Epidermal stem cells at the point of no return

The skin is our main barrier against outside harm, and it helps to maintain internal homeostasis. These functions are mostly fulfilled by the epidermis, which is the outermost layer of the skin, formed from keratinocytes that are constantly being shed and replaced through proliferation, differentiation, and subsequent delamination of tissue resident stem cells. Keratinocytes also give rise to hair follicles which go through regular cycles of growth and rest. These processes are supported by, for example, fibroblasts, immune cells, nerves and vessels, helping to maintain the protective function of the skin. The wealth of different cell types and stem cell behaviours makes skin an attractive model system for research. The overarching aim of this thesis was to study the epidermal stem cells at their onset of differentiation – to determine their point of no return beyond which they cannot revert back to a less differentiated state. To address this question, a combination of single-cell RNA-sequencing, in situ mRNA stainings and intra vital imaging methods were used, allowing for the assessment of cells’ transcriptional states, their location in the tissue and their behaviour over time. In Paper I, we present a thorough characterization of differentiation-committed basal cells in the interfollicular epidermis. We study their behaviour in living tissue and describe their transcriptional states upon commitment and delamination. Finally, we show how mitosis of committed cells is not integral to their differentiation journey. In Paper II, we analysed full thickness adult skin in the growth and resting stages of the hair cycle to create an unbiased transcriptional atlas of all major cell types. We uncover unexpected heterogeneity in the anagen hair follicle lineages and highlight how fibroblast populations can undergo transcriptional state changes during hair cycling. In Paper III, we expanded the transcriptional atlas to embryonic development, describing the transcriptional and anatomic landmarks at the time when crucial skin structures are established. We show early heterogeneity within the fibroblast population, as well as the first signs of hair follicle development and the formation of the panniculus carnosus muscle. Taken together, this thesis investigates skin stem cells molecularly, spatially, and behaviourally when they commit to specific lineages whether in the adult or developing epidermis, in the growing hair follicle or in the embryonic fibroblast compartment. Finally, we speculate on what could be the underlying cause that pushes the stem cells over the point of no return

Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here, we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts

Retrograde movements determine effective stem cell numbers in the intestine

The morphology and functionality of the epithelial lining differ along the intestinal tract, but tissue renewal at all sites is driven by stem cells at the base of crypts(1-3). Whether stem cell numbers and behaviour vary at different sites is unknown. Here we show using intravital microscopy that, despite similarities in the number and distribution of proliferative cells with an Lgr5 signature in mice, small intestinal crypts contain twice as many effective stem cells as large intestinal crypts. We find that, although passively displaced by a conveyor-belt-like upward movement, small intestinal cells positioned away from the crypt base can function as long-term effective stem cells owing to Wnt-dependent retrograde cellular movement. By contrast, the near absence of retrograde movement in the large intestine restricts cell repositioning, leading to a reduction in effective stem cell number. Moreover, after suppression of the retrograde movement in the small intestine, the number of effective stem cells is reduced, and the rate of monoclonal conversion of crypts is accelerated. Together, these results show that the number of effective stem cells is determined by active retrograde movement, revealing a new channel of stem cell regulation that can be experimentally and pharmacologically manipulated.Peer reviewe

Aktiveeritud lovastatiini ja nutliin-3 mõju tüvirakuliini H9 rakkudele

2018-06-0

Single-Cell Transcriptomics of Traced Epidermal and Hair Follicle Stem Cells Reveals Rapid Adaptations during Wound Healing

Summary: Epithelial tissues, such as the skin, rely on cellular plasticity of stem cells (SCs) from different niches to restore tissue function after injury. How these molecularly and functionally diverse SC populations respond to injury remains elusive. Here, we genetically labeled Lgr5- or Lgr6-expressing cells from the hair follicle bulge and interfollicular epidermis (IFE), respectively, and monitored their individual transcriptional adaptations during wound healing using single-cell transcriptomics. Both Lgr5 and Lgr6 progeny rapidly induced a genetic wound signature that, for Lgr5 progeny, included the remodeling of receptors to permit interactions with the wound environment, a property that Lgr6 progeny possessed even before wounding. When contributing to re-epithelialization, Lgr5 progeny gradually replaced their bulge identity with an IFE identity, and this process started already before Lgr5 progeny left the bulge. Altogether, this study reveals how different SCs respond and adapt to a new environment, potentially explaining cellular plasticity of many epithelial tissues. : Joost et al. elucidate how skin stem cells from different niches respond upon injury. Single-cell transcriptomics revealed that Lgr5 and Lgr6 progeny molecularly converge during wound healing. Instant cell adaptations of Lgr5 cells within their original niche permit interactions with the wound environment, an ability Lgr6 cells already possess before wounding. Keywords: mouse skin, Lgr5 stem cells, Lgr6 stem cells, lineage tracing, wound healing, RNA sequencing, cellular plasticity, transcriptional adaptation, receptor-ligand pairing, computational analysi

ARF suppression by MYC but not MYCN confers increased malignancy of aggressive pediatric brain tumors

Medulloblastoma, the most common malignant pediatric brain tumor, often harbors MYC amplifications. Compared to high-grade gliomas, MYC-amplified medulloblastomas often show increased photoreceptor activity and arise in the presence of a functional ARF/p53 suppressor pathway. Here, we generate an immunocompetent transgenic mouse model with regulatable MYC that develop clonal tumors that molecularly resemble photoreceptor-positive Group 3 medulloblastoma. Compared to MYCN-expressing brain tumors driven from the same promoter, pronounced ARF silencing is present in our MYC-expressing model and in human medulloblastoma. While partial Arf suppression causes increased malignancy in MYCN-expressing tumors, complete Arf depletion promotes photoreceptor-negative high-grade glioma formation. Computational models and clinical data further identify drugs targeting MYC-driven tumors with a suppressed but functional ARF pathway. We show that the HSP90 inhibitor, Onalespib, significantly targets MYC-driven but not MYCN-driven tumors in an ARF-dependent manner. The treatment increases cell death in synergy with cisplatin and demonstrates potential for targeting MYC-driven medulloblastoma.De två första författarna delar förstaförfattarskapet.</p

Molecular and spatial landmarks of early mouse skin development

A wealth of specialized cell populations within the skin facilitates its hair-producing, protective, sensory, and thermoregulatory functions. How the vast cell-type diversity and tissue architecture develops is largely unexplored. Here, with single-cell transcriptomics, spatial cell-type assignment, and cell-lineage tracing, we deconstruct early embryonic mouse skin during the key transitions from seemingly uniform developmental precursor states to a multilayered, multilineage epithelium, and complex dermal identity. We identify the spatiotemporal emergence of hair-follicle-inducing, muscle-supportive, and fascia-forming fibroblasts. We also demonstrate the formation of the panniculus carnosus muscle (PCM), sprouting blood vessels without pericyte coverage, and the earliest residence of mast and dendritic immune cells in skin. Finally, we identify an unexpected epithelial heterogeneity within the early single-layered epidermis and a signaling-rich periderm layer. Overall, this cellular and molecular blueprint of early skin development-which can be explored at https://kasperlab.org/tools-establishes histological landmarks and highlights unprecedented dynamic interactions among skin cells.Peer reviewe

OTULIN maintains skin homeostasis by controlling keratinocyte death and stem cell identity

OTULIN is a deubiquitinase for linear ubiquitin chains. Here the authors show, using genetic mouse models and single-cell RNA-sequencing, that deficiency of OTULIN in keratinocytes causes skin inflammation and verrucous carcinoma via the induction of keratinocyte death, thereby implicating a function of OTULIN in keratinocyte homeostasis. OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1 beta response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis

Data from: Signals from the brain and olfactory epithelium control shaping of the mammalian nasal capsule cartilage

Facial shape is the basis for facial recognition and categorization. Facial features reflect the underlying geometry of the skeletal structures. Here we reveal that cartilaginous nasal capsule (corresponding to upper jaw and face) is shaped by signals generated by neural structures: brain and olfactory epithelium. Brain-derived Sonic Hedgehog (SHH) enables the induction of nasal septum and posterior nasal capsule, whereas the formation of a capsule roof is controlled by signals from the olfactory epithelium. Unexpectedly, the cartilage of the nasal capsule turned out to be important for shaping membranous facial bones during development. This suggests that conserved neurosensory structures could benefit from protection and have evolved signals inducing cranial cartilages encasing them. Experiments with mutant mice revealed that the genomic regulatory regions controlling production of SHH in the nervous system contribute to facial cartilage morphogenesis, which might be a mechanism responsible for the adaptive evolution of animal faces and snouts

Retrograde movements determine effective stem cell numbers in the intestine

The morphology and functionality of the epithelial lining differ along the intestinal tract, but tissue renewal at all sites is driven by stem cells at the base of crypts1–3. Whether stem cell numbers and behaviour vary at different sites is unknown. Here we show using intravital microscopy that, despite similarities in the number and distribution of proliferative cells with an Lgr5 signature in mice, small intestinal crypts contain twice as many effective stem cells as large intestinal crypts. We find that, although passively displaced by a conveyor-belt-like upward movement, small intestinal cells positioned away from the crypt base can function as long-term effective stem cells owing to Wnt-dependent retrograde cellular movement. By contrast, the near absence of retrograde movement in the large intestine restricts cell repositioning, leading to a reduction in effective stem cell number. Moreover, after suppression of the retrograde movement in the small intestine, the number of effective stem cells is reduced, and the rate of monoclonal conversion of crypts is accelerated. Together, these results show that the number of effective stem cells is determined by active retrograde movement, revealing a new channel of stem cell regulation that can be experimentally and pharmacologically manipulated