TOB1 and TOB2 mark distinct RNA processing granules in differentiating lens fiber cells.

Differentiation of lens fiber cells involves a complex interplay of signals from growth factors together with tightly regulated gene expression via transcriptional and post-transcriptional regulators. Various studies have demonstrated that RNA-binding proteins, functioning in ribonucleoprotein granules, have important roles in regulating post-transcriptional expression during lens development. In this study, we examined the expression and localization of two members of the BTG/TOB family of RNA-binding proteins, TOB1 and TOB2, in the developing lens and examined the phenotype of mice that lack Tob1. By RT-PCR, both Tob1 and Tob2 mRNA were detected in epithelial and fiber cells of embryonic and postnatal murine lenses. In situ hybridization showed Tob1 and Tob2 mRNA were most intensely expressed in the early differentiating fibers, with weaker expression in anterior epithelial cells, and both appeared to be downregulated in the germinative zone of E15.5 lenses. TOB1 protein was detected from E11.5 to E16.5 and was predominantly detected in large cytoplasmic puncta in early differentiating fiber cells, often co-localizing with the P-body marker, DCP2. Occasional nuclear puncta were also observed. By contrast, TOB2 was detected in a series of interconnected peri-nuclear granules, in later differentiating fiber cells of the inner cortex. TOB2 did not appear to co-localize with DCP2 but did partially co-localize with an early stress granule marker (EIF3B). These data suggest that TOB1 and TOB2 are involved with different aspects of the mRNA processing cycle in lens fiber cells. In vitro experiments using rat lens epithelial explants treated with or without a fiber differentiating dose of FGF2 showed that both TOB1 and TOB2 were up-regulated during FGF-induced differentiation. In differentiating explants, TOB1 also co-localized with DCP2 in large cytoplasmic granules. Analyses of Tob1-/- mice revealed relatively normal lens morphology but a subtle defect in cell cycle arrest of some cells at the equator and in the lens fiber mass of E13.5 embryos. Overall, these findings suggest that TOB proteins play distinct regulatory roles in RNA processing during lens fiber differentiation.

Genotype, Age, Genetic Background, and Sex Influence Epha2-Related Cataract Development in Mice.

Purpose: Age-related cataract is the leading cause of blindness worldwide. Variants in the EPHA2 gene increase the disease risk, and its knockout in mice causes cataract. We investigated whether age, sex, and genetic background, risk factors for age-related cataract, and Epha2 genotype influence Epha2-related cataract development in mice. Methods: Cataract development was monitored in Epha2+/+, Epha2+/-, and Epha2-/- mice (Epha2Gt(KST085)Byg) on C57BL/6J and FVB:C57BL/6J (50:50) backgrounds. Cellular architecture of lenses, endoplasmic reticulum (ER) stress, and redox state were determined using histological, molecular, and analytical techniques. Results: Epha2-/- and Epha2+/- mice on C57BL/6J background developed severe cortical cataracts by 18 and 38 weeks of age, respectively, compared to development of similar cataract significantly later in Epha2-/- mice and no cataract in Epha2+/- mice in this strain on FVB background, which was previously reported. On FVB:C57BL/6J background, Epha2-/- mice developed severe cortical cataract by 38 weeks and Epha2+/- mice exhibited mild cortical cataract up to 64 weeks of age. Progression of cataract in Epha2-/- and Epha2+/- female mice on C57BL/6J and mixed background, respectively, was slower than in matched male mice. N-cadherin and ß-catenin immunolabeling showed disorganized lens fiber cells and disruption of lens architecture in Epha2-/- and Epha2+/- lenses, coinciding with development of severe cataracts. EPHA2 immunolabeling showed intracellular accumulation of the mutant EPHA2-ß-galactosidase fusion protein that induced a cytoprotective ER stress response and in Epha2+/- lenses was also accompanied by glutathione redox imbalance. Conclusions: Both, Epha2-/- and Epha2+/- mice develop age-related cortical cataract; age as a function of Epha2 genotype, sex, and genetic background influence Epha2-related cataractogenesis in mice.

Neural differentiation medium for human pluripotent stem cells to model physiological glucose levels in human brain.

Cortical neurospheres (NSPs) derived from human pluripotent stem cells (hPSC), have proven to be a successful platform to investigate human brain development and neuro-related diseases. Currently, many of the standard hPSC neural differentiation media, use concentrations of glucose (approximately 17.5-25 mM) and insulin (approximately 3.2 µM) that are much greater than the physiological concentrations found in the human brain. These culture conditions make it difficult to analyse perturbations of glucose or insulin on neuronal development and differentiation. We established a new hPSC neural differentiation medium that incorporated physiological brain concentrations of glucose (2.5 mM) and significantly reduced insulin levels (0.86 µM). This medium supported hPSC neural induction and formation of cortical NSPs. The revised hPSC neural differentiation medium, may provide an improved platform to model brain development and to investigate neural differentiation signalling pathways impacted by abnormal glucose and insulin levels.

Photoreceptor Degeneration in Pro23His Transgenic Rats (Line 3) Involves Autophagic and Necroptotic Mechanisms.

Photoreceptor death contributes to 50% of irreversible vision loss in the western world. Pro23His (P23H) transgenic albino rat strains are widely used models for the most common rhodopsin gene mutation associated with the autosomal dominant form of retinitis pigmentosa. However, the mechanism(s) by which photoreceptor death occurs are not well understood and were the principal aim of this study. We first used electroretinogram recording and optical coherence tomography to confirm the time course of functional and structural loss. Electroretinogram analyses revealed significantly decreased rod photoreceptor (a-wave), bipolar cell (b-wave) and amacrine cell responses (oscillatory potentials) from P30 onward. The cone-mediated b-wave was also decreased from P30. TUNEL analysis showed extensive cell death at P18, with continued labeling detected until P30. Focused gene expression arrays indicated activation of, apoptosis, autophagy and necroptosis in whole retina from P14-18. However, analysis of mitochondrial permeability changes (ΔΨm) using JC-1 dye, combined with immunofluorescence markers for caspase-dependent (cleaved caspase-3) and caspase-independent (AIF) cell death pathways, indicated mitochondrial-mediated cell death was not a major contributor to photoreceptor death. By contrast, reverse-phase protein array data combined with RIPK3 and phospho-MLKL immunofluorescence indicated widespread necroptosis as the predominant mechanism of photoreceptor death. These findings highlight the complexity of mechanisms involved in photoreceptor death in the Pro23His rat model of degeneration and suggest therapies that target necroptosis should be considered for their potential to reduce photoreceptor death.

Epha2 genotype influences ultraviolet radiation induced cataract in mice.

Age-related cataract is the major cause of blindness worldwide. Both genetic and environmental factors contribute to the disease. Genetic variation in the Ephrin type-A receptor 2 (EPHA2) gene is associated with the risk of age-related cataract in multiple populations, and exposure to ultraviolet-B (UV-B) radiation is a well-established risk factor for the disease. Epha2 knockout and UV-B radiation independently lead to cataract in mice, and UV-B radiation reportedly alters EPHA2 expression in cultured cells. We hypothesised that an interaction between UV-B radiation exposure and Epha2 signalling may influence cataract development. To test this hypothesis, 5-week-old Epha2+/+ and Epha2+/- mice (n = 8 per group) were exposed to repeated below-threshold doses of UV-B radiation (0.0125-0.05 J/cm2), before development of Epha2-mediated cataract. Cataract development was monitored after termination of exposure and at least one month later. Histological analysis of exposed and unexposed lenses was performed to assess pathological changes, and gene expression analysis to investigate the mechanism underlying cataract. Both Epha2+/+ and Epha2+/- mice developed UV-B dose-dependent anterior polar cataract; cataract severity in both genotypes of mice exposed to either 0.025 or 0.05 J/cm2 UV-B was significantly higher than that in matched unexposed mice (p < 0.05). Histological analysis of lenses of both genotypes of mice exposed to 0.025 or 0.05 J/cm2 UV-B radiation consistently revealed disruption of the lens architecture. A month after the exposure, cataract severity increased in Epha2+/+ mice treated with the highest dose of UV-B radiation (p = 0.03) but remained unchanged in Epha2+/- mice. Gene expression analysis of lenses of both genotypes of mice showed significant upregulation of the cell proliferation marker Mki67 in Epha2+/+ (p = 0.036) but not in Epha2+/- mice exposed to the highest dose of UV-B radiation compared to matched unexposed mice. In conclusion, this study suggests that repeated exposure to doses of UV-B radiation lower than the single minimum dose required for inducing cataract leads to cataract in wild-type and Epha2 heterozygous knockout mice. Furthermore, this study indicates, for the first time, a potentially favourable effect of partial Epha2 deficiency against UV radiation-induced damage in the lens.

Exposure of trophoblast cells to fine particulate matter air pollution leads to growth inhibition, inflammation and ER stress.

Ambient air pollution is considered a major environmental health threat to pregnant women. Our previous work has shown an association between exposure to airborne particulate matter (PM) and an increased risk of developing pre-eclamspia. It is now recognized that many pregnancy complications are due to underlying placental dysfunction, and this tissue plays a pivotal role in pre-eclamspia. Recent studies have shown that PM can enter the circulation and reach the human placenta but the effects of PM on human placental function are still largely unknown. In this work we investigated the effects of airborne PM on trophoblast cells. Human, first trimester trophoblast cells (HTR-8/SV) were exposed to urban pollution particles (Malmö PM2.5; Prague PM10) for up to seven days in vitro and were analysed for uptake, levels of hCGß and IL-6 secretion and proteomic analysis. HTR-8/SVneo cells rapidly endocytose PM within 30 min of exposure and particles accumulate in the cell in perinuclear vesicles. High doses of Prague and Malmö PM (500-5000 ng/ml) significantly decreased hCGß secretion and increased IL-6 secretion after 48 h exposure. Exposure to PM (50 ng/ml) for 48h or seven days led to reduced cellular growth and altered protein expression. The differentially expressed proteins are involved in networks that regulate cellular processes such as inflammation, endoplasmic reticulum stress, cellular survival and molecular transport pathways. Our studies suggest that trophoblast cells exposed to low levels of urban PM respond with reduced growth, oxidative stress, inflammation and endoplasmic reticulum stress after taking up the particles by endocytosis. Many of the dysfunctional cellular processes ascribed to the differentially expressed proteins in this study, are similar to those described in PE, suggesting that low levels of urban PM may disrupt cellular processes in trophoblast cells. Many of the differentially expressed proteins identified in this study are involved in inflammation and may be potential biomarkers for PE.

Esrp1 is a marker of mouse fetal germ cells and differentially expressed during spermatogenesis.

ESRP1 regulates alternative splicing, producing multiple transcripts from its target genes in epithelial tissues. It is upregulated during mesenchymal to epithelial transition associated with reprogramming of fibroblasts to iPS cells and has been linked to pluripotency. Mouse fetal germ cells are the founders of the adult gonadal lineages and we found that Esrp1 mRNA was expressed in both male and female germ cells but not in gonadal somatic cells at various stages of gonadal development (E12.5-E15.5). In the postnatal testis, Esrp1 mRNA was highly expressed in isolated cell preparations enriched for spermatogonia but expressed at lower levels in those enriched for pachytene spermatocytes and round spermatids. Co-labelling experiments with PLZF and c-KIT showed that ESRP1 was localized to nuclei of both Type A and B spermatogonia in a speckled pattern, but was not detected in SOX9+ somatic Sertoli cells. No co-localization with the nuclear speckle marker, SC35, which has been associated with post-transcriptional splicing, was observed, suggesting that ESRP1 may be associated with co-transcriptional splicing or have other functions. RNA interference mediated knockdown of Esrp1 expression in the seminoma-derived Tcam-2 cell line demonstrated that ESRP1 regulates alternative splicing of mRNAs in a non-epithelial cell germ cell tumour cell line.

Localization and Possible Function of P2X Receptors in Normal and Diseased Retinae.

Purines, when present in the extracellular space, can mediate fast neurotransmission in the retina and central nervous system. Over the last decade there has been emerging evidence for the expression of P2X and P2Y receptors in a range of retinal neuronal subtypes. These results have highlighted important roles for purines in modulating specific retinal circuits, including the rod pathway and amacrine cell circuits. Traditionally, synaptic release of adenosine triphosphate (ATP) involves the novel anion vesicular nucleotide transporter, VNUT, which has recently been identified in a single wide-field amacrine cell population. In addition, nontraditional, conductive mechanisms of release have also been described in the retina. In the synapse, the enzymes involved in rapid degradation of purines are present in both plexiform layers of the retina. A role for P2X receptors in retinal diseases has also emerged recently. High concentrations of ATP lead to photoreceptor loss, through mechanisms involving P2X7 receptors. In addition, activation of P2X7 receptors is associated with activation of the inflammasome, a protein complex important for the release of proinflammatory cytokines. P2X receptors, especially P2X7, are emerging as targets to combat retinal disease.

Expression analysis of Cdx2 and Pou5f1 in a marsupial, the stripe-faced dunnart, during early development.

The first lineage allocation during mouse development forms the trophectoderm and inner cell mass, in which Cdx2 and Pou5f1 display reciprocal expression. Yet Cdx2 is not required for trophectoderm specification in other mammals, such as the human, cow, pig, or in two marsupials, the tammar and opossum. The role of Cdx2 and Pou5f1 in the first lineage allocation of Sminthopsis macroura, the stripe-faced dunnart, is unknown. In this study, expression of Cdx2 and Pou5f1 during oogenesis, development from cleavage to blastocyst stages, and in the allocation of the first three lineages was analyzed for this dunnart. Cdx2 mRNA was present in late antral-stage oocytes, but not present again until Day 5.5. Pou5f1 mRNA was present from primary follicles to zygotes, and then expression resumed starting at the early unilaminar blastocyst stage. All cleavage stages and the pluriblast and trophoblast cells co-expressed CDX2 and POU5F1 proteins, which persisted until early stages of hypoblast formation. Hypoblast cells also show co-localisation of POU5F1 and CDX2 once they were allocated, and this persisted during their division and migration. Our studies suggest that CDX2, and possibly POU5F1, are maternal proteins, and that the first lineage to differentiate is the trophoblast, which differentiates to trophectoderm after shell loss one day before implantation. In the stripe-faced dunnart, cleavage cells, as well as trophoblast and pluriblast cells, are polarized, suggesting the continued presence of CDX2 in both lineages until late blastocyst stages may play a role in the formation and maintenance of polarity.

Tob1 is expressed in developing and adult gonads and is associated with the P-body marker, Dcp2.

Tob1 is a member of the BTG/TOB family of proteins with established antiproliferative function. In Danio rerio and Xenopus laevis, the Tob1 gene is expressed from the one-cell stage through to early gastrula stages, followed in later development by discrete expression in many tissues including the notochord and somites. In both mouse and human, Tob1 is expressed in many adult tissues including the testis and ovary; however, the specific cell types are unknown. We examine Tob1 gene expression in mouse in developing germ cells and in sorted male germ cells (gonocytes, spermatogonia, pachytene spermatocytes and round spermatids) by reverse transcription and droplet digital polymerase chain reaction (RT-ddPCR) and in adult ovary and testis by immunofluorescence with anti-Tob1 protein staining. By RT-ddPCR, Tob1 expression was low in developing male germ cells but was highly expressed in round spermatids. In developing female germ cells undergoing entry into meiosis, it increased 10-fold. Tob1 was also highly expressed in round spermatids and in oocytes in all stages of folliculogenesis. Notably, a marker for P-bodies, Dcp-2, was also highly expressed in round spermatids and all oocyte stages examined. The cytoplasmic presence of Tob1 protein in round spermatids and oocytes and the association of Tob1 protein with Dcp2 in both cell types suggest that Tob1 protein plays a role in post-transcriptional mechanisms.

Inner retinal change in a novel rd1-FTL mouse model of retinal degeneration.

While photoreceptor loss is the most devastating result of inherited retinal degenerations such as retinitis pigmentosa, inner retinal neurons also undergo significant alteration. Detailing these changes has become important as many vision restorative therapies target the remaining neurons. In this study, the rd1-Fos-Tau-LacZ (rd1-FTL) mouse model was used to explore inner retinal change at a late stage of retinal degeneration, after the loss of photoreceptor nuclei. The rd1-FTL model carries a mutation in the phosphodiesterase gene, Pde6b, and an axonally targeted transgenic beta galactosidase reporter system under the control of the c-fos promoter. Retinae of transgenic rd1-FTL mice and control FTL animals aged 2-12 months were processed for indirect fluorescence immunocytochemistry. At 2 months of age, a time when the majority of photoreceptor nuclei are lost, there was negligible c-fos reporter (FTL) expression, however, from 4 months, reporter expression was observed to increase within subpopulations of amacrine and ganglion cells within the central retina. These areas of inner retinal FTL expression coincided with regions that contained aberrant Müller cells. Specifically, these cells exhibited reduced glutamine synthetase and Kir4.1 immunolabelling, whilst showing evidence of proliferative gliosis (increased cyclinD1 and glial fibrillary acidic protein expression). These changes were limited to distinct regions where cone photoreceptor terminals were absent. Overall, these results highlight that distinct areas of the rd1-FTL central retina undergo significant glial alterations after cone photoreceptor loss. These areas coincide with up-regulation of the c-fos reporter in the inner retina, which may represent a change in neuronal function/plasticity. The rd1-FTL mouse is a useful model system to probe changes that occur in the inner retina at later stages of retinal degeneration.

A role for smoothened during murine lens and cornea development.

Various studies suggest that Hedgehog (Hh) signalling plays roles in human and zebrafish ocular development. Recent studies (Kerr et al., Invest Ophthalmol Vis Sci. 2012; 53, 3316-30) showed that conditionally activating Hh signals promotes murine lens epithelial cell proliferation and disrupts fibre differentiation. In this study we examined the expression of the Hh pathway and the requirement for the Smoothened gene in murine lens development. Expression of Hh pathway components in developing lens was examined by RT-PCR, immunofluorescence and in situ hybridisation. The requirement of Smo in lens development was determined by conditional loss-of-function mutations, using LeCre and MLR10 Cre transgenic mice. The phenotype of mutant mice was examined by immunofluorescence for various markers of cell cycle, lens and cornea differentiation. Hh pathway components (Ptch1, Smo, Gli2, Gli3) were detected in lens epithelium from E12.5. Gli2 was particularly localised to mitotic nuclei and, at E13.5, Gli3 exhibited a shift from cytosol to nucleus, suggesting distinct roles for these transcription factors. Conditional deletion of Smo, from ∼E12.5 (MLR10 Cre) did not affect ocular development, whereas deletion from ∼E9.5 (LeCre) resulted in lens and corneal defects from E14.5. Mutant lenses were smaller and showed normal expression of p57Kip2, c-Maf, E-cadherin and Pax6, reduced expression of FoxE3 and Ptch1 and decreased nuclear Hes1. There was normal G1-S phase but decreased G2-M phase transition at E16.5 and epithelial cell death from E14.5-E16.5. Mutant corneas were thicker due to aberrant migration of Nrp2+ cells from the extraocular mesenchyme, resulting in delayed corneal endothelial but normal epithelial differentiation. These results indicate the Hh pathway is required during a discrete period (E9.5-E12.5) in lens development to regulate lens epithelial cell proliferation, survival and FoxE3 expression. Defective corneal development occurs secondary to defects in lens and appears to be due to defective migration of peri-ocular Nrp2+ neural crest/mesenchymal cells.

Studying age-related macular degeneration using animal models.

Over the recent years, there have been tremendous advances in our understanding of the genetic and environmental factors associated with the development of age-related macular degeneration (AMD). Examination of retinal changes in various animals has aided our understanding of the pathogenesis of the disease. Notably, mouse strains, carrying genetic anomalies similar to those affecting humans, have provided a foundation for understanding how various genetic risk factors affect retinal integrity. However, to date, no single mouse strain that develops all the features of AMD in a progressive age-related manner has been identified. In addition, a mutation present in some background strains has clouded the interpretation of retinal phenotypes in many mouse strains. The aim of this perspective was to describe how animals can be used to understand the significance of each sign of AMD, as well as key genetic risk factors.

Integrin linked kinase (ILK) is required for lens epithelial cell survival, proliferation and differentiation.

While the role of growth factors in lens development has been investigated extensively, the role of extracellular matrix signalling is less well understood. The developing lens expresses predominantly laminin-binding integrins (such as α3ß1, α6ß1), which are cooperatively required in the lens epithelium during development. We investigated the role of ILK, a downstream mediator of integrin signalling in mice conditionally null for Ilk. Mutant lenses showed epithelial thinning at E17.5 with reduced proliferation and epithelial cell number and aberrant fibre differentiation. There was complete loss of the central epithelium from postnatal day (P) 2 due to cell death followed by fibre cell degeneration and death by P10 as well as rupture of the lens capsule between P10 and P21. At E17.5 there was significant inhibition (∼50%) of epithelial cell cycle progression, as shown by BrdU incorporation, cyclin D1/D2 and phospho-histone H3 immunostaining. The epithelial marker, E-cadherin, was decreased progressively from E17.5 to P2, in the central epithelium, but there was no significant change in Pax6 expression. Analyses of ERK and Akt phosphorylation indicated marked depression of MAPK and PI3K-Akt signalling, which correlated with decreased phosphorylation of FRS2α and Shp2, indicating altered activation of FGF receptors. At later postnatal stages there was reduced or delayed expression of fibre cell markers (ß-crystallin and p57(kip2)). Loss of Ilk also affected deposition of extracellular matrix, with marked retention of collagen IV within differentiating fibre cells. By quantitative RT-PCR array there was significantly decreased expression of 19 genes associated with focal adhesions, actin filament stability and MAPK and PI3K/Akt signalling. Overall, these data indicate that ILK is required for complete activation of signalling cascades downstream of the FGF receptor in lens epithelium and fibre cells during development and thus is involved in epithelial proliferation, survival and subsequent fibre differentiation.

A requirement for FGF signalling in the formation of primitive streak-like intermediates from primitive ectoderm in culture.

BACKGROUND: Embryonic stem (ES) cells hold considerable promise as a source of cells with therapeutic potential, including cells that can be used for drug screening and in cell replacement therapies. Differentiation of ES cells into the somatic lineages is a regulated process; before the promise of these cells can be realised robust and rational methods for directing differentiation into normal, functional and safe cells need to be developed. Previous in vivo studies have implicated fibroblast growth factor (FGF) signalling in lineage specification from pluripotent cells. Although FGF signalling has been suggested as essential for specification of mesoderm and endoderm in vivo and in culture, the exact role of this pathway remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using a culture model based on early primitive ectoderm-like (EPL) cells we have investigated the role of FGF signalling in the specification of mesoderm. We were unable to demonstrate any mesoderm inductive capability associated with FGF1, 4 or 8 signalling, even when the factors were present at high concentrations, nor any enhancement in mesoderm formation induced by exogenous BMP4. Furthermore, there was no evidence of alteration of mesoderm sub-type formed with addition of FGF1, 4 or 8. Inhibition of endogenous FGF signalling, however, prevented mesoderm and favoured neural differentiation, suggesting FGF signalling was required but not sufficient for the differentiation of primitive ectoderm into primitive streak-like intermediates. The maintenance of ES cell/early epiblast pluripotent marker expression was also observed in cultures when FGF signalling was inhibited. CONCLUSIONS/SIGNIFICANCE: FGF signalling has been shown to be required for the differentiation of primitive ectoderm to neurectoderm. This, coupled with our observations, suggest FGF signalling is required for differentiation of the primitive ectoderm into the germ lineages at gastrulation.

Conditional mutations of beta-catenin and APC reveal roles for canonical Wnt signaling in lens differentiation.

PURPOSE: Previous studies indicate that the Wnt/beta-catenin-signaling pathway is active and functional during murine lens development. In this study, the consequences of constitutively activating the pathway in lens during development were investigated. METHODS: To activate Wnt/beta-catenin signaling, beta-catenin (Catnb) and adenomatous polyposis coli (Apc) genes were conditionally mutated in two Cre lines that are active in whole lens (MLR10) or only in differentiated fibers (MLR39), from E13.5. Lens phenotype in mutant lenses was investigated by histology, immunohistochemistry, BrdU labeling, quantitative RT-PCR arrays, and TUNEL. RESULTS: Only intercrosses with MLR10 resulted in ocular phenotypes, indicating Wnt/beta-catenin signaling functions in lens epithelium and during early fiber differentiation. Mutant lenses were characterized by increased progression of epithelial cells through the cell cycle, as shown by BrdU labeling, and phosphohistone 3 and cyclin D1 labeling, and maintenance of epithelial phenotype (E-cadherin and Pax6 expression) in the fiber compartment. Fiber cell differentiation was delayed as shown by reduced expression of c-maf and beta-crystallin and delay in expression of the CDKI, p57(kip2). From E13.5, there were numerous cells undergoing apoptosis, and by E15.5, there was evidence of epithelial-mesenchymal transition with numerous cells expressing alpha-smooth muscle actin. Quantitative PCR analyses revealed large changes in expression of Wnt target genes (Lef1, Tcf7, T (Brachyury), and Ccnd1), Wnt inhibitors (Wif1, Dkk1, Nkd1, and Frzb) and also several Wnts (Wnt6, Wnt10a, Wnt8b, and Wnt11). CONCLUSIONS: These data indicate that the Wnt/beta-catenin pathway plays key roles in regulating proliferation of lens stem/progenitor cells during early stages of fiber cell differentiation.

Differential requirement for beta-catenin in epithelial and fiber cells during lens development.

Recent studies implicate Wnt/beta-catenin signaling in lens differentiation (Stump, R. J., et al., 2003. A role for Wnt/beta-catenin signaling in lens epithelial differentiation. Dev Biol;259:48-61). Beta-catenin is a component of adherens junctions and functions as a transcriptional activator in canonical Wnt signaling. We investigated the effects of Cre/LoxP-mediated deletion of beta-catenin during lens development using two Cre lines that specifically deleted beta-catenin in whole lens or only in differentiated fibers, from E13.5. We found that beta-catenin was required in lens epithelium and during early fiber differentiation but appeared to be redundant in differentiated fiber cells. Complete loss of beta-catenin resulted in an abnormal and deficient epithelial layer with loss of E-cadherin and Pax6 expression as well as abnormal expression of c-Maf and p57(kip2) but not Prox1. There was also disrupted fiber cell differentiation, characterized by poor cell elongation, decreased beta-crystallin expression, epithelial cell cycle arrest at G(1)-S transition and premature cell cycle exit. Despite cell cycle arrest there was no induction of apoptosis. Mutant fiber cells displayed altered apical-basal polarity as evidenced by altered distribution of the tight junction protein, ZO1, disruption of apical actin filaments and abnormal deposition of extracellular matrix, resulting in a deficient lens capsule. Loss of beta-catenin also affected the formation of adhesion junctions as evidenced by dissociation of N-cadherin and F-actin localization in differentiating fiber cells. However, loss of beta-catenin from terminally differentiating fibers had no apparent effects on adhesion junctions between adjacent embryonic fibers. These data indicate that beta-catenin plays distinct functions during lens fiber differentiation and is involved in both Wnt signaling and adhesion-related mechanisms that regulate lens epithelium and early fiber differentiation.

Extracellular matrix and integrin signaling in lens development and cataract.

During development of the vertebrate lens there are dynamic interactions between the extracellular matrix (ECM) of the lens capsule and lens cells. Disruption of the ECM causes perturbation of lens development and cataract. Similarly, changes in cell signaling can result in abnormal ECM and cataract. Integrins are key mediators of ECM signals and recent studies have documented distinct repertoires of integrin expression during lens development, and in anterior subcapsular cataract (ASC) and posterior caspsule opacification (PCO). Increasingly, studies are being directed to investigating the signaling pathways that integrins modulate and have identified Src, focal adhesion kinase (FAK) and integrin-linked kinase (ILK) as downstream kinases that mediate proliferation, differentiation and morphological changes in the lens during development and cataract formation.