Here, we provide fundamental insights into early human development by single-cell RNA-sequencing of human and mouse preimplantation embryos. We elucidate conserved transcriptional programs along with ...those that are human specific. Importantly, we validate our RNA-sequencing findings at the protein level, which further reveals differences in human and mouse embryo gene expression. For example, we identify several genes exclusively expressed in the human pluripotent epiblast, including the transcription factor KLF17. Key components of the TGF-β signalling pathway, including NODAL, GDF3, TGFBR1/ALK5, LEFTY1, SMAD2, SMAD4 and TDGF1, are also enriched in the human epiblast. Intriguingly, inhibition of TGF-β signalling abrogates NANOG expression in human epiblast cells, consistent with a requirement for this pathway in pluripotency. Although the key trophectoderm factors Id2, Elf5 and Eomes are exclusively localized to this lineage in the mouse, the human orthologues are either absent or expressed in alternative lineages. Importantly, we also identify genes with conserved expression dynamics, including Foxa2/FOXA2, which we show is restricted to the primitive endoderm in both human and mouse embryos. Comparison of the human epiblast to existing embryonic stem cells (hESCs) reveals conservation of pluripotency but also additional pathways more enriched in hESCs. Our analysis highlights significant differences in human preimplantation development compared with mouse and provides a molecular blueprint to understand human embryogenesis and its relationship to stem cells.
The peri-implantation window of mammalian development is the crucial window for primordial germ cell (PGC) specification. Whereas pre-implantation dynamics are relatively conserved between species, ...the implantation window marks a stage of developmental divergence between key model organisms, and thus potential variance in the cell and molecular mechanisms for PGC specification. In humans, PGC specification is very difficult to study
To address this, the combined use of human and nonhuman primate embryos, and stem cell-based embryo models are essential for determining the origin of PGCs, as are comparative analyses to the equivalent stages of mouse development. Understanding the origin of PGCs in the peri-implantation embryo is crucial not only for accurate modeling of this essential process using stem cells, but also in determining the role of global epigenetic reprogramming upon which sex-specific differentiation into gametes relies.
There were errors published in Development 142, 3151-3165.In the issue published online on 22 September 2015, Fig. 3 was mislabelled: panels A, B, C and D should have been B, C, D and A, ...respectively. In the legend, the text prior to ‘(A) Cytoscape enrichment map…’ should not have been included. The correct version of the figure and legend now appear online and in print.We apologise to the authors and readers for this mistake.
Mitochondrial DNA (mtDNA) mutations are maternally inherited and are associated with a broad range of debilitating and fatal diseases. Reproductive technologies designed to uncouple the inheritance ...of mtDNA from nuclear DNA may enable affected women to have a genetically related child with a greatly reduced risk of mtDNA disease. Here we report the first preclinical studies on pronuclear transplantation (PNT). Surprisingly, techniques used in proof-of-concept studies involving abnormally fertilized human zygotes were not well tolerated by normally fertilized zygotes. We have therefore developed an alternative approach based on transplanting pronuclei shortly after completion of meiosis rather than shortly before the first mitotic division. This promotes efficient development to the blastocyst stage with no detectable effect on aneuploidy or gene expression. After optimization, mtDNA carryover was reduced to <2% in the majority (79%) of PNT blastocysts. The importance of reducing carryover to the lowest possible levels is highlighted by a progressive increase in heteroplasmy in a stem cell line derived from a PNT blastocyst with 4% mtDNA carryover. We conclude that PNT has the potential to reduce the risk of mtDNA disease, but it may not guarantee prevention.
Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use ...CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.
Human Pre-gastrulation Development Wamaitha, Sissy E; Niakan, Kathy K
Current topics in developmental biology,
2018, Letnik:
128
Journal Article
Recenzirano
Understanding the progression of early human embryonic development prior to implantation is of fundamental biological importance. Greater insights into early developmental events may lead to clinical ...improvements, not only via the establishment of novel stem cell models with increased potential or more physiological relevance, but also by uncovering some underlying causes of infertility, miscarriages, and developmental disorders. The majority of human embryos available for study are those donated to research once they are surplus to family building following in vitro fertilization, though in some countries it is also possible to create embryos using donated gametes. As human embryo development is surprisingly inefficient, with only 40% reaching the blastocyst stage in vitro (French, Sabanegh, Goldfarb, & Desai, 2010; Gardner, Lane, Stevens, Schlenker, & Schoolcraft, 2000), many embryos may not develop to a stage suitable for study. Where legally permitted, the oversight of human embryo research is subject to either ethics approval from a local institutional review board (i.e., China and the United States) or both a national regulator as well as a regional research ethics committee (i.e., the United Kingdom). The study of human development has historically been by necessity comparative, relying on model organisms and stem cell lines to inform analyses. Preimplantation mouse and human embryos in particular exhibit remarkably similar gross morphologies at these early stages of development, although key differences have been identified in gene expression patterns and developmental timing. While recent advances in high-resolution transcriptomic analyses at the single cell level have improved our capability to interrogate expression patterns directly in the human embryo, we still lack an understanding of basic molecular events in the human embryo, including how the first cell lineages become specified. Here, we present a current overview of the major developmental events during human preimplantation development, from fertilization to delineation of the embryonic and extraembryonic lineages prior to implantation. Comparisons to both the mouse and alternative models are included where these have formed the basis for similar investigations in a human context.
Transcription factor-mediated reprograming is a powerful method to study cell fate changes. In this study, we demonstrate that the transcription factor Gata6 can initiate reprograming of multiple ...cell types to induced extraembryonic endoderm stem (iXEN) cells. Intriguingly, Gata6 is sufficient to drive iXEN cells from mouse pluripotent cells and differentiated neural cells. Furthermore, GATA6 induction in human embryonic stem (hES) cells also down-regulates pluripotency gene expression and up-regulates extraembryonic endoderm (ExEn) genes, revealing a conserved function in mediating this cell fate switch. Profiling transcriptional changes following Gata6 induction in mES cells reveals step-wise pluripotency factor disengagement, with initial repression of Nanog and Esrrb, then Sox2, and finally Oct4, alongside step-wise activation of ExEn genes. Chromatin immunoprecipitation and subsequent high-throughput sequencing analysis shows Gata6 enrichment near pluripotency and endoderm genes, suggesting that Gata6 functions as both a direct repressor and activator. Together, this demonstrates that Gata6 is a versatile and potent reprograming factor that can act alone to drive a cell fate switch from diverse cell types.
Development of primordial germ cells (PGCs) is required for reproduction. During PGC development in mammals, major epigenetic remodeling occurs, which is hypothesized to establish an epigenetic ...landscape for sex-specific germ cell differentiation and gametogenesis. In order to address the role of embryonic ectoderm development (EED) and histone 3 lysine 27 trimethylation (H3K27me3) in this process, we created an EED conditional knockout mouse and show that EED is essential for regulating the timing of sex-specific PGC differentiation in both ovaries and testes, as well as X chromosome dosage decompensation in testes. Integrating chromatin and whole genome bisulfite sequencing of epiblast and PGCs, we identified a poised repressive signature of H3K27me3/DNA methylation that we propose is established in the epiblast where EED and DNMT1 interact. Thus, EED joins DNMT1 in regulating the timing of sex-specific PGC differentiation during the critical window when the gonadal niche cells specialize into an ovary or testis.
Display omitted
•EED regulates the timing of PGC differentiation in the embryonic gonad•X decompensation in XY PGCs is regulated by EED•Gametogenesis promoters begin with H3K27me3 and DNA methylation in the epiblast•DNMT1 and EED interact in ESCs and EpiLCs
Lowe and Yen et al. show that promoters of germ cell differentiation genes in the mouse post-implantation epiblast are enriched in H3K27me3 and DNA methylation, with EED regulating the timing of primordial germ cell (PGC) differentiation. This expands our understanding of the epigenetic regulation of PGC differentiation in the embryonic gonad.
Our understanding of the signalling pathways regulating early human development is limited, despite their fundamental biological importance. Here, we mine transcriptomics datasets to investigate ...signalling in the human embryo and identify expression for the insulin and insulin growth factor 1 (IGF1) receptors, along with IGF1 ligand. Consequently, we generate a minimal chemically-defined culture medium in which IGF1 together with Activin maintain self-renewal in the absence of fibroblast growth factor (FGF) signalling. Under these conditions, we derive several pluripotent stem cell lines that express pluripotency-associated genes, retain high viability and a normal karyotype, and can be genetically modified or differentiated into multiple cell lineages. We also identify active phosphoinositide 3-kinase (PI3K)/AKT/mTOR signalling in early human embryos, and in both primed and naïve pluripotent culture conditions. This demonstrates that signalling insights from human blastocysts can be used to define culture conditions that more closely recapitulate the embryonic niche.
Formation of either an ovary or a testis during human embryonic life is one of the most important sex-specific events leading to the emergence of secondary sexual characteristics and sex assignment ...of babies at birth. Our study focused on the sex-specific and sex-indifferent characteristics of the prenatal ovarian stromal cells, cortical cords, and germline, with the discovery that the ovarian mesenchymal cells of the stroma are transcriptionally indistinguishable from the mesenchymal cells of the testicular interstitium. We found that first-wave pre-granulosa cells emerge at week 7 from early supporting gonadal cells with stromal identity and are spatially defined by KRT19 levels. We also identified rare transient state f0 spermatogonia cells within the ovarian cords between weeks 10 and 16. Taken together, our work illustrates a unique plasticity of the embryonic ovary during human development.
PDF
