The term "pattern formation" refers to the process by which order unfolds in development. The present thesis deals with a particular aspect of molecular pattern formation during vertebrate embryogenesis. The model system in the focus of this study is the zebrafish, Danio rerio. In the early developmental phases of the zebrafish, Fibroblast growth factors (Fgfs) are involved in the molecular patterning of various tissues, including two regions of the brain, the forebrain and the midbrain-hindbrain region, affecting cellular processes as diverse as cell proliferation, differentiation, and axonal targeting. The goal of this study was to better understand the mechanisms by which Fgf signaling regulates pattern formation and embryogenesis. I addressed this question on several levels, investigating the extent of intracellular signaling (MAPK activation) relative to sources of Fgf expression, and the transcriptional responses of cells to Fgf signaling during embryogenesis. By a macroarray analysis, I identified putative transcriptional targets of Fgf signaling in late gastrulation, providing a set of molecules that are likely to act as functional players in relaying the patterning information encoded by Fgf signals. Among those are the secreted signaling molecules Chordin and Wnt8, as well as Isthmin, a novel secreted molecule that I found capable to interfere with anterior embryonic patterning. In addition, I identified two ETS domain transcription factors, Erm and Pea3, which constitute bona fide integrators of FgfR signaling. By gain- and loss-of-function studies, I demonstrate that transcript levels of erm and pea3 are tightly regulated by Fgf signaling. Detailed analysis of the expression patterns of erm and pea3 along with other Fgf target genes also provides evidence for a differential read-out of Fgf concentration in the embryo, consistent with a role of Fgf as a vertebrate morphogen. The discovery of novel molecular components downstream of Fgf receptor activity paves a way to characterize previously unknown or underestimated developmental roles of Fgfs in the molecular patterning of the forebrain, the eye and parts of the neural crest.

The term "pattern formation" refers to the process by which order unfolds in development. The present thesis deals with a particular aspect of molecular pattern formation during vertebrate embryogenesis. The model system in the focus of this study is the zebrafish, Danio rerio. In the early developmental phases of the zebrafish, Fibroblast growth factors (Fgfs) are involved in the molecular patterning of various tissues, including two regions of the brain, the forebrain and the midbrain-hindbrain region, affecting cellular processes as diverse as cell proliferation, differentiation, and axonal targeting. The goal of this study was to better understand the mechanisms by which Fgf signaling regulates pattern formation and embryogenesis. I addressed this question on several levels, investigating the extent of intracellular signaling (MAPK activation) relative to sources of Fgf expression, and the transcriptional responses of cells to Fgf signaling during embryogenesis. By a macroarray analysis, I identified putative transcriptional targets of Fgf signaling in late gastrulation, providing a set of molecules that are likely to act as functional players in relaying the patterning information encoded by Fgf signals. Among those are the secreted signaling molecules Chordin and Wnt8, as well as Isthmin, a novel secreted molecule that I found capable to interfere with anterior embryonic patterning. In addition, I identified two ETS domain transcription factors, Erm and Pea3, which constitute bona fide integrators of FgfR signaling. By gain- and loss-of-function studies, I demonstrate that transcript levels of erm and pea3 are tightly regulated by Fgf signaling. Detailed analysis of the expression patterns of erm and pea3 along with other Fgf target genes also provides evidence for a differential read-out of Fgf concentration in the embryo, consistent with a role of Fgf as a vertebrate morphogen. The discovery of novel molecular components downstream of Fgf receptor activity paves a way to characterize previously unknown or underestimated developmental roles of Fgfs in the molecular patterning of the forebrain, the eye and parts of the neural crest.

In unseren Studien haben wir uns unter Verwendung von in vitro und in vivo Modellen des neuronalen Zelltodes mit verschiedenen neuroprotektiven Mechanismen beschäftigt. Uns interessierten Faktoren aus drei verschiedenen Komplexen, die an zellulärer Vitalität teilhaben. Im Mittelpunkt unserer Untersuchung stand ein antioxidatives Enzym und dessen Rolle bezüglich des neuronalen Zelltodes - die NAD(P)H-Quinonoxidoreduktase1. In den Sudien haben wir die Aktivität, Expression und die Lokalisation dieses Enzyms nach neuronalem Schaden untersucht. Wir konnten zeigen, dass die Quinonoxidoreduktase den neuronalen Schaden exazerbieren kann. Ein anderer Schwerpunkt unserer Arbeit bildete das Steroidhormon 17-beta-Estradiol und sein neuroprotektiver Einfluss auf neurodegenerative Prozesse. Wir deckten verschiedene Expressionen der beiden Estradiolrezeptoren alpha und beta in kortikalen, septalen und hippokampalen Nervenzellen auf und das daraus folgende unterschiedliche neuroprotektive Potenzial des Hormons. Im dritten Teil der Studien konnten wir den Einfluss des zytoskelettmodulierenden Proteins Gelsolin auf die Progression des neuronalen Zelltodes nach zellulärem Schaden charakterisieren. / In our studies we characterized different neuroprotective mechanism by in vitro and in vivo models of neuronal cell death. We were interested in factors of three different complexes wich play a role in cellulare vitality. In the centre of our studies there was an antioxidative enzyme and his role in the neuronal cell death-the NAD(P)H:quinone oxidoreductase1. We have investigated in the activity, expression and localisation of this enzyme after neuronal damage. We could show that this enzyme can exacerbate neuronal cell death. Another point of our work were the steroid hormon 17-beta-Estradiol and the neuroprotective character in neurodegeneration. We discovered different expression of the two estradiol- receptors alpha and beta in brain caused in different protective potential to cortical, septal and hippocampal structures by the hormon. The cytoskeletal structures modulate by the protein gelsolin was the third part of the studies. We could show that the modulation of the cytoskeleton were involved in the progression of neuronal cell death after cellulare damage.

In der vorliegenden Arbeit sind Untersuchungen vorgestellt, die sich mit Wirkungen und Interaktionen von zwei ubiquitär im menschlichen Körper vorkommenden Gasen befassen, i.e. Sauerstoff und Stickstoffmonoxid. Im Falle beider Substanzen ermöglicht die geringe Größe der Moleküle eine freie Diffusion über Membranen hinweg, eine Eigenschaft, die für die Funktion der Signaltransduktion geradezu prädestiniert. Aus den vorgelegten Untersuchungen lassen sich die folgenden Folgerungen ableiten: * Stickstoffmonoxid wirkt in-vitro selektiv bakteriostatisch auf Bakterien, die üblicherweise Früh- und Neugeborene besiedeln. Dabei hängt die Selektivität von den jeweiligen bakteriellen Verteidigungsmechanismen ab, die bakteriostatische Wirkung liegt in einem Konzentrationsbereich, der außerhalb desjenigen liegt, der derzeit klinisch angewendet wird. * Hyperoxie führt im Ganztiermodell der unreifen Ratte zu einer zerebralen Hochregulation von iNOS und damit zur Synthese von NO. Soweit dies anhand der Synthese von Peroxynitrit als definitivem Schädigungsmechanismus beurteilbar ist, wird trotz entsprechender iNOS-Expression wenig bis gar kein Peroxynitrit gebildet. Da das Zusammentreffen von NO und Sauerstoff sonst regelhaft zur Entstehung von Peroxynitrit führt, müssen im Gehirn der unreifen Ratte ausreichende antioxidative Schutzmechanismen präsent sein, die diese Reaktion verhindern. * Im in-vitro-Modell der Gasäquilibrierung von Nabelschnur-PMN zeigte sich unter Hyperoxie das ausgeprägteste Aktivierungsmuster aller verglichenen Sauerstoffkonzentrationen. Dies stand im Gegensatz zur Exposition adulter Zellen, hier fand sich eine größere Hyperoxietoleranz bei gleichzeitig stärkster Aktivierung unter Hypoxiebedingungen. Welche Bedeutung diesen Ergebnissen im klinischen Umgang mit Neugeborenen zukommt muß derzeit noch offen bleiben. Allerdings häufen sich Hinweise aus experimentellen Studien, die darauf hindeuten, daß ein restriktiver Umgang mit hohen Sauerstoffkonzentrationen auch im klinischen Umfeld gerechtfertigt sein könnte. / The present investigations deal with the effects and interactions of gases, which are ubiquitous in the human body i.e. oxygen and nitric oxide. Both substances are small enough to freely diffuse across biological membranes. This ability predestines both molecules for the function of signal transduction. The results of our investigations lead to conclusions as follows: * Nitric oxide has selective bacteriostatic effects in-vitro on some bacterial strains typically isolated from preterm and term newborn infants. Selectivity depends on the presence of bacterial defense mechanisms. The bacteriostatic effect takes place at concentrations above those currently used in clinical practice. * Hyperoxia leads to upregulation of iNOS and subsequent NO production in an animal model of the immature rat. Despite this upregulation of iNOS synthesis there is no increased production of peroxynitrite which is known to cause cellular and DNA damage. Since the combination of NO and high concentrations of oxygen lead to peroxynitrite formation on a regular basis, effective antioxidant mechanisms appear to prevent peroxynitrite formation in the brain of the immature rat. * The most pronounced activation of cord blood polymorphonuclear cells (PMN) during conditions of hyperoxia, normoxia, and hypoxia was found for exposure towards high oxygen concentrations in an in-vitro model of gas equilibration. As opposed to that, hypoxia was the most potent trigger for adult PMN. It remains to be determined which clinical implications must be derived from these results. However, increasing experimental evidence indicates that exposure towards high oxygen concentrations should be restricted also in clinical practice and not only in preterm infants, but also in term newborns.

The goal of a Brain-Computer Interface (BCI) consists of the development of a unidirectional interface between a human and a computer to allow control of a device only via brain signals. While the BCI systems of almost all other groups require the user to be trained over several weeks or even months, the group of Prof. Dr. Klaus-Robert Müller in Berlin and Potsdam, which I belong to, was one of the first research groups in this field which used machine learning techniques on a large scale. The adaptivity of the processing system to the individual brain patterns of the subject confers huge advantages for the user. Thus BCI research is considered a hot topic in machine learning and computer science. It requires interdisciplinary cooperation between disparate fields such as neuroscience, since only by combining machine learning and signal processing techniques based on neurophysiological knowledge will the largest progress be made.<br><br> In this work I particularly deal with my part of this project, which lies mainly in the area of computer science. I have considered the following three main points:<br><br> <b>Establishing a performance measure based on information theory:</b> I have critically illuminated the assumptions of Shannon's information transfer rate for application in a BCI context. By establishing suitable coding strategies I was able to show that this theoretical measure approximates quite well to what is practically achieveable.<br> <b>Transfer and development of suitable signal processing and machine learning techniques:</b> One substantial component of my work was to develop several machine learning and signal processing algorithms to improve the efficiency of a BCI. Based on the neurophysiological knowledge that several independent EEG features can be observed for some mental states, I have developed a method for combining different and maybe independent features which improved performance. In some cases the performance of the combination algorithm outperforms the best single performance by more than 50 %. Furthermore, I have theoretically and practically addressed via the development of suitable algorithms the question of the optimal number of classes which should be used for a BCI. It transpired that with BCI performances reported so far, three or four different mental states are optimal. For another extension I have combined ideas from signal processing with those of machine learning since a high gain can be achieved if the temporal filtering, i.e., the choice of frequency bands, is automatically adapted to each subject individually.<br> <b>Implementation of the Berlin brain computer interface and realization of suitable experiments:</b> Finally a further substantial component of my work was to realize an online BCI system which includes the developed methods, but is also flexible enough to allow the simple realization of new algorithms and ideas. So far, bitrates of up to 40 bits per minute have been achieved with this system by absolutely untrained users which, compared to results of other groups, is highly successful. / Ein Brain-Computer Interface (BCI) ist eine unidirektionale Schnittstelle zwischen Mensch und Computer, bei der ein Mensch in der Lage ist, ein Gerät einzig und allein Kraft seiner Gehirnsignale zu steuern. In den BCI Systemen fast aller Forschergruppen wird der Mensch in Experimenten über Wochen oder sogar Monaten trainiert, geeignete Signale zu produzieren, die vordefinierten allgemeinen Gehirnmustern entsprechen. Die BCI Gruppe in Berlin und Potsdam, der ich angehöre, war in diesem Feld eine der ersten, die erkannt hat, dass eine Anpassung des Verarbeitungssystems an den Menschen mit Hilfe der Techniken des Maschinellen Lernens große Vorteile mit sich bringt. In unserer Gruppe und mittlerweile auch in vielen anderen Gruppen wird BCI somit als aktuelles Forschungsthema im Maschinellen Lernen und folglich in der Informatik mit interdisziplinärer Natur in Neurowissenschaften und anderen Feldern verstanden, da durch die geeignete Kombination von Techniken des Maschinellen Lernens und der Signalverarbeitung basierend auf neurophysiologischem Wissen der größte Erfolg erzielt werden konnte.<br><br> In dieser Arbeit gehe ich auf meinem Anteil an diesem Projekt ein, der vor allem im Informatikbereich der BCI Forschung liegt. Im Detail beschäftige ich mich mit den folgenden drei Punkten:<br><br> <b>Diskussion eines informationstheoretischen Maßes für die Güte eines BCI's:</b> Ich habe kritisch die Annahmen von Shannon's Informationsübertragungsrate für die Anwendung im BCI Kontext beleuchtet. Durch Ermittlung von geeigneten Kodierungsstrategien konnte ich zeigen, dass dieses theoretische Maß den praktisch erreichbaren Wert ziemlich gut annähert.<br> <b>Transfer und Entwicklung von geeigneten Techniken aus dem Bereich der Signalverarbeitung und des Maschinellen Lernens:</b> Eine substantielle Komponente meiner Arbeit war die Entwicklung von Techniken des Machinellen Lernens und der Signalverarbeitung, um die Effizienz eines BCI's zu erhöhen. Basierend auf dem neurophysiologischem Wissen, dass verschiedene unabhängige Merkmale in Gehirnsignalen für verschiedene mentale Zustände beobachtbar sind, habe ich eine Methode zur Kombination von verschiedenen und unter Umständen unabhängigen Merkmalen entwickelt, die sehr erfolgreich die Fähigkeiten eines BCI's verbessert. Besonders in einigen Fällen übertraf die Leistung des entwickelten Kombinationsalgorithmus die beste Leistung auf den einzelnen Merkmalen mit mehr als 50 %. Weiterhin habe ich theoretisch und praktisch durch Einführung geeigneter Algorithmen die Frage untersucht, wie viele Klassen man für ein BCI nutzen kann und sollte. Auch hier wurde ein relevantes Resultat erzielt, nämlich dass für BCI Güten, die bis heute berichtet sind, die Benutzung von 3 oder 4 verschiedenen mentalen Zuständen in der Regel optimal im Sinne von erreichbarer Leistung sind. Für eine andere Erweiterung wurden Ideen aus der Signalverarbeitung mit denen des Maschinellen Lernens kombiniert, da ein hoher Erfolg erzielt werden kann, wenn der temporale Filter, d.h. die Wahl des benutzten Frequenzbandes, automatisch und individuell für jeden Menschen angepasst wird.<br> <b>Implementation des Berlin Brain-Computer Interfaces und Realisierung von geeigneten Experimenten:</b> Eine weitere wichtige Komponente meiner Arbeit war eine Realisierung eines online BCI Systems, welches die entwickelten Methoden umfasst, aber auch so flexibel ist, dass neue Algorithmen und Ideen einfach zu verwirklichen sind. Bis jetzt wurden mit diesem System Bitraten von bis zu 40 Bits pro Minute von absolut untrainierten Personen in ihren ersten BCI Experimenten erzielt. Dieses Resultat übertrifft die bisher berichteten Ergebnisse aller anderer BCI Gruppen deutlich. <br> <hr> Bemerkung:<br> Der Autor wurde mit dem <i>Michelson-Preis</i> 2005/2006 für die beste Promotion des Jahrgangs der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Potsdam ausgezeichnet.

The central theme of development, how cells are organized into functional structures and assembled into whole organisms, is addressed by developmental biology. One important feature of embryonic development is pattern formation, which is the generation of a particular arrangement of cells in three-dimensional space at a given point of time. Central to this work is the model system of the zebrafish, Danio rerio. The aim of the first part of this study was to try to understand how a distinct part of the embryonic brain called midbrain-hindbrain boundary (MHB), a region that acts as an organizer for the adjacent brain regions, is established in vertebrates. spiel-ohne-grenzen (spg) is one mutant which interferes with MHB development. Here, I addressed the role of pou2 in brain development by molecular, phenotypical and functional analysis. By genetic complementation and mapping I could elucidate the molecular nature of this mutant and found that the pou2 gene encoding the POU domain transcription factor is affected in spg mutant embryos. By chromosomal syntenic conservation, phylogenetic sequence comparison, and expression and functional data I imply that pou2 is the orthologue of the mammalian Oct4 (Pou5F1) gene. I find by detailed expression and transplantation analysis that pou2 is cell autonomously required within the neuroectoderm to activate genes of the MHB and hindbrain primordium, like pax2.1, wnt1, gbx2 or krox20. By gain-of-function experiments I demonstrate that pou2 synergizes with Fgf8 signaling in order to activate particularly the hindbrain primordium. Since pou2 is already provided to the embryo by the mother, I generated embryos which lack maternal and zygotic pou2 function (MZspg) to reveal a possible earlier than neuroectodermal role of pou2. In the second part of this work I demonstrate that pou2 is a key factor controlling endoderm differentiation. By expression and gain-of-function analysis I suggest a cell autonomous function for Pou2 in the first step of endodermal differentiation. By gain-of-function experiments involving the gene encoding the HMG transcription factor Casanova (Cas) I show that both Cas and Pou2 are necessary to activate expression of the endodermal differentiation marker sox17 in a mutually dependent way, and that the ability of Cas to ectopically induce sox17 strictly requires Pou2. I conclude that both maternal and zygotic pou2 function is necessary for commitment of endodermal progenitor cells to differentiate into endodermal precursor cells.

Developmental biology adresses how cells are organised into functional structures and eventually into a whole organism. It is crucial to understand the molecular basis for processes in development, by studying the expression and function of relevant genes and their relationship to each other. A gene function can be studied be creating loss-of-function situations, in which the change in developmental processes is examined in the absense of a functional gene product, or in gain-of-function studies, where a gene product is either intrinsically overproduced or ectopically upregulated. One approach for a loss-of-function situation is the creation of specific mutants in single genes, and the zebrafish (Danio rerio) has proven to be an excellent model organism for this purpose. In this thesis, I report on two forward genetic screens performed to find new mutants affecting brain development, in particular mutants defective in development and function of the midbrain-hindbrain boundary (MHB), an organiser region that patterns the adjacent brain regions of the midbrain and the hindbrain. In the first screen, I could identify 10 specific mutants based on morphology and the analysis of the expression patterns of lim1 and fgf8, genes functioning as early neuronal markers and as a patterning gene, respectively. Three of these mutants lacked an MHB, and by complementation studies, I identified these mutants as being defective in the spg locus. The second screen produced 35 new mutants by screening morphologically and with antibodies against acetylated Tubulin, which marks all axonal scaffolds, and anti-Opsin, which is a marker for photoreceptors in the pineal gland. According to their phenotype, I distributed the mutant lines into 4 phenotypic subgroups, of which the brain morphology group with 18 mutant lines was studied most intensively. In the last part of my thesis, I characterise one of these brain morphology mutants, broken heart. This mutant is defective in axonal outgrowth and locomotion, and shows a striking reduction of serotonergic neurons in the epiphysis and in the raphe nuclei in the hindbrain, structures involved in serotonin and melatonin production. Studies in other model organisms suggested a role of factors from the floor plate and the MHB in induction of the serotonergic neurons in the hindbrain, and using broken heart, I show that Fgf molecules such as Fgf4 and Fgf8 can restore partially the loss of serotonergic neurons in the mutant. I conclude that forward genetic screens are an invaluable tool to generate a pool of mutations in specific genes, which can be used to dissect complex processes in development such as brain development.

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