Thesis (M.S.)--Rochester Institute of Technology, 2009. / Typescript. Includes bibliographical references (leaves 132-134).

No description available.

Primary cilia are non-motile organelles having 9+0 microtubules that project from the basal body of the cell. While the main purpose of motile cilia in mammalian cells is to move fluid or mucus over the cell surface, the purpose of primary cilia has remained elusive for the most part. Primary cilia are shortened in the kidney tubules of Tg737orpk mice, which have polycystic kidney disease due to ciliary defects. The product of the Tg737 gene is polaris, which is directly involved in a microtubule-dependent transport process called intraflagellar transport (IFT). In order to determine the importance of polaris in the development of neuronal cilia, cilium length and numerical density of cilia were quantitatively assessed in six different brain regions on postnatal days 14 and 31 in Tg737orpk mutant and wildtype mice. Our results indicate that the polaris mutation leads to shortening of cilia as well as decreased percentage of ciliated neurons in all brain regions that were quantitatively assessed. Maintainance of cilia was especially affected in the ventromedial nucleus of the hypothalamus. Furthermore, the polaris mutation curtailed cilium length more severely on postnatal day 31 than postnatal day 14. These data suggests that even after ciliogenesis, intraflagellar transport is necessary in order to maintain neuronal cilia. Regional heterogeneity in the effect of this gene mutation on neuronal cilia suggests that the functions of some brain regions might be more compromised than others.

No description available.

Cilia are small microtubule based structures found on the surface of almost all mammalian cells, enclosed in a highly specialised extension of the cell membrane. Components of several key developmental signalling pathways, in particular Hedgehog (Hh) signalling, are enriched in cilia and cells with mutations in cilia structure show aberrant signalling, suggesting cilia act as “antennae” to focus these signalling cascades. A spectrum of human diseases, termed ciliopathies, are caused by problems in cilia formation or cilia function, which display wide ranging phenotypes from embryonic lethality to retinal degeneration, polydactyly to cystic kidneys. Despite recent advances in the understanding of the essential roles cilia play in mammalian development, exactly how these complex structures are put together, how they carry out their diverse functions, and how they are regulated is not well understood. In this thesis, I describe a screen for genes involved in cilia formation and function. While optimising ciliogenesis and immunofluorescence protocols for the screen, the phenotypes of two ciliary mutant cell lines were analysed. Wdr35yet/yet and Dync2h1pol/pol mouse lines were identified in an ENU screen for genes involved in early development, and shown to have gross phenotypes similar to other ciliary mutants (Mill et al. 2011). Intraflagellar transport (IFT) is the active transport of proteins up and down the ciliary axoneme. Dync2h1 is a retrograde IFT motor component, whereas Wdr35 is part of the retrograde IFT-A complex. In this thesis, the cellular phenotypes of mouse embryonic fibroblasts derived from these mutants are described, showing that despite the fact both genes are thought to be involved in retrograde IFT, they show distinct ciliary phenotypes, suggesting novel roles for Wdr35 in mouse ciliogenesis. An siRNA screen was carried out in mouse fibroblasts to identify genes involved in (i) cilia formation, assayed by immunofluorescence for ciliary markers, and (ii) cilia function, assayed by activity of a Hh responsive luciferase transgene as an indirect readout of ciliary function. Although scalable, I initially screened a small test set of thirty-six putative cilia candidates, identified by cross species transcriptomic analysis. We identified several possible hits, many of which were in the ciliome database but also importantly, several genes with no known link to ciliogenesis. Repeats, correlation of phenotype to knockdown efficiencies and localisation studies validated two hits, Ccdc63 and Azi1. Ccdc63 is a novel coiled-coil gene with no previous link to ciliogenesis; the phenotype for this gene was analysed in real time using fluorescently tagged ciliary markers. A second hit, Azi1, was followed up in more detail. The reduction in ciliogenesis upon Azi1 knockdown was confirmed with separate siRNAs, and was rescued by overexpressing siRNA insensitive Azi1-GFP, confirming the phenotype is not due to off-target effects of the siRNAs. Azi1 gene trap mutant mice were generated and confirmed to be null mutations. Surprisingly, the mice survive, showing Azi1 is not essential for mammalian ciliogenesis. However, mutant males are infertile, with highly reduced sperm count and sperm abnormalities indicative of an arrest at Stage IX of spermiogenesis, when the flagellum, a highly specialised motile cilium, forms. The small number of sperm that do get to the epididymus are immotile. We suggest Azi1 is essential to in the formation of the sperm flagella and male fertility.

Meckel-Gruber syndrome (MKS) is a recessive genetic disease that is uniformly lethal in affected children due to resultant developmental defects in the kidney and brain. 13 MKS genes have been identified, and further candidate genes have been linked to this disease, all encoding unrelated proteins. Their role is believed to be in generation and compartmentalisation of the primary cilium, a microtubule-based organelle that functions in signal transduction of developmentally-crucial pathways. However, recent evidence indicates that these proteins are also likely involved in regulation of the actin cytoskeleton. Furthermore, research is beginning to uncover roles of other ciliopathy proteins in regulation of additional subcellular structures, such as the microtubule cytoskeleton, focal adhesions and the Golgi. To begin to understand the roles of the MKS proteins beyond the cilium, I examined a number of cellular features of patient fibroblasts carrying mutations in TMEM216 (MKS2) and TMEM67 (MKS3). In this thesis, I describe the temporal appearance and nature of prominent actin bundles observed in these cells, and analyse the dependency of these on the Rho/ROCK signalling pathway. Furthermore, I identify novel alterations to the microtubule cytoskeleton and organisation of the Golgi complex in MKS patient cells, and subsequently establish a temporal order of these phenotypes, demonstrating microtubule defects as the first to occur in these cells. Finally, I connect these phenotypic defects to Rho/ROCK signalling. In contrast to the prevailing view in the ciliopathy field, I believe that a diffusion barrier at the transition zone is not the primary role of MKS proteins. Instead I propose, supported by these data, that MKS protein complexes play a dual role as effectors of Rho signalling in addition to performing a structural role with particular importance in tethering the cytoskeleton to membranes. I therefore conclude that these, and other ciliopathy protein complexes, may act as important signal transduction and structural components at multiple locations throughout the cell.

Drosophila provide an excellent model organism in which to study cilia as there are only two ciliated cell types; the sensory neurons and sperm cells. The chordotonal neuron is one such ciliated cell and is required for hearing, proprioception and gravitaxis. Mechanical manipulation of the cilium that extends from the neuronal dendrite is required for signal transduction. Chordotonal neuronal differentiation is regulated by a transcription factor cascade. Atonal begins the cascade, which is then continued by RFX and Fd3F for ciliary genes (Cachero et al 2011, Newton et al 2012). Genes expressed in developing chordotonal neurons are downstream of these transcription factors and their characterisation can further elucidate how neuronal differentiation is regulated. Ciliary genes are highly enriched in developing chordotonal cells; uncharacterised genes enriched in these cells can therefore be considered candidate ciliary genes (Cachero et al 2011). A behavioural assay was conducted to identify further genes that could have a role in ciliary formation and function. Candidate genes were identified by combining enrichment data with previous genomic, proteomic and transcriptomic studies of cilia. A climbing assay of RNAi mediated knock down of these genes identified a number of candidates for future work. One gene found to be highly enriched in developing chordotonal neurons is CG11253. CG11253EY10866 P element insertion mutant flies show a mild uncoordinated phenotype in a climbing assay consistent with reduced chordotonal organ function. Male flies are also infertile due to a lack of motile sperm. CG11253 is expressed in motile ciliated cells and is conserved in organisms with motile cilia. CG11253 expression is also regulated by RFX and Fd3F, suggesting that it is involved in cilium motility. This was confirmed by electron microscopy, which showed disruption of axonemal dynein arm localisation in chordotonal cilia and sperm flagella. A CG11253::mVenus fusion protein was found to localise mainly to the cytoplasm and to a lesser extent the cilia of chordotonal neurons. Patients with symptoms consistent with Primary Ciliary Dyskinesia (PCD), a condition caused by cilium immotility, have subsequently been found to have point mutations in ZMYND10, the human homologue of CG11253. The identification of PCD patients with ZMYND10 mutations showed that investigating cilium motility in Drosophila chordotonal neurons could identify novel PCD genes. It was thought that investigating previously uncharacterised targets of Fd3F could identify novel genes involved in cilium motility and thus candidate PCD genes. CG31320 is a gene regulated by RFX and Fd3F and conserved in organisms with motile cilia. RNAi mediated knock down of CG31320 resulted in both a mild uncoordinated phenotype and male infertility due to a lack of motile sperm. Electron microscopy showed a complete loss of axonemal dynein arms in chordotonal neuron cilia. An mVenus fusion protein of CG6971, an inner dynein arm component, was also mislocalised from the cilia in CG3132027 deletion mutant larvae. This shows that CG31320 is required for the appropriate localisation of the axonemal dynein arms and thus cilium motility. This further showed that uncharacterised genes enriched in chordotonal neurons and regulated by Fd3F could be novel ciliary genes required for cilium motility. Our collaborators and Horani et al (2012) showed that the human homologue of CG31320 (HEATR2) is mutated in patients with PCD, further confirming that this method can be used to identify PCD genes. I have identified two factors required for cilium motility. Disruption of the axonemal dynein arms in both cases results in reduced coordination, and lack of fertility due to sperm immotility. Mutations in the human homologues of these genes have been found to result in PCD. This indicates that further PCD genes could be identified from genes enriched in Drosophila chordotonal neurons that are regulated by Fd3F.

During normal activity, tendons are subjected to dynamic tensile strains of approximately 1-10%, whilst mechanical overload can lead to damage and degradation and the development of tendinopathy. The tenocytes within tendon respond to this mechanical environment although the mechanisms are poorly understood. Primary cilia consist of a slender axoneme composed of acetylated α-tubulin and are known to regulate a variety of signalling pathways including mechanosignalling. In various cell types, mechanical loading also influences primary cilia length. However relatively little is known about tendon primary cilia structure and function. This thesis set out to examine the structure and organisation of primary cilia in tendon cells and the effect of mechanical loading, both in situ and in isolated cells cultured in monolayer. Studies analysed cilia expression using confocal immunofluorescence microscopy in tendon fascicles from rat tail and isolated human tenocytes. Results demonstrated that the prevalence and orientation of primary cilia was different in the fascicular matrix (FM) and interfascicular matrix (IFM) regions of the tendon. Stress deprivation caused differential cilia elongation between the FM and IFM, associated with disruption of the surrounding extracellular matrix and alterations in tissue biomechanics. In isolated tenocytes, primary cilia were significantly longer with a greater prevalence than in situ. Cyclic tensile loading applied using the Flexcell system resulted in cilia disassembly within 8 hours with a dramatic reduction in prevalence and length. This effect was completely reversible on removal of strain. A similar response was observed in situ within both FM and IFM regions of the tendon. This mechanically-induced cilia disassembly was shown to be mediated, at least in part, by the release of TGFβ and activation of HDAC6 which causes tubulin deacetylation. These results in this thesis suggest a novel feedback mechanism through which physiological and pathological mechanical loading may regulate primary cilia signalling.

oligodendrocytes migrate from the corpus callosum into the overlying cortex. The incidence of cilia did not change markedly across age groups, and did not vary consistently with the number of processes per cell, which was used as an indication of the maturation stage of OPCs and young OLs. The mean percent of Olig1 immunopositive (Olig1+) cells having cilia across ages was 33.1% + 16.5%, with all ages combined. In O4+ cells of these mice, 56.7 + 3.6% had primary cilia. If it is the case that adult OLs do not have cilia, the point in the lineage when primary cilia are lost is still unknown. Adult mice that had been injected with cyclopamine to block cilia-dependent Shh signaling were examined to determine whether the rate of generating new OPCs was influenced. In the CC of control mice, the numerical density of Olig1+/BrdU+ cells was 1.29 + 0.07/mm2 was reduced to 0.68 + 0.38/mm2 in the cyclopamine-injected group, and the numerical density of all BrdU+ cells (including both Olig1+ and Olig1- cells) of 4.55 + 1.50/mm2 in the control group was reduced to 3.14 + 1.27/mm2 in the cyclopamine-injected group. However, there were only 2 mice in each group and the differences were not statistically significant.

No description available.