Retinal Expression of γ-Crystallins in the Mouse

Abstract

purpose. High levels of expression of a form of γ-crystallin mRNA in mouse retina have been identified. Because the six murineγ -crystallins have generally been regarded as specific to the lens, the expression of these crystallins at the mRNA and protein levels in the retina were evaluated in more detail.

methods. Expression of γE/F-crystallin mRNA was examined by northern blot analysis and reverse transcription–polymerase chain reaction (RT–PCR) analysis applied to murine retinal and lens total RNAs. Forγ A-D-crystallin mRNAs, a multiplex RT–PCR was used on total cDNAs. The detection of total γ-crystallin protein in the retina was performed using an antibody to bovine lens γ-crystallins, applied to protein extracts in immunoblot analysis and to cryostat sections of ocular tissues in immunofluorescence studies.

results. By RT–PCR, we confirmed expression of both γE- and γF-crystallin as well as all four (γA–γD) remaining crystallins at the mRNA level in the mouse retina. γ-Crystallin proteins were also detectable in murine retina by immunoblot analysis, although at a lower level than in the lens. By immunocytochemistry, γ-crystallins were localized particularly to the inner retina, outer plexiform layer, and the photoreceptors during postnatal development.

conclusions. Our findings of γ-crystallin mRNA and protein expression in the retina indicate that none of the major crystallin classes is uniquely expressed in the lens. The expression of γ-crystallins in the developing murine retina suggests a role analogous to the anti-stress properties established for the small heat-shock proteinα B-crystallin, perhaps in response to varying exposure to light.

The α-, β-, and γ-crystallins comprise the three main families of major soluble proteins ubiquitously expressed in vertebrate lenses. Together with the taxon-specific members, crystallins appear to have evolved from preexisting proteins that were recruited to new structural roles in the lens. ¹ Thus, the α-crystallins,α A and αB, resemble heat-shock proteins with chaperone functions; the β/γ superfamily shows similarities to bacterial spore coat protein and to Physarum species stress-induced proteins; and the taxon-specific crystallins are closely related or identical to certain metabolic enzymes such as lactate dehydrogenase and aldose reductase. Although extralenticular expression has been established forα - and β-crystallin families, the expression of γ-crystallins (of which there are 6, γA–γF, in the mouse) has generally been considered lens specific, based particularly on transgenic mouse studies using a γ-crystallin promoter to drive reporter gene expression. ² To date, there have been three reports providing evidence of extralenticular γ-crystallin expression. These are of the mRNAs in the developing Xenopus laevis tadpole, occurring at very low levels throughout much of the embryo, ^{3 4} and, most recently, evidence for a seventh member of the γ-crystallin family, γS-crystallin, expressed in murine lens, retina, and cornea. ⁵ Such evidence has provided initial indications that none of the crystallins classes can be characterized as lens specific. 

During the course of an analysis of retinal gene expression patterns occurring in wild-type and retinal degeneration (rd) mice, using the technique of mRNA differential display, we identified a gene showing high levels of expression in retinal RNAs. The cloned product corresponded to a partial γE-crystallin cDNA and prompted us to examine further the expression of the γ-crystallin family in the mouse retina. Our evidence indicates that none of the γ-crystallins is uniquely expressed in the lens and suggests that nonrefractive functions for these proteins may be retained in the retina. 

Materials and Methods

Tissues

C57BL/6(J) or nondegenerative rd/+ strain mice were reared in a cyclic 12-hour light/12-hour dark environment and were cared for and treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The mice were the source of tissues for RNA and protein extraction and for immunochemical analyses. Postmortem eyes, enucleated during the light phase, were either dissected to retrieve the lenses and the retinas (the latter free of any lens tissue), for subsequent storage in liquid nitrogen until RNA extraction or at −70°C for protein extraction, or fixed in fresh 4% paraformaldehyde in 0.1 M phosphate buffer overnight, followed by embedding, freezing, and cryosectioning as previously described. ⁶  

RNA Extraction and Northern Blot Analysis

Total RNA was extracted from pooled frozen tissues (a minimum of 6 retinas or lenses per time) using either the RNeasy kit (Qiagen GmbH, Hilden, Germany) or a guanidinium thiocyanate/phenol–based protocol (Clontech, Palo Alto, CA), according to the manufacturers’ instructions. For northern blot analysis, 4-μg samples were denatured, electrophoresed in 1.3% agarose gels, and blotted onto nylon membranes (Amersham International, Slough, UK). Cloned inserts from plasmids, or polymerase chain reaction (PCR) products, were labeled with α-[³²P]dCTP using the Rediprime kit (Amersham International) and hybridized to the blots in the presence of 50% formamide at 42°C overnight, followed by stringent washing and autoradiography. 

Reverse Transcription–PCR and Restriction Enzyme Digestion

Single-strand cDNA was obtained by reverse transcription (RT) of retina or lens RNA using approximately 1-μg RNA samples, incubated in 20-μl reactions with 1.25 U AMV reverse transcriptase (GIBCO–BRL, Paisley, UK) and random hexamer primers (Pharmacia Biotech, Uppsala, Sweden) for 60 to 90 minutes at 42°C, followed by heat inactivation of the enzyme. PCR amplifications were performed using primers according to the strategy of Goring et al. ⁷ For detection of γE/F-crystallin expression, primers GECRY.1: 5′-AGCCATGGGGAAGATCACCTTCTATG-3′ and GCRYR: 5′-AAGCGGTCCTGCAGGTGGGAGCAG-3′ were used, in 30-μl reactions incorporating 0.75 U Taq polymerase (GIBCO–BRL) and 2 μl cDNA in a thermal cycler (Hybaid, Teddington, UK) with the following parameters: 94°C for 3 minutes, then 35 cycles of 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 30 seconds, and a final step of 72°C for 5 minutes. Aliquots of 10 μl were cut with BglII (New England Biolabs, Beverly, MA) for a minimum of 2 hours and the products resolved on ethidium bromide–stained 1.0% agarose (Pharmacia Biotech, Uppsala, Sweden)/3.0% NuSieve GTG (FMC; Flowgen, Lichfield, UK) gels. For the detection of γA-, γB-, γC-, and γD-crystallins, primers were as described by Goring et al., ⁷ and amplification conditions were as above with the exception that the anneal temperature was reduced to 50°C. Molecular size markers were obtained from GIBCO–BRL. Verification of the amplified products was performed by probing of a Southern blot of theγ E/F-crystallin reaction products with labeled insert of the sequenced γE-crystallin clone, which confirmed cross-hybridization, and by HinfI restriction digestion of theγ A-γD-crystallin multiplex reaction products, which led to the expected reduction in size of all 4 bands and generation of a single additional small fragment, due to cleavage at a conserved site (data not shown). The primer pairs in all cases were set across an intron, but additional amplifications in the absence of reverse transcriptase were performed to confirm that the resultant products were not due to genomic DNA contamination. 

Western Blot Analysis

Equal amounts of total protein (∼5 μg) from extracts of retina, lens, heart, and liver tissue were subjected to electrophoresis under denaturing conditions and transferred to Immobilon-P (Millipore, Bedford, MA) membranes according to established protocols. ⁸ Immunodetection was with a rabbit anti-bovine lens γ-crystallin antibody as the primary antibody (used at 5μ g/ml), and peroxidase-conjugated goat anti-rabbit IgG at 1:1000 (Sigma, St. Louis, MO) as the secondary antibody, followed by development in the presence of 3,3′-diaminobenzidine and hydrogen peroxide. The analysis of retina and lens samples was repeated a minimum of 4 times. 

Immunocytochemistry

Enucleated eyes were fixed in fresh 4% paraformaldehyde in 0.1 M phosphate buffer overnight, incubated in 30% sucrose, and embedded and frozen as described previously. ⁶ Cryostat sections (10-μm–thick) were processed for immunocytochemistry using the classic immunofluorescence technique. ⁹ The primary antiserum was as described above for immunoblot analysis, used at a concentration of 2.5 μg/ml, and the secondary antibody a fluorescein isothiocyanate–coupled anti-rabbit IgG used at 1:200 (Sigma, St Louis, MO). Sections from 3 different animals were examined for each time, and typical results presented. 

Results

During an analysis of altered patterns of gene expression associated with murine retinal degeneration, we isolated a differential display PCR product, which, when used as a northern blot analysis probe, detected highly abundant transcripts of approximately 0.8-kb size in control mouse retinal RNA. Figure 1 A shows the corresponding detection of this mRNA species in postnatal day (P)20 mouse retina using the cloned version of the PCR product. Sequence analysis of this clone indicated that the insert corresponded to approximately 0.3 kb cDNA of murine γE-crystallin, one of the family of γ-crystallin genes highly expressed in vertebrate lenses but not previously reported in the retina. In the mouse, γA-, γB-,γ C-, γD-, and γE-crystallin genes form a linked tandem array on chromosome 1, with γF-crystallin 200 to 400 kbp upstream of the cluster (see ^{Ref. 7} and references therein). Sequence data have shown that the γF gene is almost identical to that of γE, and use of aγ E probe would not permit discrimination between these two genes by northern blot analysis. To establish first whether both γE- andγ F-crystallins are expressed in the retina, we adopted a strategy of using RT–PCR based on that of Goring et al. ⁷ This approach exploits the fact that by selecting appropriately sited primers the resulting PCR products can be cut by the restriction enzyme BglII within the γE but not within the γF sequence, due to a nucleotide difference at position 272 in the γE cDNA. In the selected samples tested (C57BL/6 retina, at P15, P17, and P20), we found that expression of γE and γF transcripts was detected (Fig. 1B , lanes 4–6); and while the PCR was not strictly quantitative, the levels of the two appeared similar. The pattern obtained with the retinal samples resembled that seen with the lens control (Fig. 1B , lane 7). 

To examine further whether the remaining four γ-crystallin genes were expressed in the retina, we performed a multiplex PCR on murine retinal and lens total cDNAs (Fig. 1C) . All four (γA–γD) crystallins showed detectable expression in both tissues, although the level ofγ C-crystallin expression appeared more variable as well as lower than that of the others, the latter observation in agreement with the findings of Goring et al. ⁷ on total ocular RNA. In all cases of PCR amplification, the primers were designed to span an intron, thus any genomic DNA contamination should not yield products of the sizes obtained. Additional confirmation that the RT–PCR was detecting crystallin expression was provided by the absence of products when reverse transcriptase was omitted (Fig. 1D) . 

Evidence for expression of a gene at the mRNA level does not necessarily imply the presence of translation products, however. We therefore used an antibody raised against bovine total lensγ -crystallin proteins in immunochemical assessments of expression at the protein level in the retina. Total retinal and lens extracts from mice were subjected to western blot analysis, and reactive bands of∼ 20 kDa were detected in both tissues, although at a much higher concentration in the lens (Fig. 2 : lanes 1, 2, and 5). Close inspection (and of blots from repeat experiments) revealed that multiple bands were present in each sample, most likely corresponding to the different γ-crystallin forms, although discrimination among these was not possible. No bands were detected in extracts of mouse heart or liver (Fig. 2 , lanes 3 and 4). 

Typical patterns of localization of immunoreactive γ-crystallins by fluorescence staining in the mouse retina at selected postnatal ages (P10, P16, and P18) are shown in Figures 3A 3B 3C . We found that despite some variability in fluorescence intensity during postnatal development, the main sites ofγ -crystallin protein localization were the ganglion cell layer, the outer plexiform layer, and photoreceptors, particularly in the outer segment regions. The immunostaining also extended from the inner edge of the inner nuclear layer through the inner plexiform layer and nerve fiber layer to the ganglion cell layer. At the times examined, there was no detectable immunoreactivity at the retinal pigment epithelium. Control sections lacking primary antibody (including those of retinas from P10, P16, and P18, not shown) displayed minimal background fluorescence, mainly associated with the retinal vasculature (Fig. 3D) . 

Discussion

In their analysis of the expression of the γA-γF-crystallin genes in mouse eye development, Goring et al. ⁷ assayed total ocular mRNA on the assumption that γ-crystallin expression was entirely lens specific. Our present study indicates that at least in certain cases the retinal component of ocular γ-crystallin expression can be significant and that all 6 genes are expressed in the retina and the lens. In the lens, the spatial distribution of the different crystallin proteins has been interpreted as contributing, perhaps via differing biophysical properties such as water exclusion, to the final optical characteristics of this structure. The potential function ofγ -crystallins in the neural retina is less clear. Although it is possible that the molecular packing properties of the crystallins contribute to retinal translucency, it seems more plausible that nonrefractive properties may be preserved from the evolutionary precursor proteins. From parallels with the heat-shock and stress-induced genes, γ-crystallins may, in conjunction with αB- and possibly also certain β-crystallins, constitute a family of defensive proteins, which are induced during critical periods of stress on the retina. The most obvious stressor is incident light, particularly after the opening of the eyes at about P13 to P14 in the mouse. Exposure to intense light is a well-characterized inducer of photoreceptor apoptosis and is associated with the induction of specific genes including that for the glycoprotein clusterin. ¹⁰ It will be of interest to examine the response of crystallin gene expression to light damage, and in relation to another well-established modulator of retinal gene expression, the diurnal cycle of light exposure. It remains possible that retinalγ -crystallins have no specific function and represent an adventitious form of expression, rudiments perhaps of early interactions between the developing lens placode and the optic vesicle. Whichever is the case, it is clear that the lens can no longer be regarded as the unique repository of any of the major crystallin families. 

 Supported by British Retinitis Pigmentosa Society, Guide Dogs for the Blind Association, and Iris Fund for Prevention of Blindness.

 Submitted for publication October 2, 1998; revised April 29 and June 17, 1999; accepted June 30, 1999.

 Commercial relationships policy: N.

¹  Present address: Department of Vascular Biology (H30-1-051), SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Third Avenue, Harlow, Essex, CM19 5AW, UK.

 

Figure 1.

 

Analysis of γ-crystallin mRNA expression in murine ocular tissues. (A) Northern blot analysis of C57BL/6 mouse retinal total RNA (4 μg) at P20 days using γE-crystallin cDNA probe. (B) RT–PCR analysis of γE and γF–crystallin expression. Total cDNA was subjected to PCR using primers GECRY.1 and GCRYR. Products were digested with BglII and separated on an ethidium bromide–stained agarose gel. Uncut product of 351 bp corresponds to γF transcript; cut products of 276 and 75 bp to γE transcript. Lane 1, molecular size markers (200- and 300-bp bands indicated); lane 2, uncut product, P20 retina; lane 3, uncut product, P20 lens; lanes 4 through 7, cut products: lane 4, P15 retina; lane 5, P17 retina; lane 6, P20 retina; and lane 7, P20 lens. (C) Multiplex RT–PCR analysis of γA-γD–crystallin mRNA expression. Lane 1, molecular size markers as in (B); lane 2, P15 retina; lane 3, P17 retina; lane 4, P20 retina; and lane 5, P20 lens. (D) RT–PCR control reactions using P20 retinal RNA in the presence (lanes 2, 5) or absence (lanes 3, 6) of reverse transcriptase, followed by amplification using the γE/F (lanes 2, 3) and γA-γD (lanes 5, 6) primers. Molecular size markers, lanes 1, 4; 300 bp markers indicated by arrowheads.

Figure 2.

 

Western blot analysis of γ-crystallin expression in murine tissues (∼5 μg protein/lane). Lane 1, P20 retina; lane 2, P20 lens; lane 3, P18 heart; lane 4, P18 liver; and lane 5, P20 lens (duplicate of lane 2).

Figure 3.

 

Immunofluorescence detection of γ-crystallin expression in sections of murine retinal tissue at different postnatal ages. (A) P10; (B) P16; (C) P18. (D) control section (P20) lacking primary antibody. PR, photoreceptors; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 100μ m. Results similar to those shown in (D) were obtained with additional control sections at ages P10, P16, and P18.

We thank the British Retinitis Pigmentosa Society, the Iris Fund for Prevention of Blindness, the Guide Dogs for the Blind Association, and the National Lottery Charities Board for their support for this work. We also thank Hannah Stewart for excellent technical assistance, J. Samuel Zigler, Jr. (National Eye Institute, Bethesda, MD), for providing the anti–γ-crystallin antibody, Bill Starkey for sequencing the cloned PCR products, and the Rayne Management Committee for the provision of research facilities.