CD4 Helper and CD8 Cytotoxic T Cell Differentiation.

A fundamental question in developmental immunology is how bipotential thymocyte precursors generate both CD4+ helper and CD8+ cytotoxic T cell lineages. The MHC specificity of αß T cell receptors (TCRs) on precursors is closely correlated with cell fate-determining processes, prompting studies to characterize how variations in TCR signaling are linked with genetic programs establishing lineage-specific gene expression signatures, such as exclusive CD4 or CD8 expression. The key transcription factors ThPOK and Runx3 have been identified as mediating development of helper and cytotoxic T cell lineages, respectively. Together with increasing knowledge of epigenetic regulators, these findings have advanced our understanding of the transcription factor network regulating the CD4/CD8 dichotomy. It has also become apparent that CD4+ T cells retain developmental plasticity, allowing them to acquire cytotoxic activity in the periphery. Despite such advances, further studies are necessary to identify the molecular links between TCR signaling and the nuclear machinery regulating expression of ThPOK and Runx3.

A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells.

Cellular lineage histories and their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have insufficient barcode diversity and single-cell lineage coverage for profiling tissues composed of millions of cells. Here, we developed DARLIN, an inducible Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) and 30 CRISPR target sites. DARLIN is inducible, generates massive lineage barcodes across tissues, and enables the detection of edited barcodes in ∼70% of profiled single cells. Using DARLIN, we examined fate bias within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we established a protocol for joint transcriptomic and epigenomic single-cell measurements with DARLIN and found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable the high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts.

What Is a Plant Cell Type in the Age of Single-Cell Biology? It's Complicated.

One of the fundamental questions in developmental biology is how a cell is specified to differentiate as a specialized cell type. Traditionally, plant cell types were defined based on their function, location, morphology, and lineage. Currently, in the age of single-cell biology, researchers typically attempt to assign plant cells to cell types by clustering them based on their transcriptomes. However, because cells are dynamic entities that progress through the cell cycle and respond to signals, the transcriptome also reflects the state of the cell at a particular moment in time, raising questions about how to define a cell type. We suggest that these complexities and dynamics of cell states are of interest and further consider the roles signaling, stochasticity, cell cycle, and mechanical forces play in plant cell fate specification. Once established, cell identity must also be maintained. With the wealth of single-cell data coming out, the field is poised to elucidate both the complexity and dynamics of cell states.

Transcriptional regulation of innate and adaptive lymphocyte lineages.

The lymphocyte family has expanded significantly in recent years to include not only the adaptive lymphocytes (T cells, B cells) and NK cells, but also several additional innate lymphoid cell (ILC) types. ILCs lack clonally distributed antigen receptors characteristic of adaptive lymphocytes and instead respond exclusively to signaling via germline-encoded receptors. ILCs resemble T cells more closely than any other leukocyte lineage at the transcriptome level and express many elements of the core T cell transcriptional program, including Notch, Gata3, Tcf7, and Bcl11b. We present our current understanding of the shared and distinct transcriptional regulatory mechanisms involved in the development of adaptive T lymphocytes and closely related ILCs. We discuss the possibility that a core set of transcriptional regulators common to ILCs and T cells establish enhancers that enable implementation of closely aligned effector pathways. Studies of the transcriptional regulation of lymphopoiesis will support the development of novel therapeutic approaches to correct early lymphoid developmental defects and aberrant lymphocyte function.

Quantitative fate mapping: A general framework for analyzing progenitor state dynamics via retrospective lineage barcoding.

Natural and induced somatic mutations that accumulate in the genome during development record the phylogenetic relationships of cells; whether these lineage barcodes capture the complex dynamics of progenitor states remains unclear. We introduce quantitative fate mapping, an approach to reconstruct the hierarchy, commitment times, population sizes, and commitment biases of intermediate progenitor states during development based on a time-scaled phylogeny of their descendants. To reconstruct time-scaled phylogenies from lineage barcodes, we introduce Phylotime, a scalable maximum likelihood clustering approach based on a general barcoding mutagenesis model. We validate these approaches using realistic in silico and in vitro barcoding experiments. We further establish criteria for the number of cells that must be analyzed for robust quantitative fate mapping and a progenitor state coverage statistic to assess the robustness. This work demonstrates how lineage barcodes, natural or synthetic, enable analyzing progenitor fate and dynamics long after embryonic development in any organism.

Optimization of the Irf8 +32-kb enhancer disrupts dendritic cell lineage segregation.

Autoactivation of lineage-determining transcription factors mediates bistable expression, generating distinct cell phenotypes essential for complex body plans. Classical type 1 dendritic cell (cDC1) and type 2 dendritic cell (cDC2) subsets provide nonredundant functions for defense against distinct immune challenges. Interferon regulatory factor 8 (IRF8), the cDC1 lineage-determining transcription factor, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32-kb enhancer, responsible for IRF8 autoactivation, is naturally suboptimized with low-affinity IRF8 binding sites. Introducing multiple high-affinity IRF8 sites into the Irf8 +32-kb enhancer causes a gain-of-function effect, leading to erroneous IRF8 autoactivation in specified cDC2 progenitors, redirecting them toward cDC1 and a novel hybrid DC subset with mixed-lineage phenotypes. Further, this also causes a loss-of-function effect, reducing Irf8 expression in cDC1s. These developmental alterations critically impair both cDC1-dependent and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of cDCs required for normal immune function.

Chromatin and gene-regulatory dynamics of the developing human cerebral cortex at single-cell resolution.

Genetic perturbations of cortical development can lead to neurodevelopmental disease, including autism spectrum disorder (ASD). To identify genomic regions crucial to corticogenesis, we mapped the activity of gene-regulatory elements generating a single-cell atlas of gene expression and chromatin accessibility both independently and jointly. This revealed waves of gene regulation by key transcription factors (TFs) across a nearly continuous differentiation trajectory, distinguished the expression programs of glial lineages, and identified lineage-determining TFs that exhibited strong correlation between linked gene-regulatory elements and expression levels. These highly connected genes adopted an active chromatin state in early differentiating cells, consistent with lineage commitment. Base-pair-resolution neural network models identified strong cell-type-specific enrichment of noncoding mutations predicted to be disruptive in a cohort of ASD individuals and identified frequently disrupted TF binding sites. This approach illustrates how cell-type-specific mapping can provide insights into the programs governing human development and disease.

Mouse totipotent stem cells captured and maintained through spliceosomal repression.

Since establishment of the first embryonic stem cells (ESCs), in vitro culture of totipotent cells functionally and molecularly comparable with in vivo blastomeres with embryonic and extraembryonic developmental potential has been a challenge. Here we report that spliceosomal repression in mouse ESCs drives a pluripotent-to-totipotent state transition. Using the splicing inhibitor pladienolide B, we achieve stable in vitro culture of totipotent ESCs comparable at molecular levels with 2- and 4-cell blastomeres, which we call totipotent blastomere-like cells (TBLCs). Mouse chimeric assays combined with single-cell RNA sequencing (scRNA-seq) demonstrate that TBLCs have a robust bidirectional developmental capability to generate multiple embryonic and extraembryonic cell lineages. Mechanically, spliceosomal repression causes widespread splicing inhibition of pluripotent genes, whereas totipotent genes, which contain few short introns, are efficiently spliced and transcriptionally activated. Our study provides a means for capturing and maintaining totipotent stem cells.

A cellular and spatial map of the choroid plexus across brain ventricles and ages.

The choroid plexus (ChP) in each brain ventricle produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier. Here, we construct a single-cell and spatial atlas of each ChP in the developing, adult, and aged mouse brain. We delineate diverse cell types, subtypes, cell states, and expression programs in epithelial and mesenchymal cells across ages and ventricles. In the developing ChP, we predict a common progenitor pool for epithelial and neuronal cells, validated by lineage tracing. Epithelial and fibroblast cells show regionalized expression by ventricle, starting at embryonic stages and persisting with age, with a dramatic transcriptional shift with maturation, and a smaller shift in each aged cell type. With aging, epithelial cells upregulate host-defense programs, and resident macrophages upregulate interleukin-1ß (IL-1ß) signaling genes. Our atlas reveals cellular diversity, architecture and signaling across ventricles during development, maturation, and aging of the ChP-brain barrier.

Changes in genome architecture and transcriptional dynamics progress independently of sensory experience during post-natal brain development.

Both transcription and three-dimensional (3D) architecture of the mammalian genome play critical roles in neurodevelopment and its disorders. However, 3D genome structures of single brain cells have not been solved; little is known about the dynamics of single-cell transcriptome and 3D genome after birth. Here, we generated a transcriptome (3,517 cells) and 3D genome (3,646 cells) atlas of the developing mouse cortex and hippocampus by using our high-resolution multiple annealing and looping-based amplification cycles for digital transcriptomics (MALBAC-DT) and diploid chromatin conformation capture (Dip-C) methods and developing multi-omic analysis pipelines. In adults, 3D genome "structure types" delineate all major cell types, with high correlation between chromatin A/B compartments and gene expression. During development, both transcriptome and 3D genome are extensively transformed in the first post-natal month. In neurons, 3D genome is rewired across scales, correlated with gene expression modules, and independent of sensory experience. Finally, we examine allele-specific structure of imprinted genes, revealing local and chromosome (chr)-wide differences. These findings uncover an unknown dimension of neurodevelopment.

Intrinsically disordered domain of transcription factor TCF-1 is required for T cell developmental fidelity.

In development, pioneer transcription factors access silent chromatin to reveal lineage-specific gene programs. The structured DNA-binding domains of pioneer factors have been well characterized, but whether and how intrinsically disordered regions affect chromatin and control cell fate is unclear. Here, we report that deletion of an intrinsically disordered region of the pioneer factor TCF-1 (termed L1) leads to an early developmental block in T cells. The few T cells that develop from progenitors expressing TCF-1 lacking L1 exhibit lineage infidelity distinct from the lineage diversion of TCF-1-deficient cells. Mechanistically, L1 is required for activation of T cell genes and repression of GATA2-driven genes, normally reserved to the mast cell and dendritic cell lineages. Underlying this lineage diversion, L1 mediates binding of TCF-1 to its earliest target genes, which are subject to repression as T cells develop. These data suggest that the intrinsically disordered N terminus of TCF-1 maintains T cell lineage fidelity.

The biology of recent thymic emigrants.

The generation of the TCRαß lineage of T cells occurs in the thymus through a series of orchestrated developmental events that result in a carefully selected population of CD4 or CD8 lineage-committed TCR(+) thymocytes capable of recognizing foreign antigen in the context of self MHC. T cells first exit the thymus in a phenotypically and functionally immature state and require an approximately 3-week period of post-thymic maturation before transitioning into the mature T cell compartment. A greater understanding of recent thymic emigrant biology has come with the development of methods to exclusively identify and isolate this population for further characterization. I now review current knowledge about the phenotype and function of this key but understudied population of peripheral T cells.

Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin.

Cell differentiation and function are regulated across multiple layers of gene regulation, including modulation of gene expression by changes in chromatin accessibility. However, differentiation is an asynchronous process precluding a temporal understanding of regulatory events leading to cell fate commitment. Here we developed simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq), a highly scalable approach for measurement of chromatin accessibility and gene expression in the same single cell, applicable to different tissues. Using 34,774 joint profiles from mouse skin, we develop a computational strategy to identify cis-regulatory interactions and define domains of regulatory chromatin (DORCs) that significantly overlap with super-enhancers. During lineage commitment, chromatin accessibility at DORCs precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for lineage commitment. We computationally infer chromatin potential as a quantitative measure of chromatin lineage-priming and use it to predict cell fate outcomes. SHARE-seq is an extensible platform to study regulatory circuitry across diverse cells in tissues.

Single-Cell Transcriptomics Reveals Early Emergence of Liver Parenchymal and Non-parenchymal Cell Lineages.

The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-parenchymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.

Unraveling Hematopoiesis through the Lens of Genomics.

Hematopoiesis has long served as a paradigm of stem cell biology and tissue homeostasis. In the past decade, the genomics revolution has ushered in powerful new methods for investigating the hematopoietic system that have provided transformative insights into its biology. As part of the advances in genomics, increasingly accurate deep sequencing and novel methods of cell tracking have revealed hematopoiesis to be more of a continuous and less of a discrete and punctuated process than originally envisioned. In part, this continuous nature of hematopoiesis is made possible by the emergent outcomes of vast, interconnected regulatory networks that influence cell fates and lineage commitment. It is also becoming clear how these mechanisms are modulated by genetic variation present throughout the population. This review describes how these recently uncovered complexities are reshaping our concept of tissue development and homeostasis while opening up a more comprehensive future understanding of hematopoiesis.

MorphoSeq: Full Single-Cell Transcriptome Dynamics Up to Gastrulation in a Chordate.

Single-cell RNA sequencing (scRNA-seq) provides a leap forward in resolving cellular diversity and developmental trajectories but fails to comprehensively delineate the spatial organization and precise cellular makeup of individual embryos. Here, we reconstruct from scRNA-seq and light sheet imaging data a canonical digital embryo that captures the genome-wide gene expression trajectory of every single cell at every cell division in the 18 lineages up to gastrulation in the ascidian Phallusia mammillata. By using high-coverage scRNA-seq, we devise a computational framework that stratifies single cells of individual embryos into cell types without prior knowledge. Unbiased transcriptome data analysis mapped each cell's physical position and lineage history, yielding the complete history of gene expression at the genome-wide level for every single cell in a developing embryo. A comparison of individual embryos reveals both extensive reproducibility between symmetric embryo sides and a large inter-embryonic variability due to small differences in embryogenesis timing.

An Engineered CRISPR-Cas9 Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression Profiles in Single Cells.

Tracing the lineage history of cells is key to answering diverse and fundamental questions in biology. Coupling of cell ancestry information with other molecular readouts represents an important goal in the field. Here, we describe the CRISPR array repair lineage tracing (CARLIN) mouse line and corresponding analysis tools that can be used to simultaneously interrogate the lineage and transcriptomic information of single cells in vivo. This model exploits CRISPR technology to generate up to 44,000 transcribed barcodes in an inducible fashion at any point during development or adulthood, is compatible with sequential barcoding, and is fully genetically defined. We have used CARLIN to identify intrinsic biases in the activity of fetal liver hematopoietic stem cell (HSC) clones and to uncover a previously unappreciated clonal bottleneck in the response of HSCs to injury. CARLIN also allows the unbiased identification of transcriptional signatures associated with HSC activity without cell sorting.

Defining the Design Principles of Skin Epidermis Postnatal Growth.

During embryonic and postnatal development, organs and tissues grow steadily to achieve their final size at the end of puberty. However, little is known about the cellular dynamics that mediate postnatal growth. By combining in vivo clonal lineage tracing, proliferation kinetics, single-cell transcriptomics, and in vitro micro-pattern experiments, we resolved the cellular dynamics taking place during postnatal skin epidermis expansion. Our data revealed that harmonious growth is engineered by a single population of developmental progenitors presenting a fixed fate imbalance of self-renewing divisions with an ever-decreasing proliferation rate. Single-cell RNA sequencing revealed that epidermal developmental progenitors form a more uniform population compared with adult stem and progenitor cells. Finally, we found that the spatial pattern of cell division orientation is dictated locally by the underlying collagen fiber orientation. Our results uncover a simple design principle of organ growth where progenitors and differentiated cells expand in harmony with their surrounding tissues.

A double-negative thymocyte-specific enhancer augments Notch1 signaling to direct early T cell progenitor expansion, lineage restriction and ß-selection.

T cell differentiation requires Notch1 signaling. In the present study, we show that an enhancer upstream of Notch1 active in double-negative (DN) mouse thymocytes is responsible for raising Notch1 signaling intrathymically. This enhancer is required to expand multipotent progenitors intrathymically while delaying early differentiation until lineage restrictions have been established. Early thymic progenitors lacking the enhancer show accelerated differentiation through the DN stages and increased frequency of B, innate lymphoid (IL) and natural killer (NK) cell differentiation. Transcription regulators for T cell lineage restriction and commitment are expressed normally, but IL and NK cell gene expression persists after T cell lineage commitment and T cell receptor ß VDJ recombination, Cd3 expression and ß-selection have been impaired. This Notch1 enhancer is inactive in double-positive (DP) thymocytes. Its aberrant reactivation at this stage in Ikaros mutants is required for leukemogenesis. Thus, the DN-specific Notch1 enhancer harnesses the regulatory architecture of DN and DP thymocytes to achieve carefully orchestrated changes in Notch1 signaling required for early lineage restrictions and normal T cell differentiation.

Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.

Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. Using two newly generated transgenic mice that report and trace the expression of terminal deoxynucleotidyl transferase (TdT), transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset that lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction on MPPs reflected a transcriptionally dynamic but uncommitted stage, characterized by low expression of lineage-associated genes. Single-cell CITE-seq indicated that multipotency in the TdT+ MPPs is associated with expression of the endothelial cell adhesion molecule ESAM. Stable and progressive upregulation of TdT defined the lymphoid developmental trajectory. Collectively, we here identify a new multipotent progenitor within the MPP4 compartment. Specification and commitment are defined by downregulation of ESAM which marks the progressive loss of alternative fates along all lineages.