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Program of monkey embryogenesis and germ cell formation in vitro [Bulk-seq]

ABSTRACT: Embryo development of post-implantation and gastrulation is a milestone of embryogenesis. However, the fundamental mechanisms underlying this process remain elusive as technological and ethical limitations. Here, we we improved an in vitro culture system that cynomolgus monkey embryos could develop to 20 days without any maternal tissues, which clearly reveal lineage specification, bi-laminar disc generation, amniotic and yolk sac formation, and primordial germ cell like cells (PGCLC). By applying single cell-omics analysis, unsupervised dimensionality reduction and clustering algorithm data show clear, distinct, stepwise development routes for the epiblast, primitive endoderm, trophectoderm and extra-embryonic mesenchyme lineage. We also found development path of PGCLCs, suggesting origin of PGLCs in amnion instead of EPI. Furthermore, signal pathways analysis showed existence of intimate interactions among lineages. By sequencing (scATAC-seq ) assay for transposase-accessible chromatin, we identify transcription factor grammar specifying of each cell type (EPI, PE and TE) and infer potential master transcriptional regulators. Collectively, the results provide the new insight into understanding molecular and cellular dynamics that underlie monkey post-implantation development, especially beyond E13. Our study provides a basic knowledge about regulation of primate pluripotency in vitro for cell replacement therapy.

ORGANISM(S): Macaca fascicularis

PROVIDER: GSE149974 | GEO | 2020/05/07

REPOSITORIES: GEO

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Program of monkey embryogenesis and germ cell formation in vitro [RNA-seq]

Project description:Embryo development of post-implantation and gastrulation is a milestone of embryogenesis. However, the fundamental mechanisms underlying this process remain elusive as technological and ethical limitations. Here, we we improved an in vitro culture system that cynomolgus monkey embryos could develop to 20 days without any maternal tissues, which clearly reveal lineage specification, bi-laminar disc generation, amniotic and yolk sac formation, and primordial germ cell like cells (PGCLC). By applying single cell-omics analysis, unsupervised dimensionality reduction and clustering algorithm data show clear, distinct, stepwise development routes for the epiblast, primitive endoderm, trophectoderm and extra-embryonic mesenchyme lineage. We also found development path of PGCLCs, suggesting origin of PGLCs in amnion instead of EPI. Furthermore, signal pathways analysis showed existence of intimate interactions among lineages. By sequencing (scATAC-seq ) assay for transposase-accessible chromatin, we identify transcription factor grammar specifying of each cell type (EPI, PE and TE) and infer potential master transcriptional regulators. Collectively, the results provide the new insight into understanding molecular and cellular dynamics that underlie monkey post-implantation development, especially beyond E13. Our study provides a basic knowledge about regulation of primate pluripotency in vitro for cell replacement therapy.

2020-05-07 | GSE150004 | GEO

A developmental coordinate of the spectrum of pluripotency among mice, monkeys, and humans

Project description:The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of the spectrum of pluripotent stem cells (PSCs) in vitro. To explore the ontogeny of human/primate pluripotency, we performed comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys. Here we show that after specification in the blastocysts [embryonic day (E)7], cyEPI undergoes major transcriptome changes upon implantation. Thereafter, cyEPI, while generating gastrulating cells (~E13), maintains its transcriptome relatively stably over a week, retaining a unique set of pluripotency genes while acquiring properties for âneuron differentiation.â h/cyPSCs show the highest similarity to post-implantation late cyEPI (~E17), which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI (E5.5) and epiblast-like cells (EpiLCs) in vitro. These findings not only reveal divergence/coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro. Single cell transcriptome analysis of cynomolgus monkey embryo E6-17 and mouse embryo E4.5 - E6.5 as well as of cynomogus monkey embryonic stem cells (ESCs) and mouse ESC, epiblast like cells (EpiLCs), using SC3-seq technology.

2016-08-24 | E-GEOD-74767 | biostudies-arrayexpress

Reconstituting the transcriptome and DNA methylome landscapes of human implantation

Project description:Implantation is a milestone event during mammalian embryogenesis. Implantation failure is a nonnegligible cause of human early pregnancy loss. Due to the extreme difficulty of obtaining in vivo human early post-implantation embryos, it remains elusive how the gene regulatory network and epigenetic mechanisms control human embryo implantation. Here, combining an in vitro culture system for human post-implantation development and single-cell omics sequencing technologies, over 8,000 individual cells from 65 human peri-implantation embryos were systematically analyzed. Unsupervised dimensionality reduction and clustering algorithm of the transcriptome data show stepwise implantation routes for the epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE) lineages, suggesting robust preparation for the proper establishment of a mother-to-offspring connection during implantation. Female embryos showed initiation of random X chromosome inactivation based on analysis of parental allele-specific expression of X chromosome-linked genes during implantation. Surprisingly, by the single-cell Trio-Seq (scTrio-Seq) analysis, the genome re-methylation of PE lineage was shown to be much slower than those of both EPI and TE lineages during implantation process, indicating distinct DNA methylome re-establishment features between EPI and PE although both of which were derived from inner cell mass (ICM). Collectively, our work paves the way for understanding the complex molecular mechanisms that regulate human embryo implantation, offering new insights and future efforts in early embryonic development and reproductive medicine.

2019-07-11 | GSE109555 | GEO

Reconstituting the transcriptome and DNA methylome landscapes of human implantation at single-cell resolution

Project description:Implantation is a milestone event during mammalian embryogenesis. Due to the extreme difficulty of obtaining in vivo human early post-implantation embryos, the gene regulatory network and epigenetics controlling human embryo implantation remains elusive. Here, combining an in vitro culture system for human post-implantation development and single-cell omics sequencing technologies, over 10,000 single cells at five representative stages of pre/post-implantation development were systematically analyzed. Unsupervised dimensionality reduction and clustering algorithm of the transcriptome data show stepwise implantation routes for the epiblast, primitive endoderm, and trophectoderm lineages, suggesting preparation for the establishment of a mother-to-offspring connection after implantation. Female embryos showed asynchronous progress of dosage loss of X chromosomes during implantation. Furthermore, using the single cell trio-Seq (scTrio-Seq) strategy, re-methylation of the genomes of all the three lineages was unambiguously revealed. Surprisingly, the genome re-methylation of PE lineage were much slower than both EPI and TE lineages during the implantation process, indicating distinct methylome features between EPI and PE although both of which were derived from ICM. Collectively, our work paves the way for understanding the complex molecular mechanisms that regulate human embryo implantation, informing new insights and future efforts in early embryonic development and reproductive medicine.

| EGAS00001003443 | EGA

A developmental coordinate of the spectrum of pluripotency among mice, monkeys, and humans

Project description:The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of the spectrum of pluripotent stem cells (PSCs) in vitro. To explore the ontogeny of human/primate pluripotency, we performed comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys. Here we show that after specification in the blastocysts [embryonic day (E)7], cyEPI undergoes major transcriptome changes upon implantation. Thereafter, cyEPI, while generating gastrulating cells (~E13), maintains its transcriptome relatively stably over a week, retaining a unique set of pluripotency genes while acquiring properties for “neuron differentiation.” h/cyPSCs show the highest similarity to post-implantation late cyEPI (~E17), which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI (E5.5) and epiblast-like cells (EpiLCs) in vitro. These findings not only reveal divergence/coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro.

2016-08-24 | GSE74767 | GEO

scRNA-seq analysis of in vitro cultured monkey embryos

Project description:Primates embryo implants into the maternal uterus, forms three germ layers and begin early organogenesis, such as neurulation, heart formation, during the first 4 weeks of development. Although, this stage represents a milestone in early embryonic development of primate, our understanding remains limited. Here, we optimized our previously established in vitro culture system, which enable the ex-utero culture of cynomolgus monkey up to 25 days. Morphology histological, and single-cell RNA sequencing analyses confirm that the in vitro cultured embryos recapitulate key events of in vivo development. Using this platform, we delineate monkey early-stage lineage trajectory and genetic programs accompanying the neural, mesoderm and endoderm speciﬁcation, as well as primordial germ cell–like cell (PGCLC). Thus, this culture system represents a valuable and easily accessible platform to study and manipulate early primate embryogenesis, which cannot easily accomplish in vivo.

2023-05-11 | GSE207534 | GEO

The Long Terminal Repeats of ERV6 Are Activated in Pre-Implantation Embryos of Cynomolgus Monkey

Project description:Precise temporal development is an important event during embryo development. However, transcriptional changes in the temporal development of early epiblast cells (EPI), primitive endoderm (PrE) and trophectoderm (TrB) cells remain unclear. Long terminal repeat elements (LTRs) of ERVs (Endogenous retroviruses) are dynamically expressed in blastocysts of mammalian embryos. However, the expression of LTRs in monkey blastocyst is still unknown. By analyzing publicly available single-cell RNA-sequencing (seq) data, LTRs of several ERV families, including MacERV6, MacERV3, MacERV2, MacERVK1 and MacERVK2 were highly expressed in pre-implantation embryo cells including EPIs, TrB and PrE but absent in post-implantation. We knockdown MacERV6-LTR1a in cynomolgus monkey with short hairpin RNA (shRNA) strategy to examine the potential function of MacERV6-LTR1a in early development of monkey embryo. The silence of MacERV6-LTR1a mainly postpone trophectodermal cell differentiation and cell polarity of TrB, and the genes relate to epithelial proliferation, differentiation and development were downregulated in EPI of shRNA-treated embyos. Addtionally, stem cell differentiation relate genes were downregulated in PrE when MacERV6-LTR1a was silenced. Moreover, we have comfirmed MacERV6-LTR1a can recruit ESRRB (Estrogen Related Receptor Beta). Actually, recent reports demonstrated that ESRRB plays a important role in maintenance of self-renewal and pluripotency of embryonic and trophoblast stem cells through different signaling pathways including FGF and Wnt signaling pathways. In summary, these results suggest that MacERV6-LTR1a potentially regulates the temporal development of pre-implantation embryo of cynomolgus monkey.

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2019-08-22 | GSE112906 | GEO

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