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Item A crystallin mutant cataract with mineral deposits(Elsevier, 2023) Minogue, Peter J.; Gao, Junyuan; Mathias, Richard T.; Williams, James C., Jr.; Bledsoe, Sharon B.; Sommer, Andre J.; Beyer, Eric C.; Berthoud, Viviana M.; Anatomy, Cell Biology and Physiology, School of MedicineConnexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.Item Connexin Mutants Cause Cataracts Through Deposition of Apatite(Frontiers Media, 2022-07-22) Minogue, Peter J.; Sommer, Andre J.; Williams, James C., Jr.; Bledsoe, Sharon B.; Beyer, Eric C.; Berthoud, Viviana M.; Anatomy, Cell Biology and Physiology, School of MedicineCataracts are lens opacities that are among the most common causes of blindness. It is commonly believed that cataracts develop through the accumulation of damage to lens proteins. However, recent evidence suggests that cataracts can result from calcium ion accumulation and the precipitation of calcium-containing salts. To test for the presence of precipitates and to identify their components, we studied the lenses of mice that develop cataracts due to mutations of connexin46 and connexin50. Micro-computed tomography showed the presence of radio-dense mineral in the mutant lenses, but not in wild-type lenses. Three-dimensional reconstructions of the scans showed that the distribution of the radio-dense mineral closely paralleled the location and morphology of the cataracts. The mutant lens homogenates also contained insoluble particles that stained with Alizarin red (a dye that stains Ca2+ deposits). Using attenuated total internal reflection micro–Fourier transform infrared spectroscopy, we identified the mineral as calcium phosphate in the form of apatite. Taken together, these data support the novel paradigm that cataracts are formed through pathological mineralization within the lens.Item Frs2α and Shp2 signal independently of Gab to mediate FGF signaling in lens development(Company of Biologists, 2014-02-01) Li, Hongge; Tao, Chenqi; Cai, Zhigang; Hertzler-Schaefer, Kristina; Collins, Tamica N.; Wang, Fen; Feng, Gen-Sheng; Gotoh, Noriko; Zhang, Xin; Department of Medical and Molecular Genetics, IU School of MedicineFibroblast growth factor (FGF) signaling requires a plethora of adaptor proteins to elicit downstream responses, but the functional significances of these docking proteins remain controversial. In this study, we used lens development as a model to investigate Frs2α and its structurally related scaffolding proteins, Gab1 and Gab2, in FGF signaling. We show that genetic ablation of Frs2α alone has a modest effect, but additional deletion of tyrosine phosphatase Shp2 causes a complete arrest of lens vesicle development. Biochemical evidence suggests that this Frs2α-Shp2 synergy reflects their epistatic relationship in the FGF signaling cascade, as opposed to compensatory or parallel functions of these two proteins. Genetic interaction experiments further demonstrate that direct binding of Shp2 to Frs2α is necessary for activation of ERK signaling, whereas constitutive activation of either Shp2 or Kras signaling can compensate for the absence of Frs2α in lens development. By contrast, knockout of Gab1 and Gab2 failed to disrupt FGF signaling in vitro and lens development in vivo. These results establish the Frs2α-Shp2 complex as the key mediator of FGF signaling in lens development.Item RNA-binding proteins in eye development and disease: implication of conserved RNA granule components(Wiley, 2016-07) Dash, Soma; Siddam, Archana D.; Barnum, Carrie E.; Janga, Sarath Chandra; Lachke, Salil A.; BioHealth Informatics, School of Informatics and ComputingThe molecular biology of metazoan eye development is an area of intense investigation. These efforts have led to the surprising recognition that although insect and vertebrate eyes have dramatically different structures, the orthologs or family members of several conserved transcription and signaling regulators such as Pax6, Six3, Prox1, and Bmp4 are commonly required for their development. In contrast, our understanding of posttranscriptional regulation in eye development and disease, particularly regarding the function of RNA-binding proteins (RBPs), is limited. We examine the present knowledge of RBPs in eye development in the insect model Drosophila as well as several vertebrate models such as fish, frog, chicken, and mouse. Interestingly, of the 42 RBPs that have been investigated for their expression or function in vertebrate eye development, 24 (~60%) are recognized in eukaryotic cells as components of RNA granules such as processing bodies, stress granules, or other specialized ribonucleoprotein (RNP) complexes. We discuss the distinct developmental and cellular events that may necessitate potential RBP/RNA granule-associated RNA regulon models to facilitate posttranscriptional control of gene expression in eye morphogenesis. In support of these hypotheses, three RBPs and RNP/RNA granule components Tdrd7, Caprin2, and Stau2 are linked to ocular developmental defects such as congenital cataract, Peters anomaly, and microphthalmia in human patients or animal models. We conclude by discussing the utility of interdisciplinary approaches such as the bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to prioritize RBPs for deriving posttranscriptional regulatory networks in eye development and disease. WIREs RNA 2016, 7:527-557. doi: 10.1002/wrna.1355 For further resources related to this article, please visit the WIREs website.Item The role of adaptor proteins Crk and CrkL in lens development(2016-05-04) Collins, Tamica N.; Zhang, XinCell shape changes and signaling pathways are essential for the development and function of the lens. During lens development proliferating epithelial cells will migrate down to the equator of the lens, differentiate into lens fiber cells, and begin to elongate along the lens capsule. The Fibroblast Growth Factor (FGF) signaling pathway has been extensively studied for its role in lens fiber cell differentiation and elongation. However, the main mediators of FGF stimulated lens fiber cell elongation have not been identified. Adaptor proteins Crk and CrkL are SH2- and SH3-containing proteins that transduce signals from upstream tyrosine phosphorylated proteins to downstream effectors, including Ras, Rac1 and Rap1, which are important for cell proliferation, adhesion and migration. Underlying their diverse function, these two adaptor proteins have been implicated in receptor tyrosine kinase signaling, focal adhesion assembly, and cell shape. To explore the role of Crk and CrkL in FGF signaling-dependent lens development and fiber elongation, we employed Cre/LoxP system to generate a lens specific knockout of Crk/CrkL. This led to extracellular matrix defects, disorganization of the lens fiber cells, and a defect in lens fiber cell elongation. Deletion of Crk and CrkL in the lens also mitigated the gain-of-function phenotype caused by overexpression of FGF3, indicating an epistatic relationship between Crk/CrkL and FGF signaling during lens fiber cell elongation. Further studies, revealed that the activity of Crk and CrkL in FGF signaling is controlled by the phosphatase Shp2 and the defect observed in lens fiber cell elongation can be rescued by constitutive activation of the GTPases Ras and Rac1 in the Crk and CrkL mutant lens. Interestingly, the deletion of the GTPases Rap1 in the lens showed no obvious phenotype pertaining to lens fiber cell elongation. These findings suggest that Crk and CrkL play an important role in integrating FGF signaling and mediating lens fiber cell elongation during lens development.Item Transcriptome analysis of developing lens reveals abundance of novel transcripts and extensive splicing alterations(Nature Publishing group, 2017-09-14) Srivastava, Rajneesh; Budak, Gungor; Dash, Soma; Lachke, Salil A.; Janga, Sarath Chandra; BioHealth Informatics, School of Informatics and ComputingLens development involves a complex and highly orchestrated regulatory program. Here, we investigate the transcriptomic alterations and splicing events during mouse lens formation using RNA-seq data from multiple developmental stages, and construct a molecular portrait of known and novel transcripts. We show that the extent of novelty of expressed transcripts decreases significantly in post-natal lens compared to embryonic stages. Characterization of novel transcripts into partially novel transcripts (PNTs) and completely novel transcripts (CNTs) (novelty score ≥ 70%) revealed that the PNTs are both highly conserved across vertebrates and highly expressed across multiple stages. Functional analysis of PNTs revealed their widespread role in lens developmental processes while hundreds of CNTs were found to be widely expressed and predicted to encode for proteins. We verified the expression of four CNTs across stages. Examination of splice isoforms revealed skipped exon and retained intron to be the most abundant alternative splicing events during lens development. We validated by RT-PCR and Sanger sequencing, the predicted splice isoforms of several genes Banf1, Cdk4, Cryaa, Eif4g2, Pax6, and Rbm5. Finally, we present a splicing browser Eye Splicer (http://www.iupui.edu/~sysbio/eye-splicer/), to facilitate exploration of developmentally altered splicing events and to improve understanding of post-transcriptional regulatory networks during mouse lens development.