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Epigenetic modifications, of which DNA methylation is the most stable, are a mechanism conveying environmental information to subsequent generations via parental germ lines. The paternal contribution to adaptive processes in the offspring might be crucial, but has been widely neglected in comparison to the maternal one. To address the paternal impact on the offspring's adaptability to changes in diet composition, we investigated if low protein diet (LPD) in F0 males caused epigenetic alterations in their subsequently sired sons. We therefore fed F0 male Wild guinea pigs with a diet lowered in protein content (LPD) and investigated DNA methylation in sons sired before and after their father's LPD treatment in both, liver and testis tissues. Our results point to a 'heritable epigenetic response' of the sons to the fathers' dietary change. Because we detected methylation changes also in the testis tissue, they are likely to be transmitted to the F2 generation. Gene-network analyses of differentially methylated genes in liver identified main metabolic pathways indicating a metabolic reprogramming ('metabolic shift'). Epigenetic mechanisms, allowing an immediate and inherited adaptation may thus be important for the survival of species in the context of a persistently changing environment, such as climate change.
The sequencing of the human genome in the early 2000s led to an increased interest in cheap and fast sequencing technologies. This interest culminated in the advent of next generation sequencing (NGS). A number of different NGS platforms have arisen since then all promising to do the same thing, i.e. produce large amounts of genetic information for relatively low costs compared to more traditional methods such as Sanger sequencing. The capabilities of NGS meant that researchers were no longer bound to species for which a lot of previous work had already been done (e.g. model organisms and humans) enabling a shift in research towards more novel and diverse species of interest. This capability has greatly benefitted many fields within the biological sciences, one of which being the field of evolutionary biology. Researchers have begun to move away from the study of laboratory model organisms to wild, natural populations and species which has greatly expanded our knowledge of evolution. NGS boasts a number of benefits over more traditional sequencing approaches. The main benefit comes from the capability to generate information for drastically more loci for a fraction of the cost. This is hugely beneficial to the study of wild animals as, even when large numbers of individuals are unobtainable, the amount of data produced still allows for accurate, reliable population and species level results from a small selection of individuals.
The use of NGS to study species for which little to no previous research has been carried out on and the production of novel evolutionary information and reference datasets for the greater scientific community were the focuses of this thesis. Two studies in this thesis focused on producing novel mitochondrial genomes from shotgun sequencing data through iterative mapping, bypassing the need for a close relative to serve as a reference sequence. These mitochondrial genomes were then used to infer species level relationships through phylogenetic analyses. The first of these studies involved reconstructing a complete mitochondrial genome of the bat eared fox (Otocyon megalotis). Phylogenetic analyses of the mitochondrial genome confidently placed the bat eared fox as sister to the clade consisting of the raccoon dog and true foxes within the canidae family. The next study also involved reconstructing a mitochondrial genome but in this case from the extinct Macrauchenia of South America. As this study utilised ancient DNA, it involved a lot of parameter testing, quality controls and strict thresholds to obtain a near complete mitochondrial genome devoid of contamination known to plague ancient DNA studies. Phylogenetic analyses confidently placed Macrauchenia as sister to all living representatives of Perissodactyla with a divergence time of ~66 million years ago. The third and final study of this thesis involved de novo assemblies of both nuclear and mitochondrial genomes from brown and striped hyena and focussed on demographic, genetic diversity and population genomic analyses within the brown hyena. Previous studies of the brown hyena hinted at very low levels of genomic diversity and, perhaps due to this, were unable to find any notable population structure across its range. By incorporating a large number of genetic loci, in the form of complete nuclear genomes, population structure within the brown hyena was uncovered. On top of this, genomic diversity levels were compared to a number of other species. Results showed the brown hyena to have the lowest genomic diversity out of all species included in the study which was perhaps caused by a continuous and ongoing decline in effective population size that started about one million years ago and dramatically accelerated towards the end of the Pleistocene.
The studies within this thesis show the power NGS sequencing has and its utility within evolutionary biology. The most notable capabilities outlined in this thesis involve the study of species for which no reference data is available and in the production of large amounts of data, providing evolutionary answers at the species and population level that data produced using more traditional techniques simply could not.
The movement of organisms has formed our planet like few other processes. Movements shape populations, communities, entire ecosystems, and guarantee fundamental ecosystem functions and services, like seed dispersal and pollination. Global, regional and local anthropogenic impacts influence animal movements across ecosystems all around the world. In particular, land-use modification, like habitat loss and fragmentation disrupt movements between habitats with profound consequences, from increased disease transmissions to reduced species richness and abundance. However, neither the influence of anthropogenic change on animal movement processes nor the resulting effects on ecosystems are well understood. Therefore, we need a coherent understanding of organismal movement processes and their underlying mechanisms to predict and prevent altered animal movements and their consequences for ecosystem functions.
In this thesis I aim at understanding the influence of anthropogenically caused land-use change on animal movement processes and their underlying mechanisms. In particular, I am interested in the synergistic influence of large-scale landscape structure and fine-scale habitat features on basic-level movement behaviours (e.g. the daily amount of time spend running, foraging, and resting) and their emerging higher-level movements (home range formation). Based on my findings, I identify the likely consequences of altered animal movements that lead to the loss of species richness and abundances.
The study system of my thesis are hares in agricultural landscapes. European brown hares (Lepus europaeus) are perfectly suited to study animal movements in agricultural landscapes, as hares are hermerophiles and prefer open habitats. They have historically thrived in agricultural landscapes, but their numbers are in decline. Agricultural areas are undergoing strong land-use changes due to increasing food demand and fast developing agricultural technologies. They are already the largest land-use class, covering 38% of the world’s terrestrial surface. To consider the relevance of a given landscape structure for animal movement behaviour I selected two differently structured agricultural landscapes – a simple landscape in Northern Germany with large fields and few landscape elements (e.g. hedges and tree stands), and a complex landscape in Southern Germany with small fields and many landscape elements.
I applied GPS devices (hourly fixes) with internal high-resolution accelerometers (4 min samples) to track hares, receiving an almost continuous observation of the animals’ behaviours via acceleration analyses. I used the spatial and behavioural information in combination with remote sensing data (normalized difference vegetation index, or NDVI, a proxy for resource availability), generating an almost complete idea of what the animal was doing when, why and where. Apart from landscape structure (represented by the two differently structured study areas), I specifically tested whether the following fine-scale habitat features influence animal movements: resource, agricultural management events, habitat diversity, and habitat structure.
My results show that, irrespective of the movement process or mechanism and the type of fine-scale habitat features, landscape structure was the overarching variable influencing hare movement behaviour. High resource variability forces hares to enlarge their home ranges, but only in the simple and not in the complex landscape. Agricultural management events result in home range shifts in both landscapes, but force hares to increase their home ranges only in the simple landscape. Also the preference of habitat patches with low vegetation and the avoidance of high vegetation, was stronger in the simple landscape. High and dense crop fields restricted hare movements temporarily to very local and small habitat patch remnants. Such insuperable barriers can separate habitat patches that were previously connected by mobile links. Hence, the transport of nutrients and genetic material is temporarily disrupted. This mechanism is also working on a global scale, as human induced changes from habitat loss and fragmentation to expanding monocultures cause a reduction in animal movements worldwide.
The mechanisms behind those findings show that higher-level movements, like increasing home ranges, emerge from underlying basic-level movements, like the behavioural modes. An increasing landscape simplicity first acts on the behavioural modes, i.e. hares run and forage more, but have less time to rest. Hence, the emergence of increased home range sizes in simple landscapes is based on an increased proportion of time running and foraging, largely due to longer travelling times between distant habitats and scarce resource items in the landscape. This relationship was especially strong during the reproductive phase, demonstrating the importance of high-quality habitat for reproduction and the need to keep up self-maintenance first, in low quality areas. These changes in movement behaviour may release a cascade of processes that start with more time being allocated to running and foraging, resulting into an increased energy expenditure and may lead to a decline in individual fitness. A decrease in individual fitness and reproductive output will ultimately affect population viability leading to local extinctions.
In conclusion, I show that landscape structure has one of the most important effects on hare movement behaviour. Synergistic effects of landscape structure, and fine-scale habitat features, first affect and modify basic-level movement behaviours, that can scales up to altered higher-level movements and may even lead to the decline of species richness and abundances, and the disruption of ecosystem functions. Understanding the connection between movement mechanisms and processes can help to predict and prevent anthropogenically induced changes in movement behaviour. With regard to the paramount importance of landscape structure, I strongly recommend to decrease the size of agricultural fields and increase crop diversity. On the small-scale, conservation policies should assure the year round provision of areas with low vegetation height and high quality forage. This could be done by generating wildflower strips and additional (semi-) natural habitat patches. This will not only help to increase the populations of European brown hares and other farmland species, but also ensure and protects the continuity of mobile links and their intrinsic value for sustaining important ecosystem functions and services.
High-throughput sequence data retrieved from ancient or other degraded samples has led to unprecedented insights into the evolutionary history of many species, but the analysis of such sequences also poses specific computational challenges. The most commonly used approach involves mapping sequence reads to a reference genome. However, this process becomes increasingly challenging with an elevated genetic distance between target and reference or with the presence of contaminant sequences with high sequence similarity to the target species. The evaluation and testing of mapping efficiency and stringency are thus paramount for the reliable identification and analysis of ancient sequences. In this paper, we present ‘TAPAS’, (Testing of Alignment Parameters for Ancient Samples), a computational tool that enables the systematic testing of mapping tools for ancient data by simulating sequence data reflecting the properties of an ancient dataset and performing test runs using the mapping software and parameter settings of interest. We showcase TAPAS by using it to assess and improve mapping strategy for a degraded sample from a banded linsang (Prionodon linsang), for which no closely related reference is currently available. This enables a 1.8-fold increase of the number of mapped reads without sacrificing mapping specificity. The increase of mapped reads effectively reduces the need for additional sequencing, thus making more economical use of time, resources, and sample material.
Spotlight on islands
(2018)
Groups of proximate continental islands may conceal more tangled phylogeographic patterns than oceanic archipelagos as a consequence of repeated sea level changes, which allow populations to experience gene flow during periods of low sea level stands and isolation by vicariant mechanisms during periods of high sea level stands. Here, we describe for the first time an ancient and diverging lineage of the Italian wall lizard Podarcis siculus from the western Pontine Islands. We used nuclear and mitochondrial DNA sequences of 156 individuals with the aim of unraveling their phylogenetic position, while microsatellite loci were used to test several a priori insular biogeographic models of migration with empirical data. Our results suggest that the western Pontine populations colonized the islands early during their Pliocene volcanic formation, while populations from the eastern Pontine Islands seem to have been introduced recently. The inter-island genetic makeup indicates an important role of historical migration, probably due to glacial land bridges connecting islands followed by a recent vicariant mechanism of isolation. Moreover, the most supported migration model predicted higher gene flow among islands which are geographically arranged in parallel. Considering the threatened status of small insular endemic populations, we suggest this new evolutionarily independent unit be given priority in conservation efforts.
Systems biology aims at investigating biological systems in its entirety by gathering and analyzing large-scale data sets about the underlying components. Computational systems biology approaches use these large-scale data sets to create models at different scales and cellular levels. In addition, it is concerned with generating and testing hypotheses about biological processes. However, such approaches are inevitably leading to computational challenges due to the high dimensionality of the data and the differences in the dimension of data from different cellular layers.
This thesis focuses on the investigation and development of computational approaches to analyze metabolite profiles in the context of cellular networks. This leads to determining what aspects of the network functionality are reflected in the metabolite levels. With these methods at hand, this thesis aims to answer three questions: (1) how observability of biological systems is manifested in metabolite profiles and if it can be used for phenotypical comparisons; (2) how to identify couplings of reaction rates from metabolic profiles alone; and (3) which regulatory mechanism that affect metabolite levels can be distinguished by integrating transcriptomics and metabolomics read-outs.
I showed that sensor metabolites, identified by an approach from observability theory, are more correlated to each other than non-sensors. The greater correlations between sensor metabolites were detected both with publicly available metabolite profiles and synthetic data simulated from a medium-scale kinetic model. I demonstrated through robustness analysis that correlation was due to the position of the sensor metabolites in the network and persisted irrespectively of the experimental conditions. Sensor metabolites are therefore potential candidates for phenotypical comparisons between conditions through targeted metabolic analysis.
Furthermore, I demonstrated that the coupling of metabolic reaction rates can be investigated from a purely data-driven perspective, assuming that metabolic reactions can be described by mass action kinetics. Employing metabolite profiles from domesticated and wild wheat and tomato species, I showed that the process of domestication is associated with a loss of regulatory control on the level of reaction rate coupling. I also found that the same metabolic pathways in Arabidopsis thaliana and Escherichia coli exhibit differences in the number of reaction rate couplings.
I designed a novel method for the identification and categorization of transcriptional effects on metabolism by combining data on gene expression and metabolite levels. The approach determines the partial correlation of metabolites with control by the principal components of the transcript levels. The principle components contain the majority of the transcriptomic information allowing to partial out the effect of the transcriptional layer from the metabolite profiles. Depending whether the correlation between metabolites persists upon controlling for the effect of the transcriptional layer, the approach allows us to group metabolite pairs into being associated due to post-transcriptional or transcriptional regulation, respectively. I showed that the classification of metabolite pairs into those that are associated due to transcriptional or post-transcriptional regulation are in agreement with existing literature and findings from a Bayesian inference approach.
The approaches developed, implemented, and investigated in this thesis open novel ways to jointly study metabolomics and transcriptomics data as well as to place metabolic profiles in the network context. The results from these approaches have the potential to provide further insights into the regulatory machinery in a biological system.
Objective
We investigated the potential role of indirect benefits for female mate preferences in a highly promiscuous species of live-bearing fishes, the sailfin molly Poecilia latipinna using an integrative approach that combines methods from animal behavior, life-history evolution, and genetics. Males of this species solely contribute sperm for reproduction, and consequently females do not receive any direct benefits. Despite this, females typically show clear mate preferences. It has been suggested that females can increase their reproductive success through indirect benefits from choosing males of higher quality.
Results
Although preferences for large body size have been recorded as an honest signal for genetic quality, this particular study resulted in female preference being unaffected by male body size. Nonetheless, larger males did sire more offspring, but with no effect on offspring quality. This study presents a methodical innovation by combining preference testing with life history measurements—such as the determination of the dry weight of fish embryos—and paternity analyses on single fish embryos.
Abstract
Background
The unisexual Amazon molly (Poecilia formosa) originated from a hybridization between two sexual species, the sailfin molly (Poecilia latipinna) and the Atlantic molly (Poecilia mexicana). The Amazon molly reproduces clonally via sperm-dependent parthenogenesis (gynogenesis), in which the sperm of closely related species triggers embryogenesis of the apomictic oocytes, but typically does not contribute genetic material to the next generation. We compare for the first time the gonadal transcriptome of the Amazon molly to those of both ancestral species, P. mexicana and P. latipinna.
Results
We sequenced the gonadal transcriptomes of the P. formosa and its parental species P. mexicana and P. latipinna using Illumina RNA-sequencing techniques (paired-end, 100 bp). De novo assembly of about 50 million raw read pairs for each species was performed using Trinity, yielding 106,922 transcripts for P. formosa, 115,175 for P. latipinna, and 133,025 for P. mexicana after eliminating contaminations. On the basis of sequence similarity comparisons to other teleost species and the UniProt databases, functional annotation, and differential expression analysis, we demonstrate the similarity of the transcriptomes among the three species. More than 40% of the transcripts for each species were functionally annotated and about 70% were assigned to orthologous genes of a closely related species. Differential expression analysis between the sexual and unisexual species uncovered 2035 up-regulated and 564 down-regulated genes in P. formosa. This was exemplary validated for six genes by qRT-PCR.
Conclusions
We identified more than 130 genes related to meiosis and reproduction within the apomictically reproducing P. formosa. Overall expression of these genes seems to be down-regulated in the P. formosa transcriptome compared to both ancestral species (i.e., 106 genes down-regulated, 29 up-regulated). A further 35 meiosis and reproduction related genes were not found in the P. formosa transcriptome, but were only expressed in the sexual species. Our data support the hypothesis of general down-regulation of meiosis-related genes in the apomictic Amazon molly. Furthermore, the obtained dataset and identified gene catalog will serve as a resource for future research on the molecular mechanisms behind the reproductive mode of this unisexual species.
Plants are unable to move away from unwanted environments and therefore have to locally adapt to changing conditions. Arabidopsis thaliana (Arabidopsis), a model organism in plant biology, has been able to rapidly colonize a wide spectrum of environments with different biotic and abiotic challenges. In recent years, natural variation in Arabidopsis has shown to be an excellent resource to study genes underlying adaptive traits and hybridization’s impact on natural diversity. Studies on Arabidopsis hybrids have provided information on the genetic basis of hybrid incompatibilities and heterosis, as well as inheritance patterns in hybrids. However, previous studies have focused mainly on global accessions and yet much remains to be known about variation happening within a local growth habitat. In my PhD, I investigated the impact of heterozygosity at a local collection site of Arabidopsis and its role in local adaptation. I focused on two different projects, both including hybrids among Arabidopsis individuals collected around Tübingen in Southern Germany. The first project sought to understand the impact of hybridization on metabolism and growth within a local Arabidopsis collection site. For this, the inheritance patterns in primary and secondary metabolism, together with rosette size of full diallel crosses among seven parents originating from Southern Germany were analyzed. In comparison to primary metabolites, compounds from secondary metabolism were more variable and showed pronounced non-additive inheritance patterns. In addition, defense metabolites, mainly glucosinolates, displayed the highest degree of variation from the midparent values and were positively correlated with a proxy for plant size.
In the second project, the role of ACCELERATED CELL DEATH 6 (ACD6) in the defense response pathway of Arabidopsis necrotic hybrids was further characterized. Allelic interactions of ACD6 have been previously linked to hybrid necrosis, both among global and local Arabidopsis accessions. Hence, I characterized the early metabolic and ionic changes induced by ACD6, together with marker gene expression assays of physiological responses linked to its activation. An upregulation of simple sugars and metabolites linked to non-enzymatic antioxidants and the TCA cycle were detected, together with putrescine and acids linked to abiotic stress responses. Senescence was found to be induced earlier in necrotic hybrids and cytoplasmic calcium signaling was unaffected in response to temperature. In parallel, GFP-tagged constructs of ACD6 were developed.
This work therefore gave novel insights on the role of heterozygosity in natural variation and adaptation and expanded our current knowledge on the physiological and molecular responses associated with ACD6 activation.
In this work, different strategies for the construction of biohybrid photoelectrodes are investigated and have been evaluated according to their intrinsic catalytic activity for the oxidation of the cofactor NADH or for the connection with the enzymes PQQ glucose dehydrogenase (PQQ-GDH), FAD-dependent glucose dehydrogenase (FAD-GDH) and fructose dehydrogenase (FDH). The light-controlled oxidation of NADH has been analyzed with InGaN/GaN nanowire-modified electrodes. Upon illumination with visible light the InGaN/GaN nanowires generate an anodic photocurrent, which increases in a concentration-dependent manner in the presence of NADH, thus allowing determination of the cofactor. Furthermore, different approaches for the connection of enzymes to quantum dot (QD)-modified electrodes via small redox molecules or redox polymers have been analyzed and discussed. First, interaction studies with diffusible redox mediators such as hexacyanoferrate(II) and ferrocenecarboxylic acid have been performed with CdSe/ZnS QD-modified gold electrodes to build up photoelectrochemical signal chains between QDs and the enzymes FDH and PQQ-GDH. In the presence of substrate and under illumination of the electrode, electrons are transferred from the enzyme via the redox mediators to the QDs. The resulting photocurrent is dependent on the substrate concentration and allows a quantification of the fructose and glucose content in solution. A first attempt with immobilized redox mediator, i.e. ferrocenecarboxylic acid chemically coupled to PQQ-GDH and attached to QD-modified gold electrodes, reveal the potential to build up photoelectrochemical signal chains even without diffusible redox mediators in solution. However, this approach results in a significant deteriorated photocurrent response compared to the situation with diffusing mediators. In order to improve the photoelectrochemical performance of such redox mediator-based, light-switchable signal chains, an osmium complex-containing redox polymer has been evaluated as electron relay for the electronic linkage between QDs and enzymes. The redox polymer allows the stable immobilization of the enzyme and the efficient wiring with the QD-modified electrode. In addition, a 3D inverse opal TiO2 (IO-TiO2) electrode has been used for the integration of PbS QDs, redox polymer and FAD-GDH in order to increase the electrode surface. This results in a significantly improved photocurrent response, a quite low onset potential for the substrate oxidation and a broader glucose detection range as compared to the approach with ferrocenecarboxylic acid and PQQ-GDH immobilized on CdSe/ZnS QD-modified gold electrodes. Furthermore, IO-TiO2 electrodes are used to integrate sulfonated polyanilines (PMSA1) and PQQ-GDH, and to investigate the direct interaction between the polymer and the enzyme for the light-switchable detection of glucose. While PMSA1 provides visible light excitation and ensures the efficient connection between the IO-TiO2 electrode and the biocatalytic entity, PQQ-GDH enables the oxidation of glucose. Here, the IO-TiO2 electrodes with pores of approximately 650 nm provide a suitable interface and morphology, which is required for a stable and functional assembly of the polymer and enzyme. The successful integration of the polymer and the enzyme can be confirmed by the formation of a glucose-dependent anodic photocurrent. In conclusion, this work provides insights into the design of photoelectrodes and presents different strategies for the efficient coupling of redox enzymes to photoactive entities, which allows for light-directed sensing and provides the basis for the generation of power from sun light and energy-rich compounds.
Global change threatens the maintenance of ecosystem functions that are shaped by the persistence and dynamics of populations. It has been shown that the persistence of species increases if they possess larger trait adaptability. Here, we investigate whether trait adaptability also affects the robustness of population dynamics of interacting species and thereby shapes the reliability of ecosystem functions that are driven by these dynamics. We model co‐adaptation in a predator–prey system as changes to predator offense and prey defense due to evolution or phenotypic plasticity. We investigate how trait adaptation affects the robustness of population dynamics against press perturbations to environmental parameters and against pulse perturbations targeting species abundances and their trait values. Robustness of population dynamics is characterized by resilience, elasticity, and resistance. In addition to employing established measures for resilience and elasticity against pulse perturbations (extinction probability and return time), we propose the warping distance as a new measure for resistance against press perturbations, which compares the shapes and amplitudes of pre‐ and post‐perturbation population dynamics. As expected, we find that the robustness of population dynamics depends on the speed of adaptation, but in nontrivial ways. Elasticity increases with speed of adaptation as the system returns more rapidly to the pre‐perturbation state. Resilience, in turn, is enhanced by intermediate speeds of adaptation, as here trait adaptation dampens biomass oscillations. The resistance of population dynamics strongly depends on the target of the press perturbation, preventing a simple relationship with the adaptation speed. In general, we find that low robustness often coincides with high amplitudes of population dynamics. Hence, amplitudes may indicate the robustness against perturbations also in other natural systems with similar dynamics. Our findings show that besides counteracting extinctions, trait adaptation indeed strongly affects the robustness of population dynamics against press and pulse perturbations.
WRKY23 is a component of the transcriptional network mediating auxin feedback on PIN polarity
(2018)
Auxin is unique among plant hormones due to its directional transport that is mediated by the polarly distributed PIN auxin transporters at the plasma membrane. The canalization hypothesis proposes that the auxin feedback on its polar flow is a crucial, plant-specific mechanism mediating multiple self-organizing developmental processes. Here, we used the auxin effect on the PIN polar localization in Arabidopsis thaliana roots as a proxy for the auxin feedback on the PIN polarity during canalization. We performed microarray experiments to find regulators of this process that act downstream of auxin. We identified genes that were transcriptionally regulated by auxin in an AXR3/IAA17-and ARF7/ARF19-dependent manner. Besides the known components of the PIN polarity, such as PID and PIP5K kinases, a number of potential new regulators were detected, among which the WRKY23 transcription factor, which was characterized in more detail. Gain-and loss-of-function mutants confirmed a role for WRKY23 in mediating the auxin effect on the PIN polarity. Accordingly, processes requiring auxin-mediated PIN polarity rearrangements, such as vascular tissue development during leaf venation, showed a higher WRKY23 expression and required the WRKY23 activity. Our results provide initial insights into the auxin transcriptional network acting upstream of PIN polarization and, potentially, canalization-mediated plant development.
Specialized glial subtypes provide support to developing and functioning neural networks. Astrocytes modulate information processing by neurotransmitter recycling and release of neuromodulatory substances, whereas ensheathing glial cells have not been associated with neuromodulatory functions yet. To decipher a possible role of ensheathing glia in neuronal information processing, we screened for glial genes required in the Drosophila central nervous system for normal locomotor behavior. Shopper encodes a mitochondrial sulfite oxidase that is specifically required in ensheathing glia to regulate head bending and peristalsis. shopper mutants show elevated sulfite levels affecting the glutamate homeostasis which then act on neuronal network function. Interestingly, human patients lacking the Shopper homolog SUOX develop neurological symptoms, including seizures. Given an enhanced expression of SUOX by oligodendrocytes, our findings might indicate that in both invertebrates and vertebrates more than one glial cell type may be involved in modulating neuronal activity.
Endothelial cells (ECs) are involved in a variety of cellular responses. As multifunctional components of vascular structures, endothelial (progenitor) cells have been utilized in cellular therapies and are required as an important cellular component of engineered tissue constructs and in vitro disease models. Although primary ECs from different sources are readily isolated and expanded, cell quantity and quality in terms of functionality and karyotype stability is limited. ECs derived from human induced pluripotent stem cells (hiPSCs) represent an alternative and potentially superior cell source, but traditional culture approaches and 2D differentiation protocols hardly allow for production of large cell numbers. Aiming at the production of ECs, we have developed a robust approach for efficient endothelial differentiation of hiPSCs in scalable suspension culture. The established protocol results in relevant numbers of ECs for regenerative approaches and industrial applications that show in vitro proliferation capacity and a high degree of chromosomal stability.
Plant-derived Transcription Factors for Orthologous Regulation of Gene Expression in the Yeast Saccharomyces cerevisiae
Control of gene expression by transcription factors (TFs) is central in many synthetic biology projects where tailored expression of one or multiple genes is often needed. As TFs from evolutionary distant organisms are unlikely to affect gene expression in a host of choice, they represent excellent candidates for establishing orthogonal control systems. To establish orthogonal regulators for use in yeast (Saccharomyces cerevisiae), we chose TFs from the plant Arabidopsis thaliana. We established a library of 106 different combinations of chromosomally integrated TFs, activation domains (yeast GAL4 AD, herpes simplex virus VP64, and plant EDLL) and synthetic promoters harbouring cognate cis-regulatory motifs driving a yEGFP reporter. Transcriptional output of the different driver / reporter combinations varied over a wide spectrum, with EDLL being a considerably stronger transcription activation domain in yeast, than the GAL4 activation domain, in particular when fused to Arabidopsis NAC TFs. Notably, the strength of several NAC - EDLL fusions exceeded that of the strong yeast TDH3 promoter by 6- to 10-fold. We furthermore show that plant TFs can be used to build regulatory systems encoded by centromeric or episomal plasmids. Our library of TF – DNA-binding site combinations offers an excellent tool for diverse synthetic biology applications in yeast.
COMPASS: Rapid combinatorial optimization of biochemical pathways based on artificial transcription factors
We established a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) for controlling the expression of pathway genes, and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, we equipped COMPASS with multi-locus CRISPR/Cas9-mediated modification capacity. In its current realization, COMPASS allows combinatorial optimization of up to ten pathway genes, each transcriptionally controlled by nine different ATFs spanning a 10-fold difference in expression strength. The application of COMPASS was demonstrated by generating cell libraries producing beta-carotene and co-producing beta-ionone and biosensor-responsive naringenin. COMPASS will have many applications in other synthetic biology projects that require gene expression balancing.
CaPRedit: Genome editing using CRISPR-Cas9 and plant-derived transcriptional regulators for the redirection of flux through the FPP branch-point in yeast. Technologies developed over the past decade have made Saccharomyces cerevisiae a promising platform for production of different natural products. We developed CRISPR/Ca9- and plant derived regulator-mediated genome editing approach (CaPRedit) to greatly accelerate strain modification and to facilitate very low to very high expression of key enzymes using inducible regulators. CaPRedit can be implemented to enhance the production of yeast endogenous or heterologous metabolites in the yeast S. cerevisiae. The CaPRedit system aims to faciltiate modification of multiple targets within a complex metabolic pathway through providing new tools for increased expression of genes encoding rate-limiting enzymes, decreased expression of essential genes, and removed expression of competing pathways. This approach is based on CRISPR/Cas9-mediated one-step double-strand breaks to integrate modules containing IPTG-inducible plant-derived artificial transcription factor and promoter pair(s) in a desired locus or loci. Here, we used CaPRedit to redirect the yeast endogenous metabolic flux toward production of farnesyl diphosphate (FPP), a central precursor of nearly all yeast isoprenoid products, by overexpression of the enzymes lead to produce FPP from glutamate. We found significantly higher beta-carotene accumulation in the CaPRedit-mediated modified strain than in the wild type (WT) strain. More specifically, CaPRedit_FPP 1.0 strain was generated, in which three genes involved in FPP synthesis, tHMG1, ERG20, and GDH2, were inducibly overexpressed under the control of strong plant-derived ATFPs. The beta–carotene accumulated in CaPRedit_FPP 1.0 strain to a level 1.3-fold higher than the previously reported optimized strain that carries the same overexpressed genes (as well as additional genetic modifications to redirect yeast endogenous metabolism toward FPP production). Furthermore, the genetic modifications implemented in CaPRedit_FPP 1.0 strain resulted in only a very small growth defect (growth rate relative to the WT is ~ -0.03).
Polar nuclear migration is crucial during the development of diverse eukaryotes. In plants, root hair growth requires polar nuclear migration into the outgrowing hair. However, knowledge about the dynamics and the regulatory mechanisms underlying nuclear movements in root epidermal cells remains limited. Here, we show that both auxin and Rho-of-Plant (ROP) signaling modulate polar nuclear position at the inner epidermal plasma membrane domain oriented to the cortical cells during cell elongation as well as subsequent polar nuclear movement to the outer domain into the emerging hair bulge in Arabidopsis (Arabidopsis thaliana). Auxin signaling via the nuclear AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and INDOLE ACETIC ACID7 pathway ensures correct nuclear placement toward the inner membrane domain. Moreover, precise inner nuclear placement relies on SPIKE1 Rho-GEF, SUPERCENTIPEDE1 Rho-GDI, and ACTIN7 (ACT7) function and to a lesser extent on VTI11 vacuolar SNARE activity. Strikingly, the directionality and/or velocity of outer polar nuclear migration into the hair outgrowth along actin strands also are ACT7 dependent, auxin sensitive, and regulated by ROP signaling. Thus, our findings provide a founding framework revealing auxin and ROP signaling of inner polar nuclear position with some contribution by vacuolar morphology and of actin-dependent outer polar nuclear migration in root epidermal hair cells.
Communication is key to a wide variety of animal behaviours and multiple modalities are often involved in this exchange of information from sender to receiver. The communication of African weakly electric fish, however, is thought to be predominantly unimodal and is mediated by their electric sense, in which species-specific electric organ discharges (EODs) are generated in a context-dependent and thus variable sequence of pulse intervals (SPI). While the primary function of the electric sense is considered to be electrolocation, both of its components likely carry information regarding identity of the sender. However, a clear understanding of their contribution to species recognition is incomplete. We therefore analysed these two electrocommunication components (EOD waveform and SPI statistics) in two sympatric mormyrid Campylomormyrus species. In a set of five playback conditions, we further investigated which components may drive interspecific recognition and discrimination. While we found that both electrocommunication components are species-specific, the cues necessary for species recognition differ between the two species studied. While the EOD waveform and SPI were both necessary and sufficient for species recognition in C. compressirostris males, C. tamandua males apparently utilize other, non-electric modalities. Mapped onto a recent phylogeny, our results suggest that discrimination by electric cues alone may be an apomorphic trait evolved during a recent radiation in this taxon.
DNA nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. Here, the efficacy of SWL, an ephrin-mimicking peptide that binds specifically to EphrinA2 (EphA2) receptors, increased by presenting up to three of these peptides on small DNA nanostructures in an oligovalent manner. Ephrin signaling pathways play crucial roles in tumor development and progression. Moreover, Eph receptors are potential targets in cancer diagnosis and treatment. Here, the quantitative impact of SWL valency on binding, phosphorylation (key player for activation) and phenotype regulation in EphA2-expressing prostate cancer cells was demonstrated. EphA2 phosphorylation was significantly increased by DNA trimers carrying three SWL peptides compared to monovalent SWL. In comparison to one of EphA2’s natural ligands ephrin-A1, which is known to bind promiscuously to multiple receptors, pinpointed targeting of EphA2 by oligovalent DNA-SWL constructs showed enhanced cell retraction. Overall, we show that DNA scaffolds can increase the potency of weak signaling peptides through oligovalent presentation and serve as potential tools for examination of complex signaling pathways.
Agricultural land‐use practices have intensified over the last decades, leading to population declines of various farmland species, including the European hare (Lepus europaeus). In many European countries, arable fields dominate agricultural landscapes. Compared to pastures, arable land is highly variable, resulting in a large spatial variation of food and cover for wildlife over the course of the year, which potentially affects habitat selection by hares. Here, we investigated within‐home‐range habitat selection by hares in arable areas in Denmark and Germany to identify habitat requirements for their conservation. We hypothesized that hare habitat selection would depend on local habitat structure, that is, vegetation height, but also on agricultural field size, vegetation type, and proximity to field edges. Active hares generally selected for short vegetation (1–25 cm) and avoided higher vegetation and bare ground, especially when fields were comparatively larger. Vegetation >50 cm potentially restricts hares from entering parts of their home range and does not provide good forage, the latter also being the case on bare ground. The vegetation type was important for habitat selection by inactive hares, with fabaceae, fallow, and maize being selected for, potentially providing both cover and forage. Our results indicate that patches of shorter vegetation could improve the forage quality and habitat accessibility for hares, especially in areas with large monocultures. Thus, policymakers should aim to increase areas with short vegetation throughout the year. Further, permanent set‐asides, like fallow and wildflower areas, would provide year‐round cover for inactive hares. Finally, the reduction in field sizes would increase the density of field margins, and farming different crop types within small areas could improve the habitat for hares and other farmland species.
Sex-specific differences in nutritional requirements may crucially influence the performances of the sexes, which may have implications for sexual reproduction and thus is of great ecological and evolutionary interest. In the freshwater model species Daphnia magna, essential lipid requirements have been extensively studied. Dietary deficiencies in sterols and polyunsaturated fatty acids (PUFA) have been shown to constrain somatic growth and parthenogenetic reproduction of female Daphnia. In contrast, nutrient requirements of male Daphnia have not been studied yet. Supplementation experiments were conducted to investigate differences in sterol (cholesterol) and PUFA (eicosapentaenoic acid, EPA) requirements between female and male D. magna. Thresholds for sterol-limited juvenile growth were higher in females than in males, suggesting that females are more susceptible to dietary sterol deficiencies than males. Sex-specific differences in maximum somatic growth rates were evident primarily in the presence of dietary EPA; females could not exploit their generally higher growth potential in the absence of dietary PUFA. However, the thresholds for EPA-limited growth did not differ between sexes, suggesting that both sexes have similar dietary EPA requirements during juvenile growth. During a life history experiment, the gain in body dry mass was higher in females than in males, irrespective of food treatment. In both sexes, the gain in body dry mass increased significantly upon EPA supplementation, indicating that both sexes benefited from dietary EPA supply also later in life. However, the positive effects of EPA supplementation were most pronounced for female reproduction-related traits (i.e., clutch sizes, egg dry masses, and total dry mass investment in reproduction). The high maternal investment in reproduction resulted in a depletion of nutrients in female somata. In contrast, the comparatively low paternal investment in reproduction allowed for the accumulation of nutrients in male somata. We conclude that males are generally less susceptible to dietary nutrient deficiencies than females, because they can rely more on internal body stores. Our data suggest that the performances of the sexes are differentially influenced by lipid-mediated food quality, which may have consequences for sexual reproduction and thus the production of resting eggs and the maintenance of Daphnia populations.