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.

Chromatin modifications are crucial for development, yet little is known about their dynamics during differentiation. Hematopoiesis provides a well-defined model to study chromatin state dynamics, however technical limitations impede profiling of homogeneous differentiation intermediates. We developed a high sensitivity indexing-first chromatin immunoprecipitation approach (iChIP) to profile the dynamics of four chromatin modifications across 16 stages of hematopoietic differentiation. We identify 48,415 enhancer regions and characterize their dynamics. We find that lineage commitment involves de novo establishment of 17,035 lineage-specific enhancers. These enhancer repertoire expansions foreshadow transcriptional programs in differentiated cells. Combining our enhancer catalog with gene expression profiles, we elucidate the transcription factor network controlling chromatin dynamics and lineage specification in hematopoiesis. Together, our results provide a comprehensive model of chromatin dynamics during development.

Innate lymphoid cells (ILCs) are critical modulators of mucosal immunity, inflammation, and tissue homeostasis, but their full spectrum of cellular states and regulatory landscapes remains elusive. Here, we combine genome-wide RNA-seq, ChIP-seq, and ATAC-seq to compare the transcriptional and epigenetic identity of small intestinal ILCs, identifying thousands of distinct gene profiles and regulatory elements. Single-cell RNA-seq and flow and mass cytometry analyses reveal compartmentalization of cytokine expression and metabolic activity within the three classical ILC subtypes and highlight transcriptional states beyond the current canonical classification. In addition, using antibiotic intervention and germ-free mice, we characterize the effect of the microbiome on the ILC regulatory landscape and determine the response of ILCs to microbial colonization at the single-cell level. Together, our work characterizes the spectrum of transcriptional identities of small intestinal ILCs and describes how ILCs differentially integrate signals from the microbial microenvironment to generate phenotypic and functional plasticity.

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/EBPβ and C/EBPβ-deficient mice lacked Ly6C monocytes. Mechanistically, C/EBPβ bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor.

Background: Cardiac fibroblasts (CF) have a central role in the ventricular remodeling process associated with different types of fibrosis. Recent studies have shown that fibroblasts do not respond homogeneously to heart injury. Due to the limited set of bona fide fibroblast markers, a proper characterization of fibroblast population heterogeneity in response to cardiac damage is still missing. The purpose of this study was to define the CF heterogeneity during ventricular remodeling and the underlying mechanisms that regulate their function. Methods: Collagen1α1-GFP + CF were characterized after myocardial infarction (MI) by single-cell and bulk RNA-seq, ATAC-seq and functional assays. Swine and patient samples were studied using bulk RNA-seq. Results: We identified and characterized a unique CF subpopulation that emerges after MI in mice. These activated fibroblasts exhibit a clear pro-fibrotic signature, express high levels of Collagen Triple Helix Repeat Containing 1 ( Cthrc1 ) and localize into the scar. Non-canonical TGF-β signaling and different transcription factors including SOX9 are important regulators mediating their response to cardiac injury. Moreover, the absence of CTHRC1 results in pronounced lethality due to ventricular rupture. Finally, a population of CF with a similar transcriptome was identified in a swine model of MI and in heart tissue from patients with MI and dilated cardiomyopathy. Conclusions: We report CF heterogeneity, their dynamics during the course of MI and redefine the CF that respond to cardiac injury and participate in myocardial remodeling. Our study identifies Cthrc1 as a novel regulator of the healing scar process, and as a target for future translational studies.

The derivation of induced pluripotent cells (iPSCs) from individuals suffering from genetic syndromes offers new opportunities for basic research into these diseases and the development of therapeutic compounds. iPSCs can self renew and can be differentiated to many cell types, offering a potentially unlimited source of material for study. In this review we discuss the conceptual and practical issues to consider when attempting to model genetic diseases using iPSCs.

Gaucher's disease (GD) is caused by mutations in the GBA1 gene, which encodes acid-β-glucosidase, an enzyme involved in the degradation of complex sphingolipids. While the non-neuronopathic aspects of the disease can be treated with enzyme replacement therapy (ERT), the early-onset neuronopathic form currently lacks therapeutic options and is lethal. We have developed an induced pluripotent stem cell (iPSc) model of neuronopathic GD. Dermal fibroblasts of a patient with a P.[LEU444PRO];[GLY202ARG] genotype were transfected with a loxP-flanked polycistronic reprogramming cassette consisting of Oct4, Sox2, Klf4 and c-Myc and iPSc lines derived. A non-integrative lentiviral vector expressing Cre recombinase was used to eliminate the reprogramming cassette from the reprogrammed cells. Our GD iPSc express pluripotent markers, differentiate into the three germ layers, form teratomas, have a normal karyotype and show the same mutations and low acid-β-glucosidase activity as the original fibroblasts they were derived from. We have differentiated them efficiently into neurons and also into macrophages without observing deleterious effects of the mutations on the differentiation process. Using our system as a platform to test chemical compounds capable of increasing acid-β-glucosidase activity, we confirm that two nojirimycin analogues can rescue protein levels and enzyme activity in the cells affected by the disease.

Understanding the fate of dendritic cells (DCs) after productive immune synapses (postsynaptic DCs) with T cells during antigen presentation has been largely neglected in favor of deciphering the nuances of T cell activation and memory generation. Here, we describe that postsynaptic DCs switch their transcriptomic signature, correlating with epigenomic changes including DNA accessibility and histone methylation. We focus on the chemokine receptor Ccr7 as a proof-of-concept gene that is increased in postsynaptic DCs. Consistent with our epigenomic observations, postsynaptic DCs migrate more efficiently toward CCL19 in vitro and display enhanced homing to draining lymph nodes in vivo. This work describes a previously unknown DC population whose transcriptomics, epigenomics, and migratory capacity change in response to their cognate contact with T cells.