Control vs CRB2 cKO retinas at early postnatal time points
Ontology highlight
ABSTRACT: We used microarray gene profiling to study the transcriptome of retinas lacking CRB2 during late retinal development. Unexpectedly, the retinas of newborn mice lacking CRB2 showed no changes in the transcriptome during retinal development. Comparison between Control and CRB2 null retinas at different time points (P0, P3, P6 and P10)
Project description:TaqMan low density array (TLDA) was carried out to screen of the profiles of circulating miRNAs in pooled plasma samples from healthy controls and pre-operative osteosarcoma patients. The expression changes of circulating miRNAs in osteosarcoma patients were identified. To select candidate plasma miRNAs for osteosarcoma detection and monitoring, we employed TLDA technique to screen expression levels of 739 miRNAs in pooled plasma samples from healthy controls and pre-operative osteosarcoma patients (each pooled from 10 individuals).
Project description:Recently we reported that the rat inner retina undergoes significant functional changes during maturation. Aiming to gain knowledge on additional aspects of retinal development and maturation, we used the microarray system to monitor gene expression patterns in the rat retina at ages 5, 11, and 20 weeks. The analysis revealed the expression of many well-documented retinal genes as well as a high number of nonâ??annotated genes. Quantitative realtime PCR analysis verified the microarray results in the majority of studied genes. A statistical analysis of the 4 microarray slides revealed 603 differentially expressed genes which were grouped into 6 expression clusters. A bioinformatic analysis of these clusters revealed sets of genes encoding proteins with functions that are likely to be relevant to inner retinal function (e.g. potassium, sodium, calcium, and chloride channels, synaptic vesicle transport, and axonogenesis). In addition, we performed a histological analysis of the maturing retina and studied different aspects of retinal structure. The analysis revealed a significant reduction of outer nuclear layer thickness between 11 and 19 weeks of age and a significant reduction of retinal ganglion cell number at 11 and 19 weeks comparing to 5 weeks. We identified in this study genes with differential expression pattern during retinal maturation. Some of the genes encode proteins that may be involved in the functional maturation of inner retinal cells. These data, taken together with our histological and electrophysiological data, contribute to our understanding of the developmental processes occurring in the retina of this widely-used animal model. Experiment Overall Design: Rat retinal samples at ages 5, 11, and 20 weeks were studied using 4 microarray slides in a dye-swap design: 5-11, 11-5, 5-20, and 20-5.
Project description:The histone methyltransferase complex PRC2 controls key steps in developmental transitions and cell fate choices. However, its roles in vertebrate eye development remain unknown. Here we report that in Xenopus PRC2 regulates the progression of retinal progenitors from proliferation to differentiation. We show that the PRC2 core components are enriched in retinal progenitors and downregulated with differentiation. Knockdown of the PRC2 core component Ezh2 leads to reduced retinal progenitor proliferation in part due to upregulation of the cdk inhibitor p15Ink4b. In addition, while PRC2 knockdown does not alter eye patterning, retinal progenitor gene expression or expression of the neural competence factor Sox2, it does cause suppression of proneural bHLH gene expression, indicating that PRC2 is critical for the initiation of neural differentiation in the retina. Consistent with this, knocking down or blocking PRC2 function constrains the generation of most retinal neural cell types and promotes a Mueller glial cell fate decision. We also show that Wnt/?-catenin signaling acting through the receptor Frizzled 5, but independent of Sox2, regulates expression of key PRC2 subunits in the developing retina. This is consistent with a role for this pathway in coordinating proliferation and the transition to neurogenesis in the Xenopus retina. Our data establishes PRC2 as a regulator of proliferation and differentiation during eye development. Xenopus embryos were injected at the 8-cell stage with 5ng Ezh2 ATG MO or 5ng control MO (scrambled sequence of Ezh2 ATG MO) together with 400 pg mRNA for GFP as a lineage tracer. At stage 27, GFP-positive eyes were isolated by microdissection. Pools of 20-25 eyes were used to prepare total RNA for each sample on the microarray. 4 control and 4 Ezh2 ATG MO samples were hybridized to Agilent 1-color microarrays.
Project description:Purpose. XOPS-mCFP transgenic zebrafish experience a continual cycle of rod photoreceptor development and degeneration throughout life, making them a useful model to investigate the molecular determinants of rod photoreceptor regeneration. The purpose of this study was to compare the gene expression profiles of wild type and XOPS-mCFP retinas in order to identify genes that may contribute to the regeneration of the rods. Methods. Wild type and XOPS-mCFP retinal mRNA was subjected to microarray analysis using the Agilent platform. The Ingenuity Pathway Analysis program was used to identify biologically relevant processes that were significantly represented in the dataset. Expression changes were verified by RT-PCR. Selected genes were further examined during retinal development and in adult retinas by in situ hybridization, immunohistochemistry, and using a transgenic fluorescent reporter line. Results. Over 600 genes displayed significant expression changes in XOPS-mCFP retinas compared to wild type controls. Many of the downregulated genes were associated with phototransduction, whereas upregulated genes were associated with several biological functions, including cell cycle, DNA replication and repair, cell development and cell death. RT-PCR analysis of a subset of these genes confirmed the microarray results. Three transcription factors (sox11b, insm1a, and c-myb) displaying increased expression in XOPS-mCFP retinas were also expressed throughout retinal development and in the persistently neurogenic ciliary marginal zone. Conclusions. This study identified numerous gene expression changes in response to rod degeneration in zebrafish, and further suggests a role for the transcriptional regulators sox11b, insm1a, and c-myb in both retinal development and rod photoreceptor regeneration. Two-condition experiment: wild-type vs. XOPS-mCFP retinas. Four biological replicates for each condition. RNA was prepared from one retina for each sample. Each hybridization was accompanied by a dye-swap control, for a total of eight array hybridizations.
Project description:Objective: Pathological retinal angiogenesis is vision threatening. In mouse oxygen-induced retinopathy (OIR) we sought to define mitochondrial respiration changes longitudinally during hyperoxia-induced vessel loss and with hypoxia-induced neovascularization (NV), and test interventions to address those changes to prevent NV. Methods: OIR was induced in C57BL/6J mice and retinal vasculature was examined at maximum neovessel formation. We assessed total proteome change and the ratio of mitochondrial/nuclear DNA copy numbers (mtDNA/nDNA) of OIR vs. control retinas, and mitochondrial oxygen consumption rates (OCR) in ex vivo OIR vs. control retinas (BaroFuse). Pyruvate vs. vehicle control was supplemented in OIR mice either prior to or during neovessel formation. Results: In OIR vs. control retinas proteomics identified decreased retinal mitochondrial respiration and synaptic formation pathway proteins at peak neovascularization. mtDNA/nDNA was decreased during hypoxia-induced neovessel growth (as was OCR) suggesting impaired mitochondrial respiration. Pyruvate administration during but not prior to neovessel formation (in line with compromised mitochondrial activity) suppressed NV in vivo. Conclusions: Mitochondrial energetics are suppressed during retinal NV in OIR. Appropriately timed supplementation of energy substrates (pyruvate) may be a novel approach in neovascular retinal diseases.
Project description:We report RNAseq analysis of the transcriptome of retinas from mature rod-specific Dicer1 cKO mice and control littermates lacking Cre expression in order to better understand changes in gene regulation that could lead to retinal degeneration in cKO mice. Examine retinal transcriptome of 3 biological replicates for each genotype from 4-week-old animals with tissue collected between 8:00 - 10:00AM
Project description:Photoreceptor disorders are collectively known as retinal degeneration (RD), and include retinitis pigmentosa (RP), cone-rod dystrophy and age related macular degeneration (AMD). These disorders are largely genetic in origin; individual mutations in any one of >200 genes cause RD, making mutation specific therapies prohibitively expensive. A better treatment plan, particularly for late stage disease, may involve stem cell transplants into the photoreceptor or ganglion cell layers of the retina. Stem cells from young mouse retinas can be transplanted, and can form photoreceptors in adult retinas. These cells can be grown in tissue culture, but can no longer form photoreceptors. We have used microarrays to investigate differences in gene expression between cultured retinal progenitor cells (RPCs) that have lost photoreceptor potential, postnatal day 1 (pn1) retinas and the postnatal day 5 (pn5) retinas that contain transplantable photoreceptors. We have also compared FACS sorted Rho-eGFP expressing rod photoreceptors from pn5 retinas with Rho-eGFP negative cells from the same retinas. We have identified over 300 genes upregulated in rod photoreceptor development in multiple comparisons, 37 of which have been previously identified as causative of retinal disease when mutated. It is anticipated that this research should bring us closer to growing photoreceptors in culture and therefore better treatments for RD. This dataset is also a resource for those seeking to identify novel retinopathy genes in RD patients. We extracted whole retinas from postnatal day 1 (Pn1) and postnatal day 5 (Pn5) mice, and compared them with cultured RPCs derived from pn5 retinas, using Affymetrix mouse 430A_2 arrays. We also extracted cells from Rho-eGFP Pn5 retinas and FACS sorted them. GFP+ve cells represent immature rod photoreceptors, as they express the Rho-eGFP fusion protein, which is only expressed in rods. GFP-ve cells represent all other retinal neurons. These samples were amplified and compared using Affymetrix mouse 430A_2arrays, by Source Biosciences GMBH, Berlin, Germany. Results from immature rods were then compared with those from other retinal neurons, while results from whole Pn5 retinas were compared with Pn1 retinas (which don't yet express rod specific genes), and RPCs, which are glial precursors. RPCs were also compared with Pn1 retinas. Genes which showed changed expression profiles in at least 3/4 of comparisons were prioritised for further investigation.
Project description:Background: Biological conversion of the surplus of renewable electricity to CH4 could support energy storage and strengthen the power grid. Biological methanation (BM) is closely linked to the activity of biogas-producing bacterial community and methanogenic Archaea in particular. During reactor operations, the microbiome is often subject to various changes whereby the microorganisms are challenged to adapt to the new conditions. In this study, a hydrogenotrophic-adapted microbial community in a laboratory-scale BM fermenter was monitored for its pH, gas production, conversion yields and composition. To investigate the robustness of BM regarding power oscillations, the biogas microbiome was exposed to five H2 starvations patterns for several hours.
Project description:We used microarray gene profiling to study the transcriptome of retinas lacking CRB2 during late retinal development. Unexpectedly, the retinas of newborn mice lacking CRB2 showed no changes in the transcriptome during retinal development.