Cilia are dynamic organelles projecting from the cell surface. They consist of a basal body located under the cell surface and of a projecting structure called the axoneme. The basal body is composed of a pair of centrioles embedded in the pericentriolar material (PCM). The ciliary axoneme contains nine microtubule doublets surrounded by a membrane contiguous with the plasma membrane. Cilia are classified on the basis of structure and function. The basic structure of the different types of cilia is similar although their function may be tissue-specific and may change during development, tissue morphogenesis and homeostasis.

The traditional classification of cilia into two main classes, motile 9+2 and non-motile 9+0, is insufficient to reflect the complexity of all cilia types. The most recent studies indicate that cilia can be divided into at least four main cilia types: motile 9+2, motile 9+0, non-motile 9+2 and non-motile 9+0. In the 9+2 configuration the axoneme contains a central microtubule pair surrounded by the nine microtubule doublets, which is missing in the 9+0 configuration. Moreover, motile cilia contain inner and outer dynein arms, radial spokes and nexin links (reviewed in 1). Inner and outer dynein arms on the doublets mediate axoneme motility. The radial spokes play an essential role in the control of dynein arm activity by relaying signals from the central pair of microtubules to the arms. Nexin links are the connecting links between microtubules in cilia and flagella. Radial cuts of 9+0 and 9+2 axonemal structures of non-motile and motile cilia, respectively, are illustrated in Figure 1A and 1B. Respiratory and ependymal cilia are motile 9+2 with a back-and-forth motion. In the murine embryonic node, primary cilia are an admixture of 9+2 and 9+0 cilia 2. The specific feature of the unique 9+0 motile cilium is the rotational movement generating an anti-clockwise flow of extra-embryonic fluid in the nodal area 3. Thus, the central microtubule pair seems to be required for back-and-forth movement while its absence produces a rotational movement. Non-motile 9+2 cilia are present on specialized olfactory neurons 4. Renal monocilia, photoreceptor-connecting cilia and cilia of pancreatic islets are non-motile 9+0 cilia 567. Finally, among the different cilia types, 9+4 cilia have been identified on the notochordal plate of the rabbit embryo 8.

The number of cilia can vary among cell types. Epithelial cells may possess several hundred 9+2 motile cilia while 9+0 cilia are usually solitary. Non-motile cilia are generally considered chemical or mechanical sensors and are called 'primary cilia', but recent advances have suggested that all cilia might have sensory functions. The primary cilium only assembles when cells are not in mitosis, and it is considered an organelle of cells in a quiescent or differentiated state. In fact, cell cycle re-entry is preceded by cilium reabsorption 9. The primary cilium is considered a highly dynamic organelle both because it is assembled only during a certain phase of cell life and because an active molecular transport occurs within its axoneme.

During ciliogenesis, cilia elongate from the basal body through the addition of new axonemal subunits organized in macromolecular particles to the distal tip. Intraflagellar transport (IFT) is responsible for this transfer, which can be bidirectional 10. Anterograde transport is driven by heterotrimeric kinesin 2, which is composed of two motor subunits (Kif3a and Kif3b) and a non-motor subunit 11. Retrograde transport back to the cell body is accomplished by cytoplasmic dynein 1B 1213. Ciliogenesis is also coordinated by PCM, which functions as a nucleation site for microtubules. RNA interference (RNAi) knockdown of pericentrin, a protein important for PCM organization, inhibits ciliogenesis and reduces the presence of IFT components near the centrioles 14.

Primary cilia are present on a wide variety of cell types such as the bile duct, the kidney tubule, the endocrine pancreas, the thyroid, smooth muscle cells, neurons, fibroblasts, and chondrocytes. Some examples of cells or tissues presenting primary cilia are illustrated in Figure 1C–F. For a complete list of cells and tissues containing cilia, refer to 15.

The function of primary cilia in most tissues is unknown. In the kidney they are mechanosensitive organelles that detect fluid flow through the tubule lumen 16. In the liver primary cilia are present on cholangiocytes and they function as mechano-, osmo-, and chemosensors in intrahepatic bile ducts. Mutations in genes encoding cholangiocytes' ciliary-associated proteins result in cholangiociliopathies 17.

In recent years, growing attention on cilia has stimulated the creation of numerous databases 1819 including genomic and proteomic data on cilia composition 202122.

Cilia are unique organelles, which can act as antennae for the cells. They have crucial roles in several signal transduction pathways such as Hh, Wnt, planar cell polarity (PCP) and platelet-derived growth factor (PDGF) pathways. A summary of the pathways is reported in Figure 2.

Mutations in human ciliary genes give rise to a wide spectrum of disorders named ciliopathies. These include different types of disease from embryonic lethal to less severe multisystemic disorders (reviewed in 606162). Primary cilia are multi-subunit complexes composed of hundreds of proteins, and the inactivation of only one of these may be sufficient to produce a defective cilium or a complete immature cilium, resulting in variability of phenotypic severity. Many organs, and corresponding phenotypes, may be affected in ciliopathies, suggesting a tissue-specific function of primary cilia. Furthermore, many ciliary genes are mutated in more than one ciliopathy, indicating an interaction among proteins. Ciliary proteins may also have other roles in addition to those strictly connected to cilia. All these observations together shed light on the complexity and variety of ciliopathies. A list of ciliary genes whose mutations are responsible for human genetic disorders is reported in Table 1.

Mutations in genes encoding for ciliary proteins, leading to immotile cilia or disrupted movement of cilia, are responsible of a set of human diseases named primary ciliary dyskinesia (PCD; OMIM: 242650). Patients with PCD may suffer from respiratory infections, anosmia, male infertility, otitis media and situs inversus and have been shown to have defects in ciliary structure and function (63 and reviewed in 64). Male infertility can be caused by loss of sperm flagellar motility, and some patients with PCD also display hydrocephalus. Respiratory infections are mainly caused by defective mucociliary clearance, which requires the presence of motile 9+2 cilia in respiratory epithelial cells.

PCD presents extensive locus heterogeneity 65 and only few genes, accounting for 40% of patients, have been identified to date for this disease. A successful approach for localization of putative PCD genes has been a homozygosity mapping strategy, which allows the localization of the DNAH5 gene 66. Unfortunately, large informative families are rarely available. PCD affects motile cilia that are characterized by the presence of motor molecules within the ciliary axoneme. Inner (IDAs) and outer dynein arms (ODAs), together with radial spokes and nexin links, are necessary for cilia movement. Individuals with PCD generally present with mutations in one of the above motor molecules. Mutations in DNAI1, a dynein intermediate chain, and in DNAH5, encoding for one of the ODA heavy chains, have been associated with PCD 6768. Furthermore, mutations in the DNAH11 axonemal dynein heavy chain 69 and in the DNAI2 protein have been identified 70. TXNDC3, encoding for a thioredoxin-nucleoside diphosphate kinase 71, RSPH9 and RSPH4A, encoding for radial spoke head proteins, were also found to be mutated in patients with PCD 72.

Furthermore, several dynein chain genes have been analyzed for mutational analysis in patients with PCD without success. Mutational analysis was negative for both the DNAH9 73 and the DNAL1 74 genes. DNAH1 was proposed as a candidate gene for human PCD 75 but to date no study has validated this hypothesis. PCD is classically transmitted as an autosomal recessive trait. Moore and colleagues have also demonstrated an X-linked transmission of PCD, where RPGR mutations were found in patients with a complex X-linked phenotype combining PCD and retinitis pigmentosa 76.

Meckel-Gruber syndrome (MKS; OMIM 249000) is an autosomal recessive lethal disorder characterized by renal and hepatic cysts, polydactyly, malformations in the central nervous system (CNS), and occasionally hydrocephalus. To date, mutations in five genes responsible for the disease have been identified 7778798081. Several MKS fetuses presented mutations in BBS genes, indicating possible genetic interaction between MKS and BBS genes 82. In addition, most of the MKS genes are also mutated in other ciliopathies such as Joubert syndrome, suggesting a genetic interaction between ciliary genes 78808384.

Among ciliopathies that mainly affect the kidney the PKDs, which include autosomal dominant PKD (ADPKD), autosomal recessive PKD (ARPKD) and nephronophthisis (NPHP), are worth mentioning. Correlations between genotype and phenotype in ADPKD and ARPKD were reviewed by 85. PKD1 and PKD2 proteins are multi-pass integral membrane proteins that interact to form a channel for the Ca²⁺ ion 86. Intracellular Ca²⁺ levels, which are important for cell proliferation, apoptosis and ion reabsorption rates, may contribute to renal cyst formation 87. PKHD1, the gene mutated in ARPKD (OMIM 263200), encodes for the membrane-associated receptor-like protein fibrocystin/polyductin 88. PKHD1 associates with primary cilia of epithelial cells and co-localizes with PKD2. Recently, in vivo studies have demonstrated that the two proteins may function in a common molecular pathway 89. To date, the molecular mechanisms considered responsible for kidney cysts are increased cell proliferation and/or loss of cell polarity 909192.

NPHP (OMIM 256100) is an autosomal recessive cystic renal disease (reviewed in 9394). Individuals with this disease can also suffer from situs inversus, pancreatic and hepatic fibrosis, retinal degeneration (in Senior-Løken syndrome (SLSN) and Joubert syndrome (JBTS)), complex brainstem malformation and mental retardation (JBTS). In contrast with PKD, NPHP shows normal or diminished kidney size, cysts are concentrated at the corticomedullary junction, and tubulointerstitial fibrosis is dominant.

To date, mutations in nine genes linked to NPHP have been identified 9596979899100101102103. Nephrocystins, the proteins encoded by NPHP genes, are highly conserved in evolution. Mutations in NPHP genes cause defects in signaling mechanisms, including the non-canonical Wnt signaling pathway. NPHP1 encodes for nephrocystin-1, a protein that interacts with components of cell-cell and cell-matrix signaling, including p130Cas, focal adhesion kinase 2, tensin, and filamin A and B. Mutations in this gene causes juvenile NPHP type1 100. Mutations in the Inversin gene cause NPHP type 2 96. The inv murine model presents a complete inversion of left-right asymmetry and pancreatic and renal cysts 104. Recently, Shiba and colleagues have demonstrated that the Inv protein is localized at a distinctive proximal segment of the primary cilium 105.

Mutations in NPHP3 are responsible for adolescent NPHP type 3 95. Mutations in the murine ortholog Nphp3 cause the renal cystic mouse mutant pcy. NPHP4 is mutated in NPHP type 4 101. The encoded protein, nephrocystin-4/nephroretinin, forms a complex with other proteins involved in cell adhesion and actin cytoskeleton organization, such as nephrocystin-1, p130Cas, Pyk2, tensin, filamin, and α-tubulin. NPHP5, mutated in NPHP type 5, encodes the protein nephrocystin-5 97. All patients had early onset retinitis pigmentosa (SLSN). Nephrocystin-5 contains two IQ domains, which directly interact with calmodulin and form a complex with the retinitis pigmentosa GTPase regulator, which, when defective, causes X-linked retinitis pigmentosa. Both nephrocystin-5 and retinitis pigmentosa (RP) GTPase regulator (RPGR) localize to connecting cilia of photoreceptors and in primary cilia of renal epithelial cells. The fact that connecting cilia of photoreceptors are the structural equivalents of primary cilia of renal epithelial cells may explain retinal involvement in the retinal-renal syndrome SLSN.

NPHP6/CEP290 encodes a centrosomal protein and is the cause of NPHP type 6 and JBTS type 5. Abrogation of NPHP6 function in zebrafish causes PCP (convergent extension) defects and recapitulates the human phenotype of NPHP type 6, including renal cysts, RP, and cerebellar defects. Nephrocystin-6 is expressed in the centrosomes and mitotic spindle in a cell-cycle-dependent manner. Its identification establishes a link between centrosome function and tissue architecture in the pathogenesis of cystic kidney disease, RP, and CNS development. Mutations in NPHP6/CEP290 have been confirmed to cause JBTS with or without renal involvement 99. It is interesting that a 300-amino acid in-frame deletion of NPHP6/CEP290 caused retinal degeneration alone, without renal or cerebellar involvement, in the rds16 mouse model. This is in accordance with the recent finding that a hypomorphic mutation of NPHP6/CEP290 represents the most frequent cause of Leber's congenital amaurosis. The seventh gene identified in NPHP is GLIS2, which encodes for a transcription factor 98. The murine model of Glis2 presented severe renal atrophy and fibrosis of the kidney. The essential role of Glis2 is the maintenance of renal tissue architecture through prevention of apoptosis and fibrosis 98. Recently, mutations in the gene RPGRIP1L 102 and NEK8 were found in patients affected by NPHP 103.

JBTS is a ciliopathy characterized by extensive genetic heterogeneity and variability in phenotypic severity (reviewed in 106). To date, five genes linked to JBTS have been identified 8084107108109110111. Children with JBTS appear to have a characteristic facial appearance, delayed language, autism, polydactyly, renal cysts, microcephaly and ocular abnormalities. JBST1 and JBST2 loci have been mapped but no causative genes have yet been identified. The gene responsible for JBTS type 3 is AHI1, which encodes for jouberin that contains an N-terminal coiled domain 108. The gene for JBTS type 4 was identified in patients presenting with juvenile NPHP 107. The gene mutated in patients with JBTS type 5 encodes for the centrosomal protein CEP290 110.

Cilia also have a major role in the retina. Defects in their function can cause retinal ciliopathies (reviewed in 112). Photoreceptors are composed of an outer and an inner segment connected by a highly specialized 9+0 cilium called 'connecting cilium'. The two proteins RP1 and RPGR associated with RP both localize predominantly to the photoreceptor connecting cilium 113114. The microtubule-associated protein RP1 is mutated in RP type 1 (OMIM 180100) 115116117. Mutations in RPGR are associated with RP type 3 (OMIM 180100) 118. Moreover, in ciliopathies retinal degeneration is often one of the major phenotypic features. In particular, in SLSN (OMIM 266900), a rare autosomal recessive disorder, patients are affected by NPHP and progressive eye disease. The genes responsible for SLSN are NPHP1, NPHP4, NPHP5/IQCB1 and CEP290, which are the same genes mutated in other ciliopathies such as NPHP and JBTS 9799101119.

Oral-facial-digital type I syndrome (OFDI; OMIM 311200) is an X-linked dominant developmental disorder with lethality in males. Female patients present malformations of the oral cavity, face, digits and CNS malformations. OFD1, the gene responsible for this genetic disorder, encodes a protein localized at the centrosome/basal body 120121. Ofd1-knockout animals reproduce the main features of the human disease, albeit with increased severity, possibly due to differences of X-inactivation patterns between human and mouse 122. Inactivation of the gene indicated that OFD1 is required for primary cilia formation and left-right axis specification 43. About 50% of patients with OFDI present CNS abnormalities (agenesis of corpus callosum, intracerebral cysts/porencephaly, gray matter heterotopias, and cerebellar malformations). Interestingly, about 50% of patients present mental retardation, which in some cases is not accompanied by gross structural abnormality, suggesting a role for primary cilia in the maintenance of adult organs.

BBS (OMIM 209900) is a genetically heterogeneous pleiotropic disorder with symptoms including kidney and gonadal abnormalities, mental retardation, retinal degeneration, obesity, diabetes, and polydactyly. To date, fourteen BBS genes have been identified whose mutations are associated with the syndrome 123124125126127128129130131132133134135. BBS exhibits a complex pattern of inheritance, the triallelic inheritance, in which three mutations at two loci simultaneously are necessary and sufficient in some families to manifest the phenotype 136. The genetic and cellular characteristics of BBS have recently been extensively discussed 137. Most of the BBS proteins localize to the cilia/basal body/centrosome complex. BBS proteins interact in a multi-subunit complex that is proposed to regulate RAB8-dependent vesicular trafficking of membrane proteins from the Golgi to the ciliary membrane 138. Moreover, studies conducted both in humans and in several other model systems have indicated that BBS proteins act in microtubule-based cellular processes 139140141142. Studies in Caenorhabditis elegans revealed that BBS-7 and BBS-8 are required to keep IFT particles intact 139. Morpholino knock-down of BBS genes in zebrafish resulted in a delay of retrograde intracellular transport of melanosomes with melanophores 140. RNAi silencing of BBS4 causes PCM1 mis-localization, de-anchoring of microtubules at the centrosome and arrested cell division 141. Patients with BBS can suffer from anosmia and, interestingly, olfactory cilia of Bbs1 and Bbs4 mutant mice are depleted of stable microtubules 142.

Almström syndrome (ALMS; OMIM 203800) is a disorder similar to BBS since patients are affected by obesity, diabetes and retinal degeneration. In addition they show sensorineural deafness, cardiomyopathy, liver dysfunction and kidney dysfunction, but do not have polydactyly. To date, one gene has been identified as responsible for this disease 143144. ALMS1 encodes for a protein that localizes both to the basal body and centrosomes 145. In vitro studies have demonstrated that ALMS1 is important for ciliogenesis and that it has a role in mechanosensation, since inactivation of the gene resulted in the prevention of Ca²⁺ influx into the cytosol 146.

All the pathways described, together with primary cilia function, have crucial roles in developmental processes (reviewed in 62147). Cilia may have diverse cellular functions during both development and adult tissue homeostasis. Among cilia developmental defects, intraflagellar transport proteins play crucial roles and they contribute to the establishment of left-right asymmetry. The transgene insertion of the Ift88 (Tg737 or polaris) allele in homozygosity caused PKD and preaxial polydactyly 26. The hypomorphic mutants Ift88^orpk died within two weeks of birth, showed growth defects and were affected by hydrocephalus 26. Complete inactivation of the Ift88 allele caused lethality at midgestation 148. The mutant embryo showed randomized left-right asymmetry associated with loss of cilia in the node.

Furthermore, inactivation of the Tbx6 gene led to randomized turning and heart looping. The mutation had a severe effect on the morphology and motility of nodal cilia, demonstrating that Tbx6 is essential for correct left/right axis determination in the mouse and acts through effects on Notch signaling around the node as well as through an effect on the morphology and motility of nodal cilia 149.

The Ift122 protein is a component of IFT particle A. Ift122 null embryos show multiple developmental defects that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively 150.

A mouse mutation, hennin (hnn), caused coupled defects in cilia structure and Shh signaling 151. The hnn murine mutant model showed defects in the neural tube with a Shh-independent expansion of the domain of motor neuron progenitors. The hnn mutation is a null allele of Arl13b, a small GTPase of the Arf/Arl family, and the Arl13b protein is localized to cilia 151.

Primary cilia are required for cerebellar, hippocampus and forebrain development. Specific inactivation in the CNS of Tg737 and Kif3a causes severe cerebellar hypoplasia and foliation abnormalities, primarily attributable to a failure of expansion of the neonatal granule cell progenitor population 152. In addition, recent studies have demonstrated that Kif3a is essential for Shh-dependent expansion of cerebellar progenitors 153. Conditional ablation of the gene in cells derived from Cre-expressing cells under the human glial fibrillary acidic protein promoter resulted in loss of primary cilia in cerebellar granule cell precursors (GCPs). In this animal model, GCPs were specified, but a severe defect in late embryonic and early postnatal expansion of GCPs resulted in atrophied cerebella 153. The same animal model was analyzed for production of adult neural stem cells in the hippocampus and also revealed the absence of primary cilia in the developing dentate gyrus. Primary cilia and Shh signaling are essential for the expansion and establishment of granule neuron precursors in the post-natal dentate gyrus 154.

Mutant mice for Stumpy lack cilia and have evident abnormalities in post-natal developing brain regions, including a hypoplasic hippocampus characterized by a primary deficiency in astrocyte-like neural precursors 155.

Cobblestone is a hypomorphic allele of the IFT gene Ift88. Cobblestone mutants show both severe defects in the formation of dorsomedial telencephalic structures, such as the choroid plexus, cortical hem and hippocampus, and also a relaxation of both dorsal-ventral and rostral-caudal compartmental boundaries. In this animal model, Gli3 proteolytic processing is reduced and an upregulation of canonical Wnt signaling in the neocortex and in the caudal forebrain has been observed. These results indicate a critical role for ciliary function in the developing forebrain 156. In addition, the inactivation of Ift172 revealed that it is required in the patterning of the mammalian brain, and it plays a crucial role in primary cilia formation during development 157.

On the basis of all these studies, the role of cilia in development is important in defining the structure of the organism. In fact, impairment in cilia function leads to structural defects (polydactyly, brain abnormalities, left-right asymmetry). Several ciliopathies such as Bardet-Biedl, Almström syndrome, Joubert and oral-facial-digital syndrome type I are pleiotropic disorders, which include limb abnormalities, renal cystic disease, CNS abnormalities including mental retardation, and/or obesity. In several cases, however, mental retardation is not associated with CNS structural abnormalities. Obesity and mental retardation, not associated with structural defects, can be considered behavioral defects. This observation suggests that cilia may have an important role in organ maintenance and function, yet to be defined, besides the well-established role during development.

For this reason, an intriguing issue is the role of cilia in adult life, when all the structures are already defined. To date, little is known about the role and importance of cilia during post-natal life. It would be fascinating to speculate about developmental defects in congenital ciliopathies and defects of adult-onset ciliopathies. The role of the ciliary gene Pkd1 during post-natal life has been investigated 158. Inactivation of Pkd1 in mice before day 13 resulted in severe polycystic kidney, while inactivation at day 14 and later resulted in slow-onset cystic kidney disease. These studies revealed a temporal breakpoint for cyst formation in the kidney and may reflect the different function of Pkd1 and cilia in the adult organ with respect to the developing one 158.

Moreover, recent studies have shown that inactivation of two ciliary genes Tg737 and Kif3a in adult life leads to obesity and slow-onset cystic kidney disease 159. In order to better investigate the role of cilia in obesity, Davenport and colleagues 159 produced two additional mouse models, disrupting Kif3a in the neurons of the CNS and in the hypothalamus. Interestingly, loss of cilia on neurons resulted in hyperphagia and obesity, indicating that neuronal cilia have a main role in satiety responses 159.

Deletion of Tg737 from cells in the ovary by Prx1-Cre activity, which is detected in the follicle cells of the ovary, demonstrated that primary cilia have an essential role in ovarian function. Mutant mice showed abnormalities in the estrous cycle, alterations in ovulation, and a delay in mammary gland development, characterized by a lack of terminal end buds 160.

Recently, expression studies of components of the Hh pathway in the developing pancreas and in adult cancer pancreatic cell lines have demonstrated that the onset of Hh signaling from human embryogenesis to fetal development is associated with accumulation of Smo and Gli2 in duct primary cilia and with reduction of Gli3 in the duct epithelium 161. Smo, Ptc1, and Gli2 localized to primary cilia of two pancreatic cancer cell lines, which may maintain high levels of non-stimulated Hh pathway activity. These findings indicate that primary cilia are involved in pancreatic development and post-natal tissue homeostasis 161.

Obesity in adult life is a main feature of the BBS human phenotype and BBS mouse models. Inactivation of Bbs2, Bbs4 and Bbs6 genes leads to obesity associated with increased food intake 162163164 and with hyperleptinemia and leptin resistance 165. Recently, studies have demonstrated that BBS proteins are required for LepR signaling 166 and that a peripheral primary dysfunction of adipogenesis contributes to the pathogenesis of obesity in BBS 167.

The world of cilia is very complex and the studies carried out up to this point have demonstrated that cilia may have different roles in space and time. Further research is needed to dissect the role of cilia during adult life and to fill the gap in knowledge concerning adult-onset phenotypes.

Remarkable advances in the understanding of cilia function have been made in a relatively short amount of time. Recent studies have provided key insights into the mechanisms involving cilia. These organelles appear to be involved in many cellular processes, such as proliferation, signal transduction and differentiation. The studies on primary cilia are revealing a complex scenario and their role may be organ-specific and even cell type-specific. This could explain the heterogeneity and complexity of phenotypes associated with mutations in ciliary genes.

To date, much attention has been focused on the role of primary cilia during development; recent studies are now shedding light on their function in post-natal life. Obesity and mental retardation seem to be prominent features of several ciliopathies. Understanding primary cilia function in adult life could help to elucidate the mechanisms responsible for such phenotypes, and further studies are needed in this direction.

Although the growing body of literature on primary cilia is promising, further studies are necessary to dissect and understand the primary cilium, a complex and dynamic organelle.

The authors declare that they have no competing interests.

AD and BF wrote the review manuscript. The authors read and approved the final manuscript.