Thesis (M.Res.(Med.))--University of Hong Kong, 2007.

Thesis (M.S.)--Ohio State University, 2009. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 41-46).

Thesis (Ph.D.)--University of Melbourne, Dept. of Paediatrics, The Bruce Lefroy Centre for Genetic Health Research, The Murdoch Childrens Research Institute, 2009. / Typescript. Includes bibliographical references (leaves 183-207)

Primary cilia are cellular organelles that protrude into the extracellular space, acting as antennas. They detect a wide range of chemical cues, including SHH and PDGF, as well as fluid flow, and they modulate downstream signalling systems, such as WNT and ERK. Due to this cue-sensing ability and the close association of the primary cilium with the centrosome the organelle is able to influence both cell cycle progression and cell migration. This work investigated the effect of mutations on two genes associated with primary cilia: Arl13b and Ift172. The effects of the HNN genotype of Arl13b and the WIM genotype of Ift172 on cell migration were assessed uniquely within the context of direct current electric fields. Both cell lines showed a decreased migratory response when compared to WT cells, despite no clear involvement of cilia in sensing the direction of the electric field. This corroborated with previous data of in vivo Arl13b cellular migration. Through the use of in utero electroporation the migratory deficits of IFT172 knock down were then confirmed in vivo in the developing mouse neocortex. Further in vitro investigation revealed a slower proliferation rate of HNN and WIM cells, though this was not confirmed in vivo after IFT172 knock down using a standard BrDU protocol. Nevertheless, further in vitro investigations revealed a wide variety of cell cycle and intracellular changes within both cell lines. The commonalities included lower numbers of cells in the S-phase and lower MAPK3 phosphorylation compared to WT, and differences such as GSK3β phosphorylation on Ser9. This work showed for the first time that ciliopathies affect galvanotaxis, and revealed fundamental commonalities in cell migration and proliferation between various ciliary mutations, as well as differences in specific signalling pathways. This will hopefully aid in developing future therapeutic interventions for ciliary diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / Obesity is a growing pandemic that claims close to three hundred thousand lives per year in the United States alone. Despite strong interest and investment in potential treatments, obesity remains a complex and challenging disorder. In the study of obesity, mouse models have been excellent tools that help in understanding the function of different genes that contribute to this disease of energy homeostasis. However, it was surprising when disfunction in primary cilia was found to be linked to syndromic obesity. To understand the role of primary cilia in obesity, a growing subset of GPCRs have been identified to selectively localize to the organelle. Several of which have known roles in energy homeostasis. In some examples, ciliary GPCRs appear to dynamically localize to the organelle; such is the case of GPR161 and smoothened in the hedgehog signaling pathway. Thus, we were interested to see if other GPCRs dynamically localize to the primary cilia as part of their regulation of energy homeostasis. For example, the GPCR MCHR1 selectively localizes to the cilia and is involved in energy homeostasis. Although much is known about the expression of the receptor in the brain, how its ciliary subcellular localization impacts its roles in energy homeostasis is unknown. Observing neuronal cilia in vivo remains a difficult task as some of the available tools such as tagged alleles rely on overexpression of ciliary protein which may impact function. Additionally, most of the work is done in vitro, leaving much to be discovered about neuronal cilia in vivo. In this thesis, we show that using a newly constructed reporter allele mCherryMCHR1, we can see ciliary expression of MCHR1 in the brain of developing and adult mice; more specifically in the ARC and PVN. Subsequently, using a novel Artificial intelligence analysis approach, we measured the length and composition of MCHR1 positive cilia under physiological conditions associated with MCHR1 function. Although in this work we are reporting no changes in dynamic localization of MCHR1 in the hypothalamus specifically, we are not excluding the potential for changes in other regions of the brain or under other conditions; and we are suggesting that pharmacological approaches may help highlight potential ciliary GPCR dynamic localization.

Mammalian skin is a dynamic organ that constantly undergoes self-renewal during homeostasis and regenerates in response to injury. Crucial for the skin’s self-renewal and regenerative capabilities is the epidermis and its stem cell populations. Here we have interrogated the role of primary cilia and Intraflagellar Transport 20 (Ift20) in epidermal development as well as during homeostasis and wound healing in postnatal, adult skin. Using a transgenic mouse model with fluorescent markers for primary cilia and basal bodies, we characterized epidermal primary cilia during embryonic development as well as in postnatal and adult skin and find that both the Interfollicular Epidermis (IFE) and hair follicles (HFs) are highly ciliated throughout development as well as in postnatal and adult skin. Leveraging this transgenic mouse, we also developed a technique for live imaging of epidermal primary cilia in ex vivo mouse embryos and discovered that epidermal primary cilia undergo ectocytosis, a ciliary mechanism previously only observed in vitro. We also generated a mouse model for targeted ablation of Ift20 in the hair follicle stem cells (HF-SCs) of adult mice. We find that loss of Ift20 in HF-SCs inhibits ciliogenesis, as expected, but strikingly it also inhibits hair regrowth. Closer examination of these mice reveals that Ift20 is crucial in maintaining HF-SC identity. Specifically, ablation of Ift20 in HF-SCs results in loss of SOX9 expression in HF-SCs and results in ectopic expression of the IFE marker KLF5 in HF-SCs. Additionally, ectopic differentiation is observed in HF-SCs following loss of Ift20. Finally, using both in vitro and in vivo models, we also characterize the role of primary cilia and Ift20 in epidermal wound healing. We find that loss of Ift20 slows collective keratinocyte migration in vitro and also slows HF-SC migration in vivo during wound repair. Interestingly our data suggests that Ift20 regulates keratinocyte migration in a primary cilia-independent manner. Instead, we find that Ift20 mediates focal adhesion (FA) turnover during keratinocyte migration. Specifically, Ift20 together with Rab5, regulates recycling of FA integrins and loss of Ift20 inhibits proper return of integrins to the keratinocyte surface. Overall, we demonstrate that the epidermis is highly ciliated throughout development and in postnatal skin. We show that Ift20 is crucial in maintaining HF-SC identity and the telogen to anagen transition in HFs. We finally demonstrate that Ift20 regulates keratinocyte migration independent of its function in ciliogenesis and instead regulates recycling of FA integrins through a Rab5 dependent mechanism.

Adult female cats (2.4-2.8 kg) underwent surgical removal of the left ciliary ganglion under pentobarbital anesthesia. Twenty-one, 560 and 616 days later, pupil size of both left and right (control) eyes was measured in response to progressively increasing doses of carbachol applied topically. By 21 days after denervation, ganglionectomized eyes showed marked supersensitivity to the miotic effects of pilocarpine and carbachol. By 560 days, however, responsiveness of the denervated eyes to lower and intermediate doses of carbachol was the same as that of contralateral control eyes, while responsiveness to higher doses was significantly reduced. Responsiveness to both lower and higher doses of carbachol was significantly less than that of the controls on the 616th day. Ganglionectomized eyes showed no pupillary response to light 14, 562, or 620 days after surgery. Histochemical analysis of iris tissue collected from eyes of these cats 720 days after ganglion removal revealed an almost complete absence of acetylcholinesterase-containing nerve fibers on the denervated side. These findings indicate that the return to normal or lower sensitivity to carbachol of denervated eyes at prolonged periods after ciliary ganglion removal is not due to significant cholinergic reinnervation of the iris sphincter muscle.

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