Transcriptional and epigenetic control of cell fate decisions in early embryos

Mammalian embryo development is characterized by regulative mechanisms of lineage segregation and cell specification. A combination of carefully orchestrated gene expression networks, signalling pathways and epigenetic marks define specific developmental stages that can now be resolved at the single-cell level. These new ways to depict developmental processes have the potential to provide answers to unresolved questions on how lineage allocation and cell fate decisions are made during embryogenesis. Over the past few years, a flurry of studies reporting detailed single-cell transcription profiles in early embryos has complemented observations acquired using live-cell imaging following gene editing techniques to manipulate specific genes. The adoption of this newly available toolkit is reshaping how researchers are designing experiments and how they view animal development. This review presents an overview of the current knowledge on lineage segregation and cell specification in mammals, and discusses some of the outstanding questions that current technological advances can help scientists address, like never before

Conserved features of non-primate bilaminar disc embryos and the germline.

Post-implantation embryo development commences with a bilaminar disc in most mammals, including humans. Whereas access to early human embryos is limited and subject to greater ethical scrutiny, studies on non-primate embryos developing as bilaminar discs offer exceptional opportunities for advances in gastrulation, the germline, and the basis for evolutionary divergence applicable to human development. Here, we discuss the advantages of investigations in the pig embryo as an exemplar of development of a bilaminar disc embryo with relevance to early human development. Besides, the pig has the potential for the creation of humanized organs for xenotransplantation. Precise genetic engineering approaches, imaging, and single-cell analysis are cost effective and efficient, enabling research into some outstanding questions on human development and for developing authentic models of early human development with stem cells

Epigenetic reprogramming in the porcine germ line

<p>Abstract</p> <p>Background</p> <p>Epigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.</p> <p>Results</p> <p>Our results show loss of DNA methylation in PGC colonizing the genital ridges. Analysis of <it>IGF2-H19 </it>regulatory region showed a gradual demethylation between E22-E42. In contrast, DMR2 of <it>IGF2R </it>was already demethylated in male PGC by E22. In females, <it>IGF2R </it>demethylation was delayed until E29-31, and was de novo methylated by E42. DNA repeats were gradually demethylated from E25 to E29-31, and became de novo methylated by E42. Analysis of histone marks showed strong H3K27me3 staining in migratory PGC between E15 and E21. In contrast, H3K9me2 signal was low in PGC by E15 and completely erased by E21. Cell cycle analysis of gonadal PGC (E22-31) showed a typical pattern of cycling cells, however, migrating PGC (E17) showed an increased proportion of cells in G2.</p> <p>Conclusions</p> <p>Our study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding of the sequential reprogramming of PGC in the pig will facilitate the derivation of embryonic germ cells in this species.</p

Actin depolymerization is associated with meiotic acceleration in cycloheximide-treated ovine oocytes

Oocytes treated with the protein synthesis inhibitor cycloheximide (CHX) arrest at the germinal vesicle (GV) stage and undergo accelerated GV breakdown (GVBD) after CHX is removed. However, little is known about the underlying mechanism of accelerated meiotic maturation. Here, we investigated this mechanism and found that oocytes released from CHX arrest have higher amounts of cyclin B1 (CCNB1) and phosphorylated mitogen-activated protein kinase (pMAPK) proteins. Increased levels of these factors were not associated with mRNA polyadenylation or increased transcription rates of CCNB1 and MOS (Moloney murine sarcoma viral oncogene homolog) during CHX arrest. We found that treatment of CHX-arrested oocytes with the actin filament-stabilizing agent Jasplakinolide (Jasp) delayed GVBD following release from CHX arrest and that this was correlated with reduced maturation-promoting factor (MPF) activity. These results suggest that CCNB1 mRNAs released from actin filaments during CHX arrest increase CCNB1 transcripts available for translation after release from CHX arrest, leading to the precocious activation of MPF and accelerated meiotic progression

Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer

Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs

Germline competent mesoderm: the substrate for vertebrate germline and somatic stem cells?

In vitro production of tissue-specific stem cells [e.g. haematopoietic stem cells (HSCs)] is a key goal of regenerative medicine. However, recent efforts to produce fully functional tissue-specific stem cells have fallen short. One possible cause of shortcomings may be that model organisms used to characterize basic vertebrate embryology (Xenopus, zebrafish, chick) may employ molecular mechanisms for stem cell specification that are not conserved in humans, a prominent example being the specification of primordial germ cells (PGCs). Germ plasm irreversibly specifies PGCs in many models; however, it is not conserved in humans, which produce PGCs from tissue termed germline-competent mesoderm (GLCM). GLCM is not conserved in organisms containing germ plasm, or even in mice, but understanding its developmental potential could unlock successful production of other stem cell types. GLCM was first discovered in embryos from the axolotl and its conservation has since been demonstrated in pigs, which develop from a flat-disc embryo like humans. Together these findings suggest that GLCM is a conserved basal trait of vertebrate embryos. Moreover, the immortal nature of germ cells suggests that immortality is retained during GLCM specification; here we suggest that the demonstrated pluripotency of GLCM accounts for retention of immortality in somatic stem cell types as well. This article has an associated Future Leaders to Watch interview with the author of the paper

NANOG controls testicular germ cell tumour stemness through regulation of MIR9-2

BackgroundTesticular germ cell tumours (TGCTs) represent a clinical challenge; they are most prevalent in young individuals and are triggered by molecular mechanisms that are not fully understood. The origin of TGCTs can be traced back to primordial germ cells that fail to mature during embryonic development. These cells express high levels of pluripotency factors, including the transcription factor NANOG which is highly expressed in TGCTs. Gain or amplification of the NANOG locus is common in advanced tumours, suggesting a key role for this master regulator of pluripotency in TGCT stemness and malignancy.MethodsIn this study, we analysed the expression of microRNAs (miRNAs) that are regulated by NANOG in TGCTs via integrated bioinformatic analyses of data from The Cancer Genome Atlas and NANOG chromatin immunoprecipitation in human embryonic stem cells. Through gain-of-function experiments, MIR9-2 was further investigated as a novel tumour suppressor regulated by NANOG. After transfection with MIR9-2 mimics, TGCT cells were analysed for cell proliferation, invasion, sensitivity to cisplatin, and gene expression signatures by RNA sequencing.ResultsFor the first time, we identified 86 miRNAs regulated by NANOG in TGCTs. Among these, 37 miRNAs were differentially expressed in NANOG-high tumours, and they clustered TGCTs according to their subtypes. Binding of NANOG within 2 kb upstream of the MIR9-2 locus was associated with a negative regulation. Low expression of MIR9-2 was associated with tumour progression and MIR9-2-5p was found to play a role in the control of tumour stemness. A gain of function of MIR9-2-5p was associated with reduced proliferation, invasion, and sensitivity to cisplatin in both embryonal carcinoma and seminoma tumours. MIR9-2-5p expression in TGCT cells significantly reduced the expression of genes regulating pluripotency and cell division, consistent with its functional effect on reducing cancer stemness.ConclusionsThis study provides new molecular insights into the role of NANOG as a key determinant of pluripotency in TGCTs through the regulation of MIR9-2-5p, a novel epigenetic modulator of cancer stemness. Our data also highlight the potential negative feedback mediated by MIR9-2-5p on NANOG expression, which could be exploited as a therapeutic strategy for the treatment of TGCTs

In vitro culture of ovine embryos up to early gastrulating stages

Developmental failures occurring shortly after blastocyst hatching from the zona pellucida constitute a major cause of pregnancy losses in both humans and farm ungulates. The developmental events occurring following hatching in ungulates include the proliferation and maturation of extra-embryonic membranes - trophoblast and hypoblast - and the formation of a flat embryonic disc, similar to that found in humans, which initiates gastrulation prior to implantation. Unfortunately, our understanding of these key processes for embryo survival is limited because current culture systems cannot sustain ungulate embryo development beyond hatching. Here, we report a culture system that recapitulates most developmental landmarks of gastrulating ovine embryos: trophoblast maturation, hypoblast migration, embryonic disc formation, disappearance of the Rauber's layer, epiblast polarization and mesoderm differentiation. Our system represents a highly valuable platform for exploring the cell differentiation, proliferation and migration processes governing gastrulation in a flat embryonic disc and for understanding pregnancy failures during the second week of gestation. This article has an associated 'The people behind the papers' interview

Pluripotency and X chromosome dynamics revealed in pig pre-gastrulating embryos by single cell analysis.

High-resolution molecular programmes delineating the cellular foundations of mammalian embryogenesis have emerged recently. Similar analysis of human embryos is limited to pre-implantation stages, since early post-implantation embryos are largely inaccessible. Notwithstanding, we previously suggested conserved principles of pig and human early development. For further insight on pluripotent states and lineage delineation, we analysed pig embryos at single cell resolution. Here we show progressive segregation of inner cell mass and trophectoderm in early blastocysts, and of epiblast and hypoblast in late blastocysts. We show that following an emergent short naive pluripotent signature in early embryos, there is a protracted appearance of a primed signature in advanced embryonic stages. Dosage compensation with respect to the X-chromosome in females is attained via X-inactivation in late epiblasts. Detailed human-pig comparison is a basis towards comprehending early human development and a foundation for further studies of human pluripotent stem cell differentiation in pig interspecies chimeras

Epigenetic reprogramming of breast cancer cells with oocyte extracts

<p>Abstract</p> <p>Background</p> <p>Breast cancer is a disease characterised by both genetic and epigenetic alterations. Epigenetic silencing of tumour suppressor genes is an early event in breast carcinogenesis and reversion of gene silencing by epigenetic reprogramming can provide clues to the mechanisms responsible for tumour initiation and progression. In this study we apply the reprogramming capacity of oocytes to cancer cells in order to study breast oncogenesis.</p> <p>Results</p> <p>We show that breast cancer cells can be directly reprogrammed by amphibian oocyte extracts. The reprogramming effect, after six hours of treatment, in the absence of DNA replication, includes DNA demethylation and removal of repressive histone marks at the promoters of tumour suppressor genes; also, expression of the silenced genes is re-activated in response to treatment. This activity is specific to oocytes as it is not elicited by extracts from ovulated eggs, and is present at very limited levels in extracts from mouse embryonic stem cells. Epigenetic reprogramming in oocyte extracts results in reduction of cancer cell growth under anchorage independent conditions and a reduction in tumour growth in mouse xenografts.</p> <p>Conclusions</p> <p>This study presents a new method to investigate tumour reversion by epigenetic reprogramming. After testing extracts from different sources, we found that axolotl oocyte extracts possess superior reprogramming ability, which reverses epigenetic silencing of tumour suppressor genes and tumorigenicity of breast cancer cells in a mouse xenograft model. Therefore this system can be extremely valuable for dissecting the mechanisms involved in tumour suppressor gene silencing and identifying molecular activities capable of arresting tumour growth. These applications can ultimately shed light on the contribution of epigenetic alterations in breast cancer and advance the development of epigenetic therapies.</p