Comparative analysis of transcriptional changes in zebrafish cep290 and bbs2 mutants by RNA-seq reveals upregulation of inflammatory and stress-related pathways.

Acute injury to the adult zebrafish retina triggers the release of pro-inflammatory cytokines and growth factors that stimulate multiple gene regulatory networks, which ultimately stimulate Müller glia to proliferate and regenerate neurons. In contrast, zebrafish carrying mutations in cep290 or bbs2 undergo progressive loss of cone photoreceptors and exhibit signs of microglia activation and inflammation, but the mutants fail to stimulate a regeneration response. To identify transcriptional changes that occur in zebrafish mutants undergoing progressive photoreceptor degeneration, RNA-seq transcriptional profiling was performed on cep290-/- and bbs2-/- retinas. The PANTHER Classification System was used to identify biological processes and signaling pathways that were differentially expressed between mutants and wild-type siblings during degeneration. As expected, genes associated with phototransduction were downregulated in cep290 and bbs2 mutants compared to wild-type siblings. Although both cep290 and bbs2 mutants undergo proliferation of rod precursors in response to retinal degeneration, the process of negatively regulating proliferation is enriched for upregulated genes, and this negative regulation may restrict proliferation of Müller glia and inhibit regeneration. A total of 815 differentially expressed genes (DEGs) were shared by cep290 and bbs2 retinas. Genes in pathways associated with inflammation, apoptosis, stress response, and PDGF signaling were overrepresented. Identifying the genes and biological pathways that are common in zebrafish models of inherited retinal degeneration provides a foundation for future studies on the mechanisms that regulate cell death as well as processes that prohibit Müller cell reprogramming or proliferation in a model capable of retinal regeneration. The pathways will provide targets for future interventions that may promote successful regeneration of lost photoreceptors.

Zebrafish models of inherited retinal dystrophies.

Inherited retinal degenerations (IRDs) cause permanent vision impairment or vision loss due to the death of rod and cone photoreceptors. Animal models of IRDs have been instrumental in providing knowledge of the pathological mechanisms that cause photoreceptor death and in developing successful approaches that could slow or prevent vision loss. Zebrafish models of IRDs represent an ideal model system to study IRDs in a cone-rich retina and to test strategies that exploit the natural ability to regenerate damaged neurons. This review highlights those zebrafish mutants and transgenic lines that exhibit adult-onset retinal degeneration and serve as models of retinitis pigmentosa, cone-rod dystrophy, and ciliopathies.

Notch Inhibition Promotes Regeneration and Immunosuppression Supports Cone Survival in a Zebrafish Model of Inherited Retinal Dystrophy.

Photoreceptor degeneration leads to irreversible vision loss in humans with retinal dystrophies such as retinitis pigmentosa. Whereas photoreceptor loss is permanent in mammals, zebrafish possesses the ability to regenerate retinal neurons and restore visual function. Following acute damage, Müller glia (MG) re-enter the cell cycle and produce multipotent progenitors whose progeny differentiate into mature neurons. Both MG reprogramming and proliferation of retinal progenitor cells require reactive microglia and associated inflammatory signaling. Paradoxically, in zebrafish models of retinal degeneration, photoreceptor death does not induce the MG to reprogram and regenerate lost cells. Here, we used male and female zebrafish cep290 mutants to demonstrate that progressive cone degeneration generates an immune response but does not stimulate MG proliferation. Acute light damage triggered photoreceptor regeneration in cep290 mutants but cones were only restored to prelesion densities. Using irf8 mutant zebrafish, we found that the chronic absence of microglia reduced inflammation and rescued cone degeneration in cep290 mutants. Finally, single-cell RNA-sequencing revealed sustained expression of notch3 in MG of cep290 mutants and inhibition of Notch signaling induced MG to re-enter the cell cycle. Our findings provide new insights on the requirements for MG to proliferate and the potential for immunosuppression to prolong photoreceptor survival.SIGNIFICANCE STATEMENT Inherited retinal degenerations (IRDs) are genetic diseases that lead to the progressive loss of photoreceptors and the permanent loss of vision. Zebrafish can regenerate photoreceptors after acute injury by reprogramming Müller glia (MG) into stem-like cells that produce retinal progenitors, but this regenerative process fails to occur in zebrafish models of IRDs. Here, we show that Notch pathway inhibition can promote photoreceptor regeneration in models of progressive degeneration and that immunosuppression can prevent photoreceptor loss. These results offer insight into the pathways that promote MG-dependent regeneration and the role of inflammation in photoreceptor degeneration.

Vision and sensorimotor defects associated with loss of Vps11 function in a zebrafish model of genetic leukoencephalopathy.

Genetic Leukoencephalopathies (gLEs) are heritable white matter disorders that cause progressive neurological abnormalities. A founder mutation in the human endolysosomal trafficking protein VPS11 has been identified in Ashkenazi Jewish patients manifesting classic gLE symptoms of hypomyelination, developmental delay, motor and systemic deficits. In this study, we characterized the visual and sensorimotor function of two zebrafish vps11 mutant lines: the previously reported vps11(plt), and a new vps11(-/-) null mutant line, using behavioral analysis to track larval motor responses to visual and acoustic stimuli. We found that mutant larvae from both vps11(plt) and vps11(-/-) lines were able to visually distinguish light and dark, but showed a progressive loss of a normal sensorimotor response to visual stimuli from 5 days post fertilization (dpf) to 7dpf. Additionally, optokinetic response analysis performed at 5dpf indicated that the mutants were significantly visually impaired. Both mutant lines also displayed a progressively lower sensorimotor response to a singular acoustic stimulus from 5-7dpf. Next, we tested the habituation response of the mutant lines to series of acoustic taps. We found both mutant lines habituated faster than their siblings, and that vps11(plt) mutants habituated faster than the vps11(-/-) mutants. Together, these data suggest that loss of Vps11 function results in progressive visual and sensorimotor abnormalities in the zebrafish vps11(plt) and vps11(-/-) mutant lines. This is the first study to characterize behavioral deficits in a vertebrate model of Vps11-dependent gLE. The mutants and behavioral assays described here could be a valuable model system in which to test potential pharmacological interventions for gLE.

PI(3,4)P2-mediated cytokinetic abscission prevents early senescence and cataract formation.

Cytokinetic membrane abscission is a spatially and temporally regulated process that requires ESCRT (endosomal sorting complexes required for transport)­dependent control of membrane remodeling at the midbody, a subcellular organelle that defines the cleavage site. Alteration of ESCRT function can lead to cataract, but the underlying mechanism and its relation to cytokinesis are unclear. We found a lens-specific cytokinetic process that required PI3K-C2α (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2α), its lipid product PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate), and the PI(3,4)P2­binding ESCRT-II subunit VPS36 (vacuolar protein-sorting-associated protein 36). Loss of each of these components led to impaired cytokinesis, triggering premature senescence in the lens of fish, mice, and humans. Thus, an evolutionarily conserved pathway underlies the cell type­specific control of cytokinesis that helps to prevent early onset cataract by protecting from senescence.

Knockout of the CEP290 gene in human induced pluripotent stem cells.

CEP290 is a principal component of the primary cilium and is important for the proper function of ciliated cells. CEP290 mutations have been linked to numerous ciliopathies, with a wide range of phenotypic severities, but with poor genotype:phenotype correlation. Here we have used CRISPR/Cas9 technology to target the CEP290 gene and generate a line of induced pluripotent stem cells that lack detectable CEP290 expression, but retain a normal karyotype and differentiation potential. This line of cells will be useful for the study of disorders resulting from CEP290 mutations.

Cone Photoreceptor Degeneration and Neuroinflammation in the Zebrafish Bardet-Biedl Syndrome 2 (bbs2) Mutant Does Not Lead to Retinal Regeneration.

Bardet-Biedl syndrome (BBS) is a heterogeneous and pleiotropic autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, renal dysfunction, and mental retardation. BBS results from defects in primary and sensory cilia. Mutations in 21 genes have been linked to BBS and proteins encoded by 8 of these genes form a multiprotein complex termed the BBSome. Mutations in BBS2, a component of the BBSome, result in BBS as well as non-syndromic retinal degeneration in humans and rod degeneration in mice, but the role of BBS2 in cone photoreceptor survival is not clear. We used zebrafish bbs2-/- mutants to better understand how loss of bbs2 leads to photoreceptor degeneration. Zebrafish bbs2-/- mutants exhibited impaired visual function as larvae and adult zebrafish underwent progressive cone photoreceptor degeneration. Cone degeneration was accompanied by increased numbers of activated microglia, indicating an inflammatory response. Zebrafish exhibit a robust ability to regenerate lost photoreceptors following retinal damage, yet cone degeneration and inflammation was insufficient to trigger robust Müller cell proliferation. In contrast, high intensity light damage stimulated Müller cell proliferation and photoreceptor regeneration in both wild-type and bbs2-/- mutants, although the bbs2-/- mutants could only restore cones to pre-damaged densities. In summary, these findings suggest that cone degeneration leads to an inflammatory response in the retina and that BBS2 is necessary for cone survival. The zebrafish bbs2 mutant also represents an ideal model to identify mechanisms that will enhance retinal regeneration in degenerating diseases.

A Functional Binding Domain in the Rbpr2 Receptor Is Required for Vitamin A Transport, Ocular Retinoid Homeostasis, and Photoreceptor Cell Survival in Zebrafish.

Dietary vitamin A/all-trans retinol/ROL plays a critical role in human vision. ROL circulates bound to the plasma retinol-binding protein (RBP4) as RBP4-ROL. In the eye, the STRA6 membrane receptor binds to circulatory RBP4 and internalizes ROL. STRA6 is, however, not expressed in systemic tissues, where there is high affinity RBP4 binding and ROL uptake. We tested the hypothesis that the second retinol binding protein 4 receptor 2 (Rbpr2), which is highly expressed in systemic tissues of zebrafish and mouse, contains a functional RBP4 binding domain, critical for ROL transport. As for STRA6, modeling and docking studies confirmed three conserved RBP4 binding residues in zebrafish Rbpr2. In cell culture studies, disruption of the RBP4 binding residues on Rbpr2 almost completely abolished uptake of exogenous vitamin A. CRISPR-generated rbpr2-RBP4 domain zebrafish mutants showed microphthalmia, shorter photoreceptor outer segments, and decreased opsins, which were attributed to impaired ocular retinoid content. Injection of WT-Rbpr2 mRNA into rbpr2 mutant or all-trans retinoic acid treatment rescued the mutant eye phenotypes. In conclusion, zebrafish Rbpr2 contains a putative extracellular RBP4-ROL ligand-binding domain, critical for yolk vitamin A transport to the eye for ocular retinoid production and homeostasis, for photoreceptor cell survival.

Genomic non-redundancy of the mir-183/96/182 cluster and its requirement for hair cell maintenance.

microRNAs are important regulators of gene expression. In the retina, the mir-183/96/182 cluster is of particular interest due to its robust expression and studies in which loss of the cluster caused photoreceptor degeneration. However, it is unclear which of the three miRNAs in the cluster are ultimately required in photoreceptors, whether each may have independent, contributory roles, or whether a single miRNA from the cluster compensates for the loss of another. These are important questions that will not only help us to understand the role of these particular miRNAs in the retina, but will deepen our understanding of how clustered microRNAs evolve and operate. To that end, we have developed a complete panel of single, double, and triple mir-183/96/182 mutant zebrafish. While the retinas of all mutant animals were normal, the triple mutants exhibited acute hair cell degeneration which corresponded with impaired swimming and death at a young age. By measuring the penetrance of this phenotype in each mutant line, we determine which of the three miRNAs in the cluster are necessary and/or sufficient to ensure normal hair cell development and function.

Ciliary genes arl13b, ahi1 and cc2d2a differentially modify expression of visual acuity phenotypes but do not enhance retinal degeneration due to mutation of cep290 in zebrafish.

Mutations in the gene Centrosomal Protein 290 kDa (CEP290) result in multiple ciliopathies ranging from the neonatal lethal disorder Meckel-Gruber Syndrome to multi-systemic disorders such as Joubert Syndrome and Bardet-Biedl Syndrome to nonsyndromic diseases like Leber Congenital Amaurosis (LCA) and retinitis pigmentosa. Results from model organisms and human genetics studies, have suggest that mutations in genes encoding protein components of the transition zone (TZ) and other cilia-associated proteins can function as genetic modifiers and be a source for CEP290 pleiotropy. We investigated the zebrafish cep290fh297/fh297 mutant, which encodes a nonsense mutation (p.Q1217*). This mutant is viable as adults, exhibits scoliosis, and undergoes a slow, progressive cone degeneration. The cep290fh297/fh297 mutants showed partial mislocalization of the transmembrane protein rhodopsin but not of the prenylated proteins rhodopsin kinase (GRK1) or the rod transducin subunit GNB1. Surprisingly, photoreceptor degeneration did not trigger proliferation of Müller glia, but proliferation of rod progenitors in the outer nuclear layer was significantly increased. To determine if heterozygous mutations in other cilia genes could exacerbate retinal degeneration, we bred cep290fh297/fh297 mutants to arl13b, ahi1, and cc2d2a mutant zebrafish lines. While cep290fh297/fh297 mutants lacking a single allele of these genes did not exhibit accelerated photoreceptor degeneration, loss of one alleles of arl13b or ahi1 reduced visual performance in optokinetic response assays at 5 days post fertilization. Our results indicate that the cep290fh297/fh297 mutant is a useful model to study the role of genetic modifiers on photoreceptor degeneration in zebrafish and to explore how progressive photoreceptor degeneration influences regeneration in adult zebrafish.

Mutant Ahi1 Affects Retinal Axon Projection in Zebrafish via Toxic Gain of Function.

Joubert syndrome (JBTS) is an inherited autosomal recessive disorder associated with cerebellum and brainstem malformation and can be caused by mutations in the Abelson helper integration site-1 (AHI1) gene. Although AHI1 mutations in humans cause abnormal cerebellar development and impaired axonal decussation in JBTS, these phenotypes are not robust or are absent in various mouse models with Ahi1 mutations. AHI1 contains an N-terminal coiled-coil domain, multiple WD40 repeats, and a C-terminal Src homology 3 (SH3) domain, suggesting that AHI1 functions as a signaling or scaffolding protein. Since most AHI1 mutations in humans can result in truncated AHI1 proteins lacking WD40 repeats and the SH3 domain, it remains unclear whether mutant AHI1 elicits toxicity via a gain-of-function mechanism by the truncated AHI1. Because Ahi1 in zebrafish and humans share a similar N-terminal region with a coiled-coil domain that is absent in mouse Ahi1, we used zebrafish as a model to investigate whether Ahi1 mutations could affect axonal decussation. Using in situ hybridization, we found that ahi1 is highly expressed in zebrafish ocular tissues, especially in retina, allowing us to examine its effect on retinal ganglion cell (RGC) projection and eye morphology. We injected a morpholino to zebrafish embryos, which can generate mutant Ahi1 lacking the intact WD40 repeats, and found RGC axon misprojection and ocular dysplasia in 4 dpf (days post-fertilization) larvae after the injection. However, ahi1 null zebrafish showed normal RGC axon projection and ocular morphology. We then used CRISPR/Cas9 to generate truncated ahi1 and also found similar defects in the RGC axon projection as seen in those injected with ahi1 morpholino. Thus, the aberrant retinal axon projection in zebrafish is caused by the presence of mutant ahi1 rather than the loss of ahi1, suggesting that mutant Ahi1 may affect axonal decussation via toxic gain of function.

Developmental expression of the zebrafish Arf-like small GTPase paralogs arl13a and arl13b.

Members of the Arf-like (Arl) family of small GTP-binding proteins regulate a number of cellular functions and play important roles in cilia structure and signaling. The small GTPase Arl13a is a close paralog to Arl13b, a small GTPase required for normal cilia formation that causes Joubert Syndrome when mutated. As mutation of arl13b causes a slow retinal degeneration in zebrafish (Song et al., 2016), we hypothesized that expression of arl13a may provide functional redundancy. We determined the expression domains of arl13a and arl13b during zebrafish development and examined subcellular localization by expression of fluorescence fusion proteins. Both genes are widely expressed during early cell division and gastrulation and Arl13a and Arl13b both localize to microtubules in ciliated and dividing cells of the early zebrafish embryo. Between 2 and 5 days post fertilization (dpf), arl13b is expressed in neural tissues while expression of arl13a is downregulated by 2 dpf and restricted to craniofacial structures. These results indicate that arl13a and arl13b have evolved different roles and that arl13a does not function in the zebrafish retina.

Pregnancy-Associated Plasma Protein-aa Regulates Photoreceptor Synaptic Development to Mediate Visually Guided Behavior.

To guide behavior, sensory systems detect the onset and offset of stimuli and process these distinct inputs via parallel pathways. In the retina, this strategy is implemented by splitting neural signals for light onset and offset via synapses connecting photoreceptors to ON and OFF bipolar cells, respectively. It remains poorly understood which molecular cues establish the architecture of this synaptic configuration to split light-onset and light-offset signals. A mutant with reduced synapses between photoreceptors and one bipolar cell type, but not the other, could reveal a critical cue. From this approach, we report a novel synaptic role for pregnancy-associated plasma protein aa (pappaa) in promoting the structure and function of cone synapses that transmit light-offset information. Electrophysiological and behavioral analyses indicated pappaa mutant zebrafish have dysfunctional cone-to-OFF bipolar cell synapses and impaired responses to light offset, but intact cone-to-ON bipolar cell synapses and light-onset responses. Ultrastructural analyses of pappaa mutant cones showed a lack of presynaptic domains at synapses with OFF bipolar cells. pappaa is expressed postsynaptically to the cones during retinal synaptogenesis and encodes a secreted metalloprotease known to stimulate insulin-like growth factor 1 (IGF1) signaling. Induction of dominant-negative IGF1 receptor expression during synaptogenesis reduced light-offset responses. Conversely, stimulating IGF1 signaling at this time improved pappaa mutants' light-offset responses and cone presynaptic structures. Together, our results indicate Pappaa-regulated IGF1 signaling as a novel pathway that establishes how cone synapses convey light-offset signals to guide behavior.SIGNIFICANCE STATEMENT Distinct sensory inputs, like stimulus onset and offset, are often split at distinct synapses into parallel circuits for processing. In the retina, photoreceptors and ON and OFF bipolar cells form discrete synapses to split neural signals coding light onset and offset, respectively. The molecular cues that establish this synaptic configuration to specifically convey light onset or offset remain unclear. Our work reveals a novel cue: pregnancy-associated plasma protein aa (pappaa), which regulates photoreceptor synaptic structure and function to specifically transmit light-offset information. Pappaa is a metalloprotease that stimulates local insulin-like growth factor 1 (IGF1) signaling. IGF1 promotes various aspects of synaptic development and function and is broadly expressed, thus requiring local regulators, like Pappaa, to govern its specificity.

Intraflagellar transport proteins are involved in thrombocyte filopodia formation and secretion.

Intraflagellar transport (IFT) proteins are vital for the genesis and maintenance of cilia. Our identification of ift122 transcripts in zebrafish thrombocytes that lack primary cilia was unexpected. IFT proteins serve transport in cilia, whose narrow dimensions may have necessitated the evolution of IFT from vesicular transport in ancestral eukaryotes. We hypothesized that IFTs might also facilitate transport within the filopodia that form when thrombocytes are activated. To test this possibility, we knocked down ift122 expression by injecting antisense Morpholino oligonucleotides (MOs) into zebrafish embryos. Laser-induced arterial thrombosis showed prolonged time to occlusion (TTO) of the vessel, as would be expected with defective thrombocyte function. Acute effects in adult zebrafish were evaluated by Vivo-Morpholino (Vivo-MO) knockdown of ift122. Vivo-MO morphants showed a prolonged time to thrombocyte aggregation (TTA) in the plate tilt assay after thrombocyte activation by the following agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in thrombocyte function.

The Ciliopathy Gene ahi1 Is Required for Zebrafish Cone Photoreceptor Outer Segment Morphogenesis and Survival.

Purpose: Joubert syndrome (JBTS) is an autosomal recessive ciliopathy with considerable phenotypic variability. In addition to central nervous system abnormalities, a subset of JBTS patients exhibit retinal dystrophy and/or kidney disease. Mutations in the AHI1 gene are causative for approximately 10% of all JBTS cases. The purpose of this study was to generate ahi1 mutant alleles in zebrafish and to characterize the retinal phenotypes. Methods: Zebrafish ahi1 mutants were generated using transcription activator-like effector nucleases (TALENs). Expression analysis was performed by whole-mount in situ hybridization. Anatomic and molecular characterization of photoreceptors was investigated by histology, electron microscopy, and immunohistochemistry. The optokinetic response (OKR) behavior assay was used to assess visual function. Kidney cilia were evaluated by whole-mount immunostaining. Results: The ahi1lri46 mutation in zebrafish resulted in shorter cone outer segments but did not affect visual behavior at 5 days after fertilization (dpf). No defects in rod morphology or rhodopsin localization were observed at 5 dpf. By 5 months of age, cone degeneration and rhodopsin mislocalization in rod photoreceptors was observed. The connecting cilium formed normally and Cc2d2a and Cep290 localized properly. Distal pronephric duct cilia were absent in mutant fish; however, only 9% of ahi1 mutants had kidney cysts by 5 dpf, suggesting that the pronephros remained largely functional. Conclusions: The results indicate that Ahi1 is required for photoreceptor disc morphogenesis and outer segment maintenance in zebrafish.

The adult zebrafish retina: In vivo optical sectioning with Confocal Scanning Laser Ophthalmoscopy and Spectral-Domain Optical Coherence Tomography.

Non-invasive imaging is an invaluable diagnostic tool in ophthalmology. Two imaging devices, the scanning laser ophthalmoscope (SLO) and spectral domain optical coherence tomography (SDOCT), emerged from the clinical realm to provide research scientists with a real-time view of ocular morphology in living animals. We utilized these two independent imaging modalities in a complementary manner to perform in vivo optical sectioning of the adult zebrafish retina. Due to the very high optical power of the zebrafish lens, the confocal depth of field is narrow, allowing for detailed en face views of specific retinal layers, including the cone mosaic. Moreover, we demonstrate that both native reflectance, as well as fluorescent features observed by SLO, can be combined with axial in-depth information obtained by SDOCT. These imaging approaches can be used to screen for ocular phenotypes and monitor retinal pathology in a non-invasive manner.

Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish.

PURPOSE: Mutations in the gene ARL13B cause the classical form of Joubert syndrome, an autosomal recessive ciliopathy with variable degrees of retinal degeneration. As second-site modifier alleles can contribute to retinal pathology in ciliopathies, animal models provide a unique platform to test how genetic interactions modulate specific phenotypes. In this study, we analyzed the zebrafish arl13b mutant for retinal degeneration and for epistatic relationships with the planar cell polarity protein (PCP) component vangl2. METHODS: Photoreceptor and cilia structure was examined by light and electron microscopy. Immunohistochemistry was performed to examine ciliary markers. Genetic interactions were tested by pairwise crosses of heterozygous animals. Genetic mosaic animals were generated by blastula transplantation and analyzed by fluorescence microscopy. RESULTS: At 5 days after fertilization, photoreceptor outer segments were shorter in zebrafish arl13b-/- mutants compared to wild-type larvae, no overt signs of retinal degeneration were observed by light or electron microscopy. Starting at 14 days after fertilization (dpf) and continuing through 30 dpf, cells lacking Arl13b died following transplantation into wild-type host animals. Photoreceptors of arl13b-/-;vangl2-/- mutants were more compromised than the photoreceptors of single mutants. Finally, when grown within a wild-type retina, the vangl2-/- mutant cone photoreceptors displayed normal basal body positioning. CONCLUSIONS: We show that arl13b-/- mutants have shortened cilia and photoreceptor outer segments and exhibit a slow, progressive photoreceptor degeneration that occurs over weeks. The data suggest that loss of Arl13b leads to slow photoreceptor degeneration, but can be exacerbated by the loss of vangl2. Importantly, the data show that Arl13b can genetically and physically interact with Vangl2 and this association is important for normal photoreceptor structure. The loss of vangl2, however, does not affect basal body positioning.

Mutation of wrb, a Component of the Guided Entry of Tail-Anchored Protein Pathway, Disrupts Photoreceptor Synapse Structure and Function.

PURPOSE: Tail-anchored (TA) proteins contain a single hydrophobic domain at the C-terminus and are posttranslationally inserted into the ER membrane via the GET (guided entry of tail-anchored proteins) pathway. The role of the GET pathway in photoreceptors is unexplored. The goal of this study was to characterize the zebrafish pinball wizard mutant, which disrupts Wrb, a core component of the GET pathway. METHODS: Electroretinography, optokinetic response measurements (OKR), immunohistochemistry, and electron microscopy analyses were employed to assess ribbon synapse function, protein expression, and ultrastructure in 5-day-old zebrafish larvae. Expression of wrb was investigated with real-time qRT-PCR and in situ hybridization. RESULTS: Mutation of wrb abolished the OKR and greatly diminished the ERG b-wave, but not the a-wave. Ribeye and SV2 were partially mislocalized in both photoreceptors and hair cells of wrb mutants. Fewer contacts were seen between photoreceptors and bipolar cells in wrb-/- mutants. Expression of wrb was observed throughout the nervous system and Wrb localized to the ER and synaptic region of photoreceptors. Morpholino knockdown of the cytosolic ATPase trc40, which targets TA proteins to the ER, also diminished the OKR. Overexpression of wrb fully restored contrast sensitivity in mutants, while overexpression of mutant wrbR73A, which cannot bind Trc40, did not. CONCLUSIONS: Proteins Wrb and Trc40 are required for synaptic transmission between photoreceptors and bipolar cells, indicating that TA protein insertion by the TRC pathway is a critical step in ribbon synapse assembly and function.