Transcription profiling of mouse to identify strain-specific variation and the onset of gap junction mutation-induced cataractsc
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ABSTRACT: Disruption of the mouse gene encoding the gap junction subunit alpha3 connexin 46 (Cx46) results in the formation of lens cataracts. The timing of the onset of this lens opacity is affected by the genetic background, i.e. the mouse strain. To elucidate the mechanism by which cataracts form in the 129Sv/Jae strain earlier than in the C57BL/6J strain, global gene expression was quantitated in the lenses of these strains. Lens cDNAs were analyzed by hybridization to DNA microarrays and with real time-PCR. Theories are proposed based on the observed higher level of expression of the stress-response genes in the C57BL/6J strain and variations in the expression levels of genes involved in protein synthesis, metabolism, catabolism and cell proliferation. How these variations in gene expression might affect the response of lens fiber cells to the increased calcium, caused by lack of alpha3Cx46, is considered. The possibility that the proteins coded by the strain-variable genes might influence the cataract-associated proteolysis of gamma-crystallin is also addressed. Experiment Overall Design: To determine differences in transcript expression between the lenses of two mouse wild type strains (129 SvJae and C57BL/6J), as well as between alpha3Cx46 KO and wild-type mice. The former comparison may lead to identification of potential candidate(s) genes that prevent (or promote) cataract formation, whereas the latter comparison may provide insights into the mechanism by which cataract formation occurs in the alpha3Cx46 KO mice. Total 8 samples were used ( two separate samples for each of the following 4 types of mice: 129SvJae wild type, 129SvJae alpha3Cx46 KO, C57BL/6J wild type and C57BL/6J alpha3Cx46 KO)
Project description:Disruption of the mouse gene encoding the gap junction subunit alpha3 connexin 46 (Cx46) results in the formation of lens cataracts. The timing of the onset of this lens opacity is affected by the genetic background, i.e. the mouse strain. To elucidate the mechanism by which cataracts form in the 129Sv/Jae strain earlier than in the C57BL/6J strain, global gene expression was quantitated in the lenses of these strains. Lens cDNAs were analyzed by hybridization to DNA microarrays and with real time-PCR. Theories are proposed based on the observed higher level of expression of the stress-response genes in the C57BL/6J strain and variations in the expression levels of genes involved in protein synthesis, metabolism, catabolism and cell proliferation. How these variations in gene expression might affect the response of lens fiber cells to the increased calcium, caused by lack of alpha3Cx46, is considered. The possibility that the proteins coded by the strain-variable genes might influence the cataract-associated proteolysis of gamma-crystallin is also addressed. Keywords: Lens, cataract, microarray, connexin 46, gene knock out
Project description:We report the application of RNA-sequencing technology for the high-throughput profiling of mammalian lens gene expression at embryonic day 15.5. The lens has a particularly biased transcriptome, with the top 50 genes encoding approximately 90% of the protein content. Using RNA-Seq, we have shown that there are over 7,700 genes being expressed in the lens at embryonic day 15.5. As expected, the crystallins and many structural genes were amoung the most highly expressed; however, numerous genes expressed at lower transcript abundance were also identified. This study provides a framework for the application of RNA-Seq technology towards characterization of the mammalian lens transcriptome during development. Using high-throughput RNA-sequencing, gene expression in the mammalian lens was compared between an inbred C56Bl/6<har> strain and a mix background strain at E15.5. This analysis identifies almost 2,000 genes being differentially expressed between the inbred and mixed background lenses, ranging from 6.5 fold upregulated to 5.2 fold downregulated in the mixed background compared to the inbred strain. This list does not include unknown/predicted genes or pseudogenes which are known to change between strains. Further, it appears that approximately 98% of these genes are altered at levels less than 2.5 fold. This study therefore provides a fold change threshold cutoff (2.5 fold) to use in the analysis of differentially expressed lens genes at E15.5 using RNA-Seq technology as it takes into account genetic variation due to background strain differences. SIP1 encodes a DNA-binding transcription factor that regulates multiple developmental processes as highlighted by the pleiotropic defects observed in Mowat-Wilson Syndrome, which results from mutations in this gene. Further, in adults, dysregulated SIP1 expression has been implicated in both cancer and fibrotic diseases where it functionally links TGFb signaling to the loss of epithelial preferred gene expression. In the ocular lens, an epithelial tissue important for vision, Sip1 is co-expressed with epithelial markers such as E-cadherin, and is required for the complete separation of the lens vesicle from the head ectoderm during early ocular morphogenesis. However, the function of Sip1 after early lens morphogenesis is still unknown. Here, we conditionally deleted Sip1 from the developing mouse lens shortly after lens vesicle closure, leading to defects in coordinated fiber cell tip migration, defective suture formation and cataract. Interestingly, RNA-Sequencing analysis on Sip1 knockout lenses identified 190 differentially expressed genes, all of which are distinct from previously described Sip1 target genes involved in EMT/cancer. Furthermore, 34% of the upregulated genes in the Sip1 knockout lenses are normally downregulated as the lens transitions from the lens vesicle to early lens, while 49% of the genes downregulated in the Sip1 knockout lenses are normally upregulated during early lens development. Overall, these data imply that Sip1 plays a major role in reprogramming the lens vesicle away from a surface ectoderm cell fate towards that necessary for the development of a transparent lens and demonstrate that Sip1 regulates distinctly different sets of genes in different cellular contexts. RNA-Seq of inbred background wild type lenses at E15.5 RNA-Seq comparison of mixed background wild type controls and inbred wild type (C57Bl/6<har>) lenses at E15.5 RNA-Seq comparison of mixed background wild type controls and Sip1 conditional knockout lenses at E15.5
Project description:Whole lenses were dissected from E15.5, P1, or P30 wild-type C57BL/6J mice or from K6W-Ub transgenic mice, which develop congenital cataracts. Lens homogenates were subjected to quantitative proteomic analysis using tandem mass tag (TMT) isobaric labeling.
Project description:Although majority of the genes linked to pediatric cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. Here, we identify small Maf transcription factors MafG and MafK as regulators of several non-crystallin human cataract genes in fiber cells and establish their significance to cataract. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify MafG and its co-regulators in the lens, and generated various null-allelic combinations of MafG:MafK mouse mutants for phenotypic and molecular analysis. By age 4-months, MafG-/-:MafK+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of MafG-/-:MafK+/- lens reveals severe defects in fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of MafG-/-:MafK+/- lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to human cataract. This analysis prioritizes 36 highly promising candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes elevation of oxidative stress. These data identify MafG and MafK as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to mouse and human cataract. Microarray comparision of lenses from mixed background (129Sv/J, C57BL/6J, and ICR) control (MafG+/-:MafK+/-; no-cataract) and compound (MafG-/-:MafK+/-; cataract) mouse mutants
Project description:Differential expression of HSF4 in null newborn mouse and wildtype lenses was examined to identify putative downstream targets of HSF4. To examine roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific aA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in E15.5 embryonic wild type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous, and Hsf4 homozygous lenses identified multiple genes co-regulated by Brg1, Hsf4 and Pax6. Among them DNase IIb, a key enzyme required for lens fiber cell denucleation, was found downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation and indirectly for retinal development but was not essential for lens lineage formation. Keywords: Differential mRNA Expression Three biological replicate experiments were performed with HSF null and wildtype lenses.
Project description:Genome-wide approach to identify the cell-autonomous role of Brg1 in lens fiber cell terminal differentiation. To examine roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific alphaA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in E15.5 embryonic wild type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous, and Hsf4 homozygous lenses identified multiple genes co-regulated by Brg1, Hsf4 and Pax6. Among them DNase IIbeta, a key enzyme required for lens fiber cell denucleation, was found downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was required for lens fiber cell differentiation and indirectly for retinal development but was not essential for lens lineage formation. Wild type and dnBrg1 transgenic lenses, 4 biological replicates each
Project description:Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors has not been previously investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, and cataracts. Notch2 mutants also had a persistent lens stalk phenotype at E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2) and Trp63 (p63) that negatively regulates Wnt signaling. Although removal of Notch2 phenocopied the increased proportion of fiber cells of Rbpj and Jag1 conditional mutant lenses, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. Instead, we found that the Notch2 normally blocks progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation. We have compared gene expression of ocular lenses of mice that are lens specific conditional mutants of Notch2 gene to that of littermate controls that had no ablation of Notch2 gene in the lens. Two lenses of each of the three conditional mutants and controls were pooled together and total RNA was harvested from embryonic day 19.5 (E19.5) lenses. Gene expression changes caused by absence of Notch2 gene in the lens were analyzed.
Project description:Deficiency of the small Maf proteins Mafg and Mafk cause multiple defects, namely, progressive neuronal degeneration, cataract, thrombocytopenia and mid-gestational/perinatal lethality. Previous data shows Mafg-/-:Mafk+/- compound knockout (KO) mice exhibit cataracts age 4-months onward. Strikingly, Mafg-/-:Mafk-/- double KO mice develop lens defects significantly early in life, during embryogenesis, but the pathobiology of these defects is unknown, and is addressed here. At embryonic day (E)16.5, the epithelium of lens in Mafg-/-:Mafk-/- animals appears abnormally multilayered as demonstrated by E-cadherin and nuclear staining. Additionally, Mafg-/-:Mafk-/- lenses exhibit abnormal distribution of F-actin near the “fulcrum” region where epithelial cells undergo apical constriction prior to elongation and reorientation as early differentiating fiber cells. To identify the underlying molecular changes, we performed high-throughput RNA-sequencing of E16.5 Mafg-/-:Mafk-/- lenses and identified a cohort of differentially expressed genes that were further prioritized using stringent filtering criteria and validated by RT-qPCR. Several key factors associated with the cytoskeleton, cell cycle or extracellular matrix (e.g. Cdk1, Cdkn1c, Camsap1, Col3A1, Map3k12, Sipa1l1) were mis-expressed in Mafg-/-:Mafk-/- lenses. Further, the congenital cataract-linked extracellular matrix peroxidase Pxdn was significantly overexpressed in Mafg-/-:Mafk-/- lenses, which may cause abnormal cell morphology. These data also identified the ephrin signaling receptor Epha5 to be reduced in Mafg-/-:Mafk-/- lenses. This likely contributes to the Mafg-/-:Mafk-/- multilayered lens epithelium pathology, as loss of an ephrin ligand, Efna5 (ephrin-A5), causes similar lens defects. Together, these 35 findings uncover a novel early function of Mafg and Mafk in lens development and identify their new downstream regulatory relationships with key cellular factors.
Project description:This is a comparative microarray analysis of LE-AP-2a mutants vs. wild-type P0 littermate lenses. This analysis revealed differential gene expression of 415 mRNAs, including reduced expression of genes important for maintaining lens epithelial cell phenotype, such as E-cadherin. Experiment Overall Design: Biological triplicates of mouse lenses were analyzed (three wild-type lens samples and three Le-AP-2 mutant samples)
Project description:Mutations/deficiency of TDRD7, which encodes a tudor domain containing protein involved in post-transcriptional gene expression control, causes early-onset cataract in humans and animal models. While Tdrd7 is implicated in the control of key lens mRNAs, the impact of Tdrd7 deficiency on microRNAs (miRNAs), and how this contributes to cataracts, is undefined. Here, we address this critical knowledge-gap by investigating Tdrd7-targeted knockout (Tdrd7-/-) mice that exhibit fully penetrant juvenile cataracts. To gain global insights into miRNA profile alterations, we performed Affymetrix miRNA 3.0 microarray analysis on Tdrd7-/- mouse lenses at postnatal day (P) 4, because this stage precedes cataract formation and thus informs on early miRNA misexpression while minimizing detection of secondary changes.