Expression of phototransduction cascade genes in the ground ...

Expression of Phototransduction Cascade Genes in theGround Squirrel Retina

Malcolm von Schantz,*-f Agoston Szel,*% Theo van Veen,* and Debora B. Farberf

Purpose. This study describes the expression and distribution of phototransduction cascadegene products in the cone-dominant retina of the ground squirrel Spermophilus tridecemlinea-tus.

Methods. Messenger RNA expression was studied by blot hybridization, and the distribution ofthe gene products was investigated by immunocytochemistry.

Results. RNA blot hybridization showed messages for the a2, /31, and /53 subunits of transducinbut was negative for rhodopsin, a 1-transducin, and the a, /3, and 7 subunits of cyclic guanosinemonophosphate (cGMP) phosphodiesterase. Immunocytochemical labeling indicated that theapproximate ratio of the photoreceptor types in ground squirrel retina is 90.6% for greencones, 6.3% for rod-like cells, and 3.1% for blue cones. Rod-like cells were immunopositive forrhodopsin and blue opsin. All photoreceptor elements were labeled by antibodies againsta 1-transducin (which recognizes both the al and a2 isoforms), /33-transducin, and the rod 7subunit of phosphodiesterase, whereas no cells were labeled by antibodies against the rod aand /? subunits of phosphodiesterase or against the rod cGMP-gated cation channel. Rod-likecells and blue cones were stained by antibodies against /?1 -transducin.

Conclusions. The authors demonstrate new cone-like traits in the biochemical make-up ofrod-like cells, and a distribution of the transducin /? subunit in the ground squirrel is differentfrom that found in other mammals. Invest Ophthalmol Vis Sci. 1994;35:2558-2566.

A he 13-lined ground squirrel Spermophilus tridecem-lineatus (previously called Citellus tridecemlineatus) is asmall, diurnal rodent that has a cone-dominant retinacontaining a small percentage of rod-like cells.Whereas the cones carry the normal features of theircounterparts in retinas of other mammals, the rod-likecells have both rod and cone traits in morphology aswell as in synaptic organization.1 Outer segments ofthe different photoreceptor types are, however, re-

From the * Department of Zoology, University of Goteborg, Sweden; the fJules SteinEye Institute, University of California at Los Angeles School of Medicine; and theXSecond Department of Anatomy, Histology, and Embryology, SemmelweisUniversity of Medicine, Budapest, Hungary.Supported by grants from the Paul and Marie Berghaus and the Count Knut Possefunds (MvS), the Hungarian Ministry of Welfare grant T-243, the HungarianScience Research Fund grant 1134 (AS), the Swedish Natural Science Researchcouncil grant 4644-311 (TvV), the Retinitis Pigmentosa Foundation FightingBlindness (TvV and DBF), and the National Institutes of Health grant EY02651(DBF). DBF is the recipient of a Research to Prevent Blindness Senior ScientificInvestigator Award.Submitted for publication September 23, 1993; revised November 29, 1993;accepted November 30, 1993.Proprietary interest category: N.Reprint requests: Debora B. Farber, Jules Stein Eye Institute, UCLA School ofMedicine, 100 Stein Plaza, Los Angeles, CA 90024.

newed in the manners typical of cone and rod photo-receptors,2 respectively.

The rod-like cells of the ground squirrel exhibitboth rod and cone traits in their spectral mechanisms,3

as well as an interesting epitope dimorphism in theirphotopigments.4 Electroretinogram (ERG) measure-ments suggest that 5. tridecemlineatus has a scotopicspectral sensitivity similar to that of a 502-nm rhodop-sin.5 However, the dynamic behavior of this photopig-ment is reminiscent of the normal cone signal in thatthe b2 component of the ERG, with an absorptionmaximum of 502 nm, has a dark-adaptation curve thatis identical with that of the bj curve.6 An interestingfeature of the incomplete photoreceptor duplicity wasfound in the immunocytochemical heterogeneity dis-played in the rod-like cells of the European 5. citellus.4

In the retina of this species, half of the rod-like cellswere recognized solely by a rhodopsin antibody,whereas the other half were also recognized by a blueopsin antibody.

Electrophysiologic investigations performed in 5.tridecemlineatus and the Mexican ground squirrel 5.

2558Investigative Ophthalmology & Visual Science, April 1994, Vol. 35, No. 5Copyright © Association for Research in Vision and Ophthalmology

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Phototransduction Cascade in the Ground Squirrel 2559

mexicanus,1 as well as in 5. citellus,8 indicated the pres-ence of two photopigments, one with an absorptionpeak of 440 nm and the other one with a maximum at520 to 525 nm. Single photoreceptor recordings madein the golden-mantled ground squirrel S. lateralis havegiven similar results.9 The latter photopigment wasalso partially purified from the Siberian species 5. un-dulatus, and found to have an absorption maximum of518 to 520 nm.10 These two spectral peaks can becorrelated morphologically with a cone dimorphismreported in the California ground squirrel, 5.beecheyi.11 In this species, 10% of the cones containlarge ellipsoids. The hypothesis that these cells repre-sent blue-sensitive cones was confirmed histochemi-cally in S. citellus.i2 The same species was used for astudy of the photoreceptors with antibodies raisedagainst different photopigments, which showed thatgreen- and blue-sensitive cones constitute 92% and5%, respectively, of the total photoreceptor popula-tion.4

The phototransduction cascade in rod cells of dif-ferent species has been characterized in great detailand the genes for most of the involved proteins havebeen identified, cloned and sequenced. These includerhodopsin, the ctl, j81, and 7I subunits of transducin,the a, /3, and 7 subunits of cyclic guanosine monophos-phate (cGMP) phosphodiesterase, guanylate cyclase,and the cGMP-dependent cation channel.

The phototransduction cascade in cones is notcompletely characterized, but in most ways appears tomirror that of rods, although it operates at higher lev-els of light and has different response kinetics.13 Eachcone type has its own photopigment, which activates atransducin complex that contains the cone-specificsubunits a2,14 j83,15 and 72.16 The cone phosphodies-terase complex contains two cone-specific a' subunits,lacks a /3 subunit, but may contain up to three 7 sub-units.17

Because the ground squirrel has a cone-dominantretina, it is a very interesting model for biochemicalstudies on cone phototransduction. In contrast torod-dominant retinas, the ground squirrel retina ex-hibits no measurable decrease in cGMP concentrationafter exposure to light.18 This is probably due to arapid cGMP turnover rate, which prevents the detec-tion of changes in its concentration. However, cGMPdoes act as a second messenger for the light signal inisolated cones, as demonstrated in the larval tiger sala-mander.19 The ground squirrel rod-like cells, whichmight have been expected to display a light-dependentdecrease in cGMP, are so few that they may not make asignificant contribution to the total cGMP content. Inthe ground squirrel species 5. undulatus, the presenceof a cGMP phosphodiesterase with a ratio of approxi-mately 5 molecules per 100 molecules of cone opsinhas been reported.10

In the current study, we have used a panel ofcDNA probes and antibodies to investigate which pho-totransduction cascade genes are expressed in thecone-dominant retina of the ground squirrel 5. tride-cemlineatus. Our aim was to investigate which agentsthe various types of cone and rod-like cells use in theirphototransduction machinery, and where these pro-teins are localized. Part of this investigation has beenpresented previously in abstract form.20

METHODS

Animals and Tissue Preparation

All animals were maintained and sacrificed in accor-dance with the ARVO Statement for the Use of Ani-mals in Ophthalmic and Vision Research.

Wild ground squirrels (5. tridecemlineatus) weretrapped by commercial vendors in Illinois and Wiscon-sin, shipped to our laboratory, and maintained on agrain/nut diet. Light-adapted animals were anesthe-tized with carbon dioxide, and then decapitated. ForRNA analysis, eyes were enucleated, and the retinadissected and kept at — 80°C until RNA extraction wasperformed. For immunocytochemistry, animals wereeither perfusion-fixed with formaldehyde before enu-cleation, or enucleated within minutes post mortem.For orientation, the cornea was marked with a radialline with a hot needle.

Mice (C57B1/6J) were maintained under normallaboratory conditions, and killed by cervical disloca-tion. After enucleation, the retinas were dissected andkept at -80°C until RNA extraction.

A donor human eye was enucleated 1 hour and 45minutes post mortem and kept at —80°C until thawedand dissected for RNA extraction. This was performedfollowing the tenets of the Declaration of Helsinki,with informed consent, and approval by the institu-tional human experimentation committee.

RNA Analysis

Total RNA was purified as described earlier.21 Poly-ARNA was obtained using a messenger RNA (mRNA)purification kit (Pharmacia LKB Biotechnology, Upp-sala, Sweden). 10 )Lig of total RNA or 1.0 /xg of poly-ARNA was separated in a 1.2% agarose-formaldehydegel. RNA was blotted overnight onto Hybond N+membranes (Amersham, UK) in 20 X SSC. Crosslink-ing of RNA to membranes and stripping of probe be-tween hybridizations were performed according to themanufacturer's instructions. The following cDNAclones encoding photoreceptor genes were used asprobes for hybridization: pHL1301, a 1.19-kb Eco RIfragment encoding human a-tubulin22; bd 20, a 1.64Eco RI fragment encoding bovine rhodopsin23; pML7,a 2.19-kb Nco I fragment encoding bovine a 1-trans-


2560 Investigative Ophthalmology & Visual Science, April 1994, Vol. 35, No. 5

ducin24; a 1.6-kb Nco I/Bam H I fragment encodingbovine a2-transducin25; XTB12, a 1.47-kb Eco Rlfragment encoding bovine /31-transducin26; B3R, a1.05-kb fragment of bovine /33-transducin27; JN69B, a1.0-kb Eco Rl fragment encoding human rod a-phos-phodiesterase (Viczian A, Farber DB, unpublisheddata); MBP3, a 1.15-kb PCR-amplified fragment en-coding rod j8-phosphodiesterase28; and a 0.48-kb EcoRl fragment encoding murine rod y-phosphodiester-ase.29 DNA (50 ng) was labeled30 using the Klenowfragment of DNA polymerase (United States Biochemi-cal Corp., Cleveland, OH) with a-32P-dCTP (NEN Re-search Products, Boston, MA, or ICN Biomedicals, Ir-vine, CA). Overnight hybridization was carried out in ashaking water bath at 65 °C in a phosphate-buffered1% BSA solution containing 7% SDS. Blots werewashed 2 X 1 5 minutes in 2 X SSC/0.2% SDS at 47°C,and 2 X 1 5 minutes in 0.2 X SSC/0.2% SDS at 51 °C,and then visualized with Hyperfilm MP (Amersham)kept at —80°C with intensifying screens for the appro-priate time (until clear bands were detected in the hu-man, murine, and, when applicable, ground squirrellanes). In the cases where the latter was negative, thiswas confirmed by overexposing the film for twice aslong a period. As a control for quality and quantity ofthe RNA, each blot was initially hybridized with a tu-bulin probe.

Immunocytochemistry

Enucleated eye cups were immediately immersed in4% paraformaldehyde and 0.1% glutaraldehyde dis-solved in 0.1 M Sorensen phosphate buffer (pH 7.2)for 4 hours. The material was embedded in one of twoalternative ways. Eyecups were dehydrated and em-bedded either in Araldite (Durcupan ACM, Fluka,Buchs, Switzerland), or diethylene glycol distearatewax (DGD, Polysciences, Inc., Warrington, PA). Thearaldite and DGD blocks were sectioned on an ultra-microtome (1.5 fim) with glass knives. Serial tangentialand radial sections were obtained.

Before the immunoreaction, the araldite sectionswere etched with sodium methoxide.31 The DGD wax

was removed from the sections with toluene. Bothtypes of sections were preincubated for 10 minutes in0.1 M phosphate buffered saline (PBS), pH 7.25, con-taining 2% bovine serum albumin (BSA). The primaryantibodies (Table 1) were diluted in the same buffer,and the sections were incubated overnight at roomtemperature. The bound antibodies were detected bythe avidin-biotin-peroxidase system (Vectastain, Vec-tor, Burlingame, CA). All steps of the immunocyto-chemistry were carried out with the addition of 0.25%Triton X-100 to the PBS.

After visualization with diaminobenzidine in thepresence of hydrogen peroxide, sections were dehy-drated and covered with Permount. The results weredocumented with an Axiophot photomicroscope(Zeiss, Oberkochen, Germany), using Nomarski op-tics. For demonstration of colocalization of photore-ceptor-specific proteins, corresponding areas of adja-cent tangential sections were photographed.

RESULTSThe qualitative results of RNA blot hybridization andimmunocytochemistry are summarized in Table 2, andthe transcript sizes of the different detected messagesare given in Table 3.

RNA blot hybridization using a rhodopsin cDNAprobe (Fig. 1) showed five transcripts in murine, andthree transcripts in human retina. No hybridizationwas detected in the ground squirrel. With a rod a-transducin cDNA probe, one murine and two humantranscripts were observed (Fig. 2A). Again, no hybrid-ization to ground squirrel retinal RNA could be de-tected. In contrast, a cone a-transducin probe hybrid-ized to two murine mRNA transcripts, and to a veryabundant transcript in ground squirrel (Fig. 2B). Thesize and number of mRNA bands in human retinacould not be interpreted due to nonspecific binding ofthe probe. cDNA encoding jSl-transducin hybridizedto two mRNA transcripts in all three species, with amarkedly weaker intensity in the ground squirrel (Fig.2C), whereas the /33-transducin probe labeled oneband in each species with a stronger signal in ground

TABLE l. Antibodies

Designation Antigen Origin Host Type Dilution Source

AOOS-2COS-1306LAP-636LAP-63811-2-84PDEi(73-87)PHclDl

OpsinBlue opsinGreen opsina-transducinj81-transducin/33-transducinRod a/?-phosphodiesteraseRod 7-phosphodiesteraseRod cGMP-dependent channel

BovineChickenChickenBovineHumanHumanBovineMouseBovine

RatMouseMouseRabbitRabbitRabbitRabbitRabbitMouse

PMMPPPPPM

1:10,0001:5,0001:5001:2,0001:3001:3001:2501:3501:50

Produced by Szel and Rohlich39



Gift from Allen Spiegel34

Gift from Bernard K.-K. Fung15


Produced by DB Farber40


Gift from Robert S. Molday41

P = polyclonal; M = monoclonal.



TABLE 2. Qualitative Results of RNA Blot Hybridizationand Immunocytochemistry

GeneDNAProbes

N/EN/E

Rod-like Cells

Antibodies

Blue Cones

1 +

1

Green Cones

—RhodopsinBlue opsinGreen opsinal-transducina2-transducin/31-transducin/33-transducinRod a-phosphodiesteraseRod /3-phosphodiesteraseRod 7-phosphodiesterasecGMP-gated cation channel

+*+*

+*

+*+*

+*N/E

+*

+*

N/E = not examined.* Antibody does not appear to discriminate between rod and cone isoenzymes.

squirrel RNA than in the other samples (Fig. 2D).Probes for the a, /?, and 7 subunits of rod cGMP phos-phodiesterase hybridized to one murine mRNA tran-script each. Hybridization to human RNA was specificonly with the rod 7-phosphodiesterase probe, whichbound to one transcript. None of the phosphodiester-ase probes hybridized to ground squirrel RNA (datanot shown).

Analysis of consecutive sections derived from thecentral part of the ground squirrel retina allowed us todistinguish different photoreceptor types: AO, a poly-clonal antiserum against rhodopsin, recognized rod-like cells, with the typical rod-like outer segmentshape, which constituted 6.3% of the analyzed photor-eceptors. The antibody COS-1 stained an abundantgreen-sensitive cone population (90.6%), whereas theblue opsin antibody OS-2 stained a smaller populationof cones with a relatively larger diameter (3.1%), aswell as all rod-like cells (Fig. 3). All photopigment anti-bodies labeled the photoreceptor outer segments.

The antiserum against a-transducin (which recog-nizes both the al and the a2 isoforms) was found tolabel all photoreceptors (Fig. 4). The labeling was mostintense in the inner segments and the pedicles, but was

also present in outer segments, and the cytoplasm ofthe cell body. The antibody against /31-transducin la-beled rod-like cells and blue cones, with the strongestlabeling concentrated to the membrane (Figs. 5, 6A),whereas the /53-transducin antibody labeled the outersegments of green cones, and the inner and outer seg-ments of rod-like cells and blue cones (Fig. 6B).

No immunoreaction was observed in any retinalcell type with an antiserum against the rod a, j8 cata-lytic complex of cGMP-phosphodiesterase (notshown); however, the antibody against rod 7-phospho-diesterase labeled the whole cell body of all photore-ceptors, with the strongest reaction in the inner seg-ments and the pedicles (Fig. 7). In addition, both rod-like cells and cones were immunonegative for thecGMP-dependent channel antibody (not shown).

For all the antibodies used, the immunocytochemi-cal staining pattern was the same with light- and dark-adapted animals.

DISCUSSION

The first point that must be addressed to enable aninterpretation of the data from RNA and antibody hy-

TABLE 3. Molecular Weights (kb) of mRNA Species Hybridizing With DifferentPhotoreceptor DNA Probes

Gene

Rhodopsinal-transducina2-transducin/31-transducinj83-transducinRod a-phosphodiesteraseRod /3-phosphodiesteraseRod 7-phosphodiesterase

M. musculus

4.6,3.4,2.7, 1.9, 1.42.32.3, 1.63.4, 1.61.24.83.30.7

H. sapiens

2.8, 1.9, 1.52.7, 1.2No specific signal3.6, 2.71.4No specific signalNo specific signal0.9

S. tridecemlineatus

No hybridizationNo hybridization2.33.3, 1.71.4No hybridizationNo hybridizationNo hybridization


2562 Investigative Ophthalmology 8c Visual Science, April 1994, Vol. 35, No. 5

• IFIGURE 1. (A) Blot of retinal total RNA probed with a rho-dopsin cDNA. Left lane, murine RNA; middle lane, humanRNA; right lane, ground squirrel RNA. (B) Hybridization ofthe same blot with a tubulin probe, demonstrating the pres-ence of similar levels of RNA in all lanes.

bridization experiments is the significance of negativeresults, in particular with regard to which genes areexpressed in rod-like cells. The relative scarcity of rod-like cells cannot account for negative results in immu-nocytochemical labeling. However, the absence of adetectable signal in a blot of RNA from the whole ret-ina may signify (1) the actual absence of a message, (2)a message present in levels below the detection limit,or (3) insufficient interspecific crossreactivity of theprobe. We have found that although the human retinacontains only 5% cones,32 and the mouse retina 3%cones,?3 cone-specific messages such as the a2 and /33subunits of transducin were easily detectable in retinalRNA from these species. As we have found, S. tride-cemlineatus contains approximately 6% rod-like cells.However, no hybridization was detected in ground

squirrel retinal RNA with a probe for rhodopsin, themajor mRNA constituent in rods. Nonetheless, allrod-like cells were labeled by antibodies against rho-dopsin as well as blue opsin. This leads us to hypothe-size that the photopigment in the rod-like cells of theground squirrel, although sharing its spectral quali-ties, may have significant structural differences fromrhodopsin.

The finding that all rod-like cells in S. tridecemlin-eatus are positive for rhodopsin and blue opsin anti-bodies indicates that the previously reported heteroge-neity in S. citellus4 is not a characteristic feature of thegenus. A possible interpretation is that the blue opsin-negative rod-like cells (type 2 rods) in 5. citellus are infact typical rods. ERG measurements seem to excludethe possibility of a colocalization of rhodopsin andblue opsin in 5. tridecemlineatus.3 Because the molecu-lar weights of all photopigments fall within the samerange, this question will have to be addressed by clon-ing and sequencing of the pigment gene rather than byWestern blotting.

We were also unable to find a detectable messagein the ground squirrel retina for other rod-specificproteins such as the al subunit of transducin and thea, /?, and y subunits of rod cGMP-phosphodiesterase.

A R:'T^ ^ D."

FIGURE 2. RNA blots probed with cDNA fragments for (A)a 1-transducin (total RNA), (B) a2-transducin (mRNA), (C)01 -transducin (total RNA), and (D) 03-transducin (totalRNA). All blots: left lane, murine RNA; middle lane, humanRNA; right lane, ground squirrel RNA. (B) A composite,showing the much more intense ground squirrel signal in an8-hour exposure, and the mouse lane in a 2-day exposure.Note that jSl-transducin gives a relatively weaker and /33-transducin a relatively stronger signal in the ground squirrellane.



FIGURE 3. Consecutive tangential sections reacted with anti-bodies against (A) rhodopsin, (B) blue opsin, and (C) greenopsin. (D) Diagram showing the localization and antigenicityof the individual photoreceptor cells. The sections are cut atthe outer segment level, but at lower edges some inner seg-ments are also encountered. The retinal pigment epitheliumsurrounds each outer segment, giving the section a honey-comb appearance. The majority of outer segments arestained by COS-1 (green cones, A). The remaining photore-ceptors all are recognized by OS-2 (thick and thin arrows,B). Some of them, however, are also labeled by anti-opsin(rod-like cells; thin arrows, C, These cells are also markedwith thin arrows in B.). Note that the outer segments that arepositive with OS-2, but not with AO, exhibit a relativelylarger diameter (blue cones; thick arrows, B). (D) Drawn us-ing a copy of (A): black, blue cones; white, rod-like cells; andgray, green cones. (Magnification X900.)

BFIGURE 4. Distribution of a-transducin in the groundsquirrel retina in (A) radial and (B) tangential sections. Insection (A), the outer limiting membrane (OLM) and theretinal pigment epithelium (RPE) are marked with an openarrow and asterisk, respectively. The reaction is strongest inthe inner segments (arrowheads) and in the pedicles of thephotoreceptors (white arrow), but the outer segments (blackarrows) and the cytoplasm of the cell bodies are also stained.Section (B), taken at the outer segment level, shows that allphotoreceptors are stained by the antibody. The right side ofthe section represents a more vitreal level, which is why nu-merous inner segments are not stained. (Magnification (A)X820, (B) X550.)

On the translational level, our antisera against a-transducin and rod 7-PDE labeled rod-like cells andcones alike, whereas both the a/?-phosphodiesteraseand the rod cGMP-dependent cation channel antibod-ies gave negative immunoreactions. The most likelyreason for the labeling observed with the a-transducinand rod 7-phosphodiesterase antisera is a crossreactiv-ity with the corresponding cone enzymes. Somecrossreactivity between the former antibody (whichwas produced before the identification of distinct rodand cone subunits) and cone photoreceptors has al-ready been reported.34 The 7-PDE antibody was raisedagainst a synthetic peptide identical with part of therod-specific subunit.35 Rod and cone a-transducin



FIGl'RF. 5. T.ingciili.il s tat ion of tlit* g round squirrel retina

rc.K led with an antibody against fj\ -(ransdiu in. The section

is derived from the same series as shown in Figure li, so that

all photoreceplors can he identified with those of Figure 3.

A lew pholorecep lor profiles are stained, hut the majority of

the i ells are left unlabeled. All of the elements that are

stained with either OS-2 (Fig. 'Mi, thick and thin arrows) or

anli- ihodopsin (Fig. !Ui. thin arrows) are also rccogni /ed by

the ji\ -transdiu in antihodv. (Magnification X900.)

have a 78% amino acid sequence identity," whereasthe only cone 7-phosphodiesterase sequence reportedso far has an 80% identity to its rod counterpart'" (allsequences from cattle). Just as in the case of rhodop-sin, the specificity issue cannot be resolved with West-ern blotting due to the similarity in molecular weights.It can thus not be determined whether the rod-likecells and the two cone types contain identical or differ-ent proteins; however, the RNA blot hybridization ex-periments gave unambiguously negative results in allcases. Thus, our investigation confirms that antibodyprobe hybridization is less discriminatory towardminor sequence differences than DNA probe hybrid-ization.

In agreement with previously described results,we found two 111RNA transcripts (1.2 and 2.7 kb) for

mmfB

FlCilRF. 6. Consecutive tangential I)("•!) sections of theground stjiiirrel retina reacted with antibodies against the(A) \i\ and (B) [i'S sulxmits ol transdiuin. (A) Only a fewpholoreceplois are stained b\ the 0\ antihodv (shortarrows). (B) I he same elements of the pholoreceptor mosaicare also stained hy the fi'.i antihodv (short arrows). Both theouter and inner segment membranes ol these cells are la-beled. I he majority of the pholorei eptoi s are left unlabeledbv the fi\ antibody (A, long allows). The rf3 antibody (B).however, recognizes the outer segments of all cells (longarrows). (Magnification X900.)

BFIGURE 7. Distribution of 7-phosphodiesterase in theground squirrel retina in (A) radial and (B) tangential sec-tions. In section (A), the OI.M and the Rl'F". are marked withopen arrow and asterisk, respectively. The reaction is stron-gest in the pedicles (white arrow) and in the myoid of theinner segments (arrow heads). A few cells are also labeled inthe inner nuclear layer. Section (B), cut at the outer segmentlevel, shows that all photoreceptors are stained hy the anti-body. (Magnification (A) X820, (B) X550.)



al-transducin in human retina,37 and one in murineretina.38

However, we detected two (1.6- and 2.3-kb) a2-transducin transcripts in the mouse. Because only onetranscript is present in ground squirrel, it appears veryimprobable that the two mouse transcripts representdifferent isoenzymes specific for short- and middle-wavelength cones.

It has been reported that /?l-transducin immuno-reactivity is found in bovine rod outer and inner seg-ments, as well as in their synaptic terminals.15 In thesame investigation, /33-transducin was detected incones, whereas rods were left completely unstained.Essentially the same results were observed in retinalsections from monkey, in a study that used double la-beling with photopigment antibodies to confirm thatall cone types were stained by the antibody against/33-transducin.16 In our work with the ground squirrel,/31-transducin antiserum labeled the photoreceptorplasma membrane of rod-like cells and blue cones, and/33-transducin antiserum stained the outer segmentsof green cones and both the outer and inner segmentsof blue cones and rod-like cells. The occurrence of/31-transducin not only in rod-like cells but also in bluecones should be noted, as blue cones are reported tobe immunonegative for this protein in monkey ret-ina.16 On the other hand, the colocalization of bothj81- and the otherwise cone-specific /?3-transducin torod-like cells also is a novel finding and emphasizes thefact that these cells carry both rod- and cone-liketraits. The results of the RNA blot hybridization corre-spond well with these findings, with a /31-transducinsignal in the ground squirrel retina that is weaker thanthe one found in the rod-dominant human and murineretinas, and a correspondingly stronger signal for (33-transducin.

The rod-like cells of ground squirrel share mor-phologic and electrophysiologic features of both rodsand cones. We have postulated that 5. tridecemlineatusmay have a 502 nm photopigment with structural dif-ferences from the rhodopsin molecule in other mam-mals. As we could not demonstrate the presence of rodtransducin, rod phosphodiesterase, or the rod cGMP-gated channel in the ground squirrel retina, we alsosuggest that the rod-like cells of the ground squirrelmay be equipped with a phototransduction machineryconsisting of molecules that deviate from those foundin rods, whether they be specific for rod-like cells oridentical to those found in cones.

The cone is a phylogenetically older photorecep-tor than the rod. As rods may have evolved from coneson several different occasions, it remains an open andinteresting question if the rod-like cells in the groundsquirrel are a result of a relatively recent de novo rodphotoreceptor development from cone precursor

cells, or if they have evolved from rod cells that haveresumed some of their cone-like characteristics.

Key Words

cone photoreceptors, rod-like photoreceptors, Spertnophilustridecemlineatus, RNA blot analysis, immunocytochemistry

Acknowledgments

The authors thank Bernard K.-K. Fung, Rehwa H. Lee,Robert S. Molday, Jeremy Nathans, Winston A. Salser, To-shimishi Shinohara, Melvin I. Simon, and Allen Spiegel forthe gift of DNA probes and antibodies. The authors alsothank Clyde K. Yamashita for his invaluable help with theground squirrel colony and with the handling and dissectionof retinas; Katharina Ryden for technical assistance; and An-drea Viczian for sharing the human retinal RNA sample.

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