May 2011
Volume 52, Issue 6
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Retina  |   May 2011
Knockdown of the Zebrafish Ortholog of the Retinitis Pigmentosa 2 (RP2) Gene Results in Retinal Degeneration
Author Affiliations & Notes
  • Xinhua Shu
    From the Departments of Biological and Biomedical Sciences and
    Vision Sciences, Glasgow Caledonian University, Glasgow, United Kingdom;
    MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom; and
  • Zhiqiang Zeng
    MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom; and
  • Philippe Gautier
    MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom; and
  • Alan Lennon
    MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom; and
  • Milica Gakovic
    MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom; and
  • Michael E. Cheetham
    UCL Institute of Ophthalmology, London, United Kingdom.
  • E. Elizabeth Patton
    MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom; and
  • Alan F. Wright
    MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom; and
  • *Each of the following is a corresponding author: Xinhua Shu, Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow, United Kingdom; xinhua.shu@gcu.ac.uk. Alan Wright, MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Edinburgh, United Kingdom; alan.wright@hgu.mrc.ac.uk
Investigative Ophthalmology & Visual Science May 2011, Vol.52, 2960-2966. doi:10.1167/iovs.10-6800
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      Xinhua Shu, Zhiqiang Zeng, Philippe Gautier, Alan Lennon, Milica Gakovic, Michael E. Cheetham, E. Elizabeth Patton, Alan F. Wright; Knockdown of the Zebrafish Ortholog of the Retinitis Pigmentosa 2 (RP2) Gene Results in Retinal Degeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(6):2960-2966. doi: 10.1167/iovs.10-6800.

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

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Abstract

Purpose.: The authors investigated the expression and function of the zebrafish ortholog of the retinitis pigmentosa 2 (RP2) gene.

Methods.: Zebrafish RP2 (ZFRP2) cDNA was isolated from adult eye mRNA by reverse transcription–polymerase chain reaction (RT-PCR). Gene expression was examined by RT-PCR. The deduced peptide sequence was aligned with RP2 orthologues from different species. Translational suppression (knockdown) of zebrafish RP2 was carried out by antisense morpholino-injection. The phenotype of ZFRP2 knockdown morphants was characterized by immunohistology and histology. Human wild-type and mutant RP2 mRNAs were coinjected with ZFRP2 morpholinos to test whether human RP2 mRNA could rescue ZFRP2 knockdown phenotypes.

Results.: ZFRP2 encodes a protein of 376 amino acids containing an N-terminal tubulin folding cofactor C-like domain and a C-terminal nucleoside diphosphate kinase-like domain. It shares 63% to 65% amino acid identity with human, mouse and bovine RP2. RP2 is expressed at the earliest stages of zebrafish development and persists into adulthood. Knockdown of RP2 in zebrafish causes a curved body axis and small eye phenotype, associated with increased cell death throughout the retina. Human wild-type RP2 mRNA could rescue the body curvature phenotype of ZFRP2 morphants, and the eye size of the resultant morphants was significantly increased over that of morphants in which ZFRP2 had been depleted.

Conclusions.: Zebrafish RP2 is widely expressed throughout development. ZFRP2 knockdown caused retinal degeneration in zebrafish. Human RP2 could partially rescue the small eye phenotype of ZFRP2 morphants.

Retinitis pigmentosa (RP; Mendelian Inheritance in Man #268000) is one of the most heterogeneous genetic disorders known in humans. 1 Most cases are presumed to result from a mutation in one or more genes showing autosomal dominant, recessive, X-linked, or mitochondrial inheritance, although about half of all cases appear to be sporadic. X-linked RP (XLRP) is one of the most severe forms of human retinal degeneration, as determined by age of onset and progression, and it accounts for 6% to 20% of all RP cases. 2 At least six XLRP loci—RP2, RP3, RP6, RP23, RP24, and RP34 (http:www.sph.uth.tmc.edu/retnet/)—have been mapped, but only two major genes have been identified: RPGR, accounting for 70% to 80% of XLRP, and RP2, accounting for up to 15% of XLRP. 3 5 More than 53 mutations have been reported in RP2, most of which are premature stop or frame-shift truncation mutations, which account for two-thirds of pathogenic RP2 variants. Point mutations are clustered in the tubulin-specific chaperone protein cofactor (TBCC) homology domain. 5 7  
The human RP2 gene encompasses five exons, encoding a 350-amino acid protein. The RP2 protein is widely expressed, though at low levels in most human tissues. The N terminus of RP2 shares homology with TBCC, whereas the C terminus consists of a domain with similarity to nucleoside diphosphate kinases. 5,8 RP2 has been reported to be targeted to the cytoplasmic face of the plasma membrane by myristoylation and palmitoylation of its N-terminal glycine 2 and cysteine 3 amino acids, respectively. 9 RP-causing mutations at the N terminus have been shown to abolish plasma membrane localization of RP2, 9 11 indicating that posttranslational modifications are vital for the correct localization and function of the protein. The first identified interaction partner is the ADP ribosylation factor (Arf)-like protein Arl3, a member of the Arf subfamily of small G proteins. RP2 binds to the GTP-bound, but not to the GDP-bound, form of Arl3 and was initially thought to be an effector, but detailed structural and biochemical analyses showed that RP2 functions as a GTPase activating protein for Arl3. 8,12 Recent work has demonstrated that RP2 also localizes to the basal body, the Golgi complex, and the periciliary ridge of photoreceptors and that targeting of RP2 to the ciliary base is dependent on N-terminal myristoylation. 13 Depletion of RP2 in cell lines results in the dispersal of vesicles that appear to be cycling cargo from the Golgi complex to the cilium, suggesting a role for RP2 in maintaining photoreceptor pericentriolar traffic. 13,14  
Because of their rapid and external development, coupled with their optical clarity during embryogenesis, zebrafish have been widely used as a model to provide fundamental insight into the development of vertebrate animals and, more specifically, to understand the mechanisms of visual disorders. 15,16 To use zebrafish as a model for investigating RP2 function, we cloned the zebrafish RP2 ortholog and examined its expression during development and in adult tissues. We also characterized the phenotype of morphants after antisense morpholino knockdown (translational suppression) of RP2
Materials and Methods
Animals and Tissues
Zebrafish of the AB strain were maintained at 28.5°C on a 14-hour light/10-hour dark cycle. Fertilized eggs were obtained and grown in incubators, and embryos were staged as described. 17 Brain, eye, kidney, liver, heart, muscle, skin, ovary, and testis were dissected from adult zebrafish. Experimental procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Total RNA Extraction and RT-PCR
RNAs from zebrafish embryos at different stages of development or from different tissues of adult zebrafish were extracted using a purification kit (RNeasy Mini Kit; Qiagen, Valencia, CA) according to the manufacturer's instruction. Two micrograms of the resultant RNA sample was reverse transcribed with random primers using a cDNA synthesis kit (Transcriptor High Fidelity; Roche, Indianapolis, IN). The cDNA was then amplified using recombinant polymerase (Platinum Taq DNA; Invitrogen, Carlsbad, CA) as previous described. 18 PCR primers were manufactured by Sigma-Aldrich (St. Louis, MO). 
Identification of Zebrafish RP2 Orthologs
The human RP2 amino acid sequence was compared with the translated protein sequences from zebrafish genomic sequence data using a basic local alignment search tool (BLAST). Gene prediction was made using GeneWise with the human RP2 protein sequence as template. The prediction was subsequently manually curated and refined using other sources of information available: Ensembl gene prediction models, de novo GenScan prediction, expressed sequence tags, cDNA and protein homologies. On the basis of the resultant sequences, primers were designed to amplify the complete sequence of the zebrafish RP2 gene. Primer sequences are available on request. PCR products were analyzed by agarose gel electrophoresis and sequenced. 
Immunohistochemistry and Histology
Eyes from adult fish were fixed in 4% paraformaldehyde in PBS overnight at 4°C, washed with PBS twice, then cryoprotected in 5% sucrose/PBS at room temperature for 4 hours and in 20% sucrose/PBS at 4°C overnight. Eyes were embedded, and 10-μm-thick sections were cut. Sections were blocked in 10% FCS, 2% BSA, and 0.1% Triton X-100 in PBS for 1 hour, then incubated for 1 hour with primary antibodies diluted in blocking solution. After washing, the sections were incubated with secondary antibodies. Sections were mounted in medium (Vectashield; Vector Laboratories, Burlingame, CA) containing DAPI (1 μg/mL). Images were captured using a fluorescence microscope (Axioplan II; Zeiss, Thornwood, NY) and analyzed using imaging software (IPLab; Spectra Services, Ontario, NY). 
For rhodopsin or Zpr-1 staining, control and zebrafish RP2 morpholino-injected morphants (72 hours postfertilization [hpf]) were fixed in 4% paraformaldehyde in PBS, dehydrated, embedded in paraffin, and sectioned at 7-μm thickness. The sections were dewaxed, blocked with 2% BSA-PBS, incubated with anti–rhodopsin antibody or anti–Zpr-1 antibody, and subsequently incubated with Texas Red-conjugated secondary antibody. Sections were mounted, and images were captured and analyzed (IPLab software; Spectra Services). Hematoxylin and eosin staining for those sections from the 72-hpf embryos was performed using standard conditions. 
Measure of Eye Size
Embryos were dechorionated and anesthetized with Tracine. The phenotypes were recorded by photographing, and prints used for eye measurement. Eye measurements were taken from the anterior to the posterior edge. Significant differences between groups were analyzed by Student's t-test (Prism; GraphPad, San Diego, CA). 
Morpholino and mRNA Injection
Morpholino antisense oligonucleotides (MOs) targeted to the translational start site (MO_ATG) or to a splicing site (MO_Spl) were used. MO_Spl was targeted to the splice site at the boundary of exon2 and intron2, designed and synthesized by Gene Tools (Philomath, OR). The sequence for the MO_ATG was 5′-TGGAGAAGAAGCACCCCATTTATGG-3′; for the MO_Spl, the sequence was 5′ GCG TCA CAA ATA AGT TCT AAC CTC A 3′; for the standard control MO (CMO), the sequence was 5′-CCTCTTACCTCAGTTACAATTTATA-3′. All MOs were solubilized in water and diluted to the required concentration for embryo microinjections. For the phenotypic rescue, cDNAs including human wild-type RP2, a rare RP2 variant, and RP2 mutants causing XLRP (see Fig. 6A) were cloned into the pCS2+ vector. In vitro transcription of synthetic capped mRNA was performed using a capped RNA transcription kit (SP6 mMESSAGE mMACHINE; Ambion, Austin, TX) according to the manufacturer's instruction. Two nanoliters of MO or mixed MO/mRNA was injected into each one- to two-cell-stage embryo. The final concentrations of MO and mRNA were 125 μM and 400 ng/μL, respectively. 
Detection of Cell Death
Apoptotic cells in live embryos up to age 48 hpf were detected by incubating the embryos in acridine orange solution (5 μg/mL) 19 for 30 minutes at room temperature, and then were washed briefly five times in embryo medium. Cells loaded with dye were visualized immediately using a fluorescence microscope (Axioplan II; Zeiss). Apoptotic cells in the retina of 72 hpf morphants were detected by a fluorometric TUNEL system (DeadEND; Promega, Madison, WI). Sections from 4% paraformaldehyde-fixed and paraffin-embedded morphants were washed with PBS, permeabilized with 20 μg/mL proteinase K, fixed again with 4% paraformaldehyde in PBS, and then equilibrated with equilibration buffer. The sections were labeled with TdT reaction mix for 60 minutes at 37°C, and the reactions were stopped with 2× SSC. After washing with PBS three times (5 minutes each), the specimens were mounted in medium (Vectashield; Vector Laboratories) containing DAPI. Images were captured using a fluorescence microscope (Axioplan II; Zeiss) and analyzed using imaging software (IPLab; Spectra Services). 
Results
Cloning and Sequence Analysis of Zebrafish RP2 Ortholog
A BLAST search of the Ensembl zebrafish databases was performed using the human RP2 protein sequence (NP_008846) to identify zebrafish RP2. It showed there is one copy of this gene on chromosome 6. The genomic sequence around the putative zebrafish RP2 gene was used to identify the predicted zebrafish protein and coding sequence with human RP2 protein as a template in the GeneWise program. On the basis of these predictions, specific primers were designed to clone zebrafish RP2 cDNA by RT-PCR. The zebrafish RP2 (ZFRP2) transcript encodes an open reading frame of 376 amino acids and consists of at least five exons, spanning approximately 10.2 kb. Alignment of the ZFRP2 protein sequence with orthologs from a range of species shows that the deduced amino acid sequence is highly homologous to the Fugu fish (81% identity) and stickleback (78% identity), followed by possum, chicken, frog (66% identity), human, macaque (65% identity), cow, rabbit, lizard (64% identity), and mouse (63% identity). There is relatively low identity between the zebrafish and invertebrate orthologs of RP2 (48% identity with sea anemone, 38% identity with Ciona, and 25% identity with worm; Fig. 1). 
Figure 1.
 
Conservation of RP2 across different species. Accession numbers (Ensembl Ids where accession numbers are unavailable) for the amino acid sequence of each species are: HQ641392, zebrafish (Danio rerio); NP_008846, human (Homo sapiens); ENSMMUP 00000030304, macaque (Macaca mulatta); ENSOCUP00000004897, rabbit (Oryctolagus cuniculus); AAI53223.1, cow (Bos taurus); NP_598430, mouse (Mus musculus); XP_001362637, possum (Monodelphis domestica); NP_001008680, chick (Gallus gallus); ENSACAP 00000011842, lizard (Anolis carolinensis); XP_002937023, frog (Xenopus tropicalis); ENSGACP00000018275, stickleback (Gasterosteus aculeatus); ENSTRUP00000027655, fugu (Takifugu rubripes); XP_001628286, sea anemone (Nematostella vectensis); XP_002121234, ciona (Ciona intestinalis); NP_500127, nematode (Caenorhabditis elegans).
Figure 1.
 
Conservation of RP2 across different species. Accession numbers (Ensembl Ids where accession numbers are unavailable) for the amino acid sequence of each species are: HQ641392, zebrafish (Danio rerio); NP_008846, human (Homo sapiens); ENSMMUP 00000030304, macaque (Macaca mulatta); ENSOCUP00000004897, rabbit (Oryctolagus cuniculus); AAI53223.1, cow (Bos taurus); NP_598430, mouse (Mus musculus); XP_001362637, possum (Monodelphis domestica); NP_001008680, chick (Gallus gallus); ENSACAP 00000011842, lizard (Anolis carolinensis); XP_002937023, frog (Xenopus tropicalis); ENSGACP00000018275, stickleback (Gasterosteus aculeatus); ENSTRUP00000027655, fugu (Takifugu rubripes); XP_001628286, sea anemone (Nematostella vectensis); XP_002121234, ciona (Ciona intestinalis); NP_500127, nematode (Caenorhabditis elegans).
The proposed motif for myristoylation and palmitoylation at the N-terminal of RP2 is conserved in the predicted ZFRP2 sequence and in all species except for nematodes, which have serine rather than glycine at residue 2. Serine 6, which was deleted by the pathogenic mutation ΔS6 and disrupted acyl-mediated targeting of RP2 to the plasma membrane, was conserved across all species examined. Most residues affected by XLRP disease-associated RP2 missense mutations reported to date 6,7 are conserved across species; the exceptions are those occurring at residues G2, C108, L188, and R211. Residues G2 and L188 are conserved in all species except the worm, C108 is conserved in all species except the fish, and R282 is not conserved beyond human, macaque, and rabbit. In contrast to a single report suggesting R282W to be a pathogenic mutation, 5 the R282W change appears to be a polymorphism 2,20,21 present at an allele frequency of 3.3% in Europeans (Hapmap snp rs1805147). 
Expression of Zebrafish RP2
The temporal and spatial expression pattern of the ZFRP2 gene during embryogenesis was examined by RT-PCR, using primers spanning exons 1 and 2. Total RNA was prepared from oocytes, fertilized eggs, blastula-, gastrula-, segmentation-, pharyngula-stage embryos, and larvae. The ZFRP2 mRNA was readily detected at the time of fertilization and persisted during gastrulation and through the tail bud and larval stages (Fig. 2A). ZFRP2 expression in adult tissues was examined by RT-PCR in total RNA isolated from kidney, muscle, ovary, liver, intestine, brain, eye, heart, skin and testis. RP2 expression was readily detected in all tissues and was not enriched in the retina, which is consistent with early reports of RP2 expression in mouse and human adult tissues. 5,9 To investigate the expression of ZFRP2 protein in adult zebrafish eyes, we carried out immunostaining with a polyclonal antibody (HPA000234–100UL; Sigma-Aldrich) raised against a human RP2 peptide, whose sequence is conserved between human and zebrafish RP2. Specific immunolabeling was detected in both rod and cone photoreceptors extending from the tip of the outer segment to the inner segment and synapses of the outer nuclear layer (ONL), with some weak labeling in the outer plexiform layer (OPL) and inner plexiform layer (IPL; Figs. 2C, 2D). The results are consistent with the localization of human and mouse RP2 protein in retina. 14,22  
Figure 2.
 
Expression and localization of zebrafish RP2 (ZFRP2). (A) Temporal expression of ZFRP2 gene in the zebrafish development detected by RT-PCR. (B) ZFRP2 expression in different tissues detected by RT-PCR. (C, D) ZFRP2 localization in adult zebrafish photoreceptor, labeled with anti–RP2 antibody (C, D, green). The outer segments of rods were labeled with anti–rhodopsin antibody (C, red), and cone cells were labeled with anti–ZPR-1 antibody (D, red). Nuclear layers were labeled with DAPI (C, D, blue). Magnification, ×20.
Figure 2.
 
Expression and localization of zebrafish RP2 (ZFRP2). (A) Temporal expression of ZFRP2 gene in the zebrafish development detected by RT-PCR. (B) ZFRP2 expression in different tissues detected by RT-PCR. (C, D) ZFRP2 localization in adult zebrafish photoreceptor, labeled with anti–RP2 antibody (C, D, green). The outer segments of rods were labeled with anti–rhodopsin antibody (C, red), and cone cells were labeled with anti–ZPR-1 antibody (D, red). Nuclear layers were labeled with DAPI (C, D, blue). Magnification, ×20.
Phenotype of Morpholino-Mediated Zebrafish RP2 Knockdown
To evaluate whether the loss of ZFRP2 function caused a retinal defect, we designed antisense morpholinos to disrupt protein translation by targeting the initiating methionine in ZFRP2. Morpholinos were injected into one- to two-cell-stage embryos. As negative controls, either a standard negative control morpholino or a mispaired control morpholino was used at the same concentration as other morpholinos. Neither of the control morpholinos produced any morphologic defect. We checked for evidence of a defect in the visual system at 24 hpf; results showed that the eye area of ZFRP2 morpholino-injected embryos displayed dark brown granules, suggesting cell death. 23,24 In addition, the boundary between the lens and the neural retina was not clear (Fig. 3A). By 48 hpf, ZFRP2 morpholino-suppressed zebrafish embryos were shorter, and they had slim yolk extensions, small eyes, and small heads with slightly curved bodies and tails (Figs. 3B, 3C). Knockdown embryos were also less pigmented (Fig. 3B). Some morphants with a severe phenotype had very small eyes and heads with markedly curved body axes and short tails. Severe morphants had enlarged brain ventricles, hydrocephalus, and, occasionally, pericardial effusion. MO-Spl morpholino–injected morphants exhibited a phenotype similar to that of MO-ATG–injected morphants (data not shown). 
Figure 3.
 
Knockdown of ZFRP2 by morpholinos. (A) At 24 hpf, the eyes of ZFRP2 morphants displayed a dark brown and unclear boundary between lens and neural retina compared with control morphants. (B) At 48 hpf, ZFRP2 MO-injected embryos exhibited a small eye phenotype with slightly curved tail (RP2_MO a) or severe curved tail (RP2_MO b). ZFRP2 morphants also showed slim yolk extension. (C) The measurement of diameter (eye size) of the eyes from CMO-injected (control) morphants and ZFRP2 MO-injected morphants. Mean eye size was 245.5 μm for control morphants (65 embryos) and 164.9 μm for ZFRP2 morphants (62 embryos).
Figure 3.
 
Knockdown of ZFRP2 by morpholinos. (A) At 24 hpf, the eyes of ZFRP2 morphants displayed a dark brown and unclear boundary between lens and neural retina compared with control morphants. (B) At 48 hpf, ZFRP2 MO-injected embryos exhibited a small eye phenotype with slightly curved tail (RP2_MO a) or severe curved tail (RP2_MO b). ZFRP2 morphants also showed slim yolk extension. (C) The measurement of diameter (eye size) of the eyes from CMO-injected (control) morphants and ZFRP2 MO-injected morphants. Mean eye size was 245.5 μm for control morphants (65 embryos) and 164.9 μm for ZFRP2 morphants (62 embryos).
Knockdown of Zebrafish RP2 Leads to Cell Death
We carried out histologic analysis of the retina from control and ZFRP2-deficient morphants at 72 hpf and found that the control morphants displayed normal retinal lamination with discrete ganglion cell layer (GCL), inner nuclear layer (INL), and ONL. By this stage, the outer segments of photoreceptors are normally formed. In the ZFRP2-deficient morphants, lamination was defective because the three cell layers (GCL, INL, and ONL) were absent (Fig. 4A) and the outer segments of rod photoreceptors (detected with antirhodopsin; data not shown) and cone photoreceptors (detected with anti–Zpr-1 antibody) were both absent (Fig. 4B). 
Figure 4.
 
Eye histology in control (CMO) and ZFRP2 morpholino-injected embryos at 72 hpf. (A) Control morphants showed normal retinal lamination with three cell layers (GCL, INL, and ONL), In ZFRP2 morphants, lamination did not occur, and the three layers were not visible. The abnormal lens of ZFRP2 morphants was possibly caused by a general retardation of ocular development because of ZFRP2 knockdown. Magnification, ×20. (B) Immunostaining with anti–ZPR-1 antibody identified cone photoreceptors in control morphants, which were absent from ZFRP2-deficient morphants. Some nonspecific staining was seen in the lens (L) of CMO and RP2_MO morphants. Magnification, ×20. R, retina; L, lens.
Figure 4.
 
Eye histology in control (CMO) and ZFRP2 morpholino-injected embryos at 72 hpf. (A) Control morphants showed normal retinal lamination with three cell layers (GCL, INL, and ONL), In ZFRP2 morphants, lamination did not occur, and the three layers were not visible. The abnormal lens of ZFRP2 morphants was possibly caused by a general retardation of ocular development because of ZFRP2 knockdown. Magnification, ×20. (B) Immunostaining with anti–ZPR-1 antibody identified cone photoreceptors in control morphants, which were absent from ZFRP2-deficient morphants. Some nonspecific staining was seen in the lens (L) of CMO and RP2_MO morphants. Magnification, ×20. R, retina; L, lens.
ZFRP2 morphants exhibited abnormal dark brown and nontransparent eyes as early as 24 hpf. Later, at 48 and 72 hpf, the eyes and heads of morphants were small, and the boundaries between eye and brain, and occasionally between hindbrain and midbrain, were not clear. These phenotypes have previously been suggested to indicate ongoing neurodegeneration as a result of cell death, 23 25 which was therefore investigated further. Acridine orange is a vital dye reported to stain apoptotic but not necrotic cells in Drosophila 26 and is widely used to detect cell death in vivo in zebrafish. 19,24,27 Control and ZFRP2-deficient embryos at 48 hpf incubated in acridine orange solution showed that the lack of ZFRP2 protein led to an increase in acridine orange accumulation in the retina (Fig. 5). Cell death in the retinas of ZFRP2-deficient and control morphants at 72 hpf was also examined by TUNEL staining, which again showed that the depletion of ZFRP2 caused a nearly three-fold increase in cell death in the retina (data not shown). 
Figure 5.
 
Increased cell death in ZFRP2 morpholino injected embryos (RP2_MO), as indicated by acridine orange accumulation in the retina compared with control embryos (CMO).
Figure 5.
 
Increased cell death in ZFRP2 morpholino injected embryos (RP2_MO), as indicated by acridine orange accumulation in the retina compared with control embryos (CMO).
Rescue of Zebrafish RP2 Morphant Phenotype
To test whether the phenotypes described above are relevant to human RP2 function, we coinjected mRNA encoding human wild-type, or mutant RP2 (RP2c.257G>A, C86Y; RP2c.358C>T, R120X; RP2c.414A>G, E138G; RP2c.449G>A, W150X; RP2c.759T>G, L253R) or RP2 rare variant containing a polymorphism (RP2c.846C>T, R282W; Fig. 6A), together with a ZFRP2 morpholino, and scored the phenotypes at 48 hpf. The body axis curvature phenotype was rescued with human wild-type RP2 mRNA (Fig. 6B). The small eye phenotype was also partially rescued by human RP2 mRNA because the eye size of the rescued embryos was significantly increased compared with that of ZFRP2 morphants (P < 0.0001; Figs. 6B, 6D). The rescued embryos at 72 hpf exhibited normal lamination with three discrete cell layers (GCL, INL, and ONL), but in unrescued embryos lamination was defective with the three layers absent (Fig. 6C). In contrast, only 1 of 5 proposed disease-causing missense mutations in RP2 (RP2c.759T>G, L253R) and one polymorphism (RP2c.846C>T, R282W) were able to partially rescue the small eye phenotype by significantly increasing eye size compared with the ZFRP2 knockdown morphants (Fig. 6D). 
Figure 6.
 
Rescue of the ZFRP2-deficient phenotype using human RP2 mRNA. (A) Schematic representation of the human RP2 mRNA, showing the missense changes. (B) Rescue of the overall ZFRP2 MO phenotype at 72 hpf by human wild-type RP2 mRNA. The rescued phenotypes showed increased eye size, body size, and curved tail. (C) Rescued embryos displayed normal lamination with three discrete layers (GCL, INL, and ONL), but lamination was defective in unrescued embryos. Magnification, ×20. (D) Eye sizes of each group with coinjected human wild-type or amino acid substitution mRNA. Compared with ZFRP2 morpholino-only injected morphants, human wild-type, one mutant (L253R), and one polymorphism (R282W) partially rescued the small eye phenotype with significantly increased eye size, though the partially rescued eyes were still smaller than those in control morphants. **P < 0.0001; *P < 0.005.
Figure 6.
 
Rescue of the ZFRP2-deficient phenotype using human RP2 mRNA. (A) Schematic representation of the human RP2 mRNA, showing the missense changes. (B) Rescue of the overall ZFRP2 MO phenotype at 72 hpf by human wild-type RP2 mRNA. The rescued phenotypes showed increased eye size, body size, and curved tail. (C) Rescued embryos displayed normal lamination with three discrete layers (GCL, INL, and ONL), but lamination was defective in unrescued embryos. Magnification, ×20. (D) Eye sizes of each group with coinjected human wild-type or amino acid substitution mRNA. Compared with ZFRP2 morpholino-only injected morphants, human wild-type, one mutant (L253R), and one polymorphism (R282W) partially rescued the small eye phenotype with significantly increased eye size, though the partially rescued eyes were still smaller than those in control morphants. **P < 0.0001; *P < 0.005.
Discussion
To gain new insight into the function of the XLRP protein RP2, the zebrafish ortholog of the human RP2 gene was identified and analyzed by morpholino knockdown in early zebrafish development. It appears that a single ZFRP2 gene encodes a protein of 376 amino acids, which is highly homologous to the RP2 transcripts found in other vertebrate species (63%–81% amino acid identity), but shows lower homology to RP2 transcripts identified in invertebrate species (25%–48% identity). ZFRP2 expression was analyzed during zebrafish development and was found to be expressed in oocytes and early cleavage stage embryos and at later stages of development. This result is consistent with the findings of a recent paper by Hurd et al., 28 who also identified a single zebrafish RP2 gene that was expressed during embryogenesis. 
The function of ZFRP2 was studied by morpholino knockdown. The main finding in ZFRP2 morphants was that the eyes were smaller than in controls at both 24 hpf and 72 hpf, with the latter showing both defective retinal lamination and abnormal photoreceptors, lacking outer segments, which appeared by 60 hpf (Figs. 4A, 4B). 29 The defects in retinal development were further investigated by staining with acridine orange, which stains apoptotic but not necrotic cells. 19,23,24,27 The number of apoptotic retinal neurons was significantly increased in ZFRP2-depleted morphants (Fig. 5). Cell death did not appear to affect other ocular structures, suggesting that it might be a consequence of abnormal retinal differentiation. Defects in retinal lamination or differentiation with increased cell death have been observed in the context of genetic defects affecting transcription factors (e.g., math5, NR2E3), 30 32 cell cycle regulators (e.g., Cdkn1b/c, cdk5), 33 and the intraflagellar transport protein IFT88. 34 Mutations in the nuclear receptor factor, NR2E3, result in abnormal retinal lamination and differentiation. 31,32 A homozygous mutation in the mouse IFT88 gene causes abnormal photoreceptor differentiation, with reduced and disorganized outer segment but normal lamination. 34 Cell death resulting from ZFRP2 knockdown could result from abnormal retinal development, as seen in Nr2e3 31,32 or Sdkn1b/c mutants. 33 Alternatively, abnormal retinal development could itself result from severe and early retinal degeneration, as appears likely with smarca5 and IFT88 mutations. 34,35 The small eye phenotype of ZFRP2-depleted morphants appears to be caused by both abnormal retinal development and extensive retinal cell death. 
Ciliopathies are a group of disorders characterized by dysfunction of the primary cilia. Many ciliopathy-associated genes encode functional or structural components of primary cilia or basal bodies. Patients carrying mutations in those genes commonly display renal cystic diseases, retinal degeneration, liver fibrosis, or polydactyly. RP2 is also a component of the basal bodies and binds the N-ethylmaleimide–sensitive factor, which supports a role in vesicular traffic and fusion between the Golgi and the cilium. 14,36 Depletion of RP2 results in dispersal of vesicle cycling cargoes from the Golgi complex to the cilium, including the intraflagellar protein IFT20. 14 IFT proteins are thought to mediate the translocation of proteins to the cilium. Disruption of protein translocation between photoreceptor inner and outer segments could be a cause of the observed cell death and consequent retinal degeneration. A premature stop mutation in zebrafish ift88 mutants causes abnormal retinal differentiation and lack of outer segment formation. 37 The observed retinal defect and the occurrence of various nonocular developmental defects, such as abnormal body curvature, displayed in ZFRP2 knockdowns are also observed in zebrafish embryos with an ift88 mutation 37 and with the depletion of other ciliary proteins, 38,39 consistent with a ciliary function for RP2. These phenotypes were partially rescued by human RP2 but not by 4 of 5 human RP2 loss-of-function mutants associated with retinal dystrophies. The exception is L253R, which occurs at a moderately well-conserved residue in vertebrates (but which is not conserved in invertebrates; Fig. 1). These results are consistent with ZFRP2 as the ortholog of human RP2
Hurd et al. 28 identified a zebrafish RP2 ortholog (although the protein sequence was not reported) and showed that morpholino-mediated knockdown caused a ciliary dysfunction phenotype very similar to the one observed here, which was partially rescued by injecting wild-type zebrafish RP2. They also showed that the RP2 protein interacts with polycystin 2 (PKD2), another ciliary protein. The study noted small eyes but did not examine the eyes in detail, instead focusing on the renal phenotype, which consisted of cystic kidneys in approximately 20% of morphants. In addition, they observed left-right asymmetry defects, similar to those seen in zebrafish pkd2 mutants. The present study extends this work by reporting the full cDNA and protein sequences of ZFRP2 and showing that morpholino knockdown is associated with a small eye phenotype that appears to result from defective retinal lamination and subsequent degeneration, similar to that observed with knockdown of another ciliary protein, RPGR. 38 The mechanism underlying the occurrence of apoptotic cell death after delayed or defective retinal lamination and differentiation is unclear and merits further study. The reason for the absence of a renal phenotype in human patients with X-linked retinitis pigmentosa resulting from RP2 mutations is also unclear, though only a subgroup of morphants showed a renal cystic phenotype. The availability of a zebrafish model of RP2 disease should facilitate both the elucidation of its proposed functions in ciliary trafficking and the disease process in human RP2 patients. 
Footnotes
 Supported by the Medical Research Council (AFW, EEP), British Retinitis Pigmentosa Society (XS, AFW, MEC), Royal Society (XS), TENOVUS Scotland (XS), Visual Research Trust (XS), Fight for Sight (XS, AFW), Macular Vision Research Foundation (AFW), EVI-GENORET Grant FP6 512-036 (AFW), and Association of International Cancer Research Grant 07-0421 (EEP).
Footnotes
 Disclosure: X. Shu, None; Z. Zeng, None; P. Gautier, None; A. Lennon, None; M. Gakovic, None; M.E. Cheetham, None; E.E. Patton, None; A.F. Wright, None
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Figure 1.
 
Conservation of RP2 across different species. Accession numbers (Ensembl Ids where accession numbers are unavailable) for the amino acid sequence of each species are: HQ641392, zebrafish (Danio rerio); NP_008846, human (Homo sapiens); ENSMMUP 00000030304, macaque (Macaca mulatta); ENSOCUP00000004897, rabbit (Oryctolagus cuniculus); AAI53223.1, cow (Bos taurus); NP_598430, mouse (Mus musculus); XP_001362637, possum (Monodelphis domestica); NP_001008680, chick (Gallus gallus); ENSACAP 00000011842, lizard (Anolis carolinensis); XP_002937023, frog (Xenopus tropicalis); ENSGACP00000018275, stickleback (Gasterosteus aculeatus); ENSTRUP00000027655, fugu (Takifugu rubripes); XP_001628286, sea anemone (Nematostella vectensis); XP_002121234, ciona (Ciona intestinalis); NP_500127, nematode (Caenorhabditis elegans).
Figure 1.
 
Conservation of RP2 across different species. Accession numbers (Ensembl Ids where accession numbers are unavailable) for the amino acid sequence of each species are: HQ641392, zebrafish (Danio rerio); NP_008846, human (Homo sapiens); ENSMMUP 00000030304, macaque (Macaca mulatta); ENSOCUP00000004897, rabbit (Oryctolagus cuniculus); AAI53223.1, cow (Bos taurus); NP_598430, mouse (Mus musculus); XP_001362637, possum (Monodelphis domestica); NP_001008680, chick (Gallus gallus); ENSACAP 00000011842, lizard (Anolis carolinensis); XP_002937023, frog (Xenopus tropicalis); ENSGACP00000018275, stickleback (Gasterosteus aculeatus); ENSTRUP00000027655, fugu (Takifugu rubripes); XP_001628286, sea anemone (Nematostella vectensis); XP_002121234, ciona (Ciona intestinalis); NP_500127, nematode (Caenorhabditis elegans).
Figure 2.
 
Expression and localization of zebrafish RP2 (ZFRP2). (A) Temporal expression of ZFRP2 gene in the zebrafish development detected by RT-PCR. (B) ZFRP2 expression in different tissues detected by RT-PCR. (C, D) ZFRP2 localization in adult zebrafish photoreceptor, labeled with anti–RP2 antibody (C, D, green). The outer segments of rods were labeled with anti–rhodopsin antibody (C, red), and cone cells were labeled with anti–ZPR-1 antibody (D, red). Nuclear layers were labeled with DAPI (C, D, blue). Magnification, ×20.
Figure 2.
 
Expression and localization of zebrafish RP2 (ZFRP2). (A) Temporal expression of ZFRP2 gene in the zebrafish development detected by RT-PCR. (B) ZFRP2 expression in different tissues detected by RT-PCR. (C, D) ZFRP2 localization in adult zebrafish photoreceptor, labeled with anti–RP2 antibody (C, D, green). The outer segments of rods were labeled with anti–rhodopsin antibody (C, red), and cone cells were labeled with anti–ZPR-1 antibody (D, red). Nuclear layers were labeled with DAPI (C, D, blue). Magnification, ×20.
Figure 3.
 
Knockdown of ZFRP2 by morpholinos. (A) At 24 hpf, the eyes of ZFRP2 morphants displayed a dark brown and unclear boundary between lens and neural retina compared with control morphants. (B) At 48 hpf, ZFRP2 MO-injected embryos exhibited a small eye phenotype with slightly curved tail (RP2_MO a) or severe curved tail (RP2_MO b). ZFRP2 morphants also showed slim yolk extension. (C) The measurement of diameter (eye size) of the eyes from CMO-injected (control) morphants and ZFRP2 MO-injected morphants. Mean eye size was 245.5 μm for control morphants (65 embryos) and 164.9 μm for ZFRP2 morphants (62 embryos).
Figure 3.
 
Knockdown of ZFRP2 by morpholinos. (A) At 24 hpf, the eyes of ZFRP2 morphants displayed a dark brown and unclear boundary between lens and neural retina compared with control morphants. (B) At 48 hpf, ZFRP2 MO-injected embryos exhibited a small eye phenotype with slightly curved tail (RP2_MO a) or severe curved tail (RP2_MO b). ZFRP2 morphants also showed slim yolk extension. (C) The measurement of diameter (eye size) of the eyes from CMO-injected (control) morphants and ZFRP2 MO-injected morphants. Mean eye size was 245.5 μm for control morphants (65 embryos) and 164.9 μm for ZFRP2 morphants (62 embryos).
Figure 4.
 
Eye histology in control (CMO) and ZFRP2 morpholino-injected embryos at 72 hpf. (A) Control morphants showed normal retinal lamination with three cell layers (GCL, INL, and ONL), In ZFRP2 morphants, lamination did not occur, and the three layers were not visible. The abnormal lens of ZFRP2 morphants was possibly caused by a general retardation of ocular development because of ZFRP2 knockdown. Magnification, ×20. (B) Immunostaining with anti–ZPR-1 antibody identified cone photoreceptors in control morphants, which were absent from ZFRP2-deficient morphants. Some nonspecific staining was seen in the lens (L) of CMO and RP2_MO morphants. Magnification, ×20. R, retina; L, lens.
Figure 4.
 
Eye histology in control (CMO) and ZFRP2 morpholino-injected embryos at 72 hpf. (A) Control morphants showed normal retinal lamination with three cell layers (GCL, INL, and ONL), In ZFRP2 morphants, lamination did not occur, and the three layers were not visible. The abnormal lens of ZFRP2 morphants was possibly caused by a general retardation of ocular development because of ZFRP2 knockdown. Magnification, ×20. (B) Immunostaining with anti–ZPR-1 antibody identified cone photoreceptors in control morphants, which were absent from ZFRP2-deficient morphants. Some nonspecific staining was seen in the lens (L) of CMO and RP2_MO morphants. Magnification, ×20. R, retina; L, lens.
Figure 5.
 
Increased cell death in ZFRP2 morpholino injected embryos (RP2_MO), as indicated by acridine orange accumulation in the retina compared with control embryos (CMO).
Figure 5.
 
Increased cell death in ZFRP2 morpholino injected embryos (RP2_MO), as indicated by acridine orange accumulation in the retina compared with control embryos (CMO).
Figure 6.
 
Rescue of the ZFRP2-deficient phenotype using human RP2 mRNA. (A) Schematic representation of the human RP2 mRNA, showing the missense changes. (B) Rescue of the overall ZFRP2 MO phenotype at 72 hpf by human wild-type RP2 mRNA. The rescued phenotypes showed increased eye size, body size, and curved tail. (C) Rescued embryos displayed normal lamination with three discrete layers (GCL, INL, and ONL), but lamination was defective in unrescued embryos. Magnification, ×20. (D) Eye sizes of each group with coinjected human wild-type or amino acid substitution mRNA. Compared with ZFRP2 morpholino-only injected morphants, human wild-type, one mutant (L253R), and one polymorphism (R282W) partially rescued the small eye phenotype with significantly increased eye size, though the partially rescued eyes were still smaller than those in control morphants. **P < 0.0001; *P < 0.005.
Figure 6.
 
Rescue of the ZFRP2-deficient phenotype using human RP2 mRNA. (A) Schematic representation of the human RP2 mRNA, showing the missense changes. (B) Rescue of the overall ZFRP2 MO phenotype at 72 hpf by human wild-type RP2 mRNA. The rescued phenotypes showed increased eye size, body size, and curved tail. (C) Rescued embryos displayed normal lamination with three discrete layers (GCL, INL, and ONL), but lamination was defective in unrescued embryos. Magnification, ×20. (D) Eye sizes of each group with coinjected human wild-type or amino acid substitution mRNA. Compared with ZFRP2 morpholino-only injected morphants, human wild-type, one mutant (L253R), and one polymorphism (R282W) partially rescued the small eye phenotype with significantly increased eye size, though the partially rescued eyes were still smaller than those in control morphants. **P < 0.0001; *P < 0.005.
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