remodeling of the cone photoreceptor mosaic during …web.stanford.edu/group/fernaldlab/pubs/2006...

Fax +41 61 306 12 34E-Mail [email protected]

Original Paper

Brain Behav Evol 2006;68:241–254 DOI: 10.1159/000094705

Remodeling of the Cone Photoreceptor Mosaic during Metamorphosis of Flounder (Pseudopleuronectes americanus)

Kim L. Hoke a Barbara I. Evans b Russell D. Fernald a, c

a Program in Neurosciences, Stanford University School of Medicine, Stanford, Calif. , b School of Biological Sciences, Lake Superior State University, Sault Sainte Marie, Mich. , c Department of Biological Sciences, Stanford University, Stanford, Calif. , USA

Introduction

A major issue facing developmental biologists is how undifferentiated cells give rise to adult patterns with mul-tiple different cell types. Retinal development has been a focus for studies on cell differentiation because photore-ceptors form precise arrays or mosaics. These regularly spaced arrangements of cells have predictable cell sub-types relative to neighboring cell types. Photoreceptor phenotypes within a mosaic can be distinguished by morphology and by expression of the light-sensitive op-sin photopigments. Two primary hypotheses have emerged to explain photoreceptor mosaic patterning in vertebrates: one postulating that a series of sequential in-ductive events specify cell fate based on preexisting mo-saic position as found in Drosophila photoreceptor devel-opment [Raymond et al., 1995; Stenkamp and Cameron, 2002; Raymond and Barthel, 2004]; and a second hypoth-esis proposing that differential adhesion between cell subtypes establishes position within the mosaic based on previously determined cell fate [Galli-Resta, 2001; Mo-chizuki, 2002; Reese and Galli-Resta, 2002; Tohya et al., 2003]. These two hypotheses generate distinct predic-tions as to the order of mosaic formation and cell differ-entiation. The rapidity of retinal development in most retinal model systems makes distinguishing the timing of developmental events difficult. Studies of retinal devel-opment in these model systems are complemented by

Key Words Retina � Opsins � Differentiation � Cell division � Development � Patterning � Cone � Photoreceptor � Mosaic � Teleost

Abstract The retinal cone mosaic of the winter flounder, Pseudopleu-ronectes americanus , is extensively remodeled during meta-morphosis when its visual system shifts from monochromat-ic to trichromatic. Here we describe the reorganization and re-specification of existing cone subtypes in which larval cones alter their spatial arrangement, morphology, and op-sin expression to determine whether mechanisms control-ling cell birth, mosaic position, and opsin selection are coor-dinated or independent. We labeled dividing cells with tritiated ( 3 H) thymidine prior to mosaic remodeling to deter-mine whether existing cone photoreceptors change pheno-type. We also used in situ hybridization to identify mosaic type and opsin expression in transitional retinas to under-stand the sequence of transformation. Our data indicate that in the winter flounder retina the choice of new opsin species and the cellular rearrangement of the mosaic proceed inde-pendently. The production of the precise cone mosaic ar-rangement is not due to a stereotyped series of sequential cellular inductions, but rather might be the product of a set of distinct, flexible processes that rely on plasticity in cell phenotype. Copyright © 2006 S. Karger AG, Basel

Received: October 26, 2005 Returned for revision: December 20, 2005 Accepted after revision: May 26, 2006 Published online: July 20, 2006

Kim HokeUniversity of Texas at Austin1 University Station C0930Austin, TX 78712 (USA)E-Mail [email protected]

© 2006 S. Karger AG, Basel

Accessible online at:www.karger.com/bbe

http://dx.doi.org/10.1159%2F000094705

Hoke /Evans /Fernald

Brain Behav Evol 2006;68:241–254242

work in vertebrates in which protracted periods of retinal patterning allow greater resolution of the temporal rela-tionships in photoreceptor differentiation.

Species that move to a new habitat as part of their life history typically encounter changes in their visual envi-ronment and remodel their retinas to accommodate such new visual conditions [Beaudet and Hawryshyn, 1999; Evans, 2004]. Because vision can be affected by photore-ceptor arrangement and spectral sensitivities, remodel-ing these features might improve the match between vi-sual function and habitat, as described extensively in a variety of teleost fish species. Teleost retinal remodeling includes several possible developmental changes: (1) new photoreceptor classes may be added by new cell addition

or by changes in existing cone morphology, opsin chro-mophore, or opsin subtype [Evans and Fernald, 1993; Ev-ans et al., 1993; Archer et al., 1995; Hope et al., 1998; Shand et al., 1999, 2002; Novales Flamarique, 2000; Zhang et al., 2000; Haacke et al., 2001; Chinen et al., 2003; Cheng and Novales Flamarique, 2004; Mader and Cam-eron, 2004; Takechi and Kawamura, 2005]; (2) cell class-es may be lost by cell death [Bowmaker and Kunz, 1987; Beaudet et al., 1993; Novales Flamarique and Hawryshyn, 1996; Novales Flamarique, 2000; Deutschlander et al., 2001; Allison et al., 2003], or (3) cells might move in rela-tion to one another to form different arrangements [Ev-ans and Fernald, 1993; Shand et al., 1999; Haacke et al., 2001; Helvik et al., 2001]. In the winter flounder, Pseudo-pleuronectes americanus , photoreceptors change the peak wavelength sensitivity and physical arrangement when larvae settle into deeper water, providing an excellent model for examining the relationship between photore-ceptor cell birth, opsin expression, and physical posi-tion.

P. americanus progresses from a pelagic bilaterally symmetric fish to a benthic flatfish approximately two months after hatching. During this metamorphosis, im-portant changes in photoreceptor phenotypes occur as fish transition from a monochromatic to trichromatic vi-sual system [Evans and Fernald, 1993]. Premetamorphic larval retinas contain one photoreceptor type arranged in a hexagonal array ( fig. 1 ). Photoreceptors in larvae are middle-wavelength-sensitive cones ( � max 519 nm) [Evans et al., 1993] that express photopigment RH2 opsin [Ma-der and Cameron, 2004]. In contrast, retinas of post-metamorphic flounder contain rod photoreceptors as well as single and double cones with new opsin types ex-pressed [Evans and Fernald, 1993]. Rods appear during metamorphosis and are inserted among cones without an obvious pattern, as is typical for teleosts retinas. The cones in a postmetamorphic retina form a square mosaic, which is a repeating array of double cones surrounding one single cone ( fig. 1 ). The single cones absorb blue light ( � max 457 nm) [Evans et al., 1993] and express SWS2 op-sin mRNA [Mader and Cameron, 2004]. One partner in the double cone is maximally sensitive to green light ( � max 531 nm) [Evans et al., 1993] and expresses RH2 op-sin gene [Mader and Cameron, 2004], whereas the other member of the pair can be maximally sensitive to either the same ( � max 531 nm) or longer ( � max 547 nm) wave-lengths [Evans et al., 1993] and express LWS opsin mRNA [Mader and Cameron, 2004]. Why the pre- and post-metamorphic cones that contain RH2 opsin have differ-ent spectral sensitivities is unknown [Mader and Cam-

Fig. 1. We categorized individual cone photoreceptors as mem-bers of either hexagonal or square mosaic regions by analyzing their nearest neighbors within a unit A1 – A 3 combined with the identity of adjacent mosaic units B1, B2. In these schematic illus-trations, circles represent single cone photoreceptors. A1 A com-plete unit of the hexagonal mosaic characteristic of premetamor-phic flounder retina has a central cone (drawn in gray) surround-ed by six uniformly spaced single cones. A2 A complete square mosaic unit characteristic of postmetamorphic flounder retina consists of a central single cone (drawn in gray) surrounded by four pairs of regularly spaced double cones. Note that we refer to this as a complete square mosaic unit for simplicity, although the ratio of double to single cones is actually 2: 1 in the retina. A3 The gray circle indicates half of a double cone in a square mosaic, which is positioned very closely to one other cone (drawn in black above the gray circle) and more distantly from other neighbors. B1 Two adjacent hexagonal mosaic units are shown as found in pre-transitional flounder retina, with the two central cones marked by the gray circles. B2 Two adjacent square mosaic units, characteristic of postmetamorphic flounder retina, differ from the adjacent hexagonal units in B1 in that the central cones of the two mosaics, drawn in gray, are not nearest neighbors.

Remodeling of the Cone Photoreceptor Mosaic

Brain Behav Evol 2006;68:241–254 243

eron, 2004]. Neither microspectrophotometric study found evidence for mixing opsin types nor for changes in chromophore usage [Evans et al., 1993; Mader and Cam-eron, 2004]. Furthermore, no other RH2 opsin sequences or alternative splice forms were identified by PCR from larval and postmetamorphic tissue [Mader and Camer-on, 2004; this paper and unpubl. results using four degen-erate primer pairs]. Thus, during metamorphosis the hexagonal array of single cones becomes a square array of single and double cones with new opsin genes ex-pressed, each with distinct absorbance peaks.

How does this dramatic retinal transformation occur? Postmetamorphic mosaics may be formed from existing larval cones or from new cones. Mosaic patterning could rely on mechanisms such as opsin expression directing mosaic position, mosaic position determining opsin sub-type, or independent regulation of opsin expression and mosaic patterning. Based on light and electron micro-scopic analysis in metamorphic retina of another flatfish, Shand et al. [1999] found that individual cones fuse to form postmetamorphic double cones, but no studies have linked new cell addition, mosaic arrangement, and pho-topigment expression during metamorphosis. Here we analyze the retinas of flounder as the cone mosaic in their retina is transformed during metamorphosis from a hex-agonal array with a single cone type to a square array with multiple cone types. For brevity we will refer to these as ‘transitional retinas’. We labeled dividing cells with triti-ated ( 3 H) thymidine prior to mosaic remodeling to deter-mine whether existing cone photoreceptors change phe-notype. We examined morphology and used in situ hy-bridization to identify mosaic type and opsin expression in transitional retinas to understand the sequence of transformation and determine whether mechanisms controlling cell birth, mosaic position, and opsin selec-tion are coordinated or independent.

Materials and Methods

Animals Treatment of animals was in compliance with protocols ap-

proved at the institutions providing the animals (University of Oregon, Eugene, Oreg.; Stanford University, Stanford, Calif.; Ma-rine Biological Laboratory (MBL), Woods Hole, Mass., USA; In-stitute for Marine Bioscience, Halifax, Nova Scotia, Canada; Ma-rine Science Center, New Brunswick, Canada). Flounder used for autoradiography and morphology were reared at Woods Hole Oceanographic Institute (WHOI). For cloning opsin cDNA, we used tissue from larval flounder reared in captivity (8–15 days post hatch (dph); a gift of S. Douglas, Institute for Marine Biosci-ences, National Research Council, Halifax, Nova Scotia) and from

wild-caught postmetamorphic flounder (MBL). For in situ hy-bridization experiments, flounder were reared in captivity (a gift from M. Costello and L. Lush, Huntsman Marine Science Centre, St. Andrews, New Brunswick).

We studied animals at several developmental stages to inves-tigate changes in retinal structure during metamorphosis. The developmental stage in winter flounder is most accurately pre-dicted by animal size rather than true chronological age [Wil-liams, 1902; Chambers and Leggett, 1987]. We used lens diameter to reflect animal size because it is straightforward to measure and provides a reliable estimate of body size, and hence developmental stage, in two independent analyses (data not shown). We mea-sured the largest lens diameter in each series of retinal sections using previously calibrated microscope images (Image 1.5, Wayne Rasband, NIH) and checked this estimate by multiplying the number of serial sections that included the lens by the section thickness, which gave the same result (not shown).

Cone Mosaic Patterns and Morphology We assessed cone morphology to determine the location of

square mosaics and double cones within transitional retina. Wild-caught winter flounder from the Woods Hole, Mass. area were kept at the Environmental Systems Lab at WHOI until gravid. Gametes were taken and eggs fertilized, then eggs developed in hatching trays in running seawater (7–10 ° C) on a 14 h light:10 h dark cycle. After hatching, larvae were transferred to black plastic rearing tanks (10–20 l) and fed protozoa, rotifers, copepods or brine shrimp. We observed several cohorts of larvae through the stages of hatching until metamorphosis was complete and fixed the animals for 2–3 days in Bouin’s fluid [Humason, 1979] prior to tissue processing (lens diameters 68–150 � m, 1–120 dph, n = 102 of all stages; n = 20 transitional retina, 120–140 � m lens di-ameters).

Fixed flounder were dehydrated through an ethanol series, embedded in resin (Immunobed, Polysciences, Warminster, Pa., USA) and sectioned on a microtome at 2–3 � m through the dor-soventral axis. The lack of skeletal calcification in flounder larvae allowed sections to be made through the entire animal, simplify-ing tissue orientation, which can be complex due to eye migration at metamorphosis. We made coronal sections from the nasal to the temporal pole of the eye. Depending on where the parallel sec-tions cut through the eyecup ( fig. 2 ), tissue sections provided ei-ther tangential views in which cone mosaic patterns (i.e., hexago-nal or square mosaic arrangements) were visible, or, more cen-trally, radial views in which cone morphology (presence of single or double cones) was assessed. In flounder eyes used to analyze mosaic patterns and for alkaline phosphatase in situ hybridiza-tion (described below), we determined nasotemporal and dorso-ventral position of tissue sections by charting the progressive ap-pearance of both landmarks within the eye (e.g., optic nerve) as well as external structures such as the brain or nose. We took pho-tomicrographs with a Nikon Eclipse E600 Microscope (Nikon, Melville, N.Y., USA) and Micropublisher 5.0 RTV camera (QIm-aging, Burnaby, B.C., Canada) and calibrated them (QCapture-Pro, QImaging).

Autoradiography To discover whether cones present in larvae are retained after

metamorphosis as members of double cones in square mosaics, we labeled dividing cells prior to the metamorphic transition and


Brain Behav Evol 2006;68:241–254 245

assessed the fate of these pre-transition cones following meta-morphosis. We labeled proliferating cells by treating larvae with tritiated thymidine, which is incorporated into the DNA of mi-totically active cells. We dehydrated 3 H-thymidine (1 g/l, 1 mCi/ml; ICN Pharmaceuticals, Costa Mesa, Calif., USA) and then re-hydrated with an equal volume of seawater. Larvae were im-mersed in the 3 H-thymidine labeled seawater for 1 h, and then given two 30-min rinses in non-radioactive thymidine (1 g/l in seawater, Sigma, St. Louis, Mo., USA) to clear any residual isotope. We sampled two animals immediately in each experiment, trans-ferred the remainder to a separate container of fresh seawater, and reared them through metamorphosis (n = 11 labeled pre-transi-tion and reared until transition).

We processed resin-embedded sections taken from radioac-tively labeled tissue (prepared as described above) for autoradiog-raphy by dipping the slides in photographic emulsion (Kodak

NTB-2, VWR, Westchester, Pa., USA) and exposing the emulsion to radioactive emissions from the incorporated 3 H-thymidine for several weeks in the dark at 4 ° C. Slides were developed (Kodak D-19 developer, VWR), fixed (Kodak Rapid fix, VWR), and stained with cresyl violet, a non-specific cellular stain.

We viewed retinal tissue sections at high magnification (Nikon Eclipse E600 Microscope and Micropublisher 5.0 RTV camera) and identified cells dividing at the time of radiolabeling by the presence of black silver grains in the emulsion above the cell body. We considered a cell labeled if it had at least 5 times the number of silver grains over the nucleus as compared with background areas. Retinal tissue taken from animals sampled immediately following radiolabeling showed the number and location of cells undergoing mitosis at the time of 3 H-thymidine treatment. Reti-nal tissue from animals that survived for extended periods fol-lowing 3 H-thymidine incorporation had cells that may or may not have retained the label. For example, cells that stopped dividing and differentiated shortly after 3 H-thymidine incorporation would have retained their label, whereas cells that underwent sev-eral mitotic divisions would have diluted the radiolabel to unob-servable levels because the label was divided among all daughter cells. Thus radioactively labeled cells observed at long survival times represented the subset of dividing cells that differentiated shortly after 3 H-thymidine exposure.

Winter Flounder Opsin cDNA Sequences Because flounder opsin cDNA sequences were not available

when we began these experiments, we isolated opsin cDNA se-quences in larval and postmetamorphic flounder. We later found these to be 99% identical to published sequences in regions of overlap (GenBank accession numbers AY631037, AY631038, AY631039), and we follow the published nomenclature through-out [Mader and Cameron, 2004]. We identified additional nucle-otide sequences from the 3 � untranslated region for LWS (Gen-Bank accession number DQ225171).

To isolate opsin cDNA sequences, we performed degenerate reverse transcription polymerase chain reaction (RT-PCR) on mRNA extracted from retina of postmetamorphic flounder and from whole larvae. Larvae were flash-frozen whole in liquid ni-trogen then thawed and stored for subsequent mRNA extraction (RNAlater; Ambion, Austin, Tex., USA). We anesthetized post-metamorphic flounder in 0.3% MS-222 (Sigma) prior to rapid cervical transection. Eyes were removed, lenses dissected out, and complete eyecups frozen in liquid nitrogen for mRNA isolation. Larval and postmetamorphic tissue was disrupted in liquid nitro-gen in a mortar and pestle. We then added lysis solution and ex-tracted total RNA (MicroPoly(A)Pure kit; Ambion).

We designed PCR primers by first aligning fish cone opsin se-quences [Clustal W; Chenna et al., 2003 and BlockMaker], then designing degenerate primers [CODEHOP; Rose et al., 1998; http://blocks.fhcrc.org/blocks/codehop.html]. We synthesized single-stranded cDNA (SUPERSCRIPT First-Strand Synthesis System for RT-PCR; Invitrogen, Carlsbad, Calif., USA) and per-formed PCR (upper primers 5 � TCACCGCCCAGCACAARA-ARYTNMG or GGCATGCAGTGCTCCTGYGGNCCNGA with lower primer 5 � TGCTTGTTCATCAGCACGTAGATNAYN GGRTT). We amplified RT-PCR reactions in a thermal cycler (PTC-100; MJ Research, Boston, Mass., USA; 35 cycles of 94 ° C30 s denaturation, 55 ° C 1.5 min annealing, and 72 ° C 3 min ex-tension). PCR products were purified (GENECLEAN; Qbiogene,

Fig. 2. Square cone mosaic arrangements occur near the periph-eral margin in metamorphic flounder (lens diameter 139 � m) as the photoreceptor array changes from hexagonal cone mosaics typical of premetamorphic animals to square mosaics character-istic of postmetamorphic animals. Schematic illustration of eyes indicating location and planes of sections pictured in B– E . A Planes of section near the edge of the eye produce tangential views of the photoreceptor mosaic arrangements (B– D ), whereas more centrally located planes in the same eye show the photore-ceptor layer in radial section (E ). B Low magnification view of a section near the nasal margin showing brain in relation to the retina. D = Dorsal; V = ventral. Scale bar = 25 � m. C High-mag-nification photomicrograph of the section in B at the level of the cone photoreceptors. The dorsal portion (top) has cones arranged in a hexagonal mosaic as in premetamorphic flounder (fig. 1 ; A1), whereas the ventral portion (bottom) has cones arranged in a square mosaic as in the postmetamorphic retina (fig. 1 ; A2). The dashed line indicates a transitional zone between square and hex-agonal mosaics. Scale bar = 10 � m. D Section through the tempo-ral margin of the same retina shows cones arranged in a square mosaic (top), whereas more ventrally the cones are in a hexagonal mosaic (bottom). The dashed line indicates transitional zone be-tween square and hexagonal mosaics. Scale bar = 10 � m. E This radial section shows a double cone (arrow) close to dorsal margin (asterisk). Other double cones are visible in this region, however, more central photoreceptors in the right half of image are single cones (arrowhead indicates one example). Scale bar = 10 � m. F Higher magnification view of square cone mosaic region on left of D . Top shows photomicrograph of cones in square mosaic for-mation, with individual cones marked on the image below, and two square mosaic units outlined. Scale bar = 10 � m. G Higher magnification view of double cone region in E . Double cone la-beled in E is again indicated by black arrow. Note the nucleus and inner segment of labeled cone are thicker compared to the single cones (H ) and have a dorsoventral division marking paired cones. Scale bar = 10 � m. H Higher magnification view of single cone region in E . Single cone labeled in E is indicated by white arrow-head. Cone morphology is slender and regular compared to mix of double and single cones in H . Scale bar = 10 � m.



Vista, Calif.), cloned into a vector (TOPO TA Cloning Kit for se-quencing; Invitrogen), and then sequenced (Biotech Core, Moun-tain View, Calif., USA).

We cloned and sequenced the 3 � untranslated region of all cone opsins to produce in situ hybridization probes to less con-served regions of the opsin gene to avoid cross-hybridization be-tween opsin types. We used single-stranded cDNA templates in PCR amplifications with one specific opsin primer designed from our degenerate PCR sequences and an oligo(dT) adapter using the conditions described above. Amplified products were purified (GENECLEAN) and either sequenced directly or cloned for sub-sequent sequencing (TOPO TA cloning). Note that PCR amplifi-cations from larval and postmetamorphic tissue spanning the complete RH2 opsin sequence yielded no differences in coding or untranslated regions between these stages of fish in which cone absorption spectra differ [Evans et al., 1993; Mader and Cameron, 2004]. Opsin probes used for in situ hybridization ranged in length from 300–600 bp.

In situ Hybridization: Alkaline Phosphatase Visualization We performed in situ hybridization on retinal tissue from

flounder in metamorphic transition to describe the general pat-tern of individual opsin expression throughout metamorphosing retinas. We generated specific RNA probes for in situ hybridiza-tion for all opsin sequences from TOPO TA templates purified using Qiaprep Spin Miniprep Kit (Qiagen, Chattsworth, Calif., USA). Plasmids were linearized using restriction enzymes, puri-fied with GENECLEAN, then used as template in MAXIscript In vitro Transcription Kit (Ambion) with either digoxigenin- or flu-orescein-labeled UTP (Roche Molecular Biochemicals (Roche), Indianapolis, Ind., USA). RNA probes were precipitated prior to partial alkaline hydrolysis in 60 m M sodium carbonate and 40 m M sodium bicarbonate pH 10.2 at 60 ° C. We targeted an aver-age size of 200 bp probe fragments for greater access to the target RNA in the tissue. We again precipitated probes and measured yield by comparison with RNA mass standards, then diluted probes in 1! hybridization buffer (Sigma) for long-term stor-age.

We fixed whole flounder (lens diameters 132–143 � m, 42–56 dph) in formalin for 24 h prior to storage in saline, placed tissue in 30% sucrose at 4 ° C overnight for cryoprotection, and sectioned embedded tissue (Tissue-Tek O.C.T. Compound; Sakura Finetek, Torrance, Calif., USA) in a cryostat (Microm, Zeiss, Thornwood, N.Y., USA) at 10–16 � m. After sectioning, slides were heated at 40 ° C or dried in a vacuum chamber for 1 h and processed imme-diately as follows. After rehydration in 3.7% formaldehyde, we digested tissue in Proteinase K (Ambion; 10 � g/ml) for 10 min at room temperature. Slides were re-fixed in 3.7% formaldehyde in PBS, rinsed in 2 ! SSC, then dipped in 70% ethanol and allowed to dry. Slides were hybridized overnight at 55 ° C with probes di-luted to 1 ng/ � l in hybridization buffer. We washed slides in de-creasing SSC concentrations to a final concentration of 0.1 ! SSC at 55 ° C for 20 min. Nonspecific antibody binding was blocked (5% heat inactivated normal goat serum (Pel-Freez, Rogers, Ariz., USA), 2% Boehringer Blocking Reagent (Roche), 0.1% Tween-20 in Maleic Acid Buffer (MAB)) for 1 h at room temperature. We then applied sheep alkaline phosphatase-conjugated anti-digoxi-genin antibody (Roche: 1: 1,000 in blocking solution) overnight at 4 ° C or at room temperature for 3–4 h. Slides were washed in MAB with 0.3% Tween-20, rinsed in AP development buffer (0.1 M Tris

pH 9.5, 50 m M MgCl 2 , 0.1 M NaCl, 0.1% Tween 20), and incubated in BM Purple (Roche) for 4–24 h. When sufficient purple color developed in the outer retina to be visible under a dissecting scope, we coverslipped slides in Fluoromount-G (Southern Bio-technologies Associates, Birmingham, Ala., USA).

We viewed tissue under 400 ! or 1,000 ! total magnification using brightfield illumination (Axioskop, Zeiss) and captured digital images using a SPOT digital camera and SPOT Advanced software (Diagnostic Instruments, Sterling Heights, Mich., USA). Preliminary experiments confirmed expression of RH2, SWS2, and LWS opsin mRNA in postmetamorphic flounder in the same pattern as proposed by Mader and Cameron [2004].

In situ Hybridization: Double-Label Fluorescent Visualization To localize RH2, LWS, and SWS2 opsin mRNAs simultane-

ously, we used double in situ hybridization on transitional retinal tissue sections cut in varying planes to allow tangential views of the orderly array of photoreceptors. To increase the number of locations in the eye in which photoreceptor mosaics were sec-tioned tangentially, we rotated the eyecup several times during sectioning such that portions of each eye were cut coronally and others longitudinally. This sectioning method was biased toward the nasal and central retina and away from the less accessible tem-poral pole. In contrast to the other tissue sections used for mor-phology, autoradiography, and alkaline phosphatase in situ hy-bridization (described above), we could not reconstruct the dor-soventral or nasotemporal position of each mosaic type within these retinas due to the varying section planes.

After sectioning, slides were heated at 40 ° C or dried in a vac-uum chamber for 1 h and subsequently processed. We digested tissue in Proteinase K, re-fixed, rinsed, and dehydrated as above. Slides were hybridized overnight at 55 ° C as above with pooled digoxigenin- (antisense LWS and SWS2) and fluorescein-labeled (antisense RH2) probes diluted to 0.25 or 1 ng/ � l in hybridization buffer. We removed nonspecific hybridization in decreasing SSC concentrations to a final concentration of 0.1 ! SSC at 55 ° C for 20 min. Between steps in the fluorescent detection protocol, we rinsed slides in PBS with 0.3% Tween-20. We blocked nonspe-cific antibody binding for 1 h in 1% blocking buffer in PBS (PBSB; NEN, Boston, Mass.) and incubated overnight at 4 ° C or for 3–4 h at room temperature in sheep anti-digoxigenin antibody conju-gated with horseradish peroxidase (HRP) (Roche; 1: 250 diluted in PBSB). We applied biotinyl tyramide diluted 1: 50 in Amplifica-tion Diluent (TSA kit, NEN) for 5–7 min and incubated in avidin-conjugated Alexa546 (1: 1,000 in PBS; Molecular Probes, Eugene, Oreg., USA) for 1.5 h at room temperature. We quenched peroxi-dase remaining from the initial probe detection (15 min, 0.3% hydrogen peroxide in PBS) and blocked free biotins remaining after incubation in the avidin conjugate using Avidin-Biotin blocking kit (Vector), then applied HRP-conjugated anti-fluores-cein antibody (Roche, 1: 250 in PBSB) for 3–4 h at room tempera-ture. Biotinyl-tyramide treatment (TSA kit, NEN) was followed by avidin-conjugated fluorescein (1: 1,000 in PBS, Vector) and coverslipping in Fluoromount-G.

The density and small size of the cones required confocal mi-croscopy to minimize overlap of signals from cells in different planes of focus. We used 1- � m optical sections to analyze fluo-rescently labeled sections using a confocal microscope (Zeiss LSM510, Thornwood, N.Y., USA or Molecular Dynamics Multi-Probe 2010 Amersham Biosciences, Sunnyvale, Calif., USA).


Brain Behav Evol 2006;68:241–254 247

Preliminary experiments verified that the double label proto-col did not produce any cross-reaction between digoxigenin and fluorescein detection in postmetamorphic flounder retinas. Omission of digoxigenin-labeled probes, anti-digoxigenin anti-body, the initial biotinyl tyramide step, or biotinylated Alexa546 eliminated fluorescence in the red channel without influencing fluorescein label. Conversely, omitting components important for fluorescein detection preserved Alexa546 localization while re-moving green fluorescence. In addition, we performed pairwise combinations of in situ hybridization probes to identify co-local-ization of any opsins in cones of transitional or postmetamorphic fish. RH2 and LWS opsin probes were never co-localized in the same cell with each other or with SWS2 opsin expression (not shown).

Localization of Opsin Expression within Cone Photoreceptor Mosaics Because retinas from animals in metamorphic transition have

some regions with hexagonal mosaics and some with square mo-saics, we classified the mosaic type using tangential sections of transitional retinas and identified opsins expressed within each mosaic type based on confocal sections of retinas processed with double label fluorescent in situ hybridization. Images containing separate fluorescent signals were converted to false color (Adobe Photoshop 5, Adobe Systems., Inc., San Jose, Calif., USA) and merged to combine information on positions of cones expressing opsins typical of postmetamorphic flounder (pooled LWS and SWS2 probes) and opsins expressed in both pre- and postmeta-morphic animals (RH2) [Mader and Cameron, 2004]. We con-verted images to grayscale for mosaic classification, and marked the locations of individual cells with circles. We then categorized mosaic type by analyzing the circles alone without the original photomicrograph using criteria described below. After mosaic classification, we compared the mosaic pattern with the original color image to determine where the opsins LWS/SWS2 (post-metamorphic) and RH2 (pre- and postmetamorphic) were ex-pressed within the mosaic.

We based mosaic classifications on the distinct spatial differ-ences between square and hexagonal mosaics as shown in fig-ure 1 . In an ideal hexagonal mosaic, all cone cells are equidistant from one another, and each cone can be considered as a central cone surrounded by six equally spaced neighbors ( fig. 1 , A1; one hexagonal ‘mosaic unit’). In a square mosaic on the other hand, cone cells have one of two possible positions: cones can be central, surrounded by four pairs of double cones ( fig. 1 , A2; one square mosaic unit), or they can be part of a double cone, in contact with its twin and more distant from the central cone or neighboring double cones ( fig. 1 , A3; one double cone).

Based on these differences in cone neighbor positions, we classified mosaics in transitional f lounder retinal tissue pro-cessed for double in situ hybridization into four categories: hex-agonal, partial hexagonal, partial square, and square. Distortions in sectioning caused an imperfect recreation of mosaics, so clas-sification was based on both the single mosaic components ( fig. 1 , A1–A3) and on the presence of neighboring mosaic units (hex-agonal, fig. 1 , B1, and square, fig. 1 , B2) using the following cri-teria:

Hexagonal Two adjacent hexagonal mosaic units ( fig. 1 , B1) No double cones ( fig. 1 , A3)

Partial No full square mosaic units ( fig. 1 , A2) hexagonal One or two hexagonal mosaic units ( fig. 1 , A1)

Pairs of closely associated cones (putative double cones; fig. 1 , A3)

Partial One or two full square mosaics ( fig. 1 , A2) square Many pairs of closely associated cones

(putative double cones; fig. 1 , A3) Square Two adjacent full square mosaic units ( fig. 1 , B2)

No hexagonal mosaic units ( fig. 1 , A1)

These classifications did not vary with confocal plane. In four cases we independently determined mosaic type in two or three different confocal planes through the same regions of a single eye, and in every case the duplicates yielded identical classifica-tions.

Results

Locating Square Mosaic Initiation in Transitional Retina To discover how re-patterning in the retina proceeds,

we classified mosaic type in tangential and radial views of retinas from transitional animals to compare the first regions of the retina that become organized into square mosaics with the first regions to show opsin expression characteristic of a postmetamorphic retina (described below). We used tangential views through cone inner and outer segments to assign mosaics as either square or hex-agonal, and radial views through the same eye to confirm single or double cone morphology. We examined transi-tional retinas and in all cases found both regions of the retina with hexagonal mosaics and areas of square mo-saic containing both double and single cones ( fig. 2 ). Sin-gle cones arrayed in hexagonal mosaics were visible in the central retina, whereas square mosaics and correspond-ing double cones occurred near the margins of the eyes. Figure 2 includes photomicrographs of square mosaics near the nasal and temporal margins (square mosaics present at dorsal and ventral margins are not shown). Premetamorphic fish (lens diameters less than 115 � m) had pure hexagonal mosaics.

Cell Addition in Transitional Retina To determine the origin of cells in the square mosaic

in transitional eyes, we identified dividing cells using tri-tiated thymidine before retinal transition (68–120 � m lens diameters) and examined mosaic structure in cohort siblings either within one day or up to 84 days after label. Just after uptake of label, cells containing 3 H-thymidine were restricted to the periphery of the retina, where pre-cursor cells divide in the marginal germinal zone ( fig. 3 A).



Fig. 3. Photomicrographs taken from a cohort of flounder ex-posed to 3 H-thymidine that survived for either 1 day (A ), 11 days (B– D ), or 57 days (F– H ) demonstrate that the square cone mosaic formation includes photoreceptors added before retinal transfor-mation. Panel A is a composite image (Image-Pro Express Ver-sion5) combining 4 focal planes; all other panels are unmanipu-lated. Scale bars = 10 � m. A Newly divided cells in the retinas of flounders that survived for 1 day are restricted to the peripheral margin of the retina, where the only cells labeled with 3 H-thymi-dine are found (asterisk). The central retina has only single cones organized into hexagonal mosaics. We found no portions of the retina that had begun the transformation associated with meta-morphosis. B , C For flounder surviving 11 days, newly differenti-ated cells labeled with 3 H-thymidine (between dashed and solid lines) are visible adjacent to the peripheral margin (asterisk). Ret-inal transformation has just begun, with a small number of double cones visible central to the zone of 3 H-thymidine labeling (out-lined by box) that differentiated prior to labeling, and therefore

prior to retinal transformation. The image in B is focused on the photoreceptors whereas that in C shows the overlying silver grains indicating the 3 H-thymidine label. D , E Higher magnifica-tion image of box in B . For clarity, an example of a double cone is outlined in E in an otherwise identical image. F For animals sur-viving 57 days, postmitotic cells with 3 H-thymidine label are seen in a band of cells (white arrowhead) central to the dividing mar-ginal cells (asterisk). Black arrow marks the location of a double cone central to the band of 3 H-thymidine labeling, an example of a double cone formed from cells that differentiated prior to retinal transformation. G , H Higher magnification photomicrograph of the retinal section shown in F. The micrograph in G shows the photoreceptors whereas that in H is focused on the silver grains of the 3 H-thymidine label. Double cones (black arrow marks one example) can be seen in retina that is central to the 3 H-thymidine label in the outer nuclear layer (white arrowhead). Gray arrow-heads indicate the location of the 3 H-thymidine band in the inner nuclear layer and ganglion cell layer.


Brain Behav Evol 2006;68:241–254 249

Note that no double cones were present in these pre-tran-sition eyes. By 57 days after label, a band of 3 H-thymidine labeling was visible at a distance of100 � m from the mar-gin, comprised of cells that differentiated within a few cell divisions after the 3 H-thymidine labeling ( fig. 3 B). Given that new cell addition occurs at the margin, cells peripheral to this band were born after labeling, whereas more central cells were born prior to labeling. We found unlabeled double cones central to this labeled band ( fig. 3 C, D) and thus concluded that these double cones likely differentiated as single cones prior to retinal trans-formation and subsequently altered their morphology and mosaic position as the retina reorganized.

Localization of Opsin Gene Expression in Transitional Retina To determine whether onset of opsin genes expressed

in postmetamorphic flounder (SWS2 and LWS) occurs with the same spatial pattern as the square mosaic forma-tion, we localized RH2, LWS, and SWS2 opsin mRNA in transitional flounder retina using alkaline phosphatase in situ hybridization. We examined both eyes from two fish at three stages to generate a series of images showing the distribution of opsin mRNA expression throughout the transitional eye. Figure 4 shows representative adja-cent sections from the youngest transitional fish. Fish at all ages showed the same pattern. RH2 opsin mRNA was present throughout the photoreceptor layer. In contrast, LWS and SWS2 opsin expression was more restricted. LWS and SWS2 opsin mRNA was expressed in two patch-es in the retina, one in the ventronasal region ( fig. 4 ) and one in a more dorsal and temporal position (not shown). Note that LWS and SWS2 are absent from the temporal half in the ventral retina ( fig. 4 ) including the region near the peripheral margin.

Double in situ Hybridization to Localize Opsin Gene Expression within the Cone Mosaic To determine whether opsin expression changes be-

fore or after mosaic rearrangement, we classified mosaic type in tangential sections through transitional retinas

Fig. 4. These representative sections through the retina of a tran-sitional flounder show SWS2 and LWS opsin expression in similar locations in the ventronasal retina. RH2, SWS2, and LWS opsin mRNAs were localized in the photoreceptor layer using alkaline phosphatase in situ hybridization. The ventronasal patch of retina with LWS and SWS2 opsin expression is indicated by arrows. Note that the temporal retina, including the region near the peripheral margin, lacks LWS and SWS2 opsin expression. Schematic at bot-

tom illustrates extent of the ventronasal region of LWS and SWS2 opsin expression as well as a second patch of LWS and SWS2 opsin expression in the dorsotemporal retina (not shown), both of which were visible in all ages of metamorphic flounder examined. Sec-tions through more regions dorsal or ventral to these patches con-tained only RH2 opsin expression (not shown). ON = Optic nerve head; T = temporal; N = nasal. Scale bar = 100 � m.



( fig. 1 ) and identified the opsin genes expressed in each mosaic type. RH2 opsin-expressing cones are found in pre- and postmetamorphic flounder, but LWS and SWS2 opsin genes are expressed only in postmetamorphic flounder retina [Mader and Cameron, 2004]. Based on

the alkaline phosphatase expression patterns described above, we expected that LWS and SWS2 opsin gene ex-pression would be found in overlapping regions. There-fore, we combined probes for LWS and SWS2 opsin mRNAs and considered the combined expression indica-

Fig. 5. Postmetamorphic opsin mRNA is expressed within larval-like mosaic regions of retinal tissue from transitional flounder. A1 , B1, and C1 are confocal micrographs of tangential retinal sec-tions showing opsin mRNA expression. Red indicates expression of LWS or SWS2 opsin mRNA (pooled probes), and green label indicates RH2 opsin mRNA localization. A2, B2, and C2 show schematics used to classify the mosaic types present in the confo-cal micrographs. The brightly labeled red cells in A1, B1, and C1 are indicated by red dots for clarity; opsin subtype expression was not considered in mosaic classification. These isolated red cells

expressing LWS or SWS2 opsin are in regions of RH2-expressing cells that form a hexagonal ( A , B ) or partial hexagonal ( C ) mosaic. A3 and B3 show the outlines of three adjacent hexagonal units in each section (units outlined in blue, turquoise, or purple) required for categorization of these regions as hexagonal mosaics. In C3, one hexagonal mosaic unit is outlined in blue, but note the paired cones outlined in green. The occasional paired cones in this re-gion led to classification of the mosaic in C as partial hexagonal rather than hexagonal.


Brain Behav Evol 2006;68:241–254 251

tive of postmetamorphic opsin localization. Each transi-tional retina had both regions that were classified as square or partially square (similar to postmetamorphic mosaics) as well as regions that were hexagonal or par-tially hexagonal (similar to premetamorphic retina). We predicted that if opsin expression switches prior to mo-saic formation, we would find hexagonal arrays with sin-gle cones expressing LWS and SWS2 opsin genes.

We compared mosaic type with opsin expression in 16 planes of section through 6 transitional eyes to determine whether opsin type changes before or after mosaic reor-ganization. Square or partial square mosaics had regular spacing of cones expressing LWS or SWS2 opsin mRNA (n = 8, not shown). Partial hexagonal mosaics had regu-larly spaced (n = 2; not shown) or scattered cones (n = 1; fig. 5 C) with LWS or SWS2 opsin mRNA. One hexagonal mosaic contained only RH2-expressing cells (not shown) whereas others contained a single LWS- or SWS2-labeled cone in an otherwise RH2-containing region (n = 4; fig. 5 A, B).

Discussion

We examined cone photoreceptor cell birth, mosaic arrangement, and opsin mRNA expression during reti-nal transformation in the metamorphic winter flounder to understand how cell birth and differentiation are co-ordinated to produce precise cone photoreceptor mosaics characteristic of adults. We found that cone mosaic trans-formation was initiated near the peripheral margins of the retina, and these square mosaics included cones pro-duced prior to the onset of mosaic transformation. We also showed that expression of opsin mRNA types pres-ent in postmetamorphic but not premetamorphic retinas arose in two distinct patches in the retina. The expression of new opsin types occurred, in part, within cone mosaic regions that had not yet transformed to the square mo-saic characteristic of postmetamorphic retinas. We con-clude that retinal changes during metamorphosis are in-dependently regulated; existing cone photoreceptors re-organize into new mosaic arrangements and switch their opsin expression with distinctly different spatiotemporal patterns ( fig. 6 ).

We present our data in the framework of an emerging model of photoreceptor mosaic formation in which cone cell fate determines both initial opsin protein content and cone morphology as cells differentiate, but these features are plastic and may be changed [Szel et al., 1994; Archer et al., 1995; Hope et al., 1998; Shand et al., 1999; Zhang et

al., 2000; Helvik et al., 2001; Cheng and Novales Fla-marique, 2004; Cornish et al., 2004; Takechi and Kawamura, 2005]. Cell spacing in mosaics might proceed via tangential cell movement, perhaps employing differ-ential adhesion between neighboring cells based on cell type to produce square or hexagonal mosaics [Mochizu-ki, 2002; Reese and Galli-Resta, 2002; Tohya et al., 2003]. Unincorporated or misplaced cells might be induced ei-ther to switch fate again or be eliminated by cell death, although we did not test these possibilities. We argue be-low that our results do not favor an alternative model in which nearest neighbor interactions determine cone cell fate [Raymond and Barthel, 2004].

Newly Formed Square Mosaics Incorporate Existing Cone Photoreceptors Transformation of the cone mosaic from a hexagonal

mosaic with one cone type in premetamorphic flounder to a square mosaic with three different cone types in post-metamorphic flounder could in principle occur by two mechanisms: reorganization of existing cones that switch phenotypes, or insertion of new cones throughout the retina. Phenotype switching would entail coordination of both morphological changes (formation of double cones, alteration in photoreceptor shape) and molecular chang-es (shifts in opsin gene expression). Individual photore-ceptors alter opsin content throughout development in other fish [Hope et al., 1998; Cheng and Novales Fla-marique, 2004], although we do not demonstrate opsin switching directly in this paper. Previous research in an-other flatfish favored the hypothesis that larval cones re-organize to form the square mosaic arrangement: chang-es in cone morphology, including appearance of chains of cones connected by subsurface cisternae, precede square mosaic formation in the sea bream [Shand et al., 1999]. Our data complement this evidence suggesting that double cones form by fusion of existing single cones. We labeled dividing cells using 3 H-thymidine prior to retinal transformation then examined retinas after trans-formation using 3 H-thymidine presence to identify cells that underwent terminal division shortly after labeling. These 3 H-thymidine-labeled cells form a ring around the retina (a band in radial section) indicating the position of the peripheral margin, where cell division occurs, at the time of label. In radial sections through the retina, cones peripheral to this band are younger than the labeled cells as they divided after the incorporation of 3 H-thymidine, whereas cones central to this band are older because they likely stopped dividing before exposure to 3 H-thymidine label. The unlabeled double cones we found central to the



3 H-thymidine band in these postmetamorphic fish had most likely differentiated first as single cones prior to ret-inal transformation, and later changed from single cones in a hexagonal mosaic to double cones in a square mo-saic.

Our data are most consistent with a reorganization of existing cones during metamorphosis of flatfish to pro-duce the postmetamorphic square mosaic, congruent with the conclusions of Shand et al. [1999] in another metamorphosing fish species and the generally accepted mechanism for retinal remodeling. There are two less likely alternative explanations for our data. First, it is pos-sible that cells born in the periphery during retinal trans-formation move towards the central retina where they express SWS2 or LWS opsin mRNA and become double cones. This explanation is unlikely because a large num-ber of cones would have to migrate long distances and because we would not expect the 3 H-thymidine-labeled cells to form the compact band we observe if these cells had moved through this ring. Second, new cells could be born throughout the retina during metamorphosis and

differentiate in situ as double cones containing LWS and RH2 opsins or as single cones expressing SWS2 opsin mRNA. We do see 3 H-thymidine-positive cells in the out-er nuclear layer, but these centrally dividing cells are re-stricted to the ventral retina and have the morphology of rods not cones [B. Evans and R. Fernald, unpubl. observ.], consistent with the addition of rods in the central retina of in adult teleosts [Johns and Fernald, 1981]. We con-clude that the precise square mosaic pattern in the post-metamorphic flounder results from reorganization of ex-isting larval cones during metamorphosis, a reorganiza-tion which necessitates changes in opsin expression, morphological changes such as the fusion of single cones to produce double cones, and short-distance migrations to align disorganized cells.

Photopigment Expression and Cell Position Are Independently Regulated Are the reorganization of the cone mosaic and the

changes in opsin expression during metamorphosis co-ordinated or are these events independently regulated?

Fig. 6. Schematic illustration summarizing the patterns of mo-saic reorganization and opsin changes expected under two hy-potheses: (1) mosaic change regulates changes in opsin expression or (2) independent regulation of mosaic type and opsin expres-sion. The second hypothesis was supported by our data. In both panels, the observed restriction of square mosaics to the periph-eral part of the eye is shown in light gray. Predicted extent of ex-pression of LWS and SWS2 opsin mRNA characteristic of square mosaics in postmetamorphic flounder is shown in dark gray. A If mosaic position dictates opsin expression, we would expect ex-pression of LWS and SWS2 opsin mRNA only in regions that have

already transformed from hexagonal to square mosaics. In this case, the dark band indicating predicted LWS/SWS2 opsin local-ization should occur as a subset of the light band indicating square mosaic extent. B If mosaic position and opsin selection are inde-pendently regulated, we would expect that LWS and SWS2 opsin expression (shown here as two dark patches) might have no rela-tionship to the regions of the retina with cones arranged in square mosaics (light gray). Our experiments localizing opsins within serial radial sections indicated two patches of expression of LWS and SWS2 opsin mRNA as pictured here.


Brain Behav Evol 2006;68:241–254 253

We present two lines of evidence consistent with the hy-pothesis that cone photoreceptor opsin expression and cone mosaic arrangement occur independently during retinal transition. First, the earliest cones expressing LWS and SWS2 opsin mRNA occur in two patches in the ven-tronasal and dorsotemporal retina in all transitional ani-mals. In contrast, square mosaic formation occurred in a continuous ring close to the retinal margin throughout the retina in flounder of equivalent developmental stages based on lens diameter. These distinct spatial patterns suggest that the onset of postmetamorphic cone opsin expression and square mosaic formation occur indepen-dently ( fig. 6 ). Second, we show LWS/SWS2 opsin expres-sion in regions of the retina containing cones arrayed in hexagonal patterns, a mosaic type which in early pre-metamorphic flounder only contains RH2 opsin mRNA [Evans et al., 1993; Mader and Cameron, 2004]. This ob-servation precludes the possibility that square mosaic formation is a necessary prerequisite to LWS and SWS2 opsin expression, and thus does not support the idea that cone mosaic position dictates opsin protein expression. Conversely, the absence of LWS and SWS2 opsin expres-sion in the ventrotemporal retina ( fig. 4 ), or in the far ventral or dorsal retina (not shown) suggests that the square cone mosaics in these regions express only RH2 opsin mRNA, indicating that LWS and SWS2 opsin selec-tion are unlikely to direct mosaic organization. Because our tangential sectioning was biased toward the central and nasal portions of the retina and confocal analyses biased toward regions with LWS or SWS2 expression, we did not directly demonstrate square mosaics expressing only RH2 opsin mRNA. Moreover, the absence of detect-able in situ hybridization signal using LWS or SWS2 probes in a region does not prove the absence of low levels of opsin proteins in this region, and we did not examine

whether earlier markers of early cone fate might be pres-ent. Thus, we conclude that cone cell fate could play a role in mosaic formation, but that opsin expression and mo-saic position likely have separate regulatory influences.

Overall, our data suggest that the precise square mo-saic of cones in postmetamorphic flounder arises without a rigid sequential series of cell-cell inductions such as that responsible for Drosophila ommatidia formation as pro-posed by previous researchers [e.g., Stenkamp and Cam-eron, 2002; Raymond and Barthel, 2004]. Not only do we support the conclusion that cells shift opsin content and alter cone type during metamorphic reorganization of the cone mosaic, but we also provide evidence that cell position within the mosaic is not the cue guiding opsin selection. Shand et al. [1999] similarly conclude that cone morphological development proceeds independently from cone mosaic formation in the black bream, and Hel-vik et al. [2001] show that opsins restricted to double cones in postmetamorphic halibut appear in single cones of larvae. Vertebrate cone mosaic formation, in contrast to the series of fixed near-neighbor inductions in fly om-matidial development, might require individual cones to alter gene expression while jostling for position in the crystalline array of receptors on the retina.

Acknowledgements

We thank Mark Costello, Lynn Lush, Susan Douglas, Scott Gallager, Laura McInnery, Michael Cummings, Seung Choi and Karen Beck for help with experiments, and Molly Cummings, Anna Greenwood, Rachel Henderson, Kathleen Lynch, Rex Ro-bison, and Ryan Taylor for comments on the manuscript. Sup-ported by a Grass Foundation Fellowship to K.L.H., NSF MRI-0116086 to B.I.E., and NIH EY 05051 to R.D.F.

References

Allison WT, Dann SG, Helvik JV, Bradley C, Moyer HD, Hawryshyn CW (2003) Ontoge-ny of ultraviolet-sensitive cones in the retina of the rainbow trout (Oncorhynchus mykiss) . J Comp Neurol 461: 294–306.

Archer SN, Hope AJ, Partridge JC (1995) The molecular basis for the green-blue sensitivity shift in the rod visual pigments of the Euro-pean eel. Proc R Soc Lond B 262: 289–295.

Beaudet L, Hawryshyn CW (1999) Ecological as-pects of vertebrate visual ontogeny. In: Adap-tive Mechanisms in the Ecology of Vision (Archer SN, Djamgoz MBA, Loew ER, Par-tridge JC, Vallerga S, eds), pp 413–437. Dor-drecht: Kluwer.

Beaudet L, Browman HI, Hawryshyn CW (1993) Optic nerve response and retinal structure in rainbow trout of different sizes. Vision Res 33: 1739–1746.

Bowmaker JK, Kunz YW (1987) Ultraviolet pho-toreceptors, tetrachromatic colour vision, and retinal mosaics in the brown trout (Salmo trutta) : age dependent changes. Vi-sion Res 27: 2101–2108.

Chambers RC, Leggett WC (1987) Size and age at metamorphosis in marine fishes: an anal-ysis of laboratory-reared winter f lounder (Pseudopleuronectes americanus) with a re-view of variation in other species. Can J Fish Aqua Sci 44: 1936–1947.

Cheng CL, Novales Flamarique I (2004) New mechanism for modulating colour vision. Nature 428: 279.

Chenna R, Sugawara H, Koike T, Lopez R, Gib-son TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clus-tal series of programs. Nucleic Acids Res 31: 3497–3500.

Chinen A, Hamaoka T, Yamada Y, Kawamura S (2003) Gene duplication and spectral diver-sification of cone visual pigments of zebra-fish. Genetics 163: 663–675.



Cornish EE, Xiao M, Yang Z, Provis JM, Hen-drickson AE (2004) The role of opsin expres-sion and apoptosis in determination of cone types in human retina. Exp Eye Res 78: 1143–1154.

Deutschlander ME, Greaves DK, Haimberger TJ, Hawryshyn CW (2001) Functional map-ping of ultraviolet photosensitivity during metamorphic transitions in a salmonid fish, Oncorhynchus mykiss . J Exp Biol 204: 2401–2413.

Evans BI (2004) A fish’s eye view of habitat change. In: The Senses of Fishes: Adapta-tions for the Reception of Natural Stimuli (von der Emde G, Mogdans J, Kapoor BG, eds), pp 1–30. New Delhi: Narosa Publishing House.

Evans BI, Fernald RD (1993) Retinal transfor-mation at metamorphosis in the winter f lounder (Pseudopleuronectes americanus) . Vis Neurosci 10: 1055–1064.

Evans BI, Harosi FI, Fernald RD (1993) Photore-ceptor spectral absorbance in larval and adult winter f lounder (Pseudopleuronectes americanus) . Vis Neurosci 10: 1065–1071.

Galli-Resta L (2001) Assembling the vertebrate retina: global patterning from short-range cellular interactions. Neuroreport 12:A103–106.

Haacke C, Hess M, Melzer RR, Gebhart H, Smo-la U (2001) Fine structure and development of the retina of the grenadier anchovy Coilia nasus (Engraulididae, Clupeiformes). J Mor-phol 248: 41–55.

Helvik JV, Drivenes O, Harboe T, Seo HC (2001) Topography of different photoreceptor cell types in the larval retina of Atlantic halibut (Hippoglossus hippoglossus) . J Exp Biol 204: 2553–2559.

Hope AJ, Partridge JC, Hayes PK (1998) Switch in rod opsin gene expression in the European eel, Anguilla anguilla (L.). Proc R Soc Lond B 265: 869–874.

Humason GL (1979) Animal Tissue Techniques, 4 Edition. San Francisco: W. H. Freeman & Co.

Johns PR, Fernald RD (1981) Genesis of rods in teleost fish retina. Nature 293: 141–142.

Mader MM, Cameron DA (2004) Photoreceptor differentiation during retinal development, growth, and regeneration in a metamorphic vertebrate. J Neurosci 24: 11463–11472.

Mochizuki A (2002) Pattern formation of the cone mosaic in the zebrafish retina: a cell re-arrangement model. J Theor Biol 215: 345–361.

Novales Flamarique I (2000) The ontogeny of ul-traviolet sensitivity, cone disappearance, and regeneration in the sockeye salmon On-corhynchus nerka . J Exp Biol 203: 1161–1172.

Novales Flamarique I, Hawryshyn CW (1996) Retinal development and visual sensitivity of young pacific sockeye salmon (Oncorhyn-chus nerka) . J Exp Biol 199: 869–882.

Raymond PA, Barthel LK (2004) A moving wave patterns the cone photoreceptor mosaic ar-ray in the zebrafish retina. Int J Dev Biol 48: 935–945.

Raymond PA, Barthel LK, Curran GA (1995) De-velopmental patterning of rod and cone pho-toreceptors in embryonic zebrafish. J Comp Neurol 359: 537–550.

Reese BE, Galli-Resta L (2002) The role of tan-gential dispersion in retinal mosaic forma-tion. Prog Retin Eye Res 21: 153–168.

Rose TM, Schultz ER, Henikoff JG, Pietrokovski S, McCallum CM, Henikoff S (1998) Con-sensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res 26: 1628–1635.

Shand J, Archer MA, Collin SP (1999) Ontoge-netic changes in the retinal photoreceptor mosaic in a fish, the black bream, Acan-thopagrus butcheri . J Comp Neurol 412: 203–217.

Shand J, Hart NS, Thomas N, Partridge JC (2002) Developmental changes in the cone visual pigments of black bream Acanthopagrus butcheri . J Exp Biol 205: 3661–3667.

Stenkamp DL, Cameron DA (2002) Cellular pat-tern formation in the retina: retinal regen-eration as a model system. Mol Vis 8: 280–293.

Szel A, van Veen T, Rohlich P (1994) Retinal cone differentiation. Nature 370: 336.

Takechi M, Kawamura S (2005) Temporal and spatial changes in the expression pattern of multiple red and green subtype opsin genes during zebrafish development. J Exp Biol 208: 1337–1345.

Tohya S, Mochizuki A, Iwasa Y (2003) Differ-ence in the retinal cone mosaic pattern be-tween zebrafish and medaka: cell-rearrange-ment model. J Theor Biol 221: 289–300.

Williams SR (1902) Changes accompanying the migration of the eye and observations on the tractus opticus and tectum opticum in Pseu-dopleuronectes americanus . Bull Mus Comp Zool Harvard 40: 1–57.

Zhang H, Futami K, Horie N, Okamura A, Utoh T (2000) Molecular cloning of fresh water and deep-sea rod opsin genes from Japanese eel Anguilla japonica and expressional anal-yses during sexual maturation. FEBS Lett 469: 39–43.

remodeling of the cone photoreceptor mosaic during …web.stanford.edu/group/fernaldlab/pubs/2006...

Documents