Human Molecular Genetics, 2002, Vol. 11, No. 16 1879-1886
© 2002 Oxford University Press
Mfrp, a gene encoding a frizzled related protein, is mutated in the mouse retinal degeneration 6
1The Jackson Laboratory, Bar Harbor, ME 04609, USA, 2Department of Ophthalmology, Akita University School of Medicine, Akita 010-8543, Japan and 3Jules Stein Eye Institute/UCLA, Los Angeles, CA 90095, USA
Received April 19, 2002; Revised June 3, 2002; Accepted June 4, 2002
DDBJ/EMBL/GenBank accession no.
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ABSTRACT |
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The autosomal recessive mouse mutation retinal degeneration 6 (rd6) causes small, white retinal spots and progressive photoreceptor degeneration similar to that observed in human flecked retinal diseases. Using a positional cloning approach, we determined that rd6 mice carry a splice donor mutation in the mouse homolog of the human membrane-type frizzled-related protein (Mfrp) gene that results in the skipping of exon 4. We found that mRNA of Mfrp is predominantly expressed in the eye, and at a lower level in the brain. To determine where in the eye Mfrp is expressed, in situ hybridization was done and showed that Mfrp is expressed specifically in the retinal pigment epithelium (RPE) and ciliary epithelium of the eye. The deduced amino acid sequence of MFRP contains a region with similarities to the cysteine-rich domain (CRD) of frizzled, a gene originally found in Drosophila that controls tissue polarity. The CRD is essential for Wnt binding and signaling. Wnt signaling has been shown to be involved in the control of gene expression, cell adhesion, planar polarity, proliferation and apoptosis. We also observed the localization of Wnt family proteins in the apical membrane of the RPE. Our results provide genetic evidence for an involvement of the Mfrp gene expressed by RPE in the degeneration of photoreceptors.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Several human disorders are characterized by scattered white retinal dots or lesions. Such inherited retinal disorders were originally classified as flecked retinal diseases (1). The main disorders described within this category are retinitis punctata albescens (RPA), fundus albipunctatus (FA) and Stargardt's disease (1,2). RPA is a progressive disorder that causes visual loss similar to that observed in patients with retinitis pigmentosa, whereas FA is a form of congenital stationary night blindness which tends to be more stable over time. Stargardt's disease is characterized by progressive loss of central vision and progressive atrophy of the RPE overlying the macula.
Animal models with spontaneous retinal degeneration have been used for many years to provide insight into the etiologies of retinal degeneration and tissue to study disease progression and pathology. Many of these animal models have come from screening mice from genetically independent mouse strains and stocks at The Jackson Laboratory by indirect ophthalmoscopy and electroretinography (ERG) (3–6). Retinal degeneration 6 (rd6), which arose on the C3HfB/GaCas1b strain, is one of the animal models identified by this method (7). The inheritance pattern of rd6 is autosomal recessive. Fundus examination of mice homozygous for rd6 shows discrete dots distributed across the retina. Histological examination shows slowly progressive photoreceptor degeneration from the normal 12- to 14-cell-layer thickness to 4–5 layers at 4.5 months, 2–4 layers by 7 months, and 1 layer by 24 months. ERGs of eyes from mice homozygous for the rd6 mutation show a slow progressive retinal dysfunction of both rods and cones, beginning at about 1 month of age. ERG response is extinguished by 70 weeks of age. Both rod and cone systems are affected by the degenerative process and diminish at a similar rate (7).
Identification of disease-causing genes in animal models is extremely important. Knowledge of genes such as rd6 may lead to an understanding of pathways that are critical in maintaining normal function and physiology of the eye and, perhaps, may identify therapeutic targets for prevention of vision loss. A positional cloning approach was used to identify the gene responsible for the photoreceptor degeneration in rd6, and here we show a mutation in the Mfrp gene, a gene predominantly expressed in the eye and at a lower level in the brain.
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Genetic mapping of rd6
Previous genetic mapping localized rd6 to mouse chromosome 9,
24 cM from the centromere (7). To map rd6 more precisely, we constructed a high-resolution genetic map of the region. In total, 657 F2 progeny from a (B6.C3Ga-rd6/rd6xCAST) F1 intercross were genotyped for the flanking markers D9Mit329 and D9Mit23 and phenotyped using indirect ophthalmoscopy. The maximum estimated genetic size for the chromosome 9 segment containing rd6 was 0.46±0.19 cM (6 recombinants/1314 meiosis; Fig. 1). A physical map of this area was assembled using bacterial artificial chromosome (BAC) clones. Polymorphic markers were generated from a BAC end sequence and from intron sequences of genes located within this region in order to narrow the genetic interval further. Two new simple sequence length polymorphic (SSLP) markers, 82D20S and Ptd009, refined the minimal interval containing rd6 to 0.15±0.11 cM, a region covered by four overlapping BAC clones (Fig. 1). The availability of ordered draft sequence for human chromosome 11q23.3, the region homologous to the rd6 region, allowed us to identify transcripts in the interval using comparative in silico analyses.
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Mfrp is mutated in rd6
Eighteen transcripts were identified within the region, and 14 were screened for mutations by sequencing of products from reverse transcription—polymerase chain reactions (RT–PCR) or by northern blot analysis. Northern analysis using a probe from the Mfrp gene revealed a difference in transcript size between mRNAs extracted from eyes of rd6 and control C57BL/6J (B6) mice. A shorter Mfrp transcript was observed in rd6 mice (Fig. 2A). Since the full-length mouse cDNA encoding MFRP was not reported in the public database, we used oligonucleotide primers designed from the human MFRP cDNA (8) to amplify the entire coding region of Mfrp. Sequencing of RT–PCR products revealed a 1752 bp open reading frame (ORF), encoding a 584 amino acid MFRP protein (Fig. 2B). The ORF assignment is supported by high amino acid sequence similarity throughout the proposed coding sequences between human and mouse (Fig. 2B) (8). To determine the molecular basis for the size difference between mutant and control transcripts observed in the northern blots, we performed RT–PCR analysis. We found that exon 4 was absent in the cDNA obtained from rd6 mice (Fig. 2C). This exon skipping does not cause a frameshift. However, the skipping results in deletion of 58 amino acids from the MFRP polypeptide. To determine the cause of the exon skipping, we sequenced the exon–intron boundaries in genomic DNA from rd6 mice and controls and found a 4 bp deletion in the splice donor sequence of intron 4 in rd6 genomic DNA (Fig. 2D). The four nucleotides deleted in the rd6 genomic DNA are well conserved in splice donor sites of vertebrates, and most mutations affecting splicing have been identified in the first six bases of splice donor site and the last two bases of splice acceptor site sequences (9). Comparison of the sequences surrounding this donor splice site in standard inbred strains from historically independent lineages (129X1/SvJ, CBA, NOD, CAST/Ei, SPR/Ei, A/J) and C3H substrains (C3H/HeJ, C3H/HeSnJ) showed complete conservation of the B6-like sequence, suggesting that the nucleotide changes are not a normal allelic variation but a mutation leading to the abnormal transcript. Also, since northern blot analysis of the B6 control did not show smaller transcripts, the transcript found in rd6 is unlikely to be a shorter isoform of Mfrp.
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Mfrp is expressed as a dicistronic transcript with C1qtnf5
In examining the genomic structure of Mfrp, we noted that the complement-C1q tumor necrosis factor-related protein (C1qtnf5) gene was located immediately adjacent to Mfrp. Hybridization of probes from the coding regions of both Mfrp and C1qtnf5 to the multiple tissue northern blots identified mRNA transcripts of 4.4 kb in the eye with lower expression levels in the brain (Fig. 2E and 3A). Since hybridization using either probe identified the same pattern of expression for the 4.4 kb transcript, we hypothesized that Mfrp and C1qtnf5 formed a dicistronic transcript. To test this hypothesis, we performed RT–PCR analysis using a forward primer from the coding region of Mfrp and a reverse primer derived from the coding region of C1qtnf5 (Fig. 3B), and amplified a transcript containing both Mfrp and C1qtnf5 coding sequences. The length of the entire cDNA was 4220 bp and the transcript and corresponding genomic structure are shown in Fig. 3C. The deduced amino acid sequences of MFRP for both mouse and human contain regions with similarities to the cysteine-rich domain (CRD) of frizzled and the CUB domain found in complement subcomponent C1r/C1s, Uegf and bone morphogenetic protein-1 (Fig. 3D and E) (8). The 10 cysteines of the frizzled CRD which are highly conserved in the frizzled protein family (10) are also conserved in MFRP. We also found a similarity between MFRP and the ligand-binding domain of the low-density lipoprotein receptor (LDLR) (Fig. 3F). The C1QTNF5 protein contains a region similar to the C1q domain that is found in complement subcomponent C1q (Fig. 3G).
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Mfrp is expressed in the retinal pigment epithelium (RPE) and ciliary epithelium
The spatial expression of Mfrp in the eye was determined by in situ hybridization. Mfrp mRNA is highly expressed in the RPE and the ciliary body (Fig. 4A–H). In the ciliary body, Mfrp mRNA is expressed in both non-pigmented and pigmented epithelial cells. However, despite the early focal disorganization and shortening of the outer segments of the photoreceptor cells observed in rd6 mice, Mfrp was not expressed in cells of the neural retina. To test the potential for interaction between MFRP and Wnt proteins in the RPE, we performed immunohistochemical analyses against Wnt proteins for which commercial antibodies were available to determine if they localized to the RPE cells. Two antibodies recognizing two different antigenic sites for each of the Wnt proteins 1, 2, 3, 4 and 10b, respectively, were tested to assess their specificity. Wnt-2 did not stain in the RPE at all and staining for Wnt-1 and Wnt-10b was the same for the two antibodies tested, whereas Wnt-3 and Wnt-4 showed different staining patterns with the two antibodies used. The latter suggests that the antibodies for Wnt-3 and Wnt-4 are not specific. The specificity of antibodies for their respective antigenic site was confirmed by peptide neutralization assay. Wnt-1 and Wnt-10b staining was observed in the apical membrane of the RPE (Fig. 4I and J). However, no staining differences between mutant and control were observed for these antibodies. Additional studies to examine all of the Wnt family members need to be conducted. However, our results suggest that MRFP could potentially interact with Wnt family proteins, as they are expressed in the same cells.
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DISCUSSION |
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Mfrp is found as a dicistronic transcript
Mfrp is expressed on a dicistronic transcript, which also encodes C1qtnf5. Polycistronic transcripts are well described in prokaryotes, where they commonly encode proteins involved in the same functional pathway, thereby constituting an operon (11). Several dicistronic transcripts have been reported in mammals, and most of those genes encode functionally related proteins (12–18). MFRP and C1QTNF5 proteins also share the functionally related CUB and C1q domains. The CUB domain is found in complement subcomponent C1r/C1s, and the C1q domain is found in complement subcomponent C1q. C1 is the complex modular protease that triggers the classical pathway of complement in response to the formation of antigen–antibody complexes (19). CUB domains retain the ability to mediate Ca2+-dependent protein–protein interactions to form C1r/C1s tetramers, and C1q provides a framework for the tetramer (20). This suggests that MFRP and C1QTNF5 may also interact in vivo.
Mfrp has a domain similar to CRD of frizzled
The deduced amino acid sequence of MFRP contains regions with similarities to the CRD of frizzled, including the 10 cysteines which are highly conserved in this family of proteins (10). The first member of the frizzled family was identified as a Drosophila tissue polarity-determining gene (21). Subsequently, the frizzled family of proteins was confirmed to comprise receptors for the Wnt family of proteins (22). At least 10 frizzled family members have since been identified, and all members contain an extracellular CRD domain and seven transmembrane domains (23). A group of secreted frizzled-related proteins (SFRPs) has also been described that contain an N-terminal signal peptide and a frizzled-related CRD but lack any transmembrane domains (24). To date, five different mammalian SFRP genes have been identified (24,25). The CRD has been described to be sufficient and necessary for binding of Wingless (Wg), a member of the Wnt family of proteins, and thus it appears to constitute a ligand-binding domain (22). Wnt signaling has been shown to be involved in the control of gene expression, cell adhesion, planar polarity, proliferation and apoptosis (26). Recently, increased expression of SFRP1, 2, 3 and 5 mRNA has been found in the retinas of patients with inherited retinal degenerations such as retinitis pigmentosa (RP) (27,28). Since SFRPs have been demonstrated to interact with the Wnt/Frizzled signaling pathway (29,30), it is proposed that altered patterns of Wnt signal transduction may be involved in the photoreceptor degeneration process in RP patients (27). SFRP5 is expressed predominantly in RPE (25), the cell type in which Mfrp is also expressed. One of the important functions of the RPE is to support photoreceptor cell integrity through maintenance of retinal adhesion. Thus it is possible that an altered pattern of Wnt signal transduction within the RPE caused by a mutation in Mfrp is involved in the photoreceptor degeneration pathway through the interaction between RPE and photoreceptor cells.
Relevance to human disease
Animal models are widely used for investigating human retinal diseases. Mutations within the genes encoding peripherin, rod cGMP phosphodiesterase ß-subunit, myosin VIIa and Mertk were first identified as causative for retinal degeneration in the mouse and rat models retinal degeneration slow, retinal degeneration 1, shaker 1 and the RCS rat, respectively (31–34). Once the mutant genes in rodent models were identified, the similarity of phenotypes and knowledge of homologous chromosomal locations between mouse, rat and humans led to the identification of mutations in orthologous genes in humans (35–39). The availability of the mouse model also allows for the intensive investigation of the role of the gene in normal and pathophysiologic states. Human MFRP has been mapped to chromosome 11q23.3 (8). To our knowledge, no retinal degeneration locus has yet been linked to this region, but the extensive genetic heterogeneity of the condition in humans and the mutation we have characterized suggests the possibility that mutations in MFRP may be responsible for human photoreceptor degeneration, especially flecked retinal diseases.
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MATERIALS AND METHODS |
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Animals and clinical retinal evaluation
The mice used in this study were bred and maintained in standardized conditions in the Research Animal Facility at The Jackson Laboratory. The rd6 stock used in this study has been maintained as a B6 congenic as previously described (7). They were maintained in a 12 h light–dark cycle facility that was monitored regularly to maintain a specific pathogen-free environment. All experiments were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Pupils were dilated with 1% atropine ophthalmic drops and evaluated by indirect ophthalmoscopy with a 60 diopter lens. Signs of retinal degeneration, such as vessel attenuation, alterations in the RPE, and presence or absence of retinal spots, were noted.
cDNA cloning and mutation analysis
We performed RT–PCR analysis to obtain full-length Mfrp cDNAs. Wild-type mRNAs were obtained from whole-eye tissues of B6 mice with the TRIzol reagent (Gibco BRL, Grand Island, NY, USA), and template cDNA was generated with oligo(dT) primers. We designed exon-spanning primer pairs and used them to amplify the coding region. RT–PCR was performed in an MJ Research PTC-200 using the following program: initial denaturation at 94°C for 2 min; then 35 cycles of 94°C for 30 s, 56°C for 30 s, and 68°C for 2 min. The resulting product was electrophoresed on a 1% agarose gel and isolated using a NucleoSpin Extraction Kit (Clontech, Palo Alto, CA, USA). The recovered DNA was subcloned into the pCR-XL-TOPO vector (Invitrogen, Carlsbad, CA, USA) by using the TOPO XL PCR Cloning Kit (Invitrogen). Plasmid DNA from three positive clones was purified using the Qiagen Plasmid Purification Kit (Qiagen, Valencia, CA, USA). The purified DNA was sequenced using Big Dye Terminator Cycle Sequencing Chemistry on a PE Applied Biosystems 377 with M13 universal and reverse primers. Multiple sequence alignments were carried out with the ClustalW program (40).
Northern blot analysis
For northern blot analysis, total RNA was isolated from organs of B6 mice using TRIzol Reagent (Gibco BRL). Poly(A)+ RNA was purified from total RNA using BioMag Oligo (d)T (Polysciences Inc., Warrington, PA, USA). One microgram of poly(A)+ RNA was fractionated on 1% agarose–formaldehyde gels and transferred to Hybond N+ nylon membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). Hybridization was carried out overnight with 32P-random-labeled probes derived from a cDNA fragment of Mfrp (nt 909–1564) or C1qtnf5 (nt 3097–3848) at 65°C in Rapid-Hyb Buffer (Amersham Pharmacia Biotech). Blots were washed at 65°C in 2xSodium chloride/sodium citrate (SSC)/0.1% sodium dodecyl sulphate (SDS) for 15 min, followed by 0.5x SSC/0.1% SDS for 15 min and 0.1x SSC/0.1% SDS for 15 min.
In situ hybridization
For in situ hybridization, eyes from 3-week-old B6 mice were enucleated and postfixed in 4% paraformaldehyde in phosphate buffered saline (PBS) overnight, dehydrated, and embedded in paraffin. A minimum of three eyes from three different animals were used for this experiment. Sections of 6 µm thickness were cut and mounted on slides pretreated with Vectabond (Vector Laboratories, Burlingame, CA, USA). Sections were deparaffinized in xylene and rehydrated through a graded series of alcohol and PBS. Sections were then treated with proteinase K (20 µg/ml) for 7 min at room temperature (RT), acetylated in 0.1 M triethanolamine (pH 8.0)–0.25% acetic anhydride solution, dehydrated, and air dried. [
-33P]UTP-labeled sense and antisense riboprobes (105 cpm/µl) were generated from plasmids containing cDNA fragments from Mfrp (nt 909–1564). Hybridization solution [50% formamide, 0.3 M NaCl, 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 10 mM NaPO4 (pH 8.0), 10% dextran sulfate, 1x Denhardt's solution, and 0.5 mg/ml yeast tRNA] containing 50 000 cpm/µl labeled probe was applied to each slide. Sections were coverslipped and incubated overnight at 65°C in a humidified chamber. The slides were washed in a solution of 50% formamide–2x SSC at 65°C for 30 min, rinsed twice in NTE [0.5 m NaCl, 10 mM Tris-HCl (pH 7.5), 5 mM EDTA] at 37°C for 15 min, treated with RNase A (1 µg/ml) at 37°C for 15 min, and rinsed in NTE for another 15 min. The slides were then washed in a solution of 50% formamide–2x SSC at 65°C for 20 min, in 2x SSC at RT for 15 min, and in 0.1x SSC at RT for 15 min and dehydrated. The slides were coated with NTB-2 emulsion (Eastman Kodak) and, after exposure for 7 days at 4°C, they were developed (D19; Eastman Kodak) and counterstained with hematoxylin.
Immunohistochemistry
Eyes from 2-week-old rd6 and B6 mice were enucleated and postfixed in 1 : 3 acetic acid–methanol overnight and embedded in paraffin. After blocking with 2% normal horse serum in PBS, sections of 6 µm thickness were incubated overnight with anti-Wnt-1 (G-19, A-20), -Wnt2 (H-20), -Wnt3 (C-15, N-15), -Wnt-4 (R-19, M-70) and -Wnt-10b (N-19, C-19) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), at a dilution of 1 : 200. Binding was detected using biotin conjugated anti-goat antibody (1 : 200, Vector Laboratories) and fluorescein isothiocyanate (FITC)–Avidin D (1 : 200 Vector Laboratories). Sections were also stained with 4',6-Diamidino-2-phenylindole (DAPI) (5 µg/ml). Images from sections were collected on a Leica DMRXE fluorescent microscope equipped with a SPOT CCD camera using an appropriate bandpass filter for the fluorochrome. For the peptide neutralization assay, we combined antibodies with a 50-fold excess of blocking peptide in 500 µl of PBS and incubated overnight at 4°C prior to use on eye sections.
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ACKNOWLEDGEMENTS |
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We thank Cindy Avery and Wanda Hicks for expert animal care, and the JAX Microchemistry Service for help in assembling the BAC clones and in DNA sequencing. These data were generated through the use of GenBank, Celera Discovery System and Celera Genomics' associated databases. This study was supported by grants from the National Eye Institute (EY11996 and EY12093) and an Institutional core grant (CA34196).
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 2072886383; Fax: +1 2072886077; Email: pmn{at}aretha.jax.org
AF469650 (cDNA sequence of Mfrp and C1qtnf5).
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