Das Nervensystem von Drosophila melanogaster entwickelt sich aus dem Neuroectoderm entlang der anterior-posterioren Axe des Embryos. Dieses Gewebe unterteilt sich in drei Expressionsdomänen, die von ventral nach dorsal durch Expression der drei Homöobox-Transkriptionsfaktoren vnd, ind und msh charakterisiert sind. Vnd und Ind wurden als notwendig und ausreichend beschrieben, um die Zellidentitäten ihrer jeweiligen Gewebe zu determinieren. Um zu untersuchen, wie diese Transkriptionsfaktoren agieren, um ihre jeweiligen Zelltypen hervorzubringen, habe ich die genomweiten Genexpressionsmuster dieser verwandten Gewebe in Wildtyp-Embryos und in einer vnd-Mutante bestimmt. Ich fand heraus, dass anstelle eines Gewebes, das der IC-Domäne ähnelt, die Vnd-Mutante ein Gewebe hervorbringt, das neuronale Charakteristika zum grossen Teil verloren hat. Ich habe gefunden, dass der Transkiptionsfaktor "eyeless" bei ektopischer Expression in der VC den Vnd-Nervensystemphänotyp phänokopiert und ein ähnliches DNA-Bindemuster aufweist wie Vnd. Unsere Daten lassen vermuten, das Vnd sowohl als direkter Aktivator von VC-spezifischen Genen als auch als direkter Repressor von nicht-neuronalen Genen wirkt. Um die zeitlich-örtliche Auflösung weiter zu erhöhen, habe ich mit Nikolaos Karaiskos kollaboriert, um Einzelzell-Transkriptome von Embryos einer einzigen embryonalen Stufe zu generieren und einen Genexpressions-Atlas des frühen Drosophila-Embryos mit Einzelzellauflösung zu erstellen der die nahezu genomweite Genexpression fast jeder Zelle zu rekapitulieren. Um die Nützlichkeit der Plattform zu demonstrieren, untersuchten wir Expressionsmuster von Genen, die an der Hippo-Signalkaskade beteiligt sind. Wir prognostizierten Hippo-Signalaktivität in bestimmten Bereichen des Embryos und zeigten, dass der Hippo-Signalweg die synchronisierte Zellteilung in diesen Bereichen unterbricht. / The embryonic nervous system of Drosophila melanogaster derives from the neurogenic ectoderm, which is subdivided into three distinct domains. Several key transcription factors show expression exclusive to individual columns and endow their expression domains with characteristic identities. The genes vnd and ind, for example, are homeodomain transcription factors expressed in two columns. Both have been argued to be necessary and sufficient to confer the ventral column or intermediate column fates. To address the question how these transcription factors confer identities to their expression domains I determined the transcriptomic profile of these tissues in wild type and a Vnd mutant embryos. In order to do this, I established a protocol that allowed me to sequence the transcriptomes in developing embryos with spatio-temporal resolution. I found that upon knockout of Vnd, the ventral column largely looses its neurogenic identity rather than converting its fate, as models would predict. I identified Eyeless as a novel candidate transcription factor that shapes early nerve cord identities. Furthermore the data indicates that in Vnd acts as both, activator and repressor. Excessive co-binding with the GAGA-factor GAF suggests a mechanism by which this activation might be achieved. To push spatio-temporal resolution towards the single cell level, I collaborated with Nikolaos Karaiskos to extract single cell transcriptomes of a single developmental stage. This has allowed us to establish a digital single-cell resolved transcriptomic map of a single developmental stage. We used this map to predict expression patterns of thousands of genes with striking accuracy. We identified the Hippo signaling pathway as a spatially regulated pathway in early embryos and showed that it directs the interruption of cell cycle synchronicity in specific areas of the embryo.

Les spondyloarthrites (SpA) sont des maladies inflammatoires chroniques articulaires qui se caractérisent par des atteintes de la colonne vertébrale et des articulations périphériques, en particulier des enthèses, souvent associées à des manifestations extra-articulaires telles que le psoriasis, l’uvéite, ou l’inflammation intestinale. Ces maladies complexes possèdent une forte composante génétique dominée par l'antigène HLA-B27 du complexe majeur d'histocompatibilité de classe I (CMH-I), présent chez plus de 80% des patients atteints de SpA. Découverte il y a 45 ans, l'association entre HLA-B27 et le développement des SpA reste inexpliquée. Plusieurs hypothèses ont été proposées pour expliquer cette association au niveau moléculaire. Cependant, la plupart se heurtent à des incohérences expérimentales qui semblent les invalider. Pour élucider les mécanismes moléculaires pathogènes liés au HLA-B27, nous avons utilisé une nouvelle approche. Drosophila melanogaster est un puissant modèle génétique qui a permis des avancées considérables dans la compréhension de nombreuses fonctions des cellules de métazoaires, ainsi que dans la description des processus cellulaires et moléculaires de nombreuses pathologies humaines. Nous avons établi plusieurs lignées de drosophiles transgéniques pour des formes d’HLA-B associées aux SpA ou pour une forme non associée à la maladie, ainsi que pour la chaîne invariante du CMH-I, la β2m humaine (hβ2m). L'expression des formes associées à la maladie, exclusivement en présence de la hβ2m, dans l'aile et dans l'œil de la drosophile conduit à l'apparition de deux phénotypes spécifiques. Mes résultats ont permis de mettre en évidence que le phénotype de perte des veines transversales de l’aile était associé à une perturbation de la signalisation par la voie des Bone Morphogenetic Protein (BMP). Cette perturbation est associée à une co-localisation de HLA-B27 avec le récepteur BMP de type I, Sax. Nos résultats préliminaires obtenus dans les cellules de patients atteints de SpA suggèrent l’existence d’une co-localisation analogue d’HLA-B27 avec le récepteur ALK2, orthologue de Sax. L'ensemble de nos résultats plaide en faveur d’un rôle pathogène de HLA-B27 passant par une dérégulation de la voie BMP à l’intersection des voies de l’ossification et de l’inflammation et pourrait donc s’appliquer à la physiopathologie des SpA. / Spondyloarthritis (SpA) is a chronic inflammatory rheumatic disorder characterized by joint manifestations affecting the spine, peripheral joints and entheses, as well as extra-articular manifestations such as psoriasis, uveitis, or intestinal inflammation. This complex disorder has a strong genetic component dominated by the HLA-B27 antigen of the major histocompatibility complex class I (MHC-I), which is present in more than 80% of SpA patients. Discovered 45 years ago, the association between HLA-B27 and SpA development remains unexplained. Several hypotheses have been proposed to explain this association at the molecular level, but all face experimental inconsistencies that seem to invalidate them. Therefore, it appeared to us essential to elaborate new and yet unexplored approaches in order to better understand the molecular role of HLA-B27 in SpA development. Drosophila melanogaster is a powerful genetic model that has led to considerable advances in understanding numerous functions of metazoan cells, as well as in describing the cellular and molecular processes of many human pathologies. To elucidate the molecular pathogenic mechanisms associated with HLA-B27, we have established several transgenic Drosophila lines for SpA-associated and non-associated of HLA-B alleles, as well as for the MHC-I invariant chain, the human 2-microglobulin (hβ2m). Expression of the HLA-B27 alleles, in the presence of hβ2m, in the Drosophila wing and eye led to two specific phenotypes. The crossveinless wing phenotype is due to a disturbance in the Bone Morphogenetic Protein (BMP) signaling pathway. Interestingly, this misregulation is associated with a co-localization of HLA-B27 and the BMP type I receptor named Sax. Our preliminary results obtained in SpA patient cells suggest that HLA-B27 also colocalizes with ALK2 receptor, which is ortholog to Sax. Altogether, our results suggest that the pathogenic role of HLA-B27 in SpA may depend on a BMP signaling misregulation at the crosstalk between ossification and inflammation.

Anisogami och sexuella konflikter kan vara grunden till flera fall av könslig dimorfism. Intralokus sexuell konflikt uppstår när alleler vid ett genetiskt lokus har en antagonistisk effekt på fitness hos hanar och honor. Helgenomstudier har pekat ut flera sexuellt antagonistiska kandidatgener hos Drosophila melanogaster. En av dem, CG15170, har återskapats med hjälp av genediteringstekniken CRISPR/Cas9. Den här genen är intressant då populationer med en viss allel, A1, inte förlorar balanser kromosomen, förmodligen på grund av negativ fitnesspåverkan från den återskapade allelen A1. I den här studien undersöktes om fertiliteten hos honor och ägg-till-vuxen överlevnaden är negativt påverkad. Detta gjordes genom att antalet lagda ägg och proportionen av lagda ägg som utvecklades till vuxna individer hos honor av D. melanogaster homozygota för A1 eller A2 alleler mättes. Resultatet från de statistiska tester som utfördes visade ingen signifikant skillnad mellan honor med A1 och A2 alleler varken för antalet lagda ägg eller proportionen av ägg som utvecklades till vuxna. Däremot upptäcktes en signifikant skillnad mellan de två allelerna i antalet vuxna honor som överlevde parningen i den första delen av försöket. Denna studie kan inte påvisa att fekunditeten hos honorna och den tidiga överlevnadsgraden hos deras avkommor skulle vara olika för honor med A1 respektive A2 alleler. Det bör i stället vara någon annan del av livscykeln som påverkas av att A1 inte förlorar balanser kromosomen men vilken kan ej fastställas utifrån den här studien. / Anisogamy and sexual conflict may be the basis of several cases of sexual dimorphism. Intralocus sexual conflict occurs when alleles at a genetic locus have an antagonistic effect on the fitness of males and females. Genome wide association studies have pointed out several candidate genes for sexual antagonism in Drosophila melanogaster. One of them, CG15170, has been recreated with the gene editing technique CRISPR/Cas9. This gene is interesting because populations with one particular allele, A1, do not lose the balancer chromosome, probably because of negative effects off the allele on fitness. In this study, the fertility and egg-to-adult survival of eggs laid by females was investigated to see if these traits were adversely affected by the recreated allele A1.  This was done by measuring the number of eggs laid by females of D. melanogaster, homozygous for A1 or A2 alleles, and also by measuring the proportion of eggs that developed to adults. The results of the statistical tests that were performed showed no significant difference between females with A1 and A2 alleles, not for the number of eggs laid nor the proportion of eggs that developed into adults. However, a significant difference was detected between the two alleles in the number of adult females that survived the first courtship and mating part of the experiment. Probably some other part of the life cycle is affected by A1 not losing the balancer chromosome, but which cannot be determined by this study.

Bananflugan (Drosophila melanogaster) är vanlig i den genetiska forskningen då deras genom är känt sedan länge, vi vet även att det finns stora könskonflikter som driver på utvecklingen av skillnader mellan könen. Det finns goda bevis för att sexuella antagonistiska gener spelar en stor roll i utvecklingen av könsskillnader hos många olika organismer. Jag har tittat på om det finns en längdskillnad mellan hanar av A1 och A2 hos CG3598 då det tidigare har bekräftats att det finns en skillnad i fitness mellan dessa. Jag har även undersökt om det finns en skillnad i fitness mellan honor av samma grupper vilket inte är bevisat. För att se om det fanns en längdskillnad mellan hanar av A1 och A2 har deras thoraxlängd mäts med ett okulärmikroskop. Honornas fitness testades genom att de antingen konkurrerade inom sin genotyp eller med honor av en annan genotyp, därefter räknades avkommorna och grupperna jämfördes. Resultatet tyder på att det inte fanns någon signifikant längdskillnad mellan hanar av A1 och A2 men de var i genomsnitt 85,3% så stora som honorna. För honorna fanns det ingen skillnad i fitness mellan A1 och A2, oberoende av om de konkurrerat inom genotypen eller med en annan. När larverna konkurrerade inom genotypen fick A1 och A2 fler avkommor per hona än om larverna konkurrerade med en annan genotyp. Slutsatserna blir att vi fortfarande inte vet varför A2 hanar får fler avkommor än A1 men vi vet att honor av A1 och A2 inte verkar ha några skillnader i fitness. Det behövs mer forskning inom området och det är viktigt att man väljer rätt konkurrenter när man testar fitness. / Drosophila melanogaster is common in genetic research as their genome has been known for a long time, we also know that there are major gender conflicts that drive the development of differences between the sexes. There is good evidence that sexually antagonistic genes play a major role in the development of sex differences in many different organisms. I have studied if there is a length difference between males of A1 and A2 in CG3598 as it has previously been confirmed that there is a fitness difference between these. I have also investigate whether there is a fitness difference between females of the same groups, which has not been proven. To see if there was a length difference between males of A1 and A2, their thorax length was measured with an ocular microscope. The females fitness was tested by either competing within their genotype or with females of another genotype, then the offspring was counted and the groups compared. The results indicated no significant difference in length between males of A1 and A2 but they were on average 85.3% as large as the females. For the females, there was no difference in fitness between A1 and A2, regardless of whether they competed within the genotype or with another. When the larvae competed within the genotype, A1 and A2 produced more offspring per female than if the larvae competed with another genotype. The conclusions are that we still don’t know why A2 males produce more offspring than A1 but we do know that females of A1 and A2 do not appear to have any differences in fitness. More research is needed in the field and it is important to choose the right competitors when testing fitness.

Einzelzellomics bieten unvoreingenommene Einblicke in Transkriptionsprogramme und Genom-Zugänglichkeiten auf zellulärer Ebene, auch wenn der zelluläre Kontext verloren geht. Wir haben einen virtuellen Multi-omic Embryo der Drosophila melanogaster erstellt, basierend auf den Datentypen RNA (Transkriptom) und ATAC (Zugänglichkeit der DNA), welche gleichzeitig auf Einzelzell Ebene erhoben wurden. Mithilfe des Tools novoSpaRc, welches den räumlichen Ursprung der Zellen rekonstruiert, konnte ein regulatorischen Bauplan erstellt werden, der die Genexpression und die Zugänglichkeit von Enhancern widerspiegelt. Diese Ressource hilft beim Verständnis der regulatorischen Dynamik in der Entwicklung. Bei der Untersuchung von ATAC-Peaks konnten wir Überschneidungen zwischen den Mustern der Chromatin Zugänglichkeit und der Aktivität unabhängiger getesteter Enhancer feststellen, was die Bedeutung der Zugänglichkeit unterstreicht. Die nicht-negative Matrixfaktorisierung identifizierte Archetypen der Genexpression und der Chromatin-Zugänglichkeit. Archetypen, die möglicherweise durch Transkriptionsfaktoren (TFs) reguliert werden, wurden einer Motiv-Anreicherungsanalyse für Archetyp-assoziierte CRMs unterzogen. Ein Ansatz zur Vorhersage von Enhancern, ordnete die Enhancer den Genen auf der Grundlage partieller Ähnlichkeit der Muster zu. Zusammenfassend dient unser multimodaler virtueller Embryo als Ressource und präsentiert zum ersten Mal räumliche Chromatin-Zugänglichkeiten für genomische Regionen für einen ganzen Organismus. Die Ergebnisse geben Aufschluss über die Prinzipien der Genregulation und zeigen den regulatorischen Einfluss von Transkriptionsfaktoren auf den Chromatinzustand von Enhancern. / Single-cell-omics techniques provide unbiased insights into transcriptional programs and genomic accessibility patterns at the cellular level despite sacrificing spatial information. We created a multi-omic virtual Drosophila melanogaster stage 6 embryo by simultaneously assessing genome accessibility and transcriptional states in individual cells. Using novoSpaRc, a spatial mapping tool, we accurately reconstructed the spatial origin of cells, yielding a regulatory blueprint reflecting gene expression and enhancer accessibilities. This resource aids in understanding developmental regulatory dynamics. Examining ATAC-peaks, we observed overlapping chromatin accessibility patterns with the activity of independently testes enhancers, emphasizing accessibility's importance. Non-negative matrix factorization identified archetypes in gene expression and chromatin accessibility. Accessibility archetypes, potentially regulated by transcription factors (TFs), were subjected to motif enrichment analysis for archetype-associated CRMs. An enhancer prediction approach, utilizing a generalized linear model, assigned enhancers to genes based on partial pattern similarity. In summary our multi-modal virtual embryo serves as a resource and presents for the first time single-cell chromatin accessibilities for genomic regions reconstructed in space for a whole organism in a single developmental stage. The results shed light on gene regulatory principles, highlighting the regulatory impact of TFs on chromatin states of enhancers.

Eukaryotische DNA ist in Chromatin verpackt, einem Komplex aus DNA und Proteinen. Das ermöglicht Transkriptionsregulation von Genen durch Modulation ihrer Zugänglichkeit. Histone sind evolutionär konservierte Chromatinproteine, die durch posttranslationale Modifikationen (PTMs) modifiziert sind. Das legt nahe, dass deren Primärstruktur und ihre PTMs funktionellen Beschränkungen unterliegen. Genetische Ansätze zur Entschlüsselung von Struktur-Funktions-Beziehungen für Histone waren auf Einzeller und D. melanogaster beschränkt. Das Histon-Ersatz-System in D. melanogaster, bei dem Histontransgene verwendet werden, um endogene Histone zu ersetzen, war nicht für systematische Untersuchung dieser Beziehungen ausgelegt. In meiner Arbeit habe ich die Funktion von Threonin 11 in Histon H3 (H3T11) untersucht, das phosphoryliert werden kann. Ich analysierte zwei Mutationen in H3T11 (H3T11A und H3T11E) und stellte fest, dass beide zur Derepression von Transposons führen. H3T11E hat in Gegenwart von Wildtyp-Histon H3 einen dominanten Phänotyp mit transkriptomweiten Folgen. Dazu gehören Induktion von Immun-Genen und Unterdrückung von mit DNA-Stoffwechsel in Verbindung stehenden Genen. Die Mutationen wurden unter der Prämisse charakterisiert, ein Analyse-Schema für eine große Anzahl von Histon-Ersatz-Stämmen zu entwickeln und Probleme zu identifizieren, die die Analyse beeinträchtigen. Dabei habe ich das Verfahren zur Erzeugung von Histon-Ersatz-Stämmen optimiert. Dazu gehören die optionale Verwendung größerer Histon-Transgene und zuverlässigere Produktion und optimierte Rekombinations-Strategie dieser. Ich habe das Klonierungsverfahren gestrafft und eine Plasmid-Bibliothek erstellt, die es erlaubt, 178 verschiedene mutierte Histon-H3-Transgene zu erzeugen. Mit den Änderungen am Produktionsschema, ist diese Bibliothek eine wertvolle Ressource und wird dazu beitragen, die Funktion von Histon H3 und seiner PTMs während der Entwicklung eines Vielzellers besser zu verstehen. / Eukaryotic DNA is packaged into chromatin, a complex composed of DNA and proteins. This enables transcriptional regulation of genes through modulation of their accessibility. Histones are chromatin proteins, modified by post-translational modifications (PTMs) and their sequences are conserved in evolution. This suggests functional constraints for the primary structure of histones and their PTMs. Genetic approaches to decipher structure-function relationships for histone proteins were restricted to unicellular organisms and D. melanogaster. The histone replacement system in D. melanogaster, which uses histone transgenes to replace endogenous histones, was not adapted for systematic interrogation of such relationships. Here, I investigated the function of threonine 11 in histone H3 (H3T11), which can be phosphorylated. I analyzed two mutations in H3T11 (H3T11A and H3T11E) and found that both lead to de-repression of transposable elements. I also found that H3T11E, has a dominant phenotype in the presence of wildtype histone H3 with transcriptome-wide consequences. These include induction of immune-related genes and repression of genes associated with DNA metabolism. I characterized both mutations under the premise of establishing an analysis scheme suitable for a large set of histone replacement strains and identifying problems that interfere with this analysis. As a consequence, I optimized the procedure to generate histone replacement strains. These include an option to incorporate larger histone transgenes, a more reliable production of transgenes and an optimized strategy to recombine them. I streamlined the cloning procedure and created a plasmid library allowing for the generation of 178 distinct mutant histone H3 transgenes. Together with my amendments to the production scheme, this library provides a valuable resource to the field and will help to better understand the function of histone H3 and its PTMs during the development of a multicellular organism.

Not much is known about the mechanisms affecting the genetic composition of populations of different species. To investi­gate one of these potential mechanisms, mating behaviour, the fruit fly Drosophila melanogaster, was chosen as an experimen­tal animal. To quantify mating behaviour in easily measurable parameters, it was subdivided into several distinct components; mating activity, mating time, mating competition ability and male mating capacity. As behavioural components to a great extent are influenced by environmental conditions all experiments were performed under controlled temperature and humidity. All components of mating behaviour were estimated by introducing females and males into mating chambers. Mating behaviour seems to be one of the major factors affect­ing the genetic composition of Drosophila melanogaster popula­tions. The experiments performed showed that differences in mating properties led to a substantial sexual selection among the genotypes. This selection was of a stabilizing type with regard to characters associated to bristle number and Y chromo­somal chromatin. This selection situation seems to warrant the retention of intermediate phenotypes in a population and will therefore contribute to the genetic variation present. Differences in mating properties were also shown to be able to maintain a balanced polymorphism for allozyme variants in populations. This maintenance was obtained through different forms of balancing selection as heterozygous superiority in sexual activity and balancing selection between female and male genotypes. Heterozygous superiority or overdominance in fitness always leads to balanced polymorphism through segre­gation of individuals with lowered fitness. The balancing selection between the female and male genotypes is best looked upon as a form of marginal overdominance, conferring the aver­aged highest fitness to the heterozygous genotype, thereby maintaining the polymorphism of the population. / <p>Härtill 5 uppsatser</p> / digitalisering@umu

Multicellular organisms need to control their size throughout development and adult life in the face of challenges such as rapid growth. Unraveling the mechanisms that regulate tissue growth in epithelial tissues, in order to generate organs of correct size and proportion, remains a crucial goal of developmental biology. A suitable epithelial tissue for studying tissue growth in Drosophila, is the proliferative monolayer epithelial sheet of imaginal wing discs, which gives rise to the adult wing. The Hippo signaling pathway regulates tissue growth in wing development. There are several observations that link tissue growth/Hippo signaling with cell polarity and the actin cytoskeletal organization. The aim of this thesis was the study of the interplay between cell polarity, cytoskeletal organization and tissue growth. To gain further insight into how apical polarity proteins regulate tissue growth, an enhancer/suppressor screen that was previously conducted in our lab by Linda Nemetschke, was used. The screen was based on the modification of a dominant smaller wing phenotype induced upon overexpression of CrbextraTM-GFP. One of the enhancers identified in this screen is a gene called big bang (bbg). The absence of bbg results in smaller wings with a slower cell cycle and increased apoptosis in wing discs. bbg encodes a protein expressed in the apical cortex in wing disc cells and is required for the proper localization of apical proteins, like Crb, in wing disc epithelia. Bbg is also in the same complex with Spaghetti Squash (Sqh) in the apical cortex of the wing disc epithelia. sqh encodes an actin-binding protein that has actin cross-linking and contractile properties. Bbg stabilizes Sqh in the apical compartment of the cell. It is reported that both Crb and Sqh regulate tissue growth through the Hippo signaling pathway. In conclusion, Bbg regulates wing tissue growth, acting as a scaffolding molecule, through the proper localization of apical components of the cells like Crb and the cytoskeletal component Sqh.

In many different insect species the highly conserved neuropil regions known as the central complex or central body complex have been shown to be important in behaviours such as locomotion, visual memory and courtship conditioning. The aim of this project is to generate accurate quantitative neuroanatomy of the central complex in the fruit fly Drosophila melanogaster. Much of the authoritative neuroanatomy of the fruit fly from past literature has been derived using Golgi stains, and in important cases these data are available only from 2D camera lucida drawings of the neurons and linguistic descriptions of connectivity. These cannot easily be mapped onto 3D template brains or compared directly to our own data. Using GAL4 driver and reporter constructs, some of the findings within these studies could be visualized using immunohistochemistry and confocal microscopy. A range of GAL4 driver lines were selected that particularly had prominent expression in the fan-shaped body. Images of brains from these lines were archived using a web-based 3D image stack archive developed for the sharing and backup of large confocal stacks. This is also the platform which we use to publish the data, so that other researchers can reuse this catalogue and compare their results directly. Each brain was annotated using desktop-based tools for labelling neuropil regions, locating landmarks in image stacks and tracing fine neuronal processes both manually and automatically. The development of the tracing and landmark annotation tools is described, and all of the tools used in this work are available as free software. In order to compare and aggregate these data, which are from many different brains, it is necessary to register each image stack onto some standard template brain. Although this is a well-studied problem in medical imaging, these high resolution scans of the central fly brain are unusual in a number of respects. The relative effectiveness of various methods currently available were tested on this data set. The best registrations were produced by a method that generates free-form deformations based on B-splines (the Computational Morphometry Toolkit), but for much faster registrations, the thin plate spline method based on manual landmarks may be sufficient. The annotated and registered data allows us to produce central complex template images and also files that accurately represent the possible central complex connectivity apparent in these images. One interesting result to arise from these efforts was evidence for a possible connection between the inferior region of the fan-shaped body and the beta lobe of the mushroom body which had previously been missed in these GAL4 lines. In addition, we can identify several connections which appear to be similar to those described in [Hanesch et al., 1989], the canonical paper on the architecture of the Drosophila melanogaster central complex, and describe for the first time their variation statistically. This registered data was also used to suggest a method for classifying layers of expression within the fan-shaped body.

The robust behavioural courtship ritual displayed by Drosophila melanogaster males is governed by their underlying nervous system (NS). Two key genes of the Sex Determination Hierarchy, fruitless (fru) and doublesex (dsx), determine most neuronal substrates for sexual behaviour. In this study we aim to better understand the role fru plays in determining these neural substrates, as a means of better understanding the relationships between brain, behaviour and genes, and thus how the development of neuronal networks shape innate and species-specific behaviours. Fru has two major functions: control of male sexual behaviour, and viability in both sexes. Alternative splicing of fru produces transcription factors driven by four promoters: P1 transcripts are sex-specifically spliced (only viable in the male), and P2-4 transcripts are crucial to both sexes survival. The resulting proteins contain a BTB protein-protein interaction domain at the N-terminus, and one of four C-terminal zinc-finger (ZnF) DNA binding domains. Male-specific proteins (FruM) contain an additional 101 amino acid N-terminal domain, and one of three alternative C2H2 ZnF domains (FruMA, FruMB, FruMC). These male-specific isoforms are expressed in the central nervous system (CNS) beginning in the late stage larvae (L3), peaking during pupation and on into adulthood. Little is known, however, about the roles of the individual isoforms, and no clear transcriptional targets have been identified. The central aims of this thesis are to document the wild-type expression patterns of Fru isoforms throughout development in the CNS, create and characterise isoform-specific mutants, and to identify and evaluate putative transcriptional targets of Fru. Combining these findings will lead to a better understanding of the underlying molecular functions of individual FruM isoforms, as a means to understanding their roles in sexual behaviour. Expression analysis of FruM isoforms throughout development in the NS is described. To further characterise the role of individual FruM isoforms, isoform-specific mutants in fruA and fruB exons were generated using site-specific homologous recombination (HR). These novel mutants were validated by PCR and Fru isoform-specific antibody stainings. Mutants were analysed in fruM- and fru-null genetic backgrounds, to distinguish the roles of sex-specific vs. common isoforms. These analyses included: fertility, viability and morphology. FruA was found to have a role in wing extension with possible repercussions for song production. FruB was found to be developmentally lethal, in addition to having defects in male courtship behaviour. To understand the role of fru in the NS, downstream transcriptional targets of FruM isoforms were identified. DNA adenine methyltransferase identification (DamID) was used to identify putative transcription targets of FruM isoforms. The Dam protein methylates DNA in Drosophila in a sequence-specific manner allowing targets of Fru to be isolated. Candidate genes were identified using computational analysis (including gene ontology, peak analysis and motif analysis) along with a biologically significant connection with fru. The relationship between fru and six candidate genes were characterised using RNAi. The results of these studies advance our knowledge of how FruM isoforms influence the development and physiology of the NS underlying male sexual behaviour in Drosophila.