Research Article 611 Functional interactions between the - associated Meckel syndrome 1 (MKS1) and two novel MKS1-related (MKSR)

Nathan J. Bialas1,*, Peter N. Inglis1,*, Chunmei Li1,*, Jon F. Robinson2, Jeremy D. K. Parker1, Michael P. Healey1, Erica E. Davis2, Chrystal D. Inglis1, Tiina Toivonen3, David C. Cottell3, Oliver E. Blacque4, Lynne M. Quarmby1, Nicholas Katsanis2,5,6 and Michel R. Leroux1,‡ 1Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada 2McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA 3Electron Microscopy Laboratory and 4School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland 5Department of Molecular Biology and Genetics, and 6Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA *These authors contributed equally to this work ‡Author for correspondence (e-mail: [email protected])

Accepted 20 October 2008 Journal of Cell Science 122, 611-624 Published by The Company of Biologists 2009 doi:10.1242/jcs.028621

Summary Meckel syndrome (MKS) is a ciliopathy characterized by mks/mksr mutants do not display overt defects in ciliary encephalocele, cystic renal disease, liver fibrosis and . structure, or chemosensation. However, An identifying feature of MKS1, one of six MKS-associated we find genetic interactions between all double mks/mksr proteins, is the presence of a B9 domain of unknown function. mutant combinations, manifesting as an increased lifespan Using phylogenetic analyses, we show that this domain occurs phenotype, which is due to abnormal insulin–IGF-I signaling. exclusively within a family of three proteins distributed widely Our findings therefore demonstrate functional interactions in ciliated organisms. Consistent with a ciliary role, all between a novel family of proteins associated with basal bodies Caenorhabditis elegans B9-domain-containing proteins, MKS- or cilia, providing new insights into the molecular etiology of a 1 and MKS-1-related proteins 1 and 2 (MKSR-1, MKSR-2), pleiotropic human disorder. localize to transition zones/basal bodies of sensory cilia. Their subcellular localization is largely co-dependent, pointing to a

Journal of Cell Science functional relationship between the proteins. This localization Supplementary material available online at is evolutionarily conserved, because the human orthologues also http://jcs.biologists.org/cgi/content/full/122/5/611/DC1 localize to basal bodies, as well as cilia. As reported for MKS1, disrupting human MKSR1 or MKSR2 causes ciliogenesis Key words: Meckel syndrome, Cilia, , Ciliopathy, Insulin, defects. By contrast, single, double and triple C. elegans Signaling

Introduction disorders that collectively affect the renal, cardiac, hepatic, Primary cilia, the hair-like microtubule-based organelles found on pancreatic, skeletal, visual, nervous, olfactory and auditory systems the majority of human cell types, have gained attention recently (Badano et al., 2006; Bisgrove and Yost, 2006; Tan et al., 2007). owing to their involvement in a multitude of sensory processes, Numerous cilia-associated disorders () have been signaling pathways and genetic disorders (Davis et al., 2006; described, including Bardet-Biedl syndrome (BBS), Meckel Davenport and Yoder, 2005; Marshall and Nonaka, 2006; Satir and syndrome (MKS) and polycystic kidney disease (PKD) (reviewed Christensen, 2007; Singla and Reiter, 2006). These non-motile cilia by Pazour and Rosenbaum, 2002; Badano et al., 2006; Bisgrove are now implicated in most physiological sensory modalities, and Yost, 2006; Blacque and Leroux, 2006; Christensen et al., 2007; including chemosensation, olfaction, mechanosensation, Hildebrandt and Otto, 2005). photoreception and thermosensation (Davis et al., 2006; Davenport Meckel syndrome is a rare autosomal recessive disorder and Yoder, 2005; Perkins et al., 1986; Satir and Christensen, 2007; characterized by central nervous system malformations Tan et al., 2007). Primary cilia not only capture and transduce (encephalocele), polydactyly, renal cysts and hepatic ductal environmental stimuli, but also have key roles in the transduction dysplasia and cysts (Alexiev et al., 2006; Badano et al., 2006). of Wnt, Hedgehog and PDGFRαα signaling pathways (Christensen Numerous lines of evidence have defined MKS as a ciliopathy, et al., 2007; Eggenschwiler and Anderson, 2007; Gerdes et al., 2007; although the nature of the ciliary defect is unclear. The recently Breunig et al., 2008). Moreover, ciliary assembly and disassembly identified MKS1 protein (Kyttälä et al., 2006), for example, are coordinated intimately with the cell cycle (Pan and Snell, 2007; localizes to basal bodies, which are the centriolar structures required Quarmby and Parker, 2005). In humans, defects in the sensory and for nucleating eukaryotic cilia. Together with a second identified signaling functions of primary cilia lead to numerous developmental MKS-associated protein, MKS3/meckelin (Smith et al., 2006), 612 Journal of Cell Science 122 (5)

MKS1 is reported to have a role in basal body migration to the Williams et al. (Williams et al., 2008) recently reported on the apical membrane, and thus, ciliogenesis (Dawe et al., 2007). A third analysis of the three B9-domain-containing proteins found in C. protein implicated in MKS, NPHP6/CEP290/MKS4/BBS14, also elegans. Using GFP-tagged variants, they observed interdependent localizes to basal bodies, but its molecular function is unclear (Baala localization of the proteins to ciliary transition zones (akin to basal et al., 2007; den Hollander et al., 2006; Leitch et al., 2008; Sayer bodies), and as such, named the proteins encoded by Y38F2AL.2 et al., 2006; Valente et al., 2006). RPGRIP1L (MKS5), also and K03E6.4 ‘ciliary transition zone associated’ protein-1 and implicated in Joubert syndrome, is a basal body protein that protein-2 (TZA-1 and TZA-2), respectively. Disruption of any of interacts with the nephronophthisis-associated NPHP-4 protein; the three respective proteins did not result in overt changes to ciliary although it is not required for formation, it has an important morphology nor in any specific behavioral or sensory abnormalities, (and presumably cilium-based) role in Hedgehog signaling (Arts with the exception of a subtle foraging behavior phenotype. et al., 2007; Delous et al., 2007; Vierkotten et al., 2007). Most Interestingly, unlike disruption of stumpy in mammals, C. elegans recently, two other proteins, NPHP3 (Bergmann et al., 2008), and transition zone positioning and ciliogenesis was only impaired by CC2D2A (MKS6), whose function is unknown but is associated further disruption of nphp-1 or nphp-4, suggesting a genetic with the formation of cilia (Tallila et al., 2008), were found to be redundancy in the system. disrupted in MKS patients. In the present study, we investigated the molecular basis of The MKS1 protein contains no domains of recognized function. Meckel syndrome by characterizing in further detail the three C. Nonetheless, it does harbor a predicted so-called ‘B9’ domain of elegans and human B9-domain-containing proteins. Our undetermined function (Kyttälä et al., 2006). Two additional highly phylogenetic analyses demonstrate that B9-domain-containing conserved proteins containing B9 domains, referred to in mammals proteins are invariably absent from non-ciliated organisms but co- as B9D1 and B9D2, can also be identified. We previously reported occur as a family of three different proteins with orthologues in that all three putative B9 protein orthologues in the nematode nearly all ciliated species. We show that the C. elegans MKS-1, Caenorhabditis elegans (R148.1/xbx-7, K03E6.4 and Y38F2AL.2) MKSR-1 and MKSR-2 proteins localize to transition zones/basal are found solely in ciliated sensory neurons and possess X-box bodies in a largely interdependent manner, and that the subcellular sequences in the upstream promoter sequences of their associated localization to basal bodies is evolutionarily conserved for the that are regulated by the ciliogenic transcription factor DAF- human B9 protein counterparts. Knockdown of the human MKSR1 19 (Blacque et al., 2005; Efimenko et al., 2005). These observations and MKSR2 genes using RNA interference (RNAi) leads to a are consistent with comparative genomic analyses of ciliated versus ciliogenesis defect, which is similar to that reported for MKS1 by non-ciliated organisms (Avidor-Reiss et al., 2004; Li et al., 2004a), Dawe et al. (Dawe et al., 2007). By contrast, rigorous analysis of as well as the recent discovery that two of the Drosophila IFT or cilia ultrastructure by electron microscopy for each of the melanogaster B9-domain-containing genes are also X-box regulated single, double and triple C. elegans mks/mksr mutants showed (Laurençon et al., 2007), collectively implicating all three proteins no clear defects compared with the wild-type animals. However, in ciliary function(s). This notion is also supported by the recent all double mks/mksr gene mutant combinations revealed genetic finding that the murine B9D2 gene is abrogated in the stumpy interactions between the different family members that are mutant, which is characterized by impaired ciliogenesis, cystic manifested by an increased lifespan dependent on the DAF-2

Journal of Cell Science kidneys and hydrocephalus (Town et al., 2008). Disruption of stumpy (insulin–IGF-I receptor)–DAF-16 (FOXO transcription factor) in this mouse model was further shown to be required for the pathway. Together, our data reveal that a highly conserved family proliferation and neurogenesis of astrocyte-like neural precursor of three proteins functionally interact at basal bodies or cilia to (ALNP) cells, probably as a result of dramatically downregulated support ciliogenesis in human cells and a cilium-associated cilium-dependent sonic hedgehog (Shh) signaling (Breunig et al., signaling process in C. elegans. Based on our present study, we 2008). propose a unified nomenclature that captures the evolutionary and In C. elegans, defects in proteins implicated in cilia structure functional relatedness of the three proteins, namely Meckel and/or function have been associated with various sensory syndrome 1 (MKS-1) and MKS1-related proteins 1 and 2 (MKSR- phenotypes (Bae and Barr, 2008; Inglis et al., 2007; Perkins et 1 and MKSR-2). al., 1986; Scholey, 2008). For example, abrogation of the C. elegans orthologues of two proteins associated with the transition Results zone/basal body and linked to nephronophthisis, NPHP-1 and An evolutionarily conserved family of B9-domain-containing NPHP-4, leads to chemosensation, male mating, and various proteins in ciliated organisms axonemal and intraflagellar transport (IFT) defects (Jauregui and The identification of C. elegans xbx-7/R148.1, K03E6.4 and Barr, 2005; Jauregui et al., 2008; Winkelbauer et al., 2005; Wolf Y38F2AL.2 as genes expressed exclusively in ciliated cells initially et al., 2005); disruption of BBS proteins results in partially provided evidence that they might encode proteins with important truncated cilia as well as chemo- and thermo-sensory phenotypes ciliary functions (Blacque et al., 2005; Efimenko et al., 2005). (Blacque et al., 2004; Ou et al., 2005; Tan et al., 2007). Notably, Moreover, the presence of a single B9 domain of unknown function cilium- and/or basal-body-associated sensory inputs are transduced in each of these three proteins hinted at the potential significance by at least one major signaling pathway in the nematode, namely of this protein motif, a notion that was strongly reinforced following the insulin–IGF-I pathway, which regulates longevity (Apfeld and the cloning of the Meckel-syndrome-associated MKS1 gene, the Kenyon, 1999). Hence, many basal body or cilium mutants, human orthologue of xbx-7/R148.1 (Kyttälä et al., 2006). On this including nphp-1 and nphp-4, as well as strains with defects in basis, we conducted comprehensive searches of sequence databases the IFT process required for building all cilia (e.g. osm-5, che- to identify all possible proteins harboring the B9 domain. Without 11, ifta-2, etc.), display increased lifespans, indicative of impaired exception, we failed to find B9-domain-containing proteins in ciliary signaling (Apfeld and Kenyon, 1999; Schafer et al., 2006; prokaryotes or in eukaryotes lacking cilia, such as Saccharomyces Winkelbauer et al., 2005). cerevisiae, Dictyostelium discoideum and Arabidopsis thaliana Meckel syndrome 1 and related proteins 613 Journal of Cell Science

Fig. 1. Phylogenetic analysis showing that B9-domain-containing proteins from ciliated organisms belong to a family of proteins consisting of three clades or family members, namely Meckel Syndrome 1 protein (MKS-1), MKS-1-related protein 1 (MKSR-1) and MKS-1-related protein 2 (MKSR-2), and sequence comparisons of different B9 domains. (A) Phylogenetic tree of B9-domain-containing proteins from several diverse ciliated eukaryotes. Support for nodes (posterior probabilities) are indicated by blue (1.0), green (>0.9) or red (>0.8) circles. The MKS-1, MKSR-1 and MKSR-2 protein families are shown in blue, green and red, respectively. Scale bar denotes 0.1 substitutions per site. Bd, Batrachochytrium dendrobatidis; Ce, Caenorhabditis elegans; Cb, Caenorhabditis briggsae; Cr, Chlamydomonas reinhardtii; Dm, Drosophila melanogaster; Dr, Danio rerio; Hs, Homo sapiens; Mm, Mus musculus; Sp, Strongylocentrotus purpuratus; Tb, Trypanosoma brucei; Thaps, Thalassiosira pseudonana; Xl, Xenopus laevis. Supplementary material Table S1 provides the full listing of proteins (with accession numbers) considered in the analysis. (B) Multiple amino acid sequence alignment of 22 B9 protein domains from eight different species. MKS-1, MKSR-1 and MKSR-2 protein sequence names are colored blue, green and red, respectively. Dark blue highlights signify sequences that match the consensus sequence. Light blue sequences do not match the consensus sequence, but have a positive Blosum62 score. Conservation values for each residue are calculated based on identities and conserved physicochemical properties between different amino acid residues in each column. Quality is a measurement of the likelihood of mutations in each column; lower quality signifies greater likelihood of mutations, if any, present between sequences. Species abbreviations are as above except for Tt, Tetrahymena thermophila.

(supplementary material Table S1). In the vast majority of fully family member that probably arose from an independent gene sequenced ciliated organisms queried (21 in total), however, we duplication (supplementary material Table S1). uncovered three different proteins containing a single B9 domain. Using amino acid sequence alignments of B9 domains obtained The only exceptions were the moss Physcomitrella patens, the from a comprehensive list of species chosen to represent the major parasitic species Giardia lamblia and the apicomplexan Plasmodium eukaryotic groupings outlined previously (Keeling et al., 2005), we falciparum, which have no recognizable B9-domain-containing derived phylogenetic trees of B9-domain-containing proteins. The proteins, and the free-living Tetrahymena thermophila, Paramecium neighbour-joining algorithm of ClustalW (Higgins et al., 1994) and tetraurelia and Chlamydomonas reinhardtii, which contain a fourth the Bayesian phylogeny inference program MrBayes (Huelsenbeck 614 Journal of Cell Science 122 (5)

and Ronquist, 2001) were used independently on the protein acid sequence alignment of 22 B9 domains from MKS-1, MKSR- alignments to generate the trees. Each method produced essentially 1 and MKSR-2 proteins across six different species, shown in Fig. identical phylogenies that group the sequences into three clades, 1B, reveals sequence conservation throughout the ~115 residue which we named MKS-1, MKSR-1 and MKSR-2 (the Bayesian domain, where some residues are invariant in >90% of the tree is shown in Fig. 1A). These analyses demonstrate that proteins sequences. containing B9 domains are evolutionarily ancient; specifically, the diversity of organisms represented in each clade shown in the tree All three B9-domain-containing proteins localize to basal suggests that the gene duplications that led to the three clades bodies and/or cilia preceded the speciation events resulting in the emergence of major Three human proteins associated to date with Meckel syndrome eukaryotic lineages, the last common ancestor of which is inferred (MKS1, CEP290/NPHP6/MKS4 and RPGRIP1L) localize to the to be ciliated (Richards and Cavalier-Smith, 2005). basal body at the base of the cilium, and a fourth, MKS3/Meckelin, We found that although the MKSR-1 and MKSR-2 family is distributed along the length of the cilium (Arts et al., 2007; Dawe members typically consist of little more than the B9 domain, et al., 2007; Delous et al., 2007; Keller et al., 2005; Sayer et al., 2006; members of the MKS-1 clade were larger in size, with poorly Valente et al., 2006; Vierkotten et al., 2007). These findings, which conserved regions outside of the B9 domain that sometimes contain are consistent with MKS being a ciliopathy, led us to examine the other domains (for example, in the Drosophila and Chlamydomonas subcellular localization of all three human B9-domain-containing MKS-1 proteins). These observations suggest that the B9 domain proteins in a ciliated mouse cell line (IMCD3) derived from the inner is critically important for the function(s) of the proteins. An amino medullary collecting duct of the kidney. We confirmed the localization Journal of Cell Science

Fig. 2. MKS-1, MKSR-1 and MKSR-2 proteins localize to centrosomes or basal bodies in human cells and transition zones in C. elegans. (A,B) IMCD3 cells transiently expressing constructs encoding V5 epitope-tagged human MKS1, MKSR1 (EPPB9/B9D1) and MKSR2 (LOC80776/B9D2), are shown in the top, middle and bottom panels, respectively. In ciliated IMCD3 cells (A), the V5-tagged MKS1, MKSR1 and MKSR2 proteins (red) colocalize with the centrosomal γ-tubulin marker (green), as seen in the merged images (yellow denotes overlap in signals). Cilia are labeled with an against acetylated tubulin (also green). In ciliated IMCD3 cells stably expressing GFP-tagged versions of the MKS1, MKSR1 and MKSR2 proteins (green) (B), colocalization is observed with the acetylated tubulin antibody (red), which highlights the ciliary . Arrows denote centrosomes or basal bodies; brackets show the ciliary axoneme. (C) GFP-tagged C. elegans MKS-1, MKSR-1 and MKSR-2 localize specifically to transition zones (akin to basal bodies) in ciliated sensory neurons. Transition zone staining near the tip of the head (left panels) at the base of amphid cilia and near the tail of the animal at the base of phasmid cilia (right panels) are indicated with arrowheads and are shown enlarged in the insets. The relative positions of cilia, transition zones and dendrites are shown in some of the images. Scale bar: 5 μm (insets magnified ϫ3). Meckel syndrome 1 and related proteins 615

of a transiently-expressed, V5-epitope-tagged version of MKS1 to Interdependent localization of MKS-1, MKSR-1 and MKSR-2 the basal body in ciliated cells (Fig. 2A, top panels), and to to basal bodies centrosomes in non-ciliated IMCD3 cells (supplementary material On the basis that all three C. elegans B9-domain-containing proteins Fig. S1) by co-staining with γ-tubulin (a centriolar marker) alone or can be observed at transition zones/basal bodies, we hypothesized in combination with acetylated α-tubulin (a ciliary marker). The that the proteins form a functional complex and that their localization previously uncharacterized human MKSR1 (B9D1) and MKSR2 might be co-dependent. To test this possibility, we examined the (B9D2) proteins, also tagged with the V5 epitope and expressed localization of the three individual C. elegans GFP-tagged MKS- transiently, showed the same localization to basal bodies in ciliated 1, MKSR-1 and MKSR-2 proteins in each of their two IMCD3 cells (Fig. 2A, middle and bottom panels, respectively), and complementary mks/mksr mutant backgrounds. to centrosomes in non-ciliated IMCD3 cells (supplementary material To perform this study, we first obtained from the National Fig. S1). The localization to centrosomes in non-ciliated cells is BioResource Project (NBRP, University of Tokyo, Japan) strains consistent with the proposed pre-ciliogenic function(s) of MKS1 in with deletions in each of the respective mks-1/xbx-7, mksr-1/tza- centriolar migration (Dawe et al., 2007), and the recent differential 2 and mksr-2/tza-1 genes; the three gene models and their lesions, localization of the murine stumpy protein (MKSR2) to basal bodies as deduced by RT-PCR analysis of the transcripts, are shown in or cilia (Town et al., 2008). Interestingly, in contrast to the transiently supplementary material Fig. S2A,B. In the Y38F2AL.2/mksr- transfected IMCD3 cells, versions of all three GFP-tagged B9 2(tm2452) strain, the mutant allele encodes a protein roughly half proteins produced from stably transfected IMCD3 cells localized to the size of the wild type, resulting from a significant truncation the ciliary (Fig. 2B). These results suggest, along with the (approximately 50 residues) within the B9 domain. In the case of findings of Town et al. (Town et al., 2008), that the B9 proteins can the xbx-7/mks-1(tm2705) mutant strain, the deletion results in the localize differentially to the basal body alone or to the ciliary axoneme. splicing of exon 2 with exon 5, ultimately generating a protein To investigate whether the colocalization of the three B9-domain- lacking approximately 70 residues in the N-terminal region, but containing proteins at basal bodies or cilia is evolutionarily conserved, does not abrogate any of the B9 domain. Finally, in the and to initiate a functional analysis of the three proteins in a K03E6.4/mksr-1(tm3083) strain, mis-splicing results in the genetically tractable system, we generated transgenic C. elegans inclusion of nucleotides 1032-1057 into the transcript, which then strains harboring GFP-tagged versions of the respective protein fuse in-frame to nucleotide 1276 of exon 3 (at the start of the 3Ј orthologues. The three expression constructs included the endogenous flanking sequence of the deletion). The predicted MKSR-1 protein promoter for each gene (encompassing the X-box regulatory element), encoded by this allele is one amino acid larger than the wild type, as well as the entire coding region fused in-frame to GFP at the C- and possesses a distinct 18 amino acid alteration to the B9 domain terminus (see supplementary material Fig. S2A for the gene (the sequences of which are shown in supplementary material Fig. structures). Similar to our previous findings using promoter-GFP S2B). Although the possibility exists that all three deletion mutants (transcriptional) fusion constructs (Efimenko et al., 2005; Blacque et (tm2452, tm2705, tm3083) are hypomorphs rather than null al., 2005), the three mks/mksr transgenes were expressed specifically mutants, in two cases (mksr-1 and mksr-2) the B9 domain is in most, if not all ciliated cells, including the amphid and phasmid disrupted, and we confirm below that all alleles are associated with sensory neurons. More notable, however, was that the three GFP- observable protein mislocalization and/or lifespan and signaling

Journal of Cell Science tagged proteins (MKS-1, MKSR-1 and MKSR-2) localized phenotypes. specifically to transition zones at the base of cilia (Fig. 2C), which By carrying out standard genetic crosses, we generated strains are akin to the basal bodies of other species (Perkins et al., 1986). carrying all six possible combinations of MKS-1, MKSR-1 and For each amphid bundle, we were able to observe up to ~12 transition MKSR-2 GFP-tagged proteins present in the two complementary zones as fluorescent ‘spots’ near the head of the animal; for the mks/mksr gene mutant backgrounds. Visualization of the MKSR- phasmid sensory neurons situated near the tail, two pairs of transition 1::GFP and MKSR-2::GFP proteins in the mks-1 mutant background zones could be seen (individual pairs are shown in Fig. 2C). This revealed no significant change in localization to the transition zones localization pattern is equivalent to that observed for the mammalian compared with that of the wild-type strain, and no unusual or orthologues (supplementary material Fig. S1) except that the latter prominent accumulations along the dendrites (Fig. 3C,E). By proteins could also associate with the ciliary axonemes (Fig. 2B) contrast, the MKSR-1::GFP protein showed consistently reduced (Town et al., 2008). Of note, our findings confirm the localization of or unclear localization to transition zones in the amphid (head) and the C. elegans proteins recently reported by Williams et al. (Williams phasmid (tail) neurons of mksr-2 mutant animals, and pronounced et al., 2008); furthermore, the C. elegans NPHP-1 and NPHP-4 accumulations in the dendrites (Fig. 3D). Similarly, MKSR-2::GFP proteins, whose respective genes interact with the mks/mksr genes, showed reduced and less distinct signals at the transition zones of also localize specifically to transition zones (Jauregui et al., 2005; mksr-1 mutants (compared with wild-type animals), although in Winkelbauer et al., 2005; Williams et al., 2008). many cases the protein could be observed at or near the transition Together, our findings establish that all B9-domain-containing zones and thus is likely to be only partly mislocalized (Fig. 3F). proteins associate with, and presumably function at, basal bodies The GFP-tagged MKS-1 protein was similarly mislocalized in the in two highly divergent species (humans and nematodes); in mksr-1 and mksr-2 mutants (Fig. 3A,B); specifically, the protein humans, ciliary localization is also observed, suggesting two was less apparent at the transition zones and was found consistently potentially distinct regions of localization and thus, function. These along the dendrites in a manner never observed for any of the MKS- data, combined with our phylogenetic analysis revealing an 1, MKSR-1 and MKSR-2 proteins in wild-type animals (compare essentially strict co-occurrence of the three proteins in ciliated Fig. 3A,B with Fig. 2C). All analyses were performed at least three organisms and absence from non-ciliated species (Fig. 1A; times using >50 worms/strain and blind to the genotype and the supplementary material Table S1), raise the distinct possibility that identity of the GFP-tagged protein. all MKS-1, MKSR-1 and MKSR-2 proteins share a common ciliary Our data are comparable, but not identical, to those recently function at the base of the organelle and within the cilium itself. reported by Williams et al. (Williams et al., 2008). Unlike the 616 Journal of Cell Science 122 (5)

previous study, which used the ok2092 allele of mksr-1, we were hypothesized that all of the B9 proteins might be required for able to observe at least partial mislocalization of MKSR-2 in the cilium formation in C. elegans, and perhaps more specifically, mksr-1(tm3083) strain. Taken together, these data further regulation of the IFT process. To investigate these possibilities, demonstrate that the proper localization of the MKS-1, MKSR-1 we first tested each of the mks-1, mksr-1 and mksr-2 single and MKSR-2 proteins to transition zones in C. elegans is co- mutants for an inability to take up a fluorescent dye, a dye-filling dependent, suggesting that they interact directly as a hetero- (Dyf) phenotype that is observed in all bbs and IFT gene mutants oligomeric complex or indirectly as part of a larger, functional multi- (Inglis et al., 2007). None of the mutant strains displayed a Dyf subunit complex. phenotype (Fig. 4A; supplementary material Fig. S3A), suggesting that all possess full-length, environmentally exposed The C. elegans MKS-1, MKSR-1 and MKSR-2 proteins are not cilia. To examine the structure of the amphid and phasmid cilia essential for transition-zone positioning, cilium formation or IFT in the mutants directly, we introduced into these strains several function GFP-tagged ciliary markers (the anterograde IFT motor To date, the majority (but not all) of C. elegans genes regulated components OSM-3-kinesin and the kinesin-associated protein by the X-box-binding DAF-19 transcription factor encode KAP-1, the IFT protein CHE-2/IFT80 and the light chain ciliogenic proteins, meaning that their disruption causes cilia component XBX-2 which is essential for driving retrograde IFT). structure defects; these include Bardet-Biedl syndrome genes Consistent with the results of the Dyf assays, each of the ciliary (bbs-1, bbs-7 and bbs-8), and genes encoding core intraflagellar proteins localize correctly to the transition zones and cilia, with transport (IFT) components (e.g. che-2, osm-5, osm-6 and che- no overt accumulations seen in dendrites leading to the transition 11), that are collectively required for proper ciliogenesis in zones or within the cilia; taken together, the data provide strong nematodes (Ansley et al., 2003; Swoboda et al., 2000). Because evidence that the relative positions of the transition zones, and the three C. elegans mks/mksr genes are X-box regulated the ciliary structures, are not different from those of wild-type (supplementary material Fig. S2A) (Blacque et al., 2005; animals (Fig. 4B shows the CHE-2::GFP results; data for OSM- Efimenko et al., 2005), and MKS1 was first shown to be required 3-kinesin, KAP-1 and XBX-2 are presented in supplementary for ciliogenesis in mammalian cells (Dawe et al., 2006), we material Fig. S4A). In addition, all GFP-tagged proteins appeared Journal of Cell Science

Fig. 3. Interdependent localization of C. elegans MKS-1, MKSR-1 and MKSR-2 proteins to transition zones. (A-F) The localization patterns of GFP-tagged MKS-1, MKSR-1 and MKSR-2 proteins in the indicated mks-1, mksr-1 or mksr-2 mutant strains are shown in both amphid (head) and phasmid (tail) sensory neurons. Arrowheads indicate representative (individual) transition zones, and asterisks highlight protein accumulations not normally observed for the GFP-tagged MKS-1, MKSR-1 or MKSR-2 proteins in wild-type animals (see Fig. 2C). The orientations of the animals are the same for all, i.e. the head is up and the tail is down. Scale bar: 5 μm. Meckel syndrome 1 and related proteins 617 Journal of Cell Science

Fig. 4. Single, double and triple C. elegans mks/mksr mutants exhibit normal transition zone positioning, ciliary axonemal structures, intraflagellar transport and chemosensory behaviors. (A) The mks/mksr single, double and triple mutants display normal filling of amphid and phasmid neurons with the fluorescent dye diI, indicating that the cilium structure is probably intact and that the ciliary endings are properly exposed to the external environment; representative images for N2 (wild-type), bbs-8 mutant (dye-filling defective), mks-1;mksr-1;mksr-2 (triple) mutant, and mks-1;mksr-1;mksr-2;bbs-8 quadruple mutant animals are shown. Filled and hollow arrowheads indicate amphid and phasmid neurons that took up dye, respectively. (B) The GFP-tagged CHE-2 (IFT80) intraflagellar transport protein localizes normally to the transition zones (TZ; arrowhead in each panel) and ciliary axonemes (labeled cilia) in both the head and tail sensory neurons of N2 (wild- type), and mks/mksr single, double and triple mutants, as indicated. All panels are oriented with the head up and tail down. Scale bar: 5 μm. (C) The single, double and triple mks/mksr mutants exhibit normal intraflagellar transport. Kymographs of N2 and mutants (mks-1;mksr-1;mksr-2 triple mutant and mks-1;mksr-1;mksr- 2;bbs-8 quadruple mutant) are shown for the ciliary middle segment (MS) and distal segment (DS) in the amphid (head) cilia using CHE-2::GFP in intraflagellar transport assays. For each strain, fluorescent images of phasmid cilia and the corresponding kymographs (actual kymographs and schematics/traces of particle movement) for the MS and DS are shown; in the first fluorescent image, the transition zones (TZ) as well as MS and DS are labeled. The table shows the measurement of CHE-2::GFP rates (in μm/second) in the MS and DS for the indicated strains. n, number of IFT particles measured. Note that the rates of CHE- 2::GFP movement in the mks/mksr mutants do not deviate statistically from N2; in the bbs-8 background, CHE-2::GFP moves at the fast unitary rate of OSM-3 kinesin in both the MS and DS (Ou et al., 2005). The asterisks in the image indicate CHE-2::GFP accumulations normally observed in the bbs-8 mutant background (Blacque et al., 2004). Scale bar: 5 μm. (D) IMCD3 cells cotransfected with GFP and short-hairpin RNAi constructs for MKSR1/B9D1 and MKSR2/B9D2 each show a significant reduction in the number of cilia, as assessed by staining with an acetylated tubulin antibody, in comparison with those transfected with GFP alone (control). *P<0.05. (E) The mks/mksr single, double and triple mutants show no statistically significant differences compared with wild- type animals with respect to chemotaxis towards a volatile attractant (isoamyl alcohol) or avoidance of a high-osmolarity solution (8 M glycerol). The osm-5 ciliary mutant, defective in both chemotaxis and osmoavoidance, is included as a positive control. The chemotaxis and osmoavoidance indices are calculated as described in the Materials and Methods. *P<0.05. 618 Journal of Cell Science 122 (5)

to display normal IFT; this was confirmed by observing, using time-lapse microscopy, the expected (wild-type) rates of movement of CHE-2::GFP in the middle (~0.7 μm/second) and distal (~1.2 μm/second) segments of the cilia in the single mks/mksr mutant animals (Fig. 4C) (reviewed by Blacque et al., 2008; Inglis et al., 2007; Scholey, 2008). These data are similarly supported using the OSM-3, KAP-1, and XBX-2 markers (supplementary material Fig. S4B). The presence of a common B9 protein domain in the three proteins raises the possibility that their functions might be at least partially redundant, where one or more mks/mksr genes may mask the effect of a disruption in another (mutated) mks/mksr gene. To address this possibility, we generated all possible double mutant combinations, as well as the triple mutant. Similar to our observations for each individual mutant, the double and triple mks/mksr mutant strains had no observable defects in dye-filling (supplementary material Fig. 3A; the representative triple mutant strain is shown in Fig. 4A), or in transition zone positioning, ciliary structure or IFT, as deduced from observing GFP-tagged CHE-2 and XBX-2 protein localization and transport behavior in live animals (Fig. 4B,C; supplementary material Fig. S4). Because of the apparent phenotypic and genotypic overlap between the Bardet-Biedl and Meckel syndromes (Karmous- Benailly et al., 2005; Leitch et al., 2008), we sought to test for a possible genetic interaction between the three mks/mksr genes and a bbs gene. We therefore generated a quadruple mutant (triple mks/mksr mutant in a bbs-8 mutant genetic background) harboring a CHE-2::GFP protein ciliary (IFT) marker and looked for cilia structure and IFT defects. We found that the ciliary structures of the quadruple mutant, based on the CHE-2::GFP marker, were indistinguishable from those of the bbs-8 mutant alone, with nearly full-length cilia and accumulations at the middle or distal segment midpoint and at the tip of the truncated cilia (Fig. 4C) (Blacque et Fig. 5. Genetic interactions between the C. elegans mks-1, mksr-1 and mksr-2 al., 2004). By itself, the bbs-8 mutation has the effect of separating genes revealed by an increased lifespan phenotype. (A) The mean lifespans of the individual mks-1, mksr-1 and mksr-2 mutant strains (as indicated) are Journal of Cell Science the IFT machinery into two independently moving subassemblies indistinguishable from that of the wild-type (N2) strain. (B) All combinations consisting of kinesin-II–IFT subcomplex A and OSM-3– kinesin– of double mutant animals (mks-1;mksr-1, mks-1;mksr-2, and mksr-1;mksr-2) IFT subcomplex B (Ou et al., 2005; Ou et al., 2007). In the mks- exhibit statistically significant increases in lifespan relative to N2 animals 1;mksr-1;mksr-2;bbs-8 quadruple mutant strain, the localization and (indicated by <0.0001*). The lifespan of the triple mutant is not statistically behavior of CHE-2::GFP was indistinguishable from that of the different from that of the N2 strain. (C) mks/mksr double mutant animals have enhanced lifespans comparable with those of nphp-1 and nphp-4 animals bbs-8 mutant, wherein it was transported along the length of the (Winkelbauer et al., 2005) but shorter than that of the long-lived strain with a proximal and (partially truncated) distal segments at the fast unitary defect in the CHE-11 intraflagellar transport protein. All graphs show rate of OSM-3-kinesin (~1.2 μm/second) (Fig. 4C). representative experiments, and the tables present mean lifespans ± s.e. Recently, Jauregui et al. (Jauregui et al., 2008) demonstrated that Experiments were repeated at least twice and the results were found to be abrogation of the transition zone-specific protein NPHP-4 results reproducible. *P<0.0001 compared with the wild type. in relatively subtle yet significant changes to the microtubule architecture of ciliary axonemes. We therefore sought to determine whether similar ultrastructural changes were present in a similar to those observed for the knockdown of the MKS1 gene representative (mks-1;mksr-1) double mutant, which we demonstrate (Dawe et al., 2007). Hence, it appears that in C. elegans, disruption (below) has a lifespan/signaling defect. Consistent with the results of mks/mksr genes is less detrimental to cilium formation until a of our GFP-based analyses of amphid cilia, transmission electron ‘second-hit’ mutation in nphp-4 or nphp-1 occurs, where transition microscopy (TEM) observation of cross-sections through the heads zone/basal body and ciliogenesis defects become apparent (Williams of the mks-1;mksr-1 mutants revealed no significant differences in et al., 2008). the axonemal structures of amphid cilia compared with wild-type Altogether, our data indicate that in contrast to the mammalian worms (supplementary material Fig. S5). Specifically, the double B9-domain-containing proteins, the C. elegans counterparts are mutant exhibited well-formed transition zones, intact doublet unlikely to be directly implicated in the positioning of transition microtubules in the middle segments, and intact singlet microtubules zones/basal bodies, or the biogenesis of sensory cilia, or have an in the distal segments. essential role in intraflagellar transport. This raises the possibility By contrast, we observed that disruption of the mammalian that the C. elegans MKS-1, MKSR-1 and MKSR-2 proteins MKSR1/B9D1 and MKSR2/B9D2 genes by RNAi in IMCD3 cells participate in other aspect(s) of basal body and/or cilia function that caused ciliogenesis defects, namely a smaller proportion of ciliated relate not to the building of, but rather, the sensory/signaling roles cells compared with the control cells (Fig. 4D). These results are of the ciliary organelle. Meckel syndrome 1 and related proteins 619

The C. elegans mks/mksr genes control lifespan via the insulin signaling pathway To investigate the possibility that the C. elegans MKS-1, MKSR- 1 and MKSR-2 proteins have roles in the sensory functions of cilia, we first tested the three single mks/mksr mutant strains for defects in their ability to sense and move towards a volatile attractant (isoamyl alcohol) using an established chemotaxis assay. Although bbs and IFT mutant animals show clear sensory phenotypes in these assays (Blacque et al., 2004; Inglis et al., 2007; Perkins et al., 1986), no significant differences were observed between wild-type (N2) animals and the single mks/mksr gene mutants (Fig. 4E, light gray bars). Similarly, we assayed for the ability of the mks/mksr mutant animals to recognize and avoid a solution of high osmolarity (8 M glycerol). In this assay, where bbs and IFT gene mutants show osmoavoidance defects (Inglis et al., 2007; Perkins et al., 1986) (our unpublished data), the mks/mksr mutant strains exhibited wild- type osmoavoidance (Fig. 4E, dark gray bars). Given the possibility of functional overlap between the mks/mksr genes, we also tested combinations of the double and triple mks/mksr mutant animals; no defects in chemotaxis or osmoavoidance were observed (Fig. 4E). Likewise, another assay that can reveal cilia function defects in the nematode – Nile Red staining to determine lipid content (Li et al., 2008; Mak et al., 2006) – revealed no differences between control animals and the mks-1, mksr-2 and mks-1;mksr-2 mutants (supplementary material Fig. S3B). These data are reminiscent of other C. elegans transition zone/basal body proteins whose deletion does not overtly or severely affect cilium formation or IFT, but might instead still be required for specific sensory and/or signaling capacities of cilia. Two examples are the homologues of the nephronophthisis- associated NPHP-1 and NPHP-4 proteins. Their disruption causes Fig. 6. MKS-1, MKSR-1 and MKSR-2 proteins appear to function upstream subtle ciliary structure anomalies, as well as male mating defects of DAF-2 and DAF-16 in the insulin–IGF-I signaling pathway to regulate and an increased lifespan phenotype, the latter two often being lifespan. (A) The lifespan of mksr-1;mksr-2;daf-2 triple mutant animals is the associated with cilia dysfunction (Jauregui et al., 2005; Winkelbauer same as that of the long-lived daf-2 mutants, supporting the notion that B9- domain-containing proteins function upstream of the DAF-2 insulin–IGF-I Journal of Cell Science et al., 2005; Wolf et al., 2005). Similarly, disruption of IFTA-2, a signaling pathway. *P<0.0001 compared with daf-2. (B) The lifespan of all protein associated with the IFT machinery, does not appear to result mks/mksr double mutants is shortened when introduced into a daf-16 mutant in cilia structure or IFT defects but causes an increased lifespan background, suggesting that the MKS-1, MKSR-1 and MKSR-2 proteins phenotype (Schafer et al., 2006). function upstream of the DAF-16 FOXO transcription factor in the We therefore tested all single and double mks/mksr mutants, as insulin–IGF-I signaling pathway. *P<0.0001 compared with daf-16. (C) The well as the triple mutant, in ageing assays. None of the single or mks/mksr double mutants have increased levels of DAF-16::GFP in the nucleus, consistent with their increased lifespan compared with wild-type (N2) triple mutants display alterations to lifespan compared with wild- animals and their corresponding single mutants (mks-1 and mksr-1). The osm-5 type animals (Fig. 5A,B). By contrast, all three double mutant ciliary mutant positive control is impaired in DAF-2 signaling and has an combinations (mks-1;mksr-1, mks-1;mksr-2 and mksr-1;mksr-2) increased level of nuclear DAF-16::GFP. Animals were categorized as having displayed statistically significant increases in lifespan when mainly cytoplasmic DAF-16::GFP (white bars), intermediate localization between the and nucleus (light gray bars), or mainly nuclear compared with the single or triple mutants, or wild-type animals localization (dark gray bars); examples of these three localization patterns are (Fig. 5B). Interestingly, the lifespan increases seen in the mks/mksr shown on the right. *P<0.05 compared with N2. double mutants were comparable to those of the ifta-2 (data not shown) (see Schafer et al., 2006) and nphp-1 or nphp-4 (Fig. 5C) (see also Winkelbauer et al., 2005) ciliary mutants, but were not as pronounced as those exhibited by some IFT gene mutants, such that regulates longevity in C. elegans (Kenyon et al., 1993; Kimura as che-11 (Fig. 5C) (see also Apfeld and Kenyon, 1999). Thus, et al., 1997). Lifespan assays demonstrate that the mksr-1;mksr- our genetic and functional analyses of the mks/mksr genes suggest 2;daf-2 triple mutant exhibits the same longevity as that of the that, similarly to the nephrocystin proteins NPHP-1 and NPHP- extremely long-lived daf-2 mutant (Fig. 6A), implying that the B9 4, which localize to the transition zones at the base of cilia, the proteins function upstream of DAF-2 in the insulin-signaling MKS-1, MKSR-1 and MKSR-2 proteins perform function(s) pathway. To confirm this result, we tested the epistatic relationships relevant to longevity control, suggesting a role in cilia-associated between all three mks/mksr double mutants and daf-16(mu86), the signaling. gene encoding the FOXO transcription factor required for To support this hypothesis, we next tested for an epistatic downstream DAF-2–insulin–IGF-I-receptor-mediated signaling interaction between a representative double mutant (mksr-1;mksr- (Ogg et al., 1997). All mks/mksr double gene mutants, when 2) and daf-2(e1370). The daf-2 gene encodes the lone receptor combined by genetic crossing with the daf-16 mutation, displayed responsible for the well-established insulin–IGF-I signaling pathway lifespans that were shorter than the mks/mksr double mutants and 620 Journal of Cell Science 122 (5)

were indistinguishable from that of the short-lived daf-16 mutant IFT and BBS proteins altogether. These observations suggest that (Fig. 6B). These data support the notion that the B9 proteins function at least in some organisms, the MKS-1, MKSR-1 and MKSR-2 upstream of DAF-16 in the insulin–IGF-I signaling pathway. proteins, similarly to the BBS proteins, are not absolutely required Consistent with the above observations, we observed a direct to build motile or non-motile cilia. In addition, MKS-1, MKSR-1 effect of disrupting the B9 proteins and the activity of the DAF-16 and MKSR-2 proteins might have more specific roles relating to protein. Within a population of worms at 20°C, DAF-16 is usually the sensory and/or signaling functions of cilia. phosphorylated in response to DAF-2 signaling, which leads to its nearly complete exclusion from the nucleus; however, in long-lived MKS1, MKSR1 and MKSR2 are associated with basal bodies mutants such as daf-2(e1370) or cilia mutants such as osm-5, the and cilia proportion of animals showing nuclear localization increases Human MKS1 was previously found to localize to basal bodies significantly as a result of inhibited daf-2 signaling (reviewed by (Dawe et al., 2007), as with the Chlamydomonas orthologue POC12 Mukhopadhyay et al., 2006). We observed that GFP-tagged DAF- (Proteome of Centriole protein 12) (Keller et al., 2005). We have 16 in control animals normally shows ~0.8% localization to the confirmed the basal body or centrosomal localization of a transiently nucleus, which is similar to that of the mks-1 and mksr-1 single expressed, V5-epitope-tagged version of MKS1 (Fig. 2A; mutant animals, which have a normal lifespan (Fig. 6C). By contrast, supplementary material Fig. S1), and further showed the same the mks-1;mksr-1, mks-1;mksr-2 and mksr-1;mksr-2 double mutants subcellular localization for the other two human B9 domain- all displayed a statistically significant increase in GFP-tagged DAF- containing proteins (Fig. 2A; supplementary material Fig. S1). In 16 nuclear localization (4-4.8%), and a corresponding decrease in addition, we found that GFP-tagged variants of all B9-domain- cytosolic localization, as expected from their increased lifespan containing proteins localized, in stably transfected cells, to the ciliary phenotypes (Fig. 6C). In conclusion, our findings strongly support axoneme (Fig. 2B). This dual-localization pattern (basal body and the notion that the C. elegans MKS-1, MKSR-1 and MKSR-2 cilia) is comparable to that of the mouse protein stumpy (MKSR- proteins functionally interact at the base of cilia to support a process 2) (Town et al., 2008; Breunig et al., 2008). Importantly, we that is required upstream of the DAF-2/DAF-16 insulin–IGF-I confirmed that the localization of the MKS-1, MKSR-1 and MKSR- signaling pathway to specify longevity. 2 proteins to basal bodies is evolutionarily conserved by observing – as recently reported by Williams et al. (Williams et al., 2008) – Discussion that the C. elegans MKS-1, MKSR-1 and MKSR-2 proteins reside A wealth of genomic, transcriptomic and proteomic data have at ciliary transition zones (Fig. 2C), which are akin to basal bodies identified numerous basal body and ciliary proteins, several of which (Perkins et al., 1986). The MKS-1, MKSR-1 and MKSR-2 protein are implicated in ciliopathies (Gherman et al., 2006; Inglis et al., family therefore joins an increasing number of ciliopathy-associated 2006; Keller et al., 2005). In this study, we report that B9-domain- proteins that concentrate at the base of cilia or to the ciliary axoneme, containing proteins uncovered in some of these studies belong to including among others the Meckel syndrome, Joubert syndrome three separate phylogenetic clades – MKS-1, MKSR-1 and MKSR- or nephrocystin proteins, RPGRIP1L, CEP290/NPHP6, NPHP1 and 2 – that are found exclusively in ciliated species. The three human NPHP4. proteins and C. elegans orthologues localize to basal bodies/

Journal of Cell Science transition zones, and, in the case of the mammalian proteins, to the Function of the MKS-1, MKSR-1 and MKSR-2 proteins in ciliary axoneme as well. We further demonstrate that the C. elegans ciliogenesis and cilium-associated signaling proteins function cooperatively at the base of cilia to support the The notion that MKS1 is implicated in cilia function received strong proper function of the insulin–IGF-I signal transduction pathway support from a recent study by Dawe et al. (Dawe et al., 2007), required for the specification of lifespan. which demonstrated that knockdown of mammalian MKS1, similarly to the disruption of MKS3, is associated with basal body Evolutionary conservation of B9-domain-containing proteins in positioning and thus ciliogenesis defects. The reason for the basal ciliated organisms body position phenotype remains unclear, but the downstream The potential significance of the B9 protein domain was highlighted effects on renal tubule formation were pronounced. Similarly, very recently when one of the first two genes linked to Meckel syndrome little is known about the functions of the other two B9-domain- MKS1 was identified by Kyttälä et al. (Kyttälä et al., 2006). The containing proteins, MKSR1 and MKSR2. Ponsard et al. (Ponsard 559-residue human MKS1 protein lacks known protein motif(s) or et al., 2007) showed that knockdown of ICIS-1 (orthologue of C. other recognizable features, but harbors an approximately 115- elegans mksr-2) by RNAi in Paramecium tetraurelia results in residue B9 protein domain of unknown function (Fig. 1B). We defects in cilia stability or formation. Another recent study identified in nearly all ciliated organisms, two more members of demonstrated that stumpy (MKSR-2) is required for the proper the MKS1/B9 protein family, which we name MKS1-related biogenesis or morphology of cilia in a mouse knockout model (Town proteins 1 and 2 (MKSR-1 and MKSR-2). The mutual presence of et al., 2008), and is essential for a Shh-based signaling pathway in three B9-domain-containing proteins in ciliated organisms, and neural stem cells (Breunig et al., 2008). Finally, the Yoder laboratory complete absence from organisms devoid of cilia (Fig. 1A; (Williams et al., 2008) reported genetic interactions between the supplementary material Table S1), suggests that these proteins B9-domain genes and the nphp-4 gene (discussed further below). perform common cilia-associated function(s). It is notable that the Using C. elegans to dissect the relationship between, and MKS-1, MKSR-1 and MKSR-2 proteins are absent from at least functions of, the MKS-1, MKSR-1 and MKSR-2 proteins, we three groups of organisms that possess cilia (the moss discovered that the proper localization of the proteins was largely Physcomitrella patens, the Diplomonad Giardia lamblia and the co-dependent. Specifically, in mksr-1 mutant animals, the MKS-1 Apicomplexan Plasmodium falciparum). Interestingly, G. lamblia and MKSR-2 proteins did not reproducibly show clear, wild-type has a much reduced complement of Bardet-Biedl syndrome proteins localization to the transition zones, and displayed accumulations in compared with most ciliated organisms, and P. falciparum lacks the dendrites (Fig. 3A,F). Similarly, in the mksr-2 mutant strain, Meckel syndrome 1 and related proteins 621

the MKS-1 and MKSR-1 proteins were also not tightly associated It is intriguing that lifespan phenotypes are observed in double with the transition zones (Fig. 3B,D). Nevertheless, some partial but not single mks/mksr mutants given that the abrogation of either localization to, or near, transition zones was often observed, MKSR-1 or MKSR-2 causes the improper localization of the others suggesting either that the disrupted genes encode aberrant truncated (Fig. 3). However, our data indicate that in most cases, the co- proteins that retain some function or that the proteins can localize dependency of localization reflects an effect on the efficiency of independently, but with lower efficiency. This might be the case localization rather than an essential aspect of correct trafficking (Fig. for the mks-1 mutant, where we observe essentially wild-type 3); thus, at least partial function might be retained in the incorrectly localization of MKSR-1 and MKSR-2 proteins (Fig. 3C,E). localized proteins. Nevertheless, it remains perplexing that the mks- Altogether, these data, which are comparable to those recently 1;mksr-1;mksr-2 triple mutant does not exhibit a longevity reported (Williams et al., 2008), suggest that the three B9-domain- phenotype. Although it is unclear why abrogation of the third containing proteins either interact directly, such that the disruption mks/mksr gene restores normal lifespan, it provides further evidence of one can lead to the mislocalization of the other, or that they are of a functional (genetic) interaction between all three mks/mksr functionally associated via other transition-zone-localized proteins. genes, since the triple mutant phenotype is reproducibly distinct We favour the first possibility, given that a genome-wide C. elegans from that of the three double mutant phenotypes. It might also study uncovered a yeast two-hybrid interaction between K03E6.4 indicate a complex regulation – which probably includes NPHP-1 (MKSR-1) and Y38F2AL.2 (MKSR-2) (Li et al., 2004b). The and NPHP-4 proteins (Williams et al., 2008) – of the various ciliary interdependent localization of the C. elegans MKS-1, MKSR-1 and signals modulating lifespan at the transition zone. MKSR-2 proteins provides evidence of a functional relationship Longevity in C. elegans is specifically controlled by cilia- between this family of proteins. dependent sensory inputs and the insulin–IGF-I signaling pathway Interestingly, we found that the mks-1, mksr-1 and mksr-2 mutant (Apfeld and Kenyon, 1999). We show by epistasis analyses, that strains have no obvious defects in transition zone positioning, ciliary this is also the case for the MKS-1, MKSR-1 and MKSR-2 structures, intraflagellar transport, chemo- and osmo-sensation or proteins, which appear to function upstream of both the lipid accumulation (Fig. 4A-C,E; supplementary material Figs S3- insulin–IGF-I receptor DAF-2 and the FOXO transcription factor S5). As the mks/mksr gene functions could be partly redundant, we (DAF-16), which transduces insulin-like signals to regulate generated the three double mutant combinations as well as a triple lifespan (Fig. 6). But what are the function(s) of the MKS-1, mutant, and tested them for the same ciliary phenotypes; we could MKSR-1 and MKSR-2 proteins in this pathway? Given our results, not, however, detect any difference from wild-type animals in these it is possible that components of the insulin–IGF-I signaling assays (Fig. 4A-C,E; supplementary material Figs S3-S5). It is cascade might be improperly trafficked in the mks/mksr double unclear whether the gene deletion mutants available for these studies mutants; given that the MKS-1, MKSR-1 and MKSR-2 proteins represent hypomorphs instead of null mutants, although the localize at the base of cilia in C. elegans, they might cooperate conserved B9 domains of the mksr-1(tm3083) and mksr-2(tm2452) with other basal body proteins (such as NPHP-1, NPHP-4 or gene mutants are disrupted (supplementary material Fig. S2) and Meckel syndrome-associated RPGRIP1L, for which there is a C. probably disrupt the function of the proteins. Nevertheless, all elegans orthologue, C09G5.8) in vesicle or protein trafficking (or mutant alleles give an observable longevity phenotype when docking at the base of cilia) prior to incorporation into cilia (e.g.

Journal of Cell Science combined (see below), and we propose that the simultaneous by intraflagellar transport). disruption of the three mks/mksr genes is highly likely to impair Although the functions of the MKS-1, MKSR-1 and MKSR-2 their collective function. In light of our data, it seems possible or proteins appear to not be crucial for ciliogenesis in C. elegans even likely that unlike in mammalian cells (Dawe et al., 2007; Town (and perhaps other organisms), they are essential for establishing et al., 2008) (Fig. 4D), in C. elegans, the MKS-1, MKSR-1 and important signaling cascade(s). Such a ‘non-essential’ function MKSR-2 proteins do not perform an essential ciliogenic role (see might explain why some ciliated organisms, such as Plasmodium also Williams et al., 2008). and Giardia, lack the MKS-1, MKSR-1 and MKSR-2 proteins However, the possibility remained that the C. elegans mks/mksr altogether. In other systems, for example mammalian cells, the genes are specifically implicated in one or more ciliary signaling MKS1, MKSR1 and MKSR2 proteins might carry out similar pathways; we were able to test for this possibility in the absence trafficking or docking roles at basal bodies, but perhaps functional of potentially confounding major cilia structure defects by interaction(s) with their cargo or binding partners are absolutely analysing the strains for an increased lifespan phenotype that is necessary for establishing the positioning of the basal body at the typical of ciliary gene mutants, including nphp-1, nphp-4, ifta- membrane (Dawe et al., 2007), and thus, ciliogenesis. Once cilia 2, and most core IFT-associated mutants (Apfeld and Kenyon, formation has occurred in normal (wild-type) situations, the 1999; Schafer et al., 2006; Winkelbauer et al., 2005). Although mammalian MKS1, MKSR1 and MKSR2 proteins might then also none of the single mks/mksr mutants differed from wild-type be necessary for the targeting or trafficking of ciliary cargo animals (Fig. 5A), all double mutant combinations (mks-1;mksr- required for the sensory/signaling functions of the primary cilia 1, mks-1;mksr-2, and mksr-1;mksr-2) displayed statistically (e.g. Shh and perhaps other signaling pathways) (Breunig et al., significant expansions in lifespan (Fig. 5B). This is reminiscent 2008). In C. elegans, disruption of an MKS-1, MKSR-1 and of the single nphp-1 or nphp-4 mutants, which show no overt MKSR-2 protein together with the NPHP-1 or NPHP-4 protein cilia-dependent male mating defects, whereas the nphp-1;nphp- causes essentially the same defects in basal body positioning and 4 double mutant animals possess pronounced mating defects ciliogenesis (Williams et al., 2008), potentially suggesting greater (Jauregui and Barr, 2005). Also of note, the enhanced lifespan functional redundancy in the nematode ciliary system. This is of mks/mksr double mutants was comparable to that observed particularly insightful, given that the NPHP-1 and NPHP-4 when the nphp-1 and nphp-4 genes were individually disrupted, proteins appear to be important regulators for the proper but less than when ciliary structures were severely abrogated (e.g. localization or function of the IFT/BBS ciliary proteins (Jauregui as in a che-11 mutant) (Fig. 5C). et al., 2008). 622 Journal of Cell Science 122 (5)

MKSR1 and MKSR2 as potential Meckel syndrome gene (10 minutes), washed, and blocked with 5.5% FBS (Gemini) for 1 hour. Cells were candidates stained with primary at 4°C overnight (rabbit anti-GFP, 1:1000, Invitrogen A11122; mouse anti-γ-tubulin, 1:1000, Sigma T6557; goat anti-acetylated-tubulin, Our functional data strongly suggest that, in addition to the six 1:1000, Sigma), washed in PBS, then incubated with secondary antibodies [goat anti- presently known human Meckel syndrome-associated genes, namely mouse (A21206) and donkey anti-rabbit IgG conjugated to Alexa Fluor 488 (A11001) MKS1, MKS3, CEP290/NPHP6/MKS4, RPGRIP1L/MKS5, NPHP3 or Alexa Fluor 594 (A11005), 1:1000, Invitrogen]. Cells where then incubated with DAPI (1:5000, 500 mg/ml stock) at 25°C for 10 minutes, washed, and mounted with and CC2D2A, the two MKS1-related genes MKSR1 and MKSR2 Vectashield. Images were recorded using an epifluorescence microscope at ϫ64 are excellent Meckel syndrome, Joubert syndrome and Leber magnification. congenital amaurosis gene candidates. The genetic locations of For the C. elegans MKS-1, MKSR-1 and MKSR-2 proteins, we generated MKSR1 (B9D1; 17p11.2) and MKSR2 (B9D2; 19q13.2d) are clearly translational constructs containing the natural promoter of each gene and the entire coding region fused in-frame to EGFP, and generated transgenic lines harboring these outside the uncloned MKS2 , which has been mapped to constructs as reported previously (Blacque et al., 2004). The subcellular localization 11q13 by Roume et al. (Roume et al., 1998); thus, of the GFP-tagged proteins was assessed by fluorescence microscopy in either wild- MKSR1 and MKSR2 might represent additional disease loci. type (N2) animals or in the indicated mks/mksr mutant backgrounds. Mislocalization Understanding the roles of basal bodies and ciliary axonemes in phenotypes were assayed blind to the genotype and expressed GFP-tagged MKS-1, MKSR-1 and MKSR-2 protein, on at least 50 different animals for each strain. various sensory and signaling processes, and their implications in various ciliopathies, will require the identification and analysis of Mammalian RNAi key, conserved components of the basal body-ciliary organelle. Here, Mouse IMCD3 cells were grown in a 10 cm dish and co-transfected with short-hairpin we have uncovered and characterized three B9-domain-containing constructs for either murine MKSR1/B9D1 or MKSR2/B9D2, and GFP as a transfection control using Fugene6 (Invitrogen) as directed. After 48-72 hours, the cells were proteins, MKS-1, MKSR-1 and MKSR-2, as a family of basal fixed in paraformaldehyde and co-stained with anti-acetylated tubulin and anti-GFP. body/ciliary components whose functional interactions are required The cells expressing GFP, which coexpressed the short-hairpins, were then scored for proper ciliary function/signaling in C. elegans, and are needed for the percentage of cilia in comparison with cells expressing GFP alone. The MKSR1/B9D1 short-hairpin construct was purchased from Open BioSystems. The for ciliogenesis in mammalian cells. Further analyses of these target sequence for the RNAi Consortium (TRC), TRC-Mm1.0 (Mouse) is not proteins in C. elegans and other model organisms, as well as published. The B9D1 short-hairpin construct is expressed from the p.KLO.1 vector exploration of their possible involvement in cilia-associated and has the following TRC ID: TRCN0000198329. The target sequence for diseases, will provide important insights into the physiological MKRSR2/B9D2 used in the pSuper-Basic vector (Oligoengine Cat. No. VEC-PBS- 0001/0002) is: GAACAGTTGGCACGGGCTT. The primers designed for cloning functions of cilia and the molecular etiologies of ciliopathies such of the short-hairpin into pSuper.basic are: 5Ј-GATCCCCGAACAGTTGG - as Meckel syndrome. CACGGGCTTTTCAAGAGAAAGCCCGTGCCAACTGTTCTTTTTA-3Ј and 3Ј- GGGCTTGTCAACCGTGCCCGAAAAGTTCTCTTTCGGGCACGGTTGACAA- Ј Materials and Methods GAAAAATTCGA-5 . C. elegans strains and genetic crosses Phenotypic assays for ciliary structure, chemosensation and lipid All strains were maintained and cultured at 20°C. Strains carrying deletions in the C. elegans mks-1/xbx-7, mksr-1/tza-2, mksr-2/tza-1 genes, R148.1(tm2705), content K03E6.4(tm3083) and Y38F2AL.2(tm2452) respectively, were obtained from the Chemotaxis assays using isoamyl alcohol as an attractant were performed as National Bioresource Project (http://shigen.lab.nig.ac.jp/c.elegans/index.jsp) and described (Blacque et al., 2006). Osmoavoidance assays were performed essentially outcrossed to wild-type (N2) at least five times. Standard mating procedures were as described (Culotti and Russell, 1978) using a ring of 8 M glycerol as the source used to introduce GFP-tagged protein constructs into different genetic backgrounds of high osmolarity. The filling of environmentally exposed ciliated sensory neurons and to make the different combinations of double mutants or the triple mutant. Single- with DiI was assessed as described in Blacque et al. (Blacque et al., 2004). Lipid

Journal of Cell Science worm PCR reactions were used to genotype the three mks/mksr mutants. The other content measurements were performed essentially as described using Nile Red (Mak strains used were bbs-8(nx77), nphp-1(ok500), nphp-4(tm925), che-11(e1810), osm- et al., 2006). Experiments were done blind to the genotype and repeated at least three 5(p813), daf-2(e1370), and daf-16(mu86). times.

Characterization of the C. elegans mks-1, mksr-1 and mksr-2 Lifespan assays alleles Lifespan assays were similar to that described (Apfeld and Kenyon, 1999). Animals N2 and mks/mksr cDNAs were initially isolated by RT-PCR. Briefly, following were grown for one generation at 20°C before eggs were collected by treatment with suspension of worms in Trizol reagent (Invitrogen) and purification with RNeasy sodium hypochlorite. At the L4 molt, worms were transferred to NGM plates μ (Qiagen), first-strand cDNAs were generated using the Superscript First-Strand containing 16 m FUDR to prevent progeny growth and kept at 20°C for the duration Synthesis System for RT-PCR (Invitrogen). PCR amplifications specific to each of the assay. 100 worms were picked for each of the indicated strains, with 10 worms mks/mksr transcript were then performed to isolate appropriate double-stranded cDNA on each plate. Plates were scored every 1-2 days for live or dead worms. Individual sequences. PCR products were then incorporated into the pGEM-T Easy Vector animals were considered dead when they no longer responded to harsh touch (prodding (Promega) and sequenced. with platinum wire). Worms that exploded or crawled off the plate were censored. All assays were performed at least twice with consistent results. Subcellular localization of the human and C. elegans MKS-1, MKSR-1 and MKSR-2 proteins DAF-16 nuclear localization analyses To assess the localization of the three human B9 proteins using transient expression DAF-16 localization assays were performed essentially as described (Schafer et al., of V5-tagged proteins, the respective cDNAs (MKS1/BC010061.2, EPPB9/ 2006). Briefly, the various mks/mksr single or double mutant strains were crossed BC002944.2/MKSR1 and LOC80776/NM_030578.2/MKSR2 (Invitrogen Ultimate into the integrated DAF-16::GFP strain {TJ356; N2(zIs356)[DAF-16::GFP+pRF4(rol- ORF clones IOH12254, IOH5726, and IOH4997, respectively) were cloned into the 6)]}. To observe the localization, healthy, well-fed worms grown at 20°C were mammalian Gateway pcDNA6.2/c-Lumio vector (Invitrogen), which contains a C- mounted on an agar pad containing 20 mM sodium azide and quantified immediately. terminal V5 epitope tag. Murine IMCD3 cells were plated on glass coverslips and To quantify localization, DAF-16::GFP was determined to be either nuclear, transfected with both the expression vector and a pUC12-TAG tRNA suppressor intermediate or cytosolic as reported (Oh et al., 2005). Control animals (TJ356 strain) (Invitrogen) at 70% confluency using FuGENE6 (Roche). 48 hours after transfection, were assayed in parallel. For each strain analyzed, at least 200 worms were assayed cells were subjected to immunofluorescence analysis (below). To generate stable in triplicate. IMCD3 cell lines harboring GFP-tagged MKS1, MKSR1/B9D1 and MKSR2/B9D2, cells were transfected with each GFP-fusion construct, split 1:5 after 24 hours and Intraflagellar transport assays grown 24 hours later in the presence of geneticin (750 μg/ml); cells were kept under Transgenic animals harboring GFP-tagged proteins were mounted on 1% agarose selection for 7 days, after which they were split 1:10 and several colonies were picked pads and immobilized with 100 mM levamisole. Amphid and phasmid cilia were to produce independent lines. Cells were grown to confluency and maintained for at examined with a 100ϫ 1.35 NA objective and an ORCA AG CCD camera mounted least 48 hours before immunofluorescence analysis. on an Zeiss Axioskop 2 mot plus microscope, with time-lapse images being acquired For immunofluorescence analysis, cells were fixed in PFA at 4°C for 1 hour, and at 300-500 mseconds/frame, depending on the specific marker used. Images and subsequently fixed in ice-cold methanol at –20°C for 10 minutes. After rinsing with movies were obtained in Openlab version 5.02 (Improvision). Kymographs were PBS, cells were permeabilized using 0.1% Triton-X (American Bioanalytical) in PBS generated using the MultipleKymograph ImageJ plug-in (http://www.embl- Meckel syndrome 1 and related proteins 623

heidelberg.de/eamnet/html/body_kymograph.html). Rates from middle and distal elegans BBS-7 and BBS-8 protein function results in cilia defects and compromised segments were obtained essentially as described (Ou et al., 2007). intraflagellar transport. Genes Dev. 18, 1630-1642. Blacque, O. E., Perens, E. A., Boroevich, K. A., Inglis, P. N., Li, C., Warner, A., Khattra, Bioinformatic and phylogenetic analyses J., Holt, R. A., Ou, G., Mah, A. K. et al. (2005). Functional genomics of the cilium, B9-domain-containing proteins were identified using the NCBI Conserved Domain a sensory organelle. Curr. Biol. 15, 935-941. Database (Marchler-Bauer et al., 2005) with human MKS1 as query. This dataset Blacque, O. E., Li, C., Inglis, P. N., Esmail, M. A., Ou, G., Mah, A. K., Baillie, D. L., Scholey, J. M. and Leroux, M. R. (2006). The WD repeat-containing protein IFTA-1 was simplified by removal of duplicate sequences from species not of interest. is required for retrograde intraflagellar transport. Mol. Biol. Cell 17, 5053-5062. Sequences from species not shown here (but retrieved by the CDD) were manually Blacque, O. E., Cevik, S. and Kaplan, O. I. (2008). Intraflagellar transport: from molecular removed. Sequences from additional species were identified and/or confirmed using characterisation to mechanism. Front. Biosci. 13, 2633-2652. BLAST (Altschul et al., 1997) with sequences from species closely related on the Breunig, J. J., Sarkisian, M. R., Arellano, J. I., Morozov, Y. M., Ayarb, A. E., Sojitra, eukaryotic tree [as defined by Keeling et al. (Keeling et al., 2005)] as queries at the S., Wang, B., Flavell, R. A., Rakic, P. and Town, T. (2008). Primary cilia regulate individual genome sites of each species. Sequences were retained only if reciprocal hippocampal neurogenesis by mediating sonic hedgehog signaling. Proc. Natl. Acad. BLAST identified B9-domain-containing proteins as the top hits. Bayesian analysis Sci. USA 105, 13127-13132. of phylogenies was carried out using MrBayes 3.1.2 (Huelsenbeck and Ronquist, Christensen, S. T., Pedersen, L. B., Schneider, L. and Satir, P. (2007). Sensory cilia and 2001) as previously described (Parker et al., 2007), except that whole protein sequences integration of signal transduction in human health and disease. Traffic 8, 97-109. were aligned using the Muscle algorithm (Edgar, 2004). Trees were visualized with Culotti, J. G. and Russell, R. L. (1978). Osmotic avoidance defective mutants of the TreeView (Page, 1996) or Phylodrendrum (http://iubio.bio.indiana.edu/treeapp/ nematode Caenorhabditis elegans. Genetics 90, 243-256. treeprint-form.html). An additional analysis (not shown) was carried out on a slightly Davenport, J. R. and Yoder, B. K. (2005). An incredible decade for the primary cilium: different protein dataset using the neighbor-joining algorithm of ClustalW (Higgins a look at a once-forgotten organelle. Am. J. Physiol. Renal Physiol. 289, F1159-F1169. et al., 1994). Davis, E. E., Brueckner, M. and Katsanis, N. (2006). The emerging complexity of the vertebrate cilium: new functional roles for an ancient organelle. Dev. Cell 11, 9-19. Dawe, H. R., Smith, U. M., Cullinane, A. R., Gerrelli, D., Cox, P., Badano, J. L., Blair- We would like to thank the C. elegans Genetics Center (CGC) and Reid, S., Sriram, N., Katsanis, N., Attie-Bitach, T. et al. (2007). The Meckel-Gruber Shohei Mitani (National BioResource Project, University of Tokyo, Syndrome proteins MKS1 and meckelin interact and are required for primary cilium Japan) for providing C. elegans strains used in this study, and the formation. Hum. Mol. Genet. 16, 173-186. Delous, M., Baala, L., Salomon, R., Laclef, C., Vierkotten, J., Tory, K., Golzio, C., WestGrid computer cluster for phylogenetic analyses. This research is Lacoste, T., Besse, L., Ozilou, C. et al. (2007). The ciliary gene RPGRIP1L is mutated funded by grants from the March of Dimes (M.R.L.), NSERC (L.M.Q.), in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome. grant R01HD04260 from the National Institute of Child Health and Nat. Genet. 39, 875-881. den Hollander, A. I., Koenekoop, R. K., Yzer, S., Lopez, I., Arends, M. L., Voesenek, Development, R01DK072301 and R01DK075972 from the National K. E., Zonneveld, M. N., Strom, T. M., Meitinger, T., Brunner, H. G. et al. (2006). Institute of Diabetes, and Digestive and Kidney Disorders (N.K), and Mutations in the CEP290 (NPHP6) gene are a frequent cause of Leber congenital the Science Foundation Ireland PIYRA (O.E.B.). M.R.L. holds scholar amaurosis. Am. J. Hum. Genet. 79, 556-561. awards from Canadian Institutes of Health Research and Michael Smith Edgar, R. C. (2004). MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113. Foundation for Health Research (MSFHR). E.E.D. acknowledges an Efimenko, E., Bubb, K., Mak, H. Y., Holzman, T., Leroux, M. R., Ruvkun, G., Thomas, NRSA fellowship (F32 DK079541-01) and a doctoral fellowship from J. H. and Swoboda, P. (2005). Analysis of xbx genes in C. elegans. Development. 132, the Visual Neuroscience Training Program (National Eye Institute). 1923-1934. P.N.I. and M.P.H. acknowledge MSFHR scholarships; P.N.I. also holds Eggenschwiler, J. T. and Anderson, K. V. (2007). Cilia and developmental signaling. Annu. Rev. Cell Dev. Biol. 23, 345-373. an NSERC doctoral research award. Deposited in PMC for release after Gerdes, J. M., Liu, Y., Zaghloul, N. A., Leitch, C. C., Lawson, S. S., Kato, M., Beachy, 12 months. P. A., Beales, P. L., DeMartino, G. N., Fisher, S. et al. (2007). Disruption of the basal body compromises proteasomal function and perturbs intracellular Wnt response. Nat. Genet. 39, 1350-1360. References Gherman, A., Davis, E. E. and Katsanis, N. (2006). The ciliary proteome database: an Alexiev, B. A., Lin, X., Sun, C. C. and Brenner, D. S. (2006). Meckel-Gruber syndrome: integrated community resource for the genetic and functional dissection of cilia. Nat. Journal of Cell Science pathologic manifestations, minimal diagnostic criteria, and differential diagnosis. Arch. Genet. 38, 961-962. Pathol. Lab. Med. 130, 1236-1238. Higgins, D., Thompson, J., Gibson, T., Thompson, J. D., Higgins, D. G. and Gibson, Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein alignment through sequence weighting,position-specific gap penalties and weight matrix database search programs. Nucleic Acids Res. 25, 3389-3402. choice. Nucl. Acids Res. 22, 4673-4680. Ansley, S. J., Badano, J. L., Blacque, O. E., Hill, J., Hoskins, B. E., Leitch, C. C., Kim, Hildebrandt, F. and Otto, E. (2005). Cilia and centrosomes: a unifying pathogenic concept J. C., Ross, A. J., Eichers, E. R., Teslovich, T. M. et al. (2003). Basal body dysfunction for cystic kidney disease? Nat. Rev. Genet. 6, 928-940. is a likely cause of pleiotropic Bardet-Biedl syndrome. Nature 425, 628-633. Huelsenbeck, J. P. and Ronquist, F. (2001). MRBAYES: Bayesian inference of phylogeny. Apfeld, J. and Kenyon, C. (1999). Regulation of lifespan by sensory perception in Bioinformatics 17, 754-755. Caenorhabditis elegans. Nature 402, 804-809. Inglis, P. N., Boroevich, K. A. and Leroux, M. R. (2006). Piecing together a ciliome. Arts, H. H., Doherty, D., van Beersum, S. E., Parisi, M. A., Letteboer, S. J., Gorden, Trends Genet. 22, 491-500. N. T., Peters, T. A., Marker, T., Voesenek, K., Kartono, A. et al. (2007). Mutations Inglis, P. N., Ou, G., Leroux, M. R. and Scholey, J. M. (2007). The sensory cilia of in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, Caenorhabditis elegans. WormBook 8, 1-22. cause Joubert syndrome. Nat. Genet. 39, 882-888. Jauregui, A. R. and Barr, M. M. (2005). Functional characterization of the C. elegans Avidor-Reiss, T., Maer, A. M., Koundakjian, E., Polyanovsky, A., Keil, T., nephrocystins NPHP-1 and NPHP-4 and their role in cilia and male sensory behaviors. Subramaniam, S. and Zuker, C. S. (2004). Decoding cilia function: defining specialized Exp. Cell Res. 305, 333-342. genes required for compartmentalized cilia biogenesis. Cell 117, 527-539. Jauregui, A. R., Nguyen, K. C., Hall, D. H. and Barr, M. M. (2008). The Caenorhabditis Baala, L., Audollent, S., Martinovic, J., Ozilou, C., Babron, M. C., Sivanandamoorthy, elegans nephrocystins act as global modifiers of cilium structure. J. Cell Biol. 180, 973- S., Saunier, S., Salomon, R., Gonzales, M., Rattenberry, E. et al. (2007). Pleiotropic 988. effects of CEP290 (NPHP6) mutations extend to Meckel syndrome. Am. J. Hum. Genet. Karmous-Benailly, H., Martinovic, J., Gubler, M. C., Sirot, Y., Clech, L., Ozilou, C., 81, 170-179. Auge, J., Brahimi, N., Etchevers, H., Detrait, E. et al. (2005). Antenatal presentation Badano, J. L., Mitsuma, N., Beales, P. L. and Katsanis, N. (2006). The ciliopathies: an of Bardet-Biedl syndrome may mimic Meckel syndrome. Am. J. Hum. Genet. 76, 493- emerging class of human genetic disorders. Annu. Rev. Genomics Hum. Genet. 7, 125- 504. 148. Keeling, P. J., Burger, G., Durnford, D. G., Lang, B. F., Lee, R. W., Pearlman, R. E., Bae, Y. K. and Barr, M. M. (2008). Sensory roles of neuronal cilia: cilia development, Roger, A. J. and Gray, M. W. (2005). The tree of eukaryotes. Trends Ecol. Evol. 20, morphogenesis, and function in C. elegans. Front. Biosci. 13, 5959-5974. 670-676. Bergmann, C., Fliegauf, M., Brüchle, N. O., Frank, V., Olbrich, H., Kirschner, J., Keller, L. C., Romijn, E. P., Zamora, I., Yates, J. R. r. and Marshall, W. F. (2005). Schermer, B., Schmedding, I., Kispert, A., Kränzlin, B. et al. (2008). Loss of Proteomic analysis of isolated Chlamydomonas centrioles reveals orthologs of ciliary- nephrocystin-3 function can cause embryonic lethality, Meckel-Gruber-like syndrome, disease genes. Curr. Biol. 15, 1090-1098. situs inversus, and renal-hepatic-pancreatic dysplasia. Am. J. Hum. Genet. 82, 959-970. Kenyon, C., Chang, J., Gensch, E., Rudner, A. and Tabtiang, R. (1993). A C. elegans Bisgrove, B. W. and Yost, H. J. (2006). The roles of cilia in developmental disorders and mutant that lives twice as long as wild type. Nature 366, 461-464. disease. Development 133, 4131-4143. Kimura, K. D., Tissenbaum, H. A., Liu, Y. and Ruvkun, G. (1997). daf-2, an insulin Blacque, O. E. and Leroux, M. R. (2006). Bardet-Biedl syndrome: an emerging receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans. pathomechanism of intracellular transport. Cell Mol. Life Sci. 63, 2145-2161. Science 277, 942-946. Blacque, O. E., Reardon, M. J., Li, C., McCarthy, J., Mahjoub, M. R., Ansley, S. J., Kyttälä, M., Tallila, J., Salonen, R., Kopra, O., Kohlschmidt, N., Paavola-Sakki, P., Badano, J. L., Mah, A. K., Beales, P. L., Davidson, W. S. et al. (2004). Loss of C. Peltonen, L. and Kestila, M. (2006). MKS1, encoding a component of the flagellar 624 Journal of Cell Science 122 (5)

apparatus basal body proteome, is mutated in Meckel syndrome. Nat. Genet. 38, 155- Ponsard, C., Skowron-Zwarg, M., Seltzer, V., Perret, E., Gallinger, J., Fisch, C., Dupuis- 157. Williams, P., Caruso, N., Middendorp, S. and Tournier, F. (2007). Identification of Laurençon, A., Dubruille, R., Efimenko, E., Grenier, G., Bissett, R., Cortier, E., Rolland, ICIS-1, a new protein involved in cilia stability. Front. Biosci. 12, 1661-1669. V., Swoboda, P. and Durand, B. (2007). Identification of novel regulatory factor X Quarmby, L. M. and Parker, J. D. (2005). Cilia and the cell cycle? J. Cell Biol. 169, (RFX) target genes by comparative genomics in Drosophila species. Genome Biol. 8, 707-710. R195. Richards, T. A. and Cavalier-Smith, T. (2005). Myosin domain evolution and the primary Leitch, C. C., Zaghloul, N. A., Davis, E. E., Stoetzel, C., Diaz-Font, A., Rix, S., Al- divergence of eukaryotes. Nature 436, 1113-1118. Fadhel, M., Lewis, R. A., Eyaid, W., Banin, E. et al. (2008). Hypomorphic mutations Roume, J., Genin, E., Cormier-Daire, V., Ma, H. W., Mehaye, B., Attie, T., Razavi- in syndromic encephalocoele genes are associated with Bardet-Biedl syndrome. Nat. Encha, F., Fallet-Bianco, C., Buenerd, A., Clerget-Darpoux, F. et al. (1998). A gene Genet. 40, 443-448. for Meckel syndrome maps to chromosome 11q13. Am. J. Hum. Genet. 63, 1095-1101. Li, C., Inglis, P. N., Leitch, C. C., Efimenko, E., Zaghloul, N. A., Mok, C. A., Davis, Satir, P. and Christensen, S. T. (2007). Overview of structure and function of mammalian E. E., Bialas, N. J., Healey, M. P., Héon, E. et al. (2008). An essential role for DYF- cilia. Annu. Rev. Physiol. 69, 377-400. 11/MIP-T3 in assembling functional intraflagellar transport complexes. PLoS Genet. 4, Sayer, J. A., Otto, E. A., O’Toole, J. F., Nurnberg, G., Kennedy, M. A., Becker, C., e1000044. Hennies, H. C., Helou, J., Attanasio, M., Fausett, B. V. et al. (2006). The centrosomal Li, J. B., Gerdes, J. M., Haycraft, C. J., Fan, Y., Teslovich, T. M., May-Simera, H., protein nephrocystin-6 is mutated in Joubert syndrome and activates transcription factor Li, H., Blacque, O. E., Li, L., Leitch, C. C. et al. (2004a). Comparative genomics ATF4. Nat. Genet. 38, 674-681. identifies a flagellar and basal body proteome that includes the BBS5 human disease Schafer, J. C., Winkelbauer, M. E., Williams, C. L., Haycraft, C. J., Desmond, R. A. gene. Cell 117, 541-552. and Yoder, B. K. (2006). IFTA-2 is a conserved cilia protein involved in pathways Li, S., Armstrong, C. M., Bertin, N., Ge, H., Milstein, S., Boxem, M., Vidalain, P. O., regulating longevity and dauer formation in Caenorhabditis elegans. J. Cell Sci. 119, Han, J. D., Chesneau, A., Hao, T. et al. (2004b). A map of the interactome network 4088-4100. of the metazoan C. elegans. Science 303, 540-543. Scholey, J. M. (2008). Intraflagellar transport motors in cilia: moving along the cell’s Mak, H. Y., Nelson, L. S., Basson, M., Johnson, C. D. and Ruvkun, G. (2006). Polygenic antenna. J. Cell Biol. 180, 23-29. control of Caenorhabditis elegans fat storage. Nat. Genet. 38, 363-368. Singla, V. and Reiter, J. F. (2006). The primary cilium as the cell’s antenna: signaling at Marchler-Bauer, A., Anderson, J. B., Cherukuri, P. F., DeWeese-Scott, C., Geer, L. a sensory organelle. Science 313, 629-633. Y., Gwadz, M., He, S., Hurwitz, D. I., Jackson, J. D., Ke, Z. et al. (2005). CDD: a Smith, U. M., Consugar, M., Tee, L. J., McKee, B. M., Maina, E. N., Whelan, S., Conserved Domain Database for protein classification. Nucleic Acids Res. 33, D192- Morgan, N. V., Goranson, E., Gissen, P., Lilliquist, S. et al. (2006). The transmembrane D196. protein meckelin (MKS3) is mutated in Meckel-Gruber syndrome and the wpk rat. Nat. Marshall, W. F. and Nonaka, S. (2006). Cilia: tuning in to the cell’s antenna. Curr. Biol. Genet. 38, 191-196. 16, R604-R614. Swoboda, P., Adler, H. T. and Thomas, J. H. (2000). The RFX-type transcription factor Mukhopadhyay, A., Oh, S. W. and Tissenbaum, H. A. (2006). Worming pathways to DAF-19 regulates sensory neuron cilium formation in C. elegans. Mol. Cell 5, 411-421. and from DAF-16/FOXO. Exp. Gerontol. 41, 928-934. Tan, P. L., Barr, T., Inglis, P. N., Mitsuma, N., Huang, S. M., Garcia-Gonzalez, M. A., Ogg, S., Paradis, S., Gottlieb, S., Patterson, G. I., Lee, L., Tissenbaum, H. A. and Bradley, B. A., Coforio, S., Albrecht, P. J., Watnick, T. et al. (2007). Loss of Bardet Ruvkun, G. (1997). The Fork head transcription factor DAF-16 transduces insulin-like Biedl syndrome proteins causes defects in peripheral sensory innervation and function. metabolic and longevity signals in C. elegans. Nature 389, 994-999. Proc. Natl. Acad. Sci. USA 104, 17524-17529. Oh, S. W., Mukhopadhyay, A., Svrzikapa, N., Jiang, F., Davis, R. J. and Tissenbaum, Tallila, J., Jakkula, E., Peltonen, L., Salonen, R. and Kestila, M. (2008). Identification H. A. (2005). JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear of CC2D2A as a Meckel syndrome gene adds an important piece to the ciliopathy puzzle. translocation of forkhead transcription factor/DAF-16. Proc. Natl. Acad. Sci. USA 102, Am. J. Hum. Genet. 82, 1361-1367. 4494-4499. Town, T., Breunig, J. J., Sarkisian, M. R., Spilianakis, C., Ayoub, A. E., Liu, X., Ou, G., Blacque, O. E., Snow, J. J., Leroux, M. R. and Scholey, J. M. (2005). Functional Ferrandino, A. F., Gallagher, A. R., Li, M. O., Rakic, P. et al. (2008). The stumpy coordination of intraflagellar transport motors. Nature 436, 583-587. gene is required for mammalian ciliogenesis. Proc. Natl. Acad. Sci. USA 105, 2853-2858. Ou, G., Koga, M., Blacque, O. E., Murayama, T., Ohshima, Y., Schafer, J. C., Li, C., Valente, E. M., Silhavy, J. L., Brancati, F., Barrano, G., Krishnaswami, S. R., Castori, Yoder, B. K., Leroux, M. R. and Scholey, J. M. (2007). Sensory ciliogenesis in M., Lancaster, M. A., Boltshauser, E., Boccone, L., Al-Gazali, L. et al. (2006). Caenorhabditis elegans: assignment of IFT components into distinct modules based on Mutations in CEP290, which encodes a centrosomal protein, cause pleiotropic forms of transport and phenotypic profiles. Mol. Biol. Cell 18, 1554-1569. Joubert syndrome. Nat. Genet. 38, 623-625. Page, R. D. (1996). TreeView: an application to display phylogenetic trees on personal Vierkotten, J., Dildrop, R., Peters, T., Wang, B. and Ruther, U. (2007). Ftm is a novel computers. Comput. Appl. Biosci. 12, 357-358. basal body protein of cilia involved in Shh signalling. Development 134, 2569-2577. Pan, J. and Snell, W. (2007). The primary cilium: keeper of the key to cell division. Cell Williams, C. L., Winkelbauer, M. E., Schafer, J. C., Michaud, E. J. and Yoder, B. K.

Journal of Cell Science 129, 1255-1257. (2008). Functional redundancy of the B9 proteins and nephrocystins in Caenorhabditis Parker, J. D., Bradley, B. A., Mooers, A. O. and Quarmby, L. M. (2007). Phylogenetic elegans ciliogenesis. Mol. Biol. Cell 19, 2154-2168. analysis of the Neks reveals early diversification of ciliary-cell cycle kinases. PLoS ONE Winkelbauer, M. E., Schafer, J. C., Haycraft, C. J., Swoboda, P. and Yoder, B. K. 24, e1076. (2005). The C. elegans homologs of nephrocystin-1 and nephrocystin-4 are cilia transition Pazour, G. J. and Rosenbaum, J. L. (2002). Intraflagellar transport and cilia-dependent zone proteins involved in chemosensory perception. J. Cell Sci. 118, 5575-5587. diseases. Trends Cell Biol. 12, 551-555. Wolf, M. T., Lee, J., Panther, F., Otto, E. A., Guan, K. L. and Hildebrandt, F. (2005). Perkins, L. A., Hedgecock, E. M., Thomson, J. N. and Culotti, J. G. (1986). Mutant Expression and phenotype analysis of the nephrocystin-1 and nephrocystin-4 homologs sensory cilia in the nematode Caenorhabditis elegans. Dev. Biol. 117, 456-487. in Caenorhabditis elegans. J. Am. Soc. Nephrol. 16, 676-687.