The exocyst is required for photoreceptor ciliogenesis and retinal development

Abstract

We previously have shown that the highly conserved eight-protein exocyst trafficking complex is required for ciliogenesis in kidney tubule cells. We hypothesized here that ciliogenic programs are conserved across organs and species. To determine whether renal primary ciliogenic programs are conserved in the eye, and to characterize the function and mechanisms by which the exocyst regulates eye development in zebrafish, we focused on exoc5, a central component of the exocyst complex, by analyzing both exoc5 zebrafish mutants, and photoreceptor-specific Exoc5 knock-out mice. Two separate exoc5 mutant zebrafish lines phenocopied exoc5 morphants and, strikingly, exhibited a virtual absence of photoreceptors, along with abnormal retinal development and cell death. Because the zebrafish mutant was a global knockout, we also observed defects in several ciliated organs, including the brain (hydrocephalus), heart (cardiac edema), and kidney (disordered and shorter cilia). exoc5 knockout increased phosphorylation of the regulatory protein Mob1, consistent with Hippo pathway activation. exoc5 mutant zebrafish rescue with human EXOC5 mRNA completely reversed the mutant phenotype. We accomplished photoreceptor-specific knockout of Exoc5 with our Exoc5 fl/fl mouse line crossed with a rhodopsin-Cre driver line. In Exoc5 photoreceptor-specific knock-out mice, the photoreceptor outer segment structure was severely impaired at 4 weeks of age, although a full-field electroretinogram indicated a visual response was still present. However, by 6 weeks, visual responses were eliminated. In summary, we show that ciliogenesis programs are conserved in the kidneys and eyes of zebrafish and mice and that the exocyst is necessary for photoreceptor ciliogenesis and retinal development, most likely by trafficking cilia and outer-segment proteins. We previously have shown that the highly conserved eight-protein exocyst trafficking complex is required for ciliogenesis in kidney tubule cells. We hypothesized here that ciliogenic programs are conserved across organs and species. To determine whether renal primary ciliogenic programs are conserved in the eye, and to characterize the function and mechanisms by which the exocyst regulates eye development in zebrafish, we focused on exoc5, a central component of the exocyst complex, by analyzing both exoc5 zebrafish mutants, and photoreceptor-specific Exoc5 knock-out mice. Two separate exoc5 mutant zebrafish lines phenocopied exoc5 morphants and, strikingly, exhibited a virtual absence of photoreceptors, along with abnormal retinal development and cell death. Because the zebrafish mutant was a global knockout, we also observed defects in several ciliated organs, including the brain (hydrocephalus), heart (cardiac edema), and kidney (disordered and shorter cilia). exoc5 knockout increased phosphorylation of the regulatory protein Mob1, consistent with Hippo pathway activation. exoc5 mutant zebrafish rescue with human EXOC5 mRNA completely reversed the mutant phenotype. We accomplished photoreceptor-specific knockout of Exoc5 with our Exoc5 fl/fl mouse line crossed with a rhodopsin-Cre driver line. In Exoc5 photoreceptor-specific knock-out mice, the photoreceptor outer segment structure was severely impaired at 4 weeks of age, although a full-field electroretinogram indicated a visual response was still present. However, by 6 weeks, visual responses were eliminated. In summary, we show that ciliogenesis programs are conserved in the kidneys and eyes of zebrafish and mice and that the exocyst is necessary for photoreceptor ciliogenesis and retinal development, most likely by trafficking cilia and outer-segment proteins. Cilia are thin rod-like microtubule-based organelles, which are found on most mammalian cell types. Cilia can be classified as either motile or non-motile (more commonly referred to as primary) cilia. Motile cilia function mainly as motor organelles, and primary cilia are mainly sensory organelles (1.Badano J.L. Mitsuma N. Beales P.L. Katsanis N. The ciliopathies: an emerging class of human genetic disorders.Annu. Rev. Genomics Hum. Genet. 2006; 7: 125-148Crossref PubMed Scopus (886) Google Scholar, 2.Bhogaraju S. Engel B.D. Lorentzen E. Intraflagellar transport complex structure and cargo interactions.Cilia. 2013; 2: 10Crossref PubMed Scopus (80) Google Scholar3.Cardenas-Rodriguez M. Badano J.L. Ciliary biology: understanding the cellular and genetic basis of human ciliopathies.Am. J. Med. Genet. C Semin. Med. Genet. 2009; 151C: 263-280Crossref PubMed Scopus (120) Google Scholar). Dysfunction of primary cilia results in human disorders, termed ciliopathies. Ciliopathies, such as Bardet-Biedl, Joubert, Meckel-Gruber, and Senior-Loken syndromes, affect multiple organs, resulting in central nervous system malformation, cystic kidney disease, and retinal dystrophy (4.Baker K. Beales P.L. Making sense of cilia in disease: the human ciliopathies.Am. J. Med. Genet. C Semin. Med. Genet. 2009; 151C: 281-295Crossref PubMed Scopus (246) Google Scholar, 5.Blacque O.E. Leroux M.R. Bardet-Biedl syndrome: an emerging pathomechanism of intracellular transport.Cell. Mol. Life Sci. 2006; 63: 2145-2161Crossref PubMed Scopus (151) Google Scholar6.Smyth B.J. Snyder R.W. Balkovetz D.F. Lipschutz J.H. Recent advances in the cell biology of polycystic kidney disease.Int. Rev. Cytol. 2003; 231: 51-89Crossref PubMed Scopus (12) Google Scholar). Zebrafish models for several ciliopathies, including cep290, cc2d2a, inpp5e, ift57, ift88, and ift172, have kidney and retina phenotypes that suggest a common mechanism underlying these defects (7.Bachmann-Gagescu R. Phelps I.G. Stearns G. Link B.A. Brockerhoff S.E. Moens C.B. Doherty D. The ciliopathy gene cc2d2a controls zebrafish photoreceptor outer segment development through a role in Rab8-dependent vesicle trafficking.Hum. Mol. Genet. 2011; 20: 4041-4055Crossref PubMed Scopus (87) Google Scholar8.Baye L.M. Patrinostro X. Swaminathan S. Beck J.S. Zhang Y. Stone E.M. Sheffield V.C. Slusarski D.C. The N-terminal region of centrosomal protein 290 (CEP290) restores vision in a zebrafish model of human blindness.Hum. Mol. Genet. 2011; 20: 1467-1477Crossref PubMed Scopus (51) Google Scholar, 9.Luo N. Lu J. Sun Y. Evidence of a role of inositol polyphosphate 5-phosphatase INPP5E in cilia formation in zebrafish.Vision Res. 2012; 75: 98-107Crossref PubMed Scopus (26) Google Scholar10.Sukumaran S. Perkins B.D. Early defects in photoreceptor outer segment morphogenesis in zebrafish ift57, ift88, and ift172 intraflagellar transport mutants.Vision Res. 2009; 49: 479-489Crossref PubMed Scopus (69) Google Scholar). An open question is whether primary ciliogenic programs, such as exocyst-related ciliary trafficking, are conserved across organs and species. Here, we build on our previous data on exocyst-dependent renal ciliopathy and extend this to retinal ciliopathy, comparing zebrafish and mice. The vertebrate retina is organized into three distinct laminae: the outer nuclear layer (ONL), 2The abbreviations used are: ONL, outer nuclear layer; E, embryonic day; ERG, electroretinography; hpf, hours post-fertilization; INL, inner nuclear layer; MDCK, Madin-Darby canine kidney; P, postnatal; PKD, polycystic kidney disease; OPL, outer plexiform layer; RPE, retina pigmented epithelium; TEM, transmission electron microscopy; TGN, trans-Golgi network; dpf, days post-fertilized; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)- propane-1,3-diol; ANOVA, analysis of variance; PNA, peanut agglutinin. 2The abbreviations used are: ONL, outer nuclear layer; E, embryonic day; ERG, electroretinography; hpf, hours post-fertilization; INL, inner nuclear layer; MDCK, Madin-Darby canine kidney; P, postnatal; PKD, polycystic kidney disease; OPL, outer plexiform layer; RPE, retina pigmented epithelium; TEM, transmission electron microscopy; TGN, trans-Golgi network; dpf, days post-fertilized; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)- propane-1,3-diol; ANOVA, analysis of variance; PNA, peanut agglutinin. the inner nuclear layer (INL), and the ganglion cell layer. The photoreceptor cells in the ONL have a specialized morphology consisting of an inner and outer segment that is linked by a connecting cilium and are highly polarized and light-sensitive cells. The inner segment, where protein synthesis occurs, is connected by the cilium to the outer segment, which consists of a microtubule-based axoneme and membrane disc stacks required for K. transport and retinal a conserved pathway common to motile and sensory Med. 2003; PubMed Scopus Google Scholar, Zhang sensory cilia and retinal Med. PubMed Scopus Google M. J. of photoreceptor development and function in J. PubMed Scopus Google Scholar). The photoreceptor outer segment is to be a cilium Zhang sensory cilia and retinal Med. PubMed Scopus Google Scholar, M. J. of photoreceptor development and function in J. PubMed Scopus Google Early retinal development in the zebrafish, and electron PubMed Scopus Google Scholar). Zebrafish are and zebrafish eyes are that are to the eyes of including morphogenesis in the zebrafish at and the is by hpf, most of the retina is into Early retinal development in the zebrafish, and electron PubMed Scopus Google Scholar). The connecting cilia and in the inner segment are observed at hpf, and the outer segment is by The visual responses can be hpf, and photoreceptor cells by Early retinal development in the zebrafish, and electron PubMed Scopus Google Scholar, R. The development of in the zebrafish, PubMed Scopus Google Scholar). In mice, eye development at embryonic and of retinal development is at the photoreceptor cells can be with the of at In are and these photoreceptor the of the retina a of cells. the have into the ONL, which of the of the outer segment, and the of the outer plexiform layer of the OPL, a layer of of and cells the inner nuclear and photoreceptor the ONL, is by J. PubMed Scopus Google Scholar). cells in the retina are here we to photoreceptor cells. In photoreceptor that are for the outer segment the trans-Golgi to the of the connecting cilium transport D. N. M. K. in retinal in transport and in outer segment Sci. PubMed Google Scholar, D. is with a that the trans-Golgi to the of outer segment formation in retinal Sci. PubMed Google J. D. and of and cell of PubMed Scopus Google Scholar). The connecting cilium a role as the the inner and outer of the central in trafficking the to the cilium in is the Zhang J. Slusarski D.C. Sheffield V.C. complex of with the to ciliary membrane PubMed Scopus Google Scholar). of in photoreceptor cells vesicle trafficking and results in abnormal of at the of the connecting through such as the a highly conserved protein trafficking complex D. N. M. K. in retinal in transport and in outer segment Sci. PubMed Google Scholar, J. D. and of and cell of PubMed Scopus Google Scholar). We previously that the exocyst is required for ciliogenesis in canine renal tubule to role in and ciliary X. Lipschutz J.H. The exocyst protein is necessary for primary ciliogenesis and in 2009; 20: PubMed Scopus Google Scholar). We also that the exocyst to the primary cilium and and with exocyst complex Exoc5 as X. Lipschutz J.H. The is necessary for primary ciliogenesis in renal 2011; PubMed Scopus Google Scholar). we found in zebrafish, that of exoc5 in eyes X. Lipschutz J.H. The exocyst protein with and Genet. 2011; 7: PubMed Scopus Google and of to eyes and of photoreceptor cilia S. X. Lipschutz J.H. ciliary and cystic 2013; PubMed Scopus Google and that exoc5 and to in and retinal development X. J.L. Lipschutz J.H. and are required for retinal development in Sci. PubMed Scopus Google Scholar). Here, we the role of exoc5 in eye development mutant exoc5 zebrafish, photoreceptor-specific Exoc5 mouse and transmission electron data show that a conserved ciliogenic across and We previously that of exoc5 as in zebrafish is with ciliary including and as as eyes and increased cell in the the that exoc5 a role in retinal cell development and X. Lipschutz J.H. The exocyst protein with and Genet. 2011; 7: PubMed Scopus Google Scholar). the of in to characterize the role of exoc5 in retinal cell development and we an exoc5 mutant zebrafish line the Zebrafish exoc5 of the mutants, we that a at to a and which previously shown to in a and Exoc5 we observed that the of exoc5 was to zebrafish or morphants with defects in cilia formation and function R. K. E. J. M. M. M. in a role for in PubMed Scopus Google Scholar, S. S. J. K. S. in a ciliopathy and the 7: PubMed Scopus Google Scholar). dpf, exoc5 an and and with C and The was observed in of the of as be for of a with exoc5 to at 4 we that Exoc5 protein was in exoc5 mutant with The exocyst is to as a and we previously that of Exoc5 in cells in the of the exocyst and of exocyst complex most likely by in the as the exocyst complex in the absence of Exoc5 X. Lipschutz J.H. The exocyst protein is necessary for primary ciliogenesis and in 2009; 20: PubMed Scopus Google Scholar). To this in we for protein in the exoc5 of In knockout of exoc5 in of protein knockout of exoc5 in an in phosphorylation consistent with of the Hippo pathway and a pathway to be in in and 2009; PubMed Scopus Google Scholar, E. Hippo in polycystic kidney 2011; PubMed Scopus Google Scholar). 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The exocyst protein with and Genet. 2011; 7: PubMed Scopus Google Scholar). data a role for the exocyst in renal the question of whether this pathway was conserved across organs and in The exocyst is to function as a that and the trans-Golgi and to the PubMed Scopus Google Scholar). which is of to polycystic kidney disease, the most common ciliopathy, and the most common genetic to affect B.J. Snyder R.W. Balkovetz D.F. Lipschutz J.H. Recent advances in the cell biology of polycystic kidney disease.Int. Rev. Cytol. 2003; 231: 51-89Crossref PubMed Scopus (12) Google Scholar). We have shown in the kidney that a role of the exocyst is to ciliary such as to the primary cilium X. Lipschutz J.H. The exocyst protein is necessary for primary ciliogenesis and in 2009; 20: PubMed Scopus Google X. Lipschutz J.H. The is necessary for primary ciliogenesis in renal 2011; PubMed Scopus Google Scholar, X. Lipschutz J.H. The exocyst protein with and Genet. 2011; 7: PubMed Scopus Google S. X. 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