Structure, Chromosomal Location, and Tissue-Specific Expression of The

Structure, Chromosomal Location, and Tissue-Speciﬁc Expression of the Mouse Opticin Gene

Masamine Takanosu,1 Tanya C. Boyd,1 Magali Le Goff,2 Stephen P. Henry,3 Youwen Zhang,1 Paul N. Bishop,2 and Richard Mayne1

3 PURPOSE. To determine the structure, location, and tissue-spe- prepared from this tissue. Studies in human tissue have also cific expression of the mouse opticin gene (Optc) and to shown the presence of opticin in brain by Western blot anal- compare expression in the eye with that of Prelp, collagen II, ysis3 and in ligament and skin by RT-PCR,1 but opticin expres- and collagen IX. sion has not been detected in several other tissues that have METHODS. Expressed sequence tags (ESTs) to mouse opticin been investigated. were identified and the full-length sequence obtained after PCR The SLRPs are characterized by tandem leucine-rich repeats, which may form a horseshoe-shaped solenoid-like struc- reactions using a 15-day-postconception (dpc) whole-mouse 4–6 embryo cDNA library. The mouse chromosomal localization of ture. They are divided into three classes according to the Optc was determined by radiation hybrid mapping and its number of leucine-rich repeats, the spacing of a four-cysteine genomic structure determined using an Optc-containing BAC cluster located N-terminal to the leucine-rich repeats, and the number of exons that encode the gene. Opticin is a class III clone. Tissue-specific expression of opticin, PRELP, collagen II, 7–9 and collagen IX mRNAs was investigated by in situ hybridiza- member, along with epiphycan and osteoglycin/mimecan. tion and by dot blot hybridization for opticin. Most of the SLRPs are proteoglycans, but some of them (including opticin and PRELP) are not, and opticin is unique in RESULTS. The Optc gene was localized to mouse chromosome 1 that it is glycosylated with sialylated O-linked oligosaccha- at 74.3 cM and consisted of seven exons spanning 10 kb. The rides.1 Recently, it was reported that the genes for the SLRPs Optc gene was less than 4 kb from the Prelp gene. In situ keratocan, decorin, lumican, and epiphycan are present as a hybridization localized opticin mRNA exclusively to the pre- cluster on human chromosome 12q22-23.10 The human opti- sumptive ciliary body during development and to the nonpig- cin gene (OPTC) has been localized to 1q31-q322,3 and two mented ciliary epithelium of the adult mouse eye. Expression other SLRP genes, PRELP11,12 and fibromodulin (FMOD),13 of Prelp was also detected in the nonpigmented ciliary epithe- have been localized to 1q32 in the order FMOD, PRELP, and lium of the adult eye. However, expression of collagen types II OPTC.9 and IX was detected largely in the developing mouse eye, with The biological function of the SLRPs in vivo is still some- type IX expression confined primarily to the presumptive cil- what unclear, although several members of the SLRP family iary body. bind to collagen fibrils through their leucine-rich repeat do- CONCLUSIONS. The Optc, Prelp, and fibromodulin (Fmod) genes mains and appear to regulate fibril diameter and spacing.14 form a cluster on mouse chromosome 1. Opticin may repre- Knockout models of mice without decorin,15 fibromodulin,16 sent a marker for ciliary body differentiation. Continued ex- or lumican17 all support this hypothesis, in that the mice show pression of opticin in the adult mouse eye suggests functions irregular forms of collagen fibrils primarily in the dermis, ten- other than that of putative regulator of vitreous collagen don, and cornea, respectively. The vitreous gel contains a fibrillogenesis. (Invest Ophthalmol Vis Sci. 2001;42: dilute network of thin collagen fibrils (composed of collagen 2202–2210) types II, IX, and V/XI) that are essential to maintain its structure.18 It therefore appears likely that opticin is involved in pticin is a member of the family of extracellular matrix fibrillogenesis of collagen molecules to form the vitreous gel Osmall leucine-rich repeat proteins (SLRPs) and was origi- and is potentially involved in maintaining the spacing between nally discovered associated with the collagen fibrils of bovine the collagen fibrils of the tissue.19 vitreous.1 Subsequently, opticin cDNAs were demonstrated in In this study, we determined the gene structure of mouse libraries from adult human iris,2,3 and opticin cDNA was found Optc, its chromosomal localization, and its relationship to the to be one of the most common transcripts (0.7%) in a library Prelp and Fmod genes. We also investigated mRNA expression in developing and adult mouse eye and other tissues and compared expression with PRELP, collagen type II, and all From the 1Department of Cell Biology, University of Alabama at three chains of collagen type IX. Birmingham; the 2Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, and Research Group in Eye and Vision Sciences, School of Medicine, University of Manchester, United King- MATERIALS AND METHODS dom; and the 3Center for Extracellular Matrix Biology, Texas A&M University System Health Science Center, Institute of Biosciences and cDNA Cloning of Mouse Opticin Technology, Houston. Supported in part by Grant R37 AR30481 from the National Human opticin cDNA (AJ133790) was used to search GenBank (pro- Institutes of Health and by a grant from Fight for Sight. PNB is a vided by the National Institutes of Health and available in the public Wellcome Trust Senior Research Fellow in Clinical Science. domain at http://www.ncbi.nlm.nih.gov) for mouse ESTs to opticin. Submitted for publication February 15, 2001; revised April 27, Based on the sequence of several EST clones, specific primers were 2001; accepted May 15, 2001. designed to mouse opticin both in sense and antisense directions. PCR Commercial relationships policy: N. was performed using a 15-day-postconception (dpc) mouse embryo The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- cDNA library (Clontech, Palo Alto, CA) with vector primers. PCR ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. products were subcloned into a vector (pGEM-T Easy; Promega, Mad- Corresponding author: Richard Mayne, Department of Cell Biol- ison, WI) and sequenced manually by using a kit (Thermosequenase; ogy, University of Alabama at Birmingham, VH G82C6 Box 302, 1530 Amersham Pharmacia Biotech, Piscataway, NJ). To establish the poten- Third Avenue S, Birmingham, AL 35294-0019. [email protected] tial full-length cDNA of mouse opticin, including 5Ј- and 3Ј-untrans-

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TABLE 1. Probes Used for In Situ Hybridization of Mouse Tissues

Gene Primer Sequence Length (bp) Location

Optc OP2 5Ј-AGCTTGGTGCTGCAGAAGGCA-3Ј 620 Exon 2–4 OP11 5Ј-CAGCCGATTCAGGCGGACGT-3Ј Prelp PRELP-S 5Ј-GACACACGCAGACAGGCACCAACTGGGAGAC-3Ј 550 Exon 1–2 PRELP-A 5Ј-GAATTCGGTTGTCCAGGTTGACCCACCTCAGG-3Ј Col2a1 2A1-S 5Ј-ACACACTGGTAAGTGGGGCA-3Ј 435 3Ј-UTR 2A1-A 5Ј-TGGGGCTGGGAACAGTCACT-3Ј Col9a1 9A1-S 5Ј-GTGCTCTTGGCTTAAGAGGA-3Ј 400 3Ј-UTR 9A1-A 5Ј-TGATGTCAGAGGTGAAACCT-3Ј Col9a2 9A2-S 5Ј-AGCAACCAGCCAGGACAGAG-3Ј 470 3Ј-UTR 9A2-A 5Ј-TACACAAAGGCCAGAGTGGT-3Ј Col9a3 9A3-S 5Ј-CTTCAGTAGGAAATGGCTCC-3Ј 500 3Ј-UTR 9A3-A 5Ј-CAGATGGTGCAGTGTAGTTC-3Ј S, sense; A, antisense.

lated regions (UTRs), the cDNA sequences determined by PCR reac- Thick sections (10 ␮m) were cut in a cryotome at a parasagittal angle tions were compared with the mouse EST database. Several EST clones that passed through the eye. The sections were fixed in 4% parafor- including 5Ј-or3Ј-UTRs were found, and the full-length cDNA se- maldehyde and digested with 0.2 ␮g/ml proteinase K for 5 minutes at quence of mouse opticin was assembled from the sequences of PCR room temperature. After acetylation with 0.25% acetic anhydride, products and EST clones. The putative transcriptional start site was sections were dehydrated with a graded series of ethanol. Hybridiza- designated from the longest available 5Ј EST sequence (AA223016). tion was performed using a 33P-UTP-labeled riboprobe overnight at 50°C. Sections were washed in 50% formamide in 2ϫ SSC for 30 Annotation of a BAC Clone Containing Fmod, minutes at 55°C, followed by digestion with RNase A. Sections were PRELP, and Optc washed again under highly stringent conditions (0.1ϫ SSC for 60 minutes at 60°C) and then dehydrated with ethanol. Sections were A mouse BAC clone (control number 24885, clone address 164[K13]) exposed in emulsion (Hypercoat LM-1; Amersham Pharmacia Biotech) containing the Optc gene was obtained after library screening by Incyte for 3 to 6 days at 4°C and developed (D-19 developer; Eastman Kodak, Genomics (St. Louis, MO) using the following mouse opticin primers: Rochester, NY). Sections were visualized, and photomicrographs were OP17, 5Ј-CTG GAT TCC ATC CCT GGG CCT-3Ј, and OP29, 5Ј-ACA taken under a dark-field microscope (model DC200; Leica, Deerfield, GGG AGC CGT GGC AGG CA-3Ј. To analyze the genomic structure of IL) and processed using image management software (Photoshop; mouse Optc, primers designed from the cDNA sequence were used for Adobe, San Jose, CA). intronic bridging PCR reactions. PCR products were subcloned into a vector (pGEM-T Easy; Promega) and sequenced on an automated se- quencer (model 377; Perkin Elmer-Applied Biosystems, Inc., Foster Dot Blot Hybridization City, CA) using vector primers. A mouse BAC clone that contains the A mouse multiple-tissue expression array (Clontech) was used for dot Fmod, Prelp, and Optc genes is present in GenBank (AC026760), but blot hybridization. An 870-bp PCR product (forward primer: 5Ј-CAA the annotation is presently not complete. Similarly, human BAC clones CCA CTC GTC CTC TCC CT-3Ј; reverse primer: 5Ј-CAG CCG ATT CAG containing the PRELP and OPTC genes (AC022000, AL391817) were GCG GAC GT-3Ј) obtained from a 15-dpc embryo cDNA (Marathon- identified together with a third human BAC clone that contains FMOD Ready; Clontech) was used as a probe. The probe was labeled with and PRELP (AL359837). The human BAC clones were further anno- 32P-dCTP (3000 Ci/mmol; Random Primed DNA Labeling Kit; Roche tated by using cDNA sequences of PRELP and opticin, and the remain- Molecular Biochemicals, Indianapolis, IN). Hybridization was per- ing gaps were filled in by performing bridging PCR reactions on BAC formed at 65°C overnight in hybridization solution (ExpressHyb; Clon- DNAs. In addition, PCR reactions were performed to determine the tech). The membrane was washed with 2ϫ SSC and 1% SDS at 65°C distance between the mouse Prelp and Optc genes by using AC026760 and 0.1ϫ SSC and 0.1% SDS at 50°C. The membrane was exposed to with the following primers: PR1, 5Ј-CGT CCC TTC GTG AGG ATG x-ray film (XAR-5; Kodak) with intensifying screens for 2 days at Ϫ80°C GT-3Ј, and OP14, 5Ј-GGG GAC AGC TAG ACA CCC CCA-3Ј. The PCR before the film was developed. product was ligated to the vector (pGEM-T Easy; Promega) and sequenced. Radiation Hybrid Mapping of the Mouse Optc Gene In Situ Hybridization The mouse Optc gene was mapped by screening a mouse/hamster RH Probes used for in situ hybridization were as described in Table 1. All panel (Research Genetics, Huntsville, AL) using the following primers: probes were amplified by PCR from a 15-dpc embryo cDNA prepara- I-1, 5Ј-ATA GGT CTC GCA TGG GCC AA-3Ј, and I-4, 5Ј-TGG CTC TTT tion (Marathon-Ready; Clontech). PCR products were subcloned into a CTA GGA CCT CT-3Ј. The data were analyzed by computer (Auto- vector (pBluescript-SK; Stratagene, La Jolla, CA). Antisense and sense RHMAPPER, provided by Whitehead Institute/Massachusetts Institute riboprobes were transcribed by T3 and T7 RNA polymerase by a of Technology Center for Genome Research radiation hybrid map of commercial system (In Vitro Transcription System; Promega) in the the mouse genome and available in the public domain at http://www. presence of 33P-UTP (1000 Ci/mmol). genome.wi.mit.edu/cgi-bin/mouse_rh/rhmap-auto/rhmapper.cgi). The Albino BALB/c or 129SV mice of agouti color were used for in situ mouse Optc gene was mapped at 74.3 cM on mouse chromosome 1 hybridization. In this manner, the detailed morphology of ocular struc- and placed 5.23 cR from D1Mit348 (lod Ͼ3.0). tures could be determined from the presence or absence of pigmented cells in the uveal tract. All animals were treated humanely in accordance with the ARVO Statement for the Use of Animals in Ophthalmic RESULTS and Vision Research. Coitus was confirmed by the presence of a cDNA Sequence of Mouse Opticin vaginal plug the next morning and designated as 0.5 dpc. Whole selected embryos (12.5, 15.5, and 17.5 dpc) together with whole eyes The full-length sequence of mouse opticin cDNA was 1901 bp. dissected from adult mice were embedded in optimal cutting temper- Mouse opticin cDNA had a 290-bp UTR at the 5Ј end and a ature (OCT) compound and frozen in dry ice-acetone immediately. 618-bp UTR at the 3Ј end, both of which were longer than

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reported for human opticin cDNA (GenBank AF333980 for the SignalP/). The alignment was performed on computer (Clust- mouse was submitted with this manuscript, AJ133790 for hu- alW ver.1.81; SGI, Mountain View, CA). mans). The open reading frame (ORF) of human and mouse sequences was almost the same length, 999 bp and 986 bp, Structure of the Optc Gene respectively. Alignment of the human and mouse cDNA sequences showed that the sequence of opticin was well con- The genomic structure of mouse Optc was elucidated by exon- served (Fig. 1). Identity of the ORF was 77% at the nucleotide bridging PCR reactions using a BAC clone (AC026760) that was level and 73% at the amino acid level. The seven leucine-rich found to contain the whole Optc gene but was not annotated. repeat domains and the position of all cysteine residues were PCR products were cloned and subsequently sequenced to conserved in both species. The signal peptide cleavage site was complete the annotation of the Optc gene (Fig. 2A). The mouse determined with sequence analysis software (SignalP ver. 1.1; Optc gene had seven exons and the total size of the gene was provided in the public domain by the Center for Biological approximately 10 kb. (The complete sequence of the mouse Sequence Analysis, Technical University of Denmark, Lyngby, Optc gene was submitted to GenBank AF333981). The human Denmark, and available at http://www.cbs.dtu.dk/services/ OPTC gene was shown to have eight exons (Fig. 2A). Exons 1

FIGURE 1. The alignment of human and mouse cDNA of opticin and de- duced protein sequences. Lowercase letters:5Ј- and 3Ј-UTR sequences; up- percase letters: ORFs; box: transla- tion initiation codon and termination codon; underline: leucine-rich repeat domains; highlight: conserved cysteine residues; double underline: polyadenylation signal; arrow: signal peptide cleavage site.

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to 6 of the mouse Optc gene correspond to the human OPTC the PRELP and OPTC genes, together with a gap within the gene, but the last exon in the 3Ј-UTR is different. In human OPTC gene, by using human BAC clone (AC022000). The OPTC, exon 8 contains the polyadenylation signal and is lo- distance from the 3Ј end of the longest transcript of PRELP to cated 5 kb downstream of exon 7 (Fig. 2A). In mouse Optc, the 5Ј end of OPTC was found to be 4.5 kb in humans and was exon 8 does not exist, and a longer exon 7 contains the completely resequenced. polyadenylation signal (Fig. 1). Exon 7 of mouse Optc includes In the case of the mouse, we attempted to annotate a mouse a3Ј-UTR of 618 bp. The consensus sequences for each exon– BAC clone (AC026760) to elucidate the relationship of each intron boundary of both human OPTC and mouse Optc are gene at this locus. Although this BAC clone clearly contained shown in Figure 2B. all three genes, it was not possible to annotate the orientation In humans, the OPTC gene is clearly present in a gene of Fmod and its distance from Prelp and Optc. The published cluster with FMOD and PRELP on chromosome 1q32 (Gen- cDNA sequence of mouse PRELP did not contain the full length Bank NT_004523) with FMOD considered to be located 128 kb of the 3Ј-UTR (GenBank AF261886), and the downstream se- upstream of PRELP (GenBank Chromosome 1 Contig Map). quence from mouse BAC clone AC026760 was therefore used However, some gaps still existed in the available sequence. to survey the EST database. Several ESTs were identiﬁed that We, therefore, performed PCR reactions to ﬁll the gap between overlapped with the previously published sequence of exon 3

FIGURE 1. (Continued)

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FIGURE 2. (A) Genomic organization of OPTC and PRELP in humans and the mouse. The PRELP and OPTC genes have been mapped on chromosome 1q32 for humans. The cluster of human genes was derived from annotated sequences (Gen- Bank NT_004523). Because sequencing of the cluster is presently not complete, each intron for human OPTC is not represented as an exact size (gap marked by vertical dashed line). The genomic structure of human PRELP was determined by annotation of a BAC clone (AC022000) and with an EST clone to the longest 3Ј-UTR (AI584095). Although the FMOD gene is clearly upstream of PRELP, it was not included in the diagram, because the exact orientation and location of the gene is still unknown. (B) Exon–intron boundary of the human and mouse OPTC genes. Upper- case letters: exon sequences; lowercase letters: ﬂanking intronic sequences. The sequences for human were derived from BAC clone AC022000 and for mouse were determined by sequencing of PCR products from BAC clone AC026760.

of mouse Prelp. The longest EST identified that contained a embryo, opticin expression was observed specifically in the polyadenylation signal at the correct location was determined region of the anterior tip of the developing retina (Figs. 3C, to be 2.5 kb by comparison with BAC clone AC026760. The 3D). No significant signal for opticin was detected in any other longest mRNA of mouse Prelp was therefore predicted to be structure in the eye, including the lens and cornea. In the 4.5 kb, and this prediction was confirmed by Northern blot 17.5-dpc embryonic eye, a strong signal was observed only in analysis (Henry SP, unpublished observations, 2000). PCR re- the region of the developing ciliary body (Figs. 3E, 3F). During actions were performed to bridge from the 3Ј end of mouse development, at 12.5, 15.5, and 17.5 dpc no clear signal for Prelp to the Optc gene, assuming the same relationship be- opticin expression was detected in any other tissue of the tween these genes as occurs in humans. PCR products were whole embryo (data not shown). In the adult eye, the signal for cloned and sequenced. The results show that the mouse Prelp opticin was detected only in the nonpigmented epithelium of gene is located approximately 3.5 kb upstream of the Optc the ciliary body in both the BALB/c albino mouse (Figs. 4A, 4B) gene and that they are arranged on the same DNA strand in a and the agouti mouse (Fig. 4C). No other structures showed a head-to-tail manner. The Prelp and Optc genes therefore form positive signal for opticin mRNA. No signal was observed on a very similar gene cluster in both the human and the mouse. the slide with the use of a sense control in the agouti mouse, We were unable to annotate the exact location and orientation in which the pigment is clearly observed (Fig. 4D). of the mouse Fmod gene, but preliminary results suggest that The expression of PRELP mRNA could not be detected it, as in the human, lies at a considerable distance upstream of during development of the eye at 12.5, 15.5, or 17.5 dpc Prelp. There are no apparent intervening genes, in either the (data not shown), but PRELP expression was seen in other human or mouse, between FMOD and PRELP. tissues during development, including cartilaginous compo- Expression of mRNA for Opticin, PRELP, and nents of the vertebrae and ribs, the cochlea, and the neuro- hypophysis (Figs. 5A–D). However, in the adult eye, PRELP Type IX Collagen during Eye Development mRNA was detected in the nonpigmented epithelium of the in the Mouse ciliary body in a pattern of expression that resembled that of The eye from a 12.5-dpc embryo did not show any signal of opticin mRNA (Figs. 4E, 4F) although the signal was much opticin by in situ hybridization (Figs. 3A, 3B). In the 15.5-dpc weaker.

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SLRP family. No other tissue-speciﬁc RNA present on the blot showed a positive signal.

DISCUSSION In this study, we present the gene structure of mouse Optc and investigate mRNA expression in the developing and adult mouse eye. Expression of opticin was ﬁrst detected in the 15.5-dpc embryonic eye at the anterior tip of the developing neuroretina. However, it was impossible to distinguish mor- phologically a particular cell type expressing opticin mRNA in the hematoxylin-eosin–stained sections at this stage. In the

FIGURE 3. Messenger RNA expression of opticin during development of the mouse eye: (A, B) 12.5-dpc, (C, D) 15.5-dpc, and (E, F) 17.5-dpc mouse embryonic eyes. (A, C, and E) Bright-ﬁeld views; (B, D, and F) dark-ﬁeld views. (A, B) No positive signal was observed in the 12.5-dpc embryonic eye. (C, D) The small regions at the tip of the neuroretina showed a signal in the 15.5-dpc embryonic eye (D, arrowhead). (E, F) A strong signal was observed only in the small areas between the retina and iris in the 17.5-dpc embryonic eye (F, arrowhead). Scale bar, (A, B) 150 ␮m; (C, D) 300 ␮m; (E, F) 400 ␮m.

In the 17.5-dpc embryonic eye, mRNA of COL9A1 was expressed in the retina and developing ciliary body (Figs. 6A, 6B). A weak signal was also detected in the cornea, but not in the lens or sclera, confirming earlier results.20,21 COL9A2 mRNA was detected only in the developing ciliary body, similar to opticin mRNA (Figs. 6C, 6D). No positive signal was observed in the retina, cornea, lens, iris, or sclera. COL9A3 mRNA was weakly detected in the ciliary body (Fig. 6E and arrowhead in Fig. 6F), and, unexpectedly, was also present in the choroid. In the adult eye, mRNA of COL9A1 was detected in the ciliary body and inner nuclear layer of the retina (Figs. 4G, 4H). Expression of COL9A2 and COL9A3 was also detected in the ciliary body, but the signal was extremely weak (data not shown). The expression of COL2A1 was detected in the devel- FIGURE 4. Messenger RNA expression of opticin, PRELP, and COL9A1 oping cornea, retina, and sclera of the embryonic eye at 17.5 in the adult mouse eye. All micrographs show only the region in the dpc (Figs. 6G, 6H). In the adult eye, COL2A1 expression was vicinity of the ciliary body. (A, B) Bright- and dark-field views of the ciliary body of an albino mouse. Opticin messenger RNA was specifi- detected in the lens and cornea as well as in the retina, sclera, cally expressed in the nonpigmented epithelial cells of the ciliary body and ciliary body. These expression patterns for COL2A1 were 22,23 (B, arrowhead). (C, D) Dark-field, high-magnification views of the similar to those previously observed by Savontaus et al. In ciliary body of a pigmented mouse. Section in (C) was processed with all experiments, sense control probes for each gene were also an antisense probe and in (D) with a sense probe. The pigmented cells processed and no signal was detected above background. in the retina, ciliary body, and iris are red-brown. Positive signal was Dot blot hybridization was performed to survey the expres- clearly seen in the nonpigmented epithelium of the ciliary body (C, sion of opticin in adult tissues. The probe used for these arrowhead). A sense control did not show any signal (D). (E, F) Bright- experiments was longer than that used for in situ hybridization and dark-field views of expression of PRELP. A relatively weak signal is but included the same sequence. A strong signal was detected seen in the nonpigmented epithelium of the ciliary body (F, arrowhead). (G, H) Bright- and dark-field views showing expression of for adult eye mRNA after dot blot hybridization (Fig. 7), and a COL9A1. A signal was observed in the nonpigmented epithelium of the weak signal was also detected for heart, brain, testis, thyroid, ciliary body and inner nuclear layer of the retina (H, arrowhead). c, and epididymis. It is uncertain whether the weak signals rep- cornea; cb, ciliary body; gl, ganglion layer; i, iris; in, inner nuclear layer; resent a low level of opticin expression or cross-reactivity with on, outer nuclear layer, vb, vitreous body. Scale bar, (A, B, E–H) 200 the mRNA of other molecules, especially other members of the ␮m; (C, D) 100 ␮m.

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derived from other eye tissues both in the mouse20,22,23 and chicken.25 Why type IX collagen and opticin should be expressed only in the nonpigmented layer of the presumptive ciliary body is not clearly understood, nor is it understood whether they are coordinately regulated. In previous work, expression of ␣1(IX) was detected in the optic cup as early as 10.5 dpc and began to be concentrated in the anterior part of the retina as early as 13.5 dpc.20 However, type IX collagen expression appeared to be decreased in the adult mouse (Fig. 4H), whereas opticin expression appeared to be maintained (Fig. 4B). This suggests that opticin may perform other functions and is being continuously released into the vitreous cavity of the adult mouse. However, the adult mouse has very little if any vitreous gel, and it therefore appears unlikely that opticin is necessary to maintain the spacing between the collagen ﬁbrils within the gel. The data provided by studying the expression of the indi- vidual genes encoding the three type IX collagen chains

FIGURE 5. Messenger RNA expression of PRELP in the 15.5-dpc mouse embryo. Signal was seen in the vertebral column (A) and ribs (B). The signal was strong in the peripheral region of the cartilage in both vertebrae and ribs. Signal was also seen in the cartilaginous component of the cochlea (C) and the pituitary gland (D). The neuro- hypophysis (D, arrow) and a part of the forebrain were positive. CH, cochlea; SB, sphenoid bone; AH, adenohypophysis; N, neurohypoph- ysis; R, rib; S, spinal cord; V, vertebral column. Scale bar, (A, B) 275 ␮m; (C, D) 400 ␮m.

17.5-dpc embryonic eye, opticin mRNA was positive in cells developing between the iris and neuroretina. Because expression of opticin mRNA was specifically restricted to the nonpigmented epithelium of the ciliary body in the adult, the positive cells in the 15.5- and 17.5-dpc embryonic eyes probably are committed to become this cell type. A previous study showed that the developing ciliary body could be distinguished mor- phologically in the mouse embryonic eye at approximately 16 dpc.24 Our in situ hybridization data indicate that functional differentiation of the ciliary body had started by 15.5 dpc and that opticin mRNA may represent a marker for ciliary body differentiation. In humans, cDNA transcripts for opticin have been observed at high levels in adult iris libraries.3 In addition, most of the human opticin ESTs that can be identified in GenBank are reportedly derived from retinal cDNA. By contrast, in the current study opticin expression was observed by in situ hybridization only in the ciliary body. It is possible that the human tissues used to make these cDNA libraries were con- taminated by ciliary body or that the pattern of opticin expression is different in humans. We are currently undertaking further studies to resolve this issue. Although opticin expression was observed only by in situ hybridization in the ciliary body and dot blot analysis revealed a strong opticin mRNA signal only from the eye, several mouse opticin ESTs were found in GenBank from nonocular tissues, including brain, kidney, mammary gland, uterus, urinary blad- der, pineal gland, and cerebellum. Taken together, these data suggest that opticin expression occurs only at high levels in the FIGURE 6. Messenger RNA expression of chains of type IX collagen in eye, but low-level expression may be present in many other the 17.5-dpc embryonic eye (A–F) and type II collagen (G, H). (A, C, tissues, such as parts of the brain. E, and G) Bright-field views; (B, D, F, and H) dark-field views. (A, B) The functions of opticin in the eye remain uncertain. The Expression of COL9A1 mRNA. The ciliary body showed a strong signal. nonpigmented epithelium of the ciliary body is assumed to be In addition, a positive signal was seen in the cornea and outer nuclear the source of opticin in the vitreous cavity. In humans, opticin layer of the retina. (C, D) Expression of COL9A2 mRNA. A positive signal was seen only in the presumptive ciliary body. (E, F) Expression has been shown to be closely associated with the collagen 1 of COL9A3 mRNA. The presumptive ciliary body showed a weak signal fibrils of vitreous suggesting a role in vitreous collagen fibril- for COL9A3 mRNA (F, arrowhead). For unknown reasons, a strong logenesis. In mice, we have confirmed that the ciliary body signal was seen in the choroid. (G, H) Expression of COL2A1 mRNA. 20–22 expresses type IX collagen as well as opticin, whereas the A positive signal was seen in the developing cornea, retina, and sclera. type II collagen of the vitreous appears to be partially or totally Scale bar, 400 ␮m.

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FIGURE 7. A mouse multiple tissue expression array was used for dot blot hybridization to investigate expression of opticin mRNA in adult tissues. The eye showed a strong signal for opticin. Weak signals were detected in the heart, brain, testis, thyroid, and epididymis.

(Col9a1, Col9a2, and Col9a3) in the 17.5-dpc embryonic References mouse eye was intriguing. Although, all three genes were preferentially expressed in the ciliary body, Col9a1 was also 1. Reardon AJ, Le Goff M, Briggs MD, et al. Identification in vitreous expressed by the retina. By contrast, the Col9a2 gene was and molecular cloning of opticin, a novel member of the family of leucine-rich repeat proteins of the extracellular matrix. J Biol apparently expressed in the choroid, but Col9a1 and Col9a3 Chem. 2000;275:2123–2129. were not. The significance of these differential expression 2. Friedman JS, Ducharme R, Raymond V, Walter MA. Isolation of a patterns remains uncertain. Expression of the three genes novel iris-specific and leucine-rich repeat protein (oculoglycan) would be expected to be in a 1:1:1 ratio to allow stoichiomet- using differential selection. Invest Ophthalmol Vis Sci. 2000;41: 26,27 ric assembly of the three chains of type IX collagen. 2059–2066. PRELP expression has been demonstrated in tissues in- 3. Hobby P, Wyatt MK, Gan W, et al. Cloning, modeling, and chro- cluding cartilage and lung,12 but expression in the eye is a mosomal localization for a small leucine-rich repeat proteoglycan novel finding. By contrast, high-level expression of opticin is (SLRP) family member expressed in human eye. Mol Vision. 2000; found in the eye, but expression levels are low in other 6:72–78. tissues and expression could not be demonstrated in carti- 4. Scott JE. Proteodermatan and proteokeratocan sulfate (decorin, lage or lung.1 It is possible that PRELP is secreted by the lumican/fibromodulin) proteins are horseshoe shaped: implica- ciliary body and has a function in the vitreous humor, tions for their interactions with collagen. Biochemistry. 1996;35: although to date we have not been able to demonstrate the 8795–8799. presence of any members of the SLRP family apart from 5. Weber IT, Harrison RW, Iozzo RV. Model structure of decorin and opticin in the bovine vitreous humor. Alternatively, it is implications for collagen fibrillogenesis. J Biol Chem. 1997;271: 31767–31770. possible that, because the Prelp and Optc genes are so close 6. Keene DR, San Antonio JD, Mayne R, et al. Decorin binds near the ( 4 kb), a regulatory element that drives opticin expression Ͻ C terminus of type I collagen. J Biol Chem. 2000;275:21801– also moderately affects PRELP expression. 21804. In conclusion, OPTC is a well-conserved gene between 7. Hocking AM, Shinomura T, McQuillan DJ. Leucine-rich repeat humans and mice and forms part of a gene cluster with FMOD glycoproteins of the extracellular matrix. Matrix Biol. 1998; and PRELP. Expression of opticin in the adult mouse eye is 17:1–19. specifically localized to the nonpigmented epithelium of the 8. Iozzo RV. Matrix proteoglycans: from molecular design to cellular ciliary body. The high level of opticin expression in the adult function. Annu Rev Biochem. 1998;67:609–652. eye suggests that it has an important, as yet undetermined role 9. Henry SP, Takanosu M, Boyd TC, et al. Expression pattern and in maintaining homeostasis within the eye. gene characterization of asporin: a newly discovered member of

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