A tissue injury sensing and repair pathway distinct from host pathogen defense.

Pathogen infection and tissue injury are universal insults that disrupt homeostasis. Innate immunity senses microbial infections and induces cytokines/chemokines to activate resistance mechanisms. Here, we show that, in contrast to most pathogen-induced cytokines, interleukin-24 (IL-24) is predominately induced by barrier epithelial progenitors after tissue injury and is independent of microbiome or adaptive immunity. Moreover, Il24 ablation in mice impedes not only epidermal proliferation and re-epithelialization but also capillary and fibroblast regeneration within the dermal wound bed. Conversely, ectopic IL-24 induction in the homeostatic epidermis triggers global epithelial-mesenchymal tissue repair responses. Mechanistically, Il24 expression depends upon both epithelial IL24-receptor/STAT3 signaling and hypoxia-stabilized HIF1α, which converge following injury to trigger autocrine and paracrine signaling involving IL-24-mediated receptor signaling and metabolic regulation. Thus, parallel to innate immune sensing of pathogens to resolve infections, epithelial stem cells sense injury signals to orchestrate IL-24-mediated tissue repair.

Adaptive Immune Resistance Emerges from Tumor-Initiating Stem Cells.

Our bodies are equipped with powerful immune surveillance to clear cancerous cells as they emerge. How tumor-initiating stem cells (tSCs) that form and propagate cancers equip themselves to overcome this barrier remains poorly understood. To tackle this problem, we designed a skin cancer model for squamous cell carcinoma (SCC) that can be effectively challenged by adoptive cytotoxic T cell transfer (ACT)-based immunotherapy. Using single-cell RNA sequencing (RNA-seq) and lineage tracing, we found that transforming growth factor ß (TGF-ß)-responding tSCs are superior at resisting ACT and form the root of tumor relapse. Probing mechanism, we discovered that during malignancy, tSCs selectively acquire CD80, a surface ligand previously identified on immune cells. Moreover, upon engaging cytotoxic T lymphocyte antigen-4 (CTLA4), CD80-expressing tSCs directly dampen cytotoxic T cell activity. Conversely, upon CTLA4- or TGF-ß-blocking immunotherapies or Cd80 ablation, tSCs become vulnerable, diminishing tumor relapse after ACT treatment. Our findings place tSCs at the crux of how immune checkpoint pathways are activated.

Impaired Epidermal to Dendritic T Cell Signaling Slows Wound Repair in Aged Skin.

Aged skin heals wounds poorly, increasing susceptibility to infections. Restoring homeostasis after wounding requires the coordinated actions of epidermal and immune cells. Here we find that both intrinsic defects and communication with immune cells are impaired in aged keratinocytes, diminishing their efficiency in restoring the skin barrier after wounding. At the wound-edge, aged keratinocytes display reduced proliferation and migration. They also exhibit a dampened ability to transcriptionally activate epithelial-immune crosstalk regulators, including a failure to properly activate/maintain dendritic epithelial T cells (DETCs), which promote re-epithelialization following injury. Probing mechanism, we find that aged keratinocytes near the wound edge don't efficiently upregulate Skints or activate STAT3. Notably, when epidermal Stat3, Skints, or DETCs are silenced in young skin, re-epithelialization following wounding is perturbed. These findings underscore epithelial-immune crosstalk perturbations in general, and Skints in particular, as critical mediators in the age-related decline in wound-repair.

Ras drives malignancy through stem cell crosstalk with the microenvironment.

Squamous cell carcinomas are triggered by marked elevation of RAS-MAPK signalling and progression from benign papilloma to invasive malignancy1-4. At tumour-stromal interfaces, a subset of tumour-initiating progenitors, the cancer stem cells, obtain increased resistance to chemotherapy and immunotherapy along this pathway5,6. The distribution and changes in cancer stem cells during progression from a benign state to invasive squamous cell carcinoma remain unclear. Here we show in mice that, after oncogenic RAS activation, cancer stem cells rewire their gene expression program and trigger self-propelling, aberrant signalling crosstalk with their tissue microenvironment that drives their malignant progression. The non-genetic, dynamic cascade of intercellular exchanges involves downstream pathways that are often mutated in advanced metastatic squamous cell carcinomas with high mutational burden7. Coupling our clonal skin HRASG12V mouse model with single-cell transcriptomics, chromatin landscaping, lentiviral reporters and lineage tracing, we show that aberrant crosstalk between cancer stem cells and their microenvironment triggers angiogenesis and TGFß signalling, creating conditions that are conducive for hijacking leptin and leptin receptor signalling, which in turn launches downstream phosphoinositide 3-kinase (PI3K)-AKT-mTOR signalling during the benign-to-malignant transition. By functionally examining each step in this pathway, we reveal how dynamic temporal crosstalk with the microenvironment orchestrated by the stem cells profoundly fuels this path to malignancy. These insights suggest broad implications for cancer therapeutics.

Publisher Correction: Mechanics of a multilayer epithelium instruct tumour architecture and function.

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

Mechanics of a multilayer epithelium instruct tumour architecture and function.

Loss of normal tissue architecture is a hallmark of oncogenic transformation1. In developing organisms, tissues architectures are sculpted by mechanical forces during morphogenesis2. However, the origins and consequences of tissue architecture during tumorigenesis remain elusive. In skin, premalignant basal cell carcinomas form 'buds', while invasive squamous cell carcinomas initiate as 'folds'. Here, using computational modelling, genetic manipulations and biophysical measurements, we identify the biophysical underpinnings and biological consequences of these tumour architectures. Cell proliferation and actomyosin contractility dominate tissue architectures in monolayer, but not multilayer, epithelia. In stratified epidermis, meanwhile, softening and enhanced remodelling of the basement membrane promote tumour budding, while stiffening of the basement membrane promotes folding. Additional key forces stem from the stratification and differentiation of progenitor cells. Tumour-specific suprabasal stiffness gradients are generated as oncogenic lesions progress towards malignancy, which we computationally predict will alter extensile tensions on the tumour basement membrane. The pathophysiologic ramifications of this prediction are profound. Genetically decreasing the stiffness of basement membranes increases membrane tensions in silico and potentiates the progression of invasive squamous cell carcinomas in vivo. Our findings suggest that mechanical forces-exerted from above and below progenitors of multilayered epithelia-function to shape premalignant tumour architectures and influence tumour progression.

Distinct modes of cell competition shape mammalian tissue morphogenesis.

Cell competition-the sensing and elimination of less fit 'loser' cells by neighbouring 'winner' cells-was first described in Drosophila. Although cell competition has been proposed as a selection mechanism to optimize tissue and organ development, its evolutionary generality remains unclear. Here, by using live imaging, lineage tracing, single-cell transcriptomics and genetics, we identify two cell competition mechanisms that sequentially shape and maintain the architecture of stratified tissue during skin development in mice. In the single-layered epithelium of the early embryonic epidermis, winner progenitors kill and subsequently clear neighbouring loser cells by engulfment. Later, as the tissue begins to stratify, the basal layer instead expels losers through upward flux of differentiating progeny. This cell competition switch is physiologically relevant: when it is perturbed, so too is barrier formation. Our findings show that cell competition is a selective force that optimizes vertebrate tissue function, and illuminate how a tissue dynamically adjusts cell competition strategies to preserve fitness as its architectural complexity increases during morphogenesis.

Translation from unconventional 5' start sites drives tumour initiation.

We are just beginning to understand how translational control affects tumour initiation and malignancy. Here we use an epidermis-specific, in vivo ribosome profiling strategy to investigate the translational landscape during the transition from normal homeostasis to malignancy. Using a mouse model of inducible SOX2, which is broadly expressed in oncogenic RAS-associated cancers, we show that despite widespread reductions in translation and protein synthesis, certain oncogenic mRNAs are spared. During tumour initiation, the translational apparatus is redirected towards unconventional upstream initiation sites, enhancing the translational efficiency of oncogenic mRNAs. An in vivo RNA interference screen of translational regulators revealed that depletion of conventional eIF2 complexes has adverse effects on normal but not oncogenic growth. Conversely, the alternative initiation factor eIF2A is essential for cancer progression, during which it mediates initiation at these upstream sites, differentially skewing translation and protein expression. Our findings unveil a role for the translation of 5' untranslated regions in cancer, and expose new targets for therapeutic intervention.

CTCF maintains differential methylation at the Igf2/H19 locus.

Genomic imprinting relies on establishing and maintaining the parental-specific methylation of DNA elements that control the differential expression of maternal and paternal alleles. Although the essential DNA methyltransferases have been discovered, proteins that regulate the sequence-specific establishment and maintenance of allelic methylation have not been identified. One candidate regulator of methylation, the zinc-finger protein CTCF, binds to the imprinting control region (ICR) of the genes Igf2 (encoding insulin-like growth factor 2) and H19 (fetal liver mRNA; refs. 1,2). The unmethylated maternal ICR is a chromatin boundary that prevents distant enhancers from activating Igf2 (refs. 3-6). In vitro experiments have suggested that CTCF mediates boundary activity of the maternal ICR, and that methylation of the paternal ICR abolishes this activity by preventing CTCF binding. Using mice with point mutations in all four CTCF sites in the ICR, we show that maternally transmitted mutant ICRs in neonatal mice acquire a substantial but heterogeneous degree of methylation. Mutant ICRs in oocytes and blastocysts are not methylated, however, indicating that binding of CTCF is not required to establish the unmethylated ICR during oogenesis. We also show that the mutant ICR lacks enhancer-blocking activity, as the expression of Igf2 is activated on mutant maternal chromosomes. Conversely, maternal H19 expression is reduced, suggesting a positive role for CTCF in the transcription of that gene. This study constitutes the first in vivo demonstration of the multiple functions of CTCF in an ICR.

Stem cells tightly regulate dead cell clearance to maintain tissue fitness.

Macrophages and dendritic cells have long been appreciated for their ability to migrate to and engulf dying cells and debris, including some of the billions of cells that are naturally eliminated from our body daily. However, a substantial number of these dying cells are cleared by 'non-professional phagocytes', local epithelial cells that are critical to organismal fitness. How non-professional phagocytes sense and digest nearby apoptotic corpses while still performing their normal tissue functions is unclear. Here, we explore the molecular mechanisms underlying their multifunctionality. Exploiting the cyclical bouts of tissue regeneration and degeneration during the hair cycle, we show that stem cells can transiently become non-professional phagocytes when confronted with dying cells. Adoption of this phagocytic state requires both local lipids produced by apoptotic corpses to activate RXRα, and tissue-specific retinoids for RARγ activation. This dual factor dependency enables tight regulation of the genes requisite to activate phagocytic apoptotic clearance. The tunable phagocytic program we describe here offers an effective mechanism to offset phagocytic duties against the primary stem cell function of replenishing differentiated cells to preserve tissue integrity during homeostasis. Our findings have broad implications for other non-motile stem or progenitor cells which experience cell death in an immune-privileged niche.

Characterization of multidrug resistance 1a/P-glycoprotein knockout rats generated by zinc finger nucleases.

The development of zinc finger nuclease (ZFN) technology has enabled the genetic engineering of the rat genome. The ability to manipulate the rat genome has great promise to augment the utility of rats for biological and pharmacological studies. A Wistar Hannover rat model lacking the multidrug resistance protein Mdr1a P-glycoprotein (P-gp) was generated using a rat Mdr1a-specific ZFN. Mdr1a was completely absent in tissues, including brain and small intestine, of the knockout rat. Pharmacokinetic studies with the Mdr1a P-gp substrates loperamide, indinavir, and talinolol indicated that Mdr1a was functionally inactive in the blood-brain barrier and intestine in Mdr1a(-/-) rats. To identify possible compensatory mechanisms in Mdr1a(-/-) rats, the expression levels of drug-metabolizing enzyme and transporter-related genes were compared in brain, liver, kidney, and intestine of male and female Mdr1a(-/-) and control rats. In general, alterations in gene expression of these genes in Mdr1a(-/-) rats seemed to be modest, with more changes in female than in male rats. Taken together, our studies demonstrate that the ZFN-generated Mdr1a(-/-) rat will be a valuable tool for central nervous system drug target validation and determining the role of P-gp in drug absorption and disposition.

Dissecting self-renewal in stem cells with RNA interference.

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells.

Liquid-liquid phase separation drives skin barrier formation.

At the body surface, skin's stratified squamous epithelium is challenged by environmental extremes. The surface of the skin is composed of enucleated, flattened surface squames. They derive from underlying, transcriptionally active keratinocytes that display filaggrin-containing keratohyalin granules (KGs) whose function is unclear. Here, we found that filaggrin assembles KGs through liquid-liquid phase separation. The dynamics of phase separation governed terminal differentiation and were disrupted by human skin barrier disease-associated mutations. We used fluorescent sensors to investigate endogenous phase behavior in mice. Phase transitions during epidermal stratification crowded cellular spaces with liquid-like KGs whose coalescence was restricted by keratin filament bundles. We imaged cells as they neared the skin surface and found that environmentally regulated KG phase dynamics drive squame formation. Thus, epidermal structure and function are driven by phase-separation dynamics.

Progenitors oppositely polarize WNT activators and inhibitors to orchestrate tissue development.

To spatially co-exist and differentially specify fates within developing tissues, morphogenetic cues must be correctly positioned and interpreted. Here, we investigate mouse hair follicle development to understand how morphogens operate within closely spaced, fate-diverging progenitors. Coupling transcriptomics with genetics, we show that emerging hair progenitors produce both WNTs and WNT inhibitors. Surprisingly, however, instead of generating a negative feedback loop, the signals oppositely polarize, establishing sharp boundaries and consequently a short-range morphogen gradient that we show is essential for three-dimensional pattern formation. By establishing a morphogen gradient at the cellular level, signals become constrained. The progenitor preserves its WNT signaling identity and maintains WNT signaling with underlying mesenchymal neighbors, while its overlying epithelial cells become WNT-restricted. The outcome guarantees emergence of adjacent distinct cell types to pattern the tissue.

Clock regulatory elements control cyclic expression of Lunatic fringe during somitogenesis.

Somitogenesis requires a segmentation clock and Notch signaling. Lunatic fringe (Lfng) expression in the presomitic mesoderm (PSM) cycles in the posterior PSM, is refined in the segmenting somite to the rostral compartment, and is required for segmentation. We identify distinct cis-acting regulatory elements for each aspect of Lfng expression. Fringe clock element 1 (FCE1) represents a conserved 110 bp region that is necessary to direct cyclic Lfng RNA expression in the posterior PSM. Mutational analysis of E boxes within FCE1 indicates a potential interplay of positive and negative transcriptional regulation by cyclically expressed bHLH proteins. A separable Lfng regulatory region directs expression to the prospective rostral aspect of the condensing somite. These independent Lfng regulatory cassettes advance a molecular framework for deciphering somite segmentation.

An RNAi screen unravels the complexities of Rho GTPase networks in skin morphogenesis.

During mammalian embryogenesis, extensive cellular remodeling is needed for tissue morphogenesis. As effectors of cytoskeletal dynamics, Rho GTPases and their regulators are likely involved, but their daunting complexity has hindered progress in dissecting their functions. We overcome this hurdle by employing high throughput in utero RNAi-mediated screening to identify key Rho regulators of skin morphogenesis. Our screen unveiled hitherto unrecognized roles for Rho-mediated cytoskeletal remodeling events that impact hair follicle specification, differentiation, downgrowth and planar cell polarity. Coupling our top hit with gain/loss-of-function genetics, interactome proteomics and tissue imaging, we show that RHOU, an atypical Rho, governs the cytoskeletal-junction dynamics that establish columnar shape and planar cell polarity in epidermal progenitors. Conversely, RHOU downregulation is required to remodel to a conical cellular shape that enables hair bud invagination and downgrowth. Our findings underscore the power of coupling screens with proteomics to unravel the physiological significance of complex gene families.

Stem cell-driven lymphatic remodeling coordinates tissue regeneration.

Tissues rely on stem cells (SCs) for homeostasis and wound repair. SCs reside in specialized microenvironments (niches) whose complexities and roles in orchestrating tissue growth are still unfolding. Here, we identify lymphatic capillaries as critical SC-niche components. In skin, lymphatics form intimate networks around hair follicle (HF) SCs. When HFs regenerate, lymphatic-SC connections become dynamic. Using a mouse model, we unravel a secretome switch in SCs that controls lymphatic behavior. Resting SCs express angiopoietin-like protein 7 (Angptl7), promoting lymphatic drainage. Activated SCs switch to Angptl4, triggering transient lymphatic dissociation and reduced drainage. When lymphatics are perturbed or the secretome switch is disrupted, HFs cycle precociously and tissue regeneration becomes asynchronous. In unearthing lymphatic capillaries as a critical SC-niche element, we have learned how SCs coordinate their activity across a tissue.

Functional characterization of a novel Ku70/80 pause site at the H19/Igf2 imprinting control region.

The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequence-specific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg(2+), implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.

Temporal Layering of Signaling Effectors Drives Chromatin Remodeling during Hair Follicle Stem Cell Lineage Progression.

Tissue regeneration relies on resident stem cells (SCs), whose activity and lineage choices are influenced by the microenvironment. Exploiting the synchronized, cyclical bouts of tissue regeneration in hair follicles (HFs), we investigate how microenvironment dynamics shape the emergence of stem cell lineages. Employing epigenetic and ChIP-seq profiling, we uncover how signal-dependent transcription factors couple spatiotemporal cues to chromatin dynamics, thereby choreographing stem cell lineages. Using enhancer-driven reporters, mutagenesis, and genetics, we show that simultaneous BMP-inhibitory and WNT signals set the stage for lineage choices by establishing chromatin platforms permissive for diversification. Mechanistically, when binding of BMP effector pSMAD1 is relieved, enhancers driving HF-stem cell master regulators are silenced. Concomitantly, multipotent, lineage-fated enhancers silent in HF-stem cells become activated by exchanging WNT effectors TCF3/4 for LEF1. Throughout regeneration, lineage enhancers continue reliance upon LEF1 but then achieve specificity by accommodating additional incoming signaling effectors. Barriers to progenitor plasticity increase when diverse, signal-sensitive transcription factors shape LEF1-regulated enhancer dynamics.

Functional characterization of a testis-specific DNA binding activity at the H19/Igf2 imprinting control region.

The DNA methylation state of the H19/Igf2 imprinting control region (ICR) is differentially set during gametogenesis. To identify factors responsible for the paternally specific DNA methylation of the ICR, germ line and somatic extracts were screened for proteins that bind to the ICR in a germ line-specific manner. A specific DNA binding activity that was restricted to the male germ line and enriched in neonatal testis was identified. Its three binding sites within the ICR are very similar to the consensus sequence for nuclear receptor extended half sites. To determine if these binding sites are required for establishment of the paternal epigenetic state, a mouse strain in which the three sites were mutated was generated. The mutated ICR was able to establish a male-specific epigenetic state in sperm that was indistinguishable from that established by the wild-type ICR, indicating that these sequences are either redundant or have no function. An analysis of the methylated state of the mutant ICR in the soma revealed no differences from the wild-type ICR but did uncover in both mutant and wild-type chromosomes a significant relaxation in the stringency of the methylated state of the paternal allele and the unmethylated state of the maternal allele in neonatal and adult tissues.