Rhodopsin promoter-EGFP fusion transgene expression in photoreceptor neurons of retina and pineal complex in mice

Neuroscience Letters 379 (2005) 138–143

Rhodopsin promoter-EGFP fusion transgene expression in photoreceptor neurons of retina and pineal complex in mice Andi Muhammad Ichsana,b , Ichiro Katoa,c,∗ , Toshiko Yoshidad , Kumi Takasawaa , Seiji Hayasakab,c , Koichi Hiragaa a

Department of Biochemistry, Toyama Medical and Pharmaceutical University, School of Medicine, Sugitani 2630, Toyama 930-0194, Japan b Department of Ophthalmology, Toyama Medical and Pharmaceutical University, School of Medicine, Toyama 930-0194, Japan c COE Program in the 21st Century, Toyama Medical and Pharmaceutical University, School of Medicine, Toyama 930-0194, Japan d Department of Anatomy, Toyama Medical and Pharmaceutical University, School of Medicine, Toyama 930-0194, Japan Received 23 September 2004; received in revised form 3 December 2004; accepted 21 December 2004

Abstract Light detection in vertebrate eyes is mediated through retinal photoreceptor rod and cone cells that transduce light signals into electrical responses. The differentiation and synaptogenesis of photoreceptor cells are especially important since these cells are the main targets of degeneration in retinitis pigmentosa. We produced transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the mouse rhodopsin gene promoter. In Western blot analyses of transgenic retinal homogenates, expression of the endogenous rhodopsin gene was detected from post-natal day (P)8; however, EGFP expression in transgenic retinas was initially detected at P12, indicating delayed expression of the transgene. At P14, fluorescence microscopy showed a weak expression of EGFP in the transgenic retina. In the adult transgenic mice, the strongest EGFP expression was observed in the outer nuclear layer, and to a lesser extent in the outer plexiform layer as well as in the inner and outer segments. EGFP expression was also observed in the pineal stalk. The rhodopsin promoter-EGFP transgenic mice will be not only useful to assess rhodopsin gene promoter activity in vivo, but also for retinal transplant studies as a source of functional photoreceptor cells that are fluorescent green. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Transgenic mice; EGFP; Photoreceptor; Rhodopsin; Promoter; Retina

The retina has been the target of many developmental studies, because of interest in using the retina as a model for the central nervous system development. Histological studies of mammalian retinal neurogenesis have revealed the processes of neuroepithelial cell development into the highlyorganized, multilayered retina [24]. Cell lineage tracing in mammals revealed that retinal progenitor cells are multipotent and retain their ability to generate various cell types such as ganglion cells, horizontal cells, cone-photoreceptors, amacrine cells, rod-photoreceptors, bipolar cells and M¨uller glia cells [17,28]. Many recent studies are focusing on the transcription factors required for retinal cell diversification ∗

Corresponding author. Tel.: +81 76 434 7227; fax: +81 76 434 5014. E-mail address: [email protected] (I. Kato).

0304-3940/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.12.072

[1,3,7,10,18,19,23,25,26]. In many cases of human retinal degenerative disease, photoreceptor cells are the targets of neurodegeneration, while the neurons of the inner retina are well preserved [24]. Rhodopsin, which is present in mature rod-photoreceptor cells, is a 40-kDa transmembrane apoprotein that transduces light signals into a G-protein mediated signal cascade [16]. A point mutation in exon 1 of the human opsin gene has been shown to cause one form of autosomal dominant retinitis pigmentosa [8]. To date, more than 100 mutations in opsin gene which can lead to retinitis pigmentosa are reported (see for example tables in RetNet: http://www.sph.uth.tmc.edu/Retnet/home.htm). Interestingly, rhodopsin-null mice almost completely lose their photoreceptor cells within 3 months after birth [11]. These

A.M. Ichsan et al. / Neuroscience Letters 379 (2005) 138–143

results indicate that rhodopsin is a key molecule in the organization and maintenance of rod photoreceptor cells. Therefore, rhodopsin gene expression in vivo can be a good marker to assess the maturation process as well as the viability of photoreceptor cells. Since the cDNA cloning of green fluorescent protein (GFP) in 1992, this molecule has been used as a valuable biomarker in a large number of biological systems. Unlike other previously developed markers such as Escherichia coli ␤-galactosidase, GFP can act as a powerful chromophore without additional cofactors [5]. When excited by blue or ultraviolet light, GFP emits green fluorescence at a wavelength of 510 nm. In the present study, we have for the first time produced a transgenic mouse carrying the genuine enhanced green fluorescent protein (EGFP) gene controlled by the mouse rhodopsin gene promoter and characterized the developmental and spatial expression of EGFP in the transgenic mice. Mouse rhodopsin promoter DNA was cloned by polymerase chain reaction (PCR) of embryonic stem cell-derived high molecular weight genomic DNA. Primers used in the PCR were 5 -CCGTCGACGTCGAGGCTCAGAGAGGAATACTTC-3 and 5 -CCAAGCTTCGTAGACAGAGACCAAGGCTGC-3 ; these sequences correspond to the nucleotides −1322 to −1298 and +65 to +41 of mouse opsin gene (EMBL data bank accession number M55171; Ref. [2]) and contain SalI and HindIII sites (underlined sequences), respectively. The cDNA encoding EGFP was amplified by PCR with primers, 5 -AAAAGCTTTCCACCGGTCGCCACCATGGT-3 and 5 -AAACTAGTTTACTTGTACAGCTCGTCCA-3 , using pEGFP-N1 (Clontech) as a template. These sequences contain HindIII and SpeI sites (underlined sequences), respectively. The amplified products were confirmed by subcloning and sequencing. Retinas were homogenized with a polytron (20 s, 3000 rpm) in a lysis buffer containing 10 mM Tris–HCl pH 7.4, 1 mM ethylenediaminetetraacetic acid, 250 mM sucrose, pepstatin A (5 ␮g/ml), antipain (5 ␮g/ml), leupeptin (5 ␮g/ml) and 0.25 mM PMSF. Protein amount was quantified with a protein assay kit (Bio-Rad). Protein samples (25 ␮g each) were electrophoresed under reducing conditions on 12% polyacrylamide gels in the presence of SDS and transferred onto Hybond-C nitrocellulose membrane (Amersham Biosciences). The membrane was incubated with monoclonal mouse anti-EGFP antibody (1:5000, Chemicon International) or rabbit anti-rhodopsin (1:2000, Cosmo Bio) for 1 h at room temperature. Blots were then incubated with horseradish peroxidase-linked anti-mouse or anti-rabbit IgG conjugates (1:5000, Amersham Biosciences) for 1 h at room temperature. After washing, proteins were visualized with the ECL detection system (Amersham Biosciences). The mouse rhodopsin promoter-EGFP hybrid gene (RhodEGFP; see Fig. 1A) was microinjected into fertilized eggs as described [13]. Seven out of 22 newborn mice were found to carry the transgene, as detected by PCR analyses using

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primers for the EGFP and SV40 genes. Founder mice were mated with ICR mice (Charles River) to obtain littermates. Two lines (14 and 35) of mice expressing the transgene in the retina were finally selected. All data represented in this paper were derived from line-14 transgenic mice, however, the reproducibility of the data was confirmed using line-35 mice. All procedures with mice were performed in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. To clarify the developmental regulation of EGFP expression, retinal extracts prepared from transgenic mice of various ages (P6–P20) were examined by Western blot analyses. In the transgenic retina, a band corresponding to EGFP was first detected at P12 and gradually increased to P20 (Fig. 1B). Since the promoter activity of the rhodopsin gene is strong, by introducing an extra copy of the promoter, expression of endogenous rhodopsin may be downregulated. Since a potential downregulation of endogenous rhodopsin gene expression may alter the physiology of the retina and make the mouse less useful for the future studies, we tested for rhodopsin expression by Western blot analysis and compared expression level with wild-type mice. In wild-type retinal homogenates, endogenous rhodopsin was initially detected at P8 and gradually increased until P20 (Fig. 1B). The developmental course of endogenous rhodopsin expression in the transgenic retinas was essentially similar to that in the wild-type retinas. Next, rhodopsin expression in the retinas of aged mice were examined (Fig. 1C). At P49 and P98, there were no significant differences in the levels of rhodopsin expression between transgenic and wild-type retinas. In addition, EGFP expression in the transgenic retina was not attenuated in the transgenic mice of P49 and P98. In order to determine the cellular localization of EGFP expression, whole mounted retinas of transgenic mice were examined by fluorescence microscopy. At P7 in the hematoxylin–eosin staining (Fig. 2A), retinal layers were still not clear and retina was essentially composed of neuroblastic cell mass. At this stage, EGFP fluorescence was not observed in any regions of the transgenic retina (Fig. 2B). At P14, the outer and inner segments of rod cells, outer nuclear, outer plexiform, inner nuclear, inner plexiform and ganglion cell layers were clearly distinguished (Fig. 2C), indicating that differentiation of the retinal cells was essentially completed. At this stage, a faint fluorescence by EGFP was already detectable in the transgenic retinal layers (Fig. 2D). At P28 (Fig. 2E) and P42 (Fig. 2F), the strongest EGFP fluorescence was observed in the outer nuclear layer where the rod-photoreceptor cell bodies are densely located. This is also in good agreement with the fact that 97.2% of the cells in the outer nuclear layer are composed of rod cells in mouse retina [12]. EGFP, a cytosolic small protein of 27 kDa, can readily diffuse into any spaces where the cytosolic connections exist [5]. Consistent with this, EGFP fluorescence was observed also in the outer plexiform layer, outer and inner segments where cytosolic connections with rod-photoreceptor cell bodies are maintained

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Fig. 1. (A) Schematic representation of the mouse rhodopsin promoter-EGFP fusion gene (Rhod-EGFP) used for microinjection. The 1.4-kbp SalI–HindIII fragment of the mouse rhodopsin promoter, the 0.75-kbp HindIII–SpeI fragment of EGFP cDNA, and the 1.6-kbp BglII–EcoRI fragment of the SV40 intron/polyadenylation signal were ligated sequentially in the correct orientation. The resultant hybrid gene (3.75 kbp) separated from the pBlueScript SK minus (Stratagene) by SalI and NotI was used for microinjection. Positions of PCR primers for mouse screening are indicated by arrows. (B) Western blot analyses of retinal protein extracts from transgenic (Tg) or wild-type (W) mice of age P6–P20 using anti-EGFP antibody (upper) or anti-rhodopsin antibody (middle and lower). (C) Western blot analyses of retinal protein extracts from wild-type (W) or transgenic (Tg) mice of age P49 and P98 using anti-rhodopsin antibody (upper) or anti-EGFP antibody (lower).

(Fig. 2E and F). From the results of fluorescence microscopy, it appears that the 1.4-kbp fragment of the mouse rhodopsin promoter correctly drives EGFP expression in rhodopsinproducing neurons. Since photoreceptor-specific transgene expression often leads to retinal degeneration [20], we next examined retinal morphology of old transgenic mice. The retinal layers of P540 transgenic mice were not altered as compared with their nontransgenic littermate (Fig. 2G and H), indicating that long-term, high levels of EGFP expression have no deleterious effect on the mouse retina. Similar results in the beta-actin promoter-driven EGFP transgenic mice were recently reported [21]. In addition, EGFP expression in the retinas of P540 transgenic mice were still strong (Fig. 2I). Several lines of evidence have indicated that extraretinal photoreception by the pineal complex influences the circadian rhythm in many vertebrates. It has been reported that the mouse pineal organ, especially pinealocytes, contains authentic rhodopsin mRNA and proteins [4]. Therefore, we next investigated the EGFP expression in pineal regions of the transgenic mice. Fluorescence microscopy of frozen brain section at the level of the pineal region showed EGFP expression in the pineal stalk, but not in the pineal body nor in the habenular commissure (Fig. 3). This is not surprising, since EGFP, a cytosolic small protein, could easily diffuse from neuronal cell bodies (pinealocytes) to the pineal stalk where its neural axons are densely located.

Rhodopsin, the G-protein-coupled light receptor, is expressed specifically in the rod photoreceptor cells of the retina and plays an essential role in visual function [8,11,16]. Photoreceptor-specific transcription of the rhodopsin gene is mediated by multiple cis-acting elements in the proximal promoter region [19]. Although many transcription factors may be involved in the rhodopsin gene expression, NRL (neural retina leucine zipper) and CRX (cone rod homeobox) proteins are currently thought to play central roles in the photoreceptor cell-specific expression of the rhodopsin gene; NRL and CRX both positively regulate the rhodopsin gene expression, and when expressed together they synergistically activate rhodopsin gene transcription [7,19,23]. The importance of NRL and CRX proteins in visual function was confirmed by the findings of missense mutations of human NRL and CRX genes in autosomal dominant retinitis pigmentosa, cone-rod dystrophy-2 and Leber congenital amaurosis, all of which ultimately lead to the loss of vision [3,25,26]. Furthermore, NRL- and CRX-knockout mouse studies [10,18] have clearly shown that NRL and CRX proteins are both essential for rhodopsin gene expression in photoreceptor cells as well as photoreceptor cell development in vivo. The 1.4kbp fragment of the mouse rhodopsin gene promoter used in the present study contains the DNA sequence elements that bind to NRL and CRX proteins. It is, therefore, reasonable to assume that NRL and CRX proteins act synergistically to induce EGFP gene expression in the photoreceptor cells of Rhod-EGFP transgenic mice.

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Fig. 2. Morphology and fluorescence images of mouse retinas. The eyeballs were embedded in Tissue-Tek (Sakura Finetechnical) and frozen in hexane with dry ice-acetone. Cryostat sections of 6-␮m thickness were stained with hematoxylin and eosin. For evaluation of the fluorescent image of EGFP, the serial sections were examined by fluorescence microscope (LEITZ DMR, Leica) with light at 488 nm for excitation. Hematoxylin–eosin staining from transgenic mice of P7 (A), P14 (C), P540 (H) and from P540 wild-type mouse (G). The other micrographs show EGFP fluorescence (green) from transgenic mice of P7 (B), P14 (D), P28 (E), P42 (F) and P540 (I). Retinal layers indicated are: neuroblastic cell mass (NB), outer segment (OS) and inner segment (IS) of photoreceptor cells, outer nuclear layer (ON), outer plexiform layer (OP), inner nuclear layer (IN), inner plexiform layer (IP) and ganglion cell layer (GC).

In Western blot analysis of retinal homogenates, the endogenous rhodopsin was initially detected at P8, however, EGFP was initially detected at P12 (Fig. 1B). The reason for this significant delay in the detection of the EGFP as compared with the endogenous rhodopsin is at present not clear. A possible explanation for this is that the 1.4-kb mouse rhodopsin promoter lacks 5 -upstream cis-acting DNA elements that are necessary for very early expression of the gene. Although delayed, fluorescent microscopy showed proper spatial patterns of EGFP expression at least until P540. In transgenic Xenopus and zebrafish, GFP had been expressed by their own rhodopsin gene promoters. In transgenic Xenopus, GFP was localized most strongly to the inner segment but also in the outer segment and synaptic terminal [15]. In transgenic zebrafish, EGFP was specifically localized to the outer nuclear layer and rod outer segments [14]. These results are in good agreement with the present transgenic mouse results in which EGFP was localized to the outer nuclear layer, the outer plexiform layer, the inner and outer segments.

In the transgenic mice expressing GFP under the control of the human red/green opsin gene 5 -promoter sequences, the fluorescent retinal cells were visualized as early as on embryonic day (E)15 and increased dramatically at P6–P7 [9]. The time course of GFP expression by the red/green opsin gene promoter is much earlier as compared with our transgenic mice. This phenomenon can be explained by much earlier developmental nature of cone cells (peak at E14–E15) as compared with that of rod cells (peak at P3–P4) [17]. Until now a considerable number of transgenic mouse studies that utilize the rhodopsin gene promoter for rodspecific gene expression have been reported and characterized [20,27,29,30]. However, no transgenic mice has been reported which expresses genuine EGFP controlled by the rhodopsin promoter. Very recently, the knock-in mice whose native rhodopsin gene has been replaced with the corresponding human gene modified to encode an EGFP fusion at the C terminus of rhodopsin [6]. The knocked-in human rhodopsinGFP fusion gene faithfully mimicked the expression and dis-

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Acknowledgments We thank E. Furuichi and E. Takado for technical assistance, and Y. Kojima for her kind help. This work was supported by a Grant-in-Aid for the 21st Century COE Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References

Fig. 3. Plain (A) and fluorescence (B) images of sagittal brain section of the transgenic mouse at the level of the pineal complex. EGFP fluorescence (arrows) was detected in the pineal stalk (PS) but not in the pineal body (PB) nor in the habenular commissure (HC). Scale bar indicates 250 ␮m.

tribution of wild-type rhodopsin in heterozygotes and served as a sensitive reporter of rod-cell structure and integrity. However, in homozygous knock-in mice the gene induced progressive retinal degeneration bearing many of the hallmarks of recessive retinitis pigmentosa, suggesting certain toxicity of the rhodopsin-GFP fusion protein. This is in contrast to our results that EGFP alone showed no toxic effects after the long-term, high levels of expression in the mouse retina. This transgenic mouse has several practical advantages. Since the rhodopsin gene expression closely influences photoreceptor cell function as well as cell viability [11], rhodopsin gene promoter activity in vivo can be a good marker for assessing cellular functions and the viability of retinal photoreceptor neurons. In retinal transplant studies in which donor cells should be distinguished from recipient cells, this transgenic mouse will be useful as a source of functional photoreceptor cells that are fluorescent green. In addition, multipotent cells such as bone marrow and nerve stem cells are now widely used in differentiation studies to generate functional photoreceptor cells in vitro and in vivo [22]. If stem cells from the Rhod-EGFP mice are employed, the expression of EGFP in the differentiating cells would provide presumptive evidence for differentiation along a rod pathway.

[1] T. Akagi, T. Inoue, G. Miyoshi, Y. Bessho, M. Takahashi, J.E. Lee, F. Guillemot, R. Kageyama, Requirement of multiple basic helix–loop–helix genes for retinal neuronal subtype specification, J. Biol. Chem. 279 (2004) 28492–28498. [2] M.R. Al-Ubaidi, S.J. Pittler, M.S. Champagne, J.T. Triantafyllos, J.F. McGinnis, W. Baehr, Mouse opsin, J. Biol. Chem. 265 (1990) 20563–20569. [3] D.A.R. Bessant, A.M. Payne, K.P. Mitton, Q.-L. Wang, P.K. Swain, C. Plant, A.C. Bird, D.J. Zack, A. Swaroop, S.S. Bhattacharya, A mutation in NRL is associated with autosomal dominant retinitis pigmentosa, Nat. Genet. 21 (1999) 355–356. [4] S. Blackshaw, S.H. Snyder, Developmental expression pattern of phototransduction components in mammalian pineal implies a lightsensing function, J. Neurosci. 17 (1997) 8074–8082. [5] M. Chalfie, Y. Tu, G. Euskirchen, W.W. Ward, D.C. Prasher, Green fluorescent protein as a marker for gene expression, Science 263 (1994) 802–805. [6] F. Chan, A. Bradley, T.G. Wensel, J.H. Wilson, Knock-in human rhodopsin-GFP fusions as mouse models for human disease and targets for gene therapy, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 9109–9114. [7] S. Chen, Q.-L. Wang, Z. Nie, H. Sun, G. Lennon, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, D.J. Zack, Crx, a novel Otx-like pairedhomeodomain protein, binds to and transactivates photoreceptor cellspecific genes, Neuron 19 (1997) 1017–1030. [8] T.P. Dryja, T.L. McGee, E. Reichel, L.B. Hahn, G.S. Cowley, D.W. Yandell, M.A. Sandberg, E.L. Berson, A point mutation of the rhodopsin gene in one form of retinitis pigmentosa, Nature 343 (1990) 364–366. [9] Y. Fei, Development of the cone photoreceptor mosaic in the mouse retina revealed by fluorescent cones in transgenic mice, Mol. Vis. 9 (2003) 31–42. [10] T. Furukawa, E.M. Morrow, T. Li, F.C. Davis, C.L. Cepko, Retinopathy and attenuated circadian entrainment in Crx-deficient mice, Nat. Genet. 23 (1999) 466–470. [11] M.M. Humhpries, D. Rancourt, G.J. Farrar, P. Kenna, M. Hazel, R.A. Bush, P.A. Sieving, D.M. Sheils, N. McNally, P. Creighton, A. Erven, A. Boros, K. Gulya, M.R. Capecchi, P. Humphries, Retinopathy induced in mice by targeted disruption of the rhodopsin gene, Nat. Genet. 15 (1997) 216–219. [12] C.-J. Jeon, E. Strettoi, R.H. Masland, The major cell populations of the mouse retina, J. Neurosci. 18 (1998) 8936–8946. [13] I. Kato, S. Takasawa, A. Akabane, O. Tanaka, H. Abe, T. Takamura, Y. Suzuki, K. Nata, H. Yonekura, T. Yoshimoto, H. Okamoto, Regulatory role of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) in insulin secretion by glucose in pancreatic beta cells, J. Biol. Chem. 270 (1995) 30045–30050. [14] B.N. Kennedy, T.S. Vihtelic, L. Checkley, K.T. Vaughan, D.R. Hyde, Isolation of a zebrafish rod opsin promoter to generate a transgenic zebrafish line expressing enhanced green fluorescent protein in rod photoreceptors, J. Biol. Chem. 276 (2001) 14037–14043. [15] B.E. Knox, C. Schlueter, B.M. Sanger, C.B. Green, J.C. Besharse, Transgene expression in Xenopus rods, FEBS Lett. 423 (1998) 117–121.

A.M. Ichsan et al. / Neuroscience Letters 379 (2005) 138–143 [16] B. K¨onig, A. Arendt, J.H. McDowell, M. Kahlert, P.A. Hargrave, K.P. Hofmann, Three cytoplasmic loops of rhodopsin interact with transducin, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 6878–6882. [17] T. Marquardt, P. Gruss, Generating neuronal diversity in the retina: one for nearly all, Trends Neurosci. 25 (2002) 32–37. [18] A.J. Mears, M. Kondo, P.K. Swain, Y. Takada, R.A. Bush, T.L. Saunders, P.A. Sieving, A. Swaroop, Nrl is required for rod photoreceptor development, Nat. Genet. 29 (2001) 447–452. [19] K.P. Mitton, P.K. Swain, S. Chen, S. Xu, D.J. Zack, A. Swaroop, The leucine zipper of NRL interacts with the CRX homeodomain, J. Biol. Chem. 275 (2000) 29794–29799. [20] K. Mori, P. Gehlbach, A. Ando, G. Dyer, E. Lipinsky, A.G. Chaudhry, S.F. Hackett, P.A. Campochiaro, Retina-specific expression of PDGF-B versus PDGF-A: vascular versus nonvascular proliferative retinopathy, Invest. Ophthalmol. Vis. Sci. 43 (2002) 2001–2006. [21] M. Nour, A.B. Quiambao, M.R. Al-Ubaidi, M.I. Naash, Absence of functional and structural abnormalities associated with expression of EGFP in the retina, Invest. Ophthalmol. Vis. Sci. 45 (2004) 15–22. [22] S. Rafii, D. Lyden, Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration, Nat. Med. 9 (2003) 702–712. [23] A. Rehemtulla, R. Warwar, R. Kumar, X. Ji, D.J. Zack, A. Swaroop, The basic motif-leucine zipper transcription factor Nrl can positively regulate rhodopsin gene expression, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 191–195.

143

[24] R.K. Sharma, T.E. O’Leary, C.M. Fields, D.A. Johnson, Development of the outer retina in the mouse, Dev. Brain Res. 145 (2003) 93–105. [25] A. Swaroop, Q.-L. Wang, W. Wu, J. Cook, C. Coats, S. Xu, S. Chen, D.J. Zack, P.A. Sieving, Leber congenital amaurosis caused by a homozygous mutation (R90W) in the homeodomain of the retinal transcription factor CRX: direct evidence for the involvement of CRX in the development of photoreceptor function, Hum. Mol. Genet. 8 (1999) 299–305. [26] R.T. Tzekov, Y. Liu, M.M. Sohocki, D.J. Zack, S.P. Daiger, J.R. Heckenlively, D.G. Birch, Autosomal dominant retinal degeneration and bone loss in patients with a 12-bp deletion in the CRX gene, Invest. Ophtalmol. Vis. Sci. 42 (2001) 1319–1327. [27] H. Yamada, E. Yamada, A. Ando, N. Esumi, N. Bora, J. Saikia, C.H. Sung, D.J. Zack, P.A. Campochiaro, Fibroblast growth factor-2 decreases hyperoxia-induced photoreceptor cell death in mice, Am. J. Pathol. 159 (2001) 1113–1120. [28] R.W. Young, Cell differentiation in the retina of the mouse, Anat. Rec. 212 (1985) 199–205. [29] D.J. Zack, J. Bennett, Y. Wang, C. Davenport, B. Klaunberg, J. Gearhart, J. Nathans, Unusual topography of bovine rhodopsin promoter-lacZ fusion gene expression in transgenic mouse retinas, Neuron 6 (1991) 187–199. [30] T. Zhang, Y.H. Tan, J. Fu, D. Lui, Y. Ning, F.R. Jirik, S. Brenner, B. Venkatesh, The regulation of retina specific expression of rhodopsin gene in vertebrates, Gene 313 (2003) 189–200.