The regulation of neural precursor cells (NPCs), which encompass neural progenitor and neural stem cells (NSCs), is fundamental for proper brain development and function. These cells are regulated by orchestrated signalling within their local environment. Aberrant aspects of cell proliferation, differentiation, survival, or integration have been linked to various neurological diseases including Fragile X syndrome (FXS)—a disorder characterized by intellectual and social changes due to the silencing of the gene encoding FMRP. The biology of hippocampal NPCs in FXS during early postnatal development has not been studied, despite high FMRP expression levels in the hippocampus at the end of the first postnatal week. In this thesis, the Fmr1-knockout (KO) mouse model was used to study hippocampal cell biology during early postnatal development. A tissue culture assay, used to study the effect of astrocyte-secreted factors on the proliferation of NSCs, indicated that astrocyte secreted factors from Fmr1-KO brains enhanced the proliferation of wild type, but not Fmr1-KO NSCs (Chapter 3). Next, the proliferation and cell cycle profiles of NPCs in vitro and in vivo studied with immunocytochemistry, Western blotting, and flow cytometry revealed decreased proliferation of NPCs in the Fmr1-KO hippocampus (Chapter 4). Finally, cells isolated from the P7 dentate gyrus and characterized by flow cytometry, showed a reduced proportion of NSCs and an increased proportion of neuroblasts—neuronal committed progenitors—in Fmr1-KO mice. Together, these results indicate that hippocampal NPCs show aberrant proliferation and neurogenesis during early postnatal development. This could indicate stem-cell depletion, increased quiescence, or a developmental delay in relation to lack of FMRP and uncovers a new role for FMRP in the early postnatal hippocampus. In turn, elucidating the mechanisms that underlie FXS will aid in the development of targeted treatments. / Thesis / Doctor of Philosophy (PhD) / Fragile X syndrome is the leading inherited cause of intellectual impairment and autism spectrum disorder. The syndrome is caused by a defect in one gene. This gene has been suggested to play a role in regulating the birth of new brain cells termed neural precursor cells. The importance of neural precursor cells stems from their ability to generate neurons and glia, the main cells in the brain. In this thesis, I focus on studying neural precursor cells from the hippocampus, a brain region important for learning and memory. A mouse model was used to compare neural precursor cells from healthy and Fragile X mice during early postnatal development. I found that neural precursor cells do not divide as much as they should in the Fragile X mouse hippocampus. The results help to determine the causes for learning and memory deficits in Fragile X and potentially open avenues for intervention.

Down syndrome (DS) is caused by triplication of human chromosome 21 (HSA21) and is the most common genetic form of intellectual disability. It is unknown precisely how triplication of HSA21 results in the intellectual disability, but it is thought that the global transcriptional dysregulation caused by trisomy 21 perturbs multiple aspects of neurodevelopment that cumulatively contribute to its etiology. While the characteristics associated with DS can arise from any of the genes triplicated on HSA21, in this work we focus on oligodendrocyte transcription factor 2 (OLIG2). The progeny of neural progenitor cells (NPCs) expressing OLIG2 are likely to be involved in many of the cellular changes underlying the intellectual disability in DS. To explore the fate of OLIG2+ neural progenitors, we took advantage of two distinct models of DS, the Ts65Dn mouse model and induced pluripotent stem cells (iPSCs) derived from individuals with DS. Our results from these two systems identified multiple perturbations in development in the cellular progeny of OLIG2+ NPCs. In Ts65Dn, we identified alterations in neurons and glia derived from the OLIG2 expressing progenitor domain in the ventral spinal cord. There were significant differences in the number of motor neurons and interneurons present in the trisomic lumbar spinal cord depending on age of the animal pointing both to a neurodevelopment and a neurodegeneration phenotype in the Ts65Dn mice. Of particular note, we identified changes in oligodendrocyte (OL) maturation in the trisomic mice that are dependent on spatial location and developmental origin. In the dorsal corticospinal tract, there were significantly fewer mature OLs in the trisomic mice, and in the lateral funiculus we observed the opposite phenotype with more mature OLs being present in the trisomic animals. We then transitioned our studies into iPSCs where we were able to pattern OLIG2+ NPCs to either a spinal cord-like or a brain-like identity and study the OL lineage that differentiated from each progenitor pool. Similar to the region-specific dysregulation found in the Ts65Dn spinal cord, we identified perturbations in trisomic OLs that were dependent on whether the NPCs had been patterned to a brain-like or spinal cord-like fate. In the spinal cord-like NPCs, there was no difference in the proportion of cells expressing either OLIG2 or NKX2.2, the two transcription factors whose co-expression is essential for OL differentiation. Conversely, in the brain-like NPCs, there was a significant increase in OLIG2+ cells in the trisomic culture and a decrease in NKX2.2 mRNA expression. We identified a sonic hedgehog (SHH) signaling based mechanism underlying these changes in OLIG2 and NKX2.2 expression in the brain-like NPCs and normalized the proportion of trisomic cells expressing the transcription factors to euploid levels by modulating the activity of the SHH pathway. Finally, we continued the differentiation of the brain-like and spinal cord-like NPCs to committed OL precursor cells (OPCs) and allowed them to mature. We identified an increase in OPC production in the spinal cord-like trisomic culture which was not present in the brain-like OPCs. Conversely, we identified a maturation deficit in the brain-like trisomic OLs that was not present in the spinal cord-like OPCs. These results underscore the importance of regional patterning in characterizing changes in cell differentiation and fate in DS. Together, the findings presented in this work contribute to the understanding of the cellular and molecular etiology of the intellectual disability in DS and in particular the contribution of cells differentiated from OLIG2+ progenitors.

Neurons are generated from unspecialized neural precursor cells (NPCs) in a process termed neurogenesis. This neuronal development continues throughout life in the ventricular zone (VZ) of the lateral ventricles, and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. NPCs undergo a complex and highly regulated set of behaviours in order to ultimately integrate into the existing brain circuitry as fully functional neurons. Recently the pannexin (Panx) large-pore channel proteins were discovered. One family member, Panx1 is expressed in the nervous system in mature neurons, and acts as an ATP release channel in various cell types throughout the body. Post-natal NPCs are responsive to ATP via activation of purinergic receptors, which modulate a variety of NPC behaviours. I therefore investigated the hypothesis that Panx1 was expressed in post-natal VZ NPCs, where it functioned as an ATP release channel and regulated neuronal development. In the course of my studies, I found that Panx1 positively regulated NPC proliferation and migration, and negatively regulated neurite outgrowth in vitro. Using an NPC-specific Panx1 knock-out strategy, I showed that Panx1 expression was required for maintenance of a consistent population of VZ NPCs in vivo in both healthy and injured brain. Together these data indicated that Panx1 directed NPC behaviours associated with neuronal development both in vitro and in vivo. To further understand the molecular underpinnings of this regulation, I examined the Panx1 interactome, and uncovered a novel association with collapsin response mediator protein 2 (Crmp2). Functional studies suggested that this interaction likely was at least in part responsible for Panx1’s negative impact on neurite outgrowth. Overall, my results represent important novel findings that contribute to our understanding of post-natal neuronal development and the molecular function of Panx1 within the brain. / Graduate / 0317 / 0379 / leighws@uvic.ca

Schizophrenia is a major psychiatric disorder that affects 1% of the world population and is among the 10 leading worldwide causes of disability. Disrupted-In- Schizophrenia (DISC1) is one of the most studied risk genes for mental illness and is disrupted by a balanced translocation between chromosomes 1 and 11 that co-segregates with major mental illness in a single large Scottish family. DISC1 is a scaffold protein with numerous interactors and has been shown to hold key roles in neuronal progenitor proliferation, migration, cells signalling and synapse formation and maintenance. The studies herein provide the platform in order to investigate the molecular and cellular consequences of the t(1;11) translocation using induced pluripotent stem cells (iPSCs)-derived neural precursor cells and neurons from within-family carriers and controls. Towards this end, several iPSC lines have been converted into neural progenitor cells (NPCs) and differentiated into physiologically active forebrain neurons following well-characterised protocols. These cells were characterised in terms of basic marker expression at each developmental stage. Inter-line variation was observed in all subsequent experiments but overall t(1;11) lines did not generate less neuronal or less proliferating cells compared to control lines. Furthermore, the expression pattern of genes disrupted by the t(1;11) translocation was investigated by RT-qPCR. DISC1 was reduced by ~50% in the translocation lines, both neural precursors and neurons. This observation corresponds to previous findings in lymphoblastoid cell lines (LBCs) derived from members of the same family. Moreover, DISC1 expression was found to increase as neural precursors differentiation to neurons. Two other genes are disrupted by the t(1;11) translocation;DISC2 and DISC1FP1. Their expression was detectable, but below the threshold of quantification. Similarly, DISC1/DISC1FP1 chimeric transcripts corresponding to such transcripts previously identifies in LBCs from the family were detectable, but not quantifiable. A fourth gene, TSNAX, was also investigated because it is located in close proximity to, and undergoes intergenic splicing with, DISC1. Interestingly, TSNAX was found to be altered in some but not all time points studied, in the translocation carriers compared to control lines. In addition to breakpoint gene expression profiling, iPSC-derived material was used to investigate neuronal differentiation. There seemed to be attenuation in BIII-TUBULIN expression at two weeks post-differentiation, while NESTIN, MAP2 and GFAP expression was similar between translocation carrier and control lines at all time points studied. I also had access to targeted mice designed to mimic the derived chromosome 1 of the t(1;11) balanced translocation. Using RT-qPCR Disc1 expression was found to be 50% lower in heterozygous mice compared to wild types, and I detected a similar profile of chimeric transcript expression as detected in translocation carrier-derived LBCs. These observations support my gene expression studies of the human cells and indicate that the iPSC-derived neural precursors and neurons can be studied in parallel with the genome edited mice to obtain meaningful insights into the mechanism by which the t(1;11) translocation confers substantially elevated risk of major mental illness. In conclusion, the studies described in this thesis provide an experimental platform for investigation of the effects of the t(1;11) translocation upon function and gene and protein expression in material derived from translocation carriers and in brain tissue from a corresponding mouse model.

Adult neural precursor cells (NPCs) are a heterogeneous population of mitotically active, self-renewing multipotent cells of both adult and developing CNS. They can be expanded in vitro in the presence of mitogens. The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks) and significant PC loss (24 weeks). A stereological count demonstrates that mice with significant cell loss exhibit highest survival of grafted NPCs and migration to the vicinity of PCs as compared to wt and younger grafted animals. These animals showed improved motor skills as compared to sham animals. Confocal analysis and profiling shows that many of implanted cells present in the cerebellar cortex have formed gap junctions with host PCs and express connexin43. Grafted cells did not adopt characteristics of PCs, but stereological and morphometric analysis of the cerebellar cortex revealed that grafted animals had more surviving PCs and a better preserved morphology of these cells than the control groups. Perforated patch clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. In this study, evidence for a neuroprotective effect came, in addition to motor behavior improvement, from stereological and electrophysiological analyses and suggest that timing of stem cell delivery is important to determine its therapeutic effect. In a brain stem cell niche, NSCs reside in a complex cellular and extracellular microenvironment comprising their own progeny, ependymal cells, numerous blood vessels and various extracellular matrix molecules. Recently, it was reported that blood vessel ECs-NSCs crosstalk plays an important role in tissue homeostasis. Bloodstream offers a natural delivery vehicle especially in case of diffuse neurodegenerative diseases which require widespread distribution of exogenous cells. As NSCs are confronted with blood-brain barrier endothelial cells (BBB-ECs) before they can enter into brain parenchyma, we investigated their interaction using primary cultures in an in vitro BBB model. We isolated human fetal neural precursor cells (hfNPCs) from aborted fetal brain tissues and expanded in vitro. We showed that in an in vitro model, human BBB endothelium induces the rapid differentiation of hfNPCs and allows them to cross the endothelial monolayer, with the differentiated progeny remaining in close contact with endothelial cells. These results are not reproduced when using a non-BBB endothelium and are partly dependent on the cytokine MCP1. Our data suggest that, in the presence of attractive signals released by a damaged brain, intravascularly administered NPCs can move across an intact BBB endothelium and differentiate in its vicinity. Overall, our findings have implications for the development of cellular therapies for cerebellar degenerative diseases and understanding of the brain stem cell niche.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

Die Inzidenz von neurodegenerativen Erkrankungen und Schlaganfällen steigt in Folge der Überalterung der westlichen Gesellschaft immer weiter an. Die Behand-lung von Schlaganfall-, Alzheimer und Parkinsonpatienten ist bisher aber meist unbefriedigend bzw. weitgehend erfolglos. Ein neues Modell in der Schlaganfallforschung wurde daher am Schaf entwickelt. In diesem wird auch der in den letzten zwei Jahrzehnten verstärkt verfolgte zelltherapeutische Ansatz untersucht (BOLTZE et al. 2011, DREYER et al. 2012). Neurale Vorläuferzellen gelten dabei, auf Grund ihrer wichtigen Rolle bei den endogenen Reparaturmechanismen nach einem Schlaganfall, als besonders vielversprechend. Die Gewinnung dieser Zellen für eine autologe Transplantation ist jedoch aufwendig und nur eingeschränkt möglich. Im Vergleich zu Nervengewebe stellt die Haut eine sowohl beim Tier als auch beim Menschen leicht zugängliche und in ausreichendem Maß verfügbare Quelle verschiedener Stamm- und Vorläuferzellen dar. Bei verschiedenen Spezies wurde die Isolation spezieller, dermaler Vorläuferzellen beschrieben, die als skin-derived precursor cells (SKPs) bezeichnet werden. SKPs wiesen dabei ein ähnliches Differenzierungspotential auf wie neurale Vorläuferzellen (TOMA et al. 2001, FERNANDES et al. 2006). Ein Einsatz der SKPs in der Schlaganfalltherapie wäre somit denkbar, muss aber zunächst im Schafmodell erforscht werden. SKPs wurden jedoch noch nicht bei der Spezies Schaf isoliert. Ziel der vorliegenden Arbeit war es daher, ein Isolationsprotokoll für SKPs aus der ovinen Dermis zu etablieren und diese morphologisch und immunzytologisch zu charakterisieren. Im Rahmen dieser Arbeit wurden verschiedene in der Literatur beschriebene Isolati-onsverfahren an ovinen Hautproben getestet und modifiziert. Es wurden verschiedene Körperregionen auf ihre Eignung zur Probenentnahme und zur anschließenden Isolierung untersucht. Des Weiteren wurde der Effekt einer Rasur eine Woche vor Exzision des Hautareals auf die Sphäroidbildung überprüft. Der Einsatz von Enzymen in Kombinationslösungen oder singulär wurde variiert und eine unterschiedlich intensive mechanische Aufbereitung der Proben durchgeführt. Der Erfolg der zwei vielversprechendsten Isolationsprotokolle wurde statistisch validiert. Außerdem wurde der Effekt einer initialen Fibronektinbeschichtung analysiert. Die von den isolierten Zellen gebildeten sphärenartigen Zellaggregate wurden unter morphologischen Gesichtspunkten sechs und neun Wochen nach Isolation ausgewertet. Dabei wurden die Anzahl der Sphäroide/cm², die Größe und die Form berücksichtigt. Des Weiteren erfolgte eine immunzytologische Analyse der Sphäroide mit Fokus auf das in der Literatur beschriebene Expressionsmuster von SKPs und neuralen Vorläuferzellen. Für die Isolation von ovinen SKPs erwies sich die Regio nasofrontalis als das geeignetste Hautareal. Dabei war die Isolation eine Woche nach Rasur des beprobten Areals zuverlässiger als ohne diese. Bei vergleichender Betrachtung der Methoden erwies sich ein enzymatisch orientiertes Isolationsverfahren modifiziert nach FERNANDES und MILLER (2009) als zielführend. Neben einer hohen Anzahl an isolierten Zellen erfolgte in jedem Versuchsdurchgang eine Zusammenlagerung der Zellen in frei flotierenden Aggregaten. Diese waren im Median 70,97 µm groß. Auf Grund ihrer Geometrie ist es korrekter sie als Sphäroide und nicht, wie bei anderen Spezies üblich, als Sphären zu bezeichnen. Eine anfängliche Beschichtung der Zellkulturplatten mit Fibronektin hatte keinen fördernden Effekt auf die Bildung und die Größe der Sphäroide. Lediglich eine anfänglich höhere Proliferationsrate war bemerkbar. Immunzytologisch konnte gezeigt werden, dass in den Sphäroiden eine heterogene Zellpopulation vorlag. Die Sphäroide wurden überwiegend von Zellen gebildet, in denen neben mesenchymalen Markern auch klassische Vorläuferantigene wie Nestin und Sox2 nachgewiesen wurden. Das immunzytologische Expressionsmuster ist damit vergleichbar mit dem von SKPs anderer Spezies. Außerdem wurden in unterschiedlicher Ausprägung Antigene detektiert, die typischerweise in neuralen Vorläuferzellen der ventrikulären und subventrikulären Zone vorkommen. Dies konnte auch in den Positivkontrollen für das ovine Gehirn bestätigt werden. Die Anzahl proliferierender Zellen in den Sphäroiden war relativ gering und die Anzahl an kokultivierter Keratinozyten minimal. Die Zusammenfassung der heterogenen Vorläuferzellpopulation unter dem Begriff skin-derived precursor cells ist auf Grund ihres dermalen Ursprungs und ihrer morphologischen und immunzytologischen Eigenschaften gerechtfertigt. Somit ist es in dieser Arbeit gelungen, zum ersten Mal SKPs aus der ovinen Dermis zu isolieren und über neun Wochen zu kultivieren. Es wurde ein Isolationsprotokoll entwickelt, das eine Sphäroidbildung reproduzierbar ermöglicht und an die Gegebenheiten beim Schaf angepasst ist. Bevor eine autologe Transplantation von diesen SKPs etwa im Schlaganfallmodell am Schaf vorgenommen werden kann, ist eine intensivere Untersuchung der isolierten Zellen etwa mittels PCR durchzuführen und eine fluoreszenzbasierte Zellsortierung der heterogenen Vorläuferzellen zu entwickeln. / In consequence of the demographic changes in modern western society, the inci-dence of neurodegenerative diseases and stroke is increasing. Unfortunately, there is still no successful or at least satisfactory treatment available for patients who suffer from stroke Alzheimer’s or Parkinson’s disease. Therefore, a new animal model in stroke research has been established in sheep (BOLTZE et al. 2011, DREYER et al. 2012). First cell therapy studies have already been performed in this model. Especially neural precursor cells seem to be promising as they play an important role in endogenous repair processes in the brain after stroke. However, the extraction of these cells prior to an autologous transplantation is elaborate and of limited success. Compared to neural tissue, skin is an easily accessible and sufficiently available source of a variety of stem and precursor cells in animals as well as in humans. Thus, the isolation of a specific type of dermal precursor cells, called skin-derived precursor cells (SKPs), seems to be easier compared to neural precursor cells and in vitro SKPs are capable of neural differentiation as well (TOMA et al. 2001, FERNANDES et al. 2006). According to these findings, a therapeutic application of SKPs after stroke seems to be promising. Prior to that, however, intensive studies in the ovine stroke model are necessary. Thus, SKPs have to be isolated from the dermis of sheep for an autologous transplantation. Therefore, the aim of this dissertation has been the establishment of an optimal isolation protocol for SKPs from the ovine dermis as well as the morphological and by immunocytochemical characterisation of those cells. Within this study, several previously described isolation protocols were modified for ovine skin. Skin samples were taken from several body regions to assess the local suitability for excision and isolation. Additionally, the effect of shaving the areas one week before sampling on spheroid forming was tested. A variety of enzymes was used alone and in combination. Furthermore, the effectiveness of an isolation protocol using enhanced mechanical treatment was analysed. The two most promising protocols were evaluated statistically and compared to each other. In these experiments, the influence of an initial fibronectin coating was determined as well. The isolated cells formed spheroids, which were assessed after six and nine weeks of cultivation considering the amount of spheroids per cm², their size and form. Moreover, immunocytochemical tests were conducted, focusing on expression patterns described for SKPs and neural precursor cells. According to these experiments, it is advisable to take skin samples from the naso-frontal region one week after shaving. Comparing all tested protocols, a predominantly enzymatic isolation protocol modified according to FERNANDES and MILLER (2009) was most successful. A high cell yield was achieved and free-floating spheroids formed spontaneously in all test runs. The median diameter of these spheroids was 70.97 µm. Due to their three-dimensional shape, it is more correct to use the term “spheroid” instead of the commonly used term “sphere”. Growing the isolated cells initially on fibronectin coated culture plates does not support both formation and size of the spheroids. Only a higher cell proliferation at the beginning of cultivation can be noticed. Immunocytochemical assays demonstrated that the formed spheroids consisted of a heterologous cell population. Besides mesenchymal antigens the cells in the spheroids expressed characteristic antigens of precursor cells, like Nestin and Sox2. Thus, the immunocytochemical expression pattern is comparable to SKPs isolated from other species. Furthermore, common markers of neural precursor cells of the ventricular and subventricular zone, whose existence in the ovine brain was also proven in this study, were detected in the spheroid forming cells. There were only a few proliferating cells and a minimal amount of keratinocytes in the spheroids. Due to the dermal origin and the given morphological and immunocytochemical characteristics, the heterogeneous cell population can be addressed by the term “skin-derived precursor cells”. In conclusion, in this study ovine SKPs were isolated for the first time and cultured successfully over nine weeks. An isolation protocol was established, which guarantees reproducible formation of spheroids in cell isolates from ovine dermis. Further intensive examinations of the isolated cells, for example using PCR, have to be conducted before SKPs can be applied in autologous transplantation in the ovine stroke model. Additionally, the usage of fluorescence-activated cell sorting of the heterogeneous precursor cells should be considered.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

Gene mit gerigem Einfluss auf einen untersuchten Phänotyp können durch den Ein- schluss einer genetischen Variation im Tierversuch untersucht werden. Adulte Neuro- genese, der Prozess der Neubildung und Integration von funktionellen Neuronen in das existierende neurale Netzwerk, wird von vielen solchen Genen mit geringem Effekt beeinflusst. All diese Gene im lebenden Tier zu untersuchen wäre mit einem hohen Arbeitsaufwand verbunden, und würde hohe Tierzahlen erfordern. Bereits publizierte Ergebnisse zeigen, dass diese Gene auch in der Zellkultur unter Verwendung von Zelllinien genetisch rekombinanter Tiere untersucht werden können (Kannan et al., 2016). Die hier verwendeten, ingezüchteten Mausstämme des so genannten BXD Panels stellen die Nachkommen der Kreuzung der beiden Mausstämme C57BL/6J und DBA/2J dar (Peirce et al., 2004), die sich in der Ausprägung von unterschiedlichen Neurogenese bezogenen Phänotypen bereits deutlich unterscheiden (Kempermann et al., 2006). Durch die Verwendung der BXD Tiere wird hierbei die Aussagekraft der genetischen Variation mit dem Zellkultursystem verbunden. Die Aussagekraft dieser Studie ist jedoch darin limitiert, dass aufgrund des verwendeten Protokolls nur eine Zelllinie pro Mausstamm generiert werden konnte. Daher präsentiere ich hier ein neues Protokoll welches es erlaubt eine Zelllinie aus nur einem einzelnen Tier zu generieren. Diese Methode kombiniert zwei bestehende Zellkultursysteme, die Neurosphärenkultur und die Monolayerkultur. Es stellte sich heraus, dass die Überlebensrate der einzelnen Zelllinien vom biologischen Hintergrund der Zellen beeinflusst wird. So ist die Überlebensrate von Zellen der DBA/2J Mäuse deutlich schlechter als die der C57BL/6J oder die der F1 Generation aus der Verpaarung der beiden Stämme. Es zeigte sich allerdings, dass diese Überlebensrate nicht ausschließlich von der vorhandenen Anzahl proliferierender Zellen abhängt, da B6D2F1 (F1 Generation mit einem C57BL/6J Muttertier) signifikant weniger proliferierende (Ki67 positive) Zellen in vivo aufweisen, jedoch keine geringere Überlebensrate der Zelllinien haben. Eine hoch standardisierte, umfangreiche Analyse der Zelllinien aller vier Mausstämme (C57BL/6J, DBA/2J, und die zwei reziproken F1 Nachkommen BDF1 und DBF1) zeigte eine hohe Varianz innerhalb genetisch identischer Linien, was die Be- stimmung eines Effektes, der durch den genetischen Hintergrund der Linien verursacht wird, beeinträchtigte. Die Zelllinien werden signifikant von äußeren Faktoren beeinflusst, wie z.B. durch das Einfrieren der Zellen. Dies gibt Hinweise darauf, dass Untersuchungen in der Zellkultur genau geplant, kritisch hinterfragt, sowie möglichst alle potentiellen Einflussfaktoren gleich gehalten werden müssen. Nur so können valide, aussagekräftige Ergebnisse mit der Zellkultur gewonnen werden. Automatische Zellkultursysteme, neue Mikroskopieverfahren, sowie besser definierte Langzeitstudien werden unser Verständnis von Zellen in der Zellkultur deutlich verbessern und dabei ihren Wert, sowie bestehende Limitationen, endgültig klären.:List of Figures I List of Tables II List of Abbreviations III List of Publications V 1. Introduction 1 1.1 Genetic variation in animal research 2 Recombinant inbred strains 3 The BXD panel 4 The Gene Network 5 Genetic modifications 5 
 1.2 Adult hippocampal neurogenesis 6 History 7 Clinical relevance 8 The BXD panel and adult hippocampal neurogenesis 9 
 1.3 Developmental stages of neural precursor cells 9 
 1.4 Studying adult neurogenesis in vitro 11 Culturing hippocampal precursor cells 11 A mouse cell culture genetic reference panel 13 
 1.5 Tracking 13 
 1.6 Objectives 15 
 2. Materials and Methods 16 2.1 Components and equipment 16 
 2.2 Antibodies 20 
 2.3 Recipes 21 
 General buffers and solutions 21 Cell culture solutions 21 Immunocytochemistry solutions 23 Immunohistochemistry solutions 24 
 2.4 Experimental animals 25 
 2.5 Cell culture 25 Coating of cell culture vessels 25 Fire-polished pipettes 25 Dentate gyrus isolation 26 Neurosphere assay 26 Monolayer culture 27 
 2.6 Immunocytochemistry 29 BrdU staining preparations 29 Staining protocol 30 Imaging and counting 30 
 2.7 Immunohistochemistry 30 Sample preparation 30 Staining protocol 31 Cell counting 31 
 2.8 Tracking 32 Cell preparation and imaging setup 34 Image processing 35 Data analysis 35 
 2.9 Generation of CRISPR/Cas mediated knock-out lines 36 Construct design and cloning 36 E. coli Top10 transformation and plasmid isolation 37 Transfection of neural precursor cells and expansion of knock-out lines 38 Genotyping of the generated cell lines 39 Agarose gel electrophoresis 40 
 2.10 Statistical analysis 40 2.11 Data visualization 40 3. Results 41 3.1 Single animal monolayer cultures41 The three phenotypes of the neurosphere assay 44 Neurosphere assay phenotypes could not predict the survival of a cell line 45 The genetic background had an influence on all three phenotypes of the neurosphere assay 46 Significantly less proliferating cells in vivo but no difference in the neurosphere assay of BDF1 compared to BL6 animals 48 BDF1 cells could not be activated to form more spheres but sphere size could be increased using KCl 49 
 3.2 A new cell line phenotyping standard operation procedure and its application 50 Line generation data 52 Marker staining 54 Cell tracking 55 
 3.3 Cell culture – a system with limitations 59 Freezing effect 60 Cell culture data - technical variance hinders the analysis of small effects 62 3.4 Migration speed and GFAP 63 The strength of the BXD panel – cumulative data 65 
 3.5 Other applications of the tracking procedure 68 
 Tracking labeled cells in an embryonic zebrafish xenograft model 68 Cell tracking in mouse retina explants 68 4. Discussion 70 4.1 Single animal monolayer cultures – a new protocol 70 
 4.2 A new phenotyping pipeline 74 
 4.3 Semi-automated (user-supervised) cell tracking 77 
 4.4 A possible correlation between migration speed and differentiation 79 4.5 CRISPR/Cas knock-out lines - an ill-conceived system with high potential 82 4.6 The problem of the validity of cell culture experiments - a comment 83 4.7 Conclusion 84 
 Bibliography 88 A Single animal cell line generation protocol 106 
 B Cell line characterization SOP 112 
 C R Scripts 117 / Uncovering gene loci that assert only small effects onto a phenotype of interest, can be achieved by including genetic variation in animal research. Adult hippocampal neurogenesis, the process of the formation of new neurons and their functional integration into existing circuitry, is influenced by a broad range of such small effect genes. Analyzing all of these genes in vivo would be laborious and require a high number of animals. Previously published data merged the power of genetic variation with a cell culture system by using cell lines generated from the BXD recombinant inbred mouse strains (Kannan et al., 2016). These strains are inbred progeny of F2 crosses originating from the two mouse strains C57BL/6J and DBA/2J (Peirce et al., 2004), which already differ quite extensively in neurogenesis related phenotypes (Kempermann et al., 2006). As previous studies were limited by the number of strains that could be generated due to the demand for high numbers of animals, I developed a new method that allows the generation of a cell line from one single animal. For this new method, I combined the neurosphere culture with a subsequent monolayer culture. The survival of the resulting cell lines, is thereby greatly influenced by the genetic background. The survival rate of cell lines derived from DBA/2J animals is much lower as compared to C57BL/6J-derived lines or lines from the F1 generation of crossing the two strains. Whether or not a cell line survived did not seem to be solely influenced by the number of proliferating cells in vivo, as B6D2F1 (F1 progeny with a C57BL/6J mother) showed significantly less proliferative (Ki67 positive) cells in vivo while exhibiting a survival rate that exceeded both parental strains. An extensive study of the cell lines gained from all four mouse strains (C57BL/6J, DBA/2J, and the two reciprocal F1 progeny B6D2F1 and D2B6F1) in a highly standardized manner showed that the individual difference between single cell lines was rather high, hampering the successful detection of in-between strain differences. The standardized characterization of the generated cell lines, further allowed the identification of external factors, influencing the cells, as for example the freezing of the cells. This indicates that cell culture experiments need to be thoroughly planned and critically scrutinized, while all external factors should be kept as constant as possible to ensure the validity of the resulting data. Automated cell handling, new imaging technologies, as well as more defined long-term studies will greatly improve the understanding of cells in culture and thereby show their true values and limitations.:List of Figures I List of Tables II List of Abbreviations III List of Publications V 1. Introduction 1 1.1 Genetic variation in animal research 2 Recombinant inbred strains 3 The BXD panel 4 The Gene Network 5 Genetic modifications 5 
 1.2 Adult hippocampal neurogenesis 6 History 7 Clinical relevance 8 The BXD panel and adult hippocampal neurogenesis 9 
 1.3 Developmental stages of neural precursor cells 9 
 1.4 Studying adult neurogenesis in vitro 11 Culturing hippocampal precursor cells 11 A mouse cell culture genetic reference panel 13 
 1.5 Tracking 13 
 1.6 Objectives 15 
 2. Materials and Methods 16 2.1 Components and equipment 16 
 2.2 Antibodies 20 
 2.3 Recipes 21 
 General buffers and solutions 21 Cell culture solutions 21 Immunocytochemistry solutions 23 Immunohistochemistry solutions 24 
 2.4 Experimental animals 25 
 2.5 Cell culture 25 Coating of cell culture vessels 25 Fire-polished pipettes 25 Dentate gyrus isolation 26 Neurosphere assay 26 Monolayer culture 27 
 2.6 Immunocytochemistry 29 BrdU staining preparations 29 Staining protocol 30 Imaging and counting 30 
 2.7 Immunohistochemistry 30 Sample preparation 30 Staining protocol 31 Cell counting 31 
 2.8 Tracking 32 Cell preparation and imaging setup 34 Image processing 35 Data analysis 35 
 2.9 Generation of CRISPR/Cas mediated knock-out lines 36 Construct design and cloning 36 E. coli Top10 transformation and plasmid isolation 37 Transfection of neural precursor cells and expansion of knock-out lines 38 Genotyping of the generated cell lines 39 Agarose gel electrophoresis 40 
 2.10 Statistical analysis 40 2.11 Data visualization 40 3. Results 41 3.1 Single animal monolayer cultures41 The three phenotypes of the neurosphere assay 44 Neurosphere assay phenotypes could not predict the survival of a cell line 45 The genetic background had an influence on all three phenotypes of the neurosphere assay 46 Significantly less proliferating cells in vivo but no difference in the neurosphere assay of BDF1 compared to BL6 animals 48 BDF1 cells could not be activated to form more spheres but sphere size could be increased using KCl 49 
 3.2 A new cell line phenotyping standard operation procedure and its application 50 Line generation data 52 Marker staining 54 Cell tracking 55 
 3.3 Cell culture – a system with limitations 59 Freezing effect 60 Cell culture data - technical variance hinders the analysis of small effects 62 3.4 Migration speed and GFAP 63 The strength of the BXD panel – cumulative data 65 
 3.5 Other applications of the tracking procedure 68 
 Tracking labeled cells in an embryonic zebrafish xenograft model 68 Cell tracking in mouse retina explants 68 4. Discussion 70 4.1 Single animal monolayer cultures – a new protocol 70 
 4.2 A new phenotyping pipeline 74 
 4.3 Semi-automated (user-supervised) cell tracking 77 
 4.4 A possible correlation between migration speed and differentiation 79 4.5 CRISPR/Cas knock-out lines - an ill-conceived system with high potential 82 4.6 The problem of the validity of cell culture experiments - a comment 83 4.7 Conclusion 84 
 Bibliography 88 A Single animal cell line generation protocol 106 
 B Cell line characterization SOP 112 
 C R Scripts 117