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Corene Ollig
Mutations in eyes shut homolog (EYS), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive retinal disorder. Due to the absence of EYS in several rodent species and its retina-specific expression, still little is known about the exact function of EYS and the pathogenic mechanism underlying EYS-associated RP. We characterized eys in zebrafish, by RT-PCR analysis on zebrafish eye-derived RNA, which led to the identification of a 8,715 nucleotide coding sequence that is divided over 46 exons. The transcript is predicted to encode a 2,905-aa protein that contains 39 EGF-like domains and five laminin A G-like domains, which overall shows 33% identity with human EYS. To study the function of EYS, we generated a stable eysrmc101/rmc101 mutant zebrafish model using CRISPR/Cas9 technology. The introduced lesion is predicted to result in premature termination of protein synthesis and lead to loss of Eys function. Immunohistochemistry on retinal sections revealed that Eys localizes at the region of the connecting cilium and that both rhodopsin and cone transducin are mislocalized in the absence of Eys. Electroretinogram recordings showed diminished b-wave amplitudes in eysrmc101/rmc101 zebrafish (5 dpf) compared to age- and strain-matched wild-type larvae. In addition, decreased locomotor activity in response to light stimuli was observed in eys mutant larvae. Altogether, our study shows that absence of Eys leads to a disorganized retinal architecture and causes visual dysfunction in zebrafish.
Citation: Messchaert M, Dona M, Broekman S, Peters TA, Corral-Serrano JC, Slijkerman RWN, et al. (2018) Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish. PLoS ONE 13(7): e0200789.
Eyes shut homolog is an ortholog of the Drosophila spacemaker (spam) protein, which plays a major role in the development of photoreceptors and the maintenance of the photoreceptor morphology [7]. Since the composition of the Drosophila eye is substantially different compared to that of the human eye, it remains questionable whether Drosophila can serve as a good model to study EYS-associated RP. Extensive bio-informatic analysis showed that Eys is absent from several rodent genomes (e.g. mice) [6], which also excludes these animals for being used as a model system to study Eys function. In the zebrafish genome, several gene predictions encoding EGF-like domains and LamG domains are predicted on chromosome 13, including Chr13.1401, Chr13.1402, Chr13.1403, ENSDART00000108504 and ENSDART00000122834. This indicates that eys probably is present in zebrafish, although its complete sequence was not characterized. Furthermore, the zebrafish retina is morphologically similar to that of the human retina, in the sense that all the major cell layers found in humans are also present in zebrafish [8], thereby providing a promising model to study the function of EYS.
For staining with boron-dipyrromethene (BODIPY), zebrafish larvae or eyes were fixed in 4% PFA for 2 hours, cryoprotected in 10% sucrose in PBS for 30 minutes, embedded in OCT, snap frozen in melting isopentane and cryosectioned. BODIPY was applied to the cryosections at room temperature for 20 minutes.
Electroretinography measurements were performed on isolated larval eyes (5 dpf) as previously described [14]. Larvae were dark-adapted for a minimum of 30 minutes prior to the measurements, and subsequently handled under dim red illumination. The isolated eye was positioned to face the light source. Under visual control via a standard microscope (Stemi SV8, Zeiss) equipped with red illumination (KL1500 electronic, Zeiss), the recording electrode with an opening of approximately 15 μm at the tip was placed against the center of the cornea with a micromanipulator. This electrode was filled with E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl, and 0.33 mM MgSO4). A custom-made stimulator was invoked to provide light pulses of 100 ms duration, with a 100% light intensity of 6000 lux. It uses a ZEISS XBO 75W light source and a fast shutter (custom made) driven by the self-developed NI Labview program. Electronic signals were amplified 1,000 times by a pre-amplifier (P55 A.C. Preamplifier, Astro-Med. Inc, Grass Technology) with a band pass between 0.1 and 100 Hz, digitized by DAQ Board NI BNC-2090 (National Instruments) and displayed via the self-developed NI Labview program. All experiments were performed at room temperature.
The complete zebrafish eys sequence was not completely identified, however, in the UCSC genome browser, several gene predictions for zebrafish eys were present. On zebrafish chromosome 13, genes encoding EGF-like domains and LamG domains were predicted, including Chr13.1401, Chr13.1402, Chr13.1403, ENSDART00000108504 and ENSDART00000122834 (S1 Fig). Based on these gene predictions, we hypothesized that eys is present in zebrafish. We performed RT-PCR using primers targeting eys on RNA derived from adult zebrafish eyes as a template. This resulted in the identification of a 8,715 nucleotide long transcript encompassing 46 exons. Most of these exons were previously predicted by several gene prediction programs, although also a number of new exons were identified (S1A Fig). The resulting eys transcript translates into a protein of 2,905 amino acids that is predicted to harbor 39 EGF-like domains and five LamG domains. This domain organization is similar to what is observed for the human and Drosophila EYS proteins (S1B Fig). Interestingly, the zebrafish Eys protein appears to lack a so called low-complexity region, as seen for the human and Drosophila protein, in which no domains are predicted. In human, this region is completely encoded by exon 26, which is lacking in zebrafish eys.
Mutations in eyes shut homolog (EYS), a secreted extracellular matrix protein containing multiple laminin globular (LG) domains, and in protein O-mannose β1, 2-N-acetylglucosaminyl transferase 1 (POMGnT1), an enzyme involved in O-mannosyl glycosylation, cause retinitis pigmentosa (RP), RP25 and RP76, respectively. How EYS and POMGnT1 regulate photoreceptor survival is poorly understood. Since some LG domain-containing proteins function by binding to the matriglycan moiety of O-mannosyl glycans, we hypothesized that EYS interacted with matriglycans as well. To test this hypothesis, we performed EYS Far-Western blotting assay and generated pomgnt1 mutant zebrafish. The results showed that EYS bound to matriglycans. Pomgnt1 mutation in zebrafish resulted in a loss of matriglycan, retention of synaptotagmin-1-positive EYS secretory vesicles within the outer nuclear layer, and diminished EYS protein near the connecting cilia. Photoreceptor density in 2-month old pomgnt1 mutant retina was similar to the wild-type animals but was significantly reduced at 6-months. These results indicate that EYS protein localization to the connecting cilia requires interaction with the matriglycan and that O-mannosyl glycosylation is required for photoreceptor survival in zebrafish. This study identified a novel interaction between EYS and matriglycan demonstrating that RP25 and RP76 are mechanistically linked in that O-mannosyl glycosylation controls targeting of EYS protein.
Retinitis pigmentosa (RP) is, genetically, a very heterogeneous group of blinding diseases resulting from photoreceptor degeneration. It affects roughly 1 in 4,000 people worldwide with no effective therapy, according to the National Eye Institute1. Like usherin (USH2A), mutations in the extracellular matrix protein eyes shut homolog (EYS)2,3 are a common cause of the autosomal recessive form of the disease4,5,6,7,8,9,10, retinitis pigmentosa 25 (RP25, OMIM#612424). Some patients with EYS mutations exhibit cone-rod dystrophy11. The EYS gene encodes a large secreted protein comprised of two types of structural domains, laminin globular (LG) domains and EGF repeats. The functions of these domains in EYS are unknown. Interestingly, some mammalian clades, including mouse, have lost the EYS locus2, limiting our ability to investigate how EYS contributes to photoreceptor survival in the rodent. Thus, the zebrafish, which has an EYS locus, has been used to explore EYS function in the retina12,13,14. EYS-deficient zebrafish exhibits photoreceptor degeneration, confirming that EYS is essential for photoreceptor survival. Previous studies indicate that EYS protein is highly enriched near the connecting cilium/transition zone (CC/TZ) and is required for maintaining the structural integrity of the ciliary pocket12. However, the mechanisms that control its specific localization are unclear.
For eyes shut homolog Far-Western assay on zebrafish samples, muscle lysate proteins were separated on SDS-PAGE and transferred to PVDF membrane. Far-Western was performed as above but the blotting signal was detected with DyLight Fluor secondary antibodies and visualized with Odyssey CLx (Li-Cor Biosciences).
A recent news report about a woman who accidentally glued one of her eyes shut when she mistook Super Glue (cyanoacrylate adhesive) for her eye drops is a reminder that the potential for this mix-up is real. The Associated Press reported that a woman who had cataract surgery a year ago was reaching for what she thought was one of her half-dozen eye medications but picked up a nearby super glue container in error. A burning sensation immediately indicated that something was seriously wrong, so she went to the hospital where doctors worked on getting her eye open.
There is one time where it is acceptable for your chameleon to have their eyes closed during the day. And that is when they are settling in to go to sleep. In the wild, the transition between day and night is gradual and the chameleon has the warning and time to find a good place to sleep. In captivity they must adjust to a sudden switch from day to night as the lights go out. Many chameleons adjust to the by finding their sleeping spot before the lights go out. And, yes, as long as your schedule is consistent, your chameleon will have a feel for when it is time to find their sleeping spot even though the light is still on.
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