Mutations in CSPP1, Encoding a Core Centrosomal Protein,Cause a Range of Ciliopathy Phenotypes in Humans

Ciliopathies are characterized by a pattern of multisystem involvement that is consistent with the developmental role of the primary cilium. Within this biological module, mutations in genes that encode components of the cilium and its anchoring structure, the basal body, are the major contributors to both disease causality and modification. However, despite rapid advances in this field, the majority of the genes that drive ciliopathies and the mechanisms that govern the pronounced phenotypic variability of this group of disorders remain poorly understood. Here, we show that mutations in CSPP1, which encodes a core centrosomal protein, are disease causing on the basis of the independent identification of two homozygous truncating mutations in three consanguineous families (one Arab and two Hutterite) affected by variable ciliopathy phenotypes ranging from Joubert syndrome to the more severe Meckel-Gruber syndrome with perinatal lethality and occipital encephalocele. Consistent with the recently described role of CSPP1 in ciliogenesis, we show that mutant fibroblasts from one affected individual have severely impaired ciliogenesis with concomitant defects in sonic hedgehog (SHH) signaling. Our results expand the list of centrosomal proteins implicated in human ciliopathies.     

Generation of mice with functional inactivation of talpid3, a gene first identified in chicken

Specification of digit number and identity is central to digit pattern in vertebrate limbs. The classical talpid(3) chicken mutant has many unpatterned digits together with defects in other regions, depending on hedgehog (Hh) signalling, and exhibits embryonic lethality. The talpid(3) chicken has a mutation in KIAA0586, which encodes a centrosomal protein required for the formation of primary cilia, which are sites of vertebrate Hh signalling. The highly conserved exons 11 and 12 of KIAA0586 are essential to rescue cilia in talpid(3) chicken mutants. We constitutively deleted these two exons to make a talpid3(-/-) mouse. Mutant mouse embryos lack primary cilia and, like talpid(3) chicken embryos, have face and neural tube defects but also defects in left/right asymmetry. Conditional deletion in mouse limb mesenchyme results in polydactyly and in brachydactyly and a failure of subperisoteal bone formation, defects that are attributable to abnormal sonic hedgehog and Indian hedgehog signalling, respectively. Like talpid(3) chicken limbs, the mutant mouse limbs are syndactylous with uneven digit spacing as reflected in altered Raldh2 expression, which is normally associated with interdigital mesenchyme. Both mouse and chicken mutant limb buds are broad and short. talpid3(-/-) mouse cells migrate more slowly than wild-type mouse cells, a change in cell behaviour that possibly contributes to altered limb bud morphogenesis. This genetic mouse model will facilitate further conditional approaches, epistatic experiments and open up investigation into the function of the novel talpid3 gene using the many resources available for mice.     

NEK1 mutations cause short-rib polydactyly syndrome type majewski

Defects of ciliogenesis have been implicated in a wide range of human phenotypes and play a crucial role in signal transduction and cell-cycle coordination. We used homozygosity mapping in two families with autosomal-recessive short-rib polydactyly syndrome Majewski type to identify mutations in NEK1 as an underlying cause of this lethal osteochondrodysplasia. NEK1 encodes a serine/threonine kinase with proposed function in DNA double-strand repair, neuronal development, and coordination of cell-cycle-associated ciliogenesis. We found that absence of functional full-length NEK1 severely reduces cilia number and alters ciliar morphology in vivo. We further substantiate a proposed digenic diallelic inheritance of ciliopathies by the identification of heterozygous mutations in NEK1 and DYNC2H1 in an additional family. Notably, these findings not only increase the broad spectrum of ciliar disorders, but suggest a correlation between the degree of defective microtubule or centriole elongation and organization and the severity of the resulting phenotype.     

The Zn Finger protein Iguana impacts Hedgehog signaling by promoting ciliogenesis

Hedgehog signaling is critical for metazoan development and requires cilia for pathway activity. The gene iguana was discovered in zebrafish as required for Hedgehog signaling, and encodes a novel Zn finger protein. Planarians are flatworms with robust regenerative capacities and utilize epidermal cilia for locomotion. RNA interference of Smed-iguana in the planarian Schmidtea mediterranea caused cilia loss and failure to regenerate new cilia, but did not cause defects similar to those observed in hedgehog(RNAi) animals. Smed-iguana gene expression was also similar in pattern to the expression of multiple other ciliogenesis genes, but was not required for expression of these ciliogenesis genes. iguana-defective zebrafish had too few motile cilia in pronephric ducts and in Kupffer's vesicle. Kupffer's vesicle promotes left-right asymmetry and iguana mutant embryos had left-right asymmetry defects. Finally, human Iguana proteins (dZIP1 and dZIP1L) localize to the basal bodies of primary cilia and, together, are required for primary cilia formation. Our results indicate that a critical and broadly conserved function for Iguana is in ciliogenesis and that this function has come to be required for Hedgehog signaling in vertebrates.     

A ciliopathy complex builds distal appendages to initiate ciliogenesis

Cells inherit two centrioles, the older of which is uniquely capable of generating a cilium. Using proteomics and superresolved imaging, we identify a module that we term DISCO (distal centriole complex). The DISCO components CEP90, MNR, and OFD1 underlie human ciliopathies. This complex localizes to both distal centrioles and centriolar satellites, proteinaceous granules surrounding centrioles. Cells and mice lacking CEP90 or MNR do not generate cilia, fail to assemble distal appendages, and do not transduce Hedgehog signals. Disrupting the satellite pools does not affect distal appendage assembly, indicating that it is the centriolar populations of MNR and CEP90 that are critical for ciliogenesis. CEP90 recruits the most proximal known distal appendage component, CEP83, to root distal appendage formation, an early step in ciliogenesis. In addition, MNR, but not CEP90, restricts centriolar length by recruiting OFD1. We conclude that DISCO acts at the distal centriole to support ciliogenesis by restraining centriole length and assembling distal appendages, defects in which cause human ciliopathies.     

CYLD mediates ciliogenesis in multiple organs by deubiquitinating Cep70 and inactivating HDAC6

Cilia are hair-like organelles extending from the cell surface with important sensory and motility functions. Ciliary defects can result in a wide range of human diseases known as ciliopathies. However, the molecular mechanisms controlling ciliogenesis remain poorly defined. Here we show that cylindromatosis (CYLD), a tumor suppressor protein harboring deubiquitinase activity, plays a critical role in the assembly of both primary and motile cilia in multiple organs. CYLD knockout mice exhibit polydactyly and various ciliary defects, such as failure in basal body anchorage and disorganization of basal bodies and axenomes. The ciliary function of CYLD is partially attributed to its deconjugation of the polyubiquitin chain from centrosomal protein of 70 kDa (Cep70), a requirement for Cep70 to interact with γ-tubulin and localize at the centrosome. In addition, CYLD-mediated inhibition of histone deacetylase 6 (HDAC6), which promotes tubulin acetylation, constitutes another mechanism for the ciliary function of CYLD. Small-molecule inhibitors of HDAC6 could partially rescue the ciliary defects in CYLD knockout mice. These findings highlight the importance of protein ubiquitination in the modulation of ciliogenesis, identify CYLD as a crucial regulator of this process, and suggest the involvement of CYLD deficiency in ciliopathies.     

Mutations of CEP83 Cause Infantile Nephronophthisis and Intellectual Disability

Ciliopathies are a group of hereditary disorders associated with defects in cilia structure and function. The distal appendages (DAPs) of centrioles are involved in the docking and anchoring of the mother centriole to the cellular membrane during ciliogenesis. The molecular composition of DAPs was recently elucidated and mutations in two genes encoding DAPs components (CEP164/NPHP15, SCLT1) have been associated with human ciliopathies, namely nephronophthisis and orofaciodigital syndrome. To identify additional DAP components defective in ciliopathies, we independently performed targeted exon sequencing of 1,221 genes associated with cilia and 5 known DAP protein-encoding genes in 1,255 individuals with a nephronophthisis-related ciliopathy. We thereby detected biallelic mutations in a key component of DAP-encoding gene, CEP83, in seven families. All affected individuals had early-onset nephronophthisis and four out of eight displayed learning disability and/or hydrocephalus. Fibroblasts and tubular renal cells from affected individuals showed an altered DAP composition and ciliary defects. In summary, we have identified mutations in CEP83, another DAP-component-encoding gene, as a cause of infantile nephronophthisis associated with central nervous system abnormalities in half of the individuals.     

Basal body stability and ciliogenesis requires the conserved component Poc1

Centrioles are the foundation for centrosome and cilia formation. The biogenesis of centrioles is initiated by an assembly mechanism that first synthesizes the ninefold symmetrical cartwheel and subsequently leads to a stable cylindrical microtubule scaffold that is capable of withstanding microtubule-based forces generated by centrosomes and cilia. We report that the conserved WD40 repeat domain–containing cartwheel protein Poc1 is required for the structural maintenance of centrioles in Tetrahymena thermophila. Furthermore, human Poc1B is required for primary ciliogenesis, and in zebrafish, DrPoc1B knockdown causes ciliary defects and morphological phenotypes consistent with human ciliopathies. T. thermophila Poc1 exhibits a protein incorporation profile commonly associated with structural centriole components in which the majority of Poc1 is stably incorporated during new centriole assembly. A second dynamic population assembles throughout the cell cycle. Our experiments identify novel roles for Poc1 in centriole stability and ciliogenesis.     

TCTN3 Mutations Cause Mohr-Majewski Syndrome

Orofaciodigital syndromes (OFDSs) consist of a group of heterogeneous disorders characterized by abnormalities in the oral cavity, face, and digits and associated phenotypic abnormalities that lead to the delineation of 13 OFDS subtypes. Here, by a combined approach of homozygozity mapping and exome ciliary sequencing, we identified truncating TCTN3 mutations as the cause of an extreme form of OFD associated with bone dysplasia, tibial defect, cystic kidneys, and brain anomalies (OFD IV, Mohr-Majewski syndrome). Analysis of 184 individuals with various ciliopathies (OFD, Meckel, Joubert, and short rib polydactyly syndromes) led us to identify four additional truncating TCTN3 mutations in unrelated fetal cases with overlapping Meckel and OFD IV syndromes and one homozygous missense mutation in a family with Joubert syndrome. By exploring roles of TCTN3 in human ciliary related functions, we found that TCTN3 is necessary for transduction of the sonic hedgehog (SHH) signaling pathway, as revealed by abnormal processing of GLI3 in patient cells. These results are consistent with the suggested role of its murine ortholog, which forms a complex at the ciliary transition zone with TCTN1 and TCTN2, both of which are also implicated in the transduction of SHH signaling. Overall, our data show the involvement of the transition zone protein TCTN3 in the regulation of the key SHH signaling pathway and that its disruption causes a severe form of ciliopathy, combining features of Meckel and OFD IV syndromes.     

Disruption of a Ciliary B9 Protein Complex Causes Meckel Syndrome

Nearly every ciliated organism possesses three B9 domain-containing proteins: MKS1, B9D1, and B9D2. Mutations in human MKS1 cause Meckel syndrome (MKS), a severe ciliopathy characterized by occipital encephalocele, liver ductal plate malformations, polydactyly, and kidney cysts. Mouse mutations in either Mks1 or B9d2 compromise ciliogenesis and result in phenotypes similar to those of MKS. Given the importance of these two B9 proteins to ciliogenesis, we examined the role of the third B9 protein, B9d1. Mice lacking B9d1 displayed polydactyly, kidney cysts, ductal plate malformations, and abnormal patterning of the neural tube, concomitant with compromised ciliogenesis, ciliary protein localization, and Hedgehog (Hh) signal transduction. These data prompted us to screen MKS patients for mutations in B9D1 and B9D2. We identified a homozygous c.301A>C (p.Ser101Arg) B9D2 mutation that segregates with MKS, affects an evolutionarily conserved residue, and is absent from controls. Unlike wild-type B9D2 mRNA, the p.Ser101Arg mutation failed to rescue zebrafish phenotypes induced by the suppression of b9d2. With coimmunoprecipitation and mass spectrometric analyses, we found that Mks1, B9d1, and B9d2 interact physically, but that the p.Ser101Arg mutation abrogates the ability of B9d2 to interact with Mks1, further suggesting that the mutation compromises B9d2 function. Our data indicate that B9d1 is required for normal Hh signaling, ciliogenesis, and ciliary protein localization and that B9d1 and B9d2 are essential components of a B9 protein complex, disruption of which causes MKS.     

The centriolar satellite proteins Cep72 and Cep290 interact and are required for recruitment of BBS proteins to the cilium

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Centriolar satellites are centrosome-associated structures, defined by the protein PCM1, that are implicated in centrosomal protein trafficking. We identify Cep72 as a PCM1-interacting protein required for recruitment of the ciliopathy-associated protein Cep290 to centriolar satellites. Loss of centriolar satellites by depletion of PCM1 causes relocalization of Cep72 and Cep290 from satellites to the centrosome, suggesting that their association with centriolar satellites normally restricts their centrosomal localization. We identify interactions between PCM1, Cep72, and Cep290 and find that disruption of centriolar satellites by overexpression of Cep72 results in specific aggregation of these proteins and the BBSome component BBS4. During ciliogenesis, BBS4 relocalizes from centriolar satellites to the primary cilium. This relocalization occurs normally in the absence of centriolar satellites (PCM1 depletion) but is impaired by depletion of Cep290 or Cep72, resulting in defective ciliary recruitment of the BBSome subunit BBS8. We propose that Cep290 and Cep72 in centriolar satellites regulate the ciliary localization of BBS4, which in turn affects assembly and recruitment of the BBSome. Finally, we show that loss of centriolar satellites in zebrafish leads to phenotypes consistent with cilium dysfunction and analogous to those observed in human ciliopathies.     

WDR34 Mutations that Cause Short-Rib Polydactyly Syndrome Type III/Severe Asphyxiating Thoracic Dysplasia Reveal a Role for the NF-κB Pathway in Cilia

Short-rib polydactyly (SRP) syndrome type III, or Verma-Naumoff syndrome, is an autosomal-recessive chondrodysplasia characterized by short ribs, a narrow thorax, short long bones, an abnormal acetabulum, and numerous extraskeletal malformations and is lethal in the perinatal period. Presently, mutations in two genes, IFT80 and DYNC2H1, have been identified as being responsible for SRP type III. Via homozygosity mapping in three affected siblings, a locus for the disease was identified on chromosome 9q34.11, and homozygosity for three missense mutations in WDR34 were found in three independent families, as well as compound heterozygosity for mutations in one family. WDR34 encodes a member of the WD repeat protein family with five WD40 domains, which acts as a TAK1-associated suppressor of the IL-1R/TLR3/TLR4-induced NF-κB activation pathway. We showed, through structural modeling, that two of the three mutations altered specific structural domains of WDR34. We found that primary cilia in WDR34 mutant fibroblasts were significantly shorter than normal and had a bulbous tip. This report expands on the pathogenesis of SRP type III and demonstrates that a regulator of the NF-κB activation pathway is involved in the pathogenesis of the skeletal ciliopathies.     

DCDC2 Mutations Cause a Renal-Hepatic Ciliopathy by Disrupting Wnt Signaling

Nephronophthisis-related ciliopathies (NPHP-RC) are recessive diseases characterized by renal dysplasia or degeneration. We here identify mutations of DCDC2 as causing a renal-hepatic ciliopathy. DCDC2 localizes to the ciliary axoneme and to mitotic spindle fibers in a cell-cycle-dependent manner. Knockdown of Dcdc2 in IMCD3 cells disrupts ciliogenesis, which is rescued by wild-type (WT) human DCDC2, but not by constructs that reflect human mutations. We show that DCDC2 interacts with DVL and DCDC2 overexpression inhibits β-catenin-dependent Wnt signaling in an effect additive to Wnt inhibitors. Mutations detected in human NPHP-RC lack these effects. A Wnt inhibitor likewise restores ciliogenesis in 3D IMCD3 cultures, emphasizing the importance of Wnt signaling for renal tubulogenesis. Knockdown of dcdc2 in zebrafish recapitulates NPHP-RC phenotypes, including renal cysts and hydrocephalus, which is rescued by a Wnt inhibitor and by WT, but not by mutant, DCDC2. We thus demonstrate a central role of Wnt signaling in the pathogenesis of NPHP-RC, suggesting an avenue for potential treatment of NPHP-RC.     

Loss of cilia causes embryonic lung hypoplasia,liver fibrosis,and cholestasis in the talpid3 ciliopathy mutant

Sonic hedgehog plays an essential role in maintaining hepatoblasts in a proliferative non-differentiating state during embryogenesis. Transduction of the Hedgehog signaling pathway is dependent on the presence of functional primary cilia and hepatoblasts, therefore, must require primary cilia for normal function. In congenital syndromes in which cilia are absent or non-functional (ciliopathies) hepatorenal fibrocystic disease is common and primarily characterized by ductal plate malformations which underlie the formation of liver cysts, as well as less commonly, by hepatic fibrosis, although a role for abnormal Hedgehog signal transduction has not been implicated in these phenotypes. We have examined liver, lung and rib development in the talpid³ chicken mutant, a ciliopathy model in which abnormal Hedgehog signaling is well characterized. We find that the talpid³ phenotype closely models that of human short-rib polydactyly syndromes which are caused by the loss of cilia, and exhibit hypoplastic lungs and liver failure. Through an analysis of liver and lung development in the talpid³ chicken, we propose that cilia in the liver are essential for the transduction of Hedgehog signaling during hepatic development. The talpid³ chicken represents a useful resource in furthering our understanding of the pathology of ciliopathies beyond the treatment of thoracic insufficiency as well as generating insights into the role Hedgehog signaling in hepatic development.     

Aberrant FGF signaling, independent of ectopic hedgehog signaling, initiates preaxial polydactyly in Dorking chickens

The formation of supernumerary digits, or polydactyly, is a common congenital malformation. Although mutations in a number of genes have been linked to polydactyly, the molecular etiology for a third of human disorders with polydactyly remains unknown. To increase our understanding of the potential causes for polydactyly, we characterized a spontaneous chicken mutant, known as Dorking. The hindlimbs of Dorkings form a preaxial supernumerary digit. During the early stages of limb development, ectopic expression of several genes, including Sonic Hedgehog (Shh) and Fibroblast Growth Factor 4 (Fgf4), was found in Dorking hindlimbs. In addition to ectopic gene expression, a decrease in cell death in the anterior of the developing Dorking hindlimb was observed. Further molecular investigation revealed that ectopic Fgf4 expression was initiated and maintained independent of ectopic Shh. Additionally, inhibition of Fgf signaling but not hedgehog signaling was capable of restoring the normal anterior domain of cell death in Dorking hindlimbs. Our data indicates that in Dorking chickens, preaxial polydactyly is initiated independent of Shh.     

The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly

We have identified Talpid3/KIAA0586 as a component of a CP110-containing protein complex important for centrosome and cilia function. Talpid3 assembles a ring-like structure at the extreme distal end of centrioles. Ablation of Talpid3 resulted in an aberrant distribution of centriolar satellites involved in protein trafficking to centrosomes as well as cilia assembly defects, reminiscent of loss of Cep290, another CP110-associated protein. Talpid3 depletion also led to mislocalization of Rab8a, a small GTPase thought to be essential for ciliary vesicle formation. Expression of activated Rab8a suppressed cilia assembly defects provoked by Talpid3 depletion, suggesting that Talpid3 affects cilia formation through Rab8a recruitment and/or activation. Remarkably, ultrastructural analyses showed that Talpid3 is required for centriolar satellite dispersal, which precedes the formation of mature ciliary vesicles, a process requiring Cep290. These studies suggest that Talpid3 and Cep290 play overlapping and distinct roles in ciliary vesicle formation through regulation of centriolar satellite accretion and Rab8a.     

Ofd1 Controls Dorso-Ventral Patterning and Axoneme Elongation during Embryonic Brain Development

Oral-facial-digital type I syndrome (OFDI) is a human X-linked dominant-male-lethal developmental disorder caused by mutations in the OFD1 gene. Similar to other inherited disorders associated to ciliary dysfunction OFD type I patients display neurological abnormalities. We characterized the neuronal phenotype that results from Ofd1 inactivation in early phases of mouse embryonic development and at post-natal stages. We determined that Ofd1 plays a crucial role in forebrain development, and in particular, in the control of dorso-ventral patterning and early corticogenesis. We observed abnormal activation of Sonic hedgehog (Shh), a major pathway modulating brain development. Ultrastructural studies demonstrated that early Ofd1 inactivation results in the absence of ciliary axonemes despite the presence of mature basal bodies that are correctly orientated and docked. Ofd1 inducible-mediated inactivation at birth does not affect ciliogenesis in the cortex, suggesting a developmental stage-dependent role for a basal body protein in ciliogenesis. Moreover, we showed defects in cytoskeletal organization and apical-basal polarity in Ofd1 mutant embryos, most likely due to lack of ciliary axonemes. Thus, the present study identifies Ofd1 as a developmental disease gene that is critical for forebrain development and ciliogenesis in embryonic life, and indicates that Ofd1 functions after docking and before elaboration of the axoneme in vivo.     

Acute Versus Chronic Loss of Mammalian Azi1/Cep131 Results in Distinct Ciliary Phenotypes

Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1^Gt/Gt). Surprisingly, Azi1^Gt/Gt MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease.