50 results on '"Courtney J, Haycraft"'
Search Results
2. ATXN10 Is Required for Embryonic Heart Development and Maintenance of Epithelial Cell Phenotypes in the Adult Kidney and Pancreas
- Author
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Melissa R. Bentley-Ford, Reagan S. Andersen, Mandy J. Croyle, Courtney J. Haycraft, Kelsey R. Clearman, Jeremy B. Foote, Jeremy F. Reiter, and Bradley K. Yoder
- Subjects
ataxin 10 (ATXN10) ,epithelial-to-mesenchymal transition (EMT) ,cilia ,acinar-to-ductal metaplasia (ADM) ,heart ,kidney ,Biology (General) ,QH301-705.5 - Abstract
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.
- Published
- 2021
- Full Text
- View/download PDF
3. Transcriptomic characterization of signaling pathways associated with osteoblastic differentiation of MC-3T3E1 cells.
- Author
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Louis M Luttrell, Moahad S Dar, Diane Gesty-Palmer, Hesham M El-Shewy, Katherine M Robinson, Courtney J Haycraft, and Jeremy L Barth
- Subjects
Medicine ,Science - Abstract
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.
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- 2019
- Full Text
- View/download PDF
4. CD206+ resident macrophages are a candidate biomarker for renal cystic disease in preclinical models and patients with ADPKD
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Zhang, Li, Kurt A, Zimmerman, Sreelakshmi, Cherakara, Phillip, Chumley, James F, Collawn, Jun, Wang, Courtney J, Haycraft, Cheng J, Song, Teresa, Chacana, Reagan S, Andersen, Mandy J, Croyle, Ernald J, Aloria, Raksha P, Hombal, Isis N, Thomas, Hanan, Chweih, Kristin L, Simanyi, James F, George, John M, Parant, Michal, Mrug, and Bradley K, Yoder
- Abstract
Although renal macrophages have been shown to contribute to cystic kidney disease in PKD animal models, it remains unclear if there is a specific macrophage subpopulation involved. Here we analyze changes in macrophage populations during renal maturation in association with cystogenesis rates in conditional Pkd2 mutant mice. We demonstrate that CD206+ resident macrophages are minimal in a normal adult kidney but accumulate in cystic areas in adult-induced Pkd2 mutants. Using Cx3cr1 null mice, we reduced macrophage number, including CD206+ macrophages, and show this significantly reduces cyst severity in adult-induced Pkd2 mutant kidneys. We also found that the number of CD206+ resident macrophage-like cells increases in kidneys and in the urine from ADPKD patients relative to the rate of renal functional decline. These data indicate a direct correlation between CD206+ resident macrophages and cyst formation and demonstrate that the CD206+ resident macrophages in urine may serve as a biomarker for renal cystic disease activity in preclinical models and ADPKD patients.
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- 2022
5. A transgenic <scp>Alx4‐CreER</scp> mouse to analyze anterior limb and nephric duct development
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Courtney J. Haycraft, Kathryn M Brewer, Devan M Rockwell, Melissa R Bentley-Ford, Amber K. O'Connor, Mandy J. Croyle, Nicolas F. Berbari, Bradley K. Yoder, and Robert A. Kesterson
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0301 basic medicine ,Transgene ,Mice, Transgenic ,Hindlimb ,Biology ,Article ,Mesonephric duct ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Fate mapping ,Ciliogenesis ,medicine ,Animals ,Hedgehog Proteins ,Transgenes ,Homeodomain Proteins ,Integrases ,Mesonephros ,Extremities ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Ureteric bud ,Forelimb ,030217 neurology & neurosurgery ,Transcription Factors ,Developmental Biology - Abstract
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.
- Published
- 2021
6. A mouse model of BBS identifies developmental and homeostatic effects of BBS5 mutation and identifies novel pituitary abnormalities
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Mandy J. Croyle, Reagan S Andersen, Nicolas F. Berbari, Staci E. Engle, Addison Rains, Kelsey R. Clearman, Bradley K. Yoder, Melissa R Bentley-Ford, and Courtney J. Haycraft
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Male ,0301 basic medicine ,BBSome ,BBS5 ,Biology ,Ciliopathies ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Genetics ,medicine ,Animals ,Allele ,Bardet-Biedl Syndrome ,Molecular Biology ,Genetics (clinical) ,Cilium ,General Medicine ,Phosphate-Binding Proteins ,medicine.disease ,Null allele ,Cell biology ,Cytoskeletal Proteins ,Disease Models, Animal ,Ciliopathy ,Phenotype ,030104 developmental biology ,Membrane protein ,Pituitary Gland ,Mutation ,General Article ,030217 neurology & neurosurgery - Abstract
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.
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- 2021
7. Evolutionarily conserved genetic interactions between nphp-4 and bbs-5 mutations exacerbate ciliopathy phenotypes
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Courtney J. Haycraft, Mandy J. Croyle, Reagan S Andersen, Cameron LaFayette, Mikyla Scott, Bradley K. Yoder, Melissa R Bentley-Ford, Melissa LaBonty, Holly R. Thomas, and John M. Parant
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Genetics ,Investigation ,BBSome ,Cilium ,Mutant ,Mutagenesis (molecular biology technique) ,Biology ,medicine.disease ,biology.organism_classification ,Phenotype ,Ciliopathy ,medicine ,Animals ,Allele ,Caenorhabditis elegans ,Zebrafish - Abstract
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.
- Published
- 2021
8. Evolutionarily conserved genetic interactions between nphp-4 and bbs-5 mutations exacerbate ciliopathy phenotypes
- Author
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Melissa R Bentley-Ford, Holly R. Thomas, Mandy J. Croyle, Courtney J. Haycraft, Mikyla Scott, Bradley K. Yoder, Melissa LaBonty, Cameron LaFayette, and John M. Parant
- Subjects
Genetics ,Ciliopathy ,BBSome ,biology ,Cilium ,Mutant ,medicine ,Mutagenesis (molecular biology technique) ,Allele ,biology.organism_classification ,medicine.disease ,Zebrafish ,Phenotype - Abstract
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 C. 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 mice 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, viable Nphp4;Bbs5 double mutant mice were not obtained and there were fewer mice than expected carrying three mutant alleles. Additionally, postnatal loss of Bbs5 in mice using a conditional allele compromised survival when combined with a Nphp4 allele. As cilia are 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.
- Published
- 2021
9. ABbs5mouse model reveals pituitary cilia contributions to developmental abnormalities
- Author
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Nicolas F. Berbari, Addison Rains, Mandy J. Croyle, Reagan S Andersen, Staci E. Engle, Melissa R. Bentley, Courtney J. Haycraft, Bradley K. Yoder, and Kelsey R. Clearman
- Subjects
Ciliopathy ,BBSome ,Membrane protein ,Cilium ,medicine ,BBS5 ,Biology ,medicine.disease ,Ciliopathies ,Phenotype ,Neuroscience ,Ventriculomegaly - Abstract
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 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 congenital null and conditional allele ofBbs5.Bbs5null mice develop a complex phenotype including craniofacial defects, skeletal shortening, ventriculomegaly, infertility, and pituitary anomalies. Utilizing the conditional allele, we show that the male fertility defects, ventriculomegaly, and pituitary abnormalities are only found whenBbs5is mutated prior to P7 indicating a developmental origin. In contrast, mutation ofBbs5results in obesity independent of the age ofBbs5loss. Compared to other animal models of BBS,Bbs5mutant mice exhibit pathologies that suggest a specialized role for Bbs5 in ciliary function.
- Published
- 2020
10. A disintegrin and metalloenzyme (ADAM) 17 activation is regulated by α5β1 integrin in kidney mesangial cells.
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Pal Gooz, Yujing Dang, Shigeki Higashiyama, Waleed O Twal, Courtney J Haycraft, and Monika Gooz
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Medicine ,Science - Abstract
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.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.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.
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- 2012
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11. A Novel Mouse Model for Cilia‐Associated Cardiovascular Anomalies with a High Penetrance of Total Anomalous Pulmonary Venous Return
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Tara A. Burns, Aimee L. Phelps, John Bullard, Russell A. Norris, Emilye Hiriart, Katelynn Toomer, Andy Wessels, Raymond N. Deepe, and Courtney J. Haycraft
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Male ,0301 basic medicine ,Cell type ,Histology ,Penetrance ,Heart Septal Defects, Atrial ,Article ,Total anomalous pulmonary venous return ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Intraflagellar transport ,Double outlet right ventricle ,Animals ,Medicine ,Cilia ,Atrioventricular Septal Defect ,Ecology, Evolution, Behavior and Systematics ,Mice, Knockout ,Heart development ,MEF2 Transcription Factors ,business.industry ,Tumor Suppressor Proteins ,Cilium ,Scimitar Syndrome ,Anatomy ,medicine.disease ,Disease Models, Animal ,Collagen Type III ,030104 developmental biology ,Pulmonary Veins ,business ,030217 neurology & neurosurgery ,Biotechnology - Abstract
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.
- Published
- 2018
12. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function.
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Courtney J Haycraft, Boglarka Banizs, Yesim Aydin-Son, Qihong Zhang, Edward J Michaud, and Bradley K Yoder
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Genetics ,QH426-470 - Abstract
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.
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- 2005
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13. Transcriptomic characterization of signaling pathways associated with osteoblastic differentiation of MC-3T3E1 cells
- Author
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Moahad Dar, Louis M. Luttrell, Hesham M. El-Shewy, Diane Gesty-Palmer, Jeremy L. Barth, Katherine M. Robinson, and Courtney J. Haycraft
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0301 basic medicine ,Cell signaling ,Microarrays ,Cellular differentiation ,Gene Expression ,Datasets as Topic ,Signal transduction ,Extracellular matrix ,Mice ,0302 clinical medicine ,Animal Cells ,Bone Density ,Osteogenesis ,Medicine and Health Sciences ,Gene Regulatory Networks ,Connective Tissue Cells ,Oligonucleotide Array Sequence Analysis ,Regulation of gene expression ,0303 health sciences ,Multidisciplinary ,Wnt signaling pathway ,Signaling cascades ,Cell Differentiation ,Osteoblast ,3T3 Cells ,Osteoblast Differentiation ,Extracellular Matrix ,Cell biology ,Autocrine Communication ,Bioassays and Physiological Analysis ,medicine.anatomical_structure ,RANKL ,Connective Tissue ,030220 oncology & carcinogenesis ,Medicine ,Bone Remodeling ,Cellular Types ,Anatomy ,Network Analysis ,Research Article ,musculoskeletal diseases ,Computer and Information Sciences ,Science ,030209 endocrinology & metabolism ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Paracrine signalling ,Paracrine Communication ,Genetics ,medicine ,Animals ,Gene Regulation ,Autocrine signalling ,030304 developmental biology ,Osteoblasts ,Gene Expression Profiling ,Mesenchymal stem cell ,Biology and Life Sciences ,Signaling Networks ,Gene expression profiling ,Biological Tissue ,030104 developmental biology ,TGF-beta signaling cascade ,biology.protein ,Transcriptome ,Developmental Biology - Abstract
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 known to be 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 trea™ent of conditions characterized by low bone mineral density.
- Published
- 2019
14. Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage
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Zihai Li, Radhika Gudi, Courtney J. Haycraft, Chenthamarakshan Vasu, and P. Darwin Bell
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Proteasome Endopeptidase Complex ,Centriole ,Cell division ,Microtubule-associated protein ,Ubiquitination ,Cell Cycle Proteins ,Cell Biology ,Cell cycle ,Biology ,Biochemistry ,Centriole elongation ,Cell Line ,Up-Regulation ,respiratory tract diseases ,Cell biology ,Microtubule ,Centrosome ,Proteolysis ,Humans ,Microtubule-Associated Proteins ,Molecular Biology ,Mitosis ,Gene Deletion ,Centrioles - Abstract
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.
- Published
- 2015
15. Primary cilia mediate mechanotransduction through control of ATP‐induced Ca2+signaling in compressed chondrocytes
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A.K. Wann, Ning Zuo, Susan R. McGlashan, C. Anthony Poole, Cynthia G. Jensen, Courtney J. Haycraft, and Martin M. Knight
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Compressive Strength ,Mice, Transgenic ,Biology ,Mechanotransduction, Cellular ,Biochemistry ,Chondrocyte ,Research Communications ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Adenosine Triphosphate ,Chondrocytes ,0302 clinical medicine ,Genetics ,medicine ,Animals ,Calcium Signaling ,Cilia ,Mechanotransduction ,education ,Molecular Biology ,Cells, Cultured ,Aggrecan ,030304 developmental biology ,Calcium signaling ,0303 health sciences ,education.field_of_study ,Cilium ,Purinergic receptor ,Extracellular Matrix ,Cell biology ,medicine.anatomical_structure ,Polycystin 2 ,chemistry ,Calcium ,Stress, Mechanical ,Adenosine triphosphate ,030217 neurology & neurosurgery ,Biotechnology - Abstract
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 IFT88orpk (ORPK) mutant chondrocytes that lack primary cilia and assessed intracellular Ca2+ 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 Ca2+ 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 Ca2+ response to exogenous ATP even though WT and ORPK cells express similar levels of purine receptors. We suggest that purinergic Ca2+ 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.—Wann, A. K. T., Zuo, N., Haycraft, C. J., Jensen, C. G., Poole, C. A., McGlashan, S. R., Knight, M. M. Primary cilia mediate mechanotransduction through control of ATP-induced Ca2+ signaling in compressed chondrocytes.
- Published
- 2012
16. Loss of Primary Cilia Upregulates Renal Hypertrophic Signaling and Promotes Cystogenesis
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Brad Yoder, Lisa M. Guay-Woodford, Sandra K. Gilley, Wayne R. Fitzgibbon, Kelli M. Sas, Ryan J. Reichert, P. Darwin Bell, Mehmet Bilgen, May Y. Amria, Courtney J. Haycraft, Amber Houston, John J. Bissler, Peter Cheng Te Chou, Gene P. Siegal, Brian J. Siroky, and Antine E. Stenbit
- Subjects
Nephrology ,medicine.medical_specialty ,Kidney ,Pathology ,Renal Hypertrophy ,Cilium ,General Medicine ,Biology ,urologic and male genital diseases ,medicine.disease ,Muscle hypertrophy ,medicine.anatomical_structure ,Internal medicine ,medicine ,Polycystic kidney disease ,PI3K/AKT/mTOR pathway ,Renal stem cell - Abstract
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.
- Published
- 2011
17. The Primary Cilium as a Complex Signaling Center
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Bradley K. Yoder, Courtney J. Haycraft, Amber K. O'Connor, and Nicolas F. Berbari
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Sensation ,Regulator ,Biology ,Mechanotransduction, Cellular ,Ciliopathies ,Article ,Olfactory Receptor Neurons ,General Biochemistry, Genetics and Molecular Biology ,Organelle ,Animals ,Humans ,Compartment (development) ,Basal body ,Hedgehog Proteins ,Photoreceptor Cells ,Cilia ,Mechanotransduction ,Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,Cilium ,Cell biology ,Wnt Proteins ,sense organs ,Signal transduction ,General Agricultural and Biological Sciences ,Signal Transduction - Abstract
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.
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- 2009
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18. Intraflagellar transport is essential for endochondral bone formation
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Rosa Serra, Courtney J. Haycraft, Peter J. Detloff, Bradley K. Yoder, Qihong Zhang, Walker S. Jackson, and Buer Song
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medicine.medical_specialty ,Indian hedgehog ,Neural tube patterning ,Mesenchyme ,Mice, Transgenic ,Ectoderm ,Biology ,Mice ,Pregnancy ,Intraflagellar transport ,Internal medicine ,medicine ,Animals ,Limb development ,Hedgehog Proteins ,Cilia ,Molecular Biology ,Process (anatomy) ,In Situ Hybridization ,Body Patterning ,Mice, Knockout ,Bone Development ,Tumor Suppressor Proteins ,Cilium ,Intracellular Signaling Peptides and Proteins ,Gene Expression Regulation, Developmental ,biology.organism_classification ,Mice, Mutant Strains ,Cell biology ,Polydactyly ,Phenotype ,Endocrinology ,medicine.anatomical_structure ,Flagella ,Mutation ,Female ,sense organs ,Carrier Proteins ,Developmental Biology - Abstract
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.
- Published
- 2007
19. Caenorhabditis elegansDYF-2, an Orthologue of Human WDR19, Is a Component of the Intraflagellar Transport Machinery in Sensory Cilia
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Jonathan M. Scholey, Courtney J. Haycraft, Guangshuo Ou, Evgeni Efimenko, Peter Swoboda, Oliver E. Blacque, Bradley K. Yoder, and Michel R. Leroux
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Transcription, Genetic ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Mutant ,Sequence Homology ,Helminth genetics ,Biology ,Models, Biological ,Fluorescence ,Mice ,Intraflagellar transport ,Animals ,Humans ,Cilia ,Cloning, Molecular ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,Genes, Helminth ,Regulation of gene expression ,Base Sequence ,Gene Expression Profiling ,Cilium ,Intracellular Signaling Peptides and Proteins ,Proteins ,Exons ,Articles ,Cell Biology ,biology.organism_classification ,Phenotype ,Transport protein ,Cell biology ,Cytoskeletal Proteins ,Protein Transport ,Gene Expression Regulation ,Flagella ,Mutation ,Mutant Proteins ,sense organs ,Carrier Proteins - Abstract
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.
- Published
- 2006
20. TheC. eleganshomologs of nephrocystin-1 and nephrocystin-4 are cilia transition zone proteins involved in chemosensory perception
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Courtney J. Haycraft, Bradley K. Yoder, Peter Swoboda, Marlene E. Winkelbauer, and Jenny C. Schafer
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Cystic kidney ,Axoneme ,Time Factors ,Cilium ,Ciliary transition zone ,Cell Biology ,Biology ,medicine.disease ,biology.organism_classification ,Cell biology ,Cystic kidney disease ,Gene Expression Regulation ,Nephronophthisis ,Ciliogenesis ,Mutation ,medicine ,Animals ,Cilia ,Neurons, Afferent ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Transcription Factors - Abstract
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.
- Published
- 2005
21. Comparative Genomics Identifies a Flagellar and Basal Body Proteome that Includes the BBS5 Human Disease Gene
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Jane Green, Michel R. Leroux, Bradley K. Yoder, Lisa M. Guay-Woodford, Helen May-Simera, Haitao Li, Susan K. Dutcher, Jin Billy Li, William S. Davidson, Courtney J. Haycraft, Tanya M. Teslovich, Gary D. Stormo, Jantje M. Gerdes, Philip L. Beales, Nicholas Katsanis, Yanli Fan, Linya Li, Oliver E. Blacque, Carmen C. Leitch, Patrick S. Parfrey, and Richard A. Lewis
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Male ,DNA, Complementary ,Proteome ,Centriole ,DNA Mutational Analysis ,Molecular Sequence Data ,Arabidopsis ,Flagellum ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Microtubule ,Intraflagellar transport ,Sequence Homology, Nucleic Acid ,Animals ,Humans ,Basal body ,Cilia ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Bardet-Biedl Syndrome ,030304 developmental biology ,Genomic Library ,0303 health sciences ,Undulipodium ,Sequence Homology, Amino Acid ,Biochemistry, Genetics and Molecular Biology(all) ,Cilium ,Chlamydomonas ,Proteins ,Ciliary transition zone ,Phosphate-Binding Proteins ,Pedigree ,Cell biology ,Cytoskeletal Proteins ,Flagella ,Chromosomes, Human, Pair 2 ,Mutation ,Female ,RNA Interference ,030217 neurology & neurosurgery ,Transcription Factors - Abstract
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.
- Published
- 2004
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22. Identification of CHE-13, a novel intraflagellar transport protein required for cilia formation
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Patrick D. Taulman, Courtney J. Haycraft, Qihong Zhang, Jenny C. Schafer, and Bradley K. Yoder
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Axoneme ,DNA, Complementary ,Amino Acid Motifs ,Molecular Sequence Data ,Mutant ,Nerve Tissue Proteins ,Biology ,Intraflagellar transport ,Sequence Homology, Nucleic Acid ,Ciliogenesis ,Animals ,Cilia ,Neurons, Afferent ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Promoter Regions, Genetic ,Process (anatomy) ,Transcription factor ,Cells, Cultured ,Sequence Homology, Amino Acid ,Cilium ,Neuropeptides ,Cell Biology ,Cell Compartmentation ,Cell biology ,DNA-Binding Proteins ,Protein Transport ,B vitamins ,Flagella ,Mutation ,sense organs ,Carrier Proteins ,Transcription Factors - Abstract
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.
- Published
- 2003
23. Fibulin-1 is required for bone formation and Bmp-2-mediated induction of Osterix
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Sakamuri V. Reddy, Marion A. Cooley, W. Scott Argraves, Sloan F. Miler, Keerthi Harikrishnan, James A. Oppel, Jeremy L. Barth, and Courtney J. Haycraft
- Subjects
medicine.medical_specialty ,Histology ,Physiology ,Endocrinology, Diabetes and Metabolism ,Bone Morphogenetic Protein 2 ,Real-Time Polymerase Chain Reaction ,Bone morphogenetic protein 2 ,Article ,Extracellular matrix ,Mice ,Osteogenesis ,Internal medicine ,medicine ,In Situ Nick-End Labeling ,Animals ,Endochondral ossification ,Mice, Knockout ,Osteoblasts ,Chemistry ,Calcium-Binding Proteins ,Osteoblast ,Cell Differentiation ,Immunohistochemistry ,Fibulin ,FBLN1 ,Skull ,medicine.anatomical_structure ,Frontal bone ,Endocrinology ,Sp7 Transcription Factor ,Transcriptome ,Transcription Factors - Abstract
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. Micro CT (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.
- Published
- 2014
24. Development of sterile ovules on bisexual cones of Gnetum gnemon (Gnetaceae)
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Courtney J. Haycraft and Jeffrey S. Carmichael
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biology ,Gnetum ,Pollination ,Welwitschia ,Embryo ,Plant Science ,biology.organism_classification ,Botany ,Genetics ,Gnetum gnemon ,Ploidy ,Ovule ,Ecology, Evolution, Behavior and Systematics ,Whorl (botany) - Abstract
Angiosperms and Gnetales (Ephedra, Gnetum, Welwitschia ) represent the only seed plants that regularly produce bisexual cones. Unfortunately, the fertility and function of ovules formed on bisexual cones of Gnetales have remained unclear. Some reports indicate that the ovules are sterile while others indicate that they may develop into seeds. This study demonstrates three different developmental patterns of ovules formed on bisexual cones of Gnetum gnemon.Type I ovules did not develop at all after pollination and represented the majority of ovules on each cone. Type II ovules enlarged slightly after pollination due to the enlargement of nucellar tissue. Type III ovules were typically found on the terminal whorl and developed into seed-like structures. The enlargement was due to proliferation of megagametophyte tissue. Sectioned material revealed that megagametophytes show altered development compared to those found in functional female ovules. None of the ovules studied contained embryos, and thus all were sterile. Densitometry of 49,6-diamidino2-phenylindole (DAPI)- stained sections revealed that megagametophyte nuclei formed in the sterile ovules are unreduced (diploid) and thus do not form viable female gametes.
- Published
- 2001
25. The C. elegans homolog of the murine cystic kidney disease gene Tg737 functions in a ciliogenic pathway and is disrupted in osm-5 mutant worms
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Bradley K. Yoder, Courtney J. Haycraft, Peter Swoboda, James H. Thomas, and Patrick D. Taulman
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Repetitive Sequences, Amino Acid ,Axoneme ,Molecular Sequence Data ,Mutant ,Nerve Tissue Proteins ,Biology ,Flagellum ,Green fluorescent protein ,Mice ,Intraflagellar transport ,Animals ,Tissue Distribution ,Amino Acid Sequence ,Cilia ,Neurons, Afferent ,Cloning, Molecular ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,Genes, Helminth ,Polycystic Kidney, Autosomal Recessive ,Regulation of gene expression ,Sequence Homology, Amino Acid ,Tumor Suppressor Proteins ,Cilium ,fungi ,Gene Expression Regulation, Developmental ,Proteins ,Helminth Proteins ,Sequence Analysis, DNA ,biology.organism_classification ,Cell biology ,Protein Transport ,Mutation ,Transcription Factors ,Developmental Biology - Abstract
Cilia and flagella are important organelles involved in diverse functions such as fluid and cell movement, sensory perception and embryonic patterning. They are devoid of protein synthesis, thus their formation and maintenance requires the movement of protein complexes from the cytoplasm into the cilium and flagellum axoneme by intraflagellar transport (IFT), a conserved process common to all ciliated or flagellated eukaryotic cells. We report that mutations in the Caenorhabditis elegans gene Y41g9a.1 are responsible for the ciliary defects in osm-5 mutant worms. This was confirmed by transgenic rescue of osm-5(p813) mutants using the wild-type Y41g9a.1 gene. osm-5 encodes a tetratricopeptide repeat (TPR)-containing protein that is the homolog of murine polaris (Tg737), a protein associated with cystic kidney disease and left-right axis patterning defects in the mouse. osm-5 is expressed in ciliated sensory neurons in C. elegans and its expression is regulated by DAF-19, an RFX-type transcription factor that governs the expression of other genes involved in cilia formation in the worm. Similar to murine polaris, the OSM-5 protein was found to concentrate at the cilium base and within the cilium axoneme as shown by an OSM-5::GFP translational fusion and immunofluorescence. Furthermore, time-lapse imaging of OSM-5::GFP fusion protein shows fluorescent particle migration within the cilia. Overall, the data support a crucial role for osm-5 in a conserved ciliogenic pathway, most likely as a component of the IFT process. Movies available on-line: http://www.biologists.com/Development/movies/dev3342.html
- Published
- 2001
26. Polaris, a Protein Involved in Left-Right Axis Patterning, Localizes to Basal Bodies and Cilia
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Patrick D. Taulman, Courtney J. Haycraft, Bradley K. Yoder, and Daniel F. Balkovetz
- Subjects
Male ,Ependymal Cell ,Population ,Biology ,Antibodies ,Epithelium ,Article ,Cell Line ,Mice ,Dogs ,Polaris ,Pregnancy ,Testis ,Polycystic kidney disease ,medicine ,Animals ,Basal body ,Tissue Distribution ,Cilia ,Axis specification ,education ,Lung ,Molecular Biology ,Body Patterning ,Organelles ,education.field_of_study ,Tumor Suppressor Proteins ,Cilium ,Brain ,Proteins ,Cell Biology ,Apical membrane ,medicine.disease ,Spermatozoa ,Mice, Mutant Strains ,Cell biology ,Sperm Maturation ,Kidney Tubules ,Flagella ,Mutation ,Female ,Hydrocephalus - Abstract
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 Tg737orpkbrains and by the lack of node cilia inTg737Δ2-3βGalmutants.
- Published
- 2001
27. Simvastatin Inhibits Lipopolysaccharide-Induced Osteoclastogenesis and Reduces Alveolar Bone Loss in Experimental Periodontal Disease
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Junfei Jin, Hong Yu, Qiyan Li, Courtney J. Haycraft, Xiaoming Zhang, Zhongyang Lu, Maria F. Lopes-Virella, Yan Huang, Keith L. Kirkwood, and Yanchun Li
- Subjects
Lipopolysaccharides ,Simvastatin ,Statin ,Lipopolysaccharide ,medicine.drug_class ,Acid Phosphatase ,Alveolar Bone Loss ,Anti-Inflammatory Agents ,Gingiva ,Osteoclasts ,Inflammation ,Pharmacology ,Reductase ,Aggregatibacter actinomycetemcomitans ,Article ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,Gingivitis ,Medicine ,Animals ,cardiovascular diseases ,Dental alveolus ,Periodontal Diseases ,biology ,business.industry ,Tartrate-Resistant Acid Phosphatase ,Toll-Like Receptors ,nutritional and metabolic diseases ,X-Ray Microtomography ,biology.organism_classification ,Maxillary Diseases ,Rats ,Isoenzymes ,Disease Models, Animal ,chemistry ,Matrix Metalloproteinase 9 ,Immunology ,Periodontics ,Cytokines ,lipids (amino acids, peptides, and proteins) ,medicine.symptom ,Hydroxymethylglutaryl-CoA Reductase Inhibitors ,Inflammation Mediators ,business ,medicine.drug - Abstract
Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase and have anti-inflammatory effects independent of cholesterol lowering. Recent clinical studies have indicated that statin intake has a beneficial effect on periodontal disease. However, the underlying mechanisms have not been well understood. In the current study, we employed a rat model with lipopolysaccharide (LPS)-induced periodontal disease and determined the effect of simvastatin, a commonly prescribed statin, on osteoclastogenesis, gingival inflammation and alveolar bone loss.Sprague-Dawley rats were injected with Aggregatibacter actinomycetemcomitans LPS in periodontal tissue three times per week for 8 wk and part of the rats with LPS injection were also given simvastatin via gavage. After the treatments, the rat maxillae were scanned by microcomputed tomography and the images were analyzed to determine alveolar bone loss. To explore the underlying mechanisms, the effect of simvastatin on osteoclastogenesis and gingival expression of proinflammatory cytokines were also determined by tartrate-resistant acid phosphatase staining and real-time polymerase chain reaction assays, respectively.Results showed that LPS treatment markedly increased bone loss, but administration of simvastatin significantly alleviated the bone loss. Results also showed that LPS treatment stimulated osteoclastogenesis and the expression of inflammatory cytokines, but simvastatin significantly modulates the stimulatory effect of LPS on osteoclastogenesis and cytokine expression.This study demonstrated that simvastatin treatment inhibits LPS-induced osteoclastogenesis and gingival inflammation and reduces alveolar bone loss, indicating that the intake of simvastatin may hinder the progression of periodontal disease.
- Published
- 2013
28. Erratum to: the buccohypophyseal canal is an ancestral vertebrate trait maintained by modulation in sonic hedgehog signaling
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Roman H Khonsari, Maisa Seppala, Alan Pradel, Hugo Dutel, Gaël Clément, Oleg Lebedev, Sarah Ghafoor, Michaela Rothova, Abigael Tucker, John G Maisey, Chen-Ming Fan, Atsushi Ohazama, Paul Tafforeau, Brunella Franco, Jill Helms, Courtney J Haycraft, Albert David, Philippe Janvier, Martyn T Cobourne, and Paul T Sharpe
- Subjects
Agricultural and Biological Sciences(all) ,Biochemistry, Genetics and Molecular Biology(all) ,Physiology ,Structural Biology ,Correction ,Cell Biology ,Plant Science ,General Agricultural and Biological Sciences ,General Biochemistry, Genetics and Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Developmental Biology ,Biotechnology - Published
- 2013
29. The buccohypophyseal canal is an ancestral vertebrate trait maintained by modulation in sonic hedgehog signaling
- Author
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Gaël Clément, Oleg V. Lebedev, Alan Pradel, Courtney J. Haycraft, Paul T. Sharpe, Brunella Franco, Maisa Seppala, Jill A. Helms, Albert David, Philippe Janvier, Michaela Rothova, Roman Hossein Khonsari, Paul Tafforeau, Sarah Ghafoor, Hugo Dutel, Atsushi Ohazama, Chen-Ming Fan, John G. Maisey, Abigael Tucker, Martyn T. Cobourne, Kings Coll London, Inst Dent, Comprehens Biomed Res Ctr, Dept Craniofacial Dev & Stem Cell Res, London WC2R 2LS, England, Kings Coll London, Guys Hosp, Inst Dent, Dept Orthodont, London WC2R 2LS, England, American Museum of Natural History (AMNH), Centre de recherche sur la Paléobiodiversité et les Paléoenvironnements (CR2P), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Mécanismes adaptatifs : des organismes aux communautés, Muséum national d'Histoire naturelle (MNHN)-Centre National de la Recherche Scientifique (CNRS), Russian Acad Sci, Inst Paleontol, Moscow V71, Russia, Acad Sci Czech Republic, Inst Expt Med, Prague, Czech Republic, Carnegie Institution for Science [Washington], European Synchrotron Radiation Facility (ESRF), Univ Naples Federico II, Dept Pediat, Naples, Italy, Stanford University, Medical University of South Carolina [Charleston] (MUSC), Hôtel-Dieu de Nantes, Khonsari, Rh, Seppala, M, Pradel, A, Dutel, H, Clément, G, Lebedev, O, Ghafoor, S, Rothova, M, Tucker, A, Maisey, Jg, Fan, Cm, Kawasaki, M, Ohazama, A, Tafforeau, P, Franco, Brunella, Helms, J, Haycraft, Cj, David, A, Janvier, P, Cobourne, Mt, Sharpe, Pt, Carnegie Institution for Science, University of Naples Federico II = Università degli studi di Napoli Federico II, Centre de Recherche en Paléontologie - Paris (CR2P), and Muséum national d'Histoire naturelle (MNHN)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Pituitary gland ,Coelacanth ,Midline ,Physiology ,[SDV]Life Sciences [q-bio] ,Cell Cycle Proteins ,Ectoderm ,Plant Science ,Shh ,Mice ,Neural crest ,0302 clinical medicine ,Primary cilia ,Structural Biology ,Sonic hedgehog ,[SDV.BDD]Life Sciences [q-bio]/Development Biology ,ComputingMilieux_MISCELLANEOUS ,Phylogeny ,0303 health sciences ,Agricultural and Biological Sciences(all) ,Fossils ,Endoderm ,Fishes ,Gene Expression Regulation, Developmental ,Vertebrate ,Anatomy ,Hedgehog signaling pathway ,medicine.anatomical_structure ,Pituitary Gland ,Vertebrates ,embryonic structures ,General Agricultural and Biological Sciences ,Research Article ,Signal Transduction ,Biotechnology ,animal structures ,Buccohypophyseal canal ,Biology ,Extinction, Biological ,GPI-Linked Proteins ,Chondrichthyans ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Anterior pituitary ,biology.animal ,Notochord ,medicine ,Animals ,Hedgehog Proteins ,Cilia ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Mouth ,Neuroectoderm ,Biochemistry, Genetics and Molecular Biology(all) ,Skull ,Cell Biology ,Knock-out mouse ,Jaw ,Mutation ,biology.protein ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
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. 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. 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.
- Published
- 2013
30. NIP45 Negatively Regulates RANK Ligand Induced Osteoclast Differentiation
- Author
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Sakamuri V. Reddy, William L. Ries, Courtney J. Haycraft, and Srinivasan Shanmugarajan
- Subjects
musculoskeletal diseases ,Blotting, Western ,Down-Regulation ,Osteoclasts ,Biology ,Biochemistry ,Bone resorption ,Article ,Small hairpin RNA ,Mice ,Osteoclast ,medicine ,Animals ,Immunoprecipitation ,Molecular Biology ,Transcription factor ,Cells, Cultured ,TNF Receptor-Associated Factor 6 ,Microscopy, Confocal ,NFATC Transcription Factors ,RANK Ligand ,Intracellular Signaling Peptides and Proteins ,Signal transducing adaptor protein ,Nuclear Proteins ,NFAT ,Cell Differentiation ,Cell Biology ,medicine.anatomical_structure ,RANKL ,biology.protein ,Cancer research ,Signal Transduction - Abstract
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.
- Published
- 2012
31. A Disintegrin and Metalloenzyme (ADAM) 17 Activation Is Regulated by α5β1 Integrin in Kidney Mesangial Cells
- Author
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Shigeki Higashiyama, Monika Gooz, Yujing Dang, Courtney J. Haycraft, Pal Gooz, and Waleed O. Twal
- Subjects
lcsh:Medicine ,Proximity ligation assay ,CD49c ,Biochemistry ,Collagen receptor ,Receptors, G-Protein-Coupled ,Rats, Sprague-Dawley ,0302 clinical medicine ,Molecular Cell Biology ,Membrane Receptor Signaling ,lcsh:Science ,Cells, Cultured ,0303 health sciences ,Multidisciplinary ,Mesangial cell ,Integrin beta1 ,Mechanisms of Signal Transduction ,3. Good health ,Cell biology ,Extracellular Matrix ,Protein Transport ,Integrin alpha M ,Nephrology ,030220 oncology & carcinogenesis ,Mesangial Cells ,Cytochemistry ,Medicine ,Intercellular Signaling Peptides and Proteins ,Integrin, beta 6 ,Research Article ,Signal Transduction ,Heparin-binding EGF-like Growth Factor ,Integrin alpha5beta1 ,Protein Binding ,Integrin ,Thiophenes ,Biology ,ADAM17 Protein ,03 medical and health sciences ,Disintegrin ,Cell Adhesion ,Animals ,030304 developmental biology ,lcsh:R ,Molecular biology ,Rats ,Enzyme Activation ,ADAM Proteins ,Multiprotein Complexes ,biology.protein ,lcsh:Q - Abstract
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.
- Published
- 2012
32. MKP-1 Regulates Cytokine mRNA Stability Through Selectively Modulation Subcellular Translocation of AUF1
- Author
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Yuhao Sun, Keith L. Kirkwood, Viswanathan Palanisamy, Courtney J. Haycraft, and Hong Yu
- Subjects
MAPK/ERK pathway ,medicine.medical_treatment ,Immunology ,Blotting, Western ,RNA-binding protein ,Enzyme-Linked Immunosorbent Assay ,Biology ,Real-Time Polymerase Chain Reaction ,Biochemistry ,Article ,Cell Line ,Mice ,Western blot ,GTP-Binding Proteins ,medicine ,Immunology and Allergy ,Animals ,Heterogeneous Nuclear Ribonucleoprotein D0 ,RNA, Messenger ,Heterogeneous-Nuclear Ribonucleoprotein D ,Molecular Biology ,Mice, Knockout ,Gene knockdown ,Messenger RNA ,medicine.diagnostic_test ,Dual Specificity Phosphatase 1 ,Hematology ,Molecular biology ,Rats ,Blot ,Protein Transport ,Cytokine ,Cytokines ,Tumor necrosis factor alpha ,Subcellular Fractions - Abstract
MAPK phosphatase-1 (MKP-1)/dual specificity protein phosphatase-1 (DUSP-1) is a negative regulator of the host inflammatory response to infection. However, the mechanisms underlying the regulation of cytokine expression by MKP-1, especially at the post-transcriptional level, have not been fully delineated. In the current study, MKP-1 specifically dephosphorylated activated MAPK responses and attenuated LPS-induced IL-6, IL-10, and TNF-α expression. In addition, MKP-1 was important in destabilizing cytokine mRNAs. In LPS-stimulated rat macrophages with overexpressed MKP-1, half-lives of IL-6, IL-10 and TNF-α mRNAs were significantly reduced compared to controls. Conversely, half-lives of IL-6, IL-10, and TNF-α mRNAs were significantly increased in bone marrow macrophages derived from MKP-1 knock out (KO) mice compared with macrophages derived from MKP-1 wild type (WT) mice. Furthermore, MKP-1 promoted translocation of RNA-binding protein (RNA-BP) ARE/poly-(U) binding degradation factor 1 (AUF1) from the nucleus to the cytoplasm in response to LPS stimulation as evidenced by Western blot and immunofluorescent staining. Knockdown AUF1 mRNA expression by AUF1 siRNA in MKP-1 WT bone marrow macrophages significantly delayed degradation of IL-6, IL-10 and TNF- α mRNAs compared with controls. Finally, AUF1 was immunoprecipitated with the RNA complex in cellular lysates derived from bone marrow macrophages of MKP-1 KO vs. WT mice, which had increased AUF1-bound target mRNAs, including IL-6, IL-10, and TNF-α in WT macrophages compared with MKP-1 KO macrophages. Thus, this work provides new mechanistic insight of MKP-1 signaling and regulation of cytokine mRNA stability through RNA binding proteins in response to inflammatory stimuli.
- Published
- 2011
33. GMAP210 and IFT88 are present in the spermatid golgi apparatus and participate in the development of the acrosome-acroplaxome complex, head-tail coupling apparatus and tail
- Author
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Rosa M. Ríos, Laura L. Tres, Bradley K. Yoder, Courtney J. Haycraft, Abraham L. Kierszenbaum, Michel Bornens, Eugene Rivkin, and National Institutes of Health (US)
- Subjects
Male ,Outer dense fiber ,Intramanchette transport ,Immunoblotting ,Golgi Apparatus ,Biology ,Microtubules ,Article ,Mice ,symbols.namesake ,chemistry.chemical_compound ,Microtubule ,Microtubes ,medicine ,Animals ,Fluorescent Antibody Technique, Indirect ,Acrosome ,Brefeldin A ,Spermatid ,Reverse Transcriptase Polymerase Chain Reaction ,Nocodazole ,Tumor Suppressor Proteins ,Nuclear Proteins ,Golgi apparatus ,Immunohistochemistry ,Spermatids ,Actins ,Rats ,Cell biology ,Cytoskeletal Proteins ,Microscopy, Electron ,medicine.anatomical_structure ,Acroplaxome ,chemistry ,Intraflagellar transport ,symbols ,Developmental Biology - Abstract
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. Developmental Dynamics 240:723-736, 2011. © 2011 Wiley-Liss, Inc., NIH. Grant Numbers: HD36477, DK065655, HD37282.
- Published
- 2011
34. Telomerase immortalization of principal cells from mouse collecting duct
- Author
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Courtney J. Haycraft, Monika Gooz, Lisa M. Guay-Woodford, Hai Yao, Stacy L. Steele, Robert J. Kolb, Yongren Wu, P. Darwin Bell, and Kent T. Keyser
- Subjects
Pathology ,medicine.medical_specialty ,Telomerase ,medicine.diagnostic_test ,Physiology ,Reverse Transcriptase Polymerase Chain Reaction ,Cell Culture Techniques ,Biology ,Immunofluorescence ,Viral vector ,Flow cytometry ,Cell biology ,Cell Line ,Mice ,Cell culture ,Genetically Engineered Mouse ,medicine ,Innovative Methodology ,Animals ,Ectopic expression ,Telomerase reverse transcriptase ,Kidney Tubules, Collecting - Abstract
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.
- Published
- 2010
35. EGFR augments cell proliferation in polycystic kidney disease through activation of a novel ion channel
- Author
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Monika Gooz, Aleksander Baldys, Wen-Feng Chu, Courtney J. Haycraft, Zhi-Ren Zhang, Binlin Song, Robert J Kolb, John R. Raymond, Bernd Nilius, Stacy L. Steele, Grzegorz Owsianik, Peter Komlosi, Amber Houston, and P. D. Bell
- Subjects
medicine.medical_specialty ,business.industry ,Cell growth ,medicine.disease ,Biochemistry ,Endocrinology ,Internal medicine ,Genetics ,Polycystic kidney disease ,Medicine ,business ,Molecular Biology ,Ion channel ,Biotechnology - Published
- 2009
36. Primary cilia regulate Shh activity in the control of molar tooth number
- Author
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Courtney J. Haycraft, Hervé Lesot, Paul T. Sharpe, Maisa Seppala, James Blackburn, Sarah Ghafoor, Chen-Ming Fan, Atsushi Ohazama, Martyn T. Cobourne, Renata Peterkova, Bradley K. Yoder, and David C. Martinelli
- Subjects
Molar ,Mesenchyme ,Context (language use) ,Cell Cycle Proteins ,Mice, Transgenic ,Biology ,GPI-Linked Proteins ,Mice ,Downregulation and upregulation ,stomatognathic system ,Intraflagellar transport ,medicine ,Animals ,Hedgehog Proteins ,Cilia ,Molecular Biology ,Cilium ,Diastema ,Gene Expression Regulation, Developmental ,Membrane Proteins ,Research Reports ,Anatomy ,Epithelium ,Cell biology ,stomatognathic diseases ,medicine.anatomical_structure ,Phenotype ,Mutation ,Tooth ,Developmental Biology - Abstract
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. Gas1mutants 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.
- Published
- 2009
37. Cilia involvement in patterning and maintenance of the skeleton
- Author
-
Courtney J, Haycraft and Rosa, Serra
- Subjects
Bone Development ,Animals ,Cilia ,Article ,Body Patterning - Abstract
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.
- Published
- 2009
38. Role for primary cilia in the regulation of mouse ovarian function
- Author
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Ellen T. Johnson, Kevin Roarty, Rosa Serra, Courtney J. Haycraft, Teodora Nicola, and Bradley K. Yoder
- Subjects
Ovulation ,medicine.medical_specialty ,medicine.drug_class ,media_common.quotation_subject ,Mammary gland ,Ovary ,Estrous Cycle ,Biology ,Mice ,Mammary Glands, Animal ,Intraflagellar transport ,Internal medicine ,medicine ,Animals ,Cilia ,media_common ,Estrous cycle ,Homeodomain Proteins ,Granulosa Cells ,Estradiol ,Integrases ,Cilium ,Tumor Suppressor Proteins ,Biological Transport ,Estrogens ,Phenotype ,Mice, Mutant Strains ,Cell biology ,Endocrinology ,medicine.anatomical_structure ,Estrogen ,Female ,Developmental Biology - Abstract
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.
- Published
- 2008
39. THM1 negatively modulates mouse sonic hedgehog signal transduction and affects retrograde intraflagellar transport in cilia
- Author
-
Annick Turbe-Doan, Courtney J. Haycraft, Haiyan Qiu, David R. Beier, Rolf W. Stottmann, Allyson L. Chesebro, Pamela V. Tran, Bradley K. Yoder, Bruce J. Herron, Paul Scherz, Tatiana Y. Besschetnova, and Jagesh V. Shah
- Subjects
Yellow fluorescent protein ,Male ,Alkylating Agents ,Blotting, Western ,Molecular Sequence Data ,Genes, Recessive ,Biology ,Zinc Finger Protein GLI1 ,Article ,Mice ,Intraflagellar transport ,Genetics ,Animals ,Hedgehog Proteins ,Amino Acid Sequence ,Cilia ,Sonic hedgehog ,Cloning, Molecular ,Hedgehog ,Caenorhabditis elegans ,Cells, Cultured ,In Situ Hybridization ,Adaptor Proteins, Signal Transducing ,Mice, Knockout ,Oncogene Proteins ,Sequence Homology, Amino Acid ,Cilium ,Tumor Suppressor Proteins ,Biological Transport ,Fibroblasts ,biology.organism_classification ,Cell biology ,Luminescent Proteins ,Spinal Cord ,Mutagenesis ,Ethylnitrosourea ,biology.protein ,Trans-Activators ,Female ,sense organs ,Signal transduction ,Smoothened ,Signal Transduction - Abstract
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.
- Published
- 2008
40. Altered Regulation of Ion Channel Activity by EGF in Polycystic Kidney Disease
- Author
-
Courtney J. Haycraft, Wen-Feng Chu, Zhi-Ren Zhang, P. D. Bell, and Peter Komlosi
- Subjects
medicine.medical_specialty ,Endocrinology ,Ion channel activity ,business.industry ,Internal medicine ,Genetics ,medicine ,Polycystic kidney disease ,business ,medicine.disease ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2008
41. Cytosolic Calcium‐Dependence of Elevated cAMP in Polycystic Kidney Disease
- Author
-
Wen-Feng Chu, Courtney J. Haycraft, Zhi-Ren Zhang, Peter Komlosi, and P. D. Bell
- Subjects
medicine.medical_specialty ,Endocrinology ,business.industry ,Internal medicine ,Genetics ,Polycystic kidney disease ,Medicine ,business ,medicine.disease ,Molecular Biology ,Biochemistry ,Biotechnology ,Cytosolic calcium - Published
- 2008
42. Chapter 11 Cilia Involvement in Patterning and Maintenance of the Skeleton
- Author
-
Courtney J. Haycraft and Rosa Serra
- Subjects
Body Patterning ,Mechanosensation ,ved/biology ,Cilium ,ved/biology.organism_classification_rank.species ,Anatomy ,Biology ,biology.organism_classification ,Phenotype ,Cell biology ,Organelle ,Basal body ,Model organism ,Zebrafish - Abstract
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.
- Published
- 2008
43. Articular cartilage and growth plate defects are associated with chondrocyte cytoskeletal abnormalities in Tg737orpk mice lacking the primary cilia protein polaris
- Author
-
CA Poole, Susan R. McGlashan, Bradley K. Yoder, Cynthia G. Jensen, and Courtney J. Haycraft
- Subjects
Cartilage, Articular ,medicine.medical_specialty ,Chondrocyte hypertrophy ,Mice, Transgenic ,Biology ,Chondrocyte ,Mice ,Chondrocytes ,Tubulin ,Internal medicine ,Ciliogenesis ,medicine ,Animals ,Cilia ,Growth Plate ,Cytoskeleton ,Molecular Biology ,Endochondral ossification ,Cell Proliferation ,Cilium ,Cartilage ,Tumor Suppressor Proteins ,Actins ,Cell biology ,Extracellular Matrix ,medicine.anatomical_structure ,Endocrinology ,Gene Expression Regulation ,Epiphysis ,Collagen Type X - Abstract
Primary cilia are highly conserved organelles found on almost all eukaryotic cells. Tg737(orpk) (orpk) mice carry a hypomorphic mutation in the Tg737 gene resulting in the loss of polaris, a protein essential for ciliogenesis. Orpk mice have an array of skeletal patterning defects and show stunted growth after birth, suggesting defects in appositional and endochondral development. This study investigated the association between orpk tibial long bone growth and chondrocyte primary cilia expression using histomorphometric and immunohistochemical analysis. Wild-type chondrocytes throughout the developing epiphysis and growth plate expressed primary cilia, which showed a specific orientation away from the articular surface in the first 7-10 cell layers. In orpk mice, primary cilia were identified on very few cells and were significantly shorter. Orpk chondrocytes also showed significant increases in cytoplasmic tubulin, a likely result of failed ciliary assembly. The growth plates of orpk mice were significantly smaller in length and width, with marked changes in cellular organization in the presumptive articular cartilage, proliferative and hypertrophic zones. Cell density at the articular surface and in the hypertrophic zone was significantly altered, suggesting defects in both appositional and endochondral growth. In addition, orpk hypertrophic chondrocytes showed re-organization of the F-actin network into stress fibres and failed to fully undergo hypertrophy, while there was a marked reduction in type X collagen sequestration. These data suggest that failure to form a functional primary cilium affects chondrocyte differentiation and results in delayed chondrocyte hypertrophy within the orpk growth plate.
- Published
- 2006
44. Development of the post-natal growth plate requires intraflagellar transport proteins
- Author
-
Buer Song, Bradley K. Yoder, Rosa Serra, Hwa-Seon Seo, and Courtney J. Haycraft
- Subjects
Tg737 ,Ihh ,Kinesins ,Mice ,0302 clinical medicine ,Primary cilia ,Cell Movement ,Aggrecans ,Growth Plate ,In Situ Hybridization ,0303 health sciences ,Ptc1 ,Reverse Transcriptase Polymerase Chain Reaction ,Cilium ,Cell Differentiation ,Immunohistochemistry ,Cell biology ,medicine.anatomical_structure ,Kinesin ,IFT88 ,Rotation ,Mice, Transgenic ,Biology ,Kif3a ,Chondrocyte ,Article ,Polaris ,03 medical and health sciences ,Chondrocytes ,Microtubule ,Intraflagellar transport ,medicine ,Animals ,KIF3A ,Cilia ,Molecular Biology ,Process (anatomy) ,Actin ,030304 developmental biology ,DNA Primers ,FAK ,Cell Biology ,Actin cytoskeleton ,Radiography ,Cartilage ,sense organs ,030217 neurology & neurosurgery ,Developmental Biology ,Collagen Type X - Abstract
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.
- Published
- 2006
45. Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function
- Author
-
Edward J. Michaud, Bradley K. Yoder, Yesim Aydin-Son, Boglarka Banizs, Courtney J. Haycraft, and Qihong Zhang
- Subjects
Patched ,Cancer Research ,animal structures ,lcsh:QH426-470 ,Neural tube patterning ,Kruppel-Like Transcription Factors ,Nerve Tissue Proteins ,Zinc Finger Protein Gli2 ,Biology ,Models, Biological ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Zinc Finger Protein Gli3 ,Intraflagellar transport ,GLI3 ,Genetics ,Animals ,Hedgehog Proteins ,Sonic hedgehog ,Molecular Biology ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Mice, Inbred BALB C ,0303 health sciences ,Tumor Suppressor Proteins ,Ciliary transition zone ,Extremities ,Hedgehog signaling pathway ,Cell biology ,Mice, Inbred C57BL ,lcsh:Genetics ,Flagella ,embryonic structures ,Trans-Activators ,biology.protein ,Smoothened ,030217 neurology & neurosurgery ,Transcription Factors ,Research Article - Abstract
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., Synopsis Cilia are small projections extending from the surface of most cells. Research has shown that they are important in diseases such as cystic kidney diseases as well as during the development of many tissues including the limb. More recently, proteins such as Polaris, which is required to build cilia, have been demonstrated to be essential for the regulation of Sonic hedgehog (Shh) signaling, although the mechanism has remained elusive. Precise regulation of Shh signal transduction is important for the proper development of many tissues. Excessive activation of the Shh pathway results in severe developmental defects and has been implicated in certain types of cancer. In the limb, Shh signaling is involved in digit development, and excess signaling leads to the formation of extra digits. The main targets of Shh signaling are the Glioma (Gli) family of transcription factors, and Gli3 has been shown to be processed to a shortened repressor form when Shh signaling is repressed. The localization of the Gli transcription factors and Suppressor of fused, a protein involved in the regulation of Gli protein function, to cilia suggests that the cilia may be an important site for regulation of Shh signal transduction by modulating Gli protein function.
- Published
- 2005
46. Cilia-driven fluid flow in the zebrafish pronephros, brain and Kupffer's vesicle is required for normal organogenesis
- Author
-
Bradley K. Yoder, Albrecht Kramer-Zucker, Felix Olale, Alexander F. Schier, Iain A. Drummond, and Courtney J. Haycraft
- Subjects
Central Nervous System ,congenital, hereditary, and neonatal diseases and abnormalities ,Embryo, Nonmammalian ,Organogenesis ,Molecular Sequence Data ,Oligonucleotides ,Motility ,Nerve Tissue Proteins ,Kidney ,Kidney cysts ,Cystic kidney disease ,medicine ,Animals ,Cilia ,Cloning, Molecular ,Molecular Biology ,Zebrafish ,In Situ Hybridization ,Body Patterning ,DNA Primers ,Microscopy, Video ,biology ,Base Sequence ,Cilium ,Organizers, Embryonic ,Dyneins ,Anatomy ,Sequence Analysis, DNA ,medicine.disease ,biology.organism_classification ,Immunohistochemistry ,Cell biology ,Pronephros ,Body Fluids ,Situs inversus ,Microscopy, Electron ,Microscopy, Fluorescence ,medicine.symptom ,NODAL ,Developmental Biology - Abstract
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.
- Published
- 2005
47. Disruption of IFT results in both exocrine and endocrine abnormalities in the pancreas of Tg737(orpk) mutant mice
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Bradley K. Yoder, Qihong Zhang, James R. Davenport, Mandy J. Croyle, and Courtney J. Haycraft
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Male ,medicine.medical_specialty ,Glucose uptake ,Apoptosis ,Mice, Transgenic ,Biology ,Pathology and Forensic Medicine ,Islets of Langerhans ,Mice ,Intraflagellar transport ,Internal medicine ,Ciliogenesis ,medicine ,Glucose homeostasis ,Endocrine system ,Animals ,Cilia ,Fluorescent Antibody Technique, Indirect ,Molecular Biology ,Pancreas ,Cell Proliferation ,Cilium ,Tumor Suppressor Proteins ,Pancreatic Ducts ,Cell Biology ,Glucose Tolerance Test ,beta-Galactosidase ,Hedgehog signaling pathway ,Mice, Mutant Strains ,Pancreas, Exocrine ,Disease Models, Animal ,Endocrinology ,medicine.anatomical_structure ,Animals, Newborn ,Female - Abstract
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.
- Published
- 2004
48. XBX-1 encodes a dynein light intermediate chain required for retrograde intraflagellar transport and cilia assembly in Caenorhabditis elegans
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James H. Thomas, Jenny C. Schafer, Peter Swoboda, Courtney J. Haycraft, and Bradley K. Yoder
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Axoneme ,Cilium ,Molecular Motor Proteins ,Dynein ,Dyneins ,Cell Biology ,Articles ,Flagellum ,Biology ,Cell biology ,Animals, Genetically Modified ,B vitamins ,Gene Expression Regulation ,Intraflagellar transport ,Flagella ,Genes, Reporter ,Axoplasmic transport ,Kinesin ,Animals ,sense organs ,Cilia ,Caenorhabditis elegans ,Molecular Biology ,Sequence Deletion - Abstract
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.
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- 2003
49. Cilia/IFT in mammalian limb patterning
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Buer Song, Courtney J. Haycraft, Qihong Zhang, Bradley K. Yoder, and Rosa Serra
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Cilium ,fungi ,Cell Biology ,Biology ,Molecular Biology ,Cell biology ,Developmental Biology - Published
- 2006
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50. An inducible CiliaGFP mouse model for in vivo visualization and analysis of cilia in live tissue
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Amber K. O'Connor, Bradley K. Yoder, P. Darwin Bell, Courtney J. Haycraft, Mandy J. Croyle, Nicolas F. Berbari, Peter Hohenstein, Robert A. Kesterson, and Erik B. Malarkey
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0303 health sciences ,Cell type ,Research ,Cilium ,Somatostatin receptor 3 ,Cell Biology ,Biology ,respiratory system ,In vivo cilia labeling ,Ciliopathies ,Inducible transgene ,Cell biology ,Intravital microscopy ,03 medical and health sciences ,0302 clinical medicine ,Intraflagellar transport ,Motile cilium ,Developmental biology ,ROSA26 locus ,030217 neurology & neurosurgery ,Function (biology) ,030304 developmental biology - Abstract
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.
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