Advances in whole-genome and whole-transcriptome amplification have permitted the sequencing of the minute amounts of DNA and RNA present in a single cell, offering a window into the extent and ...nature of genomic and transcriptomic heterogeneity which occurs in both normal development and disease. Single-cell approaches stand poised to revolutionise our capacity to understand the scale of genomic, epigenomic, and transcriptomic diversity that occurs during the lifetime of an individual organism. Here, we review the major technological and biological breakthroughs achieved, describe the remaining challenges to overcome, and provide a glimpse into the promise of recent and future developments.
Single-cell sequencing provides information that is not confounded by genotypic or phenotypic heterogeneity of bulk samples. Sequencing of one molecular type (RNA, methylated DNA or open chromatin) ...in a single cell, furthermore, provides insights into the cell's phenotype and links to its genotype. Nevertheless, only by taking measurements of these phenotypes and genotypes from the same single cells can such inferences be made unambiguously. In this review, we survey the first experimental approaches that assay, in parallel, multiple molecular types from the same single cell, before considering the challenges and opportunities afforded by these and future technologies.
Single-cell RNA sequencing (scRNA-seq) has become an established and powerful method to investigate transcriptomic cell-to-cell variation, thereby revealing new cell types and providing insights into ...developmental processes and transcriptional stochasticity. A key question is how the variety of available protocols compare in terms of their ability to detect and accurately quantify gene expression. Here, we assessed the protocol sensitivity and accuracy of many published data sets, on the basis of spike-in standards and uniform data processing. For our workflow, we developed a flexible tool for counting the number of unique molecular identifiers (https://github.com/vals/umis/). We compared 15 protocols computationally and 4 protocols experimentally for batch-matched cell populations, in addition to investigating the effects of spike-in molecular degradation. Our analysis provides an integrated framework for comparing scRNA-seq protocols.
The major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, ...we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation.
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•Oct4 and Sox2 targets such as Sox21 are highly heterogeneous in the 4-cell embryo•Depleting Sox21 levels leads to upregulation of Cdx2 and extra-embryonic fate•Sox21 expression responds to the histone H3R26 methyltransferase CARM1•Heterogeneous gene expression initiates cell-fate decisions at the 4-cell stage
Heterogeneous gene expression in 4-cell stage in mouse embryos contributes to cell-fate decisions, with lower levels of Sox21 promoting extra-embryonic fate over pluripotency.
Heterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have ...been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system.
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•Comparing HSCs purified with four methods identifies key functional molecules•Index sorting links single-cell RNA-seq with single-cell transplantation•EPCRhiCD48−CD150+Scahi purifies HSCs with durable self-renewal•Single-cell biology links mammalian stem cell function with markers and pathways
Wilson et al. combine single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays to reveal gene expression programs of HSCs with durable self-renewal potential in transplantation assays. They also demonstrate the broader applicability of this approach for linking key molecules with defined stem cell functions.
Single-cell transcriptomic approaches have revolutionised the study of complex biological systems, with the routine measurement of gene expression in thousands of cells enabling construction of ...whole-organism cell atlases. However, the transcriptome is just one layer amongst many that coordinate to define cell type and state and, ultimately, function. In parallel with the widespread uptake of single-cell RNA-seq (scRNA-seq), there has been a rapid emergence of methods that enable multiomic profiling of individual cells, enabling parallel measurement of intercellular heterogeneity in the genome, epigenome, transcriptome, and proteomes. Linking measurements from each of these layers has the potential to reveal regulatory and functional mechanisms underlying cell behaviour in healthy development and disease.
To understand intercellular heterogeneity within an organism, it is essential to make coordinated measurements linking the genome and its epigenetic regulation to gene and protein expression at the single-cell level.Rapid advances in single-cell multiomics approaches have enabled analysis of multiple molecular modalities from the same single cell.Methods incorporating several modalities now exist, although several challenges remain with regard to resolution, data integration, and scale.Further developments in multiomics approaches will provide unique insights into the regulatory processes governing how individual cells function collectively to produce whole-organism phenotypes in development, health, and disease.
The transcriptional programs that govern hematopoiesis have been investigated primarily by population-level analysis of hematopoietic stem and progenitor cells, which cannot reveal the continuous ...nature of the differentiation process. Here we applied single-cell RNA-sequencing to a population of hematopoietic cells in zebrafish as they undergo thrombocyte lineage commitment. By reconstructing their developmental chronology computationally, we were able to place each cell along a continuum from stem cell to mature cell, refining the traditional lineage tree. The progression of cells along this continuum is characterized by a highly coordinated transcriptional program, displaying simultaneous suppression of genes involved in cell proliferation and ribosomal biogenesis as the expression of lineage specific genes increases. Within this program, there is substantial heterogeneity in the expression of the key lineage regulators. Overall, the total number of genes expressed, as well as the total mRNA content of the cell, decreases as the cells undergo lineage commitment.
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•Single-cell RNA-sequencing reveals the continuous nature of thrombocyte development•Coordinated transcriptional programs govern progression of differentiation•Number of genes expressed and mRNA content per cell decrease during differentiation•Zebrafish thrombocytes remain transcriptionally active in circulation
Computational reconstruction of the thrombocyte’s developmental chronology from scRNA-seq data reveals the continuous nature of the differentiation process. Macaulay et al. show that a highly coordinated transcriptional program characterizes the progression of cells along this continuum. Within this program, there is substantial heterogeneity in the expression of key lineage regulators.
In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system ...are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis.
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•Embryonic lymphoid lineage restriction initiates prior to emergence of definitive HSC•Embryonic lymphoid-primed progenitors have immune-restricted lymphomyeloid potential•Embryonic lymphoid-primed progenitors contribute to myelo- and lymphopoiesis in utero•Lympho-myelo immune restriction is an early-fate decision in embryonic hematopoiesis
An immune-restricted lymphomyeloid-primed progenitor with the capacity to contribute to both myeloid and lymphoid lineages in the developing embryo emerges prior to definitive HSCs.
Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed ...protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ∼3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.
The foundations of mammalian development lie in a cluster of embryonic epiblast stem cells. In response to extracellular matrix signalling, these cells undergo epithelialization and create an apical ...surface in contact with a cavity, a fundamental event for all subsequent development. Concomitantly, epiblast cells transit through distinct pluripotent states, before lineage commitment at gastrulation. These pluripotent states have been characterized at the molecular level, but their biological importance remains unclear. Here we show that exit from an unrestricted naive pluripotent state is required for epiblast epithelialization and generation of the pro-amniotic cavity in mouse embryos. Embryonic stem cells locked in the naive state are able to initiate polarization but fail to undergo lumenogenesis. Mechanistically, exit from naive pluripotency activates an Oct4-governed transcriptional program that results in expression of glycosylated sialomucin proteins and the vesicle tethering and fusion events of lumenogenesis. Similarly, exit of epiblasts from naive pluripotency in cultured human post-implantation embryos triggers amniotic cavity formation and developmental progression. Our results add tissue-level architecture as a new criterion for the characterization of different pluripotent states, and show the relevance of transitions between these states during development of the mammalian embryo.
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