Thymic epithelial cells (TECs) are indispensable for the development of T cells in the thymus. Two subtypes of TECs exist in the thymus, medullary mTECs and cortical cTECs. Both mTECs and cTECs originate from endodermal thymic epithelial progenitor cells (TEPCs) in the embryo, but how the differentiation of TEPCs is regulated is not well understood. The aims of this thesis were to establish the role of Notch signalling in TEPC differentiation, and how it interacts with known regulators such as FOXN1 and the NFκB pathway. Gene expression data showed that Notch is active in TEPCs and exhibits a correlation with the mTEC lineage. Loss of Notch function led to a significant reduction in the number of mTECs in the thymus, and this can be attributed to aberrant mTEC specification. Furthermore, the duration of Notch activity in determining mTEC number appears limited to the early phase of organogenesis, and precedes RANK/NFκB mediated mTEC proliferation. Gain of Notch function resulted in a considerable shift to a primitive, TEPC-like phenotype, and subsequently a latent increase in mTEC frequency. Finally, transcriptomic and functional analyses pointed to a cross-repressive mechanism between Notch and FOXN1 in TEPCs. Taken together, these results identified Notch as a novel regulator of mTEC specification, likely through maintaining the potency of fetal TEPCs, a prerequisite for mTEC lineage commitment.

Delta-like homologue 1 (Dlk1) and Dlk2 encode vertebrate-specific transmembrane proteins belonging to the Jagged/Delta/Serrate family of Notch ligands. Murine Dlk1 is widely expressed during embryonic development and targeted deletion results in defects in numerous developmental processes, such as adipogenesis, haematopoiesis, neurogenesis and skeletal muscle formation. However, the mechanisms by which DLK1 regulates these processes remains unclear. The purpose of this project is to examine the function of these genes using zebrafish as an in vivo model, allowing insight to the ancestral functions of these genes. We have strong evolutionary evidence that dlk2 is the ancestral version of the gene from which dlk1 is derived; therefore, the thesis focuses primarily on the role of dlk2 in the zebrafish system. I initially examine the expression of zebrafish dlk1 and dlk2 during embryonic development and in the adult brain, determining similarities and differences between mouse and zebrafish. In particular, dlk1 and dlk2 in the fish exhibit a pattern that is more reminiscent of Dlk2 in the mouse. This developmental expression pattern is essential for the interpretation of the modulation of Dlk2 in later chapters, and is aided by the generation of a mammalian Dlk2 antibody that cross-reacts with zebrafish. We obtained a dlk2 mutant and used this line to examine the role of the DLK2 protein in development and in the adult brain. I demonstrate that, in the absence of DLK2, a population of neural precursor cells appear to over-proliferate early in zebrafish development. Later, by larval stages, these cells are absent, suggesting a premature activation and subsequent depletion of the progenitor cell pool in the mutant, reminiscent of the Dlk1 mutant in mouse. Associated with this phenotype are larval behavioral defects in motor response. In this thesis, it will be shown that in the adult dlk2 mutant zebrafish, the radial glial cell population in the telencephalon is completely depleted. These radial glial cells are thought to be responsible for adult neural regeneration in zebrafish, and our characterization of a mutant completely lacking this cell population provides a rich model to further examine and understand the functions of this well-studied but poorly understood cell population. These findings have both functional and evolutionary implications for the relative roles of these two vertebrate specific atypical Notch ligands.

A paracoccidioidomicose (PCM) é uma micose sistêmica de natureza profunda e granulomatosa, que afeta preferencialmente o tecido pulmonar causada pelo Paracoccidioides brasiliensis, um fungo que exibe dimorfismo térmico. O P. brasiliensis interage com células apresentadoras de antígenos (APCs), alterando suas principais funções biológicas. Entre as APCs, os macrófagos são células que desempenham um papel importante na indução e regulação da resposta imune e/ou inflamatória. São células do sistema fagocítico mononuclear que podem discriminar entre o que é próprio do organismo e os patógenos, através da expressão de receptores de reconhecimento padrão (PRR) que reconhecem padrões moleculares associados à patógenos (PAMPs), sendo células importantes no processo de fagocitose controlando o crescimento destes patógenos. Recentemente tem sido demonstrada a importância dos receptores Notch na regulação da atividade de macrófagos e do sistema imune. Os ligantes de Notch estão envolvidos nas locais inflamatórios de infecção devido ao aumento da expressão dos mesmos na superfície de células envolvidas neste processo. Considerando que a sinalização Notch pode estar envolvida na modulação da função de macrófagos, nós avaliamos se P. brasiliensis tem a capacidade de modular a ativação desta via e interferir na produção de citocinas pró-inflamatórias. Para isso, macrófagos J774, pré-estimulados com LPS ou não, foram interagidos com leveduras do fungo, seguido por análise de PCR em tempo real e citometria de fluxo, dosagem de citocinas e índice de fagocitose. Nossos dados revelaram que na presença do fungo existe aumento dos níveis de transcrição do receptor Notch 1 e diminuição da transcrição do ligante Delta 4 em macrófagos pré-estimulados com LPS. Entretanto, verificamos que o fungo sozinho não é capaz de induzir a transcrição de NF-κB, nem na presença do LPS, mas quando os macrófagos são estimulados com LPS e sofrem inibição da via de Notch existe aumento dos níveis de transcritos após interação com o fungo, sugerindo que esse fator é ativado na ausência de Notch. Nesse contexto de inibição de Notch, evidenciamos que a fagocitose de leveduras do fungo por macrófagos tornou-se mais eficiente, visto que houve aumento do índice de fagocitose na ausência de Notch. Foi possível verificar que o fungo tem a capacidade de promover a produção de IL-6 via TLR-Notch, fazendo-nos supor que essa citocina seja importante para o estabelecimento da doença ao ser benéfica para o fungo e prejudicar o hospedeiro. E concomitantemente ao aumento de IL-6 existe diminuição da produção de TNF-α. Com base nesses resultados, podemos sugerir que o P. brasiliensis utiliza a via de sinalização Notch como um mecanismo de escape. A interação entre as leveduras do fungo e os macrófagos promove a ativação dessa via, através do receptor Notch 1, induzindo maior produção de IL-6, citocina importante para o crescimento do fungo no hospedeiro, conjuntamente com a diminuição de TNF-α prejudicando a atividade fungicida dos macrófagos. / Paracoccidioidomycosis (PCM) is a systemic mycosis and deep granulomatous in nature, which affects mainly the lung tissue caused by Paracoccidioides brasiliensis, a fungus that exhibits thermal dimorphism. The P. brasiliensis interacts with antigen presenting cells (APCs), changing its main biological functions. Among the APCs, macrophages are cells that play an important role in the induction and regulation of the immune response and/or inflammatory response. They are cells of the mononuclear phagocytic system that can discriminate between what is characteristic of the organisms and pathogens, by expression of pattern recognition receptors (PRR) that recognizes the pathogen-associated molecular pattern (PAMPs), and are considered cells important in phagocytosis for controlling the growth of these pathogens. It has been recently demonstrated the importance of the Notch receptor in regulating the activity of macrophages and of the immune system. The ligands of Notch are involved in inflammatory sites of infection because there are increased expression of these ligands on cell surface involved in this process. Whereas the Notch signaling may be involved in modulating macrophage function, we evaluated whether P. brasiliensis has the ability to modulate the activation of this pathway and interfere with the production of pro-inflammatory cytokines. For this, J774 macrophages, pre-stimulated with LPS or not, are interacted with yeast fungus, followed by Real Time PCR analysis and flow cytometry, cytokine and phagocytosis index. Our data showed that the presence of the fungus exists increased levels of transcription of the Notch 1 receptor, and a decrease in ligand Delta 4 transcription on macrophages pre-stimulated with LPS. However, we found that the fungus itself is not able to induce transcription NF-κB, even in the presence of LPS, but when macrophages are stimulated with LPS and suffer inhibition of the Notch signaling, exists increased levels of transcripts after interaction with the fungus, suggesting that this factor is activated in the absence of Notch. Within the context of inhibition of Notch, we found that phagocytosis of yeasts by macrophages become more efficient, since the increased rate of phagocytosis in the absence of Notch. It was verified that the fungus has the ability to promote the production of IL-6 via TLR-Notch, making us suppose that this cytokine is important for the establishment of the disease to be beneficial for the fungus and damage the host. And concurrently with increased IL-6 there is decreased production of TNF-α. Based on these results, we suggest that P. brasiliensis uses the Notch signaling pathway as an escape mechanism. The interaction between the yeasts with macrophages promotes the activation of this pathway, by means of a Notch 1 receptor, inducing increased production of IL-6 cytokine important for the growth of fungus on host, together with a reduction of TNF-α, contributing with a damaging fungicidal activity of macrophages.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18905号 / 医科博第61号 / 新制||医科||4(附属図書館) / 31856 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 河本 宏, 教授 武田 俊一, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

NOTCH1 est un récepteur transmembranaire qui, suite à la liaison de son ligand, subit une série de clivages protéolytiques libérant un fragment intracellulaire actif NIC1. Une fois libéré, NIC1 transloque au noyau où il s’associe à des cofacteurs transcriptionnels tels CSL et MAML1 afin d’initier la transcription de gènes cibles dont HES1. La voie Notch joue un rôle dans différents processus cellulaires, dont la prolifération, la mort cellulaire et la différenciation cellulaire. En conséquence, une mauvaise régulation ou une perte d’activité de cette voie de signalisation mène à des maladies humaines telles que des pathologies du développement et le cancer. De ce fait, la voie Notch est grandement régulée à divers niveaux. Entre autres, NIC1 subit des modifications post-traductionnelles qui régulent sa stabilité et par conséquent influence la durée du signal NOTCH1. L’activité transcriptionnelle de NIC1 est également régulée via le recrutement coordonné de cofacteurs transcriptionnels. Malgré tout, très peu de choses sont connues quant aux modifications post-traductionnelles pouvant moduler spécifiquement l’activité du complexe transcriptionnel formé par NIC1. De plus, les études jusqu’à présent n’ont pas permis d’identifier tous les partenaires d’interaction se retrouvant dans le complexe transcriptionnel NIC1/CSL/MAML1 et pouvant moduler son activité. Ainsi, l’objectif de cette étude était d’établir un système permettant l’identification de nouveaux partenaires transcriptionnels de NIC1. Nous avons exprimé une forme tronquée et constitutivement clivée de Notch1 et, suivant des analyses par spectrométrie de masse, nos résultats suggèrent l’interaction possible de NIC1 avec des membres du complexe médiateur et d’autres protéines impliquées dans la régulation génique. Nous avons également exprimé un NIC1-GFP et nos travaux ont permis de montrer qu’il était principalement localisé au noyau, où il s’associe à l’ARN polymérase II, et qu’il est dégradé par le protéasome. Suivant des analyses par spectrométrie de masse, nos résultats suggèrent l’interaction possible de NIC1 avec plusieurs régulateurs transcriptionnels et des protéines impliqués dans l’ubiquitination/Sumoylation. Bref, les analyses protéomiques sont un bon système d’exploitation pour l’identification de nouveaux partenaires d’interaction de NIC1. Ainsi, notre étude permettra une meilleure compréhension de la signalisation Notch d’apparence simple, mais plutôt complexe.

Methylmercury (MeHg) is a ubiquitous environmental toxin. Exposure to MeHg in humans occurs primarily through the consumption of contaminated seafood. MeHg has been shown to act most strongly during neural development. Epidemiological data on the effect MeHg exposure through seafood has on children and fetuses is conflicted, with large cohort studies showing both presence and absence of MeHg-induced deficits in achieving developmental milestones. Because of this uncertainty in the literature it is important that we come to understand the mechanisms of MeHg toxicity so that we might advise the public more accurately on the risks of MeHg exposure. Research into the mechanisms of MeHg toxicity has found a number of cellular and molecular effects including disruptions of microtubule formation, Ca2+ homeostasis, and glutamate signaling. However, none of these effects of MeHg fully explains its neurodevelopmental specificity. Previous work in Drosophila neural-derived cell lines has shown that MeHg causes upregulation of the canonical Notch response gene E(spl)m . The Notch pathway is crucial to neural development and perturbation of a Notch target may explain the developmental specificity of MeHg. In this dissertation I describe experiments I performed to test the hypothesis that the observed upregulation of E(spl)m plays an important role in MeHg toxicity in Drosophila. I first describe experimental evidence that E(spl)m is upregulated by MeHg treatment in vivo in Drosophila embryos in addition to cells, as has previously been shown. By contrasting the effects of the toxic inorganic mercurial HgCl2 with MeHg I show that the E(spl)m expression response to MeHg is not simply a stress response and is a likely specific activity of MeHg. I also show that the effect of MeHg on E(spl)m expression is not simply due to a developmental delay induced by the toxin. I also identify two neural phenotypes of MeHg toxicity in Drosophila embryos, in the outgrowth of the intersegmental and segmental motor nerves. Genetic manipulation causing overactivity of the Notch pathway in neurons can mimic these phenotypes. However, induced expression of E(spl)m in neurons does not cause a failure of motor nerve outgrowth. Upon further examination I demonstrate that endogenous expression of E(spl)m occurs in the muscle. Induced E(spl)m expression in the muscle causes a segmental nerve phenotype similar to MeHg treatment, indicating a role for E(spl)m in MeHg toxicity in this system. MeHg treatment and E(spl)m overexpression in the muscle causes a failure of normal muscle development. Yet, this gross developmental abnormality only partially explains the observed motor nerve phenotype. E(spl)m is unique among the E(spl) genes in its ability to cause these muscle and motor nerve phenotypes as shown by contrasting genetic manipulation of the closely related E(spl)m . Overall my findings support the hypothesis that MeHg toxicity in Drosophila is mediated in part by E(spl)m . They also suggest that E(spl)m plays an important role in the formation of the muscle during embryonic development, contributing to the literature describing disparate functions for E(spl) genes despite structural similarities. Finally, my findings suggest that MeHg may be able to impact neural development through toxicity in supporting tissues rather than neurons themselves. This final finding has implications for the study of MeHg toxicity in humans, and is supported by previous findings that describe a role of glia in modulating MeHg neurotoxicity.

Chronic liver disease remains a significant cause of morbidity and mortality globally. Transplantation is the only effective treatment for end-stage disease but is limited by organ availability, surgical complications and risks of long term immunosuppression. Novel therapies for advanced disease are therefore required. The liver has a remarkable capacity to regenerate through division of mature hepatocytes, however in chronic or severe disease hepatocyte replication fails, senescence occurs and liver failure ensues. Ductular reactions (DRs), containing hepatic progenitor cells capable of repopulating the parenchyma, arise in chronic liver injury when hepatocyte regeneration is impaired. Enhancing this endogenous repair mechanism is a key therapeutic goal. Notch and Wnt are key signals required for liver regeneration, however to date they have principally been characterised in end-point disease and the temporal kinetics of these signalling pathways not known. I sought to identify if these signals control expansion of DRs after hepatocyte injury and whether they can be therapeutically manipulated. I examined the dynamics of Notch and Wnt activity using a genetic model of hepatocellular injury and ductular-mediated regeneration whereby induction of injury could be timed, synchronising the regenerative response. Using lineage tracing, small molecules, blocking antibodies and genetic loss of function experiments I defined distinct time-sensitive Notch and Wnt signatures where early regeneration is driven by Notch and the later response by Wnt. I demonstrated that inhibition of Notch1 and Notch3 but not Notch2 reduces the generation of DRs. I identified that DRs were a source of potent growth hormone IGF1 and this production was Wnt driven. Notch driven expression of IGF1-receptor within DRs identified this axis as a node for cooperation between Notch and Wnt signals. Blocking the IGF1 axis prevented DR expansion, which conversely could be enhanced by administration of recombinant IGF1. Here, I functionally defined complex temporal dynamics controlling of DRs and identified therapeutic pathways to enhance liver regeneration.

In the mid-gestation embryo, the first definitive transplantable hematopoietic stem cells (dHSCs) emerge by embryonic day E10.5-E11 in the aorta-gonadomesonephros (AGM) region, as a result of a step-wise maturation of precursors called pre-HSCs. The analysis of several Notch mutants suggests that Notch signalling is essential for the execution of the definitive hematopoietic programme in the AGM. Mouse embryos deficient for Notch1, RBP-Jk or Jagged1 cannot efficiently generate intra-embryonic hematopoeitic progenitors. It has also been reported that knockdown of Notch target genes (Hes1, Hes5) results in hematopoietic impairment. However a clear picture of the role of Notch pathway in HSC development is still missing. In this work we characterised precise stages and cell types during HSC development in which Notch signalling is involved. First we used a Hes1-dEGFP reporter mouse line that allowed us to monitor Notch pathway activity in a narrow window of time. The results suggest that the level of Notch activity fluctuates in HSC lineage in the AGM region and is down-regulated in dHSCs in the foetal liver (where dHSCs migrate after generation in the AGM region). By using transplantation assay, we further showed that fluctuations of Notch activity are essential for HSC development, and that this pattern in the HSC lineage might work as a switch between maturation and proliferation of PreHSC1, PreHSC2 and dHSC, in which temporary decrease might be required to mature from one type to another, both in vitro and in vivo. These findings might need to be taken into consideration for in vitro generation of haematopoietic stem cells, where a fine tuning of Notch signalling activity could greatly improve their emergence.

Effect of Manipulation of Notch Signaling Pathway on Neural Stem Cell Proliferation in the Hippocampus Following Traumatic Brain Injury By Seung L. Kim A thesis statement submitted for degree requirement in Mater of Science Virginia Commonwealth University, 2019 Advisor: Dong Sun, MD. PhD. Department of Anatomy & Neurobiology The Notch signaling pathway is known as a core signaling system in maintaining neural stem cells (NSCs) in embryonic development and adulthood including cell proliferation, maturation, and cell fate decision. Proliferation of NSCs persists throughout lifespan in neurogenic niches and is often upregulated following neurological insults including traumatic brain injury (TBI). Therefore, NSCs are viewed as the brain’s endogenous source for repair and regeneration. We speculate Notch signaling pathway is also involved in injury-induced cell proliferation in the neurogenic niche following TBI. TBI, which is a leading cause of death and disability, has been a huge burden to our society. Many efforts have been made in attempt to treat and manage TBI. In this study, we examined the involvement of Notch signaling pathway in injury induced NSC proliferation in the neurogenic niche, by administering exogenous Notch ligands including, Notch agonist or antagonist. Adult rats were intraventricularly infused with Notch1 receptor agonists (anti-Notch1 antibody at the dose of 0.5, 2 or 4μg/ml), Notch1 receptor antagonist (recombinant Jagged1 fusion protein at the dose of 25, 50 or 100μg/ml) or vehicle for 7 days following TBI. 5-bromo-2-deoxyuridine (BrdU) was administered single daily via intraperitoneal injection to label proliferating cells for 7 days post injury. The animals were sacrificed on the 7th day at 2 hours after the last BrdU injection. Sequential vibratome sliced coronal brain sections were processed for proliferation marker BrdU, Ki67 or immature neuronal marker DCX staining. BrdU, Ki67 or DCX-labeled cells in the dentate gyrus of the hippocampus were quantified using unbiased stereological method. We found TBI in the form of moderate lateral fluid percussion injury (LFPI) induced cell proliferation was further augmented by 7-day infusion of Notch agonist (Notch1-2μg/ml) as shown by BrdU and Ki67 labeling. Further, 7-day infusion of Notch antagonist (Jagged1-50μg/ml) post-injury greatly reduced the number of BrdU+ cells. However, ambiguous dose related responses were also observed where 7-day infusion of higher dose of Notch agonist (Notch1-4μg/ml) resulted in reduced cell proliferation. No major changes in the numbers of newly generated neurons were observed across the animals, except a slight reduction in Notch agonist (Notch1-2μg/ml) and Notch antagonist (Jagged1-50μg/ml) infused animals as shown by DCX labeling. Infusion of Notch agonist or antagonist affects NSC proliferation following TBI suggesting the involvement of Notch signaling pathway in regulating post-TBI NSC proliferation in the neurogenic niche. For the unexpected opposite results of higher dosing of Notch 1 agonist, the presence of other Notch receptors regulating NSC in the neurogenic niche should be considered. Future studies involving selective manipulation of these Notch receptors and their downstream effectors would clear some results.

Die Neuralentwicklung wird durch eine Vielzahl von Genen reguliert. Hierbei scheint der Notch-Signaltransduktionsweg eine wichtige Rolle zu spielen. Die primären Zielgene der Notch-Signalkaskaskade sind die Hes- und Hey-Gene. Die genaue Bedeutung der Signalkaskade, der Hes- und insbesondere der Hey-Gene für den Differenzierungsprozess neuraler Stamm- und Vorläuferzellen ist noch nicht bekannt. Ziel dieser Arbeit war es, die Aufgaben von Notch und der Hey-Gene beim Differenzierungsprozess neuraler Stamm- und Vorläuferzellen genauer zu untersuchen. Da das gezielte Ausschalten eines Gens eine aussagekräftige Methode zur Erforschung seiner Funktion ist, wurden zunächst neurale Stamm- und Vorläuferzellen in Form von sogenannten Neurosphären von Hey1-/- und Hey2-/- Mäuseembryonen mit denen von Wildtyp-Mäuseembryonen verglichen. Dabei differenzierten bei den Hey1-/- Neurosphärenkulturen ca. 6,6% (bei den Kontrollen 6,6%) und bei den Hey2-/- Kulturen 10,9% (Kontrollen 8,7%) der Zellen zu Neuronen. Eine komplette Inhibierung der Notch-Signalkaskade wurde durch das Etablieren von RBP-Jκ-/- Kulturen erreicht. RBP-Jκ-/- Zellen waren jedoch während der Differenzierung nur noch zu einem kleinen Teil in der Lage zu adhärieren, was weitere Experimente mit den Zellen unmöglich machte. Als nächstes sollten Neurosphären mit einer Überexpression von Hey1 mit Wildtyp-Neurosphären verglichen werden. Es gelang allerdings aufgrund einer schon hohen Ausgangsexpression von Hey1 in Neurosphären nur, die Expression zu verdreifachen, was für weitere Experimente nicht ausreichend schien. Um die Prozesse, die auf Genebene während der Differenzierung neuraler Stamm- und Vorläuferzellen ablaufen, besser zu verstehen, wurden die Expression von Genen, die in der Neuralentwicklung und in der Notch-Signalkaskade eine Rolle spielen, in Wildtyp-Neurosphärenzellen mittels qRT-PCR zu verschiedenen Differenzierungszeitpunkten quantifiziert. Die Genexpression von Hes1 und Hes5, sowie Hey2 wurde während der Differenzierung zum Teil deutlich herunterreguliert. Die Gene Hes3, Hey1 und Id4 hingegen stiegen zunächst bis Tag 3 stark an, um dann an Tag 7 und 14 etwa wieder den Ausgangswert zu erreichen. Die Regulation der Gene war insgesamt recht uneinheitlich und nicht immer nachvollziehbar. Da diese teils widersprüchlichen Ergebnisse auf die vorgegebene Heterogenität einer Neurosphärenkultur zurückzuführen sein könnten, wurde nach einer Alternative zur Neurosphärenkultur gesucht. Deshalb wurde im Weiteren versucht homogene Monolayerkulturen nach einem Protokoll von Conti et al. (2005) zu etablieren. Das Protokoll musste aber an einigen Stellen angepasst werden, um adäquate Kulturen zu erhalten. Da die Monolayerkulturen auf Gelatine nicht gut hafteten wurde auf eine Polyornithin-Beschichtung umgestellt. Außerdem wurde das NS-A Medium mit N-2 Zusatz aufgrund schlechter Proliferation der Zellen auf Neurobasalmedium mit B-27 Zusatz umgestellt. Färbungen dieser Monolayerkulturen zeigten, dass sich fast alle Zellen mit dem Stammzellmarker Nestin anfärben ließen und keine Zellen Tuj1+ (Neuronen) und nur einige wenige Zellen Gfap+ (Astrozyten) waren. Es ist deshalb wahrscheinlich, dass die Zellen in einer Monolayerkultur unter den oben beschriebenen Bedingungen hauptsächlich undifferenziert sind und neuralen Stamm- bzw. Vorläuferzellcharakter haben. / Neural development go on in three sequential processes. First expansion of neural stem and precursor cells, then neurogenesis and last the gliogenesis take place. There are different methods to analyse this development in vitro. Cultivation of neurospheres and monolayers are two important possibilities. The cultivation is followed by differentiation to neurons, astrocytes and oligodendrocytes. Many protocols for cultivation and differentiation of neurospheres and monolayers were tested. But no method did work adequately. So an own procedure was established. Monolayer culturing on polyornithine with Neurobasalmedium®(Gibco) supplemented with B27®(Gibco), FGF and EGF and passaging with Accutase®(PAA) worked best. Differentiation with NeuroCult® NSC Differentiation Supplement(StemCell Technologies) added to Neurobasalmedium showed best results. The neural development is regulated by a lot of genes. Especially the Notch signaling cascade seams to play a central role. The exact role of this cascade and particularly of Hey and Hes genes is not known yet. To analyse the importance of these genes for the differentiation process, the expression of some of these and other genes were quantified by qRT-PCR. The results revealed, that Blbp and Hes5 were downregulated massively. Several differentiated Hey1-/- and Hey2-/- were compared with wildtype lineages. No significant differences in number of neurons (about 8%) and astrocytes (about 85%) were seen. A complete inihibtion of the Notch signaling cascade was reached through establishment of RBP-Jκ-/- lineages. RBP-Jκ-/- cells were no longer able to attach, but didn’t die, after differentiation was initiated. So RBP-Jκ may play a role for the attachment of differentiating neural stem and precursor cells.