Distinct enhancer signatures in the mouse gastrula delineate progressive cell fate continuum during embryo development.

Primary germ layers have the potential to form all tissues in the mature organism, and their formation during gastrulation requires precise epigenetic modulation of both proximal and distal regulatory elements. Previous studies indicated that spatial and temporal patterns of gene expression in the gastrula predispose individual regions to distinct cell fates. However, the underlying epigenetic mechanisms remain largely unexplored. Here, we profile the spatiotemporal landscape of the epigenome and transcriptome of the mouse gastrula. We reveal the asynchronous dynamics of proximal chromatin states during germ layer formation as well as unique gastrula-specific epigenomic features of regulatory elements, which have strong usage turnover dynamics and clear germ layer-specific signatures. Importantly, we also find that enhancers around organogenetic genes, which are weakly expressed at the gastrulation stage, are frequently pre-marked by histone H3 lysine 27 acetylation (H3K27ac) in the gastrula. By using the transgenic mice and genome editing system, we demonstrate that a pre-marked enhancer, which is located in the intron of a brain-specific gene 2510009E07Rik, exhibits specific enhancer activity in the ectoderm and future brain tissue, and also executes important function during mouse neural differentiation. Taken together, our study provides the comprehensive epigenetic information for embryonic patterning during mouse gastrulation, demonstrates the importance of gastrula pre-marked enhancers in regulating the correct development of the mouse embryo, and thus broadens the current understanding of mammalian embryonic development and related diseases.

Dissecting the transcriptome landscape of the human fetal neural retina and retinal pigment epithelium by single-cell RNA-seq analysis.

The developmental pathway of the neural retina (NR) and retinal pigment epithelium (RPE) has been revealed by extensive research in mice. However, the molecular mechanisms underlying the development of the human NR and RPE, as well as the interactions between these two tissues, have not been well defined. Here, we analyzed 2,421 individual cells from human fetal NR and RPE using single-cell RNA sequencing (RNA-seq) technique and revealed the tightly regulated spatiotemporal gene expression network of human retinal cells. We identified major cell classes of human fetal retina and potential crucial transcription factors for each cell class. We dissected the dynamic expression patterns of visual cycle- and ligand-receptor interaction-related genes in the RPE and NR. Moreover, we provided a map of disease-related genes for human fetal retinal cells and highlighted the importance of retinal progenitor cells as potential targets of inherited retinal diseases. Our findings captured the key in vivo features of the development of the human NR and RPE and offered insightful clues for further functional studies.

Maintenance of Nucleolar Homeostasis by CBX4 Alleviates Senescence and Osteoarthritis.

CBX4, a component of polycomb repressive complex 1 (PRC1), plays important roles in the maintenance of cell identity and organ development through gene silencing. However, whether CBX4 regulates human stem cell homeostasis remains unclear. Here, we demonstrate that CBX4 counteracts human mesenchymal stem cell (hMSC) aging via the maintenance of nucleolar homeostasis. CBX4 protein is downregulated in aged hMSCs, whereas CBX4 knockout in hMSCs results in destabilized nucleolar heterochromatin, enhanced ribosome biogenesis, increased protein translation, and accelerated cellular senescence. CBX4 maintains nucleolar homeostasis by recruiting nucleolar protein fibrillarin (FBL) and heterochromatin protein KRAB-associated protein 1 (KAP1) at nucleolar rDNA, limiting the excessive expression of rRNAs. Overexpression of CBX4 alleviates physiological hMSC aging and attenuates the development of osteoarthritis in mice. Altogether, our findings reveal a critical role of CBX4 in counteracting cellular senescence by maintaining nucleolar homeostasis, providing a potential therapeutic target for aging-associated disorders.

Single-cell multiomics sequencing and analyses of human colorectal cancer.

Although genomic instability, epigenetic abnormality, and gene expression dysregulation are hallmarks of colorectal cancer, these features have not been simultaneously analyzed at single-cell resolution. Using optimized single-cell multiomics sequencing together with multiregional sampling of the primary tumor and lymphatic and distant metastases, we developed insights beyond intratumoral heterogeneity. Genome-wide DNA methylation levels were relatively consistent within a single genetic sublineage. The genome-wide DNA demethylation patterns of cancer cells were consistent in all 10 patients whose DNA we sequenced. The cancer cells' DNA demethylation degrees clearly correlated with the densities of the heterochromatin-associated histone modification H3K9me3 of normal tissue and those of repetitive element long interspersed nuclear element 1. Our work demonstrates the feasibility of reconstructing genetic lineages and tracing their epigenomic and transcriptomic dynamics with single-cell multiomics sequencing.

Single-Cell RNA Sequencing Analysis Reveals Sequential Cell Fate Transition during Human Spermatogenesis.

Spermatogenesis generates mature male gametes and is critical for the proper transmission of genetic information between generations. However, the developmental landscapes of human spermatogenesis remain unknown. Here, we performed single-cell RNA sequencing (scRNA-seq) analysis for 2,854 testicular cells from donors with normal spermatogenesis and 174 testicular cells from one nonobstructive azoospermia (NOA) donor. A hierarchical model was established, which was characterized by the sequential and stepwise development of three spermatogonia subtypes, seven spermatocyte subtypes, and four spermatid subtypes. Further analysis identified several stage-specific marker genes of human germ cells, such as HMGA1, PIWIL4, TEX29, SCML1, and CCDC112. Moreover, we identified altered gene expression patterns in the testicular somatic cells of one NOA patient via scRNA-seq analysis, paving the way for further diagnosis of male infertility. Our work allows for the reconstruction of transcriptional programs inherent to sequential cell fate transition during human spermatogenesis and has implications for deciphering male-related reproductive disorders.

Publisher Correction: Single-cell multi-omics sequencing of human early embryos.

In the version of this Resource originally published, owing to a technical error an incorrect file was used for Supplementary Table 2; this has now been replaced with the correct file.

Single-cell multi-omics sequencing of human early embryos.

DNA methylation, chromatin states and their interrelationships represent critical epigenetic information, but these are largely unknown in human early embryos. Here, we apply single-cell chromatin overall omic-scale landscape sequencing (scCOOL-seq) to generate a genome-wide map of DNA methylation and chromatin accessibility at single-cell resolution during human preimplantation development. Unlike in mice, the chromatin of the paternal genome is already more open than that of the maternal genome at the mid-zygote stage in humans, and this state is maintained until the 4-cell stage. After fertilization, genes with high variations in DNA methylation, and those with high variations in chromatin accessibility, tend to be two different sets. Furthermore, 1,797 out of 5,155 (35%) widely open chromatin regions in promoters closed when transcription activity was inhibited, indicating a feedback mechanism between transcription and open chromatin maintenance. Our work paves the way for dissecting the complex, yet highly coordinated, epigenetic reprogramming during human preimplantation development.

Single-cell RNA-seq analysis unveils a prevalent epithelial/mesenchymal hybrid state during mouse organogenesis.

BACKGROUND: Organogenesis is crucial for proper organ formation during mammalian embryonic development. However, the similarities and shared features between different organs and the cellular heterogeneity during this process at single-cell resolution remain elusive. RESULTS: We perform single-cell RNA sequencing analysis of 1916 individual cells from eight organs and tissues of E9.5 to E11.5 mouse embryos, namely, the forebrain, hindbrain, skin, heart, somite, lung, liver, and intestine. Based on the regulatory activities rather than the expression patterns, all cells analyzed can be well classified into four major groups with epithelial, mesodermal, hematopoietic, and neuronal identities. For different organs within the same group, the similarities and differences of their features and developmental paths are revealed and reconstructed. CONCLUSIONS: We identify mutual interactions between epithelial and mesenchymal cells and detect epithelial cells with prevalent mesenchymal features during organogenesis, which are similar to the features of intermediate epithelial/mesenchymal cells during tumorigenesis. The comprehensive transcriptome at single-cell resolution profiled in our study paves the way for future mechanistic studies of the gene-regulatory networks governing mammalian organogenesis.

ATF6 safeguards organelle homeostasis and cellular aging in human mesenchymal stem cells.

Loss of organelle homeostasis is a hallmark of aging. However, it remains elusive how this occurs at gene expression level. Here, we report that human mesenchymal stem cell (hMSC) aging is associated with dysfunction of double-membrane organelles and downregulation of transcription factor ATF6. CRISPR/Cas9-mediated inactivation of ATF6 in hMSCs, not in human embryonic stem cells and human adipocytes, results in premature cellular aging, characteristic of loss of endomembrane homeostasis. Transcriptomic analyses uncover cell type-specific constitutive and stress-induced ATF6-regulated genes implicated in various layers of organelles' homeostasis regulation. FOS was characterized as a constitutive ATF6 responsive gene, downregulation of which contributes to hMSC aging. Our study unravels the first ATF6-regulated gene expression network related to homeostatic regulation of membrane organelles, and provides novel mechanistic insights into aging-associated attrition of human stem cells.

Single-cell DNA methylome sequencing of human preimplantation embryos.

DNA methylation is a crucial layer of epigenetic regulation during mammalian embryonic development 1-3 . Although the DNA methylome of early human embryos has been analyzed 4-6 , some of the key features have not been addressed thus far. Here we performed single-cell DNA methylome sequencing for human preimplantation embryos and found that tens of thousands of genomic loci exhibited de novo DNA methylation. This finding indicates that genome-wide DNA methylation reprogramming during preimplantation development is a dynamic balance between strong global demethylation and drastic focused remethylation. Furthermore, demethylation of the paternal genome is much faster and thorough than that of the maternal genome. From the two-cell to the postimplantation stage, methylation of the paternal genome is consistently lower than that of the maternal genome. We also show that the genetic lineage of early blastomeres can be traced by DNA methylation analysis. Our work paves the way for deciphering the secrets of DNA methylation reprogramming in early human embryos.

Single-cell multi-omics sequencing of mouse early embryos and embryonic stem cells.

Single-cell epigenome sequencing techniques have recently been developed. However, the combination of different layers of epigenome sequencing in an individual cell has not yet been achieved. Here, we developed a single-cell multi-omics sequencing technology (single-cell COOL-seq) that can analyze the chromatin state/nucleosome positioning, DNA methylation, copy number variation and ploidy simultaneously from the same individual mammalian cell. We used this method to analyze the reprogramming of the chromatin state and DNA methylation in mouse preimplantation embryos. We found that within < 12 h of fertilization, each individual cell undergoes global genome demethylation together with the rapid and global reprogramming of both maternal and paternal genomes to a highly opened chromatin state. This was followed by decreased openness after the late zygote stage. Furthermore, from the late zygote to the 4-cell stage, the residual DNA methylation is preferentially preserved on intergenic regions of the paternal alleles and intragenic regions of maternal alleles in each individual blastomere. However, chromatin accessibility is similar between paternal and maternal alleles in each individual cell from the late zygote to the blastocyst stage. The binding motifs of several pluripotency regulators are enriched at distal nucleosome depleted regions from as early as the 2-cell stage. This indicates that the cis-regulatory elements of such target genes have been primed to an open state from the 2-cell stage onward, long before pluripotency is eventually established in the ICM of the blastocyst. Genes may be classified into homogeneously open, homogeneously closed and divergent states based on the chromatin accessibility of their promoter regions among individual cells. This can be traced to step-wise transitions during preimplantation development. Our study offers the first single-cell and parental allele-specific analysis of the genome-scale chromatin state and DNA methylation dynamics at single-base resolution in early mouse embryos and provides new insights into the heterogeneous yet highly ordered features of epigenomic reprogramming during this process.

Single-Cell RNA-Seq Analysis Maps Development of Human Germline Cells and Gonadal Niche Interactions.

Human fetal germ cells (FGCs) are precursors to sperm and eggs and are crucial for maintenance of the species. However, the developmental trajectories and heterogeneity of human FGCs remain largely unknown. Here we performed single-cell RNA-seq analysis of over 2,000 FGCs and their gonadal niche cells in female and male human embryos spanning several developmental stages. We found that female FGCs undergo four distinct sequential phases characterized by mitosis, retinoic acid signaling, meiotic prophase, and oogenesis. Male FGCs develop through stages of migration, mitosis, and cell-cycle arrest. Individual embryos of both sexes simultaneously contain several subpopulations, highlighting the asynchronous and heterogeneous nature of FGC development. Moreover, we observed reciprocal signaling interactions between FGCs and their gonadal niche cells, including activation of the bone morphogenic protein (BMP) and Notch signaling pathways. Our work provides key insights into the crucial features of human FGCs during their highly ordered mitotic, meiotic, and gametogenetic processes in vivo.

MR-seq: measuring a single cell's transcriptome repeatedly by RNA-seq.

We described a novel single-cell RNA-seq technique called MR-seq (measure a single-cell transcriptome repeatedly), which permits statistically assessing the technical variation and identifying the differentially expressed genes between just two single cells by measuring each single cell twice. We demonstrated that MR-seq gave sensitivity and reproducibility similar to the standard single-cell RNA-seq and increased the positive predicate value. Application of MR-seq to early mouse embryos identified hundreds of candidate intra-embryonic heterogeneous genes among mouse 2-, 4- and 8-cell stage embryos. MR-seq should be useful for detecting differentially expressed genes among a small number of cells.

DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells.

Chromatin remodeling is important for the epigenetic reprogramming of human primordial germ cells. However, the comprehensive chromatin state has not yet been analyzed for human fetal germ cells (FGCs). Here we use nucleosome occupancy and methylation sequencing method to analyze both the genome-wide chromatin accessibility and DNA methylome at a series of crucial time points during fetal germ cell development in both human and mouse. We find 116 887 and 137 557 nucleosome-depleted regions (NDRs) in human and mouse FGCs, covering a large set of germline-specific and highly dynamic regulatory genomic elements, such as enhancers. Moreover, we find that the distal NDRs are enriched specifically for binding motifs of the pluripotency and germ cell master regulators such as NANOG, SOX17, AP2γ and OCT4 in human FGCs, indicating the existence of a delicate regulatory balance between pluripotency-related genes and germ cell-specific genes in human FGCs, and the functional significance of these genes for germ cell development in vivo. Our work offers a comprehensive and high-resolution roadmap for dissecting chromatin state transition dynamics during the epigenomic reprogramming of human and mouse FGCs.

Tracing the expression of circular RNAs in human pre-implantation embryos.

BACKGROUND: PolyA- RNAs have not been widely analyzed in human pre-implantation embryos due to the scarcity of materials. In particular, circular RNA (circRNA), a novel type of polyA- RNA, has not been characterized during human pre-implantation development. RESULTS: We systematically analyze polyA+ messenger RNAs (mRNAs) and polyA- RNAs in individual human oocytes and pre-implantation embryos using SUPeR-seq. We de novo identify 10,032 circRNAs from 2974 hosting genes. Most of these circRNAs are developmentally stage-specific and dynamically regulated. Many of them are maternally expressed, implying their potentially important regulatory functions in oogenesis and the formation of totipotent zygotes. Comparison between human and mouse embryos reveals both high conservation and clear distinction between these two species. Human pre-implantation embryos generate more types of circRNA compared with mouse embryos and this is associated with a striking increase of the length of the circRNA flanking introns in humans. We also perform RNA de novo assembly and identify novel transcript units, many of which are potentially novel long non-coding RNAs. CONCLUSIONS: This study reports the first analysis of the whole transcriptome comprising both polyA+ mRNAs and polyA- RNAs including circRNAs during human pre-implantation development. It provides an invaluable resource for analyzing the unique function and complex regulatory mechanisms of circRNAs during this process.

BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes.

The mRNAs stored in oocytes undergo general decay during the maternal-zygotic transition (MZT), and their stability is tightly interconnected with meiotic cell-cycle progression. However, the factors that trigger decay of maternal mRNA and couple this event to oocyte meiotic maturation remain elusive. Here, we identified B-cell translocation gene-4 (BTG4) as an MZT licensing factor in mice. BTG4 bridged CNOT7, a catalytic subunit of the CCR4-NOT deadenylase, to eIF4E, a key translation initiation factor, and facilitated decay of maternal mRNA. Btg4-null females produced morphologically normal oocytes but were infertile, owing to early developmental arrest. The intrinsic MAP kinase cascade in oocytes triggered translation of Btg4 mRNA stored in fully grown oocytes by targeting the 3' untranslated region, thereby coupling CCR4-NOT deadenylase-mediated decay of maternal mRNA with oocyte maturation and fertilization. This is a key step in oocyte cytoplasmic maturation that determines the developmental potential of mammalian embryos.

Single-cell triple omics sequencing reveals genetic, epigenetic, and transcriptomic heterogeneity in hepatocellular carcinomas.

Single-cell genome, DNA methylome, and transcriptome sequencing methods have been separately developed. However, to accurately analyze the mechanism by which transcriptome, genome and DNA methylome regulate each other, these omic methods need to be performed in the same single cell. Here we demonstrate a single-cell triple omics sequencing technique, scTrio-seq, that can be used to simultaneously analyze the genomic copy-number variations (CNVs), DNA methylome, and transcriptome of an individual mammalian cell. We show that large-scale CNVs cause proportional changes in RNA expression of genes within the gained or lost genomic regions, whereas these CNVs generally do not affect DNA methylation in these regions. Furthermore, we applied scTrio-seq to 25 single cancer cells derived from a human hepatocellular carcinoma tissue sample. We identified two subpopulations within these cells based on CNVs, DNA methylome, or transcriptome of individual cells. Our work offers a new avenue of dissecting the complex contribution of genomic and epigenomic heterogeneities to the transcriptomic heterogeneity within a population of cells.

Epigenomic Landscape of Human Fetal Brain, Heart, and Liver.

The epigenetic regulation of spatiotemporal gene expression is crucial for human development. Here, we present whole-genome chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) analyses of a wide variety of histone markers in the brain, heart, and liver of early human embryos shortly after their formation. We identified 40,181 active enhancers, with a large portion showing tissue-specific and developmental stage-specific patterns, pointing to their roles in controlling the ordered spatiotemporal expression of the developmental genes in early human embryos. Moreover, using sequential ChIP-seq, we showed that all three organs have hundreds to thousands of bivalent domains that are marked by both H3K4me3 and H3K27me3, probably to keep the progenitor cells in these organs ready for immediate differentiation into diverse cell types during subsequent developmental processes. Our work illustrates the potentially critical roles of tissue-specific and developmental stage-specific epigenomes in regulating the spatiotemporal expression of developmental genes during early human embryonic development.