Otx2 Homeobox Gene Induces Photoreceptor-Specific Phenotypes in Cells Derived from Adult Iris and Ciliary Tissue

Abstract

purpose. It remains unclear which gene induction effectively generates photoreceptor-specific phenotypes from nonretinal tissues. The purpose of this study was to determine whether Crx and Otx2—homeobox genes related to photoreceptor development—can induce the generation of these phenotypes in cells derived from adult ciliary and iris tissue and in mesencephalon-derived neural stem cells.

methods. Crx and Otx2 were transferred into adult rat ciliary- and embryonic mesencephalon-derived neurospheres and adult rat iris-derived cells with the aid of a recombinant retrovirus. The presence of photoreceptor-specific phenotypes was confirmed by immunocytochemistry and Western blot analysis.

results. More than 90% of the Crx- and Otx2-transfected ciliary- and iris-derived cells exhibited rod opsin immunoreactivity, whereas few of the similarly transfected mesencephalon-derived neural stem cells expressed rod opsin. At least two additional key components of the phototransduction cascade, recoverin and G∂t1, were expressed by Crx- and Otx2-transfected iris-derived cells.

conclusions. Crx and Otx2 effectively induced the generation of photoreceptor-specific phenotypes from ciliary- and iris-derived cells. That both Crx and Otx2 induced phenotype generation in cells derived from iris or ciliary tissue may suggest an approach to photoreceptor cell preparation for retinal transplantation.

Photoreceptor cells of the retina constitute a group of light-sensitive neurons that originate from a pool of multipotent progenitors during retinal development. ^{1 2} It has been shown that both intrinsic and extrinsic factors participate in determining photoreceptors’ fate. ^{3 4 5 6} A loss-of-function study of Crx has demonstrated that the Crx homeobox gene is essential for terminal differentiation of photoreceptors. ⁷ Otx2 is a homeobox gene with a high degree of amino acid sequence homology to Crx, ⁸ and complete loss of Otx2 results in absence of the forebrain and is lethal to the embryo. ^{9 10 11} In a recent study in an Otx2 conditional knockout mouse line, in which the Otx2 genes were inactivated under control of the Crx promoter, it was found that Otx2 is a key regulatory gene for retinal photoreceptor cells. ¹² Thus, both Crx and Otx2 are believed to be important intrinsic factors of photoreceptor generation. 

The ciliary margin of the adult mammalian retina has been shown in both in vitro ^{13 14} and in vivo ¹⁵ studies to contain a population of retinal stem cells. It has also been reported that cultured iris pigmented epithelium has the potential to produce neuronal cells and the capacity to express photoreceptor-specific phenotypes as a result of Crx induction. ¹⁶ These findings suggest that ciliary- and iris-derived cells would be donor cells for retinal transplantation if they generated the needed retinal neurons. 

In the study presented herein, we conducted cell cultures of ciliary- and iris-derived cells of adult rat and of mesencephalon-derived neural stem cells of embryonic rat. We then analyzed the functions of Crx and Otx2 gene transfer in these different types of cells. 

Materials and Methods

Animals

Three-week-old female Dark Agouti (DA) rats and embryonic day (E)18 pregnant Wistar rats were obtained from Shimizu Laboratory Supplies (Kyoto, Japan). In all experimental procedures, the animals were treated according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Guidelines for Animal Experiments of Kyoto University. All animal experiments were conducted with the approval of the Animal Research Committee, Graduate School of Medicine, Kyoto University. 

Preparation of Tissue and Cell Culture

Rat iris and ciliary tissues were prepared from 3- to 4-week-old female DA rats. The procedure for iris cell culture has been described. ¹⁶ Briefly, after the iris with the cornea was separated from the other eye tissue at sufficient distance by careful incision, the iris tissue, treated with 1000 IU/mL dispase and 0.05% EDTA, was plated on a laminin-coated chamber slide. To prepare ciliary-derived cells, the neural retina was first peeled from the retinal pigmented epithelium (RPE) after removal of the cornea, iris tissue, and lens, not including neural retinal cells. Next, the pigmented epithelium of the ciliary margin (including the RPE) was separated by careful incision and treated with the Papain dissociation system (Worthington Biochemical Corp., Lakewood, NJ). Briefly, the pigmented epithelium was treated with Earle’s balanced salt solution (EBSS) containing papain and DNase I (DNase) for 40 minutes at 37°C and then centrifuged at 1000 rpm for 5 minutes and dissociated into small pieces by gentle trituration with a micropipette in EBSS containing DNase and albumin inhibitor. Then, the cell suspension was layered on top of EBSS containing albumin-inhibitor in a centrifuge tube and centrifuged at 800 rpm for 6 minutes. The supernatant was discarded, and the pelleted cells were resuspended in culture medium. The cells were dissociated into single cells by gentle trituration with a micropipette, and the pieces of undissociated tissues were removed. The dissociated ciliary-derived cells were cultured in 100-mm culture dishes containing serum-free culture medium at a cell density of 5 to 10 × 10⁴/mL. Dissociated ciliary-derived cells were cultured for 5 days to 2 weeks, to generate ciliary-derived sphere colonies. The iris tissue- and ciliary-derived cells were maintained at 37°C in Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12; Invitrogen-Gibco, Grand Island, NY) with B27 supplement (Invitrogen-Gibco) and 40 ng/mL basic fibroblast growth factor (bFGF; Genzyme/Techne, Minneapolis, MN). As with ciliary-derived cells, bFGF was added without medium change, or half the culture medium was changed every 2 days. The ciliary-derived cell cultures were passaged every 6 to 8 days. The medium of iris-derived cell culture was changed every 2 days. For cell differentiation, 1 day after gene induction the cells were cultured for 14 days in DMEM/F12/B27 with 1% fetal bovine serum (FBS) and 10 ng/mL bFGF. 

For preparation of brain-derived neural stem cells, tissue blocks of E18.5 Wistar rat ventral mesencephalon were resected, collected in cold phosphate-buffered saline (PBS), and dissociated into single cells by gentle trituration with a fire-polished Pasteur pipette, as described previously. ^{17 18} The cells were then cultured at 37°C in DMEM/F12 with N2 supplement (Invitrogen-Gibco) and 20 ng/mL bFGF. After 3 to 5 days of culture, the mesencephalic cells formed floating neurospheres. bFGF was added, or half the culture medium was changed every 2 days. The cultures were passaged every 8 to 10 days. For cell differentiation in the case of gene induction, the cells were first plated on a laminin-coated chamber slide and then cultured for 14 days under the same culture conditions as the ciliary- and iris-derived cells. 

Preparation of Recombinant Retrovirus

For construction of CLIG-Crx and CLIG-Otx2, cDNAs for these factors were cloned into the EcoRI site of pCLIG, which directs expression of the cloned genes, together with enhanced green fluorescent protein (GFP) from the upstream long terminal repeat (LTR) promoter with cytomegalovirus enhancer. ¹⁹ To intensify the efficiency of the infection, viral preparations were pseudotyped with a vesicular stomatitis virus (VSV-G) coat protein. Retroviral DNAs and pcDNA-VSV-G (kindly provided by Tal Kafri, University of North Carolina), which are env expression vectors, were transfected (Lipofectamine; Invitrogen, Paisley, UK) into 293GP (BD-Clontech, Palo Alto, CA), the producer cells expressing gag and pol. ^{20 21} The supernatant was collected as described previously. ¹⁹  

Immunocytochemistry

After fixation with 4% paraformaldehyde, the samples were preincubated 1 hour with a blocking solution (5% normal goat serum and 0.1% Triton X-100 in PBS) and then incubated 3 hours at room temperature or overnight at 4°C in 1% goat serum and 0.1% Triton X-100, with the following antibodies: rabbit anti-GFP (1:500; Molecular Probes, Eugene, OR), mouse anti-rod opsin RET-P1 (1:2500; Sigma-Aldrich, St. Louis, MO), mouse anti-βIII-tubulin (TuJ1, 1:500; Sigma-Aldrich), mouse anti-nestin (1:1000; BD-PharMingen, San Diego, CA), and rabbit anti-glial fibrillary acidic protein (GFAP; 1:500; Dako, Glostrup, Denmark). Cell nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI, 1:1000; Molecular Probes) or Cytox blue (1:500 in distilled water; Molecular Probes). Fluorescence was visualized by microscope (Leica Microsystems, Bannockburn, IL). Cultured cells were quantified by calculating the number of marker-positive cells as a percentage of immunopositive cells per ocular grid area at ×40 magnification (four random areas per sample). 

Western Blot Analysis

After cultured iris-derived cells and neural retina were washed three times with cold PBS, they were solubilized in 100 to 300 μL lysis buffer (50 mM Tris [pH 7.5], 0.5 M NaCl, 1% NP-40, 1% sodium deoxycholate monohydrate, 2 mM EDTA, and 0.1% SDS). After centrifugation at 10,000 rpm for 10 minutes, protein extracts were diluted with sample buffer (126 mM Tris HCl [pH 6.8] containing 20% glycerol, and 4% SDS, 0.005% bromophenol blue, and 5% 2-mercaptoethanol) at a 1:1 ratio and boiled for 3 minutes. Protein extracts were then subjected to 4% to 20% Tris-glycine gel electrophoresis and transferred electrically to a polyvinylidene difluoride (PVDF) membrane (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was then soaked for 1 hour at room temperature in a blocking buffer (TBS containing 0.2% Tween-20 and 5% skim milk) and incubated overnight at 4°C with the primary antibody: rabbit anti-recoverin (donated by James F. McGinnis and Rajesh J. Elias, University of Oklahoma Health Sciences Center, Oklahoma City, OK), ²² at a dilution of 1:8000, or rabbit anti-G∂t1 (Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:500. The antibody was followed by the addition of horseradish-peroxidase–conjugated anti-mouse or rabbit Ig antibodies (Amersham Pharmacia Biotech) for 1 hour at room temperature. The membranes were then washed with TBS containing 0.2% Tween-20, and the signals detected with a Western blot analysis system (ECL; Amersham Pharmacia Biotech). 

Results

Differentiation of Adult Ciliary-Derived Cells into a Photoreceptor-Specific Phenotype

To determine the effects of gene induction, we first prepared a replication-incompetent retrovirus, CLIG, which controls the expression of GFP as a marker of the upstream LTR promoter. For CLIG-Crx and CLIG-Otx2, cDNAs for Crx and Otx2 were inserted upstream of the internal ribosomal entry site (IRES) so that these genes were expressed bicistronically with GFP (Fig. 1) . 

It has been reported, with the neurosphere culture method, that adult mammalian eyes contain retinal stem cells in the ciliary margin. ^{13 14} To examine the effects of induction of Crx and Otx2 on ciliary-derived cells, we prepared ciliary-derived spheres from adult rats. Most of these cells were divided into single cells, and then, after 5 days, some of them formed spherical structures containing pigmented and nonpigmented progeny (Fig. 2A) . These results are consistent with those previously reported. ¹³ The spheres continued to grow slowly in serum-free medium containing bFGF, and many cells migrated from the spheres after they had been plated on a laminin-coated chamber slide (Fig. 2B) . Most of these cells (85.7% ± 1.41%, 262/306) immunostained with nestin, a marker for neural stem cells, a day after being plated. Table 1 shows the cell counts in the different types of cells studied. 

We next examined whether the ciliary-derived cells would respond to the forced expression of Crx and Otx2. The retrovirus was applied to the ciliary-derived cells 3 days after they had been plated on a laminin-coated chamber slide in serum-free medium, and the cells were transferred into an environment that promotes retinal cell differentiation. ^{13 16} As a negative control, 1.50% ± 1.37% of the CLIG-infected cells expressed rod opsin, suggesting that these ciliary-derived cells had rod-photoreceptor–producing capability without gene transfer (Figs. 2C 2D 2E 3) . In contrast, 91.7% ± 3.06% of the CLIG-Crx–infected and 92.3% ± 3.14% of the CLIG-Otx2–infected ciliary-derived cells had rod opsin immunoreactivity, suggesting that Crx and Otx2 induces the differentiation of ciliary-derived cells into photoreceptor-specific phenotypes, whereas only part of the CLIG-infected cells were rod opsin positive (Figs. 2F 2G 2H 2I 2J 2K 3) . 

Induction of Expression of a Photoreceptor-Specific Phenotype in Rat Iris Tissue

Iris-derived cells were prepared as described previously. ¹⁶ The virus was applied to the iris-derived cells 4 to 5 days after culturing in serum-free medium, and the culture conditions were changed to an environment that promotes retinal cell differentiation 1 day after transfection. ^{13 16} After culturing for 14 days, 97.9% ± 1.50% of the CLIG-Crx–infected, iris-derived cells expressed rod opsin, whereas the iris-derived cells without gene transfer showed no rod opsin immunoreactivity, as has been reported (Figs. 3 4D 4E 4F) . ¹⁶ To examine whether Otx2 induces iris-derived cells to differentiate into a photoreceptor-specific phenotype, we applied CLIG-Otx2 to the cells, transferred them into an retinal cell-differentiation–promoting environment 1 day after transfection, and cultured them for 14 days. Of the CLIG-Otx2–infected, iris-derived cells, 95.7% ± 2.17% were immunopositive for rod opsin, demonstrating that Otx2 induction may lead the iris-derived cells to express rod opsin, whereas no CLIG-infected, iris-derived cells displayed immunoreactivity to rod opsin (Figs. 3 4A 4B 4C 4G 4H 4I) . The rod opsin-expressing cells misexpressed by Crx or Otx2 had small, round shapes, characteristic of rod photoreceptors in monolayer culture. Otx2 thus has the capacity to produce photoreceptor-like cells from iris-derived cells in the same manner as Crx, although it has not been determined whether Otx2 and Crx operate at the same hierarchical level to establish the identity of photoreceptors. 

Responsiveness of Mesencephalon-Derived Neural Stem Cells to Crx and Otx2 Induction

To determine whether rod opsin expression responding to Crx- and Otx2-inductions were relatively specific for iris and ciliary-derived cells, we prepared neurospheres derived from E18.5 rat ventral mesencephalon (Figs. 5A 5B) . The mesencephalon-derived neurospheres proliferated in serum-free medium containing bFGF, and most of these cells were positive for nestin (Figs. 5D 5E 5F) . After being transferred to differentiation-promoting conditions, neurons (βIII-tubulin-positive: 4.5% ± 0.41%, 106/2364) and glial cells (GFAP positive: 80.5% ± 0.40%, 1903/2364) were produced from the same neurospheres, indicating that these neurospheres have the characteristics of neural stem cells (Fig. 5C) . 

To examine the effects of Crx and Otx2 induction on these neural stem cells, we applied CLIG, CLIG-Crx, and CLIG-Otx2 to the neurospheres and cultured them under differentiation-promoting conditions 1 to 3 days after plating them on laminin-coated chamber slides. The pseudotyped retrovirus effectively infected the cells of the neurospheres. No mesencephalic neural stem cells infected with CLIG were rod-opsin immunopositive, and only 2.88% ± 3.61% of the CLIG-Crx–infected cells and 7.0% ± 4.76% of the CLIG-Otx2–infected cells expressed rod opsin, suggesting that few of them acquired rod-opsin immunoreactivity, although transfection of the Crx and Otx2 genes into the neural stem cells was successful (Figs. 3 5G 5H 5I 5J 5K 5L 5M 5N 5O) . Most of the rod-opsin–negative cells misexpressed by Crx or Otx2 did not assume small, round shapes. This suggests that the mesencephalic neural stem cells may be intrinsically restricted in their response to Crx or Otx2. 

Immunoblot Analysis of Photoreceptor-Specific Antigens in Iris-Derived Cells after Gene Transfer

Immunocytochemical analysis showed that the Crx- and Otx2-transfected rat iris-derived cells were labeled with a monoclonal antibody against rod opsin, RETP1, whereas the iris-derived cells without gene delivery did not express rod opsin (Figs. 3 4A 4B 4C 4D 4E 4F 4G 4H 4I) . To determine whether the iris-derived cells with Crx- or Otx2-gene transfer expresses any other phototransduction components, anti-recoverin and anti-α subunits of transducin (G∂t1) antibodies were tested by means of Western blot. Total protein extract from the rat retina (1.0 μg) was used as a positive control (Figs. 6A 6B ; lane 4). Recoverin is a calcium-binding protein that is involved in the inactivation of phototransduction, and G∂t1 is a photoreceptor-specific G-protein molecule that activates cGMP-specific phosphodiesterase (PDE). ²³ We recognized similar patterns in the extracts (40 μg/lane) of the Crx- and Otx2-transfected, iris-derived cells that expressed recoverin and G∂t1, although we did not detect bands of the same size in equal amounts of proteins prepared from the iris-derived cells infected with CLIG as we did in the corresponding retinal extracts (Figs. 6A 6B ; lanes 1–3). These results indicated that the Crx- and Otx2-transfected rat iris-derived cells express at least two additional key components of the phototransduction cascade: recoverin and G∂t1. 

Discussion

In this study, both Otx2 and Crx inductions effectively led ciliary- and iris-derived cells to differentiate into a photoreceptor-specific phenotype. These photoreceptor-like cells induced by Otx2 and Crx misexpressions displayed photoreceptor-specific morphology in vitro. A recently conducted extensive experiment, using an Otx2 conditional knockout mouse line in which the Otx2 genes were inactivated under control of the Crx promoter, proved that Otx2 is a key regulatory gene for photoreceptor development. ¹² In our study, Otx2 induction effectively led ciliary- and iris-derived cells to differentiate into a photoreceptor-specific phenotype. However, the mechanism of the photoreceptor generation remains obscure, in that Otx2 expression is not restricted to the photoreceptor cells, but covers most of the forebrain and midbrain, including the eye domain in the developmental stage. ²⁴ Furthermore, Otx2 expression in the eye has been demonstrated in several cell types, including bipolar cells, ganglion cells, photoreceptor cells, and retinal pigment epithelial cells. ^{12 25 26} In addition, Otx2 has been shown to be a direct upstream regulator of Crx, so that Crx is also thought to be misexpressed in Otx2-induced cells. ¹² Crx appears to be capable of inducing photoreceptor-specific phenotypes in ciliary- and iris-derived cells, although further studies are needed to examine the differences between Crx- and Otx2-induced cells in terms of other photoreceptor-specific characteristics, including photoresponsiveness, which may provide the key to producing genuine functional photoreceptors. 

The pigmented cells of the adult mouse ciliary marginal zone contain a population of retinal stem cells. ^{13 14} We cultured pigmented cells of the ciliary margin and successfully obtained neurospheres; however, without gene induction the level of expression of rod opsin by the differentiated cells in our experiment was very small compared with the a previously report. ¹³ This low rod photoreceptor-generating capability is possibly due to different culture conditions, including dissociation and culture media. Another possible cause is the difference in species—that is, mouse and rat. ¹³ Although the culture conditions for ciliary-derived cells need improvement, Crx or Otx2 induction helped increase the differentiation efficiency of photoreceptor cells dramatically. 

It has been reported that cultured human ciliary epithelial cells have the potential to express components of phototransduction, ²⁷ whereas iris tissue also reportedly to has the potential to respond to light in vitro and in vivo by following a non-neural pathway. ^{28 29 30 31 32} The iris-derived cells in our study, however, did not differentiate into cells expressing rod opsin, recoverin, and G∂t1 without appropriate gene induction, which means that rat iris tissue does not normally express components of phototransduction. The possible explanation for this is that iris-derived cells normally have the potential to express these components, including rhodopsin, but that their expression levels are too low to be detectable by immunocytochemistry. 

We have reported that Crx does not induce rhodopsin immunoreactivity in hippocampus-derived neural stem cells. ¹⁶ In the current study, some cell populations responded to Crx or Otx2 induction in neurospheres derived from the embryonic mesencephalon, although only very few did so. This lack of uniformity may be explained in terms of the different characteristics between the long-cultured neural stem cells and the freshly obtained neural progenitor cells. Further studies are needed to find an explanation for the inhibitory effects of other gene products, which may result in gaining information as to how to produce photoreceptor cells from other tissues. 

In conclusion, forced expression of the Otx2 gene, in the same manner as the Crx gene, effectively induced the cells derived from the adult iris and ciliary margin to become photoreceptor-like cells, whereas most of the mesencephalon-derived neural stem cells did not respond to Otx2. Furthermore, the Crx- and Otx2-transfected rat iris-derived cells expressed at least two additional key components of the phototransduction cascade. Thus, the Otx2 gene is one of the key regulators of both normal photoreceptor development and, by means of gene delivery, photoreceptor production. 

 Supported by a grant from the Research Committee on Chorioretinal Degeneration and Optic Atrophy, The Ministry of Health, Welfare and Labor of the Japanese Government and the 21st Century Center of Excellence Program of the Japanese Ministry of Education, Culture, Sports, Science and Technology.

 Submitted for publication June 15, 2004; revised August 11, 2004; accepted August 17, 2004.

 Disclosure: T. Akagi, None; M. Mandai, None; S. Ooto, None; Y. Hirami, None; F. Osakada, None; R. Kageyama, None; N. Yoshimura, None; M. Takahashi, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Viral constructs used to express Crx and Otx2. CLIG was designed to express a marker gene, GFP, via an IRES sequence and another gene under the control of the LTR promoter.

Figure 2.

 

The potential of adult rat ciliary-derived cells to express photoreceptor specific phenotypes. (A, B) Phase-contrast micrographs of rat ciliary-derived cells. Neurospheres cultured in serum-free medium containing bFGF (A). Placed on a chamber slide, the cells proliferated and migrated from the sphere as a monolayer of cells (B). (C–K) Immunocytochemical analysis with anti-GFP and anti-rod opsin antibodies after infection with CLIG (C–E), CLIG-Crx (F–H), or CLIG-Otx2 (I–K). Most of the ciliary-derived cells infected with the control retrovirus CLIG showed no rod opsin immunoreactivity (C–E). (C–E, arrowheads) Rod opsin–positive cells infected with CLIG. Most of the ciliary-derived cells infected with CLIG-Crx (F–H) and CLIG-Otx2 (I–K) expressed rod opsin. Virally infected cells are shown in green (C, E, F, H, I, K), rod opsin-positive cells in red (D, E, G, H, J, K), and nuclei stained with DAPI in blue (E, H, K). Scale bar: (A, B) 100 μm; (C–K) 50 μm.

Table 1.

 

Cell Count Data in Different Types of Cells

Cell Type	Retrovirus	RETP1/GFP	%
Ciliary-derived cell	CLIG	15/996	1.50 ± 1.37
	CLIG-Crx	966/1051	91.7 ± 3.06
	CLIG-Otx2	951/1027	92.3 ± 3.14
Iris-derived cell	CLIG	0/1951	0
	CLIG-Crx	3562/3620	97.9 ± 1.50
	CLIG-Otx2	916/960	95.7 ± 2.17
Mesencephalon-derived cell	CLIG	0/824	0
	CLIG-Crx	34/1148	2.88 ± 3.61
	CLIG-Otx2	62/986	7.0 ± 4.76

 The total number of counted and RETP1-positive cells infected with CLIG, CLIG-Crx, and CLIG-Otx2 and the RETP1-positive percentages are shown.

Figure 3.

 

The ratio of rod-opsin–positive cells to GFP-positive cells in ciliary-, iris-, and mesencephalon-derived cells with gene induction. Three independent samples of the ciliary- and iris-derived cells infected with CLIG, CLIG-Crx, and CLIG-Otx2 were tested. The data for the mesencephalon-derived cells were obtained with four samples in independent experiments. The total number of each cell type is shown in Table 1 . Results are shown as the mean ± SEM. Shown are statistically significant differences (*a: P = 0.00000040; *b: P = 0.0000014; *c: P = 0.00000014; and *d: P = 0.00000084; Student’s t-test) in the production of rod-opsin–positive cells compared with the mesencephalon-derived cells with Crx (*a,*c) or Otx2 induction (*b,*d).

Figure 4.

 

Induction of photoreceptor-specific phenotypes from adult rat iris-derived cells. After viral infection of the iris-derived cells, they were subjected to immunocytochemistry when they had been cultured in a differentiating environment for 14 days. Whereas iris-derived cells infected with CLIG showed no rod opsin immunoreactivity (A–C), the cells infected with CLIG-Crx (D–F) and CLIG-Otx2 (G–I) expressed rod opsin. Virally infected cells are shown in green (A, C, D, F, G, I), rod opsin-positive cells in red (B, C, E, F, H, I), and nuclei stained with DAPI in blue (C, F, I). Scale bar, 50 μm.

Figure 5.

 

Crx and Otx2 induction into mesencephalon-derived neural stem cells. (A, B) Phase-contrast micrographs of the rat mesencephalon-derived neurospheres when cultured in serum-free medium containing bFGF (A). The cells were arranged as a monolayer of cells outside of the neurosphere a day after placement on a chamber slide (B). (C) βIII-tubulin-positive neuronal (green) and GFAP-positive glial cells (red) were generated from the mesencephalon-derived neurosphere. (D–F) Confocal images of immunostaining showed that most cells of the neurospheres were positive for nestin: (D, red) Nestin, (E, blue) nuclei in cells stained with Cytox blue, (F) and combined images. Virally infected cells are shown in green (G, I, J, L, M, O), and rod-opsin–positive cells in red (H, I, K, L, N, O). The nuclei are counterstained blue with DAPI (C, I, L, O). Scale bar: (A–F) 100 μm; (G–O) 50 μm.

Figure 6.

 

Western blot analysis of photoreceptor-specific antigens in iris-derived cells with gene transfer. (A, B) Cell lysates (40 μg per lane) from CLIG-Crx- (lane 1), CLIG- (lane 2), and CLIG-Otx2-infected iris-derived cells (lane 3) and from normal rat neural retinal protein extracts (1 μg per lane) (lane 4) were probed with recoverin (A) and G∂t1 antibodies (B). Arrows: electrophoretic mobility for recoverin (A) and G∂t1 (B). (A, B, left) Molecular mass.

The authors thank Constance L. Cepko (Harvard Medical School, Boston, MA) and Takahisa Furukawa (Osaka Bioscience Institute, Osaka, Japan) for their generous gift of Crx cDNA.