Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole–cilium interface and facilitates proper cilium formation and function

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1^−/− cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1^−/− cells.     

Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development

Mucociliary epithelia are essential for homeostasis of many organs and consist of mucus-secreting goblet cells and ciliated cells. Here, we present the ciliated epidermis of Xenopus embryos as a facile model system for in vivo molecular studies of mucociliary epithelial development. Using an in situ hybridization-based approach, we identified numerous genes expressed differentially in mucus-secreting cells or in ciliated cells. Focusing on genes expressed in ciliated cells, we have identified new candidate ciliogenesis factors, including several not present in the current ciliome. We find that TTC25-GFP is localized to the base of cilia and to ciliary axonemes, and disruption of TTC25 function disrupts ciliogenesis. Mig12-GFP localizes very strongly to the base of cilia and confocal imaging of this construct allows for simple visualization of the planar polarity of basal bodies that underlies polarized ciliary beating. Knockdown of Mig12 disrupts ciliogenesis. Finally, we show that ciliogenesis factors identified in the Xenopus epidermis are required in the midline to facilitate neural tube closure. These results provide further evidence of a requirement for cilia in neural tube morphogenesis and suggest that genes identified in the Xenopus epidermis play broad roles in ciliogenesis. The suites of genes identified here will provide a foundation for future studies, and may also contribute to our understanding of pathological changes in mucociliary epithelia that accompany diseases such as asthma.     

Mutations in DDX59 Implicate RNA Helicase in the Pathogenesis of Orofaciodigital Syndrome

Orofaciodigital syndrome (OFD) is a recognized clinical entity with core defining features in the mouth, face, and digits, in addition to various other features that have been proposed to define distinct subtypes. The three genes linked to OFD—OFD1, TMEM216, and TCTN3—play a role in ciliary biology, a finding consistent with the clinical overlap between OFD and other ciliopathies. Most autosomal-recessive cases of OFD, however, remain undefined genetically. In two multiplex consanguineous Arab families affected by OFD, we identified a tight linkage interval in chromosomal region 1q32.1. Exome sequencing revealed a different homozygous variant in DDX59 in each of the two families, and at least one of the two variants was accompanied by marked reduction in the level of DDX59. DDX59 encodes a relatively uncharacterized member of the DEAD-box-containing RNA helicase family of proteins, which are known to play a critical role in all aspects of RNA metabolism. We show that Ddx59 is highly enriched in its expression in the developing murine palate and limb buds. At the cellular level, we show that DDX59 is localized dynamically to the nucleus and the cytoplasm. Consistent with the absence of DDX59 representation in ciliome databases and our demonstration of its lack of ciliary localization, ciliogenesis appears to be intact in mutant fibroblasts but ciliary signaling appears to be impaired. Our data strongly implicate this RNA helicase family member in the pathogenesis of OFD, although the causal mechanism remains unclear.     

Development of cilia in embryos of the turbellarian Macrostomum

Electron microscopy of Macrostomum hystricinum raised in culture shows that ciliogenesis in the worm's epidermal blastomeres begins in embryos 39–41 h old with kinetosomal and de novo genesis of presumptive basal bodies, which are morphologically distinguishable from centrioles of the mitotic apparatus, and proceeds by the migration of basal bodies to the apical plasma membrane of the cells and their production there of ciliary axonemes by an age of 51–53 h when the bastomeres emerge between yolk cells on the embryo's surface. Ciliogenesis continues throughout development with the addition of cilia virtually one by one to the expanding epidermal cells' surfaces. At no time in ciliogenesis are stages seen that might show derivation of these multiciliated cells from the primitive monociliated cell type presumably present in the ancestors of the Turbellaria.     

Defective ciliogenesis, embryonic lethality and severe impairment of the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling.     

CEP290 is essential for the initiation of ciliary transition zone assembly

Cilia play critical roles during embryonic development and adult homeostasis. Dysfunction of cilia leads to various human genetic diseases, including many caused by defects in transition zones (TZs), the “gates” of cilia. The evolutionarily conserved TZ component centrosomal protein 290 (CEP290) is the most frequently mutated human ciliopathy gene, but its roles in ciliogenesis are not completely understood. Here, we report that CEP290 plays an essential role in the initiation of TZ assembly in Drosophila. Mechanistically, the N-terminus of CEP290 directly recruits DAZ interacting zinc finger protein 1 (DZIP1), which then recruits Chibby (CBY) and Rab8 to promote early ciliary membrane formation. Complete deletion of CEP290 blocks ciliogenesis at the initiation stage of TZ assembly, which can be mimicked by DZIP1 deletion mutants. Remarkably, expression of the N-terminus of CEP290 alone restores the TZ localization of DZIP1 and subsequently ameliorates the defects in TZ assembly initiation in cep290 mutants. Our results link CEP290 to DZIP1-CBY/Rab8 module and uncover a previously uncharacterized important function of CEP290 in the coordination of early ciliary membrane formation and TZ assembly.

Dysfunction of cilia leads to various human genetic diseases, including many caused by defects in transition zones (TZs), the “gates” of cilia. A study in Drosophila reveals that the cilia TZ core protein CEP290 coordinates early ciliary membrane formation and TZ assembly; the N-terminus of CEP290 recruits DZIP1, which in turn recruits Rab8 and CBY to promote early ciliary membrane formation.     

The TBC1D31/praja2 complex controls primary ciliogenesis through PKA‐directed OFD1 ubiquitylation

The primary cilium is a microtubule‐based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X‐linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin‐proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G‐protein‐coupled receptor (GPCR)‐cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2‐UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non‐phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders.     

An essential role for DYF-11/MIP-T3 in assembling functional intraflagellar transport complexes

MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia 1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. elegans MIP-T3 ortholog DYF-11 is an intraflagellar transport (IFT) protein that plays a critical role in assembling functional kinesin motor-IFT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the IFT machinery, including Kinesin-II, as well as IFT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that DYF-11 functions as a novel component of IFT subcomplex B. Consistent with an evolutionarily conserved cilia-associated role, mammalian MIP-T3 localizes to basal bodies and cilia, and zebrafish mipt3 functions synergistically with the Bardet-Biedl syndrome protein Bbs4 to ensure proper gastrulation, a key cilium- and basal body-dependent developmental process. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis and further highlight the emerging role of this organelle in vertebrate development.     

A non-canonical Hedgehog pathway initiates ciliogenesis and autophagy

Primary cilia function as critical signaling hubs whose absence leads to severe disorders collectively known as ciliopathies; our knowledge of ciliogenesis remains limited. We show that Smo induces ciliogenesis through two distinct yet essential noncanonical Hh pathways in several cell types, including neurons. Surprisingly, ligand activation of Smo induces autophagy via an LKB1-AMPK axis to remove the satellite pool of OFD1. This is required, but not sufficient, for ciliogenesis. Additionally, Smo activates the Gα_i-LGN-NuMA-dynein axis, causing accumulation of a portion of OFD1 at centrioles in early ciliogenesis. Both pathways are critical for redistribution of BBS4 from satellites to centrioles, which is also mediated by OFD1 centriolar translocation. Notably, different Smo agonists, which activate Smo distinctly, activate one or the other of these pathways; only in combination they recapitulate the activity of Hh ligand. These studies provide new insight into physiological stimuli (Hh) that activate autophagy and promote ciliogenesis and introduce a novel role for the Gα_i-LGN-NuMA-dynein complex in this process.     

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.     

Two appendages homologous between basal bodies and centrioles are formed using distinct Odf2 domains

Ciliogenesis is regulated by context-dependent cellular cues, including some transduced through appendage-like structures on ciliary basal bodies called transition fibers and basal feet. However, the molecular basis for this regulation is not fully understood. The Odf2 gene product, ODF2/cenexin, is essential for both ciliogenesis and the formation of the distal and subdistal appendages on centrioles, which become basal bodies. We examined the effects of Odf2 deletion constructs on ciliogenesis in Odf2-knockout F9 cells. Electron microscopy revealed that ciliogenesis and transition fiber formation required the ODF2/cenexin fragment containing amino acids (aa) 188–806, whereas basal foot formation required aa 1–59 and 188–806. These sequences also formed distal and subdistal appendages, respectively, indicating that the centriole appendages are molecularly analogous to those on basal bodies. We used the differential formation of appendages by Odf2 deletion constructs to study the incorporation and function of molecules associated with each appendage type. We found that transition fibers and distal appendages were required for ciliogenesis and subdistal appendages stabilized the centrosomal microtubules.     

Cluap1 localizes preferentially to the base and tip of cilia and is required for ciliogenesis in the mouse embryo

Qilin is one of several genes in zebrafish whose mutation results in cystic kidney. We have now studied the role of its mouse ortholog, Cluap1, in embryonic development by generating Cluap1 knockout (Cluap1^−/−) mice. Cluap1^−/− embryos died mid-gestation manifesting impairment of ciliogenesis in various regions including the node and neural tube. The basal body was found to be properly docked to the apical membrane of cells in the mutant, but the axoneme failed to grow. Cluap1 is a ciliary protein and is preferentially localized at the base and tip of cilia. Hedgehog signaling, as revealed with a Pacthed1-lacZ reporter gene, was lost in Cluap1^−/− embryos at embryonic day (E) 8.5 but was ectopically expanded at E9.0. The Cluap1 knockout embryos also failed to manifest left–right asymmetric expression of Nodal in the lateral plate, most likely as a result of the loss of Hedgehog signaling in node crown cells that in turn leads to pronounced down-regulation of Gdf1 expression in these cells. Crown cell-specific restoration of Cluap1 expression rescued Gdf1 expression in crown cells and left-sided Nodal expression in the lateral plate of mutant embryos. Our results suggest that Cluap1 contributes to ciliogenesis by regulating the intraflagellar transport (IFT) cycle at the base and tip of the cilium.     

TMEM231, mutated in orofaciodigital and Meckel syndromes,organizes the ciliary transition zone

The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS.     

Redistribution of the kinesin-II subunit KAP from cilia to nuclei during the mitotic and ciliogenic cycles in sea urchin embryos

KAP is the non-motor subunit of the heteromeric plus-end directed microtubule (MT) motor protein kinesin-II essential for normal cilia formation. Studies in Chlamydomonas have demonstrated that kinesin-II drives the anterograde intraflagellar transport (IFT) of protein complexes along ciliary axonemes. We used a green fluorescent protein (GFP) chimera of KAP, KAP-GFP, to monitor movements of this kinesin-II subunit in cells of sea urchin blastulae where cilia are retracted and rebuilt with each mitosis. As expected if involved in IFT, KAP-GFP localized to apical cytoplasm, basal bodies, and cilia and became concentrated on basal bodies of newly forming cilia. Surprisingly, after ciliary retraction early in mitosis, KAP-GFP moved into nuclei before nuclear envelope breakdown, was again present in nuclei after nuclear envelope reformation, and only decreased in nuclei as ciliogenesis reinitiated. Nuclear transport of KAP-GFP could be due to a putative nuclear localization signal and nuclear export signals identified in the sea urchin KAP primary sequence. Our observation of a protein involved in IFT being imported into the nucleus after ciliary retraction and again after nuclear envelope reformation suggests KAP115 may serve as a signal to the nucleus to reinitiate cilia formation during sea urchin development.     

Ttc21b is required to restrict sonic hedgehog activity in the developing mouse forebrain

Organizing centers in the developing brain provide an assortment of instructive patterning cues, including Sonic hedgehog (Shh). Here we characterize the forebrain phenotype caused by loss of Ttc21b, a gene we identified in an ENU mutagenesis screen as a novel ciliary gene required for retrograde intraflagellar transport. The Ttc21b mutant has defects in limb, eye and, most dramatically, brain development. We show that Shh signaling is elevated in the rostral portion of the mutant embryo, including in a domain in or near the zona limitans intrathalamica. We demonstrate here that ciliary defects seen in the Ttc21b mutant extend to the embryonic brain, adding forebrain development to the spectrum of tissues affected by defects in ciliary physiology. We show that development of the Ttc21b brain phenotype is modified by lowering levels of the Shh ligand, supporting our hypothesis that the abnormal patterning is a consequence of elevated Shh signaling. Finally, we evaluate Wnt signaling but do not find evidence that this plays a role in causing the perturbed neurodevelopmental phenotype we describe.     

Sept6 Is Required for Ciliogenesis in Kupffer's Vesicle,the Pronephros,and the Neural Tube during Early Embryonic Development

Septins are conserved filament-forming GTP-binding proteins that act as cellular scaffolds or diffusion barriers in a number of cellular processes. However, the role of septins in vertebrate development remains relatively obscure. Here, we show that zebrafish septin 6 (sept6) is first expressed in the notochord and then in nearly all of the ciliary organs, including Kupffer''s vesicle (KV), the pronephros, eye, olfactory bulb, and neural tube. Knockdown of sept6 in zebrafish embryos results in reduced numbers and length of cilia in KV. Consequently, cilium-related functions, such as the left-right patterning of internal organs and nodal/spaw signaling, are compromised. Knockdown of sept6 also results in aberrant cilium formation in the pronephros and neural tube, leading to cilium-related defects in pronephros development and Sonic hedgehog (Shh) signaling. We further demonstrate that SEPT6 associates with acetylated α-tubulin in vivo and localizes along the axoneme in the cilia of zebrafish pronephric duct cells as well as cultured ZF4 cells. Our study reveals a novel role of sept6 in ciliogenesis during early embryonic development in zebrafish.     

Heterotrimeric Kinesin-II Is Required for the Assembly of Motile 9+2 Ciliary Axonemes on Sea Urchin Embryos

Heterotrimeric kinesin-II is a plus end– directed microtubule (MT) motor protein consisting of distinct heterodimerized motor subunits associated with an accessory subunit. To probe the intracellular transport functions of kinesin-II, we microinjected fertilized sea urchin eggs with an anti–kinesin-II monoclonal antibody, and we observed a dramatic inhibition of ciliogenesis at the blastula stage characterized by the assembly of short, paralyzed, 9+0 ciliary axonemes that lack central pair MTs. Control embryos show no such defect and form swimming blastulae with normal, motile, 9+2 cilia that contain kinesin-II as detected by Western blotting. Injection of anti–kinesin-II into one blastomere of a two-cell embryo leads to the development of chimeric blastulae covered on one side with short, paralyzed cilia, and on the other with normal, beating cilia. We observed a unimodal length distribution of short cilia on anti–kinesin-II–injected embryos corresponding to the first mode of the trimodal distribution of ciliary lengths observed for control embryos. This short mode may represent a default ciliary assembly intermediate. We hypothesize that kinesin-II functions during ciliogenesis to deliver ciliary components that are required for elongation of the assembly intermediate and for formation of stable central pair MTs. Thus, kinesin-II plays a critical role in embryonic development by supporting the maturation of nascent cilia to generate long motile organelles capable of producing the propulsive forces required for swimming and feeding.