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.

Fusionspeptid-, Redox-, Viruscore- und sonstige Proteine. Alle analysierten Single ORF (SO)-VVOV Rekombinanten vermittelten einen signifikanten Schutz vor einer tödlichen Belastung mit Aujeszky-Virus. Zwei der Rekombinanten (SO 93-, SO 94-VVOV) enthalten ORFs, die für ATI/Fusionspeptid-Proteine kodieren. In SO 19- und SO 70-VVOV sind dagegen für Redoxproteine kodierende ORFs integriert. Weiterführende Untersuchungen zeigten, dass SO 94- und SO 19-VVOV in zwei weiteren Modellsystemen immunstimulatorisch aktiv sind. Im Baculo-Virussystem exprimierte Proteine waren nur in Kombination mit Vaccinia Lister-Virus (VV) wirksam. Dabei zeigten jeweils Virus-Protein-Gemische mit dem geringsten Proteinanteil den stärksten immunstimulatorischen Effekt. Proben in denen VV durch bovines Herpes-Virus-1 ersetzt wurde, sind dagegen nicht wirksam. Dies lässt auf eine Beteiligung VV-spezifischer Faktoren schließen. Übereinstimmend mit diesen Ergebnissen führte eine Frameshift-Mutation in ORF 94r von SO 94mut-VVOV nur zur Abschwächung und nicht zum vollständigen Verlust der immunstimulatorischen Wirkung. Beide in Schizosaccharomyces pombe exprimierten Proteine, sp-ORF19 und sp-ORF94r, induzierten keinen signifikanten Schutz im Aujeszky Maus Modell. Mit der Identifikation einzelner immunstimulatorisch aktiver PPVO-Komponenten ist es erstmals gelungen, den paramunisierenden Effekt von Parapox-Viren einzelnen viralen Genen zu zuordnen. Insbesondere stellen SO 94- und SO 19-VVOV viel versprechende Kandidaten für die prophylaktische bzw. therapeutische Anwendung in verschiedenen Indikationen als auch für weitere Untersuchungen des Wirkmechanismus dar.

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.