Alternatives Spleißen ist von großer Bedeutung für die Steuerung der Genexpression und Proteindiversität. Die Cdc2-like Kinasen (Clk) und DNA Topoisomerase I (DNA topo I) steuern alternatives Spleißen über die Phosphorylierung von Serine/Arginin-reichen (SR) Proteinen. Tissue Factor (TF) und die endotheliale Stickstoffmonoxidsynthase (eNOS) beeinflussen maßgeblich die Endothelfunktion. Alternatives Spleißen führt zur Bildung von Isoformen von TF („full length“ (fl)TF, alternativ gespleißter humaner (asH)TF) und eNOS (eNOS, eNOS 13A, B und C). Wie das alternative Spleißen von TF und eNOS gesteuert wird und welchen Einfluss dies auf die Endothelfunktion hat ist unbekannt und wurde in dieser Arbeit untersucht. Die TNF-a-Stimulation der HUVEC erhöhte die Expression beider TF-Isoformen. Die Clk-Inhibition reduzierte die mRNA-Expression von flTF und asHTF. Die DNA topo I-Inhibition erhöhte die asHTF-Expression und verringerte flTF. Die Inhibition der DNA topo I reduzierte ebenfalls die Expression des flTF-Proteins aber nicht von asHTF, verbunden mit einer reduzierten TF-Aktivität. Die Clk-Inhibition reduzierte das flTF-Protein und die TF-Aktivität nur leicht aber asHTF vollständig. Die Inhibition der Kinasen beeinflusste das SR Protein-Phosphorylierungsmuster. Die Inhibition von SF2/ASF und SRp75 mittels siRNAs veränderte die Expression und die Aktivität von TF in HUVEC. Die TNF-a-Stimulation von HUVEC induzierte ferner die mRNA-Expression von eNOS 13A, B und C, nicht aber von eNOS. Dies führte zu einer Reduktion der eNOS-Aktivität. Die Inhibition der DNA topo I, nicht aber der Clks hob diese Effekte auf. Diese Daten zeigen, dass die DNA topo I sowie die Clks an der Regulation der endothelialen TF-Expression und Aktivität beteiligt sind, während die eNOS-Isoformexpression und Aktivität nur durch die DNA topo I beeinflusst wurde.Diese Ergebnisse eröffnen neue Einblicke in die Regulation des alternativen Spleißens von TF und eNOS sowie dessen Einfluss auf die Endothelfunktion. / Alternative splicing is an important mechanism to control gene expression and protein diversity. The Cdc2-like kinases (Clk) and DNA topoisomerase I (DNA topo I) control alternative splicing by regulating the phosphorylation state of serine/arginine-rich (SR) proteins. Tissue Factor (TF) and the endothelial nitric oxide synthase (eNOS) are crucial for the endothelial function. Alternative splicing lead to the formation of different isoforms of TF („full length“ (fl)TF, alternatively spliced human (asH)TF) and eNOS (eNOS, eNOS 13A, B and C). How alternative splicing of TF and eNOS is regulated as well as the impact on endothelial function is still unknown and was investigated in this study. The stimulation of HUVEC with TNF-a induced the expression of both TF isoforms. The inhibition of Clks reduced the mRNA expression of flTF and asHTF. DNA topo I inhibition increased asHTF and reduced flTF mRNA expression. Inhibition of DNA topo I also reduced flTF but not asHTF on protein level in stimulated cells leading to a reduced TF activity. Clk-inhibition slightly reduced flTF protein and TF activity, whereas asHTF was completely blocked. The inhibition of both kinases altered the phosphorylation pattern of SR proteins. The siRNA-mediated inhibition of SF2/ASF and SRp75 modified the TF isoform expression and TF activity in TNF-a-induced HUVEC. Moreover, stimulation of HUVEC with TNF-a induced the mRNA expression of eNOS 13A, B and C, but not of eNOS. This led to a reduction of eNOS activity. The inhibition of DNA topo I but not of Clks abolished these TNF-a-mediated effects in HUVEC. These data indicate DNA topo I and the Clks to be involved in the regulation of endothelial TF expression and activity, whereas, eNOS isoform expression and activity was influenced by DNA topo I, but not Clks in TNF-a-stimulated HUVEC. In conclusion, this study reveals new insights into the regulation of alternative splicing of TF and eNOS and the impact of these processes on endothelial function.

The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

To form complex organisms characterized by different tissues with specialized functions, cells must acquire distinct identities during development. Yet, all the cells of an organism are equipped with the same genomic information. Elucidating the mechanisms that regulate the determination of a cell identity, i.e. the cell-fate commitment, is a main purpose in developmental biology. Numerous studies focused on genes that are activated or repressed at each stage of differentiation, identifying several key regulators of development. However, this approach ignores the transcript variability derived from alternative splicing, the transcriptional process by which different gene coding segments, i.e. exons, are combined giving rise to multiple transcripts and proteins from the same gene. With the advent of novel sequencing technologies, it is becoming clear that alternative splicing is widespread in higher organisms, regulates several processes and presents tissue- and cell-specificity. In mammals, the brain shows the highest degree of alternative splicing, with neurons expressing a high variety of splice variants. In this project I investigated whether and how alternative splicing could regulate cell-fate determination in the context of the embryonic development of the mouse neocortex, a highly complex structure presenting several different neuronal subtypes generated at specific time points. For this purpose, I analyzed transcriptome data of cells of the neurogenic lineage isolated from the developing mouse neocortex at subsequent stages of differentiation. I showed that the expression pattern of the proteins regulating splicing, i.e. the splicing factors, changes during neocortical development. By employing several bioinformatic tools, I described the splicing profile that characterizes each differentiation stage and, for the first time, I identified the splicing events that mark cell-fate commitment to a neurogenic identity. Alternative splicing mostly involved genes with a role in nervous system development, cell growth and signaling, mainly leading to the production of alternative protein isoforms. Splicing choices taken during the neurogenic commitment were kept throughout neurogenesis. Thus, exons that start to be included during cell-fate determination are always included in post-mitotic neurons. Exons gained during neurogenic commitment were characterized by strong features in their upstream intron, presented a general short length with an overrepresentation of microexons in the 3-27 nucleotides length range and showed an enrichment for binding motifs of the neural splicing factor nSR100. In vivo manipulation in the embryonic mouse neocortex highlighted isoform-specific effects on neocortical development, strongly suggesting a causal relationship between alternative splicing choices and cell-fate commitment. Moreover, the higher cell-specificity offered by the present dataset, compared to similar studies, allowed a better understanding of previously identified splicing events that characterize the nervous system and the relationships between neural-specific splicing factors.:Table of Contents Abstract I Zusammenfassung III Table of Contents V List of Figures VII List of Tables IX Abbreviations X Gene abbreviations XII 1 Introduction 1 1.1 Neurogenesis during embryonic development 2 1.1.1 Formation and patterning of the neural tube 2 1.1.2 Neural progenitors in the dorsal telencephalon 6 1.1.3 Neurogenesis 8 1.1.4 Regulation of neurogenesis 10 1.1.5 A novel tool to investigate cell-fate determination in the central nervous system: the Btg2RFP/Tubb3GFP mouse line 13 1.2 Alternative splicing: an additional level of genomic regulation 15 1.2.1 The splicing reaction 16 1.2.2 What makes splicing alternative? 18 1.2.3 Regulation of alternative splicing 19 1.2.4 The challenge to detect splicing 23 1.2.5 New sequencing technologies reveal a high transcriptome complexity 29 1.2.6 Splicing in nervous system development 31 1.2.7 Aims of the project 36 2 Materials and methods 38 2.1 Materials 38 2.1.1 Bacteria, cells, mouse strains 38 2.1.2 Vector 38 2.1.3 Primers 38 2.1.4 Chemicals and buffers 41 2.1.5 Antibodies 42 2.1.6 Kits and enzymes 42 2.2 Methods 43 2.2.1 Animal experiments 43 2.2.2 Molecular biology 44 2.2.3 Immunohistochemistry 46 2.2.4 Bioinformatics 47 3 Results 53 3.1 Splicing factors are differentially expressed during neurogenic commitment and neurogenesis 53 3.2 Detection of alternative splicing 55 3.2.1 Isoform-switching 55 3.2.2 Exon usage and splicing events 57 3.3 Validation 62 3.3.1 The isoform switching method has a poor validation rate 62 3.3.2 Analysis at the exon level has a high rate of validation 65 3.4 Pattern and representation of splicing events 67 3.4.1 Splicing choices during neurogenic commitment define the splicing profiles of neurons 67 3.4.2 Splicing events: microexon inclusion characterizes neurogenic commitment 69 3.5 Alternative splicing changes the protein output of genes involved in neurogenesis 75 3.5.1 Spliced genes are involved in neurogenesis and signaling 75 3.5.2 Impact of alternative splicing on the proteome 77 3.6 Splicing regulation: neural exon features and splicing factor binding 79 3.6.1 Included neural exons are short and preceded by strong exon-definition features 79 3.6.2 Early included exons are enriched for nSR100 binding sites 85 3.7 The Btg2RFP/Tubb3GFP mouse line outperforms previous models for the study of cell-type-specific splicing in the brain 88 3.8 In vivo manipulation of splice variants 90 4 Discussion 94 4.1 The combination of different bioinformatic approaches allows an accurate identification of splicing events at the exon-level 95 4.2 Splicing choices during neurogenic commitment establish a neural signature characterized by microexon inclusion 97 4.3 Splicing during neocortical development leads to the generation of alternative protein isoforms in genes involved in neurogenesis and signaling 98 4.4 Neural exons are short and present strong features facilitating inclusion 101 4.5 Neural exons are prevalently regulated by nSR100 during neurogenic commitment 102 4.6 In vivo overexpression of splice variants highlights isoform-specific functions in neurogenic commitment 105 4.7 Concluding remarks and future perspectives 108 5 Supplementary figures 110 6 References 118 Acknowledgments 137 Anlange I 138 Anlange II 139

During evolution, the mammalian cerebral cortex has undergone a considerable increase in size and complexity. The emergence of the cortical structure begins during embryonic development when neural stem cells initially undergo proliferative division to expand their pool and then switch to neurogenic division, generating differentiating progenitors that will give rise to neurons. Although the intrinsic molecular mechanisms instructing the switch from proliferative to neurogenic division have been well-studied, most work to date has focused on gene expression. However, as a consequence of transcriptional and post-transcriptional regulation, different transcripts can arise from a single gene. In particular, the process of alternative splicing occurs at a high frequency in the nervous system and can lead to changes in protein output regardless of gene expression. In the past years, the role of post- transcriptional mechanisms in neuronal maturation and function have been extensively investigated, mostly focusing on the function of specific isoforms or RNA binding proteins. Yet, the role of alternative splicing in generating transcript and protein diversity during neurogenic commitment is still unknown. Therefore, I used a combination of different RNA sequencing technologies and bioinformatic tools to reveal the transcript and protein diversity of proliferating progenitors, differentiating progenitors, and neurons isolated from double reporter mouse line. I identified widespread isoform diversity and many novel transcripts amongst expressed genes in the developing cortex. To date, this analysis represents the most comprehensive characterization of full-length transcript diversity at different stages of the neurogenic lineage in the developing mouse cortex. The resulting transcriptome annotation was used to quantify changes in exon inclusion across cells of the neurogenic lineage and identified alternative splicing events potentially involved in neurogenic commitment. These alternative splicing events were enriched in the coding sequence of isoforms, indicating that they might be relevant for protein diversity generation in the developing cortex. During neurogenesis, alternative splicing events were less enriched in regions that could disrupt or strongly affect protein structure and function, such as transmembrane regions, active sites, and domains. Interestingly, my results indicated that alternative splicing enables increased functional diversity by modulating protein-protein interaction sites and signaling properties of proteins. Still, further studies are required to delineate the causal relationship between these alternative splicing choices and cell-fate commitment. Applying a similar approach to other mammalian species, including humans, has the potential to uncover species-specific innovations and conserved features that underlie evolutionary cortex expansion. Moreover, understanding the function of isoforms during neural development could provide important insights into the molecular mechanisms involved in the onset of neurodevelopmental disorders. Therefore, the higher cell-specificity offered by the present dataset, compared to similar studies, allowed not only a better understanding of transcript and protein diversity generated by alternative splicing in the nervous system and highlighted potential functional consequences, but also shed light on the advantages of applying such strategy to address different biological questions.

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Durch Immunregulation und -modulation sorgt das Immunsystem dafür, daß von außen in den Organismus gelangende Agentien nicht zu dauerhaften Schäden führen. Wesentliche Funktionen des Immunsystems stützen sich dabei auf die zelluläre Immunität. Gerät dieses komplizierte Regelwerk aus dem Gleichgewicht, können schwere Erkrankungen autoimmuner oder atopischer Genese resultieren. Der erste Teil der Arbeit befaßt sich mit zwei Aspekten der zellulären Immunantwort. Allergische Immunantworten sind durch die Typ 2 T Zellantwort charakterisiert. Für die Induktion einer Typ 2 Antwort wird Interleukin-4 benötigt, dessen Herkunft nicht geklärt ist. NK1.1 positive T Zellen als Quelle des initialen IL-4 konnten durch in vivo und in vitro Messung von allergie-spezifischen Parametern ausgeschlossen werden. Der MHC-Komplex präsentiert T Zellen Antigene in Form von Peptiden. Pathologische T Zellantworten können durch fortwährende Antigenpräsentation unterhalten werden. Durch Untersuchungen zur molekularen Charakteristik der MHC - Peptid Interaktion ließen sich Bindungsmotive so verfeinern, daß Peptide mit sehr starker Bindung an den MHC ohne gleichzeitige Erkennungssequenz für den T Zellrezeptor entwickelt werden konnten. Peptide dieser Art könnten zur Blockierung einer pathologischen T Zellantwort genutzt werden. Für viele immunologische Erkrankungen ist die Beteiligung genetischer Faktoren beschrieben worden. Der zweite Teil der Arbeit befaßt sich mit der Bedeutung genetischer Disposition bei Allergien im Mausmodell. Homozygote Inzuchtstämme konnten als High- und Low-Responder für den Phänotyp "allergische Soforttypreaktion der Haut" gegenüber Birkenpollenextrakt definiert werden. Die Phänotypisierung der F1 Generation wies auf dominante Vererbung hin, die informative Rückkreuzung auf die Beteiligung von mindestens zwei Genen für die Ausprägung des Merkmals. Wie die Analyse MHC congener Mäuse zeigte, entspricht einer dieser Loci dem MHC Komplex. Durch eine genomweite Kartierung mit Mikrosatelliten wurde als weiterer Kandidat der IL-5 Rezeptor identifiziert. Die detaillierte Analyse des Gens in High-und Low-Respondern weist auf eine Anzahl funktionell bedeutsamer Polymorphismen hin. Dabei imponiert das Low-Responder Allel als Suszeptibilitätsallel. Die Unterschiede haben Auswirkung auf Transkription/Translation und Spleißvorgänge, die zu quantitativer Differenz der Genprodukte führt. Die Daten weisen damit auf einen regulatorischen Mechanismus hin, da die Proteinstruktur des Rezeptors bei High- und Low-Respondern identisch ist. / Immune deviations can lead to serious autoimmune or atopic disorders. Two possible candidates for such pathological immune responses have been investigated: (i) the MHC class I allele HLA-B27, which is strongly linked to ankylosing spondylitis and reactive arthritis. Its presentation of potentially pathogenic peptides and therapeutical modifications of peptidic ligands have been studied. (ii) Interleukin-4 (IL-4), which is the key player in the induction and maintenance of allergic immune responses. A subpopulation of natural killer (NK1.1) cells have been discussed as a source of initial IL-4. Through experiments with NK1.1 deficient mice we could demonstrate, that such immune resonses are not dependent on the presence of NK1.1 cells. Both, autoimmunity and atopy belong to the large group of multifactorial diseases, i. e. genetic and environmental factors influence the expression of the various phenotypes. To dissect the genetics of allergic diseases systematically, a mouse model of immmediate cutaneous hypersensitivity (ICHS) upon birch pollen sensitization has been etablished. Phenotyping of the F1 progeny of high- and low-responder mice revealed ICHS as a dominant trait with incomplete penetrance. Mice from a backcross to the low-responder strain did split into the three classes of high-, intermediate and low-response. Genotyping of these mice revealed a strong candidate gene on chromosome 6: the interleukin-5 receptor (IL-5R). Analysis of the IL-5R gene resulted in the detection of genetic variance of the high- and low-responder allele in non-coding regions with functional relevance. Additional regulatory variance is implicated through differential alternative splicing of the three receptor isoforms. A second gene cluster contributes to the expression of the allergic phenotype: MHC class II alleles, as has been demonstrated for other immunologic disorders in mice and humans.