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Research Report

Regulation of photoreceptor gene expression byCrx-associated transcription factor network☆

Anne K. Henniga,1, Guang-Hua Penga,1, Shiming Chena,b,⁎aDepartment of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USAbDepartment of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110, USA

A R T I C L E I N F O

☆ Per guidelines of the Human Gene Nomenletters, while only the first letter is capitalizedreferences to protein products are not italiciz⁎ Corresponding author. Department of Ophth

Avenue, Campus Box 8096, St. Louis, MO 631E-mail address: chen@vision.wustl.edu (S

1 These two authors contributed equally to

0006-8993/$ – see front matter © 2007 Elsevidoi:10.1016/j.brainres.2007.06.036

A B S T R A C T

Article history:Accepted 20 June 2007Available online 30 June 2007

Rod and cone photoreceptors in themammalian retina are special types of neurons that areresponsible for phototransduction, the first step of vision. Development andmaintenance ofphotoreceptors require precisely regulated gene expression. This regulation ismediated by anetwork of photoreceptor transcription factors centered on Crx, an Otx-like homeodomaintranscription factor. The cell type (subtype) specificity of this network is governed by factorsthat are preferentially expressed by rods or cones or both, including the rod-determiningfactors neural retina leucine zipper protein (Nrl) and the orphan nuclear receptor Nr2e3; andcone-determining factors, mostly nuclear receptor family members. The best-documentedof these include thyroid hormone receptor β2 (Trβ2), retinoid related orphan receptor Rorβ,and retinoid X receptor Rxrγ. The appropriate function of this network also depends ongeneral transcription factors and cofactors that are ubiquitously expressed, such as the Spzinc finger transcription factors and STAGA co-activator complexes. These cell type-specificand general transcription regulators form complex interactomes; mutations that interferewith any of the interactions can cause photoreceptor development defects or degeneration.In this manuscript, we review recent progress on the roles of various photoreceptortranscription factors and interactions in photoreceptor subtype development. We alsoprovide evidence of auto-, para-, and feedback regulation among these factors at thetranscriptional level. These protein–protein and protein–promoter interactions provideprecision and specificity in controlling photoreceptor subtype-specific gene expression,development, and survival. Understanding these interactions may provide insights to moreeffective therapeutic interventions for photoreceptor diseases.

© 2007 Elsevier B.V. All rights reserved.

Keywords:CrxRetina developmentCone and rod photoreceptorTranscription factor networkNuclear receptorHomeodomain

clature Committee, the names of the human genes and proteins are represented by capitalfor those from other species. Locus, gene, and nucleotide sequence names are in italics anded.almology and Visual Sciences, Washington University School of Medicine, 660 South Euclid10, USA. Fax: +1 314 747 4211.. Chen).this work.

er B.V. All rights reserved.

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1. Introduction

Photoreceptors in the vertebrate retina carry out phototrans-duction, the conversion of light into a neuronal signal thatinitiates the visual process. In rodents, about 73% of retinalneurons are photoreceptor cells (Young, 1985). Rods andcones, the two types of photoreceptors in the retina, show aspecies-specific ratio and spatial distribution. Rods areresponsible for vision in dim light, while cones are responsiblefor color vision in bright light. Both rods and cones haveunique cellular structures called outer segments, containinghighly compact membrane discs where the visual pigmentopsins and other machinery involved in phototransductionare densely packed. At themolecular level, photoreceptor cellspreferentially express a set of genes that are essential for theirfunction, so called photoreceptor-specific genes. Mutations inmany of the photoreceptor specific genes are known to causeretinal degeneration diseases in humans. [For reviews, see(Hartong et al., 2006); and Retnet: http://www.sph.uth.tmc.edu/Retnet/]. Furthermore, the expression levels of thesephotoreceptor genes need to be precisely regulated. Increasedor decreased expression levels of a wild-type photoreceptorgene can also lead tophotoreceptor degeneration (Olsson et al.,1992; Humphries et al., 1997).

Precisely regulated photoreceptor gene expression is also adriving force for photoreceptor development/differentiation.Lineage tracing and birth dating experiments demonstratedthat all of the neuronal cell types in the retina are derived froma common multi-potent progenitor cell (Turner and Cepko,1987; Wetts and Fraser, 1988). For photoreceptor cells, conesare usually born (exit from themitotic cycle and commit to thephotoreceptor lineage) earlier than rods. In rodents, cones areborn on embryonic days E11.5–E18.5, while rods are born in alonger period from E12.5 to postnatal day 7 (P7) with a peak atP0 (Carter-Dawson and LaVail, 1979; Young, 1985). However,there is a significant delay, several days in rodents, for newlyborn photoreceptor precursors to begin expressing the specifictype of opsin and other genes that confer the maturephenotype (Watanabe and Raff, 1990; Cepko, 1996). Duringthis lag time, the photoreceptor precursors appear to be“plastic” and can be induced to differentiate into differentphotoreceptor subtypes, depending on intrinsic and extrinsicregulatory factors (Nishida et al., 2003; Cheng et al., 2006;MacLaren et al., 2006; Roberts et al., 2006). The intrinsic factorsmainly consist of transcription factors of homeodomain, bZIP,and nuclear receptor families. In this manuscript, we reviewthe recent progress in understanding these photoreceptortranscription factors, provide some evidence for the presenceof network interactions among the major players, and presenta model of how these interactions determine photoreceptorgene expression and development.

Photoreceptor transcription factors are the transcriptionregulators preferentially expressed by post-mitotic photore-ceptor precursors and/or mature photoreceptors. Table 1 liststhe major factors that are known to be important forphotoreceptor development and maintenance, mostly basedon in vivo loss-of-function studies. It is well established thatmembers of bHLH and homeodomain transcription factorfamilies play important roles in specifying various neuronal

cell types in the retina (for review, see Hatakeyama andKageyama, 2004; Yan et al., 2005), including photoreceptorprecursors. Recently, though, much progress has been madein elucidating the roles of members of the nuclear receptor,bZIP, and homeodomain families of transcription factors inspecifying rod and cone photoreceptor subtypes. Below wewill focus on these new findings and discuss some key factorsin detail.

1.1. Factors specifying the photoreceptor lineage—Otx2and Crx

The role of Otx homeodomain transcription factors in eyedevelopment originally came from studies of Drosophilaorthodenticle (Otd), a paired-type homeodomain proteinthat is required for the formation of anterior brain, eye andantenna in the fly (Finkelstein and Boncinelli, 1994). Sub-sequent studies showed that Otd plays an essential role inDrosophila photoreceptor development (Vandendries et al.,1996) by regulating the expression of opsin genes (Tahayato etal., 2003). Mammals have three Otd orthologs, Otx1, Otx2, andCrx, which is equivalent to Otx5 in fish, amphibians and chick(Plouhinec et al., 2003). The function of these Otd orthologshas diverged over time with Crx dedicated specifically to thedevelopment and maintenance of retinal photoreceptors andpinealocytes in the pineal gland involved in circadianregulation (see below). In terms of the protein sequence,the three mammalian Otd homologs share 87–88% homologyin the homeodomain near the N-terminus and high homol-ogy in several discrete regions in the C-terminal portion,including a glutamine-rich region and the Otx-tail (Chen etal., 1997; Furukawa et al., 1997; Fig. 1). Their homeodomainbelongs to the K50 (lysine at position 50) paired-like class,similar to that of Drosophila bicoid protein, which, based onstructure and functional studies, prefers to bind to DNAmotifs with TAATCC or TAAGCT sequences (Treisman et al.,1989; Furukawa et al., 1997; Baird-Titus et al., 2006). Thesesequence motifs are widely present in the promoter orenhancer regions of many photoreceptor genes, includingthe opsin genes (Chen and Zack, 1996; Furukawa et al., 1997;Yu et al., 2006).

1.1.1. Otx2 specifies photoreceptor lineage by regulating theexpression of Crx and other photoreceptor genesOtx2 is expressed in the forebrain and midbrain neuroepithe-lium during development, including the eye domain. Duringdevelopment and in adults, Otx2 is expressed in several eyetissues, including neural retina and retinal pigmented epithe-lium (RPE) (Bovolenta et al., 1997). Otx2 is known to be requiredfor the development andmaintenance of the RPE by regulatingthe expression of RPE-specific transcription factors and genes(Martinez-Morales et al., 2001, 2003). In the neural retina, Otx2expression is seen in post-mitotic neuroblast cells that havethe potential to develop into various cell types, includingganglion cells, bipolar cells, and photoreceptor cells (Bovo-lenta et al., 1997; Baas et al., 2000). Nishida et al. (2003) carriedout a parallel in situ hybridization analysis of Otx2 and CrxmRNA expression in developing mouse retina and showedthat Otx2 expression is initially seen at E11.5. Its expressionincreases at E12.5 together with an induction of Crx expression,

Table 1 – Transcription regulators for photoreceptor gene expression, development and/or maintenance

Factors Expression in photoreceptors Functiona/targetsb Key references

Subtype Periodc

Photoreceptor-enrichedHomeodomainOtx2 dev ph precursors E10.5-P6 act/Crx, Rbp3, etc. Nishida et al., 2003Crx dev/ad rods/cones E12.5-ad act/opsins, reg. factors Chen et al., 1997; Furukawa et al., 1997; 1999Rax dev/ad ph P10.5-ad act/Rho, Arr, Rbp3 Zhang et al., 2000; Kimura et al., 2000Qrx/RaxL dev/ad ph ad(Bo/Hu/Ch/Xe) act/Rho Chen and Cepko, 2002; Wang et al., 2004

bZIPNrl dev/ad rods E13.5-ad act/Rho, Nr2e3, Pde6b, etc. Liu et al., 1996; Mears et al., 2001

bHLHNeuroD1 dev rods E12.5-P3 ph survival/unclear Morrow et al., 1999; Pennesi et al., 2003Mash1 dev rods E13.5-P3 act/Rho Guillemot and Joyner, 1993; Ahmad, 1995Math5 dev precursors E11.5-birth rep/NeuroD1, Neurog2 Brown et al., 1998; Le et al., 2006Neurog2 dev precursors E10-birth act?/NeuroD1 Ma and Wang, 2006; Yan et al., 2005

Nuclear receptorsTrβ2 dev cones E16-ad dual/S- and M-opsin Yanagi et al., 2002; Roberts et al., 2006Rxrγ dev cones E14.5-ad rep/S-opsin Roberts et al., 2005Rorβ dev cones E12.5-ad act/S-opsin Chow et al., 1998; Srinivas et al., 2006Nr1d1(Rev-erb-α) dev ph P0-ad dual?/circadian genes Cheng et al., 2004Nr2e1(Tlx) dev cones Turned on E8 dual/S-opsin, Pax2, Rar Zhang et al., 2006Nr2e3 dev/ad rods E18-ad dual/all opsins Kobayashi et al., 1999; Peng et al., 2005

Ubiquitously expressedRb1 Rods P12-P21 act?/Nrl Zhang et al., 2004Sp1, Sp4 dev/ad ph E12.5-ad act/Pde6b, Rho Lerner et al., 2001ataxin-7 dev/ad ph E12.5-ad coact/opsins Palhan et al., 2005Gcn5 dev/ad ph E12.5-ad coact/opsins Palhan et al., 2005Cbp/p300 dev/ad ph E12.5-ad coact/opsins Yanagi et al., 2000

Abbreviations: act—activator; ad—adult; coact—co-activator; dev—developmental; dual—activator and repressor; ph—photoreceptors; reg.—regulatory; rep—repressor; Arr—rod arrestin; Neurog2—neurogenin 2.a Based on loss-of-function studies.b Direct targets if known (based on protein-DNA binding assays).c In mice or in species as noted: Bo—bovine, Hu—human, Ch—chicken, Xe—Xenopus.

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coinciding with early cone development. At E17.5, Otx2expression is highly intensified in the outer part of theneuroblastic layer, where a Crx expression zone is established.After birth, when Crx expression reaches a peak and rodmaturation begins around P5–6, Otx2 expression is down-regulated in the presumptive photoreceptor cell layer but up-regulated in the inner nuclear layer where bipolar and Mullerglia cells are developing. The Otx2 spatial and temporalexpression patterns suggest that Otx2 could play an essentialrole in photoreceptor development.

1.1.1.1. Human genetic studies. The human OTX2 genemaps to 14q21–q22, in an interval associated with micro-phthalmia and pituitary insufficiency. Using a candidate geneapproach, Ragge et al. (2005) identified eight heterozygousOTX2mutations from 333 patients with ocularmalformations.The ocular phenotypes of these patients vary from severebilateral anophthalmia to unilateral microphthalmia withLeber's congenital amaurosis (LCA). In vitro biochemicalanalysis (Ragge et al., 2005; Chatelain et al., 2006) suggeststhat these OTX2 mutations are likely to cause disease by aloss-of-function (haplo-insufficiency) mechanism, as many ofthe mutations reduce the ability of OTX2 to bind to DNA and/or activate the target gene promoter RBP3 in transfected HeLacells.

1.1.1.2. Animal studies. Homozygous Otx2 knockout in themouse is embryonic lethal due to defects in gastrulation andlack of rostral brain (Acampora et al., 1995; Matsuo et al., 1995;Ang et al., 1996). The heterozygous Otx2 knockout mouse(Otx2+/−) showed multiple ocular defects, including micro-phthalmia, hyperplastic retina and RPE, and lack of lens,cornea and iris (Matsuo et al., 1995). To understand the role ofOtx2 in photoreceptor development, Nishida et al. (2003)generated a conditional Otx2 knockout in developing photo-receptors using a Cre transgene under control of the mouseCrx promoter. This Otx2 deficiency converts developingphotoreceptors into amacrine-like cells in the retina, andcompletely blocks the formation of pinealocytes in the pinealgland. Thus, Otx2 is required for photoreceptor cell fatedetermination and pineal gland development. Otx2's role inphotoreceptor development is also demonstrated by Otx2over-expression studies (Nishida et al., 2003). Forced Otx2expression in P0 rat retina using a retroviral vector results in asignificant increase in the number of rod photoreceptors at theexpense of bipolar, amacrine, and Muller glia cells, suggestingthat Otx2 promotes photoreceptor cell fate (Nishida et al.,2003). Given the observation that Otx2 expression switches tobipolar cells after the peak of photoreceptor development,these results also suggest that Otx2may be involved in bipolardevelopment as well. Forced Otx2 expression in adult iris- and

Fig. 1 – Schematic diagram of photoreceptor-specific transcription factors. The domain structures of photoreceptor-specifictranscription factors discussed in this paper are presented in scale. Conserved domains for classifying families oftranscription factors are indicated in black, other regions of homology conserved among different familymembers are indicatedby stippling. Functional regions are indicated above the box representing the factor; sites of mutations discussed in the text areindicated by arrowheads below the box. N- and C-terminals are indicated, and the number below the C-terminal endindicates the number of amino acids in the human protein. HOMEO, homeodomain; b, basic domain; L Zipper, leucine zipperdomain; Zn F, zinc finger domain.

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ciliary-derived “stem” cells of rat origin is sufficient to inducethe differentiation of photoreceptor-like cells (Akagi et al.,2004), consistent with Otx2 having a key role in specifyingphotoreceptor cell fate.

1.1.1.3. Mechanisms of action. Otx2 target genes in thephotoreceptors are being studied by microarray analysis inDr. Furukawa's laboratory in Japan. Although the microarrayresults remain to been seen, two direct Otx2 target genes areknown. One is Rbp3 (Bobola et al., 1999; Fong and Fong, 1999), aCrx-independent gene (Furukawa et al., 1999) expressed byboth rods and cones, and the other is Crx. Crx expression isabolished in the Otx2 conditional knockout mouse retina(Nishida et al., 2003). Otx2 significantly enhances Crx promoteractivity in transient cotransfection assays. Chromatin immu-noprecipitation analysis showed that Otx2 binds to thepromoter region of Crx in vivo, further supporting the Crxgene as a direct target of Otx2 (see Results and Discussion andFig. 3). Otx2 has also been reported to bind to and auto-activateits own promoter (Martinez-Morales et al., 2003). Furthermore,we have shown that Otx2 also binds to the promoter/enhancerregion of several other known Crx targets, including rod andcone opsins, in the presence or absence of Crx. In transientlytransfected HEK293 cells, Otx2 is also able to activate rhodopsinand M-cone opsin promoter activity, although less potentlythan Crx (Peng and Chen, 2005). These results suggest thatOtx2 acts by directly regulating the expression of the keytranscription factor Crx and its target genes.

1.1.2. Crx directly regulates the expression of manyphotoreceptor genesCrx was identified by three laboratories independently. UsingRT-PCR with degenerate primers corresponding to the paired-like homeodomain, Furukawa et al. cloned a murine Crx gene,which shows a photoreceptor-specific expression pattern(Furukawa et al., 1997). Chen et al. reported cloning bovineCrx using a yeast one-hybrid assay with a rhodopsin promoterelement, Ret4, as bait (Chen et al., 1997) and demonstratedthat Crx can bind to three target sites in the rhodopsin promoteras well as targets in several other rod gene promoters.Furthermore, Crx acts as a transcription activator andsynergizes with the bZIP transcription factor Nrl in activatingrhodopsin-reporter gene expression, suggesting for the firsttime that a high level of rhodopsin expression requires thefunction of at least two photoreceptor transcription factors.Using in situ hybridization analysis, Crx expression was foundin both rods and cones, and in their precursors, starting atembryonic day 12.5 in mouse, coinciding with cone cell birth.Expression peaks at P5, correlating with the onset of rodphotoreceptor maturation when rod-specific gene expressionis turned up. BrdU incorporation assays confirmed that Crxexpressing cells are post-mitotic photoreceptor precursorsderived from those cells that have just exited the cell cycle andexpress Otx2 but not Pax6 (Garelli et al., 2006). Crx is theearliest expressed photoreceptormarker in the retina. It is alsoexpressed in pinealocytes in the pineal gland and regulatesphotoentrainment (Furukawa et al., 1999) and expression of

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genes involved in synthesizing the circadian hormone mela-tonin in mice (Li et al., 1998). Interestingly, using immunohis-tochemistry studies, we have also found that Crx is expressedin rod bipolar cells that co-stain with the bipolar cell markerPKCα in bothmouse and human retinas (Wang et al., 2002; anddata not shown), suggesting a possible role of Crx in bipolarcell function.

1.1.2.1. Human genetic studies. The first piece of evidencefor Crx's role in development and maintenance of photo-receptors came from genetic studies performed by Freund etal. (1997), who cloned the human CRX gene based on itshomology in the homeodomain to another retinal home-odomain protein, Chx10. The human CRX gene maps to19q13.3, within a cone-rod dystrophy (CORD2) locus. Subse-quent genetic screens not only identified CRX mutations inautosomal dominant cone-rod dystrophy (Freund et al., 1997),but also in autosomal dominant retinitis pigmentosa (adRP)(Sohocki et al., 1998) and Leber's congenital amaurosis (LCA)(Freund et al., 1998; Rivolta et al., 2001b). Most CRX mutationsare inherited in an autosomal dominant manner or occur denovo, particularly in LCA cases (Rivolta et al., 2001a). Manymutations are nucleotide insertions or deletions resulting information of a premature stop codon 3′ of the mutated sites,which produce C-terminal truncated forms of CRX. Others aremissense mutations, several of which are located in thehomeodomain (Rivolta et al., 2001a; see Fig. 1). In vitrofunctional analysis demonstrated that many of the disease-linked mutations altered the ability of CRX to bind to DNA(homeodomain mutations) and/or activate transcription ofthe rhodopsin gene (Chen et al., 2002). Thus, CRX mutationsmay cause disease by impairing CRX-mediated transcrip-tional regulation of photoreceptor genes. However, in vitrobiochemical studies have not found a clear correlationbetween disease severity and the degree of biochemicalabnormality, and it is not clear why CRX mutations causedominant disease.

1.1.2.2. Animal studies. The second piece of evidence forCrx function came from knockout mouse studies. Homozy-gous Crx knockout mice (Crx−/−) are blind at birth withoutdetectable photoreceptor function, resembling the phenotypeof LCA. Their photoreceptors never develop the outer seg-ments critical for phototransduction, and subsequentlydegenerate (Furukawa et al., 1999). Serial analysis of geneexpression (SAGE) performed on Crx−/− retinae before theonset of photoreceptor degeneration showed that 46% ofphotoreceptor-enriched genes are Crx-dependent (Blackshawet al., 2001), particularly the opsin genes, providing convincingevidence that altered photoreceptor gene expression is aprimary cause of the Crx deficient phenotype. HeterozygousCrx knockout (Crx+/−) mice, on the other hand, have normalphotoreceptor function at the ages of 3 months or older.However, a delay in development of photoreceptor functionwas detected by electroretinogram (ERG) measures, despitenormal appearance of the retina at 1 month of age (Furukawaet al., 1999). No photoreceptor degeneration was observed inCrx+/− mice, raising the possibility that human diseasesassociated with CRX mutations could involve a dominant-negative effect on the Crx regulatory pathway.

The third piece of evidence for Crx function came fromectopic expression studies. Forced expression of recombinantCrx in P0 rat retina using a retroviral vector (Furukawa et al.,1997) induces rod differentiation, although less potent thanforced Otx2 expression. As observed with Otx2, forced Crxexpression leads to a reduction in the number of amacrine andMuller glia cells. However, the number of bipolar cells isunchanged. These results suggest that, like Otx2, Crx is animportant factor for photoreceptor cell fate determination. Instem cell studies, forced Crx expression in adult rat iris- andciliary-derived cells is sufficient to induce the formation ofrhodopsin-expressing cells as potently as Otx2 (Haruta et al.,2001; Akagi et al., 2004). Similar experiments with primatestem cells, however, require both Crx and NeuroD1 to inducethe photoreceptor phenotype (Akagi et al., 2005). Thesefindings suggest that interaction with other photoreceptortranscription factors is important for Crx function.

1.1.2.3. Mechanisms of action. Crx is a trans-activator formany photoreceptor genes, based on gene expression profilestudies. Using chromatin immunoprecipitation assays, wehave shown that Crx activates transcription by directlybinding to the promoter and/or enhancer regions of the targetgenes in photoreceptor cells (Peng and Chen, 2005). However,in transient cell transfection assays with target promoter-luciferase reporters, Crx alone has only a moderate transacti-vating activity (two to fivefold enhancement), even with therhodopsin promoter, a well-known Crx target (Chen et al.,1997). Thus, one mechanism for Crx to activate transcriptionis to interact with other transcription regulators. Functionalinteractions with numerous other proteins have beenreported. These include photoreceptor-specific transcriptionfactors [Nrl and Nr2e3, discussed below; Qrx (Wang et al.,2004)] and general transcription factors [Sp family members(Lerner et al., 2001); and nuclear receptor Ror isoforms(Srinivas et al., 2006)]. Crx also interacts with chromatinremodeling factors [ataxin-7 (La Spada et al., 2001), HMG I/Y(Chau et al., 2000), Baf (Wang et al., 2002)], the transcriptionco-activators Cbp and p300 (Yanagi et al., 2000), and theSTAGA co-activator/chromatin remodeling complex (Palhanet al., 2005). STAGA is a highly conserved multi-proteincomplex present from yeast (SAGA) to man (Martinez et al.,2001). One key component of STAGA is the histone acetyl-transferase (HAT) Gcn5 that catalyzes acetylation of histones,a chromatin modification often associated with transcrip-tional activation (Martinez et al., 2001). Crx interacts withSTAGA via ataxin-7, a 110-kDa protein in the STAGA complex.Expansion of the poly-glutamine tract of ataxin-7 is asso-ciated with a dominant neurological disorder, spinocerebellarataxia type 7 (SCA7), which features cone-rod dystrophy-likeretinal degeneration similar to the pathology linked to CRXmutations. Animal model studies and in vitro functionalanalysis suggest that Crx is a STAGA-dependent transcriptionactivator (La Spada et al., 2001; Chen et al., 2004; Palhan et al.,2005). A polyglutamine-expanded ataxin-7 disrupts Gcn5 HATactivity, resulting in hypoacetylation of histones on Crx targetgenes and transcription impairment in a SCA7 transgenicmouse model. Thus, one possible mechanism for Crxtranscription activation is to promote chromatin remodelingby recruiting STAGA or other HAT-containing co-activators to

Table 2 – P14 retinalmRNA levels relative toWT (qRT-PCRanalysis)

Genes Mouse strains

Crx−/− Nrl−/− Nr2e3−/−

RegulatorsOtx2 2.50±0.12* 1.00±0.12 1.00±0.12Crx 0.05±0.06* 1.00±0.06 1.03±0.06Nrl 0.67±0.06* 0.06±0.06* 1.00±0.06Nr2e3 0.61±0.05* 0.04±0.15* 0.04±0.17*Trβ2 0.46±0.15* 1.02±0.15 1.02±0.17Rxrγ 4.32±0.10* 1.57±0.15* 1.32±0.10*Rorβ 2.22±0.15* 1.78±0.06* 1.28±0.10*NeuroD1 0.75±0.15* 1.01±0.15 1.00±0.15

TargetsSop 0.09±0.06* 2.14±0.10* 1.23±0.10*Mop 0.06±0.06* 1.17±0.06* 1.19±0.10*Rho 0.12±0.06* 0.07±0.06* 0.84±0.06*Rbp3 1.00±0.10 1.00±0.15 1.00±0.15

Results of quantitative RT-PCR are presented as the ratio of theknockout to wild-type expression level of each gene (see Experi-mental procedures for calculations). The numbers represent themean value±standard deviation from three repeats. *Pb0.05 basedon paired Student's t-test.

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its target genes. This possibility is further supported by thefindings that Crx also interacts with two other co-activatorswith intrinsic HAT activity, Cbp and p300 (Yanagi et al., 2000;and data not shown).

One question related to Crx function is why the Crxdeficient retina develops photoreceptor cells in the firstplace if Crx is critical for the expression ofmany photoreceptorgenes. A possible explanation is that another Otd/Otx familymember plays a redundant role with Crx in specifyingphotoreceptor cell fate, therefore partially compensating forCrx function in Crx−/− mice. Indeed, the closely related familymember Otx2 is expressed in developing photoreceptors in theretina and up-regulated in the Crx-/- mouse retina (Furukawaet al., 1999; Table 2). Apparently, Otx2 and Crx have redundantbut indispensable roles in photoreceptor development andmaintenance. These roles might be contributed by protein–protein or protein–promoter interactions between the twofactors.

1.2. Factors for rod development—Nrl and Nr2e3

1.2.1. Nrl specifies rod lineage in photoreceptor precursorsThe neural retina leucine zipper protein (Nrl) is a basic-leucinezipper (bZIP) transcription factor belonging to the Mafsubfamily (Swaroop et al., 1992). The Nrl cDNA was originallycloned from an adult human retina library by subtractivehybridization (Swaroop et al., 1992). The recombinant Nrlprotein was subsequently found to bind to and regulate therhodopsin promoter via NRE, an AP-1 like element located inthe proximal rhodopsin promoter region (Kumar et al., 1996;Rehemtulla et al., 1996). RT-PCR and in situ hybridizationanalysis demonstrated that Nrl is highly expressed in theretina, although expression was also detected in the develop-ing brain and lens (Liu et al., 1996). In the neural retina, native

Nrl transcripts were seen at E14.5 (Liu et al., 1996) and stay onthrough the developmental period and into adulthood. Line-age tracing using a GFP transgene driven by the Nrl promoterand BrdU pulse-chase experiments in mouse retina showedthat Nrl mRNA can be detected as early as E12.5 in those cellsjust completing terminal mitosis (Akimoto et al., 2006). TheseNrl+ cells subsequently develop into rod photoreceptors. Inaddition, Nrl is also highly expressed in the pineal gland of thebrain (Akimoto et al., 2006), suggesting a role in pineal glanddevelopment. At the protein level, Nrl in the nuclear fractionof human and mouse retinal extracts consists of multipledifferentially phosphorylated isoforms ranging from 29 to35 kDa on SDS-PAGE/Western blots (Swain et al., 2001; Kandaet al., 2007). The function of the phosphorylated Nrl isoformsremains to be determined, but they are more prominent at thepeak of rod differentiation and are altered by some humanNRL mutations (Kanda et al., 2007; see below). Immunostain-ing of Nrl in the human and mouse retina showed that Nrl islocalized in the nucleus of rods but not cones (Swain et al.,2001). These results suggest that Nrl plays a role in roddevelopment and maintenance.

1.2.1.1. Human genetic studies. The role of Nrl in rodfunction was first demonstrated by human genetic studies.The human NRL gene maps to chromosome 14q11.2 (Farjo etal., 1997). Subsequent mutation analysis of a large pedigreewith autosomal dominant retinitis pigmentosa identified aS50T missense mutation in NRL that cosegregates with thedisease (Bessant et al., 1999). AlthoughNRLmutations are rare,additional missense mutations linked to adRP have beenidentified, with hot spots at residues S50 and P51 (Martinez-Gimeno et al., 2001; DeAngelis et al., 2002; Nishiguchi et al.,2004). Some of these hot spot mutations result in mutantforms of NRL that demonstrate reduced phosphorylation buthyperactivity in activating the rhodopsin promoter with CRX invitro (Bessant et al., 1999; Nishiguchi et al., 2004; Kanda et al.,2007). This suggests that these are gain-of-function muta-tions. It is notable that the function of both rods and cones ismore severely affected in patients with heterozygous NRLmutations than in patients with the RHODOPSIN mutationP23H (DeAngelis et al., 2002), raising the possibility that NRLmutations could actively affect cone function in humans.RecessiveNRLmutations were also found in patients sufferingclumped pigmentary retinal degeneration (Nishiguchi et al.,2004) with loss of rod, but normal blue cone function. Thesemutations are likely to be loss-of-function mutations asshown by in vitro function studies (Nishiguchi et al., 2004).

1.2.1.2. Animal studies. The most direct evidence for Nrlfunction in rod fate determination comes from Nrl knockoutmouse studies (Mears et al., 2001). Knockout of Nrl results inloss of rod photoreceptor function based on ERG measures.However, the function of cones, especially S-cones, is sig-nificantly enhanced compared to that in wild-type mice,resembling the phenotype of enhanced S-cone syndrome(ESCS) in humans and rd7 in mice, both associated with Nr2e3mutations (see below). Cone function in Nrl knockout mice ispreserved as late as 31 weeks (Mears et al., 2001). Single-cellelectrophysiology showed responses driven by both S- and M-opsin in all cells tested fromNrl knockout mice (Nikonov et al.,

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2005), as seen for the wild-type cones that co-express bothopsins (Applebury et al., 2000; Nikonov et al., 2006; see Conesubtypes and gradients). Although the dorsal/ventral M/S-cone gradient is preserved in Nrl−/− mice, more S-opsinsensitivity is seen in Nrl−/− cones than in wild-type conesfrom comparable dorsal/ventral levels of the retina (Nikonovet al., 2006). Consistent with the above electrophysiologicalmeasures, the ultrastructural analysis showed that the Nrl−/−

retina has much shorter cone-like outer segments withdisruptedmorphology and cone-like nuclei with decondensedchromatin (Mears et al., 2001; Daniele et al., 2005). Whorls androsettes are seen at early ages and thinning of the outernuclear layer occurs later on (Mears et al., 2001), similar toretinal histopathology in rd7 mice. The inner neurons of therod pathway, specifically rod bipolar cells, horizontal cells,and amacrine cells, make connections with these “trans-differentiated” cones (Strettoi et al., 2004). At the molecularlevel, the Nrl−/− retina has no detectable expression of rod-specific genes, but a much higher level of S-opsin andmoderately increased M-opsin expression (Mears et al., 2001;see Results and Discussion and Table 2). Thus, knockout of Nrlessentially turns a rod dominant retina into an all-cone (orrodless) retina based on morphological, physiological, andmolecular criteria, suggesting that Nrl is required for rod cellfate determination.

To determine if Nrl is sufficient to induce the rodphenotype, Oh et al. (2007) studied transgenic mice thatexpress Nrl in all photoreceptor precursors under the controlof a Crx promoter. This ectopic Nrl expression converts theretina of either wild-type (WT) or Nrl−/− mice to an all-rodretina. The inner neurons that ordinarily contact cones, suchas ON cone bipolar and horizontal cells, make connectionswith rods in these mice. No cone-specific gene products weredetected by RT-PCR or immunohistochemistry (Oh et al., 2007).Thus, Nrl is sufficient to guide photoreceptor precursors to arod lineage. However, conditional expression of Nrl laterduring S-cone differentiation, using the S-cone promoter onthe Nrl−/− background, generated hybrid cells that co-expressed both rhodopsin and S-opsin. Although lineage-tracingexperiments showed that some of the S-cones might haveswitched to a rod lineage, no rod functionwas detected by ERGin these mice (Oh et al., 2007), suggesting that differentiatedphotoreceptors may have limited plasticity, but requireappropriate transcriptional regulation for maintenance.Another possible explanation, however, is that Nrl repressesexpression of S-cone genes, resulting in inactivation of cellsalready committed to the S-cone lineage.

1.2.1.3. Mechanisms of action. Gene expression profile stu-dies demonstrated that Nrl is required for the expression ofmany rod genes, including the photoreceptor nuclear receptorNr2e3 (Mears et al., 2001) that is linked to enhanced S-conesyndrome (ESCS) (see below). Thus, the phenotype of Nrl−/−

mice is in part contributed by the loss of Nr2e3 expression.This was further demonstrated by mouse studies showingthat ectopic expression of Nr2e3 in photoreceptor precursorsof Nrl−/− mice can transform developing cones to rod-likephotoreceptors (Cheng et al., 2006). Microarray analyses of thedeveloping and mature retina of wild-type and Nrl−/− micehave identified clusters of Nrl target genes (Yoshida et al.,

2004; Yu et al., 2004; Akimoto et al., 2006). These not onlyinclude genes coding for phototransduction and structural ormembrane associated proteins as expected, but also transcrip-tional regulators, intracellular transport proteins, and compo-nents of known signaling pathways. The Bmp/Smad pathwayis repressed in the Nrl−/− retina, as Bmp (Bmp2, 4 and Bmpr1a)and Smad (Smad 1, 5, and 4) genes are down-regulated (Yu etal., 2004). Chromatin immunoprecipitation assays demon-strated that Nrl binds to the promoter of Bmp2 and Bmp4,suggesting they are direct targets of Nrl. In contrast, manycomponents of the Wnt/Ca2+ signaling pathway showedaltered (either up- or down-regulated) expression in Nrl−/−

retina. These results suggest that Nrl is also important formaintaining rod function and homeostasis by integratingvarious signaling pathways.

In the Nrl−/− mouse, the expression of all rod-specificgenes is lost but cone genes are up-regulated, suggesting thatNrl represses cone genes either directly or indirectly. Chro-matin immunoprecipitation assays showed that Nrl candirectly bind to cone gene promoters, including S-opsin, M-opsin, cone arrestin (Arr3), and the cone transcription factorTrβ2 (Peng and Chen, 2005; Oh et al., 2007; Fig. 3), suggestingthat Nrl may directly regulate cone gene expression in rods.On the other hand, transient cotransfection assays withluciferase reporter constructs driven by either M-cone or S-cone opsin promoters showed that Nrl actually activates coneopsin promoters in vitro (Peng et al., 2005). It is known thatthe retinoid X receptor gamma (Rxrγ) that represses S-coneexpression (see below) is up-regulated in the Nrl−/− mouseretina. Furthermore, the expression of cone genes is up-regulated in rd7 mouse retina where Nr2e3 protein is missing,but Nrl is normally expressed (Peng et al., 2005). Thesefindings suggest that Nrl itself may not act as a transcriptionrepressor but rather indirectly repress cone genes via thefunction of Rxrγ, Nr2e3, or other transcription repressors(see below).

Nrl is known to interact with several transcriptionalregulators, in addition to the possibility of forming hetero-dimers with other bZIP family members expressed in theretina, such as Jun/Fos family members (He et al., 1998). Nrlwas the first protein identified that acts synergistically withCrx to activate the rhodopsin promoter (Chen et al., 1997;Mitton et al., 2000). This synergy, however, is not observed forcone opsin promoters (Peng and Chen, 2005). Nrl and Crxinteract through the leucine zipper and homeodomain,respectively (Mitton et al., 2000). Two mutations in the CRXhomeodomain identified in human patients (R41W and R90W)decreased this interaction (Mitton et al., 2000). Nrl alsointeracts with the TATA-binding protein, Tbp, through adifferent domain near the N-terminal that is conservedamong Maf family members (Friedman et al., 2004). Thus,Nrl may function by recruiting or stabilizing Tbp, which thenrecruits the general transcription complex (Friedman et al.,2004). Nrl was also reported to interact with Fiz1, a zinc fingerprotein that complexes with Flt3 receptor tyrosine kinase(Mitton et al., 2003). Fiz1 potentiates Nrl or Crx/Nrl activity onrhodopsin and PDE6B promoters (Mali et al., 2007), furtherimplicating Nrl's involvement in coordinating the intrinsicphotoreceptor developmental program with extracellularsignaling pathways.

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1.2.2. Nr2e3 is a dual transcription regulator required forterminal differentiation of rodsThe nuclear orphan receptor Nr2e3 (photoreceptor-specificnuclear receptor, PNR) was originally identified by its homol-ogy to the orphan nuclear receptor tailless (Tlx, Nr2e1) initiallyfound in Drosophila, and by its specific expression in retinalphotoreceptor cells (Kobayashi et al., 1999). Similar to othermembers of the Tlx nuclear receptor family, Nr2e3 has a zinc-finger DNA-binding domain near the N-terminus and a ligand-binding domain in the C-terminus for a ligand yet to beidentified (Kobayashi et al., 1999; Fig. 1). Nr2e3 is expressed inretinal photoreceptor cells beginning around E18 in themouse, peaking during rod differentiation, and persistinginto adulthood at a decreased level (Kobayashi et al., 1999;Takezawa et al., 2007). Expression appears primarily localizedto the outer nuclear layer (Kobayashi et al., 1999; Haider et al.,2000, 2001). This persistent expression, mainly in rods inmammals (Bumsted O'Brien et al., 2004; Chen et al., 2005; Penget al., 2005), suggests that Nr2e3 plays a major role in roddifferentiation and maintenance. On the other hand, someearly cone precursors appear to transiently express Nr2e3, atleast in lower vertebrates (Chen et al., 2005) and Nr2e3expression has been reported in other retinal cell types(Chen et al., 1999) including mouse cones (Haider et al., 2006;see below), so it may also be involved in other developmentalprocesses.

1.2.2.1. Human genetic studies. Mutations in human NR2E3cause enhanced S-cone syndrome (ESCS), an autosomalrecessive disease featuring hyperfunction of blue cones,defective function of rods, and blindness in the late stages(Haider et al., 2000; Jacobson et al., 2004; Wright et al., 2004).Histopathological studies showed excess S-cones in theretina, some of which express both S- and M-cone opsins(Milam et al., 2002), which is unusual in humans (Lukats et al.,2005; Peichl, 2005).

1.2.2.2. Animal studies. The rd7 mouse, a naturally occur-ring mutant strain, resembles the phenotype of humanESCS. Genetic analysis revealed that these mice carry ahomozygous 380-bp deletion in the coding region of theNr2e3 cDNA (Akhmedov et al., 2000; Haider et al., 2001), dueto mRNA splicing defects resulting from an L1-retrotran-sposon insertion (Chen et al., 2006). This leads to a frame-shift with a premature termination. Subsequent studiesdemonstrated that the rd7 mouse does not produce Nr2e3protein, and therefore represents a bona fide null mutant ofNr2e3 (Nr2e3-/-) (Peng et al., 2005; Haider et al., 2006). The rd7mouse retina contains whorls and rosettes in the photo-receptor layer at early ages, followed by slow photoreceptordegeneration. Similar to ESCS in humans, the rd7 retina hasexcess cones that express mostly S-cone opsin (Haider et al.,2001). Rod and cone function measured by electroretino-graphy (ERG) is near normal in young adults but signifi-cantly declines in older mice as a result of degeneration ofboth rods and cones (Akhmedov et al., 2000; Ueno et al.,2005; Haider et al., 2006). These phenotypes of human ESCSand rd7 mice support a role for Nr2e3 in rod/conedevelopment by demonstrating how its mutations lead todisease.

1.2.2.3. Mechanisms of action. The phenotype of Nr2e3mutants suggests two possible hypotheses for Nr2e3 functionin vivo: (1) Nr2e3 mutations cause defects in cell-fatedetermination resulting in transdifferentiation of developingrods into cones, or (2) Nr2e3 mutations result in abnormalcone proliferation leading to an increase in the absolutenumber of photoreceptors as well as disrupting the rod/coneratio. Several pieces of evidence strongly support the firsthypothesis: (a) As a downstream target of the rod-specifictranscription factor Nrl, Nr2e3 is predominantly expressed inpost-mitotic developing and mature rods. In Nrl−/− mice,enhanced S-cones arise from postmitotic rod precursors andNr2e3 expression is completely abolished (Mears et al.,2001). (b) Morphological and microarray analysis of the rd7retina indicates that the majority of photoreceptors exhibit ahybrid rod-cone phenotype, i.e. expressing both rod and conegenes (Chen et al., 2005; Corbo and Cepko, 2005). (c) Directtarget gene studies suggest that Nr2e3 is a dual transcriptionregulator for both rod and cone genes. In vitro protein-DNAbinding assays initially showed that Nr2e3 recognizes aconsensus DNA sequence with a direct repeat and binds asa homodimer (Kobayashi et al., 1999; Chen et al., 2005). Thisbinding appears to mediate transcriptional repression (Chenet al., 2005) rather than activation as previously observed forthe rhodopsin promoter (Cheng et al., 2004). The consensusNr2e3 DNA recognition sequence has actually not been foundin native opsin promoters or other known target genes,although the half site is present (Peng et al., 2005). Chromatinimmunoprecipitation assays subsequently demonstratedthat the Nr2e3 protein is indeed associated with both rodand cone opsin gene promoters in the retina of wild-type and“coneless” mice (Peng et al., 2005; Peng and Chen, 2005).However, this association depends on the Crx protein, as itdoes not occur in the Crx−/− retina in spite of the presence ofNr2e3 protein (Peng et al., 2005). Furthermore, in transienttransfection assays, Nr2e3 alone has limited regulatedactivity on target opsin gene promoters. In the presence ofCrx and Nrl, however, Nr2e3 potentiates Crx/Nrl activation ofrhodopsin (Cheng et al., 2004; Peng et al., 2005), but repressestheir activity on the cone opsin promoters (Chen et al., 2005;Peng et al., 2005). In addition, four genetically identifiedNR2E3 missense mutations demonstrated altered dual reg-ulatory activity (Peng et al., 2005). Consistent with these invitro results, quantitative RT-PCR analysis demonstrated thatthe rd7 retina exhibits down-regulated rod gene expression,but up-regulated cone gene expression during photoreceptordifferentiation (Peng et al., 2005). These suggest that Nr2e3acts as a dual regulator to promote rod phenotype differ-entiation and suppress cone gene expression in developingrods by modulating Crx/Nrl activity on rod and conepromoters. (d) Gain-of-function studies have shown thatectopic expression of Nr2e3 using a Crx promoter in Nrl−/−

retina is sufficient for guiding photoreceptor precursors todevelop into rod-like cells that express rhodopsin but not coneopsins (Cheng et al., 2006). Ectopic expression of Nr2e3 drivenby the S-opsin promoter is also sufficient to transformdifferentiating S-cones into rod-like cells. Altogether, thesefindings suggest that Nr2e3 acts downstream of Nrl and Crxto reinforce the development of the rod phenotype bysuppressing the cone pathway.

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These findings, however, do not necessarily rule out thesecond possibility that Nr2e3 has a function in limitingproliferation of early S-cone precursors (Haider et al., 2001;Yanagi et al., 2002). As seen in zebrafish retina (Chen et al.,2005), using more sensitive immunostaining assays with GFP-labeled cones, Nr2e3 was recently found to be expressed indeveloping and mature M/S-cones (Haider et al., 2006). SomeNr2e3 positive cells in E18 mouse retina also express Ki67, amitotic marker, suggesting that Nr2e3 is expressed in mitoticcells of the developing retina. BrdU incorporation assays in rd7retina at late developmental stages, including P14 and P30,demonstrated prolonged proliferation of ectopic retinal pro-genitors that subsequently develop into S-cones. No S-coneswere observed to re-enter the cell cycle, however. Increasedapoptosis is also seen in late developing stages of rd7 retina.These results support a role of Nr2e3 in suppressing coneproliferation.

The molecular mechanisms by which Nr2e3 plays a dualregulatory role in rod vs. cone gene expression remain to bedetermined, but are expected to involve interactions withco-activators and co-repressors. Nr2e3 is known to interactwith another nuclear receptor, Nr1d1 (Rev-erb-α) (Cheng etal., 2004), forming a complex with Crx/Nrl that potentiatesrhodopsin promoter activity. Nr1d1 is a member of thecircadian clock (Yin et al., 2006) involved in regulatingdiurnal variations in gene expression. It is also known thatthe expression of some photoreceptor genes such as rho-dopsin is under circadian regulation (Bowes et al., 1988; vonSchantz et al., 1999). Thus the Nr2e3/Nr1d1 interaction mayplay a role in regulating the diurnal expression pattern ofthese genes. Nr2e3 has also been reported to interact withRarα and Rxrβ (Chen et al., 1999). As for possible co-repressors, its closely related family member Nr2e1 (Tlx),was reported to interact with the co-repressor atrophin1(Atn1) in the retina (Zhang et al., 2006), raising thepossibility that Nr2e3 might also use Atn1 as a co-repressor.Interestingly, Nr2e1 plays a role in modulating retinalprogenitor cell proliferation and cell cycle re-entry byinhibiting the expression of Pten, a negative regulator ofneural stem cell proliferation (Zhang et al., 2006). An Nr2e1null mutation also results in enhanced S-cone syndrome inmice (Zhang et al., 2006). Takezawa et al. (2007) identifieda novel cell cycle-dependent Nr2e3 co-repressor named Ret-CoR, which is preferentially expressed in the developingretina and brain as well as Y79 retinoblastoma cells. Ret-CoR expression is down-regulated in mature retina, butclearly present in the photoreceptor nuclear layer whereNr2e3 is expressed. As reported for the other nuclear co-repressors NCoR/SMRT, Ret-CoR forms a multiprotein com-plex containing histone deacetylases (HDAC 1/2 and 3),NCoR, and Rb/p107 that are known to regulate cell cycleprogression. Nr2e3 appears to recruit Ret-CoR to thepromoter of Cyclin D1, which is required for proliferationof retinal progenitor cells, and repress its expression. Thisnew finding supports a possible role of Nr2e3 in regulatingcell proliferation as discussed above. Altogether, thesefindings suggest that Nr2e3 promotes rod differentiationby bi-directionally regulating rod vs. cone gene expres-sion and possibly inhibiting the proliferation of developingcones.

1.3. Factors for cone development

1.3.1. Cone subtypes and gradientsCones are less sensitive to light than rods, but they providevisual acuity, the ability to distinguish features of the visualenvironment. Consequently, their distribution across theretina is not random, but varies among species to reflecteach species' visual needs (Peichl, 2005; Fig. 2). Most verte-brates have at least two different cone subtypes, producingopsins with different spectral sensitivities. In addition, thecone outputs are subjected to more processing in the retinathan rod signals in order to extractmore visual information, socones make synaptic connections with a larger number andgreater variety of inner retina cells than rods. Conversely, inorder to preserve spatial resolution, cone signals are notsummed by converging on a small number of output cells theway rod signals are (Peichl, 2005). The result is that more innerretina and central visual pathway neurons are involved withprocessing cone signals than rod signals. This has implica-tions for understanding retina development, and should alsobe taken into account in experiments in which photoreceptorfate is genetically manipulated.

Most studies aimed at understanding the genetic mechan-isms underlying human cone development have beenperformed in mice, which have two subtypes of cones. Micedo not have a cone-rich area in their retina like primates do,but the two cone subtypes are distributed in inversegradients across the retina (Figs. 2A and B). The density ofcones expressing short-wavelength sensitive S-opsin is high-est in the ventral retina, and cones expressing longerwavelength sensitive M-opsin are most dense in the dorsalretina. In the region where these two gradients overlap,many cones express both photopigments (Rohlich et al., 1994;Applebury et al., 2000; Lukats et al., 2005). As in mostvertebrates, there is a region of higher visual sensitivityalong the equator of the retina that corresponds with adisproportionately increased area of representation in thevisual cortex (Luo et al., 2006). In the mouse retina, S-conesdifferentiate first, beginning shortly after birth, with mostdifferentiating cells localized to the ventral retina. M-conesbegin differentiating about a week later, mostly in the dorsalretina and coinciding with a decrease in the number of cellsexpressing S-opsin (Cepko, 1996; Roberts et al., 2006).Increasing evidence suggests that the cone gradients aregenerated during development by the action of diffusiblegrowth factors or hormones (extrinsic signals) and theirreceptors, mainly nuclear receptor family transcriptionfactors (intrinsic program). These receptors exist as familiesof related genes that have apparently diverged from acommon ancestral precursor (Germain et al., 2006). In thefollowing section, we will focus on recent progress on therole of nuclear receptors in cone development and discussthe possible action of their ligands.

1.3.2. Factors that influence S-cone differentiation

1.3.2.1. Extrinsic signal—to be determined. The regulatoryfactors that are responsible for triggering cone progenitors tobegin differentiating are still notwell understood. Retinoic acidor related compoundsare tempting candidates, since theyhave

Fig. 2 – Distribution of cones and opsin expression in mouse and human retina. (A) In the mouse, cones are scatteredthroughout the retina, withM-cones (green) predominating in the dorsal retina and S-cones (blue) predominating in the ventralretina. Many cones express both opsins. (B) M-opsin and S-opsin are expressed in complementary dorsal (D) to ventral (V)gradients across the mouse retina, likely in response to a gradient of thyroid hormone (TH), which is established between P4and P10, and is highest in the dorsal retina as indicated. (C) This graph shows the spatial density of rods (blue) and cones(orange) in a horizontal strip of human retina across the fovea (centered at position 0) and optic disc. Cones are concentrated inthe fovea, and rods are excluded from this region. Elsewhere in the retina, rods predominate and cones are sparse. (D) Withinthe fovea, cones expressing either green or red opsin predominate, with cones expressing blue opsin found sparsely aroundthe peripheral fovea region. Human cones only express a single type of opsin. Panels A and B are from Applebury et al. (2000),reprinted by permission fromCell Press, with TH gradient added fromRoberts et al. (2006). Panel C is fromRodieck (1998), pg. 43,reprinted by permission from Sinauer Associates, Inc. Panel D is from Cepko (2000), Fig. 2, reprinted by permission fromMacmillan Publishers, Ltd.: Nature 24: 99–100, copyright 2000.

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been shown to induce expression of photoreceptor-specificgenes and to influence cell fate choices in vitro (Hyatt andDowling, 1997). It is also known that during early eye develop-ment in rodents, the equator region of the retina expressesretinoic acid degrading enzymes, while the rest of the retinaexpresses enzymes that convert Vitamin A to retinoic acid,thus establishing a discontinuous gradient of retinoic acidsignaling across the retina (Luo et al., 2006). However, there iscurrently no compelling evidence that retinoic acid or relatedcompounds influence cone photoreceptor distribution in vivo,although these studies led to interesting findings regardingthe receptors for these compounds.

1.3.2.2. Rxrγ and Trβ2 are negative regulators for S-cones.Retinoic acid and related compounds work by diffusingthrough cellular membranes and binding to nuclear recep-tors, which associate with specific regulatory elements inthe DNA of gene promoter and enhancer regions. Thesereceptors exist as families of related genes. The retinoic acidreceptor (Rar) and retinoid-related receptor (Rxr) familiesboth consist of three genes, A, B, and C, producing the α, β,and γ isoforms, respectively, of the receptors. Extensiveknockout mouse studies have been performed to try todetermine which isoforms are expressed in particular tissuesand mediate signals for particular processes or sets of genes.

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Most isoforms are expressed in the retina (Janssen et al.,1999; Mori et al., 2001), but the Rxrγ receptor is of particularinterest because it is localized to developing cone photo-receptors in a number of species. Knockout of Rxrγ in micedestroys the gradient of S-cone distribution and results in S-opsin expression in all cones in the retina (Roberts et al.,2005), indicating that its likely role in S-cones is inhibitoryrather than inductive. Rxrs are unique among nuclearreceptors because they heterodimerize with members ofseveral other nuclear receptor families, including Nr2e3(Chen et al., 1999) and thyroid hormone receptors (Szantoet al., 2004). Thyroid hormone receptor Trβ2 is likely to bethe heterodimerization partner involved in suppressing S-cones (see below).

1.3.2.3. Rorβ2 is a positive regulator for S-cones. Retinoid-related orphan receptors (Ror) are another family of geneti-cally related receptors with homology to the receptors forretinoids, but whose actual ligands have not been identified.One isoform in particular, Rorβ2, is expressed in photorecep-tors as well as other cells in regions of the brain involved inregulating circadian rhythm (Andre et al., 1998). This isoformis expressed early in retinal progenitor cells (beginning atE13.5 in rat) and appears to increase their proliferation (Chowet al., 1998). The other product of this gene, Rorβ1, is notproduced in the retina (Azadi et al., 2002). As developmentproceeds, expression of Rorβ2 continues at a lower level inphotoreceptor cells, as well as in amacrine and ganglion cells(Chow et al., 1998). Rorβ2 has also been shown to synergizewith Crx in vitro to activate the S-opsin gene (Srinivas et al.,2006). Mice that are homozygous for knockout of Rorβ fail toexpress S-opsin at the appropriate developmental time,although M-opsin expression is unaffected. However, inthese mice the outer nuclear layer is disorganized andphotoreceptor outer and inner segments are not produced,indicating that Rorβ2 may have several additional roles indifferentiation of both rod and cone photoreceptors (Srinivaset al., 2006). Yanagi et al. (2002) suggest that the S-conephenotype is a default state, to explain why disruption ofeither rod-inducing factors (Nrl or Nr2e3) or factors involved incone differentiation (Rxrγ or Trβ2) results in over-productionof S-cones. The discovery of this positive regulator of S-opsin,however, suggests that the S-cone phenotype might beactively selected, arguing against the hypothesis of a defaultpathway.

1.3.3. Factors that influence M-cone differentiation

1.3.3.1. Extrinsic signal—thyroid hormone. M-cones developlater than S-cones in rodents, but the mechanisms involvedare currently more completely understood. The M-conegradient (Figs. 2A and B) is most likely established by thyroidhormone (TH). This hormone is produced by the thyroidgland and distributed by blood circulation to the tissues,where it is partially deiodinated to the active form (3,5,3′ tri-iodothyronine, or T3) (Forrest et al., 2002). Its involvement inmediating the visual changes that occur during amphibianmetamorphosis has long been known (Hoskins, 1990), and itwas also shown to influence chick and rat retinal progenitordevelopment. Harpavat and Cepko (2003) reviewed the role

of TH in retinal development in 2003. TH is distributeduniformly across the retina at birth, but during the periodof M-cone differentiation between P4 and P10 forms a gra-dient with higher concentrations in the dorsal than ventralretina (Roberts et al., 2006; Fig. 2B). This implicates TH as theextrinsic signal responsible for establishing the M-conegradient.

1.3.3.2. Trβ2 is a positive regulator for M-cones. Vertebrateshave two genes for TH receptors, and each produces severalprotein isoforms (reviewed in Forrest et al., 2002; Eckey et al.,2003). The thyroid hormone receptor Trβ2, a splice variant ofthe thyroid hormone receptor B (Thrb) gene, is implicated inphotoreceptor development in chick and mouse, based on itsexpression in retinal progenitor cells and developing photo-receptors. In mouse eyes, expression of Trβ2 begins about E16,peaks around E18 as cone photoreceptors begin differentiat-ing, then decreases (Ng et al., 2001; Yanagi et al., 2002), but theexpression is uniform across the retina (Ng et al., 2001; Robertset al., 2005, 2006). However, during the time M-cones aredeveloping, its ligand TH becomes distributed in a gradientwith higher concentrations in the dorsal than ventral retina(Roberts et al., 2006; Fig. 2B).

1.3.3.2.1. In vitro and animal studies. Transient transfec-tion studies showed that Trβ2, in the presence of TH,activated M-opsin transcription and inhibited Crx-mediatedtranscription of S-opsin (Yanagi et al., 2002). Addition ofexogenous TH also promoted M-opsin expression andinhibited S-opsin in embryonic retina explant cultures fromwild-type (WT) but not Trβ2 knockout mice (Roberts et al.,2006). Furthermore, daily injection of TH into WT mousepups beginning on P0 drastically decreased the number ofS-cones found in all parts of the retina 3 days later. Nodecrease in S-cone numbers was seen in Trβ2 knockoutmice treated similarly (Roberts et al., 2006). These resultsshowed that Trβ2 induced M-opsin expression and concur-rently inhibited S-cone production in a ligand-dependentmanner. Knockout of the photoreceptor-specific Trβ2 iso-form of the Thrb gene converted all cones to the S-phenotype, resulting in loss of both M-opsin expressionand the S-cone gradient in vivo (Ng et al., 2001). Thisphenotype is also reproduced in a mouse with a knockinmutation in the DNA binding domain of Thrb that abolishesspecific DNA sequence binding without affecting ligandbinding or cofactor interactions (Shibusawa et al., 2003).These findings indicate that both M-opsin induction and theestablishment of the S-cone gradient depend on Trβ2 DNAbinding. The role of Trβ2 thus appears to induce a subset ofdeveloping cones to further specialize as M-cones (Yanagi etal., 2002) by suppressing expression of S-opsin (and possiblyother S-cone genes) but promoting expression of M-opsin(and possibly other M-cone genes).

1.3.3.2.2. Mechanisms of action. TH nuclear receptorsare reported to exert their effects as heterodimers incombination with retinoid (usually Rxr) receptors (Mangels-dorf and Evans, 1995). The Rxrγ receptor mentionedpreviously is a likely candidate for the Trβ2 dimerizationpartner in developing cones. Rxrγ itself is not involved inM-opsin expression, however, since the M-cone gradientforms normally in Rxrγ knockout mice (Roberts et al., 2005).

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Roberts et al. (2006) hypothesize that a Trβ2:Rxrγ hetero-dimer is responsible for suppressing S-opsin expression,while binding of TH induces dissociation of Rxrγ andformation of Trβ2 homodimers, which activate M-opsinexpression. This would also explain the inhibition of S-opsin expression in the dorsal retina prior to P6. ExogenousTH could overcome this inhibition if it induced dissociationof receptor heterodimers prematurely, before the gradient ofthyroid hormone becomes established. Thyroid hormonereceptors mediate repression (usually in the absence ofligand) by interacting with nuclear co-repressors NCoR and/or SMRT (Eckey et al., 2003; Makowski et al., 2003; Havis etal., 2006). In fact, a growing body of evidence indicates thatheterodimers of thyroid hormone and Rxr receptors adoptdifferent configurations based on information provided bythe DNA binding site with which they associate, thatfacilitate or block interactions with co-activators or co-repressors (Harvey et al., 2007 and references cited therein;Ghosh et al., 2002; Diallo et al., 2007). Thus, the action of anuclear receptor heterodimer can be influenced by eachtarget promoter sequence to fine-tune interactions withligands and cofactors. Since S-cones develop several daysbefore M-cones appear in mice but are less prevalent in thedorsal retina, a repressive mechanism must exist for

Fig. 3 – ChIP analysis demonstrating that network transcriptioAntibodies against Crx, Nrl, Nr2e3, Trβ2, and NeuroD1 were ufrom wild-type (WT), Crx−/−, Nrl−/−, or Nr2e3rd7/rd7 (“Nr2e3−/−”) relisted on the left (Peng and Chen, 2005; Table 3) were used to dimmunoprecipitates by PCR. A band indicates that the transcripis bound to the promoter region of the indicated regulator orM-opsin (Mop), rhodopsin (Rho), and interphotoreceptor bindingGluR6, which is expressed in bipolar cells but not photoreceptoaddition, PCR reactions using primers against DNA sequences imconfirming regulatory region-specific binding. Control immunoyielded no specific promoter sequences from WT (second lanesof retina homogenates (“input”) from WT (far right lanes) andfor PCR.

suppressing S-cones (or at least expression of S-conegenes) in regions where M-cones will predominate. Increas-ing evidence therefore supports a dual role for Trβ2, inconjunction with Rxrγ, in mediating this repression.

The actual role of Trβ2 on S-opsin expression may bemore complex than the simple inhibition implied above.Findings from a study designed to map expression quanti-tative trait loci (eQTL) in the rat showed that pointmutations affecting a conserved serine and another residuein the N-terminal domain of Trβ2 (Ser56Asn and His58Arg)correlated with decreases in S-opsin expression levels of asmuch as 30% in homozygotes (Scheetz et al., 2006). Theaffected residues fall within a poorly characterized ligand-independent transactivation domain that can interact withcofactors and is the target of post-translational modificationin some nuclear receptors (Germain et al., 2006). Trβ2 bindsdirectly to the S-opsin promoter (Fig. 3) and has beenreported to interact with the basal transcription machineryas well as co-activators and co-repressors to exert complexregulatory effects on target genes (Eckey et al., 2003). Themutations identified by Scheetz et al. (2006) might thereforealter one or more of these interactions, making activation ofthe S-opsin gene less efficient in the presence of the mutatedreceptor.

n factors bind to their own and each other's promoters.sed to immunoprecipitate the bound chromatin fragmentstinae. Primers specific to the promoter regions of the genesetect the presence of the candidate promoter regions in thetion factor recognized by the immunoprecipitating antibodytarget gene. Target genes examined include S-opsin (Sop),protein (Rbp3), all of which are expressed in photoreceptors.rs, serves as a control for photoreceptor specificity. Inmediately 3′ of each gene gave no bands (data not shown),

precipitates using purified non-specific rabbit or goat IgGfrom the right) or knockout (data not shown) mice. Samplesknockout (data not shown) mice serve as positive controls

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2. Results and discussion

2.1. Photoreceptor transcription factors form a network toregulate the expression of themselves and each other

During the course of reviewing and studying the above rod andcone transcription factors, we have observed evidence of cross-talk and feedback regulation among these factors at thetranscriptional level.Wehypothesized that eachof these factorsregulates its own expression and that of the other factors bydirect binding of the transcription factor protein to promoterelements in the DNA. Such interaction and feedback regulationcould be important for regulating development and mainte-nance of each of the photoreceptor phenotypes. To test thishypothesis, we performed chromatin immunoprecipitation(ChIP) and quantitative RT-PCR analysis of five of thesetranscription factors: the photoreceptor lineage determinantCrx, rod-lineage determinants Nrl and Nr2e3, the cone determi-nation factor Trβ2, and the HLH factor NeuroD1 that has beenshown to be important for photoreceptor survival (Morrowet al.,1999; Pennesi et al., 2003). Chromatin immunoprecipitation(ChIP) assays were performed on the retinas of wild-type, Crx−/−,Nrl−/−, and Nr2e3rd7/rd7 (labeled as “Nr2e3−/−”) mice at the age ofP14 when these factors are all expressed but before photo-receptor degeneration begins in the mutant mice. The ChIPresults were analyzed using PCR with primers spanning thepromoter region of each regulatory factor; their dependenttarget genes, rod and cone opsins; and their independent geneRbp3 (Fig. 3). PCR assays with primers spanning 3′ regionsimmediately downstream of each gene were also performed ascontrols for the transcription factors' binding specificity to theregulatory regions (data not shown). To correlate the ChIPresults with transcriptional regulation, we also performedquantitative RT-PCR for each of the regulatory factors in theretinaeof the fourstrains (Table2).These results, combinedwithwhat we have learned from the literature, are discussed below.

2.1.1. Opposing regulation of subtype-specific genesIt is poorly understood how each photoreceptor subtypeexpresses the genes that determine its own identity butshuts off expression of genes specific to other subtypes. Fig. 3shows that in wild-type retinae (“WT” lanes) all five photo-receptor-specific transcription factors bind to both rod andcone target genes, regardless of subtype association. Each ofthe transcription factors assayed is found on the promoters ofrhodopsin (Rho), cone opsins (Sop and Mop), and Rbp3, genesexpressed in photoreceptors, but not the control gene GluR6that is not expressed in photoreceptors. This suggests thateach subtype-specific factor could play opposing roles on theexpression of its own subtype-specific genes vs. genes specificto other photoreceptor subtypes. The best-understood exam-ple of this is Nr2e3, which is known to activate rhodopsin butrepress cone opsin genes (Cheng et al., 2004; Peng et al., 2005).This is reflected in Fig. 3 in the “Nr2e3” panel by the presence ofNr2e3 on all three opsin promoters, and in Table 2 by a decreasein Rhodopsin expression (0.84 timesWT) and increases in S- andM-opsin expression (1.23 and 1.19 timesWT, respectively) in theNr2e3−/− mouse compared withWT. Similarly, Nrl also directlybinds to both rod and cone gene promoters in rods (Fig. 3, “Nrl”

panel), although a conventional Nrl target binding site has notbeen reported in the cone opsin regulatory sequences. Thisbinding is independent of Crx, since it still occurs in the Crx−/−

retina. Whether Nrl binds to cone promoters through interac-tion with other proteins or as a result of the presence of lowaffinity binding site(s) in the cone promoters remains to bedetermined. In any case, the presence of Nrl on thesepromoters suggests that Nrl is involved in regulating both rodand cone genes in the same cell (Peng and Chen, 2005).Consistentwith this, in theNrl−/− retina, Rho gene expression isdecreased (0.07 times WT) while cone opsin gene expression isincreased (2.14 and 1.17 times WT; Table 2). Thus, Nrl exertsopposing effects on rod and cone gene expression in vivo.However, its repressive effect on cone genes is likely mediatedby indirect mechanisms, as Nrl (alone or in combination withCrx) does not repress M- or S-opsin promoter activation intransiently transfected HEK293 cells (Peng et al., 2005).

The cone transcription factor Trβ2 binds to both M-opsinand S-opsin promoters in WT retinae (Fig. 3, “Trβ2” panel) toactivate M-opsin but repress S-opsin expression (Ng et al., 2001;Yanagi et al., 2002). Trβ2 also binds to the rhodopsin promoter(Fig. 3) and several other rod genes (data not shown) in a Crx-dependentmanner, suggesting that it could also be involved inregulating (likely repressing) the expression of rod genes incone cells. Although no rod abnormalities have been reportedin several different genetically engineered mice with disrup-tions of Thrb/Trβ2, this would be an interesting hypothesis totest. Theoutcome is likely to dependon interactionswith othernuclear receptors and the availability of ligands and cofactors.

2.1.2. Auto-, para-, and feedback regulationFig. 3 also shows that each transcription factor binds to its ownpromoter as well as those of the other regulators examined(“WT” lanes in each panel), suggesting that each factorregulates its own expression (auto-regulation), regulates theother factors acting in parallel or downstream (para-regula-tion), and feeds back regulatory information to the promotersof the upstream factors that induced it (feedback regulation).The best example of this is Crx. First, the Crx protein directlybinds to its own promoter (Fig. 3; Furukawa et al., 2002) andauto-activates its own expression. The strength of Crxactivation depends on the amount of Crx present. In Crxknockout mice that have low (Crx+/−) or no (Crx−/−) Crx proteinas a result of replacement of the Crx coding sequence of one orboth alleles, respectively, transgenic LacZ reporter genesdriven by Crx promoter sequences are only expressed at 57%or 31% of wild-type levels, respectively (Furukawa et al., 2002).Thus, auto-activation substantially increases expressionlevels. Second, Crx also binds to the promoter of otherphotoreceptor transcription factors Nrl, Nr2e3, Trβ2, Rxrγ,Rorβ, and NeuroD1 (Fig. 3, “Crx” panel) and regulates theirexpression in para-regulatory fashion (Table 2; Furukawa,1999; Blackshaw et al., 2001). In Crx−/− mice, expression of therod factors Nrl and Nr2e3 is significantly reduced but notabolished (0.67 and 0.61 times WT; Table 2), consistent withthe dramatic reduction in rhodopsin expression (0.12 timesWT).Crx is also required for the expression of the M-cone factorTrβ2 by binding to the promoter of the Trβ2 gene (Fig. 3).Trβ2 transcription in the Crx-/- retina is only half (Table 2) ofthe wild-type level, consistent with lack of Trβ2 binding on

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target cone genes (Fig. 3, “Trβ2” panel) and defective coneopsin transcription (Table 2) in the Crx−/− retina. Likewise,Crx may activate the expression of NeuroD1 by binding to itspromoter (Fig. 3), as NeuroD1 expression is also decreased(0.75 of WT level) in Crx−/− mice (Table 2; Blackshaw et al.,2001). NeuroD1 has been implicated in the survival andmaintenance of both rods and cones (Morrow et al., 1999;Pennesi et al., 2003), and it may play this role by regulatingthe expression of rod/cone genes and their transcriptionregulators as suggested by the results shown in Fig. 3.Although the reduced (0.75 times WT; Table 2) levels ofNeuroD1 apparently do not dramatically affect its binding totarget genes in the Crx−/− mice (Fig. 3, “NeuroD1” panel), ourresults suggest that insufficient NeuroD1 might contribute tothe photoreceptor degeneration in these mice. Interestingly,Rxrγ and Rorβ2 mRNA levels are elevated in the Crx−/− retina(Table 2; Blackshaw et al., 2001). This suggests that Crx plays anegative regulatory role on expression of these two factors,although the mechanism for this remains to be determined.Finally, Otx2 has been shown to act upstream of Crx bybinding to and activating the Crx promoter (Nishida et al.,2003; Nishida, 2005). Crx also binds to the Otx2 promoter torepress rather than activate Otx2 expression, since Otx2transcript levels are increased more than twofold in Crx−/−

mice (Table 2). This is consistent with the fact that Otx2expression is down-regulated in the photoreceptor cell layerwhen Crx expression reaches a high level during normalretinal development (Nishida et al., 2003). Thus, at the centerof the photoreceptor transcription factor network, Crxregulates expression of itself (auto-regulation), other mem-bers acting downstream or in parallel (para-regulation) andits upstream inducer (feedback regulation).

Similar auto- and para-regulation may also apply to photo-receptor subtype-specific regulatory factors. For example, the rodfactorNrl binds to its ownpromoter and auto-activates it, as only40% of WT reporter transcript levels are seen in Nrl−/− micecarrying a transgene under the control of the Nrl promoter(Yoshida et al., 2004). Besides regulating the expression of Nr2e3in the rod pathway, Nrl also binds to the Trβ2, Rxrγ, and Rorβpromoters in the cone pathway (Fig. 3). This likely results inrepression of Rxrγ, and Rorβ, as they are increased in Nrl−/−

retinae (Table 2; Yoshida et al., 2004). Althoughwe did not detectsignificant changes in Trβ2 expression levels or in Trβ2 proteinbinding to target promoters in Nrl−/− or Nr2e3−/− mice, underphysiological conditions in the wild-type background Nrl couldplay a role in repressing Trβ2 expression either directly orindirectly. For the cone pathway factors, Trβ2 binds to its ownand other cone and rod regulator genes, raising the possibilitythat it could also mediate repression of these regulators toreinforce the M-cone pathway. Trβ2 also binds to the promotersof upstream regulators Crx and Otx2, which could mediatefeedback regulation of these factors. It would be interesting toevaluate this hypothesis by examining expression of theseupstream regulators in Trβ2 knockout mice.

2.1.3. Protein–promoter interactions can be affected byprotein–protein interactions and accessibility of individualpromotersTheChIP results presented in Fig. 3 andexpression level data inTable 2 also suggest crosstalk between protein–promoter

interactions and protein–protein interactions for the photo-receptor transcription factors. As an example, Nr2e3 binding toits target genes, including both opsins and transcriptionregulators, appears to depend on its interacting partner Crx,as this binding does not occur in Crx−/−mice. This lack of Nr2e3target binding cannot be fully explained by the moderatereduction in Nr2e3 expression (60% of wild-type level) in Crx−/−

retina, as rd7 heterozygous mice that produce half the normalamount of Nr2e3 protein have normal Nr2e3 function.Furthermore, in homozygous Crx−/− mice, Nr2e3 still binds tothe Crx-independent gene Rbp3. Thus, Crx-dependent genesrequire either a high dose of Nr2e3 to bind to their promotersor else the presence of Crx to recruit Nr2e3 binding andregulation. Similarly, Trβ2 also appears to show such Crx-dependency in binding and regulating target genes (Fig. 3,“Trβ2” panel), although no direct Trβ2/Crx interaction hasbeen reported. In Crx−/− mice, consistent with the reductionof Trβ2 mRNA shown in Table 2, the Trβ2 protein level is alsomoderately reduced on Western blots (data not shown). Thisreduction cannot fully account for the lack of Trβ2 binding torod or cone target genes in Crx−/− mice, suggesting that Trβ2binding to target genes depends on Crx or other Crx-regulated factor(s). Since many nuclear receptor consensusbinding sites in photoreceptor gene promoters appear not tofavor binding of nuclear receptor homodimers, these resultsraised the possibility that other nuclear receptors known toregulate photoreceptor genes might also function in a similarway, i.e. interacting with Crx/Otx2 to bind and regulatephotoreceptor genes. In contrast, Nrl does not appear to berequired for binding of the nuclear receptors to photoreceptorgenes, as Trβ2 binds to its targets well in Nrl−/− mice (Fig. 3).No Nr2e3 target binding is detected in Nrl−/− mice becauseNr2e3 is not made in this genetic background (Mears et al.,2001; Table 2). We have not yet observed dramatic changes intarget binding for Nrl and Crx in any of the mutant strainstested, suggesting their binding is independent of the otherfactors examined.

One exception for Crx-dependent binding of nuclearreceptors to target genes is the Rbp3 gene. Rbp3 is known tobe a target of Otx2 (Fong and Fong, 1999) and is expressed earlyduring retina development, at the time Crx is turned on (Bibbet al., 2001). Thus, Otx2might assist Nr2e3 and Trβ2 binding tothe Rbp3 promoter in Crx−/− mice. Another possible explana-tion is that the Rbp3 promoter is more accessible to regulatoryfactors so that a low concentration of a nuclear receptor in thepresence of Otx2 is sufficient for Rbp3 promoter binding. Itwould be interesting to compare histone modification andother chromatin configuration markers between the Rbp3promoter and the other promoters, to determine what makesRbp3 more accessible to regulatory factors.

3. Conclusion

Taken together, the recent progress in understanding themolecular mechanisms controlling photoreceptor subtypedevelopment and the results of the combined ChIP-expressionanalysis presented above suggest that the photoreceptortranscription factors form two types of network: protein–protein interaction and protein–promoter interaction. The

Fig. 4 – Model for transcription factor network regulation ofphotoreceptor subtype development. Photoreceptorsubtypes develop from photoreceptor precursors derivedfrom multi-potent progenitors via three major pathways(thick arrows). Photoreceptor transcription factors that play amajor role in this process are listed based on their epistaticrelationship as determined by in vivo and/or in vitrofunctional studies. Thin lines show protein–promoterinteractions; solid lines show interactions reported hereand/or previously; dotted lines are from unpublished data.Arrows indicate positive regulation, while blocked linesrepresent inhibition/suppression. Absence of lines indicatesthat the relationship remains to be determined.

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information for these two networks is just beginning toemerge. Fig. 4 shows our current model for the protein–promoter interaction network. Although this model is stillmissing components and connections, it does offer an over-view of how this protein–promoter interaction networkcoordinates the auto-, para-, and feedback regulation amongphotoreceptor transcription factors that determine general orsubtype-specific photoreceptor lineages. This regulatory net-

Table 3 – PCR primers for ChIP and quantitative RT-PCR

Genes Assays Sen

Trβ2 ChIP (−321/−53)a 5′-ACCTGCCTGCCAqRT-PCRb 5′-GCACATCTCCCT

NeuroD1 ChIP (−1504/−1336) 5′-TCCAGCCACTCAqRT-PCR 5′-CGCTCAGCATCA

Otx2 qRT-PCR 5′-ACTTGCCAGAATCrx qRT-PCR 5′-TGTCCCATACTCNrl qRT-PCR 5′-TTCTGGTTCTGANr2e3 qRT-PCR 5′-AGTCCCAGGTGARxrγ ChIP (−362/−109) 5′-AAAGGGCTCTG

qRT-PCR 5′-CAATGCTCTTGGRorβ ChIP (−471/−96) 5′-AAAGAGACAGA

qRT-PCRb 5′-AAGGGATTCTTC

a For ChIP primers, the numbers in parentheses indicate the position relb Note that qRT-PCR primers for Trβ2 recognize only the β2 isoform, whi

work is essential for precisely controlling spatial and temporalphotoreceptor gene expression, development, and mainte-nance. Therefore, perturbing any of the components, either bymutations or changes in expression levels of factors, couldpotentially disturb the balance of the network and result indevelopmental defects or degeneration of particular photo-receptor subtypes. Understanding this network is importantfor future therapeutic interventions to treat those diseasesassociated with photoreceptor transcription factors.

4. Experimental procedures

4.1. Animals

All experimental procedures were pre-approved by theInstitutional Animal Care and Use Committee of WashingtonUniversity School of Medicine, and conformed to the guide-lines of the Association for Research in Vision and Ophthal-mology for the use of live animals in vision research. Micewere bred and maintained in barrier facilities at WashingtonUniversity School of Medicine under a 12-h light, 12-h darkcycle. C57Bl/6J and rd7 mice were originally purchased fromthe Jackson Laboratory. Nrl−/− mice were obtained from AnandSwaroop at the University of Michigan. Crx−/−micewere kindlyprovided by Connie Cepko at Harvard Medical School.

4.2. Chromatin immunoprecipitation

The protocol used for chromatin immunoprecipitation hasbeen published (Peng and Chen, 2005). Briefly, DNA andchromatin in pooled nuclear extracts from 6 retinae werecross-linked with formaldehyde prior to immunoprecipitationwith specific antibodies. Antibodies used in the work pre-sented here that are not referenced in Peng and Chen (2005)include: rabbit anti-Nrl (Chemicon; see Swain et al., 2001 forspecificity details), rabbit anti-Trβ2 (Upstate; see Srinivas etal., 2006; Yen et al., 1992), and goat anti-NeuroD1 (Santa Cruzsc-1084; see Acharya et al., 1997; Cissell et al., 2003). Antibodyspecificity was confirmed by Western blotting and immuno-histochemistry on retinal sections, comparing WT and theappropriate knockout mouse retinae; or the use of antibodies

se Anti-sense

TTTTCCC-3′ 5′-ATTTGCCAGCCCCCTGAAC-3′GAAGAAAAGC-3′ 5′-TCCCCACACACTACACAGAGC-3′ACCCTGAC-3′ 5′-GAGGAGGAGGAGGAATGGTG-3′GCAACTC-3′ 5′-CTTGTCTGCCTCGTGTTCC-3′CCAGGGTG-3′ 5′-TGAGCCAGCATAGCCTTGAC-3′AAGTGCCC-3′ 5′-TGCTGTTTCTGCTGCTGTCG-3′CAGTGACTACG-3′ 5′-AAGGCTCCCGCTTTATTTC-3′TGCTAAGC-3′ 5′-TTCTAAGATGTGCTGCCCC-3′

TTCTCTCTTGG-3′ 5′-CGGGTGGCACAATCTATTAGC-3′CTCTCCG-3′ 5′-ATCTTTGTTATCCCGACAGGTG-3′GGAGAGAGGGG-3′ 5′-CAGTTAGAGGATGCTGGGTGC-3′AGGAGGAGC-3′ 5′-CCGCTGCTTCTTGGACATC-3′

ative to the transcription start site as +1.le Rorβ primers recognize both β1 and β2 isoforms.

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from different sources, which gave identical results (data notshown). The results of ChIP assays were analyzed usingcandidate gene-based PCR with primers spanning the promo-ter region of each gene (listed in Peng et al., 2005 or shown inTable 3). PCR assays with primers spanning 3′ regionsimmediately downstream of each gene were also performedas controls for factors' binding specifically in the regulatoryregions (Peng and Chen, 2005). Results shown are representa-tive of at least three separate experiments. Controls includethe use of normal rabbit/goat IgG (Santa Cruz) in immunopre-cipitation reactions (negative controls) and input (without ip)as positive controls in PCR reactions.

4.3. Quantitative real-time PCR

The protocol used for quantitative RT-PCR has been published(Peng and Chen, 2005). Sequences for additional primers usedin this study are shown in Table 3. Briefly, cDNA reverse-transcribed from 1 μg total RNA was diluted 10-fold andquantified by real-time PCR analysis in triplicate on an iCyclerPCR machine (Bio-Rad), using SYBR Green JumpStart Ready-Mix (Sigma). β-Actin was used as a loading control. Relativeexpression levels were normalized to the β-actin levels for eachsample according to standard methodology (http://www.openlink.org/dorak), as follows:

DCT ¼ CTðtestÞ � CTðb−actinÞ

where CT, the threshold cycle, is the cycle number (in theexponential phase) at which enough amplified product hasaccumulated to yield a detectable fluorescent signal that issignificantly above the baseline fluorescence level. Results arepresented as the ratio of ΔCTknockout /ΔCTWT. Mean values andstandard deviation (STDEV) were calculated for each experi-ment from three replicates, and statistical significance wasdetermined using the paired Student's t-test.

Acknowledgments

The authors wish to thank Jianfeng Liu and Hui Wang fortechnical assistance. This work was supported by NIH grantsEY12543 (to SC) and EY02687 (to Washington UniversityDepartment of Ophthalmology and Visual Sciences Core) andan unrestricted grant from Research to Prevent Blindness, Inc.

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