TMEM216 Deletion Causes Mislocalization of Cone Opsin and Rhodopsin and Photoreceptor Degeneration in Zebrafish

Purpose Mutations in TMEM216, a ciliary transition zone tetraspan transmembrane protein, are linked to Joubert syndrome and Meckel syndrome. Photoreceptor degeneration is a prominent phenotype in Joubert syndrome. How TMEM216 contributes to photoreceptor health is poorly understood. Methods We have generated tmem216 knockout zebrafish by CRISPR genome editing. The impact of TMEM216 deletion on photoreceptors was evaluated by immunofluorescence staining and electron microscopy. Results Homozygous tmem216 knockout zebrafish died before 21 days after fertilization. Their retina exhibited reduced immunoreactivity to rod photoreceptor outer segment marker 4D2 and cone photoreceptor outer segment marker G protein subunit α transducin 2 (GNAT2). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) revealed an increase in TUNEL-positive nuclei in the knockout retina, indicating photoreceptor degeneration. The tmem216 mutation resulted in shortened photoreceptor ciliary axoneme, as revealed by acetylated α-tubulin immunostaining. Photoreceptors in knockout zebrafish exhibited mislocalization of outer segment proteins such as rhodopsin, GNAT2, and red opsin to the inner segment and cell bodies. Additionally, electron microscopy revealed that the mutant photoreceptors elaborated outer segment with abnormal disc morphology such as shortened discs and vesicles/vacuoles within the outer segment. Conclusions Our results indicate that TMEM216 is essential for normal genesis of outer segment disc structures, transport of outer segment materials, and survival of photoreceptors in zebrafish. These tmem216 knockout zebrafish will be useful in studying how transition zone proteins regulate photoreceptor outer segment formation and maintenance.

TMEM216 is a small protein with four hydrophobic putative transmembrane domains predicted to form two extracellular loops and one intracellular loop. 12 TMEM216 knockdown resulted in a reduction in ciliogenesis. 12,25 TMEM216 is a member of the transition zone tectonic complex. 12,26 This protein-interacting complex consists of a group of Meckel and Joubert syndrome-related proteins including the secreted protein TCTN1, transmembrane proteins TCTN2, TCTN3, meckelin (TMEM67), and TMEM216, as well as intracellular proteins B9 domain-containing protein 1 (B9D1), CEP290, Meckel syndrome type 1 protein (MKS-1), and coiled-coil and C2 domain-containing protein 2A (CC2D2A). 26 Similarly, an experiment aimed to identify proteins interacting with B9D1 found a similar set of proteins at the transition zone B9 complex including TCTN1, TCTN2, TMEM231, B9D1, MKS1, CC2D2A, and Jouberin. 27 The tectonic/B9 complex is involved in the formation of cilia, regulates localization of ciliary membrane proteins such as Arl13b, and limits plasma membrane proteins in cilia. 26,27 How these proteins contribute to photoreceptor survival is poorly understood because deleting some of these proteins in the mouse, such as cc2d2a, 28 MKS1, 29,30 TMEM67, 26 and TCTN1 and 2, 26 results in premature lethality before or shortly after birth, precluding detailed evaluation on photoreceptor survival. CEP290 mutant mice survive up to weaning, and photoreceptors show no outer segments growth with severely shortened inner segments. 31 To evaluate the roles of TMEM216 in photoreceptor survival, we generated tmem216 knockout zebrafish using CRISPR. Deletion of TMEM216 did not affect photoreceptor generation but resulted in their eventual degeneration. Photoreceptor degeneration is correlated with shortened cilia, mislocalization of outer segment proteins, abnormal organization of F-actin, and outer segment morphological defects.

Zebrafish Maintenance
AB/Tubingen strain zebrafish were purchased from Zebrafish International Resource Center (Eugene, OR, USA). They were housed in a recirculating water system (pH 6.6-7.4) at 26°C to 28.5°C with a daily cycle of 14 hours light:10 hours dark. Zebrafish were fed once a day with Gemma Micro (Skretting, Tooele, UT, USA). Protocols for all experimental handling of the animals were approved by the Upstate Medical University Institutional Animal Care and Use Committee and were in accordance to National Institute of Health guidelines and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

In Situ Hybridization
Riboprobes for in situ hybridization were synthesized using reverse transcription-polymerase chain reaction (RT-PCR) to amplify a 483-base pair (bp) sequence in the zebrafish tmem216 mRNA transcript. Primers were designed to insert T3 and T7 promoter sequences, as well as EcoRI and NotI cleavage sites on each end of the amplicon. The following RT-PCR primers were generated through Eurofins Genomics (Ebersberg, Germany): forward primer T3EcoR1TMEM216rtf6 (5 -AATTAACCCTCACTAAAGAATTCGTTTCATTTGAACGGCT-GGT-3 ) and reverse primer T7Not1TMEM216rtr6 (5 -TAATACGACTCACTATAGGCGGCCGCGGTACTTGTCAGCT-TTATGTTTAATTG-3 ). The RT-PCR product was analyzed by gel electrophoresis and was extracted and purified using the QIAquick Gel Extraction Kit (28706; Qiagen, Hilden, Germany). The purified RT-PCR product was subsequently amplified by polymerase chain reaction (PCR) and digested with EcoRI (R0101S; New England Biolabs, Ipswich, MA, USA) or NotI-HF (R3189L; New England Biolabs), according to manufacturer suggestions. In vitro transcription was performed using T3 RNA polymerase (EP0101; Ther-moFisher, St. Louis, MO, USA) to synthesize the sense riboprobe and T7 RNA polymerase (EP0111; ThermoFisher) to synthesize the anti-sense riboprobe. The 10x DIG RNA labeling mix (11277073910; Sigma-Aldrich, St. Louis, MO, USA) was used in the in vitro transcription reactions to label each probe. Probes were purified using lithium chloride precipitation.
For in situ hybridization, zebrafish larvae were collected and fixed in 4% paraformaldehyde for 45 minutes, trans-ferred to 20% sucrose for two hours and frozen in optimal cutting temperature (OCT) medium. Blocks were cryosectioned and collected on Fisherbrand Superfrost Plus Microscope Slides (Fisher Scientific, Waltham, MA, USA). Slides were washed in phosphate-buffered saline solution (PBS) for five minutes at room temperature, 100% methanol for 10 minutes at room temperature, and PBS and 0.1% Tween-20 three times for five minutes/wash. Slides were then treated with Proteinase K for 30 seconds and re-fixed in 0.2% glutaraldehyde/4% paraformaldehyde for 10 minutes. In situ hybridization was performed as described. 32,33

Generation of tmem216 Knockout Zebrafish
We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to generate tmem216 knockout zebrafish. Three gRNAs targeting the coding region in exons 3 and 4 of zebrafish tmem216 locus respectively, TCCTGTTTCATTTGAACGGCTGG and CCCACAAGATAATCTGATATTGG, were designed with E-CRISP online gRNA design tool (http://www.e-crisp.org/E-CRISP/designcrispr.html) and generated at Genscript (Piscataway, NJ, USA). A 25-μL microinjection mix consisting of 50 pmol gRNA and 50 pmol Cas9 protein (New England Biolabs) in TE buffer (10 mmol/L Tris-HCl pH 7.4 and 0.1 mmol/L ethylenediamine tetra-acetic acid) was prepared; 1 nL of this mix was then injected into zebrafish embryos at one-to two-cell stage. Screening of F0 for mutant zebrafish was carried out by PCR with forward primer CACTTTTGCAGGAAGACAACC and reverse primer GCATCGTCAAACACTGCTTC, followed by gel electrophoresis to identify the presence of amplicons of sizes bigger or smaller than the wildtype. F0 zebrafish that yielded shorter bands were used to cross with the wildtype zebrafish to obtain F1 zebrafish. F1 zebrafish were genotyped by PCR and PCR bands shorter than the wildtype (577bp) were purified and sequenced to identify mutations. This effort yielded two tmem216 knockout lines (Fig. 2). The data in this study were collected from F2 and F3 generations.
Fluorescence intensities of GNAT2, 1D4, and 4D2 were quantified using images taken with a ×40 objective lens on a Zeiss Axioskop epifluorescence microscope. Two images near the central plane of the retina were taken per animal. Images were imported to ImageJ software (National Insti-tutes of Health) for quantification. Using ImageJ, the outer segment region of the retina was outlined, and the average fluorescence intensity for that region was calculated. Average background fluorescence was subtracted from the average fluorescence intensity to generate the reported adjusted average fluorescence intensity. Student's t-tests were performed to assess significance.
The length and number of acetylated α-tubulin axonemes were quantified using images taken with a Zeiss LSM780 confocal microscope with a ×40 oil objective lens. Two images near the central plane of the retina were taken per animal. Images were imported to ImageJ software to measure axoneme count and length. Student's t-tests were performed to assess significance.
Blot images were exported to ImageJ software for quantification. Each image was color-inverted, and the band was manually selected, in software, using the polygon selection tool. GNAT2 and β-actin band intensities were measured, and the ratio of GNAT2:β-actin intensity was calculated.

Transmission Electron Microscopy (EM)
Zebrafish fry heads were fixed in 2% paraformaldehyde and 2% glutaraldehyde in phosphate buffer. Transmission EM was carried out as we previously described. 34,35 For quantification of outer segment disruptions, photoreceptors with visible connecting cilia were selected for imaging spanning from the basal body to the outer segment at magnification × 40,000 or 60,000. Photoreceptors exhibiting shortened discs and vacuoles in the outer segment were counted. Fisher's exact test was performed to test for significance.

Expression of tmem216 in Zebrafish
To evaluate tissue expression of tmem216, we performed in situ hybridization with tmem216 antisense probe (Figs. 1A-1D) using sense probe as a control (Figs. 1E-1H). At 3dpf, in situ hybridization with antisense probe resulted in widely distributed signal in multiple organs including the eye, pronephros, brain, liver, intestine, and muscle (Figs. 1A and 1E). Within the retina, expression of tmem216 was observed in all cell layers of the neural retina including the outer nuclear layer, inner nuclear layer, and the ganglion cell layer (Figs. 1B, 1C, 1F, and 1G). The tmem216 expression was maintained in the 7-dpf zebrafish neural retina (Figs. 1D and 1H). RT-PCR showed that TMEM216 mRNA was detected in freshly laid eggs, 7-dpf larvae, and adult eye, brain, and skeletal muscle (Fig. 1I). These data suggest that tmem216 is widely expressed in zebrafish.

Generation of TMEM216-Deficient Zebrafish
To study the roles of TMEM216 in photoreceptor survival, we used CRISPR/Cas9 genome editing to generate tmem216 knockout zebrafish. This effort yielded two knockout zebrafish lines, tmem216 sny 175 , and tmem216 snyR8 60 ( Fig.  2A). The tmem216 sny 175 mutation harbors two deletions, a 172-bp deletion from exon 3 to exon 4 and a 3-bp deletion within exon 4. The tmem216 snyR8 60 mutation had an 8-bp duplication (CAGATCCT) within exon 3 and deletion of two fragments, a 56-bp deletion within exon 3, and a 4bp deletion within exon 4. All of these mutations disrupted the reading frame and were expected to result in the loss of the majority of the TMEM216 protein, starting at the first of the four transmembrane domains and were thus predicted to be null mutations (Fig. 2B). RT-PCR revealed that homozygous knockout animals expressed only mutant mRNA whereas heterozygous animals expressed both wildtype and mutant mRNA (Fig. 2C). Number of homozygous zebrafish for both mutations were expected from Mendelian ratios at one week after fertilization but decreased after two weeks (Table 1). Only one homozygous zebrafish survived to three weeks after fertilization. No homozygous animals were found in ages older than four weeks after fertilization. At the time of sacrifice at 3-dpf, 7-dpf, and 14-dpf, no obvious gross morphologic abnormalities, including edema, pericardial effusion, hydrocephalus, or kidney cysts, were observed in tmem216 snyR8 60 and tmem216 sny 175 homozygous zebrafish. The phenotypes observed in the retina of both homozygous knockout animals were indistinguishable. Thus the data from these knockout zebrafish are combined.

Mislocalization of GNAT2 and 1D4 Reactivity in Photoreceptors of TMEM216-Deficient Zebrafish
GNAT2 protein is highly enriched in cone outer segment with little expression in the inner segment and cell bodies.
In light-adapted wildtype animals, GNAT2 is found in the outer segment of cones (Fig. 4A, see arrow for example). In the tmem216 snyR8 60 homozygous retina, however, GNAT2 immunoreactivity is fragmented, with signal observed in the cell bodies around the nuclei (Fig. 4C, arrowheads). 1D4 antibody recognizes red opsin of long double cones outer segment in zebrafish. 37 In the wildtype animals, 1D4 immunoreactivity is strongly observed in the outer segment layer (Fig. 4B, arrow). In the tmem216 snyR8 60 homozygous retina, 1D4 immunoreactivity appeared fragmented and many 1D4 immunoreactive puncta were observed around the cell bodies (Fig. 4D, arrowhead). These results indicated that there is mislocalization of cone outer segment materials to the inner segments and cell bodies of photoreceptors in tmem216 knockout fish.
In the wildtype animals (Figs. 5A-5F), 4D2 immunoreactivity was observed between the outer nuclear layer and the retinal pigment epithelium throughout the retina. In the tmem216 sny 175 homozygous animals (Figs. 5G-5L), 4D2 immunoreactivity was sparsely present. Fluorescence intensity of 4D2 reactivity was significantly reduced in tmem216 sny 175 homozygous zebrafish at three, seven, and 14 days after fertilization when compared with the wildtype animals (Figs. 5M-5O). These results suggested that rod photoreceptors were significantly reduced in tmem216 knockout zebrafish.

Mislocalization of 4D2 Immunoreactivity and Disorganization of F-Actin in Photoreceptors of TMEM216-Deficient Zebrafish
Rhodopsin staining with 4D2 antibody revealed that immunofluorescence activity is highly enriched in the outer segment layer of wildtype zebrafish (Fig. 6A, arrow). In tmem216 sny 175 homozygous retina, high 4D2 reactivity in the outer nuclear layer was often observed (Fig. 6C, arrowheads). We measured 4D2 immunofluorescence intensity in the outer segment layer and outer nuclear layer and calculated the ratio of fluorescence intensity of cell body layer/outer segment layer. Although the ratio in the wildtype was 0.70 ± 0.0084 (mean ± standard error of the mean [SEM]), the knockout was 1.82 ± 0.288 (mean ± SEM) (P = 0.018, Student's t-test). These results indicated mislocalization of rhodopsin to the inner segments and cell bodies of photoreceptors in tmem216 knockout zebrafish.
The actin cytoskeleton is involved in translocation of proteins, such as arrestin, transducin, and cyclic nucleotidegated channel α-subunit (CNGA1), within photoreceptors. Because several outer segment proteins were found to be mislocalized in the photoreceptors of tmem216 sny 175 homozygous zebrafish, we used phalloidin-RITC (Sigma-Aldrich) to examine F-actin in tmem216 sny 175 homozygous zebrafish. In wildtype zebrafish retina, phalloidin staining showed that polymerized F-actin was arranged in a characteristic organization, parallel to the basalapical radiation axis of photoreceptors (Fig. 6B, arrows). However, in tmem216 sny 175 homozygous retina, F-actin reactivity was thinner or fragmented with some oriented at an angle relative to the basal-apical axis (Fig. 6D, arrowheads).

TUNEL-Positive Nuclei Were Increased in TMEM216-Deficient Zebrafish
To determine photoreceptors loss by programmed cell death, we carried out terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as described. 38 TUNEL-positive nuclei in the outer nuclear layer of tmem216 snyR8 60 homozygous zebrafish were frequently observed (arrows, Figs. 7B and 7C) and significantly increased over the wildtype animals (Fig. 7D). These results indicate that apoptotic cell death in photoreceptors were increased in tmem216 knockout zebrafish.
The tmem216 mutation in zebrafish may cause ER stress, which might result in photoreceptor cell death. To evaluate this, we stained 7-dpf wildtype and knockout retina with anti-CCAAT/-enhancer-binding protein homologous protein (CHOP). In the wildtype and tmem216 snyR8 60 homozygous zebrafish, CHOP labeling was present throughout all layers of the neural retina, including the photoreceptor inner  segments and cell bodies (Figs. 7E and 7F). However, we did not observe an increase in CHOP expression in the knockout retina, suggesting a lack of significant ER stress in TMEM216deficient photoreceptors.

Photoreceptor Axonemes Were Shorter in TMEM216-Deficient Zebrafish
TMEM216 is a member of the tectonic complex highly enriched at the transition zone of primary cilia. 26,27,39 Tctn1, a member of this complex, is required for ciliogenesis of some but not all cell types. We therefore evaluated whether TMEM216 deletion affected localization of other transition zone proteins, CC2D2A (Figs. 8A and 8B) and TMEM231 (Figs. 8C and 8D), by immunofluorescence staining of 7-dpf zebrafish. In the wildtype animals, CC2D2A and TMEM231 immunoreactivity (green fluorescence) was localized to the basal end of the acetylated α-tubulin (Figs. 8A and 8C, arrowheads), an axoneme marker. Expression of both CC2D2A and TMEM231 at the basal end of acetylated α-tubulin was not affected in tmem216 sny 175 homozygous animals (arrowheads in Figs. 8B and 8D), indicating that CC2D2A and TMEM231 was not affected by TMEM216 deletion. Similarly, localization of EYS, an extracellular matrix protein expressed near the connecting cilia was not affected by TMEM216 deletion (green fluorescence, Figs. 8E-8F, 8J, 8K).
With regard to acetylated α-tubulin-labeled axonemes, they were observed throughout the outer nuclear layer and outer segment layer at 7-dpf in the wildtype retina (Fig. 8E). In tmem216 sny 175 homozygous zebrafish, axonemes were also observed throughout these layers at 7-dpf ( Fig. 8F) with similar numbers as the wildtype (Fig. 8G). However, the length of acetylated α-tubulin-positive ciliary axoneme was reduced in tmem216 sny 175 homozygous zebrafish retina at 7-dpf (Figs. 8F, 8H, 8I). These results indicated that TMEM216 knockout photoreceptors generate cilia, but the length of photoreceptor cilia was shorter than wildtype animals. At 14-dpf, density of acetylated α-tubulin in tmem216 sny 175 homozygous zebrafish was reduced when compared to the wildtype retina (Figs. 8K, 8L). Similar to the 7-dpf zebrafish knockout axonemes, the average axoneme   length in the 14-dpf tmem216 sny 175 homozygous zebrafish was shorter than the wildtype (Figs. 8K, 8M, 8N). Reduced number of acetylated α-tubulin in tmem216 knockout retina at 14-dpf was expected as a result of photoreceptor degeneration. These results indicate that photoreceptor primary cilia were formed in tmem216 knockout retina but were shortened compared with the wildtype.

TMEM216 Is Required for Normal Genesis of Outer Segment
To determine the impact of TMEM216 deletion on the ultrastructure of photoreceptors, we carried out transmission electron microcopy analysis of tmem216 snyR8 60 homozygous zebrafish at 7-dpf. Photoreceptors with elaborated outer segments were observed for both rods and cones in wildtype and tmem216 snyR8 60 homozygous zebrafish (Figs. 9A, 9E). Since acetylated α-tubulin staining showed similar pattern of immunoreactivity at 7-dpf in the knockout retina, it is expected that ciliogenesis of photoreceptors was not affected at the ultrastructural level. Indeed, newly elaborated cilia with clear basal bodies (arrows), ciliary pockets (arrowheads), and axonemes (asterisks) in tmem216 snyR8 60 homozygous retina (Fig. 9F) were phenotypically similar to the wildtype retina (Fig. 9B). However, multiple abnormalities were observed in the outer segment in tmem216 snyR8 60 homozygous animals. In the wildtype retina, outer segment of rods (Figs. 9B, 9C) and cones (Fig. 9D) with uniform discs were observed at 7-dpf. However, in tmem216 snyR8 60 homozygous photoreceptors, some outer segments exhibit scrambled disc structures (Fig. 9F), with large vacuoles within the outer segment (asterisks in Figs. 9G, 9H), vacuoles near the base of outer segment/apical end of inner segment (asterisks, Fig. 9I), and uneven disc sizes with apparent shortened discs that do not span the width of the photoreceptor outer segment (arrows, Fig. 9G). We counted outer segment exhibiting normal disc morphology, shortened discs, and presence of vacuoles from images focused on photoreceptors showing the connecting cilia (Table 2) from 3 wild-type and 3 tmem216 snyR8 60 homozygous zebrafish. The majority of the outer segment in TMEM216 knockout photoreceptors exhibited outer segment disc defects (P < 0.0001, Fisher's exact test). These data indicate that loss of TMEM216 does not affect photoreceptor ciliogenesis but, rather, outer segment genesis.

DISCUSSION
We deleted TMEM216 in zebrafish with CRISPR-mediated genome editing. Homozygous tmem216 knockout animals died before three weeks after fertilization. Although photoreceptors were generated in TMEM216-deficient animals and elaborated cilia, the ciliary length was shortened. Loss of photoreceptors was observed at 3-, 7and 14-dpf. This was accompanied by mislocalization of outer segment proteins including GNAT2, cone opsin, and rhodopsin. F-actin morphology was altered in the knockout photoreceptors. EM analysis revealed that tmem216 knockout photoreceptors elaborated outer segment with ultrastructural morphological defects including disorganized discs, presence of large vacuoles within the outer segment or at the base of outer segment, un-uniform disc sizes. These results indicate that TMEM216 is required for normal outer segment disc morphogenesis and photoreceptor survival in zebrafish.
The transition zone Tectonic/B9D1 complex proteins play critical roles in ciliary trafficking and genesis. 26,27,37 Tectonic complex protein member TCTN1 is required for cilia formation in select types of cells such as those in the node and neural tube cells but not in the limb bud and perineural mesenchyme. 23 As a member of the Tectonic/B9D1 complex proteins, TMEM216 is also involved in ciliogenesis. Tmem216 mutations in patient fibroblasts result in impaired ciliogenesis and centrosomal docking. 12 Morpholino-mediated knockdown of tmem216 in zebrafish reveal ciliopathy-associated phenotypes, such as curved/kinked tail, pericardial effusion and gastrula-tion defects. 12,25 The effect of morpholino knockdown of tmem216 on the zebrafish retina was not reported. Here, we show that loss of TMEM216 does not affect localization of tectonic complex proteins CC2D2A and TMEM231. The tmem216 knockout zebrafish exhibit normal number of photoreceptor axonemes at seven days after fertilization, indicating that a normal number of photoreceptor cilia were generated. However, the axoneme length was reduced in tmem216 knockout zebrafish. Immunostaining for rod and cone outer segment markers showed significantly reduced reactivity in tmem216 knockout retinas. These results indicated that TMEM216 was not absolutely required for elaboration of the connecting cilia in photoreceptors, though these cilia were shortened.
Mutations in tectonic complex genes often cause severe retinal phenotypes. Besides cystic kidney and cerebellar vermis hypoplasia, 40,41 Ahi1-null mice lack photoreceptor outer segments and are reported to have severe retinal degeneration at early ages. 42,43 Loss of TCTN2 or TCTN3 results in microphthalmia in mice. 44,45 A frameshift mutation in Cep290 in the rdAc Abyssinian cat has been reported to result in rod-cone dystrophy, with complete blindness occurring between 3-5 years of age. [46][47][48][49] EM studies of the rdAc cat revealed accumulation of vesicles near the rod connecting cilium. 49 Similar to the rdAc cat, loss of Cep290 in mice, results in loss of photoreceptor outer segments as well as a shortening of photoreceptor inner segments. 31 Tmem67/mks3 is crucial in photoreceptor health, because tmem67-null rat photoreceptors do not develop outer segments. 50 CC2D2A mutant zebrafish exhibits disruptions of rod and cone outer segments. 21,43 Photoreceptors in CC2D2A-deficient zebrafish can grow cilia but outer segment genesis is defective, with accumulation of vesicles in the photoreceptor inner segments. 43 In tmem216 knockout zebrafish, photoreceptor outer segment exhibited multiple defects in disc morphology, with the presence of vacuoles throughout the photoreceptor outer segment, as well as shortened outer segment discs. Together with mislocalization of outer segment protein in tmem216 knockout photoreceptors, these data indicated that TMEM216 plays critical roles in photoreceptor disc morphogenesis and extension, supporting an essential role of Tectonic/B9 complex proteins in morphogenesis of the outer segment.