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: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: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:To identify more targets in soybean, particularly specific targets of Cd-stress-responsive miRNAs, high-throughput degradome sequencing was used. In total, we obtained 8913111 raw reads from the library which was constructed from a mixture of four samples (HX3-CK, HX3-Cd-treatment, ZH24-CK and ZH24-Cd-treatment). After removing the reads without the CAGAG adaptor, 5430126 unique raw-reads were obtained. The unique sequences were aligned to the G. max genome database, and 6516276 reads were mapped to the genome. The mapped reads from the libraries represented 51481 annotated G. max genes. Identification of miRNA targerts in soybean roots
Project description:We investigated the transcriptome of the eye imaginal discs and retinae in wild type (GMR/+) and DeIF6 overexpressing (GMR>DeIF6) flies at the third instar larva stage and pupa 40 hours after puparium formation.
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: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:Obesity is well recognized as a risk factor for coronary heart disease and mortality. The relationship between abdominal obesity and ischemic stroke remains less clear. Previous publication showed the obesity is an independent, potent risk factor for ischemic stroke in all race-ethnic groups. It is a stronger risk factor than BMI and has a greater effect among younger persons. The goal of this experiment was to compare genome wide enrichment of H3K9ac histone mark profile of white blood cells of healthy controls, patients with obesity and/or stroke in order to understand the histone modifications differences behind the different phenotypes. There were 3 subjects in each group.