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Normal Ciliogenesis Requires Synergy between the Cystic Kidney Disease MKS-3 and NPHP-4

Corey L. Williams, Svetlana V. Masyukova, and Bradley K. Yoder

Department of Cell Biology, University of Alabama at Birmingham Medical Center, Birmingham, Alabama

ABSTRACT Cilia dysfunction contributes to renal cyst formation in multiple human syndromes including nephron- ophthisis (NPHP), Meckel-Gruber syndrome (MKS), Joubert syndrome (JBTS), and Bardet-Beidl syn- drome (BBS). Although genetically heterogeneous, these diseases share several loci that affect cilia and/or , but the functions and interactions of these products are incompletely understood. Here, we report that the ciliated sensory neurons (CSNs) of C. elegans express the putative transmembrane MKS-3, which localized to the distal end of their dendrites and to the base but not to the cilium itself. Localization of MKS-3 and other known MKS and NPHP proteins partially overlapped. By analyzing mks-3 mutants, we found that ciliogenesis did not require MKS-3; instead, cilia elongated and cilia-mediated chemoreception was abnormal. Genetic analysis indicated that mks-3 functions in a pathway with other mks genes. Furthermore, mks-1 and mks-3 genetically interacted with a separate pathway (involving nphp-1 and nphp-4) to influence proper positioning, orientation, and formation of cilia. Combined disruption of nphp and mks pathways had cell nonautonomous effects on C. elegans sensilla. Taken together, these data demonstrate the importance of mutational load on the presentation and severity of and expand the understanding of the interactions between genes.

J Am Soc Nephrol 21: 782–793, 2010. doi: 10.1681/ASN.2009060597

Ciliopathies, or diseases associated with cilia dys- important insights into cilia biology and have function, display a diverse array of clinical features. helped identify conserved cilia genes. Homologs of Meckel-Gruber syndrome (MKS) is a severe cili- several ciliopathy-associated proteins concentrate opathy characterized by renal cystic dysplasia, poly- at the base of the cilium in C. elegans. Examples dactyly, occipital encephalocele, and perinatal include the C. elegans homologs of human nephro- death.1 MKS is an autosomal recessive, genetically cystin-1 (NPHP-1), nephrocystin-4 (NPHP-4), heterogeneous disorder with at least six associated MKS-1 (MKS-1/XBX-7), and multiple BBS pro- loci (MKS1 through MKS6). The five identified teins.12–14 Analysis of C. elegans nphp-1 and nphp-4 MKS genes encode cilia and/or basal body pro- mutants revealed involvement of these genes in cil- teins.2–7 Interestingly, mutations in several of the ia-mediated signaling responses such as chemoat- MKS genes have been found in relatively milder cil- traction, male mating behavior, foraging behavior, iopathies such as nephronophthisis (NPHP), Jou- and lifespan.12,13,15 Although cilia morphology ap- bert syndrome (JBTS), and Bardet-Biedl syndrome pears overtly normal in nphp-1 and nphp-4 single (BBS). For example, some MKS1 mutations mani- fest BBS- or NPHP-like phenotypes, and mutations Received June 9, 2009. Accepted December 18, 2009. in MKS3 were identified as causing JBTS or Published online ahead of print. Publication date available at NPHP.8–11 Such findings suggest that these diseases www.jasn.org. represent a spectrum of phenotypes resulting from Correspondence: Dr. Bradley K. Yoder, 1918 University Boule- a common underlying etiology. vard, MCLM 689, Birmingham, AL 35294. Phone: 205-934-0995; Studies conducted in both Chlamydomonas rein- Fax: 205-934-0950; E-mail: [email protected] hardtii and Caenorhabditis elegans have provided Copyright ᮊ 2010 by the American Society of Nephrology

782 ISSN : 1046-6673/2105-782 J Am Soc Nephrol 21: 782–793, 2010 www.jasn.org BASIC RESEARCH and double mutants, electron micrographs show occasional branching morphogenesis in the kidney, centriole migration microtubule axonemal defects in some cilia.16 Mutations in and duplication, and endoplasmic-reticulum-associated deg- mks-1 or either of two genes encoding proteins structurally radation.9,19,21 The homolog of MKS3 in C. elegans, F35D2.4 related to MKS-1 (mksr-1/tza-2 and mksr-2/tza-1) caused no (hereafter referred to as MKS-3), has not been described. discernable defects in cilia morphology.12,17 However, we BLAST analysis indicates that the nematode mks-3 gene prod- found that worms with a mutation in mks-1 (or either mksr uct is 30% identical and 46% similar to the human protein. On gene) and either nphp-1 or nphp-4 (e.g., mks-1;nphp-4 double the basis of computational analysis, it also contains each of the mutants) had severe defects in cilia formation, positioning, seven transmembrane domains predicted in human MKS-3, a and orientation.12 On the basis of these data, we proposed that cysteine-rich region near the N-terminus, and a highly con- the mks family of genes (mks-1, mksr-1, and mksr-2) participate served region in the C-terminal tail (Supplementary Figure 1A in one genetic pathway affecting cilia whereas nphp-1 and and Supplementary Figure 2). nphp-4 participate in a second, separate but at least partially redundant, pathway; disruption of either pathway alone has no DAF-19 Regulates Expression of mks-3 in CSNs overt effect on cilia morphology whereas disruption of both The C. elegans MKS3 homolog (mks-3) was first identified as a pathways is detrimental to cilia formation and/or mainte- candidate cilia gene in a computational search for X-box pro- nance. moter elements, which are utilized by the ciliogenic RFX-type Herein, we demonstrate that the transmembrane protein transcription factor DAF-19c to drive gene expression in MKS-3 (F35D2.4), the homolog of the human ciliopathy pro- CSNs.22–24 A putative X-box motif was identified in the pro- tein MKS-3//meckelin, localizes to two distinct do- moter of mks-3 at position Ϫ159 relative to the predicted mains in C. elegans ciliated sensory neurons (CSNs), one at the translational start. We generated an mks-3::GFP reporter com- distal end of the dendrite (dendritic tip) and the second at the prised of a 320-bp promoter segment and a portion of the first cilium base. Localization at the cilium base overlaps that of exon fused to green fluorescent protein (GFP). In wild-type MKS-1. Mimicking the elongation of cilia resulting from dis- worms we observed expression of mks-3::GFP in the amphid ruption of MKS-3 in rodents,18,19 C. elegans mks-3 mutants and labial neurons in the head (Figure 1A) and phasmid neu- form cilia that are increased in length. Our data indicate a rons of the tail (data not shown). This confirmed similar mks-3 genetic interaction between mutations in mks-3 and nphp-4 promoter analyses performed by Efimenko et al.22 mks-3::GFP that causes cilia and sensilla morphology defects. These phe- expression was nearly abolished in homozygous daf-19(m86) notypes are not seen in worms with combined mutations af- mutant background but could be restored by backcrossing fecting mks-3 and any of the other mks-1 gene family members, with daf-19(ϩ/ϩ) worms (Figure 1, B and C). These data sug- although cilia function is further impaired in mks-3;mks-1; gest mks-3 is regulated by the DAF-19 transcription factor. nphp-4 triple mutants. Intriguingly, our analysis also revealed that mks-3;nphp-4 double mutants exhibit cell nonautono- MKS-3 Concentrates at the Base of the Cilium in C. mous defects in the connections between sheath and socket elegans cells, which, along with the CSNs, comprise the C. elegans sen- Reported colocalization of endogenous mouse MKS3 with sory organs (sensilla). Together, the localization of MKS-3 and acetylated ␣-tubulin in mammalian inner medullar collecting genetic interaction data indicate that mks-3 can be functionally duct cells indicates that in mammals MKS3 is present along the assigned to the mks-1/mksr-1/mksr-2 genetic pathway. More ciliary .9 To determine if C. elegans MKS-3 also local- importantly, this report provides further insight into the inter- izes to the cilium, we generated an MKS-3 translational fusion play of the ciliopathy proteins in the influence on cilia function construct comprised of the 320-bp promoter segment and the and reflects the role of genetic background in the severity of entire coding region of mks-3 fused in frame with GFP disease. (MKS-3::GFP). MKS-3::GFP, coexpressed in wild-type worms with a cilium axoneme marker [XBX-1::tdTomato, light intermediate chain involved in retrograde intraflagellar RESULTS transport (IFT)], localized to the base of cilia, and remarkably was not evident along the cilium axoneme (Figure 2B through mks-3 Encodes a Predicted Seven Transmembrane- 2D, Supplementary Movie 1). MKS-3::GFP was present at its Spanning Protein highest levels at the cilium base (Supplementary Figure 3, A Recently, tmem67 was identified as the gene responsible for and AЈ, region 3) and was also localized to the dendritic tips of phenotypes in the Wistar polycystic kidney rat, the bilateral CSNs (Supplementary Figure 3, A and AЈ, region 1). Because polycystic kidney mouse, and also as a disease for human MKS-3 possesses putative transmembrane domains, we com- MKS and JBTS patients.5,18 MKS3 is a predicted seven trans- pared localization of MKS-3::GFP with a known membrane- membrane-spanning protein that colocalizes with acetylated associated protein, retinitis pigmentosa 2 (RP2::GFP).25,26 In ␣-tubulin along the cilium axoneme in mammalian inner single confocal sections of phasmid neurons, RP2::GFP and medullar collecting duct cells.9,20 In vivo and in vitro data im- MKS-3:GFP concentrated around the edges of dendritic tips plicate roles of MKS3 in ciliogenesis and cilium length control, (Figure 3). Membrane association of MKS-3::GFP was not ob-

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frameshift resulting in a premature stop (Supplementary Fig- ure 1B). tm2547 would therefore encode a truncated protein of only 152 amino acids (compared with 897 amino acid full length) lacking all of the transmembrane domains. Thus, tm2547 likely represents a null allele (Supplementary Figure 1, B and C).

mks-3 Is Required for Normal Chemotaxis but Not Ciliogenesis In C. elegans, direct exposure to the environment via pores in the cuticle allows the membrane of amphid and phasmid cilia in the head and tail of the worm, respectively, to contact and absorb fluorescence hydrophobic dye (DiI). mks-3(tm2547) mutant worms absorbed DiI normally into amphid and phas- mid neurons (Figure 4B), indicating that at this level of analy- sis, cilia are properly formed in this mutant background. This is consistent with the presence of cilia on rodent and human cells with MKS3 mutations.18,19 mks-3(tm2547) mutants were next analyzed for possible cilia-mediated signaling defects including altered chemotactic response to volatile chemical attractants, avoidance of regions of high osmolarity, and foraging behavior in the presence of food. We previously reported that a mutation in mks-1 did not affect any of these behaviors.12 Similarly, we observed no de- Figure 1. Expression and DAF-19 regulation of mks-3.(A fects in foraging or osmotic avoidance as a result of the mks- through C) Confocal fluorescence images of worms coexpressing 3(tm2547) mutation (data not shown). However, mks- mks-3::GFP. (A) mks-3::GFP was strongly expressed in amphid 3(tm2547) mutants had decreased chemotactic response (arrowhead) and labial (arrow) sets of neurons of the head, which toward the attractant benzaldehyde (Figure 5). This deficiency both extend dendritic processes to the (double arrowhead) ante- was less severe than that observed in retrograde IFT che-11 rior of the worm where they project sensory cilia. (B) mks-3 mutants, in which cilia are severely malformed (Figure 5). Be- transgene expression was almost completely abolished in worms cause benzaldehyde sensation occurs specifically through the crossed into daf-19(m86) mutant background. mks-3::GFP was amphid wing C (AWC) neuron, we examined whether mks- faintly detected only in labial neurons (arrow). (C) Outcrossing 3(tm2547) mutants had cilium morphologic defects in this cell transgenic worms from daf-19(m86)todaf-19(ϩ) restored expres- 27,28 sion of mks-3::GFP. type. In mks-3(tm2547) mutant worms expressing GFP driven by the AWC-specific str-2 promoter, no alterations of served in any other regions of the CSN dendrites (data not the wing-like structure of the AWC ciliated endings were ob- shown). served (Supplementary Figure 4). These data indicate that Because mutations in MKS1 and MKS3 result in similar MKS-3 is not necessary for cilia-mediated dye uptake or for disease phenotypes in humans, and the two associated proteins AWC wing formation but is required for normal cilia-initiated are reportedly found in complex in mammalian cells, we com- sensory signaling. pared localization of MKS-3::GFP to MKS-1::tdTomato in C. elegans (Figure 2E, Supplementary Movie 2). MKS-1::tdTomato mks-3 Functions as Part of the mks-1 Genetic Pathway localization was tightly restricted to the base of the cilium in a The mks related genes (mks-1, mksr-1, and mksr-2) and the domain that overlapped with a portion of MKS-3::GFP (Sup- nphp genes (nphp-1/nphp-4) act in two distinct but function- plementary Figure 3B). However, MKS-1::tdTomato was not ally redundant genetic pathways that are required for proper C. found in adjacent dendritic tips where MKS-3::GFP diffusely elegans sensilla morphology. Because MKS3 mutations have localized (Supplementary Figure 3, B and BЈ, region 1). been identified in MKS and NPHP patients, we assessed whether mks-3 functions as part of either the mks-1/mksr-1/ Characterization of an mks-3 Deletion Allele mksr-2 or nphp-1/nphp-4 genetic pathways.11 For this analysis To analyze mks-3 gene function in C. elegans, we obtained a we first performed chemotaxis assays on mks-3;mks-1 and mks- genetic mutant FX2547 mks-3(tm2547) from the National 3;nphp-4 double mutants to determine whether this would ex- Bioresource Project (Japan). The tm2547 allele contains a large acerbate the mks-3(tm2547) chemotaxis deficiency. Compared internal genomic deletion of 949 nucleotides. Reverse-tran- with mks-3(tm2547) mutants, in which chemoattraction to- scriptase PCR analysis of the mutant transcript revealed that ward benzaldehyde was reduced, neither addition of the mks- the deletion fuses exons 4 and 7 and creates a translational 1(tm2705) nor nphp-4(tm925) mutations resulted in alter-

784 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 782–793, 2010 www.jasn.org BASIC RESEARCH

ations of chemotactic response (Figure 5). Combination of the mks-1(tm2705) and nphp-4(tm925) mutations also did not re- duce chemotaxis in comparison to nphp-4(tm925) single mu- tants (Figure 5). However, in mks-3;mks-1;nphp-4 triple mu- tant animals, chemotaxis was diminished in comparison to any of the single or double mutants and resembled the defect seen in IFT che-11 mutants, indicating a genetic interaction between mks-3, mks-1, and nphp-4 in chemosensory responses. To further examine the relationship between mks-3 and the mks-1/mksr-1/mksr-2 and nphp-1/nphp-4 genetic path- ways, we analyzed mks-3;mksr-2 and mks-3;nphp-4 mutant strains for potential defects in amphid and phasmid cilio- genesis. Cilia were labeled using a transgene expressing the IFT complex B protein CHE-13::YFP (IFT57).29 We ob- served no overt cilia morphology defects in mks-3;mksr-2 mutants (Figure 6, A and B). In contrast, severe cilia and dendrite abnormalities were apparent in mks-3;nphp-4 mu- tants (Figure 6C). As previously seen in mks-1;nphp-4 mu- tants, defects in mks-3;nphp-4 double mutants included loss of amphid cilia fasciculation in the head and shortened mis- oriented cilia projecting from malformed phasmid den- drites in the tail (Figure 6C).12 Quantification of phasmid cilia length confirmed that mks-3;nphp-4 mutants had sig- nificantly shorter cilia than wild-type worms and mks- 3(tm2547) mutants (Table 1). IFT movement was still evi- dent in the shortened cilia of mks-3;nphp-4 mutants (Supplementary Movie 3). As part of this analysis we uncov- ered that phasmid cilia in mks-3(tm2547) single mutants were consistently longer than wild-type worms (Table 1). Because overexpression of the IFT protein OSM-6 was ob- served by others to synthetically inhibit ciliogenesis in nphp- 4(tm925) mutants, and because utilization of an IFT protein as a marker for cilia morphology may complicate assessment of cilia morphology, we also analyzed cilia in mks-3 single and compound mutants using the ␤-tubulin protein TBB-4.16 As observed with CHE-13::YFP, phasmid cilia length measure- ments based on TBB-4::GFP fluorescence indicated longer cilia in mks-3(tm2547) mutants compared with wild type (Supple- Figure 2. MKS-3 concentrates at the base of cilia in C. el- mentary Figure 4D, Table 1). In contrast, phasmid egans. (A) An illustration depicting the anatomical positions of (left) amphid cilia bundles in the head and (right) phasmid cilia were shortened in mks-3;nphp-4 mutants although not in mks- bundles in the tail with respect to (middle) a differential inter- 3;mks-1 mutants (Supplementary Figure 4, E and F, Table 1). ference contrast image of the full body of an adult hermaph- These data indicate that mks-3(tm2547) and mks-3;nphp-4 rodite worm. Red represents cilia axonemes, green represents mutants have altered cilium morphology reflected at the mi- bases of cilia, and blue represents the dendritic processes from crotubule level. which the cilia project. Short labial cilia surrounding the distal Consistent with cilia morphology defects, mks-3;nphp-4 region of the amphid bundles and the degenerate PQR cilium mutants were hindered in the ability to uptake DiI (Figure 7 near the phasmid bundles are not shown. (B through D) Con- and Table 2). Although 89.0% of mks-3;nphp-4 mutant am- focal fluorescence images of wild-type transgenic worms co- phid neuron bundles dye-filled, intensity of dye-filling was expressing MKS-3::GFP (driven by its endogenous promoter) consistently less than wild-type controls. Furthermore, phas- and XBX-1/dynein light intermediate chain::tdTomato (driven mid neurons rarely stained with DiI (Figure 7 and Table 2). In by the osm-5 promoter). With respect to XBX-1, which freely entered the cilia (arrowheads), MKS-3::GFP concentrated spe- cifically at the base of cilia (arrows) on (B and D, left column) MKS-1::tdTomato (driven by its endogenous promoter). With re- sensory neurons in the head and (C and D, right column) spect to MKS-3::GFP, MKS-1::GFP localized only to the proximal sensory neurons in the tail. (E) Confocal fluorescence images of end of cilia where MKS-3::GFP concentrated most intensely (ar- wild-type transgenic worms coexpressing MKS-3::GFP and rows). Scale ϭ 5 ␮m.

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Figure 4. mks-3(tm2547) mutants dye-fill normally. The ability to uptake DiI was examined in wild-type worms and mks-3(tm2547) mutants. Confocal fluorescence images of DiI staining are shown. Indicative of properly formed cilia, (left) wild-type and (right) Figure 3. Localization of MKS-3 in the dendritic tip resembles mks-3(tm2547) worms consistently absorbed DiI into CSNs in the that of the membrane-associated protein RP2. Single-section con- head (large panels) and tail (insets). The four phasmid cell bodies focal fluorescence images of wild-type transgenic worms express- are outlined in the insets. Scale ϭ 7 ␮m. ing (A) RP2::GFP (driven by its endogenous promoter) or (B) MKS-3::GFP. One phasmid neuronal ciliated ending is shown in each panel. RP2::GFP accumulated around the edges of the dendritic tip (dt), indicative of membrane association, but was not present at the cilium base (cb). MKS-3::GFP also accumulated around the edges of the dendritic tip in addition to concentrating at the cilium base. Scale ϭ 5 ␮m. comparison, amphid dye-filling at some level was only ob- served in 27.6% of mks-3;mks-1;nphp-4 triple mutants. We were able to partially rescue the mks-3;nphp-4 phasmid dye- filling defect by generating mutant worms coexpressing wild- type MKS-3::tdTomato and TBB-4::GFP (Figure 7, Supple- mentary Figure 4G). Phasmid cilia length, as determined via TBB-4::GFP fluorescence, was also partially restored in rescue animals (Supplementary Figure 4G, Table 1). In C. elegans, pores through which amphid and phasmid Figure 5. mks-3 mutants exhibit reduced chemotaxis toward cilia extend consist of a socket cell-derived channel that asso- benzaldehyde. Compared with wild type, mks-3(tm2547) and nphp-4(tm925) single mutants were mildly but significantly defec- ciates with the cuticle of the worm and a sheath support cell tive in response to a volatile attractant. Severity of mks-3;mks-1; surrounding the cilia bundles, altogether forming the amphid nphp-4 triple mutant chemotaxis deficiency was similar to retro- 30,31 and phasmid sensilla (Figure 8, A and D). Previously, we grade IFT che-11(e1810) mutants, which were used as a negative observed in mksr-2;nphp-4 mutants in which cilia were short control. Each group was assayed at least four times with 50 to 120 and phasmid dendrites were malformed that sheath cells re- worms per run. Error bars represent SD. No significant difference main associated as part of the channels despite detachment of was observed in groups represented by bars of matching color. the dendrites/cilia from the channels.12 To determine whether P Ͻ 0.05 was deemed significant. this was also true in mks-3;nphp-4 mutants, we generated worms coexpressing XBX-1::tdTomato to visualize dendrite/ visualize cilia/dendrite positioning and endogenous promoter- cilia positioning and f16f9.3::GFP to visualize sheath cells.12 driven MAGI-1::GFP (membrane-associated guanylate kinase in- Interestingly, in contrast to results with mksr-2;nphp-4,inmks- verted), which we have found localizes around the channel por- 3;nphp-4 mutants phasmid sheath cells remained in associa- tion of phasmid sockets (Figure 8G).32 Compared with misplaced tion with misplaced cilia instead of extending normally to form phasmid cilia in mks-3;nphp-4 mutants, socket channels re- part of the channels (Figure 8F). No overt sheath cell morphol- mained in association with the cuticle where cilia would normally ogy alterations were observed in the head sensilla of mks-3; extend into the external environment (Figure 8, G and H). To- nphp-4 mutants, likely because of the presence of properly ex- gether, these data indicate that combined loss of mks-3 and tended amphid dendrites (Figure 8C). nphp-4 in CSNs results in cell nonautonomous alterations of To determine whether this apparent detachment of the den- phasmid sensilla architecture. drites and sheath cells from the phasmid channels also affected the Overall, the genetic interactions between mks-3 and nphp-4 sockets, we generated worms coexpressing XBX-1::tdTomato to with regards to cilia assembly are identical to that obtained

786 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 782–793, 2010 www.jasn.org BASIC RESEARCH

Figure 6. mks-3;nphp-4 double mutants have short and incorrectly positioned cilia. Cilia morphology was analyzed in confocal fluorescence images of wild-type, mks-3(tm2547), mks-3;mksr-2, and mks-3;nphp-4 worms expressing the transgenic IFT particle B protein CHE-13::YFP. (A and B) Cilia morphology appeared overtly normal in wild-type (top), mks-3(tm2547) (middle), and mks-3;mksr-2 (bottom) strains. Arrowheads, distal ends of cilia; double arrowheads, cilia bases. (AЈ and CЈ) 1.66ϫ magnification of amphid bundles in A and C, respectively. Arrows indicate the shorter labial cilia that are not part of the amphid channel organs. (B) Compared with wild type (top), mutant strains mks-3(tm2547) and mks-3;mksr-2 exhibited no abnormalities in the extension of dendrites (dotted lines) from the phasmid cell bodies (arrows) nor in the projection of full-length cilia (double-headed arrow) from the distal tips of the dendrites. (C) Amphid and (D) phasmid cilia arrangement and morphology was abnormal in mks-3;nphp-4 double mutants. Compared with wild type, some amphid cilia were positioned posterior with the respect to the rest of the sensory organ (CЈ, arrowhead), and other amphid cilia often altogether lacked axonemes (arrow). (D) Phasmid dendrites often failed to properly extend from the cell bodies and projected stunted cilia. Scale ϭ 5 ␮m. when we conducted similar analysis of mks-1 and nphp-4 (Ta- caused by a separate homozygous mutation in another ciliopa- ble 2).12 mks-3;nphp-1 mutants were not generated because the thy gene in the same individual.10 Additionally, morpholino genes are closely linked in the genome. Together, our data in- knockdown of multiple cilia-related disease genes in zebrafish dicate that mks-3 functions as an additional member of the can have synergistic effects on developmental phenotypes.10 mks-1/mksr-1/mksr-2 genetic pathway influencing cilia orien- These complex interactions between ciliopathy genes are also tation, cilia positioning, and ciliogenesis in conjunction with observed in C. elegans. Combination of an mks-1 mutation the nphp-1/nphp-4 pathway. with either an nphp-1 or nphp-4 mutation caused ciliogenesis defects not evident in any of the single mutants.12 These obser- vations suggest that genetic background will have a major in- DISCUSSION fluence on the severity of cilia-associated phenotypes and in- dicate that studies in C. elegans will be useful in elucidating the Although the number of identified cystic kidney disease genes molecular basis of these phenotypes. has rapidly increased, our basic understanding of their inter- In human patients and in rodent models, genetic mutations connected functions has lagged. This is an important issue be- affecting MKS3 did not inhibit ciliogenesis. Rather, disruption cause in human ciliopathies, heterozygous mutations in one of MKS3 was found to cause centrosome duplication and mul- disease-related gene can enhance the severity of phenotypes ticiliation as well as cilia elongation, the latter of which we also

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Table 1. Phasmid cilia length measurements Length (␮m) P Value Compared with P Value Compared with Length (␮m) ؎ SEM Strain ؎ SEM Wild Type mks-3 TBB-4::GFP Wild Type mks-3 mks-3; CHE-13::YFP (tm2547) (tm2547) nphp-4 Wild type 7.06 Ϯ 0.11 6.77 Ϯ 0.13 mks-3(tm2547) 7.42 Ϯ 0.11 0.02a 7.27 Ϯ 0.20 0.04a mks-3;mksr-2 7.23 Ϯ 0.13 0.33 0.2 mks-3;mks-1 7.39 Ϯ 0.17 0.007a 0.64 mks-3;nphp-4 3.63 Ϯ 0.19 Ͻ0.0001a Ͻ0.0001a 4.59 Ϯ 0.29 Ͻ0.0001a Ͻ0.0001a mks-3;nphp-4 ϩ Ex1 5.98 Ϯ 0.29 0.002a (MKS-3::tdTomato) aP Ͻ 0.05 was deemed significant. found here in C. elegans mks-3 mutants.18,19 This observation is not without precedent, because Nphp3pcy/ko, jck (Nphp9/ Nek8), and Bbs4 mutant mice have elongated renal epithelial cilia as well as cystic kidneys.33–35 Additionally, elongation of male-specific CEM cilia in nphp-1 and nphp-4 mutant C. el- egans is sometimes observed.16 Not until we combined the mks-3(tm2547) mutation with the nphp-4(tm925) mutation did obvious ciliogenesis defects arise. Because of this genetic interaction and our observation that ciliogenesis was not impaired when the mks-3(tm2547) mutation was combined with mutations in any of the three mks-1 family of genes, we conclude that mks-3 is likely an ad- ditional member of the mks-1/mksr-1/mksr-2 genetic pathway, functioning redundantly with the nphp-1/nphp-4 pathway for proper C. elegans ciliogenesis and sensilla assembly (Supple- mentary Figure 6). Our finding that the combination of mks-3 and nphp-4 mutations did not additively impair chemotactic response beyond the defect observed in mks-3 single mutants would suggest that mks-3 functions upstream of nphp-4 in a chemosensation pathway. However, the interpretation of this result is complicated by the relatively mild nphp-4 mutant che- motaxis phenotype and the SD observed in the mks-3 and mks- 3;nphp-4 mutants. Functional differences between mks-3 and mks-1 within the mks-1/mksr-1/mksr-2/mks-3 genetic pathway were highlighted by our finding that combination of the mks-1 and mks-3 mutations together with the nphp-4 mutation re- sulted in further impairment of chemotaxis and ciliogenesis Figure 7. Partial restoration of phasmid dye-filling in mks-3; beyond that observed in mks-1;nphp-4 and mks-3;nphp-4 mu- nphp-4 double mutants by transgene rescue. The ability to tants (Supplementary Figure 6). This observation suggests that uptake DiI was examined in wild-type worms, mks-3(tm2547); mks-1 and mks-3 contributions to the mks-1/mksr-1/mksr-2/ nphp-4(tm925) double mutants, and transgenic mks- mks-3 pathway are similar but not identical, which could be 3(tm2547);nphp-4(tm925) double mutants coexpressing reflective of how MKS-3 patients have some clear phenotypic MKS-3::tdTomato and the coelomocyte marker UNC-122::GFP differences from MKS-1 patients.36 from a nonintegrated extrachromosomal array (Ex1). Phasmid The mechanisms by which these genes and pathways influ- dye-filling was regularly observed in wild-type worms but rarely ence cilia morphogenesis are not entirely clear. Ultrastructural seen in mks-3(tm2547);nphp-4(tm925) double mutants. Prog- data indicate that the nphp-1/nphp-4 pathway may be respon- eny of mks-3(tm2547);nphp-4(tm925) double mutants coex- pressing MKS-3::tdTomato and UNC-122::GFP were exposed sible for maintaining the integrity of the ciliary axoneme on the to DiI and then siblings were segregated as transgenic versus basis of observations of axonemal microtubule defects in these nontransgenic on the basis of the presence or absence of 16 mutants. Ultrastructural analysis of mks-1 gene family mu- UNC-122::GFP signal, respectively. Partial rescue of phasmid tants uncovered no microtubule defects in C. elegans cilia, in- dye-filling was observed in transgenic double mutants com- dicating this pathway may influence cilia morphology in a sep- pared with nontransgenic siblings. At least 100 worms in each arate manner from the nphp-1/nphp-4 pathway.17 Because category were analyzed. Error bars represent SEM.

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Table 2. Summary of mks and nphp mutant phenotypes a Dendrite Percent Dye-Filling Benzaldehyde Cilia Lengtha Strain Extensiona Chemotaxisa (Phasmid) Amphid Phasmid (Phasmid) Wild type 100 100 ϩϩϩ mks-1(tm2705) 100 100 ϩϩϩ mks-3(tm2547) 100 100 ϪϪ ϩϩ ϩ mks-3;mks-1 100 100 ϪϪ ϩϩ ϩ nphp-4(tm925) 100 100 Ϫϩϩ mks-1;nphp-4 94.3 52.7 ϪϪϪ mks-3;nphp-4 89 14.3 ϪϪ Ϫ Ϫ mks-3;mks-1;nphp-4 27.6 Ͻ1 ϪϪϪ ND ND aData combined from study presented here and reference 12. ϩ, similar to wild type; ϩϩ, greater than wild type; Ϫ, slightly defective; ϪϪ, moderately defective; ϪϪϪ, severely defective, similar to IFT mutant; ND, not determined.

MKS-3 is a transmembrane protein that concentrates at the that most, if not all, roles for cilium-associated signaling are cilium base, we speculate a partial role of the mks-1/mks-3 ge- eroded, thus leading to the extreme clinical features observed netic pathway in the biology of the cilium membrane, as op- in this syndrome. posed to the axoneme. Potential functions of the MKS proteins Elucidating the function of genes associated with cystic kid- at the cilium base could include processes such as tethering of ney disease is essential to the understanding of renal and extra- transition fibers to the membrane/basal body anchoring, reg- renal pathologies arising in ciliopathy patients. This should ulating the ciliary entry and/or exit of other proteins, or medi- include analysis of complex genetic interactions between cilio- ating vesicular trafficking of proteins at the cilium base. Thus, cystic genes and the determination of how genetic modifiers the combination of microtubule defects conferred by disrup- can influence disease severity. Herein, we uncovered a role of tion of the nphp-1/nphp-4 pathway coupled with some form of mks-3 in mediating a subset of cilia signaling activities and cilium membrane dysregulation conferred by disruption of the demonstrated a redundant requirement of mks-3 and nphp-4 mks-1/mks-3 pathway would together be incompatible with in ciliogenesis and C. elegans sensilla morphology. These anal- sustaining cilium and sensillum integrity. yses demonstrate the utility of the C. elegans system for explo- Our data indicate that disruption of mks-3 and nphp-4 in C. ration of the complex genetic interactions between the NPHP, elegans can cause changes in the morphology of cells in which MKS, and other ciliopathy genes and how these interactions these genes are not expressed. It has recently been shown that a may affect disease pathogenesis and presentation in human secreted tectorin-like protein matrix anchors CSN dendritic ciliopathy patients. Understanding how the MKS and NPHP tips at the initiation point of cell body migration during sen- proteins function in cilia formation and/or signaling will pro- sillum development.37 We can envision a role for membrane- vide insight into why their disruption results in systemic devel- bound proteins such as MKS-3 in mediating interactions be- opmental abnormalities in higher organisms. tween the membrane of CSNs and the surrounding matrix and/or sheath/socket cells to help maintain dendritic tip an- choring. Although difficult to predict how MKS-3 could func- CONCISE METHODS tion in membrane interactions in the context of a renal epithe- lial cell, it should be noted that most primary cilia are located in General Molecular Biology Methods pockets or depressions in the cell membrane. These pockets are Molecular biology procedures were performed according to standard sites of active vesicular transport, thus it would be interesting protocols.40 C. elegans genomic DNA, C. elegans cDNA, or cloned C. to evaluate whether MKS-3 in mammalian cells may have an elegans DNA was used for PCR amplifications, for direct sequencing, analogous role and mediate the interaction between the mem- or for subcloning.40 All PCR for cloning was performed with Phusion brane of the cilium and the cell membrane. high-fidelity DNA polymerase (Finnzymes, Espoo, Finland). Clones, How specific proteins are shuttled into and out of the ciliary primer sequences, and PCR conditions are available upon request. compartment has become a very active area of research be- DNA sequencing was performed at the University of Alabama at Bir- cause of a current paucity of data and the direct relevance to mingham Genomics Core Facility of the Heflin Center for Human disease. It is interesting to note that the BBsome, a complex of Genetics. several BBS proteins, is involved in establishing and regulating the cilium membrane and has been implicated in the traffick- DNA and Protein Sequence Analysis ing of cilia-specific receptors.38,39 However, the clinical fea- Genome sequence was obtained from the National Center for Bio- tures of BBS are relatively mild when compared with those technology Information (http://www.ncbi.nlm.nih.gov). Gene and observed in MKS. It is enticing to speculate that perhaps in protein sequences were identified using the database Wormbase and MKS the cilium membrane is more fundamentally altered such references therein (http://www.wormbase.org). Searches to identify

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homologs in human, mouse, and Strongylocentrtus purpuratus were performed using the National Center for Biotechnology Information BLAST service (http://www.ncbi.nlm.nih.gov/BLAST). Protein se- quence analyses were performed using Phobius (http://phobius. cbr.su.se).

Strains Worm strains were obtained from the Caenorhabditis Genetics Center, C. elegans Knock-out Consortium, and National BioResource Project in Ja- pan. Strains were grown using standard growth methods at 22°C unless otherwise stated.41 The wild-type strain was N2 Bristol. The following mutations were used: JT6924 daf-19(m86)II;daf-12(sa204)X, FX2705 mks-1(tm2705)III, FX2452 mksr-2/tza-1(tm2452)IV, FX925 nphp- 4(tm925)V, JT204 daf-12(sa204)X, FX2547 mks-3(tm2547)II, YH499 mks-3(tm2547)II;mks-1(tm2705)III, YH576 mks-3(tm2547)II;nphp- 4(tm925)V, YH582 mks-3(tm2547)II;mksr-2/tza-1(tm2452)IV, and YH850 mks-3(tm2547)II;mks-1(tm2705)III;nphp-4(tm925)V. FX2705, FX2547, FX2452, and FX925 were outcrossed at least three times and genotyped by PCR before analysis.

Generation of Constructs and Strains Vectors for generating transcriptional and translational fusion constructs were modified from pPD95.81 (a gift from A. Fire). pCJF37 (CHE-13::YFP) was generated as described previously.13 pPD95.81::tdTomato was generously provided by M. Barr (Rut- gers University). pCP41 (Pf16f9.3::GFP) was generously provided by S. Shaham (The Rockefeller University). pA597 (MAGI-1::GFP) was generously provided by A. Stetak (University of Basel, Switzerland).32 Pstr-2::GFP construct was generously provided by C. Bargmann (The Rockefeller University). cGV1 (pPD95.81::Gateway::GFP) and cGV6 were generated by blunt cloning the Gateway cassette c.1 into the Figure 8. Cell nonautonomous perturbation of phasmid sensilla SmaI site of pPD95.81 and pPD95.81::tdTomato, respectively (In- morphology in mks-3;nphp-4 double mutants. Illustrations of (A) am- vitrogen, Carlsbad, CA). cGV7.1 (Posm-5::Gateway::GFP) was gener- phid and (D) phasmid sensilla showing attachment of cuticle to socket ated by transferring the 250-bp promoter of osm-5 from pCJF3 to channels, connections between socket channels and sheath cells (dou- cGV1 at HindIII/SphI sites. p329.1 (mks-3::GFP) was generated by ble arrowheads), and connections between sheath cells and dendritic tips (den) (arrowheads). Scale ϭ 1 ␮m. Modified with permission from inserting pPD95.81 with a 300-bp promoter fragment amplified from Perkins et al.31 (B, C, E, and F) Confocal fluorescence images of worms N2 genomic DNA corresponding to the region immediately upstream coexpressing f16f9.3::GFP to mark sheath cells and XBX-1::tdTomato to and one codon downstream of the mks-3 ATG. p330.1 (MKS-3::GFP) mark cilia. Compared with (B) wild type, amphid sensory neurons in and p367.1 (MKS-3::tdTomato) were generated by amplifying the (C) mks-3;nphp-4 double mutants projected malformed cilia that re- 300-bp promoter and entire coding region of mks-3 from N2 genomic mained in association with the surrounding sheath cells. Despite appar- DNA and inserting the fragment into cGV1 or cGV6, respectively, via ent detachment from the phasmid channels (dotted lines mark pre- Gateway technology. p341 (RP2::GFP) was generated by amplifying dicted location of channels), phasmid sheath cells remained in the 1.0-kb promoter and entire coding region of k08d12.2 from N2 association with the misplaced ciliated endings of phasmid neurons genomic DNA and inserting the fragment in cGV7.1 via Gateway (arrowheads) in (F) mks-3;nphp-4 double mutants. (G, H) Confocal flu- technology. p344 (NPHP1::tdTomato) was generated by amplifying orescence images of worms coexpressing MAGI-1::GFP to mark the 300-bp promoter and entire coding region of nphp-1 from N2 socket channels and XBX-1::tdTomato to mark cilia. In (G) wild-type worms, socket cell-expressed MAGI-1::GFP localizes around the distal- genomic DNA and inserting the fragment in cGV6 via Gateway tech- most segments of phasmid cilia (arrows), consistent with the location of nology. p346.1 (MKSR-1::tdTomato) was generated by amplifying the socket-derived portion of the phasmid channels. In (H) mks-3; the 1.7-kb promoter and entire coding region of mksr-1 from N2 nphp-4 double mutants, socket channels were present. (Left) A pair of genomic DNA and inserting the fragment in cGV6 via Gateway tech- short phasmid cilia positioned normally with respect to the socket nology. p368 (Posm-5::TBB-4::GFP) was generated by amplifying the channel. (Right) Socket channels were most often present (arrow) de- entire coding region of tbb-4 from N2 genomic DNA and inserting the spite misplacement of cilia, although on rare occasions no socket- fragment in cGV7.1 via Gateway technology. p327.1 (MKS-1::tdTomato) expressed MAGI-1::GFP was observed (arrowhead). Scale of all fluores- was generated by subcloning the 3.2 kb mks-1 genomic fragment from cence images ϭ 5 ␮m. p318 to pPD95.81::tdTomato.12 p328.1 (Posm-5::XBX-1::tdTomato) was

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generated by subcloning the 700-bp tdTomato fragment from pPD95.81 worms at the control zone divided by the total number of worms on to pCJF17.1 (Posm-5::XBX-1::YFP).42 Transgenic worms were generated the plate. as described previously.43 A total of 56 independent extrachromosomal arrays containing MKS-3::GFP or MKS-3::tdTomato were generated, 55 of which expressed MKS-3 in the pattern reported here. A single array ACKNOWLEDGEMENTS appeared to greatly overexpress MKS-3::tdTomato, resulting in extensive aggregation of the fusion protein in dendritc tips (data not shown). We thank M. Croyle, V. Roper, and E. Brown for technical assistance and N. Berbari and A. O’Connor for review and comments of the Imaging manuscript. The C. elegans Genome Sequencing Consortium pro- Worms were anesthetized using 10 mM Levamisole and immobilized vided sequence information, and the Caenorhabditis Genetics Center, on 2% agar pads for imaging. Confocal analysis was performed on a which is funded by the National Institutes of Health, provided some Nikon 2000U inverted microscope (Melville, KY) outfitted with a of the C. elegans strains used in this study. We thank the National PerkinElmer UltraVIEW ERS 6FE-US spinning disk laser apparatus BioResource Project in Japan for the mks-3(tm2547) deletion mu- (Shelton, CT). Confocal images were processed with Volocity 5 (Im- tants. This work was supported by National Institutes of Health grants provision Inc., Waltham, MA). Image processing was performed us- R01 DK065655 and R01 DK62758 to B.K.Y. and T32 DK007545-22 to ing Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA). D.J.B.

DAF-19 Regulation To asses DAF-19 regulation in vivo, the transgenic line YH588 was DISCLOSURES generated by injection of mks-3::GFP into N2. This was then crossed None. to JT204 and subsequently to JT6924 to achieve daf-19(m86) back- ground. The strain contained a mutation in daf-12(sa204)X to sup- press the Daf-c phenotype of daf-19(m86)II. A backcross with JT204 REFERENCES was then used to assess mks-3::GFP expression in daf-19(Ϯ) back- ground. 1. Alexiev BA, Lin X, Sun, C. C. Brenner DS: Meckel-Gruber syndrome: Reverse-Transcriptase PCR Pathologic Manifestations, Minimal diagnostic criteria, and differential diagnosis. Arch Pathol Lab Med 130: 1236–1238, 2006 RNA was isolated as described previously from mks-3(tm2547) mu- 2. Baala L, Audollent S, Martinovic J, Ozilou C, Babron MC, Sivanan- 29 tants. Complementary DNA was generated using Superscript II re- damoorthy S, Saunier S, Salomon R, Gonzales M, Rattenberry E, verse transcriptase (Invitrogen, Carlsbad, CA) using the manufactur- Esculpavit C, Toutain A, Moraine C, Parent P, Marcorelles P, Dauge er’s instructions. Fragments spanning the deleted region of each MC, Roume J, Le Merrer M, Meiner V, Meir K, Menez F, Beaufrere AM, respective gene were amplified by PCR and sequenced. Francannet C, Tantau J, Sinico M, Dumez Y, MacDonald F, Munnich A, Lyonnet S, Gubler MC, Genin E, Johnson CA, Vekemans M, Encha- Razavi, F. Attie-Bitach, T.: Pleiotropic effects of CEP290 (NPHP6) Assays mutations extend to Meckel syndrome. Am J Hum Genet 81: 170– Dye-filling using DiI (Molecular Probes, Carlsbad, CA) was per- 179, 2007 formed as described previously.44 Chemotaxis assays to volatile at- 3. Delous M, Baala L, Salomon R, Laclef C, Vierkotten J, Tory K, Golzio tractants (benzaldehyde) were performed as described previously.45 C, Lacoste T, Besse L, Ozilou C, Moutkine I, Hellman NE, Anselme I, Silbermann F, Vesque C, Gerhardt C, Rattenberry E, Wolf MT, Gubler Briefly, 10-cm chemotaxis plates containing 2% Difco agar Noble, 5 MC, Martinovic J, Encha-Razavi F, Boddaert N, Gonzales M, Macher mM potassium phosphate, pH 6.0, 1 mM CaCl2, and 1 mM MgSO4 MA, Nivet H, Champion G, Bertheleme JP, Niaudet P, McDonald F, were poured 12 to 24 hours before experiments. Plates were marked at Hildebrandt F, Johnson CA, Vekemans M, Antignac C, Ruther U, the center and at opposite sides, 0.5 cm from the edge. A zone was Schneider-Maunoury S, Attie-Bitach T, Saunier S: The ciliary gene drawn at each side 1.5 cm from each of these spots representing the RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syn- drome type B) and Meckel syndrome. Nat Genet 39: 875–881, 2007 chemoattractant zone and the control zone. To the spots in each zone 4. Roume J, Genin E, Cormier-Daire V, Ma HW, Mehaye B, Attie T, ␮ 0.2 l of 1 M sodium azide was added as an anesthetic. In the che- Razavi-Encha F, Fallet-Bianco C, Buenerd A, Clerget-Darpoux F, Mun- moattractant zone, 1 ␮l of chemoattractant (diluted 1:100 in 95% nich A, Le Merrer M: A gene for Meckel syndrome maps to chromo- ethanol) was added, and at the opposite control zone 1 ␮lof95% some 11q13. Am J Hum Genet 63: 1095–1101, 1998 ethanol alone was added immediately before assays. Worms were 5. Smith UM, Consugar M, Tee LJ, McKee BM, Maina EN, Whelan S, Morgan NV, Goranson E, Gissen P, Lilliquist S, Aligianis IA, Ward CJ, grown at room temperature until young adult stage and then were Pasha S, Punyashthiti R, Malik Sharif S, Batman PA, Bennett CP, washed three times with M9 medium (0.3% KH2PO4, 0.6% Woods CG, McKeown C, Bucourt M, Miller CA, Cox P, Algazali L,

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43. Mello CC, Kramer JM, Stinchcomb D, Ambros V: Efficient gene trans- Plasterk RH, Baillie DL, Katsanis N, Quarmby LM, Wicks SR, Leroux fer in C. elegans: Extrachromosomal maintenance and integration of MR: Loss of C. elegans BBS-7 and BBS-8 protein function results in transforming sequences. EMBO J 10: 3959–3970, 1991 cilia defects and compromised intraflagellar transport. Genes Dev 18: 44. Starich TA, Herman RK, Kari CK, Yeh WH, Schackwitz WS, Schuyler 1630–1642, 2004 MW, Collet J, Thomas JH, Riddle DL: Mutations affecting the chemo- sensory neurons of Caenorhabditis elegans. Genetics 139: 171–188, 1995 45. Blacque OE, Reardon MJ, Li C, McCarthy J, Mahjoub MR, Ansley SJ, See related editorial, “Synergistic Interaction between Ciliary Genes Reflects Badano JL, Mah AK, Beales PL, Davidson WS, Johnsen RC, Audeh M, the Importance of Mutational Load in Ciliopathies,” on pages 724–726.

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