A human embryonic limb cell atlas resolved in space and time.

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

Dynamic transformations of genome-wide epigenetic marking and transcriptional control establish T cell identity.

T cell development comprises a stepwise process of commitment from a multipotent precursor. To define molecular mechanisms controlling this progression, we probed five stages spanning the commitment process using RNA-seq and ChIP-seq to track genome-wide shifts in transcription, cohorts of active transcription factor genes, histone modifications at diverse classes of cis-regulatory elements, and binding repertoire of GATA-3 and PU.1, transcription factors with complementary roles in T cell development. The results highlight potential promoter-distal cis-regulatory elements in play and reveal both activation sites and diverse mechanisms of repression that silence genes used in alternative lineages. Histone marking is dynamic and reversible, and though permissive marks anticipate, repressive marks often lag behind changes in transcription. In vivo binding of PU.1 and GATA-3 relative to epigenetic marking reveals distinctive factor-specific rules for recruitment of these crucial transcription factors to different subsets of their potential sites, dependent on dose and developmental context.

Antigen-specific age-related memory CD8 T cells induce and track Alzheimer's-like neurodegeneration.

Cerebral (Aß) plaque and (pTau) tangle deposition are hallmarks of Alzheimer's disease (AD), yet are insufficient to confer complete AD-like neurodegeneration experimentally. Factors acting upstream of Aß/pTau in AD remain unknown, but their identification could enable earlier diagnosis and more effective treatments. T cell abnormalities are emerging AD hallmarks, and CD8 T cells were recently found to mediate neurodegeneration downstream of tangle deposition in hereditary neurodegeneration models. The precise impact of T cells downstream of Aß/pTau, however, appears to vary depending on the animal model. Our prior work suggested that antigen-specific memory CD8 T ("hiT") cells act upstream of Aß/pTau after brain injury. Here, we examine whether hiT cells influence sporadic AD-like pathophysiology upstream of Aß/pTau. Examining neuropathology, gene expression, and behavior in our hiT mouse model we show that CD8 T cells induce plaque and tangle-like deposition, modulate AD-related genes, and ultimately result in progressive neurodegeneration with both gross and fine features of sporadic human AD. T cells required Perforin to initiate this pathophysiology, and IFNγ for most gene expression changes and progression to more widespread neurodegenerative disease. Analogous antigen-specific memory CD8 T cells were significantly elevated in the brains of human AD patients, and their loss from blood corresponded to sporadic AD and related cognitive decline better than plasma pTau-217, a promising AD biomarker candidate. We identify an age-related factor acting upstream of Aß/pTau to initiate AD-like pathophysiology, the mechanisms promoting its pathogenicity, and its relevance to human sporadic AD.

Spatiotemporal DNA methylome dynamics of the developing mouse fetus.

Cytosine DNA methylation is essential for mammalian development but understanding of its spatiotemporal distribution in the developing embryo remains limited1,2. Here, as part of the mouse Encyclopedia of DNA Elements (ENCODE) project, we profiled 168 methylomes from 12 mouse tissues or organs at 9 developmental stages from embryogenesis to adulthood. We identified 1,808,810 genomic regions that showed variations in CG methylation by comparing the methylomes of different tissues or organs from different developmental stages. These DNA elements predominantly lose CG methylation during fetal development, whereas the trend is reversed after birth. During late stages of fetal development, non-CG methylation accumulated within the bodies of key developmental transcription factor genes, coinciding with their transcriptional repression. Integration of genome-wide DNA methylation, histone modification and chromatin accessibility data enabled us to predict 461,141 putative developmental tissue-specific enhancers, the human orthologues of which were enriched for disease-associated genetic variants. These spatiotemporal epigenome maps provide a resource for studies of gene regulation during tissue or organ progression, and a starting point for investigating regulatory elements that are involved in human developmental disorders.

The changing mouse embryo transcriptome at whole tissue and single-cell resolution.

During mammalian embryogenesis, differential gene expression gradually builds the identity and complexity of each tissue and organ system1. Here we systematically quantified mouse polyA-RNA from day 10.5 of embryonic development to birth, sampling 17 tissues and organs. The resulting developmental transcriptome is globally structured by dynamic cytodifferentiation, body-axis and cell-proliferation gene sets that were further characterized by the transcription factor motif codes of their promoters. We decomposed the tissue-level transcriptome using single-cell RNA-seq (sequencing of RNA reverse transcribed into cDNA) and found that neurogenesis and haematopoiesis dominate at both the gene and cellular levels, jointly accounting for one-third of differential gene expression and more than 40% of identified cell types. By integrating promoter sequence motifs with companion ENCODE epigenomic profiles, we identified a prominent promoter de-repression mechanism in neuronal expression clusters that was attributable to known and novel repressors. Focusing on the developing limb, single-cell RNA data identified 25 candidate cell types that included progenitor and differentiating states with computationally inferred lineage relationships. We extracted cell-type transcription factor networks and complementary sets of candidate enhancer elements by using single-cell RNA-seq to decompose integrative cis-element (IDEAS) models that were derived from whole-tissue epigenome chromatin data. These ENCODE reference data, computed network components and IDEAS chromatin segmentations are companion resources to the matching epigenomic developmental matrix, and are available for researchers to further mine and integrate.

An atlas of dynamic chromatin landscapes in mouse fetal development.

The Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP-seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC-seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

Author Correction: An atlas of dynamic chromatin landscapes in mouse fetal development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Expanded encyclopaedias of DNA elements in the human and mouse genomes.

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

Day-One Pain Reductions After Hip and Knee Replacement When Buprenorphine-Clonidine-Dexamethasone Is Added to Bupivacaine Nerve/Plexus Blocks: A Randomized Clinical Trial.

OBJECTIVE: To compare pain outcome reports of patients undergoing hip or knee replacement who received single-injection nerve/plexus blocks with plain bupivacaine (BPV) with those of patients who received injections of buprenorphine-clonidine-dexamethasone (BCD) admixed with BPV. DESIGN: Prospective, parallel-arm, randomized, double-blind trial. SETTING: A single veterans' hospital. SUBJECTS: Ninety-eight veterans scheduled for total hip or knee replacement surgery with spinal as the primary anesthetic. METHODS: Participants were randomized to BPV-BCD or plain BPV groups. They underwent nerve/plexus blocks in the L2-L4 and L4-S3 distributions in advance of joint replacement surgery. The primary outcome was change in pain from baseline during the postoperative day, as assessed by the total pain score on the short-form McGill Pain Questionnaire-v2 (SF-MPQ-2). Secondary outcomes were pain during movement, pain interference, range of motion, mobility, and quality of recovery. RESULTS: On postoperative day one, the SF-MPQ-2 total score for the BPV-BCD group demonstrated greater pain reduction than that of the plain BPV group (mean difference 1.8 points, 95% confidence interval 0.6 to 3.0, P = 0.003). The BPV-BCD group also had larger reductions in pain during movement in the surgical joint and less pain interference, along with increased range of hip and knee flexion, compared with the plain BPV group. Outcomes of mobility and quality of recovery were not different between groups. CONCLUSIONS: Preoperative BPV-BCD blocks in the L2-L4 and L4-S3 nerve distributions for hip and knee replacements led to less pain on postoperative day one and increased knee and hip range of motion, compared with plain BPV blocks. TRIAL REGISTRATION: ClinicalTrials.gov ID NCT02891798.

Pioneering, chromatin remodeling, and epigenetic constraint in early T-cell gene regulation by SPI1 (PU.1).

SPI1 (also known as PU.1) is a dominant but transient regulator in early T-cell precursors and a potent transcriptional controller of developmentally important pro-T-cell genes. Before T-lineage commitment, open chromatin is frequently occupied by PU.1, and many PU.1 sites lose accessibility when PU.1 is later down-regulated. Pioneering activity of PU.1 was tested in this developmentally dynamic context by quantitating the relationships between PU.1 occupancy and site quality and accessibility as PU.1 levels naturally declined in pro-T-cell development and by using stage-specific gain- and loss-of-function perturbations to relate binding to effects on target genes. PU.1 could bind closed genomic sites, but rapidly opened many of them, despite the absence of its frequent collaborator, CEBPA. RUNX motifs and RUNX1 binding were often linked to PU.1 at open sites, but highly expressed PU.1 could bind its sites without RUNX1. The dynamic properties of PU.1 engagements implied that PU.1 binding affinity and concentration determine its occupancy choices, but with quantitative trade-offs for occupancy between site sequence quality and stage-dependent site accessibility in chromatin. At nonpromoter sites, PU.1 binding criteria were more stringent than at promoters, and PU.1 was also much more effective as a transcriptional regulator at nonpromoter sites where local chromatin accessibility depended on the presence of PU.1. Notably, closed chromatin presented a qualitative barrier to occupancy by the PU.1 DNA-binding domain alone. Thus, effective pioneering at closed chromatin sites also depends on requirements beyond site recognition, served by non-DNA-binding domains of PU.1.

Reconstructing a metazoan genetic pathway with transcriptome-wide epistasis measurements.

RNA-sequencing (RNA-seq) is commonly used to identify genetic modules that respond to perturbations. In single cells, transcriptomes have been used as phenotypes, but this concept has not been applied to whole-organism RNA-seq. Also, quantifying and interpreting epistatic effects using expression profiles remains a challenge. We developed a single coefficient to quantify transcriptome-wide epistasis that reflects the underlying interactions and which can be interpreted intuitively. To demonstrate our approach, we sequenced four single and two double mutants of Caenorhabditis elegans From these mutants, we reconstructed the known hypoxia pathway. In addition, we uncovered a class of 56 genes with HIF-1-dependent expression that have opposite changes in expression in mutants of two genes that cooperate to negatively regulate HIF-1 abundance; however, the double mutant of these genes exhibits suppression epistasis. This class violates the classical model of HIF-1 regulation but can be explained by postulating a role of hydroxylated HIF-1 in transcriptional control.

A comparative encyclopedia of DNA elements in the mouse genome.

The laboratory mouse shares the majority of its protein-coding genes with humans, making it the premier model organism in biomedical research, yet the two mammals differ in significant ways. To gain greater insights into both shared and species-specific transcriptional and cellular regulatory programs in the mouse, the Mouse ENCODE Consortium has mapped transcription, DNase I hypersensitivity, transcription factor binding, chromatin modifications and replication domains throughout the mouse genome in diverse cell and tissue types. By comparing with the human genome, we not only confirm substantial conservation in the newly annotated potential functional sequences, but also find a large degree of divergence of sequences involved in transcriptional regulation, chromatin state and higher order chromatin organization. Our results illuminate the wide range of evolutionary forces acting on genes and their regulatory regions, and provide a general resource for research into mammalian biology and mechanisms of human diseases.

Extended Perineural Analgesia After Hip and Knee Replacement When Buprenorphine-Clonidine-Dexamethasone Is Added to Bupivacaine: Preliminary Report from a Randomized Clinical Trial.

OBJECTIVE: We tested the hypothesis that buprenorphine-clonidine-dexamethasone (BCD) extends perineural analgesia compared with plain bupivacaine (BPV) nerve blocks used for hip and knee replacement surgery. DESIGN: Prospective, parallel-arms, randomized, double-blind trial. SETTING: A single veterans' hospital. SUBJECTS: Seventy-eight veterans scheduled for total hip or knee replacement with plans for spinal as the primary anesthetic. METHODS: Participants underwent nerve/plexus blocks at L2-L4 and L4-S3 in advance of hip or knee joint replacement surgery. Patients were randomized to receive BPV-BCD or plain BPV in a 4:1 allocation ratio. Patients answered four block duration questions (listed below). Time differences between treatments were analyzed using the t test. RESULTS: Significant (P < 0.001) prolongation of the time parameters was reported by patients after the BPV-BCD blocks (N = 62) vs plain BPV (N = 16). The time until start of postoperative pain was 26 vs 11 hours (mean difference = 15 hours, 95% CI = 8 to 21). The time until no pain relief from the blocks was 32 vs 15 hours (mean difference = 17 hours, 95% CI = 10 to 24). The time until the numbness wore off was 37 vs 21 hours (mean difference = 16 hours, 95% CI = 8 to 23). The time until the worst postoperative pain was 39 vs 20 hours (mean difference = 19 hours, 95% CI = 11 to 27). CONCLUSIONS: BPV-BCD provided 26-39 hours of perineural analgesia in the L2-L4 and L4-S3 nerve distributions after hip/knee replacement surgery, compared with 11-21 hours for plain BPV.

Landscape of transcription in human cells.

Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell's regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene.

From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing.

Single-cell RNA-seq mammalian transcriptome studies are at an early stage in uncovering cell-to-cell variation in gene expression, transcript processing and editing, and regulatory module activity. Despite great progress recently, substantial challenges remain, including discriminating biological variation from technical noise. Here we apply the SMART-seq single-cell RNA-seq protocol to study the reference lymphoblastoid cell line GM12878. By using spike-in quantification standards, we estimate the absolute number of RNA molecules per cell for each gene and find significant variation in total mRNA content: between 50,000 and 300,000 transcripts per cell. We directly measure technical stochasticity by a pool/split design and find that there are significant differences in expression between individual cells, over and above technical variation. Specific gene coexpression modules were preferentially expressed in subsets of individual cells, including one enriched for mRNA processing and splicing factors. We assess cell-to-cell variation in alternative splicing and allelic bias and report evidence of significant differences in splice site usage that exceed splice variation in the pool/split comparison. Finally, we show that transcriptomes from small pools of 30-100 cells approach the information content and reproducibility of contemporary RNA-seq from large amounts of input material. Together, our results define an experimental and computational path forward for analyzing gene expression in rare cell types and cell states.