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Although renal macrophages have been shown to contribute to cyst development in polycystic kidney disease (PKD) animal models, it remains unclear whether there is a specific macrophage subpopulation involved. Here, we analyzed changes in macrophage populations during renal maturation in association with cystogenesis rates in conditional Pkd2 mutant mice. We observed that CD206+ resident macrophages were minimal in a normal adult kidney but accumulated in cystic areas in adult-induced Pkd2 mutants. Using Cx3cr1 null mice, we reduced macrophage number, including CD206+ macrophages, and showed that this significantly reduced cyst severity in adult-induced Pkd2 mutant kidneys. We also found that the number of CD206+ resident macrophage-like cells increased in kidneys and in the urine from autosomal-dominant PKD (ADPKD) patients relative to the rate of renal functional decline. These data indicate a direct correlation between CD206+ resident macrophages and cyst formation, and reveal that the CD206+ resident macrophages in urine could serve as a biomarker for renal cystic disease activity in preclinical models and ADPKD patients. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests M.M. reports grants and consulting fees outside the submitted work from Otsuka Pharmaceuticals, Sanofi, Palladio Biosciences, Reata, Natera, Chinook Therapeutics, Goldilocks Therapeutics and Carraway Therapeutics., (© 2023. Published by The Company of Biologists Ltd.)

The nonmotile primary cilium is a sensory structure found on most mammalian cell types that integrates multiple signaling pathways involved in tissue development and postnatal function. As such, mutations disrupting cilia activities cause a group of disorders referred to as ciliopathies. These disorders exhibit a wide spectrum of phenotypes impacting nearly every tissue. In the kidney, primary cilia dysfunction caused by mutations in polycystin 1 (Pkd1), polycystin 2 (Pkd2), or polycystic kidney and hepatic disease 1 (Pkhd1), result in polycystic kidney disease (PKD), a progressive disorder causing renal functional decline and end-stage renal disease. PKD affects nearly 1 in 1000 individuals and as there is no cure for PKD, patients frequently require dialysis or renal transplantation. Pkd1, Pkd2, and Pkhd1 encode membrane proteins that all localize in the cilium. Pkd1 and Pkd2 function as a nonselective cation channel complex while Pkhd1 protein function remains uncertain. Data indicate that the cilium may act as a mechanosensor to detect fluid movement through renal tubules. Other functions proposed for the cilium and PKD proteins in cyst development involve regulation of cell cycle and oriented division, regulation of renal inflammation and repair processes, maintenance of epithelial cell differentiation, and regulation of mitochondrial structure and metabolism. However, how loss of cilia or cilia function leads to cyst development remains elusive. Studies directed at understanding the roles of Pkd1, Pkd2, and Pkhd1 in the cilium and other locations within the cell will be important for developing therapeutic strategies to slow cyst progression., (Copyright © 2023. Published by Elsevier Inc.)

Background: Genetic tools to study gene function and the fate of cells in the anterior limb bud are very limited., Results: We describe a transgenic mouse line expressing CreER T2 from the Aristaless-like 4 (Alx4) promoter that induces recombination in the anterior limb. Cre induction at embryonic day 8.5 revealed that Alx4-CreER T2 labeled cells using the mTmG Cre reporter contributed to anterior digits I to III as well as the radius of the forelimb. Cre activity is expanded further along the AP axis in the hindlimb than in the forelimb resulting in some Cre reporter cells contributing to digit IV. Induction at later time points labeled cells that become progressively restricted to more anterior digits and proximal structures. Comparison of Cre expression from the Alx4 promoter transgene with endogenous Alx4 expression reveals Cre expression is slightly expanded posteriorly relative to the endogenous Alx4 expression. Using Alx4-CreER T2 to induce loss of intraflagellar transport 88 (Ift88), a gene required for ciliogenesis, hedgehog signaling, and limb patterning, did not cause overt skeletal malformations. However, the efficiency of deletion, time needed for Ift88 protein turnover, and for cilia to regress may hinder using this approach to analyze cilia in the limb. Alx4-CreER T2 is also active in the mesonephros and nephric duct that contribute to the collecting tubules and ducts of the adult nephron. Embryonic activation of the Alx4-CreER T2 in the Ift88 conditional line results in cyst formation in the collecting tubules/ducts., Conclusion: Overall, the Alx4-CreER T2 line will be a new tool to assess cell fates and analyze gene function in the anterior limb, mesonephros, and nephric duct., (© 2021 American Association of Anatomists.)

Primary cilia are sensory and signaling hubs with a protein composition that is distinct from the rest of the cell due to the barrier function of the transition zone (TZ) at the base of the cilium. Protein transport across the TZ is mediated in part by the BBSome, and mutations disrupting TZ and BBSome proteins cause human ciliopathy syndromes. Ciliopathies have phenotypic variability even among patients with identical genetic variants, suggesting a role for modifier loci. To identify potential ciliopathy modifiers, we performed a mutagenesis screen on nphp-4 mutant Caenorhabditis elegans and uncovered a novel allele of bbs-5. Nphp-4;bbs-5 double mutant worms have phenotypes not observed in either individual mutant strain. To test whether this genetic interaction is conserved, we also analyzed zebrafish and mouse mutants. While Nphp4 mutant zebrafish appeared overtly normal, Bbs5 mutants exhibited scoliosis. When combined, Nphp4;Bbs5 double mutant zebrafish did not exhibit synergistic effects, but the lack of a phenotype in Nphp4 mutants makes interpreting these data difficult. In contrast, Nphp4;Bbs5 double mutant mice were not viable and there were fewer mice than expected carrying three mutant alleles. In addition, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with an Nphp4 allele. As cilia are still formed in the double mutant mice, the exacerbated phenotype is likely a consequence of disrupted ciliary signaling. Collectively, these data support an evolutionarily conserved genetic interaction between Bbs5 and Nphp4 alleles that may contribute to the variability in ciliopathy phenotypes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Atxn10 is a gene known for its role in cytokinesis and is associated with spinocerebellar ataxia (SCA10), a slowly progressing cerebellar syndrome caused by an intragenic pentanucleotide repeat expansion. Atxn10 is also implicated in the ciliopathy syndromes nephronophthisis (NPHP) and Joubert syndrome (JBTS), which are caused by the disruption of cilia function leading to nephron loss, impaired renal function, and cerebellar hypoplasia. How Atxn10 disruption contributes to these disorders remains unknown. Here, we generated Atxn10 congenital and conditional mutant mouse models. Our data indicate that while ATXN10 protein can be detected around the base of the cilium as well as in the cytosol, its loss does not cause overt changes in cilia formation or morphology. Congenital loss of Atxn10 results in embryonic lethality around E10.5 associated with pericardial effusion and loss of trabeculation. Similarly, tissue-specific loss of ATXN10 in the developing endothelium (Tie2-Cre) and myocardium (cTnT-Cre) also results in embryonic lethality with severe cardiac malformations occurring in the latter. Using an inducible Cagg-CreER to disrupt ATXN10 systemically at postnatal stages, we show that ATXN10 is also required for survival in adult mice. Loss of ATXN10 results in severe pancreatic and renal abnormalities leading to lethality within a few weeks post ATXN10 deletion in adult mice. Evaluation of these phenotypes further identified rapid epithelial-to-mesenchymal transition (EMT) in these tissues. In the pancreas, the phenotype includes signs of both acinar to ductal metaplasia and EMT with aberrant cilia formation and severe defects in glucose homeostasis related to pancreatic insufficiency or defects in feeding or nutrient intake. Collectively, this study identifies ATXN10 as an essential protein for survival., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bentley-Ford, Andersen, Croyle, Haycraft, Clearman, Foote, Reiter and Yoder.)

Primary cilia are critical sensory and signaling compartments present on most mammalian cell types. These specialized structures require a unique signaling protein composition relative to the rest of the cell to carry out their functions. Defects in ciliary structure and signaling result in a broad group of disorders collectively known as ciliopathies. One ciliopathy, Bardet-Biedl syndrome (BBS; OMIM 209900), presents with diverse clinical features, many of which are attributed to defects in ciliary signaling during both embryonic development and postnatal life. For example, patients exhibit obesity, polydactyly, hypogonadism, developmental delay and skeletal abnormalities along with sensory and cognitive deficits, but for many of these phenotypes it is uncertain, which are developmental in origin. A subset of BBS proteins assembles into the core BBSome complex, which is responsible for mediating transport of membrane proteins into and out of the cilium, establishing it as a sensory and signaling hub. Here, we describe two new mouse models for BBS resulting from a targeted LacZ gene trap allele (Bbs5-/-) that is a predicted congenital null mutation and conditional (Bbs5flox/flox) allele of Bbs5. Bbs5-/- mice develop a complex phenotype consisting of increased pre-weaning lethality craniofacial and skeletal defects, ventriculomegaly, infertility and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly and pituitary abnormalities are only present when Bbs5 is disrupted prior to postnatal day 7, indicating a developmental origin. In contrast, mutation of Bbs5 results in obesity, independent of the age of Bbs5 loss., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Bone remodeling involves the coordinated actions of osteoclasts, which resorb the calcified bony matrix, and osteoblasts, which refill erosion pits created by osteoclasts to restore skeletal integrity and adapt to changes in mechanical load. Osteoblasts are derived from pluripotent mesenchymal stem cell precursors, which undergo differentiation under the influence of a host of local and environmental cues. To characterize the autocrine/paracrine signaling networks associated with osteoblast maturation and function, we performed gene network analysis using complementary "agnostic" DNA microarray and "targeted" NanoString nCounter datasets derived from murine MC3T3-E1 cells induced to undergo synchronized osteoblastic differentiation in vitro. Pairwise datasets representing changes in gene expression associated with growth arrest (day 2 to 5 in culture), differentiation (day 5 to 10 in culture), and osteoblast maturation (day 10 to 28 in culture) were analyzed using Ingenuity Systems Pathways Analysis to generate predictions about signaling pathway activity based on the temporal sequence of changes in target gene expression. Our data indicate that some pathways involved in osteoblast differentiation, e.g. Wnt/β-catenin signaling, are most active early in the process, while others, e.g. TGFβ/BMP, cytokine/JAK-STAT and TNFα/RANKL signaling, increase in activity as differentiation progresses. Collectively, these pathways contribute to the sequential expression of genes involved in the synthesis and mineralization of extracellular matrix. These results provide insight into the temporal coordination and complex interplay between signaling networks controlling gene expression during osteoblast differentiation. A more complete understanding of these processes may aid the discovery of novel methods to promote osteoblast development for the treatment of conditions characterized by low bone mineral density., Competing Interests: The authors have declared that no competing interests exist.

Primary cilia are small organelles projecting from the cell surface of many cell types. They play a crucial role in the regulation of various signaling pathway. In this study, we investigated the importance of cilia for heart development by conditionally deleting intraflagellar transport protein Ift88 using the col3.6-cre mouse. Analysis of col3.6;Ift88 offspring showed a wide spectrum of cardiovascular defects including double outlet right ventricle and atrioventricular septal defects. In addition, we found that in the majority of specimens the pulmonary veins did not properly connect to the developing left atrium. The abnormal connections found resemble those seen in patients with total anomalous pulmonary venous return. Analysis of mutant hearts at early stages of development revealed abnormal development of the dorsal mesocardium, a second heart field-derived structure at the venous pole intrinsically related to the development of the pulmonary veins. Data presented support a crucial role for primary cilia in outflow tract development and atrioventricular septation and their significance for the formation of the second heart field-derived tissues at the venous pole including the dorsal mesocardium. Furthermore, the results of this study indicate that proper formation of the dorsal mesocardium is critically important for the development of the pulmonary veins. Anat Rec, 302:136-145, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Microtubule-based centrioles in the centrosome mediate accurate bipolar cell division, spindle orientation, and primary cilia formation. Cellular checkpoints ensure that the centrioles duplicate only once in every cell cycle and achieve precise dimensions, dysregulation of which results in genetic instability and neuro- and ciliopathies. The normal cellular level of centrosomal protein 4.1-associated protein (CPAP), achieved by its degradation at mitosis, is considered as one of the major mechanisms that limits centriole growth at a predetermined length. Here we show that CPAP levels and centriole elongation are regulated by centrobin. Exogenous expression of centrobin causes abnormal elongation of centrioles due to massive accumulation of CPAP in the cell. Conversely, CPAP was undetectable in centrobin-depleted cells, suggesting that it undergoes degradation in the absence of centrobin. Only the reintroduction of full-length centrobin, but not its mutant form that lacks the CPAP binding site, could restore cellular CPAP levels in centrobin-depleted cells, indicating that persistence of CPAP requires its interaction with centrobin. Interestingly, inhibition of the proteasome in centrobin-depleted cells restored the cellular and centriolar CPAP expression, suggesting its ubiquitination and proteasome-mediated degradation when centrobin is absent. Intriguingly, however, centrobin-overexpressing cells also showed proteasome-independent accumulation of ubiquitinated CPAP and abnormal, ubiquitin-positive, elongated centrioles. Overall, our results show that centrobin interacts with ubiquitinated CPAP and prevents its degradation for normal centriole elongation function. Therefore, it appears that loss of centrobin expression destabilizes CPAP and triggers its degradation to restrict the centriole length during biogenesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The extracellular matrix protein Fibulin-1 (Fbln1) has been shown to be involved in numerous processes including cardiovascular and lung development. Here we have examined the role of Fbln1 in bone formation. Alizarin red staining of skulls from Fbln1-deficient mice showed reduced mineralization of both membranous and endochondral bones. MicroCT (μCT) analysis of the calvarial bones (i.e., frontal, parietal and interparietal bones collectively) indicated that bone volume in Fbln1 nulls at neonatal stage P0 were reduced by 22% (p=0.015). Similarly, Fbln1 null frontal bones showed a 16% (p=0.035) decrease in bone volume, with a reduction in the interfrontal bone, and a discontinuity in the leading edge of the frontal bone. To determine whether Fbln1 played a role in osteoblast differentiation during bone formation, qPCR was used to measure the effects of Fbln1 deficiency on the expression of Osterix (Osx), a transcription factor essential for osteoblast differentiation. This analysis demonstrated that Osx mRNA was significantly reduced in Fbln1-deficient calvarial bones at developmental stages E16.5 (p=0.049) and E17.5 (p=0.022). Furthermore, the ability of Bmp-2 to induce Osx expression was significantly diminished in Fbln1-deficient mouse embryo fibroblasts. Together, these findings indicate that Fbln1 is a new positive modulator of the formation of membranous bone and endochondral bone in the skull, acting as a positive regulator of Bmp signaling., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The mechanisms for the development of bronchiectasis and airway hyperreactivity have not been fully elucidated. Although genetic, acquired diseases and environmental influences may play a role, it is also possible that motile cilia can influence this disease process. We hypothesized that deletion of a key intraflagellar transport molecule, IFT88, in mature mice causes loss of cilia, resulting in airway remodeling. Airway cilia were deleted by knockout of IFT88, and airway remodeling and pulmonary function were evaluated. In IFT88(-) mice there was a substantial loss of airway cilia on respiratory epithelium. Three months after the deletion of cilia, there was clear evidence for bronchial remodeling that was not associated with inflammation or apparent defects in mucus clearance. There was evidence for airway epithelial cell hypertrophy and hyperplasia. IFT88(-) mice exhibited increased airway reactivity to a methacholine challenge and decreased ciliary beat frequency in the few remaining cells that possessed cilia. With deletion of respiratory cilia there was a marked increase in the number of club cells as seen by scanning electron microscopy. We suggest that airway remodeling may be exacerbated by the presence of club cells, since these cells are involved in airway repair. Club cells may be prevented from differentiating into respiratory epithelial cells because of a lack of IFT88 protein that is necessary to form a single nonmotile cilium. This monocilium is a prerequisite for these progenitor cells to transition into respiratory epithelial cells. In conclusion, motile cilia may play an important role in controlling airway structure and function.

Background: Cilia are found on nearly every cell type in the mammalian body, and have been historically classified as either motile or immotile. Motile cilia are important for fluid and cellular movement; however, the roles of non-motile or primary cilia in most tissues remain unknown. Several genetic syndromes, called the ciliopathies, are associated with defects in cilia structure or function and have a wide range of clinical presentations. Much of what we know about the formation and maintenance of cilia comes from model systems like C. elegans and Chalmydomonas. Studies of mammalian cilia in live tissues have been hampered by difficulty visualizing them., Results: To facilitate analyses of mammalian cilia function we generated an inducible CiliaGFP mouse by targeting mouse cDNA encoding a cilia-localized protein somatostatin receptor 3 fused to GFP (Sstr3::GFP) into the ROSA26 locus. In this system, Sstr3::GFP is expressed from the ubiquitous ROSA26 promoter after Cre mediated deletion of an upstream Neo cassette flanked by lox P sites. Fluorescent cilia labeling was observed in a variety of live tissues and after fixation. Both cell-type specific and temporally regulated cilia labeling were obtained using multiple Cre lines. The analysis of renal cilia in anesthetized live mice demonstrates that cilia commonly lay nearly parallel to the apical surface of the tubule. In contrast, in more deeply anesthetized mice the cilia display a synchronized, repetitive oscillation that ceases upon death, suggesting a relationship to heart beat, blood pressure or glomerular filtration., Conclusions: The ability to visualize cilia in live samples within the CiliaGFP mouse will greatly aid studies of ciliary function. This mouse will be useful for in vivo genetic and pharmacological screens to assess pathways regulating cilia motility, signaling, assembly, trafficking, resorption and length control and to study cilia regulated physiology in relation to ciliopathy phenotypes.

Background: The pituitary gland is formed by the juxtaposition of two tissues: neuroectoderm arising from the basal diencephalon, and oral epithelium, which invaginates towards the central nervous system from the roof of the mouth. The oral invagination that reaches the brain from the mouth is referred to as Rathke's pouch, with the tip forming the adenohypophysis and the stalk disappearing after the earliest stages of development. In tetrapods, formation of the cranial base establishes a definitive barrier between the pituitary and oral cavity; however, numerous extinct and extant vertebrate species retain an open buccohypophyseal canal in adulthood, a vestige of the stalk of Rathke's pouch. Little is currently known about the formation and function of this structure. Here we have investigated molecular mechanisms driving the formation of the buccohypophyseal canal and their evolutionary significance., Results: We show that Rathke's pouch is located at a boundary region delineated by endoderm, neural crest-derived oral mesenchyme and the anterior limit of the notochord, using CD1, R26R-Sox17-Cre and R26R-Wnt1-Cre mouse lines. As revealed by synchrotron X-ray microtomography after iodine staining in mouse embryos, the pouch has a lobulated three-dimensional structure that embraces the descending diencephalon during pituitary formation. Polaris(fl/fl); Wnt1-Cre, Ofd1(-/-) and Kif3a(-/-) primary cilia mouse mutants have abnormal sonic hedgehog (Shh) signaling and all present with malformations of the anterior pituitary gland and midline structures of the anterior cranial base. Changes in the expressions of Shh downstream genes are confirmed in Gas1(-/-) mice. From an evolutionary perspective, persistence of the buccohypophyseal canal is a basal character for all vertebrates and its maintenance in several groups is related to a specific morphology of the midline that can be related to modulation in Shh signaling., Conclusion: These results provide insight into a poorly understood ancestral vertebrate structure. It appears that the opening of the buccohypophyseal canal depends upon Shh signaling and that modulation in this pathway most probably accounts for its persistence in phylogeny.

Receptor activator of NF-κB ligand (RANKL)-RANK receptor signaling to induce NFATc1 transcription factor is critical for osteoclast differentiation and bone resorption. RANK adaptor proteins, tumor necrosis factor receptor-associated factors (TRAFs) play an essential role in RANKL signaling. Evidence indicates that NIP45 (NFAT interacting protein) binds with TRAFs and NFATc2. We therefore hypothesized that NIP45 regulates RANKL induced osteoclast differentiation. In this study, we demonstrate that RANKL treatment down regulates NIP45 expression in mouse bone marrow derived pre-osteoclast cells. Lentiviral (pGIPZ) mediated shRNA knock-down of NIP45 expression in RANKL stimulated pre-osteoclast cells resulted in increased levels of NFATc1, NFATc2, and TRAF6 but not TRAF2 expression compared to control shRNA transduced cells. Also, NIP45 suppression elevated p-IκB-α levels and NF-κB-luciferase reporter activity. Confocal microscopy demonstrated NIP45 colocalized with TRAF6 in the cytosol of osteoclast progenitor cells. In contrast, RANKL stimulation induced NIP45 nuclear translocation and colocalization with NFATc2 in these cells. Coimmuneprecipitation assay demonstrated NIP45 binding with NFATc2 but not NFATc1. We further show that shRNA knock-down of NIP45 expression in pre-osteoclast cells significantly increased RANKL induced osteoclast differentiation and bone resorption activity. Taken together, our results indicate that RANKL signaling down regulates NIP45 expression and that NIP45 is a negative regulator of osteoclast differentiation., (© 2011 Wiley Periodicals, Inc.)

We investigated the role of the chondrocyte primary cilium in mechanotransduction events related to cartilage extracellular matrix synthesis. We generated conditionally immortalized wild-type (WT) and IFT88(orpk) (ORPK) mutant chondrocytes that lack primary cilia and assessed intracellular Ca(2+) signaling, extracellular matrix synthesis, and ATP release in response to physiologically relevant compressive strains in a 3-dimensional chondrocyte culture system. All conditions were compared to unloaded controls. We found that cilia were required for compression-induced Ca(2+) signaling mediated by ATP release, and an associated up-regulation of aggrecan mRNA and sulfated glycosaminosglycan secretion. However, chondrocyte cilia were not the initial mechanoreceptors, since both WT and ORPK cells showed mechanically induced ATP release. Rather, we found that primary cilia were required for downstream ATP reception, since ORPK cells did not elicit a Ca(2+) response to exogenous ATP even though WT and ORPK cells express similar levels of purine receptors. We suggest that purinergic Ca(2+) signaling may be regulated by polycystin-1, since ORPK cells only expressed the C-terminal tail. This is the first study to demonstrate that primary cilia are essential organelles for cartilage mechanotransduction, as well as identifying a novel role for primary cilia not previously reported in any other cell type, namely cilia-mediated control of ATP reception.

Background: The disintegrin and metalloenzyme ADAM17 participates in numerous inflammatory and proliferative diseases, and its pathophysiological role was implicated in kidney fibrosis, polycystic kidney disease and other chronic kidney diseases. At present, we have little understanding how the enzyme activity is regulated. In this study we wanted to characterize the role of α5β1 integrin in ADAM17 activity regulation during G protein-coupled receptor (GPCR) stimulation., Methodology/principal Findings: We showed previously that the profibrotic GPCR agonist serotonin (5-HT) induced kidney mesangial cell proliferation through ADAM17 activation and heparin-binding epidermal growth factor (HB-EGF) shedding. In the present studies we observed that in unstimulated mesangial cell lysates α5β1 integrin co-precipitated with ADAM17 and that 5-HT treatment of the cells induced dissociation of α5β1 integrin from ADAM17. Using fluorescence immunostaining and in situ proximity ligation assay, we identified the perinuclear region as the localization of the ADAM17/α5β1 integrin interaction. In cell-free assays, we showed that purified α5β1 integrin and β1 integrin dose-dependently bound to and inhibited activity of recombinant ADAM17. We provided evidence that the conformation of the integrin determines its ADAM17-binding ability. To study the effect of β1 integrin on ADAM17 sheddase activity, we employed alkaline phosphatase-tagged HB-EGF. Overexpression of β1 integrin lead to complete inhibition of 5-HT-induced HB-EGF shedding and silencing β1 integrin by siRNA significantly increased mesangial cells ADAM17 responsiveness to 5-HT., Conclusions/significance: Our data show for the first time that β1 integrin has an important physiological role in ADAM17 activity regulation. We suggest that regulating α5β1 integrin binding to ADAM17 could be an attractive therapeutic target in chronic kidney diseases.

Primary cilia dysfunction alters renal tubular cell proliferation and differentiation and associates with accelerated cyst formation in polycystic kidney disease. However, the mechanism leading from primary ciliary dysfunction to renal cyst formation is unknown. We hypothesize that primary cilia prevent renal cyst formation by suppressing pathologic tubular cell hypertrophy and proliferation. Unilateral nephrectomy initiates tubular cell hypertrophy and proliferation in the contralateral kidney and provides a tool to examine primary cilia regulation of renal hypertrophy. Conditional knockout of the primary cilia ift88 gene leads to delayed, adult-onset renal cystic disease, which provides a window of opportunity to conduct unilateral nephrectomy and examine downstream kinetics of renal hypertrophy and cyst formation. In wild-type animals, unilateral nephrectomy activated the mTOR pathway and produced appropriate structural and functional hypertrophy without renal cyst formation. However, in ift88 conditional knockout animals, unilateral nephrectomy triggered increased renal hypertrophy and accelerated renal cyst formation, leading to renal dysfunction. mTOR signaling also increased compared with wild-type animals, suggesting a mechanistic cascade starting with primary ciliary dysfunction, leading to excessive mTOR signaling and renal hypertrophic signaling and culminating in cyst formation. These data suggest that events initiating hypertrophic signaling, such as structural or functional loss of renal mass, may accelerate progression of adult polycystic kidney disease toward end-stage renal disease., (Copyright © 2011 by the American Society of Nephrology)

We describe the localization of the golgin GMAP210 and the intraflagellar protein IFT88 in the Golgi of spermatids and the participation of these two proteins in the development of the acrosome-acroplaxome complex, the head-tail coupling apparatus (HTCA) and the spermatid tail. Immunocytochemical experiments show that GMAP210 predominates in the cis-Golgi, whereas IFT88 prevails in the trans-Golgi network. Both proteins colocalize in proacrosomal vesicles, along acrosome membranes, the HTCA and the developing tail. IFT88 persists in the acrosome-acroplaxome region of the sperm head, whereas GMAP210 is no longer seen there. Spermatids of the Ift88 mouse mutant display abnormal head shaping and are tail-less. GMAP210 is visualized in the Ift88 mutant during acrosome-acroplaxome biogenesis. However, GMAP210-stained vesicles, mitochondria and outer dense fiber material build up in the manchette region and fail to reach the abortive tail stump in the mutant. In vitro disruption of the spermatid Golgi and microtubules with Brefeldin-A and nocodazole blocks the progression of GMAP210- and IFT88-stained proacrosomal vesicles to the acrosome-acroplaxome complex but F-actin distribution in the acroplaxome is not affected. We provide the first evidence that IFT88 is present in the Golgi of spermatids, that the microtubule-associated golgin GMAP210 and IFT88 participate in acrosome, HTCA, and tail biogenesis, and that defective intramanchette transport of cargos disrupts spermatid tail development., (Copyright © 2011 Wiley-Liss, Inc.)

Recently, the use of overexpression of telomerase reverse transcriptase (TERT) has led to the generation of immortalized human cell lines. However, this cell immortalization approach has not been reported in well-differentiated mouse cells, such as renal epithelial cells. We sought to establish and then characterize a mouse collecting duct cell line, using ectopic expression of mTERT. Isolated primary cortical collecting duct (CCD) cell lines were transduced with mouse (m)TERT, using a lentiviral vector. mTERT-negative cells did not survive blasticidin selection, whereas mTERT-immortalized cells proliferated in selection media for over 40 subpassages. mTERT messenger RNA and telomerase activity was elevated in these cells, compared with an SV40-immortalized cell line. Flow cytometry with Dolichos biflorus agglutinin was used to select the CCD principal cells, and we designated this cell line mTERT-CCD. Cells were well differentiated and exhibited morphological characteristics typically found in renal epithelial cells, such as tight junction formation, microvilli, and primary cilia. Further characterization using standard immunofluorescence revealed abundant expression of aquaporin-2 and the vasopressin type 2 receptor. mTERT-CCD cells exhibited cAMP-stimulated/benzamil-inhibited whole cell currents. Whole cell patch-clamp currents were also enhanced after a 6-day treatment with aldosterone. In conclusion, we have successfully used mTERT to immortalize mouse collecting duct cells that retain the basic in vivo phenotypic characteristics of collecting duct cells. This technique should be valuable in generating cell lines from genetically engineered mouse models.

Respect for the primary cilium has undergone a remarkable renaissance over the past decade, and it is now thought to be an essential regulator of numerous signaling pathways. The primary cilium's functions range from the movement of cells and fluid, to sensory inputs involved with olfaction and photoreception. Disruption of cilia function is involved in multiple human syndromes collectively called 'ciliopathies'. The cilium's activities are mediated by targeting of receptors, channels, and their downstream effector proteins to the ciliary or basal body compartment. These combined properties of the cilium make it a critical organelle facilitating the interactions between the cell and its environment. Here, we review many of the recent advances contributing to the ascendancy of the primary cilium and how the extraordinary complexity of this organelle inevitably assures many more exciting future discoveries.

Primary cilia mediate Hh signalling and mutations in their protein components affect Hh activity. We show that in mice mutant for a cilia intraflagellar transport (IFT) protein, IFT88/polaris, Shh activity is increased in the toothless diastema mesenchyme of the embryonic jaw primordia. This results in the formation of ectopic teeth in the diastema, mesial to the first molars. This phenotype is specific to loss of polaris activity in the mesenchyme since loss of Polaris in the epithelium has no detrimental affect on tooth development. To further confirm that upregulation of Shh activity is responsible for the ectopic tooth formation, we analysed mice mutant for Gas1, a Shh protein antagonist in diastema mesenchyme. Gas1 mutants also had ectopic diastema teeth and accompanying increased Shh activity. In this context, therefore, primary cilia exert a specific negative regulatory effect on Shh activity that functions to repress tooth formation and thus determine tooth number. Strikingly, the ectopic teeth adopt a size and shape characteristic of premolars, a tooth type that was lost in mice around 50-100 million years ago.

Ift88 is a component of the intraflagellar transport complex required for formation and maintenance of cilia. Disruption of Ift88 results in depletion of cilia. The goal of the current study was to determine the role of primary cilia in ovarian function. Deletion of Ift88 in ovary using Cre-Lox recombination in mice resulted in a severe delay in mammary gland development including lack of terminal end bud structures, alterations in the estrous cycle, and impaired ovulation. Because estrogen drives the formation of end buds and Cre was expressed in the granulosa cells of the ovary, we tested the hypothesis that addition of estradiol to the mutant mice would compensate for defects in ovarian function and rescue the mammary gland phenotype. Mammary gland development including the formation of end bud structures resumed in mutant mice that were injected with estradiol. Together the results suggest that cilia are required for ovarian function., (Copyright (c) 2008 Wiley-Liss, Inc.)

Characterization of previously described intraflagellar transport (IFT) mouse mutants has led to the proposition that normal primary cilia are required for mammalian cells to respond to the sonic hedgehog (SHH) signal. Here we describe an N-ethyl-N-nitrosourea-induced mutant mouse, alien (aln), which has abnormal primary cilia and shows overactivation of the SHH pathway. The aln locus encodes a novel protein, THM1 (tetratricopeptide repeat-containing hedgehog modulator-1), which localizes to cilia. aln-mutant cilia have bulb-like structures at their tips in which IFT proteins (such as IFT88) are sequestered, characteristic of Chlamydomonas reinhardtii and Caenorhabditis elegans retrograde IFT mutants. RNA-interference knockdown of Ttc21b (which we call Thm1 and which encodes THM1) in mouse inner medullary collecting duct cells expressing an IFT88-enhanced yellow fluorescent protein fusion recapitulated the aln-mutant cilial phenotype, and live imaging of these cells revealed impaired retrograde IFT. In contrast to previously described IFT mutants, Smoothened and full-length glioblastoma (GLI) proteins localize to aln-mutant cilia. We hypothesize that the aln retrograde IFT defect causes sequestration of IFT proteins in aln-mutant cilia and leads to the overactivated SHH signaling phenotype. Specifically, the aln mutation uncouples the roles of anterograde and retrograde transport in SHH signaling, suggesting that anterograde IFT is required for GLI activation and that retrograde IFT modulates this event.

Although the expression of cilia on chondrocytes was described over 40 years ago, the importance of this organelle in skeletal development and maintenance has only recently been recognized. Primary cilia are found on most mammalian cells and have been shown to play a role in chemosensation and mechanosensation. A growing number of human pleiotropic syndromes have been shown to be associated with ciliary or basal body dysfunction. Skeletal phenotypes, including alterations in limb patterning, endochondral bone formation, craniofacial development, and dentition, have been described in several of these syndromes. Additional insights into the potential roles and mechanisms of cilia action in the mammalian skeleton have been provided by research in model organisms including mouse and zebrafish. In this article we describe what is currently known about the localization of cilia in the skeleton as well as the roles and underlying molecular mechanisms of cilia in skeletal development.

In the post-natal growth plate, chondrocytes are arranged in columns parallel to the long axis of the bone. Chondrocytes divide perpendicular to this axis and then move into position one on top of another in a process called "rotation" that maintains columnar organization. Primary cilia are non-motile microtubule base appendages extending from the surface of almost all vertebrate cells. Primary cilia were described on chondrocytes almost 40 years ago but the function of these structures in cartilage biology is not known. Intraflagellar transport (IFT) is the process by which primary cilia are generated and maintained. This study tested the hypothesis that IFT plays an important role in post-natal skeletal development. Kif3a, a subunit of the Kinesin II motor complex, that is required for intraflagellar transport and the formation of cilia, was deleted in mouse chondrocytes via Col2a-Cre-mediated recombination. Disruption of IFT resulted in subsequent depletion of cilia and post-natal dwarfism due to premature loss of the growth plate likely a result of reduced proliferation and accelerated hypertrophic differentiation of chondrocytes. Cell shape and columnar orientation in the growth plate were also disrupted suggesting a defect in the process of rotation. Alterations in chondrocyte rotation were accompanied by disruption of the actin cytoskeleton and alterations in the localization of activated FAK to focal adhesion-like structures on chondrocytes. This is the first report indicating a role for IFT and primary cilia in the development of the post-natal growth plate. The results suggest a model in which IFT/cilia act to maintain the columnar organization of the growth plate via the process of chondrocyte rotation.

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.

The intraflagellar transport (IFT) machinery required to build functional cilia consists of a multisubunit complex whose molecular composition, organization, and function are poorly understood. Here, we describe a novel tryptophan-aspartic acid (WD) repeat (WDR) containing IFT protein from Caenorhabditis elegans, DYF-2, that plays a critical role in maintaining the structural and functional integrity of the IFT machinery. We determined the identity of the dyf-2 gene by transgenic rescue of mutant phenotypes and by sequencing of mutant alleles. Loss of DYF-2 function selectively affects the assembly and motility of different IFT components and leads to defects in cilia structure and chemosensation in the nematode. Based on these observations, and the analysis of DYF-2 movement in a Bardet-Biedl syndrome mutant with partially disrupted IFT particles, we conclude that DYF-2 can associate with IFT particle complex B. At the same time, mutations in dyf-2 can interfere with the function of complex A components, suggesting an important role of this protein in the assembly of the IFT particle as a whole. Importantly, the mouse orthologue of DYF-2, WDR19, also localizes to cilia, pointing to an important evolutionarily conserved role for this WDR protein in cilia development and function.

Defects in cilia are associated with diseases and developmental abnormalities. Proper cilia function is required for sonic hedgehog and PDGFRalpha signaling in mammals and for insulin-like growth factor (IGF) signaling in Caenorhabditis elegans. However, the role of cilia in these pathways remains unknown. To begin addressing this issue, we are characterizing putative cilia proteins in C. elegans that are predicted to have regulatory rather than structural functions. In this report, we characterized the novel cilia protein T28F3.6 (IFTA-2, intraflagellar transport associated protein 2), which is homologous to the mammalian Rab-like 5 protein. We found that, unlike the intraflagellar transport (IFT) genes, disruption of ifta-2 does not result in overt cilia assembly abnormalities, nor did it cause chemotaxis or osmotic avoidance defects typical of cilia mutants. Rather, ifta-2 null mutants have an extended lifespan phenotype and are defective in dauer formation. Our analysis indicates that these phenotypes result from defects in the DAF-2 (insulin-IGF-1-like) receptor signaling pathway in ciliated sensory neurons. We conclude that IFTA-2 is not a ciliogenic protein but rather is a regulator of specific cilia signaling activities. Interestingly, a mammalian IFTA-2 homolog is also found in cilia, raising the possibility that its function has been conserved during evolution.

Nephronophthisis (NPH) is a cystic kidney disorder that causes end-stage renal failure in children. Five nephrocystin (nephrocystin-1 to nephrocystin-5) genes, whose function is disrupted in NPH patients, have been identified and data indicate they form a complex at cell junctions and focal adhesions. More recently, the nephrocystin proteins have also been identified in cilia, as have multiple other cystic kidney disease related proteins. Significant insights into this cilia and cystic kidney disease connection have come from analyses in simpler eukaryotic organisms such as Caenorhabditis elegans. In this regard, we became interested in the C. elegans homologs of nephrocystin-1 (nph-1) and nephrocystin-4 (nph-4) from a database screen to identify genes coordinately regulated by the ciliogenic transcription factor DAF-19. Here we show that expression of nph-1 and nph-4 is DAF-19 dependent, that their expression is restricted to ciliated sensory neurons, and that both NPH-1 and NPH-4 concentrate at the transition zones at the base of the cilia, but are not found in the cilium axoneme. In addition, NPH-4 is required for the localization of NPH-1 to this domain. Interestingly, nph-1 or nph-4 mutants have no obvious cilia assembly defects; however, they do have abnormalities in cilia-mediated sensory functions as evidenced by abnormal chemotaxis and lifespan regulation. Our data suggest that rather than having a ciliogenic role, the NPH proteins play an important function as part of the sensory or signaling machinery of this organelle. These findings suggest that the defects in human NPH patients may not be the result of aberrant ciliogenesis but abnormal cilia-sensory functions.

Intraflagellar transport (IFT) proteins are essential for cilia assembly and have recently been associated with a number of developmental processes, such as left-right axis specification and limb and neural tube patterning. Genetic studies indicate that IFT proteins are required for Sonic hedgehog (Shh) signaling downstream of the Smoothened and Patched membrane proteins but upstream of the Glioma (Gli) transcription factors. However, the role that IFT proteins play in transduction of Shh signaling and the importance of cilia in this process remain unknown. Here we provide insights into the mechanism by which defects in an IFT protein, Tg737/Polaris, affect Shh signaling in the murine limb bud. Our data show that loss of Tg737 results in altered Gli3 processing that abrogates Gli3-mediated repression of Gli1 transcriptional activity. In contrast to the conclusions drawn from genetic analysis, the activity of Gli1 and truncated forms of Gli3 (Gli3R) are unaffected in Tg737 mutants at the molecular level, indicating that Tg737/Polaris is differentially involved in specific activities of the Gli proteins. Most important, a negative regulator of Shh signaling, Suppressor of fused, and the three full-length Gli transcription factors localize to the distal tip of cilia in addition to the nucleus. Thus, our data support a model where cilia have a direct role in Gli processing and Shh signal transduction., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Cilia, as motile and sensory organelles, have been implicated in normal development, as well as diseases including cystic kidney disease, hydrocephalus and situs inversus. In kidney epithelia, cilia are proposed to be non-motile sensory organelles, while in the mouse node, two cilia populations, motile and non-motile have been proposed to regulate situs. We show that cilia in the zebrafish larval kidney, the spinal cord and Kupffer's vesicle are motile, suggesting that fluid flow is a common feature of each of these organs. Disruption of cilia structure or motility resulted in pronephric cyst formation, hydrocephalus and left-right asymmetry defects. The data show that loss of fluid flow leads to fluid accumulation, which can account for organ distension pathologies in the kidney and brain. In Kupffer's vesicle, loss of flow is associated with loss of left-right patterning, indicating that the 'nodal flow' mechanism of generating situs is conserved in non-mammalian vertebrates.

While relatively ignored for years as vestigial, cilia have recently become the focus of intense interest as organelles that result in severe pathologies when disrupted. Here, we further establish a connection between cilia dysfunction and disease by showing that loss of polaris (Tg737), an intraflagellar transport (IFT) protein required for ciliogenesis, causes abnormalities in the exocrine and endocrine pancreas of the Tg737(orpk) mouse. Pathology is evident late in gestation as dilatations of the pancreatic ducts that continue to expand postnatally. Shortly after birth, the acini become disorganized, undergo apoptosis, and are largely ablated in late stage pathology. In addition, serum amylase levels are elevated and carboxypeptidase is abnormally activated within the pancreas. Ultrastructural analysis reveals that the acini undergo extensive vacuolization and have numerous 'halo-granules' similar to that seen in induced models of pancreatitis resulting from duct obstruction. Intriguingly, although the acini are severely affected in Tg737(orpk) mutants, cilia and Tg737 expression are restricted to the ducts and islets and are not detected on acinar cells. Analysis of the endocrine pancreas in Tg737(orpk) mutants revealed normal differentiation and distribution of cell types in the islets. However, after fasting, mutant blood glucose levels are significantly lower than controls and when challenged in glucose tolerance tests, Tg737(orpk) mutants exhibited defects in glucose uptake. These findings are interesting in light of the recently proposed role for polaris, the protein encoded by the Tg737 gene, in the hedgehog pathway and hedgehog signaling in insulin production and glucose homeostasis.

Cilia and flagella are microtubule-based structures nucleated by modified centrioles termed basal bodies. These biochemically complex organelles have more than 250 and 150 polypeptides, respectively. To identify the proteins involved in ciliary and basal body biogenesis and function, we undertook a comparative genomics approach that subtracted the nonflagellated proteome of Arabidopsis from the shared proteome of the ciliated/flagellated organisms Chlamydomonas and human. We identified 688 genes that are present exclusively in organisms with flagella and basal bodies and validated these data through a series of in silico, in vitro, and in vivo studies. We then applied this resource to the study of human ciliation disorders and have identified BBS5, a novel gene for Bardet-Biedl syndrome. We show that this novel protein localizes to basal bodies in mouse and C. elegans, is under the regulatory control of daf-19, and is necessary for the generation of both cilia and flagella.

Intraflagellar transport (IFT) is a process required for flagella and cilia assembly that describes the dynein and kinesin mediated movement of particles along axonemes that consists of an A and a B complex, defects in which disrupt retrograde and anterograde transport, respectively. Herein, we describe a novel Caenorhabditis elegans gene, xbx-1, that is required for retrograde IFT and shares homology with a mammalian dynein light intermediate chain (D2LIC). xbx-1 expression in ciliated sensory neurons is regulated by the transcription factor DAF-19, as demonstrated previously for genes encoding IFT complex B proteins. XBX-1 localizes to the base of the cilia and undergoes anterograde and retrograde movement along the axoneme. Disruption of xbx-1 results in cilia defects and causes behavioral abnormalities observed in other cilia mutants. Analysis of cilia in xbx-1 mutants reveals that they are shortened and have a bulb like structure in which IFT proteins accumulate. The role of XBX-1 in IFT was further confirmed by analyzing the effect that other IFT mutations have on XBX-1 localization and movement. In contrast to other IFT proteins, retrograde XBX-1 movement was detected in complex A mutants. Our results suggest that the DLIC protein XBX-1 functions together with the CHE-3 dynein in retrograde IFT, downstream of the complex A proteins.

Cilia are present on cells of many eukaryotic organisms and recent data in the mouse suggest that ciliary defects can cause severe developmental abnormalities and disease. Studies across eukaryotic systems indicate that cilia are constructed and maintained through a highly conserved process termed intraflagellar transport (IFT), for which many of the proteins involved have yet to be identified. IFT describes the movement of large protein particles consisting of an A and a B complex along the cilia axoneme in anterograde and retrograde directions. Herein we describe a novel C. elegans gene, F59C6.7/9, that is required for cilia assembly and whose function is disrupted in che-13 ciliogenic mutants. As previously shown for all IFT complex B genes identified to date, expression of che-13 (F59C6.7/9) is regulated by the RFX-type transcription factor DAF-19, suggesting a conserved transcriptional pathway in ciliogenesis. Fluorescent-tagged CHE-13 protein concentrates at the base of cilia and moves along the axoneme as expected for an IFT protein. Furthermore, loss of che-13 differentially affects the localization of two known IFT complex B proteins, OSM-5 and OSM-6, implying that CHE-13 functions as part of this complex. Overall, our data confirm that CHE-13 is an IFT protein and further that the IFT particle assembles in an ordered process through specific protein-protein interactions.