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.

No description available.

No description available.

Calcium imaging has been used as a promising technique to monitor the dynamic activity of neuronal populations. However, the calcium trace is temporally smeared which restricts the extraction of quantities of interest such as spike trains of individual neurons. To address this issue, spike reconstruction algorithms have been introduced. One limitation of such reconstructions is that the underlying models are not informed about the biophysics of spike and burst generations. Such existing prior knowledge might be useful for constraining the possible solutions of spikes. Here we describe, in a novel Bayesian approach, how principled knowledge about neuronal dynamics can be employed to infer biophysical variables and parameters from fluorescence traces. By using both synthetic and in vitro recorded fluorescence traces, we demonstrate that the new approach is able to reconstruct different repetitive spiking and/or bursting patterns with accurate single spike resolution. Furthermore, we show that the high inference precision of the new approach is preserved even if the fluorescence trace is rather noisy or if the fluorescence transients show slow rise kinetics lasting several hundred milliseconds, and inhomogeneous rise and decay times. In addition, we discuss the use of the new approach for inferring parameter changes, e.g. due to a pharmacological intervention, as well as for inferring complex characteristics of immature neuronal circuits.

Die asymmetrische Verteilung von mRNA und Proteinen innerhalb einer Zelle definiert die Polarität. Dies ermöglicht eine strikte Regulierung der Genexpression in Raum und Zeit. Ich habe in dieser Arbeit untersucht, wie das Soma und die Neuriten in induzierten Neuronen sich hinsichtlich ihres Transkriptoms und Translatoms unterscheiden. Eine räumliche ribosomale Profilanalyse ergab, dass die Hälfte des lokalen Proteoms durch die mRNA-Lokalisierung und der lokalen Translation definiert wird. Dies sind Prozesse, die durch die synergistische Aktivität von trans- und cis-agierenden Elementen durchgeführt werden. In dieser Arbeit konzentrierte ich mich auf MOV10 als trans-agierendes Element und die alternativen 3′UTRs als cis-agierende Elemente, um ihre Rolle in der Asymmetrie zu untersuchen. MOV10 ist eine RNA-Helikase, welche an vielen Aspekten des RNA-Metabolismus beteiligt ist. Mit den Methoden RIP und PAR-CLIP konnte ich zeigen, dass sowohl MOV10-Ziele als auch MOV10 selbst in den Neuriten lokalisiert sind. Aus ̈erdem ist MOV10 möglicherweise an der translationalen Repression mitinvolviert. In der Tat konnte ich unter den MOV10-Protein-Interaktoren mehrere Proteine identifizieren, welche an der translationalen Repression beteiligt sind, wie z.Bsp. AGO2, FMR1, und TRIM71. Für die Identifizierung der cis-agierenden Elemente führte ich das "Mapping" von alternativen 3′UTRs durch. Diese Analyse zeigte mehrere Gene, die differentiell lokalisierte 3′UTR-Isoformen exprimieren. Insbesondere habe ich mich auf Cdc42 konzentriert. Ich konnte beweisen, dass die beiden Isoformen von Cdc42 auf mRNA-Ebene unterschiedlich lokalisiert sind und dass die 3′UTR der entscheidende Faktor für die mRNA- und Proteinlokalisierung ist. Darüber hinaus habe ich mehrere RBPs identifiziert, die an der Cdc42-Lokalisierung beteiligt sind. Diese Analyse zeigt, dass für die differenzierte Lokalisierung von funktional unterschiedlichen alternativen Protein-Isoformen die Verwendung von alternativen 3′UTR Isoformen als neu-entdeckter Mechanismus eine entscheidende Rolle spielt. / Asymmetric distribution of mRNA and proteins inside a cell defines polarity, which allow tight regulation of gene expression in space and time. In this thesis I investigated how asymmetric distribution characterizes the somatic and neuritic compartments of in induced neurons, in terms of transcriptome and translatome. Spatial ribosome profiling analysis revealed that half of the local proteome is defined by mRNA localization and local translation. These, are processes accomplished by the synergistic activity of trans- and cis-acting elements. I focused on MOV10 as trans-acting element, and on alternative 3′UTRs as cis-elements, to investigate their role in asymmetry. MOV10 is an RNA helicase which participates to many aspects of RNA metabolism. With RIP and PAR-CLIP I showed that MOV10 targets are localized to the neurites, consistently with MOV10-neuritic localization, and that MOV10 might be involved in translational repression. Indeed, among MOV10 protein interactors, I identified several proteins involved in translational repression, i.e. AGO2, FMR1, and TRIM71. On the side of cis-elements, I performed mapping of alternative 3′UTRs. This analysis identified several genes expressing differentially localized 3′UTR isoforms. In particular, I focused on Cdc42. I showed that the two isoforms of Cdc42 are differentially localized at mRNA level, and that the 3′UTR is the driver of mRNA and protein localization. Moreover, I identified several RBPs that might be involved in Cdc42 localization. This analysis points to usage of alternative 3′UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms.

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.

In dieser Arbeit werden mithilfe der Fokker-Planck-Gleichung die Statistiken, vor allem die Leistungsspektren, von Punktprozessen berechnet, die von mehrdimensionalen Integratorneuronen [Engl. integrate-and-fire (IF) neuron], Netzwerken von IF Neuronen und Entscheidungsfindungsmodellen erzeugt werden. Im Gehirn werden Informationen durch Pulszüge von Aktionspotentialen kodiert. IF Neurone mit radikal vereinfachter Erzeugung von Aktionspotentialen haben sich in Studien die auf Pulszeiten fokussiert sind als Standardmodelle etabliert. Eindimensionale IF Modelle können jedoch beobachtetes Pulsverhalten oft nicht beschreiben und müssen dazu erweitert werden. Im erste Teil dieser Arbeit wird eine Theorie zur Berechnung der Pulszugleistungsspektren von stochastischen, multidimensionalen IF Neuronen entwickelt. Ausgehend von der zugehörigen Fokker-Planck-Gleichung werden partiellen Differentialgleichung abgeleitet, deren Lösung sowohl die stationäre Wahrscheinlichkeitsverteilung und Feuerrate, als auch das Pulszugleistungsspektrum beschreibt. Im zweiten Teil wird eine Theorie für große, spärlich verbundene und homogene Netzwerke aus IF Neuronen entwickelt, in der berücksichtigt wird, dass die zeitlichen Korrelationen von Pulszügen selbstkonsistent sind. Neuronale Eingangströme werden durch farbiges Gaußsches Rauschen modelliert, das von einem mehrdimensionalen Ornstein-Uhlenbeck Prozess (OUP) erzeugt wird. Die Koeffizienten des OUP sind vorerst unbekannt und sind als Lösung der Theorie definiert. Um heterogene Netzwerke zu untersuchen, wird eine iterative Methode erweitert. Im dritten Teil wird die Fokker-Planck-Gleichung auf Binärentscheidungen von Diffusionsentscheidungsmodellen [Engl. diffusion-decision models (DDM)] angewendet. Explizite Gleichungen für die Entscheidungszugstatistiken werden für den einfachsten und analytisch lösbaren Fall von der Fokker-Planck-Gleichung hergeleitet. Für nichtliniear Modelle wird die Schwellwertintegrationsmethode erweitert. / This thesis is concerned with the calculation of statistics, in particular the power spectra, of point processes generated by stochastic multidimensional integrate-and-fire (IF) neurons, networks of IF neurons and decision-making models from the corresponding Fokker-Planck equations. In the brain, information is encoded by sequences of action potentials. In studies that focus on spike timing, IF neurons that drastically simplify the spike generation have become the standard model. One-dimensional IF neurons do not suffice to accurately model neural dynamics, however, the extension towards multiple dimensions yields realistic behavior at the price of growing complexity. The first part of this work develops a theory of spike-train power spectra for stochastic, multidimensional IF neurons. From the Fokker-Planck equation, a set of partial differential equations is derived that describes the stationary probability density, the firing rate and the spike-train power spectrum. In the second part of this work, a mean-field theory of large and sparsely connected homogeneous networks of spiking neurons is developed that takes into account the self-consistent temporal correlations of spike trains. Neural input is approximated by colored Gaussian noise generated by a multidimensional Ornstein-Uhlenbeck process of which the coefficients are initially unknown but determined by the self-consistency condition and define the solution of the theory. To explore heterogeneous networks, an iterative scheme is extended to determine the distribution of spectra. In the third part, the Fokker-Planck equation is applied to calculate the statistics of sequences of binary decisions from diffusion-decision models (DDM). For the analytically tractable DDM, the statistics are calculated from the corresponding Fokker-Planck equation. To determine the statistics for nonlinear models, the threshold-integration method is generalized.

No description available.

Neuronen sind bei der Informationsübertragung des zentralen Nervensystems von entscheidender Bedeutung. Ihre Aktivität liegt der Signalverarbeitung und höheren kognitiven Prozessen zugrunde. Neuronen sind in den extrazellulären Raum eingebettet, der mehrere Teilchen, darunter auch Ionen, enthält. Ionenkonzentrationen sind nicht statisch. Intensive neuronale Aktivität kann intrazelluläre und extrazelluläre Ionenkonzentrationen verändern. In dieser Arbeit untersuche ich das Wechselspiel zwischen neuronaler Aktivität und der Dynamik der Ionenkonzentrationen. Dabei konzentriere ich mich hauptsächlich auf extrazelluläre Kalium- und intrazelluläre Natriumkonzentrationen. Mit Hilfe der Theorie dynamischer Systeme zeige ich, wie moderate Änderungen dieser Ionenkonzentrationen die neuronale Aktivität qualitativ verändern können, wodurch sich möglicherweise die Signalverarbeitung verändert. Dann modelliere ich ein leitfähigkeitsbasiertes neuronales Netzwerk mit Spikes. Das Modell sagt voraus, dass eine moderate Änderung der Konzentrationen, die einen Mikroschaltkreis von Neuronen umgeben, die Leistungsspektraldichte der Populationsaktivität verändern könnte. Insgesamt unterstreicht diese Arbeit die Bedeutung der Dynamik der Ionenkonzentrationen für das Verständnis neuronaler Aktivität auf langen Zeitskalen und liefert technische Erkenntnisse darüber, wie das Zusammenspiel zwischen ihnen modelliert und analysiert werden kann. / Neurons are essential in the information transfer mechanisms of the central nervous system. Their activity underlies both basic signal processing, and higher cognitive processes. Neurons are embedded in the extracellular space, which contains multiple particles, including ions which are vital to their functioning. Ionic concentrations are not static, intense neuronal activity alters the intracellular and extracellular ionic concentrations which in turn affect neuronal functioning. In this thesis, I study the interplay between neuronal activity and ionic concentration dynamics. I focus specifically on the extracellular potassium and intracellular sodium concentrations. Using dynamical systems theory, I illustrate how moderate changes in these ionic concentrations can qualitatively change neuronal activity, potentially altering signal processing. I then model a conductance-based spiking neural network. The model predicts that a moderate change in the concentrations surrounding a microcircuit of neurons could modify the power spectral density of the population activity. Altogether, this work highlights the need to consider ionic concentration dynamics to understand neuronal activity on long time scales and provides technical insights on how to model and analyze the interplay between them.

Koordinierte Phosphorylierung der C-terminale Domäne von RNA Polymerase II (RNAPII) ist essentiell für eine effiziente Kupplung von naszierender RNA Synthese und co-transkriptionalem RNA Prozessierens. Zirkuläre RNAs (circRNAs) sind eine neue Klasse von RNA Molekülen mit hoher Prävalenz in neuronalen Zelltypen. Die Biogenese von circRNAs ist noch ungeklärt, insbesondere die Frage warum das Intron upstream der circRNA während der Transkription des circRNA Exons zurückbehalten wird um Rück-Spleißen zu ermöglichen. Verschiede Belege suggerieren, dass unzulängliche Rekrutierung des Spleiceosoms zur circRNA Formation führen kann. In dieser Arbeit untersuche ich die Mechanismen die zu Defekten in der Erkennung und des Spleißens des Introns upstream der circRNA führen. Mit diesem Ziel erfasste ich die genomweite Verteilung von chromatinassoziierter RNAPII mit verschiedenen Phosphorylierungen, sowie Spleißfaktoren und Transkriptionsreglern mittels ChIP-seq in neuronaler Differenzierung von murinen embryonalen Stammzellen zu dopaminergen und Motoneuronen. Während der gesamten Differenzierung, aber insbesondere in den differenzieren Neuronen, konnten circRNAs detektiert werden. In meiner Arbeit finde ich, dass circRNAs detektiert werden, wenn Gene hohe Levels an mRNA exprimieren und, dass die Produktion von circRNA mit einer Dysbalance zwischen dem Laden der RNA-Polymerase II auf die DNA und dem Rekruitieren der Splice-Maschinerie zusammen hängt. Um funktionell mit den Pausier-Mechanismen der RNA-Polymerase II zu interferieren, habe ich einen ''promotor-proximal-pausing'' Faktor depletiert. Dabei stellte ich fest, dass diese Depletion genügt, um die circRNA Levels in embryonalen Stamzellen zu erhöhen. Die Ergebnisse die in dieser Arbeit gezeigt werden, beschreiben die Beteiligung des Pausierens der RNA-Polymerase II and der Formierung von circRNAs. / Coordinated phosphorylation of RNA polymerase II (RNAPII) C-terminal domain is essential for efficient coupling of nascent RNA synthesis with co-transcriptional RNA processing events. Circular RNAs (circRNAs) are a novel class of RNAs whose biogenesis remains ill understood, namely why the upstream intron is not spliced before the circRNA-exon is fully transcribed. Indirect evidence suggests that altered spliceosome recruitment can lead to circRNA formation. To investigate the mechanisms that may be involved in deficient recognition and splicing of introns upstream of exons included in circRNAs, I mapped the chromatin occupancy of RNAPII phosphorylated forms, splicing factors, and transcription regulators by ChIP-seq during mouse ESC differentiation to dopaminergic and spinal motor neurons. CircRNAs are detected throughout differentiation, peaking in differentiated neurons, as expected. I found that circRNAs are detected when genes express high levels of mRNA, and that circRNA production is associated with an imbalance between RNAPII loading and recruitment of the splicing machinery. To mechanistically interfere with pausing mechanisms, I depleted an RNAPII promoter-proximal pausing factor, and found that it was sufficient to increase the formation of circRNAs in stem cells. Results shown in this work implicate RNAPII regulation mechanisms in the formation of circRNAs.