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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.

A comparison framework applied to 15 single-cell RNA-seq protocols reveals differences in accuracy and sensitivity and discusses the utility of RNA spike-in standards. 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.

Exit of epiblasts from an unrestricted naive pluripotent state is required for epithelialization and generation of the pro-amniotic cavity in mouse embryos and for amniotic cavity formation in human embryos and human embryonic stem cells. Cells stem pluripotent state to grow During mammalian development, embryonic pluripotent stem cells form a cavitated epithelium at the time of implantation. Magdalena Zernicka-Goetz and colleagues show that, in spheroids made from mouse embryonic stem cells, the cells must leave their unrestricted naive pluripotent state for the events leading to cavity formation. The transcription factor Oct4 activates this exit and the expression of genes that code for proteins involved in lumenogenesis. The authors also show that these events are conserved in spheroids from human embryonic stem cells. 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 1 , 2 , a fundamental event for all subsequent development. Concomitantly, epiblast cells transit through distinct pluripotent states 3 , 4 , before lineage commitment at gastrulation. These pluripotent states have been characterized at the molecular level 5 , 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.

G&T-seq enables sequencing of DNA and mRNA from the same single cell, allowing the effects of genomic variation on transcription to be studied. It is compatible with any WGA method and so can be tailored to specific applications. 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.

An early stage in mouse blood development is reconstructed from gene expression data on thousands of single cells. Reconstruction of the molecular pathways controlling organ development has been hampered by a lack of methods to resolve embryonic progenitor cells. Here we describe a strategy to address this problem that combines gene expression profiling of large numbers of single cells with data analysis based on diffusion maps for dimensionality reduction and network synthesis from state transition graphs. Applying the approach to hematopoietic development in the mouse embryo, we map the progression of mesoderm toward blood using single-cell gene expression analysis of 3,934 cells with blood-forming potential captured at four time points between E7.0 and E8.5. Transitions between individual cellular states are then used as input to develop a single-cell network synthesis toolkit to generate a computationally executable transcriptional regulatory network model of blood development. Several model predictions concerning the roles of Sox and Hox factors are validated experimentally. Our results demonstrate that single-cell analysis of a developing organ coupled with computational approaches can reveal the transcriptional programs that underpin organogenesis.

Cellular decision-making is mediated by a complex interplay of external stimuli with the intracellular environment, in particular transcription factor regulatory networks. Here we have determined the expression of a network of 18 key haematopoietic transcription factors in 597 single primary blood stem and progenitor cells isolated from mouse bone marrow. We demonstrate that different stem/progenitor populations are characterized by distinctive transcription factor expression states, and through comprehensive bioinformatic analysis reveal positively and negatively correlated transcription factor pairings, including previously unrecognized relationships between Gata2 , Gfi1 and Gfi1b . Validation using transcriptional and transgenic assays confirmed direct regulatory interactions consistent with a regulatory triad in immature blood stem cells, where Gata2 may function to modulate cross-inhibition between Gfi1 and Gfi1b . Single-cell expression profiling therefore identifies network states and allows reconstruction of network hierarchies involved in controlling stem cell fate choices, and provides a blueprint for studying both normal development and human disease. Gottgens and colleagues have analysed the expression of 18 haematopoietic factors in single primary blood and progenitor cells from mouse bone marrow. They delineate distinct states of expression for these transcription factors and identify regulatory relationships between the key factors Gata2, Gfi1 and Gfi2.

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the “universal” genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel uncultured ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3’UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids.

Hematopoietic stem cells (HSCs) residing in specialized niches in the bone marrow are responsible for the balanced output of multiple short-lived blood cell lineages in steady-state and in response to different challenges. However, feedback mechanisms by which HSCs, through their niches, sense acute losses of specific blood cell lineages remain to be established. While all HSCs replenish platelets, previous studies have shown that a large fraction of HSCs are molecularly primed for the megakaryocyte-platelet lineage and are rapidly recruited into proliferation upon platelet depletion. Platelets normally turnover in an activation-dependent manner, herein mimicked by antibodies inducing platelet activation and depletion. Antibody-mediated platelet activation upregulates expression of Interleukin-1 (IL-1) in platelets, and in bone marrow extracellular fluid in vivo. Genetic experiments demonstrate that rather than IL-1 directly activating HSCs, activation of bone marrow Lepr + perivascular niche cells expressing IL-1 receptor is critical for the optimal activation of quiescent HSCs upon platelet activation and depletion. These findings identify a feedback mechanism by which activation-induced depletion of a mature blood cell lineage leads to a niche-dependent activation of HSCs to reinstate its homeostasis. Hematopoietic stem cells (HSCs) replenish blood cells. Here, Luis et al., identify a feedback mechanism by which IL-1 secreted by activated platelets signals through niche Lepr+ cells to activate HSCs and restore platelet homeostasis.

Single-cell approaches have revealed that the haematopoietic hierarchy is a continuum of differentiation, from stem cell to committed progenitor, marked by changes in gene expression. However, many of these approaches neglect isoform-level information and thus do not capture the extent of alternative splicing within the system. Here, we present an integrated short- and long-read single-cell RNA-seq analysis of haematopoietic stem and progenitor cells. We demonstrate that over half of genes detected in standard short-read single-cell analyses are expressed as multiple, often functionally distinct, isoforms, including many transcription factors and key cytokine receptors. We observe global and HSC-specific changes in gene expression with ageing but limited impact of ageing on isoform usage. Integrating single-cell and cell-type-specific isoform landscape in haematopoiesis thus provides a new reference for comprehensive molecular profiling of heterogeneous tissues, as well as novel insights into transcriptional complexity, cell-type-specific splicing events and consequences of ageing. Single-cell transcriptomics using long-read sequencing achieves both gene and isoform expression in haematopoietic stem and progenitor cells, providing novel insights into the understanding of alternative splicing in cellular differentiation.

Analysis of the transcriptome of more than 1,200 cells from gastrulating mouse embryos using single-cell sequencing, gathering unexpected insights into early mesoderm formation during gastrulation. Single-cell analysis during mammalian gastrulation Differentiation to the three major lineages that make up mammalian bodies occurs during gastrulation, with the ingression of cells towards the inside of the embryo. The molecular events directing gastrulation are still unclear as only very few cells are specified at a time, making the process difficult to analyse. It is thought that examining these events at the single-cell level would help understand how organs are formed. Berthold Göttgens and colleagues have analysed the transcriptome of more than a thousand cells from gastrulating mouse embryos using single-cell sequencing, gathering unexpected insights into early mesoderm formation during gastrulation. In mammals, specification of the three major germ layers occurs during gastrulation, when cells ingressing through the primitive streak differentiate into the precursor cells of major organ systems. However, the molecular mechanisms underlying this process remain unclear, as numbers of gastrulating cells are very limited. In the mouse embryo at embryonic day 6.5, cells located at the junction between the extra-embryonic region and the epiblast on the posterior side of the embryo undergo an epithelial-to-mesenchymal transition and ingress through the primitive streak. Subsequently, cells migrate, either surrounding the prospective ectoderm contributing to the embryo proper, or into the extra-embryonic region to form the yolk sac, umbilical cord and placenta. Fate mapping has shown that mature tissues such as blood and heart originate from specific regions of the pre-gastrula epiblast 1 , but the plasticity of cells within the embryo and the function of key cell-type-specific transcription factors remain unclear. Here we analyse 1,205 cells from the epiblast and nascent Flk1 + mesoderm of gastrulating mouse embryos using single-cell RNA sequencing, representing the first transcriptome-wide in vivo view of early mesoderm formation during mammalian gastrulation. Additionally, using knockout mice, we study the function of Tal1, a key haematopoietic transcription factor, and demonstrate, contrary to previous studies performed using retrospective assays 2 , 3 , that Tal1 knockout does not immediately bias precursor cells towards a cardiac fate.