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While the bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery) effectively identifies human cataract-associated genes, it is currently based on just transcriptome data, and thus it is necessary to include protein-level information to gain greater confidence in gene prioritization. Here we expand iSyTE through development of a novel proteome-based resource on the lens and demonstrate its utility in cataract gene discovery. We applied highthroughput tandem mass spectrometry (MS/MS) to generate a global protein expression profile of mouse lens at embryonic day (E)14.5, which identified 2371 lens-expressed proteins. A major challenge of high-throughput expression profiling is identification of high-priority candidates among the thousands of expressed proteins. To address this problem, we generated new MS/MS proteome data on mouse whole embryonic body (WB). WB proteome was then used as a reference dataset for performing "in silico WB-subtraction" comparative analysis with the lens proteome, which effectively identified 422 proteins with lens-enriched expression at ≥2.5 average spectral counts, ≥2.0 fold-enrichment (FDR <0.01) cut-off. These top 20% candidates represent a rich pool of high-priority proteins in the lens including known human cataract-linked genes and many new potential regulators of lens development and homeostasis. This rich information is made publicly accessible through iSyTE (https://research.bioinformatics.udel.edu/iSyTE/), which enables user-*
Purpose Age-related cataracts affect the majority of older adults and are a leading cause of blindness worldwide. Treatments that delay cataract onset or severity have the potential to delay cataract surgery, but require relevant animal models that recapitulate the major types of cataracts for their development. Unfortunately, few such models are available. Here, we report the lens phenotypes of aged mice lacking the critical antioxidant transcription factor Nfe2l2 (designated as Nrf2 −/−). Methods Three independent cohorts of Nrf2 −/− and wild-type C57BL/6J mice were evaluated for cataracts using combinations of slit lamp imaging, photography of freshly dissected lenses, and histology. Mice were fed high glycemic diets, low glycemic diets, regular chow ad libitum, or regular chow with 30% caloric restriction. Results Nrf2 −/− mice developed significant opacities between 11 and 15 months and developed advanced cortical, posterior subcapsular, anterior subcapsular, and nuclear cataracts. Cataracts occurred similarly in male mice fed high or low glycemic diets, and were also observed in 21-month male and female Nrf2 −/− mice fed ad libitum or 30% caloric restriction. Histological observation of 18-month cataractous lenses revealed significant disruption to fiber cell architecture and the retention of nuclei throughout the cortical region of the lens. However, fiber cell denucleation and initiation of lens differentiation was normal at birth, with the first abnormalities observed at 3 months. Conclusions Nrf2 −/− mice offer a tool to understand how defective antioxidant signaling causes multiple forms of cataract and may be useful for screening drugs to prevent or delay cataractogenesis in susceptible adults.
The transparent ocular lens in the anterior chamber of the eye is responsible for fine focusing of light onto the retina. The lens is entirely cellular with bulk of the tissue composed of fiber cells, and the anterior hemisphere of the lens is covered by a monolayer of epithelial cells. Lens epithelial cells are important for maintaining fiber cell homeostasis and for continual growth of the lens tissue throughout life. Cataracts, defined as any opacity in the lens, remain the leading cause of blindness in the world. Following cataract surgery, lens epithelial cells can undergo a process of epithelial-to-mesenchymal transition (EMT), leading to secondary cataracts due to posterior capsular opacification (PCO). Since the epithelial cells make up only a small fraction of the lens, specialized techniques are required to study lens epithelial cell biology and pathology. Studies using native lens epithelial cells often require pooling of samples to obtain enough cells to make sufficient samples for traditional molecular biology techniques. Here, we provide detailed protocols that enable the study of native mouse lens epithelial cells, including immunostaining of the native lens epithelium in flat mounts, extraction of RNA and proteins from pairs of lens epithelial monolayers, and isolation of lens epithelial cells for primary culture. These protocols will enable researchers to gain better insight on representative molecular expression and cellular structure of lens epithelial cells. We also provide comparative data between native, primary culture, and immortalized lens epithelial cells and discuss the advantages and disadvantages of each technique presented.
Defects in the development of the ocular lens can cause congenital cataracts. To understand the various etiologies of congenital cataracts, it is important to characterize the genes linked to this developmental defect and to define their downstream pathways that are relevant to lens biology and pathology. Deficiency or alteration of several RNA-binding proteins, including the conserved RBP Celf1 (CUGBP Elav-like family member 1), has been described to cause lens defects and early onset cataracts in animal models and/or humans. Celf1 is involved in various aspects of post-transcriptional gene expression control, including regulation of mRNA stability/decay, alternative splicing and translation. Celf1 germline knockout mice and lens conditional knockout (Celf1cKO) mice develop fully penetrant cataracts in early postnatal stages. To define the genome-level changes in RNA transcripts that result from Celf1 deficiency, we performed high-throughput RNA-sequencing of Celf1cKO mouse lenses at postnatal day (P) 0. Celf1cKO lenses exhibit 987 differentially expressed genes (DEGs) at cut-offs of >1.0 log2 counts per million (CPM), ≥±0.58 log2 fold-change and <0.05 false discovery rate (FDR). Of these, 327 RNAs were reduced while 660 were elevated in Celf1cKO lenses. The DEGs were subjected to various downstream analyses including iSyTE lens enriched-expression, presence in Cat-map, and gene ontology (GO) and representation of regulatory pathways. Further, a comparative analysis was done with previously generated microarray datasets on Celf1cKO lenses P0 and P6. Together, these analyses validated and prioritized several key genes mis-expressed in Celf1cKO lenses that are relevant to lens biology, including known cataract-linked genes (e.g., Cryab, Cryba2, Cryba4, Crybb1, Crybb2, Cryga, Crygb, Crygc, Crygd, Cryge, Crygf, Dnase2b, Bfsp1, Gja3, Pxdn, Sparc, Tdrd7, etc.) as well as novel candidates (e.g., Ell2 and Prdm16). Together, these data have defined the alterations in lens transcriptome caused by Celf1 deficiency, in turn uncovering downstream genes and pathways (e.g., structural constituents of eye lenses, lens fiber cell differentiation, etc.) associated with lens development and early-onset cataracts.
Whole mount imaging of the lens allows for high spatial resolution visualization of lens epithelial structures by using small molecule fluorescent probes. However, the visualization of specific proteins in lens epithelial cells within whole lenses remains a challenge as the capsule that surrounds the lens does not allow penetration of antibodies. Here we describe a whole mount imaging method that allows us to overcome this challenge by digesting the lens capsules of paraformaldehyde fixed lenses using collagenase. This method enables the penetration of antibodies for effective visualization of proteins in the epithelium of whole lenses. A limitation to lens whole mount imaging is the ability to visualize specific proteins as the collagen capsule surrounding the lens impedes the penetration of antibodies This protocol helps overcome this limitation by a light collagenase digestion of the capsule of fixed lenses prior to immunostaining This method allows for the imaging of specific proteins in the epithelium of the whole lens tissue
The lens is a transparent tissue that focuses light on the retina allowing high‐resolution vision. Loss of lens transparency is defined as cataract, the leading cause of blindness worldwide. An estimated 25–50% of congenital cataract cases arise from underlying genetic alterations, making it important to characterize cataract‐linked genes and their pathological mechanisms.Recently, our lab demonstrated that deficiency of the gene Celf1 causes cataract in fish and mouse, indicating its conserved function in vertebrate lens development. Celf1 encodes an RNA‐binding protein (RBP) that directly binds to specific mRNAs and control them on multiple “post‐transcriptional” levels, impacting their alternative splicing, decay or translation into protein. An important unresolved question is whether a post‐transcriptional regulator like Celf1 controls key transcription factors (TFs) in lens development. To address this fundamental question, I generated and characterized lens‐specific Celf1 conditional knockout mice (Celf1cKO/lacZKI). Using immunofluorescence assays, I demonstrate that Celf1cKO/lacZKI lenses exhibit abnormally high protein levels of the homeodomain‐TF Pax6. Further, by performing Celf1‐antibody coupled RNA‐immunoprecipitation (RIP) followed by RT‐qPCR, I demonstrate that Celf1 protein directly associates with Pax6 mRNA.Next, I investigated if Celf1 coordinately functions with other RBPs to exert combinatorial control over gene expression. I performed co‐immunoprecipitation to demonstrate that Celf1 is associated with another RBP, specifically HuR/Elavl1 in lens cells. Finally, by performing HuR/Elval1‐antibody coupled RIP‐RT‐qPCR assay I show that HuR/Elavl1 binds to Pax6 mRNA.These data indicate that the RBPs Celf1 and HuR/Elavl1 directly interact and bind to Pax6 mRNA to mediate combinatorial control of Pax6 protein expression in mouse lens development. Alterations of Pax6 levels are known to cause severe eye defects in human and animal models. Thus, my research uncovers a novel RBP‐based molecular mechanism for regulation of Pax6 expression in the lens, in turn, informing on a new pathway for the pathology of eye defects, including cataract.Support or Funding InformationNEI/NIH RO1 EY021505, Milton H. Stetson Memorial Undergraduate Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proper ocular lens function requires lens biomechanical flexibility which is lost in presbyopia during aging. As increasing lens size has been shown previously to correlate with lens biomechanical stiffness in aging, we tested the hypothesis that whole lens size determines gross biomechanical stiffness. We used an allometric approach to evaluate this hypothesis by comparing lenses from three rodent species (mouse, rats and guinea pigs) of varying size. While rat lenses are larger and stiffer than mouse lenses, guinea pig lenses are even larger than rat lenses but are softer than the rat lens. This indicates that lens size is not a sole determinant of lens stiffness and disproves our hypothesis. Therefore, we investigated the scaling of lens microstructural features that could potentially explain the differences in biomechanical stiffness between rat and guinea pig lenses, including lens capsule thickness, epithelial cell area, fiber cell widths, suture organization, and nuclear size. Capsule thickness, epithelial cell area, and fiber cell widths scaled with lens size (i.e., greater in guinea pig lenses than rats), indicating that sizes of these features do not correlate with the stiffness of rat lenses, while suture organization was similar between rats and guinea pigs. However, we found that the hard rat lens nucleus occupies a greater fraction of the lens than the guinea pig lens nucleus, suggesting a role for nuclear size in determining whole lens stiffness. Therefore, while many features contribute to lens biomechanical properties, the size of the lens nucleus with respect to the size of the lens could be a major determinant of lens stiffness in rats versus guinea pigs.
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