Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus

Cilia are complex organelles involved in sensory perception and fluid or cell movement. They are constructed through a highly conserved process called intraflagellar transport (IFT). Mutations in IFT genes, such as Tg737, result in severe developmental defects and disease. In the case of the Tg737orpk mutants, these pathological alterations include cystic kidney disease, biliary and pancreatic duct abnormalities, skeletal patterning defects, and hydrocephalus. Here, we explore the connection between cilia dysfunction and the development of hydrocephalus by using the Tg737orpk mutants. Our analysis indicates that cilia on cells of the brain ventricles of Tg737orpk mutant mice are severely malformed. On the ependymal cells, these defects lead to disorganized beating and impaired cerebrospinal fluid (CSF) movement. However, the loss of the cilia beat and CSF flow is not the initiating factor, as the pathology is present prior to the development of motile cilia on these cells and CSF flow is not impaired at early stages of the disease. Rather, our results suggest that loss of cilia leads to altered function of the choroid plexus epithelium, as evidenced by elevated intracellular cAMP levels and increased chloride concentration in the CSF. These data suggest that cilia function is necessary for regulating ion transport and CSF production, as well as for CSF flow through the ventricles.     

Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella

Intraflagellar transport (IFT) is a rapid movement of multi-subunit protein particles along flagellar microtubules and is required for assembly and maintenance of eukaryotic flagella. We cloned and sequenced a Chlamydomonas cDNA encoding the IFT88 subunit of the IFT particle and identified a Chlamydomonas insertional mutant that is missing this gene. The phenotype of this mutant is normal except for the complete absence of flagella. IFT88 is homologous to mouse and human genes called Tg737. Mice with defects in Tg737 die shortly after birth from polycystic kidney disease. We show that the primary cilia in the kidney of Tg737 mutant mice are shorter than normal. This indicates that IFT is important for primary cilia assembly in mammals. It is likely that primary cilia have an important function in the kidney and that defects in their assembly can lead to polycystic kidney disease.     

The cilia protein IFT88 is required for spindle orientation in mitosis

Cilia dysfunction has long been associated with cyst formation and ciliopathies. More recently, misoriented cell division has been observed in cystic kidneys, but the molecular mechanism leading to this abnormality remains unclear. Proteins of the intraflagellar transport (IFT) machinery are linked to cystogenesis and are required for cilia formation in non-cycling cells. Several IFT proteins also localize to spindle poles in mitosis, indicating uncharacterized functions for these proteins in dividing cells. Here, we show that IFT88 depletion induces mitotic defects in human cultured cells, in kidney cells from the IFT88 mouse mutant Tg737(orpk) and in zebrafish embryos. In mitosis, IFT88 is part of a dynein1-driven complex that transports peripheral microtubule clusters containing microtubule-nucleating proteins to spindle poles to ensure proper formation of astral microtubule arrays and thus proper spindle orientation. This work identifies a mitotic mechanism for a cilia protein in the orientation of cell division and has important implications for the etiology of ciliopathies.     

Polaris, a protein involved in left-right axis patterning, localizes to basal bodies and cilia

Mutations in Tg737 cause a wide spectrum of phenotypes, including random left-right axis specification, polycystic kidney disease, liver and pancreatic defects, hydrocephalus, and skeletal patterning abnormalities. To further assess the biological function of Tg737 and its role in the mutant pathology, we identified the cell population expressing Tg737 and determined the subcellular localization of its protein product called Polaris. Tg737 expression is associated with cells possessing either motile or immotile cilia and sperm. Similarly, Polaris concentrated just below the apical membrane in the region of the basal bodies and within the cilia or flagellar axoneme. The data suggest that Polaris functions in a ciliogenic pathway or in cilia maintenance, a role supported by the loss of cilia on the ependymal cell layer in ventricles of Tg737(orpk) brains and by the lack of node cilia in Tg737(Delta2-3betaGal) mutants.     

Primary cilia of inv/inv mouse renal epithelial cells sense physiological fluid flow: bending of primary cilia and Ca2+ influx

Primary cilia are hypothesized to act as a mechanical sensor to detect renal tubular fluid flow. Anomalous structure of primary cilia and/or impairment of increases in intracellular Ca2+ concentration in response to fluid flow are thought to result in renal cyst formation in conditional kif3a knockout, Tg737 and pkd1/pkd2 mutant mice. The mutant inv/inv mouse develops multiple renal cysts like kif3a, Tg737 and pkd1/pkd2 mutants. Inv proteins have been shown to be localized in the renal primary cilia, but response of inv/inv cilia to fluid stress has not been examined. In the present study, we examined the mechanical response of primary cilia to physiological fluid flow using a video microscope, as well as intracellular Ca2+ increases in renal epithelial cells from normal and inv/inv mice in response to flow stress. Percentages of ciliated cells and the length of primary cilia were not significantly different between primary renal cell cultures from normal and inv/inv mutant mice. Localization of inv protein was restricted to the base of primary cilia even under flow stress. Inv/inv mutant cells had similar bending mechanics of primary cilia in response to physiological fluid flow compared to normal cells. Furthermore, no difference was found in intracellular Ca2+ increases in response to physiological fluid flow between normal and inv/inv mutant cells. Our present study suggests that the function of the inv protein is distinct from polaris (the Tg737 gene product), polycystins (pkd1 and pkd2 gene products).     

Calreticulin reveals a critical Ca(2+) checkpoint in cardiac myofibrillogenesis

Approximately 10% of the photoreceptor outer segment (OS) is turned over each day, requiring large amounts of lipid and protein to be moved from the inner segment to the OS. Defects in intraphotoreceptor transport can lead to retinal degeneration and blindness. The transport mechanisms are unknown, but because the OS is a modified cilium, intraflagellar transport (IFT) is a candidate mechanism. IFT involves movement of large protein complexes along ciliary microtubules and is required for assembly and maintenance of cilia. We show that IFT particle proteins are localized to photoreceptor connecting cilia. We further find that mice with a mutation in the IFT particle protein gene, Tg737/IFT88, have abnormal OS development and retinal degeneration. Thus, IFT is important for assembly and maintenance of the vertebrate OS.     

Mechanosensing by the primary cilium: deletion of Kif3A reduces bone formation due to loading

Primary cilia, solitary microtubule-based structures that grow from the centriole and extend into the extracellular space, have increasingly been implicated as sensors of a variety of biochemical and biophysical signals. Mutations in primary cilium-related genes have been linked to a number of rare developmental disorders as well as dysregulation of cell proliferation. We propose that primary cilia are also important in mechanically regulated bone formation in adults and that their malfunction could play a role in complex multi-factorial bone diseases, such as osteoporosis. In this study, we generated mice with an osteoblast- and osteocyte-specific knockout of Kif3a, a subunit of the kinesin II intraflagellar transport (IFT) protein; IFT is required for primary cilia formation, maintenance, and function. These Colα1(I) 2.3-Cre;Kif3a(fl/fl) mice exhibited no obvious morphological skeletal abnormalities. Skeletally mature Colα1(I) 2.3-Cre;Kif3a(fl/fl) and control mice were exposed to 3 consecutive days of cyclic axial ulna loading, which resulted in a significant increase in bone formation in both the conditional knockouts and controls. However, Colα1(I) 2.3-Cre;Kif3a(fl/fl) mice did exhibit decreased formation of new bone in response to mechanical ulnar loading compared to control mice. These results suggest that primary cilia act as cellular mechanosensors in bone and that their function may be critical for the regulation of bone physiology due to mechanical loading in adults.     

An autosomal recessive polycystic kidney disease gene homolog is involved in intraflagellar transport in C. elegans ciliated sensory neurons

In this report, we show that the Caenorhabditis elegans gene osm-5 is homologous to the Chlamydomonas gene IFT88 and the mouse autosomal recessive polycystic kidney disease (ARPKD) gene, Tg737. The function of this ARPKD gene may be evolutionarily conserved: mutations result in defective ciliogenesis in worms [1], algae [2], and mice [2, 3]. Intraflagellar transport (IFT) is essential for the development and maintenance of motile and sensory cilia [4]. The biochemically isolated IFT particle from Chlamydomonas flagella is composed of 16 polypeptides in one of two Complexes (A and B) [5, 6] whose movement is powered by kinesin II (anterograde) and cytoplasmic dynein (retrograde) [7-9]. We demonstrate that OSM-5 (a Complex B polypeptide), DAF-10 and CHE-11 (two Complex A polypeptides), and CHE-2 [10], a previously uncategorized IFT polypeptide, all move at the same rate in C. elegans sensory cilia. In the absence of osm-5, the C. elegans autosomal dominant PKD (ADPKD) gene products [11] accumulate in stunted cilia, suggesting that abnormal or lack of cilia or defects in IFT may result in diseases such as polycystic kidney disease (PKD).     

The homodimeric kinesin, Kif17, is essential for vertebrate photoreceptor sensory outer segment development

Sensory cilia and intraflagellar transport (IFT), a pathway essential for ciliogenesis, play important roles in embryonic development and cell differentiation. In vertebrate photoreceptors IFT is required for the early development of ciliated sensory outer segments (OS), an elaborate organelle that sequesters the many proteins comprising the phototransduction machinery. As in other cilia and flagella, heterotrimeric members of the kinesin 2 family have been implicated as the anterograde IFT motor in OS. However, in Caenorhabditis elegans, OSM-3, a homodimeric kinesin 2 motor, plays an essential role in some, but not all sensory cilia. Kif17, a vertebrate OSM-3 homologue, is known for its role in dendritic trafficking in neurons, but a function in ciliogenesis has not been determined. We show that in zebrafish Kif17 is widely expressed in the nervous system and retina. In photoreceptors Kif17 co-localizes with IFT proteins within the OS, and co-immunoprecipitates with IFT proteins. Knockdown of Kif17 has little if any effect in early embryogenesis, including the formation of motile sensory cilia in the pronephros. However, OS formation and targeting of the visual pigment protein is severely disrupted. Our analysis shows that Kif17 is essential for photoreceptor OS development, and suggests that Kif17 plays a cell type specific role in vertebrate ciliogenesis.     

ICK is essential for cell type‐specific ciliogenesis and the regulation of ciliary transport

Cilia and flagella are formed and maintained by intraflagellar transport (IFT) and play important roles in sensing and moving across species. At the distal tip of the cilia/flagella, IFT complexes turn around to switch from anterograde to retrograde transport; however, the underlying regulatory mechanism is unclear. Here, we identified ICK localization at the tip of cilia as a regulator of ciliary transport. In ICK‐deficient mice, we found ciliary defects in neuronal progenitor cells with Hedgehog signal defects. ICK‐deficient cells formed cilia with mislocalized Hedgehog signaling components. Loss of ICK caused the accumulation of IFT‐A, IFT‐B, and BBSome components at the ciliary tips. In contrast, overexpression of ICK induced the strong accumulation of IFT‐B, but not IFT‐A or BBSome components at ciliary tips. In addition, ICK directly phosphorylated Kif3a, while inhibition of this Kif3a phosphorylation affected ciliary formation. Our results suggest that ICK is a Kif3a kinase and essential for proper ciliogenesis in development by regulating ciliary transport at the tip of cilia.     

Intraflagellar transport is essential for endochondral bone formation

While cilia are present on most cells in the mammalian body, their functional importance has only recently been discovered. Cilia formation requires intraflagellar transport (IFT), and mutations disrupting the IFT process result in loss of cilia and mid-gestation lethality with developmental defects that include polydactyly and abnormal neural tube patterning. The early lethality in IFT mutants has hindered research efforts to study the role of this organelle at later developmental stages. Thus, to investigate the role of cilia during limb development, we generated a conditional allele of the IFT protein Ift88 (polaris). Using the Cre-lox system, we disrupted cilia on different cell populations within the developing limb. While deleting cilia in regions of the limb ectoderm had no overt effect on patterning, disruption in the mesenchyme resulted in extensive polydactyly with loss of anteroposterior digit patterning and shortening of the proximodistal axis. The digit patterning abnormalities were associated with aberrant Shh pathway activity, whereas defects in limb outgrowth were due in part to disruption of Ihh signaling during endochondral bone formation. In addition, the limbs of mesenchymal cilia mutants have ectopic domains of cells that resemble chondrocytes derived from the perichondrium, which is not typical of Indian hedgehog mutants. Overall these data provide evidence that IFT is essential for normal formation of the appendicular skeleton through disruption of multiple signaling pathways.     

Primary cilia regulates the directional migration and barrier integrity of endothelial cells through the modulation of hsp27 dependent actin cytoskeletal organization

Cilia are mechanosensing organelles that communicate extracellular signals into intracellular responses. Altered functions of primary cilia play a key role in the development of various diseases including polycystic kidney disease. Here, we show that endothelial cells from the oak ridge polycystic kidney (Tg737(orpk/orpk) ) mouse, with impaired cilia assembly, exhibit a reduction in the actin stress fibers and focal adhesions compared to wild-type (WT). In contrast, endothelial cells from polycystin-1 deficient mice (pkd1(null/null) ), with impaired cilia function, display robust stress fibers, and focal adhesion assembly. We found that the Tg737(orpk/orpk) cells exhibit impaired directional migration and endothelial cell monolayer permeability compared to the WT and pkd1(null/null) cells. Finally, we found that the expression of heat shock protein 27 (hsp27) and the phosphorylation of focal adhesion kinase (FAK) are downregulated in the Tg737(orpk/orpk) cells and overexpression of hsp27 restored both FAK phosphorylation and cell migration. Taken together, these results demonstrate that disruption of the primary cilia structure or function compromises the endothelium through the suppression of hsp27 dependent actin organization and focal adhesion formation, which may contribute to the vascular dysfunction in ciliopathies.     

Deletion of IFT20 in the mouse kidney causes misorientation of the mitotic spindle and cystic kidney disease

Primary cilia project from the surface of most vertebrate cells and are thought to be sensory organelles. Defects in primary cilia lead to cystic kidney disease, although the ciliary mechanisms that promote and maintain normal renal function remain incompletely understood. In this work, we generated a floxed allele of the ciliary assembly gene Ift20. Deleting this gene specifically in kidney collecting duct cells prevents cilia formation and promotes rapid postnatal cystic expansion of the kidney. Dividing collecting duct cells in early stages of cyst formation fail to properly orient their mitotic spindles along the tubule, whereas nondividing cells improperly position their centrosomes. At later stages, cells lacking cilia have increased canonical Wnt signaling and increased rates of proliferation. Thus, IFT20 functions to couple extracellular events to cell proliferation and differentiation.     

Kif3a Controls Murine Nephron Number Via GLI3 Repressor,Cell Survival,and Gene Expression in a Lineage-Specific Manner

The primary cilium is required during early embryo patterning, epithelial tubulogenesis, and growth factor-dependent signal transduction. The requirement for primary cilia during renal epithelial-mesenchymal tissue interactions that give rise to nephrons is undefined. Here, we used Cre-mediated recombination to generate mice with Kif3a deficiency targeted to the ureteric and/or metanephric mesenchyme cell lineages in the embryonic kidney. Gradual loss of primary cilia in either lineage leads to a phenotype of reduced nephron number. Remarkably, in addition to cyst formation, loss of primary cilia in the ureteric epithelial cell leads to decreased expression of Wnt11 and Ret and reduced ureteric branching. Constitutive expression of GLI3 repressor (Gli3^Δ699/+) rescues these abnormalities. In embryonic metanephric mesenchyme cells, Kif3a deficiency limits survival of nephrogenic progenitor cells and expression of genes required for nephron formation. Together, our data demonstrate that Kif3a controls nephron number via distinct cell lineage-specific mechanisms.     

Deletion of kif3a in CK19 positive cells leads to primary cilia loss,biliary cell proliferation and cystic liver lesions in TAA-treated mice

Background & aimsLoss of primary cilia in epithelial cells is known to cause cystic diseases of the liver and kidney. We have previously shown that during experimental and human cirrhosis that primary cilia were predominantly expressed on biliary cells in the ductular reaction. However, the role of primary cilia in the pathogenesis of the ductular reaction is not fully understood.MethodsPrimary cilia were specifically removed in biliary epithelial cells (BECs) by the administration of tamoxifen to Kif3a^f/f;CK19^CreERT mice at week 2 of a 20-week course of TAA treatment. Biliary progenitor cells were isolated and grown as organoids from gallbladders. Cells and tissue were analysed using histology, immunohistochemistry and Western blot assays.ResultsAt the end of 20 weeks TAA administration, primary cilia loss in liver BECs resulted in multiple microscopic cystic lesions within an unaltered ductular reaction. These were not seen in control mice who did not receive TAA. There was no effect of biliary primary cilia loss on the development of cirrhosis. Increased cellular proliferation was seen within the cystic structures associated with a decrease in hepatocyte lobular proliferation. Loss of primary cilia within biliary organoids was initially associated with reduced cell passage survival but this inhibitory effect was diminished in later passages. ERK but not WNT signalling was enhanced in primary cilia loss-induced cystic lesions in vivo and its inhibition reduced the expansion of primary cilia deficient biliary progenitor cells in vitro.ConclusionsTAA-treated kif3a BEC-specific knockout mice had an unaltered progression to cirrhosis, but developed cystic lesions that showed increased proliferation.     

Kif3a interacts with Dynactin subunit p150Glued to organize centriole subdistal appendages

Formation of cilia, microtubule‐based structures that function in propulsion and sensation, requires Kif3a, a subunit of Kinesin II essential for intraflagellar transport (IFT). We have found that, Kif3a is also required to organize centrioles. In the absence of Kif3a, the subdistal appendages of centrioles are disorganized and lack p150^Glued and Ninein. Consequently, microtubule anchoring, centriole cohesion and basal foot formation are abrogated by loss of Kif3a. Kif3a localizes to the mother centriole and interacts with the Dynactin subunit p150^Glued. Depletion of p150^Glued phenocopies the effects of loss of Kif3a, indicating that Kif3a recruitment of p150^Glued is critical for subdistal appendage formation. The transport functions of Kif3a are dispensable for subdistal appendage organization as mutant forms of Kif3a lacking motor activity or the motor domain can restore p150^Glued localization. Comparison to cells lacking Ift88 reveals that the centriolar functions of Kif3a are independent of IFT. Thus, in addition to its ciliogenic roles, Kif3a recruits p150^Glued to the subdistal appendages of mother centrioles, critical for centrosomes to function as microtubule‐organizing centres.