Human Germline Cell Development: from the Perspective of Single-Cell Sequencing
2019; Elsevier BV; Volume: 76; Issue: 2 Linguagem: Inglês
10.1016/j.molcel.2019.08.025
ISSN1097-4164
Autores Tópico(s)Pluripotent Stem Cells Research
ResumoGermline cells are the beginning of new individuals in multicellular animals, including humans. Our understanding of these cell types is limited by the difficulty of analyzing the precious and heterogeneous germline tissue samples. The rapid development of single-cell sequencing technologies provides a chance for comprehensive profiling of the omics dynamics of human germline development. In this review, we discuss progress in analyzing the development of human germline cells, including preimplantation and implantation embryos, fetal germ cells (FGCs), and adult spermatogenesis by single-cell transcriptome and epigenome sequencing technologies. Germline cells are the beginning of new individuals in multicellular animals, including humans. Our understanding of these cell types is limited by the difficulty of analyzing the precious and heterogeneous germline tissue samples. The rapid development of single-cell sequencing technologies provides a chance for comprehensive profiling of the omics dynamics of human germline development. In this review, we discuss progress in analyzing the development of human germline cells, including preimplantation and implantation embryos, fetal germ cells (FGCs), and adult spermatogenesis by single-cell transcriptome and epigenome sequencing technologies. Germline cells can transmit genetic information from generation to generation in multicellular organisms (Reik and Surani, 2015Reik W. Surani M.A. Germline and Pluripotent Stem Cells.Cold Spring Harb. Perspect. Biol. 2015; 7: a019422Crossref PubMed Scopus (74) Google Scholar). They include a totipotent zygote derived from fusion of an oocyte and a sperm, the pluripotent inner cell mass (ICM) and peri-implantation epiblasts (EPIs), the unipotent fetal germ cells (FGCs), the postnatal germ cells, and finally the mature oocytes and sperms, which make a complete germline developmental circle (Figure 1). Germline cells are immortal during the millions of years of the evolutionary life of a species. Germline cells can develop into non-germline cells, but not vice versa. For example, the totipotent zygote can develop into the trophectoderm (TE) and the pluripotent EPI can develop into the ectoderm, mesoderm, and endoderm layers. During a developmental process, in contrast to somatic cells, the genomic and epigenomic information in germline cells is particularly important, because it can be transmitted to the next generation and variations in their genome serve as the driving force of genetic diversity and evolution. However, the relatively low sensitivity of early genome-wide sequencing technologies made analyzing the precious and highly heterogeneous samples of human germline cells extremely difficult. In the past ten years, improvements in single-cell sequencing technologies have allowed greater exploration of the diverse cell types that arise during human germline cell development (Table 1; Tang et al., 2009Tang F. Barbacioru C. Wang Y. Nordman E. Lee C. Xu N. Wang X. Bodeau J. Tuch B.B. Siddiqui A. et al.mRNA-Seq whole-transcriptome analysis of a single cell.Nat. Methods. 2009; 6: 377-382Crossref PubMed Scopus (1897) Google Scholar, Wen and Tang, 2016Wen L. Tang F. 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After the fusion of the oocyte and the sperm during fertilization in humans, the zygote begins cell cleavage and develops into the blastocyst comprising three lineages of cells: the TE, the primitive endoderm (PrE), and the epiblast (EPI). TE and PrE will develop into the extra embryonic tissues participating to the placenta and the yolk sac, respectively, whereas the pluripotent EPI will give rise to the whole embryo proper. One bottleneck for studying human preimplantation development is the lack of highly sensitive omics technologies for analyzing scant and precious materials. Human preimplantation development is different from that of the mouse in many aspects, including the separation of three cell lineages and X chromosome inactivation (XCI), which must be resolved at single-cell resolution. Several studies have provided single-cell RNA sequencing (scRNA-seq) data for human preimplantation embryos, with nearly 2,000 individual cells being analyzed (Blakeley et al., 2015Blakeley P. Fogarty N.M. del Valle I. Wamaitha S.E. Hu T.X. Elder K. Snell P. Christie L. Robson P. Niakan K.K. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.Development. 2015; 142: 3151-3165Crossref PubMed Scopus (299) Google Scholar, Dang et al., 2016Dang Y. Yan L. Hu B. Fan X. Ren Y. Li R. Lian Y. Yan J. Li Q. Zhang Y. et al.Tracing the expression of circular RNAs in human pre-implantation embryos.Genome Biol. 2016; 17: 130Crossref PubMed Scopus (117) Google Scholar, Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.Cell. 2016; 165: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, Stirparo et al., 2018Stirparo G.G. Boroviak T. Guo G. Nichols J. Smith A. Bertone P. Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.Development. 2018; 145 (dev158501)Crossref Scopus (0) Google Scholar, Xue et al., 2013Xue Z. Huang K. Cai C. Cai L. Jiang C.Y. Feng Y. Liu Z. Zeng Q. Cheng L. Sun Y.E. et al.Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing.Nature. 2013; 500: 593-597Crossref PubMed Scopus (642) Google Scholar, Yan et al., 2013Yan L. Yang M. Guo H. Yang L. Wu J. Li R. Liu P. Lian Y. Zheng X. Yan J. et al.Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells.Nat. Struct. Mol. Biol. 2013; 20: 1131-1139Crossref PubMed Scopus (1039) Google Scholar). These studies show that the transcriptome of preimplantation embryos is highly dynamic and cells of different stages can be distinguished, reflecting the maternal-to-zygotic transition (MZT) and the differentiation of the blastomeres into three cell lineages. Consistent with the timing of the major MZT, the scRNA-seq data show that the most remarkable gene expression change occurs between the 4- and the 8-cell stages (Yan et al., 2013Yan L. Yang M. Guo H. Yang L. Wu J. Li R. Liu P. Lian Y. Zheng X. Yan J. et al.Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells.Nat. Struct. Mol. Biol. 2013; 20: 1131-1139Crossref PubMed Scopus (1039) Google Scholar). Approximate 2,500 genes are upregulated at that time with strong enrichment for the Gene Ontology (GO) terms "RNA metabolism and translation," as well as "chromosome organization," "cell division," and "DNA packaging," indicating that the zygotic-specific machineries for transcription, translation, epigenetic, and cell division are established as zygotic genome activation (ZGA) (Yan et al., 2013Yan L. Yang M. Guo H. Yang L. Wu J. Li R. Liu P. Lian Y. Zheng X. Yan J. et al.Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells.Nat. Struct. Mol. Biol. 2013; 20: 1131-1139Crossref PubMed Scopus (1039) Google Scholar). The ZGA remains incomplete at the 8-cell stage, as exemplified by the late activation of the Y chromosome genes at the morula stage (Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.Cell. 2016; 165: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). Approximate 1,000 genes are upregulated from the 2- to the 4-cell stage, likely reflecting a minor ZGA wave (Yan et al., 2013Yan L. Yang M. Guo H. Yang L. Wu J. Li R. Liu P. Lian Y. Zheng X. Yan J. et al.Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells.Nat. Struct. Mol. Biol. 2013; 20: 1131-1139Crossref PubMed Scopus (1039) Google Scholar). The absolute counting of mRNA molecules shows that the degradation of maternal mRNA starts at the first cleavage stage and remains incomplete at the 8-cell stage, as exemplified by the continuing degradation of the maternal X chromosome genes at the morula stage (Dang et al., 2016Dang Y. Yan L. Hu B. Fan X. Ren Y. Li R. Lian Y. Yan J. Li Q. Zhang Y. et al.Tracing the expression of circular RNAs in human pre-implantation embryos.Genome Biol. 2016; 17: 130Crossref PubMed Scopus (117) Google Scholar, Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.Cell. 2016; 165: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). Mature cells of all three lineages are clearly identified and well characterized. The EPI-specific, PrE-specific, and TE-specific genes enrich for genes involved in stem cell maintenance and cell fate specification, morphogenesis of epithelium and endoderm development, and apical plasma membrane and active transmembrane transporter activity, respectively, and in accordance with their cellular functions (Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.Cell. 2016; 165: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). The identified lineage-specific marker genes include NANOG and SOX2 for EPI, GATA4 and PDGFRA for PrE, and GATA2 and GATA3 for TE. These studies have also explored the early intermediate cells and the developmental trajectory of these three lineages. The data show that the early intermediate cells are characterized by weak coexpression of lineage-specific genes (Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.Cell. 2016; 165: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar, Stirparo et al., 2018Stirparo G.G. Boroviak T. Guo G. Nichols J. Smith A. Bertone P. Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.Development. 2018; 145 (dev158501)Crossref Scopus (0) Google Scholar). In the mouse, the ICM and TE lineages are segregated first, followed by separation of the EPI and PrE lineages from the ICM (Menchero et al., 2018Menchero S. Sainz de Aja J. Manzanares M. Our First Choice: Cellular and Genetic Underpinnings of Trophectoderm Identity and Differentiation in the Mammalian Embryo.Curr. Top. Dev. Biol. 2018; 128: 59-80Crossref PubMed Scopus (6) Google Scholar, Zhang and Hiiragi, 2018Zhang H.T. Hiiragi T. Symmetry Breaking in the Mammalian Embryo.Annu. Rev. Cell Dev. Biol. 2018; 34: 405-426Crossref PubMed Scopus (30) Google Scholar). The scRNA-seq data show that the human ICM and TE lineages, as well as the EPI and PrE lineages, are distinguished at embryonic day 5 (post-fertilization, corresponding to the early blastula stage). This leads to a model of concurrent establishment of three lineages coinciding with blastocyst formation for human preimplantation development, which is different from the mouse model (Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.Cell. 2016; 165: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). Nevertheless, this model is supported by another study that integrates three human preimplantation scRNA-seq datasets (i.e., datasets from Yan et al., 2013Yan L. Yang M. Guo H. Yang L. Wu J. Li R. Liu P. Lian Y. Zheng X. Yan J. et al.Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells.Nat. Struct. Mol. Biol. 2013; 20: 1131-1139Crossref PubMed Scopus (1039) Google Scholar, Blakeley et al., 2015Blakeley P. Fogarty N.M. del Valle I. Wamaitha S.E. Hu T.X. Elder K. Snell P. Christie L. Robson P. Niakan K.K. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq.Development. 2015; 142: 3151-3165Crossref PubMed Scopus (299) Google Scholar, and Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.Cell. 2016; 165: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar) for analysis (Stirparo et al., 2018Stirparo G.G. Boroviak T. Guo G. Nichols J. Smith A. Bertone P. Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.Development. 2018; 145 (dev158501)Crossref Scopus (0) Google Scholar). An unsolved issue is the XCI in human preimplantation embryos (Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.Cell. 2016; 165: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). To acquire dosage compensation of X chromosome transcripts, the paternal X chromosome is epigenetically repressed as imprinted XCI in the mouse preimplantation embryo (Deng et al., 2014Deng Q. Ramsköld D. Reinius B. Sandberg R. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells.Science. 2014; 343: 193-196Crossref PubMed Scopus (769) Google Scholar). In contrast, human preimplantation embryos do not undergo imprinted XCI (van den Berg et al., 2011van den Berg I.M. Galjaard R.J. Laven J.S. van Doorninck J.H. XCI in preimplantation mouse and human embryos: first there is remodelling….Hum. Genet. 2011; 130: 203-215Crossref PubMed Scopus (24) Google Scholar). A model of expression dampening of both X chromosomes during human preimplantation development has been proposed by the scRNA-seq analysis (Petropoulos et al., 2016Petropoulos S. Edsgärd D. Reinius B. Deng Q. Panula S.P. Codeluppi S. Plaza Reyes A. Linnarsson S. Sandberg R. Lanner F. Single-Cell RNA-Seq Reveals Lineage and X Chromosome Dynamics in Human Preimplantation Embryos.Cell. 2016; 165: 1012-1026Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar). However, a later analysis using a novel pipeline of the same scRNA-seq dataset supports a model of random XCI during the stage (Moreira de Mello et al., 2017Moreira de Mello J.C. Fernandes G.R. Vibranovski M.D. Pereira L.V. Early X chromosome inactivation during human preimplantation development revealed by single-cell RNA-sequencing.Sci. Rep. 2017; 7: 10794Crossref PubMed Scopus (54) Google Scholar). Altogether, despite conflicts related to issues such as cell lineage differentiation and XCI, the scRNA-seq datasets provide resources for proper understanding of human preimplantation development. The human embryo implants in the uterus of the mother on day 7 of development. Our understanding of the human implanting embryos is limited because of inaccessibility of these embryos in vivo. The in vitro culture system to mimic the human embryo implantation process provides an opportunity to investigate this crucial developmental window (Deglincerti et al., 2016Deglincerti A. Croft G.F. Pietila L.N. Zernicka-Goetz M. Siggia E.D. Brivanlou A.H. Self-organization of the in vitro attached human embryo.Nature. 2016; 533: 251-254Crossref PubMed Scopus (405) Google Scholar, Shahbazi et al., 2016Shahbazi M.N. Jedrusik A. Vuoristo S. Recher G. Hupalowska A. Bolton V. Fogarty N.N.M. Campbell A. Devito L. Ilic D. et al.Self-organization of the human embryo in the absence of maternal tissues.Nat. Cell Biol. 2016; 18: 700-708Crossref PubMed Scopus (391) Google Scholar). More than 8,000 individual cells from day 6, 8, 10, 12, and 14 embryos derived from the in vitro culturing system were analyzed by scRNA-seq, and 371 additional individual cells were analyzed for both transcriptome and DNA methylome by single-cell triple omics sequencing (scTrio-seq2; Zhou et al., 2019Zhou F. Wang R. Yuan P. Ren Y. Mao Y. Li R. Lian Y. Li J. Wen L. Yan L. et al.Reconstituting the transcriptome and DNA methylome landscapes of human implantation.Nature. 2019; 572: 660-664Crossref PubMed Scopus (133) Google Scholar). Four main cell clusters were identified, corresponding to EPI, TE, PrE, and ysTE (yolk sac TE). All EPI, TE, and PrE show stepwise gene expression changes during the implantation process. For EPI, the pluripotent transition is observed with gradual downregulation of naive state-associated genes such as PRDM14 and upregulation of primed state-associated genes such as FGF2 and SOX11. For TE and PrE, the gene expression signatures show a trend of preparing for establishment of a mother-to-offspring connection. TE forms two distinct subgroups around day 10 and day 12 as cytotrophoblasts (CTBs, with the marker gene as ITGA6) and syncytiotrophoblasts (STBs, with the marker genes as CGB family genes). By distinguishing the paternal and maternal single-nucleotide polymorphisms (SNPs) in an individual cell, the results demonstrate that a random XCI is gradually strengthened from day 6 to day 12. Most cells expressed X-linked genes in a balanced way from both paternal and maternal alleles at day 6, while at day 12, many cells showed increasingly biased expression of either the paternal or the maternal allele; the biallelic expression ratio gradually decreased from day 6 to day 12. It appears that the XCI is heterogeneous among the cells such that some have completed XCI while others within the same embryo have just started XCI. The earliest FGCs, primordial germ cells (PGCs), are specified in EPIs at the beginning of gastrulation in mammals. Studies in mouse have shown that the most proximal posterior EPI cells are induced into PGCs by BMP4 secreted by extra-embryonic ectoderm (ExE, a derivative of TE), whereas the anterior visceral endoderm (AVE, a derivative of PrE) plays a role in restricting the PGC induction domain by secreting BMP4 antagonists such as Cerberus 1 (Saitou and Miyauchi, 2016Saitou M. Miyauchi H. Gametogenesis from Pluripotent Stem Cells.Cell Stem Cell. 2016; 18: 721-735Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The human PGCs (hPGCs) are thought to be specified approximately 2 weeks post-fertilization. However, understanding specification of hPGCs has been difficult because of the inaccessibility of human embryos at this stage. As a complementary approach, inducing hPGC-like cells (hPGCLCs) from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) in vitro helped in gaining insights into the specification of hPGCs (Irie et al., 2015Irie N. Weinberger L. Tang W.W. Kobayashi T. Viukov S. Manor Y.S. Dietmann S. Hanna J.H. Surani M.A. SOX17 is a critical specifier of human primordial germ cell fate.Cell. 2015; 160: 253-268Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, Sasaki et al., 2015Sasaki K. Yokobayashi S. Nakamura T. Okamoto I. Yabuta Y. Kurimoto K. Ohta H. Moritoki Y. Iwatani C. Tsuchiya H. et al.Robust In Vitro Induction of Human Germ Cell Fate from Pluripotent Stem Cells.Cell Stem Cell. 2015; 17: 178-194Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). The hPGCLCs express early PGC markers such as BLIMP1, TFAP2C, and KIT, as well as pluripotency genes such as TNAP, POU5F1 (OCT4), NANOG, and PRDM14, which are conserved to mouse PGCs. However, hPGCLCs express SOX17 and SOX15, but not SOX2, in contrast to mouse PGCs (Guo et al., 2015Guo F. Yan L. Guo H. Li L. Hu B. Zhao Y. Yong J. Hu Y. Wang X. Wei Y. et al.The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells.Cell. 2015; 161: 1437-1452Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, Irie et al., 2015Irie N. Weinberger L. Tang W.W. Kobayashi T. Viukov S. Manor Y.S. Dietmann S. Hanna J.H. Surani M.A. SOX17 is a critical specifier of human primordial germ cell fate.Cell. 2015; 160: 253-268Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). Loss-of-function experiments demonstrated that SOX17 is critical for hPGCLC specification upstream of the PGC master gene BLIMP1 (Irie et al., 2015Irie N. Weinberger L. Tang W.W. Kobayashi T. Viukov S. Manor Y.S. Dietmann S. Hanna J.H. Surani M.A. SOX17 is a critical specifier of human primordial germ cell fate.Cell. 2015; 160: 253-268Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). After induction, FGCs migrate to colonize the genital ridge in both sexes, where they undergo considerable proliferation before sexual differentiation with the female FGCs entering meiosis while the male FGCs start mitotic arrest (Saitou and Miyauchi, 2016Saitou M. Miyauchi H. Gametogenesis from Pluripotent Stem Cells.Cell Stem Cell. 2016; 18: 721-735Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Two studies provide single-cell transcriptome data for human FGCs at stages from week 4 to week 26, with nearly 2,600 individual cells being analyzed, including about 1,800 FGCs and about 800 somatic cells (Guo et al., 2015Guo F. Yan L. Guo H. Li L. Hu B. Zhao Y. Yong J. Hu Y. Wang X. Wei Y. et al.The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells.Cell. 2015; 161: 1437-1452Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, Li et al., 2017Li L. Dong J. Yan L. Yong J. Liu X. Hu Y. Fan X. Wu X. Guo H. Wang X. et al.Single-Cell RNA-Seq Analysis Maps Development of Human Germline Cells and Gonadal Niche Interactions.Cell Stem Cell. 2017; 20: 858-873.e4Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). The scRNA-seq data identified and characterized FGC populations of different stages. These include the migrating FGCs and mitotic FGCs shared by both genders; the retinoid acid (RA) signaling-responsive FGCs, the meiotic FGCs, and the primordial follicle
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