High-throughput mapping of cellular differentiation hierarchies from single-cell data promises to empower systematic interrogations of vertebrate development and disease. Here, we applied single-cell ...RNA sequencing to >92,000 cells from zebrafish embryos during the first day of development. Using a graph-based approach, we mapped a cell state landscape that describes axis patterning, germ layer formation, and organogenesis. We tested how clonally related cells traverse this landscape by developing a transposon-based barcoding approach ("TracerSeq") for reconstructing single-cell lineage histories. Clonally related cells were often restricted by the state landscape, including a case in which two independent lineages converge on similar fates. Cell fates remained restricted to this landscape in
deficient embryos. We provide web-based resources for further analysis of the single-cell data.
Time series of single-cell transcriptome measurements can reveal dynamic features of cell differentiation pathways. From measurements of whole frog embryos spanning zygotic genome activation through ...early organogenesis, we derived a detailed catalog of cell states in vertebrate development and a map of differentiation across all lineages over time. The inferred map recapitulates most if not all developmental relationships and associates new regulators and marker genes with each cell state. We find that many embryonic cell states appear earlier than previously appreciated. We also assess conflicting models of neural crest development. Incorporating a matched time series of zebrafish development from a companion paper, we reveal conserved and divergent features of vertebrate early developmental gene expression programs.
Abstract
Motivation
Single-cell gene expression profiling technologies can map the cell states in a tissue or organism. As these technologies become more common, there is a need for computational ...tools to explore the data they produce. In particular, visualizing continuous gene expression topologies can be improved, since current tools tend to fragment gene expression continua or capture only limited features of complex population topologies.
Results
Force-directed layouts of k-nearest-neighbor graphs can visualize continuous gene expression topologies in a manner that preserves high-dimensional relationships and captures complex population topologies. We describe SPRING, a pipeline for data filtering, normalization and visualization using force-directed layouts and show that it reveals more detailed biological relationships than existing approaches when applied to branching gene expression trajectories from hematopoietic progenitor cells and cells of the upper airway epithelium. Visualizations from SPRING are also more reproducible than those of stochastic visualization methods such as tSNE, a state-of-the-art tool. We provide SPRING as an interactive web-tool with an easy to use GUI.
Availability and implementation
https://kleintools.hms.harvard.edu/tools/spring.html, https://github.com/AllonKleinLab/SPRING/.
Supplementary information
Supplementary data are available at Bioinformatics online.
Haematopoiesis, the process of mature blood and immune cell production, is functionally organized as a hierarchy, with self-renewing haematopoietic stem cells and multipotent progenitor cells sitting ...at the very top. Multiple models have been proposed as to what the earliest lineage choices are in these primitive haematopoietic compartments, the cellular intermediates, and the resulting lineage trees that emerge from them. Given that the bulk of studies addressing lineage outcomes have been performed in the context of haematopoietic transplantation, current models of lineage branching are more likely to represent roadmaps of lineage potential than native fate. Here we use transposon tagging to clonally trace the fates of progenitors and stem cells in unperturbed haematopoiesis. Our results describe a distinct clonal roadmap in which the megakaryocyte lineage arises largely independently of other haematopoietic fates. Our data, combined with single-cell RNA sequencing, identify a functional hierarchy of unilineage- and oligolineage-producing clones within the multipotent progenitor population. Finally, our results demonstrate that traditionally defined long-term haematopoietic stem cells are a significant source of megakaryocyte-restricted progenitors, suggesting that the megakaryocyte lineage is the predominant native fate of long-term haematopoietic stem cells. Our study provides evidence for a substantially revised roadmap for unperturbed haematopoiesis, and highlights unique properties of multipotent progenitors and haematopoietic stem cells in situ.
A challenge in biology is to associate molecular differences among progenitor cells with their capacity to generate mature cell types. Here, we used expressed DNA barcodes to clonally trace ...transcriptomes over time and applied this to study fate determination in hematopoiesis. We identified states of primed fate potential and located them on a continuous transcriptional landscape. We identified two routes of monocyte differentiation that leave an imprint on mature cells. Analysis of sister cells also revealed cells to have intrinsic fate biases not detectable by single-cell RNA sequencing. Finally, we benchmarked computational methods of dynamic inference from single-cell snapshots, showing that fate choice occurs earlier than is detected by state-of the-art algorithms and that cells progress steadily through pseudotime with precise and consistent dynamics.
The three-dimensional structure of the genome is an important regulator of many cellular processes including differentiation and gene regulation. Recently, technologies such as Hi-C that combine ...proximity ligation with high-throughput sequencing have revealed domains of self-interacting chromatin, called topologically associating domains (TADs), in many organisms. Current methods for identifying TADs using Hi-C data assume that TADs are non-overlapping, despite evidence for a nested structure in which TADs and sub-TADs form a complex hierarchy.
We introduce a model for decomposition of contact frequencies into a hierarchy of nested TADs. This model is based on empirical distributions of contact frequencies within TADs, where positions that are far apart have a greater enrichment of contacts than positions that are close together. We find that the increase in contact enrichment with distance is stronger for the inner TAD than for the outer TAD in a TAD/sub-TAD pair. Using this model, we develop the TADtree algorithm for detecting hierarchies of nested TADs. TADtree compares favorably with previous methods, finding TADs with a greater enrichment of chromatin marks such as CTCF at their boundaries.
A python implementation of TADtree is available at http://compbio.cs.brown.edu/software/
braphael@cs.brown.edu
Supplementary data are available at Bioinformatics online.
Single-cell expression profiling reveals the molecular states of individual cells with unprecedented detail. Because these methods destroy cells in the process of analysis, they cannot measure how ...gene expression changes over time. However, some information on dynamics is present in the data: the continuum of molecular states in the population can reflect the trajectory of a typical cell. Many methods for extracting single-cell dynamics from population data have been proposed. However, all such attempts face a common limitation: for any measured distribution of cell states, there are multiple dynamics that could give rise to it, and by extension, multiple possibilities for underlying mechanisms of gene regulation. Here, we describe the aspects of gene expression dynamics that cannot be inferred from a static snapshot alone and identify assumptions necessary to constrain a unique solution for cell dynamics from static snapshots. We translate these constraints into a practical algorithmic approach, population balance analysis (PBA), which makes use of a method from spectral graph theory to solve a class of high-dimensional differential equations. We use simulations to show the strengths and limitations of PBA, and then apply it to single-cell profiles of hematopoietic progenitor cells (HPCs). Cell state predictions from this analysis agree with HPC fate assays reported in several papers over the past two decades. By highlighting the fundamental limits on dynamic inference faced by any method, our framework provides a rigorous basis for dynamic interpretation of a gene expression continuum and clarifies best experimental designs for trajectory reconstruction from static snapshot measurements.
The formation of red blood cells begins with the differentiation of multipotent haematopoietic progenitors. Reconstructing the steps of this differentiation represents a general challenge in ...stem-cell biology. Here we used single-cell transcriptomics, fate assays and a theory that allows the prediction of cell fates from population snapshots to demonstrate that mouse haematopoietic progenitors differentiate through a continuous, hierarchical structure into seven blood lineages. We uncovered coupling between the erythroid and the basophil or mast cell fates, a global haematopoietic response to erythroid stress and novel growth factor receptors that regulate erythropoiesis. We defined a flow cytometry sorting strategy to purify early stages of erythroid differentiation, completely isolating classically defined burst-forming and colony-forming progenitors. We also found that the cell cycle is progressively remodelled during erythroid development and during a sharp transcriptional switch that ends the colony-forming progenitor stage and activates terminal differentiation. Our work showcases the utility of linking transcriptomic data to predictive fate models, and provides insights into lineage development in vivo.
Bone marrow transplantation therapy relies on the life-long regenerative capacity of haematopoietic stem cells (HSCs)
. HSCs present a complex variety of regenerative behaviours at the clonal level, ...but the mechanisms underlying this diversity are still undetermined
. Recent advances in single-cell RNA sequencing have revealed transcriptional differences among HSCs, providing a possible explanation for their functional heterogeneity
. However, the destructive nature of sequencing assays prevents simultaneous observation of stem cell state and function. To solve this challenge, we implemented expressible lentiviral barcoding, which enabled simultaneous analysis of lineages and transcriptomes from single adult HSCs and their clonal trajectories during long-term bone marrow reconstitution. Analysis of differential gene expression between clones with distinct behaviour revealed an intrinsic molecular signature that characterizes functional long-term repopulating HSCs. Probing this signature through in vivo CRISPR screening, we found the transcription factor TCF15 to be required and sufficient to drive HSC quiescence and long-term self-renewal. In situ, Tcf15 expression labels the most primitive subset of true multipotent HSCs. In conclusion, our work elucidates clone-intrinsic molecular programmes associated with functional stem cell heterogeneity and identifies a mechanism for the maintenance of the self-renewing HSC state.
The main neurological manifestation of COVID-19 is loss of smell or taste. The high incidence of smell loss without significant rhinorrhea or nasal congestion suggests that SARS-CoV-2 targets the ...chemical senses through mechanisms distinct from those used by endemic coronaviruses or other common cold-causing agents. Here we review recently developed hypotheses about how SARS-CoV-2 might alter the cells and circuits involved in chemosensory processing and thereby change perception. Given our limited understanding of SARS-CoV-2 pathogenesis, we propose future experiments to elucidate disease mechanisms and highlight the relevance of this ongoing work to understanding how the virus might alter brain function more broadly.
Impairments of smell and taste perception are two of the main symptoms of COVID–19. In this issue of Neuron, Cooper et al. focus on these chemosensory symptoms and review recent hypotheses of the putative mechanisms mediating the loss of taste and smell through COVID–19.
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