Vitamin A resolves lineage plasticity to orchestrate stem cell lineage choices.

Lineage plasticity-a state of dual fate expression-is required to release stem cells from their niche constraints and redirect them to tissue compartments where they are most needed. In this work, we found that without resolving lineage plasticity, skin stem cells cannot effectively generate each lineage in vitro nor regrow hair and repair wounded epidermis in vivo. A small-molecule screen unearthed retinoic acid as a critical regulator. Combining high-throughput approaches, cell culture, and in vivo mouse genetics, we dissected its roles in tissue regeneration. We found that retinoic acid is made locally in hair follicle stem cell niches, where its levels determine identity and usage. Our findings have therapeutic implications for hair growth as well as chronic wounds and cancers, where lineage plasticity is unresolved.

Universal recording of immune cell interactions in vivo.

Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function1. To study these 'kiss-and-run' interactions directly in vivo, we previously developed LIPSTIC (labelling immune partnerships by SorTagging intercellular contacts)2, an approach that uses enzymatic transfer of a labelled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ T helper cells and antigen-presenting cells, however. Here we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T cells and identify germinal centre-resident T follicular helper cells on the basis of their ability to interact cognately with germinal centre B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalogue of the immune populations that physically interact with intestinal epithelial cells at the steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus-specific CD8+ T cells in multiple organs following systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

Beyond genetics: driving cancer with the tumour microenvironment behind the wheel.

Cancer has long been viewed as a genetic disease of cumulative mutations. This notion is fuelled by studies showing that ageing tissues are often riddled with clones of complex oncogenic backgrounds coexisting in seeming harmony with their normal tissue counterparts. Equally puzzling, however, is how cancer cells harbouring high mutational burden contribute to normal, tumour-free mice when allowed to develop within the confines of healthy embryos. Conversely, recent evidence suggests that adult tissue cells expressing only one or a few oncogenes can, in some contexts, generate tumours exhibiting many of the features of a malignant, invasive cancer. These disparate observations are difficult to reconcile without invoking environmental cues triggering epigenetic changes that can either dampen or drive malignant transformation. In this Review, we focus on how certain oncogenes can launch a two-way dialogue of miscommunication between a stem cell and its environment that can rewire downstream events non-genetically and skew the morphogenetic course of the tissue. We review the cells and molecules of and the physical forces acting in the resulting tumour microenvironments that can profoundly affect the behaviours of transformed cells. Finally, we discuss possible explanations for the remarkable diversity in the relative importance of mutational burden versus tumour microenvironment and its clinical relevance.

Understanding the microenvironment and how this controls cell fate.

The microenvironment influences cell fate. In this collection of voices, researchers from the fields of cancer and regeneration highlight approaches to establish the importance of the microenvironment and discuss future directions to understand the complex interaction between cells and their surrounding environment and how this impacts on disease and regeneration.

The pioneer factor SOX9 competes for epigenetic factors to switch stem cell fates.

During development, progenitors simultaneously activate one lineage while silencing another, a feature highly regulated in adult stem cells but derailed in cancers. Equipped to bind cognate motifs in closed chromatin, pioneer factors operate at these crossroads, but how they perform fate switching remains elusive. Here we tackle this question with SOX9, a master regulator that diverts embryonic epidermal stem cells (EpdSCs) into becoming hair follicle stem cells. By engineering mice to re-activate SOX9 in adult EpdSCs, we trigger fate switching. Combining epigenetic, proteomic and functional analyses, we interrogate the ensuing chromatin and transcriptional dynamics, slowed temporally by the mature EpdSC niche microenvironment. We show that as SOX9 binds and opens key hair follicle enhancers de novo in EpdSCs, it simultaneously recruits co-factors away from epidermal enhancers, which are silenced. Unhinged from its normal regulation, sustained SOX9 subsequently activates oncogenic transcriptional regulators that chart the path to cancers typified by constitutive SOX9 expression.

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.

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.

Designer bugs as cancer drugs?

Skin microbiota can be engineered to induce antitumor T cells.

Universal recording of cell-cell contacts in vivo for interaction-based transcriptomics.

Cellular interactions are essential for tissue organization and functionality. In particular, immune cells rely on direct and usually transient interactions with other immune and non-immune populations to specify and regulate their function. To study these "kiss-and-run" interactions directly in vivo, we previously developed LIPSTIC (Labeling Immune Partnerships by SorTagging Intercellular Contacts), an approach that uses enzymatic transfer of a labeled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ helper T cells and antigen presenting cells, however. Here, we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the cellular partners of regulatory T cells in steady state, and identify germinal center (GC)-resident T follicular helper (Tfh) cells based on their ability to interact cognately with GC B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalog of the immune populations that physically interact with intestinal epithelial cells (IECs) and find evidence of stepwise acquisition of the ability to interact with IECs as CD4+ T cells adapt to residence in the intestinal tissue. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

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.

Inflammatory memory and tissue adaptation in sickness and in health.

Our body has a remarkable ability to remember its past encounters with allergens, pathogens, wounds and irritants, and to react more quickly to the next experience. This accentuated sensitivity also helps us to cope with new threats. Despite maintaining a state of readiness and broadened resistance to subsequent pathogens, memories can also be maladaptive, leading to chronic inflammatory disorders and cancers. With the ever-increasing emergence of new pathogens, allergens and pollutants in our world, the urgency to unravel the molecular underpinnings of these phenomena has risen to new heights. Here we reflect on how the field of inflammatory memory has evolved, since 2007, when researchers realized that non-specific memory is contained in the nucleus and propagated at the epigenetic level. We review the flurry of recent discoveries revealing that memory is not just a privilege of the immune system but also extends to epithelia of the skin, lung, intestine and pancreas, and to neurons. Although still unfolding, epigenetic memories of inflammation have now been linked to possible brain disorders such as Alzheimer disease, and to an elevated risk of cancer. In this Review, we consider the consequences-good and bad-of these epigenetic memories and their implications for human health and disease.

The integrated stress response remodels the microtubule-organizing center to clear unfolded proteins following proteotoxic stress.

Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely, the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule-organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells, hence exposing a vulnerability in cancer that could be exploited therapeutically.

Lymphatics act as a signaling hub to regulate intestinal stem cell activity.

Barrier epithelia depend upon resident stem cells for homeostasis, defense, and repair. Epithelial stem cells of small and large intestines (ISCs) respond to their local microenvironments (niches) to fulfill a continuous demand for tissue turnover. The complexity of these niches and underlying communication pathways are not fully known. Here, we report a lymphatic network at the intestinal crypt base that intimately associates with ISCs. Employing in vivo loss of function and lymphatic:organoid cocultures, we show that crypt lymphatics maintain ISCs and inhibit their precocious differentiation. Pairing single-cell and spatial transcriptomics, we apply BayesPrism to deconvolve expression within spatial features and develop SpaceFold to robustly map the niche at high resolution, exposing lymphatics as a central signaling hub for the crypt in general and ISCs in particular. We identify WNT-signaling factors (WNT2, R-SPONDIN-3) and a hitherto unappreciated extracellular matrix protein, REELIN, as crypt lymphatic signals that directly govern the regenerative potential of ISCs.

Desmoplakin Maintains Transcellular Keratin Scaffolding and Protects From Intestinal Injury.

BACKGROUND & AIMS: Desmosomes are intercellular junctions connecting keratin intermediate filaments of neighboring cells. The cadherins desmoglein 2 (Dsg2) and desmocollin 2 mediate cell-cell adhesion, whereas desmoplakin (Dsp) provides the attachment of desmosomes to keratins. Although the importance of the desmosome-keratin network is well established in mechanically challenged tissues, we aimed to assess the currently understudied function of desmosomal proteins in intestinal epithelia. METHODS: We analyzed the intestine-specific villin-Cre DSP (DSPΔIEC) and the combined intestine-specific DSG2/DSPΔIEC (ΔDsg2/Dsp) knockout mice. Cross-breeding with keratin 8-yellow fluorescent protein knock-in mice and generation of organoids was performed to visualize the keratin network. A Dsp-deficient colorectal carcinoma HT29-derived cell line was generated and the role of Dsp in adhesion and mechanical stress was studied in dispase assays, after exposure to uniaxial cell stretching and during scratch assay. RESULTS: The intestine of DSPΔIEC mice was histopathologically inconspicuous. Intestinal epithelial cells, however, showed an accelerated migration along the crypt and an enhanced shedding into the lumen. Increased intestinal permeability and altered levels of desmosomal proteins were detected. An inconspicuous phenotype also was seen in ΔDsg2/Dsp mice. After dextran sodium sulfate treatment, DSPΔIEC mice developed more pronounced colitis. A retracted keratin network was seen in the intestinal epithelium of DSPΔIEC/keratin 8-yellow fluorescent protein mice and organoids derived from these mice presented a collapsed keratin network. The level, phosphorylation status, and solubility of keratins were not affected. Dsp-deficient HT29 cells had an impaired cell adhesion and suffered from increased cellular damage after stretch. CONCLUSIONS: Our results show that Dsp is required for proper keratin network architecture in intestinal epithelia, mechanical resilience, and adhesion, thereby protecting from injury.

Building and Maintaining the Skin.

The skin forms a crucial, dynamic barrier between an animal and the external world. In mammals, three stem cell populations possess robust regenerative potential to maintain and repair the body's protective surface: epidermal stem cells, which maintain the stratified epidermis; hair follicle stem cells, which power the cyclic growth of the hair follicle; and melanocyte stem cells, which regenerate pigment-producing melanocytes to color the skin and hair. These stem cells reside in complex microenvironments ("niches") comprising diverse cellular repertoires that enable stem cells to rejuvenate tissues during homeostasis and regenerate them upon injury. Beyond their niches, skin stem cells can also sense and respond to fluctuations in organismal health or changes outside the body. Here, we review these diverse cellular interactions and highlight how far-reaching signals can be transmitted at the local level to enable skin stem cells to tailor their actions to suit the particular occasion and optimize fitness.

20 years of Developmental Cell: Looking back.

In our 20th anniversary year, we reflect on how the cell and developmental biology fields have changed since the publication of Developmental Cell's first few issues. In this collection of Voices, authors who published in our early issues discuss the advances that helped shape their field over the past two decades.

Stem cells expand potency and alter tissue fitness by accumulating diverse epigenetic memories.

Immune and tissue stem cells retain an epigenetic memory of inflammation that intensifies sensitivity to future encounters. We investigated whether and to what consequence stem cells possess and accumulate memories of diverse experiences. Monitoring a choreographed response to wounds, we found that as hair follicle stem cells leave their niche, migrate to repair damaged epidermis, and take up long-term foreign residence there, they accumulate long-lasting epigenetic memories of each experience, culminating in post-repair epigenetic adaptations that sustain the epidermal transcriptional program and surface barrier. Each memory is distinct, separable, and has its own physiological impact, collectively endowing these stem cells with heightened regenerative ability to heal wounds and broadening their tissue-regenerating tasks relative to their naïve counterparts.