The normal development of lens fiber cells plays a critical role in lens morphogenesis and maintaining transparency. Factors involved in the development of lens fiber cells are largely unknown in vertebrates. In this study, we reported that GATA2 is essential for lens morphogenesis in Nile tilapia (Oreochromis niloticus). In this study, Gata2a was detected in the primary and secondary lens fiber cells, with the highest expression in primary fiber cells. gata2a homozygous mutants of tilapia were obtained using CRISPR/Cas9. Different from fetal lethality caused by Gata2/gata2a mutation in mice and zebrafish, some gata2a homozygous mutants of tilapia are viable, which provides a good model for studying the role of gata2 in non-hematopoietic organs. Our data showed that gata2a mutation caused extensive degeneration and apoptosis of primary lens fiber cells. The mutants exhibited progressive microphthalmia and blindness in adulthood. Transcriptome analysis of the eyes showed that the expression levels of almost all genes encoding crystallin were significantly down-regulated, while the expression levels of genes involved in visual perception and metal ion binding were significantly up-regulated after gata2a mutation. Altogether, our findings indicate that gata2a is required for the survival of lens fiber cells and provide insights into transcriptional regulation underlying lens morphogenesis in teleost fish.

Imaging mass spectrometry (IMS) enables targeted and untargeted visualization of the spatial localization of molecules in tissues with great specificity. The lens is a unique tissue that contains fiber cells corresponding to various stages of differentiation that are packed in a highly spatial order. The application of IMS to lens tissue localizes molecular features that are spatially related to the fiber cell organization. Such spatially resolved molecular information assists our understanding of lens structure and physiology; however, protein IMS studies are typically limited to abundant, soluble, low molecular weight proteins. In this study, a method was developed for imaging low solubility cytoskeletal proteins in the lens; a tissue that is filled with high concentrations of soluble crystallins. Optimized tissue washes combined with on-tissue enzymatic digestion allowed successful imaging of peptides corresponding to known lens cytoskeletal proteins. The resulting peptide signals facilitated segmentation of the bovine lens into molecularly distinct regions. A sharp intermediate filament transition from vimentin to lens-specific beaded filament proteins was detected in the lens cortex. MALDI IMS also revealed the region where posttranslational myristoylation of filensin occurs and the results indicate that truncation and myristoylation of filensin starts soon after filensin expression increased in the inner cortex. From intermediate filament switch to filensin truncation and myristoylation, multiple remarkable changes occur in the narrow region of lens cortex. MALDI images delineated the boundaries of distinct lens regions that will guide further proteomic and interactomic studies.

The transcription factor c-MAF is a member of the large MAF family, members of which possess transactivation and bZIP domains. c-MAF plays an important role in lens formation, T-lymphocyte differentiation, hypertrophic chondrocyte differentiation, and kidney development in mouse embryos. However, because homozygous deletion of c-Maf in C57BL/6J mice causes embryonic lethality, the functions of c-MAF in adult mice remain largely uninvestigated. To address this issue, we generated c-Maf floxed (c-Maffl/fl) C57BL/6J mice and established tamoxifen-inducible c-Maf knockout mice (c-Maffl/fl; CAG-Cre-ERTM mice, c-MafΔTAM). After tamoxifen injection, adult c-MafΔTAM mice showed successful deletion of c-Maf protein and developed severe cataracts; cataracts are also seen in human patients who have mutations in the c-MAF DNA binding domain. Furthermore, adult c-MafΔTAM mice exhibited abnormal lens structure and impaired differentiation of lens fiber cells. In summary, we established c-Maffl/fl and c-MafΔTAM C57BL/6J mice, which can be useful animal models for the investigation of c-MAF function in various developmental stages and can also be used as a disease model for cataracts.

Elevated oxidized stress contributes to lens cataracts, and gap junctions play important roles in maintaining lens transparency. As well as forming gap junctions, connexin (Cx) proteins also form hemichannels. Here, we report a new mechanism whereby hemichannels mediate transport of reductant glutathione into lens fiber cells and protect cells against oxidative stress. We found that Cx50 (also known as GJA8) hemichannels opened in response to H2O2 in lens fiber cells but that transport through the channels was inhibited by two dominant-negative mutants in Cx50, Cx50P88S, which inhibits transport through both gap junctions and hemichannels, and Cx50H156N, which only inhibits transport through hemichannels and not gap junctions. Treatment with H2O2 increased the number of fiber cells undergoing apoptosis, and this increase was augmented with dominant-negative mutants that disrupted both hemichannels formed from Cx46 (also known as GJA3) and Cx50, while Cx50E48K, which only impairs gap junctions, did not have such an effect. Moreover, hemichannels mediate uptake of glutathione, and this uptake protected lens fiber cells against oxidative stress, while hemichannels with impaired transport had less protective benefit from glutathione. Taken together, these results show that oxidative stress activates connexin hemichannels in the lens fiber cells and that hemichannels likely protect lens cell against oxidative damage through transporting extracellular reductants.

The formation and development of age-related cataract (ARC) has been demonstrated to have the involvement of defective DNA repair in lens epithelial cells (LECs). This study aimed to investigate DNA repair genes expression in human lens cortex collected from age-related cortical cataract (ARCC) and controls during surgery. The expression levels of the genes were evaluated by xx genes microarray analysis. The results were further confirmed by Quantitative Real-Time PCR (qRT-PCR). The mRNA levels of 7 genes decreased and 4 genes out of 92 genes increased in lens cortex of ARCCs compared with controls with the fold change >1.5. Using Comet assay, we found the DNA breaks in the LECs of ARCCs were obviously severer than that of controls. The present data provide a global perspective on expression of DNA repair genes that may contribute to cataract pathogenesis. The DNA damage and repair pathway might be an effective target to delay the onset of ARC.

Integrins are heterodimeric cell surface molecules that mediate cell-extracellular matrix (ECM) adhesion, ECM assembly, and regulation of both ECM and growth factor induced signaling. However, the developmental context of these diverse functions is not clear. Loss of β1-integrin from the lens vesicle (mouse E10.5) results in abnormal exit of anterior lens epithelial cells (LECs) from the cell cycle and their aberrant elongation toward the presumptive cornea by E12.5. These cells lose expression of LEC markers and initiate expression of the Maf (also known as c-Maf) and Prox1 transcription factors as well as other lens fiber cell markers. β1-integrin null LECs also upregulate the ERK, AKT and Smad1/5/8 phosphorylation indicative of BMP and FGF signaling. By E14.5, β1-integrin null lenses have undergone a complete conversion of all lens epithelial cells into fiber cells. These data suggest that shortly after lens vesicle closure, β1-integrin blocks inappropriate differentiation of the lens epithelium into fibers, potentially by inhibiting BMP and/or FGF receptor activation. Thus, β1-integrin has an important role in fine-tuning the response of the early lens to the gradient of growth factors that regulate lens fiber cell differentiation.