Adult neurogenesis occurs in proliferative zones of the brain that contain neural progenitor cell populations capable of differentiating into specific cell types. However, we remain limited in our understanding of the signals that regulate neural progenitor cell proliferation and differentiation in adults. Recently zebrafish (Danio rerio) have emerged as an excellent model for studying the molecular mechanisms behind adult neurogenesis, because sixteen proliferative zones remain active in the adult brains. Thousands of fluorescent transgenic reporter lines have been generated in zebrafish that reveal gene expression patterns of cell-cell signaling systems, some of which may regulate neurogenesis in these brain regions. Using a new tissue clearing technique and whole brain imaging with fluorescent light sheet microscopy (FLSM) we have generated the first 3-Dimensional atlas of gene expression in an intact adult zebrafish brain. So far we have created a reference brain image and have aligned the expression patterns from three transgenic lines. This work is a preliminary step in the generation of a new, open access brain atlas called the Zebrafish Adult Brain Browser (ZABB). While generating this atlas we focused on documenting the adult brain regions responsive to Sonic Hedgehog (Shh), a cell-cell signaling system known to regulate neurogenesis during embryonic development. We used two Shh-reporter lines to create another atlas comparing reporter transgene expression in whole brain and sectioned tissue to the expression of the Hedgehog (Hh) target gene ptch2 using in situ hybridization. We show that the reporter lines reveal different Hh responsive domains, but together identify fourteen Hh responsive regions in the brain, nine of which are known proliferative zones. Thus, it appears that subsets of both proliferating neural progenitors and non-proliferative cells remain Hh responsive in adult brains. Our data suggests that Hh signaling contributes to the regulation of neural progenitor cells in nine of the sixteen proliferative zones. Uncovering the molecular mechanisms behind adult neurogenesis and forming a greater understanding of adult neural stem cell regulation has the potential to influence the treatment of many neurodegenerative diseases and cancers.

Axon pathfinding is an important process required for the establishment of proper neuronal connections during development. An increasing number of secreted and membrane-anchored molecules have been identified as axon guidance cues, which can act as positive or negative factors to increase or decrease the growth of axons and influence the direction of axonal growth. These axon guidance factors present in the extracellular environment interact with receptors present on the growth cone, a structure located at the tip of the axon which functions as the motor unit for the axon. Upon binding to their receptors on the growth cone, the guidance factors then elicit an intracellular signaling cascade within the axon that ultimately influences the direction of axon growth, often through a direct, non-transcriptional mechanism. In this dissertation, we show that Sonic hedgehog (Shh) acts as an axon guidance factor for chick retinal ganglion cell (RGC) axons in a concentration-dependent manner. At a low concentration, Shh functions as a positive factor that induces axon growth and attractive turning while, at a high concentration, Shh functions as a negative factor that induces axon retraction and repulsive axon turning. We further characterized the effects of Shh on macropinocytosis, a fluid-phase type of endocytosis, in the axons. A high concentration of Shh significantly increased macropinocytosis in the axons. Macropinocytosis resulted in the generation of large, dextran-positive, clathrinindependent vesicles in the axonal growth cones, prior to growth cone collapse, axon retraction and repulsive axon turning. These vesicles were found to require dynamic F-actin, nonmuscle myosin II and dynamin for their formation but were formed independently of PI3 kinase signaling. Interestingly, a low concentration of Shh had an opposite effect on macropinocytosis. A low concentration of Shh and soluble laminin decreased macropinocytosis and additionally increased the turnover of these vesicles within the axons, suggesting positive axon guidance factors can additionally regulate downstream processing or maturation of these vesicles. The effect of Shh on regulating the motility of macropinosomes within the axons was investigated. A low concentration of Shh appeared to increase the motility of these vesicles along axonal microtubules in a cAMPdependent manner. However, a high concentration of Shh did not appear to affect the motility of the macropinosomes, suggesting that it likely plays a more predominant role in the formation of these vesicles within the growth cone. When we began this work, a large body of research existed describing the effects of guidance factors on regulating the cytoskeleton during axon motility. However, the role of membrane trafficking events during axon growth and guidance were very poorly characterized. Since we began this project, an increasing number of reports have shown that endo- and exocytosis are important for axon growth and, here, we show that macropinocytosis induced by negative axon guidance factors plays a critical role in growth cone collapse, axon retraction and repulsive axon turning. Positive axon guidance factors also affect macropinocytosis within the axons and additionally regulate their maturation, suggesting that membrane trafficking events mediated by axon guidance factors are important for regulating axon growth and pathfinding.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23464号 / 医博第4771号 / 新制||医||1053(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 林 康紀, 教授 伊佐 正, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cancers of the human skin are divided into melanoma and non-melanoma. Being among the most commonly diagnosed cancer cases globally, non-melanoma skin cancers are comprised of basal and squamous cell carcinomas. In dermato-pathology, basal cell carcinomas (BCCs) are a frequently encountered diagnosis of skin cancer, and most cases are treated with surgical excisions. While sporadic BCC tumors appear primarily due to aging and ultra-violet exposure, the development of numerous BCCs from a young age is one of the main clinical signs in Gorlin syndrome patients. The critical driver of BCC tumor formation is the sonic hedgehog (SHH) pathway, a pivotal developmental signaling pathway that regulates organ development, cell proliferation, and tissue repair. The majority of all sporadic and syndromic BCCs exhibit mutations in two key components in this pathway, the tumor suppressor gene patched 1 (PTCH1) or the proto-oncogene smoothened (SMO), which result in aberrant pathway activation and continued transcription of SHH-dependent genes. In the last decade, SHH inhibitors have emerged as a novel treatment for advanced and metastatic BCCs. Systemic treatment with vismodegib, a potent SMO inhibitor, can effectively reduce BCC tumor burden in adult Gorlin syndrome patients. However, it is associated with chemotherapy-related adverse events, and treatment cessation results in cancer recurrence and formation of a subset of drug resistant BCCs. While aberrant SHH signaling is key, mechanisms that underlie epithelial–stromal crosstalk and reprograming of tumor metabolism can potentially converge with this pathway and promote BCC tumor development. In this study, we investigated the effects vismodegib on the morphology, behavior, and energy metabolism of human BCC cells and human dermal fibroblasts, in individual cultures as well as in co-cultures, that enabled the crosstalk between these two cell types. Computer-assisted bright-field microscopy was used to characterize cell morphology and behavior. Nuclear magnetic resonance (NMR) and metabolomics were used to determine the metabolic activity of these cells. We found that continuous crosstalk between the cells and different concentrations of vismodegib led to distinct changes in cell morphology and growth, as well as consumption of glucose, pyruvate, and glutamine and secretion of acetate, lactate, and glutamate by these cells. Deciphering tumor driver mechanisms that converge with SHH pathway and contribute to changes within the tumor microenvironment are important not only for better understanding of BCC pathobiology, but also for the development of new mechanism-based BCC therapies with improved clinical outcomes. / 2023-02-22T00:00:00Z

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The physiological response to environmental cues involves complex interorgan communication via endocrine factors and hormones, but the underlying mechanisms are poorly understood. In particular, little is known about how animals coordinate systemic growth and developmental timing in response to environmental changes. The morphogen Hedgehog (Hh), which is well studied in tissue patterning and homeostasis, has only recently been implicated in the regulation of lipid and sugar metabolism. Interestingly, Hh is present in systemic circulation in both, ies and mammals. Here, we demonstrate that systemic Hh is produced in the midgut and secreted in association with the lipoprotein particle lipophorin (Lpp) into the hemolymph to mediate the interorgan communication between the midgut and two tissues, the fat body and the prothoracic gland (PG). We show that midgut hh expression is regulated by dietary sugar and amino acid levels, and RNAi-mediated knock-down of circulating Hh leads to starvation sensitivity. We demonstrate that circulating Hh is required to inhibit systemic growth and developmental progression. In insects, developmental transitions are regulated by steroid hormones, which are produced by the PG. Nutritional regulation of growth is, in part, mediated by the Drosophila fat body. Strikingly, canonical Hh pathway components are present in both tissues, the fat body and the PG. To understand the Hh-mediated function during nutritional stress, we ectopically activated or inhibited the Hh signaling pathway specifically in the fat body and the PG. Our results show that systemic Hh exerts its function through these two target tissues. Hh signaling in the fat body is required for survival during periods of nutrient deprivation, and ectopic activation of fat body Hh signaling causes an inhibition of systemic growth. Hh signaling in the PG slows down developmental progression by inhibiting steroid hormone biosynthesis. In conclusion, we propose that the midgut senses the uptake of dietary sugar and amino acids and secrets Hh in association with Lpp particles into circulation to relay information about the feeding status to the developing animal. Therefore, circulating Hh functions as a hormone and signals in an endocrine manner to the fat body and the prothoracic gland to coordinate systemic growth and developmental timing in response to changes in nutrient availability.

Basal cell carcinoma is the most common form of human malignancy, and as such there are varied methods for treating its various forms. Its more advanced and aggressive forms have required both the use of and advent of therapies which offer differing safety profiles, cost, and efficacy. Two therapies which differ substantially in these respects but have overlap in their recommended use are Mohs micrographic surgery and the pharmaceutical drug vismodegib. Few studies have sought to compare the two methods using these criteria, and as vismodegib has only received FDA approval in the past ten years, it is worthwhile to explore the limitations and advantages of each therapy. In exploring previous clinical trials and retrospective studies, the two therapies are put side by side to contrast their results with their shared intended use. The general findings were that Mohs micrographic surgery remains the gold standard for the treatment of locally advanced basal cell carcinoma, and there are few demonstrable instances in which vismodegib could be deemed a more appropriate therapy. The future of vismodegib appears to be in its use as a neoadjuvant therapy for locally advanced basal cell carcinomas for which a decrease in size by vismodegib would allow for better treatment outcomes.

The normal regulation of the Hedgehog (Hh) pathway is essential for embryonic development, stem cell maintenance, and gametogenesis for both vertebrates and invertebrates, whereas the aberrant pathway regulation can cause various developmental defects and cancers. Hence, it is important to understand the precise mechanism of how the Hh pathway is regulated. Much of our understanding of the Hh pathway comes from studies in Drosophila but applies also to vertebrates. In Drosophila, Hh signal transduction terminates with regulation of the transcriptional activator, Cubitus interruptus (Ci). In the absence of Hh signaling, Ci is 1) processed to a repressor form via the Costal2 (Cos2) complex and suppresses the transcription of Hh target genes or 2) inhibited by binding to Cos2 and Suppressor of Fused (Su(fu)). Once the cells receive Hh ligand, however, 1) Ci processing is inhibited and 2) inhibition by Su(fu), and possibly Cos2, is countered by Fused (Fu) kinase, which ultimately transforms Ci into an activator form that goes into the nucleus and induces the transcription of Hh target genes. How Fu alleviates Su(fu) inhibition and facilitates the activation of Ci is not well understood, and it was only discovered recently that Ci is the direct target of Fu phosphorylation. Many studies of Hh signaling have been conducted under artificial conditions, where proteins are often overexpressed, leading to findings that sometimes do not reflect in vivo events, where relative protein stoichiometry is important. For this dissertation, I investigated how Ci activation is regulated by Su(fu) and Fu using CRISPR/Cas to generate different Ci variants expressed at physiological levels in fly wing discs. I looked at how different regions of Ci, including known phosphorylation sites, contribute to the regulation of Ci activity. From this study, I propose that different sets of Ci phosphorylation events mediated by Fu are responsible for changes in Ci-Su(fu) interactions, by altering Ci-Su(fu) interfaces, but also by changing intramolecular Ci-Ci interactions and thereby transforming Ci to an active conformation, leading to target gene activation in response to Hh.

The liver is a multifunctional organ that regulates important processes such as lipid, glucose and xenobiotic metabolism. Most of the hepatic functions are implemented in the hepatocytes, which make up the largest cellular proportion of the liver. Various signaling pathways are required for the liver to perform its diverse metabolic tasks, two of them are the Hedgehog (HH) and mechanistic target of rapamycin (mTOR) signaling pathways. Dysregulation of these signaling pathways leads to various liver diseases, such as non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). In order to develop appropriate treatments for these disorders, it is important to understand the molecular interactions of the various signaling pathways involved in their development and progression. However, another aspect is particularly important in the treatment of hepatic diseases - the sexual dimorphism of the liver. A great variety of genes are differentially expressed in the liver of males and females. Signaling pathways affected by hepatic sexual dimorphism include lipid and drug metabolism. Although the influence of sex-specific dimorphism in the liver on diseases and drug doses is well known, there are currently only a few sex-specific treatment concepts. In order to incorporate this aspect in the development of new and more effective treatments, it is important to understand the molecular mechanism and characteristics of sexual dimorphism in model systems. Primary hepatocytes are often used for in vitro analysis of pharmaceutical studies. However, little is known about the development of sexual dimorphism during the culture of primary cells. To facilitate the development of novel sex-specific treatment approaches, the present thesis investigated the interaction of the HH and mTOR signaling pathways in hepatocytes, as well as the development of sexual dimorphism during the culture of primary hepatocytes for up to 96 h. Male and female mice with a hepatocyte-specific knock-out (KO) of the HH signaling were used to examine a possible interaction of HH and mTOR signaling. The repression of the HH signaling cascade leads to reduced mRNA expression of molecules involved in mTOR Signaling as well as decreased mitochondrial adenosine triphosphate (ATP) production in hepatocytes from female KO mice. In contrast, hepatocytes from male KO mice showed reduced phosphorylation of mTOR molecules. Furthermore, an impaired autophagy was detected in primary hepatocytes from both sexes. Consequently, the hepatocyte-specific HH-KO leads to decreased mTOR activity in males and females. However, the KO shows different points of action depending on the sex of the mice. To investigate the crosstalk of the two pathways in more detail, primary hepatocytes from male and female C57BL/6N mice were incubated with the HH inhibitor cyclopamine and the mTOR inhibitors rapamycin and Torin. Thereby, a synergistic effect of cyclopamine and rapamycin could be demonstrated on the repression of the mTOR signaling cascade and mitochondrial ATP production. Although primary hepatocytes have been isolated from male and female mice for all experiments, the further development of the sexual dimorphism of the hepatocytes in monolayer-cultivation is unclear. Therefore, cells from male and female C57BL/6N mice were isolated to conduct transcriptome, proteome and metabolome studies directly after isolation and after 24, 48, 72 and 96 h of cultivation. It was striking that the expression pattern of sexually differentiated genes developed differently during cell culture compared to the translation pattern of sexually differentiated proteins. While mRNA expression showed a large shift after 24 h, there was a feminization of the translation pattern at the protein level throughout the cultivation period. Furthermore, the study revealed a change in the sex-specific gene expression of xenobiotic metabolism with a significant decrease of female-specific expression of Cytochrome P450 (Cyp) 2b13 and Cyp2b9. Since no sex hormones or growth factors were added to the cell culture, a reduction in sex differences during the time of cell culture was to be expected. Instead, we found an increase in sex-specific gene expression for some signaling pathways, such as serotonin and melatonin degradation. For the expression of genes related to androgen signaling, beta-oxidation, hepatic steatosis and amino acid degradation, even a reversal of the sex-specific gene expression could be shown. In conclusion, this investigation reveals a connection between HH and mTOR signaling in hepatocytes. The repression of the HH pathway modulates mTOR signaling and results in impaired oxidative phosphorylation and autophagy. It is conceivable that the synergistic effect of HH and mTOR inhibition could be applied in targeted therapies. Furthermore, the culture of male and female primary hepatocytes demonstrate that sex differences change significantly over time. This should be considered when using primary hepatocytes for initial preclinical studies to analyze potential new drugs. Especially, the alterations in xenobiotic metabolism can have serious effects on pharmacodynamic processes. In addition, the enhancement or reversal of sex-specific expression of some genes in vitro suggests that there must be previously unknown mechanisms that modulate sexual dimorphism independently of the steroids and growth hormones known to date.:Table of Contents 1. Introduction 1.1 Characteristics of the liver 1.2 The hepatic sexual dimorphism 1.3 Interaction of signaling pathways in the liver 1.3.1 The Hedgehog pathway 1.3.2 The mTOR pathway 1.3.3 The role of HH and mTOR in diseases 1.4 Aim of the study 2. Publications 2.1 Cyclopamine and rapamycin synergistically inhibit mTOR signaling in mouse hepatocytes, revealing an interaction of hedgehog and mtor signaling in the liver 2.2 Sex-dependent dynamics of metabolism in primary mouse hepatocytes 33 3. Summary of the thesis 4. References 5. Attachments 5.1 List of abbreviations 5.2 Supplemental Material 5.2.1 Cyclopamine and rapamycin synergistically inhibit mTOR signaling in mouse hepatocytes, revealing an interaction of hedgehog and mtor signaling in the liver 5.2.2 Sex‑dependent dynamics of metabolism in primary mouse hepatocytes 5.3 Description of the author's own contribution 5.3.1 Cyclopamine and rapamycin synergistically inhibit mTOR signaling in mouse hepatocytes, revealing an interaction of hedgehog and mtor signaling in the liver 5.3.2 Sex‑dependent dynamics of metabolism in primary mouse hepatocytes 5.4 Statement of Authorship 5.5 Curriculum vitae 5.6 Publications 5.7 Acknowledgement

Pattern formation ensures that tissues, organs, and structures develop in the correct place and orientation within the body. Patterning processes are at the heart of morphogenesis yet remain poorly understood due to their complexity. The sea urchin larval skeleton provides a simple model to study skeletal patterning, where the skeleton-producing primary mesenchyme cells (PMCs) receive patterning cues from the overlying ectoderm. The normal skeletal patterning process requires the PMCs to migrate within the blastocoel to specific positions. While ectodermal and endodermal signals regulate PMC positioning and differentiation, additional signals act to regulate biomineralization per se in the PMCs. However, the distinction between these effects is not well understood and new efforts have been made to identify these patterning and biomineralization cues that regulate sea urchin skeletal development. Understanding the mechanism by which PMCs interpret and transduce patterning cues into a migratory bias and/or positional information will provide insight into tissue patterning and developmental plasticity both in sea urchins and, more broadly, in deuterostomes. Ethanol is a known vertebrate teratogen that causes craniofacial defects as a component of fetal alcohol syndrome. Perturbations to retinoic acid biosynthesis and the Hedgehog signaling pathway are thought to be causal for the fetal alcohol syndrome phenotype in vertebrates. We used the sea urchin embryo to gain evolutionary insight into how ethanol affects embryonic development in a basal deuterostome animal. We found that ethanol specifically perturbs skeletal patterning. When sea urchin embryos are exposed to ethanol, they exhibit conspicuously delayed development, and broad skeletal patterning defects that are potentially analogous to fetal alcohol syndrome associated facial patterning defects in vertebrates and humans. PMC transplantation experiments demonstrated that ethanol-induced defects are not specific to the PMCs, and instead reflect the perturbation of patterning cues. We also found that the expression of both patterning cues and PMC-specific genes was delayed by ethanol exposure. Surprisingly, our results indicate that retinoic acid and Hedgehog pathways are not functionally relevant for the teratogenic effects of ethanol in the larval skeletal patterning process, indicating a lack of evolutionary conservation of these pathways in ethanol-mediated teratogenesis among deuterostomes. Temporal transcriptome analysis revealed significant impacts of ethanol on signaling and metabolic gene expression and a disruption in the timing of expression for sea urchin specification gene regulatory network (GRN) genes. Surprisingly, multiple circuits with the GRN exhibit precocious expression while others are delayed. Taken together, our results suggest that the skeletal patterning perturbations in ethanol-treated sea urchin embryos arise from a loss of temporal synchrony within and between the instructive and responsive tissues during pattern formation.