Integrative transcriptomic profiling of ncRNAs and mRNAs in developing mouse lens.

In recent years, burgeoning research has underscored the pivotal role of non-coding RNA in orchestrating the growth, development, and pathogenesis of various diseases across organisms. However, despite these advances, our understanding of the specific contributions of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) to lens development remains notably limited. Clarifying the intricate gene regulatory networks is imperative for unraveling the molecular underpinnings of lens-related disorders. In this study, we aimed to address this gap by conducting a comprehensive analysis of the expression profiles of messenger RNAs (mRNAs), lncRNAs, and circRNAs at critical developmental time points of the mouse lens, encompassing both embryonic (E10.5, E12.5, and E16.5) and postnatal stages (P0.5, P10.5, and P60). Leveraging RNA-sequencing technology, we identified key transcripts pivotal to lens development. Our analysis revealed differentially expressed (DE) mRNAs, lncRNAs, and circRNAs across various developmental stages. Particularly noteworthy, there were 1831 co-differentially expressed (CO-DE) mRNAs, 150 CO-DE lncRNAs, and 13 CO-DE circRNAs identified during embryonic stages. Gene Ontology (GO) enrichment analysis unveiled associations primarily related to lens development, DNA conformational changes, and angiogenesis among DE mRNAs and lncRNAs. Furthermore, employing protein-protein interaction networks, mRNA-lncRNA co-expression networks, and circRNA-microRNA-mRNA networks, we predicted candidate key molecules implicated in lens development. Our findings underscore the pivotal roles of lncRNAs and circRNAs in this process, offering fresh insights into the pathogenesis of lens-related disorders and paving the way for future exploration in this field.

Verification of the gene and protein expression of the aquaglyceroporin AQP3 in the mammalian lens.

Transport of water is critical for maintaining the transparency of the avascular lens, and the lens is known to express at least five distinctly different water channels from the Aquaporin (AQP) family of proteins. In this study we report on the identification of a sixth lens AQP, AQP3 an aquaglyceroporin, which in addition to water also transports glycerol and H2O2. AQP3 was identified at the transcript level and protein levels using RT-PCR and Western blotting, respectively, in the mouse, rat, bovine and human lens, showing that its expression is conserved in the mammalian lens. Western blotting showed AQP3 in the lens exists as 25 kDa non-glycosylated and 37 kDa glycosylated monomeric forms in all lens species. To identify the regions in the lens where AQP3 is expressed Western blotting was repeated using epithelial, outer cortical and inner cortical/core fractions isolated from the mouse lens. AQP3 was found in all lens regions, with the highest signal of non-glycosylated AQP3 being found in the epithelium. While in the inner cortex/core region AQP3 signal was not only lower but was predominately from the glycosylated form of AQP3. Immunolabelling of lens sections with AQP3 antibodies confirmed that AQP3 is found in all regions of the adult mouse, and also revealed that the subcellular distribution of AQP3 changes as a function of fiber cell differentiation. In epithelial and peripheral fiber cells of the outer cortex AQP3 labelling was predominately associated with membrane vesicles in the cytoplasm, but in the deeper regions of the lens AQP3 labelling was associated with the plasma membranes of fiber cells located in the inner cortex and core of the lens. To determine how this adult pattern of AQP3 subcellular distribution was established, immunolabelling for AQP3 was performed on embryonic and postnatal lenses. AQP3 expression was first detected on embryonic day (E) 11 in the membranes of primary fiber cells that have started to elongate and fill the lumen of the lens vesicle, while later at E16 the AQP3 labelling in the primary fiber cells had shifted to a predominately cytoplasmic location. In the following postnatal (P) stages of lens growth at P3 and P6, AQP3 labelling remained cytoplasmic across all regions of the lens and it was not until P15 when the pattern of localisation of AQP3 changed to an adult distribution with cytoplasmic labelling detected in the outer cortex and membrane localisation detected in the inner cortex and core of the lens. Comparison of the AQP3 labelling pattern to those obtained previously for AQP0 and AQP5 showed that the subcellular distribution was more similar to AQP5 than AQP0, but there were still significant differences that suggest AQP3 may have unique roles in the maintenance of lens transparency.

Conditional Ablation of Spred1 and Spred2 in the Eye Lens Negatively Impacts Its Development and Growth.

The development and growth of the eye depends on normal lens morphogenesis and its growth. This growth, in turn, is dependent on coordinated proliferation of the lens epithelial cells and their subsequent differentiation into fiber cells. These cellular processes are tightly regulated to maintain the precise cellular structure and size of the lens, critical for its transparency and refractive properties. Growth factor-mediated MAPK signaling driven by ERK1/2 has been reported as essential for regulating cellular processes of the lens, with ERK1/2 signaling tightly regulated by endogenous antagonists, including members of the Sprouty and related Spred families. Our previous studies have demonstrated the importance of both these inhibitory molecules in lens and eye development. In this study, we build on these findings to highlight the importance of Spreds in regulating early lens morphogenesis by modulating ERK1/2-mediated lens epithelial cell proliferation and fiber differentiation. Conditional loss of both Spred1 and Spred2 in early lens morphogenesis results in elevated ERK1/2 phosphorylation, hyperproliferation of lens epithelia, and an associated increase in the rate of fiber differentiation. This results in transient microphakia and microphthalmia, which disappears, owing potentially to compensatory Sprouty expression. Our data support an important temporal role for Spreds in the early stages of lens morphogenesis and highlight how negative regulation of ERK1/2 signaling is critical for maintaining lens proliferation and fiber differentiation in situ throughout life.

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.

ATF4 May Be Essential for Adaption of the Ocular Lens to Its Avascular Environment.

The late embryonic mouse lens requires the transcription factor ATF4 for its survival although the underlying mechanisms were unknown. Here, RNAseq analysis revealed that E16.5 Atf4 null mouse lenses downregulate the mRNA levels of lens epithelial markers as well as known markers of late lens fiber cell differentiation. However, a comparison of this list of differentially expressed genes (DEGs) with other known transcriptional regulators of lens development indicated that ATF4 expression is not directly controlled by the previously described lens gene regulatory network. Pathway analysis revealed that the Atf4 DEG list was enriched in numerous genes involved in nutrient transport, amino acid biosynthesis, and tRNA charging. These changes in gene expression likely result in the observed reductions in lens free amino acid and glutathione levels, which would result in the observed low levels of extractable lens protein, finally leading to perinatal lens disintegration. These data demonstrate that ATF4, via its function in the integrated stress response, is likely to play a crucial role in mediating the adaption of the lens to the avascularity needed to maintain lens transparency.

Spatiotemporal Localisation of Heparan Sulphate Proteoglycans throughout Mouse Lens Morphogenesis.

Heparan sulphate proteoglycans (HSPGs) consist of a core protein decorated with sulphated HS-glycosaminoglycan (GAG) chains. These negatively charged HS-GAG chains rely on the activity of PAPSS synthesising enzymes for their sulfation, which allows them to bind to and regulate the activity of many positively charged HS-binding proteins. HSPGs are found on the surfaces of cells and in the pericellular matrix, where they interact with various components of the cell microenvironment, including growth factors. By binding to and regulating ocular morphogens and growth factors, HSPGs are positioned to orchestrate growth factor-mediated signalling events that are essential for lens epithelial cell proliferation, migration, and lens fibre differentiation. Previous studies have shown that HS sulfation is essential for lens development. Moreover, each of the full-time HSPGs, differentiated by thirteen different core proteins, are differentially localised in a cell-type specific manner with regional differences in the postnatal rat lens. Here, the same thirteen HSPG-associated GAGs and core proteins as well as PAPSS2, are shown to be differentially regulated throughout murine lens development in a spatiotemporal manner. These findings suggest that HS-GAG sulfation is essential for growth factor-induced cellular processes during embryogenesis, and the unique and divergent localisation of different lens HSPG core proteins indicates that different HSPGs likely play specialized roles during lens induction and morphogenesis.

Impacts of autophagy on the formation of organelle-free zone during the lens development.

The thorough degeneration of organelles in the core of the lens is certainly a hallmark event during the lens development. Organelles degradation in the terminal differentiation process of lens fiber cells to form an organelle-free zone is critical for lens maturation and transparency. Several mechanisms have been proposed to expand our understanding of lens organelles degradation, including apoptotic pathways, the participation of ribozyme, proteolytic enzyme and phospholipase A and acyltransferase, and the newly discovered roles for autophagy. Autophagy is a lysosome-dependent degradation reaction during which the "useless" cellular components are degraded and recycled. These cellular components, such as incorrectly folded proteins, damaged organelles and other macromolecules, are first engulfed by the autophagosome before being further delivered to lysosomes for degradation. Although autophagy has been recognized involving in organelle degradation of the lens, the detailed functions remain to be discovered. Recent advances have revealed that autophagy not only plays a vital role in the intracellular quality control of the lens but is also involved in the degradation of nonnuclear organelles in the process of lens fiber cell differentiation. Herein, we first review the potential mechanisms of organelle-free zone formation, then discuss the roles of autophagy in intracellular quality control and cataract formation, and finally substantially summarize the potential involvement of autophagy in the development of organelle-free zone formation.

The second extracellular domain of connexin 50 is important for in cell adhesion, lens differentiation, and adhesion molecule expression.

Connexin (Cx)-forming channels play essential roles in maintaining lens homeostasis and transparency. We showed here channel-independent roles of Cx50 in cell-cell adhesion and confirmed the second extracellular (E2) domain as a critical domain for cell adhesion function. We found that cell adhesion decreased in cells expressing chimeric Cx50 in which the E2 domain was swapped with the E2 domain of either Cx43 or Cx46. In contrast, adhesion increased in cells expressing chimeric Cx43 and Cx46 with the Cx50 (E2) domain. This function is Cx channel-independent and Cx50 E2 domain-dependent cell adhesion acting in both homotypic and heterotypic manners. In addition, we generated eight site mutations of unique residues between Cx50 and the other two lens Cxs and found that mutation of any one of the residues abolished the adhesive function. Moreover, expression of adhesive-impaired mutants decreased adhesion-related proteins, N-cadherin and ß-catenin. Expression of the adhesion-impaired Cx50W188P mutant in embryonic chick lens caused enlarged extracellular spaces, distorted fiber organization, delayed nuclear condensation, and cortical cataracts. In summary, the results from both in vitro and in vivo studies demonstrate the importance of the adhesive function of Cx50 in the lens.

The lens epithelium as a major determinant in the development, maintenance, and regeneration of the crystalline lens.

The crystalline lens is a transparent and refractive biconvex structure formed by lens epithelial cells (LECs) and lens fibers. Lens opacity, also known as cataracts, is the leading cause of blindness in the world. LECs are the principal cells of lens throughout human life, exhibiting different physiological properties and functions. During the embryonic stage, LECs proliferate and differentiate into lens fibers, which form the crystalline lens. Genetics and environment are vital factors that influence normal lens development. During maturation, LECs help maintain lens homeostasis through material transport, synthesis and metabolism as well as mitosis and proliferation. If disturbed, this will result in loss of lens transparency. After cataract surgery, the repair potential of LECs is activated and the structure and transparency of the regenerative tissue depends on postoperative microenvironment. This review summarizes recent research advances on the role of LECs in lens development, homeostasis, and regeneration, with a particular focus on the role of cholesterol synthesis (eg., lanosterol synthase) in lens development and homeostasis maintenance, and how the regenerative potential of LECs can be harnessed to develop surgical strategies and improve the outcomes of cataract surgery (Fig. 1). These new insights suggest that LECs are a major determinant of the physiological and pathological state of the lens. Further studies on their molecular biology will offer possibility to explore new approaches for cataract prevention and treatment.

A possible connection between reactive oxygen species and the unfolded protein response in lens development: From insight to foresight.

The lens is a relatively special and simple organ. It has become an ideal model to study the common developmental characteristics among different organic systems. Lens development is a complex process influenced by numerous factors, including signals from the intracellular and extracellular environment. Reactive oxygen species (ROS) are a group of highly reactive and oxygen-containing molecules that can cause endoplasmic reticulum stress in lens cells. As an adaptive response to ER stress, lens cells initiate the unfolded protein response (UPR) to maintain normal protein synthesis by selectively increasing/decreasing protein synthesis and increasing the degradation of misfolded proteins. Generally, the UPR signaling pathways have been well characterized in the context of many pathological conditions. However, recent studies have also confirmed that all three UPR signaling pathways participate in a variety of developmental processes, including those of the lens. In this review, we first briefly summarize the three stages of lens development and present the basic profiles of ROS and the UPR. We then discuss the interconnections between lens development and these two mechanisms. Additionally, the potential adoption of human pluripotent stem-cell-based lentoids in lens development research is proposed to provide a novel perspective on future developmental studies.

Beyond the Channels: Adhesion Functions of Aquaporin 0 and Connexin 50 in Lens Development.

Lens, an avascular tissue involved in light transmission, generates an internal microcirculatory system to promote ion and fluid circulation, thus providing nutrients to internal lens cells and excreting the waste. This unique system makes up for the lack of vasculature and distinctively maintains lens homeostasis and lens fiber cell survival through channels of connexins and other transporters. Aquaporins (AQP) and connexins (Cx) comprise the majority of channels in the lens microcirculation system and are, thus, essential for lens development and transparency. Mutations of AQPs and Cxs result in abnormal channel function and cataract formation. Interestingly, in the last decade or so, increasing evidence has emerged suggesting that in addition to their well-established channel functions, AQP0 and Cx50 play pivotal roles through channel-independent actions in lens development and transparency. Specifically, AQP0 and Cx50 have been shown to have a unique cell adhesion function that mediates lens development and transparency. Precise regulation of cell-matrix and cell-cell adhesion is necessary for cell migration, a critical process during lens development. This review will provide recent advances in basic research of cell adhesion mediated by AQP0 and Cx50.

Introduction to the thematic issue: Recognition of women leaders in Science.

This thematic issue of Experimental Biology and Medicine is dedicated to the incredibly important contributions made by women leaders in the biomedical sciences throughout recent history. Scientists from many disciplines have contributed papers, both original research and state of the art reviews, to demonstrate the type of work being performed every day by women leaders committed to advancing scientific knowledge in their respective fields of specialization. In this introduction, we provide readers with a brief highlight of the information to be found in the invited papers.

Defect of LSS Disrupts Lens Development in Cataractogenesis.

Congenital cataract is one of the leading causes of blindness in children worldwide. About one-third of congenital cataracts are caused by genetic defects. LSS, which encodes lanosterol synthase, is a causal gene for congenital cataracts. LSS is critical in preventing abnormal protein aggregation of various cataract-causing mutant crystallins; however, its roles in lens development remain largely unknown. In our study, we generated a mouse model harboring Lss G589S mutation, which is homologous to cataract-causing G588S mutation in human LSS. LssG589S/G589S mice exhibited neonatal lethality at postal day 0 (P0), whereas these mice showed severe opacity in eye lens. Also, we found that cataract was formed at E17.5 after we examined the opacity of embryonic lens from E13.5 to E18.5. Moreover, disrupted lens differentiation occurred at E14.5 prior to formation of the opacity of eye lens, shown as delayed differentiation of lens secondary fiber and disordered lens fiber organization. In addition, RNA-seq analysis indicated that cholesterol synthesis signaling pathways were significantly downregulated. Overall, our findings provide clear evidence that a mouse model harboring a homozygous Lss G589S mutation can recapitulate human congenital cataract. Our study points out that LSS functions as a critical determinant of lens development, which will contribute to better understanding LSS defects in cataractogenesis and developing therapies for cataracts.

Impaired GSH biosynthesis disrupts eye development, lens morphogenesis and PAX6 function.

PURPOSE: The purpose of this study was to elucidate the role and molecular consequences of impaired glutathione (GSH) biosynthesis on eye development. METHODS: GSH biosynthesis was impaired in surface ectoderm-derived ocular tissues by crossing Gclcf/f mice with hemizygous Le-Cre transgenic mice to produce Gclcf/f/Le-CreTg/- (KO) mice. Control mice included Gclcf/fand Gclcwt/wt/Le-CreTg/- mice (CRE). Eyes from all mice (at various stages of eye development) were subjected to histological, immunohistochemical, Western blot, RT-qPCR, RNA-seq, and subsequent Gene Ontology, Ingenuity Pathway Analysis and TRANSFAC analyses. PAX6 transactivation activity was studied using a luciferase reporter assay in HEK293T cells depleted of GSH using buthionine sulfoximine (BSO). RESULTS: Deletion of Gclc diminished GSH levels, increased reactive oxygen species (ROS), and caused an overt microphthalmia phenotype characterized by malformation of the cornea, iris, lens, and retina that is distinct from and much more profound than the one observed in CRE mice. In addition, only the lenses of KO mice displayed reduced crystallin (α, ß), PITX3 and Foxe3 expression. RNA-seq analyses at postnatal day 1 revealed 1552 differentially expressed genes (DEGs) in the lenses of KO mice relative to those from Gclcf/f mice, with Crystallin and lens fiber cell identity genes being downregulated while lens epithelial cell identity and immune response genes were upregulated. Bioinformatic analysis of the DEGs implicated PAX6 as a key upstream regulator. PAX6 transactivation activity was impaired in BSO-treated HEK293T cells. CONCLUSIONS: These data suggest that impaired ocular GSH biosynthesis may disrupt eye development and PAX6 function.

Role of Decorin in Posterior Capsule Opacification and Eye Lens Development.

Decorin (DCN) is involved in a variety of physiological and pathological processes. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) has been proposed as a major cause for the development of posterior capsule opacification (PCO) after cataract surgery. We investigated the plausible target gene(s) that suppress PCO. The expression of Dcn was significantly upregulated in rat PCO tissues compared to that observed in the control using a microarray-based approach. LECs treated with fibroblast growth factor (FGF) 2 displayed an enhanced level of DCN expression, while LECs treated with transforming growth factor (TGF)ß-2 showed a decrease in DCN expression. The expression of tropomyosin 1 (Tpm1), a marker of lens EMT increased after the addition of TGFß-2 in human LEC; however, upregulation of Tpm1 mRNA or protein expression was reduced in human LECs overexpressing human DCN (hDCN). No phenotypic changes were observed in the lenses of 8- and 48-week-old transgenic mice for lens-specific hDCN (hDCN-Tg). Injury-induced EMT of the mouse lens, and the expression patterns of α smooth muscle actin, were attenuated in hDCN-Tg mice lenses. Overexpression of DCN inhibited the TGFß-2-induced upregulation of Tpm1 and EMT observed during wound healing of the lens, but it did not affect mouse lens morphology until 48 weeks of age. Our findings demonstrate that DCN plays a significant role in regulating EMT formation of LECs and PCO, and suggest that for therapeutic intervention, maintenance of physiological expression of DCN is essential to attenuate EMT progression and PCO formation.

TDRD7 participates in lens development and spermiogenesis by mediating autophagosome maturation.

In humans, TDRD7 (tudor domain containing 7) mutations lead to a syndrome combining congenital cataracts (CCs) and non-obstructive azoospermia (NOA), characterized by abnormal lens development and spermiogenesis. However, the molecular mechanism underlying TDRD7's functions in eye and testicular development are still largely unknown. Here, we show that the depletion of this gene in mice and humans resulted in the accumulation of autophagosomes and the disruption of macroautophagic/autophagic flux. The disrupted autophagic flux in tdrd7-deficient mouse embryonic fibroblasts (MEFs) was caused by a failure of autophagosome fusion with lysosomes. Furthermore, transcriptome analysis and biochemical assays showed that TDRD7 might directly bind to Tbc1d20 mRNAs and downregulate its expression, which is a key regulator of autophagosome maturation, resulting in the disruption of autophagosome maturation. In addition, we provide evidence to show that TDRD7-mediated autophagosome maturation maintains lens transparency by facilitating the removal of damaged proteins and organelles from lens fiber cells and the biogenesis of acrosome. Altogether, our results showed that TDRD7 plays an essential role in the maturation of autophagosomes and that tdrd7 deletion results in eye defects and testicular abnormalities in mice, implicating disrupted autophagy might be the mechanism that contributes to lens development and spermiogenesis defects in human.Abbreviations: CB: chromatoid bodies; CC: congenital cataract; CTSD: cathepsin D; DMSO: dimethyl sulfoxide; LAMP1: lysosomal-associated membrane protein 1; LECs: lens epithelial cells; MAP1LC3/LC3/Atg8: microtubule-associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; NOA: non-obstructive azoospermia; OFZ: organelle-free zone; RG: RNA granules; SQSTM1/p62: sequestosome 1; TBC1D20: TBC1 domain family member 20; TDRD7: tudor domain containing 7; TEM: transmission electron microscopy; WT: wild type.

Characterization of embryonic surface ectoderm cell protrusions.

BACKGROUND: During embryonic development, complex changes in cell behavior generate the final form of the tissues. Extension of cell protrusions have been described as an important component in this process. Cellular protrusions have been associated with generation of traction, intercellular communication or establishment of signaling gradients. Here, we describe and compare in detail from live imaging data the dynamics of protrusions in the surface ectoderm of chick and mouse embryos. In particular, we explore the differences between cells surrounding the lens placode and other regions of the head. RESULTS: Our results showed that protrusions from the eye region in mouse embryos are longer than those in chick embryos. In addition, protrusions from regions where there are no significant changes in tissue shape are longer and more stable than protrusions that surround the invaginating lens placode. We did not find a clear directionality to the protrusions in any region. Finally, we observed intercellular trafficking of membrane puncta in the protrusions of both embryos in all the regions analyzed. CONCLUSIONS: In summary, the results presented here suggest that the dynamics of these protrusions adapt to their surroundings and possibly contribute to intercellular communication in embryonic cephalic epithelia.

The novel mutation P36R in LRP5L contributes to congenital membranous cataract via inhibition of laminin γ1 and c-MAF.

PURPOSE: The present study investigated a pathogenic mutation and its mechanism on membranous cataract in a congenital membranous cataract family. METHODS: An autosomal dominant four-generation Chinese congenital membranous cataract family was recruited and whole-exome sequencing was performed to screen for sequence variants. Candidate variants were validated using polymerase chain reaction and Sanger sequencing. Wild-type and mutant low-density lipoprotein receptor-related protein 5-like (LRP5L) plasmids were constructed and transfected into human lens epithelial cells (HLE B-3) and human anterior lens capsules. The cell lysates, nuclear and cytoplasmic proteins, and basement membrane components of HLE B-3 cells were harvested. LRP5L and laminin γ1 were knocked down in HLE B-3 cells using specific small-interfering RNA. The protein expression levels of LRP5L, laminin γ1, and c-MAF were detected using immunoblotting and immunofluorescence. RESULTS: We identified a novel suspected pathogenic mutation in LRP5L (c.107C > G, p.P36R) in the congenital membranous cataract family. This mutation was absent in 300 normal controls and 300 age-related cataract patients. Bioinformatics analysis with PolyPhen-2 and SIFT suggested that LRP5L-P36R was pathogenic. LRP5L upregulated laminin γ1 expression in the cytoplasmic proteins of HLE B-3 cells and human anterior lens capsules, and LRP5L-P36R inhibited the effects of LRP5L. LRP5L upregulated c-MAF expression in the nucleus and cytoplasm of HLE B-3 cells, and LRP5L-P36R inhibited c-MAF expression via inhibition of laminin γ1. CONCLUSION: Our study identified a novel gene, LRP5L, associated with congenital membranous cataract, and its mutant LRP5L-P36R contributed to membranous cataract development via inhibition of laminin γ1 and c-MAF.

Requirement for Crk and CrkL during postnatal lens development.

The Crk and CrkL adaptor proteins have SH2 and SH3 domains and play essential overlapping, as well as distinct, roles in many biological processes, ranging from cell structure and motility to proliferation. Conditional ablation of both Crk and CrkL in neuronal progenitor cells, using a Nestin-Cre transgene, resulted in severe defects in postnatal eye development, including progressive eye closure, lens rupture, and retinal malformation. These phenotypes were not observed in the presence of a single wild-type allele of either Crk or CrkL. We found that the lens in knockout mice started to rupture and disintegrate between postnatal days 7 and 12, although the structure of the retina was relatively well maintained. As the lens deteriorated further, the outer nuclear layer in the posterior of the retina enlarged and developed ruffles. Cre recombination occurred in the lens and retina of the knockout mice. Furthermore, the posterior lens capsule of the knockout mouse was thinner at postnatal days 0.5 and 3, suggesting that the defective lens capsule caused rupturing of the lens near the posterior pole. These results indicate that Crk and CrkL play essential overlapping roles in postnatal lens development.

Etv transcription factors functionally diverge from their upstream FGF signaling in lens development.

The signal regulated transcription factors (SRTFs) control the ultimate transcriptional output of signaling pathways. Here, we examined a family of FGF-induced SRTFs - Etv1, Etv 4, and Etv 5 - in murine lens development. Contrary to FGF receptor mutants that displayed loss of ERK signaling and defective cell differentiation, Etv deficiency augmented ERK phosphorylation without disrupting the normal lens fiber gene expression. Instead, the transitional zone for lens differentiation was shifted anteriorly as a result of reduced Jag1-Notch signaling. We also showed that Etv proteins suppresses mTOR activity by promoting Tsc2 expression, which is necessary for the nuclei clearance in mature lens. These results revealed the functional divergence between Etv and FGF in lens development, demonstrating that these SRTFs can operate outside the confine of their upstream signaling.

Many cells contain proteins known as signal-induced transcription factors, which are poised to receive messages from the environment and then react by activating genes required for the cell to respond appropriately. It is commonly thought that these transcription factors faithfully follow the instructions they receive from the external signal: for instance, if the message was to encourage the cell to grow, the transcription factors would switch on growth-related genes. As the eyes of mice and other mammals develop, a signal known as FGF is required for certain cells to specialize into lens fiber cells: these long, thin, transparent cells form the bulk of the lens, the structure that allows focused vision. Previous studies suggest that FGF activates three transcription factors known as Etv1, Etv4 and Etv5, but their precise roles in the development of the lens has remained unclear. Here, Garg, Hannan, Wang et al. confirm that FGF signaling does indeed activate all three proteins. However, mutant mice that lacked Etv1, Etv4 and Etv5 still created lens fiber cells, suggesting that the transcription factors are largely unnecessary for lens fiber cells formation. Instead, the Etv proteins participated in a cascade of molecular events involving a protein called Notch; as a result, if the transcription factors were absent, the lens fiber cells formed prematurely. In addition, deactivating Etv1, Etv4 and Etv5 also promoted the activity of a protein which interfered with the removal of internal cell compartments, a process required for lens fiber cells to mature properly. These findings reveal that the roles of Etv1, Etv4 and Etv5 deviate from and even oppose FGF signaling in the lenses of mice. Transcription factors control the ultimate fate of a cell, and there is therefore increased interest in targeting them for therapy. The work by Garg, Hannan, Wang et al. reveals an unexpected complexity in how these proteins respond to upstream signals, highlighting the importance of further dissecting these relationships.