Immunocytochemical analysis of misplaced rhodopsin-positive cells in the developing rodent retina

REGULAR ARTICLE

Immunocytochemical analysis of misplaced rhodopsin-positivecells in the developing rodent retina

Klaudia Szabó & Arnold Szabó & Anna Énzsöly &

Ágoston Szél & Ákos Lukáts

Received: 9 October 2013 /Accepted: 19 December 2013# Springer-Verlag Berlin Heidelberg 2014

Abstract During the first postnatal weeks of the developingrodent retina, rhodopsin can be detected in a number ofneuron-like cells in the inner retina. In the present study, weaim to characterize the morphology, number and stainingcharacteristics of this peculiar population. Misplacedrhodopsin-positive cells (MRCs) were analyzed on retinas offour rodent species, labeled with various rhodopsin-specificantibodies. To investigate their possible relation with non-photoreceptor cells, sections were double-stained against dis-tinct retinal cell types and proteins of the phototransductioncascade. The possibility of synapse formation and apoptosiswere also investigated. In all species studied, misplaced cellscomprised a few percent of all rhodopsin-positive elements.This ratio declined from the end of the second week andMRCs disappeared nearly completely from the retina byP24. MRCs resembled resident neurons of the inner retina,while outer segment-like processes were seen only rarely.MRCs expressed no other photopigment types and showed

no colocalization with any of the bipolar, horizontal,amacrine and ganglion cell markers used. While all MRCscolabeled for arrestin and recoverin, other proteins of thephototransduction cascade were only detectable in a minorityof the population. Only a few MRCs were shown to formsynaptic-like endings. Our results showed that, during devel-opment, some rhodopsin-expressing cells are displaced to theinner retinal layers. Although most MRCs lack morphologicalfeatures of photoreceptors, they contain some but not all,elements of the phototransduction cascade, indicating thatthey are most probably misplaced rods that failed to completedifferentiation and integrate into the photoreceptor mosaic.

Keywords Rod . Photoreceptor . Rhodopsin . Retinaldevelopment .Misplaced photoreceptor

Introduction

In primary nocturnal animals, such as most rodent species,photic information is mainly transduced by rod photoreceptorsthat constitute the dominant cell type in the retina. Rodshave been reported to comprise approximately 70 % of allretinal cells in mice, with a rod to cone ratio close to 30:1(Carter-Dawson et LaVail 1979).

During retinal maturation, rod photoreceptors are generatedthrough a series of developmental steps. All retinal cell typesarise from a common multipotent progenitor cell, from whichrestricted lineages are produced under regulation of nuclearreceptors functioning as transcription factors (Swaroop et al.2010; Forrest and Swaroop 2012). One of themost understoodof these is orthodenticle homeobox 2 protein (OTX2) thatcontrols the production of committed photoreceptor precur-sors. Rod cell fate is later determined essentially by neuralretina leucine zipper protein (NRL), which, if it reaches therequired threshold concentration, induces the expression of

Electronic supplementary material The online version of this article(doi:10.1007/s00441-013-1788-2) contains supplementary material,which is available to authorized users.

K. Szabó :A. Szabó :Á. Szél :Á. Lukáts (*)Department of Human Morphology and Developmental Biology,Semmelweis University, Tűzoltó u. 58, 1094 Budapest, Hungarye-mail: [email protected]

K. Szabóe-mail: [email protected]

A. Szabóe-mail: [email protected]

Á. Széle-mail: [email protected]

A. ÉnzsölyDepartment of Ophthalmology, Semmelweis University,Mária u. 39, 1085 Budapest, Hungarye-mail: [email protected]

Cell Tissue ResDOI 10.1007/s00441-013-1788-2

http://dx.doi.org/10.1007/s00441-013-1788-2

rod-specific genes, including rhodopsin, rod transducin andcGMP phosphodiesterase ß (Swaroop et al. 2010; Mears et al.2001). Transcriptional targets of NRL also include anotherorphan nuclear receptor (NR2E3), which can support thedifferentiation process of rods by suppressing the expressionof cone genes (Forest and Swaroop 2012).

Rod genesis is a relatively long process extending from the15th embryonic (E15) to the 12th postnatal (P12) days in rats(Rapaport et al. 2004; similar data for mouse: Young 1985).There are two distinct phases of rod development based on thetemporal window between terminalmitosis and the expressionof the rhodopsin gene. The early phase of rod formationbegins with precursors becoming postmitotic at E15–E19and prospective rods begin to express rhodopsin gene fromthe early postnatal days onward. The second wave of rodgenesis takes place after E19 (Morrow et al. 1998a, b).

Once committed, the morphologic development of rodphotoreceptor cells can be reliably followed by identifyingphotoreceptor-specific molecules with immunocytochemicallabeling. Among them, rhodopsin is the most frequently usedmarker. A panel of well-characterized antibodies raised main-ly against the C- or N-terminal region of the rhodopsin moleculeis available today (Barnstable et al. 1983; Adamus et al. 1987;Molday and MacKenzie 1983; Röhlich and Szél 1993). De-pending on the antibody, the first rhodopsin signals in rodentsare usually detectable as early as postnatal day 1 (P1) and thelabeling intensity quickly increases with retinal maturation(Hicks and Barnstable 1987; Szél et al. 2000). Differentiatingrods are integrated into the photoreceptor layer with rhodopsininitially appearing in the plasma membrane but becominggradually sequestered to the developing sensory cilium thatis transformed into the outer segment (Nir et al. 1984). Inter-estingly, however, some rhodopsin-positive cells with non-photoreceptor-like morphology also appear in the inner nucle-ar and ganglion cell layers. These cells have a neuron-likemorphology with long processes, often resembling residentcell types of the inner retinal layers. Although observed inprevious developmental studies, identified by its special rod-like nuclear morphology or by immunocytochemistry (Spiraet al. 1984; Hicks and Barnstable 1987; Araki et al. 1988;Günhan et al. 2003; Semo et al. 2007), a detailed analysis ofthese rod-like cells is still missing.

A similar but cone opsin-containing cell population in theganglion cell layer has also been reported, being present in arelatively small number in different rodent species and even inhuman (Semo et al. 2007). These cells were shown to expressrecoverin, cone transducin and cone arrestin and thereforewere assumed to function as photoreceptors. No such dataexist about the phototransduction cascade proteins ofmisplaced rhodopsin-expressing cells (MRCs).

The aim of the present report is to perform a detailed studyof these rhodopsin-positive cells of the inner retina. Themorphology and relative number of these cells were assessed,

and a series of double-labeling experiments were performed todescribe the staining characteristics of these cells. To assessthe possible functions and receptor differentiation, wescreened for the presence of different elements of thephototransduction cascade and synapse formation. Our find-ings indicate that these cells are most probably misplaced andpartially differentiated rod photoreceptors that lack some ofthe functional proteins of phototransduction.

Materials and methods

Tissue processing

All animal procedures were performed in compliance with theARVO Statement for the Use of Animals in Ophthalmic andVision Research and approved by the local ethical committee(No of approval: 22.1/1068/3/2010).

Specimens from four species of rodents, Sprague–Dawleyrat, Syrian golden hamster, Siberian hamster and C57BLmouse, obtained from Charles River Laboratories Hungary(Isaszeg, Hungary) or from local breeders (Siberian hamster),were used for the experiments. All animals were euthanizedfollowing ketamine narcosis. Eyes of early postnatal animals(P4, P7, P10, P14, P18, P21, P24 and P28 in case of rats; P7,P10 and P14 in case of the other species, when P0 indicatesthe day of birth) and adults (n=4 or higher for each stage) wereenucleated, the cornea, lens and vitreous body were removedand, following fixation (4 % paraformaldehyde, diluted in0,1 M phosphate buffer (PB), 4 °C, 2 h), the posterior eyecupswere processed for immunocytochemical examination. Theretinas were rinsed several times in PB for 24 h andcryoprotected in 10 % sucrose diluted in 0.1 M PB overnightat 4 °C. Tissues were embedded in tissue-freezing medium(Shandon Cryomatrix; Thermo Scientific, UK) and 10–15 μm tangential sections were prepared on a cryotome.

Antibodies and immunolabeling

For labeling rods in all four species investigated, we used a setof various anti-rhodopsin antibodies raised against the N- (R2-15, AO) or C-terminal (K57-142, Rho 1D4) of the molecule.S- and M/L-cone opsins were detected with monoclonal anti-bodies OS-2 and COS-1 and two commercially availablecone-specific antibodies (AB5407 and AB5405). A furtherpolyclonal antibody was used to check the presence ofmelanopsin.

For labeling distinct cell populations in the inner and outernuclear layers, we used cell-specific markers, such as antibodyagainst protein-kinase C alpha (PKC-α), recoverin, calbindin,calretinin, parvalbumin, vimentin and tyrosine hydroxylase.Ganglion cells were marked using anti-Brn-3a antibody. Forthe examination of microglia/macrophage cells, we labeled

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our specimens with antibodies to the macrophage-specificglycoprotein ED-1.

To examine if other photoreceptor-specific molecules canbe colocalized with rhodopsin in MRCs, we double-stainedthe retinas with antibodies to rod arrestin (retinal S-antigen),rhodopsin-kinase, rod transducin and additionally, conearrestin. The possibility of synapse formation was examinedby anti-synaptophysin antibody.

Double-labeling with antibodies recognizing special retinalcell types and molecules of the phototransduction cascadewas performed only in rat retinas.

Details about the applied antibodies are summarized inTable 1.

Prior to immunocytochemistry, all sections were blocked ina solution of 1 % bovine serum albumin (BSA) in 0.1 Mphosphate buffered saline (PBS), containing 0.4 % Triton-X100 (Sigma-Aldrich, Budapest, Hungary). Sections wereincubated with the primary antibodies at room temperatureovernight and the bound antisera were detected using species-specific Alexa-conjugated secondary antibodies (Alexa-488or Alexa-594 conjugates, 1:200, 2 h; Life Technologies, Carls-bad, CA, USA). Negative controls were specimens in whichthe primary antibodies were omitted. Cell nuclei were stainedwith 4,6-diamidino-2-phenylindole (DAPI).

For double-labeling, we used either AO (produced in rat) orRho 1D4 (produced in mouse) for staining rhodopsin incombination with another antibody labeling a specific cellpopulation of the retina.

The sections were examined and photographed using aBio-Rad Radiance 2100 Rainbow Confocal Laser ScanningSystem (Carl Zeiss Technika, Budaörs, Hungary) coupled toan Eclipse E800 microscope (Nikon, Tokyo, Japan) withLaserSharp 2000 6.0 software (Carl Zeiss Technika) for imageacquisition. Final montages were created by the AdobePhotoshop 7.0 (San Diego, CA, USA) program.

Cell counting

In order to approximate the number of misplaced rhodopsin-positive cells in central retinal regions, MRCs—as well as allrods of the outer nuclear layer—were counted in 15-μm-thickvertical sections going through the optic nerve in P7, P10, P14,P18, P21 and P28 rat retinas (n=3 animals, 2 sections usedfrom each specimen). Two sample photographs were taken persection, 200 μm superior and inferior from the optic nerve,respectively. Images from these confocal Z-stacks were used tocreate a 5 μm-thick combined picture, on which counting wasperformed manually over the length of 460 μm. Positivity wasdecided based on anti-rhodopsin labeling and nuclear morphol-ogy. The number of MRCs was expressed as the percentage ofall rhodopsin-positive elements.

Two different approaches were used to estimate the numberof apoptotic MRCs. First, in the region of cell counting, the

number of MRCs with pyknotic nuclei were counted. Thevalues were expressed as the total number of apoptotic MRCsper retinal length of 460 μm and also as the percentage of allMRCs in the given region.

We also performed terminal deoxynucleotidyl transferasedUTP nick end labeling (TUNEL, In situ Cell Death DetectionKit, Fluorescein; Roche Diagnostics, Mannheim, Germany)assay on sections of P07, P10, P14, P18, P21 and P28 ratretinas (n=1, 4 sections per each specimen) according to themanufacturer’s instructions. The numbers of TUNEL-positiveMRCs were counted manually on complete vertical sections.

Statistical analysis

The percentage of MRCs and the number of apoptotic MRCsestimated based on nuclear morphology were analyzed statis-tically. Testing of normality was performed using Shapiro–Wilk’s test. In case of normal distribution (number of apopto-tic MRCs) age groups were compared using one-way analysisof variance (ANOVA) and Tukey’s honest significance testwith a significance level of p=0.05. In the case of non-normaldistribution (percentage of MRCs), data were analyzed usingKruskal–Wallis one-way analysis of variance completed withTamhane’s post hoc test (p=0.05).

Results

In agreement with previous data from our laboratory aboutretinal development in rodents (Szél et al. 2000), we detectedrhodopsin immunoreactivity in the form of punctate structuresat the scleral margin of the neural retina in the early postnataldays. From this time on, labeling intensity quickly increased,delineating the differentiating rod cells (plasma membranelocalization) and concentrating to the forming outer segment.Rods reached a near-mature phenotype around the end of the3rd week. In addition to this characteristic and well-knowndistribution of rhodopsin in the photoreceptor layer, therewere anti-rhodopsin immunoreactive cells in the inner nuclearand ganglion cell layers of the developing retina in all fourrodent species studied (Figs. 1, 2). As no major difference wasdetectable in the number and morphology of these cells in thefour species, only the data from Sprague–Dawley rats arepresented in detail here.

The misplaced rhodopsin-positive cells (MRCs) did notshow the characteristic morphological features of typical rodcells but were neuron-like cells with several long processes.These cells were already present at P4, although, due to theincomplete separation of the neuroblast layer, the precise iden-tification was difficult, unless located in the differentiatingganglion cell layer (Fig. 2). From P7 to P14, the percentagesof misplaced rhodopsin-positive cells were relatively constant(2.35±1.35, 1.94±0.59 and 2.05±0.5 % at P7, P10 and P14,

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Table1

Seto

fprim

aryantib

odies

Antibody

Source

References

Host

Concentratio

nEpitope

orlabelin

gpattern

inratretina

Rhodopsin

(AO)

Produced

inourlaboratory

RöhlichetSzél1993

Ratpolyclonal

1:1,000

Rhodopsin

N-terminal

K57-142

Generousgiftof

Paul

A.H

argrave

Adamus

etal.1991

Mouse

monoclonal

1:5

Rhodopsin

C-terminal

R2-15

Generousgiftof

Grazyna

Adamus

Adamus

etal.1991

Mouse

monoclonal

1:5

Rhodopsin

N-terminal

Rho

1D4

Millipore,Billerica,MA

MoldayandMacKenzie1983

Mouse

monoclonal

1:100

Rhodopsin

C-terminal

OS-2

Produced

inourlaboratory

RöhlichandSzél1993

Mouse

monoclonal

1:5,000

S-cone

opsin,C-terminal

COS-1

Producedin

ourlaboratory

RöhlichandSzél1993

Mouse

monoclonal

1:50

M/L-coneopsin,C-terminal

AB5407


Arango-Gonzalezetal.2010

Rabbitp

olyclonal

1:1,000

S-cone

opsin

AB5405


Ngetal.2010

Rabbitp

olyclonal

1:1,000

M/L-coneopsin

Melanopsin

Affinity

Bioreagents,G

olden,CO

Lietal.2006

Rabbitp

olyclonal

1:80

Intrinsically

photosensitiv

eganglio

ncells

ED-1

Generousgiftof

Christin

eD.D

ijkstra

Dijk

stra

etal.1985

Mouse

monoclonal

1:400

Microglia/m

acrophagecelllin

eage,endothelialcells

PKC-α

SantaCruzBiotechnology,S

antaCruz,CA

Greferath

etal.1990

Mouse

monoclonal

1:200

Rod

bipolarcells

Calbindin

Swant,Marly,S

chwitzerland

Ham

anoetal.1990

Mouse

monoclonal

1:200

Horizontalcells,w

eeklabelin

gin

small

amacrine

cells

andganglio

ncells

Calretin

inMillipore,Billerica,MA

Hwangetal.2005

Polyclonalrabbit

1:2,500

Amacrine

cells

andganglio

ncells

Parvalbumin

Sigm

a-Aldrich,B

udapest,Hungary

Wässleetal.1993

Mouse

monoclonal

1:300

AIIam

acrine

cells,w

eeklabelin

gin

ganglio

ncells

Tyrosine

hydroxylase


Chang

etal.2011

Mouse

monoclonal

1:250

Dopam

inergicam

acrine

cells

Anti-Brn-3a


Nadal-N

icolás

etal.2009

Mouse

monoclonal

1:500

Ganglioncells

Vim

entin


Schnitzer

1988

Mouse

monoclonal

1:10,000

Müllerglia

Recoverin

Generousgiftof

Karl-Wilh

elm

Koch

Milanetal.1993

Rabbitp

olyclonal

1:500

Conebipolarcells,rod

andcone

photoreceptors

Rhodopsin-kinase

Generousgiftof

KrysztofPalczewsky

Zhaoetal.1998

Mouse

monoclonal

1:300

Rod

andcone

outersegm

ents

Rod

transducin

Abcam

,Cam

bridge,U

KChucairElio

ttetal.2012

Rabbitp

olyclonal

1:300

Rod

transducin

Rod

arrestin

(retinalS-antigen)

Generousgiftof

IgalGery

Mirshahietal.1985

Rabbitp

olyclonal

1:400

Rod

arrestin

Conearrestin


McG

illetal.2012

Rabbitp

olyclonal

1:1,000

Conephotoreceptor

Synaptophysin

Leica

Biosystem

s,Wetzlar,G

ermany

Al-Otaibietal.2012

Mouse

monoclonal

1:1,000

Synapticvesicleglycoprotein

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respectively) but continuously declined at later stages (0.86±0.23 and 0.64±0.14 % at P18 and P21, respectively). By theend of the 4th week, MRCs had almost completely disappearedfrom the retina (0.1±0.03 % at P28) but were occasionallydetectable in adult specimens (Fig. 3).

Morphologically, MRCs with their few long processesfrequently resembled resident neuronal cells of the inner nu-clear and ganglion cell layers (bipolar, amacrine and ganglioncell-like elements were regularly detected) (Fig. 4). Occasion-ally, terminal bulbous swellings of the processes with strongrhodopsin reactivity were found that resembled rudimentaryouter segments, characteristically at later stages of develop-ment (P14–21). In agreement with the centro-peripheral di-rection in retinal maturation, MRCs disappeared first in thecenter but were still found close to the ora serrata in laterstages (P21–28). Interestingly, ganglion cell-like MRCs were

often visible, sometimes in relatively large groups, near or atthe optic disc even at P24–28.

As demonstrated by series of double-labeling, the MRCpopulation was consistently detected with all anti-rhodopsinantibodies used, independently of whether they were raisedagainst the extracellular N-, or intracellularly localized C-terminal of the rhodopsin molecule (Electronic Supplementa-ry Material, Fig S1). In the case of antibody AO, produced inrat, blood vessels were regularly stained when applied to therat retina but no such labeling was observed in mouse orhamster retinas. This aspecific staining could not present anydifficulty in interpreting the results due to the characteristicmorphology and position of the vessels. Identification wasalso aided by DAPI staining: MRCs had round nuclei withlarge heterochromatic blocks, reminiscent of the nuclei of rodsin the outer nuclear layer.

Fig. 1 Misplaced rhodopsin-positive cells in the inner nuclearand ganglion cell layer in theretinas of different rodent species.Sections were taken from thecentral retina of P14 Syriangolden hamster (a), Siberianhamster (b) and C57BL mouse(c). Some MRCs are indicated byarrows. ONL outer nuclear layer,INL inner nuclear layer, GCLganglion cell layer. Bar 50 μm

Fig. 2 The distribution ofmisplaced rhodopsin-positivecells in the developing rat retina,as revealed byimmunocytochemistry. Sectionswere immunolabeled by apolyclonal anti-rhodopsinantibody (AO). Representativepictures were taken from centralretinal positions in P4–P18 andfrom the peripheral retina in P21.With the differentiation of theneuroblast layer (a, b, c), arelatively large population of cellsis excluded from the formingouter nuclear layer. The numberof MRCs stays relatively constantuntil P14 (d) but declinesthereafter (e, f). MRCs mostlydisappear from the central retinaby P21 but are still numerous atthe periphery (f). NBL neuroblastlayer, GCL ganglion cell layer,ONL outer nuclear layer, INLinner nuclear layer. Bar 50 μm

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In order to further characterize the MRC population, sec-tions were counterstained with various retinal cell type-specific antibodies. As the precise staining patterns of theseantisera are well characterized in rats and mice and limiteddata are available from other species, these double-labelingstudies were carried out in rats only. In the following, wepresent data obtained from P14 retinas.

Cone photopigments and melanopsin

Cone opsin expression was mostly confined to the photore-ceptor layers (Fig. 5), with time course and intensity identical

with those described previously (Szél et al. 2000; Lukáts et al.2005). In agreement with Semo et al. (2007), however, wealso regularly detected a small cell population in the ganglioncell layer that expressed S- orM/L-opsin. Although no precisequantification was attempted, we can conclude that misplacedcone opsin-positive cells were present in an extremely smallnumber and never coexpressed the rhodopsin molecule(Fig. 5d–f, inserts). Taken together, these data clearly demon-strate that this population is different fromMRCs. Using anti-melanopsin antibody, we also regularly observed, as expected,a few labeled cells in the ganglion cell layer. Also, nocolocalization was found with MRCs. As a result, ourdouble-labeling immunocytochemical studies could not con-firm the presence of any other type of photopigment thanrhodopsin in MRCs.

Microglia, Müller glia and rod bipolar cells

Rhodopsin-containing cell debris may be released from apo-ptotic rods and might be phagocytosed by members of themacrophage/microglia lineage, or Müller glia cells (Mano andPuro 1990; Bringmann et al. 2006). Therefore, double-labelingwith a microglia-specific probe was performed (ED-1), wherethe lack of double-positive elements indicated that the MRCswere not microglial cells (Fig. 6). Although the morphology ofMRCs did not resemble that of the Müller glia cells, to un-doubtedly rule out this possibility, colabeling with vimentinwas carried out but also showed no double-labeled elements.

Literature data suggest that opsin could also be taken up bybipolar cells from degenerating photoreceptors (Glösmann andPeichl 2007). The morphology of at least some of the MRCsalso resembled that of the bipolar cells; therefore, we double-labeled retinas with PKC-α, a knownmarker of rod bipolars but

Fig. 3 The percentage of MRCs of all rhodopsin-positive cells in differentstages of retinal maturation. The values are relatively constant until P14 butdecline prominently thereafter. By P28, almost all MRCs are eliminatedfrom the retina. Significant differences were found between the followingage groups: P7–P18, P7–P21, P7–P28, P10–P28 and P14–P28

Fig. 4 The most common morphological variations of MRCs in the ratretina. MRCs often resemble resident cell types of the inner retina.Bipolar (a), amacrine (b) and ganglion cell-like (c) forms were regularlydetected, while outer segment-like processes (d, arrow) were only seen ina few cases, characteristically at later stages of development. b, c are

higher magnification images of selected regions of the P4 and P7 retinaspresented in Fig. 1, while a, d were taken from P14 retinas. ONL outernuclear layer, INL inner nuclear layer, IPL inner plexiform layer, GCLganglion cell layer. Bar 20 μm

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found no double-stained elements (Fig. 6). In conclusion, thepresence of rhodopsin in the MRCs cannot be regarded asphagocytosed material derived from genuine rod cells.

Bipolar, horizontal, amacrine and ganglion cell markers

The morphology and positioning of some of the MRCs sug-gested that they might be either bipolar, amacrine, or ganglioncells; therefore, we applied a set of markers known to prefer-ably label one or a few of the mentioned cell types. Nocolocalization was found with either of the antibodies to:calbindin (primarily horizontal cells), calretinin (amacrineand ganglion cells), parvalbumin (primarily AII amacrinecells), tyrosine hydroxylase (dopaminergic amacrinecells), or the Brn-3a transcription factor (ganglion cells)(Fig. 7). Recoverin labeling (photoreceptors and conebipolar cells) has been discussed in detail amongst theproteins of the phototransduction cascade.

Proteins of the phototransduction cascade

Arrestin and recoverin are important regulatory proteinsacting on rhodopsin at the deactivation phase of thephototransduction cascade. We screened our specimens forthe presence of rod arrestin and found that all rhodopsin-positive cells (including MRCs) were labeled with the anti-body (Fig. 8). In contrast, however, none of these cells werestained by anti-cone arrestin (not shown).

Antisera against the Ca2+-binding protein recoverin areknown to label both rod and cone photoreceptors as well ascone bipolar cells in the rodent retina. When applied to ourspecimens, we found that, besides the characteristic cone bipolarcell labeling, recoverin stained all rhodopsin-containing cells ofthe retina (including MRCs), irrespective of whether they werelocated in the outer nuclear, inner nuclear, or ganglion cell layers(Fig. 8). In agreement with previous data, we also found a fewrecoverin-positive cells in the ganglion cell layer that were not

Fig. 5 MRCs do not expresscone opsins or melanopsin.Misplaced rhodopsin-positivecells do not contain S-opsin (OS-2, a–c), M/L-opsin (AB5405, d–f), or melanopsin (g–i) and do notcolocalize with the misplacedcone opsin-positive cellpopulation in the ganglion celllayer. One of these M/L-opsinpositive cells is shown at highermagnification (d–f, insert). ONLouter nuclear layer, INL innernuclear layer, GCL ganglion celllayer. Bar50 μm, (100 μm for theinserts)

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counterstained for rhodopsin. Although no double-staining wasattempted, we assume that these cells represent the S-opsin-containing population previously described (Semo et al. 2007).

In contrast to these results, when colabeling with rhodopsin-kinase or rod transducin antibody, only a small fraction of theMRCs were stained, while most elements did not contain theenzymes. As genuine rods that may be used as positive controlshowed prominent staining, the result indicates thatMRCs havealready lost or alternatively had never possessed these proteinsof the phototransduction cascade (Fig. 8).

Synapse formation and apoptosis

Anti-synaptophysin, a marker recognizing the presynap-tic elements of neural synapses was used to examine the

possibility of synapse formation by MRCs. As expected,strong immunoreactivity to synaptophysin was detect-able throughout both the inner and the outer plexiformlayers. When the processes of the MRC population wereexamined in detail, the vast majority of the cells weredevoid of synaptophysin labeling. Only a few cells on acomplete vertical section showed synaptophysin positiv-ity (1–2 cells, estimated to make up approximately 0.5–1 % of all MRCs) at the tip of cell processes. Interest-ingly, some of these processes contacted other MRCs(Fig. 9). We also detected a few synaptophysin-positiveloci per section contacting the outer surface of MRCsthat were not rhodopsin-positive, possibly coming fromother cell types. Although no other synapse-specificmolecules were tested, these results clearly demonstrate

Fig. 6 MRCs do not bindmacrophage, Müller glia and rodbipolar cell markers. Upper rowretinal sections were labeled byanti-rhodopsin (AO, a) and ED-1(b), a macrophage and endothelialcell specific antibody, cmergedimage. Colocalization is only seenin blood vessels. A labeledmicroglia is indicated by anarrow. Middle rowAO (d) andvimentin (Müller glia, e) labeling.No colocalization is visible in themerged view (f). Lower rowdouble-labeling with AO (g) andPKCα (rod bipolar cells, h). In themerged view (i), no colocalizationis detectable. ONL outer nuclearlayer, INL inner nuclear layer,GCL ganglion cell layer. Bar50 μm

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that most MRCs did not show evidence of formingsynaptic contacts.

Since almost all rhodopsin-positive cells disappeared fromthe inner retinal layers by P28, we examined the possibility ofMRCs being eliminated by apoptosis. In sections from P7 toP28 retinae, all rhodopsin-expressing cells in the inner retinallayers were identified and counted. Apoptotic MRCs wereselected according to their apoptotic nuclear morpholo-gy that was clearly different from the usual nuclearmorphology reminiscent of genuine rods. Results are

summarized in Fig. 10. The highest number of apopto-tic MRCs were found at P18, the time of decrease inthe number of misplaced cells, supporting the idea thatat least some, probably all of the cells are eliminatedby apoptosis. Significant differences between P18 andall other age groups except P21 were also testified bystatistical analysis.

In order to test if detecting nuclear morphology isspecific and sensitive enough to detect apoptotic MRCsalone, we applied TUNEL assay on a limited number of

Fig. 7 MRCs do not bind any ofthe inner retinal cell specificantibodies applied. Nocolocalization is detectable forcalbindin (a–c), calretinin (d–f),parvalbumin (g–i), tyrosinehydroxylase (j–l) and Brn-3a (m–o). Note that the pictures aretruncated and only the lowest partof the outer nuclear layer isvisible. INL inner nuclear layer,GCL ganglion cell layer. Bar50 μm

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sections. Although the number of specimen used did notallow a detailed statistical analysis, the results obtained(Electronic Supplementary Material, Fig S2) clearlyshow identical kinetics, with peak values at P18, indi-cating that the results obtained by nuclear morphologyalone were reliable.

This idea of MRCs being eliminated by apoptosis is furthersupported by the percentage values (apoptotic MRCs/allMRCs). With retinal maturation, an increase of these valueswas detectable, with approximately one-third of all MRCsbeing apoptotic by P28 (Fig. 10d).

Discussion

It was reported as early as the mid-1980s that an anti-rhodopsin antibody and 5′-nucleotidase cytochemistry appliedon developing retinas labeled a few cells with non-photoreceptor-like morphology in the inner nuclear layer(Araki et al. 1988). Similarly, cells with rod-like nuclearmorphology were identified in the inner retina during devel-opment (Spira et al. 1984), whereas, in another report,recoverin-positive ectopic cells were found in the ganglioncell layer (Günhan et al. 2003), some of them expressing

Fig. 8 The distribution ofdifferent proteins of thephototransduction cascade inMRCs of the developing ratretina. Sections from P14 ratswere reacted with anti-rhodopsinantibody (AO, a, d, g, j) and anantiserum against selectedproteins of the phototransductioncascade (middle column). Mergedimages are shown on the right. Inthe case of rod arrestin (retinal S-antigen, b, c) and recoverin (e, f)all rhodopsin-positive cells arecolabeled. Rhodopsin-kinase (h, i)stains lightly a smaller portion ofthe cells while rod transducin (k, l)is present in only a few cells persection. Insert shows one labeledcell—location indicated byarrows—in high magnification.ONL outer nuclear layer, INLinner nuclear layer, GCL ganglioncell layer. Bar 50 μm

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Fig. 9 Synapse-like ending of anMRC.Anti-rhodopsin (AO, a) and anti-synaptophysin (b) antibodies were applied consecutively on sections ofP14 rat retina; c merged image. Bulbous ending of a MRC contains

synaptophysin and contacts with another MRC. IPL inner plexiformlayer, GCL ganglion cell layer. Bar 20 μm

Fig. 10 Most MRCs areeliminated by apoptosis. Thenuclear morphology of mostMRCs resembled that of genuinerods in all stages of developmentexamined, as demonstrated byDAPI (in blue) and anti-rhodopsin(in green) colabeling (a, b, MRCsare indicated by arrows). SomeMRCs displayed pyknotic nuclei(b, arrowhead), while somepyknotic nuclei belonged todifferent cell populations (*). cThe number of pyknotic MRCsover the retinal length of 460 μm,in different stages of postnatalmaturation. Note that the highestnumbers were detected at P18,where the values weresignificantly higher than anystage examined except P21.Circlesmean, boxesmean ±standard error (SE), whiskersmean ± standard deviation (SD). dPercentage of pyknotic MRCs.By P28, almost one-third of thesenuclei were pyknotic. Bar 10 μm

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rhodopsin visual pigment. These rhodopsin- and recoverin-positive cells were reported to also appear in the inner nuclearlayer in later developmental stages but were missing from theadult retina. These cells did not colabel for any of the cell type-specific antibodies applied in the Günhan study, leaving theidentity of these cells undefined.

Semo et al. (2007) conducted a more thorough survey of therecoverin-positive cells in the ganglion cell layer and found thathey primarily contained cone opsins and only a smaller frac-tion was positive for rhodopsin. The cone opsin-positive pop-ulation was shown to form synapses containing cone arrestinand cone transducin, indicating the possibility of functioning asphotoreceptors. Although rudimentary outer segment-like struc-tures were detected by Spira et al. (1984), up to now, no datahave been published about the phototransduction proteins ofmisplaced rhodopsin-positive cells.

In this study, we aimed to conduct a more detailed survey toassess the morphology, relative number, developmental kineticsand staining characteristics of the peculiar rhodopsin-positivepopulation during the early postnatal developmental period ofthe rodent retina. MRCs were detectable in all rodent speciesexamined, in similar numbers and distribution. This cell popu-lation is not negligible, since it comprises a few percent of allrhodopsin-positive elements, which in turn make up about 70%of all cells of the rodent retina (Carter-Dawson and LaVail1979). This population is therefore much more numerous thansome of the better-known special cell types, such as, for exam-ple, dopaminergic amacrine cells (Nguyen-Legros et al. 1997).In this developmental period, MRCs also greatly outnumberdisplaced cone opsin-positive cells. Evidence that these cellsundoubtedly contain the rod visual pigment rhodopsin is thatthey could be stained by all rhodopsin-specific antibodies ap-plied, irrespective of whether they were polyclonal or monoclo-nal antibodies raised against either the C- or N-terminal residuesof the molecule. Double-labeling against the microglia/macrophage lineage, Müller glia and rod bipolar cells alsoexcluded the possibility that rhodopsin released from dying rodscould be responsible for rhodopsin staining of these cells.

Rhodopsin-expressing retinal cells are regularly and un-doubtedly classified as rods, both in in vivo and in vitroexperiments (Osakada et al. 2008). Rhodopsin immunocyto-chemistry was and still is the first choice to identify retinalrods, without any serious rival emerging. MRCs can thereforebe regarded as prospective rod cells. However, their neuron-shaped, arborized phenotype lacking an outer segment isstrikingly different from that of the genuine rods. Morpholog-ically, the MRCs rather resemble resident cell types of theinner retinal layers. To see whether or not some or all of theMRCs can be identified with neuron types of the inner retina,we conducted a series of double-labeling experiments withantibodies directed against various retinal cell types. Thenegative result of these immunocytochemical studies, likethose by Günhan et al. (2003), led us to conclude that MRCs

cannot be identical (at least at the molecular level studied)with any of the neuronal cell types in the inner retina.

Considering all these data, it seems reasonable to assumethat MRCs are rods that somehow failed to integrate into theouter nuclear layer. Sequestered in the inner retina and influ-enced by local factors specific for the given layers, theyprobably differentiate morphologically to attain bipolar,amacrine, or ganglion cell-like characteristics, while stillmaintaining their original ability to produce rhodopsin andother rod-specific molecules.

Can MRCs function as photoreceptors? According to ourresults, this possibility seems unlikely at both the molecularand cell morphological level. When staining for molecules ofthe rod phototransduction cascade, we found that (unlikedisplaced recoverin- and cone opsin-positive cells; Semoet al. 2007), most MRCs did not contain rod transducin andrhodopsin-kinase, two important members of thephototransduction pathway in rods. These cells supposedlyhave not completed functional rod differentiation, thereforetheir possible function, if any, is not photoreception.

The dendritic shape of the MRCs indicates that these cellsmust have lost their ability to form a highly differentiated,polarized cell type with the outer segment on one end and thesynaptic terminal on the other. After careful searching forouter segment-like structures, we occasionally found bulbousswellings of processes with intense rhodopsin labeling insome of the cells but these structures were sparse and couldnot be identified as primitive outer segments with certainty.Previous electron microscopic studies have also demonstratedrudimentary outer segment-like processes, belonging to cellswith rod-like nuclear morphology (Spira et al. 1984). Howev-er, full differentiation of outer segments requires functionalpigmented epithelial cells (Pinzón-Duarte et al. 2000), there-fore MRCs, sequestered in the inner layers of the retina, aremost probably unable to form such structures.

The question whether MRCs are able to establish synapticcontacts at all is an intriguing one, therefore we attempted tolocalize a synapse-specific protein, synaptophysin, in thesecells. Indeed, we observed synaptophysin-positive spots inless than 1 % of MRCs, indicating that the vast majority ofthese cells lacked synaptic connections. This is in good agree-ment with the report of Spira et al. (1984), occasionallydemonstrating presynaptic specializations of displaced recep-tor cells, without postsynaptic structures in contact.

Can these cells fulfill any other function in the differenti-ating retina? Considering their high number, their presence inall species studied and the fact that some of them survive untiladulthood, we cannot completely exclude such a possibility.In the ferret, most rods cells were shown to project processesto the inner plexiform layers during retinal development(Johnson et al. 1999). These processes were proposed to playa transitory role in directing cell migration and establishingcorrect connectivity of retinal cell types. Similar processes

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from genuine rod cells in rodents are missing, so it cannot beexcluded that MRCs, as a transitory cell population withpredominantly radially oriented processes, play similar rolesduring retinal maturation.

Questions about the further fate of these cells and why theydisappear completely from older and adult retinas are still tobe answered. Three theoretical possibilities can be considered:they die, migrate back to the outer nuclear layer, or switch offopsin production and differentiate to a yet unknown cell type.Due to the improper integration, most of these cells fail toform specific synaptic connections that are normally requiredfor neurons to survive (Spira et al. 1984; Bähr and Cellerino2000). Furthermore, being subjected to “abnormal” cell-to-cell contacts with inner retinal cells may be a source of pro-apoptotic signals that may also seriously challenge survival.Also, MRCs lack the crucial interactions with the retinalpigmented epithelium that is known to play vital roles inphotoreceptor functions by participating in outer segmentphagocytosis, retinal reizomerization and secreting a seriesof growth factors (Strauss 2005).

Lacking contacts and subjected to pro-apoptotic signals ofthe inner retina, MRCs may be eliminated by apoptosis. Whencalculating the number of apoptotic MRCs, the highest num-bers were found at P18–21, after which the number of displacedcells begins to decline significantly. Also, by P28, almost 30 %of MRCs were apoptotic. These data indicate that most—perhaps all—MRCs are eliminated by apoptosis.Wemust pointout, however, that not all MRCs are eliminated from the retina.In agreement with previous data (Spira et al. 1984; Semo et al.2007), we also detected MRCs, though in extremely smallnumbers, even in adult specimens (not shown in this report).

It is worthwhile to speculate about the time of disappear-ance of MRCs from the rat retina. The number of misplacedcells starts to decline after P14, following the time of eyeopening. This would suggest that increased light exposuremay be one of the factors involved in triggering apoptosis.Continuous background illumination is known to damagephotoreceptor cells (Noell et al. 1966). Opsin alone, in theabsence of 11-cis-retinal, is also able to activate thephototransduction cascade continuously to an extent, to causesimilar light-induced degeneration (Melia et al. 1997; Fain2006). Furthermore, KO animals, lacking arrestin orrhodopsin-kinase enzymes—from the deactivating phase ofphototransduction—are more sensible to light-induced dam-ages (Chen et al. 1999a, b). Our results demonstrated thatMRCs lacked some proteins of phototransduction, rodtransducin and rhodopsin-kinase. The absence of the latterprotein would enhance, while the former would inhibit, lightdamage of misplaced rhodopsin-positive cells and thus theseresults could not clearly support the idea that light exposure isrelated to the elimination of MRCs from the retinal mosaic.

The second theoretical option, a reintegration of MRCsinto the outer nuclear layer, is rather unlikely. By the time of

the decline of the MRC presence (between P14 and P21), thesynaptic layers of the retina are already relatively mature,thick and condensing and therefore a migration of the numer-ous MRCs with their arborized shape seems to be a difficulttask to achieve. It is also very unlikely that these cells canredifferentiate to any other cell type, since none of themcolabeled with any other cell-specific marker applied at anypostnatal stage checked.

The relatively high number of misplaced rhodopsin-expressing cells raises some interesting questions. Previousstudies using Nrl-gfp+/+ transgenic mice have shown thatNRL-positive postmitotic precursors, when transplanted tothe subretinal space of the recipients, can integrate into thephotoreceptor layer, differentiate to functional rods and estab-lish synaptic contact with the recipient’s bipolar cells(MacLaren et al. 2006). In this study, donor cells, harvestedin the period between P1 and P7 (a developmental periodoverlapping that of ours), had the best integration capacity.Interestingly, our results clearly demonstrate that, even underin vivo conditions, not all committed precursors integrate anddifferentiate to functional rod cells. After commitment, finalmaturationmust also be influenced by cell-to-cell contacts andlocal factors, which should also be taken into accountduring transplantation. Further studies conducted onMRCs may help us elucidate those factors needed forrod functional maturation.

We also cannot completely exclude the possibility thatMRCs represent a population inheritably different from gen-uine rods. We also demonstrated differences in protein expres-sion, which may either be an indicator of incomplete differ-entiation or the differences in cell fate. Consequently, if NRL(MacLaren et al. 2006) or rhodopsin (Osakada et al. 2008)expression alone is used to select rods as donor cells fortransplantation, it is possible that an additional populationwith little or no integration capacity may also be injected intothe recipient. We suggest that final differentiation should bechecked by colabeling with other molecules of thephototransduction cascade in in vitro experiments.

In summary, we here report a detailed study of a minorpopulation of rhodopsin-positive cells (MRCs) that aremisplaced towards the inner nuclear and ganglion cell layers.Although they lack all morphological resemblance to photo-receptor cells, colabeling studies indicate that they are mostprobably rods that failed to find their proper position andphotoreceptor function during development.

Acknowledgments The authors thank Paul A. Hargrave, GrazynaAdamus, Christine D. Dijkstra, Karl-Wilhelm Koch, KrysztofPalczewsky and Igal Gery for the kind donation of the antibodies. Thanksare also due to Pál Röhlich and Csaba Dávid for critical reading of themanuscript. The valuable assistance of Éva Kovácsné Dobozi andGyörgyné Vidra is highly appreciated. The work was supported by thefollowing grants: Hungarian Scientific Research Fund (OTKA #73000),TÁMOP- 4.2.1.B-09/1KMRB2010-0001.

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References

Adamus G, McDowell JH, Arendt A, Hargrave PA (1987) Structure,function and topography of rhodopsin as determined using mono-clonal antibodies. In: Ebrey TG, Frauenfelder H, Honig B,Nakanishi K (eds) Biophysical studies of retinal proteins: proceed-ings of a conference in memory of Laura Eisenstein. University ofIllinois Press, Urbana, pp 86–94

Adamus G, Zam ZS, Arendt A, Palczewski K, McDowell JH, HargravePA (1991) Anti-rhodopsin monoclonal antibodies of defined speci-ficity: characterization and application. Vis Res 31:17–31

Al-Otaibil F, Ul-Haq A, Al-Hindi H, Al Kofide A, Al Shail E (2012)Cauda equina syndrome as the initial presenting clinical feature ofmedulloblastoma: a case report. J Med Case Rep 6:135

Araki M, Hanihara T, Saito T (1988) Histochemical observations onunique rod-like cells in the developing retina of the normal rat. JNeurocytol 17:179–188

Arango-Gonzalez B, Szabó A, Pinzon-Duarte G, Lukáts Á, Guenther E,Kohler K (2010) In vivo and in vitro development of S- and M-cones in rat retina. Invest Ophthalmol Vis Sci 51:5320–5327

Bähr M, Cellerino A (2000) Apoptosis in the developing visual system.Cell Tissue Res 301:53–69

Barnstable CJ, Akagawa K, Hofstein R, Horn JP (1983) Monoclonalantibodies that label discrete cell types in the mammalian nervoussystem. Cold Spring Harb Symp Quant Biol 48:863–876

Bringmann A, Pannicke T, Grosche J, Francke M, Wiedemann P,Skatchkov SN, Osborne NN, Reichenbach A (2006) Müller cellsin the healthy and diseased retina. Prog Retin Eye Res 25:397–424

Carter-Dawson LD, LaVail MM (1979) Rods and cones in the mouseretina. I. Structural analysis using light and electron microscopy. JComp Neurol 188:245–262

Chang KK, Freestone PS, Lipski J (2011) Expression and functionalproperties of TRPM2 channels in dopaminergic neurons of thesubstantia nigra of the rat. J Neuropsychol 106:2865–2875

Chen J, Simon MI, Matthes MT, Yasumura D, LaVail MM (1999a)Increased susceptibility to light damage in an arrestin knockoutmouse model of Oguchi disease (stationary night blindness).Invest Ophthalmol Vis Sci 40:2978–2982

Chen KC, Burns ME, Spencer M, Niemi GA, Chen J, Hurley JB, BaylorDA, Simon MI (1999b) Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase. Proc NatlAcad Sci USA 96:3718–3722

Chucair-Elliott AJ, Elliott MH, Wang J, Moiseyev GP, Ma JX, Politi LE,Rotstein NP, Akira S, Uematsu S, Ash JD (2012) Leukemia inhib-itory factor coordinates the down-regulation of the visual cycle inthe retina and retinal-pigmented epithelium. J Biol Chem 287:24092–25102

Dijkstra CD, Döpp EA, Joling P, Kraal G (1985) The heterogeneity ofmononuclear phagocytes in lymphoid organs: distinct macrophagesubpopulations in the rat recognized by monoclonal antibodies EDl,ED2 and ED3. Immunology 54:589–599

Fain GL (2006)Why photoreceptors die (and why they don’t). Bioessays28:344–354

Forrest D, SwaroopA (2012)Mini review: the role of nuclear receptors inphotoreceptor differentiation and disease. Mol Endocrinol 26:905–915

GlösmannM, Peichl L (2007) Cone damage and ectopic cone opsin in thealbino rat retina induced by moderate cyclic light. InvestOphthalmol Vis Sci 48:1343, E-Abstract

Greferath U, Grünert U, Wässle H (1990) Rod bipolar cells in themammalian retina show protein kinase C-like immunoreactivity. JComp Neurol 301:433–442

Günhan E, van der List D, Chalupa LM (2003) Ectopic photoreceptorsand cone bipolar cells in the developing and mature retina. J Neursci23:1383–1389

Hamano K, Kiyama H, Emson PC, Manabe R, Nakuchi M, Tohyama M(1990) Localization of two calcium binding proteins, calbindin(28kD) and parvalbumin (12kD), in the vertebrate retina. J CompNeurol 302:417–424

Hicks D, Barnstable CJ (1987) Different rhodopsin monoclonal antibod-ies reveal different binding patterns on developing and adult ratretina. J Histochem Cytochem 35:1317–1328

Hwang IK, Yoo KY, Kim DS, Jung JY, Shin MC, Seo K, Kim KS, KangTC, Won MH (2005) Comparative study on calretinin immunore-activity in gerbil and rat retina. Anat Histol Embryol 34:129–131

Johnson PT, Williams RR, Cusato K, Reese BE (1999) Rods and conesproject to the inner plexiform layer during development. J CompNeurol 414:1–12

Li RS, ChenBY, TayBK, ChanHHL, PuML, SoKF (2006)Melanopsin-expressing retinal ganglion cells are more injury-resistant in achronic ocular hypertension model. Invest Ophthalmol Vis Sci 47:2951–2958

Lukáts Á, Szabó A, Röhlich P, Vígh B, Szél Á (2005) Photopigmentcoexpression in mammals: comparative and developmental aspects.Histol Histopathol 20:551–574

MacLaren RE, Pearson RA, MacNeil A, Douglas RH, Salt TE, AkimotoM, Swaroop A, Sowden JC, Ali RR (2006) Retinal repair bytransplantation of photoreceptor precursors. Nature 444:203–207

Mano T, Puro DG (1990) Phagocytosis by human retinal glial cells inculture. Invest Ophthalmol Vis Sci 31:1047–1055

McGill TJ, Cottam B, Lu B, Wang S, Girman S, Tian C, Huhn SL, LundRD, Capela A (2012) Transplantation of human central nervoussystem stem cells – neuroprotection in retinal degeneration. Eur JNeurosci 35:468–477

Mears AJ, Kondo M, Swan PK, Takada Y, Bush RA, Saunders TL,Sieving PA, Swaroop A (2001) Nrl is required for rod photoreceptordevelopment. Nat Genet 29:447–452

Melia TJ Jr, Cowan CW, Angleson JK, Wensel TG (1997) A comparisonof the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin. Biophys J 73:3182–3191

Milan AH, Dacey DM, Dizhoor AM (1993) Recoverin immunoreactivityin mammalian cone bipolar cells. Vis Neurosci 10:1–12

Mirshahi M, Boucheix C, Collenor G, Thillaye B, Faure JP (1985)Retinal S-antigen epitopes in vertebrate and invertebrate photore-ceptors. Invest Ophthalmol Vis Sci 26:1016–1021

Molday RS, MacKenzie D (1983) Monoclonal antibodies to rhodopsin:characterization, cross-reactivity, and application as structuralprobes. Biochemistry 22:653–660

Morrow EM, Belliveau MJ, Cepko CL (1998a) Two phases of rodphotoreceptor differentiation during rat retinal development. JNeurosci 18:3738–3748

Morrow EM, Furukawa T, Cepko CL (1998b) Vertebrate photoreceptorcell development and disease. Trends Cell Biol 8:353–358

Nadal-Nicolas FM, Jiménez-López M, Sobrado-Calvo P, Nieto-López L,Cánovas-Martínez I, Salinas-Navarro M, Vidal-Sanz M, Agudo M(2009) Brn3a as a marker of retinal ganglion cells: qualitative andquantitative time course studies in naive and optic nerve–injuredretinas. Invest Ophthalmol Vis Sci 50:3860–3868

Ng L, Lyubarsky A, Nikonov SS, Ma M, Srinivas M, Kefas B, St.Germain DL, Hernandez A, Pugh EN Jr, Forrest D (2010) Type 3deiodinase, a thyroid-hormone-inactivating enzyme, controls sur-vival and maturation of cone photoreceptors. J Neurosci 30:3347–3357

Nguyen-Legros J, Versaux-Botteri C, Savy C (1997) Dopaminergic andGABAergic retinal cell populations in mammals. Microsc Res Tech36:26–42

Nir I, Cohen D, Papermaster D (1984) Immunocytochemical localizationof opsin in the cell membrane of developing rat retinal photorecep-tors. J Cell Biol 98:1788–1795

Noell WK, Walker VS, Kang BS, Berman S (1966) Retinal damage bylight in rats. Invest Ophthalmol Vis Sci 5:450–473

Cell Tissue Res

Osakada F, Ikeda H, Mandai M, Wataya T, Watanabe K, Yoshimura N,Akaike A, Sasai Y, Takahashi M (2008) Toward the generation ofrod and cone photoreceptors from mouse, monkey and humanembryonic stem cells. Nat Biotechnol 26:215–224

Pinzón-Duarte G, Kohler K, Arango-González B, Guenther E (2000) Celldifferentiation, synaptogenesis, and influence of the retinal pigmentepithelium in a rat neonatal organotypic retina culture. Vis Res 40:3455–3465

Rapaport D, Wong LL, Wood ED, Yasumura D, Lavail MM (2004)Timing and topography of cell genesis in the rat retina. J CompNeurol 474:304–324

Röhlich P, Szél Á (1993) Binding sites of photoreceptor-specific antibod-ies COS-1, OS-2 and AO. Curr Eye Res 12:935–944

Schnitzer J (1988) Immunocytochemical studies on the development ofastrocytes, Müller (glial) cells, and oligodendrocytes in the rabbitretina. Dev Brain Res 44:59–72

Semo M, Vugler A, Jeffery G (2007) Paradoxical opsin expressing cellsin the inner retina that are augmented following retinal degeneration.Eur J Neurosci 25:2296–2306

Spira A, Hudy S, Hannah R (1984) Ectopic photoreceptor cellsand cell death in the developing rat retina. Anat Embryol169:293–301

Strauss O (2005) The retinal pigment epithelium in visual function.Physiol Rev 85:845–881

Swaroop A, Kim D, Forrest D (2010) Transcriptional regulation ofphotoreceptor development and homeostasis in the mammalianretina. Nat Rev Neurosci 11:563–576

Szél Á, Lukáts Á, Fekete T, Szepessy Z, Röhlich P (2000) Photoreceptordistribution in the retinas of subprimate mammals. J Opt Soc Am 17:568–579

Wässle H, Grünert U, Röhrenbeck J (1993) Immunocytochemical stain-ing of AII-amacrine cells in the rat retina with antibodies againstparvalbumin. J Comp Neurol 332:407–420

Young RW (1985) Cell differentiation in the retina of the mouse. AnatRec 212:199–205

Zhao X, Huang J, Khani SC, Palczewski K (1998) Molecularforms of human rhodopsin kinase (GRK1). J Biol Chem273:5124–5131

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Immunocytochemical analysis of misplaced rhodopsin-positive cells in the developing rodent retina