The central goal of this dissertation is to understand the genetic and functional aspects of how populations adapt to new or changing environments. Genetic variation within a population, either at protein coding genes or at regulatory elements, provides the substrate upon which natural selection can act to drive adaptation. There is considerable evidence that changes in gene expression account for a large proportion of morphological, physiological and behavioral variation between and within species that can contribute to adaptation and speciation. Due to the major role that gene expression changes can have in shaping phenotypes, the first three chapters of this dissertation deal with the study of how changes in gene expression can facilitate adaptation. I use Drosophila melanogaster from ancestral and derived regions of the species' range as a model system for studying local adaptation. In chapter 1, I perform high-throughput RNA-sequencing (RNA-seq) of brain tissue of flies from an ancestral (Zimbabwe) and a derived (the Netherlands) population. The whole brain transcriptome was assayed for differences in gene expression between African and European flies in order to understand how differences in brain expression may lead to local adaptation. I found over 300 candidate genes that differed significantly in expression between the populations, including a cluster of genes on chromosome arm 3R that showed reduced expression in Europe and genetic evidence for positive selection. Other candidate genes involved in stress response, olfaction and detoxification were also identified. Additionally, I compared brain gene expression between males and females and found an enrichment of sex-biased genes on the X chromosome. Chapter 2 presents a detailed study of one of the candidate genes identified in chapter 1. The metallothionein gene, MtnA, shows over four-fold higher expression in the brain of European flies than of African flies. I found a derived deletion in the 3’ untranslated region (UTR) of MtnA that segregates at high frequency within the Dutch population, but is absent from the Zimbabwean population. The presence of the deletion was perfectly associated with the observed variation in MtnA expression. When additional populations of D. melanogaster were screened for the presence of the deletion, I found that it showed a clinal distribution that was significantly correlated with latitude and temperature. Furthermore, using population genetic data and a selective sweep analysis I show that the MtnA locus is evolving under positive selection. In Chapter 3 I report a population genetic analysis of the enhancer region of CG9505, a gene that shows significantly higher expression in European than in African populations of D. melanogaster. A previous study found that there was very low nucleotide polymorphism in the enhancer region of CG9509 in flies from the Netherlands, a pattern that is consistent with a selective sweep. I analyzed an additional set of five populations from Zambia, Egypt, Malaysia, France and Germany in order to gain a better understanding of how selection has affected the evolution of this enhancer. I found that there is a depletion of nucleotide diversity in all of the non-sub-Saharan populations (Egypt, Malaysia, France and Germany), which share a common high-frequency derived haplotype. Population genetic analyses suggest that a selective sweep took place in the enhancer region of CG9509 just after D. melanogaster migrated out of sub-Saharan Africa. Finally, in chapter 4 I performed in situ hybridizations to examine the expression of tissue-specific reporter genes in the D. melanogaster testis. In the male germline of D. melanogaster, reporter genes that reside on the X chromosome show a reduction in expression relative to those located on the autosomes. This phenomenon was demonstrated by randomly inserting reporter gene constructs on the X chromosome and the autosomes. By doing in situ hybridizations on testis of flies having reporter gene insertions on the X chromosome and autosomes, I could show that the expression difference mainly occurs in meiotic and post-meiotic cells. For most constructs, expression was very low or absent in the testis apex, which is enriched with pre-meiotic cells. These results suggest that the suppression of X-linked gene expression in the Drosophila male germline occurs through a different mechanism than the MSCI (meiotic sex chromosome inactivation) known to occur in mammals.

In Zeiten des demografischen Wandels erfahren Krankheiten wie die Alzheimer-Demenz, als deren größter Risikofaktor das Alter gilt, einen rasanten Anstieg der Patientenzahl. Die Erforschung der zugrundeliegenden neurodegenerativen Mechanismen und das Einbringen daraus gewonnener Erkenntnisse in die Entwicklung von Therapieansätzen oder gar Präventionsmaßnahmen sind daher von gesamtgesellschaftlicher Bedeutung - nicht zuletzt auch unter ökonomischen Gesichtspunkten, wie den gleichsam wachsenden Kosten für das Gesundheitssystem. In der vorliegenden Arbeit wurden neuropathologische Prozesse in verschiedenen transgenen Tauopathie-Mausmodellen mittels hochauflösender Mikroskopietechniken untersucht. Der Schwerpunkt lag hierbei auf der Analyse synaptischer Veränderungen im lebenden Tier, ermöglicht durch die Zwei-Photonen-Intravitalmikroskopie. Zunächst wurden in Tau P301S-Mäusen die Auswirkungen FTDP-17-mutierten humanen Tau-Proteins auf die strukturelle Plastizität neokortikaler dendritischer Spines analysiert. Dabei wurde eine im Vergleich zu Wildtyp-Mäusen verminderte Spinedichte gemessen, welche auf eine geringere Ausbildung neuer Spines zurückzuführen war. Die verbliebenen Spines zeigten morphologische Veränderungen wie ein vergrößertes Kopfvolumen - möglicher Weise zur Kompensation des Synapsenverlusts. Ergänzend wurde eine Methode zur immunhistochemischen Synapsendichtemessung an Gehirnschnitten etabliert, welche jedoch keine Effekte der Transgenexpression auf die Dichte prä- oder postsynaptischer Spezialisierungen offenbarte. Um die Rolle inflammatorischer Prozesse in Tauopathien zu analysieren, wurde die Mauslinie Tau x CXCR erzeugt. Partielle oder vollständige genetische Fraktalkinrezeptor-Deletion in diesen Mäusen erlaubte eine gezielte Modifizierung der Kommunikation zwischen Neuronen und Mikrogliazellen. Die resultierende Aktivitätserhöhung der Mikrogliazellen hatte wider Erwarten keinen signifikanten Einfluss auf die Dichte Phospho-Tau enthaltender Zellen in den untersuchten kortikalen Gehirnregionen. Zur Modellierung der Alzheimer-Demenz wurden Tau P301S-Mäuse mit der Linie APP PS1 verpaart. Die Nachkommen wiesen Alzheimer-typische histologische Läsionen wie extrazelluläre Aβ-Plaques und intrazelluläre Tau-Ablagerungen auf. Es konnte jedoch im Vergleich zur Ursprungslinie Tau P301S keine Aβ-induzierte Verstärkung der kortikalen Tau-Pathologie gemessen werden, welche die Amyloid-Kaskaden-Hypothese suggeriert. Eine intravitalmikroskopische Analyse dendritischer Spines in Tau P301S- und Tau x APP PS1-Mäusen in unterschiedlichen Krankheitsstadien sowie in Wildtyp-Wurfgeschwistern sollte die Abgrenzung Tau-bedingter von Aβ-bedingten Effekten ermöglichen. Dabei wurden Veränderungen in der strukturellen Plastizität gefunden, beispielsweise in der Spine-Neuausbildung oder in bestimmten morphologischen Fraktionen, nicht aber in der absoluten Spinedichte. Schließlich erfolgte eine elektronenmikroskopische Untersuchung neuritischer Dystrophien in einem weiteren Alzheimer-Mausmodell, der Linie 3xTg-AD. Durch immunhistochemische Markierung konnten sowohl Aβ- als auch Tau-Ansammlungen in den pathologischen Anschwellungen nachgewiesen werden. Die präsentierten Befunde zeigen u. a. die ersten intravitalmikroskopischen Langzeitstudien dendritischer Spines in Mausmodellen mit reiner Tau-Pathologie sowie damit kombinierter Aβ-Pathologie. Sie bieten grundlegende, durch Patientenuntersuchungen nicht zu gewinnende Informationen über krankhafte synaptische Veränderungen, welche als frühe Ereignisse in der Alzheimer-Demenz betrachtet werden. / In times of demographic changes, a rapidly increasing number of people get affected by diseases such as Alzheimer’s disease. For this most common type of dementia, age is the main risk factor. Therefore, it is of great importance for the whole society to illuminate the underlying neurodegenerative mechanisms and to apply the gained knowledge to the development of therapeutic or even preventive procedures – not least for economic reasons, considering the simultaneously growing costs for the health care system. In the presented study, neuropathological processes in different transgenic tauopathy mouse models were investigated by means of high-resolution microscopy. The main focus was set on the analysis of synaptic changes in the living animal, enabled by two-photon in vivo imaging. At first, the effects of FTDP-17 mutated human tau-protein on the structural plasticity of neocortical dendritic spines were analysed in Tau P301S mice. In comparison to wildtype mice, the spine density was found to be reduced, which could be explained by a diminished fraction of newly formed spines. The remaining spines showed morphological changes such as an enlarged head volume, possibly compensating for the loss of synapses. Moreover, a method was established for immunohistochemical determination of synaptic densities on brain slices. However, no effect of transgene expression on the density of pre- or postsynaptic specializations could be measured. For studying the role of inflammatory processes in tauopathies, the mouse line Tau x CXCR was bred. Partial or complete deletion of the fractalkine receptor gene in these mice allowed a selective modification of the communication between neurons and microglia. Unexpectedly, the resulting increase of microglia activity did not have an influence on the density of phospho-tau bearing cells in the analysed brain regions. In order to create a model of Alzheimer’s disease, Tau P301S mice were further crossed with the line APP PS1. The offspring showed histological lesions typical for this kind of dementia, such as extracellular Aβ-plaques and intracellular tau-deposits. However, in comparison to the founder line Tau P301S, no Aβ-induced enhancement of cortical tau pathology could be measured, unlike suggested by the amyloid-cascade-hypothesis. An in vivo analysis of dendritic spines in Tau P301S and Tau x APP PS1 mice at different stages of the disease, as well as in wildtype littermates, allowed to distinguish effects caused by tau from those caused by Aβ. Thereby, changes in structural plasticity such as the emergence of new spines or the distribution of morphological fractions were detected, while the overall spine density was unaffected. Finally, neuritic dystrophies were analysed in another Alzheimer’s disease mouse model, the line 3xTg-AD, applying electron microscopy. By immunohistochemical labelling, Aβ- as well as tau-deposits could be detected inside the pathological swellings. The presented data show the first in vivo long-term study on dendritic spines in mouse models with tau-pathology only as well as in combination with Aβ-pathology. They provide new basic information about synaptic failure, which is considered to be an early event in Alzheimer’s disease and can not be obtained from patient studies likewise.

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The fixation of beneficial variants leaves genomic footprints characterized by a reduction of genetic variation at linked neutral sites and strong, localized allele frequency differentiation among subpopulations. In contrast, for phenotypic evolution the effect of adaptation on the genes controlling the trait is little understood. Theoretical work on polygenic selection suggests that fixations of beneficial alleles (causing selective sweeps) are less likely than small-to-moderate allele frequency shifts among subpopulations. This thesis encompasses three projects in which we have experimentally addressed the issue of selective sweeps vs. allele frequency shifts in the context of polygenic adaptation. We studied three X-linked QTL underlying variation in chill coma recovery time (CCRT), a proxy for cold tolerance, in Drosophila melanogaster from temperate (European) and tropical (African) environments. The analysis of these QTL was performed by means of selective sweep mapping and quantitative complementation tests coupled with expression assays. While the results of the selective sweep mapping approach identified a gene (CG4491) that is unlikely to be affecting CCRT, quantitative and gene expression analyses revealed two linked candidate genes (brk and CG1677) that appear to differ in their evolutionary histories. We found that the difference in expression of the gene brk between populations affects CCRT variation. Cold tolerant flies from the temperate zone have a lower expression of this gene than cold sensitive flies from the tropics. We found that a likely cause of this difference is variation in a cis-regulatory element in the brk 5’ enhancer region. Sequence variants in this element exhibit moderate frequency differences between populations from temperate and tropical environments, forming two latitudinal clines: one from the equator to the north and another one in opposite direction to the south. In contrast, the other gene within the same QTL (CG1677), which is linked to brk, showed no measurable effect on cold tolerance but is a likely target of strong positive selection leading to a selective sweep in the European population. These results are consistent with the aforementioned theoretical predictions about footprints of selection in polygenic adaptation. They are also proof of the conceptual bias incurred when identifying candidate genes within a QTL via selective sweep mapping, at least in naturally evolving populations. The challenge for the evolutionary genetics community in the coming years is to develop statistical tools that are as powerful and robust as those already available to map selective sweeps to identify sites in the genome where allele frequency shifts have occurred due to adaptive evolution at the phenotypic level. Finally, the last section of the results is a report of a new population genetics dataset. It consists of a collection of 80 inbred lines from a natural D. melanogaster population in Sweden and 19 full genome sequences derived from this sample. We hope this material will provide us with further insight into the processes underlying adaptation to novel and stressful environments.