCs) regularly acquire nonrandom genomic aberrations during culture, raising concerns about their safe therapeutic application. The International Stem Cell Initiative identified a copy number variant (CNV) amplification of chromosome 20q11.21 in 25% of hESC lines displaying a normal karyotype. By comparing four cell lines paired for the presence or absence of this CNV, we show that those containing this amplicon have higher population doubling rates, attributable to enhanced cell survival through resistance to apoptosis. Of the three genes encoded within the minimal amplicon and expressed in hESCs, only overexpression of BCL2L1 (BCL-XL isoform) provides control cells with growth characteristics similar to those of CNV-containing cells, whereas inhibition of BCL-XL suppresses the growth advantage of CNV cells, establishing BCL2L1 as a driver mutation. Amplification of the 20q11.21 region is also detectable in human embryonal carcinoma cell lines and some teratocarcinomas, linking this mutation with malignant transformation.