Amir Giladi

Transcriptional Heterogeneity and Lineage Commitment in Myeloid Progenitors

Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.

Microglia development follows a stepwise program to regulate brain homeostasis.

Microglia development follows a stepwise program Microglia are cells that defend the central nervous system. However, because they migrate into the brain during development, the changes that they undergo, including those that affect gene expression, have been difficult to document. Matcovitch-Natan et al. transcriptionally profiled gene expression and analyzed epigenetic signatures of microglia at the single-cell level in the early postnatal life of mice. They identified three stages of microglia development, which are characterized by gene expression and linked with chromatin changes, occurring in sync with the developing brain. Furthermore, they showed that the proper development of microglia is affected by the microbiome. Science, this issue p. 789 The microbiota help regulate the development of active immune defense in the central nervous system of mice. INTRODUCTION Microglia, as the resident myeloid cells of the central nervous system, play an important role in life-long brain maintenance and in pathology. Microglia are derived from erythromyeloid progenitors that migrate to the brain starting at embryonic day 8.5 and continuing until the blood-brain barrier is formed; after this, self-renewal is the only source of new microglia in the healthy brain. As the brain develops, microglia must perform different functions to accommodate temporally changing needs: first, actively engaging in synapse pruning and neurogenesis, and later, maintaining homeostasis. Although the interactions of microglia with the brain environment at steady state and in response to immune challenges have been well studied, their dynamics during development have not been fully elucidated. RATIONALE We systematically studied the transcriptional and epigenomic regulation of microglia throughout brain development to decipher the dynamics of the chromatin state and gene networks governing the transformation from yolk sac progenitor to adult microglia. We used environmental and genetic perturbation models to investigate how timed disruptions to microglia impact their natural development. RESULTS Global profiles of transcriptional states indicated that microglia development proceeds through three distinct temporal stages, which we define as early microglia (until embryonic day 14), pre-microglia (from embryonic day 14 to a few weeks after birth), and adult microglia (from a few weeks after birth onward). ATAC-seq (assay for transposase-accessible chromatin followed by sequencing) for chromatin accessibility and ChIP-seq (chromatin immunoprecipitation followed by sequencing) for histone modifications further characterized the differential regulatory elements in each developmental phase. Single-cell transcriptome analysis revealed minor mixing of the gene expression programs across phases, suggesting that individual cells shift their regulatory networks during development in a coordinated manner. Specific markers and regulatory factors distinguish each phase: For example, we identified MAFB as an important transcription factor of the adult microglia program. Microglia-specific knockout of MafB led to disruption of homeostasis in adulthood and increased expression of interferon and inflammation pathways. We found that microglia from germ-free mice exhibited dysregulation of dozens of genes associated with the adult phase and immune response. In addition, maternal immune activation, which has been linked to behavioral disorders in adult offspring, had the greatest impact on pre-microglia, resulting in a transcriptional shift toward the more advanced developmental stage. CONCLUSION Our work identifies a stepwise developmental program of microglia in synchrony with the developing brain. Each stage of microglia development has evolved distinct pathways for processing the relevant signals from the environment to balance their time-dependent role in neurogenesis with regulation of immune responses that may cause collateral damage. Genetic or environmental perturbations of these pathways can disrupt stage-specific functions of microglia and lead to loss of brain homeostasis, which may be associated with neurodevelopmental disorders. Microglia development proceeds in a stepwise manner. Microglia were isolated from mice throughout development from embryo to adult. Data from population-level RNA-seq, ChIP-seq, and ATAC-seq, as well as single-cell RNA-seq, show that microglia development proceeds through three distinct stages—early, pre-, and adult— with characteristic gene expression and functional states. Perturbations of this developmental process, such as from MafB knockout, lead to disrupted brain homeostasis by the dysregulation of adult and inflammatory genes. Tn5, transposase 5. Microglia, the resident myeloid cells of the central nervous system, play important roles in life-long brain maintenance and in pathology. Despite their importance, their regulatory dynamics during brain development have not been fully elucidated. Using genome-wide chromatin and expression profiling coupled with single-cell transcriptomic analysis throughout development, we found that microglia undergo three temporal stages of development in synchrony with the brain—early, pre-, and adult microglia—which are under distinct regulatory circuits. Knockout of the gene encoding the adult microglia transcription factor MAFB and environmental perturbations, such as those affecting the microbiome or prenatal immune activation, led to disruption of developmental genes and immune response pathways. Together, our work identifies a stepwise microglia developmental program integrating immune response pathways that may be associated with several neurodevelopmental disorders.

Single-cell spatial reconstruction reveals global division of labour in the mammalian liver

The mammalian liver consists of hexagon-shaped lobules that are radially polarized by blood flow and morphogens. Key liver genes have been shown to be differentially expressed along the lobule axis, a phenomenon termed zonation, but a detailed genome-wide reconstruction of this spatial division of labour has not been achieved. Here we measure the entire transcriptome of thousands of mouse liver cells and infer their lobule coordinates on the basis of a panel of zonated landmark genes, characterized with single-molecule fluorescence in situ hybridization. Using this approach, we obtain the zonation profiles of all liver genes with high spatial resolution. We find that around 50% of liver genes are significantly zonated and uncover abundant non-monotonic profiles that peak at the mid-lobule layers. These include a spatial order of bile acid biosynthesis enzymes that matches their position in the enzymatic cascade. Our approach can facilitate the reconstruction of similar spatial genomic blueprints for other mammalian organs.

Genomic Characterization of Murine Monocytes Reveals C/EBPβ Transcription Factor Dependence of Ly6C− Cells

Summary Monocytes are circulating, short‐lived mononuclear phagocytes, which in mice and man comprise two main subpopulations. Murine Ly6C+ monocytes display developmental plasticity and are recruited to complement tissue‐resident macrophages and dendritic cells on demand. Murine vascular Ly6C− monocytes patrol the endothelium, act as scavengers, and support vessel wall repair. Here we characterized population and single cell transcriptomes, as well as enhancer and promoter landscapes of the murine monocyte compartment. Single cell RNA‐seq and transplantation experiments confirmed homeostatic default differentiation of Ly6C+ into Ly6C− monocytes. The main two subsets were homogeneous, but linked by a more heterogeneous differentiation intermediate. We show that monocyte differentiation occurred through de novo enhancer establishment and activation of pre‐established (poised) enhancers. Generation of Ly6C− monocytes involved induction of the transcription factor C/EBP&bgr; and C/EBP&bgr;‐deficient mice lacked Ly6C− monocytes. Mechanistically, C/EBP&bgr; bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor. Graphical Abstract Figure. No Caption available. HighlightsSteady‐state Ly6C+ and Ly6C− monocytes are homogeneous populationsLy6Cint monocytes comprise a heterogeneous population expressing MHCIIC/EBP&bgr; regulates monocyte differentiation from Ly6C+ into Ly6C− cellsExpression of the monocyte survival factor Nr4a1 is regulated by C/EBP&bgr; &NA; Monocytes are circulating, short‐lived blood cells. Here, Mildner et al. (2017) use transcriptome and epigenome profiling to study murine monocyte identities and subset interrelations. They highlight the critical role of C/EBP&bgr; in monocyte conversion and reveal that while Ly6C+ and Ly6C− monocytes are homogeneous in steady state, Ly6Cint cells display heterogeneity.

Spatial reconstruction of immune niches by combining photoactivatable reporters and scRNA-seq

Spatial information from NICHE-seq Immune functions depend on the interactions of heterogeneous cells in a range of microenvironments in the body. Although information regarding immune cell function has been collected using single-cell RNA-sequencing methods, these techniques have traditionally lacked spatial information. Medaglia et al. describe NICHE-seq, a technique that allows the sorting and analysis of cells from within visually selected territories in transgenic mice that express photoactivatable green fluorescent protein. The method successfully identified T and B cell-specific niches in mouse lymph nodes and spleens after virus infection. The approach will allow us to bridge the gap between cellular and spatial information in studies of organs. Science, this issue p. 1622 NICHE-seq adds spatial information to single-cell sequencing. Cellular functions are strongly dependent on surrounding cells and environmental factors. Current technologies are limited in their ability to characterize the spatial location and gene programs of cells in poorly structured and dynamic niches. We developed a method, NICHE-seq, that combines photoactivatable fluorescent reporters, two-photon microscopy, and single-cell RNA sequencing (scRNA-seq) to infer the cellular and molecular composition of niches. We applied NICHE-seq to examine the high-order assembly of immune cell networks. NICHE-seq is highly reproducible in spatial tissue reconstruction, enabling identification of rare niche-specific immune subpopulations and gene programs, including natural killer cells within infected B cell follicles and distinct myeloid states in the spleen and tumor. This study establishes NICHE-seq as a broadly applicable method for elucidating high-order spatial organization of cell types and their molecular pathways.

Single-cell characterization of haematopoietic progenitors and their trajectories in homeostasis and perturbed haematopoiesis

The dynamics of haematopoietic stem cell differentiation and the hierarchy of oligopotent stem cells in the bone marrow remain controversial. Here we dissect haematopoietic progenitor populations at single cell resolution, deriving an unbiased reference model of transcriptional states in normal and perturbed murine bone marrow. We define the signature of the naive haematopoietic stem cell and find a continuum of core progenitor states. Core cell populations mix transcription of pre-myeloid and pre-lymphoid programs, but do not mix erythroid or megakaryocyte programs with other fates. CRISP-seq perturbation analysis confirms our models and reveals that Cebpa regulates entry into all myeloid fates, while Irf8 and PU.1 deficiency block later differentiation towards monocyte or granulocyte fates. Our transcriptional map defines a reference network model for blood progenitors and their differentiation trajectories during normal and perturbed haematopoiesis.Using a multi-tier scRNA-seq and CRISP-seq approach, Giladi et al. define a transcriptional signature for the naive haematopoietic stem cell state, and follow progenitor plasticity and fate commitment under the influence of cytokines and growth factors.

Single-cell mapping of the thymic stroma identifies IL-25-producing tuft epithelial cells

T cell development and selection are coordinated in the thymus by a specialized niche of diverse stromal populations1–3. Although much progress has been made over the years in identifying the functions of the different cell types of the thymic stromal compartment, there is no comprehensive characterization of their diversity and heterogeneity. Here we combined massively parallel single-cell RNA-sequencing4,5, spatial mapping, chromatin profiling and gene targeting to characterize de novo the entire stromal compartment of the mouse thymus. We identified dozens of cell states, with thymic epithelial cells (TECs) showing the highest degree of heterogeneity. Our analysis highlights four major medullary TEC (mTEC I–IV) populations, with distinct molecular functions, epigenetic landscapes and lineage regulators. Specifically, mTEC IV constitutes a new and highly divergent TEC lineage with molecular characteristics of the gut chemosensory epithelial tuft cells. Mice deficient in Pou2f3, a master regulator of tuft cells, have complete and specific depletion of mTEC IV cells, which results in increased levels of thymus-resident type-2 innate lymphoid cells. Overall, our study provides a comprehensive characterization of the thymic stroma and identifies a new tuft-like TEC population, which is critical for shaping the immune niche in the thymus.A comprehensive characterization of the thymic stroma identifies a tuft-cell-like thymic epithelial cell population that is critical for shaping the immune niche in the thymus.

MetaCell: analysis of single cell RNA-seq data using k-NN graph partitions

Single cell RNA-seq (scRNA-seq) has become the method of choice for analyzing mRNA distributions in heterogeneous cell populations. scRNA-seq only partially samples the cells in a tissue and the RNA in each cell, resulting in sparse data that challenge analysis. We develop a methodology that addresses scRNA-seq’s sparsity through partitioning the data into metacells: disjoint, homogenous and highly compact groups of cells, each exhibiting only sampling variance. Metacells constitute local building blocks for clustering and quantitative analysis of gene expression, while not enforcing any global structure on the data, thereby maintaining statistical control and minimizing biases. We illustrate the MetaCell framework by re-analyzing cell type and transcriptional gradients in peripheral blood and whole organism scRNA-seq maps. Our algorithms are implemented in the new MetaCell R/C++ software package.