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. Author manuscript; available in PMC: 2019 Apr 1.
Published in final edited form as: Exp Eye Res. 2018 Feb 7;169:111–121. doi: 10.1016/j.exer.2018.02.001

The klotho-related protein KLPH (lctl) has preferred expression in lens and is essential for expression of clic5 and normal lens suture formation

Jianguo Fan 1, Joshua Lerner 1, M Keith Wyatt 1, Phillip Cai 1, Katherine Peterson 1, Lijin Dong 2, Graeme Wistow 1,*
PMCID: PMC5878992  NIHMSID: NIHMS941912  PMID: 29425878

Abstract

KLPH/lctl belongs to the Klotho family of proteins. Expressed sequence tag analyses unexpectedly revealed that KLPH is highly expressed in the eye lens while northern blots showed that expression is much higher in the eye than in other tissues. In situ hybridization in mouse localized mRNA to the lens, particularly in the equatorial epithelium. Immunofluorescence detected KLPH in lens epithelial cells with highest levels in the germinative/differentiation zone. The gene for KLPH in mouse was deleted by homologous recombination. Littermate knockout (KO) and wild type (WT) mice were compared in a wide panel of pathology examinations and were all grossly normal, showing no systemic effects of the deletion. However, the lens, while superficially normal at young ages, had focusing defects and exhibited age-related cortical cataract by slit lamp examination. Whole-lens imaging showed that KO mice had disorganized lens sutures, forming a loose double-y or x instead of the tight y formation of WT. RNA-seq profiles for KO and WT littermates confirmed the absence of KLPH mRNA in KO lens and also showed complete absence of transcripts for Clic5, a protein associated with cilium/basal body related auditory defects in a mouse model. Immunofluorescence of lens epithelial flat mounts showed that Clic5 localized to cilia/centrosomes. Mice mutant for Clic5 (jitterbug) also had defective sutures. These results suggest that KLPH is required for lens-specific expression of Clic5 and that Clic5 has an important role in the machinery that controls lens fiber cell extension and organization.

Keywords: Lens, KLPH, Klotho, Clic5, cataract

1. Introduction

The transparency and refractive properties of the eye lens depend upon the organization of highly elongated, terminally differentiated lens fiber cells (Audette et al., 2017; Dawes et al., 2014; McAvoy et al., 1999; McAvoy et al., 2017). Fiber cells form by differentiation of cuboidal epithelial cells at the lens equator. The lens grows throughout life, adding new layers of fiber cells. Gradients of growth factors, particularly FGF, across the lens trigger differentiation of lens epithelium at the lens equator (Dawes et al., 2014). New fiber cells extend anteriorly with their apical tips migrating along the overlying epithelium, while the posterior ends migrate along the lens capsule (a basement membrane) (McAvoy et al., 2017). The cells elongate tremendously, extending in both directions from their origin towards the anterior and posterior poles. In normal lens, the convergence of the fiber cells forms tight suture lines, with characteristic patterns in different mammals (Kuszak et al., 2004; Maisel et al., 1981). Since the sutures form along the optical axis of the lens, their precise organization is important for light transmission (Kuszak et al., 2004).

The coordinated extension of the fiber cells, with highly polarized organization of the cilium/centrosome at the apical tips of the fibers, has been described as a planar cell polarity (PCP) mechanism, involving Wnt/Frz signaling (Chen et al., 2008; McAvoy et al., 2017). Other pathways, including signaling through Epha2, also play key roles in normal fiber cell elongation and organization (Shi et al., 2012).

Mature lens cells and their protein components do not turn over with age. Although they are remarkably resilient, age-related cataract is a major source of vision loss in humans. Stochastic processes and the accumulation of insults from oxidation and other environmental factors contribute to aging throughout the body, but there are also genetic factors. NEIBank was a project to explore the transcriptome of eye tissues in several species (Wistow, 2006; Wistow et al., 2008). It led to the discovery of several novel genes as well as providing expression profiles and sequence verified cDNA resources for ocular research. Expressed sequence tag analyses for lens in several species revealed novel transcripts, some highly expressed, such as those for lengsin (Wistow et al., 2002; Wyatt et al., 2008; Wyatt et al., 2006), and for a novel protein that was subsequently identified as KLPH (lctl) which belongs to the same superfamily as Klotho (Ito et al., 2002).

Klotho has been a subject of intense interest in aging research, since the deletion of its gene led to a premature aging syndrome in mice (Kuro-o et al., 1997; Xu and Sun, 2015). It is involved in kidney function, calcium homeostasis and regulation of receptors, including the FGF receptor (Chang et al., 2005; Urakawa et al., 2006), and has also been identified as Wnt antagonist and modulator of the Wnt/β-catenin pathway (Liu et al., 2007; Wang and Sun, 2009). Klotho is related to β-glucuronidase enzymes and the full-length protein has two glycosyl hydrolase family-1domains (Tohyama et al., 2004). Klotho has a predicted transmembrane helix but also exists as secreted factor in serum which may be truncated to a single enzyme domain (Xu and Sun, 2015). KLPH has sequence similarity to Klotho and the related β-Klotho, with a predicted membrane anchor and a single glycosyl hydrolase domain (Ito et al., 2002). KLPH expression was previously noted in kidney and skin (Ito et al., 2002), but eye expression was not examined. Here we show that KLPH is most abundantly expressed in lens and is essential for normal organization of lens fiber cells.

2. Methods

2.1. cDNA discovery

NEIBank procedures and analysis have been described before (Wistow, 2002, 2006; Wistow et al., 2002; Wistow et al., 2008). Analysis of the lens transcriptome from multiple species revealed the abundant expression of a novel transcript that was subsequently identified as KLPH/lctl (Ito et al., 2002).

2.2. Northern Blot

Mouse multi-tissue (6-10wk) Northerns were purchased from Seegene Inc (Seoul, Korea). A cDNA for mouse KLPH was identified from the NEIBank collection. The insert was excised and labelled using a prime-it II kit (Stratagene systems, La Jolla, CA) and 32P-labelled dCTP Northern blots were prehybridized in Hybrisol II (Oncor, Gaithersburg, MD) for 4 hr, followed by hybridization with the specific radiolabelled cDNA probe at 63 °C for 18 hr. After hybridization, membranes were washed in 0.2X SSC, 0.1% SDS at 63 °C and exposed to Kodak XAR film at −70 °C.

2.3. In Situ Hybridization (ISH)

Frozen tissues were cut into 10 μm sections, mounted on gelatin-coated slides and stored at −80°C. ISH was performed at Phylogeny Inc. (Columbus, OH). Slides were fixed in 4% formaldehyde (paraformaldehyde; Sigma Aldrich P6148) in phosphate-buffered saline (PBS), treated with triethanolamine/acetic anhydride, washed and dehydrated with an ethanol series.

A mouse clone for KLPH was identified from the NEIBank collection and a 750bp fragment of coding sequence was subcloned into pCR4 TOPO (Thermo Fisher Scientific, Waltham MA), This was used to generate sense and antisense cRNA using reagents from Ambion (Thermo Fisher) incorporating 35S-UTP (>1000 Ci/mmol; Amersham Bioscience, GE Healthcare, Marlborough MA). Sections were hybridized overnight at 55°C in 50% deionized formamide, 0.3 M NaCl, 20mM Tris-HCl pH 7.4, 5 mM EDTA, 10 mM NaH2PO4, 10% dextran sulfate, 1 × Denhardt’s, 50 μg/ml total yeast RNA, and 50-80,000 cpm/μl 35S-labeled cRNA probe. The tissue was subjected to stringent washing at 65°C in 50% formamide, 2 × SSC, 10 mM DTT and washed in PBS before treatment with 20 μg/ml RNAse A at 37°C for 30 minutes. Following washes in 2 × SSC and 0.1 × SSC for 10 minutes at 37°C, the slides were dehydrated, exposed to Kodak BioMaxMR x-ray film for 4 days, then dipped in Kodak NTB nuclear track emulsion and exposed for 15 days in light-tight boxes with desiccant at 4°C. Photographic development was carried out in Kodak D-19. Slides were counterstained lightly with hematoxylin and analyzed using brightfield, darkfield and Nomarski optics. Sense control cRNA probes (identical to the mRNAs) always gave background levels of hybridization signal.

2.4. Immunochemistry

A custom peptide antibody specific for KLPH was designed. The peptide Ac-QGPSYQNDRDLVELVDPC-amide was selected and used to immunize rabbits at 21st Century Biochemicals (Marlborough MA). Rabbit polyclonal anti-Clic5 antibody was obtained from Alomone Labs (Jerusalem, Israel); mouse anti-pericentrin antibody was obtained from Abcam (Cambridge, MA); goat polyclonal anti-RabIF antibody was purchased from Sigma Aldrich.

2.5. Immunofluorescence

Mouse eyes were enucleated and fixed in 4% paraformaldehyde in PBS for 2 hours, cryopreserved with 10% followed by 20% sucrose in PBS for 1 hour each. After embedding in Optimal Cutting Temperature (OCT) (Tissue-Tek, Torance, CA) with appropriate orientation to allow cutting of sagittal sections, the eye was cut into frozen sections with 8-10 μm thickness. Following permeation with 0.2% Triton X-100 in PBS, the sections were blocked with ICC buffer (1× PBS with 0.2% Tween 20, 0.5% BSA, and 0.05% sodium azide) containing10% bovine serum albumin (BSA) with or without 2% cold water fish gelatin (Sigma-Aldrich) for 3 hours at room temperature. The sections were then incubated with primary antibodies in either Signal Doctor (Solution A) (http://www.abfrontier.com) or in ICC buffer containing 10% BSA with 2% cold water fish gelatin. After washing, the sections were labeled with dye-conjugated secondary antibodies and imaged with Olympus Fluoview 1000 confocal microscope (Olympus, Waltham, MA).

2.6. Targeted deletion of the mouse KLPH/Lctl gene

The genomic region of ~5.5kb with 5′ half of the mouse KLPH/Lctl locus, including part of the promoter sequence and exons 1-7, was deleted by homologous recombination in mouse germline (Thomas and Capecchi, 1987). Briefly, a Bacteria Artificial Chromosomal (BAC) clone of SV1296 origin covering the locus of the mouse gene (RP23: purchased from BACPAC Resources, Oakland CA) was used to construct a targeting vector through ET recombination (Muyrers et al., 2004). The targeting vector contained a left arm of 3.9kb and a right arm of 4.5kb with a PGK-neo cassette in the center flanked by LoxP sites. Linearized targeting vector DNA was electroporated into the mouse R1 ES cells and homologous recombination events were selected by long range genomic PCR first and confirmed with Southern blotting. The knockout allele was successfully transmitted through germline. The Neo cassette was removed with the germline Cre allele Zp3-Cre (Lewandoski et al., 1997) followed by 5 consecutive generations of crossing with the wild type C57BL6/J mice to reach near congenic state of C57BL6/J background.

2.7 Jitterbug mice

Clic5-mutant jitterbug mice (C3H/HeJ-Clic5jbg/J) in C3H background were obtained from Dr. Mark Berryman (Ohio University Heritage College of Osteopathic Medicine, Athens, OH) and in-bred for five generations into 129S6 (Taconic, Hudson, NY).

2.8. Mouse Phenotype Survey

Three pairs of age matched adult female knockout (KO) and wild type (WT) mice were subjected to comprehensive standardized gross and histopathologic analyses including the analyses of organ weights, serum chemistries and hematology through the Division of Veterinary Resources, National Institutes of Health, Bethesda, MD USA.

2.9. Lens Imaging

Mouse eyes were enucleated and lenses with cornea/iris attached were carefully dissected in PBS buffer maintained at 37 °C by cutting along the corneo-scleral divide all around with a micro scissors. The intact lens/cornea was carefully separated from the rest of the eye and incubated in dye-free DMEM/F12 (1:1) media containing 8% fetal bovine serum and 2 μM of a fluorescent lipophilic dye FM4-64FX (Life Technologies, Carlsbad, CA) for 2 hours at 37 °C. The lens was placed posterior side up in the beveled center hole (1.7 mm in diameter) of a black 1-mm thick plastic washer submerged in the same media maintained at 37 °C in a 60-mm Petri dish and imaged at various depth by Olympus Fluoview 1000 confocal microscope equipped with a 60× water immersion objective lens.

Through-the-lens imaging to examine focusing ability of the lens was performed as described previously (Fan et al., 2012).

2.10. RNAseq

Lens transcriptomes of KO and WT mice were compared by RNAseq. DNA-free total RNA was prepared from a pool of lenses from 5 KO/KO or littermate WT mice, by use of PureLink RNA Mini Kit and TRIzol Reagent (Life Technologies: Thermo Fisher Scientific) and on-column DNase treatment with PureLink DNase (Life Technologies) according to manufacturer’s instructions. RNA integrity was evaluated by 2100 Bioanalyzer (Agilent Technologies, Santa Clara CA) with RIN no less than 7. RNA-seq was performed by NIH Intramural Sequencing Center (NISC). One paired end index library from each polyA(+)-selected RNA samples was prepared. Six samples were pooled and loaded on 2 lanes of HiSeq 2000 Sequencer (Illumina, San Diego CA), and run as paired end index 100 base reads with a total of 40 million base read pairs per sample. Quality of RNA-seq data in FASTQ format was accessed by use of FASTQC (Babraham Institute, Cambridge UK). Differential mRNA expression between knockout mice and control mice was quantified using the Tuxedo package (i.e. the Tophat-Cufflinks pipeline) (Trapnell et al., 2012) on the NIH Biowulf high performance computing platform. Alignment was performed by use of Tophat version 2.1.1 (Bowtie2) and the mouse10 reference genome (NCBI) with standard parameters. Cufflinks version 2.2.1 was used to assemble the transcriptomes and CuffDiff was used to quantify the levels of gene expression in Reads Per Kilobase of transcript per Million mapped reads (RPKM). False discovery rate (FDR) value of less than or equal to 0.1 was used as the criteria for “significant” differentially regulated genes. Data are submitted to the GEO database with accession number GSE102987.

2.11. Western blotting

Soluble proteins from the lens were extracted using Tris-buffered saline (TBS) buffer (20mM Tris, pH7.4, 150 mM NaCl, plus protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany)) and insoluble proteins were extracted with the RIPA buffer (50 mm Tris ⁄ HCl (pH 7.4), 150 mm NaCl, 0.25% deoxycholic acid, 1% NP-40, 1 mm EDTA and protease inhibitor cocktail) from the pellet obtained after extraction of the lens with TBS buffer. Protein concentrations were determined using the BCR Protein Assay (ThermoFisher Scientific, Waltham, MA). After SDS-electrophoresis, the proteins were transferred to poly(vinylidene difluoride) (PVDF) membrane, immuno-labeled with specific antibodies in Signal Doctor (Solution A) (http://www.abfrontier.com), followed by peroxidase-conjugated secondary antibodies (1 hour at room temperature). The labeled protein was visualized by detection with enhanced chemiluminescence reagents (Pico Western Detection Kit, ThermoFisher Scientific, Waltham, MA) and x-ray film.

2.12. KLPH expression in lens epithelial flat mount

Lens epithelial flat mounts were prepared according to Sugiyama and McAvoy (Sugiyama and McAvoy, 2012). After permeation with 0.2% Triton X-100 in PBS for 10 min, the lens capsule/epithelium flat mount was blocked in ICC buffer (1× PBS with 0.2% Tween 20, 0.5% BSA, and 0.05% sodium azide) containing10% bovine serum albumin (BSA) and 2% cold water fish gelatin (Sigma-Aldrich) for 3 hours at room temperature. The flat mount was then incubated with anti-KLPH antibody in ICC buffer containing 10% BSA and 2% cold water fish gelatin for 16 hours at 4 degrees Celsius. After washing, the flat mount was labeled with dye-conjugated secondary antibody and DAPI and imaged with Olympus Fluoview 1000 confocal microscope (Olympus, Waltham, MA). KLPH expression along a path across the lens epithelium flat mount was quantified with Image J software (version 1.50g, Wayne Rasband, National Institutes of Health, USA). Equivalent signal from KO lens epithelium was used as background.

2.13. Immuno-labeling of Clic5 and cilium centrosome in lens epithelial flat mount

The lens epithelial flat mounts were prepared according to Sugiyama and McAvoy (Sugiyama and McAvoy, 2012). Briefly, mouse lenses were fixed in ice-cold 100% methanol for 45 seconds and washed twice with cold PBS. The lens capsule/epithelium was removed from the lens by two fine-tipped tweezers. After permeation with 0.2% Triton X-100 in PBS for 10 min, the lens capsule/epithelium was blocked in 10% BSA in ICC buffer for 3 hours. The lens capsule was labeled with anti-clic5 antibody along with other antibodies (16 hours at 4 °C) in the buffer Signal Doctor (Solution A) followed by dye-conjugated secondary antibodies (1 hour at room temperature). The labeled protein was visualized by confocal microscopy (Olympus FluoView 1000).

2.14. Three-dimensional construction of lens epithelial flat mount z-series and cross section views

Confocal z-series scan of lens epithelial flat mounts labeled with various antibodies and DAPI were obtained with Olympus Fluoview 1000 Confocal Microscope. The image sequence of the z-series scan in all channels were imported into ImageJ software and the three-dimensional view was constructed. The maximum projections of the x-z (a) and y-z (b) cross section of the three-dimensional construction of the lens epithelial flat mount were obtained by use of Volume Viewer v2.01, a plugin of ImageJ.

2.15. Lens suture imaging

Eyes were removed from mice and intact lenses were carefully isolated by making a cut through the back of the eye. Isolated lenses were suspended in PBS and the posterior sutures were imaged on a Zeiss Stemi-2000C stereo microscopy (Gottingen, Germany) with the attached Canon A640 digital camera with side illumination.

3. Results

3.1 KLPH transcripts in the lens

Expressed sequence tag analyses of unnormalized cDNA libraries identified a novel transcript among the 20 most highly expressed in the lens of several species; for example, ranking in 10th place for a mouse lens library (https://neibank.nei.nih.gov/cgi-bin/showDataTable.cgi?lib=NbLib0113). This transcript was subsequently identified by others as KLPH/Lctl, a member of the Klotho gene family (Ito et al., 2002). No transcripts were seen in EST analyses of other tissues. A multi-tissue Northern blot for mouse showed much higher expression in whole eye than in other tissues (Fig 1A). Previous results showed expression of KLPH in kidney and skin (Ito et al., 2002), however our results suggest that expression is much higher in lens than other tissues. After this work was completed, other authors detected higher expression level for KLPH (called γ-klotho) in lens than in other human eye tissues (Zhang et al., 2017). In situ hybridization was used to localize expression within mouse eye (Fig 1B). From P1- P56, strong antisense probe labelling was detected only in lens, localized to the epithelium and the outer layers of newly extending fiber cells, with particularly strong signal in the equatorial region. No significant hybridization was apparent elsewhere in eye.

Figure 1. KLPH/Lctl expression in lens.

Figure 1

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A) Northern blot for multiple adult mouse tissues shows strong expression for KLPH/Lctl only in eye. Br: brain; He: heart; Eye; Lu: lung; Li: liver; Sp: spleen; Ki: kidney; SM: skeletal muscle; Th: thymus; Te: testis; PI: placenta; SI: small intestine.

B) In situ hybridization of mouse eye at postnatal days p1, p5, p10, p56 shows specific labelling of lens epithelium with antisense probe for KLPH/Lctl. L: lens; Re: retina; C: cornea; I: iris; Ch: choroid; ON: optic nerve. Arrows indicate regions of strongest expression in lens epithelium and superficial fibers.

3.2. KLPH in lens

Using a custom antibody, a single protein species for KLPH was detected only in the insoluble fraction of WT lens (Fig 2A). Under reducing conditions, the detected protein ran at 70kDa, consistent with a monomer of KLPH. Under non-reducing conditions, the band shifted to dimer size of 140kDa. This suggests that KLPH exists as a disulfide linked dimer associated with insoluble fractions, including membrane and cytoskeleton, in the lens.

Figure 2. Expression of KLPH protein in mouse lens.

Figure 2

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KLPH is expressed in the insoluble fraction of lens epithelium with highest expression in the equatorial periphery corresponding to mitotic, migrating and differentiating cells.

A) Western blot using anti-KLPH peptide antibody on aqueous buffer soluble (sol) and insoluble (insol) extracts from wild type (+/+) and KO (−/−) lens. Left panel without added DTT shows a band corresponding to a dimer. Right panel with DTT shows bands expected for both dimer and monomer of KLPH.

B) Immunofluorescence of mouse lens equatorial; bow region. Anti-KLPH (green), DAPI (blue). WT (+/+) (panel a and b) shows strong intracellular expression of KLPH in epithelial cells. KO (−/−) lens (panel c and d) shows no expression. Scale bar: 10 μm.

C) Flat mount of WT mouse lens epithelium labelled with DAPI and imaged with confocal microscopy (panels a and b). Distribution of nuclei shows the central epithelium, the denser nuclei of the germinative zone leading on to the transition zone and the columnar meridional rows of the cells differentiating into new fibers. White bar (panel a) is 100μm size marker. GZ: germinative zone; TZ: transition zone; MR: meridional rows. In lens epithelium flat mount from KLPH KO mice (mini panels c and d), KLPH expression was absent. Horizontal dashed lines in mini panels b and d showed the typical scan path used to quantify KLPH expression levels (see Panels D and E below).

D) Typical intensity scan for green signal across the flat mount (scan paths shown in panels C(b) and C(d)) using ImageJ software.

E) Quantitation of intensity in the same 200 μm width regions (as indicated by the purple and orange bars in D) of central and peripheral epithelium from WT lens with equivalent scan from KO lens subtracted as background. Results are shown for three independent sets of scans with standard deviations indicated by error bars.

Immunofluorescence labelling for KLPH showed expression throughout most of the lens epithelium, with the strongest signal in the equatorial cells, again there was no signal above background in the KO lens (Fig 2B). Labelling of flat mounts of complete WT lens epithelium (Fig 2C) showed evidence for a gradient of KLPH expression, with a fairly constant level of staining in the central epithelium increasing quite sharply at the equatorial germinative zone, in which epithelial cells divide, migrate and align into columnar arrays of cells which proceed to differentiate into fibers. This difference might be attributed to the increase in thickness of the epithelium from the center to the equator, but still suggests that there is more KLPH protein in the equatorial region.

3.4 Deletion of KLPH/Lctl

The mouse gene for KLPH/Lctl was deleted by homologous recombination in mouse ES cells (Fig 3). Homozygous cells were used for injection into mouse embryos and mice were screened and bred into C57Bl/6 over multiple generations to produce a uniform genetic background. Heterozygous pairs were bred to produce mixed litters to allow comparison of wild type and KO siblings. Loss of KLPH expression in the KO mice was confirmed by western blotting using a custom peptide antibody (Fig 2A) and also by RNAseq, as described below. The KO mice showed no obvious non-lens phenotypes and extensive pathological examination found no KO phenotype in any of the multiple tissues and organs tested, suggesting that KLPH has no essential systemic role in the mouse.

Figure 3. Construction of KLPH/Lctl knockout allele.

Figure 3

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Schematic diagrams show the location of the target gene on mouse chromosome 9; exon structure of the gene; the targeting vector including the selectable marker Neo cassette; the null allele; the null allele after flp recombination.

3.5 Lens phenotype

While KO lenses were normal in size and had no major opacity at young ages, slit lamp examination showed the development of cortical cataract with age (Fig 4A) while photography through isolated lenses also showed defects in lens optics, with distorted image formation (Fig 4B). These defects worsened with age.

Figure 4. Deletion of KLPH causes cataract and focusing defects.

Figure 4

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A) Slit lamp examination of typical aged matched +/+ (a and c) and −/− lenses (b and d) at 5 (a and b) and 9 months (c and d), showing age-related cortical cataract developing in the KO by 9 months. Small arrows in panel a) indicate position of cornea (right) and anterior surface of the lens (left).

B) Isolated lenses from +/+ (a-d) and −/− mice (e-h) show cataract (panels a, c, e, and g on left) and poor image formation of a picture viewed through each lens (panels b, d, f and h on the right).

Lens sutures were examined by light microscope imaging of isolated lenses at 6 weeks of age (Fig 5A). All WT lenses at this age had clear “Y” shaped posterior sutures (n=12). In contrast, all KO lenses had “X” or double “Y” shaped sutures (n=12). This defect was confirmed using confocal microscopy of isolated lenses labelled with a membrane dye (Fig 5B). This appearance is similar to that reported for a transgenic model used to disrupt Frz pathways signaling in mouse lens which produced suture defects attributed to a dysregulation of planar cell polarity (Chen et al., 2008), although with a more severe phenotype overall, and is also similar to defects in the Epha2 model (Shi et al., 2012).

Figure 5. Lens sutures are defective in KO mice.

Figure 5

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A)-D) Posterior sutures in isolated lenses from 6-week KO and WT mice. The normal “Y” suture in WT (C and D) is replaced with “X” or double “Y” patterns in KO (A and B).

E)-F) Confocal imaging of isolated 6-week old whole lens posterior sutures treated with fluorescent membrane dye shows abnormal sutures in the KO lens (E) as compared to that from wild type lens (F). Scale bars in E, F indicated 120 μm.

3.6 RNAseq and Clic5

To gain insight into the mechanism involved, RNAseq data for 6-week-old WT and KO lens were compared. Overall, there was very little differences in expression levels between WT and KO lenses: as shown in Table 1. Indeed, the results of RNAseq profiling for the most abundant components of mouse lens match quite well to results previously obtained through EST analysis for NEIBank (Wistow, 2006), with the crystallins and other lens-specific structural and membrane proteins. KLPH/Lctl itself appears in the 30 most abundant transcripts in the WT lens, but is absent from the KO. We also noticed that a few retina transcripts were detected in lens by RNAseq and showed differences between WT and KO. However, it is highly likely that these transcripts represent low level and variable contamination of the dissected lens with abundant photoreceptor transcripts. This sort of contamination could go either way, and needs to be considered in regard to the detection of lens-specific transcripts in retina (Kandpal et al., 2012).

Table 1. mRNA expression levels of most abundant genes in KLPH knockout lens and wild type controls.

FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values of the most abundant genes from lenses of KLPH knockout and wild type mice were measured by RNA-seq. Total RNAs were extracted from a pool of five wild type and knockout lenses at an age of 6 wks. Fold change of mRNA expression (FPKM values) of knockout versus wild type lens are shown.

mRNA Expression (FPKM)
Gene WT KO KO/WT
Cryaa 154150 142620 0.925
Cryba1 107184 98040.2 0.915
Crygb 64349 55231.1 0.858
Crygc 62434.5 57696.4 0.924
Crygs 52578.2 49641.2 0.944
Crybb2 46812.3 41973.8 0.897
Crygd 43811.9 39978 0.912
Cryab 26040.3 23203.4 0.891
Cryba4 25472.6 23526.6 0.924
Cryba2 20733.6 19409 0.936
Crybb3 20432.3 19585.6 0.959
Cryge 16715.8 17301.6 1.035
Crygf 15123.6 12713.5 0.841
Crybb1 14175.4 13268 0.936
Fabp5 12534.1 10380.2 0.828
Lgsn 9343.04 8064.31 0.863
Mip 5847.07 5001.19 0.855
Cryga 5493.25 6155.44 1.121
Bfsp1 5236.6 4443.06 0.848
Flad1 4936.62 4951.04 1.003
Lim2 4202.66 3882.75 0.924
Eef1a1 3321.11 3166.54 0.953
Bfsp2 2658.82 2314.69 0.871
Grifin 2267.84 2134.23 0.941
Cd24a 2123.7 2002.31 0.943
Rpl41 2059.15 2074.52 1.007
Ftl1 1683.61 1681.47 0.999
Hspa8 1490.49 1384.47 0.929
KLPH/Lctl 1460.41 0.206375 <0.001
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In addition to the complete loss of transcripts from KLPH/Lctl, RNAseq of the KO lens also showed the almost complete suppression of one other gene: Clic5. No other gene showed a major difference in expression. Clic5 belongs to a family of proteins originally named as intracellular chloride channels, but of diverse functions (Argenzio and Moolenaar, 2016). Indeed, these proteins are structurally related to glutathione S-transferases and may have activity similar to glutaredoxin (Al Khamici et al., 2015). In wild type lens, transcripts for Clic5 had a Fragments Per Kilobase of transcript per Million mapped reads (FPKM) value of 122.03 (corresponding to moderate abundance) which fell to 0.25 in the KLPH KO mouse (p value 0.00005). A spontaneous deletion mutant of Clic5, resulting in an absence of protein, produces hearing and vestibular defects in the jitterbug (jbg) mouse (Gagnon et al., 2006) and the protein is associated with stereocilia and cytoskeletal structures (Berryman and Bretscher, 2000; Salles et al., 2014). Western blot detected Clic5 in lens fiber cells in the lens, with its strongest expression in the insoluble fractions. No Clic5 was detectable in the KO lens (Fig 6A).

Figure 6. Clic5 expression and localization in lens.

Figure 6

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A) Western blot with anti-Clic5 for soluble and insoluble fractions of WT (+/+) and KO (−/−) lens extracts. Clic5 is present only in WT lens, mainly in the insoluble fraction of the lens extracts.

B) IF labelling of WT lens epithelium flat mount by confocal microscopy. Localization of Clic5 (green), pericentrin (red) and RabIF (white) and all three merged with addition of DAPI (blue). All three localize to primary cilia/centrosomes apical to the underlying epithelial sheet. Scale Bar: 20 μm.

C) Maximum projections of the x-z (a) and y-z (b) cross section of a three-dimensional construction of the lens epithelial flat mount shown in B. This shows that Clic5, pericentrin and RabIF colocalize on the anterior side of the epithelial cells (DAPI). Scale Bar: 10 μm.

D) Lens epithelial flat mount from KLPH KO labelled for Clic5, pericentrin and DAPI. Clic5 is absent from primary cilia/centrosomes. Scale Bar: 20 μm.

E) Imaging of lenses from WT and Clic5 jitterbug (jbg) mutant lenses on 129S6 background. 129S6 WT shows faint but normal Y-shaped sutures while jbg lens show no organized suture, just diffuse opacities on the optical axis.

Localization of Clic5 in the lens was challenging, however, it was detectable in flat mounts of WT mouse lens anterior epithelium peeled away from the underlying fibers, following procedures previously used to identify attached cilia/centrosomes from anterior tips of fiber cells (Chen et al., 2008). Clic5 labelled discrete structures resembling cilia/centrosomes which co-labelled with the cilium marker RabIF and the centrosome marker pericentrin (Fig 6B, C). Similar flat mounts for KLPH KO mouse lens showed no detection of Clic5 in cilim/centrosome (Fig 6D).

We examined the lens of jbg mice bred into the 129S6 genetic background and genotyped for Clic5 jbg/jbg (Fig 6E). In this background, the sutures were less easily imaged than in C57Bl/6, even in WT mice. Even so, we could see faint Y-shaped sutures in the WT lens, similar to those in Fig 5, which were absent in the Clic5 mutant mice. These instead showed diffuse opacities on the optical axis, instead of organized sutures.

4. Discussion

Expression profiling of the lens in multiple species for NEIBank revealed the unexpectedly high abundance of transcripts for the Klotho-related Klph/lctl, which had levels comparable to some crystallins. Furthermore, although KLPH had been detected in other tissues (Ito et al., 2002), our results suggest that it is has highly lens-preferred expression. Indeed, global deletion of the gene had no observable effects outside the lens. The high expression of KLPH in lens is even more remarkable as, unlike crystallins, it is not widely expressed throughout the lens but, by IF labelling, is instead quite tightly localized to the outermost layers of the lens, the epithelium and the superficial fibers. The highest levels of protein appear to be in the equatorial epithelium at the germinative zone where migrating epithelial cells begin to form organized lines as they progress towards differentiation in fibers, in this region KLPH labelling is twice as high as in the central epithelium. Fiber cells arise by differentiation of precursor epithelial cells at the lens equator which undergo tremendous elongation, reaching halfway around the lens to the anterior and posterior poles where they form well-defined suture lines (Audette et al., 2017; Kuszak et al., 2004; McAvoy et al., 2017). The mechanisms for controlling the growth and directionality of the elongating fibers are not completely understood, but this remarkable process has been described as a form of planar cell polarity (PCP), related to Wnt/Frz signaling (Chen et al., 2008).

Although the lens grows throughout life and functions well for decades in humans, age-related problems, including cataract and presbyopia are common (Glasser et al., 2001; Vrensen, 1995). Klotho first drew attention for its role in an aging syndrome in a mouse model (Kuro-o et al., 1997), indeed it was even referred to as an “anti-aging hormone” (Xu and Sun, 2015; Yamamoto et al., 2005), and it has been implicated in a wide range of processes, including regulation of Wnt activity (Liu et al., 2007). Considering its relationship with Klotho, Klph/Lctl, presented an interesting candidate for involvement in lens homeostasis and aging. In fact, deletion of Klph/Lctl does cause optical defects in mice, with subtle but important effects on lens structure throughout life that become more pronounced with age.

Deletion of Klph/Lctl results in a superficially normal, clear lens, but the lens has poor optical properties and its clarity and focusing decline with age. This is associated with defects in the organization of lens fiber cells. The cells still differentiate and elongate, but they fail to form normal tight sutures along the optical axis of the lens. Optical defects become more profound with age, possibly because of the accumulation of poorly ordered fiber cells layers.

Klotho has been shown to act as a modifier of FGF receptor function (Urakawa et al., 2006) and FGF is a key regulator of lens cell differentiation (Chamberlain and McAvoy, 1989; McAvoy and Chamberlain, 1989; McAvoy et al., 1999), however a major role for KLPH in modulating FGF response in lens seems unlikely since fiber cell differentiation occurs apparently normally in the KO lens and all major transcripts of the differentiated fiber cells show normal expression. Furthermore, IF labelling for KLPH and western blots shows a mainly intracellular localization, associated with the insoluble fraction of the cytoplasm, while FGFR in lens epithelium appears to be restricted to the plasma membrane (Lee et al., 2016).

Our results suggest that KLPH has a role in fine control of lens fiber cell organization and normal suture formation. How is this mediated? Both RNAseq and western blotting show that KLPH is required for a lens-specific mechanism for expression of Clic5 (Argenzio and Moolenaar, 2016; Berryman and Bretscher, 2000). Indeed, no other gene seems to be significantly affected in the KO lens. Clic5, which is associated with a hearing loss model in mice, has mainly been studied in auditory hair cell stereocilia where it associates with the ERM (ezrin, radixin and moesin) complex and is required for normal PCP (Salles et al., 2014). (ERM also has an important actin-related role in organization and maturation of lens fiber cells (Cheng et al., 2016)). In WT lens, Clic5 is found mainly in the insoluble fraction of fiber cells, which includes cytoskeleton. IF labelling of lens epithelial flat mounts showed that Clic5 is localized to primary cilia/centrosomes, colocalized with cilium and centrosome markers RabIF and pericentrin. Examination of lenses from mice with the jitterbug Clic5 mutation bred into the 129S6 background found that they also lacked normal Y-shaped sutures and exhibited diffuse opacity on the optical axis. This is consistent with the idea that Klph is required for expression of Clic5, while Clic5 has an important role in normal fiber cell organization and suture formation, probably related to cilium/centrosome function.

Indeed, an important role for lens cilia/centrosomes has been suggested for fiber cell extension and suture formation (Chen et al., 2008). However, a recent study has found that conditional deletion of one cilium component, IFT88, in the lens does not affect lens suture formation (Sugiyama et al., 2016). However, since both posterior and anterior ends of the fiber cells similarly form sutures, the lack of an essential role for the full primary cilium itself, which is present only at the anterior end, is perhaps not completely surprising. It may be that the mechanism of controlled extension is propagated throughout the cell through structures, such as microtubules, that interact with the cilium/centrosomal complex and do not require the complete cilium.

While KLPH is evidently necessary for Clic5 expression, it is also possible that it could have a guidance role in fiber cell extension, signaling to the growing fibers as they extend away from the lens equator towards the poles of the lens. Klotho family proteins are related to enzymes, so perhaps KLPH could be involved in production of a diffusible metabolite that could signal to the Clic5-containing complex? While many details of the mechanisms involved remain to be determined, these results show that KLPH has a particular role in lens where it is required for expression of Clic5. Clic5 is expressed in other tissues, but, remarkably, it seems to be under tissue-specific regulation in the lens. This may reflect the highly unusual, highly organized structure of the lens which is essential for its function. These results also raise the possibility that other members of the Klotho family could have similar regulatory roles in other tissues, in addition to the diverse functions already described.

Highlights.

  • KLPH has preferred expression in lens

  • Gene Deletion of KLPH produces lens suture defects

  • KLPH is essential for expression of Clic5 in lens

  • Clic5 locates to cilia/centrosomes in lens

  • Clic5 mutant mice have suture defects

Acknowledgments

This work was carried out in the Intramural program of the National Eye Institute, National Institutes of Health, Bethesda, MD, USA. We thank Dr. Mark Berryman for help in obtaining jbg mice.

Footnotes

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Conflicts of Interest

The authors have no conflicts of interest.

Author Contributions

GW supervised the project. LD constructed the KO mice. JF, JL, PC, KP performed experiments. GW and JF analyzed data and wrote the manuscript.

References

  1. Al Khamici H, Brown LJ, Hossain KR, Hudson AL, Sinclair-Burton AA, Ng JP, Daniel EL, Hare JE, Cornell BA, Curmi PM, Davey MW, Valenzuela SM. Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity. PLoS One. 2015;10:e115699. doi: 10.1371/journal.pone.0115699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Argenzio E, Moolenaar WH. Emerging biological roles of Cl- intracellular channel proteins. J Cell Sci. 2016;129:4165–4174. doi: 10.1242/jcs.189795. [DOI] [PubMed] [Google Scholar]
  3. Audette DS, Scheiblin DA, Duncan MK. The molecular mechanisms underlying lens fiber elongation. Exp Eye Res. 2017;156:41–49. doi: 10.1016/j.exer.2016.03.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berryman M, Bretscher A. Identification of a novel member of the chloride intracellular channel gene family (CLIC5) that associates with the actin cytoskeleton of placental microvilli. Mol Biol Cell. 2000;11:1509–1521. doi: 10.1091/mbc.11.5.1509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chamberlain CG, McAvoy JW. Induction of lens fibre differentiation by acidic and basic fibroblast growth factor (FGF) Growth Factors. 1989;1:125–134. doi: 10.3109/08977198909029122. [DOI] [PubMed] [Google Scholar]
  6. Chang Q, Hoefs S, van der Kemp AW, Topala CN, Bindels RJ, Hoenderop JG. The beta-glucuronidase klotho hydrolyzes and activates the TRPV5 channel. Science. 2005;310:490–493. doi: 10.1126/science.1114245. [DOI] [PubMed] [Google Scholar]
  7. Chen Y, Stump RJ, Lovicu FJ, Shimono A, McAvoy JW. Wnt signaling is required for organization of the lens fiber cell cytoskeleton and development of lens three-dimensional architecture. Dev Biol. 2008;324:161–176. doi: 10.1016/j.ydbio.2008.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheng C, Nowak RB, Fowler VM. The lens actin filament cytoskeleton: Diverse structures for complex functions. Exp Eye Res. 2016 doi: 10.1016/j.exer.2016.03.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dawes LJ, Sugiyama Y, Lovicu FJ, Harris CG, Shelley EJ, McAvoy JW. Interactions between lens epithelial and fiber cells reveal an intrinsic self-assembly mechanism. Dev Biol. 2014;385:291–303. doi: 10.1016/j.ydbio.2013.10.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fan J, Dong L, Mishra S, Chen Y, Fitzgerald P, Wistow G. A role for gammaS-crystallin in the organization of actin and fiber cell maturation in the mouse lens. FEBS J. 2012;279:2892–2904. doi: 10.1111/j.1742-4658.2012.08669.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gagnon LH, Longo-Guess CM, Berryman M, Shin JB, Saylor KW, Yu H, Gillespie PG, Johnson KR. The chloride intracellular channel protein CLIC5 is expressed at high levels in hair cell stereocilia and is essential for normal inner ear function. J Neurosci. 2006;26:10188–10198. doi: 10.1523/JNEUROSCI.2166-06.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Glasser A, Croft MA, Kaufman PL. Aging of the human crystalline lens and presbyopia. Int Ophthalmol Clin. 2001;41:1–15. doi: 10.1097/00004397-200104000-00003. [DOI] [PubMed] [Google Scholar]
  13. Ito S, Fujimori T, Hayashizaki Y, Nabeshima Y. Identification of a novel mouse membrane-bound family 1 glycosidase-like protein, which carries an atypical active site structure. Biochim Biophys Acta. 2002;1576:341–345. doi: 10.1016/s0167-4781(02)00281-6. [DOI] [PubMed] [Google Scholar]
  14. Kandpal RP, Rajasimha HK, Brooks MJ, Nellissery J, Wan J, Qian J, Kern TS, Swaroop A. Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs. Mol Vis. 2012;18:1123–1146. [PMC free article] [PubMed] [Google Scholar]
  15. Kuro-o M, Matsumura Y, Aizawa H, Kawaguchi H, Suga T, Utsugi T, Ohyama Y, Kurabayashi M, Kaname T, Kume E, Iwasaki H, Iida A, Shiraki-Iida T, Nishikawa S, Nagai R, Nabeshima YI. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature. 1997;390:45–51. doi: 10.1038/36285. [DOI] [PubMed] [Google Scholar]
  16. Kuszak JR, Zoltoski RK, Sivertson C. Fibre cell organization in crystalline lenses. Exp Eye Res. 2004;78:673–687. doi: 10.1016/j.exer.2003.09.016. [DOI] [PubMed] [Google Scholar]
  17. Lee S, Shatadal S, Griep AE. Dlg-1 Interacts With and Regulates the Activities of Fibroblast Growth Factor Receptors and EphA2 in the Mouse Lens. Invest Ophthalmol Vis Sci. 2016;57:707–718. doi: 10.1167/iovs.15-17727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lewandoski M, Wassarman KM, Martin GR. Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line. Curr Biol. 1997;7:148–151. doi: 10.1016/s0960-9822(06)00059-5. [DOI] [PubMed] [Google Scholar]
  19. Liu H, Fergusson MM, Castilho RM, Liu J, Cao L, Chen J, Malide D, Rovira II, Schimel D, Kuo CJ, Gutkind JS, Hwang PM, Finkel T. Augmented Wnt signaling in a mammalian model of accelerated aging. Science. 2007;317:803–806. doi: 10.1126/science.1143578. [DOI] [PubMed] [Google Scholar]
  20. Maisel H, Harding CV, Alcala JR, Kuszak J, Bradley R, Bloemendal H. The Morphology of the Lens. In: Bloemendal H, editor. Molecular and Cellular Biology of the Eye Lens. Wiley Interscience; New York: 1981. pp. 49–84. [Google Scholar]
  21. McAvoy JW, Chamberlain CG. Fibroblast growth factor (FGF) induces different responses in lens epithelial cells depending on its concentration. Development. 1989;107:221–228. doi: 10.1242/dev.107.2.221. [DOI] [PubMed] [Google Scholar]
  22. McAvoy JW, Chamberlain CG, dI RU, Hales AM, Lovicu FJ. Lens development. Eye. 1999;13:425–437. doi: 10.1038/eye.1999.117. [DOI] [PubMed] [Google Scholar]
  23. McAvoy JW, Dawes LJ, Sugiyama Y, Lovicu FJ. Intrinsic and extrinsic regulatory mechanisms are required to form and maintain a lens of the correct size and shape. Exp Eye Res. 2017;156:34–40. doi: 10.1016/j.exer.2016.04.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Muyrers JP, Zhang Y, Benes V, Testa G, Rientjes JM, Stewart AF. ET recombination: DNA engineering using homologous recombination in E. coli. Methods Mol Biol. 2004;256:107–121. doi: 10.1385/1-59259-753-X:107. [DOI] [PubMed] [Google Scholar]
  25. Salles FT, Andrade LR, Tanda S, Grati M, Plona KL, Gagnon LH, Johnson KR, Kachar B, Berryman MA. CLIC5 stabilizes membrane-actin filament linkages at the base of hair cell stereocilia in a molecular complex with radixin, taperin, and myosin VI. Cytoskeleton. 2014;71:61–78. doi: 10.1002/cm.21159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shi Y, De Maria A, Bennett T, Shiels A, Bassnett S. A role for epha2 in cell migration and refractive organization of the ocular lens. Invest Ophthalmol Vis Sci. 2012;53:551–559. doi: 10.1167/iovs.11-8568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sugiyama Y, McAvoy JW. Analysis of PCP defects in mammalian eye lens. Methods Mol Biol. 2012;839:147–156. doi: 10.1007/978-1-61779-510-7_12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sugiyama Y, Shelley EJ, Yoder BK, Kozmik Z, May-Simera HL, Beales PL, Lovicu FJ, McAvoy JW. Non-essential role for cilia in coordinating precise alignment of lens fibres. Mech Dev. 2016;139:10–17. doi: 10.1016/j.mod.2016.01.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987;51:503–512. doi: 10.1016/0092-8674(87)90646-5. [DOI] [PubMed] [Google Scholar]
  30. Tohyama O, Imura A, Iwano A, Freund JN, Henrissat B, Fujimori T, Nabeshima Y. Klotho is a novel beta-glucuronidase capable of hydrolyzing steroid beta-glucuronides. J Biol Chem. 2004;279:9777–9784. doi: 10.1074/jbc.M312392200. [DOI] [PubMed] [Google Scholar]
  31. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature protocols. 2012;7:562–578. doi: 10.1038/nprot.2012.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Urakawa I, Yamazaki Y, Shimada T, Iijima K, Hasegawa H, Okawa K, Fujita T, Fukumoto S, Yamashita T. Klotho converts canonical FGF receptor into a specific receptor for FGF23. Nature. 2006;444:770–774. [Google Scholar]
  33. Vrensen GF. Aging of the human eye lens–a morphological point of view. Comp Biochem Physiol A Physiol. 1995;111:519–532. doi: 10.1016/0300-9629(95)00053-a. [DOI] [PubMed] [Google Scholar]
  34. Wang Y, Sun Z. Current understanding of klotho. Ageing Res Rev. 2009;8:43–51. doi: 10.1016/j.arr.2008.10.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wistow G. A project for ocular bioinformatics: NEIBank. Mol Vis. 2002;8:161–163. [PubMed] [Google Scholar]
  36. Wistow G. The NEIBank project for ocular genomics: data-mining gene expression in human and rodent eye tissues. Prog Retin Eye Res. 2006;25:43–77. doi: 10.1016/j.preteyeres.2005.05.003. [DOI] [PubMed] [Google Scholar]
  37. Wistow G, Bernstein SL, Wyatt MK, Behal A, Touchman JW, Bouffard G, Smith D, Peterson K. Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants. Mol Vis. 2002;8:171–184. [PubMed] [Google Scholar]
  38. Wistow G, Peterson K, Gao J, Buchoff P, Jaworski C, Bowes-Rickman C, Ebright JN, Hauser MA, Hoover D. NEIBank: genomics and bioinformatics resources for vision research. Mol Vis. 2008;14:1327–1337. [PMC free article] [PubMed] [Google Scholar]
  39. Wyatt K, Gao C, Tsai JY, Fariss RN, Ray S, Wistow G. A role for lengsin, a recruited enzyme, in terminal differentiation in the vertebrate lens. J Biol Chem. 2008;283:6607–6615. doi: 10.1074/jbc.M709144200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wyatt K, White HE, Wang L, Bateman OA, Slingsby C, Orlova EV, Wistow G. Lengsin is a survivor of an ancient family of class I glutamine synthetases re-engineered by evolution for a role in the vertebrate lens. Structure. 2006;14:1823–1834. doi: 10.1016/j.str.2006.10.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Xu Y, Sun Z. Molecular basis of Klotho: from gene to function in aging. Endocrine reviews. 2015;36:174–193. doi: 10.1210/er.2013-1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yamamoto M, Clark JD, Pastor JV, Gurnani P, Nandi A, Kurosu H, Miyoshi M, Ogawa Y, Castrillon DH, Rosenblatt KP, Kuro-o M. Regulation of oxidative stress by the anti-aging hormone klotho. J Biol Chem. 2005;280:38029–38034. doi: 10.1074/jbc.M509039200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zhang Y, Wang L, Wu Z, Yu X, Du X, Li X. The Expressions of Klotho Family Genes in Human Ocular Tissues and in Anterior Lens Capsules of Age-Related Cataract. Curr Eye Res. 2017;42:871–875. doi: 10.1080/02713683.2016.1259421. [DOI] [PubMed] [Google Scholar]

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