Vision Research 51 (2011) 288–295

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Vision Research

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A mammalian melanopsin in the retina of a fresh water turtle, the red-earedslider (Trachemys scripta elegans)

James R. Dearworth Jr. ⇑, Brian P. Selvarajah, Ross A. Kalman, Andrew J. Lanzone, Abraham M. Goch,Alison B. Boyd, Laura A. Goldberg, Lori J. CooperDepartment of Biology and Neuroscience Program, Lafayette College, 311 Kunkel Hall, Easton, PA 18042, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 July 2010Received in revised form 19 October 2010Available online 2 November 2010

Keywords:Opn4mIntrinsically-photosensitive retinal ganglioncells (ipRGC)PupilIrisPhotopigment

0042-6989/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.visres.2010.10.025

⇑ Corresponding author. Fax: +1 610 330 5705.E-mail address: dearworj@lafayette.edu (J.R. Dearw

A mammalian-like melanopsin (Opn4m) has been found in all major vertebrate classes except reptile.Since the pupillary light reflex (PLR) of the fresh water turtle takes between 5 and 10 min to achieve max-imum constriction, and since photosensitive retinal ganglion cells (ipRGCs) in mammals use Opn4m tocontrol their slow sustained pupil responses, we hypothesized that a Opn4m homolog exists in the retinaof the turtle. To identify its presence, retinal tissue was dissected from seven turtles, and total RNAextracted. Reverse transcriptase-polymerase chain reactions (RT-PCRs) were carried out to amplify genesequences using primers targeting the highly conserved core region of Opn4m, and PCR products wereanalyzed by gel electrophoresis and sequenced. Sequences derived from a 1004-bp PCR product werecompared to those stored in GenBank by the basic local alignment search tool (BLAST) algorithm andreturned significant matches to several Opn4ms from other vertebrates including chicken. Quantitativereal-time PCR (qPCR) was also carried out to compare expression levels of Opn4m in different tissues.The normalized expression level of Opn4m in the retina was higher in comparison to other tissue types:iris, liver, lung, and skeletal muscle. The results suggest that Opn4m exists in the retina of the turtle andprovides a possible explanation for the presence of a slow PLR. The turtle is likely to be a useful model forfurther understanding the photoreceptive mechanisms in the retina which control the dynamics of thePLR.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

1.1. The sluggish pupillary light reflex (PLR) in the turtle

The PLR is a sensorimotor reflex that responds to a wide rangeof illumination entering the eye to change the pupil diameter andoptimize image formation by the retina (Land & Nilsson, 2002;McIlwain, 1996). In comparison to mammals (Gamlin, 2000,2005; Kardon, 2005; Loewenfeld, 1993; McDougal & Gamlin,2008), the PLR in turtle is much slower and takes as long as5 min to achieve its smallest size (Dearworth et al., 2009, 2010;Granda, Dearworth, Kittila, & Boyd, 1995). The reason for the slow-ness is poorly understood, but since efferent signals by parasympa-thetic and sympathetic pathways controlling the PLR in turtleappear to be similar to other amniotes (Dearworth & Cooper,2008; Dearworth, Cooper, & McGee, 2007; Dearworth et al., 2009,2010; Iske, 1929), the source generating the sluggishness isthought to come from a photoreceptive mechanism in the retinaor some other central processing occurring in the brain.

ll rights reserved.

orth).

1.2. Melanopsin’s role in the PLR

In mammals, the PLR was originally believed to be controlledsolely by rod and cone photoreceptors (e.g. Alpern & Campbell,1962), but now includes involvement of intrinsically-photosensi-tive retinal ganglion cells (ipRGCs) expressing melanopsin (Lucas,Douglas, & Foster, 2001). These cells and their association withmelanopsin (Berson, Dunn, & Takao, 2002; Hattar, Liao, Takao,Berson, & Yau, 2002; Provencio et al., 2000; Warren, Allen, Brown,& Robinson, 2003) have been implicated in slow sustained pupilresponses (Gamlin et al., 2007; Lall et al., 2010; Lucas et al.,2003; McDougal & Gamlin, 2010; Mure, Rieux, Hattar, & Cooper,2009; Young & Kimura, 2008; see Bailes and Lucas (2010);Markwell, Feigl, and Zele (2010) for reviews), which with the moretransient information that they receive from photoreceptors(Belenky, Smeraski, Provencio, Sollars, & Pickard, 2003; Daceyet al., 2005; Güler et al., 2008), allow them to respond over adynamic range of illumination.

1.3. Aim of study

Does turtle possess melanopsin in its retina and could this novelopsin be involved in driving the turtle’s slow PLR? To begin

J.R. Dearworth Jr. et al. / Vision Research 51 (2011) 288–295 289

addressing this question, molecular methods were used to identifywhether or not a mammalian-like melanopsin (Opn4m) is ex-pressed in the retina of the turtle.

2. Methods

2.1. Animals

All animal care and experimental procedures were carried outin accordance with the Institutional Animal Care and Use Commit-tee (IACUC) at Lafayette College. Seven red-eared slider turtles(Trachemys scripta elegans) were purchased from Kons ScientificCo. Inc. (Germantown, WI, USA) weighing from 0.80 to 1.20 kg withcarapace lengths 18–23 cm. After animals were shipped to Lafay-ette College, they were placed in a 60-gallon tank, which wasequipped with filtering systems and housed within a warm animalsuite. Bricks were positioned at the center of the tank to serve as anisland for turtles to bask under 250 W infrared lamps. A timerturned lights on at 7:00 AM and off at 7:00 PM for a 12/12 hlight/dark cycle, and an electric heater maintained temperatureof the room at 27 �C. A radiometer (model DR-2000-LED, GammaScientific, San Diego, CA) measured radiant intensity of lights at3.86 � 10�2 W cm�2 sr�1. The tank and filtering system werecleaned weekly, and a floating fish food diet (Pro-Pet, L.L.C., St.Marys, OH) was administered to the turtles ad libitum every otherday.

Fig. 1. Analysis of RT-PCR (left four lanes of gel). Skeletal muscle and retinaexpressed CTYb, but only retina expressed Opn4m. No products were amplifiedusing total RNA from retina without reverse transcriptase (-RT) or by using primerswithout template (-T). The last lane on right shows a plasmid clone with the Opn4mproduct insert and its vector, after being digested by EcoR I.

2.2. RNA isolation, cDNA synthesis, polymerase chain reaction (PCR),and tissue expression

Procedures for RNA extractions were completed during the lightcycle for the housing room. Turtles were euthanized by intraperi-toneal injection of pentobarbital (65 mg/kg) and decapitated. Eyesof animals were quickly enucleated, and retinas and irises carefullydissected from their eyes. In one animal, a 7.62 cm hole was drilledinto its plastron to harvest liver and lung tissue; skeletal musclewas also collected from a leg. To prevent RNA degradation, the tis-sues were immediately preserved with RNAlater RNA StabilizationReagent (Qiagen, Germantown, MD). Pieces of tissues (630 mg)were homogenized using a Tissue Tearor™ rotor–stator homoge-nizer (Biospec Products, Inc., Bartleville, OK), and total RNA iso-lated using an RNeasy Mini Kit (Qiagen). Single stranded cDNAwas synthesized from the RNA (2 lg) using random primers andMoloney Murine Leukemia Virus Reverse Transcriptase (M-MLVRT) according to the instructions of their manufacturer (Promega,Madison, WI).

Primers (f: 50-GAC GGT TGA TGT TCC AGA CC-30 and r: 50-AGTGGC TGG TAA CAG TGG AAC G-30), which were designed byBellingham et al. (2006) to sequence a Xenopus Opn4m (GenBankDQ384639), were used to identify a partial coding sequence forOpn4m in turtle. To confirm the fidelity of the cDNA, PCRs wereperformed using additional primers, which were designed withthe online Primer3 program (Rozen & Skaletsky, 2000). Primerswere specific for mitochondrial cytochrome b (CYTb) (GenBankAF207750) (Willmore, English, & Storey, 2001) with expectedproduct size of 392-bp. PCRs using total RNA as a template wereincluded as negative controls to exclude genomic DNA contamina-tion. Primers were commercially purchased (Integrated DNATechnologies Inc., Coralville, IA), and PCRs done using illustra™Ready-To-Go PCR Beads (GE Healthcare Bio-Sciences, Piscataway,NJ). PCRs were done in total volumes of 25 ll with 20 pmol of eachprimer, 5 nmol each of the deoxynucleotide triphosphates (dATP,dCTP, dGTP, and dTTP), and�2.5 units of puReTaq DNA polymerasedissolved in the kit’s buffer, 10 mM Tris–HCl, 50 mM KCl, and1.5 mM MgCl2. A Robcycler Gradient 96 PCR machine (Stratagene

Inc., La Jolla, CA) was programmed to carry out the reactions.Opn4m primers made products using an initial denaturation stepat 95 �C for 5 min, then 95 �C for 1 min, 60 �C for 1 min, and72 �C for 1 min for 40 cycles, followed by a final elongation at72 �C for 10 min. Conditions for making CYTb products were thesame except instead of 60 �C for the annealing temperature 57 �Cwas used.

Quantitative real-time PCR (qPCR) was done on the iQ™5 Mul-ticolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA)and analyzed using the DDCt method, which normalizes the rela-tive expression levels by different genes. Reactions were done on96 well plates in total volumes of 25 ll: 0.5 ll of cDNA templatesynthesized as describe above, 12.5 ll iQ™ SYBR� Green Supermix,and 150 pmol for each primer. To determine the normalizedexpression level of a target gene relative to a reference gene, themethod assumes that the genes are amplified by primers with100% efficiency.

New primer pairs for qPCR were designed using the online Pri-mer3 program to target three house-keeping genes for normalizingOpn4m expression: (f: 50-ACT CTC GGC CAT TCC ATA CAT TGG-30

and r: 50-TCG GGT CAG GGT TGC GT TGT-30) for cytochrome b(CYTb) (GenBank AF207750); (f: 50-AGA CCC AAT GAG CAT ACACAG GTG CT-30 and r: 50-TGT TCG GCT ATG GGT TCG TTC G-30)for NADH-ubiquinone oxidoreductase subunit 4 (Nad4) (GenBankAF206699), and (f: 50-ACG CCA TCC TCC GTC TGG ATC T-30 and r:50-CGG CTG TGG TGG TGA AGC TGT A-30) for b-actin (Actb) (Gen-Bank DQ848990). Their expected product sizes were 85, 113, and96-bp, respectively.

Two-step amplification with annealing and elongation occur-ring together during the final step was done using the followingprotocol: 95 �C for 3 min followed by 40 cycles of 95 �C for 10 sand 68 �C for 30 s. The increase in fluorescence was measured dur-ing the annealing phase, and melt curves determined after 0.5 �Cincrements from 55 to 95 �C for 81 cycles of 10 s each. Primer spec-ificities were confirmed by single peaks in their melt curves, andefficiencies verified by the plots of the amplifications versus theirdilution series. Reactions for the dilution series were done in dupli-cates. To confirm consistency for the samples, reactions were donein triplicate.

2.3. Cloning, sequencing, and phylogenetic analysis

PCR products were analyzed by electrophoresis, stained withethidium bromide, and carefully cut from gels under ultra-violet

Fig. 2. Translation of a partial coding sequence for turtle Opn4m aligned with other tetrapod homologues. The percent identities similar to turtle are at the end of thealignment. Roman numerals with horizontal bars show locations of the seven transmembrane domains predicted for chicken. Residues highlighted by gray are conservedsites, and those surrounded by black boxes are the common features for the photopigment.

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Fig. 3. Primer specificities for qPCR. The primer pair for turtle Opn4m was designed from the 1004-bp sequence (Genbank HM197714) that was previously amplified by RT-PCR: (f: 50- CCT AAT GTC CAT CAC ACA GGC TCC A-30 and r: 50-CCG CAG AAG GCA TAC AGC TCA CA-30). The new amplicon generated by qPCR was 99-bp. (A) Melting curvesand (B) amplifications in serial dilutions for each primer pair: efficiencies were 102.4% (R2 = 0.986) for Opn4m, 101.1% (R2 = 0.963) for CYTb, 102.4% (R2 = 996) for Nad4, and106.6% (R2 = 0.997) for Actb.

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light. A MinElute PCR Purification Kit (Qiagen) was used to clean-up products either for direct sequencing or for cloning into a plas-mid vector (CR�2.1-TOPO�, Invitrogen, Carlsbad, CA) using the OneShot� Chemical Transformation Protocol. Plasmids were extractedfrom colonies by alkaline lysis using the QIAprep� Miniprep kit(Qiagen) and screened for successful insertions by EcoR I digests(New England Biolabs, Beverly, MA). Inserts from multiple cloneswere sequenced and verified in both directions using forwardand reverse primers. Sequences of PCR products and plasmids weredone by Joe Bednarczyk of the Molecular Genetics Core Facility ofthe Section of Research Resources at Penn State College ofMedicine.

The sequences from products were compared to those stored inGenBank by the basic local alignment search tool (BLAST). For theOpn4m product, the nucleotide sequence was translated to aminoacids and aligned with other vertebrate melanopsins using CLUS-TAL W (1.81) (Higgins & Sharp, 1988). Further phylogenetic analy-ses were conducted using MEGA version 4 (Tamura, Dudley, Nei, &Kumar, 2007). A neighbor joining (N-J) tree (Saitou & Nei, 1987) wasgenerated by bootstrapping with 1000 replications to determinestatistical significance for each branch (Dopazo, 1994; Rzhetsky &Nei, 1992). Evolutionary distances were computed using thePoisson correction method (Zuckerkandl & Pauling, 1965).

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Fig. 4. Opn4m expression level in the retina compared to other tissue types. (A) The bar2�DDCt versus different tissue types for each house-keeping gene. (B) The geometrical m

3. Results

3.1. Expression of Opn4m and CYTb

Fig. 1 shows results of RT-PCRs on a 1.3% agarose, 1� tris–ace-tate buffer (TAE) gel. Expression of Opn4m was observed in retinaas a 1004-bp product, but not in skeletal muscle. The fidelity oftemplates for both tissues was confirmed by products of 392-bpgenerated by primers for CYTb. Negative controls for reactionsshowed no products. Successful clones of the Opn4m product(example in Fig. 1, right lane) allowed sequencing of the 1004-bpfragment expressed by the turtle retina, which was submitted toGenBank (HM197714).

3.2. Charateristics of the turtle Opn4m sequence

Sequence alignments for the 333 amino acid translation of the1004-bp fragment best matched the vertebrate family of Opn4m(Fig. 2). The sequence had highest similarity to the chicken Opn4mat 85% identity and was complete for the seven transmembranedomains of the conserved core which is predicted for chicken(Bellingham et al., 2006; Palczewski et al., 2000). The fragmentpossessed the general features for the melanopsins and the

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G-protein-coupled receptor family of proteins. Properties includetwo cysteine (C) residues at sites 143 and 221 for disulfide bond for-mation, and the possession of the aspartate/arginine/tyrosine (D167/R168/Y169) tripeptide in the third transmembrane for binding trans-ducin (Bockaert & Pin, 1999). Site positions correspond to the humansequence aligned with turtle shown in Fig. 2. Tyrosine (Y) is also pres-ent at 146 and glutamate (E) at 215 to serve as possible counterionsites (Provencio, Cooper, & Foster, 1998; Terakita, Yamashita, &Shichida, 2000), and lysine (K) is located at site 340 in the seventhdomain to form a Schiff’s base (Menon, Han, & Sakmar, 2001).

3.3. qPCR

The relative expression of Opn4m in different tissues was deter-mined by qPCR. The specificity by primers was determined first byanalysis of melting curves (Fig. 3A) and validated by efficiencieswhich were all nearly 100% (Fig. 3B). Opn4m was specific to the ret-ina with expression 10 times greater than other tissue types (Fig. 4).The normalized expression pattern for Opn4m was consistent for allthree of the house-keeping genes that were used for comparison.

4. Discussion

4.1. Phylogeny

Turtle too can now be added to the list of vertebrates possessingOpn4m in its retina (Fig. 5). The sequence matches best to chicken

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Fig. 5. Phylogenetic tree with inclusion of turtle and rooted with the melanopsin sequeStatistical significance, confidence probability multiplied by 100, is shown at each branchtree are Mus musculus (NP_001122071); Rattus norvegicus (AY07268); Spalax ehrenbecrassicaudata (ABD38715); Monodelphis domestica (XM_001377332); Gallus gallus 2L(HM197714); Xenopus laevis (ABD37674); Danio rerio m1 (BC162681), m2 (AAL82577); G(NP_001079143), Gadus morhua a (Q804X9), b (Q804Q2); Branchiostoma belcheri (Q4R1I

(Bellingham et al., 2006; Tomonari, Takagi, Akamatsu, Noji, &Ohuchi, 2007; Torii et al., 2007), followed by Xenopus (Bellinghamet al., 2006), teleost (Bellingham, Whitmore, Philp, Wells, & Foster,2002; Bellingham et al., 2006; Cheng, Tsunenari, & Yau, 2009; Grone,Sheng, Chen, & Fernald, 2007; Jenkins et al., 2003), and mammals(Avivi, Joel, & Nevo, 2007; Gooley, Lu, Chou, Scammell, & Saper,2001; Hannibal, Hindersson, Nevo, & Fahrenkrug, 2002; Hattaret al., 2002; Pires et al., 2007, 2009; Provencio et al., 2000; Semo,Munoz Llamosas, Foster, & Jeffery, 2005). Since chicken possessesmultiple isoforms found in both Op4m and Opn4x lineages(Bellingham et al., 2006), it raises the possibility that other formsare present in turtle as well. The notion is supported by findings fromthe ruin lizard (Frigato, Vallone, Bertolucci, & Foulkes, 2006), anotherreptilian representative, which shows expression not of Opn4m, butinstead Opn4x, the first ortholog discovered in Xenopus (Provencio,Jiang, De Grip, Hayes, & Rollag, 1998).

Before the fat-tailed dunnart was examined for melanopsins, itwas thought that the loss of the Opn4x in mammals occurredsomewhere before the placental/marsupial split. The data fromthe dunnart now simply show that marsupials do not appear toexpress the Opn4x gene (Pires et al., 2007). If turtle remains tobe shown to possess only Opn4m and ruin lizard only Opn4x, thena loss may have occurred at some time within the evolution ofreptiles. Complete sequencing of Opm4m in turtle, particularlythe carboxyl tail, will be necessary to arrive at a firmer conclusion.The information also should provide insight on turtle’s recentrepositioned phylogeny among the diapsids (Hedges & Poling,1999; Rieppel, 1999; Zardoya & Meyer, 1998).

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nce from amphioxus (Koyanagi, Kubokawa, Tsukamoto, Shichida, & Terakita, 2005).. Scale bar indicates genetic distance. GenBank accessions from top to bottom in thergi (CAO02487); Homo sapiens (NP_150598); Felis catus (AAR36861); Smithopsis(a) (ABX10832), 2S (b) (ABX10833), 2SS (ABX10834); Trachemys scripta elegansallus gallus 1L (ABX10830), 1S (ABX10831); Podarcis sicula (Q4U4D2); Xenopus laevis4).

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4.2. Retinal expression site

If the turtle is similar to mammal, then the expressed cellularsite for Opn4m is likely within ipRGCs (Berson et al., 2002; Gooleyet al., 2001; Hannibal et al., 2002; Hattar et al., 2002; Pires et al.,2007, 2009; Provencio et al., 2000; Semo et al., 2005). If the expres-sion of Opnm4 in the turtle, however, is more similar to the patternseen in non-mammals (teleost, Xenopus, and chicken) (Bellinghamet al., 2006), then expression sites may widely overlap with Opn4x(Bellingham et al., 2002; Chaurasia et al., 2005; Cheng et al., 2009;Drivenes et al., 2003; Grone et al., 2007; Jenkins et al., 2003;Provencio, Jiang, et al., 1998; Tomonari, Takagi, Akamatsu, Noji, &Ohuchi, 2005) with inclusion of expression sites within the innernuclear layer and outer nuclear layer of the retina as well as inthe brain and skin of some species.

Further identifications by in situ hybridization for both Opn4mand Opn4x and neural tracing by immunohistochemistry will benecessary to elucidate the precise cell sites and their retinalcircuitry in turtle. Turtle is also complicated by possession of an iristhat is intrinsically photoresponsive (Sipe et al., 2010; vonStudnitz, 1933), better known as the photomechanical response(PMR) (see Barr (1989) for review). In the current study, sincethe expression of Opn4m (Fig. 4) in the turtle iris is relativelylow compared to the retina, it suggests that it may not be the func-tional homolog. Expresssion for Opn4x, however, certainly alsoneeds to be tested for presence in the turtle iris, especially sinceXenopus highly expresses Opn4x within its iris (Provencio, Jiang,et al., 1998). Analysis of the action spectrum for the turtle PMR(Sipe et al., 2010) reveals contribution from at least two differentphotopigments, one with a peak at 410 nm and another with apeak at 480 nm, which supports this possibility. Fits by templateequations suggest that contractions are triggered by multiplephotopigments in the iris including an opsin-based visual pigment,and for turtle perhaps an Opn4x ortholog favoring incorporationwith a vitamina A2 chromophore.

4.3. The sluggish PLR in turtle: possible function involving melanopsin?

Since the PLR is so slow in the turtle (Dearworth et al., 2009,2010; Granda et al., 1995), it is still difficult to understand thefunctional significance. Indeed, because of its slowness, the PLRwas argued to be non-existent in turtle (von Studnitz, 1933; Walls,1942). The act of accommodation, the lens moving in and out of theiris, was thought instead to control pupil size. Although accommo-dation certainly influences pupil movements in turtle (Henze,Schaeffel, Wagner, & Ott, 2004), it does not appear to overridethe sluggish PLR. Perhaps the answer lies in their evolutionary his-tory and ecological constraints. It may be that turtles, similar toamphibians and fish, which also possess comparable slow PLRs(Bailes, Trezise, & Collin, 2007; Cornell & Hailman, 1984; Douglas,Collin, & Corrigan, 2002; Douglas, Harper, & Case, 1998; Henning,Henning, & Himstedt, 1991; Kuchnow, 1971), have other compen-satory factors to allow a slower photoreceptive system driven bymelanopsin to operate and control its pupil at a more efficientand less energetic cost (Cornell & Hailman, 1984; Dearworthet al., 2010). One example is possession of retinomotor responses(Ali, 1971; Drenckhahn & Wagner, 1985), and turtles certainly havea unique physiology with notable tolerate hypoxia (see Storey(2007) for review). Mammals on the other hand possess dynamicsthat appear to have evolved more recently with more transitiveproperties which although may appear dominant are still workingin coordination with a more primitive slower system. Furtherstudy is needed to understand how photoreception mechanismsin the retina and iris work together to control the slow PLR inthe turtle.

Acknowledgments

The authors thank Mrs. Paulette McKenna for her secretarialsupport, and Mr. Phil Auerbach for his technical assistance. We alsothank Drs. Laurie Caslake, Robert Kurt, Debra Walther, Wayne Lei-bel, Elaine Reynolds, Manuel Ospina-Geraldo (Department of Biol-ogy and Neuroscience Program, Lafayette College), and IgnacioProvencio (Department of Biology, University of Virginia), whoserved as consultants for the molecular methods. Funding was sup-ported through the EXCEL scholars program and by a Richard KingMellon Fellowship awarded by the Academic Research Committeeat Lafayette College.

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