KnockdownofZebrafishLumicanGene(zlum)CausesScleral ... · PDF fileKnockdownofZebrafishLumicanGene(zlum)CausesScleral ... rapid amplification of cDNA end; z, zebrafish; m, mouse; h,

Knockdown of Zebrafish Lumican Gene (zlum) Causes ScleralThinning and Increased Size of Scleral Coats*□S

Received for publication, July 19, 2009, and in revised form, May 20, 2010 Published, JBC Papers in Press, June 15, 2010, DOI 10.1074/jbc.M109.043679

Lung-Kun Yeh‡§, Chia-Yang Liu¶, Winston W.-Y. Kao¶�, Chang-Jen Huang**, Fung-Rong Hu‡‡, Chung-Liang Chien‡1,and I-Jong Wang‡‡2

From the ‡Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan, the§Department of Ophthalmology, Chang-Gung Memorial Hospital at Linko, Chang-Gung University College of Medicine,Taoyuan 333, Taiwan, the Departments of ¶Ophthalmology and �Cell Biology, Neuroscience, and Anatomy, University of CincinnatiCollege of Medicine, Cincinnati, Ohio 45267, the **Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan, and the‡‡Department of Ophthalmology, National Taiwan University Hospital, Taipei 100, Taiwan

The lumicangene (lum), which encodes oneof themajor kera-tan sulfate proteoglycans (KSPGs) in the vertebrate cornea andsclera, has been linked to axial myopia in humans. In this study,we chose zebrafish (Danio rerio) as an animalmodel to elucidatethe role of lumican in the development of axial myopia. Thezebrafish lumican gene (zlum) spans �4.6 kb of the zebrafishgenome. Like human (hLUM) andmouse (mlum), zlum consistsof three exons, two introns, and a TATA box-less promoter atthe 5�-flanking region of the transcription initiation site.Sequence analysis of the cDNA predicts that zLum encodes 344amino acids. zLum shares 51% amino acid sequence identitywith human lumican. Similar to hLUM andmlum, zlummRNAis expressed in the eye and many other tissues, such as brain,muscle, and liver as well. Transgenic zebrafish harboring anenhanced GFP reporter gene construct downstream of a 1.7-kbzlum 5�-flanking region displayed enhanced GFP expression inthe cornea and sclera, as well as throughout the body. Down-regulation of zlum expression by antisense zlum morpholinosmanifested ocular enlargement resembling axial myopia due todisruption of the collagen fibril arrangement in the sclera andresulted in scleral thinning. Administration of muscarinicreceptor antagonists, e.g. atropine and pirenzepine, effectivelysubdued the ocular enlargement caused by morpholinos in invivo zebrafish larvae assays. The observation suggests thatzebrafish can be used as an in vivo model for screening com-pounds in treating myopia.

Myopia is a very common ocular disorder, which is charac-terized by excessive elongation of the eyeball. In Taiwan, theprevalence of myopia is about 84% of schoolchildren aged16–18, and the prevalence of highmyopia (less than �6.0 D) at18 years of age is 24% in girls and 18% in boys (1). In contrast,the prevalence of high myopia is much lower inWestern coun-tries, about 1% of the general population (2). These studiesimply that genetic susceptibility of ethnic differences mayaccount for the high prevalence of myopia in Taiwanese.The sclera contains a collagen-rich extracellular matrix that

undergoes significant biochemical and biomechanical remod-eling during the development of myopia (3). Linkage studies ofhigh myopia have identified potential loci MYP1 (Xq28) andMYP3 (12q21-23); these loci are within and/or near the loci ofthe human genome containing several genes that encode smallleucine-rich proteoglycans (SLRP),3 i.e. biglycan (Xq27ter),decorin (12q21-22), lumican (12q21.3-22), and DSPG3 (12q21)(4–9). Our previous study showed that certain variations(rs3759223 (C3T)) of single nucleotide polymorphism in thelumican regulatory region may influence the promoter activi-ties of lumican and affect fibrillogenesis in myopic eyes (10).Furthermore,Majava et al. (11) found that a novel single nucle-otide polymorphism (c.893–105G3A) of the lumican genewas associated with high myopia. Recently, our prospectivecase control study also showed that genetic variation in theregulatory domains of the lumican gene (rs3759223 andrs3741834) were associated with high myopia susceptibilityamong the Han Chinese (12).Lumican, a member of the SLRP family, is one of the major

extracellular components in interstitial collagenousmatrices ofcorneal stroma, sclera, aorta, skin, skeletalmuscle, lung, kidney,bone, cartilage, and intervertebral discs (13–20). In cornealstroma, lumican is a KSPG, whereas lumican presents as anunder- or unglycanated glycoprotein in other tissues (9, 13, 14,21). It has been proposed that the horseshoe-shaped lumicancore protein binds collagen molecules to modulate collagenfibril diameter, whereas theN-linked glycosaminoglycan chains

* This work was supported, in whole or in part, by National Institutes ofHealth Grants EY12486 from NEI (to C.-Y. L.) and EY011845 (to W. W.-Y. K.). This work was also supported in part by National ScienceCouncil Grants 923112B002004, 933112B002031, 943112B002007,932314B002090, 942314B002043, 952314B002149MY3, 972314B182A059,982314B182A029MY3, and 983112B002040, Research to Prevent Blind-ness, and Ohio Lions Eye Research Foundation.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1 and 2.

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) GQ376197.

1 To whom correspondence may be addressed: Dept. of Anatomy and CellBiology, National Taiwan University, Sec. 1, Jen-Ai Rd., Taipei 100, Taiwan.Tel.: 886-2-23123456 (Ext. 88193); Fax: 886-2-23915292; E-mail:[email protected].

2 To whom correspondence may be addressed: Dept. of Ophthalmology,National Taiwan University Hospital, Chug-Shan South Rd., Taipei 100, Tai-wan. Tel.: 886-2-23123456 (Ext. 55192); Fax: 886-2-23412875; E-mail:[email protected].

3 The abbreviations used are: SLRP, small leucine-rich proteoglycan; MO, mor-pholino; KSPG, keratan sulfate proteoglycan; EGFP, enhanced GFP; RS-MO,random sequence MO; TEM, transmission electron microscope; RACE,rapid amplification of cDNA end; z, zebrafish; m, mouse; h, human; CS,corneal stroma; AS, anterior sclera; PS, posterior sclera; RPE, retinal pig-ment epithelium; hpf, h post-fertilization; dpf, days post-fertilization.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 36, pp. 28141–28155, September 3, 2010© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

SEPTEMBER 3, 2010 • VOLUME 285 • NUMBER 36 JOURNAL OF BIOLOGICAL CHEMISTRY 28141

by guest on May 18, 2018

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/cgi/content/full/M109.043679/DC1

http://www.jbc.org/

regulate fibril spacing and stromal hydration for the formationandmaintenance of transparent corneas (22–24). Thewide dis-tribution of lumican implies that lumican may have multiplefunctions in tissue morphogenesis and maintenance of tissuehomeostasis, besides serving as a regulatory molecule of colla-gen fibrillogenesis (24). Indeed, lumican plays essential roles inwound healing by modulating epithelial cell migration (25) andin epithelium-mesenchyme transition of the injured lens (26),in addition to regulating collagen fibrillogenesis (20, 25). Lumi-can-null (Lum�/�) mice and lumican- and fibromodullin-null(Lum�/�Fmod�/�) mice showed alterations of collagen fibrilarrangement in interstitial connective tissues. Lum�/�

Fmod�/� mice exhibited elongated axial lengths and thinsclera, which are characteristics of high myopia, and lumican-null mice (Lum�/�) also had a slight elongation of the eyeball(27, 28). These findings indicated that these proteoglycans maybe directly or indirectly involved in scleral development, result-ing in the pathoetiology of high myopia.The zebrafish is an excellentmodel to study vertebrate genet-

ics and development (29–31). As a disease model, transgeniczebrafish provide several advantages, such as shorter durationof embryonic development and optically transparent embryos;its development also provides easy access for observation andtreatment schemes during embryogenesis in comparison withtransgenicmousemodels. In this study, we examined the struc-ture, expression pattern, promoter activity, and function ofzlum. Our data indicated that lumican is highly conservedbetween zebrafish and mammals (e.g. human and mouse) inrespect to gene structure, expression patterns, and proteinfunction. In particular, ocular enlargement and scleral thinningwith changes in the ultrastructure of the sclera were notedwhen zLum expression was down-regulated by antisense zlummorpholinos (MO). Furthermore, in vivo zebrafish larvaeassays were performed to elucidate the potential application ofseveral muscarinic receptor antagonists to attenuate increasedscleral coats caused by zlum knockdown with MO. Ourresults suggest that zebrafish can be used as an in vivomodel forscreening compounds of treating myopia.

EXPERIMENTAL PROCEDURES

Zebrafish Husbandry—Zebrafish were raised and main-tained according to protocols established previously (32).Briefly, adult zebrafish and embryos weremaintained at 28.5 °Con a 14-h light and 10-h dark cycle. Embryos were sorted at thedifferent stages required for each experiment and stagedaccording to Kimmel et al. (33). Chorions were removed man-ually with Dumont Watchmaker’s Forceps No. 5. All proce-dures were approved by the Institutional Animal Care and UseCommittee of National Taiwan University and performed incompliance with the Association for Research in Vision andOphthalmology Statement for Use of Animals in Ophthalmicand Vision Research.Characterization of zlum—To identify the zebrafish ex-

pressed sequence tag clone encoding a putative protein sharinghigh sequence similarity with the human andmouse SLRP fam-ily proteins, we applied the Basic Local Alignment Search Tool(BLAST) analysis of the GenBankTM database using the full-length human lumican cDNA sequence. An �4.6-kb NotI/

MluI zebrafish genomic DNA fragment containing the 5� por-tion of the zebrafish lumican gene was amplified by PCR andsubcloned into the pBluescript SK vector (Stratagene, La Jolla,CA). The insert was sequenced, using T3, T7, and walk-in prim-ers, by the DNA core of the Department ofMolecular Genetics,National Taiwan University.The 5�- and 3�-ends of the zlummRNAwere amplified using

the 5�-rapid amplification of cDNA end (5�-RACE) and3�-RACE systems, respectively (Invitrogen). For the 5�-RACEexperiment, 1 �g of total RNA from zebrafish eyes was reverse-transcribed with a lumican-specific primer (5�-AAGTAGAG-GTATTTGATTCCGGTC-3�) corresponding to a sequence inexon 2 of the zlum gene. The RNA templates were degraded bytreatment with an RNase mix. A poly(dCTP) tail was added tothe 3�-end of the cDNAs with terminal deoxynucleotidyltrans-ferase. The cDNA was amplified with a second gene-specificprimer (5�-GCACAAGAAGGTGATGAAACG-3�) corre-sponding to a sequence from the junction between exon 1 and 2in conjunction with the abridged anchor primer (5�-GGCCA-CGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3�). The re-sulting PCR products were diluted 100-fold and used as tem-plates to be reamplified with a third gene-specific primer(5�-CAGACTTAGAAGTCCAGCCAAC-3�) in conjunctionwith the universal amplification primer (5�-CUACUACUAC-UAGGCCACGCGTCGACTAGTAC-3�). For 3�-RACE, PCRswere performed using a gene-specific primer (5�-GCCTCAG-AGATCATCTTTGAATAG-3�) corresponding to a sequencein exon 3 of the zlum gene. The cycling conditions were asfollows: 34 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °Cfor 3 min followed by a 10-min extension at 72 °C at the end ofthe cycles. Finally, the 5�-RACE and 3�-RACE PCR productswere gel-purified, and the sequences were determined with adideoxy sequencing protocol. The transcription initiation andtermination sites of the zlum gene were determined by asequence comparison between genomic DNA, the 5�-RACEproduct, and the 3�-RACE product, respectively.Sequence Alignment and Phylogenetic Analysis of zLum—

The amino acid sequences of the open reading frames (ORFs)were initially aligned using the ClustalW program, as describedpreviously (34). The aligned amino acid sequences used weresubsequently analyzed with the Neighbor-Joining distanceanalysis method to construct a phylogenetic tree by usingGeneious Pro software (version 4.7). Bootstrap values were cal-culated from 100 replicates, and values �50% are indicated ateach divergence point.RT-PCR—Unless otherwise specified, RT-PCR reagents used

in this procedure were purchased from Promega (Madison,WI). RevertAidTM H Minus First Strand cDNA synthesis kitwas purchased from Fermentas (St-Leon-Rot, Germany).Zebrafish cDNA was synthesized using 40 �l of 5� reversetranscription buffer, 20 �l of 0.1 M dithiothreitol, 8 �l of 25 mM

dNTPs, 10 �l of RNasin (40 units/ml), 10 �l of 50 mM randomhexamers (GE Healthcare), 10 �l of avian myeloblastosis virusreverse transcriptase (9.5 units/�l), and 1 �g of heat-denaturedcorneal poly(A)� RNAs. Diethylpyrocarbonate-treated waterwas added to bring the final reaction volume to 200 �l, and thereaction was incubated at room temperature for 10 min, 42 °Cfor 90min, 100 °C for 2 min, and 0 °C for 5 min. Twenty micro-

Zebrafish Model for Myopia

28142 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 36 • SEPTEMBER 3, 2010


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

liters of each of the above RT reactions was added to 80 �l of aPCR mixture containing the following: 8 �l of 10� PCR bufferwithoutMgCl2, 8�l of 25mMMgCl2, 10�l of 20 ng/�l primers,0.5 �l of Taq polymerase (5 units/�l), and 45.5 �l of H2O. Thecycling conditions were as follows: 35 cycles of 94 °C for 1 min,57 °C for 1min, and 72 °C for 1min followed by a 15-min exten-sion at 72 °C at the end of these cycles. Primers used were asfollows: CCGCTCGAGCGGATGTTTGCTCTGGGATCC-ATTC (forward 5�-XhoI cut site) and TCCCCGCGGGGACT-ATTCAAAGATGATCTCTGAGG (reverse 3�-SacII cut site).The PCR product was confirmed by an appropriate restrictionenzyme digestion and analyzed by electrophoresis on a 1.5%agarose gel.Generation of an Epitope-specific Anti-zebrafish Lumican

Antibody—To develop an anti-zLum antibody, an oligopeptidededuced from zLum cDNA was synthesized (N-terminal pep-tide, CNERNLKFIPIVPTGIKY). The peptides were conjugatedto keyhole limpet hemocyanin for antibody production in rab-bits. The antibodies were purified through an immune absor-bent column of the above zebrafish lumican oligopeptide conju-gated to SulfoLink gel (Pierce) according to the manufacturer’sinstructions. Fractions containing purified anti-zebrafish lumicanantibody were pooled and concentrated, and the protein concen-tration was measured by spectrophotometry at 280 nm.In Situ Hybridization and Immunohistochemistry—Embryos

were fixed in 4%paraformaldehyde in 1�PBS overnight at 4 °C,rinsed with PBS three times, transferred into 100% methanol,and stored at �20 °C until use. To prevent melanization,embryos raised to time points beyond the 24-h post-fertiliza-tion (hpf) stage were treated with 0.003% phenylthiourea.Whole mount RNA in situ hybridization was carried out asdescribed previously (34). Sense and antisense digoxigenin-la-beled oligonucleotide probes were obtained from Bio Basic Inc.(Ontario, Canada). The oligonucleotide sequence (5�-3�) wasGTTTCCATCCAAGCGCAGGGTCCTCAGTCTAGAGTA-GTTGACCGGTGAGCTAAATCTGCA. The hybridizationsignals were visualized with anti-digoxigenin antibody-alkalinephosphatase conjugates using procedures recommended byRoche Applied Science. The sections were counterstained with0.5% neutral red and mounted. Images were obtained using anAxioCam digital camera on a dissection microscope (Zeiss,Germany).Adult zebrafish corneal tissues were fixed with 4% parafor-

maldehyde in PBS and then embedded in paraffin. Paraffinizedsections (5�m) of adult zebrafish corneal tissue were placed onslides and processed for deparaffinization and immunohisto-chemistry. Before immunostaining, tissue sections were incu-bated with 0.5 unit/ml keratanase at 37 °C overnight. Afterblocking with 3% hydrogen peroxide for 30 min, the sampleswere incubated with either the primary affinity-purified anti-zebrafish lumican antibody (0.1 �g/ml) or monoclonal anti-keratan sulfate antibody, washed in PBS, and then incubatedwith a biotinylated secondary antibody (goat anti-rabbit IgG).After washing in PBS, sections were incubated with streptavi-din-HRP (DAKO, Carpinteria, CA), then washed in PBS, andincubatedwith the 3�,3-diaminobenzidine chromogen for 5–10min.Negative control sampleswere obtained using preimmunerabbit IgG.

Western Blot Analysis—Total proteins were extracted fromadult fish eyes using lysis solution (10ml containing 1ml of 200mM HEPES/KOH buffer (pH 7.5), 200 mM sucrose (0.86 g), 50mMKCl (0.04 g), 2.5 mMMgCl2 (0.005 g), and 100 �l of 100mM

DTT). To remove keratan sulfate chains, protein aliquots wereincubated with 0.1 unit/ml endo-�-galactosidase (Sigma) and 1unit/ml keratanase (Sigma) at 37 °C overnight. The zebrafishcorneal extracts were subjected to 10% SDS-PAGE and thenprobed with the anti-zebrafish lumican N-terminal peptideantibody (0.1 �g/ml) as described above. The immunocomplexwas visualized by incubation with goat anti-rabbit IgG alkalinephosphatase conjugate (1:2500 (Novagen, Madison, WI)) andWestern Blue�-stabilized substrate (Promega).Analysis of zlum Promoter by Transgenic Zebrafish—

Genomic DNA, both 1.7 and 0.48 kb from the 5�-untranslatedregion of the zlum gene,were amplifiedwith specific PCRprim-ers and inserted into the multiple cloning sites of pBluescript IISK vectors (Stratagene, La Jolla, CA) containing an EGFPsequence. PCR primers are as follows: 5�-ATAAGAATGC-GGCCGCTCCATTAATTCGACAGACCAG-3� and 5�-ATAAGAATGCGGCCGCAGGTAGACAACACGGTTA-TGT-3� (forward primer) and 5�-CGACGCGTGGCTGCAC-AACTTAAATTAAACCT-3� (reverse primer). The recombi-nant plasmids were propagated in Escherichia coli DH5� andpurified with a Qiagen plasmid purification maxi kit. Purifiedplasmid DNA was adjusted to 50 ng/�l in distilled water andmicroinjected into one-cell stage zebrafish embryos under adissecting microscope. Embryos with GFP expression wereobserved and imaged under a fluorescence microscope.zlum Knockdown by Morpholino Injection—A morpholino-

antisense oligonucleotide (Gene Tools, Philomath, OR) wasdesigned to target the 5�-untranslated and/or -flanking regions,including the translation start codon of the respective genes.The MO sequence was designed as follows: zlum-MO,5�-GATCCCAGAGCAAACATGGCTGCAC-3�. This oligo-nucleotide complemented the sequence from �8 through �17with respect to the translation initiation codon. A search of thedatabase did not identify any sequence similarity of knownzebrafish genes to zlum-MO. A random sequence MO (RS-MO) serves as a control for zlum-MO, 5�-CCTCTTACCTCA-GTTACAATTTATA-3�. This RS-MO was obtained fromGeneTools as a standard control oligonucleotidewith no targetspecificity. Solutionswere prepared and injected at the 1–4-cellstage as described by Nasevicius and Ekker (35).Transmission Electron Microscopy—Corneas of zlum-MO-

injected, RS-MO-injected, and wild type zebrafish at 7 and 12days post-fertilization (dpf) stage were fixed in 50 mM phos-phate buffer (pH 7.2) containing 2.5% glutaraldehyde and 2%paraformaldehyde for 24 h at room temperature. After re-fixa-tion in 1% osmium tetraoxide for 4 h at room temperature, thesamples were washed in phosphate buffer, dehydrated, andembedded in Epon 812 epoxy resin. Semi-thick sections (100nm) were stained with toluidine blue. Ultrathin 50-nm sectionswere collected on 75mesh copper grids and stainedwith uranylacetate and lead citrate, and images were photographed with aHitachi 7100 transmission electron microscope (TEM) (Hita-chi, Tokyo, Japan) equipped with an AMT digital camera.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Image Analysis—Collagen fibril diameters and scleral thick-ness were measured with Image-Pro Plus version 4.5. Cornealstroma, anterior sclera, and posterior sclera from six zlum-MOand six wild type zebrafish at each 7- and 12-dpf stage wereanalyzed. Six to 12 areas of collagen fibrils were analyzed foreach region in each group, generating 772–1678measurementsfor each condition, and 27–84 measurements were generatedby analysis of scleral thickness for anterior and posterior sclerain each group at the 12-dpf stage. Collagen fibers and scleralthickness (inmicrometers) are represented as themean� S.D.,and all measurements were analyzed using Student’s t testsassuming unequal variances.In Vivo Zebrafish Larvae Assays for the Eye Size Regulation

by Muscarinic Receptor Antagonists—To apply our zebrafishmodel for myopia drug screening, different muscarinic recep-tor antagonists, atropine, pirenzepine (M1), and methoctra-mine (M2) purchased from Sigma were used according to pro-tocols previously described with some modifications (36–38).Briefly, before drug screening test, the maximal sublethal con-centration of each drug was determined, and the drug concen-trationwas selected by the survival rate of embryos greater than60% at 6 dpf stage. Zebrafish larvae were placed in a 96-wellplate at 1 fish per well. In this assay, the zlum-MO-injectedlarvae were exposed to 0.5% atropine, 0.25% pirenzepine, and0.01% methoctramine starting at 3 dpf for 48 h, respectively,after which the larvae were anesthetized with Tricaine, immo-bilized in 3% methylcellulose, and observed under dissectingmicroscopy. The axial length of eyeball, diameter of scleral coat,and the ratio of RPE diameter/scleral coat diameter were mea-sured. Student’s t test was used to compare these parametersbetween control group and treated groups, respectively. Thedifference of significance was defined as p � 0.05.

RESULTS

Primary Structure of zlum and Alignment Analysis of theZebrafish Lumican Amino Acid Sequence—We identified zlumby performing a BLAST search of the publicly available

zebrafish databases with humanLUM. The zlum gene is 11 kbupstream of zkera (keratocan) (Fig.1). We have isolated clones repre-senting the full open reading frames(ORF) of zebrafish lumican. AcDNA clone encoding the lumicanORF was subcloned into the pBlue-script SK vector (Stratagene, LaJolla, CA). The ORF of the zlumgene was 1032 bp long and encoded344 amino acid residues. Theproved nucleotide sequence of thezebrafish lumican gene (zlum) genewas submitted to the GenBankTMdatabase under GenBankTM acces-sion number GQ376197. Thissequence has been scanned againstthe database and is significantlyrelated to the sequence NM_001002059.1, which is derived from

BC071347.1 and has not yet been subject to final NCBI review(39).The entire genomic DNA sequence of zlum is shown in

supplemental Fig. 1. The zlum gene spans �4.6 kb (4610 bp)and contains three exons and two introns (Fig. 1). Sequenceanalysis revealed that exon 1 contains 26 untranslated nucleo-tides, exon 2 contains 24 noncoding and 880 coding nucleo-tides, and exon 3 contains 152 bases of coding sequence and1106 bases of 3�-untranslated sequence. The transcription ini-tiation site marked �1 was determined by 5�-RACE, asdescribed under “Experimental Procedures.” The first transla-tion initiation ATG codon is located at the 844th base down-streamof the beginning of exon 1. Therewas noTATA consen-sus sequence found in the �2.58 kb of proximal 5�-flankingregion. The full-length zlum cDNA clone (�1.9 kb) contains a1032-bp ORF of 344 amino acids of zLum core protein. Likeother SLRP core proteins such as bovine lumican (40), bovinemimecan (41), and mouse keratocan (42), zebrafish lumicanconsists of three distinct domains, a highly conserved centralleucine-rich repeat region flanked by hypervariable N- andC-terminal regions. As shown in supplemental Fig. 1, after thesignal peptide (Met to Tyr, the first 20 amino acids), the nega-tively charged N-terminal domain contains a possibly sulfatedtyrosine and four possibly conserved cysteine residues thatform intramolecular disulfide bonds, followed by nine tandemleucine-rich repeats (LXXLXLXXNX(L/I)) that are similar tolumican of other species and might mediate binding to otherextracellular matrix components and C-terminal globulardomains containing two conserved cysteine residues.Sequence Alignment and Phylogenetic Analysis—For com-

parison, multiple alignment analysis of the predicted aminoacid sequences of zLum with those of other species is shown insupplemental Fig. 2. Using the sequence of zLum, a search forhomologous sequences was made with BLAST. The predictedamino acid sequences showed a high homology among lumicancore proteins of different species. Zebrafish lumican shared51% amino acid identity to human, 50% amino acid identity to

FIGURE 1. Schematic diagram showing the organization of the zlum gene. The zebrafish lumican gene is 11kb upstream from the keratocan gene. It contains three exons and two introns. The figure shows the zebrafishlumican gene drawn to scale. Blank boxes indicate the coding region of the mRNA. The translation start andstop codons are indicated by ATG and TAA, respectively.




http://ww

w.jbc.org/

Dow

nloaded from




http://www.jbc.org/

mouse, and 53% amino acid identity to chicken lumican,respectively.Human lumican shared 86% amino acid identity tomouse and 68% amino acid identity to chicken lumican.To analyze the evolutionary relationships, a bootstrapped

neighbor-joining tree using Jukes-Cantor model was used toillustrate the relationship between different 18 species (Fig. 2).The numbers at the nodes represent the statistical confidenceestimated by the bootstrap procedure. Bootstrap values werecalculated from 100 replicates, and values of �50% are indi-cated at each divergence point. The bootstrap values allowinspection of the relationships among clades with low or noambiguity. The mouse lumican appears to be more closelyrelated to the lumican of human and primates. The zebrafishand chicken lumican are closer to those of Salmo salar andTaeniopygia guttata as compared with human and other mam-malian lumican, although they displayed similar structure and�50% homology with those of other species.Spatial Distribution of Zebrafish Lumican mRNA—To fur-

ther confirm the expression patterns of zlum, RT-PCR analysiswas carried out using template cDNAs that were reverse-tran-scribed from total RNAs from various tissues. The results ofRT-PCR revealed that an �1-kb RT-PCR product was abun-dant in most tissues examined, e.g. eye, brain, liver, muscle, andfin (Fig. 3). Whole mount in situ hybridization was used toanalyze the expression of zLum mRNA during development.The results revealed that zLummRNAwas widely expressed inmany tissues throughout the fish body, and it is highly

expressed in the embryonic fore-, mid-, and hindbrain, anteriorspinal cord, and eyes at 1 and 3 dpf (Fig. 4, A–C). The corre-sponding control sense riboprobes showed negative staining insamples (Fig. 4D). Lumican could be found in the corneal stro-mal layer of adult human and mouse corneas (43). In thezebrafish eye, zLum mRNA was also expressed mainly in thecorneal stromal layer (Fig. 4, E and F) and sclera (Fig. 4, G andH), whichwas similar to other species (17, 44). The correspond-ing control sense riboprobes showed negligible hybridizationsignals in samples (Fig. 4, I–L).Characterization and Spatial Distribution of Zebrafish

Lumican Protein—An affinity-purified anti-zLum antibodyagainst a synthetic peptideN-terminal peptide (CNERNLKFIP-IVPTGIKY), corresponding to the 18 N-terminal amino acidresidues deduced from the zlum cDNA,was generated to detectzebrafish lumican. Immunohistochemistry with anti-zLumantibodies showed that similar to human and mouse lumicans,which were both found in the corneal stromal layer (25), zLumwas also present mainly in corneal stromal layers (Fig. 5, A andB) and scleral tissue (Fig. 5,C andD), andweak immunostainingsignal was also detected in the corneal epithelium and sur-rounding tissues, such as the iris and ciliary body. No immuno-reactivity could be detected in samples incubated with preim-mune rabbit IgG (Fig. 5, E–H). Tissue sections probed withanti-keratan sulfate (KS) antibody demonstrated that keratansulfate-glycosaminoglycan was primarily present in cornealstroma, and little or no immune reactivity could be seen in thecorneal epithelium (Fig. 5, I and J). Keratanase treatment abol-ished the immune reactivity seen in the stroma by the anti-KSantibody (Fig. 5, M and N). It was of interest to note that noimmune reactivity was seen in the scleral tissue by the anti-KSantibody (Fig. 5, I, K, and L). This observation indicates thatkeratan sulfate-glycosaminoglycan is not present in the scleraltissue, consistent with the notion that zLum exists as KSPG incorneal stroma and under-glycanated glycoprotein in other tis-sues, such as the corneal epithelium and sclera.To further demonstrate that the lumican in zebrafish cornea

is a proteoglycan, total lysates from zebrafish eyes were treatedwith or without keratanase or endo-�-galactosidase digestionand were then subjected to Western blotting analysis. Fig. 6

FIGURE 2. Neighbor-joining phylogenic tree of the lumican protein. Phy-logenetic tree of lumican protein from 18 different species. The numbers atthe nodes represent the statistical confidence estimates computed by thebootstrap procedure. Bootstrap values were calculated from 100 replicates,and values of �50% are indicated at each divergence point. The following arecited: Canis familiaris (dog); Equus caballus (horse); Sus scrofa (pig); Bos taurus(cow); Mus musculus (mouse); Rattus norvegicus (rat); Pan troglodytes (chim-panzee); Macaca fascicularis (macaque); Macaca mulatta (rhesus mulatta);Homo sapiens (human); Pongo abelii (Sumatran orangutan); Ornithorhynchusanatinus (platypus); Salmo salar (Atlantic salmon); Danio rerio (zebrafish); Silu-rana tropicalis (Xenopus); Taeniopygia guttata (zebra finch); Coturnix japonica(Japanese quail); Gallus gallus (chicken). The tree was constructed withGeneious Pro software (version 4.7).

FIGURE 3. zlum expression by RT-PCR analyses. RT-PCR was carried outusing total RNAs from adult fish eye (lane 1), brain (lane 2), heart (lane 3), liver(lane 4), gut (lane 5), muscle (lane 6), and fin (lane 7). Note that amplified zlumPCR product could be found not only in the eye but also in other tissues(bottom panel).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

shows that amajor band of 50 kDa andmultiple highmolecularmass bands ranging from 60 to 170 kDa were labeled by theantibodies (lane 1) in specimens prepared from eyes withoutenzyme digestion. By contrast, in specimens treated with kera-tanase (Fig. 6, lane 2) or endo-�-galactosidase digestion (lane3), the high molecular mass bands diminished, but the majorband at 50 kDa remained. These data indicated that the zLumprotein isolated from eyes exists in two forms, as a KSPG and anunder-glycanated core protein.zLum Promoter Activity in Transgenic Zebrafish—To con-

firm that the genomic DNA clone of the isolated zlum wasindeed derived from a functional allele, we examined promoteractivity of the 5�-flanking sequence of the zlum genomic DNA,using transgenic zebrafish microinjected with zlumpr1.7-EGFP-bpA and zlumpr0.5-EGFP-bpA, respectively (Fig. 7, Band C). Live transgenic EGFP-positive embryos were screenedand selected under a fluorescent microscope. In transgeniczebrafish, 86% (212:246) of the 1.7-kb zlum promoter frag-ment-injected embryos expressed the EGFP transgenethroughout the fish body, including the eyes, at 3 and 7 dpf (Fig.7,D–F). However, the 0.5-kb promoter fragment of zlum failedto drive EGFP expression in transgenic zebrafish (0:113) (Fig.7G). These results indicated that the 1.7-kb zlum promoter isable to drive EGFP expression in the transgenic fish, whereasdeletion of the 5� 1.2 kb of the sequence completely eliminates

EGFP expression, suggesting that the 1.2-kb region between the1.7- and 0.5-kb 5�-flanking sequence consists of the necessaryregulatory elements controlling lumican promoter activity forzlum expression in vivo.Effects of zlum Knockdown on Embryonic Development—To

investigate zlum function during zebrafish development, MOwas microinjected into fertilized zebrafish eggs. Western blotshowed that zLum protein decreased after zlum-MO wasinjected. (Fig. 8A). Retarded development was seen in thezlum-MO embryos as compared with wild type embryos at the22-hpf stages (Fig. 8, C and D). Significant morphological vari-ations could be readily identified between zlum-MO embryosand control embryos beginning at 6 dpf stage, although therewere no identifiable differences in phenotypes between thecontrol RS-MO and wild type embryos. About 58.6% of zlum-MO-injected 7-dpf larvae (277:473) exhibited severe majordefects, and less than 1% of the RS-MO negative control group(1:105) and 2.1% of the wild type group with mock injection ofPBS alone (9:438) had minor morphological changes. A higherdose of zlum-MO (20 ng/2 �l) produced more severe abnor-malities in embryos and led to a highermortality rate (187 deadembryos out of 211 total injected). The most common abnor-malities of the zlum MO-injected group were enlarged eyes,enlarged pericardium, and deformed body shape (Fig. 8, E andF). In particular, significantly enlarged ocular eyeballs were

FIGURE 4. In situ hybridization analyses of zLum mRNA in early zebrafish larvae (whole mount) and adult fish cornea (sections). Hybridization signalswere demonstrated with anti-digoxigenin antibody-alkaline phosphatase conjugates. A, zLum mRNA was detected in zebrafish larvae (24 hpf). B, in olderzebrafish larvae (48 hpf), the zLum mRNA was detected in the eyes and all the major subdivisions of the embryonic central nervous system, including the fore-,mid-, and hindbrain and the spinal cord. C, strong hybridization signals could be also found in corneal tissue at 48 hpf. D, no hybridization signals could bedetected in sense probe hybridization group at the 48-hpf larval stage. E, in the zebrafish eye, zLum mRNA was predominantly detected in the corneal stromallayer. F, higher magnification showed that hybridization-positive signals are observed in the corneal stromal layer. G, zLum mRNA was also detected in theperiocular extracellular matrix. H, higher magnification showed that hybridization-positive signals are observed in the scleral layer (arrow). I–L, no hybridizationsignals could be detected in the adult eye (I), cornea (J), or sclera (K and L) using a sense probe in hybridization. Sections were counterstained with fast red. (Scalebars, 100 �m, E, G, I, and K; and 50 �m, F, H, J, and L.)




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

noticed in the zlum-MO-injected group comparedwith the RS-MO-injected group and wild type group from around the 6-dpfstage. Significant increases in axial lengths were noted in thezlum-MO-injected group compared with those of RS-MO-in-jected group and wild type group (276.83 � 27.4 �m (MO)versus 237.04 � 12.8 �m (RS control) and 276.83 � 27.4 �m(MO) versus 243.32 � 10.69 �m (WT), respectively, all p �0.05) (Fig. 8G). Moreover, the zlum-MO-injected group hadlarger eyeball diameters in comparison with those of the RS-MO-injected group and wild type group (443.12 � 58.1 �m(MO) versus 337.58� 16.6 �m (control) and 443.12� 58.1 �m(MO) versus 342.57 � 14.31 �m (WT), respectively, all p �0.05) (Fig. 8H). The control group injected with RS-MO hadnormal eyemorphologies like themock-injected control group.Decreased Lumican Synthesis Results in Abnormal Collagen

Architecture of the Corneal Stroma and Scleral Tissue—To elu-cidate the function of lumican in regulating collagen fibrillo-genesis in the fish eye, eyes of zlum-MO fish were subjected to

morphological analysis withTEMat 7 and 12 dpf (n 6, in eachgroup). Fig. 9 shows the ultrastructural changes of collagenousmatrix at corneal stroma (CS), anterior sclera (AS), and poste-rior sclera (PS) area (as shown in Fig. 9A) between thezlum-MO and wild type group at 12 dpf stage. The fibril archi-tecture of corneal stroma and anterior sclera collagen of theMO-treated group differed from that of the control group andappeared irregularly, with large variations in fibril diameter andfibril spacing in comparison with WT control (compare Fig. 9,C with D and E with F). However, there was no significant dif-ference in the diameter of collagen fibrils at the posterior scleraarea between both groups (compare Fig. 9,GwithH). For sum-mary of the data of fibril diameter measurement, Fig. 9B showsthat there was significant difference in the collagen fibril diam-eter of corneal stroma and anterior sclera matrix between thewild type and zlum-MOgroup at 12 dpf stage (CS, 13.80� 2.84nm (WT) versus 22.71 � 4.46 nm (MO), p � 0.05; AS, 14.91 �3.32 nm (WT) versus 20.42 � 3.63 nm (MO), respectively, p �

FIGURE 5. Immunohistochemistry staining pattern of zebrafish eye using the epitope-specific anti-zLum antibody. A, lumican protein was found inzebrafish eye tissue. B, note that lumican protein was found mainly in the corneal stromal layer. C and D, lumican protein was also found in the scleral tissue(arrow). E–H, no immunoreactivity was detected in the negative control group. (E, whole eye; F, corneal tissue; G and H, scleral tissue.) I–P, tissue sections treatedwith (M–P) and without (I–L) keratanase and stained with an anti-keratan sulfate antibody are shown. I and J, tissue sections showed that keratan sulfate chainsexisted mainly in the corneal stromal layer. K and L, keratan sulfate chains did not exist in the scleral layer. M–P, there was nearly no immunoreactive reactionin tissue sections treated with keratanase and stained with anti-keratan sulfate antibody. (Scale bars, 200 �m, A, E, I, and M; scale bars, 100 �m, C, G, K, and O; scalebars, 50 �m, B, D, F, H, J, L, N, and P; and arrow indicates scleral tissue ins D, H, L, and P.)




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

0.05), whereas there was no significant difference in the colla-gen fibril diameter of PS matrix between the wild type andzlum-MO group at 12 dpf stage (PS, 16.34 � 4.0 nm (WT)versus 16.01 � 3.71 nm (MO), p � 0.05).Interestingly, a larger size of eyeball accompanied by thinner

sclera was noted in the zlumMO-injected larva (Figs. 8F, 9, and10). Lower magnification of electron microscopy showed thatposterior scleral tissue from the wild type group consisted of�3–4 layers of fibroblastic cells with newly formed collagenfibrils in relatively regular rearrangements at 7 dpf stage in theposterior sclera (Fig. 10A). However, there was only about onelayer of fibroblastic cells in the posterior sclera of MO-treatedlarva at 7 dpf stage (Fig. 10B), which consisted of very few andirregular collagenous matrix with large variegations in fibrildiameters and fibril spacing (Fig. 9H). Fig. 10C shows signifi-cant decrease in the posterior scleral thickness found in zlum-MO-treated group (MO) in comparison with that of controlgroup (WT) at 7 dpf stage (PS, 5.93 � 0.81 �m (WT) versus1.49 � 0.68 �m (MO), p � 0.05); in contrast, there was no

significant decrease in the thickness of AS thickness betweenboth MO and WT groups (AS, 5.64 � 1.01 �m (WT) versus6.19 � 0.60 �m (MO), p � 0.05)). At 12 dpf stage, the scleralthickness was significantly decreased at both anterior and pos-terior regions of sclera of MO-treated group (AS, 5.44 � 0.94�m (WT) versus 3.36� 0.37�m (MO); and PS, 4.87� 1.34�m(WT) versus 1.36 � 1.12 �m (MO), p � 0.05) (Fig. 10C).

In conclusion, our results demonstrated that the ultra-structural changes of scleral tissue caused by decreasedlumican synthesis with morpholinos were similar to the fea-tures found in sclera of mammalian model of high myopia.Taking the advantage that zebrafish eye development can beeasily manipulated, e.g. administration of morpholinos andanalyzed, we have attempted to develop a protocol of usingzebrafish as an experimental model to test the efficacy of

FIGURE 6. Western blot analysis of zebrafish lumican. Total proteinsextracted from adult zebrafish eyes by lysis buffer were subjected to 10%SDS-PAGE followed by Western blotting. Samples without treatment (lane 1)and with either keratanase (lane 2) or endo-�-galactosidase treatment (lane 3)were probed with a rabbit anti-zebrafish lumican antibody. The resultshowed multiple bands with a similar smearing pattern without enzymedigestion (lane 1), whereas one �50-kDa band was visualized after treatmentwith keratanase (lane 2) and endo-�-galactosidase (lane 3). These data dem-onstrate that the zebrafish lumican protein contains keratan sulfate chains.

FIGURE 7. Generation of transgenic fish harboring zlumpr1.7-EGFP SV40and zlumpr0.5-EGFPSV40. A, schematic representation of the zebrafishlumican gene. B, structure of zlumpr1.7-EGFP SV40 (3.3 kb). It contains a 1.7-kb5�-regulatory region of the zlum gene, the untranslated region of exon 1 (844bp), an SV40 polyadenylation signal, and the pEGFP vector sequence. C, struc-ture of zlumpr0.5-EGFPSV40 (2.0 kb). It contains a 0.5-kb 5�-regulatory regionof the zlum gene, the untranslated region of exon 1 (844 bp), an SV40 poly-adenylation signal, and the pEGFP vector sequence. D, EGFP expression wasobserved in the 3 dpf zebrafish after injecting linearized zlumpr1.7-EGFP SV40DNA fragment. E, under different observation view of the same fish (3 dpf),strong EGFP expression was detected in corneal tissue. F, EGFP was expressedat the 7 dpf stage. G, as a control, no EGFP was detected after injecting thelinearized zlumpr0.5-EGFP SV40 DNA fragment.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

compounds, e.g. antagonist of muscarinic receptors, whichcan potentially modulate the pathoetiology of myopia asdescribed below.Administration of Muscarinic Receptor Antagonists Reverts

the Enlargement of Scleral Coats Caused by zlum Knockdownwith Morpholinos—To evaluate if the zebrafish could be anexperimental drug screening model for treatment of inducedmyopia, we tested several different muscarinic receptor antag-onists that have been used in treating myopia. As shown in Fig.11, two drugs out of the three muscarinic receptor antagoniststested, atropine (0.5%) and pirenzepine (0.25%), producedpromising outcomes as there was significant reduction inexcessive axial elongation and enlarged scleral coats observed

in the treated zlum-MO group ascompared with those of untreatedzlum-MOgroup (Fig. 11,D, E versusC). However, no obvious improve-ment was noted in the eye of fishtreated with 0.01% methoctramine(M2 receptor antagonist) andinjected by zlum-MO at 7 dpf stage(Fig. 11F). To better evaluate theeffects of drugs, we measured thediameters of the retina and sclera ofthe experimental fish (as shown inFig. 12,A and B). Fig. 12C (1st to 5thlanes) showed a significant reduc-tion in the excessive axial elonga-tion found in the atropine- andpirenzepine-treated groups as com-pared with those of the untreatedzlum-MO group at 7 dpf stage((205.01 � 21.10 �m (atropine-treated) and 208.27 � 23.51�m (pirenzepine-treated) versus248.71 � 20.41 �m (untreated),both have a p � 0.001, n 55, 64,and 70, respectively)), and there wasno significant change in the axiallength in the methoctramine-treated group (242.05 � 21.68 �m,p� 0.05, n 56). Fig. 12C (6 to 10thlanes) showed a significant decreasein scleral diameters in the atropine-and pirenzepine-treated groups ascompared with those of theuntreated zlum-MO group at 7 dpfstage ((323.51 � 46.83 �m (atro-pine-treated) and 326.18 � 32.88�m (pirenzepine-treated) versus399.07 � 59.68 �m (untreated),both have p values �0.001, n 55,64, and 70, respectively)), whereasthere was no significant reductionof scleral diameter in methoctra-mine-treated group at 7 dpf stage(381.47 � 43.74 �m, p � 0.05,n 56).

Another index of eye enlargement is the ratio of retina diam-eter to sclera diameter (the smaller the ratio, the larger the eye).Fig. 12D showed that there was a significant decrease in theratio of the RPE/scleral coat during ocular enlargement causedby the knockdown of zlum ((97.22 � 3.86% (WT) versus56.11 � 4.64% (MO), p � 0.001)). The administration of mus-carinic receptor antagonists, 0.5% atropine and 0.25% pirenz-epine, effectively attenuate the decrease in the ratio of the RPE/scleral coat caused by zlum knockdown group ((71.29 � 8.52%(atropine-treated) and 71.33 � 9.37% (pirenzepine-treated)versus 56.11� 4.64% (MO), both have p values� 0.001, respec-tively)), whereas there was no obvious reversion of thedecreased ratio of the RPE/scleral coat in the 0.01%methoctra-

FIGURE 8. Ocular enlargement developed because of the reduction of zLum protein by morpholinomicroinjection. A, Western blot was carried out using embryo lysates from wild type embryos (1st lane) andzlum-MO-injected 3-dpf embryos (2nd lane). It showed that zLum protein decreased after zlum-MO wasinjected. B, this figure showed the definition of axial length (AL) and diameter (D) of eye in zebrafish. C, RS-MO-injected embryos showed normal phenotypes as a control group at 22 hpf stages. D, developmental delay wasfound in the zlum-MO-injected group at 22 hpf stages. E, normal phenotype was noted in the RS-MO-injectedgroup at the 7 dpf stage. F, significant ocular enlargement was noted in the zlum-MO-injected group at the 7dpf stage. G, significantly increased axial length was noted in the zlum-MO-injected group as compared withthe RS-MO-injected embryos and wild type embryos at 7 dpf (respectively, p � 0.05). H, significantly increasedeyeball diameter was also noted in the zlum-MO-injected group compared with the RS-MO-injected embryosand wild type embryos at 7 dpf (respectively, p � 0.05).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

mine-treated zlum-MO larvae (58.19 � 6.37% (methoctra-mine-treated) versus 56.11 � 4.64% (MO), p � 0.05).

DISCUSSION

In this study, we have demonstrated that zebrafish lumicanhas all the structural features of SLRPs, i.e. a central domain ofnine leucine-rich repeats flanked by N- and C-terminaldomains with conserved cysteine residues (45). Interestingly,zlum does not have a TATA box in its promoter. Unlike theTATA-less promoters of housekeeping genes, the zebrafish

lumican promoter does not have a GC-rich sequence. Asshown in Fig. 7, our results implied that zLum exists as aKSPG in corneal stroma but as unglycanated glycoprotein insclera.The sclera, a fibrous extracellular matrix, contains irregu-

larly arranged lamellae of collagen fibrils, proteoglycans,elastin, and matrix-secreting fibroblasts (46). The changes ofcollagen matrix in the sclera caused by increased ordecreased proteoglycans may lead to changes in the biome-chanical properties of the sclera and may ultimately lead to

FIGURE 9. zlum-MO knockdown induces ultrastructural changes in the CS, AS, and PS. A, WT fish at 12 dpf stage in toluidine blue staining. The figureindicates CS, AS, and PS. B, diameters of collagen fibril were analyzed in the corneal stroma, anterior and posterior sclera of 12 dpf-old-wild type, andzlum-MO-injected group. Significant increases in collagen fibril diameter of corneal stroma and anterior sclera are noted in the zlum-MO group, and thediameter of collagen fibril in the posterior sclera is not significantly different in both groups. C–H, morphological comparison of collagen fibril architecture inthe corneal stroma (C and D), anterior scleral tissue (E and F), and posterior scleral tissue (G and H) between the control group (C, E, and G) and zlum-MO-injectedgroup (D, F, and H) at the 12 dpf stage. C, TEM micrograph showing regular and smaller fibril architecture of collagen localized in the corneal stroma of the wildtype group. D, irregular arrangement and increasing collagen fibril diameter was found in the corneal stroma of the zlum-MO-injected group. E, TEM micro-graph showing relatively regular fibril architecture of collagen localized in the anterior sclera of the wild type group. F, irregular collagen fibrils with increasingfibril diameter were noted in the anterior sclera of the zlum-MO-injected group. G, top is adjacent to the retina. TEM micrograph showing fibril architecture ofcollagen localized in the posterior sclera of the wild type group. H, top is adjacent to the retina. TEM micrograph showing irregular and little collagen fibrilarchitecture was noted in the posterior sclera of the zlum-MO-injected group. (scale bar, C–H, 100 nm.)

FIGURE 10. Ultrastructure changes in scleral thinning in zlum-MO group. A, top is adjacent to the retina. Two to three layers of scleral fibroblastic cells withcollagen fibril formation between the layers was found at the posterior sclera of the WT fish at 7 dpf stage. B, top is adjacent to the retina. Only one to two layersof fibroblastic cells at the posterior sclera of the zlum-MO-injected fish at 7 dpf stage is shown. C, scleral thinning was observed obviously in the zlum-MO-injected fish at 7 dpf stage. The phenomenon was much more prominent in the zlum-MO-injected fish at 12 dpf stage. In particular, significant scleral thinningwas observed in the posterior sclera of the zlum-MO-injected fish at 7 and 12 dpf stage as compared with wild type group. (Scale bar, A and B, 1.5 �m.)




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

the alteration of the eyeball shape seen in myopic patients(46–48). Therefore, the sclera is a dynamic tissue ratherthan a static container of the eye. Moring et al. (49) foundthat biglycan and lumican mRNA levels were lowered in thesclera during experimentally induced myopia and increasedduring recovery in the tree shrew model. Lum�/� mice andLum�/�Fmod�/� mice presented altered collagen fibrildiameter in sclera (28, 29), suggesting that these proteogly-cans play an important role in the biomechanical propertiesof the sclera. Our zebrafish model indicated that the alteredcollagen fibril diameter in corneal stroma and anterior scleraby zlum knockdown with morpholinos is similar to somefeatures of the Lum�/� mice (Fig. 10). Although the collagenfibril diameter was altered in lumican-null mice, there wasno ocular enlargement found in Lum�/� mice. Young andco-workers (50) proposed that the phenotypes of Lum�/�

mice and Lum�/�Fmod�/� mice may be the possibility offalse positive results due to the “hitchhiker” effect (51). How-ever, ocular enlargement and scleral thinning resemblingcharacteristics of high myopia could be readily recognized inour zebrafish model of decreased synthesis of lumicancaused by zlum knockdown with morpholinos. This differ-

ence can be explained in part by the fact that in mice thereare more layers of lamellar organization in anterior and pos-terior sclera tissue than that of zebrafish, and thus mousesclera may be more resilient to the loss of lumican and main-tain a stable sclera structure seen in Lum�/� mice.

The changes in refractive status in developing myopia aregreatest from the elongated axial length of the eyeball ratherthan from the changes of corneal and anterior lens curvature. Inhumans, thinning of the scleral tissue, particularly at the poste-rior pole following the elongation of eyeball, is a characteristicof high myopia, which is compatible with our observations inthis zebrafish model in that the most immature collagen fibrilsare located in the posterior sclera (46). Therefore, a decreaseand/or disruptions of the arrangement of collagen fibrils in thisregion are highly probable to weaken sclera strength and integ-rity and result in ocular enlargement seen in our zebrafishmodel.In this study, we also found that the number of scleral fibro-

blasts are significantly reduced at the posterior sclera ofzlum-MO injected fish (Fig. 10B). Thus, the lumican proteinmay also affect the functions of scleral fibroblasts during earlydevelopment. Kao et al. (24, 52) has suggested that lumican is a

FIGURE 11. Zebrafish larvae assay for drug screen. A, normal phenotype of WT fish at 7 dpf stage. B, normal phenotype of RS-MO-injected embryos at 7 dpfstage. C, significantly enlarged eyeball of zlum-MO-injected fish at 7 dpf stage. D, significant decrease in ocular enlargement was noted in the zlum-MO-injectedlarvae at 7 dpf stage after treating with 0.5% atropine for 2 days. E, decrease in ocular enlargement was also found in the zlum-MO-injected larvae at 7 dpf stageafter treating with 0.25% pirenzepine (P) for 2 days. F, no obvious changes in the phenotypes of zlum-MO-injected fish at 7 dpf stage after treating with 0.01%methoctramine (M).




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

matrikine and can modulate fibroblast activities in addition toserving as a regulator of collagen fibrillogenesis.In this study, we have also established a brand new platform

of using the zlum-MO zebrafish as a screen tool for identifyingdrugs that can affect myopic progression. Recently, Barathi etal. (53) confirmed the presence of all five (M1 to M5) musca-rinic acetylcholine receptors in human andmouse scleral fibro-blasts.Muscarinic acetylcholine receptors have also been foundin the brain of the zebrafish by radioligand binding techniques(37). Liu et al. (54) demonstrated that muscarinic antagonistsacted directly on the sclera to prevent myopia developmentinduced by form deprivation in guinea pigs in which myopiaprogression was accompanied by overexpression of muscarinicacetylcholine receptors (M1 andM4). Furthermore, atropine, anonselective muscarinic antagonist, and pirenzepine, an antag-

onist specific for M1, have been found to be effective in clinicaltrials to preventmyopia progression (55–58). In experimentallyinducedmyopia, including chicks and tree shrews, severalmus-carinic antagonists have been applied to control ocular growthin different species, although the mechanism of drug effectsagainst myopia remain unclear (59–61). However, variableeffects on experimental myopia were shown by applying thesemuscarinic antagonists when administered intravitreously orby subconjunctival injection (36, 59). In this study, we chosethese muscarinic antagonists, i.e. 0.5% atropine, 0.25% pirenz-epine, and 0.01%methoctramine, according to a previous studywith minor modifications (36). In our results, we showed thatthe effect of ocular enlargement due to the reduction of zLumprotein was blocked by atropine and pirenzepine but not by theM2-selective receptor antagonist methoctramine. Therefore,

FIGURE 12. Zebrafish drug screen assay. A and B show the definition of outer margin of RPE (red color) and diameter (D) of scleral coat (green color) in zebrafish.C, significant decrease in excessive axial elongation in the zlum-MO-injected fish at the 7 dpf stage after treating with 0.5% atropine (A) and 0.25% pirenzepine(P), whereas no obvious changes in excessive axial elongation after treating with 0.01% methoctramine (M). 1st lane, WT; 2nd lane, MO � 0.5% atropine; 3rd lane,MO � 0.25% pirenzepine; 4th lane, MO � 0.01% methoctramine; 5th lane, MO. Significant decrease in the diameter of scleral coat of the zlum-MO-injected fishat the 7 dpf stage after treating with 0.5% atropine and 0.25% pirenzepine, whereas no obvious changes in the zlum-MO-injected group treated with 0.01%methoctramine. 6th lane, WT; 7th lane, MO � 0.5% atropine; 8th lane, MO � 0.25% pirenzepine; 9th lane, MO � 0.01% methoctramine; 10th lane, MO.D, significant decrease in the ratio of RPE/scleral coat (%) was noted during ocular enlargement developed due to the reduction of zLum protein. Somemuscarinic receptor antagonists (atropine and pirenzepine) attenuate the decreasing ratio of RPE/scleral coat due to the reduction of zLum protein, whereasthere was no obvious changes in the decreased ratio of RPE/scleral coat in the methoctramine-treated group. 1st lane, WT; 2nd lane, MO � 0.5% atropine; 3rdlane, MO � 0.25% pirenzepine; 4th lane, MO � 0.01% methoctramine; 5th lane, MO.




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

the observations are similar to the effects of muscarinic recep-tor antagonists in other myopic animal models and clinical tri-als. With our model, one can quickly screen drugs that mayhave therapeutic effects on controlling myopia progressionwithin a week. Another advantage of our model is it can beeasily and conveniently carried out by adding the drugs to theembryos kept in 96-well tissue culture plates without any injec-tion procedures to the experimental animals.In conclusion, we have identified and characterized the

zebrafish lumican gene. Lumican is highly conserved duringevolution. zlum shares high homology with human and mouselumican with respect to gene structure, expression pattern, andpromoter function. Moreover, we present evidence of ultra-structural changes of ocular enlargement, which are similar tothe features of high myopia in humans. We have also estab-lished a protocol of drug screening tests for the identification ofcompounds that are of potential in modulating scleral growth.Therefore, the zebrafish can serve as a potential vertebratemodel for studying the early development of corneal and scleraldiseases.

Acknowledgments—We thank Chun-Wen Chen, Yu-Ching Wu, andTing-Shuan Chiang for their excellent technical assistance through-out the course of this study and Dr. Bei-En Chang and Dr. Huey-JenTsai for their critical comments on the manuscript. We also thankHui-Chun Kung and Ya-Ling Chen for preparation of TEM studies atthe Microscope Center of Chang-Gung Memorial Hospital.

REFERENCES1. Lin, L. L., Shih, Y. F., Hsiao, C. K., Chen, C. J., Lee, L. A., and Hung, P. T.

(2001) J. Formos. Med. Assoc. 100, 684–6912. Kempen, J. H.,Mitchell, P., Lee, K. E., Tielsch, J.M., Broman, A. T., Taylor,

H. R., Ikram, M. K., Congdon, N. G., and O’Colmain, B. J. (2004) Arch.Ophthalmol. 122, 495–505

3. McBrien, N. A., Metlapally, R., Jobling, A. I., and Gentle, A. (2006) Invest.Ophthalmol. Vis. Sci. 47, 4674–4682

4. Deere, M., Johnson, J., Garza, S., Harrison, W. R., Yoon, S. J., Elder, F. F.,Kucherlapati, R., Hook,M., andHecht, J. T. (1996)Genomics 38, 399–404

5. Fisher, L. W., Heegaard, A. M., Vetter, U., Vogel, W., Just, W., Termine,J. D., and Young, M. F. (1991) J. Biol. Chem. 266, 14371–14377

6. Pulkkinen, L., Alitalo, T., Krusius, T., and Peltonen, L. (1992) Cytogenet.Cell Genet. 60, 107–111

7. Young, T. L., Ronan, S.M., Alvear, A. B.,Wildenberg, S. C., Oetting,W. S.,Atwood, L. D., Wilkin, D. J., and King, R. A. (1998)Am. J. Hum. Genet. 63,1419–1424

8. Schwartz, M., Haim, M., and Skarsholm, D. (1990) Clin. Genet. 38,281–286

9. Grover, J., Chen, X. N., Korenberg, J. R., and Roughley, P. J. (1995) J. Biol.Chem. 270, 21942–21949

10. Wang, I. J., Chiang, T. H., Shih, Y. F., Hsiao, C. K., Lu, S. C., Hou, Y. C., andLin, L. L. (2006)Mol. Vis. 12, 852–857

11. Majava, M., Bishop, P. N., Hagg, P., Scott, P. G., Rice, A., Inglehearn, C.,Hammond, C. J., Spector, T. D., Ala-Kokko, L., and Mannikko, M. (2007)Hum. Mutat. 28, 336–344

12. Chen, Z. T., Wang, I. J., Shih, Y. F., and Lin, L. L. (2009) Ophthalmology116, 1920–1927

13. Funderburgh, J. L., Funderburgh, M. L., Mann, M. M., and Conrad, G. W.(1991) J. Biol. Chem. 266, 24773–24777

14. Funderburgh, J. L., Caterson, B., and Conrad, G. W. (1987) J. Biol. Chem.262, 11634–11640

15. Iozzo, R. V., and Murdoch, A. D. (1996) FASEB J. 10, 598–61416. Knudson, C. B., and Knudson, W. (2001) Semin. Cell Dev. Biol. 12,

69–7817. Ying, S., Shiraishi, A., Kao, C. W., Converse, R. L., Funderburgh, J. L.,

Swiergiel, J., Roth, M. R., Conrad, G. W., and Kao, W. W. (1997) J. Biol.Chem. 272, 30306–30313

18. Raouf, A., Ganss, B., McMahon, C., Vary, C., Roughley, P. J., and Seth, A.(2002)Matrix Biol. 21, 361–367

19. Iozzo, R. V., and Danielson, K. G. (1999) Prog. Nucleic Acid Res. Mol. Biol.62, 19–53

20. Chakravarti, S., Magnuson, T., Lass, J. H., Jepsen, K. J., LaMantia, C., andCarroll, H. (1998) J. Cell. Biol. 141, 1277–1286

21. Funderburgh, J. L., Funderburgh, M. L., Mann, M. M., and Conrad, G. W.(1991) Biochem. Soc. Trans. 19, 871–876

22. Iozzo, R. V. (1999) J. Biol. Chem. 274, 18843–1884623. Conrad, G. W. (2000) in Carbohydrates in Chemistry and Biology (Ernst,

B., Hart, G. W., and Sinay, P., eds) pp. 717–727, Wiley Interscience, NewYork

24. Kao, W. W., Funderburgh, J. L., Xia, Y., Liu, C. Y., and Conrad, G. W.(2006) Exp. Eye Res. 82, 3–4

25. Saika, S., Shiraishi, A., Liu, C. Y., Funderburgh, J. L., Kao, C.W., Converse,R. L., and Kao, W. W. (2000) J. Biol. Chem. 275, 2607–2612

26. Saika, S., Miyamoto, T., Tanaka, S., Tanaka, T., Ishida, I., Ohnishi, Y.,Ooshima, A., Ishiwata, T., Asano, G., Chikama, T., Shiraishi, A., Liu, C. Y.,Kao, C. W., and Kao, W. W. (2003) Invest. Ophthalmol. Vis. Sci. 44,2094–2102

27. Austin, B. A., Coulon, C., Liu, C. Y., Kao, W. W., and Rada, J. A. (2002)Invest. Ophthalmol. Vis. Sci. 43, 1695–1701

28. Chakravarti, S., Paul, J., Roberts, L., Chervoneva, I., Oldberg, A., and Birk,D. E. (2003) Invest. Ophthalmol. Vis. Sci. 44, 2422–2432

29. Udvadia, A. J., and Linney, E. (2003) Dev. Biol. 256, 1–1730. Xu, Y. S., Kantorow, M., Davis, J., and Piatigorsky, J. (2000) J. Biol. Chem.

275, 24645–2465231. Soules, K. A., and Link, B. A. (2005) BMC Dev. Biol. 5, 1232. Westerfield, M. (1995) The Zebrafish Book, University of Oregon Press,

Eugene, OR33. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., and Schilling,

T. F. (1995) Dev Dyn. 203, 253–31034. Yeh, L. K., Liu, C. Y., Chien, C. L., Converse, R. L., Kao,W.W., Chen,M. S.,

Hu, F. R., Hsieh, F. J., and Wang, I. J. (2008) J. Biol. Chem. 283, 506–51735. Nasevicius, A., and Ekker, S. C. (2000) Nat. Genet. 26, 216–22036. Luft, W. A., Ming, Y., and Stell, W. K. (2003) Invest. Ophthalmol. Vis. Sci.

44, 1330–133837. Williams, F. E., and Messer, W. S. (2004) Comp. Biochem. Physiol. C.

Toxicol. Pharmacol. 137, 349–35338. Berghmans, S., Butler, P., Goldsmith, P., Waldron, G., Gardner, I., Golder,

Z., Richards, F. M., Kimber, G., Roach, A., Alderton, W., and Fleming, A.(2008) J. Pharmacol. Toxicol. Methods 58, 59–68

39. Strausberg, R. L., Feingold, E. A., Grouse, L. H., Derge, J. G., Klausner,R. D., Collins, F. S., Wagner, L., Shenmen, C. M., Schuler, G. D., Altschul,S. F., Zeeberg, B., Buetow, K. H., Schaefer, C. F., Bhat, N. K., Hopkins, R. F.,Jordan, H., Moore, T., Max, S. I., Wang, J., Hsieh, F., Diatchenko, L., Ma-rusina, K., Farmer, A. A., Rubin, G. M., Hong, L., Stapleton, M., Soares,M. B., Bonaldo, M. F., Casavant, T. L., Scheetz, T. E., Brownstein, M. J.,Usdin, T. B., Toshiyuki, S., Carninci, P., Prange, C., Raha, S. S., Loquellano,N. A., Peters, G. J., Abramson, R. D., Mullahy, S. J., Bosak, S. A., McEwan,P. J., McKernan, K. J., Malek, J. A., Gunaratne, P. H., Richards, S., Worley,K. C.,Hale, S., Garcia, A.M.,Gay, L. J., Hulyk, S.W., Villalon,D. K.,Muzny,D.M., Sodergren, E. J., Lu, X., Gibbs, R. A., Fahey, J., Helton, E., Ketteman,M.,Madan, A., Rodrigues, S., Sanchez, A.,Whiting,M.,Madan, A., Young,A. C., Shevchenko, Y., Bouffard, G. G., Blakesley, R. W., Touchman, J. W.,Green, E. D., Dickson, M. C., Rodriguez, A. C., Grimwood, J., Schmutz, J.,Myers, R. M., Butterfield, Y. S., Krzywinski, M. I., Skalska, U., Smailus,D. E., Schnerch, A., Schein, J. E., Jones, S. J., andMarra, M. A. (2002) Proc.Natl. Acad. Sci. U.S.A. 99, 16899–16903

40. Funderburgh, J. L., Funderburgh,M. L., Brown, S. J., Vergnes, J. P., Hassell,J. R., Mann, M. M., and Conrad, G. W. (1993) J. Biol. Chem. 268,11874–11880

41. Funderburgh, J. L., Corpuz, L. M., Roth, M. R., Funderburgh, M. L., Ta-sheva, E. S., and Conrad, G. W. (1997) J. Biol. Chem. 272, 28089–28095




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

42. Liu, C. Y., Shiraishi, A., Kao, C. W., Converse, R. L., Funderburgh, J. L.,Corpuz, L. M., Conrad, G. W., and Kao, W. W. (1998) J. Biol. Chem. 273,22584–22588

43. Liu, C. Y., Zhu, G., Converse, R., Kao, C. W., Nakamura, H., Tseng, S. C.,Mui, M. M., Seyer, J., Justice, M. J., Stech, M. E., Hansen, G. M., and Kao,W. W. (1994) J. Biol. Chem. 269, 24627–24636

44. Johnson, J.M., Young, T. L., and Rada, J. A. (2006)Mol. Vis. 12, 1057–106645. Iozzo, R. V. (1997) Crit. Rev. Biochem. Mol. Biol. 32, 141–17446. Rada, J. A., Shelton, S., and Norton, T. T. (2006) Exp. Eye Res. 82, 185–20047. Phillips, J. R., Khalaj, M., and McBrien, N. A. (2000) Invest. Ophthalmol.

Vis. Sci. 41, 2028–203448. Siegwart, J. T., Jr., and Norton, T. T. (1999) Vision Res. 39, 387–40749. Moring, A. G., Baker, J. R., and Norton, T. T. (2007) Invest. Ophthalmol.

Vis. Sci. 48, 2947–295650. Paluru, P. C., Scavello, G. S., Ganter, W. R., and Young, T. L. (2004)Mol.

Vis. 10, 917–92251. Morel, L. (2004) PLoS Biol. 2, E241

52. Kao, W. W. (2006) Cornea 25, S7–S1953. Barathi, V. A., Weon, S. R., and Beuerman, R. W. (2009) Mol. Vis. 15,

1277–129354. Liu, O., Wu, J., Wang, X., and Zeng, J. W. (2007)Mol. Vis. 13, 1234–124455. Shih, Y. F., Hsiao, C. K., Chen, C. J., Chang, C. W., Hung, P. T., and Lin,

L. L. (2001) Acta Ophthalmol. Scand. 79, 233–23656. Tan, D. T., Lam, D. S., Chua, W. H., Shu-Ping, D. F., and Crockett, R. S.

(2005) Ophthalmology 112, 84–9157. Chou, A. C., Shih, Y. F., Ho, T. C., and Lin, L. L. (1997) J. Ocul. Pharmacol.

Ther. 13, 61–6758. Cottriall, C. L., McBrien, N. A., Annies, R., and Leech, E. M. (1999) Oph-

thalmic Physiol. Opt. 19, 327–33559. Cottriall, C. L., andMcBrien, N. A. (1996) Invest. Ophthalmol. Vis. Sci. 37,

1368–137960. Stone, R. A., Lin, T., and Laties, A. M. (1991) Exp. Eye Res. 52, 755–75861. McBrien, N. A., Moghaddam, H. O., and Reeder, A. P. (1993) Invest. Oph-

thalmol. Vis. Sci. 34, 205–215




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Hu, Chung-Liang Chien and I-Jong WangLung-Kun Yeh, Chia-Yang Liu, Winston W.-Y. Kao, Chang-Jen Huang, Fung-Rong

Increased Size of Scleral Coats) Causes Scleral Thinning andzlumKnockdown of Zebrafish Lumican Gene (

doi: 10.1074/jbc.M109.043679 originally published online June 15, 20102010, 285:28141-28155.J. Biol. Chem.

10.1074/jbc.M109.043679Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2010/06/14/M109.043679.DC1

http://www.jbc.org/content/285/36/28141.full.html#ref-list-1

This article cites 59 references, 26 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M109.043679

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;285/36/28141&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/285/36/28141

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=285/36/28141&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/285/36/28141

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2010/06/14/M109.043679.DC1

http://www.jbc.org/content/285/36/28141.full.html#ref-list-1

http://www.jbc.org/

Documents

KnockdownofZebrafishLumicanGene(zlum)CausesScleral ... · PDF fileKnockdownofZebrafishLumicanGene(zlum)CausesScleral ... rapid amplification of cDNA end; z, zebrafish; m, mouse; h,