Comparative analysis of midline guidance and axon pathfinding in the chelicerates Achaearanea tepidariorum and Cupiennius salei

Die Neurogenese und axonale Wegfindung sind in den vergangenen Jahrzehnten Thema einer Vielzahl wissenschaftlicher Untersuchungen in den verschiedensten Organismen gewesen. Die zusammengetragenen Daten in Insekten und Crustaceen geben eine gute Übersicht darüber, wie das Nervensystem in Arthropoden aufgebaut wird. Die entwicklungsbiologischen Prozesse, die daran beteiligt sind, sind in den beiden genannten Gruppen sehr gut verstanden. In den Gruppen der Cheliceraten und Myriapoden jedoch wurden ähnliche Analysen bisher kaum durchgeführt. Das Hauptanliegen dieser Arbeit war es daher, Mechanismen in den Spinnen Achaearanea tepidariorum und Cupiennius salei, zwei Vertretern der Cheliceraten, zu untersuchen, die eine Rolle im Leitsystem der ventralen Mittellinie und bei der axonalen Wegfindung spielen. Eine Vorraussetzung hierfür sind Kenntnisse über die Architektur des Zentralnervensystems. In einem ersten Schritt beschrieb ich daher grundlegend die Morphologie des Nervensystems im Verlauf der gesamten Embryoalentwicklung. Ich konnte zeigen, dass in Spinnen ein für Arthropoden typisches Strickleiternervensystem gebildet wird. Dieses wird von segmental angelegten Neuronen geformt, wobei sowohl Gruppen von Zellen als auch einzelne Neurone daran beteiligt sind, die primären axonalen Trakte zu etablieren. Im Besonderen konnte ich eine Zelle identifizieren, die in Position, Projektionsmuster und der Expression des Markergens even-skipped vergleichbar zum PR2 Neuron in Drosophila ist, welches die posteriore Wurzel des Segmentalnervs anlegt.rnrnIn einem zweiten Ansatz untersuchte ich die ventrale Mittellinie in Spinnen im Bezug auf ihre mögliche Funktion in der axonalen Wegfindung. Es konnte gezeigt werden, dass es sich beim Epithel der Mittellinie, das die Lücke zwischen beiden Keimstreifhälften während des gesamten Prozesses der Inversion überspannt, um eine transiente Struktur handelt, die keine neuralen Zellen hervorbringt. Es ist daher vergleichbar mit der so genannten Floor plate in Vertebraten, die ebenfalls nur vorübergehend existiert. Die Untersuchung von single minded (sim) zeigte, dass es, anders als in Drosophila, wo sim ein wichtiges regulatorisches Gen für die korrekte Spezifizierung von Mittellinienzellen ist, nicht in den Zellen der Mittellinie, sondern in diesen benachbarten Zellen, exprimiert wird. Das ist vergleichbar mit Vertebraten. Zusätzlich konnte ich Expression von sim an den Basen der Gliedmassen und im Kopf nachweisen. Wie in Vertebraten könnte sim an der Musterbildung dieser Gewebe beteiligt sein. Dennoch spielt die Mittellinie in Spinnen eine wichtige Rolle als Organisator für auswachsende, kommissurale Axone. Diese Funktion teilt sie mit anderen Invertebraten und Vertebraten.rnrnDie Signaltransduktionskaskade, die an der axonalen Wegfindung an der Mittellinie beteiligt ist, ist in den verschiedensten Organismen hoch konserviert. In der vorliegenden Arbeit konnte ich sowohl in Achaearanea als auch in Cupiennius ein netrin Homolog identifizieren und eine konservierte Funktion des Wegfindungsmoleküls während der Bildung der Kommissuren aufzeigen. RNAi Experimente belegen, dass, wird die Funktion von netrin herunterreguliert, das Strickleiternervensystem nicht korrekt gebildet wird, ins Besondere die kommissuralen Faszikel. Des Weiteren konnte ich eine neue Funktion von netrin, die bisher in anderen Organsimen noch nicht beschrieben wurde, identifizieren. Neben seiner Rolle in der axonalen Wegfindung, scheint netrin auch an der epithelialen Morphogenese im zentralen Nervensystem beteiligt zu sein. In dieser Funktion scheint netrin in Gliazellen, die die epithelialen Vesikel der Invaginationsgruppen umhüllen, wichtig zu sein, um neurale Vorläuferzellen in einem undifferenzierten Zustand zu halten. Der Abbau von netrin Transkript durch RNA Interferenz führt zu einer verfrühten Segregation neuraler Vorläuferzellen aus dem epithelialen Verband der Invaginationsgruppen und zu einer Zunahme an Zellen, die den frühen Differenzierungsmarker islet exprimieren.

Investigations on formation and specification of neural precursor cells in the central nervous system of the Drosophila melanogaster embryo

Investigations on formation and specification of neural precursor cells in the central nervous system of the Drosophila melanogaster embryoSpecification of a unique cell fate during development of a multicellular organism often is a function of its position. The Drosophila central nervous system (CNS) provides an ideal system to dissect signalling events during development that lead to cell specific patterns. Different cell types in the CNS are formed from a relatively few precursor cells, the neuroblasts (NBs), which delaminate from the neurogenic region of the ectoderm. The delamination occurs in five waves, S1-S5, finally leading to a subepidermal layer consisting of about 30 NBs, each with a unique identity, arranged in a stereotyped spatial pattern in each hemisegment. This information depends on several factors such as the concentrations of various morphogens, cell-cell interactions and long range signals present at the position and time of its birth. The early NBs, delaminating during S1 and S2, form an orthogonal array of four rows (2/3,4,5,6/7) and three columns (medial, intermediate, and lateral) . However, the three column and four row-arrangement pattern is only transitory during early stages of neurogenesis which is obscured by late emerging (S3-S5) neuroblasts (Doe and Goodman, 1985; Goodman and Doe, 1993). Therefore the aim of my study has been to identify novel genes which play a role in the formation or specification of late delaminating NBs.In this study the gene anterior open or yan was picked up in a genetic screen to identity novel and yet unidentified genes in the process of late neuroblast formation and specification. I have shown that the gene yan is responsible for maintaining the cells of the neuroectoderm in an undifferentiated state by interfering with the Notch signalling mechanism. Secondly, I have studied the function and interactions of segment polarity genes within a certain neuroectodermal region, namely the engrailed (en) expressing domain, with regard to the fate specification of a set of late neuroblasts, namely NB 6-4 and NB 7-3. I have dissected the regulatory interaction of the segment polarity genes wingless (wg), hedgehog (hh) and engrailed (en) as they maintain each other’s expression to show that En is a prerequisite for neurogenesis and show that the interplay of the segmentation genes naked (nkd) and gooseberry (gsb), both of which are targets of wingless (wg) activity, leads to differential commitment of NB 7-3 and NB 6-4 cell fate. I have shown that in the absence of either nkd or gsb one NB fate is replaced by the other. However, the temporal sequence of delamination is maintained, suggesting that formation and specification of these two NBs are under independent control.

Hox genes and the regulation of programmed cell death in the embryonic central nervous system of Drosophila melanogaster

This study deals with the function and regulation of programmed cell death, or apoptosis, in the development of the embryonic central nervous system of Drosophila melanogaster. The first part provides a description of apoptosis-deficient embryos, which showed that preventing apoptosis does not cause gross morphological defects in the CNS, as it appears well organized despite the presence of too many cells. An analysis of the incidence and pattern of apoptosis over the course of development discloses a partly very orderly pattern suggesting tight spatio-temporal control, but also reveals random apoptotic cells, which suggests a certain amount of plasticity in the embryo. This analysis also allowed precise identification of some of the dying neural cells in the embryo, and establishment of single cell models for studying regulation of segment-specific apoptosis in the embryonic CNS. In the second part of the work, further investigations into mechanisms controlling segment-specific apoptosis revealed the involvement of two Hox genes, Antennapedia (Antp) and Ultrabithorax (Ubx), in this process. Hox genes control the formation of segment-specific structures in their domains of expression, but also regulate organ and tissue morphogenesis. The study presented here shows that Antp and Ubx play antagonistic roles in motoneuron survival in the embryo. Ubx expression in the CNS is strongly upregulated at a late point in development, when most cells have begun to differentiate. This upregulation shortly precedes Ubx-dependent, segment-specific apoptosis of two differentiated motoneurons. It could further be demonstrated that Antp is required for proper development of the NB7-3 lineage and for survival of the NB7-3 motoneuron in the anterior thoracic segments. In segments where Antp and Ubx expression overlaps, Ubx counteracts the anti-apoptotic function of Antp, resulting in cell death. Thus, these two Hox genes play opposing roles in the survival of differentiated neurons in the late developing nervous system. They thereby contribute to establishment of correct connections between outward-projecting neurons and their targets, which is crucial for the assembly of functional neural circuits, as these have to fulfill region-specific locomotion and sensory requirements along the antero-posterior body axis.

Detrimental effects of exuberant and restrained immune responses against the central nervous system in the context of multiple sclerosis and glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and most aggressive astrocytic tumor of the central nervous system (CNS) in adults. The standard treatment consisting of surgery, followed by a combinatorial radio- and chemotherapy, is only palliative and prolongs patient median survival to 12 to 15 months. The tumor subpopulation of stem cell-like glioma-initiating cells (GICs) shows resistance against radiation as well as chemotherapy, and has been suggested to be responsible for relapses of more aggressive tumors after therapy. The efficacy of immunotherapies, which exploit the immune system to specifically recognize and eliminate malignant cells, is limited due to strong immunosuppressive activities of the GICs and the generation of a specialized protective microenvironment. The molecular mechanisms underlying the therapy resistance of GICs are largely unknown. rnThe first aim of this study was to identify immune evasion mechanisms in GICs triggered by radiation. A model was used in which patient-derived GICs were treated in vitro with fractionated ionizing radiation (2.5 Gy in 7 consecutive passages) to select for a more radio-resistant phenotype. In the model cell line 1080, this selection process resulted in increased proliferative but diminished migratory capacities in comparison to untreated control GICs. Furthermore, radio-selected GICs downregulated various proteins involved in antigen processing and presentation, resulting in decreased expression of MHC class I molecules on the cellular surface and diminished recognition potential by cytotoxic CD8+ T cells. Thus, sub-lethal fractionated radiation can promote immune evasion and hamper the success of adjuvant immunotherapy. Among several immune-associated proteins, interferon-induced transmembrane protein 3 (IFITM3) was found to be upregulated in radio-selected GICs. While high expression of IFITM3 was associated with a worse overall survival of GBM patients (TCGA database) and increased proliferation and migration of differentiated glioma cell lines, a strong contribution of IFITM3 to proliferation in vitro as well as tumor growth and invasiveness in a xenograft model could not be observed. rnMultiple sclerosis (MS) is the most common autoimmune disease of the CNS in young adults of the Western World, which leads to progressive disability in genetically susceptible individuals, possibly triggered by environmental factors. It is assumed that self-reactive, myelin-specific T helper cell 1 (Th1) and Th17 cells, which have escaped the control mechanisms of the immune system, are critical in the pathogenesis of the human disease and its animal model experimental autoimmune encephalomyelitis (EAE). It was observed that in vitro differentiated interleukin 17 (IL-17) producing Th17 cells co-expressed the Th1-phenotypic cytokine Interferon-gamma (IFN-γ) in combination with the two respective lineage-associated transcription factors RORγt and T-bet after re-isolation from the CNS of diseased mice. Pathogenic molecular mechanisms that render a CD4+ T cell encephalitogenic have scarcely been investigated up to date. rnIn the second part of the thesis, whole transcriptional changes occurring in in vitro differentiated Th17 cells in the course of EAE were analyzed. Evaluation of signaling networks revealed an overrepresentation of genes involved in communication between the innate and adaptive immune system and metabolic alterations including cholesterol biosynthesis. The transcription factors Cebpa, Fos, Klf4, Nfatc1 and Spi1, associated with thymocyte development and naïve T cells were upregulated in encephalitogenic CNS-isolated CD4+ T cells, proposing a contribution to T cell plasticity. Correlation of the murine T-cell gene expression dataset to putative MS risk genes, which were selected based on their proximity (± 500 kb; ensembl database, release 75) to the MS risk single nucleotide polymorphisms (SNPs) proposed by the most recent multiple sclerosis GWAS in 2011, revealed that 67.3% of the MS risk genes were differentially expressed in EAE. Expression patterns of Bach2, Il2ra, Irf8, Mertk, Odf3b, Plek, Rgs1, Slc30a7, and Thada were confirmed in independent experiments, suggesting a contribution to T cell pathogenicity. Functional analysis of Nfatc1 revealed that Nfatc1-deficient CD4+ T cells were restrained in their ability to induce clinical signs of EAE. Nfatc1-deficiency allowed proper T cell activation, but diminished their potential to fully differentiate into Th17 cells and to express high amounts of lineage cytokines. As the inducible Nfatc1/αA transcript is distinct from the other family members, it could represent an interesting target for therapeutic intervention in MS.rn

Early steps in ventral nerve cord development in chelicerates and myriapods and formation of brain compartments in spiders

The central point of this work is the investigation of neurogenesis in chelicerates and myriapods. By comparing decisive mechanisms in neurogenesis in the four arthropod groups (Chelicerata, Crustacea, Insecta, Myriapoda) I was able to show which of these mechanisms are conserved and which developmental modules have diverged. Thereby two processes of embryonic development of the central nervous system were brought into focus. On the one hand I studied early neurogenesis in the ventral nerve cord of the spiders Cupiennius salei and Achaearanea tepidariorum and the millipede Glomeris marginata and on the other hand the development of the brain in Cupiennius salei.rnWhile the nervous system of insects and crustaceans is formed by the progeny of single neural stem cells (neuroblasts), in chelicerates and myriapods whole groups of cells adopt the neural cell fate and give rise to the ventral nerve cord after their invagination. The detailed comparison of the positions and the number of the neural precursor groups within the neuromeres in chelicerates and myriapods showed that the pattern is almost identical which suggests that the neural precursors groups in these arthropod groups are homologous. This pattern is also very similar to the neuroblast pattern in insects. This raises the question if the mechanisms that confer regional identity to the neural precursors is conserved in arthropods although the mode of neural precursor formation is different. The analysis of the functions and expression patterns of genes which are known to be involved in this mechanism in Drosophila melanogaster showed that neural patterning is highly conserved in arthropods. But I also discovered differences in early neurogenesis which reflect modifications and adaptations in the development of the nervous systems in the different arthropod groups.rnThe embryonic development of the brain in chelicerates which was investigated for the first time in this work shows similarities but also some modifications to insects. In vertebrates and arthropods the adult brain is composed of distinct centres with different functions. Investigating how these centres, which are organised in smaller compartments, develop during embryogenesis was part of this work. By tracing the morphogenetic movements and analysing marker gene expressions I could show the formation of the visual brain centres from the single-layered precheliceral neuroectoderm. The optic ganglia, the mushroom bodies and the arcuate body (central body) are formed by large invaginations in the peripheral precheliceral neuroectoderm. This epithelium itself contains neural precursor groups which are assigned to the respective centres and thereby build the three-dimensional optical centres. The single neural precursor groups are distinguishable during this process leading to the assumption that they carry positional information which might subdivide the individual brain centres into smaller functional compartments.rn

Translationsregulation des Myelin basischen Proteins in Gliazellen

Im zentralen Nervensystem (ZNS) myelinisieren Oligodendrozyten neuronale Axone, indem sie ihre Zellfortsätze mehrfach um axonale Segmente wickeln. Die Ausbildung dieser multilamellaren Membranstapel ermöglicht eine saltatorische und damit rasche und energie-effiziente Erregungsleitung (Nave, 2010). Eine Schädigung des Myelins beeinträchtigt die Reizweiterleitung und führt zur Degeneration der Axone, wie es zum Beispiel bei der Multiplen Sklerose der Fall ist. Das Myelin basische Protein (MBP) ist ein Hauptbestandteil des Myelin und ist essentiell für die Kompaktierung der Myelinmembran (Wood et al., 1984). Die MBP mRNA wird in hnRNP A2 enthaltenen RNA Granulen in einem translations-inaktiven Zustand zu den distalen Fortsätzen transportiert. Vermittelt durch axonale Signale wird nach axo-glialem Kontakt die Translation von MBP ermöglicht (White et al., 2008). Der genaue Mechanismus der differentiellen Genregulation des MBP Proteins ist bisher nur unzureichend aufgeklärt. In der vorliegenden Arbeit konnte eine kleine regulatorische RNA (sncRNA) identifiziert werden, welche über die seed Region mit der MBP mRNA interagieren und die Translation regulieren kann. In primären Oligodendrozyten führt die Überexpression der sncRNA-715 zu reduzierten MBP Protein Mengen und die Blockierung der endogenen sncRNA-715 führt zu einer gesteigerten MBP Synthese. Interessanterweise korreliert während der Differenzierung der Oligodendrozyten in vitro und in vivo die Synthese des MBP Proteins invers mit der Expression der sncRNA-715. In Oligodendrozyten beeinflusst eine experimentell erhöhte sncRNA-715 Menge die Zellmorphologie und induziert Apoptose. Weiterhin ist sncRNA-715 in zytoplasmatischen granulären Strukturen lokalisiert und assoziiert mit MBP mRNA in hnRNP A2 Transport- Granula. Diese Ergebnisse lassen vermuten, dass sncRNA-715 ein Bestandteil der hnRNP A2 Granula sein könnte und dort spezifisch die Translation der MBP mRNA während des Lokalisationsprozesses inhibiert. In chronischen MS Läsionen sind Olig2+-Zellen zu finden. Obwohl die MBP mRNA in diesen Läsionen nachzuweisen ist, kann kein Protein synthetisiert werden. In dieser Arbeit konnte gezeigt werden, dass in diesen Läsionen die Expression der sncRNA-715 erhöht ist. SncRNA-715 könnte die Translation von MBP verhindern und folglich als Inhibitor der Remyelinisierung während des Krankheitsverlaufs fungieren. Schwann-Zellen sind die myelinisierenden Zellen im peripheren Nervensystem (PNS). Im Zuge der Myelinisierung wird die MBP mRNA in diesen Gliazellen ebenfalls in die distalen Fortsätze transportiert und dort lokal translatiert und in die Myelinmembran eingebaut (Trapp et al., 1987). Im Gegensatz zum ZNS ist im PNS nur wenig über den Transportmechanismus der mRNA bekannt (Masaki, 2012). Es ist es sehr wahrscheinlich, dass in Schwann-Zellen und Oligodendrozyten die Lokalisation und die translationale Hemmung der MBP mRNA ähnlichen Mechanismen unterliegen. In der vorliegenden Arbeit konnte gezeigt werden, dass hnRNP A2 und sncRNA-715 in Schwann-Zellen exprimiert werden und in zytoplasmatischen Granula-ähnlichen Strukturen lokalisiert sind. Während der Differenzierung dieser Gliazellen in vivo und in vitro korreliert die Expression der sncRNA-715 invers mit der Synthese des MBP Proteins. HnRNP A2 und sncRNA-715 scheinen in Schwann-Zellen assoziiert zu sein und könnten wie in Oligodendrozyten den Transport der MBP mRNA vermitteln.