In the early embryo, the eyes form initially as relatively spherical optic vesicles (OVs) that protrude from both sides of the brain tube. Each OV grows until it contacts and adheres to the overlying surface ectoderm (SE) via an extracellular matrix (ECM) that is secreted by the SE and OV. The OV and SE then thicken and bend inward (invaginate) to create the optic cup (OC) and lens vesicle, respectively. While constriction of cell apices likely plays a role in SE invagination, the mechanisms that drive OV invagination are poorly understood. Here, we used experiments and computational modeling to explore the hypothesis that the ECM locally constrains the growing OV, forcing it to invaginate. In chick embryos, we examined the need for the ECM by (1) removing SE at different developmental stages and (2) exposing the embryo to collagenase. At relatively early stages of invagination (Hamburger–Hamilton stage HH14 $$-$$ ), removing the SE caused the curvature of the OV to reverse as it ‘popped out’ and became convex, but the OV remained concave at later stages (HH15) and invaginated further during subsequent culture. Disrupting the ECM had a similar effect, with the OV popping out at early to mid-stages of invagination (HH14 $$-$$ to HH14 $$+$$ ). These results suggest that the ECM is required for the early stages but not the late stages of OV invagination. Microindentation tests indicate that the matrix is considerably stiffer than the cellular OV, and a finite-element model consisting of a growing spherical OV attached to a relatively stiff layer of ECM reproduced the observed behavior, as well as measured temporal changes in OV curvature, wall thickness, and invagination depth reasonably well. Results from our study also suggest that the OV grows relatively uniformly, while the ECM is stiffer toward the center of the optic vesicle. These results are consistent with our matrix-constraint hypothesis, providing new insight into the mechanics of OC (early retina) morphogenesis.

Purpose: The hypoxic environment around the lens is important for maintaining lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption to assess the role of human LECs in maintaining hypoxia around the lens, as well as the impact of systemic and ocular diagnosis on these cells.Methods: Baseline cellular respiration was measured in rabbit LECs (NN1003A), canine kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and bovine corneal endothelial cells (CCEE) using a XF96 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro. Following informed written consent, lens capsule epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR was normalized to the number of cells per well using rabbit LECs as a standard. The effect of patient age, sex, race, and presence of diabetes or glaucoma on oxygen consumption was assessed by using the Mann-Whitney U test and multivariate regression analysis.Results: Primary LECs were obtained from 69 patients. The OCR from donors aged 70 and over was lower than that of those under 70 years (2.21 +/- 1.037 vs. 2.86 +/- 1.383 fmol/min/cell; p < 0.05). Diabetic patients had lower OCR than non-diabetic patients (2.02 +/- 0.911 vs. 2.79 +/- 1.332 fmol/min/cell; p < 0.05), and glaucoma patients had lower OCR than non-glaucoma patients (2.27 +/- 1.19 vs. 2.83 +/- 1.286 fmol/min/cell; p < 0.05). Multivariate regression analysis confirmed that donors aged 70 and over (p < 0.05), diabetic patients (p < 0.01), and glaucoma patients (p < 0.05) had significantly lower OCR, independent of other variables. Gender and race had no significant effect on OCR.Conclusions: The lower oxygen consumption rate of human LECs in older donors and patients with diabetes or glaucoma could contribute to cataract development. Diabetes and glaucoma are particularly important factors associated with decreased OCR, independent of age. Ongoing studies are examining pO(2) at the anterior surface of the lens in vivo and oxygen consumption in the patient's LECs. (C) 2016 Elsevier B.V. All rights reserved.

This unit describes a protocol for embedding, sectioning, and immunocytochemical analysis of pluripotent stem cell-derived 3-D organoids. Specifically, we describe a method to embed iPSC-derived retinal cups in low-melt agarose, acquire thick sections using a vibratome tissue slicer, and perform immunohistochemical analysis. This method includes an approach for antibody labeling that minimizes the amount of antibody needed for individual experiments and that utilizes large-volume washing to increase the signal-to-noise ratio, allowing for clean, high-resolution imaging of developing cell types. The universal methods described can be employed regardless of the type of pluripotent stem cell used and 3-D organoid generated. © 2016 by John Wiley & Sons, Inc.

Precise regulation of eye development is required to produce good visual acuity. Tissue interactions between the optic vesicle, surface ectoderm and neural crest and mesodermal mesenchyme cells assure alignment of the retina, lens and cornea. Coordination of ocular development depends on the deployment of nearly every type of signaling pathway known to function in embryogenesis, including classical growth factors, TGFβ superfamily molecules, Hedgehog, Notch and Wnt signaling and nuclear receptors for retinoids. These signals elicit the expression of transcription factors that guide cell type specific morphogenesis, differentiation and function, assuring the transparency of the cornea and lens, the detection of light and the processing of visual signals by the retina and the proper alignment of the eyes by the extraocular muscles. Defects in these processes or pathways can cause absence of the eyes, aberrant morphogenesis, or defective function of the ocular components, resulting in blindness or severe ocular dysfunction.

Although linked to several vitreoretinal pathologies including traumatic retinal tears, breaks, and symptomatic vitreomacular traction, the dynamic material behavior of the vitreous body in response to mechanical loads is not well understood. The purpose of this study was to evaluate spatiotemporal patterns of collagen fiber reorganization and vitreous deformation (strain) in response to tensile and compressive forces. Using thick slabs of bovine eyes we examined collagen fiber reorganization following tensile and compressive step-loading with quantitative polarized light imaging. Strains were measured from sparse marker arrays and temporal collagen behavior was estimated from creep compliance rheological tests. Results showed that under applied loads (1) collagen fibers became significantly more aligned at the vitreous base (near the pars plana and the ciliary body), (2) vitreous located directly behind the lens deformed significantly more than surrounding regions, and (3) changes in collagen fiber alignment occurred on a short (<5 s) timescale. Together these results show that, despite a homogeneous visual appearance, the vitreous body exhibits anisotropic material behavior in tension and compression. Spatiotemporal patterns of collagen rearrangement were consistent with epidemiological patterns of traumatic retinal damage and vitreoretinal topology. High strains in the vitreous corresponded with locations of lower collagen content that are prone to age-related degeneration. These data suggest that differential fiber alignment and mechanical deformation could contribute to the pathogenesis of these diseases. Computational models that incorporate these experimental data will help improve our understanding of the biomechanical mechanisms that contribute to the pathogenesis of traumatic retinal damage, vitreous degeneration, and vitreoretinal disease.

Purpose Ascorbic acid (AsA) is an important antioxidant in the eye. Ascorbic acid is usually transported by sodium-dependent AsA transporters (SVCTs), and dehydroascorbic acid (DHA) by glucose transporters (GLUTs). This study investigates these AsA-related transporters in human compared with mouse eyes. Methods Five pairs of human donor eyes and 15 pairs of mouse eyes were collected. Immunofluorescence and in situ hybridization were performed to detect SVCTs and GLUTs expression in the ciliary epithelium, retina, and lens epithelial cells (LECs). These tissues were isolated with laser microdissection followed by extraction of total RNA. Quantitative PCR (qPCR) was performed to examine the mRNA level of SVCTs and GLUTs in human and mouse ocular tissues. Results Immunofluorescence and in situ hybridization showed SVCT2 and GLUT1 expression in human ciliary epithelium with varied distributions. Sodium-dependent AsA transporter 2 is expressed only in the pigmented epithelium (PE), and GLUT1 is predominately expressed in the nonpigmented epithelium (NPE). However, SVCT2 was not identified in mouse ciliary epithelium, whereas GLUT1 expressed in both PE and NPE. Laser microdissection and qPCR revealed high levels of SVCT2 mRNA in human RPE cells and murine neural retina. Sodium-dependent AsA transporter 1 mRNA could be detected only in human and murine LECs. Glucose transporter 3 and GLUT4 mRNA could not be detected in either the human or mouse ciliary processes or in the lens epithelium. Conclusions These fundamental findings indicate AsA transporter expression in eyes of humans is significantly different compared with mice. This may explain why human aqueous and vitreous humors contain higher AsA levels compared with other animals.

Background It was recently demonstrated that deficiency of a conserved RNA binding protein (RBP) and RNA granule (RG) component Tdrd7 causes ocular defects including cataracts in human, mouse and chicken, indicating the importance of posttranscriptional regulation in eye development. Here we investigated the function of a second conserved RBP/RG component Caprin2 that is identified by the eye gene discovery tool iSyTE . Results In situ hybridization, Western blotting and immunostaining confirmed highly enriched expression of Caprin2 mRNA and protein in mouse embryonic and postnatal lens. To gain insight into its function, lens‐specific Caprin2 conditional knockout (cKO) mouse mutants were generated using a lens‐Cre deleter line Pax6GFPCre . Phenotypic analysis of Caprin2 cKO/cKO mutants revealed distinct eye defects at variable penetrance. Wheat germ agglutinin staining and scanning electron microscopy demonstrated that Caprin2 cKO/cKO mutants have an abnormally compact lens nucleus, which is the core of the lens comprised of centrally located terminally differentiated fiber cells. Additionally, Caprin2 cKO/cKO mutants also exhibited at 8% penetrance a developmental defect that resembles a human condition called Peters anomaly, wherein the lens and the cornea remain attached by a persistent stalk. Conclusions These data suggest that a conserved RBP Caprin2 functions in distinct morphological events in mammalian eye development. Developmental Dynamics 244:1313–1327, 2015 . © 2015 Wiley Periodicals, Inc.

Previous studies of mouse embryos concluded that after the optic vesicle evaginates from the ventral forebrain and contacts the surface ectoderm, signals from the ectoderm specify the distal region of the optic vesicle to become retina and signals from the optic vesicle induce the lens. Germline deletion of Bmp4 resulted in failure of lens formation. We performed conditional deletion of Bmp4 from the optic vesicle to test the function of Bmp4 in murine eye development. The optic vesicle evaginated normally and contacted the surface ectoderm. Lens induction did not occur. The optic cup failed to form and the expression of retina-specific genes decreased markedly in the distal optic vesicle. Instead, cells in the prospective retina expressed genes characteristic of the retinal pigmented epithelium. We conclude that Bmp4 is required for retina specification in mice. In the absence of Bmp4, formation of the retinal pigmented epithelium is the default differentiation pathway of the optic vesicle. Differences in the signaling pathways required for specification of the retina and retinal pigmented epithelium in chicken and mouse embryos suggest major changes in signaling during the evolution of the vertebrate eye.

551–561. Chawla, M., Safwat, A.-A., Romina, O., & Cavallo, L. (2014). Higher order structural effects stabilizing the reverse Watson–Crick guanine–cytosine base pair in functional RNAs. Nucleic Acids Research, 42, 714–726. Serra, M. J., Baird, J. D., Taraka, D., Fey, B. L., Retatagos, K., & Westhof, E. (2002). Effects of magnesium ions on the stabilization of RNA oligomers of defined structures. RNA, 8, 307–323. Réblová, K., Špačková, N., Koča, J., Leontis, N. B., & Šponer, J. (2004). Long-residency hydration, cation binding, and dynamics of loop E/helix IV rRNA-L25 protein complex. Biophysical Journal, 87, 3397–3412.

To explore how limbal niche cells (LNCs) may control quiescence, self-renewal, and corneal epithelial lineage commitment/differentiation of limbal epithelial progenitor/stem cells (LEPCs), we have established an in vitro sphere assay by reunion between the two cell types in three-dimensional Matrigel. The resultant sphere exhibits inhibition of corneal epithelial lineage commitment/differentiation and marked clonal growth of LEPCs, of which the latter is correlated with activation of canonical Wnt signaling. Herein, we have created a similar reunion assay in immobilized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3), which is purified from amniotic membrane (AM) and consists of a complex formed by hyaluronic covalently linked to heavy chain 1 of inter-α-inhibitor and noncovalently linked to pentraxin 3. The resultant spheres exhibited similar suppression of corneal epithelial lineage commitment/differentiation but upregulation of quiescence markers including nuclear translocation of Bmi-1, and negligible clonal growth of LEPCs. This outcome was correlated with the suppression of canonical Wnt but activation of noncanonical (Planar cell polarity) Wnt signaling as well as BMP signaling in both LEPCs and LNCs. The activation of BMP signaling in LNCs was pivotal because nuclear translocation of pSmad1/5/8 was prohibited in hLEPCs when reunioned with mLNCs of conditionally deleted Bmpr1a;Acvr1 DCKO mice. Furthermore, ablation of BMP signaling in LEPCs led to upregulation of cell cycle genes, downregulation of Bmi-1, nuclear exclusion of phosphorylated Bmi-1, and marked promotion of the clonal growth of LEPCs. Hence, HC-HA/PTX3 uniquely upregulates BMP signaling in LNCs which leads to BMP signaling in LEPCs to achieve quiescence, helping explain how AM transplantation is clinically useful to be used as a matrix for ex vivo expansion of LEPCs and to treat corneal blindness caused by limbal stem cells deficiency. S tem C ells 2015;33:3341–3355

PURPOSE:The goal of this study was to quantitatively identify the differentially expressed proteins in nuclear cataracts of different ages and normal lens nuclei in humans.EXPERIMENTAL DESIGN:Forty-eight human lens nucleus samples with hardness grades III, IV were obtained during cataract surgery by extracapsular cataract extraction. Seven normal transparent human lens nuclei were obtained from fresh normal cadaver eyes during corneal transplantation surgery. Lens nuclei were divided into seven groups according to age and optic axis: Group A (average age 80.8 ± 1.2 years), Group B (average age 57.0 ± 4.0 years), Group C average age 80.3 ± 4.5 years), Group D (average age 56.9 ± 4.2 years), Group E (average age 78.1 ± 2.5 years), Group F (average age 57.6 ± 3.3 years) and Group G (seven normal transparent human lenses from normal cadaver eyes, average age 34.7 ± 4.2 years). Water-soluble, water-insoluble, and water-insoluble-urea-soluble protein fractions were extracted from samples. The three-part protein fractions from the individual lenses were combined to form the total proteins of each sample. The proteomic profiles of each group were further analyzed using 8-plex iTRAQ labeling combined with 2D-LC-MS/MS. The data were analyzed with the ProteinPilot software for peptide matching, protein identification, and quantification. Differentially expressed proteins were validated by Western blotting.RESULTS:We employed biological and technical replicates and selected the intersection of the two results, which included 80 proteins. Nine proteins were differentially expressed among the 80 proteins identified using proteomic techniques. In age-related nuclear cataracts (ARNC), the expression levels of fatty acid-binding protein and pterin-4-alpha-carbinolamine dehydratase were upregulated, whereas the levels of alpha-crystallin B chain (CRYAB), GSH synthetase, phakinin, gamma-crystallin C, phosphoglycerate kinase 1, betaine-homocysteine S-methyltransferase 1 (BHMT1), and spectrin beta chain were downregulated. These proteins may be associated with abnormal protein aggregation and oxidative stress. GSH synthetase and CRYAB expression levels in the nuclear cataract decreased with age. The mass spectrometric analysis results were consistent with the Western blot validation.CONCLUSION AND CLINICAL RELEVANCE:The results indicate that CRYAB and GSH synthetase may be involved in ARNC pathogenesis. iTRAQ combined with 2D-LC-MS/MS provides new methods for future studies of pathological mechanisms and protective drug development for ARNC.

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Previous studies reported that characteristic lens opacities were present in Alzheimer Disease (AD) patients postmortem. We therefore determined whether cataract grade or lens opacity is related to the risk of Alzheimer dementia in participants who have biomarkers that predict a high risk of developing the disease. AD biomarker status was determined by positron emission tomography-Pittsburgh compound B (PET-PiB) imaging and cerebrospinal fluid (CSF) levels of Aβ42. Cognitively normal participants with a clinical dementia rating of zero (CDR = 0; N = 40) or with slight evidence of dementia (CDR = 0.5; N = 2) were recruited from longitudinal studies of memory and aging at the Washington University Knight Alzheimer's Disease Research Center. The age, sex, race, cataract type and cataract grade of all participants were recorded and an objective measure of lens light scattering was obtained for each eye using a Scheimpflug camera. Twenty-seven participants had no biomarkers of Alzheimer dementia and were CDR = 0. Fifteen participants had biomarkers indicating increased risk of AD, two of which were CDR = 0.5. Participants who were biomarker positive were older than those who were biomarker negative. Biomarker positive participants had more advanced cataracts and increased cortical light scattering, none of which reached statistical significance after adjustment for age. We conclude that cataract grade or lens opacity is unlikely to provide a non-invasive measure of the risk of developing Alzheimer dementia.

Previous results have shown that Bone Morphogenetic Protein (BMP) signaling is essential for lens specification and differentiation. How BMP signals are regulated in the prospective lens ectoderm is not well defined. To address this issue we have modulated BMP activity in a chicken embryo pre-lens ectoderm explant assay, and also studied transgenic mice, in which the type I BMP receptors, Bmpr1a and Acvr1, are deleted from the prospective lens ectoderm. Our results show that chicken embryo pre-lens ectoderm cells express BMPs and require BMP signaling for lens specification in vitro, and that in vivo inhibition of BMP signals in the mouse prospective lens ectoderm interrupts lens placode formation and prevents lens invagination. Furthermore, our results provide evidence that BMP expression is negatively auto-regulated in the lens-forming ectoderm, decreasing when the tissue is exposed to exogenous BMPs and increasing when BMP signaling is prevented. In addition, eyes lacking BMP receptors in the prospective lens placode develop coloboma in the adjacent wild type optic cup. In these eyes, Bmp7 expression increases in the ventral optic cup and the normal dorsal–ventral gradient of BMP signaling in the optic cup is disrupted. Pax2 becomes undetectable and expression of Sfrp2 increases in the ventral optic cup, suggesting that increased BMP signaling alter their expression, resulting in failure to close the optic fissure. In summary, our results suggest that negative and positive auto-regulation of BMP expression is important to regulate early eye development.

This study evaluated in vivo imaging capabilities and safety of qualitative monitoring of oxygen saturation of hemoglobin (sO(2)) of rabbit ciliary body tissues obtained with acoustic resolution (AR) photoacoustic tomography (PAT). AR PAT was used to collect trans-scleral images from ciliary body vasculature of seven New Zealand White rabbits. The PAT sO(2) measurements were obtained under the following conditions: when systemic 502 as measured by pulse oximetry was between 100% and 99% (level 1); systemic sO(2) as measured by pulse oximetry was between 98% and 90% (level 2); and systemic sO(2) as measured by pulse oximetry was less than 90% (level 3). Following imaging, histological analysis of ocular tissue was conducted to evaluate for possible structural damage caused by the AR PAT imaging. AR PAT was able to resolve anatomical structures of the anterior segment of the eye, viewed through the cornea or anterior sclera. Histological studies revealed no ocular damage. On average, sO(2) values (%) obtained with AR PAT were lower than sO(2) values obtained with pulse oximetry (all p < 0.001): 86.28 +/- 4.16 versus 99.25 +/- 0.28, 84.09 +/- 1.81 vs. 95.3 +/- 2.6, and 64.49 +/- 7.27 vs. 71.15 +/- 10.21 for levels 1, 2 and 3 respectively. AR PAT imaging modality is capable of qualitative monitoring for deep tissue sO(2) in rabbits. Further studies are needed to validate and modify the AR PAT modality specifically for use in human eyes. Having a safe, non-invasive method of in vivo imaging of sO(2) in the anterior segment is important to studies evaluating the role of oxidative damage, hypoxia and ischemia in pathogenesis of ocular diseases. Published by Elsevier Ltd.