Mouse Lens Research Articles

MAPK1 is required for establishing the pattern of cell proliferation and for cell survival during lens development

The mitogen-activated protein kinases (MAPKs; also known as ERKs) are key intracellular signaling molecules that are ubiquitously expressed in tissues and were assumed to be functionally equivalent. Here, we use the mouse lens as a model system to investigate whether MAPK1 plays a specific role during development. MAPK3 is known to be dispensable for lens development. We demonstrate that, although MAPK1 is uniformly expressed in the lens epithelium, its deletion significantly reduces cell proliferation in the peripheral region, an area referred to as the lens germinative zone in which most active cell division occurs during normal lens development. By contrast, cell proliferation in the central region is minimally affected by MAPK1 deletion. Cell cycle regulators, including cyclin D1 and survivin, are downregulated in the germinative zone of the MAPK1-deficient lens. Interestingly, loss of MAPK1 subsequently induces upregulation of phosphorylated MAPK3 (pMAPK3) levels in the lens epithelium; however, this increase in pMAPK3 is not sufficient to restore cell proliferation in the germinative zone. Additionally, MAPK1 plays an essential role in epithelial cell survival but is dispensable for fiber cell differentiation during lens development. Our data indicate that MAPK1/3 control cell proliferation in the lens epithelium in a spatially defined manner; MAPK1 plays a unique role in establishing the highly mitotic zone in the peripheral region, whereas the two MAPKs share a redundant role in controlling cell proliferation in the central region of the lens epithelium.

Are mouse lens epithelial cells more sensitive to γ-irradiation than lymphocytes?

In this pilot study we compared for the first time the radiation sensitivity of mouse lens epithelial cells (LECs) and mouse lymphocytes. We freshly prepared LECs and lymphocytes and irradiated them with γ-rays (137Cs; doses ranging from 0.25 to 2 Gy). DNA damage and repair were evaluated by alkaline comet assay and γH2AX foci assay. Using the comet assay, we observed a dose-dependent increase in DNA damage in both cell types. The faster formation of single- and double-strand breaks in LECs of C57BL/6 mice at doses below 1 Gy needs to be confirmed in other mouse strains. Immunofluorescence for γH2AX foci showed a higher degree of lesions in LECs from C57BL/6J mice compared to those of JF1 mice and to lymphocytes of both strains. Correspondingly, repair of DNA damage proceeded faster in LECs of C57BL/6J mice compared to LECs of JF1 mice and lymphocytes of both strains. It is obvious that the lymphocytes of both strains repaired DNA lesions more slowly than the corresponding LECs. In conclusion, our results demonstrate that LECs of C57Bl/6 mice show a steeper dose–response than lymphocytes in both types of experiments. It shows that both test systems are able to be used also at doses below 0.25 Gy. The observed difference in DNA repair between the LECs from C57BL/6J mice compared to the LECs from JF1 mice and to the lymphocytes of both strains warrants further experiments to identify the underlying molecular mechanisms.

Renewal versus Retention: Isotopic Composition of Intestinal Epithelium and Eye Lens

Aims: To compare isotopic signatures of contrasting (due to the structure and metabolism) organs in mice of two contrasting ages. Study Design: Cross-sectional study. Place and Duration of Study: Biology Department of Moscow State University; Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences; 2007–2011. Methodology: Mass spectrometric measurements of carbon and nitrogen isotopic ratios of jejunal epithelium and lens in 1and 22-mo mice fed a monotonous diet. Results: The lenses are enriched in carbon and nitrogen as compared with intestinal epithelium (by 5.5% and 4.5% in 1-mo mice and 8.3% and 6% in 22-mo mice, respectively). The N content is also higher in lenses than in intestinal epithelium (8.97‰ vs. 7.62‰ in 1-mo mice, and 7.40‰ vs. 6.58‰ in 22-mo mice). The C content of lenses exceeds that of intestinal epithelium in 1-mo mice (-20.27‰ vs. -21.69‰), Research Article British Journal of Medicine & Medical Research, 3(2): 210-215, 2013 211 although C content is equal in 22-mo mice (-22.56‰ vs. -22.67‰). N content is depleted in the intestinal epithelium of 22-mo mice (-1.04‰), whereas C depletion (-0.98‰) is non-significant. C and N content in lenses is also significantly decreased in 22-mo mice (-2.29‰ and -1.57‰). Conclusion: The intestinal epithelium represents a structure with short-term isotopic memory lasting a few days, whereas the events of the organism’s entire lifetime are retained in lens isotopic memory. The difference of the parameters measured is evidently determined by structural contrast, metabolic rate, and rejuvenation modes of the tissues. The N depletion in both the intestinal epithelium and lenses, as well as C depletion in lenses of 22-mo mice might be considered as a sign of ageing. In contrast, the depletion of N in lenses of 22-mo mice should be considered primarily as a result of dilution of breastmilk isotopic signature that probably obscures age-related alterations of the organ. Comparison of isotopic compositions of these contrasting organs may be useful for physiological and ecological determinations.

Verification and spatial localization of aquaporin-5 in the ocular lens

Until recently, the lens was thought to express only two aquaporin (AQP) water channels, AQP1 and AQP0. In this study we confirm lenticular AQP5 protein expression by Western blotting and mass spectrometry in lenses from a variety of species. In addition, confocal microscopy was used to map cellular distributions of AQP5 in mouse, rat and human lenses. Tandem mass spectrometry of a human lens membrane preparation revealed extensive sequence coverage (56.2%) of AQP5. Western blotting performed on total fiber cell membranes from mouse, rat, bovine and human lenses confirmed AQP5 protein expression is conserved amongst species. Western blotting of dissected lens fractions suggests that AQP5 is processed in the lens core by C-terminal truncation. Immunohistochemistry showed that AQP5 signal was most abundant in the lens outer cortex and decreased in intensity in the lens core. Furthermore, AQP5 undergoes differentiation-dependent changes in subcellular location from an intracellular localization in differentiating fiber cells to the plasma membrane of mature fiber cells upon the loss of fiber cell nuclei. Our results show that AQP5 is a significant component of lens fiber cell membranes, representing the second most abundant water channel in these cells. Together, the changes to AQP5 distribution and structure are likely to modulate the functional role of AQP5 in different regions of the lens.

The Effect of Size and Species on Lens Intracellular Hydrostatic Pressure

Previous experiments showed that mouse lenses have an intracellular hydrostatic pressure that varied from 335 mm Hg in central fibers to 0 mm Hg in surface cells. Model calculations predicted that in larger lenses, all else equal, pressure should increase as the lens radius squared. To test this prediction, lenses of different radii from different species were studied. All studies were done in intact lenses. Intracellular hydrostatic pressures were measured with a microelectrode-manometer-based system. Membrane conductances were measured by frequency domain impedance analysis. Intracellular Na(+) concentrations were measured by injecting the Na(+)-sensitive dye sodium-binding benzofuran isophthalate. Intracellular hydrostatic pressures were measured in lenses from mice, rats, rabbits, and dogs with radii (cm) 0.11, 0.22, 0.49, and 0.57, respectively. In each species, pressure varied from 335 ± 6 mm Hg in central fiber cells to 0 mm Hg in surface cells. Further characterization of transport in lenses from mice and rats showed that the density of fiber cell gap junction channels was approximately the same, intracellular Na(+) concentrations varied from 17 mM in central fiber cells to 7 mM in surface cells, and intracellular voltages varied from -45 mV in central fiber cells to -60 mV in surface cells. Fiber cell membrane conductance was a factor of 2.7 times larger in mouse than in rat lenses. Intracellular hydrostatic pressure is an important physiological parameter that is regulated in lenses from these different species. The most likely mechanism of regulation is to reduce the density of open Na(+)-leak channels in fiber cells of larger lenses.

Sulforaphane-induced transcription of thioredoxin reductase in lens: possible significance against cataract formation

PurposeSulforaphane is a phytochemically derived organic isothiocyanate 1-isothiocyanato-4-methylsulfinyl-butane present naturally in crucifers, including broccoli and cauliflower. Biochemically, it has been reported to induce the transcription of several antioxidant enzymes. Since such enzymes have been implicated in preventing cataract formation triggered by the intraocular generation of oxy-radical species, the purpose of this investigation was to examine whether it could induce the formation of antioxidant enzymes in the eye lens. Thioredoxin reductase (TrxR) was used as the target of such induction.MethodsMice lenses were cultured for an overnight period of 17 hours in medium 199 fortified with 10% fetal calf serum. Incubation was conducted in the absence and presence of sulforaphane (5 μM). Subsequently, the lenses were homogenized in phosphate-buffered saline (PBS), followed by centrifugation. TrxR activity was determined in the supernatant by measuring the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-dependent reduction of 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). Non-specific reduction of DTNB was corrected for by conducting parallel determinations in the presence of aurothiomalate. The reduction of DTNB was followed spectrophotometrically at 410 nm.ResultsThe activity of TrxR in the lenses incubated with sulforaphane was found to be elevated to 18 times of that observed in lenses incubated without sulforaphane. It was also noticeably higher in the lenses incubated without sulforaphane than in the un-incubated fresh lenses. However, this increase was much lower than that observed for lenses incubated with sulforaphane.ConclusionSulforaphane has been found to enhance TrxR activity in the mouse lens in culture. In view of the protective effect of the antioxidant enzymes and certain nutrients against cataract formation, the findings suggest that it would, by virtue of its ability to enhance the activity of such enzymes, prevent the tissue against oxidative stress that leads to cataract formation. Additional studies with the activities of other antioxidant enzymes such as quinone oxidoreductase and the levels of Nrf2 are in progress.

The LEGSKO Mouse: A Mouse Model of Age-Related Nuclear Cataract Based on Genetic Suppression of Lens Glutathione Synthesis

Age-related nuclear cataracts are associated with progressive post-synthetic modifications of crystallins from various physical chemical and metabolic insults, of which oxidative stress is a major factor. The latter is normally suppressed by high concentrations of glutathione (GSH), which however are very low in the nucleus of the old lens. Here we generated a mouse model of oxidant stress by knocking out glutathione synthesis in the mouse in the hope of recapitulating some of the changes observed in human age-related nuclear cataract (ARNC). A floxed Gclc mouse was generated and crossed with a transgenic mouse expressing Cre in the lens to generate the LEGSKO mouse in which de novo GSH synthesis was completely abolished in the lens. Lens GSH levels were reduced up to 60% in homozygous LEGSKO mice, and a decreasing GSH gradient was noticed from cortical to nuclear region at 4 months of age. Oxidation of crystallin methionine and sulfhydryls into sulfoxides was dramatically increased, but methylglyoxal hydroimidazolones levels that are GSH/glyoxalase dependent were surprisingly normal. Homozygous LEGSKO mice developed nuclear opacities starting at 4 months that progressed into severe nuclear cataract by 9 months. We conclude that the LEGSKO mouse lens mimics several features of human ARNC and is thus expected to be a useful model for the development of anti-cataract agents.

Tmod1 and CP49 Synergize to Control the Fiber Cell Geometry, Transparency, and Mechanical Stiffness of the Mouse Lens

The basis for mammalian lens fiber cell organization, transparency, and biomechanical properties has contributions from two specialized cytoskeletal systems: the spectrin-actin membrane skeleton and beaded filament cytoskeleton. The spectrin-actin membrane skeleton predominantly consists of α2β2-spectrin strands interconnecting short, tropomyosin-coated actin filaments, which are stabilized by pointed-end capping by tropomodulin 1 (Tmod1) and structurally disrupted in the absence of Tmod1. The beaded filament cytoskeleton consists of the intermediate filament proteins CP49 and filensin, which require CP49 for assembly and contribute to lens transparency and biomechanics. To assess the simultaneous physiological contributions of these cytoskeletal networks and uncover potential functional synergy between them, we subjected lenses from mice lacking Tmod1, CP49, or both to a battery of structural and physiological assays to analyze fiber cell disorder, light scattering, and compressive biomechanical properties. Findings show that deletion of Tmod1 and/or CP49 increases lens fiber cell disorder and light scattering while impairing compressive load-bearing, with the double mutant exhibiting a distinct phenotype compared to either single mutant. Moreover, Tmod1 is in a protein complex with CP49 and filensin, indicating that the spectrin-actin network and beaded filament cytoskeleton are biochemically linked. These experiments reveal that the spectrin-actin membrane skeleton and beaded filament cytoskeleton establish a novel functional synergy critical for regulating lens fiber cell geometry, transparency, and mechanical stiffness.

Regulation of Eaf2 in mouse lens cells apoptosis induced by ultraviolet radiation.

To investigate the regulation of Eaf2 protein in mouse lens cells apoptosis induced by ultraviolet (UV) radiation. An eye of Eaf2 gene knockout mice or normal control mice was exposed to UV radiation, and the other one was non-exposed. All of lenses were analyzed by TUNEL and caspase 3 activity assays to determine the difference of the apoptosis induced by UV radiation. In addition, exposed and non-exposed lenses were analyzed by quantified p53 expression and real-time reverse transcription-polymerase chain reaction (RT-PCR) of Bax, Bid, Apaf-1, Puma and Noxa, to compare Eaf2 gene knockout mice and normal control mice. UV radiation caused apoptosis of lens cells in normal control mice and Eaf2 knockout mice. Activity of caspase 3 was significantly higher in normal control mice than Eaf2 knockout mice. Expression of p53 protein was significantly higher in lenses exposed to UV radiation than nonexposed lenses, but was similar between Eaf2 gene knockout mice and normal control mice in the same UV condition. After exposing to UV radiation, the analysis of real-time RT-PCR demonstrated that mRNA levels of Puma and Noxa were significantly higher in lenses of normal control mice than Eaf2 gene knockout mice, and that mRNA levels of Bax, Bid and Apaf-1 were not significantly different between gene knockout mice and normal control mice. Eaf2 increases lens cells apoptosis induced by ultraviolet radiation. And Eaf2 up-regulates expression of the Puma and the Noxa to act on lens cells apoptosis after UV radiation.

The p53-Bak Apoptotic Signaling Axis Plays an Essential Role in Regulating Differentiation of the Ocular Lens

The tumor suppressor p53 is a master regulator of apoptosis and also plays a key role in cell cycle checking. In our previous studies, we demonstrated that p53 directly regulates Bak in mouse JB6 cells (Qin et al. 2008. Cancer Research. 68(11):4150) and that p53-Bak signaling axis plays an important role in mediating EGCG-induced apoptosis. Here, we demonstrate that the same p53-Bak apoptotic signaling axis executes an essential role in regulating lens cell differentiation. First, during mouse lens development, p53 is expressed and differentially phosphorylated at different residues. Associated with p53 expression, Bak is also significantly expressed during mouse lens development. Second, human p53 directly regulates Bak promoter and Bak expression in p53 knockout mice (p53-/-) was significantly downregulated. Third, during in vitro bFGF-induced lens cell differentiation, knockdown of p53 or Bak leads to significant inhibition of lens cell differentiation. Fourth, besides the major distribution of Bak in cytoplasm, it is also localized in the nucleus in normal lens or bFGF-induced differentiating lens cells. Finally, p53 and Bak are co-localized in both cytoplasm and nucleus, and their interaction regulates the stability of p53. Together, these results demonstrate for the first time that the p53-Bak apoptotic signaling axis plays an essential role in regulating lens differentiation.

85 Medical applications of Intravenous laser blood irradiation and LED phototherapy in sport medicine

We have investigated how connexin 46 (Cx46) regulates lens stiffness by studying different Cx46 knockout (Cx46KO) mice. A modified muscle lever system was used to determine the lens stiffness of wild-type (WT) and Cx46KO mice at the C57BL/6J (B6) and the 129SvJae (129) strain backgrounds according to total lens displacement at the point of maximum force when fresh lenses were compressed with a maximum of 2 mN of force. In comparison to B6-WT controls, young and old B6-Cx46KO lenses showed 23% and 28% reductions in lens displacement, respectively. Comparing to 129-WT controls, old 129-Cx46KO lenses showed 50% reduction in the lens displacement while young 129-Cx46KO lenses displayed similar displacement. Old B6-Cx46KO and old 129-Cx46KO lenses showed almost identical lens displacement, 128 μm versus 127 μm. Morphological data revealed unique changes of peripheral fiber cell shapes in young B6-WT lenses but not in young B6-Cx46KO, 129-WT and 129-Cx46KO lenses. This work reveals Cx46 deletion increases the lens stiffness in both young and old mice at B6 strain background but only in old mice at 129 strain background which contains intermediate filament CP49 gene deletion. Cx46 impairment increases old mouse lens stiffness and may contribute to the development of presbyopia.

A role for γS-crystallin in the organization of actin and fiber cell maturation in the mouse lens.

γS-crystallin (γS) is a highly conserved component of the eye lens. To gain insights into the functional role(s) of this protein, the mouse gene (Crygs) was deleted. Although mutations in γS can cause severe cataracts, loss of function of γS in knockout (KO) mice produced no obvious lens opacity, but was associated with focusing defects. Electron microscopy showed no major differences in lens cell organization, suggesting that the optical defects are primarily cytoplasmic in origin. KO lenses were also grossly normal by light microscopy but showed evidence of incomplete clearance of cellular organelles in maturing fiber cells. Phalloidin labeling showed an unusual distribution of F-actin in a band of mature fiber cells in KO lenses, suggesting a defect in the organization or processing of the actin cytoskeleton. Indeed, in wild-type lenses, γS and F-actin colocalize along the fiber cell plasma membrane. Relative levels of F-actin and G-actin in wild-type and KO lenses were estimated from fluorescent staining profiles and from isolation of actin fractions from whole lenses. Both methods showed a two-fold reduction in the F-actin/G-actin ratio in KO lenses, whereas no difference in tubulin organization was detected. In vitro experiments showed that recombinant mouse γS can directly stabilize F-actin. This suggests that γS may have a functional role related to actin, perhaps in 'shepherding' filaments to maintain the optical properties of the lens cytoplasm and normal fiber cell maturation.

The unfolded protein response is activated in connexin 50 mutant mouse lenses

The unfolded protein response is a set of cell signaling pathways recently recognized to be activated in the lens during both normal development and endoplasmic reticulum stress induced by either unfolded proteins or oxidative damage. While mutations in the gene for connexin 50 are known to cause autosomal dominant cataracts, it has not been previously reported whether mutant connexins can activate the unfolded protein response in the lens. Mice homozygous for the S50P or G22R mutation of connexin 50 have reduced amounts of connexin 50 protein at the cell membrane, with some intracellular staining consistent with retention in the endoplasmic reticulum. Connexin 50 mutants have elevated levels of BiP expression in both lens epithelial and fiber cells from E15.5 with the most robust elevation detected in newborns. While this elevation decreases in magnitude postnatally, BiP expression is still abnormally high in adults, particularly in the perinuclear endoplasmic reticulum of cell nuclei that are inappropriately retained in adult homozygous mutant lenses. Xbp1 splicing was elevated in lenses from both connexin mutants studied, while Atf4 and Atf6 levels were not majorly affected. Overall, these data suggest that UPR may be a contributing factor to the phenotype of connexin 50 mutant lenses even though the relatively modest extent of the response suggests that it is unlikely to be a major driver of the pathology.

Activation of the Hedgehog Signaling Pathway in the Developing Lens Stimulates EctopicFoxE3Expression and Disruption in Fiber Cell Differentiation

The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens. We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm. Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death. Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3.

Human cataract mutations in EPHA2 SAM domain alter receptor stability and function.

The cellular and molecular mechanisms underlying the pathogenesis of cataracts leading to visual impairment remain poorly understood. In recent studies, several mutations in the cytoplasmic sterile-α-motif (SAM) domain of human EPHA2 on chromosome 1p36 have been associated with hereditary cataracts in several families. Here, we have investigated how these SAM domain mutations affect EPHA2 activity. We showed that the SAM domain mutations dramatically destabilized the EPHA2 protein in a proteasome-dependent pathway, as evidenced by the increase of EPHA2 receptor levels in the presence of the proteasome inhibitor MG132. In addition, the expression of wild-type EPHA2 promoted the migration of the mouse lens epithelial αTN4-1 cells in the absence of ligand stimulation, whereas the mutants exhibited significantly reduced activity. In contrast, stimulation of EPHA2 with its ligand ephrin-A5 eradicates the enhancement of cell migration accompanied by Akt activation. Taken together, our studies suggest that the SAM domain of the EPHA2 protein plays critical roles in enhancing the stability of EPHA2 by modulating the proteasome-dependent process. Furthermore, activation of Akt switches EPHA2 from promoting to inhibiting cell migration upon ephrin-A5 binding. Our results provide the first report of multiple EPHA2 cataract mutations contributing to the destabilization of the receptor and causing the loss of cell migration activity.

Mutually inductive interactions between the lens and retina require ALK3 functions during mouse embryonic development.

To investigate the mutually inductive interactions that occur between the lens and retinal tissue during the development of the vertebrate eye. Cre-positive mice were mated with Cre-negative mice to generate 50% Cre-positive (conditional knockout, CKO) and 50% Cre-negative offspring (wild type, WT). The embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4µm. The sections were processed for hematoxylin and eosin staining. The primary antibody used for immunofluorescence detection was sc-9305 bone morphogenetic proteins (bmp7) (Santa Cruz, US). The secondary antibody was IF-0314 aG0IgG/FITC (Santa Cruz) in combination with the primary antibody. Bright-field and fluorescent images were taken. Changes in the lens and retina were associated with specific alterations to the expression of type IA BMP receptor [BMPR-IA (ALK3)], which have already been implicated in eye growth. BMPR-IA was required for lens and retinal growth, but was not essential for the formation of lens. We observed that the expression of Bmp7 in the embryonic retina was reduced in the ALK3 lens of CKO mice. This phenomenon became increasingly visible in accordance with embryo development. This apparent alteration was present at stage E15.5. ALK3 is essential for lens and retinal growth. Mutually inductive interactions between the lens and retina are present in the developing mouse eye.

Characterization of the EMT response in Crybb2Phil mutants

The most abundant protein in the adult lens fiber cells is βB2‐crystallin (Crybb2). Mutations in the Crybb2 gene cause cataracts in mice and humans. A twelve nucleotide deletion in mouse lens epithelial cells (LEC), known as the Crybb2Phil mutation, leads to cataracts associated with lens fiber cell defects as well as epithelial to mesenchymal transition (EMT) of the LEC in adulthood. However, the mechanism behind this phenotype is poorly understood. The aim of this project is to characterize the EMT response caused by the Crybb2Phil mutation.Tissues obtained from Crybb2Phil mutants were immunostained for alpha and beta integrin subunits, phospo‐SMAD3 (p‐SMAD3), transforming growth factor β‐induced (BIGH3), and EMT the markers E‐cadherin, vimentin, follistatin, and α‐smooth muscle actin (α‐SMA).Compared to the wildtype lens, β1, αV, and α5 integrins, vimentin, α‐SMA, and p‐SMAD3 were upregulated in the Crybb2Phil mice. No changes were seen in α2, α3, and α6 integrin subunits or follistatin. Consistent with EMT, E‐cadherin was downregulated in the Crybb2Phil mice.Upregulation of α‐SMA signifies that Crybb2Phil mice undergo EMT around two months of age. Furthermore, over expression of β1 and αV integrins along with p‐SMAD3 in the Crybb2Phil mutant suggests that the EMT seen in Crybb2Phil mice may be occurring via the TGFβ‐SMAD interacting pathway. This project is funded by National Eye Institute grant EY015279.

ISyTE:IntegratedSystemsTool forEye Gene Discovery

To facilitate the identification of genes associated with cataract and other ocular defects, the authors developed and validated a computational tool termed iSyTE (integrated Systems Tool for Eye gene discovery; http://bioinformatics.udel.edu/Research/iSyTE). iSyTE uses a mouse embryonic lens gene expression data set as a bioinformatics filter to select candidate genes from human or mouse genomic regions implicated in disease and to prioritize them for further mutational and functional analyses. Microarray gene expression profiles were obtained for microdissected embryonic mouse lens at three key developmental time points in the transition from the embryonic day (E)10.5 stage of lens placode invagination to E12.5 lens primary fiber cell differentiation. Differentially regulated genes were identified by in silico comparison of lens gene expression profiles with those of whole embryo body (WB) lacking ocular tissue. Gene set analysis demonstrated that this strategy effectively removes highly expressed but nonspecific housekeeping genes from lens tissue expression profiles, allowing identification of less highly expressed lens disease-associated genes. Among 24 previously mapped human genomic intervals containing genes associated with isolated congenital cataract, the mutant gene is ranked within the top two iSyTE-selected candidates in approximately 88% of cases. Finally, in situ hybridization confirmed lens expression of several novel iSyTE-identified genes. iSyTE is a publicly available Web resource that can be used to prioritize candidate genes within mapped genomic intervals associated with congenital cataract for further investigation. Extension of this approach to other ocular tissue components will facilitate eye disease gene discovery.

αA- and αB-Crystallins Interact with Caspase-3 and Bax to Guard Mouse Lens Development

The small heat shock protein, α-crystallin, exists in two isoforms, αA and αB, and displays strong ability against stress-induced apoptosis. Regarding their functional mechanisms, we and others have demonstrated that they are able to regulate members in both caspase and Bcl-2 families. In addition, we have also shown that αA and αB may display differential anti-apoptotic mechanisms under certain stress conditions. While αA-crystallin regulates activation of the AKT signaling pathway, αB negatively regulates the MAPK pathway to suppress apoptosis induced by UV and oxidative stress. Although previous studies revealed that αA and αB could regulate members in both caspase and Bcl-2 families, the molecular mechanism, especially the in vivo regulation still waits to be elucidated. In the present communication, we present both in vitro and in vivo evidence to further demonstrate the regulation of caspase-3 and Bax by αA and αB. First, Surface Plasmon Resonance (SPR) and yeast two-hybrid selection analysis demonstrate that αA and αB directly bind to caspase-3 and Bax with differential affinities. Second, immunohistochemistry reveals that αA and αB regulate caspase-3 and Bax at different developmental stages of mouse embryo. Third, coimmunoprecipitation shows that αA and αB form in vivo interacting complexes with caspase-3 and Bax. Together, our results further confirm that αA and αB regulate caspase-3 and Bax in vitro and in vivo to regulate lens differentiation.

Requirements for Jag1‐Rbpj mediated Notch signaling during early mouse lens development

During vertebrate lens development, the lens placode in the embryonic ectoderm invaginates into a lens vesicle, which then separates from the surface epithelium, followed by two waves of fiber cell differentiation. In the mouse, multiple labs have shown that Jag1-Notch signaling is critically required during the second wave of lens fiber cell formation. However, Notch signaling appears to play no obvious role during lens induction or morphogenesis, although multiple pathway genes are expressed at these earlier stages. Here, we explored functions for Notch signaling specifically during early lens development, by using the early-acting AP2α-Cre driver to delete Jag1 or Rbpj. We found that Jag1 and Rbpj are not required during lens induction, but are necessary for proper lens vesicle separation from the surface ectoderm. We conclude that precise levels of Notch signaling are essential during lens vesicle morphogenesis. In addition, AP2α-Cre-mediated deletion of Rbpj resulted in embryos with cardiac outflow tract and liver deformities, and perinatal lethality.