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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Development. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
PMCID: PMC2709064
NIHMSID: NIHMS131669

Morphogenesis and cytodifferentiation of the avian retinal pigmented epithelium require downregulation of Group B1 Sox genes

Abstract

The optic vesicle is a multipotential primordium of the retina, which becomes subdivided into the neural retina and retinal pigmented epithelium domains. Although roles of several paracrine factors in patterning the optic vesicle have been studied extensively, little is known about cell autonomous mechanisms that regulate coordinated cell morphogenesis and cytodifferentiation of the retinal pigmented epithelium. Here we demonstrate that members of the SoxB1 gene family, Sox1, Sox2 and Sox3, are all downregulated in the presumptive retinal pigmented epithelium. Constitutive maintenance of SoxB1 expression in the presumptive retinal pigmented epithelium both in vivo and in vitro resulted in the absence of cuboidal morphology and pigmentation, and in concomitant induction of neural differentiation markers. We also demonstrate that exogenous FGF4 inhibits downregulation all SoxB1 family members in the presumptive retinal pigment epithelium. These results suggest that retinal pigment epithelium morphogenesis and cytodifferentiation requires SoxB1 downregulation, which depends on absence of exposure to FGF-like signal.

Keywords: eye, retina, pigmented epithelium, electroporation, Sox genes, chick embryo

Introduction

The retina originates from bilateral evagination of the forebrain, called the optic vesicle (OV). As the overlying lens ectoderm thickens and invaginates to form the lens vesicle, the distal part of the OV begins to invaginate to form a double-layered optic cup. The inner layer of the cup forms the neural retina (NR), where six types of neurons and one type of glia are generated. On the other hand, the outer layer of the cup gives rise to the retinal pigmented epithelium (RPE) characterized as a melanin-containing simple cuboidal epithelium.

In vivo and in vitro studies have demonstrated that the early OV neuroepithelium is capable of differentiating into both of these cell types (reviewed in Moshiri et al., 2004). Patterning of the OV into RPE and NR depends on exposure to extracellular signals from periocular mesenchyme and surface ectoderm, that control the choice of the fate of OV cells. The former, presumably mediated by BMP- or activin-like signals, promotes RPE development (Furhmann et al., 2000; Hyer et al, 2003; Müller et al., 2007) whereas the latter, likely mediated by FGF signaling, inhibits it and specifies the NR domain (Guillemot and Cepko, 1992; Hyer et al., 1998; Nguyen and Arnheiter, 2000; Opas and Dziak, 1994; Park and Hollenberg, 1991; Pittack et al., 1991, 1997; Vogel-Höpker et al., 2000, Zhao et al., 2001). Misexpression of truncated FGF receptors suggest that FGF signaling is necessary for NR to undergo normal specification of retinal cell types (McFarlane et al., 1998; Zhang et al., 2003) and proliferation of precursor cells (Dias da Silva et al., 2007).

While several paracrine signals involved in OV patterning have been identified and studied extensively, little is known about transcriptional regulatory pathways that respond to paracrine signals and regulate morphogenesis and cytodifferentiation specific to each retinal subdomain. Pax6, if misexpressed, is capable of converting a pigmented RPE into a layered NR-like tissue (Azuma et al., 2005). However, its normal expression in early optic cup does not precisely delineate the NR domain (Kamachi et al., 1998; Li et al., 1994) and appears to play a role in the dorsoventral patterning (Reza et al., 2007).

Subgroup B1 Sox family genes (SoxB1 genes) encode HMG transcription factors (reviewed in Kamachi et al., 2000; Pevny and Lovell-Badge, 1997) implicated in regulating epithelial morphology and neural progenitor state (Abu-Elmagd et al., 2001; Bylund et al., 2003; Dee et al., 2008; Ferri et al., 2004; Graham et al., 2003; Ishii et al., 2001; Kamachi et al., 1998; Kamachi et al., 2001; Köster et al., 2000; Taranova et al., 2008). Although SoxB1 genes show dynamic patterns of expression in the optic cup in chick (Kamachi et al., 1998; Le Rouëdec et al., 2002; Uchikawa et al., 1999) their roles in early retinal development remains to be determined.

In the present study, we demonstrate that all SoxB1 gene members are downregulated in the presumptive RPE by the time when NR and RPE domains become evident morphologically. Using in ovo electroporation, we demonstrate that the forced maintenance of SoxB1 expression inhibits epithelial thinning and pigmentation of the RPE. These phenotypes are associated with ectopic expression of neural markers that are normally excluded from the RPE. We also demonstrate that SoxB1 genes lie downstream of FGF4-activated signaling and mediate part of inhibitory effect of FGF on RPE molecular identity. These results suggest that SoxB1 downregulation is necessary for RPE morphogenesis and cytodifferentiation and is dependent on absence of exposure to FGF-like signals.

Materials and Methods

Embryos

Chick (Gallus gallus domesticus) embryos were incubated at 38.5°C in a humidified incubator and were staged according to Hamburger and Hamilton (1951).

In situ hybridization

Sox1, [partial differential]-crystallin, Mitf, Otx2, Six3, Rx1, Chx10 and Optx2 cDNAs were amplified by RT-PCR from E3 whole chick embryo cDNA (Sox1, [partial differential]-crystallin), E3.5 NR cDNA (Rx1 and Opix2), E3.5 RPE cDNA (Otx2) or E5 whole eye cDNA (Mitf and Chx10). The following primers were used for PCR. For Sox1, 5’-CCCTTGACGCACATCTGAGCG-3’ and 5’-AACTATGTACAGTCTGGGTTCC-3’; For [partial differential]-crystallin, 5’-GAGCAAAACGTCGTCCGAAATG-3’ and 5’-CTCTGGATTAGTGAGATAAGCA-3’; for Mitf, 5’-CTTCCCACAGCAATTCCGAGC-3’ and 5’-ACACTGGGCTACCGATGAAGCAC-3’; for Otx2, 5’-GATATCCAACTTTAGCATGATGTCTTATCT-3’ and 5’-TCTAGAGTCTGAGCAGGAAATGAGTCTG-3’ (first round); 5’-GATATCCAACTTTAGCATGATGTCTTATCT-3’ and 5’-TCTAGATCACAAAACCTGGAACTTCCATGAG-3’ (second round); for Six3, 5’-ACGAAGAGTTGTCAATGTTTCAGC-3’ and 5’-TCTAGATATCATACATCACATTCCGAGTC-3’ (first round); 5’-ACGAAGAGTTGTCAATGTTTCAGC-3’ and 5’-GCTCTTTCTGTCAAACTGGAGAC-3’ (second round); for Rx1, 5’-GATATCACCAAGATGTTCCTCAATAAGTGT-3’ and 5’-TCTAGAGCGTTCATCAAATGGGCTGCCAGGT-3’ (first round); 5’-GATATCACCAAGATGTTCCTCAATAAGTGT-3’ and 5’-TCTAGATCAAATGGGCTGCCAGGTCTTGTC-3’ (second round); for Chx10, 5’-GGCTTCGGCATCCAGGAGATC-3’ and 5’-TTCTGTGATGCACTGGACTTC-3’; for Optx2, 5’-GATATCGGAACCGCCACCATGTTCCAGCT-3’ and 5’-CGGGGACAGCGATAGGCACTG-3’. Nested PCR was carried out to amplify Otx2, Six3, and Rx1. Amplified cDNA fragments were ligated into pCRII-TOPO (Invitrogen, CA) vector, sequenced and used as templates for synthesis of digoxigenin-labeled riboprobes (Hurtado and Mikawa, 2006). Plasmids used to synthesize riboprobes for Sox2 and Sox3 (Uwanogho et al., 1995) were gifts from Dr Scotting (The University of Nottingham, UK). Section in situ hybridization was performed as described previously (Ishii et al., 1998).

RT-PCR

Total RNAs were isolated from the OV at stage 10, and from separated RPE and NR segments of the optic cup at stages 19–20 and 24–25 using RNeasy Mini Kit (Qiagen, CA). Collagenase (0.03%, 5–10 minutes; Type 1; Worthington, NJ) was used to remove surrounding mesenchyme from retinal tissue. Approximately 200 ng of RNAs were reverse transcrived using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, CA). For a negative control, reactions for cDNA synthesis were carried out in parallel without reverse transcriptase. The following primers were used for PCR. For Sox1, 5’-AGAAGGTAACGGTGGCTTTACTGAC-3’ and 5’-GCGCGAGAACATCTACGGAAACTC-3’; for Sox2, 5’-GGGCTGGTTCCAGGCTAAAGTAGT-3’ and 5’-AAGGGTCTCTTCTCCGCCTCGGAT-3’; for Sox3, 5’-CGGCTCAGCAGACTCGATACTAAC-3’ and 5’-AACACAGATCAAACATCCATCC-3’; for Mitf, 5’-GGCTTCGGCATCCAGGAGATC-3’ and 5’-TTCTGTGATGCACTGGACTTC-3’; for Chx10, 5’-CTTCCCACAGCAATTCCGAGC-3’ and 5’-ACACTGGGCTACCGATGAAGCAC-3’; for Tyrosinase, 5’-TACTGCTGTGCTCTCTGGGCTCA-3’ and 5’-GTTACAAGAGGCACAAGGAGTGG-3’; for Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), 5’-CAGCCTTCACTACCCTCTTG-3’ and 5’-ACGCCATCACTATCTTCCAG-3’. PCR was carried out under the following conditions: 1 cycle of denaturing at 94°C (30 sec), 21 (Gapdh) or 29 (Sox1, Sox2, Sox3, Mitf and Tyrosinase) cycles of denaturing at 94°C (30 sec), annealing at 58°C (Gapdh), 60°C (Mitf and Tyrosinase), or 65°C (Sox1, Sox2 and Sox3) (for 45 sec), and elongation at 72°C (for 2 min). The amplified DNA fragments were separated on a 1% agarose gel containing ethidium bromide, and visualized and image captured with Foto/Analyst Investigator System (Fotodyne, WI).

Expression vectors

Full coding region of Sox1, Sox2, Sox3 and Sox9 were inserted into the dicistronic pCXIZ vector, which allows simultaneous expression of a test gene and the reporter LacZ gene (Mikawa, 1995; Das et al., 2000; Ishii et al., 2004; Ishii and Mikawa, 2005). The Sox1 fragment was excised from pCMV/SV2-cSox1 (a gift from Dr Kondoh, Osaka University) with HindIII and XhoI. The Sox3 fragment was excised from pBluescript SK-cSox3 (Uwanogho et al., 1995) with RsrII and DraI. These fragments were blunt-end ligated into the SmaI site of pBluescript KS+, excised with EcoRV and XbaI. RT-PCR was used to amplify Sox2 and Sox9 genes from E3 whole chick embryo cDNA. The following primers were used: for Sox2, 5’-AAAGATATCGGCTTGGGACTTCGCCGCCGC-3’ and 5’-GCTCTAGAGTCTTACATATGTGATAGAGG-3’; for Sox9, 5’-CTCGATATCTAACCCTTCCCCGCCCCTCAG-3’ and 5’-CGCTCTAGATTAAGGCCGGGTGAGCTGCGTG-3’. The amplified cDNAs were ligated into pCRIITOPO vector (Invitrogen, CA), sequenced and excised with EcoRV and XbaI. All the above fragments were inserted into SmaI-XbaI sites of pCXIZ. The vector used for constitutive expression of FGF4 and a repressor form of SoxB1 proteins have been published in Mima et al (1995a, 1995b) and Bylund et al. (2003), respectively.

In ovo electroporation

In ovo electroporation was carried out according to Ishii and Mikawa (2005). Briefly, after windowing the shell, 100–200 µl of ink (Black India; Rotring, Germany; diluted 1:40 in phosphate buffered saline, PBS) was injected beneath the embryo to render the embryo visible. A gold-plated wire electrode (2 mm long), which acts as anode, was placed lateral to the stage 10–11 right eye. A sharpened tungsten needle was used as a cathode, which was inserted into the lumen of the right OV. After DNA solution (2 µg/µl, 50 nl) was injected into the OV, electric pulses (5V, 50-millisecond duration, 150-millisecond interval) were applied, using a pulse generator ECM 830 (BTX, San Diego, CA). Eggs were sealed by Parafilm and reincubated. The embryos were fixed and stained by X-gal staining, in situ hybridization or immunohistochemistry.

In vitro electroporation of the RPE

The RPE at embryonic day (E) 6 were isolated together with the underlying mesodermal tissue and electroporated as described (Fukuda et al., 2000) with some modifications. A vessel made of 1% agarose gel was placed between two electrodes (Genepaddles, Harvard Apparatus, MA, 7mm distance), which were placed on a 10 mm plastic dish filled with PBS. RPE fragments were mixed with 200 ng/µl DNA solution in the gel vessel and electroporated (60V, 50 msec, 5 pulses, 150 msec interval), using ECM 830 . After electroporation, the tissues were embedded in collagen gel and cultured in Medium 199 containing 10% fetal bovine serum.

Immunostaining

Embryos were fixed in 4% paraformaldehyde in PBS, embedded in OCT compound (Sakura Finetek, CA) and sectioned in a cryostat at 12–14µm. The sections were rehydrated in PBS, permeablized in 0.2% Triton X-100 in PBS, blocked in 1% bovine serum albumin in PBS (blocking solution) and incubated with primary antibodies diluted by blocking solution. The following antibodies were used: anti-Sox2 (1:1000, rabbit polyclonal, Millipore, MA), monoclonal anti-β-galactosidase (1:500, GAL-13, Sigma, MO), rabbit polyclonal anti-β-galactosidase (1:800, 5 Prime-3 Prime, CO), anti-N-cadherin (1:1000, GC-4, Sigma, MO), anti-class-III-β-tubulin (1:500, TUJ1, Covance, CA), anti-Neurofilament (1:500; 3A10, concentrate, Developmental Studies Hybridoma Bank, IA), anti-HuC/D (1:500, 16A11, Invitrogen, CA), anti-Isl1 (1:200; 39.4D5, concentrate, Developmental Studies Hybridoma Bank, IA), anti-Visinin (1:800; 7G4, concentrate, Developmental Studies Hybridoma Bank, IA), anti-Laminin (1:200, #L9393, Sigma, MO) and HNK-1 (1:100, CD57, Becton Dickinson, CA). After overnight incubation at 4°C, the sections were washed with PBS. Primary antibodies were detected by Alexa 488- or 594-conjugated secondary antibodies (Invitrogen, CA) as described previously (Ishii et al., 2007). For detection of F-actin, Texas Red-X phalloidin (Invitrogen, CA) was added at 1:80 to the secondary antibody solution. For BrdU labeling, 30µg of Bromodeoxyuridine (BrdU) was applied on chick embryos in ovo. After 30 minutes of incubation, the embryos were fixed in 4% paraformaldehyde and frozen sectioned. The sections were treated with DNase (20 Kunits/ml; Sigma, MO), doubleimmunostained with anti-BrdU (1:100, Becton Dickinson, CA) and anti-Sox2 (1:1000; Millipore, MA), and photographed under a Zeiss 510 Meta Confocal Microscope. TUNEL staining was performed using the In Situ Cell Death Detection Kit, TMR red (Roche, IN) as per the manufacturer's instructions. All sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI).

Results

Expression patterns of subgroup B1 Sox genes in the eye primordium

To address roles of the SoxB1 gene family, we examined their expression patterns during early retinal development of avian embryos. In situ hybridization analysis of all known SoxB1 family members, Sox1, Sox2 and Sox3, revealed both member-specific and shared features in their expression patterns. At stage 10, Sox1 transcripts were detectable only in the ventral region of the forebrain (Fig. 1A), and Sox2 and Sox3 transcripts were detectable more broadly in the forebrain, including the OV (Fig. 1H,O). At stage 13, shortly after the optic stalk is constricted, all three members showed a graded expression pattern, with a strong staining in the brain and optic stalk that declines gradually toward the distal part of the OV (Fig. 1B,I,P). As the bilaminar optic cup began to form, strong signal for Sox2 remained detectable in the presumptive NR (Fig. 1J–N) whereas signals for Sox1 and Sox3 declined both in the NR and RPE (Fig. 1C,Q). Expression of Sox1 and Sox3 became detectable again in the optic cup by stage 19, but the signals were restricted to the presumptive NR (Fig. 1D–G,R–U). Importantly, none of three SoxB1 genes were detectable in the presumptive RPE after the onset of the optic cup invagination (Fig. 1C–G, J–N,Q,S–U).

Fig. 1
SoxB1 downregulation in the presumptive RPE in developing chick eye

Consistent with the in situ hybridization data, our RT-PCR analysis (Fig. 1V) detected expression of all three SoxB1 family members in the OV prior to overt RPE domain specification (stage 10), judged by the absence of detectable expression of two RPE markers, Mitf and Tyrosinase. Once the bilaminar optic cup formed, both Mift and Tyrosinase were detectable in the presumptive RPE at stages 19–20 and became even stronger by stages 24–25. Coinciding with RPE marker gene expression, expression of Sox1, Sox2 and Sox3 dramatically declined in the RPE, while their expression remained detectable in the presumptive NR through stages 19–25. These data clearly demonstrate that all three members of the SoxB1 gene family are expressed in the OV and in the NR of the optic cup, but are downregulated in the RPE.

SoxB1 expression inhibits RPE morphogenesis and differentiation

Mature RPE is a melanin-containing simple cuboidal epithelium. To address roles of downregulation of SoxB1 in RPE morphogenesis and cytodifferentiation, vectors for constitutive expression of SoxB1 gene family members were introduced into stage 10–11 OV by in ovo electroporation (Fig. 2A,B). Our di-cistronic expression vectors co-express a Sox protein and a reporter β-galactosidase (β-gal) thus allowing detection of transfected cells by X-gal staining (Fig. 2C–H), or immunostaining for β-gal (Fig. 2I,K) or Sox2 (Fig. 2I,N).

Fig. 2
Maintained expression of SoxB1 inhibits RPE morphogenesis and cytodifferentiation

Sections of X-gal stained optic cups at stage 19–20 (40 hours after electroporation) revealed that a control vector, which encodes only β-gal, causes no obvious morphological abnormality (sample number n=26; Fig. 2C,D). In contrast, a Sox1/LacZ vector dramatically increased the thickness of the presumptive RPE layer (n=11; Fig. 2E,F). A Sox2/LacZ vector (n=15; Fig. 2G–O) as well as a Sox3/LacZ vector (n=25; not shown) caused the same thickening phenotype. Co-expression of SoxB1 protein and reporter β-gal in transfected cells was confirmed by double immunostaining (Fig. 2I). Endogenous Sox2 protein was detected in nuclei of NR cells but not in β-gal-negative RPE cells, consistent with the above in situ hybridization data (Fig. 1K–N). Transfected RPE cells expressing β-gal contained Sox2-positive nuclei and exhibited a thickened morphology.

Detailed inspection of cellular morphology showed that the majority of SoxB1-transfected RPE cells were elongated across the presumptive RPE layer (Fig. 2J). Clusters of these cells were more constricted at the apical side (Fig. 2J,K), often exhibiting a local invagination (Fig. 2K). Consistent with this morphogenetic event, much denser accumulation of actin was evident at the apical side of transfected cells compared to the basal side (Fig. 2L,M). In addition, nuclei of transfected cells were distributed at different apicobasal levels in the thickened epithelial layer (Fig. 2L), in sharp contrast with nuclei in neighboring untransfected regions that were arranged in a single row at the middle of cuboidal or low columnar cells (Fig. 2M). Thus, maintained expression of SoxB1 inhibits formation of a simple cuboidal epithelium. No increase in cell proliferation (n=8, see Fig. S1A–C in supplementary material) or altered apoptosis (n=4; see Fig. S1D,E in supplementary material) was detectable in thickened SoxB1-transfected cells.

We next tested whether SoxB1 expression affects RPE cytodifferentiation by probing for pigmentation 72 hours after electroporation (stage 21–22). RPE cells electroporated with a β-gal-only vector accumulated melanin pigments (Fig. 2N; n=5) whereas the cells transfected with Sox1 (n=4; Fig. 2O), Sox2 (n= 5; Fig. 2P) or Sox3 (n=6; Fig. 2Q) vectors did not. Thus, maintained SoxB1 expression inhibits both RPE cell morphogenesis and cytodifferentiation in a cell autonomous fashion.

Maintained SoxB1 expression induces ectopic neural marker expression

Previous studies have shown that misexpression of Sox genes in the ectoderm promotes development of the lens (Kamachi et al., 2001), neural crest (Cheung et al., 2003, 2005) and neuroectoderm (Dee et al., 2008; Kishi et al., 2000; Mizuseki et al., 1998). We tested whether maintained expression of SoxB1 changes the fate of the presumptive RPE into any of these tissue types.

First, the co-presence of Sox2 and Pax6 has been shown to promote lens development in surface ectoderm (Kamachi et al., 2001). Since Pax6 is expressed in the optic vesicle (Fuhrmann et al., 2000; Kamachi et al., 1998; Vogel-Höpker et al., 2000), it was plausible that ectopic expression of SoxB1 might lead to ectopic lens development in the PRE. However, no [partial differential]-crystallin signal was detected in Sox2-transfected RPE cells, despite that an intense signal was evident in the authentic lens tissue (n=3; see Fig. S2A,B in supplementary material).

Second, overexpression of Sox9, a GroupE Sox gene can trigger ectopic neural crest-like development in the ventral spinal cord (Cheung et al., 2003, 2005). Sox2-transfected optic cup, however, showed neither cell delamination nor ectopic immunoreactivity to a neural crest marker HNK-1 (n=3; see Fig. S2C–F in supplementary material). On the other hand, Sox9 misexpression caused disruption of the basal lamina and cell delamination (n=8; see Fig. S2G–L in supplementary material). Most delaminating β-gal-positive cells were immunoreactive to HNK-1.

Third, SoxB1 members play a role in early neural fate specification in both Xenopus and zebrafish (Dee et al., 2008; Kishi et al., 2000; Mizuseki et al., 1998). Furthermore, multiple Sox-site-dependent neural enhancers of the N-cadherin (N-cad) gene have been identified (Matsumata et al., 2005). We tested whether maintained SoxB1 expression converts the identity of RPE cells to the neural fate, using a neural marker N-cad, general neuronal differentiation markers, class III β-tubulin (TuJ1; Lee et al., 1990), neurofilament (3A10; Serafini et al., 1996) and HuC/D (Marusich et al., 1994; Hyer et al., 1998), and markers for specific retinal cells, Islet-1 (Isl1, ganglion and amacrine cells, Vogel-Höpker et al., 2000) and visinin (photoreceptor cells, Bruhn and Cepko, 1996; Yamagata et al., 1990). In untransfected eyes, strong N-cad staining was detected in the central NR, lens and brain, but not in the RPE (Fig. 3A–C). In contrast, Sox2-transfected RPE cells exhibited intense apical staining for N-cad (n=12; Fig. 3D–F). Furthermore, transfected regions contained TuJ1 (n=8; Fig. 3G,H)-, 3A10 (n=4; Fig. 3I,J)-, HuC/D (n=11; Fig. 3K,L)-, Isl1 (n=6; Fig. 3M,N)- and visinin (n=6; Fig. 3O,P)-positive cells. Many of these cells were found on the basal side of the epithelium, consistent with their neuronal characteristics. Similar results were obtained with Sox1 (n=5; Fig. 3Q,R) and Sox3 (n=5; Fig. 3S,T) vectors, but not with a control LacZ vector (n=4; data not shown).

Fig. 3
Maintained expression of SoxB1 confers neural characteristics

In chick embryos, the presumptive RPE retains the potential to differentiate into the NR until E4.5 (Guillemot and Cepko, 1992; Park and Hollenberg, 1989; Pittack et al., 1991). We sought whether SoxB1 activity is capable of inducing neural characteristics in a pigmented RPE, older than E4.5. In vitro electroporation was used to achieve efficient gene transfer into these tissues, which is difficult in ovo due in part to a narrowed optic cup lumen where injected DNA can be held. RPEs were isolated from E6 embryos together with associated mesenchymal tissues. They were then electroporated and explant cultured for 2 days in vitro. Control RPEs electroporated with a LacZ-only plasmid maintained melanin pigments and thin cuboidal epithelial morphology (n=4; Fig. 4A,B). On the other hand, RPEs electroporated with a Sox2/LacZ vector exhibited an overall increase in epithelial thickness and a reduced pigmentation (n=4; Fig. 4C,D). Signals for HuC/D (Fig. 4E,F), Isl1 (Fig. 4G,H) and Visinin (Fig. 4I,J) were detected in subpopulations of transfected RPE cells (n=3). Although some of electroporated explants contained dying cells, most of these cells were located in the mesenchymal layer and were seen both in control and Sox-transfected explants (Fig. 4A,C). Thus, exogenous SoxB1 expression is capable of suppressing RPE characteristics and activating the neurogenic program even in the pigmented RPE.

Fig. 4
SoxB1 misexpression converts pigmented RPE into a neural-like tissue

SoxB1 mediates FGF-dependent inhibition of RPE development

At early stages of eye development, the optic vesicle is bipotential, able to differentiate into both NR and RPE (Lopashov, 1963). We and others have previously shown that activation of FGF signaling facilitates NR development in retinal cells (Guillemot and Cepko, 1992; Hyer et al., 1998; Nguyen and Arnheiter, 2000; Opas and Dziak, 1994; Park and Hollenberg, 1991; Pittack et al., 1991, 1997; Vogel-Höpker et al., 2000, Zhao et al., 2001). Since the above results demonstrate that SoxB1 regulates RPE development negatively and neural development positively, we asked whether SoxB1 mediates the fate conversion effects of FGF.

We first tested whether SoxB1 genes lie downstream of FGF signal by electroporating OV with a FGF4/LacZ expression vector. For easy comparison between transfected and untransfected areas, electrodes were oriented to electroporate the posterior region of the vesicles (Fig. 5A). After a 48-hour incubation, only the posterior region of the optic cup contained transfected cells, as demonstrated by immunostaining for β-gal (Fig. 5B–D), and showed an ectopic increase in epithelial thickness in the presumptive RPE (i-nr in Fig. 5D). After a 72-hour incubation (stage 21–22), β-gal was no longer detectable (data not shown), but posterior region of the presumptive RPE remained thick and was devoid of pigments, as the result of the FGF-induced RPE-to-NR conversion as reported previously (Guillemot and Cepko, 1992; Hyer et al., 1998; Park and Hollenberg, 1991). Importantly, ectopic expression of all Sox1, Sox2 and Sox3 was detected in this thickened epithelial area (n=4; Fig. 5G,H,L,M,Q,R). The expression of SoxB1 members was not detectable in untransfected region where the normal pigmentation took place (Fig. 5I,N,S). Thus, all three SoxB1 genes are downstream of FGF-activated signaling.

Fig. 5
FGF induces SoxB1 expression ectopically in the presumptive RPE

To determine the extent to which SoxB1 activity elicits the effect of FGF, we compared downstream genes of Sox2 and FGF4. Mitf and Otx2 encode transcription factors critical for RPE fate specification (Bumsted and Barnstable, 2000; Martinez-Morales et al., 2001; Martinez-Morales et al., 2003; Mochii et al., 1998a; Mochii et al., 1998b; Nakayama et al., 1998; Nguyen and Arnheiter, 2000). Other regulatory genes we examined, Six3 (Bovolenta et al., 1998; Oliver et al., 1995), Rx1 (Furukawa et al., 1997; Mathers et al., 1997; Ohuchi et al., 1999), Chx10 (Chen and Cepko, 2000; Furhmann et al., 2000; Liu et al., 1994) and Optx2 (López-Ríos et al., 1999; Jean et al., 1999; Toy et al., 1999), are expressed predominantly in the presumptive NR. Consistent with previous studies, our in situ hybridization analysis show that, in normal development, their strong signals become restricted to either RPE or NR domain as a bilayered optic cup form (see Fig. S3 in supplementary material). Sox2, when misexpressed in the presumptive RPE, downregulated Mitf and Otx2 (Fig. 6G,H,M,N), and upregulated Six3 (Fig. 6I,O). No upregulation of Rx1, Chx10 and Optx2 was detected in the transfected cells (Fig. 6J–L, P–R). FGF4 similarly downregulated Mitf and Otx2 in transfected presumptive RPE (Fig. 6S,T; i-nr), but upregulated all NR marker genes (i.e., Six3, Rx1, Chx10 and Optx2; Fig. 6U–X). Thus, while both Sox2 and FGF4 suppress critical regulators of the RPE fate, Sox2 can regulate a part but not all molecular markers associated with early NR identity.

Fig. 6
SoxB1 mediates part of the FGF effect

Sox activity in the presumptive NR is necessary to repress RPE identity

As SoxB1 expression is maintained in the NR domain, we reasoned that endogenous Sox activity might have a role in suppressing RPE identity. To address this possibility, we misexpressed a repressor form of a SoxB1 protein, where the Sox3 HMG domain is fused to the repressor domain of the D. melanogaster Engrailed protein (HMG-EnR) (Bylund et al., 2000). In these eyes ectopic expression of Mitf and Otx2 was detected in the presumptive NR (n=4; Fig. 7), implying that SoxB1 plays a role in suppressing RPE identity possively through activating transcription of downstream target genes.

Fig. 7
SoxB1-dependent transactivation plays a role in suppressing RPE marker genes

Discussion

Retinal patterning begins with partitioning the bipotential retinal primordium of the OV, into the RPE and NR. While several paracrine signals implicated in specifying RPE and NR fates in the OV have been studied extensively, little is known about underlying transcriptional regulation in this developmental process. The present study provides the first experimental evidence that developmentally regulated downregulation of SoxB1 genes is critical for normal OV patterning.

The purpose of this study was to investigate the role of SoxB1 downregulation in RPE development. Downregulation of SoxB1 in the presumptive RPE begins at a specific time window prior to the initiation of optic cup morphogenesis. Since no ideal cis-element specific for this developmental window and region is currently available, it is difficult, if not impossible, to use a whole-animal/germline transgenic approach. Electroporation, used in the present study, allows efficient introduction of exogenous genes in defined regions of the retinal primordium at this specific developmental window. Since expression of the transgene declines gradually due to the degradation and dilution of introduced DNA, our analysis was limited to early effects that became evident within 72 hours post-electroporation. Abnormalities in cell morphogenesis and molecular markers observed in this short incubation period suggest that SoxB1 downregulation play a role in early molecular events involved in RPE cell fate specification although the long-term effect of maintained SoxB1 expression on retinal patterning remains to be explored.

The SoxB1 gene family consists of three members, Sox1, Sox2 and Sox3, in multiple vertebrate species, including human, mouse, chick and Xenopus (Bowles et al., 2000; Nitta et al., 2006). Member-specific expression patterns in the eye and other organs (Collignon et al., 1996; Kamachi et al., 1998; Uchikawa et al., 1999; Wood and Episkopou, 1999; Fig. 1), as well as differential effects of SoxB1 members on cultured neural progenitor cells (Kan et al., 2004), imply that each family member may play a different role in developing eye. Our data show that, however, all the SoxB1 genes are dramatically downregulated prior to the onset of RPE differentiation and that misexpression of any single SoxB1 family member in the presumptive RPE suppressed RPE identity and promoted neural development. It is therefore likely that the absence of expression of all three SoxB1 genes is necessary for normal RPE development.

The precise mechanism underlying SoxB1 downregulation is currently unclear. Importantly, however, the present study suggests that the level of FGF signal play a critical role in this downregulation. We and others have shown previously that FGFs can act as negative regulator of RPE development in vivo, overriding BMP/activin signaling that promotes RPE development (Furhmann et al., 2000; Hyer et al., 2003; Müller et al., 2007). Ectopic expression of all SoxB1 genes in FGF4-stimulated presumptive RPE suggests that the absence of exposure to FGF-like signals is crucial for SoxB1 downregulation. A regulatory element of the Sox2 gene requires Sox2-binding site for its transactivation in the OV (Inoue et al., 2007). Given that SoxB1 members appear to share similar transactivation properties (Kamachi et al., 1999; Okuda et al., 2006), downregulation of Sox2 might depend not only on reduced activity of Sox2 itself but also on reduced activities of Sox1 and Sox3. Determining hierarchical relationship between SoxB1 members would provide a clue to resolving mechanisms of their coordinated downregulation in the presumptive RPE.

The OV cells can differentiate into either pigmented or neural cells even in the absence of the surface ectoderm (Hyer et al., 1998). Surface ectoderm organizes the placement of these cell types by instructing the fate of uncommitted bipotential OV cells, presumably through FGF or FGF-related factors (Hyer et al., 1998). Our data show that exogenous FGF4 maintains SoxB1 expression in the presumptive RPE and that maintained expression SoxB1 converts presumptive RPE into a neural-like tissue. These data are consistent with SoxB1 being downstream of FGF in a pathway that regulates this binary cell fate decision. Clear understanding of molecular mechanisms underlying this fate decision awaits further studies. Mutations in Mitf and Otx2 lead to ectopic NR-like development in the presumptive RPE (Bumsted and Barnstable, 2000; Martinez-Morales et al., 2001; Mochii et al., 1998b; Nakayama et al., 1998; Nguyen and Arnheiter, 2000). Absence of detectable expression of Mitf and Otx2 in SoxB1-transfected cells suggests that SoxB1-dependent fate conversion is mediated at least in part by suppression of these genes.

SoxB1 proteins function in both activation and repression of gene expression (Ambrosetti et al., 2000; Cavallaro et al., 2008; Kamachi et al., 2000; Kondoh et al., 2004; Mansukhani et al., 2005). Different effects of SoxB1 on RPE and NR marker genes might depend on co-DNA binding partner proteins (Kamachi et al., 1999; Kamachi et al., 2000; Kondoh et al., 2004). Distinct phenotypes caused by SoxB1 and Sox9 misexpression, demonstrated in the present study, are consistent with involvement of such protein interactions, which are likely mediated by sequences outside of the conserved HMG DNA binding domain (Kamachi et al., 1999). However, the pairing with one partner protein does not appear to fully explain how SoxB1 affects the fate of retinal cells. For example, despite the co-presence of Sox2 and Pax6 in normal and SoxB1-transfected eyes, [partial differential]-crystallin is expressed only in the lens but not in the optic cup, even though Sox2 and Pax6 directly regulate the [partial differential]-crystallin promoter (Kamachi et al., 2001). Synergistic action of Sox2 and Brn2 transactivates the Nestin neural enhancer (Tanaka et al., 2004). More recently, Otx2 has been identified as a partner factor that can interact with Sox2 to transactivate an enhancer of the Rx gene (Danno et al., 2008). Although in vivo roles of these protein interactions in retinal development are currently unknown, interactions with multiple partner proteins may underlie transcriptional activities of SoxB1 proteins on different downstream genes.

Mitf and Otx2 activities suppress NR identity and promote pigmentation both in vivo and in vitro (Tsukiji et al., 2009; Martinez-Morales et al., 2003; Mochii et al., 1998). Our data suggest that repression of SoxB1-dependent transactivation causes ectopic expression of these RPE-inducing factors, suggesting that SoxB1 expression plays a role in suppressing RPE identity. Taranova et al. (2006) have demonstrated that Sox2 dosage is critical for temporal and spatial regulation of retinal progenitor cell differentiation, using null, hypomorphic and heterozygous mutant mice. While Sox2 null mice have smaller retina lacking Notch1 expression, they still appear to retain distinct NR and RPE domains. This might be due to functional redundancy of SoxB1 members and/or to the stage when CRE-recombinase is activated. It would be interesting to test whether presumptive NR is converted into RPE in mice lacking multiple SoxB1 member genes.

Retinal regeneration occurs in variety of amphibians, some fish and embryonic chick (reviewed in Reh and Pittack, 1995). This remarkable phenotypic change involves a fate conversion of RPE cells and is promoted by FGFs (Coulombre and Coulombre, 1965; Park and Hollenberg, 1989, 1991; Yoshii et al., 2007). Our data suggest that SoxB1 genes lie downstream of FGF-activated signaling and that their activity bypasses requirement of paracrine factors and stage-dependent competence to respond to these factors. However, it is unlikely that SoxB1 mediates all effects of exogenous FGF since Sox2 activity did not induce detectable expression of Rx1, Chx10 and Optx2. It is possible that FGF-induced retinal fate conversion involves multiple branches of gene regulatory pathway and that SoxB1 mediate some, but not all, of these branches. Consistent with this view, misexpression of activated MEK-1, which activates only part of FGF signaling, inhibits RPE pigmentation but does not induce laminated NR (Galy et al., 2002). Further dissection of transcriptional regulatory pathways in the developing eye would provide insights into mechanisms whereby multiple transcriptional pathways cooperate to generate this highly organized sense organ.

Supplementary Material

fig s1

fig s2

fig s3

Acknowledgment

We would like to thank Jeannette Hyer for her critical reading of the manuscript. We also thank Hisato Kondoh for Sox1 plasmids; Paul Scotting for Sox2 and Sox3 plasmids; and Jonus Muhr for HMG-EnR expression construct. Monoclonal antibodies against Neurofilament (3A10), Isl1 (39.D5) and Visinin (7G4) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences. This work was supported in part by NIH. Y.I. was supported by fellowships from Revson Foundation and CVRI at UCSF.

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