October 2010
Volume 51, Issue 10
Free
Retinal Cell Biology  |   October 2010
In Vivo and In Vitro Development of S- and M-Cones in Rat Retina
Author Affiliations & Notes
  • Blanca Arango-Gonzalez
    From the Division of Experimental Ophthalmology, the Centre for Ophthalmology, Tübingen, Germany;
  • Arnold Szabó
    the Department of Human Morphology and Developmental Biology, Semmelweis University, Budapest, Hungary;
  • German Pinzon-Duarte
    the Department of Cell Biology, SUNY Downstate Medical Center, Brooklyn, New York; and
  • Ákos Lukáts
    the Department of Human Morphology and Developmental Biology, Semmelweis University, Budapest, Hungary;
  • Elke Guenther
    the NMI Natural and Medical Sciences Institute, the University of Tübingen, Rütlingen, Germany.
  • Konrad Kohler
    From the Division of Experimental Ophthalmology, the Centre for Ophthalmology, Tübingen, Germany;
  • *Each of the following is a corresponding author: Elke Günther, NMI Natural and Medical Sciences Institute, Dept. Cell Biology, Markwiesenstrasse 55, 72770 Rütlingen, Germany; guenther@nmi.de. Konrad Kohler, Universitätsklinikum Tübingen, Zentrum für Regenerations, Biologie und Regenerative, Medizin ZRM, Paul-Ehrlich-Strasse 15, 72076 Tübingen, Germany; konrad.kohler@uni-tuebingen.de
  • Footnotes
    2  These authors contributed equally to the work presented here and should therefore be regarded as equivalent first authors.
  • Footnotes
    6  These authors contributed equally to the work presented here and should therefore be regarded as equivalent senior authors.
Investigative Ophthalmology & Visual Science October 2010, Vol.51, 5320-5327. doi:10.1167/iovs.09-4741
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Blanca Arango-Gonzalez, Arnold Szabó, German Pinzon-Duarte, Ákos Lukáts, Elke Guenther, Konrad Kohler; In Vivo and In Vitro Development of S- and M-Cones in Rat Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(10):5320-5327. doi: 10.1167/iovs.09-4741.

      Download citation file:


      © 2016 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

Purpose.: Organ cultures of the rodent retina could provide a powerful tool in the study of cone development and differentiation. Previous attempts, however, have failed to show M-cone development in organ cultures of the mouse and rat retina. This study mimicked the in vivo dynamics of S- and M-cone development in a culturing approach for the postnatal rat retina.

Methods.: Retinas of Brown Norway rats were collected at different developmental ages (postnatal day [P]0–P270) to study cone development in vivo. For culturing, the retinas were prepared from P0 to P2 animals and allowed to develop in organ culture for 2 to 15 days. Subsequently, opsin expression was analyzed immunohistochemically and morphometrically.

Results.: In control retinas, S-opsin was already expressed at birth, whereas M-opsin was detected after P4. The maximum density of S-opsin–positive cones was reached at P10 (∼17,000 cells/mm2) and of M-opsin–positive cones, at P12 (∼14,000 cells/mm2). The number of both cone types decreased gradually thereafter to ∼1,000 S-opsin cones/mm2 and ∼4,000 M-opsin cones/mm2 in the adult. In culture, both cone types developed with dynamics of appearance comparable to those in vivo, with a peak density of ∼12,300 cones/mm2 for S-opsin and ∼7,500 cones/mm2 for M-opsin labeling.

Conclusions.: These results in rat retina showed for the first time that cone development and expression dynamics can be mimicked in organ culture. With this experimental approach, it will be possible to evaluate aspects of cone development under controlled experimental conditions and to elucidate factors crucial for proper cone differentiation.

The rod-dominated retinas of most mammalian species contain two distinct cone populations with different wavelength sensitivities. The short-wavelength–sensitive cones (S-cones) that have peak sensitivities in the blue or UV part of the spectrum are usually outnumbered by the middle-wavelength–sensitive cones (M-cones). 13 The distribution of different cone subtypes has been well studied in a variety of species; however, the factors that regulate their development at the molecular level are poorly understood. All cell types of the vertebrate retina derive from the same pool of common multipotent progenitor cells according to a strictly regulated timetable: The early born ganglion cells are followed by cone, amacrine, and horizontal cells, whereas rod, bipolar, and Müller cells are generated in the later phases. 46 During development, cell fate is determined by complex regulatory mechanisms, and the intrinsic program is altered by extrinsic signals. 711 Our present knowledge about the regulatory factors that control photoreceptor cell fate and development has been reviewed recently in several publications. 711  
Cones undergo their final mitosis in the early prenatal period, yet there is a significant delay before they express their mature phenotype; and in rodents, major events of cone development, such as outer segment formation, opsin expression, and synaptogenesis extend into the first postnatal weeks. 1215 Aside from some notable exceptions, 16 in most mammalian species studied so far, the first detectable visual pigment during development is rhodopsin in the rods, followed by the appearance of S- and finally M-opsin in the cones. 1,17 The factors that control opsin expression and the development of cone patterns are largely unknown, and despite general similarities, they may differ from one species to the next. In some species, such as the rat, where the cones are evenly distributed across the retina, 18 most of the developing cones coexpress both the S- and the M-opsin (transient dual cones) in the early postnatal life. The absence of dual cones in the adult supports the hypothesis that M-cones develop via transdifferentiation from initially S-opsin–expressing cells. 19 It is still unclear whether this developmental scheme is universal among mammals, but transient dual cones have also been identified in several other species, including the gerbil, tree shrew, and human. 1,20  
Recent experiments indicated that the differentiation of M-cones is closely related to that of S-cones and that thyroid hormone via its β2 receptor (TRβ2) has a major influence on opsin expression. 2123 In mouse-knockout experiments, it has been demonstrated that TRβ2 is necessary to activate M-opsin expression, and in cooperation with the retinoid X receptor γ (RXRγ), TRβ2 also suppresses S-opsin production. 21,24 The ligands of RXRγ in S-opsin repression are still unidentified, and whether the thyroid hormone alone can regulate opsin patterning is an open question. In addition, Crx, an Otx-like homeodomain transcription factor, and RORα and -β—members of the retinoic acid receptor-related orphan receptor family—have also been shown recently to play a role in the activation of cone-specific opsin expression. 2527  
Although organ cultures could provide a powerful tool for the study of putative regulatory factors under defined experimental conditions, all attempts so far to culture embryonic and newborn mouse retina have resulted in incomplete photoreceptor development with the complete lack of M-opsin expression. 24,2830 The fact that M-cones were detectable only when retinal explants were made from P3 or older pups indicates the possibility that soluble factors were acting during this critical period that were missing from the culturing medium. Similar results with only low levels of M-opsin mRNA and the complete absence of immunohistochemically detectable M-opsin–expressing cells were described in a rat explant culture. 31  
We present an organ culture of the postnatal rat retina that shows S- and M-opsin expression comparable to that in age-matched in vivo controls, indicating that cone transdifferentiation occurs in vitro. Our findings support the hypothesis that this process is under control of soluble factors and will help elucidate the nature of these factors and their relevance for proper cone development. 
Materials and Methods
Tissue Processing
Animal procedures were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Retinas from Brown Norway rats (Charles River, Sulzfeld, Germany) of different postnatal ages (P0, P2, P4, P6, P8, P10, P12, P14 P18, P30, P60, P120, and P270) were analyzed as wholemounts or radial sections and serve as age-matched controls for the cultured retinas. The day of birth was denoted as P0. 
After anesthesia and decapitation of the rats, the eyes were immediately enucleated and the cornea and lens were removed. For radial sections, the remaining eye cup was immersed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for 30 minutes at 4°C. After fixation, the eye cups were washed several times in 0.1 M PB, cryoprotected in 30% sucrose in 0.1 M PB at 4°C overnight, embedded in cryomatrix (Thermo Fischer Scientific, Waltham, MA), and frozen in liquid nitrogen. Sections (12 μm thick) were cut on a cryostat and collected on gelatin-coated slides. After air-drying, the sections were processed immediately or stored at −20°C. For wholemount immunohistochemistry, the retinas were carefully detached from the eye cups and fixed as just described. 
Preparation of Organ Culture
Retinal organ cultures were prepared as described elsewhere. 32 Briefly, the eyes of decapitated Brown Norway rats (P0-P2) were enucleated under sterile conditions and transferred to a Petri dish containing ice-cold sterile Ames medium (Sigma-Aldrich, St. Louis, MO) enriched with glucose (20 mM). After enzymatic digestion of the eyeball, the retina, together with the attached pigment epithelium, was dissected and four radial cuts were made to flatten it. Retinas were placed on culture plate inserts (Corning Life Sciences, Lowell, MA) with the ganglion cell layer uppermost. The explants were cultured in 1:1 DMEM/F12 medium (Invitrogen Corporation, Carlsbad, CA) and supplemented with: α-tocopherol (1 μg/mL), ascorbic acid (100 μg/mL), retinol (10 μM), retinyl acetate (10 μM), hydrocortisone (20 nM), insulin (5 μg/mL), progesterone (20 nM), putrescine (100 μM), selenium (20 nM), taurine (3 mM), transferrin (1 μg/mL), triiodothyronine (2 ng/mL) and 10% fetal calf serum (Invitrogen Corporation). All supplements were cell culture grade and obtained from Sigma-Aldrich, unless otherwise stated. The cultures were kept without shaking or rotation in an incubator with an atmosphere of 5% CO2, balanced air and 100% humidity at 37°C. The medium was changed the first day in culture and every second day thereafter. After 2 to 15 days in vitro (DIV), the cultures were fixed and analyzed as radial sections or wholemounts. The results were compared with those of age-matched control retinas. 
Immunohistochemistry
Photoreceptor phenotypes were characterized by immunostaining the cryostat sections and wholemounts of age-matched control and cultured retinas with affinity-purified JH455 and JH492 rabbit polyclonal antibodies (Table 1). 33,35 After rehydration, the radial sections were preincubated with phosphate-buffered saline (PBS; 50 mM, pH 7.4) containing 20% normal goat serum and 0.03% Triton X-100 (Sigma-Aldrich) for 2 hours at room temperature to block nonspecific antibody binding. Subsequently, the sections were incubated overnight at 4°C with specific primary antibodies. The immunoreaction was visualized with Cy3-conjugated anti-rabbit antibody (Rockland, Gilbertsville, PA) diluted 1:1000. For the visualization of cones in wholemounts, the same protocol was performed with prolonged incubation times. 
Table 1.
 
Primary Antibodies
Table 1.
 
Primary Antibodies
Antigen Host Source Working Concentration References
S-opsin (JH455) Rabbit polyclonal Gift of Jeremy Nathans 1:20,000 Wang et al. 33
M-opsin (JH492) Rabbit polyclonal Gift of Jeremy Nathans 1:20,000 Wang et al. 33
M-opsin (AB5405) Rabbit polyclonal Millipore Billerica, MA 1:1,000 Gaillard et al. 34
M-opsin (COS-1) Mouse monoclonal Gift of Ágoston Szél 1:50 Röhlich and Szél 35
For wholemounts, fixed retinas were dissected from pigmented epithelium and incubated for 2 hours in blocking solution, for 3 days in the primary antibody, and for 1 day in the secondary antibody. The specificity of M-opsin–specific reactions in culture was determined by performing double labeling experiments on retinal wholemounts using a second set of M-opsin–specific antibodies. For this, COS-1, a monoclonal hybridoma supernatant 35 and AB5405, a commercial available rabbit polyclonal antibody 34 were used (Table 1). The reactions were visualized by species-specific Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (Invitrogen Corporation), both diluted in 1:200. Stained retinas were mounted flat, with the photoreceptor layer facing upward and coverslipped. Controls for all reactions were performed by omitting the first antibody. 
Morphometric Analysis
The density of immunoreactive cells was determined in retinal wholemounts by confocal microscopy (LSM 410; Carl Zeiss Meditec, Oberkochen, Germany). The images were sampled at 1-μm intervals in a minimum of 14 and a maximum of 30 planes, with a ×40 water-immersion objective. For morphometric analysis, a wholemount was divided into four quadrants. From each quadrant, a central area of 0.1024 mm2 was selected 1 mm away from the optic disc, resulting in four areas per retina. Images were analyzed with special software (Scion Image program for Windows Beta 4.0.2; Scion Corporation, Frederick, MD). They were first converted from the gray level pixels into black or white, with the threshold set at 200, as it proved to optimize discrimination of cells from dust and scratches. The “analyze particles” function was used to export to a text file a list of parameters for each particle present on the picture. Particles with a diameter far different from that of a photoreceptor were discarded. Data were not corrected for shrinkage. 
For validation of the counting method, randomly selected images were also counted manually by an experienced investigator. Differences between the experimental groups were tested statistically by a two-tailed unpaired t-test (Prism 4 for Windows; GraphPad Software, La Jolla, CA). All data shown represent the means and standard errors of the mean from at least four separate experiments. Representative pictures were taken from the central, most differentiated area of the retina. Confocal images of double-labeled specimens were obtained by microscope (Radiance 2100 Multi Photon Imaging System [Bio-Rad, Munich, Germany] coupled to an Eclipse E800 [Nikon, Tokyo, Japan] with LaserSharp 2000 software [Bio-RD]). Image management software (Photoshop; Adobe, San Diego, CA, and Confocal Assistant [free software, copyright Todd Clark Brelje, University of Minnesota, Minneapolis, and available at http://bipl.umn.edu/downloads]) was used for primary image processing. 
Results
Postnatal Cone Development
In the control rat retinas, three distinct phases of S- and M-opsin expression were identified during the postnatal period. The first phase corresponded to the first 10 to 12 days of postnatal development. S-opsin expression was already present at birth (∼2860 cones/mm2), whereas M-opsin was detectable only after P4 (∼750 cones/mm2). In this period, the density of both types of labeled cones increased progressively, up to a peak value at P10 (∼17,000 cones/mm2) for S-opsin and P12 (∼14,000 cones/mm2) for M-opsin (Figs. 1, 2; Table 2). In this early stage of cone development, the opsin-specific antibodies labeled the whole cell (Fig. 3B, 3D). 
Figure 1.
 
Opsin expression in retinal wholemounts at different developmental stages. The retinas were treated with JH455 and JH492 antibodies to show S-opsin (left) and M-opsin (right) labeling, respectively. The photomicrographs were taken from the central, most differentiated areas. Scale bar, 100 μm.
Figure 1.
 
Opsin expression in retinal wholemounts at different developmental stages. The retinas were treated with JH455 and JH492 antibodies to show S-opsin (left) and M-opsin (right) labeling, respectively. The photomicrographs were taken from the central, most differentiated areas. Scale bar, 100 μm.
Figure 2.
 
Quantification of S- and M-opsin expression during retinal development. S-opsin expression was already present at birth, increased in number until P10, and decreased subsequently. M-opsin became detectable from P4 on and increased in number until P12. Although they also decrease in number subsequently, M-opsin–expressing cones strongly outnumber S-opsin–expressing cones in the adult.
Figure 2.
 
Quantification of S- and M-opsin expression during retinal development. S-opsin expression was already present at birth, increased in number until P10, and decreased subsequently. M-opsin became detectable from P4 on and increased in number until P12. Although they also decrease in number subsequently, M-opsin–expressing cones strongly outnumber S-opsin–expressing cones in the adult.
Table 2.
 
In Vivo and In Vitro Time Course of S-opsin– and M-opsin–Expressing Cones
Table 2.
 
In Vivo and In Vitro Time Course of S-opsin– and M-opsin–Expressing Cones
Postnatal Day Group S-opsin–Expressing Cones M-opsin–Expressing Cones
Mean ± SD P Mean ± SD P
0 P0 2,861 ± 674.6 0 ± 0.0
2 P2 7,246 ± 869.9 0 ± 0.0
4 P4 9,160 ± 854.9 0.5357 752 ± 160.7 0.0822
P2 DIV2 8,789 ± 739.1 537 ± 129.1
6 P6 11,045 ± 402.7 0.0002 2,568 ± 243.7 0.1993
P2 DIV4 9,258 ± 225.5 2,344 ± 194.0
8 P8 13,691 ± 474.4 0.0050 5,830 ± 567.3 0.0098
P2 DIV6 12,363 ± 393.9 4,648 ± 283.5
10 P10 16,914 ± 663.7 <0.0001 11,318 ± 380.6 <0.0001
P2 DIV8 9,990 ± 304.9 7,500 ± 186.0
12 P12 13,350 ± 516.6 <0.0001 13,975 ± 693.9 <0.0001
P2 DIV10 6,406 ± 264.9 7,529 ± 178.7
14 P14 10,781 ± 1,005.0 <0.0001 11,787 ± 224.1 <0.0001
P2 DIV12 5,469 ± 240.8 7,432 ± 433.1
Figure 3.
 
In the organotypic culture of the pigmented rat retina, both types of cones are present. The retinal layers are well preserved and the morphology of cones developed in culture (B, D) was comparable to that of the age-matched controls (A, C). At P10, the opsin-specific antibodies labeled the whole cone cell body. Scale bar, 50 μm.
Figure 3.
 
In the organotypic culture of the pigmented rat retina, both types of cones are present. The retinal layers are well preserved and the morphology of cones developed in culture (B, D) was comparable to that of the age-matched controls (A, C). At P10, the opsin-specific antibodies labeled the whole cone cell body. Scale bar, 50 μm.
The second phase of opsin expression was between P12 and P30. The ratio of S- to M-opsin–positive cells became inverted, and from this time, the density of S-opsin–expressing cones was lower than that of M-opsin–expressing cones. The number of labeled cells decreased rapidly during this period, but the effect was more pronounced for S-opsin–positive cells. By the end of the second postnatal week, M-opsin–positive cones clearly outnumbered S-opsin–expressing cones (Figs. 1, 2, Table 2). In this stage, the opsin-specific antibodies labeled the cone outer segments almost exclusively (data not shown). 
After P30, the density of S-opsin–positive cones decreased further to counts <1000 cells/mm2 by P270. From P60, a similar but less robust decrease was found in the number of M-opsin–expressing cones, reaching a minimum at ∼3500 cells/mm2 by P270 (Figs. 1, 2). 
In Vitro Cone Development
To analyze cone development under in vitro conditions, we examined organ cultures prepared from P0 to P2 retinas every second day, from DIV2 up to DIV14 in wholemounts or radial sections. The cultured retinas showed clear stratification, and all retinal layers developed by the end of the second postnatal week. Although cultures harvested from P0 pups had more rosettelike formations, the time of explantation had no major influence on overall retinal development and morphology. Cone inner–outer segments were easily recognizable, and their morphology was well preserved (Figs. 3, 4, 5). In contrast to previous reports, 2831 both S- and M-opsin were expressed (Figs. 4, 5), even in cultures prepared from P0 retinas (Figs. 3, 6). As in the age-matched control retinas, the opsin-specific antibodies stained the entire photoreceptor somata at younger ages (Fig. 3). Because a careful examination of cultures explanted at P0 or P2 showed no significant difference in the number of S- and M-opsin–positive cells and because of better-preserved overall morphology, cultures harvested from P2 rats were used for statistical analysis. 
Figure 4.
 
S-opsin expression in wholemounts of cultured retinas (A). Quantification of S-opsin–expressing cones (B). Despite the lower density in culture, the expression pattern of S-opsin was similar to that in the age-matched control retinas. **P < 0.01, ***P < 0.0001. Scale bar, 50 μm.
Figure 4.
 
S-opsin expression in wholemounts of cultured retinas (A). Quantification of S-opsin–expressing cones (B). Despite the lower density in culture, the expression pattern of S-opsin was similar to that in the age-matched control retinas. **P < 0.01, ***P < 0.0001. Scale bar, 50 μm.
Figure 5.
 
M-opsin expression in wholemounts of cultured retinas (A). Quantification of M-opsin–expressing cones (B). Although in present in lower number, M-opsin–expressing cones developed in the culturing approach and their quantity changed similar to that in age-matched control retinas. **P < 0.01, ***P < 0.0001. Scale bar, 50 μm.
Figure 5.
 
M-opsin expression in wholemounts of cultured retinas (A). Quantification of M-opsin–expressing cones (B). Although in present in lower number, M-opsin–expressing cones developed in the culturing approach and their quantity changed similar to that in age-matched control retinas. **P < 0.01, ***P < 0.0001. Scale bar, 50 μm.
Figure 6.
 
Double labeling for M-opsin in wholemounts of cultured rat retinas. P0 DIV7 retina (A–C) immunostained with AB5405 antibody (A) and COS-1 antibody (B); merged images (C). Only a few structures were labeled with COS-1. P0 DIV14 retina (D–F) immunostained with AB5405 antibody (D) and COS-1 antibody (E); merged images (F). Outer segments are clearly distinguishable, showing a high degree of co-localization. Scale bar, 25 μm.
Figure 6.
 
Double labeling for M-opsin in wholemounts of cultured rat retinas. P0 DIV7 retina (A–C) immunostained with AB5405 antibody (A) and COS-1 antibody (B); merged images (C). Only a few structures were labeled with COS-1. P0 DIV14 retina (D–F) immunostained with AB5405 antibody (D) and COS-1 antibody (E); merged images (F). Outer segments are clearly distinguishable, showing a high degree of co-localization. Scale bar, 25 μm.
The density of S-opsin–expressing cones in the organ culture increased to a peak of ∼12,400 cells/mm2 by day 6 in vitro and decreased to below 5,500 cells/mm2 after 12 DIV (Fig. 4, Table 2). Compared with age-matched control retinas, the peak density of S-opsin–positive cones in culture was lower and the decrease in the number of cells occurred earlier. However, the dynamic of alteration was similar (Fig. 4B). 
Likewise, the density of M-opsin–expressing cones increased during the first days in culture, reaching a plateau of ∼7500 cells/mm2 after 8 DIV (Fig. 5, Table 2). As observed for S-opsin–expressing cones, the peak density of M-opsin–positive cones in culture was lower than in age-matched control retinas, but the dynamic of alteration was similar (Fig. 5B). 
To unequivocally confirm the presence of M-opsin expression in our approach, we performed double staining on P0 DIV7 and P0 DIV14 cultures with two other antibodies with confirmed M-opsin specificity (Fig. 6, Table 1). The monoclonal COS-1 recognized M-opsin a few days later than did AB5405 and resulted in slightly weaker reactions at younger ages, but both antibodies labeled the same population of cells as demonstrated at P0 DIV14 (Fig. 6, lower right). 
Discussion
Morphogenesis and opsin expression of cones in rat retinas appear to occur in a sequential manner according to a well-defined timetable. Rhodopsin is detectable first, soon followed by S- and finally by M-opsin expression. 1,17  
Despite some important differences, our results on cone development of pigmented rats are in good agreement with observations reported earlier in the albino rat. 19 Although in albino rats the first S- and M-opsin immunoreactivities appeared only at P5 and P9, we detected S-opsin at birth and M-opsin at P4. In addition to the earlier appearance of the first opsin-expressing cells, we counted higher densities for both cone types, and the maximum densities were reached a few days earlier. As no exact isodensity maps were made in either experiment, it is worth mentioning that these values represent average densities and can therefore be used as a first approximation only. There are two possible reasons for the dissimilar findings of our study and those in a previous report 19 : First, cone densities can be remarkably different across distinct rat strains. 3638 Second, the anti-opsin antibodies used may differ in their sensitivities, leading to different cone density detection levels. 
Our analysis of cone expression during postnatal development in the pigmented rat retina has shown that the dynamic of alteration between S- and M-opsin–expressing cones was comparable but shifted in time, resulting in a 4-day delay in the expression of M-opsin. Thus, our data support the cone developmental model of transdifferentiation proposed by Szél et al., 19 in which all cones express S-opsin only during early postnatal development, but most change their phenotype and differentiate into definitive M-cones. 19,39  
Other studies also suggest that the default pathway in cone development is S-opsin expression and that switching on M-opsin expression requires developmental signals that are missing in organ culture systems. 24,2831 In cultures prepared from retinas older than P3, M-opsin expression was always detectable, independent of the culture medium. Thus, it appears that the critical period of M-cone development comprises the first two postnatal days. 
In contrast to previous organ culture approaches, we were able to demonstrate both S- and M-opsin expression in cultures prepared from retinas younger than P3. Although cone densities were lower in cultured retinas, the temporal and the spatial opsin expression patterns were similar to that of the age-matched control retinas. The expression of M-opsin was independent of the time of explantation, and no significant differences were found between retinas explanted at P0 and P2. M-opsin was detected with three different antibodies, all of which have been shown to be specific. 3335  
Taken together, our data strongly indicate that transdifferentiation of cones occurred in our organotypic culture approach and thus can be mimicked under controlled in vitro conditions, offering the possibility for the study of processes of cone development and the factors and signal pathways involved. 
At present, we do not know the factors responsible for the developmental switch from S- to M-opsin production in our culture system. The most likely explanations for the observed differences in cone transdifferentiation potential of rodent organotypic cultures may be the model animal used or, even more likely, the composition of the culturing medium. A detailed comparison of the various culture approaches for rodent retinas 24,2830,4042 will be necessary to identify these crucial factors. However, such an analysis is beyond the scope of the present study. 
One important candidate is the thyroid hormone, since its importance in cone development in the mouse is obvious, 2125,27,4347 and it may be important in the rat as well. However, the culture media used in all previous studies were either serum-supplemented 31 or contained thyroid hormone in the same or even higher concentrations than was applied in our system. 24,2830,48 Moreover, the treatment of embryonic mouse retina cultures with increasing concentrations of thyroid hormone failed to activate M-opsin in a recent report by Roberts et al. 24 Thus, it seems unlikely that this hormone alone can explain the differences; and this result emphasizes that attention must be drawn to other still unidentified extrinsic factors that are present in our system. 
The use of different animal models raises the question of whether the absence of M-cones in mouse organotypic cultures can be explained by the interspecies differences in retinal development. In the mouse, even in adults, most cones coexpress both S- and M-opsins, albeit in different ratios in different retinal locations. M-opsin dominates in dorsal cones, whereas S-opsin expression is more prominent in ventral retinal regions. 4951 This peculiar division postulates a complex regulatory process, which may be significantly different from that of the rat. However, the absence of M-cone development in a rat culture approach reported previously 31 supports our hypothesis that the crucial difference is the composition of the culture medium and the absence of important regulatory factors in other approaches. 
At present, a systematic testing of several candidates is in progress and, if successful, will result in the identification of one or several factors crucial for proper cone development and differentiation. Moreover, culturing of different species with different cone distributions 1,2 or cone-rich retinas 34,52 may help elucidate the question of whether M-cone differentiation requires different conditions in different species. 
Footnotes
 Supported by GK 794 and Charlotte and Tistou Kerstan Foundation (BA-G); EU-Marie Curie Training Site “Vision” QLGA-1999-50423 and Hungarian Scientific Research Fund OTKA K73000 (AS); Pro Retina Foundation, Germany (GP-D); and Hungarian Scientific Research Fund OTKA F61717 and OTKA K73000 (ÁL).
Footnotes
 Disclosure: B. Arango-Gonzalez, None; A. Szabó, None; G. Pinzon-Duarte, None; Á. Lukáts, None; E. Guenther, None; K. Kohler, None
The authors thank Ágoston Szél (Semmelweis University, Budapest, Hungary) for the monoclonal COS-1 antibody and for a valuable critique of the manuscript, Jeremy Nathans (Johns Hopkins University School of Medicine, Baltimore, MD) for the kind gift of JH455 and JH492 rabbit polyclonal antibodies, and Diane Blaurock for assistance in preparation of the manuscript. 
References
Lukáts Á Szabó A Röhlich P Vigh B Szél A . Photopigment coexpression in mammals: comparative and developmental aspects. Histol Histopathol. 2005;20:551–574. [PubMed]
Peichl L . Diversity of mammalian photoreceptor properties: adaptations to habitat and lifestyle? Anat Rec A Discov Mol Cell Evol Biol. 2005;287:1001–1012. [CrossRef] [PubMed]
Hunt DM Carvalho LS Cowing JA Davies WL . Evolution and spectral tuning of visual pigments in birds and mammals. Philos Trans R Soc Lond B Biol Sci. 2009;364:2941–2955. [CrossRef] [PubMed]
Carter-Dawson LD LaVail MM . Rods and cones in the mouse retina. II. Autoradiographic analysis of cell generation using tritiated thymidine. J Comp Neurol. 1979;188:263–272. [CrossRef] [PubMed]
Young RW . Cell differentiation in the retina of the mouse. Anat Rec. 1985;212:199–205. [CrossRef] [PubMed]
Rapaport DH Wong LL Wood ED Yasumura D LaVail MM . Timing and topography of cell genesis in the rat retina. J Comp Neurol. 2004;474:304–324. [CrossRef] [PubMed]
Cepko CL Austin CP Yang X Alexiades M Ezzeddine D . Cell fate determination in the vertebrate retina. Proc Natl Acad Sci U S A. 1996;93:589–595. [CrossRef] [PubMed]
Belliveau MJ Cepko CL . Extrinsic and intrinsic factors control the genesis of amacrine and cone cells in the rat retina. Development. 1999;126:555–566. [PubMed]
Livesey FJ Cepko CL . Vertebrate neural cell-fate determination: lessons from the retina. Nat Rev Neurosci. 2001;2:109–118. [CrossRef] [PubMed]
Hennig AK Peng GH Chen S . Regulation of photoreceptor gene expression by Crx-associated transcription factor network. Brain Res. 2008;1192:114–133. [CrossRef] [PubMed]
Haider NB Mollema N Gaule M . Nr2e3-directed transcriptional regulation of genes involved in photoreceptor development and cell-type specific phototransduction. Exp Eye Res. 2009;89:365–372. [CrossRef] [PubMed]
Horsburgh GM Sefton AJ . Cellular degeneration and synaptogenesis in the developing retina of the rat. J Comp Neurol. 1987;263:553–566. [CrossRef] [PubMed]
Rich KA Zhan Y Blanks JC . Migration and synaptogenesis of cone photoreceptors in the developing mouse retina. J Comp Neurol. 1997;388:47–63. [CrossRef] [PubMed]
Sharma RK O'Leary TE Fields CM Johnson DA . Development of the outer retina in the mouse. Brain Res Dev Brain Res. 2003;145:93–105. [CrossRef] [PubMed]
Young RW . Cell death during differentiation of the retina in the mouse. J Comp Neurol. 1984;229:362–373. [CrossRef] [PubMed]
Hendrickson A Troilo D Djajadi H Possin D Springer A . Expression of synaptic and phototransduction markers during photoreceptor development in the marmoset monkey Callithrix jacchus. J Comp Neurol. 2009;512:218–231. [CrossRef] [PubMed]
Szél Á Lukáts Á Fekete T Szepessy Z Röhlich P . Photoreceptor distribution in the retinas of subprimate mammals. J Opt Soc Am A Opt Image Sci Vis. 2000;17:568–579. [CrossRef] [PubMed]
Szél Á Röhlich P . Two cone types of rat retina detected by anti-visual pigment antibodies. Exp Eye Res. 1992;55:47–52. [CrossRef] [PubMed]
Szél Á van Veen T Röhlich P . Retinal cone differentiation. Nature. 1994;370:336. [CrossRef] [PubMed]
Xiao M Hendrickson A . Spatial and temporal expression of short, long/medium, or both opsins in human fetal cones. J Comp Neurol. 2000;425:545–559. [CrossRef] [PubMed]
Ng L Hurley JB Dierks B . A thyroid hormone receptor that is required for the development of green cone photoreceptors. Nat Genet. 2001;27:94–98. [PubMed]
Lu A Ng L Ma M . Retarded developmental expression and patterning of retinal cone opsins in hypothyroid mice. Endocrinology. 2009;150:1536–1544. [CrossRef] [PubMed]
Glaschke A Glosmann M Peichl L . Developmental changes of cone opsin expression but not retinal morphology in the hypothyroid Pax8 knockout mouse. Invest Ophthalmol Vis Sci. 2010;51:1719–1727. [CrossRef] [PubMed]
Roberts MR Srinivas M Forrest D Morreale de EG Reh TA . Making the gradient: thyroid hormone regulates cone opsin expression in the developing mouse retina. Proc Natl Acad Sci U S A. 2006;103:6218–6223. [CrossRef] [PubMed]
Yanagi Y Takezawa S Kato S . Distinct functions of photoreceptor cell-specific nuclear receptor, thyroid hormone receptor beta2 and CRX in one photoreceptor development. Invest Ophthalmol Vis Sci. 2002;43:3489–3494. [PubMed]
Srinivas M Ng L Liu H Jia L Forrest D . Activation of the blue opsin gene in cone photoreceptor development by retinoid-related orphan receptor beta. Mol Endocrinol. 2006;20:1728–1741. [CrossRef] [PubMed]
Fujieda H Bremner R Mears AJ Sasaki H . Retinoic acid receptor-related orphan receptor alpha regulates a subset of cone genes during mouse retinal development. J Neurochem. 2009;108:91–101. [CrossRef] [PubMed]
Söderpalm A Szél Á Caffé AR van Veen T . Selective development of one cone photoreceptor type in retinal organ culture. Invest Ophthalmol Vis Sci. 1994;35:3910–3921. [PubMed]
Wikler KC Szél Á Jacobsen AL . Positional information and opsin identity in retinal cones. J Comp Neurol. 1996;374:96–107. [CrossRef] [PubMed]
Caffé AR Ahuja P Holmqvist B . Mouse retina explants after long-term culture in serum free medium. J Chem Neuroanat. 2001;22:263–273. [CrossRef] [PubMed]
Liljekvist-Larsson I Torngren M Abrahamson M Johansson K . Growth of the postnatal rat retina in vitro: quantitative RT-PCR analyses of mRNA expression for photoreceptor proteins. Mol Vis. 2003;9:657–664. [PubMed]
Pinzon-Duarte G Kohler K Arango-Gonzalez B Guenther E . Cell differentiation, synaptogenesis, and influence of the retinal pigment epithelium in a rat neonatal organotypic retina culture. Vision Res. 2000;40:3455–3465. [CrossRef] [PubMed]
Wang Y Macke JP Merbs SL . A locus control region adjacent to the human red and green visual pigment genes. Neuron. 1992;9:429–440. [CrossRef] [PubMed]
Gaillard F Kuny S Sauve Y . Topographic arrangement of S-cone photoreceptors in the retina of the diurnal Nile grass rat (Arvicanthis niloticus). Invest Ophthalmol Vis Sci. 2009;50:5426–5434. [CrossRef] [PubMed]
Röhlich P Szél Á . Binding sites of photoreceptor-specific antibodies COS-1, OS-2 and AO. Curr Eye Res. 1993;12:935–944. [CrossRef] [PubMed]
Jacobs GH Fenwick JA Williams GA . Cone-based vision of rats for ultraviolet and visible lights. J Exp Biol. 2001;204:2439–2446. [PubMed]
Chrysostomou V Stone J Valter K . Life history of cones in the rhodopsin-mutant P23H-3 rat: evidence of long-term survival. Invest Ophthalmol Vis Sci. 2009;50:2407–2416. [CrossRef] [PubMed]
Ortin-Martinez A Jimenez-Lopez M Nadal-Nicolas FM . Automated quantification and topographical distribution of the whole population of S and L cones in the adult albino and pigmented rats. Invest Ophthalmol Vis Sci. 2010;51:3171–3183. [CrossRef] [PubMed]
Cepko C . Giving in to the blues. Nat Genet. 2000;24:99–100. [CrossRef] [PubMed]
Ogilvie JM Speck JD Lett JM Fleming TT . A reliable method for organ culture of neonatal mouse retina with long- term survival. J Neurosci Methods. 1999;87:57–65. [CrossRef] [PubMed]
Ghosh F Arner K Engelsberg K . Isolation of photoreceptors in the cultured full-thickness fetal rat retina. Invest Ophthalmol Vis Sci. 2009;50:826–835. [CrossRef] [PubMed]
Johansson K Ehinger B . Structural changes in the developing retina maintained in vitro. Vision Res. 2005;45:3235–3243. [CrossRef] [PubMed]
Applebury ML Farhangfar F Glosmann M . Transient expression of thyroid hormone nuclear receptor TRbeta2 sets S opsin patterning during cone photoreceptor genesis. Dev Dyn. 2007;236:1203–1212. [CrossRef] [PubMed]
Liu H Etter P Hayes S . NeuroD1 regulates expression of thyroid hormone receptor 2 and cone opsins in the developing mouse retina. J Neurosci. 2008;28:749–756. [CrossRef] [PubMed]
Pessoa CN Santiago LA Santiago DA . Thyroid hormone action is required for normal cone opsin expression during mouse retinal development. Invest Ophthalmol Vis Sci. 2008;49:2039–2045. [CrossRef] [PubMed]
Ng L Ma M Curran T Forrest D . Developmental expression of thyroid hormone receptor beta2 protein in cone photoreceptors in the mouse. Neuroreport. 2009;20:627–631. [CrossRef] [PubMed]
Ng L Lyubarsky A Nikonov SS . Type 3 deiodinase, a thyroid-hormone-inactivating enzyme, controls survival and maturation of cone photoreceptors. J Neurosci. 2010;30:3347–3357. [CrossRef] [PubMed]
Romijn HJ . Development and advantages of serum-free, chemically defined nutrient media for culturing of nerve tissue. Biol Cell. 1988;63:263–268. [CrossRef] [PubMed]
Szél Á Röhlich P Caffé AR Juliusson B Aguirre G van Veen T . Unique topographic separation of two spectral classes of cones in the mouse retina. J Comp Neurol. 1992;325:327–342. [CrossRef] [PubMed]
Applebury ML Antoch MP Baxter LC . The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning. Neuron. 2000;27:513–523. [CrossRef] [PubMed]
Neitz M Neitz J . The uncommon retina of the common house mouse. Trends Neurosci. 2001;24:248–250. [CrossRef] [PubMed]
Bobu C Craft CM Masson-Pevet M Hicks D . Photoreceptor organization and rhythmic phagocytosis in the Nile rat Arvicanthis ansorgei: a novel diurnal rodent model for the study of cone pathophysiology. Invest Ophthalmol Vis Sci. 2006;47:3109–3118. [CrossRef] [PubMed]
Figure 1.
 
Opsin expression in retinal wholemounts at different developmental stages. The retinas were treated with JH455 and JH492 antibodies to show S-opsin (left) and M-opsin (right) labeling, respectively. The photomicrographs were taken from the central, most differentiated areas. Scale bar, 100 μm.
Figure 1.
 
Opsin expression in retinal wholemounts at different developmental stages. The retinas were treated with JH455 and JH492 antibodies to show S-opsin (left) and M-opsin (right) labeling, respectively. The photomicrographs were taken from the central, most differentiated areas. Scale bar, 100 μm.
Figure 2.
 
Quantification of S- and M-opsin expression during retinal development. S-opsin expression was already present at birth, increased in number until P10, and decreased subsequently. M-opsin became detectable from P4 on and increased in number until P12. Although they also decrease in number subsequently, M-opsin–expressing cones strongly outnumber S-opsin–expressing cones in the adult.
Figure 2.
 
Quantification of S- and M-opsin expression during retinal development. S-opsin expression was already present at birth, increased in number until P10, and decreased subsequently. M-opsin became detectable from P4 on and increased in number until P12. Although they also decrease in number subsequently, M-opsin–expressing cones strongly outnumber S-opsin–expressing cones in the adult.
Figure 3.
 
In the organotypic culture of the pigmented rat retina, both types of cones are present. The retinal layers are well preserved and the morphology of cones developed in culture (B, D) was comparable to that of the age-matched controls (A, C). At P10, the opsin-specific antibodies labeled the whole cone cell body. Scale bar, 50 μm.
Figure 3.
 
In the organotypic culture of the pigmented rat retina, both types of cones are present. The retinal layers are well preserved and the morphology of cones developed in culture (B, D) was comparable to that of the age-matched controls (A, C). At P10, the opsin-specific antibodies labeled the whole cone cell body. Scale bar, 50 μm.
Figure 4.
 
S-opsin expression in wholemounts of cultured retinas (A). Quantification of S-opsin–expressing cones (B). Despite the lower density in culture, the expression pattern of S-opsin was similar to that in the age-matched control retinas. **P < 0.01, ***P < 0.0001. Scale bar, 50 μm.
Figure 4.
 
S-opsin expression in wholemounts of cultured retinas (A). Quantification of S-opsin–expressing cones (B). Despite the lower density in culture, the expression pattern of S-opsin was similar to that in the age-matched control retinas. **P < 0.01, ***P < 0.0001. Scale bar, 50 μm.
Figure 5.
 
M-opsin expression in wholemounts of cultured retinas (A). Quantification of M-opsin–expressing cones (B). Although in present in lower number, M-opsin–expressing cones developed in the culturing approach and their quantity changed similar to that in age-matched control retinas. **P < 0.01, ***P < 0.0001. Scale bar, 50 μm.
Figure 5.
 
M-opsin expression in wholemounts of cultured retinas (A). Quantification of M-opsin–expressing cones (B). Although in present in lower number, M-opsin–expressing cones developed in the culturing approach and their quantity changed similar to that in age-matched control retinas. **P < 0.01, ***P < 0.0001. Scale bar, 50 μm.
Figure 6.
 
Double labeling for M-opsin in wholemounts of cultured rat retinas. P0 DIV7 retina (A–C) immunostained with AB5405 antibody (A) and COS-1 antibody (B); merged images (C). Only a few structures were labeled with COS-1. P0 DIV14 retina (D–F) immunostained with AB5405 antibody (D) and COS-1 antibody (E); merged images (F). Outer segments are clearly distinguishable, showing a high degree of co-localization. Scale bar, 25 μm.
Figure 6.
 
Double labeling for M-opsin in wholemounts of cultured rat retinas. P0 DIV7 retina (A–C) immunostained with AB5405 antibody (A) and COS-1 antibody (B); merged images (C). Only a few structures were labeled with COS-1. P0 DIV14 retina (D–F) immunostained with AB5405 antibody (D) and COS-1 antibody (E); merged images (F). Outer segments are clearly distinguishable, showing a high degree of co-localization. Scale bar, 25 μm.
Table 1.
 
Primary Antibodies
Table 1.
 
Primary Antibodies
Antigen Host Source Working Concentration References
S-opsin (JH455) Rabbit polyclonal Gift of Jeremy Nathans 1:20,000 Wang et al. 33
M-opsin (JH492) Rabbit polyclonal Gift of Jeremy Nathans 1:20,000 Wang et al. 33
M-opsin (AB5405) Rabbit polyclonal Millipore Billerica, MA 1:1,000 Gaillard et al. 34
M-opsin (COS-1) Mouse monoclonal Gift of Ágoston Szél 1:50 Röhlich and Szél 35
Table 2.
 
In Vivo and In Vitro Time Course of S-opsin– and M-opsin–Expressing Cones
Table 2.
 
In Vivo and In Vitro Time Course of S-opsin– and M-opsin–Expressing Cones
Postnatal Day Group S-opsin–Expressing Cones M-opsin–Expressing Cones
Mean ± SD P Mean ± SD P
0 P0 2,861 ± 674.6 0 ± 0.0
2 P2 7,246 ± 869.9 0 ± 0.0
4 P4 9,160 ± 854.9 0.5357 752 ± 160.7 0.0822
P2 DIV2 8,789 ± 739.1 537 ± 129.1
6 P6 11,045 ± 402.7 0.0002 2,568 ± 243.7 0.1993
P2 DIV4 9,258 ± 225.5 2,344 ± 194.0
8 P8 13,691 ± 474.4 0.0050 5,830 ± 567.3 0.0098
P2 DIV6 12,363 ± 393.9 4,648 ± 283.5
10 P10 16,914 ± 663.7 <0.0001 11,318 ± 380.6 <0.0001
P2 DIV8 9,990 ± 304.9 7,500 ± 186.0
12 P12 13,350 ± 516.6 <0.0001 13,975 ± 693.9 <0.0001
P2 DIV10 6,406 ± 264.9 7,529 ± 178.7
14 P14 10,781 ± 1,005.0 <0.0001 11,787 ± 224.1 <0.0001
P2 DIV12 5,469 ± 240.8 7,432 ± 433.1
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×