Biology subjects

Watt, F. M.

Publications and source records attributed to Watt, F. M..

15 recordsLinked to original sources

bioRxiv2026

Plasticity of squamous differentiation drives drug resistance in HNSCC

A critical hallmark of carcinogenesis is the ability of cancer cells to evade the loss of self-renewal normally imposed by terminal differentiation. However, therapies directly attempting to promote differentiation have shown limited efficacy in solid tumours and the cellular mechanisms behind cancer cell persistence are poorly understood. Here we established a patient-derived orthotopic head and neck squamous cell carcinoma (HNSCC) model in vivo that recapitulates the genetic, cellular and histopathological heterogeneity of HNSCC. Experimental induction of differentiation and clonal lineage tracing by fluorescent barcoding revealed a heterogeneous response to terminal differentiation stimuli, enabling subsets of cancer cells to escape differentiation-associated loss of self-renewal. While pharmacological inhibition of ErbB-MEK1/2-ERK1/2 pathway by afatinib could induce the differentiation of patient-derived cancer cells, some highly clonogenic cells remained refractory to differentiation signals even though they were capable of differentiating. Differentiation reporter IVLmCherry further confirmed that differentiation and loss of self-renewal ability were partially uncoupled in patient-derived HNSCC cells. These findings identify differentiation-resistant clonogenic populations as a key barrier to therapeutic efficacy and provide a framework for improving differentiation-based strategies in HNSCC.

cancer biology↗

bioRxiv2025

Pathogenic Keratinocyte States and Fibroblast Niches Define the Tissue Microenvironment in Severe Hidradenitis Suppurativa

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease characterized by recurrent painful abscesses and tunnels in flexural sites. The mechanisms driving HS pathogenesis, particularly the roles of keratinocytes and fibroblasts in the HS inflammatory microenvironment, remain poorly understood. To characterise the cellular and molecular landscape of HS, we analyzed lesional skin from severe HS patients using single-cell RNA-sequencing and spatial transcriptomics to identify keratinocyte states and patterns of cellular interactions, with a focus on fibroblast-keratinocyte crosstalk. We identified a migratory S100+ pathogenic keratinocyte state enriched in HS lesions and observed spatially distinct fibroblast populations associated with different tissue compartments. COL6A5+ fibroblasts were preferentially co-localised with undifferentiated keratinocyte states, whereas APOD+ fibroblasts with enriched in immune cell-rich regions containing activated B and plasma cells consistent with tertiary lymphoid organ (TLO)-like structures. Ligand-receptor inference and spatial co-localisation analyses predicted extensive fibroblast-keratinocyte and fibroblast-immune interactions within HS lesions. Together, these data define the cellular organisation of the HS tissue microenvironment and identify spatially distinct stromal and epithelial niches associated with severe disease. These findings provide a framework for understanding how epithelial, stromal and immune cell populations coexist within HS lesions and may help to explain the limited efficacy of therapies targeting individual inflammatory pathways in the treatment of HS.

cell biology↗

bioRxiv2024

Cell volume regulates terminal differentiation of cultured human epidermal keratinocytes

Differentiation of cultured human epidermal stem cells is regulated by interactions with the underlying substrate. Whereas differentiation is typically stimulated when keratinocytes are prevented from spreading, we previously identified two micron-scale topographical substrates that regulate differentiation of spread cells. On one substrate (S1), individual cells interact with small circular topographies, and differentiation is stimulated; on the other (S2), cells interact with larger triangular topographies, and differentiation is inhibited. By scanning electron microscopy we visualised substrate interactions at higher resolution than previously and using live cell imaging we established that induction of the differentiation marker involucrin did not involve transient cell rounding on S1. Bulk gene expression profiling did not reveal any differences between cells on S1 and S2 prior to the selective upregulation of differentiation markers at 12h on S1 and cell stiffness was lower on both S1 and S2 than on flat substrates. Nevertheless, cells on S2 differed from cells on flat and S1 substrates because they exhibited reduced cell volume, prompting us to explore whether cell volume could regulate differentiation independent of culture substrate. Treatment with polyethylene glycol (PEG) reduced cell volume and inhibited differentiation regardless of whether keratinocytes were seeded on flat, S1 or S2 substrates, micropatterned islands or in suspension. Conversely, treatment with deionised water increased cell volume and stimulated differentiation of substrate adherent keratinocytes. On flat substrates treatment with the Ca2+ chelator 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester or an inhibitor of the water channel aquaporin 3 blocked induction of differentiaton by deionised water, whereas the gadolinium3+, a stretch-activated calcium channel blocker, did not. Our studies identify a new mechanism by which keratinocyte-niche interactions regulate initiation of differentiation.

cell biology↗

bioRxiv2023

Multi-scale spatial mapping of cell populations across anatomical sites in healthy human skin and basal cell carcinoma

Our understanding of how human skin cells differ according to anatomical site and tumour formation is limited. To address this we have created a multi-scale spatial atlas of healthy skin and basal cell carcinoma (BCC), incorporating in vivo optical coherence tomography, single cell RNA sequencing, spatial global transcriptional profiling and in situ sequencing. Computational spatial deconvolution and projection revealed the localisation of distinct cell populations to specific tissue contexts. Although cell populations were conserved between healthy anatomical sites and in BCC, mesenchymal cell populations including fibroblasts and pericytes retained signatures of developmental origin. Spatial profiling and in silico lineage tracing support a hair follicle origin for BCC and demonstrate that cancer-associated fibroblasts are an expansion of a POSTN+ subpopulation associated with hair follicles in healthy skin. RGS5+ pericytes are also expanded in BCC suggesting a role in vascular remodelling. We propose that the identity of mesenchymal cell populations is regulated by signals emanating from adjacent structures and that these signals are repurposed to promote the expansion of skin cancer stroma. The resource we have created is publicly available in an interactive format for the research community. Significance statementSingle cells RNA sequencing has revolutionised cell biology, enabling high resolution analysis of cell types and states within human tissues. Here, we report a comprehensive spatial atlas of adult human skin across different anatomical sites and basal cell carcinoma (BCC) - the most common form of skin cancer - encompassing in vivo optical coherence tomography, single cell RNA sequencing, global spatial transcriptomic profiling and in situ sequencing. In combination these modalities have allowed us to assemble a comprehensive nuclear-resolution atlas of cellular identity in health and disease.

cell biology↗

bioRxiv2022

Hydrogen Peroxide Production by Epidermal Dual Oxidase 1 Regulates Nociceptive Sensory Signals

Keratinocytes of the mammalian skin provide not only mechanical protection for the tissues, but also transmit mechanical, chemical, and thermal stimuli from the external environment to the sensory nerve terminals. Sensory nerve fibers penetrate the epidermal basement membrane and function in the tight intercellular space among keratinocytes. Here we show that epidermal keratinocytes produce hydrogen peroxide upon the activation of the NADPH oxidase dual oxidase 1 (DUOX1). This enzyme can be activated by increasing cytosolic calcium levels. Using DUOX1 knockout animals as a model system we found an increased sensitivity towards certain noxious stimuli in DUOX1-deficient animals, which is not due to structural changes in the skin as evidenced by detailed immunohistochemical and electron-microscopic analysis of epidermal tissue. We show that DUOX1 is expressed in keratinocytes but not in the neural sensory pathway. The release of hydrogen peroxide by activated DUOX1 alters both the activity of neuronal TRPA1 and redox-sensitive potassium channels expressed in dorsal root ganglia primary sensory neurons. We describe hydrogen peroxide, produced by DUOX1 as a paracrine mediator of nociceptive signal transmission. Our results indicate that a novel, hitherto unknown redox mechanism modulates noxious sensory signals.

physiology↗

bioRxiv2022

Perivascular macrophages collaborate to facilitate chemotherapy resistance in cancer

A subset of tumor associated macrophages (TAMs) identified by their expression of the lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) reside proximal to blood vasculature and contribute to disease progression. Using a spontaneous murine model of mammary adenocarcinoma (MMTV-PyMT), we show that Lyve-1+ TAMs, which co-express heme oxygenase-1, form coordinated multi-cellular nest structures in the perivascular niche. We show that TAM nest formation is dependent on IL-6 and a communication axis involving CCR5 and its cognate ligands CCL3/4. We demonstrate that Lyve-1+ TAM nests are associated with CD8+ T-cell exclusion from the tumor and the resistance to immune-stimulating chemotherapeutics. This study highlights an unappreciated collaboration between TAMs and uncovers a spatially driven therapeutic resistance mechanism of these cells in cancer which can be therapeutically targeted.

cancer biology↗

bioRxiv2021

Role of distinct fibroblast lineages and immune cells in dermal repair following UV radiation induced tissue damage

Solar ultraviolet radiation (UVR) is a major source of skin damage, resulting in inflammation, premature ageing and cancer. While several UVR-induced changes, including extracellular matrix reorganisation and epidermal DNA damage, have been documented, the role of different fibroblast lineages and their communication with immune cells has not been explored. We show that acute and chronic UVR exposure led to selective loss of fibroblasts from the upper dermis in human and mouse skin. Lineage tracing and in vivo live imaging revealed that repair following acute UVR is predominantly mediated by papillary fibroblast proliferation and migration. In contrast, chronic UVR exposure led to a permanent loss of papillary fibroblasts, with expansion of fibroblast membrane protrusions partially compensating for the reduction in cell number. Although UVR strongly activated Wnt-signalling in skin, stimulation of fibroblast proliferation by epidermal {beta}-catenin stabilisation did not support papillary dermis repair. Acute UVR triggered an infiltrate of neutrophils and T cell subpopulations and increased pro-inflammatory prostaglandin signalling in skin. Depletion of CD4 and CD8 positive cells resulted in increased papillary fibroblast depletion, which correlated with an increase in DNA damage and reduction in fibroblast proliferation. Conversely, topical COX-2 inhibition prevented fibroblast depletion and neutrophil infiltration after UVR. We conclude that loss of papillary fibroblasts is primarily induced by a deregulated inflammatory response, with infiltrating T cells supporting fibroblast survival upon UVR-induced environmental stress. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/447606v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@d246bborg.highwire.dtl.DTLVardef@121b5faorg.highwire.dtl.DTLVardef@324114org.highwire.dtl.DTLVardef@14e9eb7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

ERK basal and pulsatile activity are differentially regulated in mammalian epidermis to control proliferation and exit from the stem cell compartment

Fluctuation in signal transduction pathways is frequently observed during mammalian development. However, its role in regulating stem cells has not been explored. Here we tracked spatiotemporal ERK MAPK dynamics in human epidermal stem cells. While stem cells and differentiated cells were distinguished by high and low stable basal ERK activity, respectively, we also found cells with pulsatile ERK activity. Transitions from Basalhi-Pulselo (stem) to Basalhi-Pulsehi, Basalmid-Pulsehi, and Basallo-Pulselo (differentiated) cells occurred in expanding keratinocyte colonies and in response to a range of differentiation stimuli. Pharmacological inhibition of ERK induced differentiation only when cells were in the Basalmid-Pulsehi state. Basal ERK activity and pulses were differentially regulated by DUSP10 and DUSP6, leading us to speculate that DUSP6-mediated ERK pulse downregulation promotes initiation of differentiation whereas DUSP10-mediated downregulation of mean ERK activity promotes and stabilizes post-commitment differentiation. Quantification of MAPK1/3, DUSP6 and DUSP10 transcripts in individual cells demonstrated that ERK activity is controlled both transcriptionally and post-transcriptionally. When cells were cultured on a topography that mimics the epidermal-dermal interface, spatial segregation of mean ERK activity and pulses was observed. In vivo imaging of mouse epidermis revealed a patterned distribution of basal cells with pulsatile ERK activity and downregulation was linked to the onset of differentiation. Our findings demonstrate that ERK MAPK signal fluctuations link kinase activity to stem cell dynamics. SignificanceUnderstanding how intracellular signaling cascades control cell fate is a key issue in stem cell biology. Here we show that exit from the stem cell compartment in mammalian epidermis is characterised by pulsatile ERK MAPK activity. Basal activity and pulses are differentially regulated by DUSP10 and DUSP6, two phosphatases that have been shown previously to regulate differentiation commitment in the epidermis. ERK activity is controlled both transcriptionally and post-transcriptionally. Spatial segregation of mean ERK activity and pulses is observed both in reconstituted human epidermis and in mouse epidermis. Our findings demonstrate the tight spatial and temporal regulation of ERK MAPK expression and activity in mammalian epidermis.

cell biology↗

bioRxiv2018

High-throughput micro-patterning platform reveals Nodal-dependent dissection of peri-gastrulation-associated versus pre-neurulation associated fate patterning

In vitro models of post-implantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon BMP4 treatment of geometrically-confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line associated heterogeneity was found to be attributable to endogenous nodal expression, with upregulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal downregulation correlated with a neurulation-associated gene profile expression. We harness this knowledge to establish a platform of pre-neurulation-like fate patterning in geometrically confined hPSC colonies that arises due to a stepwise activation of reaction-diffusion and positional-information. Our work identifies a Nodal signalling dependent switch in peri-gastrulation versus pre-neurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of self-organized fate specification in differentiating epiblast and ectodermal tissues.

developmental biology↗

bioRxiv2018

Hair Follicle Epidermal Stem Cells Define a Niche for Tactile Sensation

The heterogeneity and compartmentalization of stem cells is a common principle in many epithelia, and is known to function in epithelial maintenance, but its other physiological roles remain elusive. Here we show transcriptional and anatomical contributions of compartmentalized epidermal stem cells (EpSCs) in tactile sensory unit formation in the hair follicle (HF). EpSCs in the follicle upper-bulge, where mechanosensory lanceolate complexes (LCs) innervate, express a unique set of extracellular matrix (ECM) and neurogenesis-related genes. These EpSCs deposit an ECM protein EGFL6 into the collar matrix, a novel ECM that tightly ensheathes LCs. EGFL6 is required for the proper patterning, touch responses, and v integrin-enrichment of LCs. By maintaining a quiescent original EpSC niche, the old bulge, EpSCs provide anatomically stable HF-LC interfaces, irrespective of the stage of follicle regeneration cycle. Thus, compartmentalized EpSCs provide a niche linking the HF and the nervous system throughout the hair cycle.

developmental biology↗

bioRxiv2018

IMMUNOMODULATORY ROLE OF KERATIN 76 IN ORAL AND GASTRIC CANCER

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.

cancer biology↗

bioRxiv2018

Identifying the genetic basis of variation in cell behaviour in human iPS cell lines from healthy donors

Large cohorts of human iPSCs from healthy donors are potentially a powerful tool for investigating the relationship between genetic variants and cellular phenotypes. Here we integrate high content imaging, gene expression and DNA sequence datasets for over 100 human iPSC lines to identify the genetic basis of inter-individual variability in cell behaviour. By applying a dimensionality reduction approach, Probabilistic Estimation of Expression Residuals (PEER), we identified genes that correlated in expression with intrinsic (genetic) and extrinsic (ECM) factors. However, variation in mRNA levels could not account for outlier cell behaviour. Instead, we identified rare, deleterious SNVs in the coding sequence of genes involved in ECM adhesion that occurred in cell lines that were outliers for one or more phenotypes such as cell spreading. These also correlated with altered germ layer differentiation on micropatterned surfaces. Our study thus establishes a strategy for integrating genetic and cell biological measurements for high-throughput analysis.

cell biology↗

bioRxiv2018

Generative adversarial networks uncover epidermal regulators and predict single cell perturbations

Recent advances have enabled gene expression profiling of single cells at lower cost. As more data is produced there is an increasing need to integrate diverse datasets and better analyse underutilised data to gain biological insights. However, analysis of single cell RNA-seq data is challenging due to biological and technical noise which not only varies between laboratories but also between batches. Here for the first time, we apply a new generative deep learning approach called Generative Adversarial Networks (GAN) to biological data. We apply GANs to epidermal, neural and hematopoietic scRNA-seq data spanning different labs and experimental protocols. We show that it is possible to integrate diverse scRNA-seq datasets and in doing so, our generative model is able to simulate realistic scRNA-seq data that covers the full diversity of cell types. In contrast to many machine-learning approaches, we are able to interpret internal parameters in a biologically meaningful manner. Using our generative model we are able to obtain a universal representation of epidermal differentiation and use this to predict the effect of cell state perturbations on gene expression at high time-resolution. We show that our trained neural networks identify biological state-determining genes and through analysis of these networks we can obtain inferred gene regulatory relationships. Finally, we use internal GAN learned features to perform dimensionality reduction. In combination these attributes provide a powerful framework to progress the analysis of scRNA-seq data beyond exploratory analysis of cell clusters and towards integration of multiple datasets regardless of origin.

genomics↗

bioRxiv2017

Epidermal Wnt signalling regulates transcriptome heterogeneity and proliferative fate in neighbouring cells

Canonical Wnt/beta-catenin signalling regulates self-renewal and lineage selection within the mouse epidermis. Although the transcriptional response of keratinocytes that receive a Wnt signal is well characterised, little is known about the mechanism by which keratinocytes in proximity to the Wntreceiving cell are co-opted to undergo a change in cell fate. To address this, we performed single-cell mRNA-Seq on mouse keratinocytes co-cultured with and without the presence of beta-catenin activated neighbouring cells. We identified seven distinct cell states in cultures that had not been exposed to the beta-catenin stimulus and show that the stimulus redistributes wild type subpopulation proportions. Using temporal single-cell analysis we reconstruct the cell fate changes induced by neighbour Wnt activation. Gene expression heterogeneity was reduced in neighbouring cells and this effect was most dramatic for protein synthesis associated genes. The changes in gene expression were accompanied by a shift from a quiescent to a more proliferative stem cell state. By integrating imaging and reconstructed sequential gene expression changes during the state transition we identified transcription factors, including Smad4 and Bcl3, that were responsible for effecting the transition in a contact-dependent manner. Our data indicate that non cell-autonomous Wnt/beta-catenin signalling decreases transcriptional heterogeneity and further our understanding of how epidermal Wnt signalling orchestrates regeneration and self-renewal.

genomics↗

bioRxiv2017

A protein phosphatase network controls temporal and spatial dynamics of differentiation commitment in human epidermis

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 for up to 12h to induce differentiation. We find that cell detachment induces a network of protein phosphatases. The pro-commitment phosphatases - including DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA - promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Their activity is antagonised by concomitant upregulation of DUSP10. Boolean network modelling of phosphatase interactions identifies commitment as an inherently unstable biological switch between the stem and differentiated cell states. Furthermore, phosphatase expression is spatially regulated both in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

developmental biology↗