The Caenorhabditis elegans nephrocystins act as global modifiers of cilium structure

Nephronophthisis (NPHP) is the most common genetic cause of end-stage renal disease in children and young adults. In Chlamydomonas reinhardtii, Caenorhabditis elegans, and mammals, the NPHP1 and NPHP4 gene products nephrocystin-1 and nephrocystin-4 localize to basal bodies or ciliary transition zones (TZs), but their function in this location remains unknown. We show here that loss of C. elegans NPHP-1 and NPHP-4 from TZs is tolerated in developing cilia but causes changes in localization of specific ciliary components and a broad range of subtle axonemal ultrastructural defects. In amphid channel cilia, nphp-4 mutations cause B tubule defects that further disrupt intraflagellar transport (IFT). We propose that NPHP-1 and NPHP-4 act globally at the TZ to regulate ciliary access of the IFT machinery, axonemal structural components, and signaling molecules, and that perturbing this balance results in cell type–specific phenotypes.


Introduction
Nephronophthisis (NPHP) is a rare autosomal recessive tubulointerstitial nephropathy characterized by cystic kidneys and renal fi brosis. NPHP extrarenal manifestations may include retinal degeneration, situs inversus, and neurological disorders. Mutations in one of nine genes ( NPHP1 -9 ) account for ‫ف‬ 35% of all NPHP cases, which suggests that other disease-causing loci exist ( Caridi at al., 2006 ;Arts et al., 2007 ;Attanasio et al., 2007 ;Delous et al., 2007 ; for review see Hildebrandt and Zhou, 2007 ;Otto et al., 2008 ). The NPHP gene products, the nephrocystins, have been localized to several regions of the cell including the cilium ( Hildebrandt et al., 1997 ;Olbrich et al., 2003 ;Otto et al., 2003Otto et al., , 2005Mollet et al., 2005 ;Sayer et al., 2006 ). Given that many cell types possess cilia, it is not surprising that NPHP presents with a variety of extrarenal pathologies that may correlate with ciliary dysfunction .
Cilia are specialized microtubule-based organelles that function in motility or sensation. Nonmotile primary cilia are found on most nondividing vertebrate cells ( Wheatley 1995 ).
In general, primary cilia have a 9 + 0 axoneme (nine outer doublet microtubules with no central pair). In the kidney, primary cilia project from the apical surfaces of epithelial cells and act as fl ow mechanosensors Spring, 2001 , 2003 ). In the eye, connecting cilia regulate protein traffi cking between inner and outer photoreceptor segments ( Marszalek et al., 2000 ;. In the mammalian embryo, nodal cilia exhibit a unique rotary motion required for left -right asymmetry ( McGrath et al., 2003 ). Defects in these types of cilia may result in polycystic kidneys, retinal degeneration, and situs inversus, respectively . In humans, perturbation of cilia formation, maintenance, morphogenesis, or function may result in a " ciliopathy " ( Badano et al., 2006 ).
The development of all cilia and fl agella requires intrafl agellar transport (IFT; Rosenbaum and Witman, 2002 ). IFT was fi rst observed in the alga Chlamydomonas reinhardtii as a rapid bidirectional movement of particles along the fl agellar length in a FLA10 kinesin-2 -dependent manner ( Kozminski et al., 1993( Kozminski et al., , 1995. The IFT kinesin-2 motor consists of two different heterodimerized motor subunits and one accessory (KAP) subunit ( Cole et al., 1993 ). The basic unicellular IFT machinery comprises heterotrimeric kinesin-2 and retrograde cytoplasmic dynein motors that move IFT particles and cargo to and from the distal tips of cilia. The IFT particle is composed of two subcomplexes (IFT-A and IFT-B) containing 16 -18 polypeptides ( Cole et al., 1998 ). In C. reinhardtii and the nematode Caenorhabditis N ephronophthisis (NPHP) is the most common genetic cause of end-stage renal disease in children and young adults. In Chlamydomonas reinhardtii , Caenorhabditis elegans , and mammals, the NPHP1 and NPHP4 gene products nephrocystin-1 and nephrocystin-4 localize to basal bodies or ciliary transition zones (TZs), but their function in this location remains unknown. We show here that loss of C. elegans NPHP-1 and NPHP-4 from TZs is tolerated in developing cilia but causes changes in localization of specifi c ciliary components and a broad range of subtle axonemal ultrastructural defects. In amphid channel cilia, nphp-4 mutations cause B tubule defects that further disrupt intrafl agellar transport (IFT). We propose that NPHP-1 and NPHP-4 act globally at the TZ to regulate ciliary access of the IFT machinery, axonemal structural components, and signaling molecules, and that perturbing this balance results in cell typespecifi c phenotypes.
The Caenorhabditis elegans nephrocystins act as global modifi ers of cilium structure molar ratio of active kinesin-II:OSM-3 bound to the IFT particles. Moreover, the basic IFT machinery is modifi ed in a cell-specifi c manner to generate diversity in ciliary shape and function ( Bae et al., 2006 ;Evans et al., 2006 ;Mukhopadhyay et al., 2007 ).
C. elegans nphp-1 and nphp-4 orthologues are expressed in the ciliated sensory nervous system and are coexpressed with the C. elegans autosomal dominant PKD genes lov-1/ PKD1 and pkd-2 ( Jauregui and Barr 2005 ;Winkelbauer et al., 2005 ;Wolf et al., 2005 ). nphp-1 and nphp-4 deletion mutants form cilia as judged by lipophilic fl uorescent dye fi lling and light microscopy, and are only slightly defective in several sensory behaviors. GFP-tagged NPHP-1 and NPHP-4 proteins localize to the ciliary TZ, with NPHP-1 requiring the presence of NPHP-4 for TZ localization ( Winkelbauer et al., 2005 ). This TZ subcellular localization is evolutionarily conserved. Human nephrocystin-1 also localizes to the TZs of renal, respiratory, and photoreceptor connecting cilia ( Schermer et al., 2005 ;Fliegauf et al., 2006 ). Moreover, both mammalian and C. reinhardtii nephrocystin-4 localize to ciliary basal bodies ( Keller et al., 2005 ;Mollet et al., 2005 ). The TZ serves as a boundary between cytoplasmic and ciliary components and has been proposed to act as a " ciliary pore complex " . How the nephrocystins act at the TZ is not known.
In this paper, we address the functions of the nephrocystins in regulating ciliary receptor localization, ciliary shape, and ciliogenesis. nphp-1 and nphp-4 are not required for the ciliary localization of the transient receptor potential vanilloid (TRPV) channel OSM-9 ( Colbert et al., 1997 ) or the TRP polycystin (TRPP) channel PKD-2 ( Barr et al., 2001 ). Rather, nphp-1 and nphp-4 are important modulators of ciliary ultrastructure, with defects resulting in a broad phenotypic spectrum. Although not localizing to the axoneme, NPHP-1 and NPHP-4 indirectly regulate the velocity distributions of most IFT components examined. In addition, a subset of GFPtagged IFT reporters (IFT52/OSM-6, OSM-3, BBS-7, and BBS-8) are mislocalized when NPHP-1 and NPHP-4 are absent from the TZ. In nphp-4 animals, overexpression of OSM-6 results in abnormal association with kinesin-II in amphid middle segments and severe ciliogenic defects, likely because of sequestered IFT components at the TZ. We propose that the nephrocystins function at the TZ to dock and organize proteins destined for the cilium and that perturbing this universal process may have cell type -specifi c consequences on cilium structure and function.
C. elegans is a powerful model system to study ciliogenesis and human ciliopathies Barr, 2005 ;Badano et al., 2006 ). In the worm, IFT builds and maintains cilia on dendritic endings of sensory neurons. The amphids, a pair of lateral sensilla in the head, are the principal sensory organs of nematodes. Amphid channel cilia are ‫ف‬ 7 m in length and can be divided into three distinct segments ( Ward et al., 1975 ;Perkins et al., 1986 ). The transition zone (TZ) is a modifi ed basal body and the place where IFT proteins accumulate for transport into the axoneme. The axoneme has middle and distal segments, consisting of nine doublet (A tubule and B tubule) and nine singlet (A tubule only) microtubules, respectively. In addition to the amphids, four cuticular sensilla (cephalic, inner/ outer labial quadrant, and outer labial lateral) are found in the head and the phasmid sensilla are located in the tail ( Ward et al., 1975 ;Ware et al., 1975 ;Hall and Russell, 1991 ). In the male, each cephalic sensillum contains an additional exposed ciliated neuron (CEM) and the tail possesses sensilla required for sexual behaviors ( Sulston et al., 1980 ;Liu and Sternberg, 1995 ;Barr and Sternberg, 1999 ). These cilia exhibit diverse morphologies and sensory properties ( Sulston et al., 1980 ;Perkins et al., 1986 ;Bargmann, 2006 ).
In C. elegans amphid channel cilia, the IFT machinery has been extensively studied using mutant analysis combined with in vivo time-lapse fl uorescence imaging ( Orozco et al., 1999 ;Blacque et al., 2004 ;Snow et al., 2004 ;Ou et al., 2005Ou et al., , 2007Pan et al., 2006 ). The following model has emerged. Amphid and phasmid channel cilia possess two IFT kinesin-2 motors: heterotrimeric kinesin-II and homodimeric OSM-3 ( Shakir et al., 1993 ;Signor et al., 1999 ). The slow kinesin-II and fast OSM-3 act cooperatively to build the middle segment, whereas OSM-3 acts alone to drive IFT in the distal segment . Consistent with in vivo velocity measurements, purifi ed mixtures of kinesin-II and OSM-3 produce intermediate motility rates in vitro ( Pan et al., 2006 ). The Bardet-Biedl syndrome (BBS) proteins have been proposed to stabilize the association of the two kinesin-2 motors with their associated IFT subcomplexes . In a bbs-1 , bbs-7 , or bbs-8 mutant background, IFT-A moves at the slow kinesin-II rate, whereas IFT-B moves at the fast OSM-3 rate. However, in a kinesin-II; bbs double mutant, IFT-A moves with OSM-3 and, likewise, in an osm-3;bbs double mutant, IFT-B moves with kinesin-II, which suggests that kinesin-II and OSM-3 mechanically compete for IFT-A and IFT-B in a BBS-dependent manner ( Pan et al. 2006 ). Variations from the canonical slow, fast, and intermediate rates are observed in some mutant backgrounds, which indicates that there is an unappreciated complexity to IFT motor regulation ( Burghoorn et al. 2007 ;Mukhopadhyay et al., 2007 ). Deviations observed from canonical rates in vivo may refl ect changes in the normal NPHP-1 AND NPHP-4 REGULATE CILIA LENGTH AND SHAPE • JAUREGUI ET AL.

nphp-4 mutants have ultrastructural defects in CEM, CEP, and OLQ cilia
We used transmission EM (TEM) to examine CEM ciliary ultrastructure of wild-type and nphp-4 males, the latter of which was missing both NPHP-1 and NPHP-4 at the TZ. Wild-type CEM TZs are similar to the CEP and OLQ TZs ( Perkins et al., 1986 ), with 9 + 0 microtubule doublets and Y-linked membrane attachments ( Fig. 4 e ). The CEM and CEP TZs are enclosed within the same sheath and socket cells. The wild-type CEM middle segment swells in diameter and displays many peripheral microtubule singlets and a few dispersed central microtubule singlets ( Fig. 4,c and d ). This is different from amphid channel cilia middle segments, which remain narrow and contain nine microtubule doublets comprised of A and B tubules (see Fig. 8 i). More anteriorly, the wild-type CEM axoneme winds around and away from the CEP cilium and OLQ distal region. CEM distal segments are narrow and contain many dark fi lled microtubule singlets ( Fig. 4 b ) reminiscent of distal segments of ray RnB cilia ( Sulston et al., 1980 ). The most distal region of the CEM curves outward and is exposed to the environment via a narrow cuticular opening ( Ward et al., 1975 ). Here, the CEM cilium typically bends by 90 ° to open laterally; thus, its microtubules are seen lengthwise in a transverse section ( Fig. 4 a ). CEP and OLQ distal segments end embedded in the cuticle at the nose tip ( Perkins et al., 1986 ).

nphp-4 regulates ciliary localization of a subset of IFT components
To determine the molecular basis for the stunted and deformed cilia, we examined the IFT machinery in nphp-1 and nphp-4 mutants. To measure IFT in CEM cilia, we used the pkd-2 promoter to express the IFT-B polypeptide OSM-5 and observed OSM-5::GFP motility in wild-type CEM cilia, nphp-4 stunted cilia, and nphp-1 misshaped/long cilia (Videos 4 -6, available at http://www.jcb.org/cgi/content/full/jcb.200707090/DC1). However, we were unable to measure IFT velocities because the CEMs curve out of the microscopic plane. As IFT has been Ultrastructural defects are also seen in the nearby CEP and OLQ cilia of nphp-4 animals. CEP cilia are enlarged and misshaped, and sometimes displaced posteriorly, elaborating their cilium within the sheath and socket without embedding in the cuticle (unpublished data). In wild-type CEP cilia, the majority of tubulin-associated material (TAM) is found in distal regions ( Fig. 4, a and b ). In nphp-4 CEP cilia, large amounts of TAM are displaced into middle regions ( Fig. 4 j ), with dense aggregations and reduced amounts of TAM in distal segments ( Fig. 4, g and h ). nphp-4 OLQ cilia are occasionally stunted ( Fig. 4 f ), distorted due to severed or missing microtubules ( Fig. 4, g and h ), or posteriorly misplaced ( Fig. 4 i ). In a few cases, the OLQ cilium may be truncated at or near the TZ. Although CEM and OLQ TZs appear normal, few CEP TZs were obtained in our slices, likely because of posterior displacement. We conclude that the TZ proteins NPHP-1 and NPHP-4 regulate ciliary length (OLQ and CEM), ciliary morphogenesis (CEM, CEP, and OLQ), and transport of axonemal components such as TAM (CEP).

NPHP-1 and NPHP-4 are not required for ciliary localization of TRP channels
NPHP-1 and/or NPHP-4 may regulate ciliary receptor localization. In wild-type CEMs, PKD-2::GFP accumulates at the  Error bars indicate ± SEM. (B) CEM cilia were divided into wild-type, curly, and stunted categories. From z stack compressions of 3D wild-type and mutant CEM cilia; lengths were measured by tracing the cilium from the base of the TZ to the end of the axoneme. Curly cilia are signifi cantly longer than the wild type. Stunted or misplaced cilia are slightly shorter than the wild type. The z distance of curls is not represented in this assay. p-values from a t test compare mutants to the wild type.
In conclusion, loss of both NPHP-1 and NPHP-4 but not NPHP-1 alone leads to the abnormal ciliary localization of the IFT-B polypeptide OSM-6, the OSM-3-kinesin, and the BBS proteins BBS-7 and BBS-8. In contrast, NPHP-1 and NPHP-4 are not required for the localization of the IFT-B polypeptides OSM-5 and CHE-2, IFT-A polypeptide CHE-11, kinesin-II, and ciliogenic proteins DYF-1 and DYF-13. . CEM, CEP and OLQ ciliary ultrastructure is abnormal in nphp-4 mutants. TEM of wild type (a -e) and nphp-4 (f -j) mutant animals in transverse thin sections. Black arrows, OLQ; white arrows, CEP; arrowheads, CEM. As depicted in the top drawings, the distal-most region of the CEM axoneme is narrow, curved outward, and exposed to the environment. Microtubule singlets are seen longitudinally near the opening (a). Traveling posteriorly, the distal segment aligns with the body axis and nears the edge of CEP socket cell (b). The CEM middle segment widens and is fi lled with microtubule outer singlets and dispersed inner singlets (c). The middle segment twists around the CEP away from the OLQ such that the CEM axoneme is furthest away from OLQ (d). CEM TZs are similar to CEP and OLQ TZs with 9 + 0 microtubule doublets (e). In nphp-4 mutants, OLQ distal segments are occasionally missing (f) but middle segments are present (g) with missing microtubules (g and h). CEM and OLQ TZs appear normal (i and j). CEP axonemes are irregular with large unidentifi ed accumulations (g -i). Panel j is from a different section of the same worm illustrating well-formed OLQ and CEM TZs but apparently missing a CEP TZ, replaced by a large mass of TAM surrounded by microtubule singlets. CEM axonemes can lack distal segments (empty channel in f with no microtubules). Drawings at bottom illustrate nphp-4 defects. Bars: (top) 100 nm; (bottom) 0.5 m. NPHP-1 AND NPHP-4 REGULATE CILIA LENGTH AND SHAPE • JAUREGUI ET AL.
However, their underlying population of velocities in the middle segment of nphp-4 and nphp-1;nphp-4 animals was significantly different from the wild type ( Table I , f-test), indicating that these IFT components are traveling at a wider range of speeds. Noncanonical IFT velocities have been observed by genetic mutations in vivo or by varying concentrations of kinesin-II and OSM-3 in vitro ( Pan et al., 2006 ;Mukhopadhyay et al., 2007 ). We propose that by genetically perturbing the composition of the IFT machinery, abnormal velocities may emerge.

nphp-4 mutants have B tubule defects in amphid channel cilia
To correlate molecular and biophysical defects in IFT at the ultrastructural level, we used TEM to examine nphp-4 amphid

Overexpression of certain elements of the IFT machinery is toxic to nephrocystin-defi cient cilia
Lipophilic fl uorescent dye fi lling of amphid and phasmid neurons is routinely used to diagnose defects in cilium structure ( Hedgecock et al., 1985 ;Inglis et al., 2006 ). IFT-A, IFT-B, and osm-3 mutants are all Dyf ( Fig. 7 c ; Perkins et al., 1986 ). In contrast, kinesin-II mutants are non-Dyf ( Fig. 7 b ) and have subtle effects on cilium formation and function but obviously affect ciliary transport at the molecular level Ou et al., 2005 ;Bae et al., 2006 ;Evans et al., 2006 ;Mukhopadhyay et al., 2007 ). Likewise, nphp-1 and nphp-4 single and double mutants have no obvious dye fi lling defects ( Jauregui and Barr, 2005 ;Winkelbauer et al., 2005 ) but subtly affect cilia formation at the ultrastructural and molecular levels (this paper). It was puzzling that nphp-4 mutants perturbed the IFT machinery and yet had no overt dye fi lling defects. We therefore reexamined dye fi lling in nphp-4 mutants using a more sensitive assay with reduced DiI concentration and shorter incubation time and observed partial Dyf defects ( Fig. 7, d and f ). Because OSM-6::GFP exhibited abnormal motility rates in middle segments and was largely absent from distal segment of nphp-4 mutants, we examined ciliary integrity of these strains Figure 6. IFT-B polypeptide OSM-6::GFP abnormally associates with kinesin-II in nphp-4 mutants. OSM-6::GFP in amphid (a -d and i -l) and phasmid (e -h and m -p) cilia in various IFT and nphp-4 mutants. The IFT-B polypeptide OSM-6::GFP enters ciliary distal segments in kap-1 and kap-1;bbs-7 single and double mutants (b and f and d and h). OSM-6::GFP localizes to middle segments of osm-3 mutants (c and g) and is absent from missing distal segments. In kap-1;nphp-4 and kap-1;bbs-7;nphp-4 animals, OSM-6::GFP severely aggregates in amphid cilia (j and l, arrowhead) and fails to enter distal segments (j and n and l and p). The arrow in panel n indicates dendritic accumulation of OSM-6::GFP. No IFT is detected, though slight amounts of OSM-6::GFP passively enter severely stunted cilia (n and p). In contrast, IFT is detected in nphp-4;osm-3 double mutants (k and o; Table I  the middle segment, with two to fi ve microtubules interrupted per ciliary axoneme, often not at the same level. Occasionally, one middle segment was missing entirely, with 9 rather than 10 cilia in the amphid channel ( Fig. 8 e ). Because true serial thin sections along the entire amphid channel were not collected, we cannot always determine which cilia are truncated, but some truncations involved cilia with unusual B tubule seam breaks in nearby portions of the middle segment (including ASJ and ASH). Two to four amphid distal segments were often missing, likely because of premature truncation of the distal segments of some cilia ( Fig. 8 d ). Intact distal segments contained normal microtubule singlets. We conclude that nphp-4 mutations do not disrupt TZ formation but do result in axonemal defects in a cell type -specifi c manner.

Discussion
NPHP is one of several human diseases that share the features of ciliary localized gene products and cystic kidneys (for review see Hildebrandt and Zhou, 2007 ). C. elegans nephrocystin-1 and nephrocystin-4 homologues have been implicated in signal transduction ( Jauregui and Barr, 2005 ;Winkelbauer et al., 2005 ;Wolf et al., 2005 ). Here, we rule out a role for the nephrocystins in ciliary receptor localization of TRPP channel PKD-2 and the TRPV channel OSM-9 ( Figs. 2 and S2). Instead, we demonstrate for the fi rst time that nephrocystin-1 and nephrocystin-4 act globally at the TZ to modulate ciliary development and morphogenesis in a cell type -specifi c manner. The human nephrocystins also localize to the TZ of renal and respiratory epithelial cells and photoreceptors ( Mollet et al., 2005 ;Otto et al., 2003Otto et al., , 2005Fliegauf et al., 2006 ), which suggests an evolutionarily conserved function. channel cilia ( Fig. 8 ). Wild-type and nphp-4 TZs appeared similar, with the characteristic 9 + 0 microtubule doublet pattern drawn together by an apical ring and attached to the membrane by Y links ( Fig. 8, c and f ). Wild-type amphid channel middle and distal segments consist of nine doublet (A tubule and B tubule; Fig. 8 j ) and nine singlet (A tubule only) microtubules, respectively. In nphp-4 middle segments, one or two doublet microtubules were occasionally missing entirely or deformed and more often prematurely reduced to singlets ( Fig. 8 e ). Some middle segments possessed abnormal microtubule singlets lacking the B tubule ( Fig. 8, e and i, arrowhead), abnormal doublets with a truncated B tubule, or open B tubules ( Fig. 8 i , arrows). In both wild-type and nphp-4 animals, ADL amphid cilia typically showed inner seam openings of all or most B tubules along most of the middle segment ( Fig. 8, g -i ). Openings at an outer seam of the B tubule were very rare, as were lateral openings of the B tubule (unpublished data). In the wild type, most surrounding amphid cilia showed few or no open B tubules, except for ASI, where more limited inner seam breaks occurred with regularity over short portions.
In nphp-4 mutant amphid channel cilia, B tubule inner seam breaks were increased in number and in linear dimension (compare Fig. 8, k -m , to the wild type in Fig. 8 j ). In nphp-4 animals, ADL cilia still showed the highest degree of inner seam breaks ( Fig. 8, g and m ), and ASI seam breaks were more common and extended over much of the middle segment ( Fig. 8 k ). ASI is one of the two neurons that does not fi ll with DiI in nphp-4 animals ( Fig. 7 f ). In nphp-4 mutants, smaller, limited inner seam breaks also occurred in ASJ ( Fig. 8, g and l ) and to a lesser extent in ASG, ASH, and ASK cilia ( Fig. 8 i ). In affected cilia other than ADL and ASI, individual microtubules generally showed small seam breaks at random along Compression of a 3D deconvoluted z stack of amphid ciliated neurons fi lled with DiI. Wild type (a) and kap-1 mutants (b) fi ll with DiI. In contrast, osm-3 animals (c) lacking distal segments of cilia fail to fi ll with DiI. Some but not all amphid neurons fi ll with DiI in nphp-4 single (d) and kap-1;nphp-4 double mutants (e). Panel f is a 2.5 × view of wild-type and nphp-4 neuron cell bodies that fi lled with dye. There is slight variability in the neurons that fail to fi ll between individual animals. The OSM-6::GFP transgene in the nphp-4 mutant background causes a SynDyf defect (j -l). The mnIs17 OSM-6::GFP transgene does not interfere with dye fi lling in the wild type (g -i) or kap-1 or nphp-1 mutants (not depicted), nor does OSM-6::GFP interfere with ciliogenesis in any published reports ( Collet et al., 1998 ;Snow et al., 2004 ;Ou et al., 2005 ). Bars: (a) 20 m; (f) 5 m. NPHP-1 AND NPHP-4 REGULATE CILIA LENGTH AND SHAPE • JAUREGUI ET AL.
that NPHP-1 and NPHP-4 contribute to ciliary development. In nphp-4 middle segments, microtubule axonemal defects include missing or incomplete B tubules, which suggests that the nephrocystins may be required for A -B microtubule stability ( Stephens, 2000 ). Interestingly, similar B tubules defects have been observed in the hennin/arl13b GTPase mutant mouse, which has defects in ciliary development and sonic hedgehog signaling ( Caspary et al., 2007 ).
Although nphp-4 ciliary ultrastructural defects vary depending on cell type, two generalities may be drawn. First, there is variable phenotypic expressivity within a type of cilium. However, the frequency of nphp-4 CEM ciliary defects ( ‫ف‬ 30%; Fig. 3 ) is similar to the frequency of nphp-4 amphid channel defects (4 of 10 distal segments are missing; Fig. 8 ). Variable phenotypic expressivity is also observed in NPHP patients. Second, nphp-4 ultrastructural defects appear not in the TZ but occur principally in the middle segment, raising the question of how the NPHP-1 The C. elegans nephrocystins act broadly to regulate cilium structure in a cell typespecifi c capacity Previous experiments using DiI as an indicator of cilium structure suggested that nphp-1 and nphp-4 were not required for ciliogenesis ( Jauregui and Barr, 2005 ;Winkelbauer et al., 2005 ). However, these dyes only fi ll amphid and phasmid cilia. A more rigorous diagnosis using fl uorescently labeled ciliary components combined with TEM ultrastructural analysis revealed a broad range of ultrastructural defects in many cilia types. For example, the CEMs of nephrocystin mutants may be excessively long and curled, which suggests that the nephrocystins regulate ciliary morphogenesis and length. In the mechanosensory CEPs, TAM is mislocalized in nphp-4 mutant cilia, which suggests a role for the transport of axonemal components. In amphid channel distal regions, nphp-4 mutants contain six to eight of the normal complement of 10 cilia, indicating Figure 8. Amphid cilia are shortened and have abnormal microtubule structure in nphp-4 mutants. TEM of amphid channel cilia in the wild type (a -c, h, and j) and nphp-4 (d -f, i, and k -m). In nphp-4 , some distal segments are shortened; panel d shows 6 out of the normal 10 distal segments that should be present. Middle segments (e, i, and m) are sometimes swollen or withered. Because of slight cilia bending, views of individual microtubules within a cilium are sometimes seen obliquely and seem distorted; when tilted in the microscope, those distortions go away. Occasionally the channel contains < 10 cilia; in panel e, nine are present, suggesting that at least one cilium is completely absent past the TZ. Some middle segments have microtubule doublets abnormally converting prematurely to singlets lacking B tubules (e and i, arrowhead) or abnormal doublets with an inner seam break of the B tubule (i, arrows), whereas others appear normal. Such inner seam breaks are also found in wild-type cilia of ADL and ASI (h, arrows) but become much more common in other cilia in nphp-4 (i, k, and l). Schematic cartoon (g) displays the region of the middle segment where seam breaks are common in ADL (wild type and mutant) and in ASJ mutant cilia, seen from a lateral aspect. In wild-type and nphp-4 ADL cilia, all microtubules tend to have long continuous seam breaks. In ASJ, shorter, discontinuous seam breaks are scattered along the middle segment, but only in nphp-4 animals. Small spurs can also be seen on the outer surface of many A tubules within the middle segment, looking vaguely similar to Y-link features of the axoneme (compare c with k -m). Panel m shows a nphp-4 ADL cilium, just distal to the axoneme, where an increased number of microtubule doublets surround the outer edge (13 rather than 8 -9), most showing B tubule seam breaks; some inner " singlets " even have partial B tubules. This represents a fused cilium (seen in 2/6 amphids in nphp-4 ); ADL normally forms two separate cilia (g). (i) An electron-dense mass that may represent jammed IFT particles. TZs are typically wellformed in nphp-4 mutants (f). Bars: (a) 0.5 m; (h) 0.5 m; (j) 100 nm.

Cilia length control and the nephrocystins
The misshaped and long CEM cilia of nphp-1 and nphp-4 mutants are unique. The nature of this defect is not known, nor do we know the reason for the higher frequency of misshaped cilia in nphp-1 animals. Marshall et al. ( 2005 ) have proposed that ciliary length is controlled by a balance between ciliary assembly and disassembly and that disrupting this balance could result in long, short, or no cilia ( Wemmer and Marshall, 2007 ). Human nephrocystin interacts with nephrocystin-2/inversin, a protein that binds to ␤ -tubulin Nurnberger et al., 2004 ), and nephrocystin-3, a protein that contains a tubulin tyrosine ligase (TTL) domain ( Olbrich et al., 2003 ). TTLs and other tubulin modifi cation enzymes catalyze tubulin posttranslational modifi cations, which regulate axonemal microtubules and perhaps IFT ( Janke et al., 2005 ;Redeker et al., 2005 ;Pathak et al., 2007 ;Stephan et al., 2007 ). In C. elegans , the ODR-3 G ␣ protein, kinesin-II, and the mitogen-activated protein kinase DYF-5 regulate ciliary morphogenesis and length in a cell typespecifi c manner ( Roayaie et al., 1998 ;Bae et al., 2006 ;Evans et al., 2006 ;Burghoorn et al., 2007 ). In mammalian cells, overexpression or constitutive activation of the Rab8 GDP/GTP exhange factor results in abnormally long cilia ( Nachury et al., 2007 ;Yoshimura et al., 2007 ). In Bbs4 Ϫ / Ϫ mice, primary cilia length is also increased ( Mokrzan et al., 2007 ). The NIMA (never in mitosis A) kinase nek8 is mutated in human NPHP9 patients and the juvenile cystic kidney ( jck ) mouse model, which has primary cilia that are signifi cantly longer than in the wild type ( Smith et al., 2006 ;Otto et al., 2008 ). Mislocalization or misregulation of these types of proteins may cause defects in ciliary length or shape in nephrocystin mutants (Fig. S4).

Genetic modifi ers in cilia development, maintenance, and function
Loss of the nephrocystins may sensitize cells to ciliogenic defects, explaining why overexpression of some IFT components in the absence of nphp-4 increases ciliary defects ( Fig. 7 , j -l ; and Fig. S4). Genetic and functional redundancy is a mechanism to ensure normal development and protect against disease. Intriguingly, genetic modifi er loci may account for extrarenal symptoms associated with NPHP ( Parisi et al., 2004 ;Tory et al., 2007 ), and oligogenicity is observed in some NPHP patients ( Hoefele et al., 2007 ). In the pcy /NPHP3 mouse, genetic background affects the cystic phenotype ( Guay-Woodford, 2003 ). In humans, NPHP has clinical phenotypes that overlap Joubert and Meckel syndromes ( Delous et al., 2007 ).
Biochemical approaches have been used to study the physical interactions of IFT components ( Cole et al., 1998 ;Cole, and NPHP-4 TZ proteins act. Rosenbaum and Witman (2002) have proposed that the transition fi bers at the base of the C. reinhardtii fl agellum serve as a " fl agellar pore complex " to restrict the access of proteins to the cilium. At the bioinformatics level, some IFT proteins are related by sequence to components of the nuclear pore complex ( Jekely and Arendt, 2006 ). At the ultrastructural level, transition fi bers form physical links between basal body microtubules and the fl agellar membrane ( Ringo, 1967 ;Weiss et al., 1977 ). At the molecular level, most of the IFT particles accumulate at the basal body ( Cole et al., 1998 ;Pazour et al., 1999 ). The C. elegans ciliary TZ also possesses links from microtubules to the membrane and accumulates IFT motors and polypeptides ( Figs. 4 e and 8 f ; Perkins et al., 1986 ). Unlike IFT components that localize to the TZ and ciliary compartment, the C. elegans nephrocystins localize to the TZ and are not detected in the cilium proper ( Fig. 1 ; Jauregui and Barr, 2005 ;Winkelbauer et al., 2005 ). Notably, immunoelectron microscopy shows that IFT52 (homologous to C. elegans OSM-6) associates at the periphery of transition fi bers, leading to the proposal that this region serves as an IFT docking site ( Deane et al., 2001 ). Our data suggests that NPHP-4 and OSM-6 functionally interact to regulate ciliogenesis ( Figs. 7 and S4).
In amphid channel neurons, nphp-4 mutations (lacking both NPHP-1 and NPHP-4 at the TZ) increased B tubule breaks in the middle segment, which in turn likely affected IFT velocity distributions ( Table I , f-test column). We propose that NPHP-1 and NPHP-4 may indirectly regulate the balance between the IFT motors, control the composition of individual IFT particles with a cilium, and/or modulate IFT in a cell type -specifi c manner that is masked by an average taken from 10 axonemes. In this respect, it is notable that a minority of cilia within the amphid bundle exhibit different assembly patterns even in the wild type, including a pronounced weakness at the inner seam junction between A and B tubules. These structural weaknesses become more general in the nphp-4 mutant cilia, now affecting the majority of all cilia.
Surprisingly, IFT-B polypeptide OSM-6::GFP moved at the slow kinesin-II rate in nphp-4 and nphp-1;nphp-4 middle segments and produced the SynDyf phenotype, refl ecting a severe defect in ciliogenesis. The former result is not predicted by the current IFT model, whereby the kinesin-II/IFT-A and OSM-3/ IFT-B units are linked by the BBS proteins . However, IFT-A may travel with OSM-3 and IFT-B may travel with kinesin-II in a bbs;kinesin-2 double mutant, indicating that genetic background infl uences IFT complex constituents in vivo ( Pan et al., 2006 ). The latter results indicate that OSM-6::GFP overexpression is toxic to nphp-4 cilia. Why OSM-6 is an outlier is not resolved. However, OSM-6 is different from other IFT polypeptides with respect to the C. elegans mutant phenotype ( Perkins et al., 1986 ;Collet et al. 1998 ) and domain structure ( Beatson and Ponting, 2004 ;Jekely and Arendt 2006 ). Although mutations in the IFT-B polypeptide encoding genes osm-1 , osm-5 , and che-13 disrupt middle and distal amphid segments, the osm-6 mutant has a less severe phenotype, primarily lacking distal segments ( Perkins et al., 1986 ). With the exception of OSM-6/IFT52 and the GTPase IFT27, IFT polypeptides contain WD40, TRP, or coiled-coil protein -NPHP-1 AND NPHP-4 REGULATE CILIA LENGTH AND SHAPE • JAUREGUI ET AL.

Dye-fi lling assay
Standard dye-fi lling assays were performed ( Perkins et al., 1986 ). Alternatively, reduced dye concentration and incubation time were used. A 2 mg/ml DiD or DiI (Invitrogen) in dimethyl formamide stock was diluted 1:1,000 in M9. Worms were incubated for < 30 min in this solution and washed three times with M9.

Microscopy
Animals were raised at 20 ° C and imaged at 25 ° C using standard C. elegans slide mounts and Plan Apochroma 63 × 1.4 oil differential interference contrast (DIC), 100 × 1.3 oil DIC, or 100 × 1.4 oil DIC objectives (Carl Zeiss, Inc.) on an imaging microscope (Axioplan 2; Carl Zeiss, Inc.). Fluorochromes used were either eGFP or tdTomato. CEM cilia were visualized using Ppkd-2::tbb-4::GFP that labels cell bodies, dendrites/axons, and cilia. Frequency of abnormally stunted or curly CEM cilia was scored for at least 50 animals scored per genotype. Wild-type animals have four CEM cilia that gently curve outward toward the cuticle. Stunted was defi ned as shortened or misplaced CEM axonemes that did not traverse the distance to the cuticle and lacked gentle outward curvature. Curly CEM cilia were defi ned as axonemes that were typically longer than the wild type and had strong curvature. Those CEM cilia that were diffi cult to image were not categorized. The score for each animal was determined by the number of abnormal CEM cilia divided by the total number of scorable CEM cilia. Percentages were then averaged to determine a population mean. To measure CEM cilia length, a z stack was taken and compressed into a single image. The CEM cilia were then traced using Metamorph software (MDS Analytical Technologies) and the length was recorded and averaged.
IFT motility was observed with an Axioplan 2 imaging microscope using a Plan APOCHROMA 100 × 1.3 or 1.4 NA oil objective (Carl Zeiss, Inc.). Motility stacks were recorded using a camera (Photometrics Cascade 512B; Roper Scientifi c), and kymographs and the particle movement rate were measured using Metamorph software. Strains were synchronized by picking L4 worms, culturing at 15 ° C overnight, and imaging within 24 h. Worms were anesthetized in a drop of M9 containing 10 mM muscimol, transferred to an agarose mount slide, and imaged immediately.
" Dim in cilia " is defi ned as signifi cantly reduced fl uorescence of IFT:: GFP reporters in cilia compared with the wild type (Fig. S3). For mutants with " dim cilia, " threshold adjustments were increased to visualize cilia. Additionally, " dim distal " is defi ned as amphid distal segments that have signifi cantly reduced fl uorescence of IFT::GFP reporters when compared with adjacent middle segments. The basis of the " dim " phenotype is likely a combination of reduced number of distal segments in the amphid channel and reduced frequency of IFT particles entering the distal segment.
For TEM, worms were fi xed in 3% glutaraldehyde in cacodylate buffer on ice. Then, heads were cut off, moved to fresh fi xative, and held in fi x 2003 ; Qin et al., 2004 ;Lucker et al., 2005 ). Genetic approaches may be exploited to identify functional relationships between ciliary proteins. In the budding yeast, functional interactions are determined by a synthetic dosage lethality phenotype, where overexpression of a normally nontoxic gene induces lethality in the context of a viable mutant ( Kroll et al., 1996 ). For example, a genome-wide synthetic dosage lethal screen identifi ed 141 nonessential deletion mutants that cannot tolerate overexpression of kinetochore genes ( Measday et al., 2005 ). We propose that SynDyf screens, in which overexpression of predicted cilioproteins (such as OSM-6::GFP) in non-Dyf or partial Dyf ciliary mutants (such as nphp-4 ) exacerbates the Dyf defect, will reveal functional rather than physical interactions. This type of strategy will allow for the systematic dissection of proteins involved in ciliogenesis and potentially identify genetic modifi er loci and candidates for human disease genes. Genetic analysis in simple animal models will continue to progress our understanding of human ciliopathies.