Evolution of the Hox gene fushi tarazu in arthropods

Homeotic (Hox) genes are important in determining regional identity in virtually all metazoans,and are conserved throughout the animal kingdom. In Drosophila melanogaster, fushi tarazu (ftz) is located within the Hox complex and contains a Hox-like DNA-binding homeodomain, but functions as a pair-rule segmentation gene. At some point(s) during evolution, ftz has undergone three specific changes thought to contribute to its new segmentation function in Drosophila: 1) The gain of an LXXLL motif allowed for interaction with a new co-factor, Ftz-F1; 2) The degeneration of the YPWM motif decreased the ability to interact with the homeotic co-factor Exd; 3) ftz expression switched from Hox-like to seven stripes in Drosophila. Here I isolated ftz sequences and examined expression from arthropods spanning 550 million years of evolutionary time to track these changes in ftz. I found that while the LXXLL motif required for segmentation was stably acquired at the base of the holometabolous insects, the YPWM motif degenerated independently many times in arthropod lineages, and these `degen-YPWMs' vary in their homeotic potential. Additionally, ftz expression in a crustacean is in a weak Hox-like pattern, suggesting a model in which different ftz variants could arise in nature and not be detrimental to organismal development. Given my findings that ftz sequence and expression is so dynamic, I investigated the features that may be preventing ftz fossilization in arthropod genomes. I tested the hypothesis that a broadly conserved role of ftz in the developing central nervous system (CNS) retains ftz in arthropod genomes. This model predicts that the homeodomain, but not variable co-factor interaction motifs, is required for Ftz CNS function. Evidence supporting this model was obtained from CNS-specific rescue experiments in Drosophila. Additionally I examined the expression and function of ftz and ftz-f1 in the short-germ beetle Tribolium castaneum. I found that both genes are expressed in pair-rule patterns, and preliminary results suggest that ftz-f1 is important for proper segmentation and cuticle deposition, and ftz function may be partially redundant with ftz-f1. Taken together, these findings show that variation of a pleiotropic transcription factor is more extensive than previously imagined, and suggest that evolutionary plasticity may be widespread among regulatory genes

Rapid isolation of gene homologs across taxa: Efficient identification and isolation of gene orthologs from non-model organism genomes, a technical report

<p>Abstract</p> <p>Background</p> <p>Tremendous progress has been made in the field of evo-devo through comparisons of related genes from diverse taxa. While the vast number of species in nature precludes a complete analysis of the molecular evolution of even one single gene family, this would not be necessary to understand fundamental mechanisms underlying gene evolution if experiments could be designed to systematically sample representative points along the path of established phylogenies to trace changes in regulatory and coding gene sequence. This isolation of homologous genes from phylogenetically diverse, representative species can be challenging, especially if the gene is under weak selective pressure and evolving rapidly.</p> <p>Results</p> <p>Here we present an approach - Rapid Isolation of Gene Homologs across Taxa (RIGHT) - to efficiently isolate specific members of gene families. RIGHT is based upon modification and a combination of degenerate polymerase chain reaction (PCR) and gene-specific amplified fragment length polymorphism (AFLP). It allows targeted isolation of specific gene family members from any organism, only requiring genomic DNA. We describe this approach and how we used it to isolate members of several different gene families from diverse arthropods spanning millions of years of evolution.</p> <p>Conclusions</p> <p>RIGHT facilitates systematic isolation of one gene from large gene families. It allows for efficient gene isolation without whole genome sequencing, RNA extraction, or culturing of non-model organisms. RIGHT will be a generally useful method for isolation of orthologs from both distant and closely related species, increasing sample size and facilitating the tracking of molecular evolution of gene families and regulatory networks across the tree of life.</p

Myo/Nog Cells Give Rise to Myofibroblasts During Epiretinal Membrane Formation in a Mouse Model of Proliferative Vitreoretinopathy.

PURPOSE: Myo/Nog cells are the source of myofibroblasts in the lens and synthesize muscle proteins in human epiretinal membranes (ERMs). In the current study, we examined the response of Myo/Nog cells during ERM formation in a mouse model of proliferative vitreoretinopathy (PVR). METHODS: PVR was induced by intravitreal injections of gas and ARPE-19 cells. PVR grade was scored by fundus imaging, optical coherence tomography, and histology. Double label immunofluorescence localization was performed to quantify Myo/Nog cells, myofibroblasts, and leukocytes. RESULTS: Myo/Nog cells, identified by co-labeling with antibodies to brain-specific angiogenesis inhibitor 1 (BAI1) and Noggin, increased throughout the eye with induction of PVR and disease progression. They were present on the inner surface of the retina in grades 1/2 PVR and were the largest subpopulation of cells in grades 3 to 6 ERMs. All α-SMA-positive (+) cells and all but one striated myosin+ cell expressed BAI1 in grades 1 to 6 PVR. Folds and areas of retinal detachment were overlain by Myo/Nog cells containing muscle proteins. Low numbers of CD18, CD68, and CD45+ leukocytes were detected throughout the eye. Small subpopulations of BAI1+ cells expressed leukocyte markers. ARPE-19 cells were found in the vitreous but were rare in ERMs. Pigmented cells lacking Myo/Nog and muscle cell markers were present in ERMs and abundant within the retina by grade 5/6. CONCLUSIONS: Myo/Nog cells differentiate into myofibroblasts that appear to contract and produce retinal folds and detachment. Targeting BAI1 for Myo/Nog cell depletion may be a pharmacological approach to preventing and treating PVR

TNF-α and NF-κB Signaling Play a Critical Role in Cigarette Smoke-induced Epithelial-mesenchymal Transition of Retinal Pigment Epithelial Cells in Proliferative Vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is characterized by the growth and contraction of cellular membranes within the vitreous cavity and on both surfaces of the retina, resulting in recurrent retinal detachments and poor visual outcomes. Proinflammatory cytokines like tumor necrosis factor alpha (TNFα) have been associated with PVR and the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells. Cigarette smoke is the only known modifiable risk factor for PVR, but the mechanisms are unclear. The purpose of this study was to examine the impact of cigarette smoke on the proinflammatory TNFα/NF-κB/Snail pathway in RPE cells to better understand the mechanisms through which cigarette smoke increases the risk of PVR. Human ARPE-19 cells were exposed to cigarette smoke extract (CSE), for 4 to 24-hours and TNFα, Snail, IL-6, IL-8, and α-SMA levels were analyzed by qPCR and/or Western blot. The severity of PVR formation was assessed in a murine model of PVR after intravitreal injection of ARPE-19 cells pre-treated with CSE or not. Fundus imaging, OCT imaging, and histologic analysis 4 weeks after injection were used to examine PVR severity. ARPE-19 cells exposed to CSE expressed higher levels of TNFα, SNAIL, IL6 and IL8 mRNA as well as SNAIL, Vimentin and α-SMA protein. Inhibition of TNFα and NF-κB pathways blocked the effect of CSE. In vivo, intravitreal injection of ARPE-19 cells treated with CSE resulted in more severe PVR compared to mice injected with untreated RPE cells. These studies suggest that the TNFα pathway is involved in the mechanism whereby cigarette smoke increases PVR. Further investigation into the role of TNFα/NF-κB/Snail in driving PVR and pharmacological targeting of these pathways in disease are warranted

SARS-CoV-2 lineage B.1.1.7 is associated with greater disease severity among hospitalised women but not men: multicentre cohort study.

BACKGROUND: SARS-CoV-2 lineage B.1.1.7 has been associated with an increased rate of transmission and disease severity among subjects testing positive in the community. Its impact on hospitalised patients is less well documented. METHODS: We collected viral sequences and clinical data of patients admitted with SARS-CoV-2 and hospital-onset COVID-19 infections (HOCIs), sampled 16 November 2020 to 10 January 2021, from eight hospitals participating in the COG-UK-HOCI study. Associations between the variant and the outcomes of all-cause mortality and intensive therapy unit (ITU) admission were evaluated using mixed effects Cox models adjusted by age, sex, comorbidities, care home residence, pregnancy and ethnicity. FINDINGS: Sequences were obtained from 2341 inpatients (HOCI cases=786) and analysis of clinical outcomes was carried out in 2147 inpatients with all data available. The HR for mortality of B.1.1.7 compared with other lineages was 1.01 (95% CI 0.79 to 1.28, p=0.94) and for ITU admission was 1.01 (95% CI 0.75 to 1.37, p=0.96). Analysis of sex-specific effects of B.1.1.7 identified increased risk of mortality (HR 1.30, 95% CI 0.95 to 1.78, p=0.096) and ITU admission (HR 1.82, 95% CI 1.15 to 2.90, p=0.011) in females infected with the variant but not males (mortality HR 0.82, 95% CI 0.61 to 1.10, p=0.177; ITU HR 0.74, 95% CI 0.52 to 1.04, p=0.086). INTERPRETATION: In common with smaller studies of patients hospitalised with SARS-CoV-2, we did not find an overall increase in mortality or ITU admission associated with B.1.1.7 compared with other lineages. However, women with B.1.1.7 may be at an increased risk of admission to intensive care and at modestly increased risk of mortality.This report was produced by members of the COG-UK-HOCI Variant substudy consortium. COG-UK-HOCI is part of COG-UK. COG-UK is supported by funding from the Medical Research Council (MRC) part of UK Research & Innovation (UKRI), the National Institute of Health Research (NIHR) and Genome Research Limited, operating as the Wellcome Sanger Institute

Investigating the role of Myo/Nog cells in proliferative vitreoretinopathy

Introduction: Myo/Nog cells are found in many adult tissues including the retina. They express the MyoD transcription factor, bone morphogenetic protein inhibitor Noggin and brain-specific angiogenesis inhibitor 1 (BAI1). It has been demonstrated that Myo/Nog are activated in response to stress and injury, migrate to wounds and differentiate into myofibroblasts that synthesize contractile proteins. After cataract surgery, Myo/Nog cells populate and deform the posterior lens capsule in a vision impairing disease called posterior capsule opacification (PCO) or secondary cataract. Myo/Nog cells also are present in membranes that form on the surface of the human retina in a condition called proliferative vitreoretinopathy (PVR) which occurs after retinal trauma or repair of a retinal detachment. Contractions of epiretinal membranes may lead to re-detachment of the retina and blindness. Objective: In this study we examined the behavior of Myo/Nog cells in a mouse model of PVR and their contributions to the progression of PVR and retinal detachment. Methods: PVR was induced in the mouse by injecting human retinal pigment epithelial cells into the vitreous. PVR was graded as 1-6 by fundus imaging, optical coherence tomography and histology. Immunofluorescence was used to view the presence of Myo/Nog cells using a confocal and epifluorescence microscope. Antibodies to BAI1, Noggin, ɑ-smooth muscle actin (ɑ-SMA), and striated myosin heavy chain were used to identify Myo/Nog cells and examine their expression of markers of muscle proteins using a double label procedure. Results: Retinas with greater PVR progression have numerous Myo/Nog cells marked by BAI1 and Noggin expression. Higher grade epiretinal membranes correlate to higher numbers of Myo/Nog cells which also express ɑ-SMA myosin. The presence of Myo/Nog cells in the PVR membrane was associated with retinal folding and retinal detachment. Conclusion: Injection of human RPE cells induces activation and expansion of the population of Myo/Nog cells. Their expression in the PVR membrane of myosin and smooth muscle actin in epiretinal membranes and their association with folds strongly suggest that their contractions lead to PVR progression and retinal detachment. Targeted elimination of Myo/Nog cells could potentially prevent re-detachment of the retina and preserve visual acuity

Salinomycin inhibits proliferative vitreoretinopathy formation in a mouse model.

Proliferative vitreoretinopathy (PVR) is a progressive disease that develops in a subset of patients who undergo surgery for retinal detachment repair, and results in significant vision loss. PVR is characterized by the migration of retinal pigment epithelial (RPE) cells into the vitreous cavity, where they undergo epithelial-to-mesenchymal transition and form contractile membranes within the vitreous and along the retina, resulting in recurrent retinal detachments. Currently, surgical intervention is the only treatment for PVR and there are no pharmacological agents that effectively inhibit or prevent PVR formation. Here, we show that a single intravitreal injection of the polyether ionophore salinomycin (SNC) effectively inhibits the formation of PVR in a mouse model with no evidence of retinal toxicity. After 4 weeks, fundus photography and optical coherence tomography (OCT) demonstrated development of mean PVR grade of 3.5 (SD: 1.3) in mouse eyes injected with RPE cells/DMSO (vehicle), compared to mean PVR grade of 1.6 (SD: 1.3) in eyes injected with RPE cells/SNC (p = 0.001). Additionally, immunohistochemistry analysis showed RPE cells/SNC treatment reduced both fibrotic (αSMA, FN1, Vim) and inflammatory (GFAP, CD3, CD20) markers compared to control RPE cells/DMSO treatment. Finally, qPCR analysis confirmed that Tgfβ, Tnfα, Mcp1 (inflammatory/cytokine markers), and Fn1, Col1a1 and Acta2 (fibrotic markers) were significantly attenuated in the RPE cells/SNC group compared to RPE/DMSO control. These results suggest that SNC is a potential pharmacologic agent for the prevention of PVR in humans and warrants further investigation

Examining the behavior of Myo/Nog cells in epiretinal membrane formation in a mouse model of Proliferative Vitreoretinopathy

INTRODUCTION: Proliferative Vitreoretinopathy (PVR) is a condition of the eye in which membranes form on the epiretinal or subretinal surface of the retina. Sometimes, fibrotic tissue may also form in the retina. These membranes contain myofibroblasts that produce a tractional force on the retina and may lead to detachment and blindness. We have previously shown that human epiretinal membranes (ERMs) contain a large population of Myo/Nog cells that had differentiated into myofibroblast. In the normal eye, small, subpopulations of Myo/Nog cells reside in the retina, lens, ciliary body and cornea. They increase in number, synthesize muscle proteins and differentiate into contractile myofibroblasts in the lens in response to cataract surgery. PURPOSE: The aim of this study is to examine the behavior of Myo/Nog cells in ERM formation in a mouse model of PVR. METHODS: Intravitreal injections of gas followed seven days later by injections of the human cell line ARPE-19 were performed to induce PVR. The eyes were monitored and graded for ERM formation one to four weeks after injection using fundus imaging and optical coherence tomography. The eyes were then collected and prepared for histology. Immunofluorescence was used to quantify Myo/Nog cells, contractile proteins, myofibroblasts and inflammatory cells. RESULTS: Myo/Nog cells increased in number throughout the eye as PVR progressed. At the highest PVR grades of 3-6, Myo/Nog cells made up the majority of the cells in the ERMs. Besides expressing Noggin and brain angiogenesis inhibitor 1 (BAI1) which specifically identify them, Myo/Nog cells also expressed the muscle proteins alpha smooth muscle actin (α-SMA) and striated muscle myosin II, markers of myofibroblast differentiation. Retinal folds and areas of detachment were overlain by large populations of Myo/Nog cells in the ERMS and the retina itself. Low numbers of cells were labeled with antibodies to the leukocyte markers, CD18, CD45 and CD68 within ERMS and the retina. Small percentages of the Myo/Nog cells expressed these leukocyte markers. Although ARPE-19 cells were found early on in the vitreous, they were rarely seen in the ERMs and most were dying as evidenced by staining with TUNEL reagents. Small populations of pigmented cells were found in some of the ERMS, and some appeared to be migrating from the retinal pigmented epithelium into the retina at higher grades of PVR. CONCLUSIONS: Myo/Nog cells increase in number and are the source of myofibroblasts in ERMS and the retina in this mouse model of PVR. Injection of human cells did not induce inflammation, and therefore, activation of Myo/Nog cells is likely mediated by the death of ARPE-19 cells, as found in other tissues. Their differentiation into myofibroblasts appears to produce the tractional force that leads to folding in the retina and eventual detachment. PVR is currently treated by surgical removal of ERMs and retinal repair. Using the BAI1 antibody to target and deplete Myo/Nog cells may prevent ERM formation and retinal detachment

A Mouse Model of Proliferative Vitreoretinopathy Induced by Intravitreal Injection of Gas and RPE Cells

Purpose: Develop a reproducible proliferative vitreoretinopathy (PVR) mouse model that mimics human PVR pathology. Methods: Mice received intravitreal injections of SF6 gas, followed by retinal pigment epithelial cells 1 week later. PVR progression was monitored using fundus photography and optical coherence tomography imaging, and histologic analysis of the retina as an endpoint. We developed a PVR grading scheme tailored for this model. Results: We report that mice that received gas before retinal pigment epithelial injection developed more severe PVR. In the 1 x 10(4) retinal pigment epithelial cell group, after 1 week, 0 of 11 mice in the no gas group developed grade 4 or greater PVR compared with 5 of 13 mice in the SF6 gas group (P = 0.02); after 4 weeks, 3 of 11 mice in the no gas group developed grade 5 or greater PVR compared with 11 of 14 mice in the SF6 gas group (P = 0.01). We were able to visualize contractile membranes both on the retinal surface as well as within the vitreous of PVR eyes, and demonstrated through immunohistochemical staining that these membranes expressed fibrotic markers alpha smooth muscle actin, vimentin, and fibronectin, as well as other markers known to be found in human PVR membranes. Conclusions: This mouse PVR model is reproducible and mimics aspects of PVR pathology reported in the rabbit PVR model and human PVR. The major advantage is the ability to study PVR development in different genetic backgrounds to further elucidate aspects of PVR pathogenesis. Additionally, large-scale experiments for testing pharmacologic agents to treat and prevent PVR progression is more feasible compared with other animal models. Translational Relevance: This model will provide a platform for screening potential drug therapies to treat and prevent PVR, as well as elucidate different molecular pathways involved in PVR pathogenesis