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Mol Biol Cell. Aug 15, 2012; 23(16): 3069–3078.
PMCID: PMC3418303
Knockdown of ttc26 disrupts ciliogenesis of the photoreceptor cells and the pronephros in zebrafish
Qi Zhang,a Qin Liu,a Chrissy Austin,b Iain Drummond,b and Eric A. Piercea1
aOcular Genomics Institute & Berman-Gund Laboratory, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114
bRenal Unit-CNY 8, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129
Erika Holzbaur, Monitoring Editor
University of Pennsylvania
1Address correspondence to: Eric Pierce (eric_pierce/at/meei.harvard.edu).
Received January 10, 2012; Revised June 7, 2012; Accepted June 12, 2012.
In our effort to understand genetic disorders of the photoreceptor cells of the retina, we have focused on intraflagellar transport in photoreceptor sensory cilia. From previous mouse proteomic data we identified a cilia protein Ttc26, orthologue of dyf-13 in Caenorhabditis elegans, as a target. We localized Ttc26 to the transition zone of photoreceptor and to the transition zone of cilia in cultured murine inner medullary collecting duct 3 (mIMCD3) renal cells. Knockdown of Ttc26 in mIMCD3 cells produced shortened and defective primary cilia, as revealed by immunofluorescence and scanning electron microscopy. To study Ttc26 function in sensory cilia in vivo, we utilized a zebrafish vertebrate model system. Morpholino knockdown of ttc26 in zebrafish embryos caused ciliary defects in the pronephric kidney at 27 h postfertilization and distension/dilation of pronephros at 5 d postfertilization (dpf). In the eyes, the outer segments of photoreceptor cells appeared shortened or absent, whereas cellular lamination appeared normal in retinas at 5 dpf. This suggests that loss of ttc26 function prevents normal ciliogenesis and differentiation in the photoreceptor cells, and that ttc26 is required for normal development and differentiation in retina and pronephros. Our studies support the importance of Ttc26 function in ciliogenesis and suggest that screening for TTC26 mutations in human ciliopathies is justified.
Inherited retinal degenerations (IRDs) are important causes of blindness (Pierce, 2001 blue right-pointing triangle). These disorders are characterized by dysfunction and death of photoreceptor cells of the retina. The light-sensitive outer segments of photoreceptor cells are specialized sensory cilia, and the importance of primary and sensory cilia in biology and disease is becoming increasingly recognized (Singla and Reiter, 2006 blue right-pointing triangle; Breunig et al., 2008 blue right-pointing triangle; Simons and Mlodzik, 2008 blue right-pointing triangle; Slough et al., 2008 blue right-pointing triangle). Primary cilia are present on most vertebrate cell types, and these structures are typically sensory organelles involved in many critical aspects of cell biology, such as signal transduction pathways and generating and detecting fluid flow (Pazour and Witman, 2003 blue right-pointing triangle; Pan et al., 2005 blue right-pointing triangle).
The rod and cone photoreceptors are among the largest of mammalian cilia (Pan et al., 2005 blue right-pointing triangle; Yang et al., 2005 blue right-pointing triangle). Like other cilia, the outer segments contain an axoneme, which begins at the basal bodies and passes through a transition zone (also called the “connecting cilium”) and into the outer segments (Beisson and Wright, 2003 blue right-pointing triangle). The photoreceptor sensory cilium (PSC) comprises the outer segment and its cytoskeleton, including the rootlet, basal body, and axoneme (Liu et al., 2007 blue right-pointing triangle). The membrane of outer segments is highly specialized for light detection, with the proteins required for phototransduction located in or associated with the membrane disks stacked in tight order at ~30 per micron along the axoneme. The transition zone in PCS is analogous to transition zones in other cilia and is where the triplet microtubule structure of the basal bodies converts to the doublet microtubule structure of the axoneme (Horst et al., 1990 blue right-pointing triangle).
The recognition of photoreceptor outer segments as cilia connects retinal degenerative disorders such as retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) to other ciliary disorders. Mutations in genes that encode cilia proteins are increasingly recognized as common causes of disease. For example, inherited renal disorders, such as polycystic kidney disease and nephronophthisis, are now understood to be ciliopathies (Hildebrandt and Otto, 2005 blue right-pointing triangle). Ciliary defects are also seen as the underlying cause of a number of inherited disorders that affect multiple organ systems. This helps explain the connection of retinal degeneration to ciliopathies, such as Bardet-Biedl syndrome (BBS), in which RP is found in association with multiple cilia-related disorders, including cystic renal disease, polydactyly, mental retardation, obesity and diabetes, gonadal malformations, and situs inversus (Satir and Christensen, 2007 blue right-pointing triangle).
Ciliogenesis involves intraflagellar transport (IFT), which mediates the delivery of ciliary components to the growing ciliary tip, the point at which new cilium assembly occurs. The transported materials include tubulin subunits, tubulin-associated proteins, cell surface receptors, and other membrane proteins. IFT proteins have been biochemically characterized in Chlamydomonas as two complexes associated with anterograde (complex B) or retrograde (complex A) transport (Cole et al., 1998 blue right-pointing triangle). Mutations in IFT complexes and other ciliary component genes have been identified in numerous ciliopathies (Pierce et al., 1999 blue right-pointing triangle; Lehman et al., 2008 blue right-pointing triangle; Louie et al., 2010 blue right-pointing triangle; Muller et al., 2010 blue right-pointing triangle; Davis et al., 2011 blue right-pointing triangle).
The pleiotropic phenotypes of ciliary disorders suggest the involvement of genes having a highly conserved, essential function in the formation and/or maintenance of cilia. The inheritance patterns of ciliopathies are mostly autosomal recessive, but with extensive genetic heterogeneity (Badano et al., 2006 blue right-pointing triangle). The ciliopathy-related mutations identified so far affect proteins residing in the basal body, transition zone, ciliary membrane, IFT, or BBSome complexes. More loci are likely to be identified.
Although some progress has been made in the characterization of IFT complexes and BBSome proteins, proteomic studies of several model systems indicate many proteins are expressed in cilia (Ostrowski et al., 2002 blue right-pointing triangle; Keller et al., 2005 blue right-pointing triangle; Pazour et al., 2005 blue right-pointing triangle; Liu et al., 2007 blue right-pointing triangle; Mayer et al., 2009 blue right-pointing triangle; Jia et al., 2010 blue right-pointing triangle). For example, we identified nearly 2000 proteins in PSC in proteomic analyses of mouse PSCs (Liu et al., 2007 blue right-pointing triangle). These data suggest ciliogenesis is a complicated process and many uncharacterized proteins are involved.
In the work reported here, we have evaluated the role of a novel cilia protein Ttc26, which we detected in the mouse PSC proteome (Liu et al., 2007 blue right-pointing triangle). Ttc26 has been classified as an IFT complex B protein based on in vitro coprecipitation with Ift88 (Follit et al., 2009 blue right-pointing triangle). In addition, the Caenorhabditis elegans homologue of Ttc26, called dyf-13, is proposed to interact with OSM-3, a homodimeric kinesin motor, to move cargo in the anterograde direction during IFT (Starich et al., 1995 blue right-pointing triangle; Blacque et al., 2005 blue right-pointing triangle). In its N-terminal portion, Ttc26 contains several tetratricopeptide repeats (TPR). The TPR motif is enriched in the ciliary proteome and may mediate its interaction with other ciliary proteins in higher-order complexes. Notably, mutations in proteins with multiple TPRs, such as fleer/DYF-1, BBS4, BBS8, and TTC21B have been reported as ciliopathy-causing genes either in animal models or in human patients (Katsanis et al., 2002 blue right-pointing triangle; Pathak et al., 2007 blue right-pointing triangle; Harville et al., 2010 blue right-pointing triangle; Riazuddin et al., 2010 blue right-pointing triangle; Davis et al., 2011 blue right-pointing triangle). The studies reported here demonstrate that Ttc26 is located in the transition zone of primary and photoreceptor cilia and is required for normal ciliogenesis in cultured cells and in vivo in zebrafish.
Ttc26 is a highly conserved protein expressed in tissues containing ciliated cells
We detected Ttc26 as a novel PSC protein in our proteomic analyses of PSCs isolated from mouse retina. In those analyses, Ttc26 was detected by eight unique peptides, suggesting that it is present in moderate abundance in mouse PSCs (Liu et al., 2007 blue right-pointing triangle). To get an indication of the evolutionary importance of Ttc26, we searched the National Center for Biotechnology Information protein sequence database and found homologues from green algae, insects, and mammals. Alignment of Ttc26 homologue protein sequences from species related to the current study (CLUSTAL W method; Supplemental Figure S1) revealed very high sequence conservation (humans and rodents: 97% identity, 99% similarity; zebrafish and humans/rodents: 80% identity, 90% similarity). Ttc26 is highly conserved across the species we examined. The sequence conservation is observed throughout the protein, not just in TPR domains.
We used northern blots to determine which mouse tissues express transcripts of Ttc26 and how abundant the transcripts are. In testis, which displayed the highest expression level, two different-sized transcripts were identified (Figure 1A). The larger transcript (4.2 kb) is consistent with the largest splice variant annotated in Ensembl (ENSMUST00000162554; CCDS51750) and was also detected in mouse retina, kidney, lung, and brain tissue. The expression level was low in liver and spleen, consistent with the low proportion of ciliated cells in these tissues (Figure 1A). The smaller transcript (2.5 kb) may represent an alternative splice variant in testis and is consistent with Ensembl transcript ENSMUST00000039394, in which exons 7–17 of the larger transcript are skipped. We also examined ttc26 developmental expression in embryonic zebrafish and found that ttc26 transcripts can be detected as early as the one-cell stage (possibly reflecting both maternal and embryonic contributions) and maintain a relatively stable level during the examined developmental stages (Figure 1B).
FIGURE 1:
FIGURE 1:
Expression of Ttc26 in cultured mIMCD3 cells, rodent tissues, and zebrafish embryos. (A) Northern blot of Ttc26 in adult mouse tissues. Ttc26 expression is high in the testis, with a 2.5-kb transcript exclusively seen in testis and a 4.2-kb transcript (more ...)
Ttc26 is localized in the transition zone of primary and photoreceptor sensory cilia
After confirming the temporal and spatial sequence of Ttc26 expression, we examined its subcellular localization by expressing a V5-tagged mouse Ttc26 fusion protein in murine inner medullary collecting duct 3 cells–somatostatin receptor 3–enhanced green fluorescent protein (mIMCD3-SSTR3-EGFP) cells. This stable cell line, derived from medullary collecting duct cells, expresses a green fluorescent protein (GFP) fused to SSTR3, a ciliary membrane receptor. By performing colocalization of Ttc26 with this ciliary marker, we showed that Ttc26 was concentrated in the ciliary base with gradually decreasing staining intensity toward the ciliary tip in interphase cells (Figure 1C). By immunostaining with antibodies to CEP164, a basal body distal appendage marker protein (Graser et al., 2007 blue right-pointing triangle), we refined the Ttc26 subcellular location at the base to the ciliary transition zone. Interestingly, Ttc26 does not localize to the distal appendage itself, but rather lies immediately adjacent to it. However, the punctate signal spreading in whole cilia (Figure 1C) suggests that Ttc26 is not restricted to the transition zone and is possibly mobile in a protein complex.
We also studied Ttc26 subcellular location in neonatal rat photoreceptor cells using an in vivo retinal electroporation approach to transfect the same plasmid DNA used in the mIMCD3 cell transfection. Using immunostaining to the V5 tag on the Ttc26 fusion protein, we were able to localize Ttc26 to the transition zone in photoreceptor cells (Figure 1D) and thus confirm it as a bona fide ciliary protein.
RNA interference (RNAi) knockdown of Ttc26 causes a defect in primary cilia in cultured kidney epithelial cells
The identification of Ttc26 as a transition zone protein suggests that it may play a functional role in ciliogenesis. To test this hypothesis, we used a ciliated renal epithelial cell model (mIMCD3) in vitro to study the effects of knockdown of Ttc26 expression. Three short hairpin RNA (shRNA) constructs directed at Ttc26 were introduced into target cells using a vector expressing GFP (pCAG-miR30-IRES-EGFP). This allowed us to identify all shRNA-transfected cells. Cotransfection of these Ttc26 shRNAs with the V5-Ttc26 cDNA in CHO cells demonstrated that all three shRNA constructs had knockdown efficiencies of over 90% compared with control shRNAs (Figure 2A). Next we tested the effect of shRNA-mediated Ttc26 knockdown on ciliogenesis in mIMCD3 cells. In this case, cilia were detected by immunostaining with antibody to acetylated α-tubulin, an axoneme marker. Transfected mIMCD3 cells (green) were observed to have shortened cilia or missing cilia (arrows in Figure 2B). We further analyzed the images by quantifying the number of transfected cells with and without cilia and by measuring the ciliary length in both transfected cells and adjacent nontransfected cells. Using this approach, we found that knockdown of Ttc26 was associated with significantly reduced ciliary length (p < 0.01; Figure 2, B and C). Nontransfected mIMCD3 cells are shown for comparison in Figure 2D. To investigate the effects of Ttc26 knockdown on ciliary morphology, we examined shRNA-transfected and nontransfected mIMCD3 cells by scanning electron microscopy (SEM) and observed that cells with Ttc26 knockdown displayed shortened cilia with enlarged ends (Figure 2E).
FIGURE 2:
FIGURE 2:
Knockdown of Ttc26 by shRNA caused shortened cilia with altered morphology in mIMCD3 cells. (A) An immunoblot assay using anti-V5 antibody verified knockdown of expressed V5-Ttc26 cotransfected with shRNA constructs in CHO cells. All three Ttc26 shRNA (more ...)
Morpholino knockdown of ttc26 disrupts photoreceptor outer segment morphogenesis in developing retina
To further assess the functional importance of Ttc26, we turned to a vertebrate model organism, the zebrafish. Over a period of 2 d, the optic cup transforms from a single neuroepithelial sheet into a functional multilayered retina (Fadool and Dowling, 2008 blue right-pointing triangle). This makes it particularly suitable for a developmental study of photoreceptor cell morphogenesis. We used knockdown by two ttc26 morpholino oligonucleotides (MO-AUG and MO-SP) to further test the hypothesis that ttc26 functions in ciliogenesis. The MO-AUG and MO-SP morpholinos were designed to interfere with translation initiation and RNA splicing, respectively. The MO-SP blocks splicing between exons 1 and 2, leading to the inclusion of intron 1 in the altered mRNA transcript. This introduces a premature termination codon 20 nucleotides into intron 1, which leads to a truncated protein and loss of function. Molecular analysis of reverse transcription PCR (RT-PCR) products from embryos injected with MO-SP revealed mis-splicing events (Figure 3, D and E) consistent with a loss-of-function phenotype similar to that caused by MO-AUG.
FIGURE 3:
FIGURE 3:
Phenotypes of ttc26 zebrafish morphant larvae. Zebrafish embryos were injected with two ttc26 MOs (MO-AUG and MO-SP) or control MO. (A) Knockdown of ttc26 results in developmental defects. Panel 1, lateral view of 120-hpf larva injected with control MO. (more ...)
Embryos injected with a control morpholino were normal in appearance (Figure 3A, 1 and 2). However, the morphant larvae exhibited body axis curvature (mostly ventral and sometimes dorsal) and kinked tails (Figure 3A, 3–10). The MO-AUG and MO-SP morpholinos led to very similar phenotypes in appearance and in histology, except the MO-SP morphants also displayed severe body edema.
The histology of 5 d postfertilization (dpf) zebrafish eye morphogenesis in control and ttc26 morphants is presented in Figure 3B. At higher magnification (60×), the five principal laminae—three cellular and two plexiform layers—can be seen to be fully formed in the retinas of both control and morphant larvae, and the proliferative marginal zones (MZ) developed normally and remained nonlaminated in both controls and morphants. A higher-magnification view of the control retina shows clear morphological differentiation of photoreceptors with well-defined inner and outer segments at 5 dpf (Figure 3B). In contrast, both types of morphants revealed a defective photoreceptor ciliogenesis with only a few outer segments visible. Outer segments in these retinas were smaller, shortened, and disorganized. Transmission electron microscopy (TEM) images of the controls show well-formed photoreceptor outer segments. In contrast, morphant eyes reveal missing and ill-formed outer segments (Figure 3C).
MO-knockdown of ttc26 results in dilated pronephric tube and duct
The observation of edema in ttc26 morphants prompted us to look at the pronephros, the embryonic kidney, of zebrafish larvae for possible defects in fluid flow and in biogenesis of cilia, including motile cilia. Indeed, as shown in Figure 4, we found that morpholino knockdown of ttc26 was associated with defects in cilia in the pronephros as early as 27 h postfertilization (hpf), with disrupted and disorganized cilia (Figure 4A). Video images taken at 2 dpf clearly show enlarged pronephric ducts and disruption of multiciliated-cell cilia beat coordination (Supplemental Movies S1, S2, and S3). The recorded video data showed the multiciliated-cell cilia beat coordination is disrupted. The overall beat rates increase from 34 beats per second (bps; control) to ~60 bps (morphants), most likely due to tubule lumen dilation (Hellman et al., 2010 blue right-pointing triangle; Figure 5). The distended and dilated pronephric tubules/ducts were observed in many morphants that exhibited precardiac and body edema at 5 dpf (Figure 4, C–E).
FIGURE 4:
FIGURE 4:
Morpholino knockdown of ttc26 disrupts motile cilia and causes tubule dilation in zebrafish pronephros. (A) Cilia in the anterior and posterior regions of the pronephric kidneys in larvae at 27 hpf. Green, cilia revealed by immunostaining of acetylated (more ...)
FIGURE 5:
FIGURE 5:
ttc26 knockdown disrupts pronephric cilia beat coordination in 2-dpf larvae. Video images on the left show the dilated pronephros ducts in ttc26 morphants (B and C) vs. control (A). Dotted lines, position of the line scan. Corresponding cilia beat amplitude (more ...)
The finding that both the MO-AUG and MO-SP morpholinos produced the same phenotypes in both eyes and pronephric kidney supports the conclusion that these results are due to genuine ttc26 knockdown and not to an off-target effect. To confirm that the morphological effects in zebrafish larvae were indeed associated with knockdown of ttc26, we used RT-PCR analysis to analyze the mRNA present in control and morphant larvae. Morpholino MO-SP treatment of zebrafish larvae partially blocks splicing of ttc26 mRNA, and thus leads to a larger-sized PCR product (Figure 3, D and E). To further demonstrate the specificity of the MO-induced knockdown phenotypes, we performed rescue experiments with coinjection of ttc26-MO and Ttc26 mRNA. Coinjection of 66 ng of mouse Ttc26 mRNA with MO-AUG reduced the percentage of abnormal larvae from 95% (21/22) in the embryos injected with MO-AUG alone to 51% (23/45) in the coinjected embryos (p < 0.001).
The data presented here indicate that Ttc26 is localized to cilia in both mouse and rat cells and is developmentally required for ciliogenesis in zebrafish photoreceptor cells and pronephric kidney. Ttc26 is localized in the primary cilia and enriched in the transition zone of the mIMCD3 and rodent photoreceptor cells. Knockdown of Ttc26 in mIMCD3 cells produced shortened and defective cilia. Furthermore, morpholino knockdown of ttc26 in zebrafish caused larvae to exhibit absent or deformed photoreceptor outer segments, but with normal lamination in the morphant retina. The morphant larvae also developed defective motile cilia in the pronephros. These morphant phenotypes demonstrate that ttc26 is required for ciliogenesis and normal ciliary function.
Related genes involved in ciliary defects
The highly conserved polypeptide sequence of Ttc26, both within its TPR domains and in other regions, suggests an important function for this protein. Furthermore, ttc26 knockdown in the eye and pronephros led to phenotypes that match those observed with defects in other ciliary genes in zebrafish larvae. These include body curvature, cardiac edema, pronephric distention and dilation, and photoreceptor outer segment defects (Malicki et al., 2011 blue right-pointing triangle). The overlap in phenotypes implies that ttc26 might function similarly to other TPR domain proteins found in ciliary protein complexes.
Ciliopathy-causal mutations have been identified in several TPR domain-containing genes, including BBS4, BBS8, TTC21B, and TTC30B (Katsanis et al., 2002 blue right-pointing triangle; Billingsley et al., 2010 blue right-pointing triangle; May-Simera et al., 2010 blue right-pointing triangle; Riazuddin et al., 2010 blue right-pointing triangle; Davis et al., 2011 blue right-pointing triangle). BBS4 and BBS8 are key scaffold components of the BBSome ciliary protein transport complex. TTC21B has been localized to centrosomes and is required for retrograde IFT, and causal mutations have been identified in human patients (Davis et al., 2011 blue right-pointing triangle). Fleer/dyf-1/TTC30B is a regulator of ciliary tubulin polyglutamylation. Mutants in fleer showed kidney cysts, randomized left–right asymmetry, hydrocephalus, and rod outer segment defects in zebrafish (Pathak et al., 2007 blue right-pointing triangle).
Photoreceptor outer segments
The outer segments or PSCs of photoreceptor cells require continuous transport of membrane and protein components due to the constant shedding and phagocytosis by retinal pigment epithelium cells of older disks at the distal ends of PSCs (Strauss, 2005 blue right-pointing triangle). Protein trafficking mediated by IFT via the transition zone is crucial for the biogenesis and maintenance of the outer segment. This makes the transition zone an important “gate” in photoreceptor cells. Indeed, numerous mutations that cause IRDs and other ciliopathies, among them RPGR, RPGRIP1, and TTC21B, have been identified in genes of transition zone components (Vervoort et al., 2000 blue right-pointing triangle; Dryja et al., 2001 blue right-pointing triangle; Davis et al., 2011 blue right-pointing triangle).
Zebrafish morphants lacking ttc26 are viable at least to the 5-dpf stage and show photoreceptor cells with short or missing PSCs. The loss of ttc26 appears to prevent or delay PSC formation during early photoreceptor cell differentiation. While outer segment disk material is generated in some photoreceptor cells, it does not appear that this can be organized into formed PSCs in most photoreceptor cells. Although the morphant photoreceptor cells displayed defects in PSC formation, they maintained the ability to laminate and differentiate normally in the retina.
Relation of kidney defects to ciliopathy and ttc26 function
A hallmark of many human ciliopathies is the formation of kidney cysts, which often begins during fetal development. The observation of edema in zebrafish ttc26 morphants prompted us to look for defects in cilia early in pronephros at 27 hpf. The kidney is a vital organ controlling homeostasis of water and electrolytes, osmoregulation of body fluids, and filtration of toxic metabolic waste products. Cilia in zebrafish pronephros have been reported to be motile and to drive fluid flow (Kramer-Zucker et al., 2005 blue right-pointing triangle). Therefore disruption of the cilia would logically be accompanied by a loss of fluid flow, leading to fluid accumulation and subsequent pronephric distension and dilation. As expected, ttc26 morphant larvae at 27 hpf exhibited disorganized and disrupted cilia, loss of cilia bundles in the pronephric duct, and distended pronephric tubules and ducts, and this would logically contribute to the disruption of kidney function and severe edema observed in zebrafish larvae after ttc26 knockdown.
In summary, our characterization of Ttc26 localization and function in mouse and rat tissue, mIMCD3 cells, and in zebrafish provides the first comprehensive studies of Ttc26 gene expression and function in primary and sensory cilia. This work demonstrates that Ttc26 plays a critical role in ciliogenesis and suggests that loss of Ttc26 function could be implicated in retinal, renal, and other disease states in humans. Further studies confirming and extending these findings in other species will help us to better understand the function of Ttc26 in cilia. In this light, it seems logical to screen human patient populations with ciliopathies for mutations of TTC26 and genes that encode other ciliary proteins involved in IFT.
Gene expression reagents
Mouse Ttc26 cDNA was amplified by RT-PCR from mouse retina, cloned into a pENTR/D-TOPO entry vector (Invitrogen, Carlsbad, CA), and fully sequence-verified. The coding sequence was moved by recombination to a Gateway destination expression vector modified to contain an N-terminal V5 epitope tag in-frame (pCAG-V5-IRES-EGFP; Hartley et al., 2000 blue right-pointing triangle; Matsuda and Cepko, 2004 blue right-pointing triangle, 2007 blue right-pointing triangle). The sequences of shRNA knockdown reagents were generated using the RNAi Central website (Cold Spring Harbor, http://hannonlab.cshl.edu). Oligonucleotides of shRNA (obtained from IDT, Coralville, IA) were PCR-amplified and cloned into a pCAG-miR30-IRES-EGFP vector, modified by our laboratory for this purpose using established methods (Hartong et al., 2006 blue right-pointing triangle). Plasmid DNA was purified using the EndoFree Plasmid Maxi kit (Qiagen, Valencia, CA).
Cell culture and transfection
An mIMCD3 stable cell line expressing SSTR3-EGFP, a ciliary membrane marker (gift of Gregory J. Pazour, University of Massachusetts Medical School), was used for Ttc26 subcellular localization. CHO-K1 and wild-type mIMCD3 cell lines were purchased from the American Type Culture Collection. The mIMCD3 cells were maintained in DMEM:F12 media supplemented with 10% fetal bovine serum (FBS) and 0.5 mM sodium pyruvate (Invitrogen). CHO cell culture was performed in F-12K medium (Invitrogen) supplemented with 10% FBS. Transfection was performed with Lipofectamine 2000 or Lipofectamine LTX reagent (Invitrogen) using 4 μg DNA per well and cells at 60–70% confluency in a six-well plate with glass coverslips (25CIR-1D; Fisher, Pittsburgh, PA). The cells were processed for immunocytochemistry at 72 h after transfection. Detection of the recombinant protein fused to the V5 epitope tag was accomplished by immunocytochemical detection or immunoblot of the V5 tag.
Antibody reagents
Monoclonal antibody (mAb) against acetylated α-tubulin (clone 6-11B-1) was obtained from Sigma-Aldrich (St. Louis, MO) and anti-V5 tag mAb from Invitrogen. Anti-CEP164 antibody (human), recognizing a basal body distal appendage marker protein, was a gift (Graser et al., 2007 blue right-pointing triangle). Anti–β-actin antibody (C-11) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All secondary antibodies conjugated with fluorophores were from Invitrogen, except the antibody used for immunoblot, which was obtained from LI-COR (Lincoln, NE). The chemical reagents were purchased from Sigma-Aldrich.
Immunofluorescence microscopy
All incubations and washes were carried out in 1× phosphate-buffered saline (PBS) at room temperature. Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min, and blocked with 1% bovine serum albumin (BSA) and 0.2% Triton X-100 in PBS for 10 min. Next the cells were incubated with antibody to the V5 expression tag (1:2500 dilution) in blocking buffer for 1 h at room temperature. After three 10-min washes in PBS, a secondary antibody incubation was carried out for 1 h in Alexa Fluor 555–conjugated goat anti–mouse immunoglobulin G (IgG; 1:1000 dilution). The cells were then washed once in PBS for 10 min, incubated with Hoechst dye (Invitrogen) at 1:1000 dilution for 5 min, and washed twice (10 min each wash). For colocalization to the basal body, cells were incubated using the above procedure with antibody against human CEP164 for 1 h (1:1000 dilution) and Alexa Fluor–conjugated goat anti–mouse IgG (1:1000 dilution; Invitrogen) for 1 h. The coverslips were mounted using Fluoromount (Electron Microscopy Sciences, Hatfield, PA). Fluorescence signals were visualized using a Nikon TE300 fluorescence microscope.
In vivo electroporation
To localize Ttc26 expression in photoreceptor cells, we used an in vivo electroporation technique in rat photoreceptor cells. Briefly, 0.5 μl of endotoxin-free pCAG-V5-Ttc26-IRES-EGFP plasmid was injected subretinally into the right eyes of neonatal rats. Injected plasmid was electroporated into retinal cells using tweezer-type electrodes as previously described (Matsuda and Cepko, 2004 blue right-pointing triangle). The plasmid was injected into four to six eyes to provide sufficient retinas with both good transfection and morphology for analysis. Animals were killed 4 wk following injection, and frozen sections were prepared from the portions of the eyecups with EGFP signal. Sections were immunostained as described previously using anti-V5 antibody followed by Alexa Fluor 555–conjugated secondary antibody. The location of V5-tagged proteins in photoreceptor cells was evaluated by three-dimensional reconstructions of the confocal image stacks generated and analyzed using Volocity 3D imaging software (Mountain View, CA).
Northern blot and immunoblot analyses
RNA expression levels were analyzed by northern blot analysis. To prepare a cDNA probe for northern blot analysis, we cloned the full-length mouse Ttc26 cDNA (NM_153600) into a pCR2-TOPO vector. The plasmid was digested with EcoRI, and the insert DNA was purified using the QIAEX II gel extraction kit (Qiagen). Northern blot analysis was performed as described previously (Zhang et al., 2000 blue right-pointing triangle). Briefly, total RNA (25 μg) was extracted with TRIzol reagent (Invitrogen), separated on a 1% agarose/0.66 M formaldehyde gel, and transferred to a Gene Screen Plus nylon membrane (Perkin Elmer-Cetus, Waltham, MA) by capillary blotting for 24 h in 10× SSC. The probe was labeled with α-dCTP (deoxycytidine triphosphate)-[32P] and hybridized with the RNA blot at 106 cpm/ml of hybridization buffer. The blots contained multiple tissues, including retinas from a wild-type adult mouse. For this reason, after decay of the cDNA probe signals, the membranes were rehybridized with an [α32P]-labeled cDNA probe for 18S RNA to assess and normalize RNA sample loading. The signal was scanned using a Typhoon 9400 phosphorimager (GE, Waukesha, WI).
Immunoblot analysis was used to verify knockdown of gene constructs expressed in CHO cells. Cells were transfected with pCAG V5-Ttc26-IRES-EGFP plasmid DNA (4 μg well) in a six-well plate. At 48 h after transfection, the plate was rinsed twice with cold 1× PBS and then the cells were lysed immediately with 2× LDS sample buffer (Invitrogen) and sonicated briefly on ice. Total protein (100 μg/well) was separated on a precast NuPAGE 4–12% Bis-Tris Gel (Invitrogen) and transferred to a low-fluorescence polyvinylidene fluoride membrane (GE, Waukesha, WI). The membrane was incubated with Odyssey blocking buffer (LI-COR, Lincoln, NE) for 1 h at room temperature. The membrane was incubated with monoclonal anti-V5 antibody (1:5000 dilution) overnight with gentle rocking in a cold room, washed 4 times with 1× PBS with 0.1% Tween-20, incubated with IRDye (LI-COR) goat anti-mouse IgG (1:10,000 dilution) for 1 h at room temperature, and washed three times with PBS with 0.1% Tween-20. After a final wash with 1× PBS, the signals were detected with an Odyssey infrared imager (LI-COR). The loading control antibody to β-actin was used at 1:1000. Protein levels (expressed in fluorescence units) were quantified with software provided by LI-COR.
MOs and zebrafish embryo injection
Wild-type zebrafish (Danio rerio) of the AB strain were obtained from the University of Pennsylvania zebrafish core facility (Philadelphia, PA). Embryos were collected in E3 solution after natural spawns and kept at 28.5°C in an incubator using standard protocols (Westerfield, 2007 blue right-pointing triangle). Morpholinos designed to block ttc26 RNA translation (MO-AUG, 5′-CTGGCTTCATCCGAGACAAGAGCAT-3′) or to block splicing (MO-SP, 5′- ATATGTTGGTTCTGATGCACCTGTT-3′) and a standard control oligo (5′-CCTCTTACCTCAGTTACAATTTATA-3′) were obtained from Gene Tools (Philomath, OR).
Embryos were injected at the one- to two-cell stages with 0.35–0.45 mM MO (3.5–4.5 ng of ttc26 MO or 4.5 ng of control MO).
Embryos were imaged with a Stemi SV II stereomicroscope (Zeiss) and photographed with an AxioCam digital camera (Zeiss), and the images were analyzed with AxioVision software (Zeiss). Embryos were prepared for flat-mounting by dissecting the yolk away from the embryo proper with a pair of fine forceps; the embryos were then mounted between two glass coverslips. Immunofluorescence detection of acetylated tubulin in the whole-mount embryos was performed according to a published protocol (Jaffe et al., 2010 blue right-pointing triangle).
High-speed video microscopy analysis has been described in detail in a recent publication (Pathak et al., 2011 blue right-pointing triangle). Larvae at 2 dpf were used for the video recording.
RT-PCR
Whole embryos (both morphant and control) collected at different developmental stages were used for total RNA extraction with TRIzol reagent (Invitrogen). Total RNA (2 μg per sample) was reverse-transcribed using the Superscript first-strand synthesis system for RT-PCR (Invitrogen). The following primer pairs were used for RT-PCR. For ttc26 developmental expression: forward, 5′-ATGCGCTCTCATTACCAGGAGGC-3′; reverse, 5′-AGAGGCCATGCACTGCCTGCCTGG-3′. For detecting morpholino splice blocking: forward (exon 1), 5′-CACCATGCTCTTGTCTCGGATGAAGC-3′, reverse (exon 2), 5′-GCCACATACACATTCAGGGCCAGAA-3′. For β-actin primers: forward, 5′-ATGGATGAGGAAATCGCTGCCCTG-3′; reverse, 5′-ACCGATCCAGACGGAGTATTTGCGC-3′.
For the phenotype rescue studies, mouse Ttc26 cDNA was amplified by PCR with a pair of primers (forward, 5′-TGCGGCCCAGACTGGCGGACGCG-3′; reverse, 5′-ATGACAACTGAGACAGGCAAGCAGG-3′) and cloned into an Sp6/T7 promoter vector (pCS2P+; Addgene, Cambridge, MA). The plasmid was linearized with SnaBI. Capped Ttc26 mRNA was synthesized in vitro using the mMESSAGE mMACHINE SP6 kit (Ambion, Austin, TX) and purified by the RNeasy Plus micro kit (Qiagen). Mouse Ttc26 mRNA (5–17 pg) was coinjected with MO-AUG (0.35 ng) into one- to two-cell stage zebrafish embryos.
Histology and SEM and TEM
Zebrafish embryos were processed for histology and TEM following a Cold Spring Harbor Protocol (http://cshprotocols.cshlp.org/content/2007/6/pdb.prot4772.abstract). For general histology, transverse semithin sections (1 μm) were stained with alkaline Toluidine blue. For TEM, thin sections (90 nm) were mounted on electron microscopy grids and stained with lead citrate and uranyl acetate. TEM images were collected on a FEI-Tecnai T12 transmission electron microscope. For SEM, cultured cells were processed using standard protocols. Briefly, mIMCD3 cells grown on 13-mm coverslips were rinsed twice with PBS, washed three times with 50 mM Na-cacodylate buffer (pH 7.3), fixed for 2 h with 2% glutaraldehyde in 50 mM Na-cacodylate buffer, and dehydrated in a graded series of ethanol concentrations over a period of 1.5 h. Dehydration in 100% ethanol was done three times. The dehydrated samples were immersed twice for 10 min in 100% hexamethyldisilazane (HMDS; Sigma-Aldrich, St. Louis, MO), which was followed by air-drying for 30 min (Braet et al., 1997 blue right-pointing triangle). The samples were mounted on stubs and sputter-coated with gold palladium. Specimens were observed and photographed using a Philips XL20 scanning electron microscope (FEI, Hillsboro, OR) at 10-kV accelerating voltage.
Statistical analyses
Statistical analyses were performed with Minitab software. A Student's t test with unequal variance or chi-square test was used for the analyses.
Supplementary Material
Supplemental Materials
Acknowledgments
We thank Gregory Pazour for providing the SSTR-3 mIMCD3 cell line and Michael Pack and Jie He of the Zebrafish Core at Penn Medicine for their assistance. This work was supported by grants from the National Institutes of Health (R01-EY012910 to E.A.P.), the Foundation Fighting Blindness USA, the F. M. Kirby Foundation, Research to Prevent Blindness, and the Rosanne Silbermann Foundation. We also acknowledge the contribution of medical editing by David Titus.
Abbreviations used:
BBSBardet-Biedl syndrome
bpsbeats per second
BSAbovine serum albumin
dpfdays postfertilization
EGFPenhanced green fluorescent protein
FBSfetal bovine serum
GFPgreen fluorescent protein
hpfhours postfertilization
IFTintraflagellar transport
IgGimmunoglobulin G
IRDinherited retinal degenerations
LCALeber congenital amaurosis
mAbmonoclonal antibody
mIMCD3murine inner medullary collecting duct 3 cells
MOmorpholino oligonucleotides
MZmarginal zones
PBSphosphate-buffered saline
PSCphotoreceptor sensory cilium
RNAiRNA interference
RPretinitis pigmentosa
RT-PCRreverse transcriptase PCR
SEMscanning electron microscopy
shRNAshort hairpin RNA
SSTR3somatostatin receptor 3
TEMtransmission electron microscopy
TPRtetratricopeptide repeats

Footnotes
This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E12-01-0019) on June 20, 2012.
  • Badano JL, Mitsuma N, Beales PL, Katsanis N. The ciliopathies: an emerging class of human genetic disorders. Annu Rev Genomics Hum Genet. 2006;7:125–148. [PubMed]
  • Beisson J, Wright M. Basal body/centriole assembly and continuity. Curr Opin Cell Biol. 2003;15:96–104. [PubMed]
  • Billingsley G, et al. Mutations in chaperonin-like BBS genes are a major contributor to disease development in a multiethnic Bardet-Biedl syndrome patient population. J Med Genet. 2010;47:453–463. [PubMed]
  • Blacque OE, et al. Functional genomics of the cilium, a sensory organelle. Curr Biol. 2005;15:935–941. [PubMed]
  • Braet F, de ZR, Wisse E. Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells. J Microsc. 1997;186:84–87. [PubMed]
  • Breunig JJ, Sarkisian MR, Arellano JI, Morozov YM, Ayoub AE, Sojitra S, Wang B, Flavell RA, Rakic P, Town T. Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling. Proc Natl Acad Sci USA. 2008;105:13127–13132. [PubMed]
  • Cole DG, Diener DR, Himelblau AL, Beech PL, Fuster JC, Rosenbaum JL. Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J Cell Biol. 1998;141:993–1008. [PMC free article] [PubMed]
  • Davis EE, et al. TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum. Nat Genet. 2011;43:189–196. [PMC free article] [PubMed]
  • Dryja TP, Adams SM, Grimsby JL, McGee TL, Hong DH, Li T, Andreasson S, Berson EL. Null RPGRIP1 alleles in patients with Leber congenital amaurosis. Am J Hum Genet. 2001;68:1295–1298. [PubMed]
  • Fadool JM, Dowling JE. Zebrafish: a model system for the study of eye genetics. Prog Retin Eye Res. 2008;27:89–110. [PMC free article] [PubMed]
  • Follit JA, Xu F, Keady BT, Pazour GJ. Characterization of mouse IFT complex B. Cell Motil Cytoskeleton. 2009;66:457–468. [PMC free article] [PubMed]
  • Graser S, Stierhof YD, Lavoie SB, Gassner OS, Lamla S, Le CM, Nigg EA. Cep164, a novel centriole appendage protein required for primary cilium formation. J Cell Biol. 2007;179:321–330. [PMC free article] [PubMed]
  • Hartley JL, Temple GF, Brasch MA. DNA cloning using in vitro site-specific recombination. Genome Res. 2000;10:1788–1795. [PubMed]
  • Hartong DT, Berson EL, Dryja TP. Retinitis pigmentosa. Lancet. 2006;368:1795–1809. [PubMed]
  • Harville HM, et al. Identification of 11 novel mutations in eight BBS genes by high-resolution homozygosity mapping. J Med Genet. 2010;47:262–267. [PMC free article] [PubMed]
  • Hellman NE, Liu Y, Merkel E, Austin C, Le CS, Beier DR, Sun Z, Sharma N, Yoder BK, Drummond IA. The zebrafish foxj1a transcription factor regulates cilia function in response to injury and epithelial stretch. Proc Natl Acad Sci USA. 2010;107:18499–18504. [PubMed]
  • Hildebrandt F, Otto E. Cilia and centrosomes: a unifying pathogenic concept for cystic kidney disease? Nat Rev Genet. 2005;6:928–940. [PubMed]
  • Horst CJ, Johnson LV, Besharse JC. Transmembrane assemblage of the photoreceptor connecting cilium and motile cilium transition zone contain a common immunologic epitope. Cell Motil Cytoskeleton. 1990;17:329–344. [PubMed]
  • Jaffe KM, Thiberge SY, Bisher ME, Burdine RD. Imaging cilia in zebrafish. Methods Cell Biol. 2010;97:415–435. [PubMed]
  • Jia Y, Xue L, Li J, Liu H. Isolation and proteomic analysis of the halotolerant alga Dunaliella salina flagella using shotgun strategy. Mol Biol Rep. 2010;37:711–716. [PubMed]
  • Katsanis N, Eichers ER, Ansley SJ, Lewis RA, Kayserili H, Hoskins BE, Scambler PJ, Beales PL, Lupski JR. BBS4 is a minor contributor to Bardet-Biedl syndrome and may also participate in triallelic inheritance. Am J Hum Genet. 2002;71:22–29. [PubMed]
  • Keller LC, Romijn EP, Zamora I, Yates JR, III, Marshall WF. Proteomic analysis of isolated Chlamydomonas centrioles reveals orthologs of ciliary-disease genes. Curr Biol. 2005;15:1090–1098. [PubMed]
  • Kramer-Zucker AG, Olale F, Haycraft CJ, Yoder BK, Schier AF, Drummond IA. Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis. Development. 2005;132:1907–1921. [PubMed]
  • Lehman JM, Michaud EJ, Schoeb TR, Aydin-Son Y, Miller M, Yoder BK. The Oak Ridge Polycystic Kidney mouse: modeling ciliopathies of mice and men. Dev Dyn. 2008;237:1960–1971. [PMC free article] [PubMed]
  • Liu Q, Tan G, Levenkova N, Li T, Pugh EN, Jr., Rux JJ, Speicher DW, Pierce EA. The proteome of the mouse photoreceptor sensory cilium complex. Mol Cell Proteomics. 2007;6:1299–1317. [PMC free article] [PubMed]
  • Louie CM, et al. AHI1 is required for photoreceptor outer segment development and is a modifier for retinal degeneration in nephronophthisis. Nat Genet. 2010;42:175–180. [PMC free article] [PubMed]
  • Malicki J, Avanesov A, Li J, Yuan S, Sun Z. Analysis of cilia structure and function in zebrafish. Methods Cell Biol. 2011;101:39–74. [PubMed]
  • Matsuda T, Cepko CL. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci USA. 2004;101:16–22. [PubMed]
  • Matsuda T, Cepko CL. Controlled expression of transgenes introduced by in vivo electroporation. Proc Natl Acad Sci USA. 2007;104:1027–1032. [PubMed]
  • Mayer U, Kuller A, Daiber PC, Neudorf I, Warnken U, Schnolzer M, Frings S, Mohrlen F. The proteome of rat olfactory sensory cilia. Proteomics. 2009;9:322–334. [PubMed]
  • May-Simera HL, Kai M, Hernandez V, Osborn DP, Tada M, Beales PL. Bbs8, together with the planar cell polarity protein Vangl2, is required to establish left-right asymmetry in zebrafish. Dev Biol. 2010;345:215–225. [PubMed]
  • Muller J, et al. Identification of 28 novel mutations in the Bardet-Biedl syndrome genes: the burden of private mutations in an extensively heterogeneous disease. Hum Genet. 2010;127:583–593. [PubMed]
  • Ostrowski LE, Blackburn K, Radde KM, Moyer MB, Schlatzer DM, Moseley A, Boucher RC. A proteomic analysis of human cilia: identification of novel components. Mol Cell Proteomics. 2002;1:451–465. [PubMed]
  • Pan J, Wang Q, Snell WJ. Cilium-generated signaling and cilia-related disorders. Lab Invest. 2005;85:452–463. [PubMed]
  • Pathak N, Austin CA, Drummond IA. Tubulin tyrosine ligase-like genes ttll3 and ttll6 maintain zebrafish cilia structure and motility. J Biol Chem. 2011;286:11685–11695. [PubMed]
  • Pathak N, Obara T, Mangos S, Liu Y, Drummond IA. The zebrafish fleer gene encodes an essential regulator of cilia tubulin polyglutamylation. Mol Biol Cell. 2007;18:4353–4364. [PMC free article] [PubMed]
  • Pazour GJ, Agrin N, Leszyk J, Witman GB. Proteomic analysis of a eukaryotic cilium. J Cell Biol. 2005;170:103–113. [PMC free article] [PubMed]
  • Pazour GJ, Witman GB. The vertebrate primary cilium is a sensory organelle. Curr Opin Cell Biol. 2003;15:105–110. [PubMed]
  • Pierce EA. Pathways to photoreceptor cell death in inherited retinal degenerations. Bioessays. 2001;23:605–618. [PubMed]
  • Pierce EA, Quinn T, Meehan T, McGee TL, Berson EL, Dryja TP. Mutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pigmentosa. Nat Genet. 1999;22:248–254. [PubMed]
  • Riazuddin SA, et al. A splice-site mutation in a retina-specific exon of BBS8 causes nonsyndromic retinitis pigmentosa. Am J Hum Genet. 2010;86:805–812. [PubMed]
  • Satir P, Christensen ST. Overview of structure and function of mammalian cilia. Annu Rev Physiol. 2007;69:377–400. [PubMed]
  • Simons M, Mlodzik M. Planar cell polarity signaling: from fly development to human disease. Annu Rev Genet. 2008;42:517–540. [PMC free article] [PubMed]
  • Singla V, Reiter JF. The primary cilium as the cell's antenna: signaling at a sensory organelle. Science. 2006;313:629–633. [PubMed]
  • Slough J, Cooney L, Brueckner M. Monocilia in the embryonic mouse heart suggest a direct role for cilia in cardiac morphogenesis. Dev Dyn. 2008;237:2304–2314. [PMC free article] [PubMed]
  • Starich TA, Herman RK, Kari CK, Yeh WH, Schackwitz WS, Schuyler MW, Collet J, Thomas JH, Riddle DL. Mutations affecting the chemosensory neurons of Caenorhabditis elegans. Genetics. 1995;139:171–188. [PubMed]
  • Strauss O. The retinal pigment epithelium in visual function. Physiol Rev. 2005;85:845–881. [PubMed]
  • Vervoort R, Lennon A, Bird AC, Tulloch B, Axton R, Miano MG, Meindl A, Meitinger T, Ciccodicola A, Wright AF. Mutational hot spot within a new RPGR exon in X-linked retinitis pigmentosa. Nat Genet. 2000;25:462–466. [PubMed]
  • Westerfield M. The Zebrafish Book, A Guide for the Laboratory Use of Zebrafish (Danio rerio), 5th ed. Eugene: University of Oregon Press. 2007
  • Yang J, Gao J, Adamian M, Wen XH, Pawlyk B, Zhang L, Sanderson MJ, Zuo J, Makino CL, Li T. The ciliary rootlet maintains long-term stability of sensory cilia. Mol Cell Biol. 2005;25:4129–4137. [PMC free article] [PubMed]
  • Zhang Q, Ray K, Acland GM, Czarnecki JM, Aguirre GD. Molecular cloning, characterization and expression of a novel retinal clusterin-like protein cDNA. Gene. 2000;243:151–160. [PubMed]
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