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Synaptic adhesion molecules (SAMs) are evolutionarily conserved proteins that play an important role in the form and function of neuronal synapses. Teneurins (Tenms) and latrophilins (Lphns) are well-known cell adhesion molecules that form a transsynaptic complex. Recent studies suggest that Tenm3 and Lphn2 (gene symbol Adgrl2) are involved in hippocampal circuit assembly via their topographical expression. However, it is not known whether other teneurins and latrophilins display similar topographically restricted expression patterns during embryonic and postnatal development. Here, we reveal the cartography of all teneurin (Tenm1-4) and latrophilin (Lphn1-3 [Adgrl1-3]) paralog expression in the mouse hippocampus across prenatal and postnatal development as monitored by large-scale single-molecule RNA in situ hybridization mapping. Our results identify a striking heterogeneity in teneurin and latrophilin expression along the spatiotemporal axis of the hippocampus. Tenm2 and Tenm4 expression levels peak at the neonatal stage when compared to Tenm1 and Tenm3, while Tenm1 expression is restricted to the postnatal pyramidal cell layer. Tenm4 expression in the dentate gyrus (DG) exhibits an opposing topographical expression pattern in the embryonic and neonatal hippocampus. Our findings were validated by analyses of multiple RNA-seq datasets at bulk, single-cell, and spatial levels. Thus, our study presents a comprehensive spatiotemporal map of Tenm and Lphn expression in the hippocampus, showcasing their diverse expression patterns across developmental stages in distinct spatial axes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Liakath-Ali et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells that facilitate cancer progression. However, our knowledge of the niches of individual TAM subsets and their development and function remain incomplete. Here, we describe a population of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-expressing TAMs, which form coordinated multi-cellular "nest" structures that are heterogeneously distributed proximal to vasculature in tumors of a spontaneous murine model of breast cancer. We demonstrate that LYVE-1 + TAMs develop in response to IL-6, which induces their expression of the immune-suppressive enzyme heme oxygenase-1 and promotes a CCR5-dependent signaling axis, which guides their nest formation. Blocking the development of LYVE-1 + TAMs or their nest structures, using gene-targeted mice, results in an increase in CD8 + T cell recruitment to the tumor and enhanced response to chemotherapy. This study highlights an unappreciated collaboration of a TAM subset to form a coordinated niche linked to immune exclusion and resistance to anti-cancer therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

At CA1→subiculum synapses, alternatively spliced neurexin-1 (Nrxn1 SS4+ ) and neurexin-3 (Nrxn3 SS4+ ) enhance NMDA-receptors and suppress AMPA-receptors, respectively, without affecting synapse formation. Nrxn1 SS4+ and Nrxn3 SS4+ act by binding to secreted cerebellin-2 (Cbln2) that in turn activates postsynaptic GluD1 receptors. Whether neurexin-Cbln2-GluD1 signaling has additional functions besides regulating NMDA- and AMPA-receptors, and whether such signaling performs similar roles at other synapses, however, remains unknown. Here, we demonstrate using constitutive Cbln2 deletions in mice that at CA1→subiculum synapses, Cbln2 performs no additional developmental roles besides regulating AMPA- and NMDA-receptors. Moreover, low-level expression of functionally redundant Cbln1 did not compensate for a possible synapse-formation function of Cbln2 at CA1→subiculum synapses. In exploring the generality of these findings, we examined the prefrontal cortex where Cbln2 was recently implicated in spinogenesis, and the cerebellum where Cbln1 is known to regulate parallel-fiber synapses. In the prefrontal cortex, Nrxn1 SS4+ -Cbln2 signaling selectively controlled NMDA-receptors without affecting spine or synapse numbers, whereas Nrxn3 SS4+ -Cbln2 signaling had no apparent role. In the cerebellum, conversely, Nrxn3 SS4+ -Cbln1 signaling regulated AMPA-receptors, whereas now Nrxn1 SS4+ -Cbln1 signaling had no manifest effect. Thus, Nrxn1 SS4+ - and Nrxn3 SS4+ -Cbln1/2 signaling complexes differentially control NMDA- and AMPA-receptors in different synapses in diverse neural circuits without regulating synapse or spine formation., Competing Interests: JD, KL, SG, TS No competing interests declared, (© 2022, Dai et al.)

Stem cell renewal and differentiation are regulated by interactions with the niche. Although multiple cell populations have been identified in distinct anatomical compartments, little is known about niche-specific molecular factors. Using skin as a model system and combining single-cell RNA-seq data analysis, immunofluorescence, and transgenic mouse models, we show that the transmembrane protein embigin is specifically expressed in the sebaceous gland and that the number of embigin-expressing cells is negatively regulated by Wnt. The loss of embigin promotes exit from the progenitor compartment and progression toward differentiation, and also compromises lipid metabolism. Embigin modulates sebaceous niche architecture by affecting extracellular matrix organization and basolateral targeting of monocarboxylate transport. We discover through ligand screening that embigin is a direct fibronectin receptor, binding to the N-terminal fibronectin domain without impairing integrin function. Our results solve the long-standing question of how embigin regulates cell adhesion and demonstrate a mechanism that couples adhesion and metabolism., Competing Interests: Declaration of interests V.J. is currently an employee at Experimentica, Kuopio, 70210, Finland. K.M.D. is currently an employee at Orion Oyj, Espoo, 02200, Finland. A.M. is currently an employee at CN Bio Innovations Cambridge, CB40XL, UK., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Five decades ago, long-term potentiation (LTP) of synaptic transmission was discovered at entorhinal cortex→dentate gyrus (EC→DG) synapses, but the molecular determinants of EC→DG LTP remain largely unknown. Here, we show that the presynaptic neurexin–ligand cerebellin-4 (Cbln4) is highly expressed in the entorhinal cortex and essential for LTP at EC→DG synapses, but dispensable for basal synaptic transmission at these synapses. Cbln4, when bound to cell-surface neurexins, forms transcellular complexes by interacting with postsynaptic DCC (deleted in colorectal cancer) or neogenin-1. DCC and neogenin-1 act as netrin and repulsive guidance molecule-a (RGMa) receptors that mediate axon guidance in the developing brain, but their binding to Cbln4 raised the possibility that they might additionally function in the mature brain as postsynaptic receptors for presynaptic neurexin/Cbln4 complexes, and that as such receptors, DCC or neogenin-1 might mediate EC→DG LTP that depends on Cbln4. Indeed, we observed that neogenin-1, but not DCC, is abundantly expressed in dentate gyrus granule cells, and that postsynaptic neogenin-1 deletions in dentate granule cells blocked EC→DG LTP, but again did not affect basal synaptic transmission similar to the presynaptic Cbln4 deletions. Thus, binding of presynaptic Cbln4 to postsynaptic neogenin-1 renders EC→DG synapses competent for LTP, but is not required for establishing these synapses or for otherwise enabling their function.

Extensive studies concluded that homophilic interactions between pre- and postsynaptic teneurins, evolutionarily conserved cell-adhesion molecules, encode the specificity of synaptic connections. However, no direct evidence is available to demonstrate that teneurins are actually required on both pre- and postsynaptic neurons for establishing synaptic connections, nor is it known whether teneurins are localized to synapses. Using super-resolution microscopy, we demonstrate that Teneurin-3 assembles into presynaptic nanoclusters of approximately 80 nm in most excitatory synapses of the hippocampus. Presynaptic deletions of Teneurin-3 and Teneurin-4 in the medial entorhinal cortex revealed that they are required for assembly of entorhinal cortex-CA1, entorhinal cortex-subiculum, and entorhinal cortex-dentate gyrus synapses. Postsynaptic deletions of teneurins in the CA1 region, however, had no effect on synaptic connections from any presynaptic input. Our data suggest that different from the current prevailing view, teneurins promote the establishment of synaptic connections exclusively as presynaptic cell-adhesion molecules, most likely via their nanomolar-affinity binding to postsynaptic latrophilins., (© 2022. The Author(s).)

Cadherins contribute to the organization of nearly all tissues, but the functions of several evolutionarily conserved cadherins, including those of calsyntenins, remain enigmatic. Puzzlingly, two distinct, non-overlapping functions for calsyntenins were proposed: As postsynaptic neurexin ligands in synapse formation, or as presynaptic kinesin adaptors in vesicular transport. Here, we show that, surprisingly, acute CRISPR-mediated deletion of calsyntenin-3 in mouse cerebellum in vivo causes a large decrease in inhibitory synapse, but a robust increase in excitatory parallel-fiber synapses in Purkinje cells. As a result, inhibitory synaptic transmission was suppressed, whereas parallel-fiber synaptic transmission was enhanced in Purkinje cells by the calsyntenin-3 deletion. No changes in the dendritic architecture of Purkinje cells or in climbing-fiber synapses were detected. Sparse selective deletion of calsyntenin-3 only in Purkinje cells recapitulated the synaptic phenotype, indicating that calsyntenin-3 acts by a cell-autonomous postsynaptic mechanism in cerebellum. Thus, by inhibiting formation of excitatory parallel-fiber synapses and promoting formation of inhibitory synapses in the same neuron, calsyntenin-3 functions as a postsynaptic adhesion molecule that regulates the excitatory/inhibitory balance in Purkinje cells., Competing Interests: ZL, MJ, KL, AS, JK, RZ, TS No competing interests declared, (© 2022, Liu et al.)

Synapses are thought to be organized by interactions of presynaptic neurexins with postsynaptic ligands, particularly with neuroligins and cerebellins. However, when a neuron forms adjacent pre- and postsynaptic specializations, as in dendrodendritic or axo-axonic synapses, nonfunctional cis neurexin/ligand interactions would be energetically favored. Here, we reveal an organizational principle for preventing synaptic cis interactions (“self-avoidance”). Using dendrodendritic synapses between mitral and granule cells in the olfactory bulb as a paradigm, we show that, owing to its higher binding affinity, cerebellin-1 blocks the cis interaction of neurexins with neuroligins, thereby enabling trans neurexin/neuroligin interaction. In mitral cells, ablating either cerebellin-1 or neuroligins severely impaired granule cell➔mitral cell synapses, as did overexpression of wild-type neurexins but not of mutant neurexins unable to bind to neuroligins. Our data uncover a molecular interaction network that organizes the self-avoidance of nonfunctional neurexin/ligand cis interactions, thus allowing assembly of physiological trans interactions.

Stem cells are characterized by their ability to self-renew and differentiate into many different cell types. Research has focused primarily on how these processes are regulated at a transcriptional level. However, recent studies have indicated that stem cell behaviour is strongly coupled to the regulation of protein synthesis by the ribosome. In this Review, we discuss how different translation mechanisms control the function of adult and embryonic stem cells. Stem cells are characterized by low global translation rates despite high levels of ribosome biogenesis. The maintenance of pluripotency, the commitment to a specific cell fate and the switch to cell differentiation depend on the tight regulation of protein synthesis and ribosome biogenesis. Translation regulatory mechanisms that impact on stem cell function include mTOR signalling, ribosome levels, and mRNA and tRNA features and amounts. Understanding these mechanisms important for stem cell self-renewal and differentiation may also guide our understanding of cancer grade and metastasis., (© 2021. Springer Nature Limited.)

Ionotropic glutamate delta receptors 1 (GluD1) and 2 (GluD2) exhibit the molecular architecture of postsynaptic ionotropic glutamate receptors, but assemble into trans-synaptic adhesion complexes by binding to secreted cerebellins that in turn interact with presynaptic neurexins 1-4 . It is unclear whether neurexin-cerebellin-GluD1/2 assemblies serve an adhesive synapse-formation function or mediate trans-synaptic signalling. Here we show in hippocampal synapses, that binding of presynaptic neurexin-cerebellin complexes to postsynaptic GluD1 controls glutamate receptor activity without affecting synapse numbers. Specifically, neurexin-1-cerebellin-2 and neurexin-3-cerebellin-2 complexes differentially regulate NMDA (N-methyl-D-aspartate) receptors and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors by activating distinct postsynaptic GluD1 effector signals. Of note, minimal GluD1 and GluD2 constructs containing only their N-terminal cerebellin-binding and C-terminal cytoplasmic domains, joined by an unrelated transmembrane region, fully control the levels of NMDA and AMPA receptors. The distinct signalling specificity of presynaptic neurexin-1 and neurexin-3 5,6 is encoded by their alternatively spliced splice site 4 sequences, whereas the regulatory functions of postsynaptic GluD1 are mediated by conserved cytoplasmic sequence motifs spanning 5-13 residues. Thus, GluDs are signalling molecules that regulate NMDA and AMPA receptors by an unexpected transduction mechanism that bypasses their ionotropic receptor architecture and directly converts extracellular neurexin-cerebellin signals into postsynaptic receptor responses.

Neurexins are presynaptic cell-adhesion molecules essential for synaptic function that are expressed in thousands of alternatively spliced isoforms. Recent studies suggested that alternative splicing at splice site 4 (SS4) of Nrxn1 is tightly regulated by an activity-dependent mechanism. Given that Nrxn1 alternative splicing at SS4 controls NMDA-receptor-mediated synaptic responses, activity-dependent SS4 alternative splicing would suggest a new synaptic plasticity mechanism. However, conflicting results confound the assessment of neurexin alternative splicing, prompting us to re-evaluate this issue. We find that in cortical cultures, membrane depolarization by elevated extracellular K + -concentrations produced an apparent shift in Nrxn1-SS4 alternative splicing by inducing neuronal but not astroglial cell death, resulting in persistent astroglial Nrxn1-SS4+ expression and decreased neuronal Nrxn1-SS4- expression. in vivo , systemic kainate-induced activation of neurons in the hippocampus produced no changes in Nrxn1-SS4 alternative splicing. Moreover, focal kainate injections into the mouse cerebellum induced small changes in Nrxn1-SS4 alternative splicing that, however, were associated with large decreases in Nrxn1 expression and widespread DNA damage. Our results suggest that although Nrxn1-SS4 alternative splicing may represent a mechanism of activity-dependent synaptic plasticity, common procedures for testing this hypothesis are prone to artifacts, and more sophisticated approaches will be necessary to test this important question., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liakath-Ali and Südhof.)

Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a ubiquitously expressed nuclear protein that regulates functions of protein serine/threonine phosphatase-1 in cell proliferation and lineage specification. The role of NIPP1 in tissue homeostasis is not fully understood. This study shows that the selective deletion of NIPP1 in mouse epidermis resulted in epidermal hyperproliferation, a reduced adherence of basal keratinocytes, and a gradual decrease in the stemness of hair follicle stem cells, culminating in hair loss. This complex phenotype was associated with chronic sterile skin inflammation and could be partially rescued by dexamethasone treatment. NIPP1-deficient keratinocytes massively expressed proinflammatory chemokines and immunomodulatory proteins in a cell-autonomous manner. Chemokines subsequently induced the recruitment and activation of immune cells, in particular conventional dendritic cells and Langerhans cells, accounting for the chronic inflammation phenotype. The data identifies NIPP1 as a key regulator of epidermal homeostasis and as a potential target for the treatment of inflammatory skin diseases., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Latrophilin-2 (Lphn2) and latrophilin-3 (Lphn3) are adhesion GPCRs that serve as postsynaptic recognition molecules in CA1 pyramidal neurons of the hippocampus, where they are localized to distinct dendritic domains and are essential for different sets of excitatory synapses. Here, we studied Lphn2 and Lphn3 in the cerebellum. We show that latrophilins are abundantly and differentially expressed in the cerebellar cortex. Using conditional KO mice, we demonstrate that the Lphn2/3 double-deletion but not the deletion of Lphn2 or Lphn3 alone suppresses parallel-fiber synapses and reduces parallel-fiber synaptic transmission by ~50% without altering release probability. Climbing-fiber synapses, conversely, were unaffected. Even though ~50% of total cerebellar Lphn3 protein is expressed in Bergmann glia, Lphn3 deletion from Bergmann glia did not detectably impair excitatory or inhibitory synaptic transmission. Our studies demonstrate that Lphn2 and Lphn3 are selectively but redundantly required in Purkinje cells for parallel-fiber synapses., Competing Interests: RZ, KL, TS No competing interests declared, (© 2020, Zhang et al.)

Myosins are molecular motors that are well known for their role in cell movement and contractile functions. Although extensively studied in muscle physiology, little is known about the function of myosins in mammalian skin. As part of the Sanger Institute Mouse Genetics Project, we have identified a role for Myo10 in pigmentation, with a phenotype unlike those of Myo5a or Myo7a. Adult mice homozygous for a disrupted Myo10 allele on a C57BL/6N background displayed a high degree of penetrance for white patches on their abdomen and dorsal surface. Forepaw syndactyly and hind paw syndactyly were also observed in these mice. Tail epidermal wholemounts showed a complete lack of melanocytes in the hair follicles and interfollicular epidermis. Myo10 has previously been implicated in human pigmentation. Our current study reveals involvement of Myo10 in murine skin pigmentation., (© 2018 The Authors. Experimental Dermatology Published by John Wiley & Sons Ltd.)

Keratin 76 (Krt76) is expressed in the differentiated epithelial layers of skin, oral cavity and squamous stomach. Krt76 downregulation in human oral squamous cell carcinomas (OSCC) correlates with poor prognosis. We show that genetic ablation of Krt76 in mice leads to spleen and lymph node enlargement, an increase in regulatory T cells (Tregs) and high levels of pro-inflammatory cytokines. Krt76 -/- Tregs have increased suppressive ability correlated with increased CD39 and CD73 expression, while their effector T cells are less proliferative than controls. Loss of Krt76 increases carcinogen-induced tumours in tongue and squamous stomach. Carcinogenesis is further increased when Treg levels are elevated experimentally. The carcinogenesis response includes upregulation of pro-inflammatory cytokines and enhanced accumulation of Tregs in the tumour microenvironment. Tregs also accumulate in human OSCC exhibiting Krt76 loss. Our study highlights the role of epithelial cells in modulating carcinogenesis via communication with cells of the immune system.

Ribosome-associated mRNA quality control mechanisms ensure the fidelity of protein translation 1,2 . Although these mechanisms have been extensively studied in yeast, little is known about their role in mammalian tissues, despite emerging evidence that stem cell fate is controlled by translational mechanisms 3,4 . One evolutionarily conserved component of the quality control machinery, Dom34 (in higher eukaryotes known as Pelota (Pelo)), rescues stalled ribosomes 5 . Here we show that Pelo is required for mammalian epidermal homeostasis. Conditional deletion of Pelo in mouse epidermal stem cells that express Lrig1 results in hyperproliferation and abnormal differentiation of these cells. By contrast, deletion of Pelo in Lgr5-expressing stem cells has no effect and deletion in Lgr6-expressing stem cells induces only a mild phenotype. Loss of Pelo results in accumulation of short ribosome footprints and global upregulation of translation, rather than affecting the expression of specific genes. Translational inhibition by rapamycin-mediated downregulation of mTOR (mechanistic target of rapamycin kinase) rescues the epidermal phenotype. Our study reveals that the ribosome-rescue machinery is important for mammalian tissue homeostasis and that it has specific effects on different stem cell populations.

Deep sequencing can detect somatic DNA mutations in tissues permitting inference of clonal relationships. This has been applied to human epidermis, where sun exposure leads to the accumulation of mutations and an increased risk of skin cancer. However, previous studies have yielded conflicting conclusions about the relative importance of positive selection and neutral drift in clonal evolution. Here, we sequenced larger areas of skin than previously, focusing on cancer-prone skin spanning five decades of life. The mutant clones identified were too large to be accounted for solely by neutral drift. Rather, using mathematical modelling and computational lattice-based simulations, we show that observed clone size distributions can be explained by a combination of neutral drift and stochastic nucleation of mutations at the boundary of expanding mutant clones that have a competitive advantage. These findings demonstrate that spatial context and cell competition cooperate to determine the fate of a mutant stem cell.

Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here, we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA - promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6 + cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.

Individual human epidermal cells differ in their self-renewal ability. To uncover the molecular basis for this heterogeneity, we performed genome-wide pooled RNA interference screens and identified genes conferring a clonal growth advantage on normal and neoplastic (cutaneous squamous cell carcinoma, cSCC) human epidermal cells. The Hippo effector YAP was amongst the top positive growth regulators in both screens. By integrating the Hippo network interactome with our data sets, we identify WW-binding protein 2 (WBP2) as an important co-factor of YAP that enhances YAP/TEAD-mediated gene transcription. YAP and WPB2 are upregulated in actively proliferating cells of mouse and human epidermis and cSCC, and downregulated during terminal differentiation. WBP2 deletion in mouse skin results in reduced proliferation in neonatal and wounded adult epidermis. In reconstituted epidermis YAP/WBP2 activity is controlled by intercellular adhesion rather than canonical Hippo signalling. We propose that defective intercellular adhesion contributes to uncontrolled cSCC growth by preventing inhibition of YAP/WBP2.

The depletion of evolutionarily conserved pelota protein causes impaired differentiation of embryonic and spermatogonial stem cells. In this study, we show that temporal deletion of pelota protein before epidermal barrier acquisition leads to neonatal lethality due to perturbations in permeability barrier formation. Further analysis indicated that this phenotype is a result of failed processing of profilaggrin into filaggrin monomers, which promotes the formation of a protective epidermal layer. Molecular analyses showed that pelota protein negatively regulates the activities of bone morphogenetic protein and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in the epidermis. To address whether elevated activities of bone morphogenetic protein and PI3K/AKT signaling pathways were the cause for the perturbed epidermal barrier in Pelo-deficient mice, we made use of organotypic cultures of skin explants from control and mutant embryos at embryonic day 15.5. Inhibition of PI3K/AKT signaling did not significantly affect the bone morphogenetic protein activity. However, inhibition of bone morphogenetic protein signaling caused a significant attenuation of PI3K/AKT activity in mutant skin and, more interestingly, the restoration of profilaggrin processing and normal epidermal barrier function. Therefore, increased activity of the PI3K/AKT signaling pathway in Pelo-deficient skin might conflict with the dephosphorylation of profilaggrin and thereby affect its proper processing into filaggrin monomers and ultimately the epidermal differentiation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The epidermis is the outermost layer of skin that acts as a barrier to protect the body from the external environment and to control water and heat loss. This barrier function is established through the multistage differentiation of keratinocytes and the presence of bioactive sphingolipids such as ceramides, the levels of which are tightly regulated by a balance of ceramide synthase and ceramidase activities. Here we reveal the essential role of alkaline ceramidase 1 (Acer1) in the skin. Acer1-deficient (Acer1(-/-) ) mice showed elevated levels of ceramide in the skin, aberrant hair shaft cuticle formation and cyclic alopecia. We demonstrate that Acer1 is specifically expressed in differentiated interfollicular epidermis, infundibulum and sebaceous glands and consequently Acer1(-/-) mice have significant alterations in infundibulum and sebaceous gland architecture. Acer1(-/-) skin also shows perturbed hair follicle stem cell compartments. These alterations result in Acer1(-/-) mice showing increased transepidermal water loss and a hypermetabolism phenotype with associated reduction of fat content with age. We conclude that Acer1 is indispensable for mammalian skin homeostasis and whole-body energy homeostasis. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Macrophages are essential for the progression and maintenance of many cancers, but their role during the earliest stages of tumor formation is unclear. To test this, we used a previously described transgenic mouse model of wound-induced skin tumorigenesis, in which expression of constitutively active MEK1 in differentiating epidermal cells results in chronic inflammation (InvEE mice). Upon wounding, the number of epidermal and dermal monocytes and macrophages increased in wild-type and InvEE skin, but the increase was greater, more rapid, and more sustained in InvEE skin. Macrophage ablation reduced tumor incidence. Furthermore, bioluminescent imaging in live mice to monitor macrophage flux at wound sites revealed that macrophage accumulation was predictive of tumor formation; wounds with the greatest number of macrophages at day 5 went on to develop tumors. Gene expression profiling of flow-sorted monocytes, macrophages, and T cells from InvEE and wild-type skin showed that as wound healing progressed, InvEE macrophages altered their phenotype. Throughout wound healing and after wound closure, InvEE macrophages demonstrated sustained upregulation of several markers implicated in alternative macrophage activation including arginase-1 (ARG1) and mannose receptor (CD206). Notably, inhibition of ARG1 activity significantly reduced tumor formation and epidermal proliferation in vivo, whereas addition of L-arginase to cultured keratinocytes stimulated proliferation. We conclude that macrophages play a key role in early, inflammation-mediated skin tumorigenesis, with mechanistic evidence suggesting that ARG1 secretion drives tumor development by stimulating epidermal cell proliferation. These findings highlight the importance of cancer immunotherapies aiming to polarize tumor-associated macrophages toward an antitumor phenotype., (©2016 American Association for Cancer Research.)

In human skin the interface between the epidermis and dermis is not flat, but undulates. The dimensions of the undulations change as a function of age and disease. Epidermal stem cell clusters lie in specific locations relative to the undulations; however, whether their location affects their properties is unknown. To explore this, we developed a two-step protocol to create patterned substrates that mimic the topographical features of the human epidermal-dermal interface. Substrates with negative patterns were first fabricated by exposing a photocurable formulation to light, controlling the topographical features (such as diameter, height and center-to-center distance) by the photomask pattern dimensions and UV crosslinking time. The negative pattern was then translated to PDMS elastomer to fabricate substrates with 8 unique surface topographies on which primary human keratinocytes were cultured. We found that cells were patterned according to topography, and that separate cues determined the locations of stem cells, differentiated cells and proliferating cells. The biomimetic platform we have developed will be useful for probing the effect of topography on stem cell behaviour.

To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development., (© 2015 The Authors.)

Permanent stop-and-shop large-scale mouse mutant resources provide an excellent platform to decipher tissue phenogenomics. Here we analyse skin from 538 knockout mouse mutants generated by the Sanger Institute Mouse Genetics Project. We optimize immunolabelling of tail epidermal wholemounts to allow systematic annotation of hair follicle, sebaceous gland and interfollicular epidermal abnormalities using ontology terms from the Mammalian Phenotype Ontology. Of the 50 mutants with an epidermal phenotype, 9 map to human genetic conditions with skin abnormalities. Some mutant genes are expressed in the skin, whereas others are not, indicating systemic effects. One phenotype is affected by diet and several are incompletely penetrant. In-depth analysis of three mutants, Krt76, Myo5a (a model of human Griscelli syndrome) and Mysm1, provides validation of the screen. Our study is the first large-scale genome-wide tissue phenotype screen from the International Knockout Mouse Consortium and provides an open access resource for the scientific community.