Project description:Mutations in Peroxidasin (PXDN) cause severe inherited eye disorders in humans, such as congenital cataract, corneal opacity, and developmental glaucoma. The role of peroxidasin during eye development is poorly understood. Here we describe the first Pxdn mouse mutant which was induced by ENU (N-ethyl-N-nitrosourea) and led to a recessive phenotype. Sequence analysis of cDNA revealed a T3816A mutation resulting in a premature stop codon (Cys1272X) in the peroxidase domain. This mutation causes severe anterior segment dysgenesis and microphthalmia resembling the manifestations in patients with PXDN mutations. The proliferation and differentiation of the lens is disrupted in association with aberrant expression of transcription factor genes (Pax6 and Foxe3) in mutant eyes. Additionally, Pxdn is involved in the consolidation of the basement membrane and lens epithelium adhesion in the ocular lens. Lens material including γ-crystallin is extruded into the anterior and posterior chamber due to local loss of structural integrity of the lens capsule as a secondary damage to the anterior segment development leading to congenital ocular inflammation. Moreover, Pxdn mutants exhibited an early-onset glaucoma and progressive retinal dysgenesis. Transcriptome profiling revealed that peroxidasin affects the transcription of developmental and eye diseases-related genes at early eye development. These findings suggest that peroxidasin is necessary for cell proliferation and differentiation and for basement membrane consolidation during eye development. Our studies provide pathogenic mechanisms of PXDN mutation-induced congenital eye diseases.
Project description:We used DNA microarrays representing ~30,000 human genes to analyze gene expression in the cornea, lens, iris, ciliary body, retina, and optic nerve. Eight whole globes (G1-G8) were harvested from autopsy donors (age range=30-85 years-old) within 24 hours of death, and the tissues were immediately stored at 4◦ C in RNAlater (Ambion). Four of the globes were from female donors (G3, G6-8) and four were from male donors (G1, 2, 4, 5). Globes 4 and 5 were harvested as a set from a single donor, as were globes 6 and 7. No ophthalmologic clinical records were available for any of the globes at the time of harvest. Seven of the globes (G1-G7) were dissected into the following components: cornea, lens, iris, ciliary body, retina, and optic nerve, while only retinal tissue was available from G8. The maculas and the peripheral retinal tissues were further dissected from several of the retinal samples. The macula was defined as the visible xanthophyll-containing tissue temporal to the optic nerve, which encompassed an approximate area of 4 mm2. For comparison purposes, three post-mortem brain specimens were analyzed. Only those tissues that yielded adequate amounts of RNA were processed on the arrays. An organism part comparison experiment design type compares tissues, regions, organs within or between organisms. Differential gene expression in anatomical compartments of the human eye. Diehn et al. Genome Biology 2005, 6:R74 Using regression correlation
Project description:Glutathione (GSH) is a critical endogenous antioxidant that protects against intracellular oxidative stress. As such, pathological alterations in GSH levels are linked to a myriad of diseases including cancer, neurodegeneration and cataract. The rate limiting step in GSH biosynthesis is catalyzed by the glutamate cysteine ligase catalytic subunit (GCLC). The high expression of GCLC in the lens supports the synthesis of millimolar concentrations of GSH in this tissue. Herein, we describe the morphological consequences of deleting (knocking out) Gclc from surface ectoderm-derived ocular tissues (using the Le-Cre transgene; Gclc KO) which includes an overt microphthalmia phenotype and severely disrupted formation of multiple ocular structures (i.e., cornea, iris, lens, retina). Controlling for the Le-Cre transgene revealed that the deletion of Gclc significantly exacerbated the microphthalmia phenotype in Le-Cre hemizygous mice and resulted in dysregulated gene expression that was unique to only the lenses of KO mice. We further characterized the impaired lens development by conducting an RNA-seq experiment on KO and Gclc control (CON) mouse lens at the day of birth. RNA-sequencing revealed significant differences between Gclc knockout (KO) and Gclc control (CON) lenses, including down-regulation of crystallins and lens fiber cell identity genes, and up-regulation of lens epithelial cell identity genes. In addition, genes related to the immune system (e.g., immune system process, inflammatory response, neutrophil chemotaxis) were upregulated, and genes related to eye/lens development were downregulated. TRANSFAC analysis of differentially expressed genes (DEGs) in the lens of Gclc KO mice implicated PAX6 as a key upstream regulator of Gclc KO sensitive genes. This was further supported by a strong positive correlation between the transcriptomes of the lenses of Gclc KO and Pax6 KO mice. Strikingly, the dysregulation of PAX6-regulated genes in Gclc KO mice was observed despite no change in the ocular localization of PAX6 or decrease in the expression of PAX6 in the lens. In vitro experiments demonstrated that suppression of intracellular GSH concentrations resulted in impairment of PAX6 transactivation activity. Taken together, the present results elucidate a novel mechanism wherein intracellular GSH concentrations may modulate PAX6 activity.
Project description:We used DNA microarrays representing ~30,000 human genes to analyze gene expression in the cornea, lens, iris, ciliary body, retina, and optic nerve. Eight whole globes (G1-G8) were harvested from autopsy donors (age range=30-85 years-old) within 24 hours of death, and the tissues were immediately stored at 4◦ C in RNAlater (Ambion). Four of the globes were from female donors (G3, G6-8) and four were from male donors (G1, 2, 4, 5). Globes 4 and 5 were harvested as a set from a single donor, as were globes 6 and 7. No ophthalmologic clinical records were available for any of the globes at the time of harvest. Seven of the globes (G1-G7) were dissected into the following components: cornea, lens, iris, ciliary body, retina, and optic nerve, while only retinal tissue was available from G8. The maculas and the peripheral retinal tissues were further dissected from several of the retinal samples. The macula was defined as the visible xanthophyll-containing tissue temporal to the optic nerve, which encompassed an approximate area of 4 mm2. For comparison purposes, three post-mortem brain specimens were analyzed. Only those tissues that yielded adequate amounts of RNA were processed on the arrays. An organism part comparison experiment design type compares tissues, regions, organs within or between organisms. Differential gene expression in anatomical compartments of the human eye. Diehn et al. Genome Biology 2005, 6:R74 Keywords: organism_part_comparison_design
Project description:Background: During eye lens development the fetal vasculature regresses leaving the lens without a direct oxygen source. Both embryonically and throughout adult life, the lens contains a decreasing oxygen gradient from the surface to the core that parallels the natural differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia in the regulation of genes required for mature lens structure and function. Since HIF1α is a master regulator of the hypoxic response, these lens properties also implicate HIF1α as a potential requirement for lens formation and homeostasis. Here, we employed a multiomics approach combining CUT&RUN, RNAseq and ATACseq analysis to establish the genomic complement of lens HIF1α binding sites, genes activated or repressed by HIF1α and the chromatin states of HIF1α-regulated genes. Results: CUT&RUN analysis revealed 8,375 HIF1α-DNA binding complexes in the chick lens genome. 1,190 HIF1α-DNA binding complexes were significantly clustered within chromatin accessible regions (χ2 test p < 1x10-55) identified by ATACseq. Formation of the identified HIF1α-DNA complexes paralleled the activation or repression of 526 genes, 116 of which contained HIF1α binding sites within 10kB of the transcription start sites. Some of the identified HIF1α genes have previously established lens functions while others have novel functions never before examined in the lens. GO and pathway analysis of these genes implicate HIF1α in the control of a wide-variety of cellular pathways potentially critical for lens formation, structure and function including glycolysis, cell cycle regulation, chromatin remodeling, Notch and Wnt signaling, differentiation, development, and transparency. Conclusions: These data establish the first functional map of genomic HIF1α-DNA complexes in the eye lens. They identify HIF1α as an important regulator of a wide-variety of genes previously shown to be critical for lens formation and function and they reveal a requirement for HIF1α in the regulation of a wide-variety of genes not yet examined for lens function. They support a requirement for HIF1α in lens development, structure and function and they provide a basis for understanding the potential roles and requirements for HIF1α in the development, structure and function of more complex tissues.
Project description:Background: During eye lens development the fetal vasculature regresses leaving the lens without a direct oxygen source. Both embryonically and throughout adult life, the lens contains a decreasing oxygen gradient from the surface to the core that parallels the natural differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia in the regulation of genes required for mature lens structure and function. Since HIF1α is a master regulator of the hypoxic response, these lens properties also implicate HIF1α as a potential requirement for lens formation and homeostasis. Here, we employed a multiomics approach combining CUT&RUN, RNAseq and ATACseq analysis to establish the genomic complement of lens HIF1α binding sites, genes activated or repressed by HIF1α and the chromatin states of HIF1α-regulated genes. Results: CUT&RUN analysis revealed 8,375 HIF1α-DNA binding complexes in the chick lens genome. 1,190 HIF1α-DNA binding complexes were significantly clustered within chromatin accessible regions (χ2 test p < 1x10-55) identified by ATACseq. Formation of the identified HIF1α-DNA complexes paralleled the activation or repression of 526 genes, 116 of which contained HIF1α binding sites within 10kB of the transcription start sites. Some of the identified HIF1α genes have previously established lens functions while others have novel functions never before examined in the lens. GO and pathway analysis of these genes implicate HIF1α in the control of a wide-variety of cellular pathways potentially critical for lens formation, structure and function including glycolysis, cell cycle regulation, chromatin remodeling, Notch and Wnt signaling, differentiation, development, and transparency. Conclusions: These data establish the first functional map of genomic HIF1α-DNA complexes in the eye lens. They identify HIF1α as an important regulator of a wide-variety of genes previously shown to be critical for lens formation and function and they reveal a requirement for HIF1α in the regulation of a wide-variety of genes not yet examined for lens function. They support a requirement for HIF1α in lens development, structure and function and they provide a basis for understanding the potential roles and requirements for HIF1α in the development, structure and function of more complex tissues.
Project description:The eye lens is composed of fiber cells, which differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. To understand the molecular mechanism of the lens cell differentiation, we compared the gene expression profiles between epithelial and cortical fiber cells of young mouse lens using a microarray analysis. Keywords: cell-type comparison
Project description:The eye lens is responsible for focusing and transmitting light to the retina. The lens does this in the absence of organelles yet maintains transparency for at least five decades before onset of age-related nuclear cataract (ARNC). It is hypothesized that oxidative stress contributes significantly to ARNC formation. It is additionally hypothesized that transparency is maintained by a microcirculation system (MCS) that delivers antioxidants to the lens nucleus and exports small molecule waste. Commonly used data-dependent acquisition (DDA) proteomics methods are hindered by the dynamic range of protein expression in the lens and provide limited context to age-related changes in the lens. In this study we utilized data-independent acquisition (DIA) mass spectrometry proteomics to analyze the urea insoluble, membrane protein fractions of 16 human lenses subdivided into three spatially distinct lens regions to characterize age-related changes, particularly concerning the lens MCS and oxidative stress response. In this pilot cohort, using the DIA approach, we measured 4,788 distinct protein groups, and 46,681 peptides, more than in any previous human lens DDA approach. Our results reveal age-related changes previously known in lens biology and expand on these findings, taking advantage of the rich dataset afforded by DIA mass spectrometry. Principally, we demonstrate that a significant proteome remodeling event occurs at approximately 50 years of age, resulting in metabolic preference for anaerobic glycolysis established with organelle degradation, decreased abundance of protein networks involved in calcium-dependent cell-cell contacts while retaining networks related to oxidative stress response. Further, we demonstrate the first identification of multiple antioxidant transporter proteins not previously shown in the human lens and describe their spatiotemporal and age-related abundance changes. Finally, we demonstrate that Aquaporin-5, among other proteins, is depleted with age. We suggest that the continued accumulation of each of these age-related outcomes in proteome remodeling contribute to decrease in fiber cell permeability and result in ARNC formation.