92 research outputs found
Single-cell RNA sequencing reveals transcriptional changes of human choroidal and retinal pigment epithelium cells during fetal development, in healthy adult and intermediate age-related macular degeneration
No full textAge-related macular degeneration (AMD) is the most prevalent cause of blindness in the developed world. Vision loss in the advanced
stages of the disease is caused by atrophy of retinal photoreceptors, overlying retinal pigment epithelium (RPE) and choroidal endothelial
cells. The molecular events that underline the development of these cell types from in utero to adult as well as the progression to
intermediate and advanced stages AMD are not yet fully understood. We performed single-cell RNA-sequencing (RNA-Seq) of human
fetal and adult RPE–choroidal tissues, profiling in detail all the cell types and elucidating cell type-specific proliferation, differentiation
and immunomodulation events that occur up to midgestation. Our data demonstrate that progression from the fetal to adult state is
characterized by an increase in expression of genes involved in the oxidative stress response and detoxification from heavy metals,
suggesting a better defence against oxidative stress in the adult RPE–choroid tissue. Single-cell comparative transcriptional analysis
between a patient with intermediate AMD and an unaffected subject revealed a reduction in the number of RPE cells and melanocytes
in the macular region of the AMD patient. Together these findings may suggest a macular loss of RPE cells and melanocytes in the
AMD patients, but given the complex processing of tissues required for single-cell RNA-Seq that is prone to technical artefacts, these
findings need to be validated by additional techniques in a larger number of AMD patients and controls
Human TBX22 expression and protein-DNA interactions
No full textCleft palate is one of the most common birth abnormalities. Figures published in 2006 by the American Centres for Disease Control and Prevention, report the incidence of those born in the United States with a cleft palate without the presence of a cleft lip (CPI) to be 6.39 for every 10000 in the three years between 1999 to 2001 and for cleft lip in association with a cleft palate (CLP) to be even greater - 10.48 per 10000 live births. In 2001, Braybrook and colleagues reported that mutations in the TBX22 gene cause X-linked cleft palate (CPX), a disease characterised by a cleft of the secondary palate and is often seen in association with ankyloglossia (tongue-tie) (Braybrook et al. 2001). A cleft of the secondary palate arises as a consequence of disturbance to correct development during palatogenesis: an anomaly in palatal shelf growth; delayed or failed shelf elevation; defective shelf fusion or a failure of medial edge epithelium cell death. This thesis reveals that the expression of TBX22 during these key developmental events in human embryos is consistent with the phenotype seen in CPX. To enable an investigation for TBX22 target genes, a DNA binding sequence is determined for the TBX22 protein. This sequence is used to generate a generic TBX22 DNA binding site, the presence of which is screened for in promoter regions, defined as 2kb upstream of transcription start sites. 132 genes were selected as candidate TBX22 targets on the basis that they underlie human disorders that include a cleft palate. The screen shows that 28 of these genes have at least one perfect or near perfect match to the generic TBX22 DNA binding site. Of these, only two both contained a perfect TBX22 generic DNA binding site and mouse mutants also had cleft palates: SUMO1 and MSX1. Interaction between SUMO1 and TBX22 has already been shown (Andreou et al. 2007). This study investigated MSX1 as a downstream target of TBX22 using a luciferase reporter gene construct in vitro. The results showed that in the presence of TBX22, the luciferase signal was reduced and support MSX1 being a downstream target gene of TBX22. These findings further the understanding of the molecular networks regulating craniofacial development. Unravelling these complex interactions is crucial to identifying the mechanisms of oro-facial clefting, important steps towards improved methods of counselling, treatment and prevention of these common birth disorders.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Embryonic cerebrospinal fluid nanovesicles carry evolutionarily conserved molecules and promote neural stem cell amplification.
No full textDuring brain development, neural stem cells (NSCs) receive on-or-off signals important for regulating their amplification and reaching adequate neuron density. However, how a coordinated regulation of intracellular pathways and genetic programs is achieved has remained elusive. Here, we found that the embryonic (e) CSF contains 10¹² nanoparticles/ml (77 nm diameter), some of which were identified as exosome nanovesicles that contain evolutionarily conserved molecules important for coordinating intracellular pathways. eCSF nanovesicles collected from rodent and human embryos encapsulate protein and microRNA components of the insulin-like growth factor (IGF) signaling pathway. Supplementation of eCSF nanovesicles to a mixed culture containing eNSCs activated the IGF-mammalian target of rapamycin complex 1 (mTORC1) pathway in eNSCs and expanded the pool of proliferative eNSCs. These data show that the eCSF serves as a medium for the distribution of nanovesicles, including exosomes, and the coordinated transfer of evolutionary conserved molecules that regulate eNSC amplification during corticogenesis
A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells
No full textPurpose
Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood.
Methods
Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs).
Results
scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks.
Conclusions
Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders
Mutations in FRMD7, a newly identified member of the FERM family, cause X-linked idiopathic congenital nystagmus.
No full textIdiopathic congenital nystagmus is characterized by involuntary, periodic, predominantly horizontal oscillations of both eyes. We identified 22 mutations in FRMD7 in 26 families with X-linked idiopathic congenital nystagmus. Screening of 42 singleton cases of idiopathic congenital nystagmus (28 male, 14 females) yielded three mutations (7%). We found restricted expression of FRMD7 in human embryonic brain and developing neural retina, suggesting a specific role in the control of eye movement and gaze stability
Disruption of embryonic blood-CSF barrier in chick embryos reveals the actual importance of this barrier to control E-CSF composition and homeostasis in early brain development
No full textIn vertebrates, early brain development takes place at the expanded anterior end of the neural tube. After closure of the anterior neuropore, the brain wall forms a physiologically sealed cavity that encloses embryonic cerebrospinal fluid (E-CSF), a complex and protein-rich fluid that is initially composed of trapped amniotic fluid. E-CSF has several crucial roles in brain anlagen development. Recently, we reported the presence of transient blood-CSF barrier located in the brain stem lateral to the ventral midline, at the mesencephalon and prosencephalon level, in chick and rat embryos by transporting proteins, water, ions and glucose in a selective manner via transcellular routes. To test the actual relevance of the control of E-CSF composition and homeostasis on early brain development by this embryonic blood-CSF barrier, we block the activity of this barrier by treating the embryos with 6-aminonicotinamide gliotoxin (6-AN). We demonstrate that 6-AN treatment in chick embryos blocks protein transport across the embryonic blood-CSF barrier, and that the disruption of the barrier properties is due to the cease transcellular caveolae transport, as detected by CAV-1 expression cease. We also show that the lack of protein transport across the embryonic blood-CSF barrier influences neuroepithelial cell survival, proliferation and neurogenesis, as monitored by neurepithelial progenitor cells survival, proliferation and neurogenesis. The blockage of embryonic blood-CSF transport also disrupts water influx to the E-CSF, as revealed by an abnormal increase in brain anlagen volume. These experiments contribute to delineate the actual extent of this blood-CSF embryonic barrier controlling E-CSF composition and homeostasis and the actual important of this control for early brain development, as well as to elucidate the mechanism by which proteins and water are transported thought transcellular routes across the neuroectoderm, reinforcing the crucial role of E-CSF for brain development
NIPBL, encoding a homolog of fungal Scc2-type sister chromatid cohesion proteins and fly Nipped-B, is mutated in Cornelia de Lange syndrome
No full textImplementation of a 9-point stencil in SOLPS-ITER and implications for Alcator C-Mod divertor plasma simulations
No full textThe SOLPS-ITER code suite is used worldwide for plasma edge modeling, the interpretation of experiments, as well as for the design of the ITER divertor. The numerical scheme of the plasma solver of the code, B2.5, is based on the assumption of perfectly field-aligned grids, while in practice grids are often strongly distorted to match divertor target shapes. Neglecting these grid distortion leads to qualitatively and quantitatively incorrect results for fluid neutral simulations, and may affect results in cold (detached) divertors even when using kinetic neutral simulations. In this contribution, we present the first results of a newly implemented 9-point stencil in B2.5 to properly handle misaligned grids. The new scheme is then applied to fluid neutral simulations of a well-diagnosed and previously modeled Alcator C-Mod discharge. Results are compared with the original 5-point scheme neglecting grid distortion effects, as well as with simulations including a full kinetic neutral model. We conclude that the 9-point stencil is essential to correctly model the transport of fluid neutrals on distorted grids, and to capture the effects of divertor closure on the fluid neutral behavior
SOLPS-ITER Modeling of the Alcator C-Mod Divertor Plasma
No full textSOLPS-ITER is a new edge code package that will be developed and maintained at the ITER Organization [X. Bonnin et al., Plasma Fusion Res. 11, 1403102 (2016)], in close collaboration with the wider SOLPS community, and will be used to support the design of the ITER divertor [A.S. Kukushkin et al., Fusion Eng. Des. 86, 2865 (2011)]. In this paper, we report on the first application of the code to the modeling of the Alcator C-Mod divertor. With its high density, high magnetic field, and strong ITER-like target shaping, C-Mod is of particular interest to ITER in terms of plasma and neutral parameters in the divertor. We show that with a fluid neutral model, we can qualitatively reproduce the observed particle fluxes to inner and outer targets under partially detached conditions. However, simulated electron temperatures in the divertor are much too low. A number of physics and numerical reasons are proposed to resolve this issue and serve as a guideline for further development of the code.sponsorship: W. Dekeyser is supported through the Monaco/ITER postdoctoral fellowship program. The views and opinions expressed herein do not necessarily reflect those of the ITER Organization. ITER is the Nuclear Facility INB-174. (Monaco/ITER postdoctoral fellowship program)status: Publishe
The Transcription Factors COUP-TFI and COUP-TFII have Distinct Roles in Arealisation and GABAergic Interneuron Specification in the Early Human Fetal Telencephalon
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