February 2001
Volume 42, Issue 2
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Retinal Cell Biology  |   February 2001
Isolation and Characterization of a Zebrafish Homologue of the Cone Rod Homeobox Gene
Author Affiliations
  • Yuhui Liu
    From the Departments of Ophthalmology,
  • Yu-Chi Shen
    Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor; and the
  • Joshua S. Rest
    Department of Biology, University of Michigan College of Literature, Science, and the Arts, Ann Arbor.
  • Pamela A. Raymond
    Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor; and the
  • Donald J. Zack
    From the Departments of Ophthalmology,
    Neuroscience, and
    Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland; the
Investigative Ophthalmology & Visual Science February 2001, Vol.42, 481-487. doi:
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      Yuhui Liu, Yu-Chi Shen, Joshua S. Rest, Pamela A. Raymond, Donald J. Zack; Isolation and Characterization of a Zebrafish Homologue of the Cone Rod Homeobox Gene. Invest. Ophthalmol. Vis. Sci. 2001;42(2):481-487.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To isolate and characterize a zebrafish Crx homologue. Mammalian Crx genes are expressed specifically in photoreceptors and pinealocytes, regulate photoreceptor gene expression, are necessary for normal photoreceptor differentiation, and when mutated cause a variety of photoreceptor degenerations.

methods. A zebrafish retinal cDNA library was screened with a human CRX cDNA probe. Radiation hybrid mapping, Northern blot analysis, in situ hybridization, and transient transfection studies were performed using standard methods.

results. Based on amino acid sequence comparisons, zebrafish crx shows 50% identity with human CRX, and 85% identity in the homeodomain. A phylogenetic analysis indicates that zebrafish crx is most closely related to the mammalian Crx proteins, and more distantly related to the Otx proteins. Zebrafish crx maps between 49.6 and 54.5 cm from the top of linkage group LG05C, a map position consistent with the location of the mouse and human CRX genes. Northern blot analysis and in situ hybridization indicate that zebrafish crx is expressed in the retina and pineal gland. In adult zebrafish, crx is expressed by both rods and cones in the outer nuclear layer, and in cells in the outer zone of the inner nuclear layer, in the region occupied by bipolar cells. Similar to mammalian Crx, zebrafish crx interacts with neural retinal leucine zipper (Nrl) to activate, although weakly, rhodopsin promoter activity.

conclusions. Based on molecular phylogeny, chromosomal location, expression pattern, and ability to activate rhodopsin promoter activity in transient transfection assays, zebrafish crx appears to be an orthologue and functional homologue of mammalian Crx.

In recent years, a number of transcription factors have been identified that appear to be important players in the regulatory gene cascade that controls retinal development. 1 2 One of these, Crx, is a member of the otd/Otx family of paired-like homeobox genes. 3 4 5 6 The available mammalian Crx sequences (human, bovine, mouse, and rat) indicate that the protein is highly conserved, with 99% overall identity and 100% identity in the paired-like homeodomain. Crx expression is largely restricted to photoreceptor and pineal cells. It is the earliest known marker of photoreceptor identity in the developing retina. Its expression increases dramatically in mice at postnatal day 3, the approximate time of initiation of rhodopsin expression and outer segment morphogenesis. Crx binds to and transactivates the promoters of rhodopsin and several other photoreceptor and pineal-specific genes. 3 5 7 8 9 It acts synergistically with the bZip transcription factor neural retina leucine zipper (Nrl). 3 10 Mutant alleles of the CRX gene are associated with a variety of retinal degenerations, including cone–rod dystrophy, Leber congenital amaurosis, and retinitis pigmentosa. 4 11 12 13  
Our understanding of retinal development has benefited from studies of a number of complementary animal model systems (for reviews see References 2 14 15 16 ). A particularly powerful and increasing popular model is the zebrafish retina. 17 18 19 20 21 22 23 24 25 26 27 28 Among the advantages of this model are the rapid rate of embryonic development, the relative ease of identifying developmental mutants, the ability to modulate gene expression, the continuation of retinal neurogenesis in adult fish, and the ability of neural retina to regenerate in the adult. 
To extend these comparative evolutionary studies and also to take advantage of zebrafish genetics and the ability to manipulate gene expression in these animals, we have been attempting to clone and characterize zebrafish homologues of retinal transcription factors that have been identified in other systems. 18 29 Here we report the cloning and initial characterization of a zebrafish crx gene. Supplemental information related to this study is available on-line at http://www.ummz.umich.edu/birds/crx/ 
Materials and Methods
Isolation of Zebrafish crx cDNA
A λZAPII adult zebrafish retinal cDNA library (kindly provided by James Hurley) was screened at low stringency with a 897-bp probe (BamHI and EcoRV fragment) derived from the open reading frame (ORF) of human CRX. Resultant clones were sequenced in both directions using dideoxy sequencing reactions and a CEQ2000 automated DNA sequencer (Beckman, Berkeley, CA). Resultant sequences were compared with the available public databases using the BLAST algorithm at the National Center for Biotechnology Information. 30  
Northern Blot Analysis and In Situ Hybridization
Zebrafish (Danio rerio) were obtained from a local pet store and kept at 28.5°C in a 14-hour light/10-hour dark cycle. To harvest retinal tissues, fish were anesthetized with 0.02% 3-aminobenzoic acid ethyl ester (Sigma, St. Louis, MO), and then chilled on ice. For Northern blot analysis, total RNA was extracted using Trizol reagent (Gibco–Life Technologies, Rockville, MD) and separated on a 1% agarose 0.7-M formaldehyde gel. Each lane contained 10 μg of total RNA, which was transferred to the nylon membrane by passive capillary transfer and probed with α 32P-labeled, random primed crx cDNA. 
Zebrafish embryos were collected shortly after spawning and maintained at 28.5°C. To prevent development of melanin pigmentation, 0.2 mM 1-phenyl-2-thiourea (PTU; Sigma) was added to the water at 12 hours past fertilization (hpf). Adult tissues and embryos at various stages were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4°C or 1 hour at room temperature. 
In situ hybridization on wholemounts and cryosections was performed as described 27 using digoxigenin (DIG)-labeled RNA sense and antisense probes. Nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Roche Molecular Biochemicals, Indianapolis, IN) was used as the color substrate. No signals were detected with the sense RNA probes. 
All procedures using fish were performed in accord with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University Committee on Use and Care of Animals in Research at the University of Michigan. 
In Vitro Transient Transfection
The pcDNA-crx expression construct was generated by cloning the crx ORF into the pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA). Varying amounts of the pcDNA-crx expression construct (0.1–1.0 μg) were cotransfected with pBR130-luc (bovine rhodopsin promoter/luciferase reporter, 5.0 μg) with and without pED-bNrl (1.0 μg). The pCMV-LacZ plasmid was included to normalize transfection efficiency. Calcium phosphate-mediated transient transfection, luciferase, andβ -galactosidase assays were performed as previously described, 31 except that transfections with glycerol shock were performed with 50% confluent, 10-cm plates of the human embryonic kidney 293 cell strain grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum, and 1% penicillin-streptomycin (Gibco) and harvested 48 hours after transfection. Each transfection experiment was performed in triplicate. 
Molecular Phylogenetic and Character Analysis
We analyzed the phylogenetic relationship of DreCrx with two Crx and six Otx genes. The genes with their GenBank accession numbers are: human: HsaCrx (AF024711); mouse: MmuCrx (U77615), MmuOtx1 (X68883), 32 and MmuOtx2 (X68884) 32 ; zebrafish: DreOtx1 (D26172), DreOtx2 (D26173), and DreOtx3 (D226174); and amphioxus, BflOtx (AF043740). The alignment 33 and details of the Wilkinson character analysis 34 can be viewed at http://www.ummz.umich.edu/birds/crx/ 
Radiation Hybrid Mapping
A zebrafish radiation hybrid panel was analyzed by the polymerase chain reaction (PCR) method, according to the directions provided by the supplier (Research Genetics, Huntsville, AL). Primers that did not cross-react with rodent genomic DNA were designed from the 3′-untranslated region of crx (forward primer: 5′-TGAAGACTGTTCCTCTGTAATCC; reverse primer: 5′CAGATTCCCCTCCAGTTATGTGC. The results were submitted for analysis to the Tübingen map of the zebrafish genome (http://www.map.tuebingen.mpg.de/). 
Results
Isolation of a Zebrafish crx Homologue
Screening an adult zebrafish retinal cDNA library with a human CRX cDNA probe under low-stringency conditions identified several cDNA clones, the longest of which was 2.4 kb. Analysis of the 2.4-kb clone revealed an 843-bp ORF that encoded a protein with a paired-like homeodomain. Database searches demonstrated that the encoded protein was a novel member of the otd/Otx/Crx family. Based on the data shown below, we named this zebrafish cDNA crx. (In accord with the convention established by the Zebrafish Nomenclature Committee[ http://zfish.uoregon.edu/index.html], zebrafish genes cloned as homologues of mammalian genes are given the same name and abbreviation, in all lowercase, italic letters.) The amino terminal end of the deduced protein sequence contains a paired-like homeodomain that is 95% identical with mammalian Otx2 proteins and 85% identical with mammalian Crx proteins (Fig. 1A ). Similar to the other known Crx and Otx2 sequences, zebrafish crx has a WSP box in the middle of the sequence, which is 55% identical with mammalian Crx (Fig. 1B) , and it shares a conserved Otx-tail (75% identity with mammalian Crx) at the carboxyl terminal (Fig. 1C) . Additional sequences (YFSGLDPYLSPM and ALSPL) that are conserved among all known Crx proteins are also found in zebrafish crx, but not in Otx2 family members (Fig. 1D)
Zebrafish crx, a Member of the Crx Gene Family
To test the hypothesis of Crx monophyly (genes that are hypothesized to be derived from a common ancestor), we used the program MEAWILK to perform a statistical evaluation (Wilkinson support) based on the amino acid alignment (available at http://www-personal.umich.edu/∼gfred/). We evaluated four alternative, hypothesized monophyletic groups: 1) DreCrx, MmuCrx, HsaCrx (95, 199, 34, 0); 2) DreCrx, MmuOtx1, DreOtx1, DreOtx3 (67, 115, 25, 0); 3) DreCrx, DreOtx2, MmuOtx2 (78, 123, 21, 0), and 4) DreCrx, BflOtx (81, 114, 24, 2.7). Numbers in parenthesis indicate the number of characters (i.e., amino acid residues) with Wilkinson support for the hypothesis, the sum of weights of those characters, their percentage of the total possible weight, and the percentage of random hypotheses with comparable statistics, respectively. These values indicate that the best-supported hypothesis places DreCrx in a monophyletic group with mouse and human Crx. 
We also developed a phylogenetic hypothesis to describe the proposed relationships of these nine members of the Otx/otd/Crx family based on the amino acids in the alignment. As shown in Figure 2 , the most parsimonious tree contains three gene clades: Otx2, Otx1/3 and Crx. Zebrafish crx (Drecrx) is monophyletic with the Crx clade, with 96% bootstrap support and seven steps until the node collapses (see http://www.ummz.umich.edu/birds/crx/ for additional explanation). Therefore, based on both Wilkinson and phylogenetic evidence, we are confident in our assessment that Drecrx is an orthologue of the mammalian Crx genes. 
Zebrafish crx Maps to Linkage Group 05
To determine whether crx might be a candidate gene for known or future zebrafish mutations, its chromosomal position was mapped by radiation hybrid analysis. This analysis on the Research Genetics/Tübingen mapping panel placed crx between 49.6 and 54.5 cM from the top of zebrafish linkage group LG05, with a lod score of 18.16. The two nearest markers were AF006488 (zebrafish deltab) and fa28d05.1/AA606175 (an EST with sequence similarity to the M chain of human creatine kinase). A comparison of linkage relationships of genes mapped on a human radiation hybrid panel (LocusLink, National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/locuslink) and the orthologous zebrafish markers located on a meiotic mapping panel 35 (John Postlethwait, personal communication) suggested that the map position of zebrafish crx on LG05 is syntenic with human CRX at 19q13.3 and mouse Crx at 7 8.5 cM. For example, human muscle creatine kinase M chain (CKMM/AA606175) maps to chromosome 19q13.2-13.3 and the orthologous mouse gene maps to 74.5 cM. Other closely linked markers on human chromosome 19 include Fc (gamma) binding protein (D84239) and vaccinia-related kinase (VRK3/AB031052) at 19q13. On a meiotic mapping panel from homozygous diploid zebrafish embryos, an expressed sequence tag (EST; fb18c01/A1416123) with sequence similarity to vrk3 and an EST (zehn1245/AI617215) with sequence similarity to Fc (gamma) binding protein are both located on linkage group 05 (John Postlethwait, personal communication). There are no currently mapped zebrafish mutations near the crx locus. 
Zebrafish crx Expressed Specifically in the Neural Retina and Epiphysis
Northern blot analysis indicated that among the five adult zebrafish tissues we tested (eye without the lens, brain, heart, liver, and skeletal muscle), the crx probe detected a single band of approximately 2.4 kb that was restricted to the eye, although when the film was overexposed as shown in Figure 3 , RNA from liver revealed a very faint band. 
We next used in situ hybridization to determine the cell-type specificity and pattern of crx expression during development and in adult zebrafish. Expression of crx was first detected in the epiphysis (primordium of the pineal gland) in embryos at 17 hpf (data not shown). Pineal expression was strong at 24 to 31 hpf (Figs. A4 4C ), and it persisted up to at least 50 hpf, the oldest age at which the brain was examined (data not shown). 
Expression of crx in the retina was first observed at 24 hpf in a few cells in the ventronasal region immediately adjacent and nasal to the choroid fissure (data not shown). At this stage, all cells in the presumptive neural retina are mitotically active retinal progenitors. 22 The site at which crx expression first appears corresponds to the location of the earliest differentiating neurons, retinal ganglion cells, which appear a few hours later, 22 28 and the first photoreceptors, which do not begin to become postmitotic until approximately 43 hpf 22 27 and do not begin to express opsin until approximately 48 hpf. 27 By 31 hpf, the expression domain of crx was confined to a localized patch of undifferentiated, retinal progenitor cells in ventronasal retina (Figs. 4B 4C) . With further development, the crx expression domain gradually expanded into the dorsotemporal retina (Figs. 4C 4D 5A ). As differentiation of the retina progressed, crx became restricted to the outer layers of the retina (Fig. 5B) , so that by 52 hpf the differentiating ganglion cell layer was devoid of staining (Fig. 5E) . In the retina of adult zebrafish, crx continued to be expressed in the outer nuclear layer by both rods and cones (Fig. 5F) , which have distinct morphologies in the zebrafish retina. 36 37 Unidentified cells in the outer zone of the inner nuclear layer, in the region occupied by bipolar cells, also express crx (Fig. 5F)
To compare the expression of crx and otx2, we examined embryos at 49 to 52 hpf and found otx2 expression in retinal pigmented epithelium (RPE), the retinal germinal zone at the ciliary margin, the inner nuclear layer (INL) of the retina, and the forebrain and midbrain (Fig. 5D) . Of particular note is that otx2 was completely absent from the developing outer nuclear layer (ONL: Fig. 5D ), in contrast to the high levels of crx expressed in those regions (Fig. 5C)
Zebrafish crx and Bovine Nrl Act Synergistically on the Proximal Rhodopsin Promoter
We showed previously that mammalian Crx acts synergistically with Nrl to transactivate the rhodopsin promoter. 3 To test whether zebrafish crx has similar activity in this assay, we transiently expressed zebrafish crx in human embryonic kidney cells (293) together with a construct containing the bovine rhodopsin proximal promoter region (RPPR) fused to a luciferase reporter gene. In these experiments, pcDNA-crx by itself demonstrated only minimal transactivating activity (less than 50% increase even at the highest concentrations; Fig. 6A ). However, when pcDNA-crx was cotransfected with pED-bNrl, reporter activity increased up to threefold compared with pED-bNrl alone, and the fold increase in stimulation showed a dose-dependent relationship with the amount of pcDNA-crx (Fig. 6B) . Although this degree of activation is small, and considerably less than that observed with mammalian Crx, it was seen reproducibly in our assay system. 
Discussion
In this article, we describe the identification and characterization of a zebrafish ortholog of Crx. The only Crx genes identified to date are mammalian (human, bovine, mouse, and rat.) 3 4 5 6 Phylogenetic molecular analysis indicates that the Crx genes are a divergent subgroup of the Otx family. 38 In contrast to the high degree of sequence conservation (96% overall identity) among mammalian Crx homeodomain proteins, zebrafish crx exhibits only moderate overall amino acid sequence homology with the mammalian proteins: 49% identity over the entire amino acid sequence and 85% identity in the homeodomain, compared with 55% overall amino acid identity to human and mouse Otx2 and 95% identity in the homeodomain. However, in specific regions outside the homeodomain zebrafish crx is more similar to mammalian Crx genes than to Otx2, and a rigorous phylogenetic analysis indicated that zebrafish crx is monophyletic with the mammalian Crx proteins. Furthermore, the zebrafish gene mapped to a location syntenic with the chromosomal loci of mouse and human Crx genes, providing additional evidence to support the designation of this gene as a zebrafish crx orthologue. 
Consistent with the proposed phylogenetic molecular relationship of zebrafish crx and mammalian Crx genes, we found that their patterns of expression were more similar to each other 3 4 5 39 40 than to any members of the Otx gene family. 41 42 In zebrafish, the onset of crx expression at 17 hpf is well after the onset of otx1, otx2, and otx3 at approximately 6 to 8 hpf, 23 43 44 and the pattern of expression of crx does not correspond to any of the otx genes. Crx is first expressed in the zebrafish retinal neuroepithelium in a small patch of progenitor cells in the ventronasal region at 24 hpf, before the onset of neural differentiation, which occurs in the same location. 22 36 45 In the mouse, Crx is first expressed in the retinal neuroepithelium at E12.5, when cone genesis is underway, 3 and human CRX was first detected in the fetal retina at 10.6 weeks after conception, before the onset of cone genesis. 40 The onset of expression of zebrafish crx in the retinal neuroepithelium at 24 hpf is also well after the initial stages in the formation of the optic primordia (at 10–11 hpf in zebrafish embryos, 18 ), but well before the first photoreceptors become postmitotic. 
Experimental evidence suggests that crx may be involved in regulation of photoreceptor differentiation. 5 Compelling evidence in support of this hypothesis is the association of mutations in the human CRX gene with several forms of retinal dystrophies that involve photoreceptor dysgenesis or degeneration. 4 11 12 13 46 47 In addition, consistent with the human studies, outer segments do not develop in the photoreceptors in Crx null mice generated by gene targeting, and the photoreceptors eventually degenerate. 48 Expression levels of some photoreceptor-specific genes are reduced in Crx /− mice, whereas other genes are upregulated, suggesting that Crx may function as both a positive and a negative regulator of gene expression. These results demonstrate that in mice, Crx is not essential for cell fate determination in photoreceptors, but it is essential for their proper differentiation. 48 Although the function of crx in the developing zebrafish retina has not yet been demonstrated, the similarities in the pattern of expression and its developmental regulation in zebrafish, compared with mammalian Crx, suggest that crx may also play a role in the commitment and differentiation of retinal photoreceptors, and perhaps other retinal neurons, in zebrafish. The strong expression of crx in the inner nuclear layer, perhaps in bipolar cells, was originally unexpected based on initial reports that mammalian Crx appears to be expressed weakly, if at all, in few INL cells. 3 However, more recent immunocytochemical data have shown substantial expression of Crx protein in the bipolar cell layer in adult mouse retinas. 39  
Zebrafish crx is also expressed in the epiphysis-pineal gland, beginning at approximately 17 hpf, earlier than its first appearance in the neural retina. In mammals, Crx is thought to be involved in circadian behavior. For example, in Crx /− mice, the gross structure of the pineal gland is normal at 1 month, but expression levels of pineal-specific genes are decreased, and circadian entrainment is affected. 48 In adult rat pineal gland, Crx mRNA in pinealocytes displays a diurnal variation with peak levels in the dark phase. 9 In contrast, Crx shows constant levels of expression in the retina of rats maintained in a 12-hour:12-hour light–dark cycle, 6 indicating that the regulation of crx mRNA expression in the retina differs from the pineal gland in this species. We did not examine the circadian expression of crx in adult zebrafish, but our Northern blot analysis showed no detectable crx message in adult brain during the light phase of the diurnal cycle. 
Mammalian Crx can activate transcription of a number of photoreceptor-specific genes. 3 5 In this study we show that zebrafish crx alone did not transactivate gene expression from a bovine rhodopsin promoter construct in a transient transfection assay. However, a small synergistic effect was observed when crx was cotransfected with the bovine Nrl transcriptional regulator, which also binds to photoreceptor-specific regulatory elements. The absence of effect with crx alone, and the relatively weak synergistic effect with Nrl, may be due to divergence in the structure of the DNA-binding and activating domains of the zebrafish and mammalian genes. Future studies are needed to determine whether the effect we observed is biologically significant. 
Taken together, the molecular phylogeny and genomic map location, the comparisons of gene expression patterns, and the ability of crx to cooperate with Nrl to activate gene expression from the rhodopsin promoter, all suggest that zebrafish crx is orthologous to the mammalian Crx genes, and the function of zebrafish crx is likely to be more similar to mammalian Crx than to the other Otx family members. 
 
Figure 1.
 
Comparison of amino acid sequences of zebrafish crx with other vertebrate Crx and Otx2 proteins. Deduced amino acid comparisons are shown for the (A) homeodomain, (B) WSP box, (C) Otx tail, and (D) Crx-specific sequences of zebrafish (Danio rerio, Dre crx, Dre otx2), human (Homo sapiens, Hsa CRX, Hsa OTX2), mouse (Mus musculus, Mmu Crx, Mmu Otx2), and Xenopus laevis (Xle otx2) proteins.
Figure 1.
 
Comparison of amino acid sequences of zebrafish crx with other vertebrate Crx and Otx2 proteins. Deduced amino acid comparisons are shown for the (A) homeodomain, (B) WSP box, (C) Otx tail, and (D) Crx-specific sequences of zebrafish (Danio rerio, Dre crx, Dre otx2), human (Homo sapiens, Hsa CRX, Hsa OTX2), mouse (Mus musculus, Mmu Crx, Mmu Otx2), and Xenopus laevis (Xle otx2) proteins.
Figure 2.
 
Hypothesized phylogeny of Crx and Otx proteins. The most parsimonious phylogenetic tree using an exhaustive search in PAUP (phylogenetic analysis using parsimony; http://www.lms.si.edu/PAUP/about.html) for nine genes from four taxa (human, Homo sapiens, Hsa; mouse, Mus musculus, Mmu; zebrafish, Danio rerio, Dre;and amphioxus, Branchiostoma floridae, Bfl). Bootstrap percentage values based on 10,000 branch and bound repetitions are given above each branch, 49 and decay indices (the number of parsimony steps before the node collapses) 50 from TreeRot, 51 are in italics below the branch. The tree was rooted with an outgroup, the amphioxus Otx protein (BflOtx). The bar indicates a scale of 10 parsimony steps.
Figure 2.
 
Hypothesized phylogeny of Crx and Otx proteins. The most parsimonious phylogenetic tree using an exhaustive search in PAUP (phylogenetic analysis using parsimony; http://www.lms.si.edu/PAUP/about.html) for nine genes from four taxa (human, Homo sapiens, Hsa; mouse, Mus musculus, Mmu; zebrafish, Danio rerio, Dre;and amphioxus, Branchiostoma floridae, Bfl). Bootstrap percentage values based on 10,000 branch and bound repetitions are given above each branch, 49 and decay indices (the number of parsimony steps before the node collapses) 50 from TreeRot, 51 are in italics below the branch. The tree was rooted with an outgroup, the amphioxus Otx protein (BflOtx). The bar indicates a scale of 10 parsimony steps.
Figure 3.
 
Northern blot analysis of crx expression in adult zebrafish. (A) Ethidium bromide–stained membrane, indicating that an approximately equal amount of RNA was present in each lane. (B) Northern blot analysis of zebrafish total RNA (10 μg/lane) extracted from lane 1: liver; lane 2: heart; lane 3: muscle; lane 4: brain; and lane 5: eye without lens.
Figure 3.
 
Northern blot analysis of crx expression in adult zebrafish. (A) Ethidium bromide–stained membrane, indicating that an approximately equal amount of RNA was present in each lane. (B) Northern blot analysis of zebrafish total RNA (10 μg/lane) extracted from lane 1: liver; lane 2: heart; lane 3: muscle; lane 4: brain; and lane 5: eye without lens.
Figure 4.
 
In situ hybridization analysis of crx expression in zebrafish embryos. Embryos were processed intact for in situ hybridization, then examined as wholemounts (C) or sectioned sagittally (A, B, and D). Nasal is to the right in all panels. (A) Expression of crx in the epiphysis (arrow) at 24 hpf. e, eye; y, yolk. (B) Expression of crx in the retina at 31 hpf (arrow); choroid fissure (*). Inset: A ventral view of crx expression as seen in a wholemount. le, lens. (C) Lateral view of an embryo at 31 hpf showing expression of crx in the ventronasal retinal patch (large arrow) and the epiphysis (small arrow). (D) Expression of crx in the nasal retina of a 34-hpf embryo. Bars, (A) 50 μm; (B) 25 μm; (C, D) 12.5 μm.
Figure 4.
 
In situ hybridization analysis of crx expression in zebrafish embryos. Embryos were processed intact for in situ hybridization, then examined as wholemounts (C) or sectioned sagittally (A, B, and D). Nasal is to the right in all panels. (A) Expression of crx in the epiphysis (arrow) at 24 hpf. e, eye; y, yolk. (B) Expression of crx in the retina at 31 hpf (arrow); choroid fissure (*). Inset: A ventral view of crx expression as seen in a wholemount. le, lens. (C) Lateral view of an embryo at 31 hpf showing expression of crx in the ventronasal retinal patch (large arrow) and the epiphysis (small arrow). (D) Expression of crx in the nasal retina of a 34-hpf embryo. Bars, (A) 50 μm; (B) 25 μm; (C, D) 12.5 μm.
Figure 5.
 
Expression of crx and otx2 at later stages of development and in adult zebrafish. Embryos were processed intact for in situ hybridization, then examined as wholemounts (A), sectioned sagittally (B), or transversely (C, D). Adult retina was first cryosectioned and then processed for in situ hybridization (E, F). (A) Ventral view of crx expression at 40 hpf. (B) Embryonic eye at 37 hpf; gcl, ganglion cell layer; le, lens. (*), Choroid fissure. (C) An embryo at 52 hpf hybridized with crx probe. Dorsal is up. (D) An embryo at 49 hpf hybridized with otx2 probe. Arrows: Germinal zone at the ciliary margin of the retina; (*), cells in the inner nuclear layer. ot, optic tectum. (E) The eye of an embryo at 52 hpf (without PTU treatment). (F) Retina of adult zebrafish; cones (c) and rods (r). Inset: ONL at higher magnification. Cone nuclei were elongated and formed a single row adjoining the RPE, whereas the rod nuclei were rounded and located vitread to the cones. All photoreceptors expressed crx. Bars, (A through D, F) 25 μm; (E) 50 μm.
Figure 5.
 
Expression of crx and otx2 at later stages of development and in adult zebrafish. Embryos were processed intact for in situ hybridization, then examined as wholemounts (A), sectioned sagittally (B), or transversely (C, D). Adult retina was first cryosectioned and then processed for in situ hybridization (E, F). (A) Ventral view of crx expression at 40 hpf. (B) Embryonic eye at 37 hpf; gcl, ganglion cell layer; le, lens. (*), Choroid fissure. (C) An embryo at 52 hpf hybridized with crx probe. Dorsal is up. (D) An embryo at 49 hpf hybridized with otx2 probe. Arrows: Germinal zone at the ciliary margin of the retina; (*), cells in the inner nuclear layer. ot, optic tectum. (E) The eye of an embryo at 52 hpf (without PTU treatment). (F) Retina of adult zebrafish; cones (c) and rods (r). Inset: ONL at higher magnification. Cone nuclei were elongated and formed a single row adjoining the RPE, whereas the rod nuclei were rounded and located vitread to the cones. All photoreceptors expressed crx. Bars, (A through D, F) 25 μm; (E) 50 μm.
Figure 6.
 
Synergistic transactivation on the rhodopsin promoter by crx and Nrl. Human embryonic kidney 293 cells were transfected with 5 μg of the bovine rhodopsin proximal promoter-luciferase construct with (left) or without (right) the Nrl expression plasmid pED-Nrl (1 μg) and with or without the indicated amount of zcrxl expression plasmid pcDNA3-zcrxl. Fold stimulation represents the ratio of the corrected luciferase activity with pED-Nrl and pcDNA3-crx to the activity of bovine rhodopsin proximal promoter-luciferase construct only. The values shown represent the means of three independent experiments performed in triplicate ± SE.
Figure 6.
 
Synergistic transactivation on the rhodopsin promoter by crx and Nrl. Human embryonic kidney 293 cells were transfected with 5 μg of the bovine rhodopsin proximal promoter-luciferase construct with (left) or without (right) the Nrl expression plasmid pED-Nrl (1 μg) and with or without the indicated amount of zcrxl expression plasmid pcDNA3-zcrxl. Fold stimulation represents the ratio of the corrected luciferase activity with pED-Nrl and pcDNA3-crx to the activity of bovine rhodopsin proximal promoter-luciferase construct only. The values shown represent the means of three independent experiments performed in triplicate ± SE.
The authors thank Anand Swaroop for providing pED-bNrl, George Estabrook and Michael Frohlich for valuable discussions and assistance with the phylogenetic analysis, and John Postlethwait for graciously providing an analysis of the syntenic regions of zebrafish, mouse and human chromosomes in the region of the crx locus. 
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Figure 1.
 
Comparison of amino acid sequences of zebrafish crx with other vertebrate Crx and Otx2 proteins. Deduced amino acid comparisons are shown for the (A) homeodomain, (B) WSP box, (C) Otx tail, and (D) Crx-specific sequences of zebrafish (Danio rerio, Dre crx, Dre otx2), human (Homo sapiens, Hsa CRX, Hsa OTX2), mouse (Mus musculus, Mmu Crx, Mmu Otx2), and Xenopus laevis (Xle otx2) proteins.
Figure 1.
 
Comparison of amino acid sequences of zebrafish crx with other vertebrate Crx and Otx2 proteins. Deduced amino acid comparisons are shown for the (A) homeodomain, (B) WSP box, (C) Otx tail, and (D) Crx-specific sequences of zebrafish (Danio rerio, Dre crx, Dre otx2), human (Homo sapiens, Hsa CRX, Hsa OTX2), mouse (Mus musculus, Mmu Crx, Mmu Otx2), and Xenopus laevis (Xle otx2) proteins.
Figure 2.
 
Hypothesized phylogeny of Crx and Otx proteins. The most parsimonious phylogenetic tree using an exhaustive search in PAUP (phylogenetic analysis using parsimony; http://www.lms.si.edu/PAUP/about.html) for nine genes from four taxa (human, Homo sapiens, Hsa; mouse, Mus musculus, Mmu; zebrafish, Danio rerio, Dre;and amphioxus, Branchiostoma floridae, Bfl). Bootstrap percentage values based on 10,000 branch and bound repetitions are given above each branch, 49 and decay indices (the number of parsimony steps before the node collapses) 50 from TreeRot, 51 are in italics below the branch. The tree was rooted with an outgroup, the amphioxus Otx protein (BflOtx). The bar indicates a scale of 10 parsimony steps.
Figure 2.
 
Hypothesized phylogeny of Crx and Otx proteins. The most parsimonious phylogenetic tree using an exhaustive search in PAUP (phylogenetic analysis using parsimony; http://www.lms.si.edu/PAUP/about.html) for nine genes from four taxa (human, Homo sapiens, Hsa; mouse, Mus musculus, Mmu; zebrafish, Danio rerio, Dre;and amphioxus, Branchiostoma floridae, Bfl). Bootstrap percentage values based on 10,000 branch and bound repetitions are given above each branch, 49 and decay indices (the number of parsimony steps before the node collapses) 50 from TreeRot, 51 are in italics below the branch. The tree was rooted with an outgroup, the amphioxus Otx protein (BflOtx). The bar indicates a scale of 10 parsimony steps.
Figure 3.
 
Northern blot analysis of crx expression in adult zebrafish. (A) Ethidium bromide–stained membrane, indicating that an approximately equal amount of RNA was present in each lane. (B) Northern blot analysis of zebrafish total RNA (10 μg/lane) extracted from lane 1: liver; lane 2: heart; lane 3: muscle; lane 4: brain; and lane 5: eye without lens.
Figure 3.
 
Northern blot analysis of crx expression in adult zebrafish. (A) Ethidium bromide–stained membrane, indicating that an approximately equal amount of RNA was present in each lane. (B) Northern blot analysis of zebrafish total RNA (10 μg/lane) extracted from lane 1: liver; lane 2: heart; lane 3: muscle; lane 4: brain; and lane 5: eye without lens.
Figure 4.
 
In situ hybridization analysis of crx expression in zebrafish embryos. Embryos were processed intact for in situ hybridization, then examined as wholemounts (C) or sectioned sagittally (A, B, and D). Nasal is to the right in all panels. (A) Expression of crx in the epiphysis (arrow) at 24 hpf. e, eye; y, yolk. (B) Expression of crx in the retina at 31 hpf (arrow); choroid fissure (*). Inset: A ventral view of crx expression as seen in a wholemount. le, lens. (C) Lateral view of an embryo at 31 hpf showing expression of crx in the ventronasal retinal patch (large arrow) and the epiphysis (small arrow). (D) Expression of crx in the nasal retina of a 34-hpf embryo. Bars, (A) 50 μm; (B) 25 μm; (C, D) 12.5 μm.
Figure 4.
 
In situ hybridization analysis of crx expression in zebrafish embryos. Embryos were processed intact for in situ hybridization, then examined as wholemounts (C) or sectioned sagittally (A, B, and D). Nasal is to the right in all panels. (A) Expression of crx in the epiphysis (arrow) at 24 hpf. e, eye; y, yolk. (B) Expression of crx in the retina at 31 hpf (arrow); choroid fissure (*). Inset: A ventral view of crx expression as seen in a wholemount. le, lens. (C) Lateral view of an embryo at 31 hpf showing expression of crx in the ventronasal retinal patch (large arrow) and the epiphysis (small arrow). (D) Expression of crx in the nasal retina of a 34-hpf embryo. Bars, (A) 50 μm; (B) 25 μm; (C, D) 12.5 μm.
Figure 5.
 
Expression of crx and otx2 at later stages of development and in adult zebrafish. Embryos were processed intact for in situ hybridization, then examined as wholemounts (A), sectioned sagittally (B), or transversely (C, D). Adult retina was first cryosectioned and then processed for in situ hybridization (E, F). (A) Ventral view of crx expression at 40 hpf. (B) Embryonic eye at 37 hpf; gcl, ganglion cell layer; le, lens. (*), Choroid fissure. (C) An embryo at 52 hpf hybridized with crx probe. Dorsal is up. (D) An embryo at 49 hpf hybridized with otx2 probe. Arrows: Germinal zone at the ciliary margin of the retina; (*), cells in the inner nuclear layer. ot, optic tectum. (E) The eye of an embryo at 52 hpf (without PTU treatment). (F) Retina of adult zebrafish; cones (c) and rods (r). Inset: ONL at higher magnification. Cone nuclei were elongated and formed a single row adjoining the RPE, whereas the rod nuclei were rounded and located vitread to the cones. All photoreceptors expressed crx. Bars, (A through D, F) 25 μm; (E) 50 μm.
Figure 5.
 
Expression of crx and otx2 at later stages of development and in adult zebrafish. Embryos were processed intact for in situ hybridization, then examined as wholemounts (A), sectioned sagittally (B), or transversely (C, D). Adult retina was first cryosectioned and then processed for in situ hybridization (E, F). (A) Ventral view of crx expression at 40 hpf. (B) Embryonic eye at 37 hpf; gcl, ganglion cell layer; le, lens. (*), Choroid fissure. (C) An embryo at 52 hpf hybridized with crx probe. Dorsal is up. (D) An embryo at 49 hpf hybridized with otx2 probe. Arrows: Germinal zone at the ciliary margin of the retina; (*), cells in the inner nuclear layer. ot, optic tectum. (E) The eye of an embryo at 52 hpf (without PTU treatment). (F) Retina of adult zebrafish; cones (c) and rods (r). Inset: ONL at higher magnification. Cone nuclei were elongated and formed a single row adjoining the RPE, whereas the rod nuclei were rounded and located vitread to the cones. All photoreceptors expressed crx. Bars, (A through D, F) 25 μm; (E) 50 μm.
Figure 6.
 
Synergistic transactivation on the rhodopsin promoter by crx and Nrl. Human embryonic kidney 293 cells were transfected with 5 μg of the bovine rhodopsin proximal promoter-luciferase construct with (left) or without (right) the Nrl expression plasmid pED-Nrl (1 μg) and with or without the indicated amount of zcrxl expression plasmid pcDNA3-zcrxl. Fold stimulation represents the ratio of the corrected luciferase activity with pED-Nrl and pcDNA3-crx to the activity of bovine rhodopsin proximal promoter-luciferase construct only. The values shown represent the means of three independent experiments performed in triplicate ± SE.
Figure 6.
 
Synergistic transactivation on the rhodopsin promoter by crx and Nrl. Human embryonic kidney 293 cells were transfected with 5 μg of the bovine rhodopsin proximal promoter-luciferase construct with (left) or without (right) the Nrl expression plasmid pED-Nrl (1 μg) and with or without the indicated amount of zcrxl expression plasmid pcDNA3-zcrxl. Fold stimulation represents the ratio of the corrected luciferase activity with pED-Nrl and pcDNA3-crx to the activity of bovine rhodopsin proximal promoter-luciferase construct only. The values shown represent the means of three independent experiments performed in triplicate ± SE.
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