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INTRODUCTION: Although the genome is often called the blueprint of an organism, it is perhaps more accurate to describe it as a parts list composed of the various genes that may or may not be used in the different cell types of a multicellular organism. Although nearly every cell in the body has essentially the same genome, each cell type makes different use of that genome and expresses a subset of all possible genes. This has motivated efforts to characterize the molecular composition of various cell types within humans and multiple model organisms, both by transcriptional and proteomic approaches. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. RATIONALE: One caveat to current approaches to make cell atlases is that individual organs are often collected at different locations, collected from different donors, and processed using different protocols. Controlled comparisons of cell types between different tissues and organs are especially difficult when donors differ in genetic background, age, environmental exposure, and epigenetic effects. To address this, we developed an approach to analyzing large numbers of organs from the same individual. RESULTS: We collected multiple tissues from individual human donors and performed coordinated single-cell transcriptome analyses on live cells. The donors come from a range of ethnicities, are balanced by gender, have a mean age of 51 years, and have a variety of medical backgrounds. Tissue experts used a defined cell ontology terminology to annotate cell types consistently across the different tissues, leading to a total of 475 distinct cell types with reference transcriptome profiles. The full dataset can be explored online with the cellxgene tool. Data were collected for the bladder, blood, bone marrow, eye, fat, heart, kidney, large intestine, liver, lung, lymph node, mammary, muscle, pancreas, prostate, salivary gland, skin, small intestine, spleen, thymus, tongue, trachea, uterus, and vasculature. Fifty-nine separate specimens in total were collected, processed, and analyzed, and 483,152 cells passed quality control filtering. On a per-compartment basis, the dataset includes 264,824 immune cells, 104,148 epithelial cells, 31,691 endothelial cells, and 82,478 stromal cells. Working with live cells, as opposed to isolated nuclei, ensured that the dataset includes all mRNA transcripts within the cell, including transcripts that have been processed by the cell’s splicing machinery, thereby enabling insight into variation in alternative splicing. The Tabula Sapiens also provided an opportunity to densely and directly sample the human microbiome throughout the gastrointestinal tract. The intestines from two donors were sectioned into five regions: the duodenum, jejunum, ileum, and ascending and sigmoid colon. Each section was transected, and three to nine samples were collected from each location, followed by amplification and sequencing of the 16S ribosomal RNA gene. CONCLUSION: The Tabula Sapiens has revealed discoveries relating to shared behavior and subtle, organ-specific differences across cell types. We found T cell clones shared between organs and characterized organ-dependent hypermutation rates among B cells. Endothelial cells and macrophages are shared across tissues, often showing subtle but clear differences in gene expression. We found an unexpectedly large and diverse amount of cell type–specific RNA splice variant usage and discovered and validated many previously undefined splices. The intestinal microbiome was revealed to have nonuniform species distributions down to the 3-inch (7.62-cm) length scale. These are but a few examples of how the Tabula Sapiens represents a broadly useful reference to deeply understand and explore human biology at cellular resolution.

p53 genes are conserved transcriptional activators that respond to stress. These proteins can also downregulate genes, but the mechanisms are not understood and are generally assumed to be indirect. Here, we investigate synthetic and native cis-regulatory elements in Drosophila to examine opposing features of p53-mediated transcriptional control in vivo. We show that transcriptional repression by p53 operates continuously through canonical DNA binding sites that confer p53-dependent transactivation at earlier developmental stages. p53 transrepression is correlated with local H3K9me3 chromatin marks and occurs without the need for stress or Chk2. In sufficiency tests, two p53 isoforms qualify as transrepressors and a third qualifies as a transcriptional activator. Targeted isoform-specific knockouts dissociate these opposing transcriptional activities, highlighting features that are dispensable for transactivation but critical for repression and for proper germ cell formation. Together, these results demonstrate that certain p53 isoforms function as constitutive tissue-specific repressors, raising important implications for tumor suppression by the human counterpart.

Skeletal stem and progenitor cell populations are crucial for bone physiology. Characterization of these cell types remains restricted to heterogenous bulk populations with limited information on whether they are unique or overlap with previously characterized cell types. Here we show, through comprehensive functional and single-cell transcriptomic analyses, that postnatal long bones of mice contain at least two types of bone progenitors with bona fide skeletal stem cell (SSC) characteristics. An early osteochondral SSC (ocSSC) facilitates long bone growth and repair, while a second type, a perivascular SSC (pvSSC), co-emerges with long bone marrow and contributes to shape the hematopoietic stem cell niche and regenerative demand. We establish that pvSSCs, but not ocSSCs, are the origin of bone marrow adipose tissue. Lastly, we also provide insight into residual SSC heterogeneity as well as potential crosstalk between the two spatially distinct cell populations. These findings comprehensively address previously unappreciated shortcomings of SSC research.

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix," as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs.

Aging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death(1). Despite rapid advances over recent years, many of the molecular and cellular processes which underlie progressive loss of healthy physiology are poorly understood(2). To gain a better insight into these processes we have created a single cell transcriptomic atlas across the life span of Mus musculus which includes data from 23 tissues and organs. We discovered cell-specific changes occurring across multiple cell types and organs, as well as age related changes in the cellular composition of different organs. Using single-cell transcriptomic data we were able to assess cell type specific manifestations of different hallmarks of aging, such as senescence(3), genomic instability(4) and changes in the organism’s immune system(2). This Tabula Muris Senis provides a wealth of new molecular information about how the most significant hallmarks of aging are reflected in a broad range of tissues and cell types.

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental and therapeutic application has been difficult for lack of agents amenable to tissue targeting. We have developed a novel agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1. This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 “switch helix”, as revealed by cryo-EM structure determination. Although this conformation likely constitutes part of the transport cycle, nanobody-trapping disrupts the cycle and prevents substrate movement through the Patched1 sterol conduit. Our conformation-selective nanobody approach provides a new route to the development of transporter-related pharmacologic agents and may be generally applicable to the study of other transporters.

Throughout the animal kingdom, p53 genes govern stress response networks by specifying adaptive transcriptional responses. The human member of this gene family is mutated in most cancers, but precisely how p53 functions to mediate tumor suppression is not well understood. Using Drosophila and zebrafish models, we show that p53 restricts retrotransposon activity and genetically interacts with components of the piRNA (piwi-interacting RNA) pathway. Furthermore, transposon eruptions occurring in the p53− germline were incited by meiotic recombination, and transcripts produced from these mobile elements accumulated in the germ plasm. In gene complementation studies, normal human p53 alleles suppressed transposons, but mutant p53 alleles from cancer patients could not. Consistent with these observations, we also found patterns of unrestrained retrotransposons in p53-driven mouse and human cancers. Furthermore, p53 status correlated with repressive chromatin marks in the 5′ sequence of a synthetic LINE-1 element. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to contain retrotransposons. Since human p53 mutants are disabled for this activity, our findings raise the possibility that p53 mitigates oncogenic disease in part by restricting transposon mobility.

SummaryHow are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

How organs maintain and restore functional integrity during ordinary tissue turnover or following injury represents a central biological problem. The maintenance of taste sensory organs in the tongue was shown 140 years ago to depend on innervation from distant ganglion neurons, but the underlying mechanism has remained unknown. Here, we show that Sonic hedgehog (Shh), which encodes a secreted protein signal, is expressed in these sensory neurons, and that experimental ablation of neuronal Shh expression causes loss of taste receptor cells (TRCs). TRCs are also lost upon pharmacologic blockade of Hedgehog pathway response, accounting for the loss of taste sensation experienced by cancer patients undergoing Hedgehog inhibitor treatment. We find that TRC regeneration following such pharmacologic ablation requires neuronal expression of Shh and can be substantially enhanced by pharmacologic activation of Hedgehog response. Such pharmacologic enhancement of Hedgehog response, however, results in additional TRC formation at many ectopic sites, unlike the site-restricted regeneration specified by the projection pattern of Shh-expressing neurons. Stable regeneration of TRCs thus requires neuronal Shh, illustrating the principle that neuronal delivery of cues such as the Shh signal can pattern distant cellular responses to assure functional integrity during tissue maintenance and regeneration.

The Tabula Muris ConsortiumWe have created a compendium of single cell transcriptome data from the model organism Mus musculus comprising more than 100,000 cells from 20 organs and tissues. These data represent a new resource for cell biology, revealing gene expression in poorly characterized cell populations and allowing for direct and controlled comparison of gene expression in cell types shared between tissues, such as T-lymphocytes and endothelial cells from distinct anatomical locations. Two distinct technical approaches were used for most tissues: one approach, microfluidic droplet-based 3’-end counting, enabled the survey of thousands of cells at relatively low coverage, while the other, FACS-based full length transcript analysis, enabled characterization of cell types with high sensitivity and coverage. The cumulative data provide the foundation for an atlas of transcriptomic cell biology.

Double duty for mammary stem cell niche The stem cell niche is a complex local signaling microenvironment that regulates stem cell activity for tissue and organ maintenance and regeneration. As well as responding locally, during puberty, the mammary gland stem cell niche also responds to systemic hormonal signals. Zhao et al. have found that Gli2, a transcriptional effector of Hedgehog signaling, coordinates the niche-signaling program and activates expression of receptors for the mammatrophic hormones estrogen and growth hormone throughout the mammary gland (see the Perspective by Robertson). Disease may result not only from stem cell defects, but also from dysregulation of the microenvironment. Science , this issue p. eaal3485 ; see also p. 250

Inflammation disrupts tissue architecture and function, thereby contributing to the pathogenesis of diverse diseases; the signals that promote or restrict tissue inflammation thus represent potential targets for therapeutic intervention. Here, we report that genetic or pharmacologic Hedgehog pathway inhibition intensifies colon inflammation (colitis) in mice. Conversely, genetic augmentation of Hedgehog response and systemic small-molecule Hedgehog pathway activation potently ameliorate colitis and restrain initiation and progression of colitis-induced adenocarcinoma. Within the colon, the Hedgehog protein signal does not act directly on the epithelium itself, but on underlying stromal cells to induce expression of IL-10, an immune-modulatory cytokine long known to suppress inflammatory intestinal damage. IL-10 function is required for the full protective effect of small-molecule Hedgehog pathway activation in colitis; this pharmacologic augmentation of Hedgehog pathway activity and stromal IL-10 expression are associated with increased presence of CD4+Foxp3+ regulatory T cells. We thus identify stromal cells as cellular coordinators of colon inflammation and suggest their pharmacologic manipulation as a potential means to treat colitis.

The stem cell niche is a complex local signaling microenvironment that sustains stem cell activity during organ maintenance and regeneration. The mammary gland niche must support its associated stem cells while also responding to systemic hormonal regulation that triggers pubertal changes. We find that

Summary Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. Video Abstract

Germline Quality Control The p53 tumor suppressor protein plays a key role in protecting organisms from aberrant cancer cells. But during evolution, animals would rarely, if ever, have lived long enough to develop cancer and so need such a function of p53. What, then, were the original functions for which p53 was selected? Lu et al. (p. 1278 ) observed a pulse of p53 activation during Drosophila development in the female germ line. In cancer, p53 is activated in response to DNA damage. Similarly, in this study, breaks in DNA that occur normally during meiosis also caused p53 activation. In animals in which resolution of DNA breaks during crossing over was inhibited, activation of p53 was prolonged; furthermore, when p53 was also lacking oogenesis was abnormal. Exactly how activation of p53 contributes to the process of chromosome recombination during meiosis remains unclear, but it may provide quality control, only allowing survival of gametes that possess intact DNA.

The selective pressures for the retention of primordial p53 genes predated the appearance of cancer. Therefore, wild-type tumour suppressive functions were probably co-opted from unrelated primordial activities. Is it possible to deduce what these early functions might have been? Evolutionary patterns indicate that primordial p53 genes predated the appearance of cancer. Therefore, wild-type tumour suppressive functions and mutant oncogenic functions that give celebrity status to this gene family were probably co-opted from unrelated primordial activities. Is it possible to deduce what these early functions might have been? And might this knowledge provide a platform for therapeutic opportunities?

We examined post-eclosion elimination of the Drosophila wing epithelium in vivo where collective “suicide waves” promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. We leveraged this phenotype in mosaic animals to discover relevant genes and establish here that homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers.

p53 is a fundamental determinant of oncogenesis, aging physiology and neurodegenerative pathologies. The complexity of the p53 regulatory network can hinder attempts to fully understand how this oncogenic protein operates within/ between cells to constrain growth potential. Orthologs of p53 in non-mammalian models, such as zebrafish, Drosophila and Caenorhabditis elegans, afford simpler models that illuminate core properties of this ancient network. The existence of p53 in short-lived organisms, where cancers do not occur, argues that tumor suppression per se was not the evolutionary pressure shaping p53. Instead, p53 as a constraint against tumor growth was probably co-opted from more ancestral, nonautonomous functions that are entirely unknown. Describing these functional properties will be essential for a comprehensive picture of the p53 regulatory network in normal and disease states. The tractable systems described here offer sophisticated genetic tools and fundamental insights that will continue to be indispensable models for achieving this goal.

Oncogenic stress provokes tumor suppression by p53 but the extent to which this regulatory axis is conserved remains unknown. Using a biosensor to visualize p53 action, we find that Drosophila p53 is selectively active in gonadal stem cells after exposure to stressors that destabilize the genome. Similar p53 activity occurred in hyperplastic growths that were triggered either by the RasV12 oncoprotein or by failed differentiation programs. In a model of transient sterility, p53 was required for the recovery of fertility after stress, and entry into the cell cycle was delayed in p53- stem cells. Together, these observations establish that the stem cell compartment of the Drosophila germline is selectively licensed for stress-induced activation of the p53 regulatory network. Furthermore, the findings uncover ancestral links between p53 and aberrant proliferation that are independent of DNA breaks and predate evolution of the ARF/Mdm2 axis. DOI: http://dx.doi.org/10.7554/eLife.01530.001, eLife digest The most common genetic change seen in cancer patients produces a faulty version of the p53 protein, which normally restricts tissue growth. This change promotes cancer because cells can now divide faster and fail to die when they should. Much remains to be learned about how p53 functions to restrain growth. As p53 is found in primitive organisms, and cancer is unlikely to have significantly influenced evolution, suppressing tumor formation was almost certainly not the original function of this gene. Furthermore, p53 works in a different way compared to many other tumour suppressors. Therefore, prevention of cancer is likely to have evolved as a side effect derived from more ancient functions. Recently, a link between p53 and stem cells has been uncovered. Stem cells are special because they can develop into many different types of cells, and they are crucial for the growth and repair of tissues. To form a particular type of cell, the stem cell divides to create two daughter cells. Commonly, one daughter cell stays in the stem state, whereas the other becomes a particular type of cell, such as a nerve cell or muscle cell. Because of this special property, scientists hypothesize that stem cells have special mechanisms to protect them from DNA damage that might partially depend on p53. This would prevent the spread of damaged genomes that would otherwise occur among daughter cells. To learn more about how p53 influences stem cells, Wylie, Lu et al. monitored its activity in the gonads of fruit flies, which are a powerful genetic model. They found that damaging DNA activates p53 in stem cells and their daughter cells, but not in other types of cells that have been damaged. In addition, p53 is activated by the uncontrolled growth and division of stem cells in the gonad, even when DNA is not damaged. This is unexpected since molecules linking inappropriate growth to p53 were thought to be present only in mammals. Therefore, it appears that the tumor-suppressing behavior of p53 in mammals was adapted from its more ancient ability to regulate stem cell growth—an ability that evolved before organisms divided into vertebrates and invertebrates. DOI: http://dx.doi.org/10.7554/eLife.01530.002

Publisher Summary Radiation exposure promotes genotoxic damage, causing wholesale chromosomal alterations that jeopardize viability and/or reproductive capacity. Consequently, all organisms have evolved sophisticated mechanisms that respond to and counteract these effects. In eukaryotes, radiation responses can be divided into three functional categories: the damage sensors, the signal transmitters, and the effectors. Sensors must have the ability to discriminate damaged from normal DNA and must show a defect in one or more damage responses when mutated. A current model for the sensors in S. pombe describes a multi-component complex that is recruited to replication forks that are stalled at damaged DNA by R17 associated replication factor C (RFC). As a metazoan, Drosophila offers fundamental insights regarding the ways in which developmental programs specify outcomes and exert constraints upon adaptive responses to radiation damage. Drosophila orthologs of these S. pombe 's genes may, likewise, be recruited to stalled replication at damaged DNA via the RFC and are reasonable candidates for damage sensors. Once DNA damage is recognized, signal transmitters including the ATM and ATR kinases are activated. In mammals, ATM responds exclusively to ionizing radiation (IR), whereas ATR responds to IR, UV radiation, and stalled replication forks. A Drosophila ATM/ATR homolog, mei-41 , was isolated as a meiotic mutant four decades ago and has been well characterized as a pleiotropic locus affecting DNA repair, chromosome instability, and defective replication control.

Bcl-2 family members are pivotal regulators of programmed cell death (PCD). In mammals, pro-apoptotic Bcl-2 family members initiate early apoptotic signals by causing the release of cytochrome c from the mitochondria, a step necessary for the initiation of the caspase cascade. Worms and flies do not show a requirement for cytochrome c during apoptosis, but both model systems express pro- and anti-apoptotic Bcl-2 family members. Drosophila encodes two Bcl-2 family members, Debcl (pro-apoptotic) and Buffy (anti-apoptotic). To understand the role of Debcl in Drosophila apoptosis, we produced authentic null alleles at this locus. Although gross development and lifespans were unaffected, we found that Debcl was required for pruning cells in the developing central nervous system. debcl genetically interacted with the ced-4/Apaf1 counterpart dark, but was not required for killing by RHG (Reaper, Hid, Grim) proteins. We found that debclKO mutants were unaffected for mitochondrial density or volume but, surprisingly, in a model of caspase-independent cell death, heterologous killing by murine Bax required debcl to exert its pro-apoptotic activity. Therefore, although debcl functions as a limited effector of PCD during normal Drosophila development, it can be effectively recruited for killing by mammalian members of the Bcl-2 gene family.

Among the seven caspases encoded in the fly genome, only dronc contains a caspase recruitment domain. To assess the function of this gene in development, we produced a null mutation in dronc . Animals lacking zygotic dronc are defective for programmed cell death (PCD) and arrest as early pupae. These mutants present a range of defects, including extensive hyperplasia of hematopoietic tissues, supernumerary neuronal cells, and head involution failure. dronc genetically interacts with the Ced4/Apaf1 counterpart, Dark , and adult structures lacking dronc are disrupted for fine patterning. Furthermore, in diverse models of metabolic injury, dronc − cells are completely insensitive to induction of cell killing. These findings establish dronc as an essential regulator of cell number in development and illustrate broad requirements for this apical caspase in adaptive responses during stress-induced apoptosis.