We recently reported that intestinal-specific overexpression of the POZ-ZF transcription factor Kaiso produced two prominent phenotypes in 1-year old mice: Kaiso transgenic (KaisoTg) mice presented with chronic intestinal inflammation, and an increase in secretory cell types – a trait typical of Notch signalling inhibition. Despite these findings however, the factor(s) responsible for Kaiso-mediated inflammation and secretory cell increases had not been elucidated. The primary goal of this thesis was to begin filling in this knowledge gap, by shedding mechanistic insight on Kaiso’s role in governing these two prominent phenotypes. First, we elucidated Kaiso’s role in the Notch signalling pathway and found that Kaiso inhibited the expression of the Notch1 receptor, and its ligand Dll-1, but promoted the expression of the Jagged-1 ligand. We postulated that the Kaiso-mediated reduction in Dll-1 might be responsible for the increase in secretory cell types, whereas Kaiso-mediated regulation of Jagged-1, which is dispensable for cell fate decisions, may be implicated in colon cancer progression. Importantly, we also found that Kaiso’s effects on Notch pathway inhibition occurred prior to the onset of chronic intestinal inflammation. Our analyses of the chronic inflammatory phenotype in KaisoTg mice demonstrated that Kaiso overexpression drives pathogenic neutrophil-specific recruitment (as evidenced by increases in neutrophil-specific enzymatic activity, the formation of crypt abscesses, and augmented expression levels of the neutrophils-specific chemokine, MIP2); an increase in the pore-forming Claudin-2; reduction of the cell adhesion protein E-cadherin; and abnormal intestinal epithelial repair mechanisms. Together, these findings imply that the pathogenesis of Kaiso-mediated intestinal inflammation is a multi-factorial process. A secondary goal of this thesis was to initiate studies to elucidate how the Kaiso binding partner, Znf131, might play a role Kaiso-mediated transcriptional regulation. We found that Znf131 indirectly associated with several Kaiso target genes, including Cyclin D1 (CCND1). Importantly, Znf131 activated a minimal CCND1 promoter previously shown to be inhibited by Kaiso. Moreover, Kaiso overexpression attenuated Znf131-mediated transcriptional activation and Znf131 expression in intestinal cells. Together, these findings hint that Znf131 and Kaiso may exert opposing biological functions, which may have implications in Kaiso-mediated intestinal homeostasis and disease. / Thesis / Doctor of Philosophy (PhD)

Man, Cheuk Him. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 219-263). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xii / List of Tables --- p.xvi / List of Publications --- p.xvii / Chapter Ch.l --- Introduction --- p.1 / Chapter 1.1 --- Aim of study --- p.1 / Chapter 1.2 --- Literature review --- p.3 / Chapter 1.2.1 --- Nasopharyngeal carcinoma (NPC) --- p.3 / Chapter 1.2.1.1 --- Structure and function of nasopharynx --- p.3 / Chapter 1.2.1.2 --- Histopathology of NPC --- p.3 / Chapter 1.2.1.3 --- Epidemiology of NPC --- p.4 / Chapter 1.2.2 --- Etiology of NPC --- p.6 / Chapter 1.2.2.1 --- Genetic factors --- p.6 / Chapter 1.2.2.2 --- Environment factors --- p.13 / Chapter 1.2.2.3 --- Epstein-Barr virus (EBV) infection --- p.14 / Chapter 1.2.3 --- Therapeutic treatment of NPC --- p.24 / Chapter 1.2.3.1 --- Radiotherapy (RT) --- p.24 / Chapter 1.2.3.2 --- Chemotherapy --- p.25 / Chapter 1.2.4 --- Notch signaling pathway --- p.26 / Chapter 1.2.4.1 --- Notch receptors and their ligands --- p.26 / Chapter 1.2.4.2 --- Activation of Notch signaling pathway --- p.29 / Chapter 1.2.4.3 --- Regulators of Notch signaling pathway --- p.32 / Chapter 1.2.4.4 --- Effectors of Notch signaling pathway --- p.32 / Chapter 1.2.5 --- Role of Notch signaling pathway in tumorigenesis --- p.33 / Chapter 1.2.5.1 --- Cell proliferation --- p.34 / Chapter 1.2.5.2 --- Cell survival --- p.35 / Chapter 1.2.5.3 --- Angiogenesis --- p.36 / Chapter 1.2.5.4 --- Cell invasion and metastasis --- p.36 / Chapter 1.2.6 --- Notch and oncogenic virus --- p.37 / Chapter 1.2.7 --- Crosstalk between Notch and other signaling pathways --- p.38 / Chapter 1.2.7.1 --- NFkB signaling pathway --- p.38 / Chapter 1.2.7.2 --- Ras signaling pathway --- p.39 / Chapter 1.2.7.3 --- Wnt signaling pathway --- p.40 / Chapter 1.2.7.4 --- Akt signaling pathway --- p.40 / Chapter 1.2.7.5 --- ErbB2 signaling pathway --- p.41 / Chapter 1.2.8 --- Notch as therapeutic target for cancer --- p.41 / Chapter Ch.2 --- Materials and Methods --- p.45 / Chapter 2.1 --- "Cell lines, xenografts and primary tumors" --- p.45 / Chapter 2.1.1 --- Cell lines --- p.45 / Chapter 2.1.2 --- Xenografts --- p.46 / Chapter 2.1.3 --- Primary tumors --- p.48 / Chapter 2.2 --- Reverse-transcription polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.2.1 --- Sample preparation for RT-PCR --- p.50 / Chapter 2.2.1.1 --- RNA extraction --- p.50 / Chapter 2.2.1.2 --- Quantitation of total RNA --- p.50 / Chapter 2.2.2 --- Conventional RT-PCR --- p.51 / Chapter 2.2.3 --- Quantitative RT-PCR --- p.51 / Chapter 2.3 --- Western immunoblot --- p.55 / Chapter 2.3.1 --- Protein extraction --- p.55 / Chapter 2.3.2 --- SDS-PAGE and immunoblotting --- p.55 / Chapter 2.4 --- Immunohistochemistry --- p.59 / Chapter 2.5 --- Cloning and plasmid DNA preparation --- p.62 / Chapter 2.5.1 --- Polymerase chain reaction (PCR) and purification of PCR products --- p.62 / Chapter 2.5.2 --- Restriction enzyme double digestion --- p.65 / Chapter 2.5.3 --- Ligation of plasmid and insert sequence --- p.65 / Chapter 2.5.4 --- Bacterial transformation --- p.66 / Chapter 2.5.5 --- Plasmid DNA extraction --- p.66 / Chapter 2.5.6 --- DNA sequencing --- p.67 / Chapter 2.6 --- Transient transfection of NPC cell lines --- p.67 / Chapter 2.7 --- Drug treatment on NPC cell lines --- p.69 / Chapter 2.8 --- Cell proliferation assays --- p.71 / Chapter 2.8.1 --- WST-1 assay --- p.71 / Chapter 2.8.2 --- BrdU assay --- p.71 / Chapter 2.9 --- Flow cytometry analysis --- p.72 / Chapter 2.9.1 --- Sample preparation --- p.72 / Chapter 2.9.2 --- Cell cycle analysis by propidium iodide staining --- p.73 / Chapter 2.9.3 --- Apoptosis analysis by AnnexinV-PI staining --- p.73 / Chapter 2.10 --- Apoptosis analysis by Caspase-3 activity assay --- p.74 / Chapter 2.11 --- RBP-Jk reporter assay --- p.75 / Chapter 2.12 --- NFKB1 reporter assay --- p.77 / Chapter 2.13 --- Dual luciferase reporter assay --- p.77 / Chapter 2.14 --- Expression array --- p.78 / Chapter 2.15 --- Statistical analysis --- p.79 / Chapter Ch.3 --- Characterization of Notch Signaling Molecules in NPC --- p.80 / Chapter 3.1 --- Introduction --- p.80 / Chapter 3.2 --- Results --- p.81 / Chapter 3.2.1 --- "Expression of Notch ligands, receptors, effectors and regulators in NPC cell lines and xenografts" --- p.81 / Chapter 3.2.2 --- "Expression of Notch ligands, receptors, regulators and effectors in NPC primary tumors" --- p.104 / Chapter 3.3 --- Discussion --- p.111 / Chapter 3.3.1 --- Overexpression of Jagl and D114 in NPC --- p.112 / Chapter 3.3.2 --- Overexpression of Notch receptors in NPC --- p.114 / Chapter 3.3.3 --- "Downregulation of Negative regulator, Numb, in NPC" --- p.116 / Chapter 3.3.4 --- Overexpression of Notch effectors in NPC --- p.117 / Chapter 3.4 --- Summary --- p.119 / Chapter Ch.4 --- Mechanisms of Activation of Notch Signaling Pathway in NPC --- p.120 / Chapter 4.1 --- Introduction --- p.120 / Chapter 4.2 --- Results --- p.122 / Chapter 4.2.1 --- EBV mediated Notch activation --- p.122 / Chapter 4.2.1.1 --- No effect of EBERs and EBNA1 on the expression of Notch Components --- p.122 / Chapter 4.2.1.2 --- LMP1 induces expression of Notch components --- p.129 / Chapter 4.2.1.3 --- LMP2A induces expression of Notch components --- p.133 / Chapter 4.2.2 --- Effect of CXCR4 on Notch signaling pathway in C666-1 --- p.137 / Chapter 4.3 --- Discussion --- p.139 / Chapter 4.3.1 --- EBV-mediated induction of Notch components --- p.139 / Chapter 4.3.2 --- Regulation of Notch expression by CXCR4 signaling pathway --- p.142 / Chapter 4.4 --- Summary --- p.145 / Chapter Ch.5 --- Investigation of the Oncogenic Role of Notch3 --- p.146 / Chapter 5.1 --- Introduction --- p.146 / Chapter 5.2 --- Results --- p.148 / Chapter 5.2.1 --- Effect of knockdown Notch 1 by siRNA on the growth of C666-1 --- p.148 / Chapter 5.2.2 --- Effect of knockdown Notch3 by siRNA on the growth of C666-1 --- p.151 / Chapter 5.2.2.1 --- Effect of knockdown Notch3 by siRNA on the RBP-Jk promoter activity of C666-1 --- p.153 / Chapter 5.2.2.2 --- Effect of knockdown Notch3 by siRNA on the proliferation of C666-1 --- p.155 / Chapter 5.2.2.3 --- Effect of knockdown Notch3 by siRNA on cell cycle progression of C666-1 --- p.158 / Chapter 5.2.2.4 --- Effect of knockdown Notch3 by siRNA on resistant to apoptosis in C666-1 --- p.160 / Chapter 5.2.3 --- Investigation of the anti-proliferation effect of therapeutic agents targeting Notch signaling pathway in NPC cells --- p.168 / Chapter 5.2.3.1 --- "Effect of DAPT on the proliferation of HEK293T, C666-1 and HK-1" --- p.168 / Chapter 5.2.3.2 --- Effect of AMD3100 on Notch signaling pathway and proliferation of NPC cells --- p.172 / Chapter 5.2.4 --- Study of downstream targets of Notch3 in NPC cells --- p.178 / Chapter 5.3 --- Discussion --- p.200 / Chapter 5.3.1 --- Oncogenic role of Notch3 in C666-1 --- p.200 / Chapter 5.3.2 --- Potential therapeutic approach in treating NPC via Notch inhibition --- p.206 / Chapter 5.3.2.1 --- "Gamma secretase inhibitor, DAPT" --- p.206 / Chapter 5.3.2.2 --- "CXCR4 antagonist, AMD3100" --- p.207 / Chapter 5.4 --- Summary --- p.209 / Chapter Ch.6 --- General Discussion --- p.210 / Chapter Ch.7 --- Conclusion --- p.217 / Reference --- p.219 / Appendices --- p.263 / Appendix 1 Summary of immunohistochemical staining results on 23 primary NPC samples --- p.264 / Appendix 2 Summary of 581 selected genes from the expression array --- p.265

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During hepatic disease the liver has the unrivalled ability to regenerate, by activating mature hepatocytes which can divide and thereby reconstitute the functional liver mass. However in the context of chronic hepatocellular disease the liver can regenerate from an endogenous population of hepatic progenitor cells (HPCs). The mechanisms which are involved in the activation and differentiation of these HPCs is not fully understood. To investigate whether there is a differential signalling requirement in HPCs acquiring a biliary versus hepatocellular fate we established in the laboratory two models of chronic liver damage and regeneration, one of which causes hepatocellular death, and results in infiltrating HPCs regenerating hepatocytes, and a second which causes biliary blockage and death, resulting in biliary regeneration. Here we describe how during biliary regeneration the Notch signalling pathway is highly expressed and activated. HPCs cells are consistently associated with a myofibroblast niche which expressed the ligand Jagged-1 at high levels. We have modulated the Notch signalling pathway in both a co-culture system and our models in vivo to demonstrate that Notch signalling is important in the specification of biliary cells, and that inhibition of this pathway both in vitro and in vivo results in the abrogation of biliary commitment. During hepatocellular regeneration we have found that the negative repressor of Notch signalling Numb is highly expressed in tandem with a low expression of the Notch pathway. We suggest that Wnt signalling maintains Numb within these HPCs at a high level and that this, along with stimulation of a hepatocellular programme allows HPCs to exit from a biliary fate and assume a hepatocellular phenotype. Finally we have found that macrophage ingestion of debris promotes the expression of Wnt, and that ablation of these cells results in a phenotypic switch between HPCs assuming a hepatocellular fate and a biliary one.

Haematopoiesis is the process to produce haematopoietic stem cells (HSCs), haematopoietic progenitors (HPCs) and terminally differentiated cell types. In the adult, HSCs resided in bone marrow while in the embryo, haematopoiesis occurred sequentially in several niches including yolk sac, aorta-gonad-mesonephros (AGM) region, placenta and fetal liver. The AGM region is the first place where HSCs arise in vivo and therefore should provide important factors to induce haematopoiesis. The mouse embryonic stem cells (mESC) system is a powerful platform to mimic the development process in vitro and is widely utilized to study the underlying mechanisms because they are pluripotent and can be genetically manipulated. A novel co-culture system has been established by culturing differentiating mESCs with primary E10.5 AGM explants and a panel of clonal stromal cell lines derived from dorsal aorta and surrounding mesenchyme (AM) in AGM region. Results of these co-culture studies suggested that the AM-derived stromal cell lines could be a potent resource of signals to enhance haematopoiesis. Molecular mechanism involved in haematopoiesis is a key research direction for understanding the regulation network of haematopoiesis and for further clinical research. A series of studies have demonstrated involvement of the Notch signalling pathway in haematopoiesis during development but with controversial conclusions because of the difference of models concerning various time windows and manipulating populations. This project aimed to investigate the role of Notch signalling pathway during haematopoiesis in the AGM environment. We analyzed the expression of Notch ligands in AGM-derived stromal cells with or without haematopoietic enhancing ability. No correlation was observed between ligand expression and haematopoietic enhancing ability in stromal cell lines or between Notch activity in EBs and haematopoietic enhancing ability. We demonstrated that inhibition of the Notch signalling pathway using the γ-secretase inhibitor could abrogate Notch activity in both mES-derived cells and the haematopoietic enhancing AM stromal cell line. To better understand the involvement of the Notch signalling pathway in a more specific spatial-temporal environment, we established a co-culture system of haemangioblast like cells (Flk1+) with one of AM region derived stromal cell lines with haematopoietic enhancing ability . We found that the AM stromal cell line could enhance Flk1+ derived haematopoiesis as assessed by haematopoietic colony formation activity and production of CD41+cKit+ progenitor cells. Based on the issue that the inhibitor could potentially affect both the ES cells and stromal cells, we carried out genetic approaches to overexpress or knock down Notch signalling pathway in this Flk1+/AM co-culture system. Interestingly, it was found that when Notch activity was enhanced in Flk1+ cells, the production of haematopoietic progenitors was inhibited and the number of cells expressing the pan-haematopoietic marker CD45 was reduced. By using the inducible dominant negative MAML1 system to knock down Notch activity, it was found that the haematopoiesis in the Flk1+/AM co-culture system was not affected, which could be accounted for the low Notch activity in this system. These results supported the hypothesis that the Notch signalling pathway plays a role in modulating Flk1+ derived haematopoietic differentiation within the AGM microenvironment.

Le manque d’outils diagnostiques et thérapeutiques efficaces font de l’adénocarcinome ductal pancréatique le cancer le plus létal pour les Canadiens, avec son taux de survie à 5 ans de 8 %. Il est caractérisé par une fréquence élevée de mutations de RAS (90 %) qui est requise pour l’initiation et le maintien de la carcinogenèse. De plus, il a été démontré qu’une activation aberrante de la voie de signalisation NOTCH coopère avec la signalisation RAS dans la promotion de la carcinogenèse pancréatique. La famille NOTCH comprend quatre récepteurs transmembranaires qui subissent une série de clivages protéolytiques suite à la liaison avec leurs ligands. Ces clivages permettent la libération du domaine intracellulaire de NOTCH (NIC) dans le cytosol. NIC transloque ensuite au noyau pour s’associer avec ses partenaires transcriptionnels CSL et MAML1 et favoriser l’expression de gènes cibles tels qu’HES1. Nous avons récemment démontré que l’activation des sérine/thréonine kinases ERK1/2, en aval de RAS, promeut l’expression d’HES1 de façon NOTCH-dépendante dans les cellules pancréatiques tumorales humaines. Bien que cela suggère une interaction entre les voies ERK et NOTCH, les mécanismes sous-jacents cette interaction restent à être identifiés. Il a été suggéré que PIN1, une prolyl-isomérase qui reconnait spécifiquement des résidus proline lorsque ceux-ci sont précédés d’une sérine ou d’une thréonine phosphorylée, interagit avec NIC1. Ainsi, nous avons émis l’hypothèse que la prolyl-isomérase PIN1 régule l’activité de la voie NOTCH et a un effet positif sur la croissance des cellules pancréatiques tumorales humaines. Pour y répondre, un modèle stable de cellules pancréatiques tumorales humaines MIAPaCa2 exprimant un shARN dirigé contre PIN1 a été généré. Nous avons démontré que PIN1 et la voie RAS/RAF/MEK/ERK régulent positivement les niveaux d’expression de NIC1. De plus, les niveaux d’expression de PIN1 régulent positivement les niveaux d’expression du récepteur NOTCH1 et du ligand DLL3 sans influencer les niveaux d’expression du récepteur NOTCH3 et des ligands JAGGED 1 et JAGGED 2. PIN1 a une influence positive sur les niveaux d’expression des gènes cibles de la voie NOTCH c-MYC et cycline D1 et une influence négative sur les niveaux d’expression d’HES1. Cette étude a également démontré que les niveaux d’expression de PIN1 influencent positivement la croissance en 2D ainsi que certains régulateurs du cycle cellulaire. Lorsque les cellules MIAPaCa2 sont ensemencées à faible densité, la taille des clones formés est influencée à la fois par les niveaux d’expression de PIN1 et par la voie RAS/RAF/MEK/ERK. Cependant, dans un contexte de croissance en indépendance d’ancrage, PIN1 influence négativement la croissance des cellules MIAPaCa2. Cette étude a démontré que PIN1 pouvait influencer le phénotype des cellules pancréatiques humaines. De plus, l’étude des mécanismes de régulation de la voie NOTCH a révélé une régulation complexe impliquant PIN1.

The polar lobe of Ilyanassa is asymmetrically partitioned into the D lineage of cells. Two of these cells, 3D and 4d, induce proper axial cell fate patterning in the embryo. Based on known embryological data in Ilyanassa, I hypothesized that Notch signaling would be required for this induction. I found that Notch signaling is required for cell fates induced by 4d and is temporally required well after 4d induction. Based on these results, I hypothesize that Notch signaling is involved in a reciprocal induction between the micromeres and the macromeres (endoderm) resulting in the maintenance of micromere fate induction and endoderm specification.Loss of the polar lobe results in the loss of cell fate induction by 3D/4d. Therefore, I hypothesized that proteins are asymmetrically bequeathed to the inducing D lineage cells by the polar lobe. To test this hypothesis, I compared global protein differences between two cell stage intact embryos, lobeless embryos, and isolated polar lobes by 2-Dimensional Electrophoresis analysis. I found several (12) quantitative differences between these samples including four spots enriched in the polar lobe isolates. I identified voltage-dependent anion-selective channel (VDAC) as one of the candidate proteins enriched in polar lobe isolates. I propose that VDAC is asymmetrically distributed by the polar lobe to the D cell and that it may function in D cell induction and mesendoderm fate specification.Lastly, I identify an acetylated tubulin antigen as a marker for cilia. I describe the pattern of cilia differentiation in the developing larvae that results in the formation of two ciliary bands, the prototroch and the metatroch, required for locomotion and feeding. These ciliary bands are conserved among annelid and mollusc larvae. Interestingly, the metatroch is derived from third quartet derivatives in the annelid Polygordius and from second quartet derivatives in the mollusc Crepidula. I provide evidence that the metatroch is derived from the first quartet derivatives in the mollusc Ilyanassa. Thus while the larval metatroch is conserved, its clonal origin is not. Based on these results, I provide support for the hypothesis that the metatroch is not homologous between annelids and molluscs or even among molluscs.

Vertebrates, annelids, and arthropods have evolved to form their body plans via segmentation. The question is whether this process stems from a common, segmented ancestor or if segmentation in these three phyla evolved from a series of independent events. To determine which of these theories is true, we look to determining whether these phyla share any pathways in the development of their segments. The Notch signaling pathway is a well-known pathway that vertebrates utilize for segmentation. Without it, somitogenesis does not occur properly as the segmentation oscillator is not functioning. Drosophila does not use this pathway for segmentation, but several other arthropods have recently been found to utilize it in the formation and maintenance of their segments (17-24). There has been debate as to whether Tribolium castaneum also uses the Notch pathway during segmentation as previous knockdowns of the Notch and Delta genes have led to a loss of segments and appendages/mouthparts (25-27). To determine this pathway’s involvement in Tribolium segmentation, I knocked down the Delta gene via eRNAi and attempted to determine Notch and Delta expression patterns via in situ hybridization. My results were inconclusive for determining the role of the Notch signaling pathway in segmentation. In the Delta dsRNA embryos, a loss of the labial segment, head and mouthpart defects, a loss of leg formation, and midline defects were seen. Future experiments need to be performed to determine whether an overexpression of mesoderm, ectoderm, or both is the cause of the defective ventral midline and whether this could lead to a loss of segments later in development. Overall, I can conclude that the Notch signaling pathway plays a role in mouthpart/leg development, the labial segment, and what I believe to be lateral inhibition between mesoderm and ectoderm determination.

In der Embryonalentwicklung von Insekten, Nematoden und Vertebraten reguliert der delta-notch-Signalweg vielfältige Zelldifferenzierungsvorgänge wie die laterale Inhibierung, Zelllinien-Entscheidungen und die Bildung von Grenzen. In Vertebraten aktivieren die transmembranen Liganden delta-like 1, 3 oder 4, bzw. jagged 1 oder 2 einen notch-Rezeptor (notch1, 2, 3 oder 4). Dessen intrazelluläre Domäne bildet im Zellkern mit rbp-j und weiteren Proteinen einen Aktivator-Komplex, der an den Promotor der notch-Zielgene bindet. Neben drei hes-Genen zählen dazu die Gene hey1, hey2 und heyL, die alle eng verwandt sind mit hairy und den Genen des Enhancer of Split-Komplexes (E(spl)) bei Drosophila melanogaster. Die hey-Gene sind in der Maus unter anderem während der Entwicklung von Niere, Arterien, Herz, Nervensystem, Thymus und Somiten spezifisch exprimiert. Um die Bedeutung des heyL-Gens für die Bildung dieser Organe zu untersuchen, wurden heyL-Knockoutmäuse generiert, bei denen jedoch keine morphologischen Veränderungen oder Erkrankungen erkennbar waren. Die durch entsprechende Verpaarung erhaltenen heyL/1-Doppelknockoutmäuse wiesen in der F9-Rückkreuzungsgeneration einen Ventrikel-Septum-Defekt (VSD) auf und verstarben größtenteils ein bis zwei Tage nach der Geburt. Ähnliche Krankheitssymptome zeigen die mit anderen Komponenten des delta-notch-Signalweges in Verbindung gebrachten Erbkrankheiten „Fallot´sche Tetralogie“ (ToF) und das Alagille Syndrom (AGS). Vergleichbare VSDs treten auch bei hey2-Knockoutmäusen auf. Es stellte sich daher die Frage, welche Zielgene des hey2-Transkriptionsfaktors an der Herzbildung beteiligt sind. Literaturbekannte HEY2-Zielgen-Kandidaten sind beim Menschen Follistatin (FST), Keratin 2-7 (KRT2-7) und das epitheliale V-ähnliche Antigen (EVA1). Durch antisense RNA in situ Hybridisierung von wildtypischen und hey2-Knockoutmausschnitten konnten jedoch keine unterschiedlichen Expressionsmuster entdeckt werden. Eigene Microarray-Analysen mit RNA aus hey2-überexprimierenden, humanen 293-Zelllinien identifizierten das Neurofilamentgen NEFL als HEY2-Zielgenkandidat. Bei der in situ Hybridisierung von wildtypischen und hey2-Knockoutmäusen mit einer Probe für das nefl-Gen konnten jedoch keine unterschiedlichen Expressionsmuster erkannt werden, so dass nefl in vivo vermutlich komplexer reguliert wird. Neben den Ventrikel-Septum-Defekten zeigen die heyL/1-Doppelknockoutmäuse einen zweiten abnormalen Phänotyp. Die embryonalen Thymi dieser Tiere sind kleiner als die der wildtypischen Mäuse. Die detaillierte Analyse der Thymozyten-Subpopulationen erbrachte, dass die niedrigere Zellzahl vor allem zu Lasten der doppelt positiven Thymozyten geht. Grund hierfür könnte eine teilweise Blockierung der Entwicklung zwischen der zweiten und dritten Phase des zuvor durchlaufenen doppelt negativen Stadiums sein, denn die absolute Zahl der DN3-Thymozyten ist um über 90 Prozent verringert. Die Blockierung des delta-notch-Signalweges durch Zugabe eines -Sekretase-Inhibitors führte ebenfalls zu einer Verringerung der DN3-Zellen in der gleichen Größenordnung, jedoch im Gegensatz zu den heyL/hey1-Doppelknockout-Thymi gleichzeitig zur Verdreifachung der absoluten Zahl der DN2-Zellen. Diese Unterschiede unterstreichen die Bedeutung weiterer notch-Zielgene wie beispielsweise hes1. Ob die beobachteten Veränderungen in der Entwicklung der Thymozyten Folgen für die Immunabwehr haben, wurde im Rahmen von Immunisierungen mit Trinitrophenyl (TNP)-Ovalbumin untersucht. Hierbei zeigte sich eine Erhöhung der IgG2a und IgG2b Werte und eine Reduktion der IgG1 Produktion bei den heyL/hey1-Doppelknockoutmäusen, während die IgM-Werte zwischen den verschiedenen Mausgenotypen keine signifikanten Unterschiede aufwiesen. Die IgG-Veränderungen deuten darauf hin, dass die T-Zellen vermehrt in die TH1-Zelldifferenzierungsrichtung getrieben werden und die TH2-Cytokinproduktion verringert ist. Bei den hey-Knockoutmäusen waren aufgrund des Expressionsprofils der hey-Gene nicht nur Veränderungen in der Herzentwicklung und im Thymus erwartet worden, sondern auch in der Somitogenese. Dies gilt im Besonderen für die bereits zuvor generierten hey2-Knockoutmäuse, denn hey2 ist im präsomitischen Mesoderm zyklisch exprimiert. Im Gegensatz zu den Knockoutmäusen des verwandten und ebenfalls zyklisch exprimierten hes1-Gens, litten jedoch weder die heyL/hey1-Doppelknockoutmäuse, noch die hey2-Knockoutmäuse an Defekten in der Somitogenese. Während die Bedeutung der Genexpression der hey-Gene in der Somitogenese weiterhin unklar sind, konnten Erkenntnisse über die Funktionen von heyL und hey1 in der Herzentwicklung und bei der Ausreifung der Thymozyten gewonnen werden. Die Identifizierung von hey-Zielgenen in diesen Entwicklungsprozessen kann das Verständnis des delta-notch-Signalweges erweitern, die Ursachen von Erbkrankheiten aufklären helfen und möglicherweise Therapiestrategien aufzeigen. / The delta-notch signaling pathway regulates numerous cell differentiation processes like lateral inhibition, cell lineage decisions and the formation of borders in the embryonic development of insects, nematodes and vertebrates. In vertebrates the transmembrane ligands delta-like 1, 3 or 4 and jagged 1 or 2 activate a notch-receptor (notch 1 – 4). In the nucleus its intracellular domain forms an activator-complex together with rbp-j and other proteins that binds to the promoter of notch target genes. Besides three hes genes these include the genes hey1, hey2 and heyL, all of which are closely related to hairy and Enhancer of Split-complex (E(spl)) genes of Drosophila melanogaster. In the mouse hey-genes are specifically expressed during the development of the kidney, arteries, heart, nervous system, thymus, and somites. In order to elucidate the significance of the heyL gene for the formation of these organs, heyL knockout mice were generated. However, these mice did not exhibited morphological changes or diseases. HeyL/1 double knockout mice, that were obtained by appropriate mating, showed ventricle septum defects (VSD) in the F9 backcross generation and most of them died within one or two days after birth. Similar symptoms are seen in the human congenital diseases Tetralogy of Fallot (ToF) and the Alagille Syndrome (AGS), which are associated with other components of the delta-notch signaling pathway. Similar ventricle septum defects also occur in hey2 knockout mice. This raises the question, which of the target genes of the hey2 transcription factor are involved in the formation of the heart? Published human HEY2-candidate target genes are Follistatin (FST), Keratin 7 (KRT2-7) and the Epithelial V-like Antigen (EVA1). Yet, antisense RNA in situ hybridisation of wildtype and hey2 knockout mouse sections did not reveal any differential expression patterns. Own micro array analysis with RNA from hey2 overexpressing human 293 cell lines identified the neurofilament gene NEFL as a HEY2-target gene candidate. However, in situ hybridisation of wildtype and heyL/1-double knockout mice with a probe for the nefl gene did not reveal differential expression patterns. Thus, the regulation of the nefl expression in vivo is presumably more complex. In addition to the VSD, heyL/1 double knockout mice show a second abnormal phenotype. The embryonic thymus of these animals was smaller than that of the wildtype mice. Detailed analysis of the thymocyte subpopulations revealed, that the low cell number is mainly due to the douple positive thymocytes. The reason for this could be a partial block of the development between the second and third phase of the previous double negative stage since the absolute number of DN3 thymocytes is reduced by over 90 percent. Blocking of the delta-notch signaling pathway by the addition of a -secretase inhibitor also led to a reduction of the DN3 cells to the same extent. Yet in contrast to the heyL/hey1 double knockout thymi, a tripling of the absolute number of DN2 cells is observed. These differences underscore the importance of other notch target genes like for example hes1. To investigate if the observed changes in the development of the thymocytes have any consequences for the immune defence, immunisations with trinitrophenyl (TNP)-ovalbumine were performed. In these experiments an elevation of the IgG2a and IgG2b values and a reduction of the IgG1 production in heyL/hey1 double knockout mice was detected. IgM values did not vary significantly between the different mouse genotypes. The IgG changes indicate that the T-cells are increasingly driven towards the TH1 cell differentiation and that TH2-cytokine production is reduced. Based on the expression profiles of hey genes, developmental changes were expected for hey-knockout mice not only during development of the heart and thymus, but also during somitogenesis. This particularly applies to the previously generated hey2 knockout mice because hey2 is expressed in the presomitic mesoderm in an oscillating manner. Yet, in contrast to the knockout mice of the related and also cyclically expressed hes1 gene, neither the heyL/hey1 double knockout mice nor the hey2 knockout mice suffered from defects during somitogenesis. While the relevance of hey gene expression during somitogenesis remains unclear, new insights in the function of heyL and hey1 during heart development and during the maturation of thymocytes were gained. The indentification of hey target genes in these developmental processes can enhance the understanding of the delta-notch signaling pathway. It may also help to explain the causes of inherited diseases and potentially point out therapeutic strategies.

Thesis advisor: Marc A. T. Muskavitch / Notch signaling is an evolutionarily conserved developmental pathway regulated by two classes of transmembrane proteins: the Notch receptors and the Delta/Serrate/LAG-2 (DSL) ligands. Notch and DSL ligands mediate cell-cell communication that results in a downstream signaling cascade that affects many aspects of metazoan development. Additional regulatory mechanisms that affect Notch signaling are being discovered continuously, and recent findings highlight the importance of endocytosis, ubiquitylation and subcellular trafficking as essential requirements for proper signaling. In order to obtain further insights into the regulation of Notch signaling, I took a two-fold approach, combining genetic and molecular techniques in <italic>Drosophila</italic>. First, I took part in a large-scale transposon-based screen in the developing <italic>Drosophila</italic> eye to identify additional genes involved in the pathway. We screened 10,447 transposon lines from the Exelixis collection for modifiers of cell fate alterations caused by overexpression of Delta, and we identified 170 distinct modifier lines that may affect up to 274 genes. I further analyzed a previously uncharacterized gene, which we have named <italic>Amun</italic>, and showed that it localizes to the nucleus and contains a putative DNA glycosylase domain. Further analyses of Amun reveal that altered levels of Amun function interfere with cell fate specification during eye and sensory organ development. Second, to investigate structural requirements for ubiquitylation of Delta, I analyzed four individual lysine residues in the Delta intracellular domain, and assessed their necessity for Delta signaling activity. I find that a conserved residue, DeltaK742, is essential for Notch signaling in the <italic>Drosophila</italic> imaginal wing disc and is apparently required for ubiquitylation of Delta by the E3 ubiquitin ligase, Mind bomb1 (one of two E3 ubiquitin ligases required for Delta signaling activity). Taken together, the findings from this thesis research contribute to the advancement of our understanding of different aspects of Notch signaling and Notch-mediated development. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.