AB3071 Sigma-AldrichAnti-Aquaporin 0 Antibody

We report analysis of the ocular lens phenotype of the recessive, larval lethal zebrafish mutant, lama1 (a69/a69). Previous work revealed that this mutant has a shortened body axis and eye defects including a defective hyaloid vasculature, focal corneal dysplasia, and loss of the crystalline lens. While these studies highlight the importance of laminin α1 in lens development, a detailed analysis of the lens defects seen in these mutants was not reported. In the present study, we analyze the lenticular anomalies seen in the lama1 (a69/a69) mutants and show that the lens defects result from the anterior extrusion of lens material from the eye secondary to structural defects in the lens capsule and developing corneal epithelium associated with basement membrane loss. Our analysis provides further insights into the role of the lens capsule and corneal basement membrane in the structural integrity of the developing eye.

Posterior capsular opacification (PCO), the most prevalent side effect of cataract surgery, occurs when residual lens epithelial cells (LECs) undergo fiber cell differentiation or epithelial-to-mesenchymal transition (EMT). Here, we used a murine cataract surgery model to investigate the role of the Zeb proteins, Smad interacting protein 1 (Sip1) and δ-crystallin enhancer-binding factor 1 (δEF1), during PCO.Extracapsular extraction of lens fiber cells was performed on wild-type and Sip1 knockout mice. Protein expression patterns were assessed at multiple time points after surgery using confocal immunofluorescence. βB1-Crystallin mRNA levels were measured using quantitative RT-PCR. We used Transfac searches to identify δEF1 binding sites in the βB1-crystallin promoter and transfection analysis to test the ability of δEF1 to regulate βB1-crystallin expression.δEF1, which, in other systems, can activate fibrotic genes (e.g., α-smooth muscle actin) and repress epithelial genes, upregulates by 48 hours after fiber cell removal. In culture, δEF1 repressed βB1-crystallin promoter activity, suggesting that it may also turn off lens gene expression following surgery, contributing to "fibrotic PCO" development. Sip1 also upregulates in LECs by 48 hours, but analysis of Sip1 knockout lenses demonstrated that Sip1 does not play a major role in EMT or fiber cell differentiation after surgery. However, Sip1 knockout LECs do express the ectodermal marker keratin 8, suggesting that Sip1 may limit the reprogramming of residual LECs to an embryonic state.Zeb transcription factors likely play important, but distinct roles in PCO development after cataract surgery.

Lens fiber formation and morphogenesis requires a precise orchestration of cell- extracellular matrix (ECM) and cell-cell adhesive changes in order for a lens epithelial cell to adopt a lens fiber fate, morphology, and migratory ability. The cell-ECM interactions that mediate these processes are largely unknown, and here we demonstrate that fibronectin1 (Fn1), an ECM component, and integrin α5, its cellular binding partner, are required in the zebrafish lens for fiber morphogenesis. Mutations compromising either of these proteins lead to cataracts, characterized by defects in fiber adhesion, elongation, and packing. Loss of integrin α5/Fn1 does not affect the fate or viability of lens epithelial cells, nor does it affect the expression of differentiation markers expressed in lens fibers, although nucleus degradation is compromised. Analysis of the intracellular mediators of integrin α5/Fn1 activity focal adhesion kinase (FAK) and integrin-linked kinase (ILK) reveals that FAK, but not ILK, is also required for lens fiber morphogenesis. These results support a model in which lens fiber cells use integrin α5 to migrate along a Fn-containing substrate on the apical side of the lens epithelium and on the posterior lens capsule, likely activating an intracellular signaling cascade mediated by FAK in order to orchestrate the cytoskeletal changes in lens fibers that facilitate elongation, migration, and compaction.

DNA methylation is one of the key mechanisms underlying the epigenetic regulation of gene expression. During DNA replication, the methylation pattern of the parent strand is maintained on the replicated strand through the action of Dnmt1 (DNA Methyltransferase 1). In mammals, Dnmt1 is recruited to hemimethylated replication foci by Uhrf1 (Ubiquitin-like, Containing PHD and RING Finger Domains 1). Here we show that Uhrf1 is required for DNA methylation in vivo during zebrafish embryogenesis. Due in part to the early embryonic lethality of Dnmt1 and Uhrf1 knockout mice, roles for these proteins during lens development have yet to be reported. We show that zebrafish mutants in uhrf1 and dnmt1 have defects in lens development and maintenance. uhrf1 and dnmt1 are expressed in the lens epithelium, and in the absence of Uhrf1 or of catalytically active Dnmt1, lens epithelial cells have altered gene expression and reduced proliferation in both mutant backgrounds. This is correlated with a wave of apoptosis in the epithelial layer, which is followed by apoptosis and unraveling of secondary lens fibers. Despite these disruptions in the lens fiber region, lens fibers express appropriate differentiation markers. The results of lens transplant experiments demonstrate that Uhrf1 and Dnmt1 functions are required lens-autonomously, but perhaps not cell-autonomously, during lens development in zebrafish. These data provide the first evidence that Uhrf1 and Dnmt1 function is required for vertebrate lens development and maintenance.

In the developing vertebrate lens, epithelial cells differentiate into fiber cells, which are elongated and flat in shape and form a multilayered lens fiber core. In this study, we identified the zebrafish volvox (vov) mutant, which shows defects in lens fiber differentiation. In the vov mutant, lens epithelial cells fail to proliferate properly. Furthermore, differentiating lens fiber cells do not fully elongate, and the shape and position of lens fiber nuclei are affected. We found that the vov mutant gene encodes Psmd6, the subunit of the 26S proteasome. The proteasome regulates diverse cellular functions by degrading polyubiquitylated proteins. Polyubiquitylated proteins accumulate in the vov mutant. Furthermore, polyubiquitylation is active in nuclei of differentiating lens fiber cells, suggesting roles of the proteasome in lens fiber differentiation. We found that an E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is involved in lens defects in the vov mutant. These data suggest that the ubiquitin proteasome system is required for cell proliferation of lens epithelium and for the differentiation of lens fiber cells in zebrafish.

Beta1-integrins are cell surface receptors that participate in sensing the cell's external environment. We used the Cre-lox system to delete beta1-integrin in all lens cells as the lens vesicle transitions into the lens. Adult mice lacking beta1-integrin in the lens are microphthalmic due to apoptosis of the lens epithelium and neonatal disintegration of the lens fibers. The first morphological alterations in beta1-integrin null lenses are seen at 16.5 dpc when the epithelium becomes disorganized and begins to upregulate the fiber cell markers beta- and gamma-crystallins, the transcription factors cMaf and Prox1 and downregulate Pax6 levels demonstrating that beta1-integrin is essential to maintain the lens epithelial phenotype. Furthermore, beta1-integrin null lens epithelial cells upregulate the expression of alpha-smooth muscle actin and nuclear Smad4 and downregulate Smad6 suggesting that beta1-integrin may brake TGFbeta family signaling leading to epithelial-mesenchymal transitions in the lens. In contrast, beta1-integrin null lens epithelial cells show increased E-cadherin immunoreactivity which supports the proposed role of beta1-integrins in mediating complete EMT in response to TGFbeta family members. Thus, beta1-integrin is required to maintain the lens epithelial phenotype and block inappropriate activation of some aspects of the lens fiber cell differentiation program.

Nance-Horan syndrome (NHS) is an X-linked disorder characterized by congenital cataracts, dental anomalies, dysmorphic features and mental retardation. A recent report suggests that the novel gene NHS1 is involved in this disorder due to the presence of point mutations in NHS patients. A possible mouse model for NHS, Xcat, was mapped to a 2.11 Mb interval on the X-chromosome. Sequence and FISH analysis of the X-chromosome region containing the Xcat mutation reveal a large insertion between exons 1 and 2 of the mouse Nhs1 gene. The insertion inhibits the expression of the Nhs1 isoform containing exon 1 and results in exclusive expression of the alternative isoform containing exon 1A. Quantitative RT-PCR of Xcat cDNA shows reduced levels of Nhs1 transcripts. The Nhs1 protein is strongly expressed within the cytoplasm of elongating lens fiber cells from wild-type neonate lens, but is significantly reduced within the Xcat lens. Transient transfection studies of CHO cells with Nhs1-GFP fusion proteins were done to determine whether the amino acids encoded by exon 1 were critical for protein localization. We found the presence of Nhs1 exon 1 critical for localization of the fusion protein to the cytoplasm, whereas fusion proteins lacking Nhs1 exon 1 are predominantly nuclear. These results indicate that the first exon of Nhs1 contains crucial information required for the proper expression and localization of Nhs1 protein. Inhibition of expression of the exon 1 containing isoform results in the abnormal phenotype of Xcat.

Major intrinsic protein (MIP, also called MP26) is the predominant fiber cell membrane protein of the ocular lens. MIP has been suggested to play a role in cell-cell communication in the lens. Its expression is tissue-specific and developmentally regulated. We have isolated and characterized the human gene encoding MIP and report here its genomic structure and entire nucleotide sequence. The gene is 3.6 kb, contains four exons separated by introns ranging in size from 0.4 to 1.6 kb, and is present in single copy per haploid human genome. Primer extension of human lens RNA indicates that transcription of the gene initiates from a single site 26 nt downstream from the TATA box. Three complete Alu repetitive elements are found in tandem in the 5'-flanking region of the gene, and a single complete Alu sequence is present in the third intron. The interspecies comparisons of the MIP gene coding sequence and homologies to other members of a putative transmembrane channel protein superfamily are also discussed.