BASIC RESEARCH www.jasn.org Intraflagellar Transport Proteins Are Essential for Cilia Formation and for Planar Cell Polarity 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 per- centage 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 developmen- tal stages. The presence of ift57 and ift172 mRNA from embryos at the 4- to 32-cell stage verified that these mRNAs are mater- nally deposited (Figure 1E). We further investigated whether IFT proteins can be detected before the mater- nal-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’).