Single cell RNA-sequencing data generated from human pluripotent stem cell-derived lens epithelial cells

Detailed transcriptomic analyses of differentiated cell populations derived from human pluripotent stem cells is routinely used to assess the identity and utility of the differentiated cells. Here we provide single cell RNA-sequencing data obtained from ROR1-expressing lens epithelial cells (ROR1e LECs), obtained via directed differentiation of CA1 human embryonic stem cells. Analysis of the data using principal component analysis, heat maps and gene ontology assessments revealed phenotypes associated with lens epithelial cells. These data provide a resource for future characterisation of both normal and cataractous human lens biology. Corresponding morphological and functional data obtained from ROR1e LECs are reported in the associated research article “A simplified method for producing human lens epithelial cells and light-focusing micro-lenses from pluripotent stem cells “ (Dewi et al., 2020)

A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts

To avoid negative environmental impacts of escapees and potential inter-breeding with wild populations, the Atlantic salmon farming industry has and continues to extensively test triploid fish that are sterile. However, they often show differences in performance, physiology, behavior and morphology compared to diploid fish, with increased prevalence of vertebral deformities and ocular cataracts as two of the most severe disorders. Here, we investigated the mechanisms behind the higher prevalence of cataracts in triploid salmon, by comparing the transcriptional patterns in lenses of diploid and triploid Atlantic salmon, with and without cataracts. We assembled and characterized the Atlantic salmon lens transcriptome and used RNA-seq to search for the molecular basis for cataract development in triploid fish. Transcriptional screening showed only modest differences in lens mRNA levels in diploid and triploid fish, with few uniquely expressed genes. In total, there were 165 differentially expressed genes (DEGs) between the cataractous diploid and triploid lens. Of these, most were expressed at lower levels in triploid fish. Differential expression was observed for genes encoding proteins with known function in the retina (phototransduction) and proteins associated with repair and compensation mechanisms. The results suggest a higher susceptibility to oxidative stress in triploid lenses, and that mechanisms connected to the ability to handle damaged proteins are differentially affected in cataractous lenses from diploid and triploid salmon.publishedVersionUnit Licence Agreemen

Express: A database of transcriptome profiles encompassing known and novel transcripts across multiple development stages in eye tissues

Advances in sequencing have facilitated nucleotide-resolution genome-wide transcriptomic profiles across multiple mouse eye tissues. However, these RNA sequencing (RNA-seq) based eye developmental transcriptomes are not organized for easy public access, making any further analysis challenging. Here, we present a new database “Express” (http://www.iupui.edu/∼sysbio/express/) that unifies various mouse lens and retina RNA-seq data and provides user-friendly visualization of the transcriptome to facilitate gene discovery in the eye. We obtained RNA-seq data encompassing 7 developmental stages of lens in addition to that on isolated lens epithelial and fibers, as well as on 11 developmental stages of retina/isolated retinal rod photoreceptor cells from publicly available wild-type mouse datasets. These datasets were pre-processed, aligned, quantified and normalized for expression levels of known and novel transcripts using a unified expression quantification framework. Express provides heatmap and browser view allowing easy navigation of the genomic organization of transcripts or gene loci. Further, it allows users to search candidate genes and export both the visualizations and the embedded data to facilitate downstream analysis. We identified total of >81,000 transcripts in the lens and >178,000 transcripts in the retina across all the included developmental stages. This analysis revealed that a significant number of the retina-expressed transcripts are novel. Expression of several transcripts in the lens and retina across multiple developmental stages was independently validated by RT-qPCR for established genes such as Pax6 and Lhx2 as well as for new candidates such as Elavl4, Rbm5, Pabpc1, Tia1 and Tubb2b. Thus, Express serves as an effective portal for analyzing pruned RNA-seq expression datasets presently collected for the lens and retina. It will allow a wild-type context for the detailed analysis of targeted gene-knockout mouse ocular defect models and facilitate the prioritization of candidate genes from Exome-seq data of eye disease patients

A transcriptomic analysis of diploid and triploid Atlantic salmon lenses with and without cataracts

To avoid negative environmental impacts of escapees and potential inter-breeding with wild populations, the Atlantic salmon farming industry has and continues to extensively test triploid fish that are sterile. However, they often show differences in performance, physiology, behavior and morphology compared to diploid fish, with increased prevalence of vertebral deformities and ocular cataracts as two of the most severe disorders. Here, we investigated the mechanisms behind the higher prevalence of cataracts in triploid salmon, by comparing the transcriptional patterns in lenses of diploid and triploid Atlantic salmon, with and without cataracts. We assembled and characterized the Atlantic salmon lens transcriptome and used RNA-seq to search for the molecular basis for cataract development in triploid fish. Transcriptional screening showed only modest differences in lens mRNA levels in diploid and triploid fish, with few uniquely expressed genes. In total, there were 165 differentially expressed genes (DEGs) between the cataractous diploid and triploid lens. Of these, most were expressed at lower levels in triploid fish. Differential expression was observed for genes encoding proteins with known function in the retina (phototransduction) and proteins associated with repair and compensation mechanisms. The results suggest a higher susceptibility to oxidative stress in triploid lenses, and that mechanisms connected to the ability to handle damaged proteins are differentially affected in cataractous lenses from diploid and triploid salmon.info:eu-repo/semantics/publishedVersio

The Tudor-domain protein TDRD7, mutated in congenital cataract, controls the heat shock protein HSPB1 (HSP27) and lens fiber cell morphology

Mutations of the RNA granule component TDRD7 (OMIM: 611258) cause pediatric cataract. We applied an integrated approach to uncover the molecular pathology of cataract in Tdrd7−/− mice. Early postnatal Tdrd7−/− animals precipitously develop cataract suggesting a global-level breakdown/misregulation of key cellular processes. High-throughput RNA sequencing integrated with iSyTE-bioinformatics analysis identified the molecular chaperone and cytoskeletal modulator, HSPB1, among high-priority downregulated candidates in Tdrd7−/− lens. A protein fluorescence two-dimensional difference in-gel electrophoresis (2D-DIGE)-coupled mass spectrometry screen also identified HSPB1 downregulation, offering independent support for its importance to Tdrd7−/− cataractogenesis. Lens fiber cells normally undergo nuclear degradation for transparency, posing a challenge: how is their cell morphology, also critical for transparency, controlled post-nuclear degradation? HSPB1 functions in cytoskeletal maintenance, and its reduction in Tdrd7−/− lens precedes cataract, suggesting cytoskeletal defects may contribute to Tdrd7−/− cataract. In agreement, scanning electron microscopy (SEM) revealed abnormal fiber cell morphology in Tdrd7−/− lenses. Further, abnormal phalloidin and wheat germ agglutinin (WGA) staining of Tdrd7−/− fiber cells, particularly those exhibiting nuclear degradation, reveals distinct regulatory mechanisms control F-actin cytoskeletal and/or membrane maintenance in post-organelle degradation maturation stage fiber cells. Indeed, RNA immunoprecipitation identified Hspb1 mRNA in wild-type lens lysate TDRD7-pulldowns, and single-molecule RNA imaging showed co-localization of TDRD7 protein with cytoplasmic Hspb1 mRNA in differentiating fiber cells, suggesting that TDRD7–ribonucleoprotein complexes may be involved in optimal buildup of key factors. Finally, Hspb1 knockdown in Xenopus causes eye/lens defects. Together, these data uncover TDRD7’s novel upstream role in elevation of stress-responsive chaperones for cytoskeletal maintenance in post-nuclear degradation lens fiber cells, perturbation of which causes early-onset cataracts

Growth factor modulation of TGF-beta (TGF-β)-induced epithelial-to-mesenchymal transition (EMT) in the eye lens

The eye lens is a transparent organ dependent on growth factors within the ocular media (e.g. FGF) to regulate its normal functionality. During stress &/or inflammation, activation of cytokine TGF-β disrupts normal lens cell processes & the epithelial cell phenotype due to epithelial reorganisation of cytoskeletal proteins (e.g. Tpm). TGF-β promotes an epithelial-mesenchymal transition (EMT) leading to lens fibrosis (cataracts) namely ASC or secondary cataracts (PCO). In situ, ASC & PCO both present differential cell types (myofibroblastic or fibre-like) that are induced/maintained by ocular growth factors. This thesis examines the interactions between FGF-2 & TGF-β2 during rodent lens EMT & targets specific molecules involved in TGF-β downstream signalling that regulate lens cell behaviour during fibrosis. We investigate the role of Tpms in cataractogenesis using in vivo postnatal lenses from wild type & an overexpressing TGF-β1 transgenic mouse line that develops ASC postnatally. We established a potential model for ASC & PCO in vitro given FGF-2+TGF-β2 cotreated rat lens epithelial cell (LEC) explants led to a heterogenous lens cell population that were differentially regulated in a dose-dependent manner. We identified several Tpms as potential markers for lens fibre differentiation &/or TGF-β-induced EMT in LECs, fibre-like cells & myofibroblasts. We characterise Tpm3.1/3.2 & Tpm4.2 deficient mouse models and use pharmacological compounds targeting Tpm3.1/3.2 to further elucidate their role during TGF-β-induced lens EMT. We examine TGF-β-activated downstream signalling protein p38α MAPK & revisit its role during lens EMT. We showed inhibition of p38α impacted morphological changes during TGF-β-induced EMT. The present study demonstrates the importance of understanding how the lens is dysregulated during lens pathology. Targeting specific signalling molecules & cytoskeletal mechanisms in rodent lenses may lead to finding new treatments for fibrotic cataract