Biology subjects

Dobie, R.

Publications and source records attributed to Dobie, R..

10 recordsLinked to original sources

bioRxiv2023

Oncogenic RAS drives rapid onset cellular plasticity and elicits a tumour-promoting neutrophil response at the inception of preneoplastic development

Oncogenic driver mutations are frequently found in normal tissues, suggesting that additional non-genetic factors are required for tumourigenesis. Phenotypic plasticity is an important gateway to malignancy and inflammation can fuel tumourigenesis, however, little is known about when and how these hallmarks first arise. Using single-cell transcriptomics and in vivo live imaging we have characterised the immediate cell intrinsic and innate immune responses during the first 24 hours following oncogenic Ras activation, in an inducible zebrafish model of HRASG12V-mediated skin tumour initiation. We found that only a subset of basal keratinocytes, but not superficial keratinocytes, are susceptible to RAS-driven phenotypic plasticity. These preneoplastic cells undergo dedifferentiation and partial EMT, resembling malignant cells observed in human squamous cell carcinoma (SCC). Strikingly, the same subset instigates the development of tumour-promoting neutrophils, which in turn enhance preneoplastic cell proliferation. Our findings demonstrate that the effects of oncogenic Ras are primarily determined by the cell of origin and reveal an association between the unlocking of phenotypic plasticity and the onset of tumour-promoting inflammation.

cancer biology↗

bioRxiv2023

Multimodal decoding of human liver regeneration

The liver has a unique ability to regenerate1,2, however in the setting of acute liver failure (ALF) this regenerative capacity is often overwhelmed and emergency liver transplantation is the only curative option3-5. To advance our understanding of human liver regeneration and to inform design of pro-regenerative therapies, we use paired single-nuclei RNA sequencing (snRNA-seq) combined with spatial profiling of healthy and ALF explant human livers to generate the first single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation which emerges during human liver regeneration, and a corollary migratory hepatocyte subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Importantly, interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. 4-D intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 expression reduces HGF-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Taken together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation which mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may open up new areas of therapeutic discovery in regenerative medicine.

cell biology↗

bioRxiv2022

Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida

The liver is a multitasking organ with essential functions for vertebrate health spanning metabolism and immunity. In contrast to mammals, our understanding of liver cellular heterogeneity and its role in regulating immunological status remains poorly defined in fishes. Addressing this knowledge gap, we generated a transcriptomic atlas of 47,432 nuclei isolated from the liver of Atlantic salmon (Salmo salar L.) contrasting control fish with those challenged with a pathogenic strain of Aeromonas salmonicida, a problematic bacterial pathogen in global aquaculture. We identified the major liver cell types and their sub-populations, revealing poor conservation of many hepatic cell marker genes utilized in mammals, while identifying novel heterogeneity within the hepatocyte, lymphoid, and myeloid lineages. This included polyploid hepatocytes, multiple T cell populations including {gamma}{delta} T cells, and candidate populations of monocytes/macrophages/dendritic cells. A dominant hepatocyte population radically remodeled its transcriptome following infection to activate the acute phase response and other defense functions, while repressing routine functions such as metabolism. These defense-specialized hepatocytes showed strong activation of genes controlling protein synthesis and secretion, presumably to support the release of acute phase proteins into circulation. The infection response further involved up-regulation of numerous genes in an immune-cell specific manner, reflecting functions in pathogen recognition and killing, antigen presentation, phagocytosis, regulation of inflammation, B cell differentiation and T cell activation. Overall, this study greatly enhances our understanding of the multifaceted role played by liver cells in immune defense and metabolic remodeling following infection and provides many novel cell-specific marker genes to empower future studies of this organ in fishes.

immunology↗

bioRxiv2022

PAX6 LIMITS THE COMPETENCE OF DEVELOPING CEREBRAL CORTICAL CELLS

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6s main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side-effects of morphogens engaged contemporaneously in other essential functions.

neuroscience↗

bioRxiv2021

Single cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation

The human endometrium experiences repetitive cycles of tissue wounding characterised by piecemeal shedding of the surface epithelium and rapid restoration of tissue homeostasis. In this study we used a mouse model of endometrial repair and three transgenic lines of mice to investigate whether epithelial cells that become incorporated into the newly formed luminal epithelium have their origins in one or more of the mesenchymal cell types present in the stromal compartment of the endometrium. Using scRNAseq we identified a novel population of PDGFRb+ mesenchymal stromal cells that developed a unique transcriptomic signature in response to endometrial breakdown/repair. These cells expressed genes usually considered specific to epithelial cells and in silico trajectory analysis suggested they were stromal fibroblasts in transition to becoming epithelial cells. To confirm our hypothesis we used a lineage tracing strategy to compare the fate of stromal fibroblasts (PDGFRa+) and stromal perivascular cells (NG2/CSPG4+). We demonstrated that stromal fibroblasts can undergo a mesenchyme to epithelial transformation and become incorporated into the re-epithelialised luminal surface of the repaired tissue. This study is the first to discover a novel population of wound-responsive, plastic endometrial stromal fibroblasts that contribute to the rapid restoration of an intact luminal epithelium during endometrial repair. These findings form a platform for comparisons both to endometrial pathologies which involve a fibrotic response (Ashermans syndrome, endometriosis) as well as other mucosal tissues which have a variable response to wounding. HighlightsO_LISingle cell RNA sequencing identified a population of PDGFR{beta}+ mesenchymal cells with a unique transcriptomic signature that arises in response to induction of a menses-like wound C_LIO_LIThe repair-specific mesenchymal cells express genes considered specific to both mesenchymal and epithelial lineages indicative of an intermediate phenotype and a mesenchymal to epithelial transition (MET) C_LIO_LIin silico trajectory analysis revealed that repair-specific cells originate from the fibroblast cell clusters, were distinct from perivascular cells, and had a predicted trans-differentiation trajectory towards definitive epithelial cells C_LIO_LIin vivo lineage tracing in adult mice provides definitive evidence that PDGFR+ endometrial fibroblasts, and not NG2+ perivascular cells, undergo MET and can become incorporated into the luminal epithelium of the post repair tissue C_LI

physiology↗

bioRxiv2020

The proportion of periportal mesenchyme to ductal epithelial cells acts as a proliferative rheostat in liver regeneration

In the homeostatic liver, ductal cells intermingle with a microenvironment of endothelial and mesenchymal cells to form the functional unit of the portal tract. Ductal cells proliferate rarely in homeostasis but do so transiently after tissue injury to replenish any lost epithelium. We have shown that liver ductal cells can be expanded as liver organoids that recapitulate several of the cell-autonomous mechanisms of regeneration, but lack the stromal cell milieu of the biliary tract in vivo. Here, we describe a subpopulation of SCA1+ periportal mesenchymal cells that closely surrounds ductal cells in vivo and exerts a dual control on their proliferative capacity. Mesenchymal-secreted mitogens support liver organoid formation and expansion from differentiated ductal cells. However, direct mesenchymal-to-ductal cell-cell contact, established following a microfluidic co-encapsulation that enables the cells to self-organize into chimeric organoid structures, abolishes ductal cell proliferation in a mesenchyme-dose dependent manner. We found that it is the ratio between mesenchymal and epithelial cell contacts that determines the net outcome of ductal cell proliferation both in vitro, and in vivo, during damage-regeneration. SCA1+ mesenchymal cells control ductal cell proliferation dynamics by a mechanism involving, at least in part, Notch signalling activation. Our findings underscore how the relative abundance of cell-cell contacts between the epithelium and its mesenchymal microenvironment are key regulatory cues involved in the control of tissue regeneration. SummaryIn the homeostatic liver, the ductal epithelium intermingles with a microenvironment of stromal cells to form the functional unit of the portal tract. Ductal cells proliferate rarely in homeostasis but do so transiently after tissue injury. We have shown that these cells can be expanded as liver organoids that recapitulate several of the cell-autonomous mechanisms of regeneration, but lack the stromal cell milieu of the portal tract in vivo. Here, we describe a subpopulation of SCA1+ periportal mesenchymal niche cells that closely surrounds ductal cells in vivo and exerts a dual control on their proliferative capacity. Mesenchymal-secreted mitogens support liver organoid formation and expansion from differentiated ductal cells. However, direct mesenchymal-to-ductal cell-cell contact, established through a microfluidic co-encapsulation method that enables the cells to self-organize into chimeric organoid structures, abolishes ductal cell proliferation in a mesenchyme-dose dependent manner. We found that it is the ratio between mesenchymal and epithelial cell contacts that determines the net outcome of ductal cell proliferation both in vitro, and in vivo, during damage-regeneration. SCA1+ mesenchymal cells control ductal cell proliferation dynamics by a mechanism involving, at least in part, Notch signalling activation. Our findings re-evaluate the concept of the cellular niche, whereby the proportions of cell-cell contacts between the epithelium and its mesenchymal niche, and not the absolute cell numbers, are the key regulatory cues involved in the control of tissue regeneration.

developmental biology↗

bioRxiv2020

Single cell RNA sequencing redefines the mesenchymal cell landscape of mouse endometrium

The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration and remodelling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear. In the current study we used the Pdgfrb-BAC-eGFP transgenic reporter mouse in combination with bulk and single cell RNA sequencing (scRNAseq) to redefine the endometrial mesenchyme. Contrary to previous reports we show that CD146 is expressed in both PDGFR{beta}+ perivascular cells as well as CD31+ endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, Abcc9). scRNAseq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFR+/CD34+ but were distinct in their expression of Spon2/Angptl7 (fibroblast 1), Smoc2/Rgs2 (fibroblast 2) and Clec3b/Col14a1/Mmp3 (fibroblast 3), with potential functions in regulation of immune responses, response to wounding and organisation of extracellular matrix respectively. In conclusion, these data are the first to provide a single cell atlas of the mesenchymal cell landscape in mouse endometrium. By identifying novel markers for subpopulations of mesenchymal cells we can use mouse models investigate their contribution to endometrial function, compare with other tissues and apply these findings to further our understanding of human endometrium. HighlightsO_LIGFP expression in the mouse endometrium, under the control of the Pdgfrb promoter, is restricted to two cell populations based on the intensity of GFP with GFPbright cells close to the vasculature C_LIO_LISingle cell RNAseq identified five subpopulations of GFP+ mesenchymal cells: pericytes, vascular smooth muscle cells (vSMC) and three closely related but distinct populations of fibroblasts C_LIO_LIBioinformatics revealed that pericytes and vSMC share functions associated with the circulatory system, actin-filament process and cell adhesion, and an apparent role for pericytes in smooth muscle cell migration and response to interferons C_LIO_LIComparisons between the fibroblast subpopulations suggest distinct roles in regulation of immune response, response to wound healing and collagen organisation. C_LI Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

physiology↗

bioRxiv2019

Neutrophils mediate the capture of peritoneal contaminants by fat-associated lymphoid clusters of the omentum

The omentum is a visceral adipose tissue rich in fat-associated lymphoid clusters (FALCs), which collects peritoneal contaminants and provides a first layer of immunological defence within the abdomen. Using single-cell RNA sequencing and spatial analysis of omental stromal cells, we reveal that the surface of FALCs are covered with specialised mesothelial cells, which we name FALC cover cells. We demonstrate that CXCL1 is expressed by FALC cover cells and that CXCL1 is critical for the retention and accumulation of neutrophils within FALCs during peritonitis. We show that protein arginine deiminase 4 mediates the formation of dense neutrophil aggregates, which are required for the neutralisation of particles present in the peritoneal cavity. Finally, we provide confirmatory evidence in humans with acute appendicitis, that the omentum is also a site of neutrophil recruitment and bacterial capture, and is thus an important component of the immunological defence against the propagation of peritoneal contaminants.

immunology↗

bioRxiv2019

Resolving the fibrotic niche of human liver cirrhosis using single-cell transcriptomics

Currently there are no effective antifibrotic therapies for liver cirrhosis, a major killer worldwide. To obtain a cellular resolution of directly-relevant pathogenesis and to inform therapeutic design, we profile the transcriptomes of over 100,000 primary human single cells, yielding molecular definitions for the major non-parenchymal cell types present in healthy and cirrhotic human liver. We uncover a novel scar-associated TREM2+CD9+ macrophage subpopulation with a fibrogenic phenotype, that has a distinct differentiation trajectory from circulating monocytes. In the endothelial compartment, we show that newly-defined ACKR1+ and PLVAP+ endothelial cells expand in cirrhosis and are topographically located in the fibrotic septae. Multi-lineage ligand-receptor modelling of specific interactions between the novel scar-associated macrophages, endothelial cells and collagen-producing myofibroblasts in the fibrotic niche, reveals intra-scar activity of several major pathways which promote hepatic fibrosis. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides the conceptual framework required to discover rational therapeutic targets in liver cirrhosis.

immunology↗

bioRxiv2018

A macrophage-pericyte axis directs tissue restoration via Amphiregulin-induced TGFβ activation

The Epidermal Growth Factor Receptor ligand Amphiregulin has a well-documented role in the restoration of tissue homeostasis following injury; however, the mechanism by which Amphiregulin contributes to wound repair remains unknown. Here we show that Amphiregulin functions by releasing bio-active TGFb from latent complexes via integrin-v activation. Using acute injury models in two different tissues, we found that by inducing TGFb activation on mesenchymal stromal cells (aka pericytes), Amphiregulin induced their differentiation into myo-fibroblasts, thereby selectively contributing to the restoration of vascular barrier function within injured tissue. Furthermore, we identified macrophages as a critical source of Amphiregulin, revealing a direct effector mechanism by which these cells contribute to tissue restoration following acute injury. Combined, these observations expose a so far under-appreciated mechanism of how cells of the immune system selectively control the differentiation of tissue progenitor cells during tissue repair and inflammation.

immunology↗