acase@tulane.edu / 1 / Forest Mannan

Proneural proteins are transcription factors of the bHLH family and have a conserved role in directing neurogenesis from invertebrate to mammals. In Drosophila, proneural proteins are required for early developmental specification of precursor cells of sense organs (SOPs). Despite considerable progress having been made in this field, it remains unknown how proneural proteins organise the well-orchestrated process that facilitates each type of SOP to acquire both generic neuronal properties and individual neuronal subtype identity during the progression from specification to differentiation. To approach this question, we investigate the gene regulatory network by proneural protein Ato by means of the microarray analysis. Ato directs the formation of the Drosophila chordotonal organs (Ch), important proprioceptive sense organs (Jarman et al., 1993b). The microarray study generated a list of candidate Ato target genes (Cachero et al., 2011). My PhD project entails the characterisation of two potential Ato target genes arising from this screen: Rfx and dila. To determine their positions in the gene regulatory network, I analysed the regulation and function of these genes. First, I demonstrated that both Rfx and dila are activated during Ch neurogenesis as direct targets of Ato. This was established by characterising their expression patterns, cis-regulation analyses and identifying the potential Ato binding sites by site-directed mutagenesis. RFX is a well-known ciliogenic regulator (Dubruille et al., 2002; El Zein et al., 2009; Swoboda et al., 2000), and its activation by Ato is consistent with Ch neurons having ciliated dendrites. However, the role of dila was completely unknown, but its sequence suggested that it may be involved in neuronal differentiation rather than gene regulation. I generated several dila mutant alleles and demonstrated that dila mutants exhibit severe uncoordination, due to a series of defects in ciliated neurons. These defects were linked to a disruption in the ciliogenesis machinery, particularly in the process known as intraflagellar transport (IFT). dila mutants also display reduced male fertility because of aberrant basal body function, which leads to a disorder in sperm individualisation. Thus DILA is required for the differentiation of all ciliated cells in Drosophila. Visualisation of tagged protein localised DILA to the basal body and transition zone of the sensory cilia. Further analysis revealed the genetic interaction between DILA and UNC (another basal body protein) during ciliogenesis. Taken together I propose that DILA regulates IFT at the base of the cilia in collaboration with UNC. Given that dila is an evolutionarily conserved gene, dila homologues could be candidate genes for human ciliopathies. Rfx is essential for ciliogenesis in both Ch and the external sense (ES) organs, which have distinctive cilia. Despite of this common role of RFX, I discovered that Rfx is expressed differently in Ch and ES lineages, which led me to hypothesise that the difference in Rfx expression modulates ciliogenesis in these two lineages. I obtained preliminary data that support this hypothesis. Overall, my study demonstrates important links between Ato and the regulation of ciliogenesis, which is an important process in Ch neuron differentiation. The data support a model in which Ato controls ciliogenesis both directly (e.g. via activating a ciliary genes like dila) and indirectly (e.g. via regulating the transcriptional factors essential for ciliogenesis, like RFX).

Primary cilia are essential in brain development, as mediators of sonic hedgehog signaling. However, their role in mature neurons remains elusive. One means to elucidate their function may be to investigate the function of the somatostatin type 3 receptor (SstR3), which is concentrated on the primary cilia of neurons. The inhibitory and anticonvulsant properties of somatostatin suggest that ciliary SstR3 might protect neurons against excitotoxicity, as seen in epileptic seizures. C57BL/6 wild type (wt) and SstR3 knockout mice were administered vehicle or epileptogenic agents kainic acid (KA) or pentylenetetrazole. Seizure behaviors were rated on seizure severity scales. KA-induced seizure behaviors were more severe in SstR3 mutants than in wt. Correspondingly, the mutants showed greater reactive gliosis, as indicated by increased numbers of GFAP immunoreactive (GFAP(+)) astrocyte processes. In addition, seizure severity was associated with a greater percentage of neural stem cells having an ACIII(+) cilium. Following injections of pentylenetetrazole, SstR3 mutants reached maximum seizure levels faster than wt. These results support the hypothesis that ciliary SstR3 are neuroprotective in mature neurons, and may provide a new avenue for the treatment of seizures.

In this thesis we developed a new mechanics model of the primary cilium and analyzed its bending behavior. The primary cilium that extends from the cell surface can detect the mechanical signals of the surrounding environment. Moreover, through its deflection and bending angle, the primary cilium can communicate with the cell regarding the extracellular. Scientists have shown that dysfunction of primary cilia can lead to many diseases as cilia are believed to play an important role in transmitting signals in cells. A good model of primary cilium can aid in the understanding of the mechanism of its bending movement. Furthermore, a good model is important for determining how the primary cilium contributes to convert mechanical signals into biochemical ones. Previous models have ignored the basal body and transition fiber that are located at the base of the primary cilium. However, it is clear that the elastic basal body and transition fibers should have a significant effect on the deformation of the whole structure. Aiming to address this issue, we established a model with a rotational spring representing the confinement induced by the basal body and transition fibers. Specially, we developed two governing equations for two different conditions, namely uniformly distributed load and spatially varying load. In addition, this model is valid for situations where the deflection is large. To obtain the results the shooting and Newton-Raphson methods are used to solve the governing equations numerically. Then, we compared the numerical results with experimental data to test the validity of the model. Comparison between our model predictions and experimental data showed that the governing equation for spatially varying load described the bending behavior of the primary cilium very well under various realistic conditions, including cases where the flow field is not uniform both spatially and temporally fluid flow with variable velocity. / published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

Thesis supervisor: Charles Yeaman. Includes bibliographic references (p. 94-100).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1981. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis--University of Wisconsin--Madison. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 152-161).

Thesis (M.S.)--University of Wisconsin--Madison. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 44-47).

Cilia are microtubule-based organelles that project into the extracellular space and have various functions including transducing sensory information, regulating developmental signalling pathways, and generating directed fluid flow, making them important regulators of vertebrate development and homeostasis. Despite their importance, there are many aspects of cilia formation and function that remain poorly understood. The planar cell polarity (PCP) pathway is a branch of Wnt signalling that provides positional information to cells and is required for polarized morphogenic cell movements. Previous studies of PCP effector proteins suggested that PCP signalling was required for cilia formation. However, these proteins are not specific to the PCP pathway and are shared with other branches of Wnt signalling. To determine the role of a core and specific PCP regulator on ciliogenesis, I examined maternal-zygotic (MZ) vangl2 zebrafish mutants using an in vivo marker of cilia, Arl13b-GFP. Analysis of MZvangl2 mutants revealed that PCP is not required for cilia formation but is required for the posterior tilting and posterior positioning of motile cilia, essential for directed fluid flow. A parallel branch of studies suggested that cilia are actually required to regulate PCP signalling because defects in PCP-mediated morphogenic movements were observed with the knockdown of certain proteins that localize at or near cilia or basal bodies. To determine whether cilia were required to establish PCP, I generated MZ-intraflagellar transport-88 (IFT88) mutants, where ciliogenesis is completely abolished. Analysis of MZift88 mutants revealed that cilia are not directly required for PCP-mediated morphogenic movements. However, I observed that MZift88 mutants had defects in oriented cell divisions (OCD) occurring during gastrulation. Remarkably, these divisions occur prior to cilia formation, suggesting a cilia-independent role for IFT proteins in cell divisions, which may have important consequences on the interpretation of the role of cilia in disease.

Cilia are microtubule-based organelles that project into the extracellular space and have various functions including transducing sensory information, regulating developmental signalling pathways, and generating directed fluid flow, making them important regulators of vertebrate development and homeostasis. Despite their importance, there are many aspects of cilia formation and function that remain poorly understood. The planar cell polarity (PCP) pathway is a branch of Wnt signalling that provides positional information to cells and is required for polarized morphogenic cell movements. Previous studies of PCP effector proteins suggested that PCP signalling was required for cilia formation. However, these proteins are not specific to the PCP pathway and are shared with other branches of Wnt signalling. To determine the role of a core and specific PCP regulator on ciliogenesis, I examined maternal-zygotic (MZ) vangl2 zebrafish mutants using an in vivo marker of cilia, Arl13b-GFP. Analysis of MZvangl2 mutants revealed that PCP is not required for cilia formation but is required for the posterior tilting and posterior positioning of motile cilia, essential for directed fluid flow. A parallel branch of studies suggested that cilia are actually required to regulate PCP signalling because defects in PCP-mediated morphogenic movements were observed with the knockdown of certain proteins that localize at or near cilia or basal bodies. To determine whether cilia were required to establish PCP, I generated MZ-intraflagellar transport-88 (IFT88) mutants, where ciliogenesis is completely abolished. Analysis of MZift88 mutants revealed that cilia are not directly required for PCP-mediated morphogenic movements. However, I observed that MZift88 mutants had defects in oriented cell divisions (OCD) occurring during gastrulation. Remarkably, these divisions occur prior to cilia formation, suggesting a cilia-independent role for IFT proteins in cell divisions, which may have important consequences on the interpretation of the role of cilia in disease.