Intraflagellar Transport Proteins Are Essential for Cilia Formation and for Planar Cell Polarity

Home , Ciliopathy, Intraflagellar transport

BASIC RESEARCH www.jasn.org

Ying Cao, Alice Park, and Zhaoxia Sun

Department of Genetics, Yale University School of Medicine, New Haven, Connecticut

ABSTRACT The highly conserved intraflagellar transport (IFT) proteins are essential for cilia formation in multiple organisms, but surprisingly, cilia form in multiple zebrafish ift mutants. Here, we detected maternal deposition of ift gene products in zebrafish and found that ciliary assembly occurs only during early developmental stages, supporting the idea that maternal contribution of ift gene products masks the function of IFT proteins during initial development. In addition, the basal bodies in multiciliated cells of the pronephric duct in ift mutants were disorganized, with a pattern suggestive of defective planar cell polarity (PCP). Depletion of pk1, a core PCP component, similarly led to kidney cyst formation and basal body disorganization. Furthermore, we found that multiple ift genes genetically interact with pk1. Taken together, these data suggest that IFT proteins play a conserved role in cilia formation and planar cell polarity in zebrafish.

J Am Soc Nephrol 21: 1326–1333, 2010. doi: 10.1681/ASN.2009091001

The cilium is a cell surface organelle that is almost In zebrafish, mutants of ift57, ift81, ift88, and ubiquitously present on vertebrate cells. Pro- ift172 have numerous defects commonly associated truding from the cell into its environment, the with ciliary abnormalities.13,14 However, cilia in cilium is involved in multiple signaling pathways, these mutants are able to form initially but degen- including the Sonic hedgehog (Shh) pathway, the erate over time, giving rise to the hypothesis that Wnt pathways, and the target of rapamycin IFT is essential for cilia maintenance rather than (TOR) pathway.1–5 Not surprisingly, defects in cilia assembly in zebrafish.14 Interestingly, products the cilium have been linked to a growing list of of many genes in zebrafish are deposited mater- human diseases, coined “ciliopathies,” ranging nally. One hypothesis for the initial formation of from laterality defects, retinal degeneration, cilia in zebrafish ift mutants is that maternal contri- polycystic kidney disease (PKD), and other hepa- bution of ift gene products masks the function of ift torenal fibrocystic disorders to obesity and dia- genes during early embryonic development. Ac- betes (for a review, see reference 6). cordingly, cilia formation is severely impaired in a Many studies have demonstrated that the for- maternal-zygotic mutant of ift88.15 In this study, we mation and maintenance of the cilium depends on demonstrate that products of ift57 and ift172 are intraflagellar transport (IFT) particles, which are indeed maternally deposited. We further show that composed of at least 17 polypeptides.7,8 These IFT particles move along microtubules in cilia and are Received September 30, 2009. Accepted April 12, 2010. thought to act as vehicles for transporting cargos Published online ahead of print. Publication date available at needed for the assembly, maintenance, and func- www.jasn.org. tion of cilia. Homologs of IFT proteins have been Correspondence: Dr. Zhaoxia Sun, Department of Genetics, Yale found in a wide spectrum of organisms including University School of Medicine, 333 Cedar Street, SHM I-329A, Caenorhabditis elegans, Drosophila, and mammals New Haven, CT 06520. Phone: 203-785-3589; Fax: 203-785- and have also been shown to be required for cilia 7227; E-mail: [email protected] formation.3,9–12 Copyright © 2010 by the American Society of Nephrology

1326 ISSN : 1046-6673/2108-1326 J Am Soc Nephrol 21: 1326–1333, 2010 www.jasn.org BASIC RESEARCH although cilia destined to form early in development show only RESULTS maintenance defects in ift57hi3417 and ift172hi2211 mutants, cilia programmed to assemble later in development fail to form, hi3417 and hi2211 are Zygotic Mutants of ift57 and providing further support for a conserved role of IFT in cilia ift172, Respectively formation in zebrafish. ift57hi3417 and ift172hi2211 were isolated from an insertional One of the most extensively studied phenotypes associ- mutagenesis screen in zebrafish.13 Both mutants show similar ated with ciliary defects is the formation of kidney cysts. morphologic phenotypes, including a ventrally curved body Both the canonical and the noncanonical Wnt pathway, or axis, kidney cyst formation in the glomerular-tubular region, planar cell polarity (PCP) pathway, have been implicated in and dilated kidney ducts (Figure 1, A and B; Supplemental kidney cyst formation.4,16–21 However, in contrast to the Figure S1; and reference 13). In both mutants, the responsible well-established role of cilia in the hedgehog pathway,2,3,22 proviral insertion is located in the 5Ј side of the corresponding the role of cilia in the Wnt pathways is unclear.4,15,23,24 In genes. Reverse transcription-polymerase chain reaction (RT- this study, we demonstrate that, in the kidney duct of PCR) with primers spanning the proviral insertion site re- ift57hi3417 and ift172hi2211 mutants, the organization of basal vealed that the wild-type transcripts are completely absent in bodies is impaired, a phenotype consistent with compro- each mutant (Figure 1, C and D). mised PCP signaling. We further show that knockdown of To further verify that inactivation of ift57 and ift172 are prickle 1 (pk1), a core PCP player, leads to disorganization of responsible for phenotypes seen in hi3417 and hi2211, respec- basal bodies and kidney cyst formation. Finally, we provide tively, we injected mRNA of each gene into embryos from het- evidence that ift57, ift88, and ift172 genetically interact with erozygous carriers of corresponding mutations. Results pk1 in convergence-extension (CE) movements during gas- showed that ift172 mRNA can reduce the frequency of pheno- trulation, a process regulated by the PCP pathway. To- typic embryo from 26 Ϯ 0to6Ϯ 3% (P ϭ 0.01, results from gether, these data support an intricate relationship between two independent experiments). Similarly, injection of ift57- IFT and the PCP pathway. GFP (GFP, green fluorescent protein) mRNA reduces the percentage of phenotypic embryos from 25 Ϯ 3to2Ϯ 2% (P Ͻ 0.001, n ϭ 3). These findings indicate that these two mutants can be rescued by overexpression of their corresponding gene. In zebrafish, zygotic transcription com- mences at around the 512-cell stage.25 However, transcripts of a number of cilia- associated genes are maternally deposited and genes can be detected before this stage. To investigate for maternal contribution, we performed RT-PCR on samples from wild-type embryos at multiple developmental stages. The presence of ift57 and ift172 mRNA from embryos at the 4- to 32-cell stage verified that these mRNAs are maternally deposited (Figure 1E). We further investigated whether IFT proteins can be detected before the maternal-zygotic transition by Western blot analysis. Results show that Ift57 can be Figure 1. hi3417 and hi2211 are zygotic loss-of-function mutants of ift57 and ift172, readily detected at the 16- to 64-cell stage respectively. (A and B) A wild-type sibling and a mutant at 4 dpf in side views. Box with (Figure 1F). Any Ift57 protein present at dashed line: cyst (in mutant) and the lack of cyst (in wild-type sibling) enlarged in insets. this stage could be maternally deposited, (C and D) Loss of wild-type ift172 and ift57 transcripts in hi2211 and hi3417 mutants at translated from maternal mRNA, or 33 hpf, respectively. Upper panels: RT-PCR with a pair of primers spanning the proviral both. Regardless of the source, the pres- insertion site. Lower panels: loading controls with a pair of elf1-specific primers using ence of an IFT protein at such an early cDNAs serially diluted 1:1, 1:10, 1:100, and 1:1000. (E) RT-PCR time course for ift172 and ift57 using wild-type samples. (F) Western blot against Ift57 (Ift57). ␤-Tub is used stage suggests that it would be difficult to as loading control. ␤-Tub, ␤-Tubulin; ctrl, uninjected embryo control; ift57MO, ift57 deplete IFT proteins completely with morphants; wt, wild type; 4-32c, 4- to 32-cell stage; 30%–27h, mixture of samples from morpholino oligos, including transla- 30% epiboly to 27 hpf; 36h, 36 hpf; 50h, 50 hfp; 5d, 5dpf; 16–64 cell, sample from tion-blocking morpholino oligos. To test embryos at the 16- to 64-cell stage; 20s, samples from embryos at the 20-somite stage. this notion more directly, we used a mor-

J Am Soc Nephrol 21: 1326–1333, 2010 Zebrafish IFT in Cilia Assembly and PCP 1327 BASIC RESEARCH www.jasn.org pholino oligo against the translation initiation site of ift57. cilia are unable to form. To test this hypothesis, we followed Indeed, a significant quantity of Ift57 protein can still be cilia formation in multiple organs in detail. detected in ift57 morphants, even when they are all pheno- Cilia in the neural tube can be detected at 24 hpf (hours typic (Figure 1F). post-fertilization) and persist thereafter. We examined cilia formation at 24 hpf in at least 30 embryos from heterozygous Cilia Formation Is Affected in ift57 and ift172 Mutants carriers for each experiment. No significant difference was seen in a Time-Dependent Manner in any of the embryos, suggesting that cilia are able to form in There are conflicting reports on the status of cilia formation in the neural tube (data not shown). By contrast, at 34 hpf, a stage zebrafish ift mutants.14,15,26 Because cilia formation starts at at which mutants can be reliably identified by their body cur- different time points in different organs, the differences ob- vature phenotype, cilia number in both mutants are drastically served may be attributed to the gradual decay of maternally reduced (Figure 2, A through B’). Interestingly the reduction of deposited gene products. Early in development, cilia are able to cilia number in ift172hi2211 mutants is more pronounced than form using maternally contributed gene products, whereas that in ift57hi3417 mutants (Figure 2, A through B’). Variations later in development, with the decay of maternal contribution, in mRNA or protein stability may account for this difference. The zebrafish pronephric duct contains both single-ciliated cells (SCCs) and multiciliated cells (MCCs).27,28 Initially, at 24 hpf, most cells in the pronephric duct display a single cilium. By 2 dpf (days postfer- tilization), MCCs can be found in the mid region of the duct, whereas SCCs can be found in both the mid and posterior region of the duct. In both ift57hi3417 and ift172hi2211 mutants, there is no detectable difference in cilia in the pronephric duct at 24 hpf (data not shown). At 2 dpf, cilia in the posterior duct still appear relatively unaffected (Figure 2, C through D’). By contrast, se- vere ciliary defects can be readily observed in the mid region of the duct (Figure 2, E through F’). A simple explanation is that IFT is specifically involved in building cilia in MCCs, but not in SCCs. Alternatively, this difference may be attributed to the decay of maternally contributed ift gene products between the time of cilia formation in SCCs and MCCs. To further clarify this picture, we investigated cilia formation in the lateral line organ, where cilia cannot be detected until 2 dpf. In both mutants, cilia formation in the lateral organ is severely affected (Figure 2, G through H’). We additionally observed disorganized basal bodies in MCCs of the pronephric Figure 2. Cilia formation is disrupted in ift172hi2211 and ift57hi3417 mutants in a time- duct. In wild-type embryos, basal bodies in dependent manner. (A through B’) Cilia in the neural tube at 34 hpf as shown by a MCC are organized into a tight array at anti-Arl13b labeling. (C through D’) Cilia in the posterior pronephric duct at 2 dpf as shown the apical side, as shown by ␥-Tubulin by anti-Arl13b labeling. (E through F’) Cilia in the MD at 2 dpf as shown by anti-Arl13b staining (Figure 2, I, J, K, and L). In both labeling. (G through H’) Cilia in the lateral line organ as shown by anti–a-Tub in green at 2 ift57hi3417 and ift172hi2211mutants, basal bod- dpf. phl labeling in red shows the presence of the LL organ. (I through L’) Basal bodies in the MD at 3 pdf as shown by ␥-Tub labeling. Anti–a-Tub in (K through L’) labeled cilia. ies appeared as disorganized clusters, no Arrows point to arrays of basal bodies, whereas arrowheads point to disorganized clusters longer tightly arrayed (Figure 2, I’, J’, K’, and of basal bodies. a-Tub, anti-acetylated Tubulin; ␥-Tub, anti-␥-Tubulin; LL, lateral line; MD, L’). We further investigated whether the api- mid region pronephric duct; NT, neural tube; PD, posterior pronephric duct; phl, Phalloi- cal localization of the basal body is affected in din; 2211ctrl, wild-type sibling of ift172hi2211; 3417ctrl, wild-type sibling of ift57hi347. these mutants by labeling with the apical

1328 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 1326–1333, 2010 www.jasn.org BASIC RESEARCH marker anti-atypical PKC. Results showed that basal bodies in nificantly enlarged in pk1 morphants as well (Figure 3, C and MCCs are still localized to the apical side of the pronephric duct in D). Moreover, similar to ift mutants, ␥-Tubulin labeling re- both mutants, as evident by clusters in side views at the edge of the vealed that basal bodies in MCCs of the pronephric duct of pk1 lumen (arrows in Supplemental Figure S2). morphants are disorganized and more loosely arranged into an oval cluster (Figure 3, E through H; Supplemental Figure S2, E Knockdown of pk1 Leads to Disorganization of Basal and F). It has recently been shown that pk1 is required for proper Bodies in the Kidney Duct and Kidney Cyst Formation apical-basal polarity of the mouse epiblast.31 We therefore further The PCP pathway has been implicated in kidney cyst forma- investigated whether the basal bodies in MCCs of pk1 morphants tion.4,16,20,21 In addition, the disorganization of basal bodies in are displaced from the apical side by colabeling with the apical ift mutants is suggestive of a PCP defect. We therefore tested marker anti-atypical PKC. Similar to ift mutants, the basal bodies whether PCP defects can lead to cyst formation directly by of pk1 morphants are still localized to the apical side, albeit in a knocking down the expression of a core PCP component, pk1, disorganized manner (Supplemental Figure S2, E and F). using a morpholino oligo.29 Injected at 2 ng, pk1 morphant showed a previously observed and typical PCP phenotype, ift57 and ift172 Genetically Interact with pk1 namely, a severely shortened body axis (Figure 3, A and B).29,30 Multiple studies provide evidence to support a critical role of the Interestingly, we additionally observed kidney cyst formation PCP pathway in kidney cyst formation.16,20 However, reports on medial to the pectoral fins, a region where the glomerulus and the role of cilia in the PCP pathway are conflicting.4,15,21 We there- the tubule are located (Figure 3, A and B). Further analysis with fore examined for potential genetic interactions between ift57, immunostaining using anti-Cadherin17 (Cdh17), an antibody ift172, and pk1. We injected suboptimal doses of the ift172 mor- that labels the basal-lateral membrane of the pronephric duct pholino (3.5 ng) and pk1 morpholino (0.6 ng), either together or and the pronephric tubule, showed that the kidney duct is sig- with a control morpholino (0.6 and 3.5 ng, respectively). Results showed that the shortened body axis phenotype was found only in embryos that were co-injected with ift172 and pk1 morpholinos (Figure 4, A through B). Similarly, although a suboptimal dose of ift57 (0.35 ng) morpholino did not cause shortened body axis, this morpholino injected in combination with the pk1 morpholino did (Supplemental Figure S3). Interestingly, maternal-zygotic mutants of ift88 lack a PCP phenotype during gastrulation,15 rais- ing the possibility that there are functional differences between ift88 and other ift genes. We therefore investigated whether ift88 genetically interacts with pk1. Results showed that a suboptimal dose (1.2 ng) of a previously published ift88 morpholino32 syner- gized with the pk1 morpholino in inducing the shortened body axis phenotype (Supplemental Figure S3), indicating that ift88 functions similarly to other ift genes in this assay. To provide more direct support for our hypothesis that ift genes genetically interact with pk1 during CE movement in gastrulation, we analyzed the morphology of paraxial cells in late gastrulae by mosaically expressing a membrane-targeted enhanced GFP (eGFP). As reported previously for wild-type embryos, control embryos injected with a suboptimal dose of either ift172 morpholino or pk1 morpholino together with a control morpholino displayed paraxial cells that were elongated along the medial-lateral axis, with a length-to-width ratio (LWR) of 2.6 Ϯ 0.8 and 2.5 Ϯ 0.6, respectively. In contrast, embryos co-injected Figure 3. Inactivation of pk1 leads to kidney cyst formation and with suboptimal doses of ift172 and pk1 morpholinos exhibited a disorganization of basal bodies. (A and B) A control embryo and LWR that was significantly reduced to 2.0 Ϯ 0.9 (Figure 4, C a pk1 morphant raised with 1-phenyl-2-thiourea at 3 dpf in side through D). Similarly, co-injection of suboptimal doses of ift57 views. Insets: enlarged areas showing a cyst (B) and the lack of and pk1 morpholino reduced the LWR to 1.7 Ϯ0.5 from 2.6 Ϯ0.8 cyst (A). (C and D) Pronephric ducts labeled with anti-Cdh17 in and 2.2 Ϯ 0.6 when compared with control embryos injected with side views. (E through H) Basal bodies as shown by anti–␥-Tub labeling and cilia by anti-a-Tub labeling. Arrows point to arrayed ift57 or pk1 together with a control morpholino, respectively (Fig- basal bodies, whereas arrowheads point to clusters of disorga- ure 4, E through F). nized basal bodies. a-Tub, acetylated Tubulin; ctrlMO, embryos Further support for IFT’s involvement in the PCP pathway injected with a standard control morpholino; ␥-Tub, ␥-Tubulin; comes from a dominant negative construct of ift172. Loss-of- pk1MO, embryos injected with pk1 morpholino. function mutants of ift172 have been identified in Chlamydo-

J Am Soc Nephrol 21: 1326–1333, 2010 Zebrafish IFT in Cilia Assembly and PCP 1329 BASIC RESEARCH www.jasn.org

the inclusion of ift172 morpholino increased the percentage of embryos with shortened body axis from 18% to 36%, indicating that GFP-N1550 is a dominant negative form of Ift172.

DISCUSSION

The Role of ift Genes in Cilia Assembly in Zebrafish In both ift57hi3417 and ift172hi2211 mutants, at 1 dpf, cilia are able to form in the neural tube and the pronephric duct and are indis- tinguishable from cilia in wild-type embryos. A simple interpretation is that ift genes are not required for cilia assembly in zebrafish. As ift genes are highly conserved for ciliary assembly in a wide spectrum of organisms including Chlamydomonas and mice,3,9–12 we considered alternative expla- nations for this difference. In zebrafish, products of many genes are deposited maternally. Therefore, a homozygous null mutant can start its life with a significant quantity of functional products of the mu- Figure 4. Interactions between ift57 or ift172 with the PCP pathway. (A through A”) tated gene provided from the mother, Embryos injected with different combinations of morpholino oligos at the 20-somite stage masking the function of that mutated gene in side views. (B) Tabulation of embryos with shortened body axis. *P Ͻ 0.05, from two independent experiments. (C through C”) and (E through E”) Paraxial cells labeled with a during early development. Accordingly, we membrane-targeting GFP in dorsal views with the anterior to the top and the middle line found transcripts of both ift57 and ift172 to the right in embryos injected with different combinations of morpholinos. (D and F) are supplied maternally. We also detected Tabulation of LWR of paraxial cells along the lateral-medial axis. P Ͻ 0.0005. (G and G’) A the presence of Ift57 protein as early as the control embryo and an embryo injected with GFP-N1550 mRNA in side views at 1 dpf. (H 16- to 64-cell stage. Interestingly, although and H’) A control embryo and an embryo injected with GFP-N1550 mRNA labeled with maternal ift57 transcripts decay by 33 hpf krox20 and myoD in situ probes in dorsal views at the 10-somite stage. Scale bars: 50 ␮m. in mutants (Figure 1D), previous studies c, a standard control morpholino; GFPN1550, embryo injected with GFP-N1550 mRNA; indicate that Ift57 protein can be detected pk1, pk1 morpholino; ift172, ift172 morpholino; ift57, ift57 morpholino. at 48 hpf, but not at 4 dpf, in ift57hi3417 mutants.26,34 Together, these results suggest monas and mouse.3,33 Both mutations are located in the C- that maternally supplied Ift57 protein or transcript can persist terminal region of Ift172 (L1615P and L1564P, respectively), for an extensive period of time. Consistent with the gradual suggesting that the C-terminal region is critical for the func- degradation of maternal gene products, cilia assembly is ini- tion of Ift172. We therefore generated a C-terminal truncation tially present on the first day of development in ift57hi3417 and of Ift172 (ift172N1550), which contains only the first 1550 ift172hi2211 mutants, but is lost at later stages of development. amino acids from the N terminus of this protein. Surprisingly, Furthermore, although zygotic ift88oval mutants are initially able overexpression of Ift172N1550 and GFP-N1550 (a GFP- to form cilia, maternal-zygotic ift88oval mutants are not capable of tagged form of Ift172N1550) caused a shortened body axis at 1 cilia assembly.15 Together, these findings suggest that the role for dpf (Figure 4, G and G’). In situ hybridization with a krox20 ift genes in cilia assembly is conserved in zebrafish. and a myoD probe at the 10-somite stage of embryos injected with GFP-N1550 mRNA revealed that both the hindbrain and The PCP Pathway and Kidney Cyst Formation the somites failed to fuse at the midline (Figure 4, H and H’), PCP refers to the coordinated polarity within a sheet of epithe- consistent with defective CE movements. To test whether lial cells. It is known that the proper establishment of PCP is GFP-N1550 is a dominant negative or dominant active form of regulated by the noncanonical Wnt pathway (for a review, see Ift172, we co-injected 150 pg of GFP-N1550 mRNA together with reference 35). Strikingly, recent studies indicate that the PCP 2ngofift172 morpholino, which by itself does not cause an obvi- pathway is critical for preventing cyst formation, which is the hall- ous body axis shortening defect. In a representative experiment, mark of polycystic kidney disease (PKD). Multiple PKD mouse

1330 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 1326–1333, 2010 www.jasn.org BASIC RESEARCH mutants show misorientation of cell division axis during kidney Cloning development before visible kidney cyst formation.16 In addition, Full-length coding sequences of ift57 and ift172 were amplified from a conditional inactivation of Four-jointed, a gene known to be in- zebrafish cDNA pool and cloned into pCS2 vector. GFP tags were volved in PCP, leads to kidney cyst formation in mouse.20 In this added by PCR cloning. ift172N1550 was generated by PCR cloning. study, we demonstrate that inactivation of pk1 can directly lead to kidney cyst formation, providing further support for the involve- Microinjection ment of the PCP pathway in preventing kidney cyst formation. mMESSAGE mMACHINE kit (Ambion) was used to synthesize capped mRNA. Morpholino oligos were purchased from Genetools. The Role of Cilia in the PCP Pathway mRNA or morpholino were injected into embryos at the 1-cell stage at Multiple lines of evidence support an intricate relationship be- indicated concentrations. 5Ј-CATCCCTCTCTCTTTCTCTTTTCAC-3Ј tween the cilium and the PCP pathway. First, a number of known was used to block the translation of ift57 and 5Ј-GACTCA- PCP pathway components, including Dishevelled, Inturned, and GGGCAGTTATAAGAACGTA-3Ј was used to block the translation Fuzzy, are required for proper ciliogenesis during Xenopus devel- of ift172.13 Previously described morpholino oligos were used to 36,37 opment. Second, multiple genes have been shown to be in- knock down the expression of pk1 (5Ј-GCCCACCGTGATTCTC- volved in both cilia-mediated processes and the PCP path- CAGCTCCAT-3Ј)29 and the expression of ift88 (5Ј-CTGGGACAA- 4,21,38 way. Most directly, inactivation of ift88 in mice leads to CE GATGCACATTCTCCAT-3Ј).32 A standard oligo (5Ј-CCTCTTAC- movement defects during cochlear duct development, a pheno- CTCAGTTACAATTTATA-3Ј) was injected as a control. type associated with PCP abnormalities.39 Furthermore, conditional knockout of Ift20 in the mouse kidney leads to misorientation of cell division axis.40 The critical role of the cilium in PKD Histologic Analysis pathogenesis, and the close association between PKD and the PCP Embryos were fixed in Bouin’s fixative overnight at room tempera- pathway, is also consistent with a role of cilia in the PCP pathway. ture, washed three times with PBS, embedded in JB-4 resin from Poly- In this study, we show that both ift57 and ift172 mutants sciences, and cut at 4 ␮m. Slides were then stained with hematoxylin display disorganized basal bodies in the kidney duct, similar to & eosin. that seen in pk1 morphants. We also demonstrate that ift57, ift88, and ift172 genetically interact with pk1 in body axis elon- gation and the alignment of paraxial cells in later gastrulae. Protein Extraction and Western Blotting Embryos were manually deyolked following a previously published Additionally, we show that overexpression of a dominant neg- protocol41 and then ground in 2ϫ SDS sample buffer (4% SDS, 200 ative form of Ift172 disrupts the convergence of somites and mM dithiothreitol, and 5% ␤-mercaptoethanol) with a disposable the hindbrain during early somitogenesis. Together, these data pestle, boiled for 5 minutes, cleared by microcentrifugation at top support a functional involvement of IFT genes in the PCP speed at room temperature for 2 minutes, and run on SDS-PAGE gels. pathway. However, a previous report has indicated that a ma- Proteins were transferred to nitrocellulose membranes. Filters were ternal-zygotic zebrafish mutant of ift88oval showed no obvious then blotted in PBS buffer with 5% instant milk, incubated sequen- defects in convergence extension during gastrulation.15 On the tially with anti-Ift57 (kindly provided by B. Perkins)34 at 1:2500 and basis of these results, we propose that during zebrafish gastru- horseradish peroxidase–conjugated secondary antibodies (Jackson lation IFT genes play a minor or redundant role that becomes ImmunoResearch Laboratories) at 1:5000 to 1:10,000. Signals were apparent only when the PCP pathway is already comprised. then detected with enhanced chemiluminescence detection using the Additionally, it remains possible that subtle PCP phenotypes Western Lightning kit from PerkinElmer Life Sciences. exist in maternal-zygotic mutants of ift genes but are not easily detectable. Intriguingly, the PCP phenotypes observed in the mouse cochlear and kidney ducts of Ift88 and Ift20 mutants39,40 Immunostaining may suggest that there exists tissue and stage specificity in the Immunostaining on fixed embryos was performed as described.42 role of IFT genes in this pathway. The genetic interaction be- Specifically, embryos were fixed in Dent’s fixative. A monoclonal anti–acety- tween multiple ift genes and a core PCP component provides lated Tubulin antibody (Sigma) was used at 1:5000. A monoclonal insight into a novel and intricate relationship between the ci- anti–␥-Tubulin antibody (Sigma) was used at 1:200. A rabbit anti- lium and planar cell polarity and furthers our understanding of aPKC (Santa Cruz Biotechnology) was used at 1:200. A custom anti- the cilium as a key signaling center for vertebrate cells. body made by Covance against the C-terminal region from amino acid 238 of Arl13b was used at 1:2000.43 Fluorescence-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were CONCISE METHODS used at 1:200. Stained embryos were manually deyolked and flat- mounted in Vectashield Hardset mounting medium (Vector Labora- Fish Husbandry tories). Images were taken using a Nikon Eclipse epifluorescence mi- Zebrafish were maintained according to standard protocols.25 All croscope and a Leica stereoscope. Global contrast and intensity of lines were from the Hopkins laboratory and maintained in TAB back- fluorescence images were adjusted using the Elements software. Im- ground.13 ages were further cropped in Photoshop.

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RT-PCR 7. Kozminski KG, Johnson KA, Forscher P, Rosenbaum JL: A motility in RNA was extracted with trizol reagent (Invitrogen) following manu- the eukaryotic flagellum unrelated to flagellar beating. Proc Natl Acad facturer’s instructions. First strand cDNA was generated with the su- SciUSA90: 5519–5523, 1993 8. Rosenbaum JL, Witman GB: Intraflagellar transport. Nat Rev Mol Cell perscript II RT-PCR system (Invitrogen) and subsequently amplified Biol 3: 813–825, 2002 with PCR. For ift57, primers 5Ј-CGGGATCCGGGATGGCGGAGGAG- 9. Han YG, Kwok BH, Kernan MJ: Intraflagellar transport is required in GAAGA-3Ј and 5Ј-GGAATTCGTGTTTCAATAAGCCTGCGCA-3Ј were Drosophila to differentiate sensory cilia but not sperm. Curr Biol 13: used. For ift172, primers 5Ј-AAACGTGAAGTATGCAGCTTAAG-3Ј 1679–1686, 2003 and 5Ј-GGGTCAGCAGGCTTTGTTGTAAA-3Ј were used. For elf1, 10. Baker SA, Freeman K, Luby-Phelps K, Pazour GJ, Besharse JC: IFT20 Ј Ј Ј links kinesin II with a mammalian intraflagellar transport complex that primers 5 -CTTCTCAGGCTGACTGTGC-3 and 5 -CCGCTAG- is conserved in motile flagella and sensory cilia. J Biol Chem 278: CATTACCCTCC-3Ј were used. 34211–34218, 2003 11. Cole DG, Diener DR, Himelblau AL, Beech PL, Fuster JC, Rosenbaum Analysis of Cell Polarity in Late Gastrulae JL: Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): A previously published protocol, with minor modifications, was fol- IFT particles contain proteins required for ciliary assembly in Caeno- rhabditis elegans sensory neurons. J Cell Biol 141: 993–1008, 1998 44 lowed. mRNA encoding a membrane-anchored GFP was injected 12. Haycraft CJ, Schafer JC, Zhang Q, Taulman PD, Yoder BK: Identifica- into one cell of embryos at the 16- to 32-cell stage, which leads to tion of CHE-13, a novel intraflagellar transport protein required for mosaic labeling of cells at later stages. Embryos at the 2- to 5-somite cilia formation. Exp Cell Res 284: 251–263, 2003 stage were imaged with a Nikon Eclipse epifluorescence microscope 13. Sun Z, Amsterdam A, Pazour GJ, Cole DG, Miller MS, Hopkins N: A and analyzed using the Element software. genetic screen in zebrafish identifies cilia genes as a principal cause of cystic kidney. Development 131: 4085–4093, 2004 14. Tsujikawa M, Malicki J: Intraflagellar transport genes are essential for differentiation and survival of vertebrate sensory neurons. Neuron 42: ACKNOWLEDGMENTS 703–716, 2004 15. Huang P, Schier AF: Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia. Development 136: 3089–3098, We thank members of the Sun Labs and members of Yale Center for 2009 PKD research for helpful discussions, Brian Perkins for anti-Ift57, 16. Fischer E, Legue E, Doyen A, Nato F, Nicolas JF, Torres V, Yaniv M, Shiaulou Yuan for his critical reading of the manuscript and the cover Pontoglio M: Defective planar cell polarity in polycystic kidney dis- image, the Reinke Lab and the Cooley Lab for use of their micro- ease. Nat Genet 38: 21–23, 2006 17. Lin F, Hiesberger T, Cordes K, Sinclair AM, Goldstein LS, Somlo S, scopes, and Nicole Semanchik for superb technical assistance. This Igarashi P: Kidney-specific inactivation of the KIF3A subunit of kine- project was supported by grants from NIDDK (RO1 DK069528 and sin-II inhibits renal ciliogenesis and produces polycystic kidney dis- P50 DK057328 (project #3)) and a research grant from PKD Founda- ease. Proc Natl Acad SciUSA100: 5286–5291, 2003 tion (to Z.S.). 18. Romagnolo B, Berrebi D, Saadi-Keddoucci S, Porteu A, Pichard AL, Peuchmaur M, Vandewalle A, Kahn A, Perret C: Intestinal dysplasia and adenoma in transgenic mice after overexpression of an activated beta-catenin. Cancer Res 59: 3875–3879, 1999 DISCLOSURES 19. Saadi-Kheddouci S, Berrebi D, Romagnolo B, Cluzeaud F, Peuchmaur None. M, Kahn A, Vandewalle A, Perret C: Early development of polycystic kidney disease in transgenic mice expressing an activated mutant of the beta-catenin gene. Oncogene 20: 5972–5981, 2001 20. Saburi S, Hester I, Fischer E, Pontoglio M, Eremina V, Gessler M, REFERENCES Quaggin SE, Harrison R, Mount R, McNeill H: Loss of Fat4 disrupts PCP signaling and oriented cell division and leads to cystic kidney 1. DiBella LM, Park A, Sun Z: Zebrafish Tsc1 reveals functional interac- disease. Nat Genet 40: 1010–1015, 2008 tions between the cilium and the TOR pathway. Hum Mol Genet 18: 21. Simons M, Gloy J, Ganner A, Bullerkotte A, Bashkurov M, Kronig C, 595–606, 2009 Schermer B, Benzing T, Cabello OA, Jenny A, Mlodzik M, Polok B, 2. Haycraft CJ, Banizs B, Aydin-Son Y, Zhang Q, Michaud EJ, Yoder BK: Driever W, Obara T, Walz G: Inversin, the gene product mutated in Gli2 and Gli3 localize to cilia and require the intraflagellar transport nephronophthisis type II, functions as a molecular switch between Wnt protein polaris for processing and function. PLoS Genet 1: e53, 2005 signaling pathways. Nat Genet 37: 537–543, 2005 3. Huangfu D, Liu A, Rakeman AS, Murcia NS, Niswander L, Anderson 22. Corbit KC, Aanstad P, Singla V, Norman AR, Stainier DY, Reiter JF: KV: Hedgehog signalling in the mouse requires intraflagellar transport Vertebrate Smoothened functions at the primary cilium. Nature 437: proteins. Nature 426: 83–87, 2003 1018–1021, 2005 4. Kishimoto N, Cao Y, Park A, Sun Z: Cystic kidney gene seahorse 23. Corbit KC, Shyer AE, Dowdle WE, Gaulden J, Singla V, Reiter JF: Kif3a regulates cilia-mediated processes and Wnt pathways. Dev Cell 14: constrains beta-catenin-dependent Wnt signalling through dual ciliary 954–961, 2008 and non-ciliary mechanisms. Nat Cell Biol 10: 70–76, 2008 5. Shillingford JM, Murcia NS, Larson CH, Low SH, Hedgepeth R, Brown 24. Ocbina PJ, Tuson M, Anderson KV: Primary cilia are not required for N, Flask CA, Novick AC, Goldfarb DA, Kramer-Zucker A, Walz G, normal canonical Wnt signaling in the mouse embryo. PLoS One 4: Piontek KB, Germino GG, Weimbs T: From the Cover: The mTOR e6839, 2009 pathway is regulated by polycystin-1, and its inhibition reverses renal 25. Westerfield M: The Zebrafish Book: a guide for the laboratory use of cystogenesis in polycystic kidney disease. Proc Natl Acad SciUSA zebrafish (Danio rerio), University of Oregon Press, Eugene, OR, 2000 103: 5466–5471, 2006 26. Lunt SC, Haynes T, Perkins BD: Zebrafish ift57, ift88, and ift172 in- 6. Hildebrandt F, Attanasio M, Otto E: Nephronophthisis: Disease mech- traflagellar transport mutants disrupt cilia but do not affect hedgehog anisms of a ciliopathy. J Am Soc Nephrol 20: 23–35, 2009 signaling. Dev Dyn 238: 1744–1759, 2009

1332 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 1326–1333, 2010 www.jasn.org BASIC RESEARCH

27. Liu Y, Pathak N, Kramer-Zucker A, Drummond IA: Notch signaling CC, Chapple JP, Munro PM, Fisher S, Tan PL, Phillips HM, Leroux MR, controls the differentiation of transporting epithelia and multiciliated Henderson DJ, Murdoch JN, Copp AJ, Eliot MM, Lupski JR, Kemp DT, cells in the zebrafish pronephros. Development 134: 1111–1122, 2007 Dollfus H, Tada M, Katsanis N, Forge A, Beales PL: Disruption of 28. Ma M, Jiang YJ: Jagged2a-notch signaling mediates cell fate choice in Bardet-Biedl syndrome ciliary proteins perturbs planar cell polarity in the zebrafish pronephric duct. PLoS Genet 3: e18, 2007 vertebrates. Nat Genet 37: 1135–1140, 2005 29. Carreira-Barbosa F, Concha ML, Takeuchi M, Ueno N, Wilson SW, 39. Jones C, Roper VC, Foucher I, Qian D, Banizs B, Petit C, Yoder BK, Tada M: Prickle 1 regulates cell movements during gastrulation and Chen P: Ciliary proteins link basal body polarization to planar cell neuronal migration in zebrafish. Development 130: 4037–4046, 2003 polarity regulation. Nat Genet 40: 69–77, 2008 30. Takeuchi M, Nakabayashi J, Sakaguchi T, Yamamoto TS, Takahashi H, 40. Jonassen JA, San Agustin J, Follit JA, Pazour GJ: Deletion of IFT20 in Takeda H, Ueno N: The prickle-related gene in vertebrates is essential the mouse kidney causes misorientation of the mitotic spindle and for gastrulation cell movements. Curr Biol 13: 674–679, 2003 cystic kidney disease. J Cell Biol 183: 377–384, 2008 31. Tao H, Suzuki M, Kiyonari H, Abe T, Sasaoka T, Ueno N: Mouse 41. Link V, Shevchenko A, Heisenberg CP: Proteomics of early zebrafish prickle1, the homolog of a PCP gene, is essential for epiblast apical- embryos. BMC Dev Biol 6: 1, 2006 basal polarity. Proc Natl Acad SciUSA106: 14426–14431, 2009 42. Drummond IA, Majumdar A, Hentschel H, Elger M, Solnica-Krezel L, 32. Kramer-Zucker AG, Olale F, Haycraft CJ, Yoder BK, Schier AF, Drummond Schier AF, Neuhauss SC, Stemple DL, Zwartkruis F, Rangini Z, Driever IA: Cilia-driven fluid flow in the zebrafish pronephros, brain and W, Fishman MC: Early development of the zebrafish pronephros and Kupffer’s vesicle is required for normal organogenesis. Development analysis of mutations affecting pronephric function. Development 125: 132: 1907–1921, 2005 4655–4667, 1998 33. Pedersen LB, Miller MS, Geimer S, Leitch JM, Rosenbaum JL, Cole DG: 43. Duldulao NA, Lee S, Sun Z: Cilia localization is essential for in vivo Chlamydomonas IFT172 is encoded by FLA11, interacts with CrEB1, and functions of the Joubert syndrome protein Arl13b/Scorpion. Develop- regulates IFT at the flagellar tip. Curr Biol 15: 262–266, 2005 ment 136: 4033–4042, 2009 34. Krock BL, Perkins BD: The intraflagellar transport protein IFT57 is re- 44. Topczewski J, Sepich DS, Myers DC, Walker C, Amores A, Lele Z, quired for cilia maintenance and regulates IFT-particle-kinesin-II dissoci- Hammerschmidt M, Postlethwait J, Solnica-Krezel L: The zebrafish ation in vertebrate photoreceptors. J Cell Sci 121: 1907–1915, 2008 glypican knypek controls cell polarity during gastrulation movements 35. Solnica-Krezel L: Gastrulation in zebrafish—all just about adhesion? of convergent extension. Dev Cell 1: 251–264, 2001 Curr Opin Genet Dev 16: 433–441, 2006 36. Park TJ, Mitchell BJ, Abitua PB, Kintner C, Wallingford JB: Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells. Nat Genet 40: 871–879, 2008 See related editorial, “More than Maintenance? A Role for IFT Genes in Planar 37. Park TJ, Haigo SL, Wallingford JB: Ciliogenesis defects in embryos Cell Polarity,” on pages 1240–1241. lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling. Nat Genet 38: 303–311, 2006 Supplemental information for this article is available online at http://www.jasn. 38. Ross AJ, May-Simera H, Eichers ER, Kai M, Hill J, Jagger DJ, Leitch org/.

J Am Soc Nephrol 21: 1326–1333, 2010 Zebrafish IFT in Cilia Assembly and PCP 1333