Ciliary biology Understanding the cellular and genetic basis of human ciliopathies.код для вставкиСкачать
American Journal of Medical Genetics Part C (Seminars in Medical Genetics) 151C:263 – 280 (2009) A R T I C L E Ciliary Biology: Understanding the Cellular and Genetic Basis of Human Ciliopathies MAGDALENA CARDENAS-RODRIGUEZ AND JOSE L. BADANO* Motile cilia have long been known to play a role in processes such as cell locomotion and fluid movement whereas the functions of primary cilia have remained obscure until recent years. To date, ciliary dysfunction has been shown to be causally linked to a number of clinical manifestations that characterize the group of human disorders known as ciliopathies. This classification reflects a common or shared cellular basis and implies that it is possible to associate a series of different human conditions with ciliary dysfunction, which allows gaining insight into the cellular defect in disorders of unknown etiology solely based on phenotypic observations. Furthermore, to date we know that the cilium participates in a number of biological processes ranging from chemo- and mechanosensation to the transduction of a growing list of paracrine signaling cascades that are critical for the development and maintenance of different tissues and organs. Consequently, the primary cilium has been identified as a key structure necessary to regulate and maintain cellular and tissue homeostasis and thus its study is providing significant information to understand the pathogenesis of the different phenotypes that characterize these human conditions. Finally, the similarities between different ciliopathies at the phenotypic level are proving to be due to their shared cellular defect and also their common genetic basis. To this end, recent studies are showing that mutations in a given ciliary gene often appear involved in the pathogenesis of more than one clinical entity, complicating their genetic dissection, and hindering our ability to generate accurate genotype–phenotype correlations. ß 2009 Wiley-Liss, Inc. KEY WORDS: cilia; cystic kidney disease; retinal dystrophy; polydactyly; obesity; Wnt; Shh; PDGF How to cite this article: Cardenas-Rodriguez M, Badano JL. 2009. Ciliary biology: Understanding the cellular and genetic basis of human ciliopathies. Am J Med Genet Part C Semin Med Genet 151C:263–280. INTRODUCTION Cilia are evolutionary conserved organelles that have been recognized for more than a 100 years [Zimmermann, 1898]. These antenna-like structures can be classified in two main types according to their ultrastructure and their capacity to move: motile and immotile/primary cilia. While cilia immotility has long been associated with distinct clinical manifestations, it has been only recently realized the role that the primary cilium is playing in the pathogenesis of several human conditions. Consistent with their broad cellular distribution, their evolutionary conservation, and their emerging role in the transduction of important paracrine signaling pathways, perturbations in the function of primary cilia are being implicated in a wide spectrum of human diseases: the ciliopathies. This classification includes a number of disorders that range from polycystic kidney disease (PKD) and nephronophthisis to broad pleiotropic syndromes [Badano et al., 2006b; Sharma et al., 2008]. Here we review the basic biology of cilia and the multiple roles that have been ascribed to these organelles to highlight how this knowledge is shedding light into our understanding of the cellular and genetic basis of this group of human disorders. THE CILIUM: BASIC BIOLOGY Structure and Classification Magdalena Cardenas-Rodriguez (MSc) is a Ph.D. student in the ‘‘Program for the development of basic sciences’’ (PEDECIBA) from the Universidad de la Republica, Uruguay. She is conducting her thesis work in Dr. Badano’s laboratory working on the functional characterization of BBS and BBS modifier proteins. Dr. Jose L. Badano holds a faculty position at the Institut Pasteur of Montevideo, Uruguay. He received his Ph.D. from the Department of Molecular and Human Genetics at Baylor College of Medicine, Houston, TX and performed his post-doctoral training with Dr. Nicholas Katsanis at Johns Hopkins University, Baltimore, MD, where he continued his work on Bardet-Biedl syndrome and other ciliopathies Grant sponsor: Genzyme Renal Innovations Program (GRIP); Grant sponsor: Agencia Nacional de Investigación e Innovación (ANII); Grant sponsor: Programa de Apoyo Sectorial a la Estrategia Nacional de Innovación - INNOVA URUGUAY; Grant number: DCI-ALA/2007/19.040. *Correspondence to: Jose L. Badano, Institut Pasteur de Montevideo, Mataojo 2020, Montevideo CP 11400, Uruguay. E-mail: firstname.lastname@example.org Received 15 June 2009; Accepted 14 September 2009 DOI 10.1002/ajmg.c.30227 Published online 27 October 2009 in Wiley InterScience(www.interscience.wiley.com) ß 2009 Wiley-Liss, Inc. Cilia and flagella extend from the cellular membrane of non-proliferating cells and are composed of a microtubule axoneme that emanates from a basal body, a structure composed of nine microtubule triplets that derives from the mother centriole of the centrosome [Rosenbaum and Witman, 2002]. In general, motile cilia axonemes are composed of nine outer microtubule doublets surrounding a central pair in a 9 þ 2 configuration. Inner and outer dynein arms are responsible for generating force 264 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) A 9+2 IFT particle 9+0 ARTICLE B G0 Kinesin-II Cytoplasmic dynein or Retrograde IFT Anterograde IFT G1 Outer dynein arm Inner dynein arm Radial spoke Ciliogenesis and M cell cycle Axoneme G2 S Centriole duplication Transition fibers Basal Body Figure 1. Basic ciliary structure. A: Schematic representation of a cilium and cross-section of a basal body composed of microtubule triplets and a ‘‘9 þ 2’’ and a ‘‘9 þ 0’’ axoneme showing the position of dynein arms and radial spokes needed for force generation and coordination. Along the outer microtubule doublets of the axoneme, molecular motors transport IFT particles. B: Ciliogenesis is tightly linked to cell cycle progression occurring in G1/G0. Mouse kidney cells (IMCD3) are shown where g- and acetylated tubulin have been stained in green showing centrioles and the axoneme. DAPI has been used to stain DNA. whereas radial spokes regulate the direction of ciliary beating. Primary cilia generally lack the central pair of microtubules (9 þ 0 configuration) and the inner dynein arms (Fig. 1A). However, this classification is simplistic and motile 9 þ 0 and immotile 9 þ 2 cilia can be found. For example, 9 þ 0 cilia in the embryonic node move in a vortical fashion to generate extra-embryonic fluid flow (see below) and the immotile kinocilium in the inner ear has a 9 þ 2 configuration. Since protein synthesis does not occur inside cilia, cells have developed a specialized mechanism named intraflagellar transport (IFT), first described in the flagellated single-celled green algae Chlamydomonas reinhardtii, necessary for the formation, maintenance and function of cilia [Kozminski et al., 1993]. Since protein synthesis does not occur inside cilia, cells have developed a specialized mechanism named intraflagellar transport (IFT), first described in the flagellated single-celled green algae Chlamydomonas reinhardtii, necessary for the formation, maintenance and function of cilia IFT requires the coordinated action of structural, adaptor, and molecular motors to move IFT particles in and out of the cilium (anterograde and retrograde movement, respectively). Anterograde movement is achieved through kinesin-II, a heterotrimeric complex formed by two motor subunits, KIF3A and KIF3B in vertebrates, and a non-motor subunit called KAP. Interestingly, it has been shown that other kinesins can also participate in the process although their exact role needs to be determined. The molecular motor responsible for retrograde transport is cytoplasmic dynein 2, which in turn is composed of heavy, intermediate and light chains [Fig. 1A; for an in depth review of IFT see Rosenbaum and Witman, 2002; Pedersen and Rosenbaum, 2008]. IFT is critical to maintain the functionality of cilia and thus its disruption or impairment is causally linked to different human phenotypes and conditions that will be discussed in following sections. One particular cell type that heavily depends on intact IFT is the photoreceptor and consequently, retinal degeneration is a characteristic phenotypic outcome of ciliary dysfunction [Table I; for an in depth review see Insinna and Besharse, 2008]. The outer segment of the photoreceptors derives from the plasma membrane of a modified primary cilium that also connects it with the inner segment. Therefore, both the formation and maintenance of the photoreceptor outer segment requires IFT and defects in this process result in photoreceptor cell death and retinal degeneration in a manner that is proportional to the disruption in IFT. For example, mutations in the IFT proteins IFT88 or IFT57 which abrogate or reduce IFT respectively, result in either lost or short outer segments [Pazour et al., 2002a; Krock and Perkins, 2008]. Similarly, conditional depletion of Kif3a in photoreceptors results in the accumulation of proteins normally transported into the outer segment, such as opsin and arrestin, leading to cell death [Marszalek et al., 2000]. ARTICLE AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) 265 TABLE I. Principal Phenotypes Observed in the Ciliopathies PKD NPHP MKKS SLSN CNS malformations Cystic kidney Diabetes Gonadal malformations Heart disease Hepatic dysfunction Mental retardation/Developmental delay Obesity Polydactyly Pulmonary dysfunction Retinal degeneration Left–right asymmetry defects Skeletal defects * * JATD * * * * * * * EVC * OFD ALMS * * * * * * * * * * * * * * * * * * JS BBS MKS * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * PKD, polycystic kidney disease; NPHP, nephronophthisis; MKKS, McKusick–Kaufman syndrome; SLSN, Senior–Løken syndrome; EVC, Ellis-van Creveld; JATD, Jeune asphyxiating thoracic dystrophy; OFD, orofaciodigital syndrome; ALMS, Alström syndrome; JS, Joubert syndrome/Cerebello-oculo-renal syndrome; BBS, Bardet–Biedl syndrome; MKS, Meckel–Gruber syndrome; CNS, Central nervous system. Ciliogenesis and Cell Cycle Cilia are post-mitotic structures that are present while cells are in G0/G1 and the beginning of the S phase, before the centrioles are needed to organize the mitotic spindle (Fig. 1B). Importantly, the tight link between cilia formation/ disassembly and cell cycle progression not only relies in the availability of centrioles but also is supported by the specific activity of centrosomal proteins participating in the control of ciliogenesis [reviewed by Santos and Reiter, Cilia are post-mitotic structures that are present while cells are in G0/G1 and the beginning of the S phase, before the centrioles are needed to organize the mitotic spindle (Fig. 1B). Importantly, the tight link between cilia formation/disassembly and cell cycle progression not only relies in the availability of centrioles but also is supported by the specific activity of centrosomal proteins participating in the control of ciliogenesis 2008]. For example, CP110 is a protein involved in centrosome duplication and cytokinesis that has been shown to inhibit ciliogenesis through an interaction with Cep97 and CEP290, a protein that is mutated in several ciliopathies [Table II; Spektor et al., 2007; Tsang et al., 2008]. Depletion of either Cep97 or CP110 uncouples the ciliary cycle and cell division leading to the formation of cilia in proliferating cells while overexpression of CP110 in serum-starved cells inhibits ciliogenesis [Spektor et al., 2007]. Cells generally reabsorb the cilium in order to divide and thus the disassembly of cilia is also tightly regulated. Aurora A (AurA), a centrosomal protein involved in the regulation of mitotic entry, has been shown to interact with the focal adhesion scaffolding protein HEF1, to facilitate ciliary disassembly by promoting deacetylation of axonemal tubulin through the histone deacetylase HDAC6 [Pugacheva et al., 2007]. Another example is the family of NIMA-related protein kinases (Nrks or Neks) which have been postulated to play a role in the coordination between cell cycle and cilia [Parker et al., 2007]. Importantly, mutations in Nek1 and Nek8 are responsible for two mouse models of cystic kidney disease, a hallmark feature of ciliary dysfunction [Table I; Upadhya et al., 2000; Liu et al., 2002]. As we will discuss in following sections, cilia can regulate cell proliferation through the modulation of different signaling cascades. In addition, several ciliary proteins appear to directly affect cell proliferation through putative extraciliary roles. For example, depletion of IFT27 in Chlamydomonas results in the expected loss of flagella but also cytokinesis defects [Qin et al., 2007]. In mammalian cell lines, overexpression of IFT88, which remains associated with the centrosome through the cell cycle, leads to cell cycle arrest regulating the G1–S transition. Furthermore, it has been shown that IFT88 inhibits the RNA polymerase II binding protein Che1 which is no longer able to suppress retinoblastoma (Rb), a 266 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) ARTICLE TABLE II. List of Selected Ciliary Genes/Proteins and Their Link to Ciliopathies Gene Protein NPHP1 NPHP2 Nephrocystin Inversin NPHP3 NPHP4 NPHP5/IQCB1 CEP290/NPHP6 NPHP7/GLIS2 RPGRIP1L/NPHP8 NPHP9/NEK8 EVC IFT80 Nephrocystin-3 Nephrocystin-4 Nephrocystin-5 CEP290 GLIS2 RPGRIP1L NEK8 EVC IFT80 BBS1 BBS2 BBS3 BBS1 BBS2 BBS3 BBS4 BBS5 BBS6/MKKS BBS4 BBS5 BBS6 BBS7 BBS8 BBS9 BBS10 BBS11 BBS7 BBS8 BBS9 BBS10 BBS11 BBS12 MKS1 MKS3 CC2D2A AHl1 ALMS1 OFD1 BBS12 MKS1 Meckelin CC2D2A Jouberin ALMS1 OFD1 OFD2 OFD2/cenexin Functional information Syndrome Basal body/cilia, cell–cell junctions. Regulate ciliary access Centrosomes/basal body/cilia, cell–cell junctions. Involved in Wnt signalling Cilia. Suggested role in Wnt signaling Centrosomes/basal body/cilia, also actin cytoskeleton Primary cilia in renal tubular epithelial cells and retinal cells Centrosome/basal body/cilia; ciliogenesis Possible role in Shh signalling Basal bodies; possible role in Shh signalling Cell cycle regulation Chondrocyte cilia; involved in Hh signaling Basal body/axoneme; IFT particle. Possible role in Shh signalling Centrosome/basal body; IFT and intracellular transport Centrosome/basal body Member of the Ras superfamily of small GTP-binding proteins; possible role in ciliary transport Centrosome/basal body. Pericentriolar organization Basal body; possible role in ciliogenesis Centrosome/basal body; group II chaperonin-like protein; possible role in cell cycle Basal body; possible role in IFT Centrosome/basal body; possible role in IFT Possible role in adipogenesis Group II chaperonin-like protein Similar to E3 ubiquitin ligase; possible role in proteasome degradation Group II chaperonin-like protein Centrosome/basal body; ciliogenesis Cilia and plasma membrane in ciliated cell-lines; ciliogenesis Basal body, ciliogenesis Centrosome/basal body/cilia; cell–cell junctions Centrosome/basal body; ciliary assembly Centrosome/basal body. Implicated in left–right axis specification and CE movements Formation of distal/subdistal appendages of mother centrioles and ciliogenesis NPHP, SLSN, JS NPHP NPHP, NPHP, NPHP, NPHP, NPHP NPHP, NPHP EVC JATD MKS-like SLSN SLSN BBS, MKS, JS MKS, JS BBS BBS, MKS-like BBS BBS, MKS-like BBS BBS, MKKS, MKS-like BBS BBS BBS BBS BBS BBS MKS, BBS MKS, JS, BBS, NPHP MKS, JS JS, NPHP ALMS OFD OFD NPHP, nephronophthisis; MKKS, McKusick–Kaufman syndrome; SLSN, Senior–Løken syndrome; EVC, Ellis-van Creveld; JATD, Jeune asphyxiating thoracic dystrophy; OFD, orofaciodigital syndrome; ALMS, Alström syndrome; JS, Joubert syndrome; BBS, Bardet–Biedl syndrome; MKS, Meckel–Gruber syndrome; CE, convergence and extension; IFT, intraflagellar transport; Hh, hedgehog; Shh, sonic hedgehog. negative regulator of the cell cycle [Robert et al., 2007]. Interestingly, ciliary dysfunction in vivo does not seem to result in marked cell proliferation defects nor it is associated with oncogenic phenotypes suggesting that there is redundancy in the system and cell cycle checkpoints are not largely affected. CILIARY DYSFUNCTION IN HUMAN DISEASE Motile Cilia Dysfunction: Primary Ciliary Dyskinesia, Hydrocephalus and Left–Right Determination Ciliary immotility in the respiratory tract and the sperm flagellum has long been associated with a defined set of human phenotypes. Afzelius  observed cilia lacking dynein arms in patients with immotile sperm and respiratory problems, characteristic phenotypes in patients with primary ciliary dyskinesia (PCD, OMIM 244400) [Afzelius, 1976]. To date we know that mutations in either of several genes ARTICLE encoding different components of the highly complex machinery required to generate and coordinate ciliary movement, can cause this condition (Fig. 1A). For example, mutations in genes encoding for dynein intermediate and heavy chains, such as DNAI1, DNAH5, and DNAH11, and other structural ciliary defects have been found in PCD patients [Pennarun et al., 1999; Bartoloni et al., 2002; Olbrich et al., 2002; Sharma et al., 2008]. While sinusitis, infertility and bronchiectasis were, to a degree, expected consequences from ciliary immotility in heavily ciliated tissues, the cellular basis of hydrocephalus, a condition also affecting PCD patients, was not fully understood. Interestingly, it is now known that ciliary beating in the ependymal cells lining the brain ventricles generates a flow of cerebrospinal fluid that is necessary to maintain an open aqueduct and mutations in the axonemal dynein heavy chain Mdnah5 in mice result in defective ependymal flow and hydrocephalus [Ibanez-Tallon et al., 2004]. Furthermore, the Chlamydomonas ortholog of Hydin, mutated in the congenital hydrocephalus hy3 mouse, is a component of the central pair of microtubules that is required for motility of the flagellum and cilia in hydin mutant mice have been shown to present structural axonemal defects and seriously compromised motility [Lechtreck and Witman, 2007; Lechtreck et al., 2008]. Another hallmark feature of ciliary dysfunction is defective determination of the left–right axis of symmetry (Table I), a characteristic feature of Kartagener syndrome (KS; OMIM 244400) which is defined by PCD and situs inversus. In vertebrates, organs are normally distributed asymmetrically between the left and right (LR) side of the body and situs inversus and heterotaxy denote a complete reversal or partial mis-positioning of organs, respectively. Interestingly, it has been shown that the first asymmetries in LR are established through the activity of motile primary cilia in the LR organizer: the embryonic node in the mouse or Kupffer’s vesicle in zebrafish for example. Using Kif3b mutant mice Nonaka and colleagues AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) demonstrated that motile primary cilia in the node are responsible for generating a leftward flow of extra-embryonic fluid, the nodal flow, which represents the earliest recognizable LR asymmetry in the developing embryo [Nonaka et al., 1998]. Furthermore, an artificially created flow applied over mouse embryos in culture is able to determine the LR axis in both wild type embryos and mutants with immotile cilia [Nonaka et al., 2002]. Additionally, mutations in the left–right dynein (lrd) gene, in the mouse model inversus viscerum (iv/iv), and in the axonemal dynein heavy chain 5 (DnaHC5), result in immotile cilia and randomization of body situs [Supp et al., 1997; Okada et al., 1999; Olbrich et al., 2002; McGrath et al., 2003]. Two main models of LR determination have been proposed although neither is sufficient to satisfactorily explain the diverse LR defects observed in patients and animal models. The first model postulates that the leftward flow is sensed by mechanosensory cilia on the right side of the node that upon bending, initiate a Ca2þ signaling cascade that is translated into differential gene expression of Nodal and Lefty [reviewed by Basu and Brueckner, 2008]. Supporting this model, it has been shown that there are two types of primary nodal cilia: a group of cilia localized in the center of the node that express the axonemal dynein lrd and can generate fluid flow through a clockwise rotational movement and a second type of non-motile cilia that sense the mechanical stimulus in a process that requires polycystin-2 (PC2), a protein mutated in human polycystic kidney disease [see below; McGrath et al., 2003]. The second model is based on a flow-generated morphogen gradient that could initiate the LR specification cascade [Okada et al., 2005]. Interestingly, fibroblast growth factor (FGF) signaling can regulate the release of nodal vesicular parcels (NVPs), membrane covered particles that are enriched for Sonic hedgehog and retinoic acid. These NVPs are transported by nodal flow and are thought to release their cargo on the left wall of the node to initiate signaling [Tanaka et al., 2005]. 267 Non-Motile Primary Cilia and Their Link to Human Disease In contrast to motile cilia, the association between the primary cilium and human disease had to wait until recent years and a key model has undoubtedly been the Oak Ridge polycystic kidney (orpk) In contrast to motile cilia, the association between the primary cilium and human disease had to wait until recent years and a key model has undoubtedly been the Oak Ridge polycystic kidney (orpk) mouse. mouse. Orpk mice bear a hypomorphic allele of Tg737 which encodes the mouse ortholog of Chlamydomonas IFT88, a protein that localizes to basal bodies and cilia and is involved in IFT. Interestingly, both motile and primary cilia in Tg737orpk animals are structurally defective and shorter than normal cilia while complete Tg737 nulls lack cilia and present with neural tube problems, LR defects and growth arrest during embryogenesis [Moyer et al., 1994; Yoder et al., 1995; Murcia et al., 2000; Pazour et al., 2000; Taulman et al., 2001]. The orpk mouse is a model of autosomal recessive polycystic kidney disease (ARPKD; OMIM 263200), a condition that presents in early childhood and is characterized by renal cysts and liver fibrosis providing a direct link between primary cilia and human disease. The Role of Cilia in the Pathogenesis of Cystic Kidney Disease Cystic diseases of the kidney (CDKs) are a group of human genetic disorders characterized by the formation of renal cysts that range from PKD to syndromes in which the formation of cysts in the kidney is a feature of a broader spectrum 268 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) of clinical manifestations. A number of mouse models and the identification of genes causing CDKs in humans have further supported the link between ciliary dysfunction and cystogenesis. For example cystin, the protein encoded by the Cys1 gene, which is mutated in the congenital polycystic kidney mouse (cpk), localizes to cilia in renal epithelial cells [Yoder et al., 2002]. Also, the absence of cilia in the renal epithelia of mice in which Kif3A has been conditionally targeted correlates with the formation of renal cysts [Lin et al., 2003]. Studies in human conditions such as autosomal dominant and recessive polycystic kidney disease (ADPKD and ARPKD) have also supported a ciliary role during cystogenesis. Mutations in two genes, PKD1 and PKD2, encoding the proteins polycystin-1 (PC1) and 2 (PC2), respectively, are the most frequent genetic alterations found in ADPKD [Consortium TEPKD, 1994; Mochizuki et al., 1996]. PC1 is a large transmembrane protein that interacts with PC2, a nonselective cation channel, to form a Ca2þ channel that localizes to primary cilia in the renal epithelium [Qian et al., 1997; Tsiokas et al., 1997; Hanaoka et al., 2000; Gonzalez-Perrett et al., 2001; Yoder et al., 2002; Pazour et al., 2002b]. ARPKD is caused by mutations in PKHD1, which also encodes a ciliary protein named polyductin/ fibrocystin [Onuchic et al., 2002; Ward et al., 2002, 2003]. Although the exact function of polyductin/fibrocystin is still not known, it has been suggested that it plays a role in collecting duct cell differentiation by interacting with PC2 and regulating its function [Mai et al., 2005; Wang et al., 2007; Kim et al., 2008]. Importantly, mutant Pkhd1 mice present with hepatic, pancreatic and renal defects and cilia in these animals are significantly shorter than in controls [Woollard et al., 2007]. In renal tubules, PC1 and PC2 have been proposed to mediate Ca2þ signaling upon ciliary bending, similarly to the putative role of cilia and PC2 in LR determination [Praetorius and Spring, 2001; Pennekamp et al., 2002; McGrath et al., 2003; Nauli et al., 2003]. Additionally, PC1 positive exosome-like vesicles (ELV), also enriched for various signaling molecules, have been found in urine and attached to renal epithelia cilia leading to the speculation that a model similar to the nodal vesicular parcel is also operating in the kidney [Pisitkun et al., 2004; Harris and Torres, 2009; Hogan et al., 2009]. However, two independent groups have shown that the cystic kidney phenotype resulting from disruption of Pkd1, Tg737, or Kif3a depends on the developmental time at which these genes are inactivated [Davenport et al., 2007; Piontek et al., 2007]. Using mice with a conditional Pkd1 allele, Piontek et al. have shown that the controlled inactivation of Pkd1 before postnatal day 13 results in cysts in 3 weeks whereas animals in which the gene is disrupted at day 14 or later develop cysts after 5 months [Piontek et al., 2007]. These data led the authors to suggest that the homeostasis of the tissue might present different requirements of ciliary mediated flow sensing during development or in the adult kidney and also that other mechanisms are likely involved in determining the onset and progression of cystic kidney disease [Davenport et al., 2007; Piontek et al., 2007]. Ciliary Dysfunction: A Unifying Defect in Cystic Kidney Disease The link between cilia and cystogenesis is not restricted to PKD but rather it has been postulated to be the unifying cellular defect underlying most if not all CDKs [Watnick and Germino, 2003; Hildebrandt and Otto, 2005]. This concept is supported by studies in nephronophthisis (NPHP; OMIM 256100), an autosomal recessive cystic kidney disease and the most frequent genetic cause of end stage renal disease in children and young individuals. It is characterized by the formation of corticomedulary cysts, interstitial fibrosis and renal insufficiency. In addition, NPHP can present associated with extra-renal phenotypes such as retinal degeneration in Senior–Løken syndrome (SLSN; OMIM 266900) and cerebellar vermis hypoplasia in Joubert syndrome (JS; OMIM 213300). NPHP is a genetically ARTICLE heterogeneous disorder for which nine genes (NPHP1-9) have been cloned to date [Hildebrandt et al., 2009 and references within]. Initially, the characterization of the proteins encoded by NPHP1 and NPHP2/INVS, nephrocystin-1 and inversin respectively, show that these proteins localize to primary cilia in renal tubular epithelial cells where they form a complex with b-tubulin [Otto et al., 2003]. Overall, the characterization of the proteins encoded by NPHP1 and NPHP2/INVS, nephrocystin-1 and inversin, respectively, show that these proteins localize to primary cilia in renal tubular epithelial cells where they form a complex with b-tubulin the characterization of all the nephrocystins has revealed a cellular localization pattern involving the primary cilium and the basal body [Table II; reviewed by Hildebrandt et al., 2009]. Ciliary Dysfunction Can Cause a Broad Range of Phenotypes In addition to cystic kidney disease, LR patterning defects and retinal degeneration, ciliary dysfunction can result in a broad spectrum of disorders that range from PKD to highly pleiotropic syndromes: the ciliopathies [Table I; Badano et al., 2006a; Sharma et al., 2008]. This classification is based in the fact that a common, or at least overlapping, cellular defect is central in the etiology of the different clinical entities. Therefore, this concept per se assigns levels of complexity not previously recognized or expected for a once thought vestigial organelle and poses the question of why alterations in a particular cellular structure can give rise to the numerous, sometimes apparently unrelated, phenotypes that define and characterize the ciliopathies. ARTICLE A first clue to answer this question comes from the observation that virtually all cell types in the human body are either ciliated or have the capacity to become so (http://www.bowserlab. org/primarycilia/ciliumpage2.htm). Thus, ciliary dysfunction will likely affect numerous tissues and organs, albeit to different degrees, depending on the specific ciliary defect, the pattern of expression of the altered ciliary gene and the functional dependency of the tissue with respect to the cilium. Second, several ciliary proteins likely play important extraciliary roles adding another layer of complexity and complicating the dissection of the specific role/s of cilia in the pathogenesis of different ciliopathy phenotypes. Finally, recent data have several ciliary proteins likely play important extraciliary roles adding another layer of complexity and complicating the dissection of the specific role/s of cilia in the pathogenesis of different ciliopathy phenotypes. shown that cilia actively participate in the transduction of key signaling cascades that participate during development and tissue homeostasis thus broadening the spectrum of phenotypes that can be caused by their dysfunction. CILIA IN SIGNAL TRANSDUCTION Understanding the Cellular Basis of Cystogenesis and Other Phenotypes: Wnt Signaling Provided the initial link between a cellular defect and a particular phenotype, the challenge becomes to understand the exact biological process that is being perturbed. Regarding the role of cilia in cystogenesis, important insight came from studies showing that AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) NPHP2/inversin is critical to regulate Wnt signaling [Simons et al., 2005]. The Wnts are a family of secreted factors that bind Frizzled receptors to activate distinct signaling cascades depending on the specific Wnt activator, the receptor and also the activity of Disheveled (Dvl), a molecular switch between signaling cascades. Canonical Wnt signaling acts through b-catenin which drives the expression of a number of TCF-LEF1 responsive genes to control proliferation, cell cycle progression, differentiation and development [Grigoryan et al., 2008] while the non-canonical planar cell polarity (PCP) signaling pathway provides cells with positional clues that are required for concerted multicellular actions such as convergence and extension (CE) movements during gastrulation and neurulation and the correct organization of tissues [Veeman et al., 2003]. Upon activation of the pathway, the subcellular localization of Dvl appears to determine the final signaling outcome whereby nuclear localization of Dvl, which represses the b-catenin destruction complex composed of GSK3b, APC and axin, is required for canonical Wnt signaling while membrane bound Dvl favors the PCP pathway [reviewed by Veeman et al., 2003; Gerdes and Katsanis, 2008]. Importantly, inversin has been shown to interact with Dvl targeting it for degradation and thus mutations in NPHP2 result in impaired control of the Wnt pathway and defective PCP [Simons et al., 2005]. Therefore, ciliary signaling, likely acting through proteins such as inversin, is thought to be required to modulate the balance between Wnt signaling pathways (Fig. 2A). These data provided important insight into the cellular basis of different phenotypes associated with ciliary dysfunction. In the mouse model inversion of embryonic turning (inv) where inversin is disrupted, nodal cilia present defective orientation, which results in abnormal movement and decreased nodal flow explaining the characteristic LR defects of these animals [Okada et al., 1999, 2005]. Importantly, the position and posterior tilt of the cilium in nodal cells, likely requiring PCP signaling, 269 have been shown to be critical for proper beating and flow generation [Nonaka et al., 2005; Okada et al., 2005]. Regarding cystic kidney disease, the Wnt pathway plays a critical role in the formation and maintenance of the kidney where it is required for the induction of the metanephric mesenchyme to develop the proximal portions of the nephron and for the regulation of cell proliferation [Simons and Walz, 2006; Bacallao and McNeill, 2009]. Interestingly, both PCP defects and hyperactivity of canonical Wnt signaling in transgenic mice overexpressing b-catenin can result in the formation of cysts [Saadi-Kheddouci et al., 2001; Simons et al., 2005]. One mechanistic model linking PCP and cystogenesis is based on the observation that a great percentage of dividing cells in the renal tubules orient their mitotic spindles parallel to the lumen and thus the net result of cell division is tubular elongation and not dilation [Fig. 2B; Fischer et al., 2006]. Importantly, misoriented mitotic spindles have been observed in both mouse and rat models of cystic kidney disease [Fischer et al., 2006]. Although the nature of the signal(s) that cilia are sensing to regulate Wnt signaling is still not entirely clear, studies on Bardet–Biedl syndrome Although the nature of the signal(s) that cilia are sensing to regulate Wnt signaling is still not entirely clear, studies on Bardet–Biedl syndrome support the role of cilia and basal bodies as critical players to maintain the correct balance between different Wnt signaling outcomes (BBS; OMIM 209900) support the role of cilia and basal bodies as critical players to maintain the correct balance between different Wnt signaling outcomes 270 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) Normal PCP B A Fluid Flow Morphogen Ligand Other? ARTICLE Mitotic spindles parallel to the lumen PC1-2 Tubule Elongation Proliferation Basal body Ca2+ Cilia-dependent signaling Inv Disrupted PCP Perturbed ciliary signaling BBS Dvl Dvl Tubule Dvl degradation Dvl relocalization Non-canonical Wnt Dilation Axin GSK APC Canonical Wnt Aberrantly positioned mitotic spindles β-catenin Canonical Wnt Axin GSK APC Non-canonical Wnt β-catenin β-catenin Degradation β-catenin TCF/LEF Non-canonical Wnt favored TCF/LEF Canonical Wnt favored Figure 2. Cilia-mediated Wnt signaling and its role in cystogenesis. A: Representation of a cilium/basal body, denoting their role in modulating the balance between canonical and non-canonical PCP signaling. In conditions of normal ciliary/basal body signaling, molecules such as Inversin (Inv) are required to favor PCP over canonical signaling by inhibiting Dvl and activating the b-catenin destruction complex composed of Axin, GSK3-b and APC. In contrast, perturbation of the basal body (i.e., mutations in the BBS proteins) or ciliamediated signal transduction results in decreased PCP and the concomitant upregulation of canonical signaling. B: Representation of the model linking perturbed PCP signaling with cystogenesis where lack of positional clues due to perturbed PCP signaling results in aberrantly positioned mitotic spindles leading to tubule dilation as opposed to extension [adapted from Germino 2005]. [Fig. 2A; review by Gerdes and Katsanis, 2008]. BBS is a disorder characterized by obesity, mental retardation, polydactyly, retinal degeneration and renal malformations including the formation of cysts [Zaghloul and Katsanis, 2009]. To date, 14 BBS genes (BBS1-12, MKS1, NPHP6/CEP290) have been identified [Stoetzel et al., 2007; Leitch et al., 2008 and references within]. The BBS proteins localize primarily to centrosomes and basal bodies and several of them can be found forming a complex, the BBSome, that localizes to the pericentriolar region and the ciliary membrane and has been implicated in ciliogenesis [Ansley et al., 2003; Fan et al., 2004; Kim et al., 2004, 2005; Li et al., 2004; Badano et al., 2006a; Nachury et al., 2007; Loktev et al., 2008]. Interestingly, depletion of different BBS proteins in mice and zebrafish result in characteristic PCP phenotypes [reviewed by Wang and Nathans, 2007]. Bbs6/, bbs1/ and bbs4/ mice present with exencephaly, misoriented stereociliary bundles in the cochlea and open eyelids, a phenotype thought to be caused by defective convergence of epithelial cells, similar to the defect underlying neural tube closure. In zebrafish, depletion of bbs proteins result in failure to achieve the concerted migration and intercalation of cells in the embryonic midline that characterize CE movements during gastrulation and thus, mutant embryos present with a shorter body axis, defective somitic definition and abnormally shaped notochords. In addition, a genetic interaction between the bbs genes and core PCP genes such as vangl2 was also demonstrated [Ross et al., 2005; Badano et al., 2006a]. More recently, it has been shown that the BBS proteins modulate the balance between canonical Wnt and PCP signaling whereby loss of BBS function leads to PCP defects and the concomitant upregulation of canonical Wnt through the stabilization of b-catenin [Gerdes et al., 2007]. Interestingly, depletion of BBS proteins leads to altered proteasome activity in Hek293 cells suggesting that protein clearance defects could at least contribute to the accumulation of b-catenin although this possibility needs further evaluation [Gerdes et al., 2007]. Importantly, this increased functional complexity associated with the BBS proteins might be a consequence of their potential ciliary and extraciliary roles. For example, depletion of BBS4 in mammalian cells results in structural and functional centrosomal defects [Kim et al., 2004]. ARTICLE Importantly, in addition to its role during cell division, the centrosome has been shown to be important in cell migration and protein clearance and thus, perturbation of the BBS proteins might affect extra-ciliary processes likely relevant to the pathogenesis of this syndrome and other ciliopathies [Badano et al., 2005]. Similarly, ALMS1, the protein mutated in Alström syndrome (ALMS; OMIM 203800), also localizes to basal bodies and centrosomes [Hearn et al., 2005]. Furthermore, complete knockdown of Alms1 in mice results in ciliogenesis defects [Li et al., 2007]. The ALMS phenotype is highly reminiscent of BBS in that is characterized by obesity, retinal dystrophy, cardiomyopathy, diabetes but present with sensorineural deafness and no polydactyly. Wnt misregulation is not exclusive of BBS and NPHP given that it is characteristic of other ciliary mutants. In Kif3a/ mice, canonical Wnt signaling is increased [Corbit et al., 2008] and in vitro reporter assays to quantify b-catenin activity showed that depletion of KIF3A and different BBS proteins results in cells that are hyper-responsive to Wnt stimuli [Gerdes et al., 2007]. Also, Ift88orpk/orpk and Ofd1/ mice present similar Wnt defects [Corbit et al., 2008]. Ofd1 is the mouse ortholog of OFD1, the gene mutated in orofaciodigital syndrome type I (OFD1; OMIM 311200), a ciliopathy characterized by malformations involving the oral cavity, face and digits that often present with central nervous system defects and cystic kidney disease [Ferrante et al., 2001]. Shh Signaling Defects in the Ciliopathies Hedgehog (Hh) signaling regulates morphogenesis, patterning and growth of different tissues and organs and therefore several ciliopathy phenotypes, including polydactyly, neural tube and brain defects Hedgehog (Hh) signaling regulates morphogenesis, AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) patterning and growth of different tissues and organs and therefore several ciliopathy phenotypes, including polydactyly, neural tube and brain defects can be consequences of alterations in this pathway. (Table I), can be consequences of alterations in this pathway. In mammals, Sonic Hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh) compose a family of Hh secreted signaling proteins that bind the Patched receptor (Ptc) to activate different signaling cascades. Upon binding of Shh, patched 1 (Ptc1) is inactivated and Smoothened (Smo) is released to block the processing of Gli3 into its repressor form (Gli3R) thus enabling 271 Gli-mediated target gene regulation [Varjosalo and Taipale, 2008]. Although the mechanism is not completely understood, the subcellular localization of the different components of the pathway is important for activity and the cilium and IFT appear as key components of the signaling apparatus (Fig. 3). Initially, a mouse mutagenesis screen uncovered two novel embryonic patterning mutants, wimple (wim) and flexo (fxo), that presented characteristic Shh defects such as open neural tube, brain and limb abnormalities. Interestingly, the wim and fxo phenotypes, also characteristic of Kif3a mutants, were shown to be caused by a mutation in IFT172 and by a novel hypomorphic allele of IFT88, respectively [Huangfu et al., 2003]. Subsequently, it has been shown that Smo translocates into the cilium upon Shh stimulation and that ciliary localization is essential for Smo activity [Corbit et al., 2005; Aanstad et al., 2009]. Furthermore, Ptc1 localizes to the primary cilium and inhibits Smo No Shh ligand Upon Shh stimulation Smo Smo Smo Smo Shh Gli3 Ptc1 Shh Smo Gli3 Ptc1 Shh Smo Ptc1 Smo Basal body Basal body Gli3R Gli3R Hedgehog responsive genes Gli Activator Gli3R Gli Activator Hedgehog responsive genes Figure 3. The cilium and sonic hedgehog (Shh) signaling. In the absence of Shh (left), the receptor patched (Ptc1) is localized to the cilium, preventing the ciliary accumulation of smoothened (Smo) and thus favoring the processing of the Gli3 transcription factor into its transcriptional repressor form (Gli3R). Upon binding of Shh (right), Ptc1 is translocated outside the ciliary compartment allowing Smo to enter it in a process that depends on the anterograde IFT machinery. The ciliary accumulation of Smo in turn inhibits the processing of Gli into Gli3R while favors the processing of Gli3 into the activator form that is able to drive the expression of different target genes. 272 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) activity by preventing its accumulation in the ciliary compartment. Upon Shh binding, Ptc1 is translocated out of the cilium and Smo is able to enter it in a process that requires b-arrestins and Kif3a [Fig. 3; Rohatgi et al., 2007; Kovacs et al., 2008]. Additionally, Gli1, Gli2, and Gli3 localize to cilia and loss of IFT88 leads to altered Gli2 and Gli3 processing [Haycraft et al., 2005]. In general, ciliogenesis and normal ciliary function appear to be critical to maintain Hh signaling as demonstrated by different IFT mutants that are characterized by severe developmental abnormalities attributable to defective Hh transduction [reviewed by Eggenschwiler and Anderson, 2007]. Briefly, mutations in IFT139, mutated in the alien mouse (aln), result in abnormal primary cilia and overactivation of Shh [Tran et al., 2008]. Mutations in the retrograde IFT dynein motor Dnchc2 and the basal body protein Ftm1 also result in abnormal Gli3 processing and mutant animals present with neural tube, LR and limb patterning defects [May et al., 2005; Vierkotten et al., 2007]. Importantly, defective Shh signaling can readily explain several of the phenotypes that characterize the ciliopathies and significant progress is being made to understand the role of this signaling cascade in their pathogenesis. Using conditional mutants to circumvent the lethality in complete ciliary nulls, it has been shown that ablation of Kif3a in the developing limb leads to aberrant Hh signaling (both Shh and Ihh) and results in polydactyly and altered digit patterning [Haycraft et al., 2007]. Similarly, depletion of Kif3a in cartilage results in skeletogenesis defects such as alterations in growth plate organization and excessive intramembranous ossification, defects that correlate with altered Hh signaling both on the level and field of expression of different components of the pathway [Koyama et al., 2007]. A skeletal dysplasia characterized by short ribs and limbs, polydactyly, and wrist bones malformations is Ellis-van Creveld syndrome (EvC; OMIM 225500). Interestingly, EVC, mutated in this condition, encodes a protein that localizes to the base of chondrocyte cilia and is required for Ihh signaling in the growth plate [Ruiz-Perez et al., 2007]. Jeune asphyxiating thoracic dystrophy (JATD; OMIM 208500) is a rare chondrodysplasia where patients present a severely constricted thoracic cage and respiratory insufficiency often lethal in the first years of life. In addition, polydactyly, kidney/liver/pancreas cysts and retinal degeneration are also present in these patients. Importantly, mutations in IFT80 and DYNC2H1 (cytoplasmic dynein 2 heavy chain 1) are found in JATD [Beales et al., 2007; Dagoneau et al., 2009]. Ablation of either Ift88 or Kif3a in different neuronal populations have demonstrated the need for intact Shh signaling to maintain neural progenitor pools both in the dentate gyrus and the cerebellum [Chizhikov et al., 2007; Han et al., 2008; Spassky et al., 2008]. In the cerebellum, it has been shown that ciliamediated Shh signaling is required to maintain and expand the pool of granule cell precursors (GCPs). Consequently, specific ablation of Ift88, Kif3a, or Smo in cerebellar GCPs results in reduced expansion of this group of cells and cerebellar hypoplasia [Chizhikov et al., 2007; Spassky et al., 2008]. Joubert syndrome (JS) is a group of disorders characterized by brain malformations that include cerebellar vermis hypoplasia and brainstem abnormalities, the molar tooth sign (MTS) in radiological examination. Patients can also present cystic renal disease, nephronophthisis, mental retardation, polydactyly, retinal degeneration, breathing problems and hypotonia among other clinical manifestations [reviewed by Parisi et al., 2007]. To date, mutations in seven genes have been causally linked to JS and ciliary dysfunction appears central in the etiology of JS as highlighted by the fact that several of the mutated proteins not only localize to basal bodies, centrosomes and cilia but also have been implicated in other ciliopathies as is the case for NPHP1, NPHP6/CEP290, MKS3/TMEM67, and NPHP8/ RPGRIP1L [Table II; Parisi et al., 2007; Sharma et al., 2008 and references within]. Therefore, the cerebellar abnormalities that characterize JS, a ARTICLE common finding among other ciliopathies (Table I), might result from altered Shh signaling and the inability to maintain and expand the population of GCPs. Other Signaling Cascades: PDGF Signaling and FGF-Mediated Control of Ciliogenesis Another signaling pathway that has been recently shown to operate through the cilium is platelet derived growth factor receptor alpha (PDGFRa). Stimulation of PDGFRaa (PDGFRa homodimer) by PDGFaa results in autophosphorylation events that lead to the activation of downstream signaling cascades mediated by AKT and Mek1/ 2-Erk1/2, that ultimately regulate cell cycle progression, survival and cell migration [reviewed by Christensen et al., 2008]. Interestingly, it has been shown that PDGFRa is upregulated during ciliogenesis and localizes to the primary cilium in NIH3T3 and mouse embryonic fibroblast arrested cells. Furthermore, the ciliary localization of PDGFRa is required for proper activation and embryonic fibroblasts derived from Ift88orpk mice failed both to upregulate the receptor and activate the pathway [Schneider et al., 2005]. While different signaling pathways require the cilium to operate, recent data indicates that other cascades might exert their actions, at least in part, through the direct regulation of ciliogenesis. Neugebauer and colleagues demonstrated that fibroblast growth factor (FGF) signaling regulates ciliogenesis, cilia length and function in epithelial cells in zebrafish and Xenopus [Neugebauer et al., 2009]. Importantly, FGF regulates developmental processes such as LR determination and convergent extension movements during gastrulation [Meyers and Martin, 1999]. Inhibition of FGF signaling through either morpholino-mediated depletion of FGF receptor 1 (Fgfr1), dominant negative mutants or pharmacological agents led to a reduction in the length of cilia and perturbed fluid flow in Kupffer’s vesicle and other ciliated cell types in zebrafish. Similarly, in Xenopus embryos, defective FGF signaling results in shorter cilia ARTICLE in the LR organizer, the gastrocoel roof plate. Interestingly, FGF signaling has been shown to regulate the prociliogenic transcription factors foxj1 and rfx2 and ift88 [Neugebauer et al., 2009]. Furthermore, Hong and Dawid  were able to show that Ier2 and Fibp1, two downstream targets of FGF signaling, regulate ciliogenesis in Kupffer’s vesicle and mutations in these genes result in LR defects in zebrafish. Thus, defective FGF can result or contribute to several of the phenotypes observed in ciliary mutants, highlighting the complexity underlying each of the biological processes regulated by the cilium where distinct signaling pathways need to interact to control biological processes in a coordinated fashion. PERTURBED CILIOGENESIS IN THE CILIOPATHIES Given the ubiquitous distribution of cilia, their involvement in multiple signaling cascades and the plethora of biological processes that these cascades regulate, it is not surprising that complete absence of cilia is generally incompatible with life. Therefore, defective ciliogenesis should result in the more severe phenotypes. One syndrome that lies at the severe end of the ciliopathy phenotypic spectrum is Meckel–Gruber syndrome (MKS; OMIM 249000; Table I). MKS is a lethal condition characterized by occipital encephalocoele, neural tube defects, cystic kidney disease, hepatic fibrosis, polydactyly and cleft palate [Alexiev et al., 2006]. Six MKS loci have been mapped to date from which five genes have been identified: MKS1, MKS3, CEP290, RPGRIP1L, and MKS6/CC2D2A [Kyttälä et al., 2006; Smith et al., 2006; Delous et al., 2007; Baala et al., 2007a; Tallila et al., 2008]. Importantly, the characterization of these genes/proteins has demonstrated that MKS is a ciliopathy that likely results from ciliogenesis defects. MKS1 and MKS3 localize to the basal body of cilia and are required for its migration to underneath the apical membrane during ciliogenesis [Dawe et al., 2007]. Interestingly, MKS1 con- AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) tains a protein motif (B9 domain) that albeit of unknown function, is shared by two other proteins, MKS1-related proteins 1 and 2 (MKSR-1 and MKSR-2) and is found exclusively in ciliated organisms. Knockdown experiments in cells have shown that similarly to MKS1, MKSR-1 and MKSR-2 are also implicated in ciliogenesis in mammalian cells [Bialas et al., 2009]. More recently, it was shown that fibroblast cells from MKS patients bearing mutations in MKS6/ CC2D2A lack cilia [Tallila et al., 2008]. Interestingly, the other two MKS genes, CEP290 and RPGRIP1L/MKS5, are also linked to cilia. CEP290 is a basal body protein also implicated in the pathogenesis of NPHP, JS and BBS [Sayer et al., 2006; Valente et al., 2006; Baala et al., 2007a; Leitch et al., 2008] and RPGRIP1L/MKS5 encodes a basal body protein mutated in JS [Delous et al., 2007] (Table II). Thus, the analysis of the different MKS proteins strongly supports a ciliary defect as the underlying cause of this disorder and also highlights the complex genetics of the ciliopathies whereby mutations in the same gene can result in seemingly distinct clinical manifestations. A COMMON GENETIC BASIS FOR DISTINCT CLINICAL ENTITIES The concept of a ciliopathy implies that different clinical entities share a common cellular defect and an overlapping set of phenotypes (Table I). Importantly, The concept of a ciliopathy implies that different clinical entities share a common cellular defect and an overlapping set of phenotypes. Importantly, the identification of typical phenotypic outcomes of ciliary dysfunction has provided a predictive tool when attempting to gain insight into 273 the cellular basis of disorders of unknown etiology. the identification of typical phenotypic outcomes of ciliary dysfunction has provided a predictive tool when attempting to gain insight into the cellular basis of disorders of unknown etiology. An example is JATD that was predicted to be a ciliopathy before causal genes were identified, based solely on clinical features that overlap with those of other known ciliopathies such as cystic kidneys, brain malformations, polydactyly, and skeletal defects [Badano et al., 2006b]. Additionally, our improved knowledge has also resulted in a positive feedback loop affecting the genetic dissection of the ciliopathies by allowing for example the possibility of filtering a list of candidate genes in a genomic locus according to whether the genes encode ‘‘ciliary proteins’’ or not. In this context, a multi-group effort has led to the identification of a number of genes/proteins involved in ciliary biology which have been integrated in a non-redundant list: the ciliary proteome [Gherman et al., 2006 and references within;http://www.ciliaproteome.org/]. For example, the categorization of BBS as a ciliopathy and the availability of the ciliary proteome has greatly facilitated the identification of several BBS genes such as for example BBS3, BBS5, and BBS12, by prioritizing candidate genes and first sequencing those included in the ciliary proteome [Fan et al., 2004; Li et al., 2004; Stoetzel et al., 2007]. Another important implication of the ciliopathy concept is that alterations in different genes can result in overlapping phenotypic features and thus a first possibility is that mutations in different genes can cause the same disorder. It is interesting to note that the majority of the ciliopathies are genetically heterogeneous disorders (Table II). In addition, it has been shown that disorders historically considered as mendelian traits can present more complex patterns of disease transmission, oligogenic inheritance, whereby mutations in more than one locus segregate 274 AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) Another important implication of the ciliopathy concept is that alterations in different genes can result in overlapping phenotypic features and thus a first possibility is that mutations in different genes can cause the same disorder. It is interesting to note that the majority of the ciliopathies are genetically heterogeneous disorders with the disease in families. For example in some BBS families, three mutant alleles involving bona fide BBS genes as well as second site modifiers, collaborate to modulate the penetrance and expressivity of the disorder [for some references see Katsanis et al., 2001; Beales et al., 2003; Badano et al., 2006a]. Similarly, mutations in more than one NPHP gene have also been detected in patients showing that in some families at least, NPHP can be inherited as an oligogenic trait [Hoefele et al., 2007]. Furthermore, it has been shown that epistatic interactions between mutant alleles in different genes can greatly influence the expressivity of the disease in NPHP patients. While alterations in NPHP1 are mainly associated with nephronophthisis, the presence of additional mutations in NPHP6 and AHI1 can determine the development of neurological symptoms associated with JS [Tory et al., 2007]. The later example introduces a second possibility in that mutations in a given gene might be related to distinct clinical entities either as causal or modifying factors (see Syndrome column in Table II). For example, RPGRIP1L contribute alleles to the pathogenesis of NPHP, JS, and MKS and it was recently shown that a specific allele of this gene is a modifier of the retinal phenotype of ciliopathy patients bearing mutations at other loci [Arts et al., 2007; Delous et al., 2007; Wolf et al., 2007; Khanna et al., 2009]. Mutations in CEP290 have been found associated with NPHP (NPHP6), JS, MKS and BBS [Sayer et al., 2006; Valente et al., 2006; Baala et al., 2007a; Frank et al., 2008; Leitch et al., 2008]. Likewise, MKS3 mutations have been identified in JS patients [Baala et al., 2007b; Brancati et al., 2009]. Another example is provided by BBS and MKS, two disorders significantly similar at the phenotypic level (Table I). Interestingly, mutations in BBS2, BBS4, and BBS6 have been found in MKS-like fetuses [Karmous-Benailly et al., 2005] and more recently, it was shown that mutations in MKS1, MKS3, and CEP290 are found in BBS patients as either causal or modifier alleles [Leitch et al., 2008]. These data raise the question of why mutations in the same gene can result in different clinical manifestations. In some cases, the specific mutated gene determines the final outcome as is the case in Senior–Løken syndrome where although different NPHP genes can cause it, mutations in NPHP5 are invariably associated with it. Importantly, nephrocystin-5 localizes to the photoreceptors and interacts with calmodulin and the retinitis pigmentosa GTPase regulator (RPGR), a gene mutated in retinitis pigmentosa [Otto et al., 2005]. In other cases, the different phenotypic outcome does not present a clear correlation with localization or pattern of expression but rather seems to depend on the type of mutation and its impact on protein function. In BBS for example, in vivo assessment of the pathogenic effect of the mutations found in the MKS genes has shown that while mutations normally associated with the lethal MKS phenotype are complete nulls, residual activity is observed in the MKS proteins with BBS-associated mutations [Leitch et al., 2008]. Recently, likely hypomorphic mutations in MKS3 have been identified in patients with NPHP and liver fibrosis [Otto et al., 2009]. Similarly, it was shown that while a hypomorphic mutation in Nphp3 in the pcy mouse model results in cystic kidney disease, complete loss of Nphp3 results in a pleiotropic lethal phenotype that includes congenital heart defects and situs inversus, and ARTICLE in humans a MKS-like phenotype [Bergmann et al., 2008]. Altogether, these data indicate that the ciliopathies, albeit they can be recognized as distinct clinical entities, represent a continuum of disease severity where the position of any given disorder is determined by the specific gene that is mutated, its biological role and pattern of expression, the total mutational load across different ciliary genes, and importantly, the type and effect of the mutations on protein function. UNDERSTANDING OTHER CILIA-ASSOCIATED PHENOTYPES In the last decade, a plethora of biological roles have been assigned to cilia including photoreception, mechanosensing, and paracrine signaling transduction. This in turn has translated into important progress in our understanding of the cellular basis of different phenotypes that characterize the ciliopathies such as for example cystic kidney disease. Other phenotypes however have proven to be more difficult to understand and dissect. One particular example is obesity, a hallmark of several ciliopathies, particularly BBS and ALMS (Table I). Bbs mutant animals show hyperphagia and present elevated leptin levels [for some references see Fath et al., 2005; Eichers et al., 2006; Zaghloul and Katsanis, 2009]. Furthermore, in Bbs2/ and Bbs4/ neurons, the ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1), which is involved in the regulation of feeding behavior, is lost [Berbari et al., 2008]. Other clues have been provided by conditional mutants of Ift88 showing that disruption of the gene in proopiomelanocortin (POMC) neurons in the hypothalamus results in mice that are obese, hyperphagic, and have elevated leptin levels [Davenport et al., 2007]. Interestingly, the BBS proteins appear to be required for leptin receptor (LepR) intracellular trafficking in hypothalamic neurons and thus it has been suggested that the LepR signaling defect observed in Bbs2/, Bbs4/, and Bbs6/ mice ARTICLE might be due to impaired targeting of the receptor to the plasma membrane or the cilium, similarly to the ciliary localization requirements of components of the Shh signaling cascade [Seo et al., 2009]. Besides this putative role of the cilium in neurons, a recent report indicates that cilia and the BBS proteins are required for proper adipocyte differentiation whereby depletion of BBS proteins favors adipogenesis suggesting that the obesity phenotype might result from defects at multiple levels [Marion et al., 2009]. A deeper understanding of the role of cilia in different tissues and the phenotypes that result from their absence or malfunction not only has provided a means to predict novel ciliopathies but has also facilitated the identification of novel phenotypes in ciliopathy patients. Given that olfactory neurons present highly specialized cilia that are important for odorant perception it was hypothesized that a cilia-related phenotype would be anosmia, a prediction that was subsequently proven in BBS patients [Kulaga et al., 2004]. Similarly, it was shown that Bbs1 and Bbs4 mutant mice present thermosensory defects due to problems with peripheral ciliated sensory neurons, a phenotype that also is present in BBS patients and was not previously recognized in the clinic [Tan et al., 2007]. Therefore, our knowledge regarding the cellular basis of these human conditions has led to a better clinical characterization of patients providing important data for diagnostic, counseling and eventually treatment purposes. CONCLUDING REMARKS The success linking ciliary function with diverse biological processes has been impressive. The challenge has become to start elucidating the exact role that the cilium is playing in each case and equally important, why tissues and organs are affected differently by defects in ciliary function. Undoubtedly one important variable to consider is the pattern of expression of the ciliary gene/s. At the same time however, the fact that a number of ciliary genes are expressed differentially between ciliated tissues AMERICAN JOURNAL OF MEDICAL GENETICS PART C (SEMINARS IN MEDICAL GENETICS) reflects the multiple and diverse roles that these organelles are playing in different contexts highlighting the need to characterize ciliary proteins in physiologically relevant settings. To this end, animal models in which the gene of interest is perturbed in a tissue specific and/or temporally regulated manner are proving to be extremely powerful and some examples have been mentioned in this review. Another likely underestimated issue that complicates the study of ciliary proteins is that many of them appear to have extraciliary roles. In addition to some of the examples provided in this review, PC2, the Ca2þ channel required for mechanosensation both in renal epithelial cells and in the embryonic node, also localizes to the plasma membrane and the endoplasmic reticulum where it operates as an intracellular Ca2þ release channel [reviewed by Tsiokas et al., 2007]. Similarly, several NPHP proteins, including NPHP2/ inversin and NPHP1, localize to basal bodies and cell–cell junctions [Nürnberger et al., 2002; Hildebrandt et al., 2009]. One question that will likely be answered as we dissect these issues is why the different signaling cascades mentioned above need the cilium in the first place. A priori, one plausible answer seems to rely on the compartmentalization that the cilium permits, allowing both the concentration and separation of different moieties, and the use of an efficient microtubule based transport mechanism to rapidly relay information from the cilium towards the centrosome, the microtubule organizing center, and into the nucleus. Considering this continuum then, it is not surprising that in general, the phenotype in ciliopathy patients or animal models appears to depend on how globally is ciliary function affected. Mutations in a protein that needs to be localized to the cilium (such as PC2) might result in a fairly restricted phenotype while global ciliary dysfunction (disrupted ciliogenesis in MKS or basal body dysfunction in BBS) probably affect, albeit to different degrees, all the biological processes that operate through the organelle resulting in highly pleiotropic or lethal pheno- 275 types. In addition, one lesson from the genetic dissection of ciliopathies is that the degree of ciliary dysfunction, and therefore the clinical manifestation, is also influenced by the number and type of mutations in different components of the system. Importantly, the availability of high-throughput mutational screening technologies, the development of reliable functional assays to test coding variants and our deeper understanding of the cellular and genetic basis of these disorders will likely have a beneficial impact on the clinical management of affected patients. ACKNOWLEDGMENTS First of all we apologize to all those scientists whose contribution to understand ciliary biology could not be properly cited in this manuscript due to space limitations. We also thank Cecilia Gascue for her critical comments on the manuscript. 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