Zebrafish Motile Cilia as a Model for Primary Ciliary Dyskinesia
Andreia PintoMargarida RasteiroCatarina BotaSara PestanaPedro SampaioClaire HoggThomas BurgoyneSusana S. Lopes
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Abstract:
Zebrafish is a vertebrate teleost widely used in many areas of research. As embryos, they develop quickly and provide unique opportunities for research studies owing to their transparency for at least 48 h post fertilization. Zebrafish have many ciliated organs that include primary cilia as well as motile cilia. Using zebrafish as an animal model helps to better understand human diseases such as Primary Ciliary Dyskinesia (PCD), an autosomal recessive disorder that affects cilia motility, currently associated with more than 50 genes. The aim of this study was to validate zebrafish motile cilia, both in mono and multiciliated cells, as organelles for PCD research. For this purpose, we obtained systematic high-resolution data in both the olfactory pit (OP) and the left-right organizer (LRO), a superficial organ and a deep organ embedded in the tail of the embryo, respectively. For the analysis of their axonemal ciliary structure, we used conventional transmission electron microscopy (TEM) and electron tomography (ET). We characterised the wild-type OP cilia and showed, for the first time in zebrafish, the presence of motile cilia (9 + 2) in the periphery of the pit and the presence of immotile cilia (still 9 + 2), with absent outer dynein arms, in the centre of the pit. In addition, we reported that a central pair of microtubules in the LRO motile cilia is common in zebrafish, contrary to mouse embryos, but it is not observed in all LRO cilia from the same embryo. We further showed that the outer dynein arms of the microtubular doublet of both the OP and LRO cilia are structurally similar in dimensions to the human respiratory cilia at the resolution of TEM and ET. We conclude that zebrafish is a good model organism for PCD research but investigators need to be aware of the specific physical differences to correctly interpret their results.Keywords:
Motile cilium
Organelle
Primary ciliary dyskinesia (PCD) is a disease that affects the function of respiratory cilia that helps protect against airway infections. Diagnosis is difficult with current methods but genetic testing may expedite the process since PCD is hereditary, however the etiology of 30% of cases remains unknown. To identify more causes of PCD, whole exome sequencing was performed on patients identifying Spindly (spdl1) as a candidate. Analysis using zebrafish indicate spdl1 is expressed in motile ciliated tissues. Spdl1 deficiency results in embryos with ventral curvatures indicating cilia dysfunction. The defects can be rescued with 100pg of WT RNA but not RNA containing one of the mutations identified. Cilia orientation and range of motion was unaffected but motile cilia length was significantly increased in mutant neural tube floorplate (p%%%%M.Sc.
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Abstract: One of the rapidly growing groups of diseases known as ciliopathies is primary ciliary dyskinesia (PCD), a rare hereditary illness of the motile cilia. Different clinical symptoms of prima-ry ciliary dyskinesia include infertility, left-right lateralization abnormalities, and chronic upper and lower respiratory tract disorders. Our knowledge of the genetics underlying primary ciliary dyskine-sia has significantly increased in recent years. Involved in the formation, shape, and operation of motile cilia are axonemal, cytoplasmic, and regulatory proteins that are encoded by a rising number of disease-associated genes and pathogenic mutations. We now have a better grasp of the clinical signs and symptoms of motile ciliopathies because of advances in our understanding of cilia genet-ics and the function of the proteins expressed. These developments have altered how we approach primary ciliary dyskinesia diagnostic testing. The clinical characteristics of primary ciliary dyskine-sia, the evolution of diagnostics, and the discovery of previously unknown genotype-phenotype connections in primary ciliary dyskinesia will all be covered in this review paper.
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Background:CEP164 encodes a centrosomal protein required for assembly of primary cilia. More recently it has been suggested it may also have a role in formation of multiple motile cilia. Pathogenic variants in CEP164 are known to cause nephronophthisis-related ciliopathies but a causative link to the motile ciliopathy primary ciliary dyskinesia (PCD) has not been proven. Aim: To assess airway cilia in a patient with a clinical history consistent with PCD, and bi-allelic variants in CEP164. Method: A patient with bronchiectasis in the UK 100,000 Genomes Project was found to have compound heterozygous stop gain variants in CEP164, and no other relevant variants. The patient underwent PCD diagnostic functional testing including high-speed video microscopy and transmission electron microscopy (TEM) of nasal epithelial cells. In addition, localisation of CEP164 protein was assessed by immunofluorescence (IF). Results: Cilia displayed a dyskinetic ciliary beat pattern, with long cilia and cilia with bulbous tips. Air liquid interface culture partially resolved dyskinesia, however an abnormal 'staggered' beat pattern was evident and the presence of long cilia persisted. Ciliary ultrastructure was normal by TEM. IF analysis demonstrated an absence of CEP164 labelling at the centriolar region. Conclusion: Supported by BEAT-PCD, we provide evidence that presence of CEP164 is vital to correct formation and function of respiratory cilia in addition to primary cilia, and that pathogenic variants in CEP164 are responsible for the PCD phenotype in this patient. We suggest CEP164 should be considered a candidate gene for PCD.
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Mucociliary clearance
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Respiratory tract
Kartagener Syndrome
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Although the basic structure of the axoneme has been highly conserved throughout evolution, the varied functions of specialized axonemes require differences in structure and regulation. Cilia lining the respiratory tract propel mucus along airway surfaces, providing a critical function to the defense mechanisms of the pulmonary system, yet little is known of their molecular structure. We have identified and cloned a dynein heavy chain that is a component of the inner dynein arm. Bronchial epithelial cells were obtained from normal donors and from a patient with primary ciliary dyskinesia (PCD) whose cilia demonstrated an absence of inner dynein arms by electron microscopy. Cilia from normal and PCD cells were compared by gel electrophoresis, and mass spectrometry was used to identify DNAH7 as a protein absent in PCD cilia. The full-length DNAH7 cDNA was cloned and shares 68% similarity with an inner arm dynein heavy chain from Drosophila. DNAH7 was induced during ciliated cell differentiation, and immunohistochemistry demonstrated the presence of DNAH7 in normal cilia. In cilia from PCD cells, DNAH7 was undetectable, whereas intracellular DNAH7 was clearly present. These studies identify DNAH7 as an inner arm component of human cilia that is synthesized but not assembled in a case of PCD.
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Background Primary ciliary dyskinesia (PCD) is characterised by an imbalance in mucociliary clearance leading to chronic respiratory infections. Cilia length is considered to be a contributing factor in cilia movement. Recently, IFT46 protein has been related to cilia length. Therefore, this work aims to study IFT46 expression in a PCD patients cohort and analyse its relationship with cilia length and function, as it was not previously described. Materials and methods The expression of one intraflagellar transport ( IFT46) and two regulating ciliary architecture ( FOXJ1 and DNAI2) genes, as well as cilia length of 27 PCD patients, were measured. PCD patients were diagnosed based on clinical data, and cilia function and ultrastructure. Gene expression was estimated by real-time RT-PCR and cilia length by electron microscopy in nasal epithelium biopsies. Results and conclusions: While IFT46 expression was only diminished in patients with short cilia, FOXJ1, and DNAI2 expression were reduced in all PCD patient groups compared to controls levels. Among the PCD patients, cilia were short in 44% (5.9 ± 0.70 µm); nine of these (33% from the total) patients’ cilia also had an abnormal ultrastructure. Cilia length was normal in 33% of patients (6.4 ± 0.39 µm), and only three patients’ biopsies indicated decreased expression of dynein.
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A child presenting with Mainzer-Saldino syndrome (MZSDS), characterized by renal, retinal and skeletal involvements, was also diagnosed with lung infections and airway ciliary dyskinesia. These manifestations suggested dysfunction of both primary and motile cilia, respectively. Targeted exome sequencing identified biallelic mutations in WDR19, encoding an IFT-A subunit previously associated with MZSDS-related chondrodysplasia, Jeune asphyxiating thoracic dysplasia and cranioectodermal dysplasia, linked to primary cilia dysfunction, and in TEKT1 which encodes tektin-1 an uncharacterized member of the tektin family, mutations of which may cause ciliary dyskinesia. Tektin-1 localizes at the centrosome in cycling cells, at basal bodies of both primary and motile cilia and to the axoneme of motile cilia in airway cells. The identified mutations impaired these localizations. In addition, airway cells from the affected individual showed severe motility defects without major ultrastructural changes. Knockdown of tekt1 in zebrafish resulted in phenotypes consistent with a function for tektin-1 in ciliary motility, which was confirmed by live imaging. Finally, experiments in the zebrafish also revealed a synergistic effect of tekt1 and wdr19. Altogether, our data show genetic interactions between WDR19 and TEKT1 likely contributing to the overall clinical phenotype observed in the affected individual and provide strong evidence for TEKT1 as a new candidate gene for primary ciliary dyskinesia.
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Abstract Primary ciliary dyskinesia (PCD) is a genetic disease of the motile cilia, and is part of a rapidly expanding collection of disorders collectively known as ciliopathies. Our understanding of the complex genetics and functional phenotypes of these conditions has rapidly advanced over the past decade. A growing number of cilia‐related genes and mutations have been identified, which segregate into genes that encode axonemal motor proteins, structural and regulatory proteins within the cilium, as well as an emerging class of cytoplasmic proteins involved in ciliary assembly. These findings have yielded unexpected insights into the processes involved in the assembly, structure and function of a cilium, which will allow us to better understand the clinical heterogeneity of disease. Moreover, these discoveries have the potential to revolutionise testing for PCD, and lead to earlier diagnosis and treatment of affected individuals. Key Concepts: Cilia are complex organelles that are on the surface of many cell types and involved in diverse cellular functions. Cilia are broadly classified as motile (motor) or primary (sensory), and the latter are important signalling organelles that sense the extracellular environment. Motile cilia are critical for the intrinsic defence of the respiratory tract, including the middle ear, paranasal sinuses, and conducting airways. Primary ciliary dyskinesia is an inherited disease that is characterised by impaired ciliary function and leads to diverse clinical manifestations, including chronic sinopulmonary disease, persistent middle ear effusions, laterality defects and infertility. Mutations in different primary ciliary dyskinesia‐associated genes that encode proteins involved in ciliary assembly, structure and function can produce similar clinical phenotypes but different ultrastructural defects.
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