Knockdown of poc1b causes abnormal photoreceptor sensory cilium and vision impairment in zebrafish

Accepted Manuscript Knockdown of poc1b causes abnormal photoreceptor sensory cilia and vision impairment in zebrafish Conghui Zhang, Qi Zhang, Fang Wang, Qin Liu PII:

S0006-291X(15)30136-4

DOI:

10.1016/j.bbrc.2015.06.083

Reference:

YBBRC 34116

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 3 June 2015 Accepted Date: 12 June 2015

Please cite this article as: C. Zhang, Q. Zhang, F. Wang, Q. Liu, Knockdown of poc1b causes abnormal photoreceptor sensory cilia and vision impairment in zebrafish, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.06.083. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Knockdown of poc1b causes abnormal photoreceptor sensory cilia and vision impairment in zebrafish Conghui Zhang1, 2, Qi Zhang2, Fang Wang*1, Qin Liu*2 1.

Department of Ophthalmology, Shanghai Tenth People’s Hospital, Tongji University, Shanghai, China.

2.

Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA,

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United States.

*Corresponding author. E-mail address: Qin Liu, [email protected]; Fang Wang, [email protected]. Address: 1. 15th floor, Building1, No.301, Yanchang Road, Shanghai, China. 200072. Tel: +86 13671886466. th

2. 5 floor, No.243, Charles Street, Boston, MA, United States. 02114. Tel: +1 6175736925.

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Abstract Defect of centriole or cilia genes were highlighted as associated with inherited retinal diseases and ciliopathy, which has a broad spectrum of phenotype including vision impairment. The mechanism may be interrupt of cilia formation. poc1b is an abundant protein in centriole proteomics, and it was found a novel gene for inherited retinal diseases. Purpose: Our aim is to study the role of poc1b in the photoreceptor sensory cilia formation and importance in vision function. Methods: Knock down of poc1b in mIMCD3 cell was used to study its role in general cilia formation. Gene blast, RT-PCR and western blot were used to study the conserved expression of poc1b. V5-poc1b and Flag-poc1b expression plasmids were constructed; and then in vitro (cell culture) and in vivo (rat sub-retinal injection) poc1b plasmid transfection were performed to detect location of poc1b in cilia and photoreceptor sensory cilia. For function study of poc1b, zebrafish gene knockdown, vision test, histology were processed. Results: poc1b was critical for cilia formation in mIMCD3 cell; poc1b was conserved expressed and was located in the basal body of mIMCD3 cell and mouse photoreceptor cilia; knockdown of poc1b leads to a spectrum phenotypes associated with cilia defect including vision impairment. Histology results show abnormal photoreceptor sensory cilia in defected larvae. Conclusion: poc1b is required for photoreceptor sensory cilia formation and normal vision in zebrafish. Key words: poc1b, photoreceptor sensory cilia (PSC), basal body, ciliopathy, zebrafish, inherited retinal diseases (IRDs).

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Introduction Inherited retinal diseases (IRDs) are one of main causes of blindness. Most of IRDs are characterized as progressed photoreceptor degeneration, including retinitis pigmentosa (RP), Cone or cone-rod dystrophy, and Leber congenital amaurosis (LCA). Numbers of IRDs are manifested as cilipathy, such as Usher syndrome, Bardet-Biedl syndrome (BBS). Cilipathy is a multi-system defect syndrome due to defect of ciliogenesis, it presents as poor visual acuity, anosmia cognitive impairments, kidney cysts, hepatic fibrosis, polydactyly, obesity, thoracic dysmorphology, genitourinary defects, cardiorespiratory abnormalities, neural tube patterning defects [1]. There are 220 IRDs genes identified, remanding about 50% genes unknown so far (Retnet, 8-8-2014). Cilia are microtubule-based organelles theta producing from the surface of the cells into the extracellular space, it can happen in virtually all cell types of human body [2]. Cilia are generally called primary cilia, which comprise of basal body, transition zone (TZ), axoneme, ciliary membrane and cilia tip [3,4], the spatial structure can be illustrated as microtubule-based axoneme anchors basal body, then extends to the cilia tip, and surrounded by ciliary membrane, which continuous from the cell membrane; the begin area of axoneme is often called transition zone [5]. Right structure and normal function of cilia are essential to cilia cell physiological function, thus abnormal of ciliogenesis or protein transport of cilia may induce lots of phenotypes mainly identifed as ciliopathy, which is cilia–related disorders either involving single organs or affecting multisystem with manifestation. Photoreceptor cilia are specialized primary cilia, and

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usually called photoreceptor sensory cilium (PSC), they are also recognized as outer segments (OS) of photoreceptor cells. The structure of the PSC is similar to the primary cilia[6] . Photoreceptor function is detecting the light signal and converting it to electrical signal with OS discs metabolism. The proteins needed for photo-transduction are located or associated with OS discs. OS can’t synthesis protein and lipids by itself, so its proteins and lipids come from the inner segment and need to be transported to OS through the transition zone. The transport mechanism is called intraflagellar transport (IFT)，which is large protein complexes carrying material along the axoneme and

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moving by motor proteins [7]. It can be concluded that PSC structure is crucial; abnormal PSC structure may impact on PSC function including photo-transduction. According to recent studies, there are 48 PSC genes were found IRDs genes, whose disease symptoms manifested as cilipathy with multi-system defect. [8] We can propose that genes that interrupt PSC formation and associate with ciliopathy may influence on vision formation and may be IRDs disease genes. poc1 gene was first found in the alga Chlamydomonas in centriole proteomics study [9], it is one of the most abundant proteins in centriole proteomics, and has a highly conserved WD 40 domain. poc1 has 2 paralogues, poc1a and poc1b. As we know, cilia generated from the mother centriole, thus disrupt of centriole may cause ciliogenesis defect. In 2009, Chad G. Pearson found that human poc1b is required for primary cilia formation based on cell culture experiment [10]. Since PSC are specialized primary cilia, the role of poc1b in PSC formation needs to be illustrated. What’s more, our study are significant since poc1b was reported a novel gene for IRD[8,11,12]. Materials and Methods

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Plasmid construct Mouse poc1b DNA was bought from open biosysterm, cloned into a pENTR/D-TOPO entry vector, and moved to a Gateway destination expression vector containing V5 and Flag epitope tag in-frame[13,14]. pCAG-V5-IRES-EGFP and pCAG-Flag-IRES-EGFP expression plasmid were generated. Three mpoc1b shRNA (EAP5141, EAP5142, EAP5143) and 1 negative control shRNA (EAP2247) were generated, cloned into a pCAG-miR30-IRES-EGFP vector, modified by our laboratory for this purpose using established methods [15] . Western blot To get the knockdown efficiency of shRNA, CHO cells were transfected with pCAG-V5-poc1b-IRES-EGFP plasmid and pCAG-miR30-shRNA-IRES-EGFP (one negative control; three mpoc1b shRNA (both 2µg/well) were transfected using lipofectamine 2000. 48 h after transfection, the cells were collected for western blot, anti-V5 tag antibody was used as primary antibody. Cell culture and Immunofluorence Wild-type mIMCD3 cell lines were purchased. A mIMCD3 stable cell line expressing

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SSTR3-EGFP, a ciliary membrane marker (gift of Gregory J. Pazour, University of Massachusetts Medical School) was used for poc1b subcellular localization. Two expression and three knockdown plasmids were transfected (Flag-poc1b in mIMCD3 cells, Flag-poc1b in SSTR3-EGFP cells, V5-poc1b in SSTR3-EGFP cells); (EAP2247, EAP5141, EAP5142, EAP5143 in mIMCD3 cells). Transfection was performed with Lipofectamine 2000 or Lipofectamine LTX reagent. The cells were processed for immunocytochemistry at 72 h after transfection. For the cell culture immunofluorence, anti-V5 tag antibody was used as primary antibody.

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RT- PCR poc1b RNA expression levels in zebrafish and mouse were tested using RT-PCR. For zebrafish, embryos (both standard control and morphant group) at different stages during 120 hours post fertilization were selected and used for total RNA extraction. Extracted RNA (2 µg per sample) was reverse-transcribed using the superscript first-strand synthesis system for RT-PCR. To get the poc1b mRNA for the rescue experiment, human poc1b cDNA was amplified by PCR, cloned into an Sp6/T7 promoter vector and linearized with SnaBI. Capped hpoc1b mRNA was synthesized in vitro using mMESSAGE mMACHINE® SP6 Transcription Kit and purified by the RNeasy Plus micro kit.

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Sub-retinal injection and Immunofluorence The rat experiments obey to the animal ethics standards. To localize poc1b expression in photoreceptor cells, in vivo electroporation technique was used in rat photoreceptor cells (10 rats, 10 right eyes). Briefly, 0.5µl of endotoxin-free pCAG-V5-poc1b-IRES-EGFP plasmid was injected sub-retinal into the right eyes of neonatal rats. Injected plasmid was electroporated into retinal cells using tweezer-type electrodes as previously described [14]. The plasmid was injected into eyes; rats were killed 4 weeks following injection, and frozen sections were prepared from the portions of the eyecups with EGFP signal. For the tissue frozen section, sections were immunostained as described above using anti-V5 antibody. Mophlino injection of zebfish embryo The zebrafish experiments obey to the animal ethics standards. Wild-type zebrafish (Danio rerio) were generated in Ocular Genomics Institute, Massachusetts Eye and Ear Infirmary. Embryos were collected in E3 solution after natural spawns and kept at 28.5°C in an incubator. Three morpholinos group were designed (MO-TB, block poc1b RNA translation, 5′-TCCTCCATTACAGACGCCATGATTC-3′; MO-SP, block splicing, 5′ACTAAATCATCTTACCATTACAGAC-3′; control, 5′-CCTCTTACCTCAGTTACAATTTATA-3′) and obtained from Gene Tools Company. Embryos were injected at the one- to two-cell stages with 0.3–0.5 mM MO. Embryos were imaged with a Stemi SV II stereomicroscope and photographed with an AxioCam digital camera. For the zebrafish larvae defect rescue, human poc1b mRNA (100-300 pg) was coinjected with MO (0.4

ACCEPTED MANUSCRIPT mM) into zebrafish embryos.

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Zebrafish larvae vision test Vision of zebrafish larvae in standard control and MO-TB groups was test on 5 days post fertilization, when normal vision should generate. The larvae were embedded in 6% pre-warmed (28°C) methylcellulose to prevent body movements with dorsal side up, and surround by a moving black-and-white wave drum at a speed of 5 rpm. The tracking was at a rate of 4 frames/second and last 1.5min with direction-changed per30s [16,17]. ImageJ and plugin (From NIH) was used to measure the position of larvae eye change per frame; and then the data were calculated and processed a graph with metalab 2013.

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Zebrafish Histology Zebrafish embryos in standard control and MO-TB groups were processed for histology following a Cold Spring Harbor Protocol[18] . For eye and heart histology, transverse semithin sections (1µm) were stained with alkaline Toluidine blue. Statistical Analysis Statistical analyses were performed with SPSS19.0. For the cilia length data analyses in mIMCD3 cell, Student's t test with unequal variance was used. For analysis the defect rate of zebrafish larvae, both Students’ test with unequal variance and chi-square test were used.

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Results

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poc1b was critical for cilia formation in mIMCD3 cell Knock down (KD) of poc1b in mIMCD3 cell cause shorten cilia. Three poc1b shRNA plasmids (EAP5141, EAP5142, EAP5143) knockdown efficiency was confirmed. Western blot result of CHO-K1 cells demonstrated that all the tree shRNA could knockdown mouse poc1b with high efficiency. Then shRNA were transfected to the mIMCD3 cells, cilia length of hundreds of shRNA-transfected cells and surrounding cells were measured. From the statistic results, it can be concluded that knockdown of poc1b in mIMCD3 cell cause shorten cilia. (Figure1) Figure1. shRNA KD of poc1b in mIMCD3 cell cause shorten cilia. (A) EAP5141, EAP5142, EAP514 KD efficiency were high, EAP2245 random oligo were negative control. (B) The mIMCD3 cell cilium became short in shRNA group compare to negative control group and non-shNNA transfected group. (The red fluoresce was acetylated a-tubulin-a cilia marker, green fluorence was EGFP expression indicating the cell was transfected with shRNA plasmids). (C) The cilia of shRNA-transfected cells were shorter than the negative control group and non-shRNA transfected one. About one hundred cilia lengths of shRNA-transfected cells were measured, and four non-transfected cell around the shRNA-transfected cell were measured, the mean cilia lengths were calculated. There is no significant distinction of the cilia lengths between non-transfected group and negative control group (p>0.05). All the three-shRNA group cilia lengths are significantly shorter than the non-transfected group and negative control group

ACCEPTED MANUSCRIPT (**p<0.05).

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poc1b was conserved expressed and was located in the basal body of mIMCD3 cell and mouse photoreceptor cilia PubMed alignment was used results revealed that: Homo sapiens/Danio rerio67%; Homo sapiens/Mus musculus77%; Homo sapiens/Rattus norvegicus84%. From Chlamydomonas to the humans, poc1b proteins have an unusual structure, including a WD40 domain in the N terminal and a coiled coil in the C terminal of the protein. Besides, WD40 was sufficient for poc1b located in the centriole. The figure elucidated poc1b was transcript in 120 hours post fertilization in zebrafish (figure 2A); meanwhile, poc1b expressed in mIMCD3 cells (figure 2B); In figure 2C, during the S Stage, poc1b located in both of two centrioles; when the cells entered M Stage, poc1b expressed in the connection of the bindles, and do not co-localized with the γ–tubulin. Both Flag-poc1b and V5-poc1b were transfected in a stable mIMCD3 cell line with a green fluorescent protein (GFP) fused to a cilia membrane receptor (SSTR3) called mIMCD3- SSTR3-EGFP, this cell line can produce the cilium green fluoresce, thus the co-localization of poc1b and cilia can be identified, poc1b located in the bottom of the mIMCD3 cell cilium (basal body). poc1b also expressed in mouse retina (figure 2D) with β-actin as the compare control. Western blot revealed poc1b expressed in mouse retina (figure 2E). Sub-retina injection of V5- hpoc1b-IRES-GFP plasmid to the new born rats revealed that the GFP protein expressed in the inner segment of photoreceptor, and poc1b expressed in the tip of the inner segment of photoreceptor. Combined the cell culture result, it can be concluded that poc1b was located in the basal body of PSC (Figure 2F). Figure2. poc1b was highly conserved and express in zebrafish, basal body of cultured mIMCD3 cell and mouse photoreceptor cilia. (A) Embryos RT-PCR result demonstrated that poc1b was transcript in 120 hours post fertilization and the transcript level was stable. (B) Expression of poc1b in mIMCD3 cell on

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the transcript level. (C) Location of poc1b in the mIMCD3 cell: Up, during the S Stage, poc1b located in both of two centrioles (red, co-localization with the centrioles marker-γ–tubulin); when the cells enter M Stage, the poc1b expressed in the connection of the bindles, and do not co-localized with the γ–tubulin.

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Down, poc1b location in the cilia cell when cell cycle exited (cilia generate): poc1b located in the basal body of mIMCD3 cell cilium (red). (D, E) Expression of poc1b in mouse cell and retina on the transcript and protein level, CHOs cell transfected with pCAG-V5-poc1b-IRES-EGFP plasmid was selected as positive control. Western result revealed that poc1b expressed in the mouse retina with 2 molecular sizes (the top 2 red bands) in accordance with the Novus antibody Information. (F) poc1b located in the basal body of mouse photoreceptor (red), the green fluoresce was EGFP expression indicating the photoreceptor was transfected with pCAG-V5-poc1b-IRES-EGFP.

KD of poc1b cause vision impairment in zebrafish There is no significant distinction in embryo survival rate between the standard control and mophlino knockdown group; but the deficiency rate was much higher in the mophlino knockdown group than the standard control one (*p<0.05)(Figure 3B), what’s more the defect can be rescued (**p<0.05) (Figure 3C). In the zebrafish larvae

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picture taken at 72 hours post fertilization, several phenotypes could be observed in knockdown group, including small eye, small body, curve tail and heart defect (Figure 3A). Larvae vision in both group were measured after 120 hours post-fertilization, when they could generate normal vision. Strike stimulation were used to calculate the saccade time of the larvae in 1:30 minutes. The results demonstrated knockdown of poc1b in zebrafish lead to low response to strike stimulation. The angle change was showed in Figure3D. From Figure 3E, both of the angle and frequency decreased in mophlino group. It can be concluded that knockdown of poc1b in zebrafish lead to vision decrease. Figure3. KD of poc1b in zebrafish cause several phenotypes in zebrafish larvae including vision impairment. (A) The zebrafish larvae picture taken at 72 hours post fertilization reveal that transcription

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mophlino knockdown of poc1b can cause several phenotypes, including small eye, small body, curve tail and heart defect. (B) The survival rate and deficiency rate of zebrafish larvae in the first 2 days. There is no significant distinction in survival rate between different groups; but the deficiency rate is much higher in both transcription blocker (TB, n=153) and splicing blocker (SB, n=40) mophlino knockdown group

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than the standard control one (control group, n=253) (*p<0.05). (C) The zebrafish larvae defect can be rescued by poc1b mRNA. The deficiency rate increase significantly in mRNA and transcription blocker co-inject group. (100mg mRNA; 200mg mRNA; 300mg mRNA; N=3) (**p<0.05). (D) The schematic of eye angle measurement: two point in the zebrafish larvae mouth which can create an vertical line of body axis was tracked; two point in the zebrafish larvae eye which can create an axis line of eye axis was tracked; eye angle were decided by these two line. (E) Both of the angle and frequency decrease in poc1b TB mophlino group, the zebrafish larvae reaction was lower in poc1b TB mophlino group when the drum

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rotation direction changed.

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KD of poc1b cause abnormal PSC From the head sections (120 hours after fertilization section), small head and brain were observed in knockdown group (Figure 4A). The eye section of 72 hours after fertilization manifested that, the larvae eye development in the mophlino injection group were delayed; they don’t have the clear retina layer development as the standard control group. The same result happened in the 120 hours after fertilization group. Further more, the out segment of photoreceptor in the mophlino injection group was much shorter than the standard control group, thus the knockdown of poc1b in zebrafish lead to abnormal PSC (Figure 4B). However, KD of poc1b cause ciliopathy related manifestations (Supplement1). Figure 4. (A) The head and brain at 5 days post fertilization became small in poc1b TB mophlino injection group compared to standard control group. (B) The eye section of 72 hours after fertilization larvae in the mophlino injection group don’t have clear retina layer development as the standard control group. 120 hours after fertilization group, out segment of photoreceptor in the mophlino injection group is much shorter than the standard control group both central and lateral retina.

Discussion Defect of PSC structure genes and IRDs

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Gene knockdown of the poc1b in zebrafish resulted visual defects and other symptoms of ciliopathies similar to those seen in Leber's congenital amaurosis, Meckel-Gruber syndrome, Joubert syndrome, Senor-Loken syndrome and Bardet-Bledl syndrome involve retinal dystrophy as well as other multi-system defect. What’s more, defect of centriole/basal body genes are also IRDs genes. Pericentrin (Pcnt) is at the basal body complex of the connecting cilium in mouse photoreceptors and associated with a range of diseases including primordial dwarfism and ciliopathies [19]. Retinitis pigmentosa gene FAM161A is found interacting with several ciliagedesis associated proteins including lebercilin, CEP290, OFD1 and SDCCAG8 [20,21] Autosomal-recessive cone-rod dystrophy (arCRD) gene rab28 is at basal body and ciliary rootlet of the photoreceptors [22]. poc1b cause shorten cilia in mIMCD3 cell, abnormal Kupffer's vesicle, thus the important of poc1b in ciliogenesis is gained.

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Photoreceptor Sensory Cilia The poc1b expression experiments reveal that poc1b has WD40 conserved domain and expresses in different species especially expresses in mouse retina. Immunochemistry results demonstrate poc1b co-located with centriole in the S Stage in mIMCD3 cell, this is in accordance with the result of Tateishi (Magali Venouxin)[23]; however, when the cells enter M Stage, the poc1b expresses in connection of the bindles, this indicate poc1b may have additional function during the M stage. poc1b locates in the centriole during the cell cycle and locates in the basal body outside cell cycle, it means poc1b is part of both centriole and basal body, and may have functions in and out of cell cycle. Our result firstly reports poc1b locates in the basal body of rat photoreceptor, which is the important of photoreceptor sensory cilia. The knockdown of poc1b results in cells and zebrafish reveal that poc1b is required for ciliogenesis, especially for photoreceptor sensory cilia formation. Previous research clarified ciliogenesis starts when cells exit the cell cycle, and initiates from the basal body. The role of poc1b may plays at the start of ciliogenesis and associates with centriole/basal body. Firstly, the mother centriole converts to basal body, in detail, distal appendages (DAs) and subdistal appendages (SAs) as two part of centriole convert to transition fibers (TFs) and basal feet (BF) as basal body respectively[24], but the mechanism remains unknown. Secondly, the basal body contacts with the membranous compartments such as ciliary vesicles (neuronal precursors) or the plasma membrane (epithelial cells), this procedure is “basal body docking”. Thirdly, axoneme extension starts from the basal feet of basal body. There are several genes of mother centriole/basal body reported essential to ciliogenesis [25,26]. An apical domain of the mother centriole named Cep164 is found taking part in docking to the membrane[27]; Cep135 specifically localizes at basal bodies and is required to stabilize basal bodies [28]; basal body protein-PtFOR20p is required to build the transition zone, a prerequisite to their maturation [29]. Distal portion of the mother centriole Nek2 compromises the ability of cells to resorb primary cilia [30]. The mechanism of poc1b function reminds studied. Signal pathways also play an important role in maintenance of the cilia; several signal pathways have been found including Wnt and Sonic hedgehog signal pathways. These

ACCEPTED MANUSCRIPT pathways can lead phenotypes like defect of heart, small brain [31,32,33,34,35,36], which are also found in our results. The function of poc1b may generate through these pathways. Knockdown of poc1b in zebrafish lead to abnormal PSC and a spectrum phenotype associated with cilia defect including vision impairment, it can be concluded that poc1b is required for photoreceptor sensory cilia formation and normal vision in zebrafish.

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[28] Bayless BA, Giddings TH Jr, Winey M, P. CG, Bld10/Cep135 stabilizes basal bodies to resist cilia-generated forces, Mol Biol Cell 23 (2012 ) 4820-4832. [29] Aubusson-Fleury A, Lemullois M, de Loubresse NG, Laligné C, Cohen J, Rosnet O, Jerka-Dziadosz M, Beisson J, K. F, The conserved centrosomal protein FOR20 is required for assembly of the transition zone and basal body docking at the cell surface, J Cell Sci 125 (2012 ) 4395-4404. [30] Spalluto C, Wilson DI, H. T, Nek2 localises to the distal portion of the mother centriole/basal body and is required for timely cilium disassembly at the G2/M transition, Eur J Cell Biol 91 (2012 ) 675-686. [31] May-Simera HL, K. MW, Cilia, Wnt signaling, and the cytoskeleton, Cilia 1 (2012 ) 7. [32] D.A.A. D'Angelo A, Avallone B, Piscopo I, Tammaro R, Studer M, Franco B, Ofd1 controls dorso-ventral patterning and axoneme elongation during embryonic brain development, PLoS One 7 (2012) e52937. [33] Abdelhamed ZA, Wheway G, Szymanska K, Natarajan S, Toomes C, Inglehearn C, J. CA, Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel-Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects, Hum Mol Genet 22 (2013 ) 1358-1372. [34] Willaredt MA, Tasouri E, T. KL, Primary cilia and forebrain development, Mech Dev 130 (2013 ) 373-380. [35] Yasunaga T, Itoh K, S. SY, Regulation of basal body and ciliary functions by Diversin, Mech Dev 128 (2011 ) 376-386. [36] Willaredt MA, Gorgas K, Gardner HA, T. KL, Multiple essential roles for primary cilia in heart development, Cilia 1 (2012) 23.

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1. Defect of centriole or cilia genes were highlighted as associated with inherited retinal diseases and ciliopathy. 2. poc1b was conserved expressed and was located in the basal body of mIMCD3 cell and mouse photoreceptor cilia. 3. Knockdown of poc1b lead to spectrum phenotypes associated with cilia defect including vision impairment. 4. poc1b is required for photoreceptor sensory cilia formation and normal vision in zebrafish.

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Supplement KD of poc1b cause ciliopathy related manifestations KD of poc1b in zebrafish caused heart defect and abnormal Kupffer's vesicle. From the zebrafish larvae picture taken at 72 hours post-fertilization, pericardial effusion was observed (Figure3A) in the transcription blocker mophlino injection group; besides, heart beat rate was really low in the taken video. In the heart section, structure of the heart was disrupted in the transcription blocker mophlino injection group while normal in the standard control group (Supplement1 A, B). From previous study, it was known that genes that impacting on primary cilia generation could disturb wnt signal, which was critical to heart formation and right formation of KV played an important role. Defect zebrafish larvae were selected and did Whole Mountain for immunochemistry of KV. The result demonstrats that the cilium in the KV become shorten after mophlino injection, and could be rescue by human poc1b mRNA (Supplement1 C, D, E). Supplement1. Staining section of zebrafish larvae heart and abnormal Kupffer's vesicle. The structure of the heart was disrupted in the transcription blocker mophlino injection group compare to the standard

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control group in different section. (A) In the standard control group, zebrafish larvae have normal atrium and ventricle, right connection between them. (B) In poc1b TB mophlino injection group, zebrafish larvae present dilate ventricle, which can’t connect with the atrium. (C) The Kupffer's vesicle cilia present normal length in standard control morphlino injection group. (D) The Kupffer's vesicle cilia were disrupted in the in poc1b TB mophlino injection group. (E) When co-inject poc1bTB and mRNA, the

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Kupffer's vesicle cilia disruption get respectively rescued.

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