The exocyst is required for photoreceptor ciliogenesis and retinal development
2017; Elsevier BV; Volume: 292; Issue: 36 Linguagem: Inglês
10.1074/jbc.m117.795674
ISSN1083-351X
AutoresGlenn P. Lobo, Diana Fulmer, Lilong Guo, Xiaofeng Zuo, Yujing Dang, Seok Hyung Kim, Yanhui Su, Kola George, Elisabeth Obert, Ben Fogelgren, Deepak Nihalani, Russell A. Norris, Bärbel Rohrer, Joshua H. Lipschutz,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoWe 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. 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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 containing opsin required for phototransduction (11.Besharse J.C. Baker S.A. Luby-Phelps K. Pazour G.J. Photoreceptor intersegmental transport and retinal degeneration: a conserved pathway common to motile and sensory cilia.Adv. Exp. Med. Biol. 2003; 533: 157-164Crossref PubMed Scopus (40) Google Scholar, 12.Liu Q. Zhang Q. Pierce E.A. Photoreceptor sensory cilia and inherited retinal degeneration.Adv. Exp. Med. Biol. 2010; 664: 223-232Crossref PubMed Scopus (30) Google Scholar13.Tsujikawa M. Malicki J. Genetics of photoreceptor development and function in zebrafish.Int. J. Dev. Biol. 2004; 48: 925-934Crossref PubMed Scopus (39) Google Scholar). The photoreceptor outer segment is considered to be a modified cilium (12.Liu Q. Zhang Q. Pierce E.A. Photoreceptor sensory cilia and inherited retinal degeneration.Adv. Exp. Med. Biol. 2010; 664: 223-232Crossref PubMed Scopus (30) Google Scholar, 13.Tsujikawa M. Malicki J. Genetics of photoreceptor development and function in zebrafish.Int. J. Dev. Biol. 2004; 48: 925-934Crossref PubMed Scopus (39) Google Scholar14.Schmitt E.A. Dowling J.E. Early retinal development in the zebrafish, Danio rerio: light and electron microscopic analyses.J. Comp. Neurol. 1999; 404: 515-536Crossref PubMed Scopus (325) Google Scholar). Zebrafish are vertebrates, and zebrafish eyes are well laminated structures that are functionally very similar to the eyes of other vertebrates, including humans. Eye morphogenesis in the zebrafish starts at 11.5 h post-fertilization (hpf), and the eyecup is well-formed by 24 hpf. By 48 hpf, most of the retina is subdivided into its characteristic sublaminae (14.Schmitt E.A. Dowling J.E. Early retinal development in the zebrafish, Danio rerio: light and electron microscopic analyses.J. Comp. Neurol. 1999; 404: 515-536Crossref PubMed Scopus (325) Google Scholar). The connecting cilia and basal body in the inner segment are observed at 50 hpf, and the outer segment is visible by 54 hpf. The first visual responses can be elicited around 70 hpf, and photoreceptor cells reach adult size by 576 hpf (24 days) (14.Schmitt E.A. Dowling J.E. Early retinal development in the zebrafish, Danio rerio: light and electron microscopic analyses.J. Comp. Neurol. 1999; 404: 515-536Crossref PubMed Scopus (325) Google Scholar, 15.Branchek T. Bremiller R. The development of photoreceptors in the zebrafish, Brachydanio rerio. I. Structure.J. Comp. Neurol. 1984; 224: 107-115Crossref PubMed Scopus (193) Google Scholar). In mice, eye development begins at embryonic day 8 (E8), and evidence of retinal development is seen at E13.5. At about the same time, E13, rod photoreceptor cells can be observed, with the peak production of rod photoreceptors occurring at postnatal day 1 (P1). In contrast, cone photoreceptors are all generated between E12 and E18. At birth, these newly formed photoreceptor precursors occupy the distal half of the retina forming a mantle of poorly differentiated cells. By P10, the differentiating photoreceptors have migrated into the ONL, after which they start their final process of maturation, the growth of the outer segment, and the establishment of functional synapses within the outer plexiform layer (OPL). Formation of the OPL, a layer of synapses between dendrites of bipolar and horizontal cells from the inner nuclear layer, and photoreceptor terminal axons from the ONL, is complete by P14 (16.Graw J. Eye development.Curr. Top. Dev. Biol. 2010; 90: 343-386Crossref PubMed Scopus (168) Google Scholar). All cells in the retina are ciliated; however, here we paid particular attention to photoreceptor cells. In photoreceptor cells, vesicles containing proteins that are destined for the outer segment traffic from the trans-Golgi network (TGN) to the base of the connecting cilium via vesicular transport (17.Deretic D. Huber L.A. Ransom N. Mancini M. Simons K. Papermaster D.S. rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis.J. Cell Sci. 1995; 108: 215-224Crossref PubMed Google Scholar, 18.Deretic D. Papermaster D.S. Rab6 is associated with a compartment that transports rhodopsin from the trans-Golgi to the site of rod outer segment disk formation in frog retinal photoreceptors.J. Cell Sci. 1993; 106: 803-813Crossref PubMed Google Scholar19.Moritz O.L. Tam B.M. Hurd L.L. Peränen J. Deretic D. Papermaster D.S. Mutant rab8 impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods.Mol. Biol. Cell. 2001; 12: 2341-2351Crossref PubMed Scopus (199) Google Scholar). The connecting cilium therefore plays a critical role as the bridge between the inner and outer segments. One of the central proteins implicated in vesicular trafficking from the TGN to the cilium in photoreceptors is the small GTPase, Rab8 (20.Nachury M.V. Loktev A.V. Zhang Q. Westlake C.J. Peränen J. Merdes A. Slusarski D.C. Scheller R.H. Bazan J.F. Sheffield V.C. Jackson P.K. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis.Cell. 2007; 129: 1201-1213Abstract Full Text Full Text PDF PubMed Scopus (1026) Google Scholar). Disruption of Rab8 in Xenopus photoreceptor cells blocks rhodopsin-bearing post-TGN vesicle trafficking and results in abnormal accumulation of rhodopsin carrier vesicles at the base of the connecting cilium. Rab proteins perform functions through downstream effectors, such as the exocyst, a highly conserved eight protein trafficking complex (17.Deretic D. Huber L.A. Ransom N. Mancini M. Simons K. Papermaster D.S. rab8 in retinal photoreceptors may participate in rhodopsin transport and in rod outer segment disk morphogenesis.J. Cell Sci. 1995; 108: 215-224Crossref PubMed Google Scholar, 19.Moritz O.L. Tam B.M. Hurd L.L. Peränen J. Deretic D. Papermaster D.S. Mutant rab8 impairs docking and fusion of rhodopsin-bearing post-Golgi membranes and causes cell death of transgenic Xenopus rods.Mol. Biol. Cell. 2001; 12: 2341-2351Crossref PubMed Scopus (199) Google Scholar). We previously demonstrated that the exocyst is required for ciliogenesis in canine renal tubule cells, due to its role in targeting and docking vesicles carrying ciliary proteins (21.Zuo X. Guo W. Lipschutz J.H. The exocyst protein Sec10 is necessary for primary ciliogenesis and cystogenesis in vitro.Mol. Biol. Cell. 2009; 20: 2522-2529Crossref PubMed Scopus (126) Google Scholar). We also showed that Cdc42, another small GTPase, localizes the exocyst to the primary cilium and biochemically and genetically interacts with exocyst complex member Exoc5 (also known as Sec10) (22.Zuo X. Fogelgren B. Lipschutz J.H. The small GTPase Cdc42 is necessary for primary ciliogenesis in renal tubular epithelial cells.J. Biol. Chem. 2011; 286: 22469-22477Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Interestingly, we found in zebrafish, using antisense morpholinos, that knockdown of exoc5 resulted in small eyes (23.Fogelgren B. Lin S.Y. Zuo X. Jaffe K.M. Park K.M. Reichert R.J. Bell P.D. Burdine R.D. Lipschutz J.H. The exocyst protein Sec10 interacts with polycystin-2 and knockdown causes PKD-phenotypes.PLoS Genet. 2011; 7: e1001361Crossref PubMed Scopus (65) Google Scholar) and knockdown of cdc42 led to small eyes and loss of photoreceptor cilia (24.Choi S.Y. Chacon-Heszele M.F. Huang L. McKenna S. Wilson F.P. Zuo X. Lipschutz J.H. Cdc42 deficiency causes ciliary abnormalities and cystic kidneys.J. Am. Soc. Nephrol. 2013; 24: 1435-1450Crossref PubMed Scopus (55) Google Scholar) and that exoc5 and cdc42 seemed to act synergistically in ocular and retinal development (25.Choi S.Y. Baek J.I. Zuo X. Kim S.H. Dunaief J.L. Lipschutz J.H. Cdc42 and sec10 are required for normal retinal development in zebrafish.Invest. Ophthalmol. Vis. Sci. 2015; 56: 3361-3370Crossref PubMed Scopus (13) Google Scholar). Here, we describe the role of exoc5 in eye development using mutant exoc5 zebrafish, photoreceptor-specific Exoc5 mouse knockouts, histology, immunohistology, and transmission electron microscopy (TEM). These data show that there exists a conserved ciliogenic program across species and organs. We previously showed that antisense morpholino knockdown of exoc5 (also known as sec10) in zebrafish is associated with ciliary defects, including upward tail curvature and cardiac malformations as well as smaller eyes and increased cell death in the retina, the latter suggesting that exoc5 plays a critical role in retinal cell development and maintenance (23.Fogelgren B. Lin S.Y. Zuo X. Jaffe K.M. Park K.M. Reichert R.J. Bell P.D. Burdine R.D. Lipschutz J.H. The exocyst protein Sec10 interacts with polycystin-2 and knockdown causes PKD-phenotypes.PLoS Genet. 2011; 7: e1001361Crossref PubMed Scopus (65) Google Scholar). Given the concerns about off-target effects of morpholinos in experiments designed to further characterize the physiologic role of exoc5 in retinal cell development and homeostasis, we utilized an exoc5 mutant zebrafish line from the Zebrafish International Resource Consortium (ZIRC), exoc5 sa23168. By sequencing of the mutants, we first confirmed that a single point mutation (CGA→TGA) at codon 377 led to a premature stop codon (Fig. 1, A and B), which previously had been shown to result in a truncated (at amino acid 376) and non-functional Exoc5 protein. By light microscopy, we observed that the gross phenotype of exoc5 mutants was similar to other zebrafish mutants or morphants with defects in cilia formation and function (26.Bizet A.A. Becker-Heck A. Ryan R. Weber K. Filhol E. Krug P. Halbritter J. Delous M. Lasbennes M.C. Linghu B. Oakeley E.J. Zarhrate M. Nitschké P. Garfa-Traore M. Serluca F. et al.Mutations in TRAF3IP1/IFT54 reveal a new role for IFT proteins in microtubule stabilization.Nat. Commun. 2015; 6: 8666Crossref PubMed Scopus (64) Google Scholar, 27.Stoetzel C. Bär S. De Craene J.O. Scheidecker S. Etard C. Chicher J. Reck J.R. Perrault I. Geoffroy V. Chennen K. Strähle U. Hammann P. Friant S. Dollfus H. A mutation in VPS15 (PIK3R4) causes a ciliopathy and affects IFT20 release from the cis-Golgi.Nat. Commun. 2016; 7: 13586Crossref PubMed Scopus (43) Google Scholar). At 3.5 dpf, all exoc5 mutants had an upward tail curvature, smaller eyes, pericardial edema, and hydrocephalus (Fig. 1, D and F), when compared with wild-type (WT) siblings (Fig. 1, C and E). The morphologic phenotype was consistently observed in ∼25% of the progeny from crosses of heterozygous parents as would be expected for Mendelian inheritance of a recessive mutation, with exoc5 mutants surviving to at least 4 dpf. By Western blot analysis, we confirmed that Exoc5 protein was undetectable in exoc5 mutant larvae, when compared with their WT siblings (Fig. 1G). The exocyst is thought to act as a holocomplex, and we previously showed that knockdown of Exoc5 in MDCK cells resulted in the disruption of the exocyst and loss of other exocyst complex members, most likely by degradation in the proteasome, as the exocyst complex disintegrates in the absence of Exoc5 (21.Zuo X. Guo W. Lipschutz J.H. The exocyst protein Sec10 is necessary for primary ciliogenesis and cystogenesis in vitro.Mol. Biol. Cell. 2009; 20: 2522-2529Crossref PubMed Scopus (126) Google Scholar). To confirm this observation in vivo, we probed for Exoc4 protein expression in the exoc5 mutants. Wild-type 3.5 dpf larvae showed strong expression of Exoc4 protein. In contrast, knockout of exoc5 resulted in significantly depleted levels of Exoc4 protein (Fig. 1G). Interestingly, knockout of exoc5 resulted in an increase in Mob1 phosphorylation (pMOB1), consistent with activation of the Hippo pathway (Fig. 1, G and H), a pathway known to be involved in restraining organ growth (28.Badouel C. Garg A. McNeill H. Herding Hippos: regulating growth in flies and man.Curr. Opin. Cell Biol. 2009; 21: 837-843Crossref PubMed Scopus (90) Google Scholar, 29.Happé H. van der Wal A.M. Leonhard W.N. Kunnen S.J. Breuning M.H. de Heer E. Peters D.J. Altered Hippo signalling in polycystic kidney disease.J. Pathol. 2011; 224: 133-142Crossref PubMed Scopus (93) Google Scholar). To confirm that the ciliary defects observed in exoc5 mutants were caused specifically by loss of exoc5, we performed rescue experiments by injecting wild-type human EXOC5 mRNA into control and exoc5 mutant embryos at the 1–2-cell stage. At 3.5 dpf, we found that low dose (100 pg) reconstitution of EXOC5 mRNA, in exoc5 mutant embryos, partially rescues the heart and eye phenotype, whereas high dose (250 pg) EXOC5 mRNA fully rescues the exoc5 mutant phenotype (Fig. 1I). To ensure that the loss of exoc5 was causing the phenotype, another exoc5 mutant, using CRISPR gene editing, was generated, and the resulting phenotype was observed to be virtually identical to the exoc5 mutant phenotype described above, as well as to our previously described exoc5 morphants (supplemental Fig. 1) (23.Fogelgren B. Lin S.Y. Zuo X. Jaffe K.M. Park K.M. Reichert R.J. Bell P.D. Burdine R.D. Lipschutz J.H. The exocyst protein Sec10 interacts with polycystin-2 and knockdown causes PKD-phenotypes.PLoS Genet. 2011; 7: e1001361Crossref PubMed Scopus (65) Google Scholar). These data show that loss of functional Exoc5 protein in zebrafish leads to gross pathology indicative of ciliopathy in the heart, brain, and eye. Histologic analysis was completed to examine retinal morphology in exoc5 mutant zebrafish larvae at 3.5 dpf. In transverse sections, all exoc5 mutants exhibited smaller eyes and abnormal retinal lamination (Fig. 2, A and B). Interestingly, the photoreceptor/ONL layer in exoc5 mutants was incompletely formed and severely disorganized (Fig. 2, A and B). Disorganization and lack of photoreceptor outer segments was more obvious by TEM of exoc5 mutant larvae at 3.5 dpf. In WT siblings, cone photoreceptor outer segments extended toward the retina pigmented epithelium (RPE) in a parallel and organized manner and exhibited well-stacked discs in the outer segments (indicated by white arrows in Fig. 2C). In contrast, the cone outer segments in exoc5 mutants were significantly fewer in number, shorter in length, and disorganized (Fig. 2D). Such abnormalities were never observed in retinal sections of WT siblings. Taken together, these results indicate that eye development, proper retinal lamination, and outer segment morphogenesis requires exoc5 function. We next used immunohistochemistry to determine whether localization and trafficking of photoreceptor outer segment proteins occurred normally in exoc5 mutants. At 3.5 dpf, rhodopsin localized normally to the rod outer segments of WT fish (Fig. 3A). In exoc5 mutants, rudimentary outer segment localization of rhodopsin was observed, together with some rhodopsin being mislocalized to the inner segments (Fig. 3B). To quantify the lengths of the outer segments, we used rhodopsin immunoreactivity as a surrogate, determining the extent of rhodopsin staining along the proximal–distal axis of the outer segments. In WT siblings, outer segments were 7.9 ± 0.45 μm in length (n = 20 embryos, 130 outer segments), whereas exoc5 mutant outer segment were 3.2 ± 0.23 μm in length (60.5% shorter, p < 0.001; n = 12 embryos, 60 outer segments) (Fig. 3G). To determine whether these shortened outer segments correlated with cilia formation, we labeled retina sections with antibodies against acetylated α-tubulin, a marker that is required and sufficient to determine the presence of photoreceptor cilia (30.Liu Q. Zuo J. Pierce E.A. The retinitis pigmentosa 1 protein is a photoreceptor microtubule-associated protein.J. Neurosci. 2004; 24: 6427-6436Crossref PubMed Scopus (123) Google Scholar). Although retina sections in WT zebrafish showed the presence of cilia, we could not detect cilia in the retina sections of exoc5 mutants (Fig. 3, C and D). We next examined cone morphology, the predominant photoreceptor cell type in the zebrafish retina, by immunolabeling with peanut agglutinin lectin (PNA-488), which labels the interphotoreceptor matrix surrounding cone outer segments and, to some extent, the retinal plexiform layers (31.Gargini C. Terzibasi E. Mazzoni F. Strettoi E. Retinal organization in the retinal degeneration 10 (rd10) mutant mouse: a morphological and ERG study.J. Comp. Neurol. 2007; 500: 222-238Crossref PubMed Scopus (374) Google Scholar). Peanut agglutinin lectin staining revealed that the exoc5 mutant cone outer segments were significantly shorter (5.8 ± 0.33 μm in WT versus 2.2 ± 0.24 μm in mutants; p < 0.001), disorganized, and misshapen (Fig. 3, E, F, and H). Additionally, the number of cone outer segments was significantly fewer in number in exoc5 mutants compared with WT siblings (Fig. 3, E and F), which suggested that loss of exoc5 results in defective cone outer segment morphogenesis. These data demonstrate that exoc5 is indispensable for cilia and subsequent photoreceptor outer segment formation in zebrafish. Photoreceptor-specific knockout of Exoc5 in mice was accomplished by crossing the Exoc5 fl/fl mouse line, which we made using embryonic stem (ES) cells from the European conditional mouse mutagenesis (EUCOMM) consortium (clone number DEPD00521_3) (32.Fogelgren B. Polgar N. Lui V.H. Lee A.J. Tamashiro K.K. Napoli J.A. Walton C.B. Zuo X. Lipschutz J.H. Urothelial defects from targeted inactivation of exocyst Sec10 in mice cause ureteropelvic junction obstructions.PLoS ONE. 2015; 10: e0129346Crossref PubMed Scopus (26) Google Scholar), with a rhodopsin-Cre driver line. The conditional target mouse line, designated as Exoc5fl/fl;Rho-Cre+, was confirmed by PCR-based genotyping (supplemental Fig. 2). Histologic analyses in frozen sections of retinas from WT and Exoc5 fl/fl;Rho-Cre+ mice revealed severe thinning of the outer retina in postnatal day 30 (P30) mice (Fig. 4B), when compared with WT animals (Fig. 4A). Disorganization and lack of photoreceptor outer segments were more obvious by transmission electron microscopy of conditional Exoc5 knock-out animals. In WT siblings at P30, photoreceptor outer segments extended toward the RPE in a parallel and organized manner, giving a palisade pattern (Fig. 5, A and C). In contrast, the photoreceptor outer segments in Exoc5 fl/fl;Rho-Cre+ mice were significantly fewer in number, shorter in length, and disorganized (Fig. 5, B and D). We further examined the structure of the photoreceptor sensory cilium components in WT and Exoc5 fl/fl;Rho-Cre+ retinas. The stacks of nascent discs were organized and were oriented perpendicular to the long axis of the axoneme that extended from the basal body and transition zone in WT mice (Fig. 5E). In contrast, we did not observe connecting cilia components in Exoc5 fl/fl;Rho-Cre+ retinas (Fig. 5F). Taken together, these results indicate that outer segment morphogenesis requires Exoc5 function, and Exoc5 is necessary for photoreceptor ciliogenesis in mice.Figure 5Ultrastructural analysis of wild-type and conditional Exoc5 knock-out mouse photoreceptors. Transmission electron microscopy provided ultrastructural views of wild-type (WT) and Exoc5 fl/fl;Rho-Cre+ mice photoreceptor cells. A and C, WT rod photoreceptors (PR) showed tightly stacked disc membranes (arrows); B and D, rod outer segments of Exoc5 fl/fl;Rho-Cre+ mouse retinas contained fewer disks and lacked proper stacking. E and F, details of the photoreceptor sensory cilium components, in retinas from P30 WT (E) and Exoc5 fl/fl;Rho-Cre+ (F) mice. The stacks of nascent discs, the axoneme, basal body, and transition zone had a normal appearance in WT mice, but no ciliary components were detected in the Exoc5 fl/fl;Rho-Cre+ mice. Scale bars, 800 nm (A and B); 400 nm (C and D); 100 nm (E and F). RPE, retinal pigmented epithelium; OS, outer segments; IS, inner segments; CC, connecting cilium; TZ, transition zone; BB, basal body; PR, photoreceptor.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In WT mice, rhodopsin was localized specifically to the outer segments by P30 (Fig. 6A). In contrast, Exoc5 fl/fl;Rho-Cre+ mouse retinas exhibited significantly decreased levels of rhodopsin, and the rhodopsin was not specifically localized to the outer segments (Fig. 6B). Indeed, in Exoc5 fl/flRho-Cre+ mice, rhodopsin was aberrantly distributed to the inner portion of the ONL (cell bodies), as well to the inner segments of photoreceptors, indicating a defect in cilia trafficking (Fig. 6B). As we did for zebrafish retinas, rhodopsin immunoreactivity along the axis of the outer segment was used to document a shortened outer segment length in mutant mice (Fig. 6G). Photoreceptor degeneration, as indicated by a thinning of the ONL, was observed in retinas from Exoc5 fl/fl;Rho-Cre+ mice by P30, a time at which the ONL was only ∼2–3 cells thick (Fig. 6, A–F). To determine whether this ONL cell loss correlated with an absence of cilia, we labeled retinal sections with antibodies against acetylated α-tubulin. Although many cilia were detected in retinal sections in WT animals throughout the ONL, no cilia were seen in the retinal sections of Exoc5 fl/fl;Rho-Cre+ mice at P30 (Fig. 6, C and D). Cone morphology was examined by immunolabeling with anti-red/green cone opsin antibody. Cone opsin staining revealed the
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