Otx2 Is Involved in the Regional Specification of the Developing Retinal Pigment Epithelium by Preventing the Expression of Sox2 and Fgf8, Factors That Induce Neural Retina Differentiation

Abstract

The retinal pigment epithelium (RPE) shares its developmental origin with the neural retina (NR). When RPE development is disrupted, cells in the presumptive RPE region abnormally differentiate into NR-like cells. Therefore, the prevention of NR differentiation in the presumptive RPE area seems to be essential for regionalizing the RPE during eye development. However, its molecular mechanisms are not fully understood. In this study, we conducted a functional inhibition of a transcription factor Otx2, which is required for RPE development, using early chick embryos. The functional inhibition of Otx2 in chick eyes, using a recombinant gene encoding a dominant negative form of Otx2, caused the outer layer of the optic cup (the region forming the RPE, when embryos normally develop) to abnormally form an ectopic NR. In that ectopic NR, the characteristics of the RPE did not appear and NR markers were ectopically expressed. Intriguingly, the repression of Otx2 function also caused the ectopic expression of Fgf8 and Sox2 in the outer layer of the optic cup (the presumptive RPE region of normally developing eyes). These two factors are known to be capable of inducing NR cell differentiation in the presumptive RPE region, and are not expressed in the normally developing RPE region. Here, we suggest that Otx2 prevents the presumptive RPE region from forming the NR by repressing the expression of both Fgf8 and Sox2 which induce the NR cell fate.

About the Authors:

Daisuke Nishihara

Affiliations Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan

Ichiro Yajima

Affiliation: Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi, Japan

Hiromasa Tabata

Affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan

Masato Nakai

Affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan

Nagaharu Tsukiji

Affiliation: Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan

Tatsuya Katahira

Affiliation: Laboratory of Developmental Neurobiology, Graduate School of Brain Science, Doshisha University, Kyotanabe, Kyoto, Japan

Kazuhisa Takeda

Affiliation: Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Miyagi, Japan

Shigeki Shibahara

Affiliation: Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai, Miyagi, Japan

Harukazu Nakamura

Affiliations Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan, Department of Molecular Neurobiology, Graduate School of Life Sciences and Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi, Japan

Hiroaki Yamamoto

* E-mail: [email protected]

Affiliation: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga, Japan

Introduction

The retinal pigment epithelium (RPE), one component of the vertebrate eye, consists of a monolayer of melanin-producing cells. Both the RPE and the neural retina (NR), which contains photoreceptors, retinal ganglion cells (RGC), horizontal cells, amacrine cells, bipolar cells and Müller glia cells, originate from the same eye primordium, called the optic vesicle (OV), which derives from the lateral wall of the forebrain. The inductive interactions between the OV and the surface ectoderm (the future lens) result in the invagination of the OV to form the bilayered optic cup (OC), in which the outer and inner layers are specified into the RPE and NR, respectively [1], [2].

The development of the RPE is promoted by several transcription factors, which are specifically expressed in the presumptive RPE region; Microphthalmia-associated transcription factor (Mitf) and Orthodenticle homeobox 1 and 2 (Otx1 and 2). Mitf promotes melanin synthesis and regulates cell proliferation in the developing RPE [3]. In mutant mice with non-functional alleles of the Mitf gene, a non-pigmented NR-like tissue is ectopically formed in the outer layer of the OC [4], [5]. The expression of Mitf in the presumptive RPE region requires the function of Otx genes [6]. Compound mutations in Otx1 and 2 (all Otx1−/−; Otx2+/− mice and 30% of Otx1+/−; Otx2+/− mice) result in the down-regulation of Mitf expression and the ectopic formation of NR-like tissue in the outer layer of the OC, although Otx1−/− mice do not display significant defects in the RPE [6]. Still, in spite of these key findings in mutant mice, it is unclear whether the loss-of-function of Otx2 affects RPE development, since the head region including the eyes is not formed in Otx2−/− mice [7], [8]. However, previous reports have pointed out the roles of Otx2 as an upstream regulator of Mitf expression and the promotion of RPE differentiation [9], [10]. In cultured quail retina cells, transfection of Otx2 induces a pigmented phenotype with Mitf expression [9]. In the chick NR, co-transfection of Otx2 and a constitutively active form of β-catenin induces the ectopic expression of Mitf [10].

While RPE development requires the functions of Mitf and Otx, NR differentiation is induced by several other transcription and growth factors, including Fibroblast growth factor 8 (Fgf8), Subgroup B1 SRY-box family genes (SoxB1) and Paired-box 6 (Pax6). When FGF8-soaked beads are placed in the vicinity of the developing RPE, cells in the RPE change their fate to differentiate into NR cells [11]. As a result, some areas of the outer layer of the OC form a non-pigmented ‘ectopic NR’ which takes on a stratified structure and displays several differentiation markers of the NR [11]. Similarly, ectopic formation of the NR can also be caused by the misexpression of SoxB1 or Pax6 in the outer layer of the OC [12], [13]. The Fgf8 and SoxB1 genes are expressed in the NR, but not in the RPE [11], [12]. Pax6 also becomes absent from the presumptive RPE, although its expression is detected in the RPE during the early stages of eye development [14], [15]. Although the expression patterns of these factors are well known, it is noteworthy that it is still unclear how these factors are restricted to the NR region and disappear from the RPE region in normally developing eyes. Unveiling how the expression domains of Fgf8, SoxB1 and Pax6 are down-regulated in the outer layer of the OC may lead to understanding the mechanism(s) involved in the regionalization of the RPE.

Since Otx2 and Mitf are specifically expressed in the outer layer of the OC (the region forming the RPE, when embryos normally develop), it is possible that these RPE-specific factors are involved in the down-regulation of Fgf8, SoxB1 and Pax6 in the outer layer of the OC. Here, we conducted a functional inhibition of Otx2 in the developing eye, using chick embryos. Chick embryos were transfected with a recombinant gene encoding a dominant negative form of Otx2 that was confirmed to repress the function of wild-type Otx2 in vitro. Functional inhibition of Otx2 in chick eyes caused the outer layer of the optic cup (the RPE region of normally developing eyes) to abnormally form an ectopic NR. In that ectopic NR, the characteristics of the RPE did not appear and NR markers were ectopically expressed. Intriguingly, the repression of Otx2 function also caused the ectopic expression of Fgf8 and Sox2 (one of the SoxB1 family members) in the outer layer of the OC, whereas the expression of Pax6 was reduced. Our data suggest that Otx2 prevents the outer layer of the OC from forming the NR by repressing the expression of Fgf8 and Sox2 which can forcedly induce NR differentiation [11], [12].

Results

Expression Pattern of Otx2 and the Dominant Negative Activity of EnR-Otx2

First, we compared the expression patterns of Otx2 with Mitf in the OV and the OC stage. In HH10 chick embryos, Otx2 was expressed in a large part of the OV (asterisks in Figure 1A), although its expression was weak in the ventral part of the OV. Mitf was not expressed in the OV in HH10 chick embryos (Figure 1B). From HH12-13, Mitf expression could be detected in the dorsal part of the OV (arrowheads in Figure 1D). At the same stages, Otx2 was highly expressed in the dorsal part of the OV (arrowheads in Figure 1C), similar to the expression pattern of Mitf. After the OC was formed, the expression of both Otx2 and Mitf was apparent in the outer layer of the OC where the RPE formed (Figure 1E and F).

[Figure omitted. See PDF.]

Figure 1. Expression pattern of Otx2 and the dominant negative activity of EnR-Otx2.

(A–F) In situ hybridization analyses of Otx2 expression and immunohistological analyses of Mitf expression. A and B are serial sections of an HH10 embryo, as well as C and D of an HH12 embryo, and E and F of an HH17 embryo. A, C and E show expression of Otx2, and B, D and F show Mitf staining. Asterisks in A and B indicate the OV. Arrows in A indicate the ventral area of the OV where Otx2 is weakly expressed. Arrowheads in C and D highlight the sites where Otx2 and Mitf are strongly expressed. Upper and lower sides of panels A–F correspond to dorsal and ventral sides of the embryos, respectively. (G) Dct promoter activity in D407 cells. The Dct promoter fused to luciferase was co-transfected with the vectors, as follows. Co-transfection with: empty vector (Lane 1), Otx2 (Lane 2), Otx2 and EnR-Otx2 (Lane 3), Otx2 and EnRΔC-Otx2 (Lane 4), EnR-Otx2 (Lane 5) and EnR-Otx2 and EnRΔC-Otx2 (Lane 6). The histogram presents means ± SD. RPE, retinal pigment epithelium. NR, neural retina. Le, lens. Scale bars: 100 µm.

https://doi.org/10.1371/journal.pone.0048879.g001

For the functional inhibition of Otx2, we used a recombinant gene encoding a dominant negative form of Otx2, called EnR-Otx2. EnR-Otx2 encodes chick Otx2 fused to the Drosophila Engrailed repressor domain (EnR). We confirmed the dominant negative activity of EnR-Otx2 with in vitro assays. Consistent with a previous study which showed that OTX2 activates the Dct gene promoter [16], chick wild-type Otx2 (wtOtx2) drove the Dct promoter (lanes 1 and 2 in Figure 1G) in D407 cultured cells. This function of wtOtx2 was blocked by EnR-Otx2 (lane 3 in Figure 1G), suggesting that EnR-Otx2 could be used for the functional inhibition of Otx2. As expected, the Dct promoter was not activated by EnR-Otx2 (lane 5 in Figure 1G). EnR-Otx2ΔC, which encodes EnR-Otx2 without its C-terminal DNA-binding domain, did not repress the activity of wtOtx2 (lane 4 in Figure 1G).

Loss of Characteristics of the RPE by EnR-Otx2 Transfection

To address how Otx2 contributes to RPE development in chick embryos, we conducted gene transfection experiments. The OV of HH9-11 chick embryos (incubated for 1.5 days) were transfected with pMiwIII-EnR-Otx2 and a GFP-expressing vector (pCAGGS-EGFP) using in ovo electroporation, carried out as described previously [3]. For controls, both the pMiwIII-empty vector and pCAGGS-EGFP were transfected. After incubation for 2 days, the transfected embryos were fixed and prepared for observation.

As in the case of normally developing eyes, the control eyes had a blackish tinge except for the lens (n = 15), since the differentiated RPE cells synthesize melanin pigment (corresponding to HH20-22 embryos, Figure 2A–C). However, the EnR-Otx2-transfected eyes revealed a highly reduced pigmentation in the GFP-positive portion (n = 23, brackets in Figure 2D–F).

[Figure omitted. See PDF.]

Figure 2. Loss of RPE characteristics and morphological changes in the chick eye following EnR-Otx2 transfection.

(A–F) Lateral views of embryos transfected with either the empty vector (A and B), or EnR-Otx2 (D and E). Illustrations in C and F correspond to B and E, respectively. Brackets in D and E indicate sites where pigmentation was reduced by EnR-Otx2. A and D indicate GFP signals (green). B and E are bright field images. (G–R) Staining of RPE markers in sections of eyes transfected with the empty vector (G–L) or EnR -Otx2 (M–R). Immunohistological analyses of Mitf expression (H and N) and in situ hybridization analyses of transcripts of Dct (J and P) or MMP115 (L and R). G, I, K, M, O and Q indicate GFP signals (green). Brackets in G and M indicate the thickness of the outer layer of the OC, which are transfected with the empty vector (G) or EnR-Otx2 (M), respectively. Dashed lines in N, P and R highlight the thickened outer layer of EnR-Otx2-transfected eyes. Arrowheads in M and N indicate sites where Mitf expression remains. In M–R, ‘RPE’ refers to the abnormally thickened outer layer caused by EnR-Otx2 transfection. G and H are the same sections, so are M and N. Each set of I and J, K and L, O and P and Q and R are serial sections. Upper and lower sides of the panels correspond to dorsal and ventral sides of embryos, respectively. RPE, retinal pigment epithelium. NR, neural retina. Le, lens. Scale bars: 100 µm.

https://doi.org/10.1371/journal.pone.0048879.g002

To address how EnR-Otx2 affects the state of differentiation and morphology of the outer layer of the OC (the presumptive RPE region of normally developing eyes), sections of EnR-Otx2-transfected or control eyes were stained for RPE differentiation markers. In the control eyes, no obvious morphological changes were observed (Figure 2G–L), and the outer layer of the OC (the presumptive RPE region) maintained the mono-layered structure (bracket in Figure 2G). In contrast, EnR-Otx2 caused an abnormally thickened tissue to be formed in the outer layer of the OC (compare brackets between Figure 2G and M). In this thickened outer layer (the areas sandwiched between the dashed lines), pigment granules were hardly detected. In addition, some of the EnR-Otx2 transfected eyes did not keep their cup-like structure to form an OV-like structure (Figure 2O–R), and the size of the lens seemed reduced in the EnR-Otx2-transfected eyes (compare the areas enclosed in the dashed lines in Figure 2M and O with Figure 2G).

In EnR-Otx2-transfected eyes (Figure 2M–R), the expression of Mitf became weakened over a large part of the thickened outer layer (Figure 2M and N), and Mitf signals were only detected in the GFP-negative areas in the thickened outer layer (arrowheads in Figure 2M and N). Differentiation markers of the RPE, dopachrome tautomerase (Dct, encoding a enzyme required for black melanin synthesis) and melanosomal matrix protein 115 (MMP115) [3], [17], [18], also could not be detected in these eyes (the areas between the dashed lines in Figure 2P and R). In contrast, Mitf, Dct and MMP115 were specifically expressed in the outer layer of the OC (the presumptive RPE region) of control eyes (Figure 2G–L). Thus, proper morphogenesis and differentiation in the outer layer of the OC (the presumptive RPE region) is disrupted by EnR-Otx2 misexpression.

Ectopic Expression of NR Markers in the Outer Layer of the OC Following EnR-Otx2 Transfection

Next, we examined the expression patterns of several NR markers. It is possible that EnR-Otx2 transfection caused the outer layer of the OC to abnormally differentiate into the NR instead of the RPE, since ectopic NR-like tissues are formed in the outer layer of the OC of Otx1 and Otx2 compound mutant mice [6]. In addition, previous studies have shown that the RPE shares a common developmental origin (OV) with the NR and also has the potential to differentiate into the NR [11], [12], [13], [19], [20], [21], [22], [23], [24], [25], [26].

For analyzing the expression of NR markers, the embryos were transfected at embryonic stages HH9-11, and then were further incubated for 2 days to reach HH20-22 embryos.

In the developing NR, the transcription factor Islet1 is detected in postmitotic ganglion cells, migrating amacrine cells [11], [12], [27], while RNA-binding protein HuC/D is expressed in the differentiated neuronal cells [21], [28], [29]. In the control eyes, those differentiation markers of the NR were expressed on the vitreal surface (the surface facing the lens, described as (v) in Figure 3C and F) of the NR (arrowheads in Figure 3B and E), but could not be detected in the outer layer of the OC/presumptive RPE region (the area between the dashed lines in Figure 3B, C, E and F). In addition, phospho histone-H3 (PHH3)-positive mitotic cells were located at the sclera surface (the surface opposite the lens, described as (s) in Figure 3I) of the NR in the control eyes (arrowheads in Figure 3H), as in the case of normally developing eyes. In these eyes, only a small number of RPE cells were positive for PHH3 (the area between the dashed lines in Figure 3H and I).

[Figure omitted. See PDF.]

Figure 3. Ectopic expression of NR markers in the outer layer of the OC following EnR-Otx2 transfection.

Immunohistological staining of NR markers in sections of eyes transfected with the empty vector (A–I) or EnR -Otx2 (J–R). A–C and J–L indicate the expression of Islet1 (magenta), and A, B, J and K are merged images with GFP (green). D–F and M–O indicate the expression of HuC/D (magenta), and D, E, M and N are merged images with GFP (green). G–I and P–R indicate signals of phospho-Histone H3 (PHH3, red) and nuclei (DAPI, blue) and G, H, P and Q are merged images with GFP (green). B and C, E and F, H and I, K and L, N and O, and Q and R are magnified images of the boxes in A, D, G, J, M and P, respectively. Dashed lines highlight the RPE of control eyes (B, C, E, F, H and I) or the thickened outer layer of EnR-Otx2-transfected eyes (K, L, N, O, Q and R). Arrowheads indicate the positions of cells which are positive for Islet1 (B and K), HuC/D (E and N) or PHH3 (H and Q). (v) and (s) in C, F, I, L, Q and R indicate the vitreal and sclera sides of the OC, respectively, across the inner and outer layers of the OC. DAPI is used to ease observation of tissues (G–I and P–R). The upper and lower sides of each image correspond to the dorsal and ventral sides of the specimen, respectively. In J-R, ‘RPE’ refers to the abnormally thickened outer layer, apparently ‘ectopic NR’. RPE, retinal pigment epithelium. NR, neural retina. Le, lens. Scale bars: 100 µm.

https://doi.org/10.1371/journal.pone.0048879.g003

In the EnR-Otx2-transfected eyes, not only the NR but also the thickened outer layer were positive for HuC/D and Islet1 (Figure 3J–Q). Ectopic signals of Islet1 or HuC/D were detected on the sclera surface (the surface opposite the lens, described as (s) in Figure 3L and Q) of the thickened outer layer (arrowheads in Figure 3K and N). Further, on the vitreal surface (the surface facing the lens, described as (v) in Figure 3R) of the thickened outer layer, most PHH3-positive cells were detected (arrowheads in Figure 3Q). The distribution of PHH3 on the opposite side of HuC/D and Islet1 in the thickened outer layer topologically mimicked the distribution of those factors in the normally developing NR. These data suggest that EnR-Otx2 misexpression results in the formation of an ‘ectopic NR’ in the outer layer of the OC.

Effects of EnR-Otx2 on Factors Involved in NR Differentiation or other Aspects of Eye Development

The ectopic NR formation in the outer layer of the OC (the presumptive RPE region) could also be induced by some transcription factors or secreted factors, which are expressed in the OC. For example, misexpression of transcription factors Sox1, 2 or 3 (SoxB1) or Pax6 causes ectopic formation of the NR in the outer layer of the OC (the presumptive RPE region) [12], [13]. The application of FGF8-soaked beads in the vicinity of the developing RPE also results in the ectopic NR formation [11].

In normally developing eyes, the expression of Sox1, 2 and 3 (SoxB1), Pax6 and Fgf8 is detected in the NR [11], [12], [14], [15]. It is noteworthy that SoxB1 and Fgf8 are not expressed in the normally developing RPE after the OC is formed [11], [12]. Although Pax6 is expressed in the normally developing RPE after the OC formation, its expression disappears from the presumptive RPE region as eye development further proceeds [14], [15].

In other words, the expression domains of SoxB1, Pax6 and Fgf8, which induce NR differentiation eventually become restricted to the presumptive NR region during normal eye development. In this point of view, we tested whether expression of these factors was induced in the ectopic NR by EnR-Otx2 transfection.

For this analysis, the embryos were transfected in embryonic stages HH9-11, and then were further incubated for 2 days to reach HH20-22 embryos.

In the control eyes, Sox2 was not expressed in a large part of the outer layer of the OC (the RPE region, the areas between the dashed lines in Figure 4B and C), but its expression was detected in the NR (Figure 4A–C). In these eyes, Sox2 was not expressed in the peripheral (arrow in Figure 4A) or central RPE, although only a small part of the proximal RPE (asterisk in Figure 4A) was positive for Sox2 signals (arrowheads in Figure 4B). In contrast, transfection of EnR-Otx2 caused the ectopic expression of Sox2 in the thickened outer layer of the OC/ectopic NR (the areas between the dashed lines in Figure 4K and L). The expression of Sox2 was detected in the peripheral, central and proximal regions of the thickened outer layer/ectopic NR following EnR-Otx2 transfection (Figure 4J–L). Moreover, unlike in the cases of Islet1 and HuC/D, Sox2 was not only expressed in the basal side but over the whole range of the ectopic NR across the apical-basal axis (Figure 4K and L).

[Figure omitted. See PDF.]

Figure 4. Alterations of expression patterns of transcription factors following EnR-Otx2 transfection.

Immunohistological staining of Sox2, Pax6 and Pax2 in sections of eyes transfected with empty vector (A–I) or EnR -Otx2 (J–R). A–C and J–L indicate the expression of Sox2 (magenta). A and J are merged images with GFP (green). DAPI (blue) in C is used to ease observation of tissue structures of the RPE and NR. D–F and M–O indicate the expression of Pax6 (magenta). D and M are merged images with GFP (green). G–I and P–R indicate the expression of Pax2 (magenta). G and P are merged images with GFP (green). C, F, I, L, O and R are magnified images of the boxes in B, E, H, K, N and Q, respectively. Dashed lines highlight the RPE of control eyes (B–I) or the thickened outer layer of EnR -Otx2-transfected eyes (K–R). Arrows and asterisks in A, J, K, M and N indicate the peripheral and proximal areas of the outer layer of the OC, respectively. The central area of the outer layer of the OC corresponds to the area between the arrow and the asterisk. Arrowheads in B highlight the Sox2-positive small area of the RPE. The upper and lower sides of each image correspond to the dorsal and ventral sides of the specimen, respectively. In J–R, ‘RPE’ refers to the abnormally thickened outer layer, apparently ‘ectopic NR’. RPE, retinal pigment epithelium. NR, neural retina. Le, lens. Scale bars: 100 µm.

https://doi.org/10.1371/journal.pone.0048879.g004

Similar to Sox2, EnR-Otx2 induced the ectopic expression of Fgf8 in the thickened outer layer of the OC/ectopic NR (Figure 5B and D–F). In the normal eyes, Fgf8 was expressed in the central part of the NR but not in the outer layer of the OC/presumptive RPE region (Figure 5A and C). Interestingly, ectopic expression of Fgf8 was also detected in the dorsal and ventral parts of the NR by EnR-Otx2, although only a few NR cells were transfected with EnR-Otx2 (Figure 5B).

[Figure omitted. See PDF.]

Figure 5. Alteration of expression pattern of Fgf8 following EnR-Otx2 transfection.

In situ hybridization analyses of the expression of Fgf8 in sections of normally developing eyes (A and C) and EnR-Otx2-transfected eyes (B and D–F). Expression of GFP is indicated as a brown signal in B and D–F. C is a highly magnified image of the box in A. D–F are highly magnified images of the boxes in B. Arrowheads in D–F indicate sites where Fgf8 and GFP signals overlap. Arrows in E and F indicate Fgf8-positive areas in which GFP signals are relatively weak. The upper and lower sides of each image correspond to the dorsal and ventral sides of the specimen, respectively. In B and D–F, ‘RPE’ refers to the abnormally thickened outer layer, apparently ‘ectopic NR’. RPE, retinal pigment epithelium. NR, neural retina. Le, lens.

https://doi.org/10.1371/journal.pone.0048879.g005

In contrast to Sox2 and Fgf8, Pax6 was strongly expressed in the outer layer of the OC (the presumptive RPE region) of control embryos, which were incubated for 2 days after electroporation (corresponding to HH20-22 embryos, Figure 4D–F). In the normally developing HH20-22 embryos, Pax6 was similarly detected in the outer layer of the OC (the presumptive RPE region). It was intriguing that EnR-Otx2 transfection resulted in the reduced expression of Pax6 in the thickened outer layer of the OC/ectopic NR (Figure 4M–O). Pax6 expression was apparently weakened in the proximal part of the thickened outer layer/ectopic NR (asterisks in Figure 4M and N), and expression of Pax6 also became weaker in the peripheral part of the thickened outer layer/ectopic NR (arrows in Figure 4M and N).

We also analyzed whether some other transcription factors changed their expression patterns in the outer layer of the OC following EnR-Otx2 transfection. We examined the expression of Six3, Lhx2 and Pax2, each of which is associated with multiple aspects of eye development.

During eye development, inactivation of Six3 causes cyclopia, small eyes or disrupted proximo-distal patterning of the OV in medaka embryos [30]. Over-expression of Six3 results in retinal hyperplasia or ectopic retinal primordia formation [31]. In the postnatal retina, Six3 is also involved in cell specification [32], [33].

In the case of eye development in Lhx2, Lhx2−/− mice have eye development that is arrested in the OV stage, and expression domains of various transcription factors are disrupted until the OV stages [34], [35]. To analyze the function of Lhx2 in OC stages, Yun et al. generated genetic mosaic mice, in which Lhx2-mutant cells exist at low frequency among the wild-type cells [35]. In the OC of these mice, Mitf, Vsx2 and Pax2 were not expressed in the Lhx2-mutant cells, although Pax6 was expressed [35].

Pax2 is required for optic fissure closure and proper projection of the optic nerve [36], [37]. Although Pax2 is not expressed in the presumptive RPE, its expression is detected in the optic stalk (OS), which is adjacent to the presumptive RPE [36], [38]. Pax2-deficient mutant mice display expansion of the RPE domain toward the OS region [36], [38]. Therefore, Pax2 is thought to repress RPE development to make a sharp boundary between the OS and the RPE [38].

In the control eyes, expression of Pax2 and Six3 was not detected in the outer layer of the OC (the presumptive RPE region) (the areas between the dashed lines in Figure 4H and I and described as ‘RPE’ in Figure 6A and B, respectively), although Lhx2 was expressed in this region (described as ‘RPE’ in Figure 6C). Similarly, the ectopic NR was negative for Pax2 and Six3 (the areas between the dashed lines in Figure 4Q and R and Figure 6E, respectively) but was positive for Lhx2 expression in the EnR-Otx2 transfected eyes (the area between the dashed lines in Figure 6F). In the control NR, Six3 and Lhx2 were expressed (Figure 6A–C), although Pax2 was highly expressed in the ventral NR but was only weakly expressed in the dorsal NR (Figure 4G–I). Similar expression patterns of Pax2, Six3 and Lhx2 were observed in the NR of EnR-Otx2 transfected eyes (Figure 4P–R and Figure 6D–F).

[Figure omitted. See PDF.]

Figure 6. Expression patterns of Six3 and Lhx2 in EnR-Otx2-transfected eyes.

In situ hybridization analyses of the expression of Six3 (B and E) and Lhx2 (C and F) in sections of eyes transfected with an empty vector (A–C) or EnR-Otx2 (D–F). A and D indicate GFP signals (green). A–C and D–F are serial sections. Dashed lines in A–C indicate boundaries between the RPE and NR. Dashed lines in D–F highlight the thickened outer layer of EnR-Otx2-transfected eyes. The upper and lower sides of each image correspond to the dorsal and ventral sides of the specimen, respectively. In D-F,‘RPE’ refers to the abnormally thickened outer layer, apparently ‘ectopic NR’. RPE, retinal pigment epithelium. NR, neural retina. Le, lens. Scale bars: 100 µm.

https://doi.org/10.1371/journal.pone.0048879.g006

Our analyses of the transcription factors and a secreted factor suggest that EnR-Otx2 induces the ectopic expression of Sox2 and Fgf8 in the thickened outer layer of the OC/ectopic NR. It is notable that these two factors share two traits; 1) being able to forcedly induce NR differentiation in the outer layer of the OC/presumptive RPE region [11], [12], and 2) being not expressed in the outer layer of the OC/presumptive RPE region but detected in the inner layer of the OC/presumptive NR region in normally developing eyes [11], [12].

Increased Cell Proliferation and Apoptosis in EnR-Otx2-transfected Eyes

Martinez-Morales et al. reported that Otx1−/−; Otx2+/− mice display increased cell proliferation and cell death in the retina [6]. Therefore, we also assessed the effects of EnR-Otx2 on cell proliferation and apoptosis in chick eyes. To analyze cell proliferation and apoptosis, anti-PHH3 and anti-single stranded DNA (ssDNA) antibodies were used, respectively.

For this analysis, the embryos were transfected in embryonic stages HH9-11, and then were further incubated for 2 days to reach HH20-22 embryos.

Although a small number of PHH3-positive or ssDNA-positive cells existed in the normal RPE and NR (Figure S1A, D, F and I), the number increased following EnR-Otx2 transfection (Figure S1B, C, E, G, H and J), as in the case of Otx mutant mice [6].

Ectopic Formation of Telencephalon-like Vesicles Following EnR-Otx2 Transfection

When embryos were incubated for about a week after EnR-Otx2 transfection, their eyes displayed a small eye phenotype (Figure S2A), as in the case of Otx1−/−; Otx2+/− mice. Intriguingly, we noticed that some vesicle-like structures were ectopically formed following EnR-Otx2 transfection (arrow in Figure S2A). The ectopic vesicles were connected to the small eyes of EnR-Otx2 transfected embryos, but were not observed in control embryos (data not shown). The ectopic vesicles lacked the characteristics of the RPE (pigmentation and a monolayered-structure, Figure S2B, C, F, G, I and J) or the NR (expression of a photoreceptor marker visinin, Figure S2C).

These ectopic vesicles were positive for markers of the developing brain, including Emx1, Nkx2.1 and Pax6 (Figure S2B and F–K), but were negative for a hindbrain marker, Gbx2 (data not shown). Emx1 is normally detected in the dorsal telencephalon [39], [40], Nkx2.1 in the hypothalamus in the ventral portion of the diencephalon and telencephalon [41] and Pax6 in the dorsal telencephalon and diencephalon. These data suggest that the ectopic vesicles induced by EnR-Otx2 transfection seem to have the characteristics of the telencephalon.

Discussion

Repression of Otx2 Function in Chick Eyes

As in the case of Otx1−/−;Otx2+/− mice [6], chick eyes displayed the formation of an “ectopic NR” in the outer layer of the OC, as a result of the severely impaired function of Otx2. The outer layer of the OC began to form an unpigmented-thick structure following EnR-Otx2 transfection. In this tissue, the expression of some RPE-specific markers (Mitf, Dct and MMP115) was reduced but, instead, the expression of several differentiation markers of the NR (HuC/D and Islet1) was detected. In addition, the expression of EnR-Otx2 also caused increases in cell proliferation and apoptosis in the developing eye, similar to Otx1−/−; Otx2+/− mice [6].

Functions of Otx2 are Associated with Regional Specification of the RPE

Both the RPE and NR are derived from the same developmental origin. As eye development proceeds, the presumptive RPE and NR regions become subdivided into the outer and inner layers of the OC, respectively. One focus of this study was to elucidate how Otx2 functions during these regional specifications of the RPE and NR.

We analyzed the expression patterns of factors contributing to NR development. Among them, Sox2 and Fgf8 were ectopically expressed in the outer layer of the OC following EnR-Otx2 transfection. In contrast, the expression of Pax6 seemed to be decreased in the ectopic NR, and there were no obvious changes in Six3 or Lhx2. Considering that Sox2 and FGF8 are known to induce NR cell fate in the outer layer of the OC (the presumptive RPE region) in vivo [11], [12], it is possible that Otx2 is required to repress the expression of these factors (Sox2 and Fgf8) in the outer layer of the OC (the presumptive RPE region). Correspondingly, in normally developing eyes, Sox2 and Fgf8 are not detected in the outer layer of the OC (the presumptive RPE region) where Otx2 is expressed [11], [12].

Considering these data, we would like to propose the following hypothesis regarding the regional specification of the RPE and NR (Figure 7). In the normally developing OC, Sox2 and Fgf8 function to induce NR differentiation. However, in the outer layer of the OC, the expression of Sox2 and Fgf8 is repressed by Otx2, and Sox2 and Fgf8 expression is restricted to and remains in the inner layer of the OC. As a result, NR differentiation is prevented in the outer layer of the OC and leads to formation of the RPE, whereas the NR is formed in the Sox2 and Fgf8-positive inner layer.

[Figure omitted. See PDF.]

Figure 7. Schematic representation of how Otx2 functions in the regional specification of the RPE and NR in the OC.

In developing chick eyes, Sox2 and Fgf8 are expressed in the OC and induce NR differentiation. However, the expression domains of Sox2 and Fgf8 are restricted to the inner layer of the OC, since Otx2 is expressed in the outer layer of the OC and represses the expression of Sox2 and Fgf8. As a result, the Sox2 and Fgf8-positive inner layer of the OC is induced to form the NR, whereas the Sox2 and Fgf8-negative outer layer is prevented from forming the NR and instead differentiates into the RPE.

https://doi.org/10.1371/journal.pone.0048879.g007

Reduction of Pax6 Expression by EnR-Otx2

Our results show that the expression of Pax6 is reduced in the thickened outer layer/ectopic NR by EnR-Otx2 transfection. However, Pax6 is known to induce ectopic NR formation in the RPE region [13], as do Sox2 and FGF8. In the case of ectopic NR formation by FGF8, Pax6 expression is initially absent, but emerges in the ectopic NR at a later stage of differentiation [11]. Therefore, we cannot exclude the possibility that Pax6 would also be expressed in the ectopic NR if the EnR-Otx2-transfected embryos were incubated for a much longer term. In fact, Martinez-Morales et al. reported that the ectopic NR of Otx1−/−; Otx2+/− mice is positive for Pax6 expression [6]. Moreover, it has been revealed that Pax6 promotes NR development [13], [42], [43], [44].

However, previous studies also indicate the requirement of Pax6 for RPE development, using chimeric mouse embryos composed of wild-type and Pax6Sey/Sey–Neu-mutant cells (both the Sey and SeyNeu alleles encode a non-functional Pax6 protein) [45]. In the outer layer of the OC of the chimera, the region occupied by Pax6-mutant cells shows an abnormally thickened-layer [45], suggesting that loss of function mutations in Pax6 cause disruption of the mono-layered structure of the RPE. Pax6 is also required to initiate Mitf expression in the developing eye, in a redundant manner with Pax2 [46]. In addition, Pax6 is expressed in the presumptive RPE region (this study and [46]), and is also detected in cultured RPE cells which are derived from embryonic stem cells [47].

To elucidate the mechanisms of OC patterning in detail, it should be unveiled how Pax6 expression is regulated and how Pax6 switches its function according to the developmental context.

Future Prospects

By incubating the embryos for a long term after EnR-Otx2 transfection, ectopic vesicles were formed near the small eyes (Figure S2). Although the ectopic vesicles were continuously connected to the EnR-Otx2 transfected small eyes, they lacked the characteristics of the RPE or NR. Instead, the expression of Emx1, Pax6 and Nkx2.1 [39], [40], [41], [48], [49] suggests that the ectopic vesicles have the characteristics of the telencephalon. Although more detailed analyses on the molecular mechanisms involved are needed, our analyses of Otx2 function bring new insights into the relationships between eye and brain development.

EnR-Otx2 may bind to the Otx1 protein, since the structures of Otx1 and Otx2 are similar in their dimerization domain (homeodomain) and are able to bind the same DNA target sequences [50]. Moreover, the replacement of Otx1 with Otx2 rescues the phenotype of Otx1 knock-out mice, at least in part [51]. Therefore, our observations reinforce the requirement for Otx genes in the development of chick eyes. Other techniques that selectively reduce Otx2 or Otx1 expression, such as RNA interference, would dissect the functional divergence of Otx1 and Otx2 in eye development.

In the developing eye, patterning the polarity of the OV along the dorsal-ventral and posterior-anterior axes is required for proper regional specification of the presumptive RPE and NR regions [52], [53], [54], [55]. In such a patterning process, BMP4 and Shh from the dorsal and ventral parts of the forebrain, respectively, are thought to be involved [56]. Moreover, Activin, BMP and Wnt from the extra-ocular mesenchyme or surface ectoderm are thought to regionalize the presumptive RPE [17], [18], [25], [26], as well as FGFs from the surface ectoderm to regionalize the presumptive NR [5], [21]. Future analyses should clarify how Otx2 mediates these signals from extra-ocular tissues to intrinsic molecular mechanisms in the OV and OC. By understanding how the expression domain of Otx2 is restricted to the presumptive RPE region, more details about the mechanisms responsible for regionalizing the RPE and NR in the developing eye will be unveiled.

Materials and Methods

Ethics Statement

All experiments involving animals were approved by the Nagahama Institute of Bio-Science and Technology (approval Id: 050).

Chick Embryos

White Leghorn chicken eggs were incubated at 38°C. Developmental stages of embryos were assigned according to Hamburger and Hamilton [57].

In situ Hybridization and Immunohistochemistry

In situ hybridization and immunohistochemistry were performed as previously described [3]. Primary antibodies used for immunohistochemistry include polyclonal antibodies against chicken Mitf (generated in our laboratory), Pax2 (COVANCE), phospho Histone-H3 (Upstate), Sox2 (MILLIPORE) and ssDNA (DAKO, Denmark), and monoclonal antibodies against HuC/D (Molecular Probes), Tuj1 (COVANCE), Islet1, neurofilaments, Pax6 and visinin (Developmental Studies Hybridoma Bank, DSHB, USA). Samples were observed using an Olympus BX51 microscope (Tokyo, Japan) with a cooled CCD camera.

Electroporation

In ovo electroporation was carried out as described previously [3] with the following modifications. White Leghorn chicken eggs were incubated at 38°C until the chick embryos reached stage 9–11, according to Hamburger and Hamilton [57]. The plasmid solution was then injected into the OV. An anode (0.5 mm in diameter, 1.0 mm in length; Unique Medical Imada, Japan) and a cathode (tungsten needle) were placed on the outside and inside of the embryo, respectively, across the OV (and surface ectoderm). Rectangular pulses (7 V, 30 ms) were then charged twice using an electroporator (CUY, Tokiwa Science, Japan).

Expression Vectors

The full-length chicken Otx2 cDNA was inserted in the pMiwIII vector. In this pMiwIII-wtOtx2 (wtOtx2) vector, Otx2 is fused to a nucleic acid encoding FLAG-tag. pMiwIII-Otx2-EnR (EnR-Otx2) is constructed by modifying pMiwIII-wtOtx2, in which the chicken Otx2 fragment with FLAG Tag is fused to a nucleic acid encoding the repressor domain of Drosophila Engrailed (corresponding to amino acids 1–298). Three to 4 µg/µl Otx2-EnR plasmids were injected into the OV for transfection.

Transient Transfection Assays

DCT promoter activity, which is driven by Otx2 (Takeda et al., 2003), was assessed by transient expression of reporter genes in D407 human RPE cells, as described previously (Takeda et al, 2003). Briefly, cells were cultured for 12–24 hr after plating in 12 well dishes, and then were transfected with pHDTL12 containing the luciferase gene under the control of the human DCT gene promoter, each expression plasmid and pRL-TK (an internal control) using the FuGENE 6 protocol (Roche Molecular Biochemicals). pRL-TK contains the herpes simplex virus thymidine kinase promoter region upstream of Renilla luciferase (Promega). Luciferase activity was measured using the Dual-LuciferaseTM Reporter Assay System (Promega). Reporter luciferase activity was normalized against Renilla luciferase activity.

Supporting Information

[Figure omitted. See PDF.]

Figure S1.

Increased cell proliferation and apoptosis in EnR-Otx2-transfected eyes. Immunohistological analyses of cell proliferation (A-E) and apoptosis (F-J) in sections of normal eyes (A, D, F and I) and EnR-Otx2-transfected eyes (B, C, E, G, H and J). A and C-E indicate PHH3-positive mitotic cells (magenta), and C and E are merged images with GFP (green). F and H-J indicate ssDNA-positive apoptotic cells (magenta), and H and J are merged images with GFP (green). D is a highly magnified image of the box in A, as well as E of C, I of F, and J of H. B and G are bright field images of C and H, respectively. Open arrows and arrowheads in A and D indicate PHH3-positive cells in the RPE and NR of the normal eye, respectively. Arrowheads and arrows in E indicate PHH3-positive cells in ectopic NR and NR of EnR-Otx2- transfected eyes, respectively. Arrows in F and I indicate ssDNA-positive cells in the normal eye. Arrows in H indicate ssDNA-positive cells which are located in the EnR-Otx2-transfected areas. Arrowheads and arrows in J indicate ssDNA-positive cells in ectopic NR and NR of EnR-Otx2-transfected eyes, respectively. RPE, retinal pigment epithelium. NR, neural retina. Le, lens. Scale bars: 100 µm in A (for A-C and F-H); 50 µm in D (for D and I); 10 µm in E (for E and J).

https://doi.org/10.1371/journal.pone.0048879.s001

(TIF)

Figure S2.

Ectopic formation of telencephalon-like vesicles following EnR-Otx2 transfection. (A) Lateral view of an embryo incubated for 1 week after EnR-Otx2 transfection. The right eye displays a ‘small eye’ compared to the untransfected-left eye. The arrow indicates a vesicle which is ectopically formed adjacent to the small eye. (B-K) Immunohistological and in situ hybridization analyses of eye and brain markers. Sections in B-K are sliced along the plane indicated by the white line in A. Sections in B, D, H and K are stained with anti-Pax6 antibody (green) and DAPI (blue). Sections in C and E are stained with anti-Visinin antibody (green) and DAPI (blue). B, C, H and K indicate the tissues around the small eye and ectopic vesicles formed by EnR-Otx2. D and E indicate parts of the normally developing eye. Sections in F and I are stained with an antisense probe for Emx1 (violet-blue). Sections in G and J are stained with an antisense probe for Nkx2.1 (violet-blue). H and K are highly magnified images of boxes in B, as well as I of F, and J of G. F, G, I and J indicate tissues around the small eye (right eye), ectopic vesicle (Etc) and normally developing untransfected eye (left eye) of an EnR-Otx2-transfected embryo. Arrowheads in H and I indicate the dorsal area of ectopic vesicles in which both Pax6 and Emx1 signals are detected. Arrowheads in J indicate the ventral areas of ectopic vesicles in which the Nkx2.1 signal is detected. RPE, retinal pigment epithelium. NR, neural retina. Etc, Ectopic vesicle. inl, inner nuclear layer. onl, outer nuclear layer. gcl, glial cell layer.

https://doi.org/10.1371/journal.pone.0048879.s002

(TIF)

Acknowledgments

We are grateful to Dr. Colin R. Goding for helpful discussions. Dr. Makoto Mochii’s gift of an antibody used in the very early stages of this study is very much appreciated.

Author Contributions

Conceived and designed the experiments: DN IY TK KT HY. Performed the experiments: DN IY HT MN KT. Analyzed the data: DN IY NT KT SS. Contributed reagents/materials/analysis tools: DN IY NT TK KT SS HN. Wrote the paper: DN IY KT SS HN HY.

Citation: Nishihara D, Yajima I, Tabata H, Nakai M, Tsukiji N, Katahira T, et al. (2012) Otx2 Is Involved in the Regional Specification of the Developing Retinal Pigment Epithelium by Preventing the Expression of Sox2 and Fgf8, Factors That Induce Neural Retina Differentiation. PLoS ONE 7(11): e48879. https://doi.org/10.1371/journal.pone.0048879

References

1. Fuhrmann S (2010) Eye morphogenesis and patterning of the optic vesicle. Curr Top Dev Biol 93: 61–84.

2. Martinez-Morales JR, Rodrigo I, Bovolenta P (2004) Eye development: a view from the retina pigmented epithelium. Bioessays 26: 766–777.

3. Tsukiji N, Nishihara D, Yajima I, Takeda K, Shibahara S, et al. (2009) Mitf functions as an in ovo regulator for cell differentiation and proliferation during development of the chick RPE. Dev Biol 326: 335–346.

4. Bumsted KM, Barnstable CJ (2000) Dorsal retinal pigment epithelium differentiates as neural retina in the microphthalmia (mi/mi) mouse. Invest Ophthalmol Vis Sci 41: 903–908.

5. Nguyen M, Arnheiter H (2000) Signaling and transcriptional regulation in early mammalian eye development: a link between FGF and MITF. Development 127: 3581–3591.

6. Martinez-Morales JR, Signore M, Acampora D, Simeone A, Bovolenta P (2001) Otx genes are required for tissue specification in the developing eye. Development 128: 2019–2030.

7. Acampora D, Mazan S, Lallemand Y, Avantaggiato V, Maury M, et al. (1995) Forebrain and midbrain regions are deleted in Otx2−/− mutants due to a defective anterior neuroectoderm specification during gastrulation. Development 121: 3279–3290.

8. Matsuo I, Kuratani S, Kimura C, Takeda N, Aizawa S (1995) Mouse Otx2 functions in the formation and patterning of rostral head. Genes Dev 9: 2646–2658.

9. Martinez-Morales JR, Dolez V, Rodrigo I, Zaccarini R, Leconte L, et al. (2003) OTX2 activates the molecular network underlying retina pigment epithelium differentiation. J Biol Chem 278: 21721–21731.

10. Westenskow PD, McKean JB, Kubo F, Nakagawa S, Fuhrmann S (2010) Ectopic Mitf in the embryonic chick retina by co-transfection of beta-catenin and Otx2. Invest Ophthalmol Vis Sci 51: 5328–5335.

11. Vogel-Hopker A, Momose T, Rohrer H, Yasuda K, Ishihara L, et al. (2000) Multiple functions of fibroblast growth factor-8 (FGF-8) in chick eye development. Mech Dev 94: 25–36.

12. Ishii Y, Weinberg K, Oda-Ishii I, Coughlin L, Mikawa T (2009) Morphogenesis and cytodifferentiation of the avian retinal pigmented epithelium require downregulation of Group B1 Sox genes. Development 136: 2579–2589.

13. Azuma N, Tadokoro K, Asaka A, Yamada M, Yamaguchi Y, et al. (2005) Transdifferentiation of the retinal pigment epithelia to the neural retina by transfer of the Pax6 transcriptional factor. Hum Mol Genet 14: 1059–1068.

14. Walther C, Gruss P (1991) Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 113: 1435–1449.

15. Kawakami A, Kimura-Kawakami M, Nomura T, Fujisawa H (1997) Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development. Mech Dev 66: 119–130.

16. Takeda K, Yokoyama S, Yasumoto K, Saito H, Udono T, et al. (2003) OTX2 regulates expression of DOPAchrome tautomerase in human retinal pigment epithelium. Biochem Biophys Res Commun 300: 908–914.

17. Fuhrmann S, Levine EM, Reh TA (2000) Extraocular mesenchyme patterns the optic vesicle during early eye development in the embryonic chick. Development 127: 4599–4609.

18. Muller F, Rohrer H, Vogel-Hopker A (2007) Bone morphogenetic proteins specify the retinal pigment epithelium in the chick embryo. Development 134: 3483–3493.

19. Spence JR, Madhavan M, Aycinena JC, Del Rio-Tsonis K (2007) Retina regeneration in the chick embryo is not induced by spontaneous Mitf downregulation but requires FGF/FGFR/MEK/Erk dependent upregulation of Pax6. Mol Vis 13: 57–65.

20. Galy A, Neron B, Planque N, Saule S, Eychene A (2002) Activated MAPK/ERK kinase (MEK-1) induces transdifferentiation of pigmented epithelium into neural retina. Dev Biol 248: 251–264.

21. Hyer J, Mima T, Mikawa T (1998) FGF1 patterns the optic vesicle by directing the placement of the neural retina domain. Development 125: 869–877.

22. Pittack C, Grunwald GB, Reh TA (1997) Fibroblast growth factors are necessary for neural retina but not pigmented epithelium differentiation in chick embryos. Development 124: 805–816.

23. Guillemot F, Cepko CL (1992) Retinal fate and ganglion cell differentiation are potentiated by acidic FGF in an in vitro assay of early retinal development. Development 114: 743–754.

24. Pittack C, Jones M, Reh TA (1991) Basic fibroblast growth factor induces retinal pigment epithelium to generate neural retina in vitro. Development 113: 577–588.

25. Fujimura N, Taketo MM, Mori M, Korinek V, Kozmik Z (2009) Spatial and temporal regulation of Wnt/beta-catenin signaling is essential for development of the retinal pigment epithelium. Dev Biol 334: 31–45.

26. Westenskow P, Piccolo S, Fuhrmann S (2009) Beta-catenin controls differentiation of the retinal pigment epithelium in the mouse optic cup by regulating Mitf and Otx2 expression. Development 136: 2505–2510.

27. Galli-Resta L, Resta G, Tan SS, Reese BE (1997) Mosaics of islet-1-expressing amacrine cells assembled by short-range cellular interactions. J Neurosci 17: 7831–7838.

28. Marusich MF, Furneaux HM, Henion PD, Weston JA (1994) Hu neuronal proteins are expressed in proliferating neurogenic cells. J Neurobiol 25: 143–155.

29. Wakamatsu Y, Weston JA (1997) Sequential expression and role of Hu RNA-binding proteins during neurogenesis. Development 124: 3449–3460.

30. Carl M, Loosli F, Wittbrodt J (2002) Six3 inactivation reveals its essential role for the formation and patterning of the vertebrate eye. Development 129: 4057–4063.

31. Loosli F, Winkler S, Wittbrodt J (1999) Six3 overexpression initiates the formation of ectopic retina. Genes Dev 13: 649–654.

32. Zhu CC, Dyer MA, Uchikawa M, Kondoh H, Lagutin OV, et al. (2002) Six3-mediated auto repression and eye development requires its interaction with members of the Groucho-related family of co-repressors. Development 129: 2835–2849.

33. Manavathi B, Peng S, Rayala SK, Talukder AH, Wang MH, et al. (2007) Repression of Six3 by a corepressor regulates rhodopsin expression. Proc Natl Acad Sci U S A 104: 13128–13133.

34. Tetreault N, Champagne MP, Bernier G (2008) The LIM homeobox transcription factor Lhx2 is required to specify the retina field and synergistically cooperates with Pax6 for Six6 trans-activation. Dev Biol. 2008 Dec 30.

35. Yun S, Saijoh Y, Hirokawa KE, Kopinke D, Murtaugh LC, et al. (2009) Lhx2 links the intrinsic and extrinsic factors that control optic cup formation. Development 136: 3895–3906.

36. Torres M, Gomez-Pardo E, Gruss P (1996) Pax2 contributes to inner ear patterning and optic nerve trajectory. Development 122: 3381–3391.

37. Viringipurampeer IA, Ferreira T, Demaria S, Yoon JJ, Shan X, et al. (2012) Pax2 regulates a fadd-dependent molecular switch that drives tissue fusion during eye development. Hum Mol Genet 21: 2357–2369.

38. Schwarz M, Cecconi F, Bernier G, Andrejewski N, Kammandel B, et al. (2000) Spatial specification of mammalian eye territories by reciprocal transcriptional repression of Pax2 and Pax6. Development 127: 4325–4334.

39. Bell E, Ensini M, Gulisano M, Lumsden A (2001) Dynamic domains of gene expression in the early avian forebrain. Dev Biol 236: 76–88.

40. Simeone A, Acampora D, Gulisano M, Stornaiuolo A, Boncinelli E (1992) Nested expression domains of four homeobox genes in developing rostral brain. Nature 358: 687–690.

41. Pera EM, Kessel M (1998) Demarcation of ventral territories by the homeobox gene NKX2.1 during early chick development. Dev Genes Evol 208: 168–171.

42. Marquardt T, Ashery-Padan R, Andrejewski N, Scardigli R, Guillemot F, et al. (2001) Pax6 is required for the multipotent state of retinal progenitor cells. Cell 105: 43–55.

43. Philips GT, Stair CN, Young Lee H, Wroblewski E, Berberoglu MA, et al. (2005) Precocious retinal neurons: Pax6 controls timing of differentiation and determination of cell type. Dev Biol 279: 308–321.

44. Oron-Karni V, Farhy C, Elgart M, Marquardt T, Remizova L, et al. (2008) Dual requirement for Pax6 in retinal progenitor cells. Development 135: 4037–4047.

45. Quinn JC, West JD, Hill RE (1996) Multiple functions for Pax6 in mouse eye and nasal development. Genes Dev 10: 435–446.

46. Baumer N, Marquardt T, Stoykova A, Spieler D, Treichel D, et al. (2003) Retinal pigmented epithelium determination requires the redundant activities of Pax2 and Pax6. Development 130: 2903–2915.

47. Vugler A, Carr AJ, Lawrence J, Chen LL, Burrell K, et al.. (2008) Elucidating the phenomenon of HESC-derived RPE: Anatomy of cell genesis, expansion and retinal transplantation. Exp Neurol. 2008 Sep 27.

48. Li HS, Yang JM, Jacobson RD, Pasko D, Sundin O (1994) Pax-6 is first expressed in a region of ectoderm anterior to the early neural plate: implications for stepwise determination of the lens. Dev Biol 162: 181–194.

49. Goulding MD, Lumsden A, Gruss P (1993) Signals from the notochord and floor plate regulate the region-specific expression of two Pax genes in the developing spinal cord. Development 117: 1001–1016.

50. Simeone A, Acampora D, Mallamaci A, Stornaiuolo A, D'Apice MR, et al. (1993) A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo. EMBO J 12: 2735–2747.

51. Acampora D, Avantaggiato V, Tuorto F, Barone P, Perera M, et al. (1999) Differential transcriptional control as the major molecular event in generating Otx1−/− and Otx2−/− divergent phenotypes. Development 126: 1417–1426.

52. Uemonsa T, Sakagami K, Yasuda K, Araki M (2002) Development of dorsal-ventral polarity in the optic vesicle and its presumptive role in eye morphogenesis as shown by embryonic transplantation and in ovo explant culturing. Dev Biol 248: 319–330.

53. Kagiyama Y, Gotouda N, Sakagami K, Yasuda K, Mochii M, et al. (2005) Extraocular dorsal signal affects the developmental fate of the optic vesicle and patterns the optic neuroepithelium. Dev Growth Differ 47: 523–536.

54. Hirashima M, Kobayashi T, Uchikawa M, Kondoh H, Araki M (2008) Anteroventrally localized activity in the optic vesicle plays a crucial role in the optic development. Dev Biol 317: 620–631.

55. Kobayashi T, Yasuda K, Araki M (2009) Generation of a second eye by embryonic transplantation of the antero-ventral hemicephalon. Dev Growth Differ 51: 723–733.

56. Kobayashi T, Yasuda K, Araki M (2010) Coordinated regulation of dorsal bone morphogenetic protein 4 and ventral Sonic hedgehog signaling specifies the dorso-ventral polarity in the optic vesicle and governs ocular morphogenesis through fibroblast growth factor 8 upregulation. Dev Growth Differ 52: 351–363.

57. Hamburger V, Hamilton HL (1992) A series of normal stages in the development of the chick embryo. 1951. Dev Dyn 195: 231–272.