L'ovaire représente à la fois un organe cible et le principal organe producteur d'estrogènes et de progestérone qui maintiennent le développement des caractères sexuels féminins et une fonction de reproduction normale. Cette production hormonale est contrôlée par les gonadotropines FSH et LH produites dans l'hypophyse, responsables dans l'ovaire de la croissance folliculaire et de l'ovulation, respectivement. Mon travail de thèse a identifié la signalisation prostaglandine D2 (PGD2), comme un nouvel élément-clé dans la signalisation des gonadotropines, contribuant à l'activation de l'expression des récepteurs FshR et LhR et des enzymes de la stéroïdogenèse SCC et StAR. La PGD2, produite dans plusieurs tissus par deux enzymes de synthèse, les prostaglandines synthases H et L-PGDS, est impliquée dans de nombreuses fonctions physiologiques et pathologiques. Comme dans l'ovaire pathologique, nous avons montré que la PGD2 avait aussi un rôle anti-prolifératif dans la cellule de granulosa de l'ovaire normal. Le cancer de l'ovaire représente la 4ème cause de mortalité par cancer chez la femme. Les mécanismes moléculaires impliqués dans le développement de ces tumeurs sont encore peu connus, bien que l'implication des estrogènes et de la Prostaglandine E2 (PGE2) dans la progression des tumeurs ovariennes épithéliales soit bien établie. D'autre part, les ovaires des souris invalidées pour les gènes codant les récepteurs aux estrogènes ou l'aromatase, possèdent des structures tubulaires contenant des cellules de Leydig et des cellules de Sertoli re-différenciées exprimant le facteur de détermination sexuelle mâle SOX9, alors qu'il n'est pas exprimé dans l'ovaire sain. Mon travail a montré que les estrogènes inhibent la transcription des gènes Sox9 et L-Pgds dans les lignées ovariennes tumorales BG1 et COV434 et que cette régulation est la résultante d'une inhibition, via le récepteur ERa et d'une activation via le récepteur ERß. Ces résultats sont en accord avec les études sur les effets prolifératifs d'ERa et le rôle anti-prolifératif d'ERß et suggèrent donc un rôle anti-prolifératif de la PGD2 dans l'ovaire tumoral et une régulation négative directe ou indirecte de l'expression de Sox9 et des Pgds par les estrogènes. / The prostaglandin D2 (PGD2) pathway is involved in numerous biological processes and while it has been identified as a partner of the embryonic sex determining male cascade, the roles it plays in ovarian function remain largely unknown. PGD2 is secreted by two prostaglandin D synthases (Pgds); the male-specific lipocalin (L)-Pgds and the hematopoietic (H)-Pgds. Here, we report the localization of H-Pgds mRNA in the granulosa cells from the primary to pre-ovulatory follicles. We used adult female mice treated with HQL-79, a specific inhibitor of H-Pgds enzymatic activity, to provide evidence of an interaction between H-Pgds-produced PGD2 signaling and FSH signaling. This leads to the activation of steroidogenic Scc and StAR gene expression through increased FshR and LhR receptor expression leading to progesterone secretion. We also identify a role whereby H-Pgds-produced PGD2 is involved in the regulation of follicular growth through inhibition of granulosa cell proliferation in the growing follicles. Indeed, we report an altered H-Pgds expression in human ovarian tumors alongside a partial or complete absence of H-Pgds protein in granulosa cell tumors, suggesting a potential association between decreased levels of H-Pgds expression and a tumoral phenotype. Together, these results show PGD2 signaling to be essential for FSH action within granulosa cells, thus identifying an important and unappreciated role for PGD2 signaling in controlling the balance of proliferation, differentiation and steroidogenic activity of these cells.

在中樞神經系統的發育過程中，神經幹細胞會先經歷神經發生 (neuro¬genesis)產生神經元，然後再通過神經膠質細胞發生 (gliogenesis)製造神經膠質細胞。這個時間順序是所有神經幹細胞分化過程中的固定模式。 Sox9是屬於一類具有 HMG (high mobility group)特徵性結構域的轉錄因子家族。以往轉基因小鼠研究證明， Sox9在脊髓和視網膜神經建構過程中，是引發神經膠質細胞新生程式的決定性主控基因。但是在小腦發育過程中，製造神經膠質細胞的調節機制仍未被界定。 / 在小鼠小腦發育過程中，室區 (ventricular zone)的神經祖細胞豐富表達 Sox9基因。因此，本實驗試圖利用條件基因剔除技術，研究 Sox9基因在小腦形成過程中的功能。結果顯示， Sox9基因在小腦被剔除後會導致包括蒲金耶氏細胞 (Purkinje cells)及 γ-氨基丁酸能中間神經元 (GABAergic interneurons)等室區衍生神經元大幅增加。與此同時，一些神經膠質細胞標記的表達亦受到影響。值得留意的是這些缺陷表型在胚胎發育後期才發生，與神經膠質細胞發生開始的時間框架一致。由於神經元和神經膠質細胞都是於共同的神經祖細胞池分化而成， Sox9基因的失活顯然影響了祖細胞池由製造神經元切換到神經膠質細胞生成的過程。進一步的微陣列基因晶片及半定量 RT-PCR分析顯示，數個參與細胞增殖、分化及細胞命運決定的基因表達量在 Sox9轉基因小鼠小腦中起了明顯的變化，而這些基因很可能與 Sox9共同調控神經膠質細胞發生的始初過程。 / 另一方面，我利用條件性 Sox9高效表達的小鼠作為動物模型及分析其表徵,希望更全面地了解 Sox9在小腦發育過程中的角色。於胚胎發育期間， Sox9基因高效表達並沒有擾亂小腦的發育；但由產後第 15周起，在小腦中持續性的 Sox9基因異位表達卻導致小鼠出現明顯的運動協調及身體平衡能力缺失。從 24 周 Sox9高效表達小鼠小腦組織分析顯示，其小腦中的貝格曼神經膠質細胞 (Bergmann glia)和蒲金耶氏細胞均出現缺陷表型，而這兩類細胞的異常變化很可能是導致條件性 Sox9高效表達小鼠運動協調缺失的主因。 / 在探究 Sox9如何調節小腦發育的同時，我發現負責分泌腦脊液及形成血腦屏障的脈絡叢 (choroid plexus)亦發生異常變化。初步分析顯示， Sox9的失活導致脈絡叢上皮細胞的凋亡率上升，而這亦解釋了為何顱內出血的情況在 Sox9基因剔除小鼠中較常見。 / 總括而言，這項研究的結果顯示 Sox9在小鼠小腦發育過程中扮演決定神經祖細胞命運的角色，在中樞神經系統發育中起著守恒的作用。而 Sox9基因的高效表達則會造成成年小鼠的運動功能障礙。此外，Sox9亦可能通過調控脈絡叢的發育和功能，以維持血腦屏障的完整性。我們需要更深入及全面的研究以了解 Sox9在小鼠小腦和脈絡叢發育中的作用及其分子機制。 / In the developing central nervous system (CNS), neural stem cells undergo a stereotypic pattern of temporal differentiation characterized by an initial wave of neurogenesis which then ceases to give way for a subsequent period of gliogenesis. Sox9 belongs to the highly conserved family of high mobility group (HMG) transcription factors, and has been shown to be the master regulator mediating the switch to the gliogenic program in several neuronal tissues including the spinal cord and the retina. While in the cerebellum, genetic control of such a developmental interval has remained poorly defined. / In the developing cerebellum, Sox9 is expressed abundantly in neural progenitors of the ventricular zone (VZ). Here, I analyzed cerebellar development of mice in which Sox9 is specifically inactivated in the cerebellum by the Cre/loxp recombination system. These mice exhibited an increased number of neuronal phenotypes, including the Purkinje cells (PCs) and GABAergic interneurons, while the expressions of several glial markers are compromised. These phenotypes occur only at late embryonic stage, a time frame which is consistent with the initiation of gliogenesis. Because neurons and glia share a common origin, the ablation of Sox9 apparently causes the progenitor pool to continue to produce neurons instead of switching to generate glial cells. Subsequent microarray and semi-quantitative RT-PCR analyses identified expression level changes in genes that have been previously implied in regulating cell fate decision and cell proliferation during development, which may possibly function in collaboration with Sox9 during the initiation of gliogenesis. / On the other hand, to comprehensively interrogate the role of Sox9 in cerebellar development, a conditional Sox9 overexpression mutant was characterized. While the ectopic expression of Sox9 did not perturb cerebellar development during embryogenesis, the continued aberrant expression of Sox9 in the cerebellum led to noticeable locomotor deficits in adult mice from 15 weeks onwards. Histological examinations at 24 weeks revealed abnormalities in both the Bergmann glia and PCs, which possibly accounted for the motor defects observed in the mutant mice. / In the course of studying the role of Sox9 in cerebellar development, noticeable abnormalities were also observed in the choroid plexus (ChP), a neurovascular tissue responsible in setting up the blood-brain barrier. Initial analysis showed that the ablation of Sox9 induced apoptosis in the ChP epithelium, which possibly explained the higher frequency of intracranial hemorrhage observed in the mutant. / In summary, the findings from this study suggest that Sox9 plays a conserved role in the developing CNS as a key molecular component in determining the neuron-glial fate choice during cerebellar development, while the ectopic expression of Sox9 could induce locomotor dysfunction in adult mice. In addition, Sox9 may also contribute to the maintenance of vascular integrity by regulating ChP development and functionality. More comprehensive investigation is required to understand the molecular mechanisms of Sox9 action during mouse cerebellar and ChP development. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Leung, Kit Ying Crystal. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 166-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / Chapter Declaration --- p.i / Chapter Abstract --- p.iii / Chapter Abstract in Chinese --- p.v / Chapter Acknowledgements --- p.vii / Chapter Table of Contents --- p.ix / Chapter List of Figures --- p.xiii / Chapter List of Tables --- p.xv / Chapter List of Abbreviations --- p.xvi / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- Preface: The developing central nervous system - Why it matters --- p.1 / Chapter 1.2 --- Development of the Mammalian Central Nervous System: An Overview --- p.3 / Chapter 1.2.1 --- Neural induction, neurulation and the formation of the neural tube --- p.3 / Chapter 1.2.2 --- Regionalization of the rostral neural tube and formation of brain vesicles --- p.4 / Chapter 1.3 --- The Cerebellum --- p.7 / Chapter 1.3.1 --- Functions of the cerebellum --- p.7 / Chapter 1.3.2 --- Disorders of the cerebellum --- p.8 / Chapter 1.3.3 --- Gross anatomy and organization of the cerebellum --- p.11 / Chapter 1.3.4 --- Cellular constituents of the cerebellum - diversity and biochemistry --- p.15 / Chapter 1.3.5 --- Neuronal circuitry of the mature cerebellum --- p.16 / Chapter 1.4 --- Development of the Cerebellum --- p.20 / Chapter 1.4.1 --- Overview of mouse early cerebellar development --- p.20 / Chapter 1.4.2 --- Germinal matrices of the cerebellar primordium --- p.22 / Chapter 1.4.3 --- Timeline of the birth of cerebellar neurons and glial cells --- p.25 / Chapter 1.4.4 --- Postnatal development of the cerebellum --- p.27 / Chapter 1.4.5 --- Genetic regulation of cerebellar development --- p.30 / Chapter 1.5 --- SOX9 and the SOX Family of Transcription Factors --- p.33 / Chapter 1.5.1 --- SOX9 as a transcription factor --- p.33 / Chapter 1.5.2 --- Molecular regulation of SOX9 action --- p.36 / Chapter 1.5.3 --- SOX9 in development and disease --- p.38 / Chapter 1.6 --- Scope of the Thesis --- p.45 / Chapter CHAPTER 2 --- Characterization of a Mouse Model with Sox9 Conditional Knockout / Chapter 2.1 --- Chapter Summary --- p.47 / Chapter 2.2 --- Introduction --- p.49 / Chapter 2.3 --- Materials and Methods --- p.54 / Chapter 2.3.1 --- Animal husbandry --- p.54 / Chapter 2.3.2 --- Breeding strategy for the generation of Sox9 conditional knockout mutants --- p.54 / Chapter 2.3.3 --- DNA extraction and genotyping --- p.55 / Chapter 2.3.4 --- Histological examination of the cerebellum --- p.57 / Chapter 2.3.5 --- β-Galactosidase staining of embryos --- p.59 / Chapter 2.3.6 --- Microarray analysis --- p.59 / Chapter 2.3.7 --- Validation of microarray data by semi-quantitative RT-PCR --- p.60 / Chapter 2.3.8 --- In situ hybridization --- p.61 / Chapter 2.3.9 --- Image acquisition and photo editing --- p.65 / Chapter 2.3.10 --- Statistical analysis --- p.66 / Chapter 2.4 --- Results -- Part I: En1[superscript Cre]- driven Sox9 Conditional Knockout --- p.67 / Chapter 2.4.1 --- Expression of Sox9 during mouse embryonic development --- p.67 / Chapter 2.4.2 --- Effective ablation of Sox9 in the cerebellum of En1[superscript Cre/]⁺; Sox9[superscript fx/fx] mutant --- p.68 / Chapter 2.4.3 --- Deficiency of Sox9 did not cause cerebellar developmental abnormalities in the Sox9 CKO mutants --- p.71 / Chapter 2.5 --- Results -- Part II: Pax2[superscript Cre]-driven Sox9 Conditional Knockout --- p.76 / Chapter 2.5.1 --- Effective ablation of Sox9 in the cerebellum of Pax2[superscript Cre/]⁺; Sox9[superscript fx/fx] mutant --- p.76 / Chapter 2.5.2 --- Sox9 deletion resulted in cerebellar malformation at late embryonic stage --- p.78 / Chapter 2.5.3 --- Loss of Sox9 caused an increased neuronal production from the ventricular zone of the Pax2[superscript Cre/]⁺; Sox9[superscript fx/fx] mutant --- p.80 / Chapter 2.5.4 --- Sox9 deletion did not alter rhombic lip-derived neurons --- p.84 / Chapter 2.5.5 --- Expression of glial markers were compromised in the Sox9 CKO mutant at late embryonic stages --- p.84 / Chapter 2.5.6 --- Comparison of cerebellar gene expression profiles between the Sox9 CKO mutant and control --- p.90 / Chapter 2.5.7 --- Expression analysis of the proto-oncogene transcription factor Prdm16 in the mouse brain --- p.93 / Chapter 2.6 --- Results -- Part III: Sox9 and the Development of the Choroid Plexus --- p.95 / Chapter 2.6.1 --- Partial loss of Sox9 in the Pax2[superscript Cre]; Sox9[superscript fx/fx] CKO mutant induced choroid plexus abnormalities and increased susceptibility to intracranial hemorrhage --- p.95 / Chapter 2.6.2 --- The mutant choroid plexus was non-cancerous --- p.98 / Chapter 2.6.3 --- Increased apoptosis in the Sox9 CKO mutant choroid plexus --- p.100 / Chapter 2.7 --- Discussion --- p.102 / Chapter 2.7.1 --- Sox9 plays an essential role in determining the neuron-glial fate choice in the developing cerebellum --- p.102 / Chapter 2.7.2 --- Potential influence of genetic background on Sox9 CKO mutant phenotypes --- p.104 / Chapter 2.7.3 --- Prdm16 as a potential candidate in a Sox9-dependent transcriptional regulatory cascade during the initiation of gliogenesis --- p.105 / Chapter 2.7.4 --- Sox9 may be important in choroid plexus development --- p.107 / Chapter 2.7.5 --- Chapter conclusion --- p.108 / Chapter CHAPTER 3 --- Characterization of a Mouse Model with Sox9 Conditional Overexpression / Chapter 3.1 --- Chapter Summary --- p.116 / Chapter 3.2 --- Introduction --- p.118 / Chapter 3.3 --- Materials and Methods --- p.120 / Chapter 3.3.1 --- Animal husbandry --- p.120 / Chapter 3.3.2 --- Breeding strategy for the generation of Sox9 overexpression mutants --- p.120 / Chapter 3.3.3 --- Genotyping --- p.120 / Chapter 3.3.4 --- Histological examination of the cerebellum --- p.121 / Chapter 3.3.5 --- Behavioral tests --- p.122 / Chapter 3.3.6 --- Image and video acquisition --- p.123 / Chapter 3.3.7 --- Video processing --- p.124 / Chapter 3.3.8 --- Statistical analysis --- p.124 / Chapter 3.4 --- Results --- p.125 / Chapter 3.4.1 --- Sox9 was overexpressed in only a subset of cells in the mutant cerebellum --- p.125 / Chapter 3.4.2 --- Overexpression of Sox9 did not cause developmental abnormalities in the cerebellum of En1[superscript Cre/]⁺; Z/Sox9 mutant embryos --- p.127 / Chapter 3.4.3 --- En1[superscript Cre/]⁺; Z/Sox9 mutants manifested locomotion deficits during adulthood --- p.132 / Chapter 3.4.4 --- Abnormal Purkinje cell dendritic arborization and Bergmann glial scaffold in adult En1[superscript Cre/]⁺; Z/Sox9 mutants --- p.138 / Chapter 3.5 --- Discussion and Chapter Conclusion --- p.143 / Chapter CHAPTER 4 --- General Discussion, Future Works and Conclusions / Chapter 4.1 --- An Evolutionary Conserved Role of Sox9 in Determining the Neuron-glial Fate Choice during Vertebrate CNS Development --- p.147 / Chapter 4.2 --- Prdm16 may be important in the transcriptional cascade during the initiation of gliogenesis in mouse cerebellar development --- p.148 / Chapter 4.3 --- A Potential Neuroprotective Role of Sox9 in the Adult Cerebellum --- p.149 / Chapter 4.4 --- Future Works --- p.150 / Chapter 4.4.1 --- Dissecting the dual roles for Sox9 in neural stem cell maintenance and gliogenesis --- p.150 / Chapter 4.4.2 --- The contribution of glutamate toxicity to the cerebellar phenotypes observed in the Sox9 CKO mutant --- p.152 / Chapter 4.4.3 --- The involvement of Prdm16 and Notch signaling in cerebellar development --- p.153 / Chapter 4.4.4 --- The molecular mechanism of Sox9-dependent neurodegenerative phenotypes in the conditional overexpression mutant --- p.153 / Chapter 4.4.5 --- The importance of Sox9 in choroid plexus development --- p.154 / Chapter 4.4.6 --- Improving the specificity of Cre deleter mouse lines --- p.155 / Chapter 4.5 --- Conclusions --- p.155 / APPENDIX / Chapter I. --- Microarray Data --- p.157 / Chapter II. --- References --- p.166

The work presented in this thesis is a study of human pancreas development. The principle goal of this work is to provide information that can be used in the development of treatments for Type 1 Diabetes and in pancreas regeneration methodologies. The transcription factor (TF) sex determining region Y homeobox gene 9 (SOX9) has been identified as a key factor in human pancreas development but its role has not been well characterized. The expression of SOX9 during early pancreas development was analyzed by immunostaining of fixed embryonic and fetal sections in the context of other developmentally important TFs. Modulators of SOX9 function, downstream targets and upstream regulatory pathways were investigated in human cell lines using coimmunoprecipitation, small interfering RNA (siRNA) knockdown, quantitative polymerase chain reaction (qPCR), luciferase assays and small molecule signaling pathway inhibitors. SOX9 was expressed in epithelial progenitors from initial human pancreas specification, but became excluded from the periphery of the epithelium and developing islet cells as differentiation proceeded. It was co-expressed with important endocrine and exocrine differentiation factors during the early stages of development. Some factors, such as Nirenberg and Kim 2, homeobox family member, drosophila, homolog of, 2 (NKX2.2) showed differing expression profile compared to murine development, while the widespread expression of endocrine factors before expression of the pro-endocrine gene neurogenin 3 (NGN3) suggested that these factors play an important role in initiating endocrine specification. Two transcription factors, GATA-binding protein 4 (GATA4) and neurogenic differentiation 1 (NEUROD1), were found to interact with SOX9 in potentially developmentally relevant complexes. This prompted the search for downstream targets of these transcriptional complexes by in silico analysis, which identified an array of novel potential downstream targets. Luciferase assay analysis of a subset of these genes showed SOX9 to activate a regulatory region of NGN3, and inhibit the regulatory regions of carboxy peptidase A6 (CPA6), v-ets avian erythroblastosis virus E26 oncogene homolog1 (ETS1) and SPONDIN1. An additional target of SOX9, osteopontin (OPN), was identified from a microarray of Sox9 knockout mouse pancreata. Investigation of SOX9 and OPN regulation by the Hedgehog signalling pathway (HH) identified both factors to be regulated by the pathway, suggesting SOX9 may act as a mediator of HH signalling. This is the first study to identify a range of SOX9 targets relevant to human pancreas development. While further characterization is required this work has provided essential clues to the function of SOX9, and provides a detailed framework of SOX9 expression and targets for future pancreatic studies to build upon.

Die Osteoarthrose ist eine degenerative Erkrankung der Gelenke. Sie zeichnet sich durch eine sukzessive Destruktion des Knorpels sowie der umliegenden Strukturen aus. Die Osteoarthrose ist die häufigste Gelenkerkrankung des Menschen und tritt insbesondere im hohen Lebensalter auf. Schätzungen zu Folge wird Osteoarthrose im Jahr 2020 zu der viert häufigsten Ursache für Erwerbsunfähigkeit aufsteigen. Neben dem Leidensdruck für den Patienten ist dies auch mit erheblichen Kosten für das Gesundheitssystem verbunden. Die natürliche Regeneration des avaskulären hyalinen Knorpels ist aufgrund der geringen Zelldichte und dem niedrigen Metabolismus der Zellen beschränkt. Es fehlt ein Perichondrium das den Knorpel stabilisiert und mit undifferenzierten Mesenchymzellen, als Quelle für frische Chondroblasten bzw. Chondrozyten, versorgt. Das fibrokartilaginäre Ersatzgewebe im erkrankten artikulären Knorpel beherbergt eine Population von Vorläuferzellen mit chondrogenen Eigenschaften. Die Herkunft dieser Zellen ist wenig erforscht. Es wird vermutet, dass diese durch Einsprossungen von Blutgefäßen aus dem subchondralen Knochen in die beschädigten Areale migrieren. Diese chondrogenen Progenitorzellen (CPCs) könnten sich als zugänglich für medikamentöse Therapien zur Stimulation ihres Reperaturpotentials erweisen. In dieser Arbeit sollten Coaktivatoren von dem chondrogenen Transkriptionsfaktor SOX9 oder Corepressoren von dem osteogenen Transkriptionsfaktor RUNX2 identifiziert werden. Dazu wurden CPCs verwendet, die durch siRNA-vermittelten Knockdown von RUNX2 in der Produktion von chondrogenen Faktoren stimuliert werden sollten. Auch der umgekehrte Versuch wurde durchgeführt. Durch den Knockdown von SOX9 sollten osteogene Faktoren stimuliert werden, die als Ziel inhibitorischer Therapien dienen könnten. Dabei konnte zunächst der antagonistische Zusammenhang zwischen SOX9 und RUNX2 in CPCs gezeigt werden. Der Knockdown von RUNX2 führte zu einer Hochregulation von SOX9 und damit zu einer Erhöhung des chondrogenen Potentials der Zellen. Umgekehrt führte der Knockdown von SOX9 zu einer Hochregulation von RUNX2 und damit zu einer Verringerung des chondrogenen Potentials der CPCs. Mithilfe massenspektrometrischer Analysen konnten aus Immunpräzipitationen und Pulldown-Isolaten von SOX9 und RUNX2 eine Vielzahl von Proteinen isoliert werden, die mit Signaltransduktionen und der Transkription in Verbindung stehen. Im Rahmen dieser Arbeit wurden einige der möglichen Coaktivatoren von SOX9 in CPCs überexprimiert und mögliche Corepressoren von SOX9 durch siRNA in ihrer Expression inhibiert. Die Überexpression von DDX5, HSPA8, RAB5C und YWHAE führte zu einer Zunahme der Genexpression von SOX9. Während HSPA8 auch eine Zunahme der Genexpression von RUNX2 bewirkte, führte YWHAE zu dessen Herabregulation und damit zu einer Erhöhung des chondrogenen Potentials von CPCs. Durch den Knockdown von LEMD2 und TMPO konnte ebenfalls eine Hochregulation von SOX9 erreicht werden. Ein Anzeichen für die Erhöhung des chondrogenen Potentials bot hierbei zusätzlich die erhöhte Expression der extrazellulären Bestandteile ACAN und die verminderte Expression von COL1A1. Darüber hinaus wurde zum ersten Mal die Interaktion zwischen SOX9 und DDX5 durch Coimmunpräzipitation untersucht. SOX9 konnte dabei copräzipitiert werden allerdings war der umgekehrte und damit der validierende Versuch nicht erfolgreich und bedarf weiterer Untersuchung, z. B. mithilfe einer anderen Methode wie Yeast-Two-Hybrid, um die tatsächliche Protein-Protein-Interaktion zu bestätigen bzw. um sie auszuschließen. Diese Ergebnisse zeigen, dass mithilfe der affnitätschromatographischen Aufreinigung von SOX9 sowie RUNX2 mögliche Interaktionspartner durch massenspektrometrische Analysen der isolierten Proteinkomplexe identifiziert werden können. Inwieweit diese Proteine direkt auf SOX9 einwirken oder Teil einer übergeordneten Transkriptionsmaschinerie sind muss in weiteren Studien untersucht werden, um die Frage zu beantworten, ob diese auch als potentielles Ziel für therapeutische Maßnahmen in Betracht kommen. Darüber hinaus muss die Frage geklärt werden, inwieweit sich die identifizierten Proteine auf die Expression von nachgeschalteten chondrogenen Faktoren auswirken. Diese Arbeiten könnten einen wichtigen Beitrag in der Entwicklung von neuen Therapeutika gegen Osteoarthrose leisten.

Sox9 est un facteur de transcription exprimé au cours du développement de l'intestin et son expression est maintenue à l'âge adulte dans trois populations cellulaires : les cellules souches, les cellules de Paneth, et les cellules tuft. L'inactivation de Sox9 dans l'épithélium intestinal embryonnaire entraîne, chez l'adulte, une hyperplasie des cryptes ainsi que l'absence de cellules de Paneth. Ce projet de thèse vise à déterminer le rôle de Sox9 dans les cellules de Paneth (dont la fonction est altérée chez les patients atteints de la maladie de Crohn), à identifier les mécanismes par lesquels Sox9 régule la prolifération et à proposer des cibles de Sox9 dans les cellules tuft. À l'aide de modèles murins d'inactivation de Sox9 au niveau de l'épithélium intestinal adulte, nous avons montré que la perte de ce facteur conduit à une augmentation de la prolifération dans les cryptes, confirmant ainsi que Sox9 régule négativement ce processus. Nos résultats indiquent que Sox9 est essentiel au maintien de l'identité des cellules de Paneth et nous proposons qu'il assure cette fonction en réprimant des gènes requis pour la différenciation des cellules de Goblet : Muc2 et Klf4. La perte de Sox9 dans les cellules de Paneth s'accompagne d'une réduction importante des molécules antimicrobiennes, ce qui entraîne une dysbiose intestinale. Dans un environnement spécifique (en présence du « mouse norovirus »), les souris déficientes en Sox9 présentent une perméabilité intestinale augmentée et une susceptibilité à l'inflammation accrue. Les dysfonctionnements des réponses antimicrobiennes et immunitaires dans notre modèle sont comparables à ceux observés chez les patients atteints de la maladie de Crohn, suggérant une implication potentielle de Sox9 dans cette pathologie. De plus, ces altérations pourraient expliquer l'augmentation de l'apparition des tumeurs observée chez les souris dont l'épithélium intestinal est déficient en Sox9, dans le contexte d'une mutation du gène suppresseur de tumeur Apc. Enfin, nous avons identifié des gènes potentiellement régulés par Sox9 qui pourraient expliquer son rôle dans le contrôle de la prolifération. Ces découvertes seront importantes pour mieux comprendre le processus du renouvellement de l'épithélium intestinal et identifier précisément le rôle de Sox9 dans le maintien de l'homéostasie et au cours du processus de la tumorigenèse intestinale. / Sox9 is a transcription factor expressed during the intestinal development and its expression is maintained throughout adult age in at least three populations of cells: stem cells, Paneth cells and tuft cells. Sox9 inactivation in the embryonic intestinal epithelium leads to crypts hyperplasia and to the loss of the Paneth cell lineage. The aim of this project is to determine Sox9 function in the adult intestinal epithelium, especially its role in Paneth cells (which function is altered in patients affected by inflammatory diseases such as Crohn disease), to identify how Sox9 controls proliferation and to propose molecular targets of Sox9 in tuft cells. Using mice models to inactivate Sox9 in adult intestinal epithelium, we could show that Sox9 is required to limit proliferation in the crypts, thus validating the hypothesis that Sox9 regulates negatively proliferation. Our results indicate that Sox9 is essential to maintain Paneth cells identity and we proposed that it ensures this function by repressing genes specific for Goblet cells differentiation: Muc2 and Klf4. Loss of Sox9 in Paneth cells is associated with a reduction of antimicrobial molecules which causes intestinal dysbiosis. In a specific environment (in presence of the « mouse norovirus »), Sox9-deficient mice have a defective intestinal permeability and are more susceptible to inflammation. The dysfunctions of the mucosal defences and of immunity responses in our model resemble those observed in Crohn patients, thus suggesting a potential implication of Sox9 in this pathology. In addition, these alterations could be responsible for the increased susceptibility of our deficient model to develop tumors in the context of a mutation of the tumor suppressor gene Apc. We started to unravel potential molecular targets of Sox9 that are involved in the control of proliferation, that will be important to better understand Sox9 function in the intestinal epithelium self-renewal and to identify precisely Sox9 function to maintain homeostasis and during intestinal tumorigenesis.

Although advances have been made toward understanding the complex mechanisms that regulate the process of DNA transcription, the specific mechanisms of activation for many individual genes remain unknown. In this study, we focus on the role the transcription factor SOX9 plays in activating cartilage-specific genes, specifically Col9a1 and Cartilage Link Protein (CRTL1). Previously, enhancers of these genes containing single SOX9 binding sites were shown to be activated through SOX9 binding. However, the hypothesis was made that in cartilage-specific genes dimeric SOX9, as opposed to monomeric SOX9, is necessary for activation. We identified a putative binding site adjacent to each of the known single SOX9 binding sites in the Col9a1 D and E enhancers and in the CRTL1 enhancer. Electrophoretic Mobility Shift Assays (EMSAs) were performed to determine whether SOX9 bound to these putative sites. Transient transfections were then performed using wild-type and mutant enhancer- reporter plasmids to determine whether these putative SOX9 binding sites were important for activation in vivo. Although dimeric SOX9 bound to each of the enhancers in vitro, several different effects were seen in vivo. In the presence of the wild-type Col9a1 D enhancer, no activation was seen. However, when the enhancer was extended to include an additional pair of newly found SOX9 binding sites, expression was increased 10-fold. When any of the four SOX9 binding sites within this enhancer were mutated, expression was completely eliminated, suggesting that interdependent dimers or a tetramer of SOX9 is necessary for the activation of transcription. The weaker Col9a1 enhancer E was found to increase gene expression minimally through binding of either dimeric or monomeric SOX9. However, dimeric SOX9 was required for the activation of gene expression by the CTRL1 enhancer. Through this study we validate the importance of not just monomeric but of dimeric and possibly tetremeric SOX9 as an activator of cartilage-specific gene expression.

Medulloblastoma (MB) is the most common malignant brain tumor affecting children. The transcription factors MYCN and SOX9 are associated with initiation, maintenance and recurrence of MB and are also connected to more aggressive tumors. In this study, a ChIP was performed to isolate DNA from genes that are transcriptionally regulated by these proteins. Identification of these target genes will reveal new potential drug targets and help us better understand the functions of MYCN and SOX9. The ChIP was not fully optimized during this project and the target genes were not sent for sequencing and identified. To study the connection between SOX9 and recurrence, cells with different levels of SOX9 were treated with drugs, after which cell viability was measured. No significant difference in resistance could be measured. Change in expression level of MYCN, SOX9 and other relevant genes after drug treatment was also studied. The results show an increase in SOX9 and HES1, suggesting that these genes are involved in tumor recurrence.

Le carcinome basocellulaire (CBC) est le cancer le plus fréquent chez l’homme et est à lui seulresponsable de plus de cas que l’incidence cumulée de tous les autres cancers réunis. Des travauxrécents qui se sont intéressés aux étapes précoces de la tumorigenèse ont fourni un supportprécieux pour l’analyse du développement tumoral précoce. Ils ont mis en évidence le rôlecentral de gènes impliqués dans les progéniteurs embryonnaires des cellules souches du folliculepileux. Dans ce travail, nous avons étudié le rôle du facteur de transcription Sox9, un gène connupour être crucial dans la spécification de ces progéniteurs et dans les cellules souches du folliculepileux. Nous avons montré que Sox9 est requis pour la progression des lésions paranéoplasiquesvers les CBC et qu’il était également requis pour la maintenance des cellules cancéreuses. Enutilisant des méthodes d’analyse génomique de pointe, nous avons montré que Sox9 agit tantcomme un activateur que comme un répresseur de la transcription. Nous avons montré qu’ilpromeut directement le renouvellement cellulaire, l’invasion ainsi que la quiescence des cellulescancéreuses.Dans la seconde partie de ce travail, nous avons découvert le mécanisme d’action de l’inhibiteurd’Hedgehog Vismodegib sur les cellules cancéreuses. En utilisant des modèles génétiques desouris, nous avons montré que l’inhibiteur empêche la reprogrammation embryonnaire qui anormalement lieu durant la formation du CBC et qu’il promeut la différentiation des cellulescancéreuses vers une identité interfolliculaire, d’infundibulum ou sébacée suivant l’origine de cescellules. Nous avons également observé qu’une population caractérisée par l’expression du gèneLgr5 ainsi que par une activation de la voie de signalisation Wnt est plus résistante au Vismodegibchez la souris et nous avons trouvé des indices suggérant qu’il pourrait en être de même chezl’humain. Enfin, nous avons pu montrer que l’inhibition combinée des voies de signalisation Wntet Hedgehog annihile cette résistance et résulte en la disparition complète des CBCs. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12802号 / 論医博第2074号 / 新制||医||1001(附属図書館) / 80846 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 戸口田 淳也, 教授 松田 秀一 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19224号 / 医博第4023号 / 新制||医||1010(附属図書館) / 32223 / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 稲垣 暢也, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM