Lens Fiber Differentiation Research Articles

Identification of Novel Gata3 Distal Enhancers Active in Mouse Embryonic Lens.

Background: The tissue‐specific transcriptional programs during normal development require tight control by distal cis‐regulatory elements, such as enhancers, with specific DNA sequences recognized by transcription factors, coactivators, and chromatin remodeling enzymes. Gata3 is a sequence‐specific DNA‐binding transcription factor that regulates formation of multiple tissues and organs, including inner ear, lens, mammary gland, T‐cells, urogenital system, and thyroid gland. In the eye, Gata3 has a highly restricted expression domain in the posterior part of the lens vesicle; however, the underlying regulatory mechanisms are unknown. Results: Here we describe the identification of a novel bipartite Gata3 lens‐specific enhancer located ∼18 kb upstream from its transcriptional start site. We also found that a 5‐kb Gata3 promoter possesses low activity in the lens. The bipartite enhancer contains arrays of AP‐1, Ets‐, and Smad1/5‐binding sites as well as binding sites for lens‐associated DNA‐binding factors. Transient transfection studies of the promoter with the bipartite enhancer showed enhanced activation by BMP4 and FGF2. Conclusions: These studies identify a novel distal enhancer of Gata3 with high activity in lens and indicate that BMP and FGF signaling can up‐regulate expression of Gata3 in differentiating lens fiber cells through the identified Gata3 enhancer and promoter elements. Developmental Dynamics 247:1186–1198, 2018. © 2018 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists

Endocytic trafficking factor VPS45 is essential for spatial regulation of lens fiber differentiation in zebrafish.

ABSTRACTIn vertebrate lens, lens epithelial cells cover the anterior half of the lens fiber core. Lens epithelial cells proliferate, move posteriorly and start to differentiate into lens fiber cells at the lens equator. Although FGF signaling promotes this equatorial commencement of lens fiber differentiation, the underlying mechanism is not fully understood. Here, we show that lens epithelial cells abnormally enter lens fiber differentiation without passing through the equator in zebrafish vps45 mutants. VPS45 belongs to the Sec1/Munc18-like protein family and promotes endosome trafficking, which differentially modulates signal transduction. Ectopic lens fiber differentiation in vps45 mutants does not depend on FGF, but is mediated through activation of TGFβ signaling and inhibition of canonical Wnt signaling. Thus, VPS45 normally suppresses lens fiber differentiation in the anterior region of lens epithelium by modulating TGFβ and canonical Wnt signaling pathways. These data indicate a novel role of endosome trafficking to ensure equator-dependent commencement of lens fiber differentiation.

Spred negatively regulates lens growth by modulating epithelial cell proliferation and fiber differentiation

Spred, like Sprouty (Spry) and also Sef proteins, have been identified as important regulators of receptor tyrosine kinase (RTK)-mediated MAPK/ERK-signaling in various developmental systems, controlling cellular processes such as proliferation, migration and differentiation. Spreds are widely expressed during early embryogenesis, and in the eye lens, become more localised in the lens epithelium with later development, overlapping with other antagonists including Spry. Given the synexpression of Spreds and Spry in lens, in order to gain a better understanding of their specific roles in regulating growth factor mediated-signaling and cell behavior, we established and characterised lines of transgenic mice overexpressing Spred1 or Spred2, specifically in the lens. This overexpression of Spreds resulted in a small lens phenotype during ocular morphogenesis, retarding its growth by compromising epithelial cell proliferation and fiber differentiation. These in situ findings were shown to be dependent on the ability of Spreds to suppress MAPK-signaling, in particular FGF-induced ERK1/2-signaling in lens cells. This was validated in vitro using lens epithelial explants, that highlighted the overlapping role of Spreds with Spry2, but not Spry1. This study provides insights into the putative function of Spreds and Spry in situ, some overlapping and some distinct, and their importance in regulating lens cell proliferation and fiber differentiation contributing to lens and eye growth.

Wnt5a Contributes to the Differentiation of Human Embryonic Stem Cells into Lentoid Bodies Through the Noncanonical Wnt/JNK Signaling Pathway.

Recent work has indicated that Wnt5a has a critical role in embryonic development. We investigate whether the Wnt5a-activated noncanonical Wnt pathway is capable of promoting embryonic lens differentiation. A "three-stage" protocol was used to induce lens differentiation of human embryonic stem cells (hESCs) in vitro, and Wnt5a levels were modified by addition of exogenous protein and RNA interference. SP600125 was adopted to inhibit JNK cascades. The number and size of lentoid bodies obtained were measured, and quantitative RT-PCR, Western blotting, and immunofluorescence were used to detect gene and protein expression. The quantity and size of lentoid bodies generated were significantly increased by addition of exogenous Wnt5a. Moreover, expression of lens-specific genes, including CRYAA, CRYAB, BFSP1, and MIP, and the lens fiber differentiation regulator PROX1 were prominently increased. We also observed activation of noncanonical Wnt signaling via upregulation of Dvl2, Rac1, and JNK. When Wnt5a-knockdown hESCs were induced to differentiate, fewer and smaller lentoid bodies resulted. In addition, expression of genes specific to lens was decreased and noncanonical Wnt/JNK pathway activity was downregulated. Accordingly, inhibition of JNK cascade suppressed the formation of lentoid bodies as well, consistent with that of Wnt5a-knockdown group. Wnt5a can promote the differentiation of hESCs into lentoid bodies through the noncanonical Wnt/JNK signaling pathway, thereby contributing to the study of human lens development and moreover the underlying etiology congenital cataracts.

Negative regulation of lens fiber cell differentiation by RTK antagonists Spry and Spred

Sprouty (Spry) and Spred proteins have been identified as closely related negative regulators of the receptor tyrosine kinase (RTK)-mediated MAPK pathway, inhibiting cellular proliferation, migration and differentiation in many systems. As the different members of this antagonist family are strongly expressed in the lens epithelium in overlapping patterns, in this study we used lens epithelial explants to examine the impact of these different antagonists on the morphologic and molecular changes associated with fibroblast growth factor (FGF)-induced lens fiber differentiation. Cells in lens epithelial explants were transfected using different approaches to overexpress the different Spry (Spry1, Spry2) and Spred (Spred1, Spred2, Spred3) members, and we compared their ability to undergo FGF-induced fiber differentiation. In cells overexpressing any of the antagonists, the propensity for FGF-induced cell elongation was significantly reduced, indicative of a block to lens fiber differentiation. Of these antagonists, Spry1 and Spred2 appeared to be the most potent among their respective family members, demonstrating the greatest block in FGF-induced fiber differentiation based on the percentage of cells that failed to elongate. Consistent with the reported activity of Spry and Spred, we show that overexpression of Spry2 was able to suppress FGF-induced ERK1/2 phosphorylation in lens cells, as well as the ERK1/2-dependent fiber-specific marker Prox1, but not the accumulation of β-crystallins. Taken together, Spry and Spred proteins that are predominantly expressed in the lens epithelium in situ, appear to have overlapping effects on negatively regulating ERK1/2-signaling associated with FGF-induced lens epithelial cell elongation leading to fiber differentiation. This highlights the important regulatory role for these RTK antagonists in establishing and maintaining the distinct architecture and polarity of the lens.

A role for Hippo/YAP-signaling in FGF-induced lens epithelial cell proliferation and fibre differentiation

Recent studies indicate an important role for the transcriptional co-activator Yes-associated protein (YAP), and its regulatory pathway Hippo, in controlling cell growth and fate during lens development; however, the exogenous factors that promote this pathway are yet to be identified. Given that fibroblast growth factor (FGF)-signaling is an established regulator of lens cell behavior, the current study investigates the relationship between this pathway and Hippo/YAP-signaling during lens cell proliferation and fibre differentiation. Rat lens epithelial explants were cultured with FGF2 to induce epithelial cell proliferation or fibre differentiation. Immunolabeling methods were used to detect the expression of Hippo-signaling components, Total and Phosphorylated YAP, as well as fibre cell markers, Prox-1 and β-crystallin. FGF-induced lens cell proliferation was associated with a strong nuclear localisation of Total-YAP and low-level immuno-staining for phosphorylated-YAP. FGF-induced lens fibre differentiation was associated with a significant increase in cytoplasmic phosphorylated YAP (inactive state) and enhanced expression of core Hippo-signaling components. Inhibition of YAP with Verteporfin suppressed FGF-induced lens cell proliferation and ablated cell elongation during lens fibre differentiation. Inhibition of either FGFR- or MEK/ERK-signaling suppressed FGF-promoted YAP nuclear translocation. Here we propose that FGF promotes Hippo/YAP-signaling during lens cell proliferation and differentiation, with FGF-induced nuclear-YAP expression playing an essential role in promoting the proliferation of lens epithelial cells. An FGF-induced switch from proliferation to differentiation, hence regulation of lens growth, may play a key role in mediating Hippo suppression of YAP transcriptional activity.

Functional role for stable microtubules in lens fiber cell elongation

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.

A New Long Noncoding RNA ALB Regulates Autophagy by Enhancing the Transformation of LC3BI to LC3BII during Human Lens Development.

Autophagy is essential in lens organelle degradation. This study aimed to seek potential autophagy-associated long noncoding RNAs (lncRNAs) and their relative mechanisms in human lens development using the “fried egg” lentoid body (LB) generation system. The expression pattern of LC3B in differentiating LBs was similar to that in developing a mouse lens in vivo. Among the massive lncRNAs expressed with a significant difference between induced pluripotent stem cells (iPSCs) and LBs, lncRNA affecting LC3B (ALB), which was predicted to have a co-relationship with the autophagy marker LC3B, was highly expressed in differentiating lens fibers in LBs. This result was consistent with its high expression in human embryonic lenses compared to those in embryonic stem cells (ESCs). Furthermore, lncRNA ALB knockdown resulted in the downregulation of LC3BII at the protein level, therefore inhibiting the autophagy process in human lens epithelial cells (HLECs). Our results identify lncRNA ALB, a potential autophagy regulator in organelle degradation during human lens development, highlighting the importance of lncRNAs in lens development.

Aqueous humour-induced lens epithelial cell proliferation requires FGF-signalling

The eye lens grows by systematic proliferation of its epithelial cells and their differentiation into fibre cells. The anterior aqueous humour regulates lens epithelial cell proliferation whereas posteriorly, the vitreous stimulates lens fibre differentiation. Vitreous-derived members of the fibroblast growth factor (FGF) family induce fibre differentiation, with added support for FGFs as putative regulators of aqueous-induced lens cell proliferation. To further characterize this, given FGFs’ known affinity for proteoglycans, we compared the effect of proteoglycan sulphation in growth factor- and aqueous-induced lens cell proliferation. Disruption of proteoglycan sulphation in lens cells specifically impacted on aqueous- and FGF-induced MAPK/ERK1/2-signalling, but not on that induced by other mitogens such as PDGF; however, cell proliferation was reduced in all treatment groups, regardless of the mitogen. Overall, by disrupting proteoglycan activity, we further highlight the significant role of FGFs in aqueous-induced ERK1/2 phosphorylation leading to lens cell proliferation.

Transcriptional dynamics, denucleation, and gene regulation in embryonic lens development

SummaryTranscription of genes occurs in apparently irregular pulses of activity termed transcriptional bursts. In contrast, cellular differentiation is marked by coordinated gene expression of batteries of genes required for cellular specialization. Ocular lens is comprised of two compartments: the lens epithelium and lens fibers. Position of individual cells within the lens is directly related to their differentiation status, marked by expression of individual crystallin genes. Another hallmark of lens fiber cell differentiation is organized degradation of their organelles. It is currently not known how transcription is terminated in the disintegrating lens fiber cell nuclei and what are the external and internal components of this process critical for lens transparency. Onset of expression of different crystallins follows the temporal pattern Cryaa > Crybb1 > Cryga in differentiating lens fibers. Surprisingly, expression of Cryaa is terminated in “normal” fiber cell nuclei while expression of Cryga persists even in highly condensed nuclei just about to be disintegrated. Differentiation influences both transcriptional burst size and frequency as well as spatial pairing of different chromosomes that carry crystallin genes.

N-myc regulates growth and fiber cell differentiation in lens development

Myc proto-oncogenes regulate diverse cellular processes during development, but their roles during morphogenesis of specific tissues are not fully understood. We found that c-myc regulates cell proliferation in mouse lens development and previous genome-wide studies suggested functional roles for N-myc in developing lens. Here, we examined the role of N-myc in mouse lens development. Genetic inactivation of N-myc in the surface ectoderm or lens vesicle impaired eye and lens growth, while "late" inactivation in lens fibers had no effect. Unexpectedly, defective growth of N-myc-deficient lenses was not associated with alterations in lens progenitor cell proliferation or survival. Notably, N-myc-deficient lens exhibited a delay in degradation of DNA in terminally differentiating lens fiber cells. RNA-sequencing analysis of N-myc-deficient lenses identified a cohort of down-regulated genes associated with fiber cell differentiation that included DNaseIIβ. Further, an integrated analysis of differentially expressed genes in N-myc-deficient lens using normal lens expression patterns of iSyTE, N-myc-binding motif analysis and molecular interaction data from the String database led to the derivation of an N-myc-based gene regulatory network in the lens. Finally, analysis of N-myc and c-myc double-deficient lens demonstrated that these Myc genes cooperate to drive lens growth prior to lens vesicle stage. Together, these findings provide evidence for exclusive and cooperative functions of Myc transcription factors in mouse lens development and identify novel mechanisms by which N-myc regulates cell differentiation during eye morphogenesis.

N-cadherin regulates signaling mechanisms required for lens fiber cell elongation and lens morphogenesis

Tissue development and regeneration involve high-ordered morphogenetic processes that are governed by elements of the cytoskeleton in conjunction with cell adhesion molecules. Such processes are particularly important in the lens whose structure dictates its function. Studies of our lens-specific N-cadherin conditional knockout mouse (N-cadcKO) revealed an essential role for N-cadherin in the migration of the apical tips of differentiating lens fiber cells along the apical surfaces of the epithelium, a region termed the Epithelial Fiber Interface (EFI), that is necessary for normal fiber cell elongation and the morphogenesis. Studies of the N-cadcKO lens suggest that N-cadherin function in fiber cell morphogenesis is linked to the activation of Rac1 and myosin II, both signaling pathways central to the regulation of cell motility including determining the directionality of cellular movement. The absence of N-cadherin did not disrupt lateral contacts between fiber cells during development, and the maintenance of Aquaporin-0 and increased expression of EphA2 at cell-cell interfaces suggests that these molecules may function in this role. E-cadherin was maintained in newly differentiating fiber cells without interfering with expression of lens-specific differentiation proteins but was not able to replace N-cadherin function in these cells. The dependence of migration of the fiber cell apical domains along the EFI for lens morphogenesis on N-cadherin provides new insight into the process of tissue development.

CRISPR/Cas9-Mediated Mutation of αA-Crystallin Gene Induces Congenital Cataracts in Rabbits.

The present study aimed to investigate the role of the αA-crystallin gene in inducing congenital cataracts in rabbits and to construct a novel animal model for characterization and pathologic analysis of congenital cataracts for future research. We generated αA-crystallin gene knockout rabbits with congenital cataracts by coinjection of Cas9 mRNA and sgRNA into zygotes. Cataract phenotypes were investigated in a repeated study of 19 F0-generation and 11 F1-generation rabbits with αA-crystallin gene mutations. Heritability was analyzed by PCR, sequencing, slim lamp, hematoxylin eosin staining, immunohistochemistry, and Western blot. We found αA-crystallin gene mutations in all 19 F0-generation pups (100%) with indel mutations in the αA-crystallin gene ranging from 3 to 52 bp. Off-target assay revealed that none of the potential off-target sites exhibited mutations, demonstrating that off-target mutagenesis was not induced by cytoplasmic microinjection of in vitro-transcribed Cas9 mRNA. Slim lamp assay revealed that 15 of 19 live pups (78.9%) exhibited typical phenotypes, including congenital cataracts, microphthalmia, obscurity, and early atrophy of the lens, and failed differentiation of lens fibers. Histologic hematoxylin and eosin staining showed that αA-crystallin gene knockout rabbits exhibited smaller lenses. Production of the αA-crystallin protein was determined to be dramatically reduced in αA-crystallin gene knockout rabbits. We induced αA-crystallin gene mutations and phenotypes in F1-generation rabbits. Our data suggest that CRISPR/Cas9-mediated mutation of the αA-crystallin gene in rabbits recapitulates phenotypes of congenital cataracts, microphthalmia, obscurity, and early atrophy of the lens, and failed differentiation of lens fibers. These findings suggest the possibility of a new animal model of congenital cataracts, which should be used to further investigate the association between mutations in αA-crystallin gene and congenital cataracts in humans.

Congenital Cataract in Gpr161vl/vl Mice Is Modified by Proximal Chromosome 15

The morphology and severity of human congenital cataract varies even among individuals with the same mutation, suggesting that genetic background modifies phenotypic penetrance. The spontaneous mouse mutant, vacuolated lens (vl), arose on the C3H/HeSnJ background. The mutation disrupts secondary lens fiber development by E16.5, leading to full penetrance of congenital cataract. The vl locus was mapped to a frameshift deletion in the orphan G protein-coupled receptor, Gpr161, which is expressed in differentiating lens fiber cells. When Gpr161vl/vl C3H mice are crossed to MOLF/EiJ mice an unexpected rescue of cataract is observed, suggesting that MOLF modifiers affect cataract penetrance. Subsequent QTL analysis mapped three modifiers (Modvl3-5: Modifier of vl) and in this study we characterized Modvl4 (Chr15; LOD = 4.4). A Modvl4MOLF congenic was generated and is sufficient to rescue congenital cataract and the lens fiber defect at E16.5. Additional phenotypic analysis on three subcongenic lines narrowed down the interval from 55 to 15Mb. In total only 18 protein-coding genes and 2 micro-RNAs are in this region. Fifteen of the 20 genes show detectable expression in the E16.5 eye. Subsequent expression studies in Gpr161vl/vl and subcongenic E16.5 eyes, bioinformatics analysis of C3H/MOLF polymorphisms, and the biological relevancy of the genes in the interval identified three genes (Cdh6, Ank and Trio) that likely contribute to the rescue of the lens phenotype. These studies demonstrate that modification of the Gpr161vl/vl cataract phenotype is likely due to genetic variants in at least one of three closely linked candidate genes on proximal Chr15.

Lens Capsule HSPG-Perlecan Regulates Lens Fibre Differentiation during Chick Embryo Development

Lens fibre differentiation is a life-long process related with lens transparency, and is particularly intense during development, being related with an FGF-2 antero-posterior gradient at the equator level as the main growth factor involved which has been related with the basal membrane of the lens anlagen known as “Lens capsule”. However the lens fibre differentiation induced by FGF2 depends, as in other biological systems, on the local bioavailability of FGF-2 regulated by their relationship with extracellular matrix molecules as Heparan Sulphate Proteoglycans. Here, we try to clarify how Perlecan (a heparan sulphate proteoglycan specific from basement membranes) is involved in lens fibre differentiation at earliest stages of eye development. Our results show that Perlecan, is a major component in the lens capsule during the earliest stages of lens development in chick embryos being present during lens plate induction, lens vesicle stage and the onset of lens fibre differentiation. In order to demonstrate a direct involvement of HSPG-Perlecan in lens fibre differentiation, we generate depleted lenses by HSPG-Perlecan synthesis disruption and specific enzymatic digestion. The HSPG-Perlecan depleted lens show a significant delay or abolition in the lens fibre differentiation which remains in an immature cells displaying DNA synthesis in the posterior epithelium and a decrease in FGF2 lens expression. These data support the hypothesis that lens capsule HSPG-Perlecan is a key molecule involved in lens fibre differentiation during development, probably by involvement in FGF-2 biodisponibility.

The αA‐crystallin gene expression in differentiating lens fiber cells, FGF signaling, and transcriptional factories

PurposeEmbryonic lens development requires intricate temporal and spatial control of gene expression that is executed through specific gene regulatory networks regulated by FGF signaling. The αA‐crystallin is the most abundant mammalian lens protein; transcriptional studies of this gene provide detailed insights into the molecular mechanisms governing lens fiber cell differentiation.MethodsDynamic changes in chromatin structure and gene expression during lens developments were evaluated by a combination of RNA‐seq and ATAC‐seq. Expression of αA‐crystallin (ch1) and other crystallins (Cryga, Crybb1, and Crybb3, localized on chromosomes 1 and 5, respectively) in individual nuclei of the developing mouse lens were analyzed by RNA‐FISH. Transgenic mice with c‐Maf promoter were generated and expression of EGFP was evaluated in mouse embryos. Localization of c‐Jun, c‐Maf, Etv5/ERM, and Pax6 was determined by ChIP using the c‐Maf and Cryaa loci and mouse lens chromatin. Co‐transfection experiments with c‐Jun and Etv5 were conducted using c‐Maf and αA‐crystallin reporters.ResultsTranscriptional factories, a subset of topologically associating domains (TADs), are formed between different crystallin loci in lens cell nuclei. These specific TADs are enriched by RNA polymerase II and other proteins. Novel FGF2‐responsive region in the c‐Maf promoter was found. Both c‐Maf and Cryaa regulatory regions contain arrays of AP‐1 and Ets‐binding sites determined by ChIP assays.ConclusionsCollectively, these studies show that the lens fiber cell nuclei spatially organize crystallin loci into TADs, and that FGF signaling up‐regulates expression of αA‐crystallin both directly and indirectly via up‐regulation of c‐Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiating lens fibers.

Identification and Ultrastructural Characterization of a Novel Nuclear Degradation Complex in Differentiating Lens Fiber Cells.

An unresolved issue in structural biology is how the encapsulated lens removes membranous organelles to carry out its role as a transparent optical element. In this ultrastructural study, we establish a mechanism for nuclear elimination in the developing chick lens during the formation of the organelle-free zone. Day 12–15 chick embryo lenses were examined by high-resolution confocal light microscopy and thin section transmission electron microscopy (TEM) following fixation in 10% formalin and 4% paraformaldehyde, and then processing for confocal or TEM as described previously. Examination of developing fiber cells revealed normal nuclei with dispersed chromatin and clear nucleoli typical of cells in active ribosome production to support protein synthesis. Early signs of nuclear degradation were observed about 300 μm from the lens capsule in Day 15 lenses where the nuclei display irregular nuclear stain and prominent indentations that sometimes contained a previously undescribed macromolecular aggregate attached to the nuclear envelope. We have termed this novel structure the nuclear excisosome. This complex by confocal is closely adherent to the nuclear envelope and by TEM appears to degrade the outer leaflet of the nuclear envelope, then the inner leaflet up to 500 μm depth. The images suggest that the nuclear excisosome separates nuclear membrane proteins from lipids, which then form multilamellar assemblies that stain intensely in confocal and in TEM have 5 nm spacing consistent with pure lipid bilayers. The denuded nucleoplasm then degrades by condensation and loss of structure in the range 600 to 700 μm depth producing pyknotic nuclear remnants. None of these stages display any classic autophagic vesicles or lysosomes associated with nuclei. Uniquely, the origin of the nuclear excisosome is from filopodial-like projections of adjacent lens fiber cells that initially contact, and then appear to fuse with the outer nuclear membrane. These filopodial-like projections appear to be initiated with a clathrin-like coat and driven by an internal actin network. In summary, a specialized cellular organelle, the nuclear excisosome, generated in part by adjacent fiber cells degrades nuclei during fiber cell differentiation and maturation.

Human βA3/A1-crystallin splicing mutation causes cataracts by activating the unfolded protein response and inducing apoptosis in differentiating lens fiber cells

βγ-Crystallins, having a uniquely stable two domain four Greek key structure, are crucial for transparency of the eye lens,. Mutations in lens crystallins have been proposed to cause cataract formation by a variety of mechanisms most of which involve destabilization of the protein fold. The underlying molecular mechanism for autosomal dominant zonular cataracts with sutural opacities in an Indian family caused by a c.215+1G>A splice mutation in the βA3/A1-crystallin gene CRYBA1 was elucidated using three transgenic mice models. This mutation causes a splice defect in which the mutant mRNA escapes nonsense mediated decay by skipping both exons 3 and 4. Skipping these exons results in an in-frame deletion of the mRNA and synthesis of an unstable p.Ile33_Ala119del mutant βA3/A1-crystallin protein. Transgenic expression of mutant βA3/A1-crystallin but not the wild type protein results in toxicity and abnormalities in the maturation and orientation of differentiating lens fibers in c.97_357del CRYBA1 transgenic mice, leading to a small spherical lens, cataract, and often lens capsule rupture. On a cellular level, the lenses accumulated p.Ile33_Ala119del βA3/A1-crystallin with resultant activation of the stress signaling pathway — unfolded protein response (UPR) and inhibition of normal protein synthesis, culminating in apoptosis. This highlights the mechanistic contrast between mild mutations that destabilize crystallins and other proteins, resulting in their being bound by the α-crystallins that buffer lens cells against damage by denatured proteins, and severely misfolded proteins that are not bound by α-crystallin but accumulate and have a direct toxic effect on lens cells, resulting in early onset cataracts.

Regulation of c-Maf and αA-Crystallin in Ocular Lens by Fibroblast Growth Factor Signaling

Fibroblast growth factor (FGF) signaling regulates a multitude of cellular processes, including cell proliferation, survival, migration, and differentiation. In the vertebrate lens, FGF signaling regulates fiber cell differentiation characterized by high expression of crystallin proteins. However, a direct link between FGF signaling and crystallin gene transcriptional machinery remains to be established. Previously, we have shown that the bZIP proto-oncogene c-Maf regulates expression of αA-crystallin (Cryaa) through binding to its promoter and distal enhancer, DCR1, both activated by FGF2 in cell culture. Herein, we identified and characterized a novel FGF2-responsive region in the c-Maf promoter (-272/-70, FRE). Both c-Maf and Cryaa regulatory regions contain arrays of AP-1 and Ets-binding sites. Chromatin immunoprecipitation (ChIP) assays established binding of c-Jun (an AP-1 factor) and Etv5/ERM (an Ets factor) to these regions in lens chromatin. Analysis of temporal and spatial expression of c-Jun, phospho-c-Jun, and Etv5/ERM in wild type and ERK1/2 deficient lenses supports their roles as nuclear effectors of FGF signaling in mouse embryonic lens. Collectively, these studies show that FGF signaling up-regulates expression of αA-crystallin both directly and indirectly via up-regulation of c-Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiated lens fibers.

Control of lens development by Lhx2-regulated neuroretinal FGFs.

Fibroblast growth factor (FGF) signaling is an essential regulator of lens epithelial cell proliferation and survival, as well as lens fiber cell differentiation. However, the identities of these FGF factors, their source tissue and the genes that regulate their synthesis are unknown. We have found that Chx10-Cre;Lhx2lox/lox mice, which selectively lack Lhx2 expression in neuroretina from E10.5, showed an early arrest in lens fiber development along with severe microphthalmia. These mutant animals showed reduced expression of multiple neuroretina-expressed FGFs and canonical FGF-regulated genes in neuroretina. When FGF expression was genetically restored in Lhx2-deficient neuroretina of Chx10-Cre;Lhx2lox/lox mice, we observed a partial but nonetheless substantial rescue of the defects in lens cell proliferation, survival and fiber differentiation. These data demonstrate that neuroretinal expression of Lhx2 and neuroretina-derived FGF factors are crucial for lens fiber development in vivo.