Knockdown of unc119c results in visual impairment and early-onset retinal dystrophy in zebrafish

Biochemical and Biophysical Research Communications 473 (2016) 1211e1217

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Knockdown of unc119c results in visual impairment and early-onset retinal dystrophy in zebrafish Nir Rainy a, Talya Etzion a, Shahar Alon a, b, Adi Pomeranz a, Yael Nisgav c, Tami Livnat c, Michael Bach d, Cecilia D. Gerstner e, Wolfgang Baehr e, f, Yoav Gothilf a, b, Hadas Stiebel-Kalish g, * a

Department of Neurobiology, George S. Wise Faculty of Life Sciences, Israel Sagol School of Neuroscience Tel Aviv University, Israel Laboratory of Eye Research, Felsenstein Medical Research Center Israel, Petah Tikva, Israel d Eye Center, Medical Center, University of Freiburg, Killianstraße 5, 79106, Freiburg, Germany e Department of Ophthalmology and Department of Neurobiology and Anatomy, University of Utah Health Science Center, Salt Lake City, UT 84132, USA f Department of Biology, University of Utah, Salt Lake City, UT 84112, USA g Department of Ophthalmology at Rabin Medical Center, Petah Tikva & Sackler Faculty of Medicine, Tel Aviv University, Israel b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 April 2016 Accepted 9 April 2016 Available online 11 April 2016

Purpose: UNC119 proteins are involved in G protein trafficking in mouse retinal photoreceptors and Caenorhabditis elegans olfactory neurons. An Unc119 null allele is associated with cone-rod dystrophy in mouse, but the mechanism leading to disease is not understood. We studied the role of Unc119 paralogs and Arl3l2 in zebrafish vision and retinal organization resulting from unc119c and arl3l2 knockdown. Methods: Zebrafish unc119c was amplified by PCR from retina and pineal gland cDNA. Its expression pattern in the eye and pineal gland was determined by whole-mount in-situ hybridization. unc119c and arl3l2 were knocked down using morpholino-modified oligonucleotides (MO). Their visual function was assessed with a quantitative optomotor assay on 6 days post-fertilization larvae. Retinal morphology was analyzed using immunohistochemistry with anti-cone arrestin (zpr-1) and anti-cone transducin-a (GNAT2) antibodies. Results: The zebrafish genome contains four genes encoding unc119 paralogs located on different chromosomes. The exon/intron arrangements of these genes are identical. Three Unc119 paralogs are expressed in the zebrafish retina, termed Unc119a-c. Based on sequence similarity, Unc119a and Unc119b are orthologs of mammalian UNC119a and UNC119b, respectively. A third, Unc119c, is unique and not present in mammals. Whole mount in-situ hybridization revealed that unc119a and unc119b RNA are ubiquitously expressed in the CNS, and unc119c is specifically expressed in photoreceptive tissues (pineal gland and retina). A Unc119 interactant, Arl3l2 also localizes to the pineal gland and the retina. As measured by the optomotor response, unc119c and arl3l2 knockdown resulted in significantly lower vision compared to wild-type zebrafish larvae and control morpholino (MO). Immunohistological analysis with anti-cone transducin and anti-cone arrestin (zpr-1) indicates that knockdown of unc119c leads to photoreceptor degeneration mostly affecting cones. Conclusions: Our results suggest that Unc119c is the only Unc119 paralog that is highly specific to the retina in zebrafish. Unc119c and Arl3l2 proteins are important for the function of cones. © 2016 Elsevier Inc. All rights reserved.

Keywords: Uncoordinated (UNC) 119 Morpholino knockdown Zebrafish retina Retina degeneration

1. Introduction

* Corresponding author. Neuro-Ophthalmology Unit, Department of Ophthalmology, Rabin Medical Center, Petah Tikva, 49100, Israel. E-mail address: [email protected] (H. Stiebel-Kalish). http://dx.doi.org/10.1016/j.bbrc.2016.04.041 0006-291X/© 2016 Elsevier Inc. All rights reserved.

Caenorhabditis elegans unc-119 was discovered based on a spontaneous mutation resulting in a nervous system defect [13]. An unc-119 ortholog was identified independently in human and rat retina (HRG4) [6], now officially termed “unc-119 lipid binding chaperone”, gene symbol UNC119. In mammalian retina, UNC119

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occurs as two paralogs, UNC119a and UNC119b [12]. UNC119a forms a stable complex with TaGTP that is capable of diffusing from the inner segment back to the outer segment following light-induced translocation [24]. UNC119 forms an immunoglobulin-like bsandwich fold which can accommodate the N-terminal acyl-chains of Ta and C. elegans olfactory G-proteins [24]. The physiological role of UNC119a is to extract Ta-GDP from inner segment membranes in the presence of GTP, forming a soluble complex. The complex was shown to play a key role in transducin's return to the outer segment following massive translocation from the outer to the inner segment in constant light [4,24]. UNC119 polypeptides are multifunctional proteins and interact with a large number of unrelated proteins e acylated Ga-subunits [24], receptor-associated src-type tyrosine kinases [3], nonreceptor protein kinases and small Arf-like GTPases (ARL proteins) [9,10]. The UNC119 acyl-binding proteins derive from a supergene family whose function has been maintained through metazoan evolution. The b-sandwich structure of UNC119a is very similar to those seen in PrBP/d [23] and RhoGDI [7]. UNC119a and UNC119b both physically interact with transducin-a as shown by pull down assays [24], but knockdown of UNC119b, but not UNC119a, prevented proper ciliary targeting of nephrocystin-3 (NPHP3) [21]. In zebrafish, three unc119 paralogs, unc119a-c, were identified [1,14,19]. Knockdown of Unc119a resulted in Kupffer's vesicle (KV) defects and reduced startle responses to bright light stimuli, while knockdown Unc119b yielded only a mild KV defect with no obvious cilia-related phenotypes function [21]. Subsequently, zebrafish pineal gland transcriptome analysis revealed a previously unknown zebrafish paralog, unc119c [1,19]. This paralog was identified as a pineal gland-enhanced transcript, initially identified as an Expressed Sequence Tag (EST, BG3055792), with the highest fold difference as compared to its expression in other tissues [1]. Also reported in these studies is the co-expression of an Arf-like (ARL) protein, Arl3l2, in the pineal gland [1], and its interaction with Unc119c by co-immunoprecipitation [19]. Moreover, it was shown that their co-expression in the pineal gland is important for proper development of the adjacent habenular commissure [20]. These previous studies focused on the role of Unc119c in the pineal gland. In this communication, we examined the role of Unc119c and Arl3l2 in the development and organization of the zebrafish retina and in its effect on vision. 2. Materials and methods 2.1. Fish and embryos Adult zebrafish (Danio rerio) were raised in a recirculation water system under 12:12 h of lightedark cycles at 28.5
C, adhering to the ARVO statement for the use of animals in Ophthalmic and Vision Research. The study was approved by the IACUC of Tel Aviv University, Israel. To produce embryos, male and female zebrafish were paired in the evening. Spawning occurred the next morning within 1 h of light. Embryos were placed in 10-cm Petri dishes with egg water (60 p.p.m. sea salts) containing methylene blue (0.3 p.p.m.) and raised in a light-controlled incubator at 28.5
C (lighting 12 W/ m2). In preparation for whole-mount in situ hybridization, 1phenyl-2-thiourea (0.2 mM) was added to the water to prevent pigmentation. 2.2. Cloning of zebrafish unc119 isoforms unc119 and arl3l2 isoforms were PCR amplified from pineal and retinal cDNA with specific sets of primers (Table 1) and subcloned in pGEM T-easy plasmid vector (Promega, Madison, WI, USA). The

identity of the resulting clones was verified by sequencing and by comparison with the zebrafish genome. These clones were used as templates to synthesize DIG-labeled antisense riboprobe for whole mount in-situ hybridization. 2.3. Whole mount in-situ hybridization (ISH) Transcripts of unc119c, unc119a, unc119b and of arl3l2 were detected by whole mount ISH using DIG-labelled antisense riboprobe RNA (DIG RNA labeling kit, Roche Diagnostics, Basel, Switzerland). Whole mount ISH analysis was carried out according to established protocol [15,18,26]. 2.4. unc119c knockdown procedure Knockdown of unc119c and of arl3l2 was performed by microinjection of morpholino modified antisense oligonucleotides (MO) (Genetools, LLC, Philomath, OR, USA) as described. unc119c MO (50 CAAGCCTGTTCAGAAGTTCAGTGTT-30 ) was designed against the intron3-exon 4 boundary, according to the manufacturer's recommendations, to interfere with splicing. arl3l2 (50 AAGCCCTGGAAACAACAACACACAC-30 ) was directed against the intron2-exon3 boundary to inhibit splicing. BLAST (Basic local alignment search tool) analysis was performed using UCSC genome browser (http:// genome.ucsc.edu/) to verify that this MO does not bind gene sequences other than its target. Standard control MO, 50 CCTCTTACCTCAGTTACAATTTATA-30 , which has not been reported to have other targets or generate any phenotypes in any known test, was injected as a negative control. Morpholinos were injected at a working concentration of 1e1.25 mM (approximately 8e10 mg/ml) in 0.1M KCL/0.05% phenol red. Approximately 2 nl were injected into the cytoplasm of one or two-cell-stage wild type zebrafish zygotes using micromanipulator and PV830 Pneumatic Pico Pump (World Precision Instruments, Sarasota, FL, USA). 100 to 400 embryos were injected using three to four different needles in each experiment. In rescue experiment, unc119c MO was co-injected with 100 ng/ml unc119c mature mRNA. Injected embryos kept at standard conditions as described above, until analyses. The effect of the unc119c MO on splicing was validated by reverse transcriptase-polymerase chain reaction (RT-PCR). RNA was extracted from pools of 60 unc119c MO and control MO embryos at 2, 3 and 6 dpf, an age at which MO is still active [2], cDNA was generated and subjected to PCR using Unc119c-specific primers directed towards exons 3 and 5 [forward 50 -TGATAGAGCTGGATGCTGGC-3'; reverse 50 -TGTCTGAGCGCGTCTCAA-3']. The PCR products were fractionated over 1.5 agarose gel, subcloned into pGEM-Teasy plasmid and sequenced. This analysis demonstrated that in addition to the normal mRNA there is a product with an additional 79 bp of intron 3 to the unc119c mRNA in unc119c MO-injected embryos, but not the control MO-injected embryos. This insertion introduced a number of premature stop codons which would lead to the production of a truncated and nonfunctional protein. 2.5. Visual testing procedure A quantitative optomotor assay was used to measure zebrafish larvae vision [18]. The optomotor response is an innate behavior of fish (and other animals) to follow visual motion of their surround in order to stabilize location or course. Here, the larvae follow the moving stripes, if they can see them, in an attempt to stabilize their location. Repeated stimulation by moving grating stimuli (256 pixels per cycle, corresponding to 0.16 cycles/degree at a distance of 8 cm, speed 144 pixels/s corresponding to 0.56
/s) was generated using a computer-generated stimulus program which is freely

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Table 1 Oligonucleotide primers used for PCR amplification of unc119 paralogs and for arl3l2.

unc119a unc119b unc119c uncarl3l2

Forward primer

Reverse primer

Length

50 -GGAACGGATAATGAGCTATTCTTG 50 -TTCCGAGAGATGAACAGTCAGA 50 -CGAGACACAGTGGACCAGAG 50 -CCAGGAGGCAAACTAAAGTCG

50 -TGGTTCTTAGGTCTGTCAGTCAA 50 -CATTCCACGCAAACACAAAC 50 -CTGCCTAATGGCGAAGAGTC 50 -CACAGTGCTGTGTGCTCTGA

857 821 923

Fig. 1. Analysis of unc119a-c sequences. A, nomenclature of zebrafish isoforms. Column 1, Unc119 isoforms labeled a-c based on their sequence similarity with mammalian isoforms. Column 2, Genbank nomenclature based on non-obsolete accessions. Column 3, chromosomal location of corresponding genes. Column 4, sequence similarity, with Unc119c set as 100%. Column 5, Genbank accession numbers. Column 6, length of Unc119 polypeptides. B, dendrogram of mammalian and Unc119 sequences. C. elegans and C. briggsiae sequences were added as a reference. Unc119a and Unc119b segregate with their mammalian counterparts. Unc119c is unique and apparently more closely related to C. elegans isoforms. C, Sequence alignment of Unc119 isoforms. Red arrows depict hydrophobic amino acids forming the hydrophobic pocket of the b-sandwich. Blue arrows identify charged amino acids inside the pocket. Black triangles mark exon/intron junctions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Whole-mound in-situ hybridization. Localization of unc119a, unc119b, unc119c and arl3l2 mRNAs in the developing zebrafish embryo, using whole mount in-situ hybridization. Embryos were probed with antisense unc119a (A), antisense unc119b (B), antisense unc119c (C), and antisense arl3l2 RNA (D). Unc119 isoform expressions are shown in 24 hpf embryo in lateral view (column 1), 48 hpf in dorsal view (column 2), 3 dpf in dorsal view (column 3) and 3 dpf in lateral view (column 4). Arrowheads point at the pineal gland and arrows points at the retina.

available at http://michaelbach.de/stim/. Altogether, including opposite motion directions, responses were recorded 24 times from four different sets of 30 zebrafish larvae. To quantify the population response to the optomotor assay, the tank was subdivided into four equal sections orthogonally to the racetracks: For each grating motion direction, the last quarter section of the computer screen and aquarium area was denoted as the ‘target area’ (left or right side). After the stimulus ran for 90 s, a digital photograph of the target area of the aquarium was taken. From this documentation the proportion of the fish in the target section was counted in relation to the total number of fish. Completely blind mutant fish are expected to exhibit no optomotor responses, thus the proportion of fish in the target area would be expected to be around 25%, corresponding to the target area, while the better the visual function, the more fish are expected to populate the target area. 2.6. Retinal histology Immunolabeling of larval cryosections was performed as

described (Zebrafish Book Protocol at https://wiki.zfin.org/display/ prot/ZFIN). Images were obtained using a LSM510 Meta confocal microscope (Zeiss) X63-oil/1.25 NA objective. The following primary antibodies were used; zpr1 (anti-cone arrestin) for doublecone staining [22], obtained from the zebrafish international resource center (ZIRC), Eugene, Oregon, USA, and used at a dilution of 1:200. Anti-cone transducin-a antibody was obtained from Dr. Sue Brockerhoff, University of Washington. Alexa Fluor 488conjugated donkey anti-mouse IgG (Thermo Fisher scientific Inc., Rockford, USA) and Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, Inc., West Grove, PA, USA) diluted to 1:200 and 1:100 respectively. Sections were counterstained with DAPI (40 , 6diamidino-2-phenylindole; 1:10,000, MP Biomedicals, LLC, Santa Ana, CA, USA) to stain nuclei. From each study group, 15 different larvae were randomly selected and screened. Four representative non-overlapping high-power fields (X100) at mid-lens sections from different animals of each study group (wild-type, unc119c knockdown and control MO) were selected.

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Fig. 3. unc119c and arl3l2 knockdown cause visual deficits. A, visual response of the unc119c knockdown (red), tested using the quantified optokinetic response assay, compared to control MO (green), rescue (blue) and wild-type zebrafish larvae (gray). Wild type larvae show strong optomotor response (80% in target area), unc119c knockdown larvae's optomotor response is markedly reduced, close to chance, largely rescued in the control MO group. Group results are depicted as boxplots: the thick line indicating the median, the notch the 95%-confidence interval for the median, the boxes are the ±25% percentiles, the antennas the ranges and the outliers (1.5* interquartile range outside the quartiles) are represented as circles. B, visual response of the arl3l2 knockdown (red), control MO (green) and wild-type zebrafish larvae (gray). C, D, wild-type controls (C) and unc119c knockdown larvae (D) are comparable in size and morphology. unc119c knockdown does not impair gross zebrafish larvae morphology or motility. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3. Results 3.1. unc119c nomenclature, phylogenetics and alignment Four distinct unc119 genes located on chromosomes 21 (unc119a), chromosome 15 (unc119 homolog A), chromosome 8 (unc119b), and chromosome 3 (unc119c) are known in the zebrafish genome (Fig. 1A). Of the four gene products, one (unc119 homolog A, acc. No. XP_695204) is predicted based on gene sequences and appears truncated at the N-terminus in exon 1. unc119a-c paralogs have been described [14,19]. unc119a (accession number NP_001035484) [14] segregates with mammalian UNC119a sharing 81% identities with human UNC119a (e-value 1e-109). The second paralog, unc119b (accession numbers AAK70467 and AF387342), segregates with human and bovine UNC119b (Fig. 1B) (71% identities, e value 1e-115). The last homolog, unc119c (accession number: XP_009297848 [19] has only 56% identities with human UNC119a (53% with UNC119b). The genes of the four unc119 isoforms have 5 exons and show identical exon/intron arrangements (Fig. 1C) suggesting that they are the product of gene duplications. The zebrafish unc119 nomenclature has seen many changes. Current and obsolete nomenclatures of unc119 paralogs are listed in Fig. 1A. The Unc119c polypeptide is distinct from its other paralogs, more closely related to C. elegans and C. briggsiae unc-119, which are ancestral to mammalian UNC119a and UNC119b (phylogenetic tree, Fig. 1B). By protein blastP analysis, Unc119c orthologs can be identified in

diverse orders of teleosts: the Mexican Tetra fish Astyanax mexicanus, a blind cave fish (accession: XP_007251104), the bicolor damselfish Stegastes partitus (accession: XP_008288458), and the spotted gar Lepisosteus oculatus. Sequence alignments show that N-terminal Unc119 sequences encoded by exon 1 are divergent, revealing little sequence similarity (Fig. 1C). Middle and C-terminal segments encoding the bsandwich structure are highly conserved. All UNC119 paralogs are predicted to form a b-sandwich structure for binding of myristoylated proteins as all amino acids forming the hydrophobic pocket (red arrows in Fig. 1C) are conserved. In particular, the charged amino acids located within the hydrophobic core involved in water binding (blue arrows in Fig. 1C) are present in the Unc119 isoforms. 3.2. In-situ hybridization reveals that Unc119c is photoreceptorspecific Temporo-spatial in-situ hybridization showed that unc119a and unc119b staining was widespread throughout the head and trunk while unc119c staining was present only in the retina and pineal gland, where primitive photoreceptors are known to be clustered (Fig. 2). At 24 h post-fertilization (pf), expression of unc119a and unc119b (Fig. 2, rows A and B) was widespread in the embryonic node, and as early as 48 h pf expression was ubiquitous in the head area (arrowheads) including the eye. Importantly, at this stage, retinal photoreceptors are not yet differentiated. In contrast, a specific expression of unc119c was found in the pineal gland of the

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Table 2 The percent of larvae within the target area after 90 s of grating motion. Experiment

Median

95%-CI

Experiment

Median

95%-CI

Unc119c kd Control MO Rescue Wild type

42.0 71.5 78.5 80.0

22.25e61.25 33.1e97.4 57.0e90.0 48.4e97.0

Arl3l2 kd control MO e Wild type

33.0 53.0 e 77.0

20.0e62.9 40.0e80.0 e 77.0e100

embryo (arrow heads Fig. 2, row C) and by 3 days pf, unc119c mRNA was first evident in the ventral patch of the retina (arrow, Fig. 2 row C), the site of differentiation of the first retinal photoreceptors. Whole-mount in-situ hybridization revealed arl3l2 also specifically located to pineal gland and retina with an expression pattern very similar to that of unc119c (Fig. 2, row D). 3.3. unc119c and arl3l2 knockdown impairs vision The unc119c morpholino oligonucleotide was designed against the intron 3-exon 4 boundary to interfere with splicing of the unc119c gene, leading to truncation of unc119c and inability to form a b-sandwich structure. Following injection of morpholinomodified antisense oligonucleotides (MO) directed to block unc119c expression, unc119c knockdown larvae displayed significantly impaired visual acuity when compared to both wild-type controls, unc119c knockdown-rescue and to control MO, as indicated by their optomotor response (Fig. 3A). Fig. 3A and Table 2 quantify the percent of larvae within the target area after 90 s of grating motion. Since the tank was divided into quarters, the random chance value of being in any quarter would be 25%. Of the control MO, unc119c knockdown-rescue and the wild-type zebrafish 6 day-old larvae, 72%, 79% and 80%, respectively, were in the target section. Of the unc119c MO-knockdown batch, the percentage of injected zebrafish following the 0.16 cycles/degree grating was 42% (95%-confidence interval (CI) 22.25e61.25%), not

significantly above the chance level of 25%. A one-way three level ANOVA found a highly significant effect (p < 0.0001) of the treatment (unc119c knockdown-WT control MO, rescue) on percentage in the target area. Fig. 3B and Table 2 display that of the arl3l2 MO-knockdown batch, the percentage of injected zebrafish following the 0.16 cycles/degree grating was 33% (95% CI 0e63%), thus including the chance level of 25%, compared to 77% of the wild-type fish (95% CI 57e100%) and 53% of Control MO (CI 40e80%). Swimming speeds and body curvature were unaffected in unc119c knockdown larvae, compared to wild-type controls (Fig. 3C, D). We followed the effect of unc119c knockdown by immunohistochemistry with mAb Zpr1 (anti-cone arrestin) is specific for double-cones [16]. WT and control retina show extensive labeling of normal double cones (Fig. 4A, B), while in the unc119c knockdown retina (Fig. 4C) double cones are stunted and appear to degenerate. Anti-cone transducin stains the cone outer segments in both WT and morpholino-control retinas (Fig. 4D, E) but staining is near absent in unc119c knockdown (Fig. 4F). The results show that knockdown of the unc119c in zebrafish larvae led to early immunohistochemical changes in the retina, most-likely because rod and cone transducin depend on Unc119c for trafficking to the outer segments.

4. Discussion In C. elegans, only one unc-119 isoform is present. unc-119 null mutants show a severe complex phenotype–defects in chemosensation, altered feeding behavior, inability to form daughter larvae [13] and excessive branching of motor neuron commissures [11]. G-protein mistrafficking to olfactory cilia is one of the causes, particularly ODR-3 mistrafficking in AWA and AWC sensory neurons causing dysosmia [17]. The Unc119/ mouse model resulted in a late visual deficit and a slow retinal degeneration [8]. The late

Fig. 4. unc119c knockdown causes cone degeneration. Immunohistochemistry of wild-type, control MO and unc119c knockdown retinas at 6 dpf. A-C, zpr1 labeling of double cones (green). D-F, double staining with zpr1 and anti-transducin (Gnat1) antibody (red). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

N. Rainy et al. / Biochemical and Biophysical Research Communications 473 (2016) 1211e1217

effect on the retina was attributed to redundant UNC119b paralogs present in the mouse retina [24]. Our study in zebrafish demonstrates that knockdown of unc119c resulted in very early onset visual impairment (Fig. 4), despite the existence of other unc119 isoforms, suggesting that Unc119c is the main Unc119 isoform involved in photoreceptor function. Visual impairment in zebrafish following unc119c knockdown is shown in this study to be an early retinal dystrophy mostly affecting cones as early as day 6pf. UNC119 has two distinct modes of interaction with other proteins. It interacts with acylated side chains of G-proteins subunits by inserting the acyl moiety into the hydrophobic pocket formed by the b-sandwich structure. Release of cargo is triggered by interaction with ARL3-GTP that binds to UNC119 through b-strand interactions [9,25]. ARL3-GTP acts as a GDF (GDI displacement factor), releasing lipidated proteins for delivery to destination membranes [9]. In zebrafish, members of the ARL gene family, arl3l1 and arl3l2, were highly enriched in the pineal gland relative to brain in microarray data [1]. Further, the results of in situ hybridization show that expression of arl3l2 is enhanced in the pineal gland and retina (Fig. 2). arl3l2 has a very similar expression pattern to that of unc119c, consistent with the evidence of Unc119-Arl3l2 interaction [19]. These results strengthen a model in which Arl3l2 might act as a zebrafish GDF, expelling cargo bound to Unc119c, as shown for ARL3 in mouse [5]. Knockout of Arl3 in mouse rod photoreceptors has profound effect on trafficking of prenylated and acylated proteins to mouse photoreceptor outer segments [5]. Knockdown of unc119c appears to affect mostly cones, as indicated by the reduced optomotor response (Fig. 4) and absence of cone outer segments in immunohistochemistry with cone specific antibodies. It is most likely that knockdown of unc119c impairs trafficking of cone transducin in zebrafish cone photoreceptors, affecting the structure of the cone outer segments and leading to cone photoreceptor degeneration. Knockdown of Arl3l2 impairs delivery of cargo bound to UNC119c to the outer segment. Further study of the UNC119c protein may add to existing knowledge gained from mice and C. elegans models on the role of Unc119 in ciliary trafficking of acylated proteins.

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Acknowledgments This study was supported by a grant from the Claire and Amedee Maratier Institute for the Study of Blindness and Visual Disorders, Sackler Faculty of Medicine, Tel-Aviv University, and by a grant from the United States-Israel Binational Science Foundation (grant number 2009/290), Jerusalem, Israel. WB was supported by NIH grants EY08123, EY019298, and EY014800-039003 (NEI core grant). WB is a recipient of a Research to Prevent Blindness Senior Investigator and a Nelson Trust Award. Parts of these results were presented as a poster at the 2013 ARVO Annual Meeting, Seattle, Washington and at the annual Israeli Society for Vision and Eye Research, 2013. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.04.041.

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