Biology subjects

Turner, S.

Publications and source records attributed to Turner, S..

27 records · Page 2Linked to original sources

bioRxiv2024

Single-cell and spatial transcriptomics of the avian embryo tailbud

Vertebrate body axis formation initiates during gastrulation and continues within the tail bud at the posterior end of the embryo. Major structures in the trunk are paired somites, which generate the musculoskeletal system, the spinal cord - forming part of the central nervous system, and the notochord, with important patterning functions. The specification of these different cell lineages by key signalling pathways and transcription factors is essential, however, a global map of cell types and expressed genes in the avian trunk is missing. Here we use single-cell RNA sequencing and RNA tomography to generate a molecular map of the emerging trunk and tailbud in the chick embryo. Single cell RNA-sequencing (scRNA-seq) identifies discrete cell lineages including somites, neural tube, neural crest, lateral plate mesoderm, ectoderm, endothelial and blood progenitors. In addition, high-throughput RNA-seq of sequential tissue sections provides a spatially resolved, genome-wide expression dataset for the avian tailbud and emerging body, comparable to other model systems. Combining the single-cell and spatial datasets, we identify spatially restricted genes, focusing on somites and early myoblasts. Thus, this high-resolution transcriptome map incorporating cell types in the embryonic trunk can expose molecular pathways involved in body axis development.

developmental biology↗

bioRxiv2023

Stricturing Crohn's disease single-cell RNA sequencing reveals fibroblast heterogeneity and intercellular interactions

BackgroundFibroblasts play a key role in stricture formation in Crohns disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. MethodsWe performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. ResultsOur integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. ConclusionA full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments.

immunology↗

bioRxiv2023

Cytokine-Mediated Degradation of the Transcription Factor ERG Impacts the Pulmonary Vascular Response to Systemic Inflammatory Challenge

BackgroundDuring infectious diseases, pro-inflammatory cytokines transiently destabilize interactions between adjacent vascular endothelial cells (ECs) to facilitate the passage of immune molecules and cells into tissues. However, in the lung the resulting vascular hyperpermeability can lead to organ dysfunction. Previous work identified the transcription factor ERG as a master regulator of endothelial homeostasis. Here we investigate whether the sensitivity of pulmonary blood vessels to cytokine-induced destabilization is due to organotypic mechanisms affecting the ability of endothelial ERG to protect lung ECs from inflammatory injury. MethodsCytokine-dependent ubiquitination and proteasomal degradation of ERG was analyzed in cultured Human Umbilical Vein ECs (HUVECs). Systemic administration of TNF or the bacterial cell wall component lipopolysaccharide (LPS) was used to cause a widespread inflammatory challenge in mice; ERG protein levels were assessed by immunoprecipitation, immunoblot, and immunofluorescence. Murine Erg deletion was genetically induced in ECs (Ergfl/fl;Cdh5(PAC)CreERT2), and multiple organs were analyzed by histology, immunostaining, and electron microscopy. ResultsIn vitro, TNF promoted the ubiquitination and degradation of ERG in HUVECs, which was blocked by the proteasomal inhibitor MG132. In vivo, systemic administration of TNF or LPS resulted in a rapid and substantial degradation of ERG within lung ECs, but not ECs of the retina, heart, liver, or kidney. Pulmonary ERG was also downregulated in a murine model of influenza infection. Ergfl/fl;Cdh5(PAC)-CreERT2 mice spontaneously recapitulated aspects of inflammatory challenges, including lung-predominant vascular hyperpermeability, immune cell recruitment, and fibrosis. These phenotypes were associated with a lung-specific decrease in the expression of Tek, a gene target of ERG previously implicated in maintaining pulmonary vascular stability during inflammation. ConclusionsCollectively, our data highlight a unique role for ERG in pulmonary vascular function. We propose that cytokine-induced ERG degradation and subsequent transcriptional changes in lung ECs play critical roles in the destabilization of pulmonary blood vessels during infectious diseases.

cell biology↗

bioRxiv2022

GSK2556286 is a novel antitubercular drug candidate effective in vivo with the potential to shorten tuberculosis treatment

As a result of a high-throughput compound screening campaign of Mycobacterium tuberculosis infected macrophages, a new preclinical drug candidate for the treatment of tuberculosis has been identified. GSK2556286 inhibits growth within human macrophages (IC50 = 0.07 {micro}M), is active against extracellular bacteria in cholesterol-containing culture media and exhibits no cross-resistance with known antitubercular drugs. In addition, it has shown efficacy in different mouse models of tuberculosis (TB) and has an adequate safety profile in two preclinical species. These features indicate a compound with a novel mode of action, although still not fully defined, that is effective against both multidrug or extensively-resistant (M/XDR) and drug-sensitive (DS) M. tuberculosis with the potential to shorten the duration of treatment in novel combination drug regimens. One Sentence SummaryGSK2556286 is a novel preclinical drug candidate for the treatment of tuberculosis with a new mode of action potentially able to contribute to the shortening of TB chemotherapy.

pharmacology and toxicology↗

bioRxiv2020

Involvement of uterine natural killer cells in the natural selection of human embryos at implantation.

Decidualizing endometrial stromal cells (EnSC) critically determine the maternal response to an implanting conceptus, triggering either menstruation-like disposal of low-fitness embryos or creating an environment that promotes further development. However, the mechanism that couples maternal recognition of low-quality embryos to tissue breakdown remains poorly understood. Recently, we demonstrated that successful transition of the cycling endometrium to a pregnancy state requires selective elimination of pro-inflammatory senescent decidual cells by activated uterine natural killer (uNK) cells. Here we report that uNK cells express CD44, the canonical hyaluronan (HA) receptor, and demonstrate that high-molecular weight HA (HMWHA) inhibits uNK cell-mediated killing of senescent decidual cells. By contrast, low-molecular weight HA (LMWHA) did not attenuate uNK cell activity in co-culture experiments. Killing of senescent decidual cells by uNK cells was also inhibited upon exposure to medium conditioned by IVF embryos that failed to implant, but not successful embryos. Embryo-mediated inhibition of uNK cell activity was reversed by recombinant hyaluronidase 2 (HYAL2), which hydrolyses HMWHA. We further report a correlation between the levels of HYAL2 secretion by human blastocysts, morphological scores, and implantation potential. Taken together, the data suggest a pivotal role for uNK cells in embryo biosensing and endometrial fate decisions at implantation.

cell biology↗

bioRxiv2020

Comparative primary paediatric nasal epithelial cell culture differentiation and RSV-induced cytopathogenesis following culture in two commercial media.

The culture of differentiated human airway epithelial cells allows the study of pathogen-host interactions and innate immune responses in a physiologically relevant in vitro model. As the use of primary cell culture has gained popularity the availability of the reagents needed to generate these cultures has increased. In this study we assessed two different media, Promocell and PneumaCult, during the differentiation and maintenance of well-differentiated primary nasal epithelial cell cultures (WD-PNECs). We compared and contrasted the consequences of these media on WD-PNEC morphological and physiological characteristics and their responses to respiratory syncytial virus (RSV) infection. We found that cultures generated using PneumaCult resulted in greater total numbers of smaller, tightly packed, pseudostratified cells. However, cultures from both media resulted in similar proportions of ciliated and goblet cells. There were no differences in RSV growth kinetics, although more ciliated cells were infected in the PneumaCult cultures. There was also significantly more IL-29/IFN{lambda}1 secreted from PneumaCult compared to Promocell cultures. In conclusion, the type of medium used for the differentiation of primary human airway epithelial cells impacts experimental results.

cell biology↗

bioRxiv2018

Gut microbiome communication with bone marrow regulates susceptibility to amebiasis

The gut microbiome provides resistance to infection. However, the mechanisms for this are poorly understood. Colonization with the intestinal bacterium Clostridium scindens provided protection from the parasite Entamoeba histolytica via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte-progenitors (GMPs) and provided neutrophil-mediated protection against subsequent challenge with E. histolytica. Adoptive transfer of bone-marrow from C. scindens colonized-mice into naive-mice protected against ameba infection and increased intestinal neutrophils. Because of the known ability of C. scindens to metabolize the bile salt cholate, we measured deoxycholate and discovered that it was increased in the sera of C. scindens colonized mice, as well as in children protected from amebiasis. Administration of deoxycholate alone (in the absence of C. scindens) increased the epigenetic mediator JMJD3 and GMPs and provided protection from amebiasis. In conclusion the microbiota was shown to communicate to the bone marrow via microbially-metabolized bile salts to train innate immune memory to provide antigen-nonspecific protection from subsequent infection. This represents a novel mechanism by which the microbiome protects from disease.\n\nOne Sentence SummaryIntroduction of the human commensal bacteria Clostridium scindens into the intestinal microbiota epigenetically alters bone marrow and protects from future parasite infection.

microbiology↗

bioRxiv2018

Paralogues of the PXY and ER receptor kinases enforce radial patterning in plant vascular tissue.

Plant cell walls do not allow cells to migrate, thus plant growth and development is entirely the consequence of changes to cell division and cell elongation. Where tissues are arranged in concentric rings, expansion of inner tissue, such as that which occurs during vascular development, must be coordinated with cell division and/or expansion of the outer tissue layers, endodermis, cortex, and epidermis, in order for tissue integrity to be maintained. Little is known of how coordination between cell layers occurs, but non-cell autonomous signalling could provide an explanation. Endodermis-derived EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) ligands have been shown to signal to the ERECTA (ER) receptor kinase present in the phloem. ER interacts with PHLOEM INTERCALLATED WITH XYLEM (PXY), a receptor present in the procambium. The PXY ligand, TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) is derived from CLE41 which is expressed in the phloem. These factors therefore represent a mechanism by which intertissue signalling could occur to control radial expansion between vascular and non-vascular tissue in plant stems. Here we show that ER regulates expression of PXY paralogues, PXL1 and PXL2, and that in turn PXY, PXL1 and PXL2 together with ER, regulate the expression of ERL1 and ERL2, genes paralogous to ER. PXY, PXL1, PXL2 and ER also regulate the expression of ER-ligands. Genetic analysis of these six receptor kinase genes demonstrated that they are required to control organisation, proliferation and cell size across multiple tissue layers. Taken together, our experiments demonstrate that ER signalling attenuates PXL expression in the stem, thus influencing vascular expansion and patterning. We anticipate that similar regulatory relationships, where tissue growth is controlled via cell signals moving across different tissue layers, will coordinate tissue layer expansion throughout the plant body.

plant biology↗

bioRxiv2017

Dissecting the genetic architecture of shoot growth in carrot (Daucus carota L.) using a diallel mating design

Crop establishment in carrot (Daucus carota L.) is limited by slow seedling growth and delayed canopy closure, resulting in high management costs for weed control. Varieties with improved growth habit (i.e. larger canopy and increased shoot biomass) may help mitigate weed control, but the underlying genetics of these traits in carrot is unknown. This project used a diallel mating design coupled with recent Bayesian analytical methods to determine the genetic basis of carrot shoot growth. Six diverse carrot inbred lines with variable shoot size were crossed in WI in 2014. F1 hybrids, reciprocal crosses, and parental selfs were grown in a randomized complete block design (RCBD) with two blocks in CA (2015, 2016) and in WI (2015). Measurements included canopy height, canopy width, shoot biomass, and root biomass. General and specific combining abilities were estimated using Griffings Model I. In parallel, additive, inbreeding, epistatic, and maternal effects were estimated from a Bayesian linear mixed model, which is more robust to dealing with missing data, outliers, and theoretical constraints than traditional biometric methods. Both additive and non-additive effects significantly influenced shoot traits, with non-additive effects playing a larger role early in the growing season, when weed control is most critical. Results suggest that early season canopy growth and root size express hybrid vigor and can be improved through reciprocal recurrent selection.\n\nArticle SummaryBreeding for improved competitive ability is a priority in carrot, which suffers yield losses under weed pressure. However, improvement and in-depth genetic studies for these traits relies on knowledge of the underlying genetic architecture. This study estimated heritable and non-heritable components of carrot shoot growth from a diallel mating design using a Bayesian mixed model. Results directly contribute to improvement efforts by providing estimates of combining ability, identifying a useful tester line, and characterizing the genetic and non-genetic influences on traits for improved competitive ability in carrot.

genetics↗