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The mouse X-linked juvenile retinoschisis cDNA: expression in photoreceptors
Gene 227 (1999) 257–266
The mouse X-linked juvenile retinoschisis cDNA: expression in photoreceptors Silvia N.M. Reid a,*, Novrouz B. Akhmedov a, Natik I. Piriev a, Christine A. Kozak b, Michael Danciger a,c, Debora B. Farber a,d a Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90095, USA b NIAID, Bethesda, MD 20892, USA c Loyola Marymount University, Los Angeles, CA 90045, USA d Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA Received 29 June 1998; received in revised form 17 September 1998; accepted 8 October 1998; Received by C.M. Kane
Abstract Retinal photoreceptor cells are particularly vulnerable to degenerations that can eventually lead to blindness. Our purpose is to identify and characterize genes expressed specifically in photoreceptors in order to increase our understanding of the biochemistry and function of these cells, and then to use these genes as candidates for the sites of mutations responsible for degenerative retinal diseases. We have characterized a cDNA, a fragment of which (SR3.1) was originally isolated by subtractive hybridization of adult, photoreceptorless rd mouse retinal cDNAs from the cDNAs of normal mouse retina. The full-length sequence of this cDNA was determined from clones obtained by screening mouse retinal and eye cDNA libraries and by using the 5∞- and 3∞-RACE methods. Both Northern blot analysis and in situ hybridization showed that the corresponding mRNA is expressed in rod and cone photoreceptors. The gene encoding this cDNA was mapped to the X chromosome using an interspecific cross. Based on the nucleotide and amino acid sequences, as well as chromosome mapping, we determined that this gene is the mouse ortholog (Xlrs1) of the human X-linked juvenile retinoschisis gene (XLRS1). Analysis of the predicted amino acid sequence indicates that the Xlrs1 mRNA may encode a secretable, adhesion protein. Therefore, our data suggest that X-linked juvenile retinoschisis originates from abnormalities in a photoreceptor-derived adhesion protein. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Retinal degeneration; Gene expression; Genetic disease; Intercellular adhesion molecules; Mu¨ller cell
1. Introduction Studies of photoreceptor-specific genes have been fruitful in identifying genetic causes of some retinal * Corresponding author. Tel.: (310) 206 6800; Fax: (310) 794 2144; E-mail: [email protected] Abbreviations: bp, base pair(s); cDNA, DNA complementary to RNA; cGMP, cyclic guanosine-5∞-monophosphate; cRNA, RNA complementary to cDNA; dATP, deoxyadenosine-5∞-triphosphate; dCTP, deoxycytidine-5∞-triphosphate; DIG, digoxigenin; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; FA58C, coagulation factor 5/8 type C domain; kb, kilobase(s) or 1000 bp; kDa, kilodalton(s); mRNA, messenger RNA; PBS, phosphate buffered saline; PDE, phosphodiesterase; RACE, rapid amplification of cDNA ends; rd, retinal degeneration; RDS, retinal degeneration slow; RNA, ribonucleic acid; rRNA, ribosomal RNA; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/0.015 M sodium citrate (pH 7.6); XLRS, X-linked juvenile retinoschisis; XLRS1, human gene for X-linked juvenile retinoschisis; Xlrs1, mouse ortholog gene for human X-linked juvenile retinoschisis; UTP, uridine-5∞-triphosphate.
diseases, and have provided a means for increasing our understanding of the mechanisms by which photoreceptors degenerate. For example, mutations have been found in the photoreceptor-specific genes encoding rhodopsin, the a-subunit of transducin, the a-subunit of the rod cGMP-gated cation channel, the a- and b-subunits of cGMP-phosphodiesterase (a- and b-PDE), guanylate cyclase, peripherin/RDS, a photoreceptor membrane protein (ROM 1), RPGR (retinitis pigmentosa GTPase regulator), the ABC (ATP-binding cassette) transporter or photoreceptor RIM protein and others (for a recent review see Farber and Danciger, 1997). To date, however, there are still many inherited retinal degenerations which cannot be accounted for by mutations in the genes that have so far been identified. Several years ago, a strategy was developed in our laboratory to clone genes specifically expressed in retinal photoreceptors. We took advantage of the rd mouse which has an autosomal recessive disease leading to a
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rapid degeneration of the photoreceptor cell layer. By subtractive hybridization of adult rd mouse retinal cDNAs from normal mouse retinal cDNAs, we obtained a pool of photoreceptor-specific cDNAs. From these cDNAs, we identified the gene encoding b-PDE and determined that it was the site of the mutation responsible for disease in the rd mouse (Bowes et al., 1989). The specific mutation was identified later (Pittler and Baehr, 1991). Many of these subtracted cDNAs have been characterized and correspond to known genes that are selectively expressed in rods or cones; a few of them are novel. Mutations in any of these novel genes may potentially be the cause of retinal disease. X-linked juvenile retinoschisis ( XLRS) is a hereditary retinal disorder that causes a type of macular dystrophy. It results from mutations in a single gene mapping to Xp22 (George et al., 1995; Pawar et al., 1996; Sauer et al., 1997). Affected individuals have a relatively normal a-wave in the electroretinogram; while the b-wave (which reflects the activity of Mu¨ller cells; Miller and Dowing, 1970) is nearly or totally absent (Peachey et al., 1987). Another hallmark of XLRS is the starshaped schisis lesion which results from the separation of the inner limiting membrane from the rest of the retina. The cavity and the remaining retina contain an amorphous material filled with filaments which blend in with the basement membrane of the Mu¨ller cells. These observations suggested that the Mu¨ller cells are defective in XLRS, and that they release the observed filaments into the extracellular space. The accumulation of these filaments eventually leads to the detachment of the inner limiting membrane (Condon et al., 1986). If the Mu¨ller cells are the site of the primary lesion in XLRS, one would expect the gene responsible for this disease to be primarily expressed in Mu¨ller cells (George et al., 1995). Recently, the human XLRS gene (XLRS1) has been identified by positional cloning (Sauer et al., 1997). Here, we report that a cDNA obtained by subtractive hybridization of rd retinal cDNAs from normal retinal cDNAs (Bowes et al., 1989) corresponds to the mouse ortholog of the XLRS1 cDNA. Surprisingly, both our Northern blot analysis and in situ hybridization data indicate that the gene is primarily expressed in photoreceptors.
2. Materials and methods
additional clones were obtained. Approximately 2/3 of the full length cDNA sequence was constructed from these clones. 5∞-RACE and 3∞-RACE (rapid amplification of cDNA ends; Gibco-BRL, Gaithersburg, MD, USA) were used to complete the full length sequence. Nucleotide sequencing was performed either using Sequenase ( US Biochemical, Cleveland, OH, USA) and [a-35S ]dATP (1000 Ci/mmol, Amersham, Arlington Heights, IL, USA) or the Taq Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). 2.2. RNA isolation, Northern blots and probes Retinas from C57BL/6J normal and rd/rd mice were used for RNA isolation. Retinas were dissected from newborn, P2 (postnatal day 2), P5, P7, P13, P21 and >P60 normal mouse eyes, and from P28, P43 and P71 rd/rd mouse eyes. Cerebral cortex, cerebellum, lung, heart, kidney, spleen, and liver were obtained from normal adult mice. Total RNA was isolated using the RNAzol B method (Tel-test, Friendswood, TX, USA), subjected to electrophoresis in 1–2% agarose gels and blotted to nylon membranes. The RNA blots were prehybridized and hybridized in 0.5 M phosphate buffer at pH 7.2, 1 mM EDTA, 7% SDS and 1% bovine serum albumin at 65°C. After hybridization, the blots were washed at a final stringency of 0.2×SSC, 0.2% SDS at 60°C and exposed to X-ray film (Hyperfilm-MP, Amersham). Probes were prepared for hybridization either by harvesting the fragments from restricted plasmid preparations or by PCR amplification, and were labeled with the multiprimer DNA labeling system (Amersham) in the presence of [a-32P]dCTP (3000Ci/mmol, Amersham). The corresponding nucleotide sequences for probes 5R, C11-short, P4, P13-1.3, and SR3.1 were −36 to 1945 bp, 1791 to 2343 bp, 3097 to 3707 bp, 3605 to 4994 bp, and 4350 to 5665 bp, respectively (Fig. 1 and Fig. 2). 2.3. Chromosome mapping The gene corresponding to the SR3.1 cDNA fragment was mapped by analysis of the progeny of the interspecific crosses (NFS/N×M. spretus)F1×C58/J or M. spretus (Adamson et al., 1991). Recombination distances were determined according to Green (1989), and loci were ordered by minimizing the number of recombinants.
2.1. Sequence analysis 2.4. In situ hybridization SR3.1, the 1.3 kb cDNA originally isolated by subtractive hybridization, was used to screen a phage mouse retinal cDNA library resulting in the identification of 12 different clones. Using two probes derived from these clones, P4 and P13-1.3 ( Fig. 1), mouse retina and mouse eye cDNA libraries were screened and four
Plasmid pcDNA I (Invitrogen, Carlsbad, CA, USA) containing C11-long (corresponding to bp 1698–4488; Fig. 1) was digested with either BamHI (sense) or NotI (antisense) to produce 5∞-protruding ends, and checked on 1.5% agarose gel to examine the completeness of
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Fig. 1. The various probes used in this study were designed to correspond to different regions of the mouse Xlrs1 mRNA. The numbers indicate the nucleotide position in the Xlrs1 mRNA sequence. A, poly(A) tail; C, poly(C ) tail; T, poly( T ) tail. 3∞ and 5∞ depict the 3∞- and 5∞-ends of the mRNA.
digestion. C11-long sense and C11-long antisense digoxigenin (DIG)-labeled cRNA probes were then synthesized with SP6 (sense) and T7 (antisense) RNA polymerases (Promega, Madison, WI, USA) in the presence of DIG-labeled-UTP (Boehringer Mannheim, Indianapolis, IN, USA). The probes were then treated with deoxyribonuclease (Boehringer Mannheim), and purified by LiCl precipitation. Two normal adult mice (C57BL/6J ) were anesthetized with Avertin (50 mg/kg body weight, i.p.) and perfused intracardially with 4% paraformaldehyde in ice-cold 0.066 M phosphate buffered saline (PBS ) at pH 7.4. After enucleation and removal of the lenses, the eye cups were postfixed overnight at 4°C. Frozen sections of 10 mm were cut on a cryostat, allowed to adhere to Superfrost Plus glass slides (Fisher Scientific, St Louis, MO, USA), air-dried overnight, and stored at −80°C until used. Sections were bleached in 0.5% H O , per2 2 meabilized with proteinase K (1 mg/ml ), treated in prehybridization solution (50% formamide, 5×SSC at pH 4.5, 1% SDS, 50 mg/ml yeast RNA, 50 mg/ml heparin) for 30 min at 70°C, and hybridized with DIGlabeled cRNA probe (1 mg/ml ) overnight at 70°C. Sections were then washed three times with 5×SSC and 1% SDS in 50% formamide at 70°C, three times with 2×SSC in 50% formamide at 65°C, and three times with Tris buffered saline–1% Tween-20 at room temperature (15 min each wash). The sections were incubated with alkaline phosphatase-conjugated sheep anti-DIG antibody (1:2000; Boehringer Mannheim) in 25 mM Tris–HCl at pH 7.4, 140 mM NaCl, 2.7 mM KCl, 1% Tween-20, 1% sheep serum. The alkaline phosphatase reaction was performed in the presence of nitro blue tetrazolium in order to visualize the SR3 mRNA. The reaction was stopped with acidic PBS containing 1% Tween-20.
3. Results 3.1. Sequence analysis and homology with the corresponding human cDNA The full length cDNA (GenBank accession number: AF073780) corresponding to the mouse ortholog of the human XLRS1 cDNA was obtained by screening cDNA libraries with probes SR3.1, P4 and P13-1.3, and employing 5∞-RACE (Fig. 1). The full length clone had a short 5∞-untranslated region (36 bp). Sequence analysis indicated that the nucleotides flanking the starting codon ATG (gagaaaATGc) are in accordance with the Kozak consensus sequence for initiating translation ( Kozak, 1996), and that the TGA termination codon is at bp 673–675 ( Fig. 2). The position of the poly(A) tail of the full length cDNA was confirmed with 3∞-RACE. cDNAs with two different poly(A) tails were identified, one preceded by a consensus polyadenylation signal (AATAAA) at bp 4879–4884, and the other by a polyadenylation-like signal (AATTAA) at bp 5646 (Fig. 2). Nucleotide sequence comparison using Genbank (BLAST search: http://www.ncbi.nlm.nih.gov/cgibin/BLAST ) shows high homology between the coding regions of the mouse and human X-linked juvenile retinoschisis mRNAs ( Fig. 2). The two mouse transcripts have very long 3∞-untranslated regions; these are approximately 4 and 5 kb, respectively vs 300 bp and 2 kb for the human mRNAs (data not shown). Most of the sequence in the 3∞-untranslated regions differs in human and mouse with the exception of the first 17 nucleotides and three segments of 45–80 bp each (932–990 bp; 1009–1085 bp; 2687–2734 bp) which share homology (Fig. 2). Translation of the 672-bp open reading frame revealed a 95% amino acid sequence homology with the human
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Fig. 2. Sequence of the full length cDNA for mouse Xlrs1 mRNA and regions of homology with the human X-linked retinoschisis transcript. The 672 bp of coding regions in both mouse and human transcripts are depicted in upper-case and the sequence homology is indicated with vertical bars. The nucleotides from both 5∞- and 3∞-untranslated regions are in lower-case. The 3∞-untranslated region is 4990 bp for the large mouse transcript and 4236 bp for the smaller one. The 3∞-end of the small transcript is marked by *. Polyadenylation and polyadenylation-like signals are boxed. The translation initiation codon (ATG) and the stop codon ( TGA) are underlined. The numbers on the left and in the middle of the human sequence correspond to the nucleotide positions.
protein encoded by the XLRS1 gene (Fig. 3; Sauer et al., 1997). 3.2. Amino acid motif analysis Analysis of the predicted amino acid sequence of the mouse Xlrs1-encoded protein ( Fig. 3) revealed that the
first 23 amino acids contain a transmembrane domain which may serve as a secretory leader sequence. This putative leader sequence is followed immediately by a peptidase cleavage site GXS at 21–23 that, when used, would result in a mature protein of 201 amino acid residues with a calculated molecular weight of 23 kDa. A search in the Prosite database (http://expasy.
Fig. 3. Predicted amino acid sequences deduced from the mouse Xlrs1 and human XLRS1 cDNAs, and sequences from some proteins from the FA58C/discoidin family. Conserved amino acids between the Xlrs1- and XLRS1-encoded proteins are in bold. These two proteins contain a FA58C1 signature (boxed) and an incomplete FA58C2 signature ([ ]). They also have a secretory leader sequence (underlined), followed immediately by a putative protease cleavage site (double underlined). Conserved amino acids with other proteins of the FA58C/discoidin family are boxed and shaded. To maximize alignment, lower cases indicate skipping amino acids and gaps are expressed by "…". The Swiss-Prot accession number is given at the far left. The corresponding amino acid positions are in parentheses. dicdi, Dictyostelium discoideum; disc, discoidin I; fa5, coagulation factor V; mfgm, milk fat globule-EGF factor 8; nrp, neuropilin; trk3, tyrosine-protein kinase receptor related 3; xenla, Xenopus laevis.
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haye.ch/sprot/prosite.html ) indicated that the predicted amino acid sequence of the Xlrs1-encoded protein contains a coagulation factor 5/8 type C domain signature 1 ( FA58C 1) at position 111–140 (Fig. 3; Kane and Davie, 1986; Johnson et al., 1993; Couto et al., 1996). Two slime mold cell adhesion protein discoidins also share a high homology with the C-terminus of the Xlrs1 protein (Fig. 3; Poole et al., 1981; Fukuzawa and Ochiai, 1996). Discoidin domains have a common C-terminal region of approx. 110 amino acids with FA58C domains (Fukuzawa and Ochiai, 1996). Discoidins and the coagulation factors V and VIII, together with eight other groups of proteins, form the FA58C/discoidin protein family ( Table 1). Mouse Xlrs1- and human XLRS1encoded proteins are new members of this family. 3.3. Gene localization on the X chromosome The mouse Xlrs1 gene was mapped to the distal region of the mouse X chromosome between Htr2c and Grpr using P4 as a probe and the progeny of the interspecific cross (NFS/N×M. spretus)F1× C58/J or M. spretus (Fig. 4). The P4 probe identified ApaI fragments of 19.0 kb in M. spretus and 23.0 kb in the inbred strains NFS/N and C58/J. This localization is consistent with the localization of the human XLRS1 to Xp22. 3.4. Tissue specificity and developmental changes In Northern blots the SR3.1 probe hybridized RNA from retina, but not from cerebral cortex, lung, heart, kidney, spleen, liver or cerebellum (Fig. 5A). Transcripts of 5 and 5.7 kb were detected by probe SR3.1 and also by probes P13-1.3, P4, C11-short and 5R (data not
Fig. 4. Genetic map location of mouse Xlrs1. The recombination fractions between adjacent loci and recombinational distances ± S.E. are indicated on the right of the map. The map locations of human homologs are on the left of the map (human loci taken from GDB). Marker loci (in italics on the right of the map) were typed as described previously ( Filie et al., 1998). The numbers in square brackets are the distances in centimorgans from the centromere according to the mouse X chromosome committee (Boyd et al., 1997).
shown). These two retinal transcripts could be accounted for by the two polyadenylation sites described earlier. In Northern blots probed with either SR3.1 or C11-short, the retinal Xlrs1 mRNA was first detected on postnatal day 7 and reached its adult level at postnatal day 21 ( Fig. 5B). However, the Xlrs1 transcripts may well be expressed earlier, since mRNA signals were detected on postnatal day 5 when X-ray film was overexposed (data not shown). Probe SR3.1 hybridized more strongly to the higher molecular weight band; while probe C11-short hybridized equally with both transcripts.
Table 1 The FA58C/discoidin protein family Secretable extracellular proteins Blood coagulation factors V and VIII Mammalian milk fat globule-EGF factor 8 (MFG-E8 or BA46 antigen) Silk moth humoral lectin hemocytin Bovine SCO-spondin Discoidins
Kane and Davie, 1986 Couto et al., 1996 Kotani et al., 1995 Gobron et al., 1996 Poole et al., 1981 Fukuzawa and Ochiai, 1996
Transmembrane proteins Neuropilin (A5 antigen) Drosophila neurexin IV Mammalian contactin associated proteins (CASPR) Epithelial discoidin domain receptor 1 ( EDDR1, CAK, DDR1, TRKE or RTK6) Neurotrophic tyrosine kinase receptor related 3 (NTRK3, TrkC, TKT or TYRO10)
Takagi et al., 1991 Baumgartner et al., 1996 Peles et al., 1997 Johnson et al., 1993 Karn et al., 1993
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Fig. 5. The Xlrs1 mRNA is retina-specific and its level increases through the juvenile stage. (A) Northern blot containing RNAs from different mouse tissues (retina, cerebral cortex, lung, heart, kidney, spleen, liver and cerebellum), probed with SR3.1. Two Xlrs1 mRNA transcripts of 5 and 5.7 kb were detected. 20 mg of total RNA from each tissue, except for retina (10 mg) were loaded in the gel. The X-ray film was exposed to the blot for 3 h at -80°C with intensifying screens. (B) Retinal Xlrs1 mRNA detected by probes SR3.1 and C11-short (C11) at different times during postnatal development. The loading was 8 mg of total RNA per lane for the blot probed with SR3.1 and 5 mg for the blot probed with C11-short. The lower panels in both (A) and (B) show the ethidium bromide-stained agarose gels before the RNAs had been transferred to the membranes. The two bands correspond to the 28S (upper band) and 18S ( lower band) rRNAs. Numbers are ages in postnatal days. Ad, Adult.
3.5. RNA expression in photoreceptors To verify that the Xlrs1 mRNA is expressed in photoreceptors, we took advantage of the rd mouse and its different times of degeneration of rods and cones. At P28, while retaining many cones (cones are approximately 5% of all photoreceptors in the mouse eye), the rd retina does not have any rods left; by adulthood
(>P60) it is devoid of both types of visual cells (CarterDawson et al., 1978). In Northern blots containing RNAs from 71-day-old retina of normal and rd mice, the Xlrs1 mRNA was detected only in the RNAs from normal retinas, suggesting that Xlrs1 mRNA is expressed in photoreceptors ( Fig. 6A). A reduced level of the two Xlrs1 transcripts was observed in the 28-day-old rd sample when compared with RNAs from normal retinas.
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Fig. 6. Xlrs1 mRNA expression in photoreceptors. (A) Northern blots containing total RNAs from normal and rd mouse retinas at different developmental ages were hybridized with either one of the two 32P-labeled probes, SR3.1 and C11-short (C11). The lower panels show the ethidium bromide-stained agarose gels with the 28S (upper) and 18S ( lower) rRNAs before RNAs were transferred to the membrane. Total retinal RNAs of normal 71- (20 mg) and 21-day-old mice (5 mg), and those of rd 71- (20 mg), 43- (10 mg) and 28-day-old mice (5 mg) were loaded on a gel. The 43-day-old rd mice signals were detected after overnight exposure of X-ray film at -80°C with intensifying screens; while 3 h of film exposure was sufficient to observe the bands from the other preparations. (B) Localization of Xlrs1 mRNA by in situ hybridization of normal adult retina with probe C11-long. (1) Retina hybridized with the C11-long sense probe. Magnification: 422×. (2) Retina hybridized with the C11-long antisense probe. Xlrs1 mRNA is visualized as a purple reaction product. Magnification: 422×. (3) Higher magnification shows the reaction product in individual photoreceptors (arrows). Magnification: 1155×. The concentration of this product in the inner segment is high and thus it appears black.
The Xlrs1 mRNA was still detectable at much lower levels at P43, a time when nearly 2/3 of the cone photoreceptors have degenerated (Carter-Dawson et al., 1978). These results suggest that the Xlrs1 transcripts are expressed in both rod and cone photoreceptors. In situ hybridization with the C11-long antisense probe further substantiated the localization of Xlrs1 mRNA; signal was most prominent in the inner segments of the photoreceptors (Fig. 6B2 and 3) but was also found in their cell bodies (outer nuclear layer of the retina) (Fig. 6B2). The reaction product was detected in individual photoreceptors (Fig. 6B3, arrows). No
signal was detected in the inner nuclear layer of the retina, where the cell bodies of Mu¨ller cells are located.
4. Discussion The results reported here indicate that the Xlrs1 cDNA, a fragment of which was originally isolated by subtractive hybridization (SR3.1), corresponds to the mouse ortholog of human XLRS1 cDNA. The expression of the Xlrs1 mRNA is developmentally regulated. It is first transcribed in the retina within a week of birth
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and increases in level through the juvenile stage. Both in situ hybridization and Northern blot analysis showed that Xlrs1 mRNA is expressed in both rods and cones. By analogy the human XLRS1 gene may also be expressed in photoreceptors, yet, up to now, X-linked juvenile retinoschisis has been thought to be caused by an abnormality in Mu¨ller cells. It is difficult to understand how a photoreceptor gene defect could lead to a Mu¨ller cell disorder. A hypothesis that we have formulated is that the presence of various mutations in the secretable XLRS1 protein prevents its interaction with Mu¨ller cells. The Xlrs1-encoded protein is a member of the FA58C/discoidin family, which is made of either secreted proteins involved in adhesion or transmembrane proteins ( Table 1). The Xlrs1 protein belongs to the former type. Many members in the FA58C/discoidin protein family have a binding protein in the extracellular matrix or on the cell surface of other cells ( Elodi and Elodi, 1983; Barondes et al., 1987; Kolodkin et al., 1997; Peles et al., 1997). In both secreted and transmembrane proteins the FA58C domains are located extracellularly, and are probably involved in binding. For example, the FA58C domain of coagulation factors has been shown to bind phospholipids (Foster et al., 1990). If the Xlrs1 mRNA encodes a cell adhesion protein, a defect in it could result in an aberrant interaction between photoreceptors and Mu¨ller cells, either at the level of the synapses or in the area of the outer limiting membrane. The testing of this hypothesis will increase our understading of the function of retinal cells and the nature of X-linked juvenile retinoschisis.
Acknowledgements The authors would like to thank Dr Xianjie Yang for sharing her mouse eye cDNA library. The authors also appreciate the assistance of Ms Jennifer Shih, Ms Tammy Rickabaugh, Mr Clyde Yamashita and Dr Danyun Zhao. This work was supported by grants from NIH ( EY08285) and The Foundation Fighting Blindness.
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(glial ) cells of mudpuppy retina: their relation to b-wave of the electroretinogram. J. Neurophysiol. 33, 323–341. Pawar, H., Bingham, E.L., Hiriyanna, K., Segal, M., Richards, J.E., Sieving, P.A., 1996. X-linked juvenile retinoschisis: localization between (DXS1195, DXS418) and AFM291wf5 on a single YAC. Hum. Here. 46, 329–335. Peachey, N.S., Fishman, G.A., Derlacki, D.J., Brigell, M.G., 1987. Psychophysical and electroretinographic findings in X-linked juvenile retinoschisis. Arch. Ophthalmol. 105, 513–516. Peles, E., Nativ, M., Lustig, M., Grumet, M., Schilling, J., Martinez, R., Plowman, G.D., Schlessinger, J., 1997. Identification of a novel contactin-associated transmembrane receptor with multiple domains implicated in protein–protein interactions. EMBO J. 16, 978–988. Pittler, S.J., Baehr, W., 1991. Identification of a nonsense mutation in
the rod photoreceptor cGMP phosphodiesterase beta subunit gene of the rd mouse. Proc. Natl. Acad. Sci. USA 88, 8322–8326. Poole, S., Firtel, R.A., Lamar, E., Rowekamp, W., 1981. Sequence and expression of the discoidin I gene family in Dictyostelium discoideum. J. Mol. Biol. 153, 273–289. Sauer, C.G., Gehrig, A., Warneke-Wittstock, R., Marquardt, A., Ewing, C.C., Gibson, A., Lorenz, B., Jurklies, B., Weber, B.H.F., 1997. Positional cloning of the gene associated with X-linked juvenile retinoschisis. Nature Genet. 17, 164–170. Takagi, S., Hirata, T., Agata, K., Mochii, M., Eguchi, G., Fujisawa, H., 1991. The A5 antigen, a candidate for the neuronal recognition molecule, has homologies to complement components and coagulation factors. Neuron 7, 295–307.
The mouse X-linked juvenile retinoschisis cDNA: expression in photoreceptors Silvia N.M. Reid a,*, Novrouz B. Akhmedov a, Natik I. Piriev a, Christine A. Kozak b, Michael Danciger a,c, Debora B. Farber a,d a Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90095, USA b NIAID, Bethesda, MD 20892, USA c Loyola Marymount University, Los Angeles, CA 90045, USA d Molecular Biology Institute, UCLA, Los Angeles, CA 90095, USA Received 29 June 1998; received in revised form 17 September 1998; accepted 8 October 1998; Received by C.M. Kane
Abstract Retinal photoreceptor cells are particularly vulnerable to degenerations that can eventually lead to blindness. Our purpose is to identify and characterize genes expressed specifically in photoreceptors in order to increase our understanding of the biochemistry and function of these cells, and then to use these genes as candidates for the sites of mutations responsible for degenerative retinal diseases. We have characterized a cDNA, a fragment of which (SR3.1) was originally isolated by subtractive hybridization of adult, photoreceptorless rd mouse retinal cDNAs from the cDNAs of normal mouse retina. The full-length sequence of this cDNA was determined from clones obtained by screening mouse retinal and eye cDNA libraries and by using the 5∞- and 3∞-RACE methods. Both Northern blot analysis and in situ hybridization showed that the corresponding mRNA is expressed in rod and cone photoreceptors. The gene encoding this cDNA was mapped to the X chromosome using an interspecific cross. Based on the nucleotide and amino acid sequences, as well as chromosome mapping, we determined that this gene is the mouse ortholog (Xlrs1) of the human X-linked juvenile retinoschisis gene (XLRS1). Analysis of the predicted amino acid sequence indicates that the Xlrs1 mRNA may encode a secretable, adhesion protein. Therefore, our data suggest that X-linked juvenile retinoschisis originates from abnormalities in a photoreceptor-derived adhesion protein. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Retinal degeneration; Gene expression; Genetic disease; Intercellular adhesion molecules; Mu¨ller cell
1. Introduction Studies of photoreceptor-specific genes have been fruitful in identifying genetic causes of some retinal * Corresponding author. Tel.: (310) 206 6800; Fax: (310) 794 2144; E-mail: [email protected] Abbreviations: bp, base pair(s); cDNA, DNA complementary to RNA; cGMP, cyclic guanosine-5∞-monophosphate; cRNA, RNA complementary to cDNA; dATP, deoxyadenosine-5∞-triphosphate; dCTP, deoxycytidine-5∞-triphosphate; DIG, digoxigenin; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; FA58C, coagulation factor 5/8 type C domain; kb, kilobase(s) or 1000 bp; kDa, kilodalton(s); mRNA, messenger RNA; PBS, phosphate buffered saline; PDE, phosphodiesterase; RACE, rapid amplification of cDNA ends; rd, retinal degeneration; RDS, retinal degeneration slow; RNA, ribonucleic acid; rRNA, ribosomal RNA; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCl/0.015 M sodium citrate (pH 7.6); XLRS, X-linked juvenile retinoschisis; XLRS1, human gene for X-linked juvenile retinoschisis; Xlrs1, mouse ortholog gene for human X-linked juvenile retinoschisis; UTP, uridine-5∞-triphosphate.
diseases, and have provided a means for increasing our understanding of the mechanisms by which photoreceptors degenerate. For example, mutations have been found in the photoreceptor-specific genes encoding rhodopsin, the a-subunit of transducin, the a-subunit of the rod cGMP-gated cation channel, the a- and b-subunits of cGMP-phosphodiesterase (a- and b-PDE), guanylate cyclase, peripherin/RDS, a photoreceptor membrane protein (ROM 1), RPGR (retinitis pigmentosa GTPase regulator), the ABC (ATP-binding cassette) transporter or photoreceptor RIM protein and others (for a recent review see Farber and Danciger, 1997). To date, however, there are still many inherited retinal degenerations which cannot be accounted for by mutations in the genes that have so far been identified. Several years ago, a strategy was developed in our laboratory to clone genes specifically expressed in retinal photoreceptors. We took advantage of the rd mouse which has an autosomal recessive disease leading to a
0378-1119/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 57 8 - 2
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rapid degeneration of the photoreceptor cell layer. By subtractive hybridization of adult rd mouse retinal cDNAs from normal mouse retinal cDNAs, we obtained a pool of photoreceptor-specific cDNAs. From these cDNAs, we identified the gene encoding b-PDE and determined that it was the site of the mutation responsible for disease in the rd mouse (Bowes et al., 1989). The specific mutation was identified later (Pittler and Baehr, 1991). Many of these subtracted cDNAs have been characterized and correspond to known genes that are selectively expressed in rods or cones; a few of them are novel. Mutations in any of these novel genes may potentially be the cause of retinal disease. X-linked juvenile retinoschisis ( XLRS) is a hereditary retinal disorder that causes a type of macular dystrophy. It results from mutations in a single gene mapping to Xp22 (George et al., 1995; Pawar et al., 1996; Sauer et al., 1997). Affected individuals have a relatively normal a-wave in the electroretinogram; while the b-wave (which reflects the activity of Mu¨ller cells; Miller and Dowing, 1970) is nearly or totally absent (Peachey et al., 1987). Another hallmark of XLRS is the starshaped schisis lesion which results from the separation of the inner limiting membrane from the rest of the retina. The cavity and the remaining retina contain an amorphous material filled with filaments which blend in with the basement membrane of the Mu¨ller cells. These observations suggested that the Mu¨ller cells are defective in XLRS, and that they release the observed filaments into the extracellular space. The accumulation of these filaments eventually leads to the detachment of the inner limiting membrane (Condon et al., 1986). If the Mu¨ller cells are the site of the primary lesion in XLRS, one would expect the gene responsible for this disease to be primarily expressed in Mu¨ller cells (George et al., 1995). Recently, the human XLRS gene (XLRS1) has been identified by positional cloning (Sauer et al., 1997). Here, we report that a cDNA obtained by subtractive hybridization of rd retinal cDNAs from normal retinal cDNAs (Bowes et al., 1989) corresponds to the mouse ortholog of the XLRS1 cDNA. Surprisingly, both our Northern blot analysis and in situ hybridization data indicate that the gene is primarily expressed in photoreceptors.
2. Materials and methods
additional clones were obtained. Approximately 2/3 of the full length cDNA sequence was constructed from these clones. 5∞-RACE and 3∞-RACE (rapid amplification of cDNA ends; Gibco-BRL, Gaithersburg, MD, USA) were used to complete the full length sequence. Nucleotide sequencing was performed either using Sequenase ( US Biochemical, Cleveland, OH, USA) and [a-35S ]dATP (1000 Ci/mmol, Amersham, Arlington Heights, IL, USA) or the Taq Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). 2.2. RNA isolation, Northern blots and probes Retinas from C57BL/6J normal and rd/rd mice were used for RNA isolation. Retinas were dissected from newborn, P2 (postnatal day 2), P5, P7, P13, P21 and >P60 normal mouse eyes, and from P28, P43 and P71 rd/rd mouse eyes. Cerebral cortex, cerebellum, lung, heart, kidney, spleen, and liver were obtained from normal adult mice. Total RNA was isolated using the RNAzol B method (Tel-test, Friendswood, TX, USA), subjected to electrophoresis in 1–2% agarose gels and blotted to nylon membranes. The RNA blots were prehybridized and hybridized in 0.5 M phosphate buffer at pH 7.2, 1 mM EDTA, 7% SDS and 1% bovine serum albumin at 65°C. After hybridization, the blots were washed at a final stringency of 0.2×SSC, 0.2% SDS at 60°C and exposed to X-ray film (Hyperfilm-MP, Amersham). Probes were prepared for hybridization either by harvesting the fragments from restricted plasmid preparations or by PCR amplification, and were labeled with the multiprimer DNA labeling system (Amersham) in the presence of [a-32P]dCTP (3000Ci/mmol, Amersham). The corresponding nucleotide sequences for probes 5R, C11-short, P4, P13-1.3, and SR3.1 were −36 to 1945 bp, 1791 to 2343 bp, 3097 to 3707 bp, 3605 to 4994 bp, and 4350 to 5665 bp, respectively (Fig. 1 and Fig. 2). 2.3. Chromosome mapping The gene corresponding to the SR3.1 cDNA fragment was mapped by analysis of the progeny of the interspecific crosses (NFS/N×M. spretus)F1×C58/J or M. spretus (Adamson et al., 1991). Recombination distances were determined according to Green (1989), and loci were ordered by minimizing the number of recombinants.
2.1. Sequence analysis 2.4. In situ hybridization SR3.1, the 1.3 kb cDNA originally isolated by subtractive hybridization, was used to screen a phage mouse retinal cDNA library resulting in the identification of 12 different clones. Using two probes derived from these clones, P4 and P13-1.3 ( Fig. 1), mouse retina and mouse eye cDNA libraries were screened and four
Plasmid pcDNA I (Invitrogen, Carlsbad, CA, USA) containing C11-long (corresponding to bp 1698–4488; Fig. 1) was digested with either BamHI (sense) or NotI (antisense) to produce 5∞-protruding ends, and checked on 1.5% agarose gel to examine the completeness of
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Fig. 1. The various probes used in this study were designed to correspond to different regions of the mouse Xlrs1 mRNA. The numbers indicate the nucleotide position in the Xlrs1 mRNA sequence. A, poly(A) tail; C, poly(C ) tail; T, poly( T ) tail. 3∞ and 5∞ depict the 3∞- and 5∞-ends of the mRNA.
digestion. C11-long sense and C11-long antisense digoxigenin (DIG)-labeled cRNA probes were then synthesized with SP6 (sense) and T7 (antisense) RNA polymerases (Promega, Madison, WI, USA) in the presence of DIG-labeled-UTP (Boehringer Mannheim, Indianapolis, IN, USA). The probes were then treated with deoxyribonuclease (Boehringer Mannheim), and purified by LiCl precipitation. Two normal adult mice (C57BL/6J ) were anesthetized with Avertin (50 mg/kg body weight, i.p.) and perfused intracardially with 4% paraformaldehyde in ice-cold 0.066 M phosphate buffered saline (PBS ) at pH 7.4. After enucleation and removal of the lenses, the eye cups were postfixed overnight at 4°C. Frozen sections of 10 mm were cut on a cryostat, allowed to adhere to Superfrost Plus glass slides (Fisher Scientific, St Louis, MO, USA), air-dried overnight, and stored at −80°C until used. Sections were bleached in 0.5% H O , per2 2 meabilized with proteinase K (1 mg/ml ), treated in prehybridization solution (50% formamide, 5×SSC at pH 4.5, 1% SDS, 50 mg/ml yeast RNA, 50 mg/ml heparin) for 30 min at 70°C, and hybridized with DIGlabeled cRNA probe (1 mg/ml ) overnight at 70°C. Sections were then washed three times with 5×SSC and 1% SDS in 50% formamide at 70°C, three times with 2×SSC in 50% formamide at 65°C, and three times with Tris buffered saline–1% Tween-20 at room temperature (15 min each wash). The sections were incubated with alkaline phosphatase-conjugated sheep anti-DIG antibody (1:2000; Boehringer Mannheim) in 25 mM Tris–HCl at pH 7.4, 140 mM NaCl, 2.7 mM KCl, 1% Tween-20, 1% sheep serum. The alkaline phosphatase reaction was performed in the presence of nitro blue tetrazolium in order to visualize the SR3 mRNA. The reaction was stopped with acidic PBS containing 1% Tween-20.
3. Results 3.1. Sequence analysis and homology with the corresponding human cDNA The full length cDNA (GenBank accession number: AF073780) corresponding to the mouse ortholog of the human XLRS1 cDNA was obtained by screening cDNA libraries with probes SR3.1, P4 and P13-1.3, and employing 5∞-RACE (Fig. 1). The full length clone had a short 5∞-untranslated region (36 bp). Sequence analysis indicated that the nucleotides flanking the starting codon ATG (gagaaaATGc) are in accordance with the Kozak consensus sequence for initiating translation ( Kozak, 1996), and that the TGA termination codon is at bp 673–675 ( Fig. 2). The position of the poly(A) tail of the full length cDNA was confirmed with 3∞-RACE. cDNAs with two different poly(A) tails were identified, one preceded by a consensus polyadenylation signal (AATAAA) at bp 4879–4884, and the other by a polyadenylation-like signal (AATTAA) at bp 5646 (Fig. 2). Nucleotide sequence comparison using Genbank (BLAST search: http://www.ncbi.nlm.nih.gov/cgibin/BLAST ) shows high homology between the coding regions of the mouse and human X-linked juvenile retinoschisis mRNAs ( Fig. 2). The two mouse transcripts have very long 3∞-untranslated regions; these are approximately 4 and 5 kb, respectively vs 300 bp and 2 kb for the human mRNAs (data not shown). Most of the sequence in the 3∞-untranslated regions differs in human and mouse with the exception of the first 17 nucleotides and three segments of 45–80 bp each (932–990 bp; 1009–1085 bp; 2687–2734 bp) which share homology (Fig. 2). Translation of the 672-bp open reading frame revealed a 95% amino acid sequence homology with the human
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Fig. 2. Sequence of the full length cDNA for mouse Xlrs1 mRNA and regions of homology with the human X-linked retinoschisis transcript. The 672 bp of coding regions in both mouse and human transcripts are depicted in upper-case and the sequence homology is indicated with vertical bars. The nucleotides from both 5∞- and 3∞-untranslated regions are in lower-case. The 3∞-untranslated region is 4990 bp for the large mouse transcript and 4236 bp for the smaller one. The 3∞-end of the small transcript is marked by *. Polyadenylation and polyadenylation-like signals are boxed. The translation initiation codon (ATG) and the stop codon ( TGA) are underlined. The numbers on the left and in the middle of the human sequence correspond to the nucleotide positions.
protein encoded by the XLRS1 gene (Fig. 3; Sauer et al., 1997). 3.2. Amino acid motif analysis Analysis of the predicted amino acid sequence of the mouse Xlrs1-encoded protein ( Fig. 3) revealed that the
first 23 amino acids contain a transmembrane domain which may serve as a secretory leader sequence. This putative leader sequence is followed immediately by a peptidase cleavage site GXS at 21–23 that, when used, would result in a mature protein of 201 amino acid residues with a calculated molecular weight of 23 kDa. A search in the Prosite database (http://expasy.
Fig. 3. Predicted amino acid sequences deduced from the mouse Xlrs1 and human XLRS1 cDNAs, and sequences from some proteins from the FA58C/discoidin family. Conserved amino acids between the Xlrs1- and XLRS1-encoded proteins are in bold. These two proteins contain a FA58C1 signature (boxed) and an incomplete FA58C2 signature ([ ]). They also have a secretory leader sequence (underlined), followed immediately by a putative protease cleavage site (double underlined). Conserved amino acids with other proteins of the FA58C/discoidin family are boxed and shaded. To maximize alignment, lower cases indicate skipping amino acids and gaps are expressed by "…". The Swiss-Prot accession number is given at the far left. The corresponding amino acid positions are in parentheses. dicdi, Dictyostelium discoideum; disc, discoidin I; fa5, coagulation factor V; mfgm, milk fat globule-EGF factor 8; nrp, neuropilin; trk3, tyrosine-protein kinase receptor related 3; xenla, Xenopus laevis.
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haye.ch/sprot/prosite.html ) indicated that the predicted amino acid sequence of the Xlrs1-encoded protein contains a coagulation factor 5/8 type C domain signature 1 ( FA58C 1) at position 111–140 (Fig. 3; Kane and Davie, 1986; Johnson et al., 1993; Couto et al., 1996). Two slime mold cell adhesion protein discoidins also share a high homology with the C-terminus of the Xlrs1 protein (Fig. 3; Poole et al., 1981; Fukuzawa and Ochiai, 1996). Discoidin domains have a common C-terminal region of approx. 110 amino acids with FA58C domains (Fukuzawa and Ochiai, 1996). Discoidins and the coagulation factors V and VIII, together with eight other groups of proteins, form the FA58C/discoidin protein family ( Table 1). Mouse Xlrs1- and human XLRS1encoded proteins are new members of this family. 3.3. Gene localization on the X chromosome The mouse Xlrs1 gene was mapped to the distal region of the mouse X chromosome between Htr2c and Grpr using P4 as a probe and the progeny of the interspecific cross (NFS/N×M. spretus)F1× C58/J or M. spretus (Fig. 4). The P4 probe identified ApaI fragments of 19.0 kb in M. spretus and 23.0 kb in the inbred strains NFS/N and C58/J. This localization is consistent with the localization of the human XLRS1 to Xp22. 3.4. Tissue specificity and developmental changes In Northern blots the SR3.1 probe hybridized RNA from retina, but not from cerebral cortex, lung, heart, kidney, spleen, liver or cerebellum (Fig. 5A). Transcripts of 5 and 5.7 kb were detected by probe SR3.1 and also by probes P13-1.3, P4, C11-short and 5R (data not
Fig. 4. Genetic map location of mouse Xlrs1. The recombination fractions between adjacent loci and recombinational distances ± S.E. are indicated on the right of the map. The map locations of human homologs are on the left of the map (human loci taken from GDB). Marker loci (in italics on the right of the map) were typed as described previously ( Filie et al., 1998). The numbers in square brackets are the distances in centimorgans from the centromere according to the mouse X chromosome committee (Boyd et al., 1997).
shown). These two retinal transcripts could be accounted for by the two polyadenylation sites described earlier. In Northern blots probed with either SR3.1 or C11-short, the retinal Xlrs1 mRNA was first detected on postnatal day 7 and reached its adult level at postnatal day 21 ( Fig. 5B). However, the Xlrs1 transcripts may well be expressed earlier, since mRNA signals were detected on postnatal day 5 when X-ray film was overexposed (data not shown). Probe SR3.1 hybridized more strongly to the higher molecular weight band; while probe C11-short hybridized equally with both transcripts.
Table 1 The FA58C/discoidin protein family Secretable extracellular proteins Blood coagulation factors V and VIII Mammalian milk fat globule-EGF factor 8 (MFG-E8 or BA46 antigen) Silk moth humoral lectin hemocytin Bovine SCO-spondin Discoidins
Kane and Davie, 1986 Couto et al., 1996 Kotani et al., 1995 Gobron et al., 1996 Poole et al., 1981 Fukuzawa and Ochiai, 1996
Transmembrane proteins Neuropilin (A5 antigen) Drosophila neurexin IV Mammalian contactin associated proteins (CASPR) Epithelial discoidin domain receptor 1 ( EDDR1, CAK, DDR1, TRKE or RTK6) Neurotrophic tyrosine kinase receptor related 3 (NTRK3, TrkC, TKT or TYRO10)
Takagi et al., 1991 Baumgartner et al., 1996 Peles et al., 1997 Johnson et al., 1993 Karn et al., 1993
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Fig. 5. The Xlrs1 mRNA is retina-specific and its level increases through the juvenile stage. (A) Northern blot containing RNAs from different mouse tissues (retina, cerebral cortex, lung, heart, kidney, spleen, liver and cerebellum), probed with SR3.1. Two Xlrs1 mRNA transcripts of 5 and 5.7 kb were detected. 20 mg of total RNA from each tissue, except for retina (10 mg) were loaded in the gel. The X-ray film was exposed to the blot for 3 h at -80°C with intensifying screens. (B) Retinal Xlrs1 mRNA detected by probes SR3.1 and C11-short (C11) at different times during postnatal development. The loading was 8 mg of total RNA per lane for the blot probed with SR3.1 and 5 mg for the blot probed with C11-short. The lower panels in both (A) and (B) show the ethidium bromide-stained agarose gels before the RNAs had been transferred to the membranes. The two bands correspond to the 28S (upper band) and 18S ( lower band) rRNAs. Numbers are ages in postnatal days. Ad, Adult.
3.5. RNA expression in photoreceptors To verify that the Xlrs1 mRNA is expressed in photoreceptors, we took advantage of the rd mouse and its different times of degeneration of rods and cones. At P28, while retaining many cones (cones are approximately 5% of all photoreceptors in the mouse eye), the rd retina does not have any rods left; by adulthood
(>P60) it is devoid of both types of visual cells (CarterDawson et al., 1978). In Northern blots containing RNAs from 71-day-old retina of normal and rd mice, the Xlrs1 mRNA was detected only in the RNAs from normal retinas, suggesting that Xlrs1 mRNA is expressed in photoreceptors ( Fig. 6A). A reduced level of the two Xlrs1 transcripts was observed in the 28-day-old rd sample when compared with RNAs from normal retinas.
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Fig. 6. Xlrs1 mRNA expression in photoreceptors. (A) Northern blots containing total RNAs from normal and rd mouse retinas at different developmental ages were hybridized with either one of the two 32P-labeled probes, SR3.1 and C11-short (C11). The lower panels show the ethidium bromide-stained agarose gels with the 28S (upper) and 18S ( lower) rRNAs before RNAs were transferred to the membrane. Total retinal RNAs of normal 71- (20 mg) and 21-day-old mice (5 mg), and those of rd 71- (20 mg), 43- (10 mg) and 28-day-old mice (5 mg) were loaded on a gel. The 43-day-old rd mice signals were detected after overnight exposure of X-ray film at -80°C with intensifying screens; while 3 h of film exposure was sufficient to observe the bands from the other preparations. (B) Localization of Xlrs1 mRNA by in situ hybridization of normal adult retina with probe C11-long. (1) Retina hybridized with the C11-long sense probe. Magnification: 422×. (2) Retina hybridized with the C11-long antisense probe. Xlrs1 mRNA is visualized as a purple reaction product. Magnification: 422×. (3) Higher magnification shows the reaction product in individual photoreceptors (arrows). Magnification: 1155×. The concentration of this product in the inner segment is high and thus it appears black.
The Xlrs1 mRNA was still detectable at much lower levels at P43, a time when nearly 2/3 of the cone photoreceptors have degenerated (Carter-Dawson et al., 1978). These results suggest that the Xlrs1 transcripts are expressed in both rod and cone photoreceptors. In situ hybridization with the C11-long antisense probe further substantiated the localization of Xlrs1 mRNA; signal was most prominent in the inner segments of the photoreceptors (Fig. 6B2 and 3) but was also found in their cell bodies (outer nuclear layer of the retina) (Fig. 6B2). The reaction product was detected in individual photoreceptors (Fig. 6B3, arrows). No
signal was detected in the inner nuclear layer of the retina, where the cell bodies of Mu¨ller cells are located.
4. Discussion The results reported here indicate that the Xlrs1 cDNA, a fragment of which was originally isolated by subtractive hybridization (SR3.1), corresponds to the mouse ortholog of human XLRS1 cDNA. The expression of the Xlrs1 mRNA is developmentally regulated. It is first transcribed in the retina within a week of birth
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and increases in level through the juvenile stage. Both in situ hybridization and Northern blot analysis showed that Xlrs1 mRNA is expressed in both rods and cones. By analogy the human XLRS1 gene may also be expressed in photoreceptors, yet, up to now, X-linked juvenile retinoschisis has been thought to be caused by an abnormality in Mu¨ller cells. It is difficult to understand how a photoreceptor gene defect could lead to a Mu¨ller cell disorder. A hypothesis that we have formulated is that the presence of various mutations in the secretable XLRS1 protein prevents its interaction with Mu¨ller cells. The Xlrs1-encoded protein is a member of the FA58C/discoidin family, which is made of either secreted proteins involved in adhesion or transmembrane proteins ( Table 1). The Xlrs1 protein belongs to the former type. Many members in the FA58C/discoidin protein family have a binding protein in the extracellular matrix or on the cell surface of other cells ( Elodi and Elodi, 1983; Barondes et al., 1987; Kolodkin et al., 1997; Peles et al., 1997). In both secreted and transmembrane proteins the FA58C domains are located extracellularly, and are probably involved in binding. For example, the FA58C domain of coagulation factors has been shown to bind phospholipids (Foster et al., 1990). If the Xlrs1 mRNA encodes a cell adhesion protein, a defect in it could result in an aberrant interaction between photoreceptors and Mu¨ller cells, either at the level of the synapses or in the area of the outer limiting membrane. The testing of this hypothesis will increase our understading of the function of retinal cells and the nature of X-linked juvenile retinoschisis.
Acknowledgements The authors would like to thank Dr Xianjie Yang for sharing her mouse eye cDNA library. The authors also appreciate the assistance of Ms Jennifer Shih, Ms Tammy Rickabaugh, Mr Clyde Yamashita and Dr Danyun Zhao. This work was supported by grants from NIH ( EY08285) and The Foundation Fighting Blindness.
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