Project description:During vertebrate retinogenesis, the precise balance between retinoblast proliferation and differentiation is spatially and temporally regulated through a number of intrinsic factors and extrinsic signaling pathways. Moreover, there are complex gene regulatory network interactions between these intrinsic factors and extrinsic pathways, which ultimately function to determine when retinoblasts exit the cell cycle and terminally differentiate. We recently uncovered a cell non-autonomous role for the intrinsic HLH factor, Id2a, in regulating retinoblast proliferation and differentiation, with Id2a-deficient retinae containing an abundance of proliferative retinoblasts and an absence of terminally differentiated retinal neurons and glia. Here, we report that Id2a function is necessary and sufficient to limit Notch pathway activity during retinogenesis. Id2a-deficient retinae possess elevated levels of Notch pathway component gene expression, while retinae overexpressing id2a possess reduced expression of Notch pathway component genes. Attenuation of Notch signaling activity by DAPT or by morpholino knockdown of Notch1a is sufficient to rescue both the proliferative and differentiation defects in Id2a-deficient retinae. In addition to regulating Notch pathway activity, through an RNA-Seq and differential gene expression analysis of Id2a-deficient retinae, we identify a number of additional intrinsic and extrinsic regulatory pathway components whose expression is regulated by Id2a. These data highlight the integral role played by Id2a in the gene regulatory network governing the transition from retinoblast proliferation to terminal differentiation during vertebrate retinogenesis. Two biological replicates for both Id2aMM and Id2aMO samples
Project description:To investigate the role of the circadian clock of the photoreceptor cells in regulation of retinal protein rhythms, we have analyzed diurnal protein expression in the photoreceptor-deficient cone-rod homeobox knock out mouse (Crx-/-). Retinal homogenates of 129/sv and Crx-/- mice were analysed by 2D-PAGE. Differentially expressed spots were in-gel digested with trypsin and identified by LC-MS/MS. Data were used to search the Swiss-Prot protein database.
Project description:To determine the role of specific cis-regulatory elements within the Sox17 endoderm-preferential TSS2 promoter, we generated Sox17mut5 mutant animals and surveyed how this mutation altered Sox17 expression. Gallbladders were isolated from P21 Sox17mut5/mut5 and Sox17+/+ (n = 4 of each genotype), RNA isolated, and bulk RNA-Seq performed.
Project description:Platelet-derived growth factor-CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades of studies on the original members of the family, PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report here a novel finding that PDGF-CC is a potent neuroprotective factor that acts by modulating glycogen synthase kinase (GSK)3beta activity. In several different animal models of neuronal injury, such as axotomy-induced neuronal death, neurotoxin-induced neuronal injury, 6-OHDA-induced Parkinson's dopaminergic neuronal death and ischemia-induced stroke, PDGF-CC protein or gene delivery protected different types of neurons from apoptosis in both the retina and brain. On the other hand, loss-of-function assays using PDGF-C null mice, neutralizing antibody or shRNA showed that PDGF-CC deficiency/inhibition exacerbated neuronal death in different neuronal tissues in vivo. Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3beta phosphorylation and expression. Our data demonstrate that PDGF-CC is critically required for neuronal survival, and may potentially be used to treat neurodegenerative diseases. Inhibition of the PDGF-CC/receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions. PDGF-C deficient mice were bred onto C57BL/6 background for more than six generations and littermates were used. After optic nerve crush (ONC), PDGF-CC protein à 1 µl of active recombinant human PDGF-CC protein 0.5 µg/µl (rhPDGF-CC) was injected into mouse vitreous once a week for two weeks. Seven days after the ONC injury and PDGF-CC protein treatment, retinae were harvested and total RNA isolated using the TRIzol reagent (Invitrogen) followed by the RNeasy Mini kit (Qiagen) purification according to the manufacturer's instructions. Microarray assay was performed using the Mouse-6 Expression BeadChips containing approximately 24,000 annotated mouse transcripts (Illumina Inc). Three biological repeats were included in the microarray assay. Two tailed Student's t-test was used for statistical analysis of gene expression data. Functional grouping of the differentially expressed genes was performed using several different tools including the WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt) and the Ingenuity Pathways Analysis (https://analysis.ingenuity.com/pa/login/login.jsp). The supplementary file 'GSE19207_non-normalized.txt' contains raw data for Samples GSM476021-GSM476026.
Project description:Retinitis pigmentosa (RP) is a photoreceptor disease that affects approximately 100,000 people in the United States. There are currently very limited treatment options and the prognosis for most patients is progressive vision loss. Unfortunately, the understanding of the molecular underpinnings of RP initiation and progression is still poorly understood. However, the development of animal models of RP, coupled with high-throughput sequencing, has provided an opportunity to study the underlying cellular and molecular changes of this disease. Using RNA-Seq, we present the first retinal transcriptome analysis of the rd10 murine model of retinal degeneration. RNA-Seq on whole-retina samples from rd10, wild-type and GFP-expressing mouse retina. Three biological replicates of each.
Project description:Background: To better understand the role DNA methylation plays in regulating gene expression in the developng heart and furthermore the role it plays in heart development we performed genome wide DNA methylation profiling of embryonic hearts at embryonic day (E)11.5 and E14.5 using methyl sensitive tiny fragment enrichment coupled with massive parallel sequencing by using the methyl-sensitive restriction enzyme HpyCH4IV, recognition site 'ACGT'. Results: We found that global methylation remains stable at analyzed 'ACGT' sites (1.64 million site) in developing hearts between E11.5 and E14.5. However, differential methylation was identified at individual loci enriched at genes involved in heart development suggesting a role for DNA methylation in the developing heart. Used Methyl Sensitive Tiny Fragment Enrichment/Massive Parallel Sequencing (MSFE/MPS) to assay methylation at 'ACGT' sites throughout the genome and generate a developmental profile of DNA methylation in the embryonic heart and to identify differences between developing mouse hearts at E11.5 and E14.5.
Project description:Cadmium is toxic and intricate pathways linked to metallothioneins (MT) drive the detoxification process. Whist the mechanisms are well understood in mammalian systems, detailed knowledge is still elusive in invertebrates (which notably differ to mammalian systems). The model nematode Caenorhabditis elegans is ideally suited for assessing metallothionein mediates detoxification in invertebrates as is relatively short-lived, can be easily exposed and its genome is fully sequenced and widely annotated. The aim of the experiment was to identify new MT mediated target genes involved in Cd toxicosis. The global transcriptome was compared in wild type and a MT double knockout strain raised in the presence or absence of 30 µM Cd.
Project description:The initial aim of this work was to understand the pathophysiology of Enhanced S-cone Syndrome (ESCS) that leads to retinal degeneration. Although ESCS was identified in humans decades ago and since then the causative genes have been elucidated, our understanding of the accompanying retinal degeneration is still poorly understood. Knockout of the Nrl transcription factor in mice produces a retina overpopulated with S-cone like photoreceptors along with absence of rod photoreceptors and recapitulates many of the phenotypic features seen in human ESCS patients. We wanted to study this murine model through a combinatorial genetic and structural approach to improve understanding of the disease process that leads to photoreceptor degeneration and blindness, potentially guiding future therapies. By using RNA-Sequencing (RNA-Seq) to examine mature wild type and Nrl-/- ocular tissues, we were able to determine global changes in their transcriptomes. The massively parallel RNA-sequencing experiment revealed new insight into the transcriptional mis-regulation in the ESCS murine model and revealed a change in gene expression in putative proteins involved in photoreceptor phagocytosis. Key photoreceptor ligands necessary for phagocyotsis, Tub and Tulp1, were down-regulated in the Nrl-/- retina. Down regulation of key retinoid metabolic genes, coupled with down-regulation of Tub and Tulp1, suggested a potential mechanism involving defective phagocytosis underlies the photoreceptor degeneration seen in ESCS. We report RNA-Seq experiments of whole eye and retinal tissues from wild type and Nrl transcription factor knockout mice on the C57BL/6 background. Examine two different ocular tissues in two mouse models of varying photoreceptor populations
Project description:The chloroplast stromal CLP protease system is essential for growth and development. It consists of a proteolytic CLP core complex that likely dynamically interacts with oligomeric rings of CLPC1, CLPC2 or CLPD AAA+ chaperones. These ATP-dependent chaperones are predicted to bind and unfold CLP protease substrates, frequently aided by adaptors (recognins), and feed them into the proteolytic CLP core for degradation. To identify new substrates and possible also new adaptors for the chloroplast CLP protease system, we generated an in vivo CLPC1 substrate trap with a C-terminal STREPII affinity tag in Arabidopsis thaliana by mutating critical glutamate residues (E374A and E718A) in the two Walker B domains of CLPC1 required for hydrolysis of ATP (CLPC1-TRAP). Based on homology to non-plant CLPB/C chaperones, it is predicted that interacting substrates are unable to be released, i.e. they are trapped. When expressed in wild-type, this CLPC1-TRAP induced a dominant visible phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered. Affinity purification of the CLPC1-TRAP resulted in a dozen proteins highly enriched compared to affinity purified CLPC1 with a C-terminal STREPII affinity tag (CLPC1-WT). These enriched proteins likely represent CLP protease substrates and/or new adaptors. Several of these trapped proteins over-accumulated in clp mutants and/or were found as interactions for the adaptor CLPS1, supporting their functional relationship to CLP function. Importantly, affinity purification of this CLPC1-TRAP also showed high enrichment of all CLPP, CLPR and CLPT subunits, indicating stabilization of the CLPC to CLP core interaction and providing direct support for their physical and functional interaction.