Embryo-scale, single-cell spatial transcriptomics Srivatsan, Sanjay R; Regier, Mary C; Barkan, Eliza ...
Science (American Association for the Advancement of Science),
07/2021, Letnik:
373, Številka:
6550
Journal Article
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Spatial patterns of gene expression manifest at scales ranging from local (e.g., cell-cell interactions) to global (e.g., body axis patterning). However, current spatial transcriptomics methods ...either average local contexts or are restricted to limited fields of view. Here, we introduce sci-Space, which retains single-cell resolution while resolving spatial heterogeneity at larger scales. Applying sci-Space to developing mouse embryos, we captured approximate spatial coordinates and whole transcriptomes of about 120,000 nuclei. We identify thousands of genes exhibiting anatomically patterned expression, leverage spatial information to annotate cellular subtypes, show that cell types vary substantially in their extent of spatial patterning, and reveal correlations between pseudotime and the migratory patterns of differentiating neurons. Looking forward, we anticipate that sci-Space will facilitate the construction of spatially resolved single-cell atlases of mammalian development.
High-throughput chemical screens typically use coarse assays such as cell survival, limiting what can be learned about mechanisms of action, off-target effects, and heterogeneous responses. Here, we ...introduce "sci-Plex," which uses "nuclear hashing" to quantify global transcriptional responses to thousands of independent perturbations at single-cell resolution. As a proof of concept, we applied sci-Plex to screen three cancer cell lines exposed to 188 compounds. In total, we profiled ~650,000 single-cell transcriptomes across ~5000 independent samples in one experiment. Our results reveal substantial intercellular heterogeneity in response to specific compounds, commonalities in response to families of compounds, and insight into differential properties within families. In particular, our results with histone deacetylase inhibitors support the view that chromatin acts as an important reservoir of acetate in cancer cells.
Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible ...pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of “chromatin hubs”—they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.
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•Cicero connects regulatory DNA elements to target genes•Co-accessible elements form chromatin hubs•Chromatin hubs are co-regulated during skeletal muscle development•Cicero can help reveal the mechanisms of cis-regulation on a genome-wide scale
Pliner et al. introduce Cicero, a software program to connect distal regulatory elements with target genes using single-cell ATAC-seq data. They find evidence that groups of co-accessible elements form chromatin hubs and undergo coordinated changes in histone marks that are predictive of changes in gene expression in skeletal muscle development.
During embryogenesis, waves of hematopoietic progenitors develop from hemogenic endothelium (HE) prior to the emergence of self-renewing hematopoietic stem cells (HSCs). Although previous studies ...have shown that yolk-sac-derived erythromyeloid progenitors and HSCs emerge from distinct populations of HE, it remains unknown whether the earliest lymphoid-competent progenitors, multipotent progenitors, and HSCs originate from common HE. In this study, we demonstrate by clonal assays and single-cell transcriptomics that rare HE with functional HSC potential in the early murine embryo are distinct from more abundant HE with multilineage hematopoietic potential that fail to generate HSCs. Specifically, HSC-competent HE are characterized by expression of CXCR4 surface marker and by higher expression of genes tied to arterial programs regulating HSC dormancy and self-renewal. Taken together, these findings suggest a revised model of developmental hematopoiesis in which the initial populations of multipotent progenitors and HSCs arise independently from HE with distinct phenotypic and transcriptional properties.
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•HSC-competent HE are characterized by CXCR4 surface expression•Multipotent progenitor potential is initially uncoupled from HSC potential•HSC-competent HE express arterial-associated dormancy and self-renewal genes
Integrating multiple single-cell approaches, Dignum et al. reveal immunophenotypic and transcriptional properties of hemogenic endothelium (HE) that are precursors to the first HSCs. They identify a population of HE with clonal multipotent hematopoietic potential distinct from HSC-competent HE and uncover arterial-associated transcriptional programs linked to HSC competence.
Recent advances in multiplexed single‐cell transcriptomics experiments facilitate the high‐throughput study of drug and genetic perturbations. However, an exhaustive exploration of the combinatorial ...perturbation space is experimentally unfeasible. Therefore, computational methods are needed to predict, interpret, and prioritize perturbations. Here, we present the compositional perturbation autoencoder (CPA), which combines the interpretability of linear models with the flexibility of deep‐learning approaches for single‐cell response modeling. CPA learns to in silico predict transcriptional perturbation response at the single‐cell level for unseen dosages, cell types, time points, and species. Using newly generated single‐cell drug combination data, we validate that CPA can predict unseen drug combinations while outperforming baseline models. Additionally, the architecture's modularity enables incorporating the chemical representation of the drugs, allowing the prediction of cellular response to completely unseen drugs. Furthermore, CPA is also applicable to genetic combinatorial screens. We demonstrate this by imputing in silico 5,329 missing combinations (97.6% of all possibilities) in a single‐cell Perturb‐seq experiment with diverse genetic interactions. We envision CPA will facilitate efficient experimental design and hypothesis generation by enabling in silico response prediction at the single‐cell level and thus accelerate therapeutic applications using single‐cell technologies.
Synopsis
The compositional perturbation autoencoder (CPA) is a deep learning model for predicting the transcriptomic responses of single cells to single or combinatorial treatments from drugs and genetic manipulations.
CPA can be trained on highly multiplexed, single‐cell experiments with thousands of conditions to predict unmeasured phenotypes (e.g., specific dose responses).
It can generalize to predict responses to small molecules never seen in the training by adding priors on chemical space.
Validations using a newly generated combinatorial drug perturbation dataset demonstrate the accuracy of CPA in predicting unseen drug combinations.
CPA is also applicable to genetic combinatorial screens, as shown by imputing in silico 5,329 missing combinations in a single‐cell perturb‐seq experiment with diverse genetic interactions.
The compositional perturbation autoencoder (CPA) is a deep learning model for predicting the transcriptomic responses of single cells to single or combinatorial treatments from drugs and genetic manipulations.
Mammalian embryogenesis is characterized by rapid cellular proliferation and diversification. Within a few weeks, a single-cell zygote gives rise to millions of cells expressing a panoply of ...molecular programs. Although intensively studied, a comprehensive delineation of the major cellular trajectories that comprise mammalian development in vivo remains elusive. Here, we set out to integrate several single-cell RNA-sequencing (scRNA-seq) datasets that collectively span mouse gastrulation and organogenesis, supplemented with new profiling of ~150,000 nuclei from approximately embryonic day 8.5 (E8.5) embryos staged in one-somite increments. Overall, we define cell states at each of 19 successive stages spanning E3.5 to E13.5 and heuristically connect them to their pseudoancestors and pseudodescendants. Although constructed through automated procedures, the resulting directed acyclic graph (TOME (trajectories of mammalian embryogenesis)) is largely consistent with our contemporary understanding of mammalian development. We leverage TOME to systematically nominate transcription factors (TFs) as candidate regulators of each cell type's specification, as well as 'cell-type homologs' across vertebrate evolution.
Single-cell RNA sequencing (scRNA-seq) offers a high-resolution molecular view into complex tissues, but suffers from high levels of technical noise which frustrates efforts to compare the gene ...expression programs of different cell types. "Spike-in" RNA standards help control for technical variation in scRNA-seq, but using them with recently developed, ultra-scalable scRNA-seq methods based on combinatorial indexing is not feasible. Here, we describe a simple and cost-effective method for normalizing transcript counts and subtracting technical variability that improves differential expression analysis in scRNA-seq. The method affixes a ladder of synthetic single-stranded DNA oligos to each cell that appears in its RNA-seq library. With improved normalization we explore chemical perturbations with broad or highly specific effects on gene regulation, including RNA pol II elongation, histone deacetylation, and activation of the glucocorticoid receptor. Our methods reveal that inhibiting histone deacetylation prevents cells from executing their canonical program of changes following glucocorticoid stimulation.
Respiratory syncytial virus (RSV) is the leading cause of hospitalisation for children under 5 years of age. We sought to engineer a viral antigen that provides greater protection than currently ...available vaccines and focused on antigenic site φ, a metastable site specific to the prefusion state of the RSV fusion (F) glycoprotein, as this site is targeted by extremely potent RSV-neutralizing antibodies. Structure-based design yielded stabilized versions of RSV F that maintained antigenic site φ when exposed to extremes of pH, osmolality, and temperature. Six RSV F crystal structures provided atomic-level data on how introduced cysteine residues and filled hydrophobic cavities improved stability. Immunization with site φ—stabilized variants of RSV F in mice and macaques elicited levels of RSV-specific neutralizing activity many times the protective threshold.
Single-cell chromatin accessibility has emerged as a powerful means of understanding the epigenetic landscape of diverse tissues and cell types, but profiling cells from many independent specimens is ...challenging and costly. Here we describe a novel approach, sciPlex-ATAC-seq, which uses unmodified DNA oligos as sample-specific nuclear labels, enabling the concurrent profiling of chromatin accessibility within single nuclei from virtually unlimited specimens or experimental conditions. We first demonstrate our method with a chemical epigenomics screen, in which we identify drug-altered distal regulatory sites predictive of compound- and dose-dependent effects on transcription. We then analyze cell type-specific chromatin changes in PBMCs from multiple donors responding to synthetic and allogeneic immune stimulation. We quantify stimulation-altered immune cell compositions and isolate the unique effects of allogeneic stimulation on chromatin accessibility specific to T-lymphocytes. Finally, we observe that impaired global chromatin decondensation often coincides with chemical inhibition of allogeneic T-cell activation.
HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here, we characterize the ...rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of ∼2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization.
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