Clinical management of esophageal adenocarcinoma (EAC) relies on radiation therapy, yet radioresistance is a pervasive challenge in this disease. The mechanisms of EAC radioresistance remain largely unknown due to a paucity of validated preclinical models. The present studies report on the development of seven primary xenograft models established from patient tumours. These models are used to interrogate the range of radiosensitivities and mechanisms of radioresistance in EAC tumours. We found that radiation enriches the tumour-initiating cell population in two xenograft lines tested. Furthermore, three tested xenograft lines respond to irradiation by upregulating Hedgehog transcripts, a pathway involved in stem cell maintenance and proliferation. Upregulation occurs in autocrine and paracrine patterns simultaneously, suggesting that Hedgehog signalling may have a complex role in the radioresponse of EAC tumours. These findings suggest that inhibiting stem cell pathways in combination with radiotherapy may have an important role in the clinical management of EAC.

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In vivo organogenesis is based on the temporal-spatial developmental processes that depend on cell behaviour, for example on their growth, migration, differentiation and intercellular interactions. Such behaviour is regulated by appropriate transient expression of various signalling molecules. Despite the significant advances in therapeutic strategies, the secret of the development of the biological replacement of a damaged or missing tooth has not yet been revealed. In this context, animal models provide a powerful tool for studying tooth normogenesis and pathogenesis in both basic and applied research. Early development of the tooth shares similar morphological and molecular features with other ectodermal organs. At the same time, these features are largely preserved also between species, which is advantageous for the use of model organisms. The dental formula of both: the human and the mouse are reduced against a common ancestor, but both groups of organisms evince simple as well as multicusped teeth. In both, structures called rudimentary were found. These structures are suppressed during ontogenetic development and generally they are not attributed to essential functions. That is why we aimed to study dental rudiments in detail and reveal their function in odontogenesis. This work presents new...

Indiana University-Purdue University Indianapolis (IUPUI) / Primary cilia are solitary cellular appendages that function as signaling centers for cells in adult energy homeostasis. Here in chapter 1, I introduce cilia and how dysfunction of these conserved organelles results in ciliopathies, such as Bardet-Biedl Syndrome (BBS), which present with childhood obesity. Furthermore, conditional loss of primary cilia from neurons in the hypothalamus leads to hyperphagia and obesity in mouse models of ciliopathies. Classically, cilia coordinate signaling often through specific G-protein coupled receptors (GPCRs) as is the case in both vision and olfaction. In addition, neurons throughout the brain including hypothalamic neurons possess primary cilia whose dysfunction contributes to ciliopathy-associated obesity. How neuronal cilia regulate the signaling of GPCRs remains unclear and many fundamental cell biology questions remain about cilia mediated signaling. For example, how cilia coordinate signaling to influence neuronal activity is unknown. To begin to address some of these cell biology questions around neuronal cilia, chapter 2, describes the development and use of a system for primary neuronal cultures from the hypothalamus. Using this system, we found that activation of the cilia regulated hedgehog pathway, which is critical in development, influenced the ability of neurons to respond to GPCR ligands. This result highlights the role of the developmentally critical hedgehog pathway on terminally differentiated hypothalamic neurons. One challenge facing the cilia field is our ability to assess cilia in large numbers without potential bias. This is especially true in tissues like the brain, where cilia appear to have region-specific characteristics. Work included in Chapter 3 describes the use of a computer-assisted artificial intelligence (Ai) approach to analyze cilia composition and morphology in a less biased and high throughput manner. Cilia length and intensities are important parameters for evaluation of cilia signaling. Evidence suggests that activation of some ciliary GPCRs results in shortening of cilia whereas deviations from normal cilia length in mutant phenotypes affects normal physiological processes such as decreased mucociliary clearance. Therefore, to analyze a large number of cilia, we describe the use of the Ai module from in vitro and in vivo samples in a reproducible manner that minimizes user bias. Using this approach, we identified that Mchr1 expression is significantly stronger in the cilia of paraventricular nucleus than that in the arcuate nucleus of adult mice. Work in Chapter 4 continues to explore the integration between hedgehog pathway and ciliary GPCR signaling in the central nervous system, and its relevance with energy homeostasis. We evaluated the hedgehog ligand in the plasma of mice in acute and long-term metabolic changes and identified that the activity of the ligand changed under altered metabolic conditions. We also developed a genetic mouse model where hedgehog signaling was constitutively active in neuronal cilia. These mice become hyperphagic and obese. These results further emphasize the potential role of the hedgehog signaling pathway in regulation of feeding behavior in adult vertebrates. Overall, results from this work will provide a better understanding of the defects not only underlying ciliopathy-associated obesity but may also reveal more common mechanisms of centrally mediated obesity. In addition, the tools I have developed will help in understanding how neuronal cilia are used for intercellular communications and ultimately how they regulate behaviors like feeding.

Glial cells maintain homeostasis that is essential to neuronal function. Injury to the nervous system leads to the activation and proliferation of glial cells and pericytes, which helps to wall off the damaged region and restore homeostatic conditions. Sonic hedgehog is a mitogen which is implicated in injury-induced proliferation of glial cells and pericytes. The mitogenic effects of sonic hedgehog require primary cilia, but the few reports on glial or pericyte primary cilia do not agree about their abundance and did not address effects of injury on these cilia. Primary cilia are microtubule-based organelles that arise from the centrosome and are retracted before cells divide. Depending on cell type, proteins concentrated in cilia can transduce several mitotic, chemosensory, or mechanosensory stimuli. The present study investigated effects of stab wound injury on the incidence and length of glial and pericyte primary cilia in the area adjacent to the injury core. Astrocytes, polydendrocytes and pericytes were classified by immunohistochemistry based on cell-type markers. In normal adult mice, Arl13b immunoreactive primary cilia were present in a majority of each cell type examined: astrocytes, 98±2%; polydendrocytes, 87±6%; and pericytes, 79±13% (mean ± SEM). Three days post-injury, cilium incidence decreased by 24% in astrocytes (p< 0.008) and 41% in polydendrocytes (p< 0.002), but there was no significant effect in pericytes. Polydendrocytes labeled with the cell cycle marker Ki67 were less likely to have cilia compared to resting, Ki67- polydendrocytes. Considering post-injury rates of proliferation for astrocytes and polydendrocytes, it appears that resorption of cilia due to cell cycle entry may account for much of the loss of cilia in polydendrocytes but was not sufficient to account for the loss of cilia in astrocytes. Under normal conditions, astrocytes rarely divide, and they maintain non-overlapping territories. However, three days after injury, there was a 7-fold increase in the number of paired mirror-image astrocytes (p< 0.018), which are most likely daughter cells from astrocytes that recently divided. Cilia incidence tended to decrease in these pairs compared to single astrocytes (p< 0.057) in injured mice. This is the first systematic investigation of cilia of astrocytes, polydendrocytes, and pericytes in the brain. Moreover, the examination of effects of brain injury on cilia adds to the understanding of injury-induced proliferation in these cells.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21257号 / 医博第4375号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 増永 慎一郎, 教授 妹尾 浩, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The Hedgehog (Hh) signaling pathway plays many important roles throughout embryonic development, including the regulation of tissue patterning, cell differentiation, proliferation, and apoptosis. The loss of SHH signaling in human development has been shown to cause holoprosencephaly. Conversely, inappropriately activated Shh signaling in adults has been implicated in many cancers. Furthermore, Shh has been found to be a key regulator of neural stem cells in the mammalian brain. To further study the roles of Hh, I have developed a transgenic zebrafish line as a tool to monitor tissues that respond to Hh signaling throughout the vertebrate life-cycle. A number of genes have been identified that are transcriptionally up-regulated by Hh signaling. Transcription of these genes is initiated through binding of activated Gli transcription factors to an identified Gli binding site (GBS) in the cis-regulatory region. This Gli binding site is largely conserved across vertebrate species. I have generated transgene constructs in which 12 GBSs have been placed upstream of a minimum promoter that drives GFP, RFP, or Kaede fluorescent proteins. These plasmid constructs are activated in embryonic regions known to be Hh responsive, such as the ventral CNS. Treatment with cyclopamine eliminates this expression, confirming that these transgenes accurately report an active Hh response. These transgenic lines will be extremely powerful tools for research into the mechanisms by which Hh signaling regulates adult cell types such as neural stem cells. These lines will also be important tools that will help understand how misregulation of Hh signaling can lead to cancer.

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Hedgehog (Hh) proteins control animal development and tissue homeostasis. They activate gene expression by regulating processing, stability, and activation of Gli/Cubitus interruptus (Ci) transcription factors. Hh proteins are secreted and spread through tissue, despite becoming covalently linked to sterol during processing. Multiple mechanisms have been proposed to release Hh proteins in distinct forms; in Drosophila, lipoproteins facilitate long-range Hh mobilization but also contain lipids that repress the pathway. Here, we show that mammalian lipoproteins have conserved roles in Sonic Hedgehog (Shh) release and pathway repression. We demonstrate that lipoprotein-associated forms of Hh and Shh specifically block lipoprotein-mediated pathway inhibition. We also identify a second conserved release form that is not sterol-modified and can be released independently of lipoproteins (Hh-N*/Shh-N*). Lipoprotein-associated Hh/Shh and Hh-N*/Shh-N* have complementary and synergistic functions. In Drosophila wing imaginal discs, lipoprotein-associated Hh increases the amount of full-length Ci, but is insufficient for target gene activation. However, small amounts of non-sterol-modified Hh synergize with lipoprotein-associated Hh to fully activate the pathway and allow target gene expression. The existence of Hh secretion forms with distinct signaling activities suggests a novel mechanism for generating a diversity of Hh responses.