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Compartmental models are the theoretical tool of choice for understanding single neuron computations. However, many models are incomplete, built ad hoc and require tuning for each novel condition rendering them of limited usability. Here, we present T2N, a powerful interface to control NEURON with Matlab and TREES toolbox, which supports generating models stable over a broad range of reconstructed and synthetic morphologies. We illustrate this for a novel, highly detailed active model of dentate granule cells (GCs) replicating a wide palette of experiments from various labs. By implementing known differences in ion channel composition and morphology, our model reproduces data from mouse or rat, mature or adult-born GCs as well as pharmacological interventions and epileptic conditions. This work sets a new benchmark for detailed compartmental modeling. T2N is suitable for creating robust models useful for large-scale networks that could lead to novel predictions. We discuss possible T2N application in degeneracy studies.
Heat stress transcription factors (Hsfs) are required for transcriptional changes during heat stress (HS) thereby playing a crucial role in the heat stress response (HSR). The target genes of Hsfs include heat shock proteins (Hsps), other Hsfs and genes involved in protection of the cell from irreversible damages due to exposure to elevated temperatures. Among 27 Hsfs in Solanum lycopersicum, HsfA1a, HsfA2 and HsfB1 constitute a functional triad which regulates important aspects of the HSR. HsfA1a is constitutively expressed and described as the master regulator of stress response and thermotolerance. Activation of HsfA1a under elevated temperatures leads to the induction of HsfA2 and HsfB1 which further stimulate the transcription of HS-responsive genes by forming highly active complexes with HsfA1a. Despite the well-established role of these three Hsfs in tomato HSR, information about functional relevance of other Hsfs is currently missing.
The heat stress inducible HsfA7 belongs alongside with HsfA2 to a phylogenetically distinct clade. Thereby the two proteins share high homology and a functional redundancy has been assumed. However, HsfA7 function and contribution to stress responses have not been investigated into detail in any plant species.
Tomato HsfA7 protein accumulates already at moderately elevated temperatures (~35°C) while HsfA2 becomes dominant at higher temperatures (>40°C). HsfA7 pre-mRNA undergoes complex and temperature-dependent alternative splicing resulting in several transcripts that encode for three protein isoforms. HsfA7-I contains a functional nuclear export signal (NES) and shows nucleocytoplasmic shuttling while HsfA7-II and HsfA7-III have a truncated NES which leads to the strong nuclear retention of the protein. Differences in the nucleocytoplasmic equilibrium have a major impact on the stability of protein isoforms, as nuclear retention is associated with increased protein turnover. Consequently, HsfA7-I shows a higher stability and can be detected even after 24 hours of stress attenuation, while HsfA7-II is rapidly degraded. The degradation of these factors is mediated by the ubiquitin-proteasome pathway.
HsfA7 can physically interact with HsfA1a and HsfA3 and form co-activator (“superactivator”) complexes with a very high transcriptional activity as shown on different HS-inducible promoters. In order for the complex to be successfully transferred to the nucleus and confer its activity it needs a functional nuclear localization signal (NLS) of HsfA7. In contrast, the activator (AHA) motif of HsfA7 is not essential for its co-activator function. Interestingly, while interaction of HsfA7 with either HsfA3 or HsfA1a stabilizes HsfA7 isoforms, concomitantly this leads to an increased turnover of HsfA1a and HsfA3. In contrast, HsfA2 has a stabilizing effect on the master regulator HsfA1a.
Thus, HsfA7 knockout mutants generated by CRISPR/Cas9 gene editing, show increased HsfA1a levels and a stronger induction of HS-related genes at 35°C compared to wild-type plants and HsfA2 knockout mutants. Consequently, HsfA7 knockout seedlings exhibit increased thermotolerance as shown by the enhanced hypocotyl elongation under a prolonged mild stress treatment at 35°C. In summary, these results highlight the importance of HsfA7 in regulation of cellular responses at elevated temperatures. Under moderately elevated temperatures, the accumulation of HsfA7 and its subsequent interaction with HsfA1a, leads to increased turnover of the latter, thereby ensuring a milder transcriptional activation of temperature-responsive genes like Hsps. In turn, in response to further elevated temperatures, HsfA2 becomes the dominant stress-induced Hsf. HsfA2 forms co-activator complexes with HsfA1a which in contrast to HsfA7, allows the stabilization of the master regulator, leading to the stronger expression of HS-responsive genes required for survival. Thereby, this study uncovers a new regulatory mechanism, where the temperature-dependent competitive interaction of HsfA2 and HsfA7 with HsfA1a control the fate of the master regulator and consequently the activity of temperature-responsive networks.
In mammalian species, including humans, the hippocampal dentate gyrus (DG) is a primary region of adult neurogenesis. Aberrant adult hippocampal neurogenesis is associated with neurological pathologies. Understanding the cellular mechanisms controlling adult hippocampal neurogenesis is expected to open new therapeutic strategies for mental disorders. Microglia is intimately associated with neural progenitor cells in the hippocampal DG and has been implicated, under varying experimental conditions, in the control of the proliferation, differentiation and survival of neural precursor cells. But the underlying mechanisms remain poorly defined. Using fluorescent in situ hybridization we show that microglia in brain express the ADP-activated P2Y13 receptor under basal conditions and that P2ry13 mRNA is absent from neurons, astrocytes, and neural progenitor cells. Disrupting P2ry13 decreases structural complexity of microglia in the hippocampal subgranular zone (SGZ). But it increases progenitor cell proliferation and new neuron formation. Our data suggest that P2Y13 receptor-activated microglia constitutively attenuate hippocampal neurogenesis. This identifies a signaling pathway whereby microglia, via a nucleotide-mediated mechanism, contribute to the homeostatic control of adult hippocampal neurogenesis. Selective P2Y13R antagonists could boost neurogenesis in pathological conditions associated with impaired hippocampal neurogenesis.
The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. Bfunction is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.
Modellierung der klimatischen Habitateignung verschiedener krankheitsübertragender Vektorarten
(2018)
Der Klimawandel hat einen starken Einfluss auf die Verbreitungsgebiete von Arten. Infolgedessen kann sich das Verbreitungsgebiet von Arten verschieben, einschränken oder ausweiten. Bei thermophilen Arten wird vermutet, dass sie von den klimatischen Änderungen profitieren und sie sich wahrscheinlich ausbreiten werden. Eine solche Ausbreitung, wozu auch die Einwanderung von gebietsfremden Arten zählt, hätte nicht nur zahlreiche Konsequenzen für diese Ökosysteme, sondern könnte sich auch zu einem ernsten Gesundheitsrisiko entwickeln, wenn es sich bei den einwandernden Neobiota um Vektorarten handelt.
Stechmücken und Sandmücken, als blutsaugende Insekten, zählen zu den bekanntesten Vektorarten. Sie sind in der Lage, eine Vielzahl von Infektionskrankheiten wie das Denguefieber oder das Gelbfieber, aber auch protozoische Parasiten wie "Leishmania"-Arten zu übertragen. Als thermophile Arten sind viele dieser Vektoren aktuell in ihrer Verbreitung weitgehend auf tropische und subtropische Gebiete beschränkt. Eine Einwanderung in gemäßigtere Gebiete kann zu einer Einschleppung der durch sie übertragenden Erreger führen und damit zum Ausbruch von Infektionskrankheiten. Aufgrund der medizinischen Relevanz dieser Arten ist es essentiell, die räumliche Verbreitung, sowie die abiotischen Ansprüche der Vektorarten zu kennen, um deren mögliche Ausbreitung nachzuvollziehen.
Vor diesem Hintergrund beschäftigte sich die vorliegende kumulative Dissertation mit den klimawandelinduzierten Änderungen der Habitateignung verschiedener medizinisch relevanter Vektorarten. Dabei wurden die zwei invasiven Stechmückenarten "Aedes albopictus" (I-III) und "Aedes japonicus" (III), sowie zehn in Europa bereits vorkommende Sandmückenarten der Gattung "Phlebotomus" (IV), untersucht. Die Arbeit basiert auf vier (ISI-) Publikationen. Unter Verwendung ökologischer Nischenmodellierung wurden geeignete Gebiete unter aktuellen und zukünftigen Klimabedingungen bestimmt. Um dabei sowohl räumliche als auch zeitliche Aspekte zu berücksichtigen, wurden mehrere räumliche Skalen (Deutschland und Europa), sowie Zeitperioden (2030, 2050 und 2070) betrachtet. Des Weiteren wurden verschiedene Ansätze (einzelne Algorithmen und Ensemble-Modelle) zur Modellierung der Habitateignung verwendet.
Die Ergebnisse dieser Dissertation zeigen eine zukünftige klimawandelbedingte Ausweitung der geeigneten Gebiete für viele der betrachteten Vektorarten. So konnte gezeigt werden, dass die Habitateignung für "Aedes albopictus" in Deutschland (I) und in Europa (III) zukünftig deutlich zunimmt. Auch für die Sandmückenarten "Phlebotomus alexandri", "Phlebotomus neglectus", "Phlebotomus papatasi", "Phlebotomus perfiliewi" und "Phlebotomus tobbi" konnte eine deutliche Zunahme der klimatisch geeigneten Gebieten projiziert werden (IV).
Lediglich Arten, wie die Asiatische Buschmücke "Aedes japonicus" (III) und auch kältetolerantere Sandmücken, wie "Phlebotomus ariasi" und "Phlebotomus mascittii" (IV) scheinen weniger von diesen klimatischen Veränderungen zu profitieren und könnten in Zukunft sogar aktuell geeignete Gebiete verlieren (klimawandelinduzierte Arealverkleinerung). Bei "Aedes japonicus" konnte dies auf eine engeren Nische mit einem Optimum bei vergleichsweise niedrigen Temperaturen zurückgeführt werden (III).
Am Beispiel von "Aedes albopictus" wurden ferner Umweltfaktoren identifiziert, die die Verbreitung der Art limitieren (II). Als wärmeliebende Art spielen bei "Aedes albopictus" in Mitteleuropa insbesondere die niedrigen Temperaturen eine Rolle, während in Zukunft die Sommertrockenheit in Südeuropa zunehmend eine Rolle spielen könnte.
Nischenmodellierung stellt trotz ihrer vereinfachenden Annahmen und Unsicherheiten, eine hilfreiche Methode zur Untersuchung klimawandelinduzierter Arealverschiebungen dar. Mit Hilfe der Modellierungsergebnisse konnten Gebiete mit einem hohen Etablierungsrisiko für die Vektorarten identifiziert werden, welche daher im Fokus künftiger Überwachungsprogramme stehen sollten. In Zukunft könnten mehr Vektorarten geeignete Bedingungen in Mitteleuropa finden, wodurch die Vektordiversität zunehmen wird. Dadurch könnte auch das Risiko für einen Ausbruch der durch die Vektoren übertragenen Krankheiten steigen.
Auch wenn das Vorhandensein eines kompetenten Vektors eine unerlässliche Voraussetzung für den Ausbruch einer Infektionskrankheit darstellt, gibt es noch weitere Faktoren, wie das Vorhandensein des Erregers. In Bezug auf die Risikoabschätzung vektorassoziierter Krankheiten sollten neben der Verbreitung des Vektors und des Erregers auch die abiotischen Bedingungen für die Entwicklung des Erregers berücksichtigt werden. Neben neu eingewanderten Arten sollten zudem auch die heimischen Arten in Bezug auf ihre Vektorkompetenz untersucht werden, da diese ebenfalls als potentielle Vektoren dienen und somit das Gesundheitsrisiko weiter erhöhen könnten.
Truffles (Tuber spp.) are the fruiting bodies of symbiotic fungi, which are prized food delicacies. The marked aroma variability observed among truffles of the same species has been attributed to a series of factors that are still debated. This is because factors (i.e. genetics, maturation, geographical location and the microbial community colonizing truffles) often co-vary in truffle orchards. Here, we removed the co-variance effect by investigating truffle flavour in axenic cultures of nine strains of the white truffle Tuber borchii. This allowed us to investigate the influence of genetics on truffle aroma. Specifically, we quantified aroma variability and explored whether strain selection could be used to improve human-sensed truffle flavour. Our results illustrate that aroma variability among strains is predominantly linked to amino acid catabolism through the Ehrlich pathway, as confirmed by 13C labelling experiments. We furthermore exemplified through sensory analysis that the human nose is able to distinguish among strains and that sulfur volatiles derived from the catabolism of methionine have the strongest influence on aroma characteristics. Overall, our results demonstrate that genetics influences truffle aroma much more deeply than previously thought and illustrate the usefulness of strain selection for improving truffle flavour.
Regulation of protein turnover allows cells to react to their environment and maintain homeostasis. Proteins can show different turnover rates in different tissue, but little is known about protein turnover in different brain cell types. We used dynamic SILAC to determine half-lives of over 5100 proteins in rat primary hippocampal cultures as well as in neuron-enriched and glia-enriched cultures ranging from <1 to >20 days. In contrast to synaptic proteins, membrane proteins were relatively shorter-lived and mitochondrial proteins were longer-lived compared to the population. Half-lives also correlate with protein functions and the dynamics of the complexes they are incorporated in. Proteins in glia possessed shorter half-lives than the same proteins in neurons. The presence of glia sped up or slowed down the turnover of neuronal proteins. Our results demonstrate that both the cell-type of origin as well as the nature of the extracellular environment have potent influences on protein turnover.
Echolocation allows bats to orientate in darkness without using visual information. Bats emit spatially directed high frequency calls and infer spatial information from echoes coming from call reflections in objects (Simmons 2012; Moss and Surlykke 2001, 2010). The echoes provide momentary snapshots, which have to be integrated to create an acoustic image of the surroundings. The spatial resolution of the computed image increases with the quantity of received echoes. Thus, a high call rate is required for a detailed representation of the surroundings.
One important parameter that the bats extract from the echoes is an object’s distance. The distance is inferred from the echo delay, which represents the duration between call emission and echo arrival (Kössl et al. 2014). The echo delay decreases with decreasing distance and delay-tuned neurons have been characterized in the ascending auditory pathway, which runs from the inferior colliculus (Wenstrup et al. 2012; Macías et al. 2016; Wenstrup and Portfors 2011; Dear and Suga 1995) to the auditory cortex (Hagemann et al. 2010; Suga and O'Neill 1979; O'Neill and Suga 1982).
Electrophysiological studies usually characterize neuronal processing by using artificial and simplified versions of the echolocation signals as stimuli (Hagemann et al. 2010; Hagemann et al. 2011; Hechavarría and Kössl 2014; Hechavarría et al. 2013). The high controllability of artificial stimuli simplifies the inference of the neuronal mechanisms underlying distance processing. But, it remains largely unexplored how the neurons process delay information from echolocation sequences. The main purpose of the thesis is to investigate how natural echolocation sequences are processed in the brain of the bat Carollia perspicillata. Bats actively control the sensory information that it gathers during echolocation. This allows experimenters to easily identify and record the acoustic stimuli that are behaviorally relevant for orientation. For recording echolocation sequences, a bat was placed in the mass of a swinging pendulum (Kobler et al. 1985; Beetz et al. 2016b). During the swing the bat emitted echolocation calls that were reflected in surrounding objects. An ultrasound sensitive microphone traveling with the bat and positioned above the bat’s head recorded the echolocation sequence. The echolocation sequence carried delay information of an approach flight and was used as stimulus for neuronal recordings from the auditory cortex and inferior colliculus of the bats.
Presentation of high stimulus rates to other species, such as rats, guinea pigs, suppresses cortical neuron activity (Wehr and Zador 2005; Creutzfeldt et al. 1980). Therefore, I tested if neurons of bats are suppressed when they are stimulated with high acoustic rates represented in echolocation sequences (sequence situation). Additionally, the bats were stimulated with randomized call echo elements of the sequence and an interstimulus time interval of 400 ms (element situation). To quantify neuronal suppression induced by the sequence, I compared the response pattern to the sequence situation with the concatenated response patterns to the element situation. Surprisingly, although the bats should be adapted for processing high acoustic rates, their cortical neurons are vastly suppressed in the sequence situation (Beetz et al. 2016b). However, instead of being completely suppressed during the sequence situation, the neurons partially recover from suppression at a unit specific call echo element. Multi-electrode recordings from the cortex allow assessment of the representation of echo delays along the cortical surface. At the cortical level, delay-tuned neurons are topographically organized. Cortical suppression improves sharpness of neuronal tuning and decreases the blurriness of the topographic map. With neuronal recordings from the inferior colliculus, I tested whether the echolocation sequence also induced neuronal suppression at subcortical level. The sequence induced suppression was weaker in the inferior colliculus than in the cortex. The collicular response makes the neurons able to track the acoustic events in the echolocation sequence. Collicular suppression mainly improves the signal-to-noise ratio. In conclusion, the results demonstrate that cortical suppression is not necessarily a shortcoming for temporal processing of rapidly occurring stimuli as it has previously been interpreted.
Natural environments are usually composed of multiple objects. Thus, each echolocation call reflects off multiple objects resulting in multiple echoes following the calls. At present, it is largely unexplored how neurons process echolocation sequences containing echo information from more than one object (multi-object sequences). Therefore, I stimulated bats with a multi-object sequence which contained echo information from three objects. The objects were different distances away from each other. I tested the influence of each object on the neuronal tuning by stimulating the bats with different sequences created from filtering object specific echoes from the multi-object sequence. The cortex most reliably processes echo information from the nearest object whereas echo information from distant objects is not processed due to neuronal suppression. Collicular neurons process less selectively echo information from certain objects and respond to each echo.
For proper echolocation, bats have to distinguish between own biosonar signals and the signals coming from conspecifics. This can be quite challenging when many bats echolocate adjacent to each other. In behavioral experiments, the echolocation performance of C. perspicillata was tested in the presence of potentially interfering sounds. In the presence of acoustic noise, the bats increase the sensory acquisition rate which may increase the update rate of sensory processing. Neuronal recordings from the auditory cortex and inferior colliculus could strengthen the hypothesis. Although there were signs of acoustic interference or jamming at neuronal level, the neurons were not completely suppressed and responded to the rest of the echolocation sequence.
The marine hydrocarbonoclastic bacterium Alcanivorax borkumensis is well known for its ability to successfully degrade various mixtures of n-alkanes occurring in marine oil spills. For effective growth on these compounds, the bacteria possess the unique capability not only to incorporate but also to modify fatty intermediates derived from the alkane degradation pathway. High efficiency of both these processes provides better competitiveness for a single bacteria species among hydrocarbon degraders. To examine the efficiency of A. borkumensis to cope with different sources of fatty acid intermediates, we studied the growth rates and membrane fatty acid patterns of this bacterium cultivated on diesel, biodiesel and rapeseed oil as carbon and energy source. Obtained results revealed significant differences in both parameters depending on growth substrate. Highest growth rates were observed with biodiesel, while growth rates on rapeseed oil and diesel were lower than on the standard reference compound (hexadecane). The most remarkable observation is that cells grown on rapeseed oil, biodiesel, and diesel showed significant amounts of the two polyunsaturated fatty acids linoleic acid and linolenic acid in their membrane. By direct incorporation of these external fatty acids, the bacteria save energy allowing them to degrade those pollutants in a more efficient way. Such fast adaptation may increase resilience of A. borkumensis and allow them to strive and maintain populations in more complex hydrocarbon degrading microbial communities.
The taxanes are effective microtubule-stabilizing chemotherapy drugs that inhibit mitosis, induce apoptosis, and produce regression in a fraction of cancers that arise at many sites including the ovary. Novel therapeutic targets that augment taxane effects are needed to improve clinical chemotherapy response in CCNE1-amplified high grade serous ovarian cancer (HGSOC) cells. In this study, we conducted an siRNA-based kinome screen to identify modulators of mitotic progression in CCNE1-amplified HGSOC cells that may influence clinical paclitaxel response. PLK1 is overexpressed in many types of cancer, which correlates with poor prognosis. Here, we identified a novel synthetic lethal interaction of the clinical PLK1 inhibitor BI6727 and the microtubule-targeting drug paclitaxel in HGSOC cell lines with CCNE1-amplification and elucidated the underlying molecular mechanisms of this synergism. BI6727 synergistically induces apoptosis together with paclitaxel in different cell lines including a patient-derived primary ovarian cancer culture. Moreover, the inhibition of PLK1 reduced the paclitaxel-induced neurotoxicity in a neurite outgrowth assay. Mechanistically, the combinatorial treatment with BI6727/paclitaxel triggers mitotic arrest, which initiates mitochondrial apoptosis by inactivation of anti-apoptotic BCL-2 family proteins, followed by significant loss of the mitochondrial membrane potential and activation of caspase-dependent effector pathways. This conclusion is supported by data showing that BI6727/paclitaxel-co-treatment stabilizes FBW7, a component of SCF-type ubiquitin ligases that bind and regulate key modulators of cell division and growth including MCL-1 and Cyclin E. This identification of a novel synthetic lethality of PLK1 inhibitors and a microtubule-stabilizing drug has important implications for developing PLK1 inhibitor-based combination treatments in CCNE1-amplified HGSOC cells.
Im Rahmen dieser Arbeit wurden sRNAs des halophilen Archaeons Haloferax volcanii hinsichtlich ihrer biologischen und ihrer regulatorischen Funktion charakterisiert.
Um einen Überblick über die biologischen Funktionen archaealer sRNAs zu erhalten, wurde eine umfassende phänotypische Charakterisierung von 27 sRNA-Deletionsmutanten im Vergleich zum Wildtyp ausgewertet. Im Zuge dieser phänotypischen Charakterisierungen wurden zehn verschiedene Wachstumsbedingungen, morphologische Unterschiede und Veränderungen in der Zellmotilität untersucht. Hierbei zeigten nahezu alle Deletionsmutanten unter mindestens einer der getesteten Bedingungen phänotypische Unterschiede. Durch den Verlust von sRNAs wurden sowohl sogenannte Gain-of-function als auch Loss-of-function Phänotypen beobachtet. Haloarchaeale sRNAs spielen eine wichtige Rolle beim Wachstum mit verschiedenen Salzkonzentrationen, mit verschiedenen Kohlenstoffquellen und beim Schwärmverhalten, sind jedoch weniger in die Adaptation an diverse Stressbedingungen involviert.
Zur näheren Charakterisierung der regulatorischen Funktion archaealer sRNAs wurden sRNA362, sRNAhtsf468 und sRNA479 mittels molekulargenetischer Methoden wie Northern Blot-Analyse und DNA-Mikroarray sowie bioinformatischer in silico-Analyse untersucht. Das Expressionslevel von sRNA362 konnte bestimmt und potentielle Zielgene für sRNAhtsf468 und sRNA479 identifiziert werden.
Eine vorangegangene Studie zeigte den Einfluss von sRNA30 unter Hitzestress und führte zur Identifikation differentiell produzierter Proteine in Abwesenheit der sRNA. In dieser Arbeit wurde mittels Northern Blot-Analysen die Expression der sRNA30 charakterisiert. Das Wachstum in An- und Abwesenheit von sRNA30 wurde bei 42°C und 51°C phänotypisch charakterisiert und der regulatorische Einfluss der sRNA auf die mRNA differentiell regulierter Proteine durch Northern Blot-Analyse überprüft. Eine Transkriptomanalyse mittels DNA-Mikroarray nach Hitzeschock-Induktion führte zur Identifikation differentiell regulierter Gene involviert in Transportprozesse, Metabolismus, Transkriptionsregulation und die Expression anderer sRNAs. Die differentielle Regulation des Proteoms nach Hitzeschockinduktion in An- und Abwesenheit von sRNA30 konnte bestätigt werden.
Desweiteren wurde in dieser Arbeit sRNA132 und deren phosphatabhängige Regulation der Ziel-mRNA HVO_A0477-80 näher charakterisiert. Eine Induktionskinetik nach Phosphatentzug bestätigte die Bedeutung von sRNA132 für die verstärkte Expression des Operons HVO_A0477-80 unter Phosphatmangel-Bedingungen und verwies auf die Existenz weiterer Regulationsmechanismen. Während vor und nach Phosphatentzug kein Unterschied bezüglich der Zellmorphologie von Wildtyp und Deletionsmutante zu erkennen war, führte das Wachstum mit einem starken Phosphatüberschuss von 5 mM zu einer Zellverlängerung der Deletionsmutante. Die Kompetition der nativen 3‘-UTR des Operons HVO_A0477-80 mit einer Vektor-kodierten artifiziellen 3‘-UTR legt eine Regulation über die Bindung von sRNA132 an die 3‘-UTR nahe. Der Transkriptomvergleich nach Phosphatentzug in An- und Abwesenheit von sRNA132 führte zur Identifikation des Phosphoregulons der sRNA. Zu diesem Phosphoregulon gehören unter anderem zwei Glycerinphosphat-Dehydrogenasen, Transkriptionsregulatoren, eine Polyphosphatkinase und eine Glycerolphosphodiesterase. Zudem waren die Transkriptlevel der beiden ABC-Transporter HVO_A0477-80 und HVO_2375-8 für anorganisches Phosphat und des Transporters HVO_B0292-5 für Glycerinaldehyd-3-Phosphat in Abwesenheit der sRNA verringert. Die beiden ABC-Transportsysteme für anorganisches Phosphat wurden im Rahmen dieser Arbeit deletiert und weiter charakterisiert. Es konnte gezeigt werden, dass das ABC-Transportsystem HVO_2375-8 bei geringen Phosphatkonzentrationen leicht induziert wird und das Transkriptlevel in Anwesenheit von sRNA132 erhöht ist. Wachstumsversuche der jeweiligen Deletionsmutante in direkter Konkurrenz mit dem Wildtyp zeigten, dass keiner der beiden ABC-Transporter den anderen vollständig ersetzen kann und der Wildtyp mit beiden intakten ABC-Transportern unter phosphatlimitierenden Bedingungen einen Wachstumsvorteil besitzt. In silico-Analysen der Promotorbereiche von sRNA und ABC-Transporter legen zudem die Existenz von P-Boxen nahe.
Background: Root and tuber crops are a major food source in tropical Africa. Among these crops are several species in the monocotyledonous genus Dioscorea collectively known as yam, a staple tuber crop that contributes enormously to the subsistence and socio-cultural lives of millions of people, principally in West and Central Africa. Yam cultivation is constrained by several factors, and yam can be considered a neglected “orphan” crop that would benefit from crop improvement efforts. However, the lack of genetic and genomic tools has impeded the improvement of this staple crop.
Results: To accelerate marker-assisted breeding of yam, we performed genome analysis of white Guinea yam (Dioscorea rotundata) and assembled a 594-Mb genome, 76.4% of which was distributed among 21 linkage groups. In total, we predicted 26,198 genes. Phylogenetic analyses with 2381 conserved genes revealed that Dioscorea is a unique lineage of monocotyledons distinct from the Poales (rice), Arecales (palm), and Zingiberales (banana). The entire Dioscorea genus is characterized by the occurrence of separate male and female plants (dioecy), a feature that has limited efficient yam breeding. To infer the genetics of sex determination, we performed whole-genome resequencing of bulked segregants (quantitative trait locus sequencing [QTL-seq]) in F1 progeny segregating for male and female plants and identified a genomic region associated with female heterogametic (male = ZZ, female = ZW) sex determination. We further delineated the W locus and used it to develop a molecular marker for sex identification of Guinea yam plants at the seedling stage.
Conclusions: Guinea yam belongs to a unique and highly differentiated clade of monocotyledons. The genome analyses and sex-linked marker development performed in this study should greatly accelerate marker-assisted breeding of Guinea yam. In addition, our QTL-seq approach can be utilized in genetic studies of other outcrossing crops and organisms with highly heterozygous genomes. Genomic analysis of orphan crops such as yam promotes efforts to improve food security and the sustainability of tropical agriculture.
Background: The ideal biofuel should not only be a regenerative fuel from renewable feedstocks, but should also be compatible with the existing fuel distribution infrastructure and with normal car engines. As the so-called drop-in biofuel, the fatty alcohol 1-octanol has been described as a valuable substitute for diesel and jet fuels and has already been produced fermentatively from sugars in small amounts with engineered bacteria via reduction of thioesterase-mediated premature release of octanoic acid from fatty acid synthase or via a reversal of the β-oxidation pathway.
Results: The previously engineered short-chain acyl-CoA producing yeast Fas1R1834K/Fas2 fatty acid synthase variant was expressed together with carboxylic acid reductase from Mycobacterium marinum and phosphopantetheinyl transferase Sfp from Bacillus subtilis in a Saccharomyces cerevisiae Δfas1 Δfas2 Δfaa2 mutant strain. With the involvement of endogenous thioesterases, alcohol dehydrogenases, and aldehyde reductases, the synthesized octanoyl-CoA was converted to 1-octanol up to a titer of 26.0 mg L−1 in a 72-h fermentation. The additional accumulation of 90 mg L−1 octanoic acid in the medium indicated a bottleneck in 1-octanol production. When octanoic acid was supplied externally to the yeast cells, it could be efficiently converted to 1-octanol indicating that re-uptake of octanoic acid across the plasma membrane is not limiting. Additional overexpression of aldehyde reductase Ahr from Escherichia coli nearly completely prevented accumulation of octanoic acid and increased 1-octanol titers up to 49.5 mg L−1. However, in growth tests concentrations even lower than 50.0 mg L−1 turned out to be inhibitory to yeast growth. In situ extraction in a two-phase fermentation with dodecane as second phase did not improve growth, indicating that 1-octanol acts inhibitive before secretion. Furthermore, 1-octanol production was even reduced, which results from extraction of the intermediate octanoic acid to the organic phase, preventing its re-uptake.
Conclusions: By providing chain length control via an engineered octanoyl-CoA producing fatty acid synthase, we were able to specifically produce 1-octanol with S. cerevisiae. Before metabolic engineering can be used to further increase product titers and yields, strategies must be developed that cope with the toxic effects of 1-octanol on the yeast cells.
The fruit fly Drosophila melanogaster is one of the most important biological model organisms, but only the comparative approach with closely related species provides insights into the evolutionary diversification of insects. Of particular interest is the live imaging of fluorophores in developing embryos. It provides data for the analysis and comparison of the threedimensional morphogenesis as a function of time. However, for all species apart from Drosophila, for example the red flour beetle Tribolium castaneum, essentially no established standard operation procedures are available and the pool of data and resources is sparse. The goal of my PhD project was to address these limitations. I was able to accomplish the following milestones:
- Development of the hemisphere and cobweb mounting methods for the non-invasive imaging of Tribolium embryos in light sheet-based fluorescence microscopes and characterization of most crucial embryogenetic events.
- Comprehensive documentation of methods as protocols that describe (i) beetle rearing in the laboratory, (ii) preparation of embryos, (ii) calibration of light sheet-based fluorescence microscopes, (iv) recording over several days, (v) embryo retrieval as a quality control as well as (vi) data processing.
- Adaption of the methods to record and analyze embryonic morphogenesis of the Mediterranean fruit fly Ceratitis capitata and the two-spotted cricket Gryllus bimaculatus as well as integration of the data into an evolutionary context.
- Further development of the hemisphere method to allow the bead-based / landmark-based registration and fusion of three-dimensional images acquired along multiple directions to compensate the shadowing effect.
- Development of the BugCube, a web-based computer program that allows to share image data, which was recorded by using light sheet-based fluorescence microscopy, with colleagues.
- Invention and experimental proof-of-principle of the (i) AGameOfClones vector concept that creates homozygous transgenic insect lines systematically. Additionally, partial proof-of-principle of the (ii) AClashOfStrings vector concept that creates double homozygous transgenic insect lines systematically, as well as preliminary evaluation of the (iii) AStormOfRecords vector concept that creates triple homozygous transgenic insect lines systematically.
- Creation and performance screening of more than fifty transgenic Tribolium lines for the long-term imaging of embryogenesis in fluorescence microscopes, including the first Lifeact and histone subunit-based lines.
My primary results contribute significantly to the advanced fluorescence imaging approaches of insect species beyond Drosophila. The image data can be used to compare different strategies of embryonic morphogenesis and thus to interpret the respective phylogenetic context. My technological developments extend the methodological arsenal for insect model organisms considerably.
Within my perspective, I emphasize the importance of non-invasive long-term fluorescence live imaging to establish speciesspecific morphogenetic standards, discuss the feasibly of a morphologic ontology on the cellular level, suggest the ‘nested linearly decreasing phylogenetic relationship’ approach for evolutionary developmental biology, propose the live imaging of species hybrids to investigate speciation and finally outline how light sheet-based fluorescence microscopy contributes to the transition from on-demand to systematic data acquisition in developmental biology.
During my PhD project, I wrote a total of ten manuscripts, six of which were already published in peer-reviewed scientific journals. Additionally, I supervised four Master and two Bachelor projects whose scientific questions were inspired by the topic of my PhD work.
The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent ApcMin/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.
Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo‐tomography
(2017)
We employed electron cryo‐tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation‐arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria.
Quercetin is a flavonoid that is ubiquitously found in vegetables and fruits. Like other flavonoids, it is active in balancing cellular reactive oxygen species (ROS) levels and has a cyto-protective function. Previously, a link between ROS balancing, aging, and the activity of O-methyltransferases was reported in different organisms including the aging model Podospora anserina. Here we describe a role of the S-adenosylmethionine-dependent O-methyltransferase PaMTH1 in quercetin-induced lifespan extension. We found that effects of quercetin treatment depend on the methylation state of the flavonoid. Specifically, we observed that quercetin treatment increases the lifespan of the wild type but not of the PaMth1 deletion mutant. The lifespan increasing effect is not associated with effects of quercetin on mitochondrial respiration or ROS levels but linked to the induction of the PaMth1 gene. Overall, our data demonstrate a novel role of O-methyltransferase in quercetin-induced longevity and identify the underlying pathway as part of a network of longevity assurance pathways with the perspective to intervene into mechanisms of biological aging.
Inhibition of midbrain dopamine (DA) neurons codes for negative reward prediction errors, and causally affects conditioning learning. DA neurons located in the ventral tegmental area (VTA) display two-fold longer rebound delays from hyperpolarizing inhibition in comparison to those in the substantia nigra (SN). This difference has been linked to the slow inactivation of Kv4.3-mediated A-type currents (IA). One known suppressor of Kv4.3 inactivation is a splice variant of potassium channel interacting protein 4 (KChIP4), KChIP4a, which has a unique potassium channel inactivation suppressor domain (KISD) that is coded within exon 3 of the KChIP4 gene. Previous ex vivo experiments from our lab showed that the constitutive knockout of KChIP4 (KChIP4 KO) removes the slow inactivation of IA in VTA DA neurons, with marginal effects on SN DA neurons. KChIP4 KO also increased firing pauses in response to phasic hyperpolarization in these neurons. Here I show, using extracellular recordings combined with juxtacellular labeling in anesthetized mice, that KChIP4 KO also selectively changes the number and duration spontaneous firing pauses by VTA DA neurons in vivo. Pauses were quantified with two different statistical methods, including one developed in house. No other firing parameter was affected, including mean frequency and bursting, and the activity of SN DA neurons was untouched, suggesting that KChIP4 gene products have a highly specific effect on VTA DA neuron responses to inhibitory input.
Following up on this result, I developed a new mouse line (KChIP4 Ex3d) where the KISD-coding exon 3 of KChIP4 is selectively excised by cre-recombinase expressed under the dopamine transporter (DAT) promoter, therefore disrupting the expression of KChIP4a only in midbrain DA neurons. I show that these mice have a highly selective behavioral phenotype, displaying a drastic acceleration in extinction learning, but no changes in acquisition learning, in comparison to control littermates. Computational fitting of the behavioral data with a modified Rescorla-Wagner model confirmed that this phenotype is congruent with a selective increase in learning from negative prediction errors. KChIP4 Ex3d also had normal open field exploration, novel object preference, hole board exploration and spontaneous alternation in a plus maze, indicating that exploratory drive, responses to novelty, anxiety, locomotion and working memory were not affected by the genetic manipulation. Furthermore semi-quantitative IHC revealed that KChIP4 Ex3d mice have increased Kv4.3 expression in TH+ neurons, suggesting that the absence of KChIP4a increases the binding of other KChIP variants, which known to increase surface expression of Kv4 channels.
Furthermore, in the course of my experimental study I identified that the most used mouse line where cre-recombinase is expressed under the DAT promoter (DAT-cre KI) has a different behavioral phenotype during conditioning in relation to WT littermate controls. These animals displayed increased responding during the initial trials of acquisition and delayed response latency extinction, consistent with an increase in motivation, which is in line with a decrease in DAT function.
I propose a working model where the disruption of KChIP4a expression in DA neurons leads to an increase in binding of other KChIP variants to Kv4.3 subunits, promoting their increased surface expression and increasing IA current density; this then increases firing pauses in response to synaptic inhibition, which in behaving animals translates to an increase in negative prediction error-based learning.
Understanding the spatial and temporal dynamics of species assemblages is a main challenge in ecology. The mechanisms that shape species assemblages and their temporal fluctuations along tropical elevational gradients are particularly poorly understood. Here, we examined the spatio-temporal dynamics of bird assemblages along an elevational gradient in Ecuador. We conducted bird point counts at three elevations (1000, 2000 and 3000 m) on 18 1-ha plots and repeated the sampling eight times over two years (216 hours in total). For each plot, we obtained data of monthly temperatures and precipitation and recorded the overall resource availability (i.e., the sum of flower, fruit, and invertebrate resources). As expected, bird richness decreased from low to high elevations. Moreover, we found a significant decrease in bird abundance and richness and an increase in evenness between the most and least humid season at each of the three elevations. Climatic factors were more closely related to these temporal fluctuations than local resource availability. While temperature had significant positive effects on the abundance of birds at mid and high elevations, precipitation negatively affected bird abundance at low and mid elevations. Our study highlights that bird assemblages along tropical elevational gradients can show pronounced seasonal fluctuations. In particular, low temperatures and high precipitation seem to impose important constraints on birds. We conclude that potential changes in climate, due to global warming, are likely to affect the spatio-temporal dynamics of bird assemblages along tropical elevational gradients.
Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear.
Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA.
Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors.
Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.
Endangered species of hosts are coupled with endangered species of parasites, which share the risk of co-extinction. Conservation efforts sometimes include breeding of rare species in captivity. Data on parasites of captive populations of endangered species is scarce and the ability of small numbers of captive host individuals to support the biodiversity of native parasites is limited. Examination of ectosymbionts of the critically endangered Philippine eagles and the endangered Mindanao Hawk-Eagle kept at the Philippine Eagle Center, Philippines, revealed three feather mite species despite regular treatment with insecticide powder. No other ectosymbiont taxa were detected. Studies in morphology and molecular phylogeny of these feather mites based on mitochondrial and nuclear DNA markers indicate that species found were typical for Accipitridae. Three new pterolichoid feather mite species (Acari: Pterolichoidea) were described from two species of eagles (Accipitriformes: Accipitridae) endemic to the Philippines: Hieracolichus philippinensis sp. n. (Gabuciniidae) and Pseudalloptinus pithecophagae sp. n. (Pterolichidae) from the Great Philippine Eagle Pithecophaga jefferyi Ogilvie-Grant, 1896, and Pseudogabucinia nisaeti sp. n. (Kramerellidae) from the Mindanao Hawk-Eagle Nisaetus pinskeri Gould, 1863. The presence of H. philippinensis on P. jefferyi supports the recent finding that the Great Philippine Eagle belongs to the lineage of serpent eagles (Circaetinae) rather than to the Harpy and other eagles.
Background: Molecular hydrogen (H2) is an attractive future energy carrier to replace fossil fuels. Biologically and sustainably produced H2 could contribute significantly to the future energy mix. However, biological H2 production methods are faced with multiple barriers including substrate cost, low production rates, and low yields. The C1 compound formate is a promising substrate for biological H2 production, as it can be produced itself from various sources including electrochemical reduction of CO2 or from synthesis gas. Many microbes that can produce H2 from formate have been isolated; however, in most cases H2 production rates cannot compete with other H2 production methods.
Results: We established a formate-based H2 production method utilizing the acetogenic bacterium Acetobacterium woodii. This organism can use formate as sole energy and carbon source and possesses a novel enzyme complex, the hydrogen-dependent CO2 reductase that catalyzes oxidation of formate to H2 and CO2. Cell suspensions reached specific formate-dependent H2 production rates of 71 mmol g protein −1 h−1 (30.5 mmol g CDW −1 h−1) and maximum volumetric H2 evolution rates of 79 mmol L−1 h−1. Using growing cells in a two-step closed batch fermentation, specific H2 production rates reached 66 mmol g CDW −1 h−1 with a volumetric H2 evolution rate of 7.9 mmol L−1 h−1. Acetate was the major side product that decreased the H2 yield. We demonstrate that inhibition of the energy metabolism by addition of a sodium ionophore is suitable to completely abolish acetate formation. Under these conditions, yields up to 1 mol H2 per mol formate were achieved. The same ionophore can be used in cultures utilizing formate as specific switch from a growing phase to a H2 production phase.
Conclusions: Acetobacterium woodii reached one of the highest formate-dependent specific H2 productivity rates at ambient temperatures reported so far for an organism without genetic modification and converted the substrate exclusively to H2. This makes this organism a very promising candidate for sustainable H2 production and, because of the reversibility of the A. woodii enzyme, also a candidate for reversible H2 storage.
Mannitol is the major compatible solute, next to glutamate, synthesized by the opportunistic human pathogen Acinetobacter baumannii under low water activities. The key enzyme for mannitol biosynthesis, MtlD, was identified. MtlD is highly similar to the bifunctional mannitol‐1‐phosphate dehydrogenase/phosphatase from Acinetobacter baylyi. After deletion of the mtlD gene from A. baumannii ATCC 19606T cells no longer accumulated mannitol and growth was completely impaired at high salt. Addition of glycine betaine restored growth, demonstrating that mannitol is an important compatible solute in the human pathogen. MtlD was heterologously produced and purified. Enzyme activity was strictly salt dependent. Highest stimulation was reached at 600 mmol/L NaCl. Addition of different sodium as well as potassium salts restored activity, with highest stimulations up to 41 U/mg protein by sodium glutamate. In contrast, an increase in osmolarity by addition of sugars did not restore activity. Regulation of mannitol synthesis was also assayed at the transcriptional level. Reporter gene assays revealed that expression of mtlD is strongly dependent on high osmolarity, not discriminating between different salts or sugars. The presence of glycine betaine or its precursor choline repressed promoter activation. These data indicate a dual regulation of mannitol production in A. baumannii, at the transcriptional and the enzymatic level, depending on high osmolarity.
Hematopoietic differentiation is driven by transcription factors, which orchestrate a finely tuned transcriptional network. At bipotential branching points lineage decisions are made, where key transcription factors initiate cell type-specific gene expression programs. These programs are stabilized by the epigenetic activity of recruited chromatin-modifying cofactors. An example is the association of the transcription factor RUNX1 with protein arginine methyltransferase 6 (PRMT6) at the megakaryocytic/erythroid bifurcation. However, little is known about the specific influence of PRMT6 on this important branching point. Here, we show that PRMT6 inhibits erythroid gene expression during megakaryopoiesis of primary human CD34+ progenitor cells. PRMT6 is recruited to erythroid genes, such as glycophorin A. Consequently, a repressive histone modification pattern with high H3R2me2a and low H3K4me3 is established. Importantly, inhibition of PRMT6 by shRNA or small molecule inhibitors leads to upregulation of erythroid genes and promotes erythropoiesis. Our data reveal that PRMT6 plays a role in the control of erythroid/megakaryocytic differentiation and open up the possibility that manipulation of PRMT6 activity could facilitate enhanced erythropoiesis for therapeutic use.
Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.
The mammalian thalamocortical system generates intrinsic activity reflecting different states of excitability, arising from changes in the membrane potentials of underlying neuronal networks. Fluctuations between these states occur spontaneously, regularly, and frequently throughout awake periods and influence stimulus encoding, information processing, and neuronal and behavioral responses. Changes of pupil size have recently been identified as a reliable marker of underlying neuronal membrane potential and thus can encode associated network state changes in rodent cortex. This suggests that pupillometry, a ubiquitous measure of pupil dilation in cognitive neuroscience, could be used as an index for network state fluctuations also for human brain signals. Considering this variable may explain task-independent variance in neuronal and behavioral signals that were previously disregarded as noise.
Cytokine regulation of high-output nitric oxide (NO) derived from inducible NO synthase (iNOS) is critically involved in inflammation biology and host defense. Herein, we set out to characterize the role of type I interferon (IFN) as potential regulator of hepatic iNOS in vitro and in vivo. In this regard, we identified in murine Hepa1-6 hepatoma cells a potent synergism between pro-inflammatory interleukin-β/tumor necrosis factor-α and immunoregulatory IFNβ as detected by analysis of iNOS expression and nitrite release. Upregulation of iNOS by IFNβ coincided with enhanced binding of signal transducer and activator of transcription-1 to a regulatory region at the murine iNOS promoter known to support target gene expression in response to this signaling pathway. Synergistic iNOS induction under the influence of IFNβ was confirmed in alternate murine Hepa56.1D hepatoma cells and primary hepatocytes. To assess iNOS regulation by type I IFN in vivo, murine acetaminophen (APAP)-induced sterile liver inflammation was investigated. In this model of acute liver injury, excessive necroinflammation drives iNOS expression in diverse liver cell types, among others hepatocytes. Herein, we demonstrate impaired iNOS expression in type I IFN receptor-deficient mice which associated with diminished APAP-induced liver damage. Data presented indicate a vital role of type I IFN within the inflamed liver for fine-tuning pathological processes such as overt iNOS expression.
Motivation: Arabidopsis thaliana is a well-established model system for the analysis of the basic physiological and metabolic pathways of plants. Nevertheless, the system is not yet fully understood, although many mechanisms are described, and information for many processes exists. However, the combination and interpretation of the large amount of biological data remain a big challenge, not only because data sets for metabolic paths are still incomplete. Moreover, they are often inconsistent, because they are coming from different experiments of various scales, regarding, for example, accuracy and/or significance. Here, theoretical modeling is powerful to formulate hypotheses for pathways and the dynamics of the metabolism, even if the biological data are incomplete. To develop reliable mathematical models they have to be proven for consistency. This is still a challenging task because many verification techniques fail already for middle-sized models. Consequently, new methods, like decomposition methods or reduction approaches, are developed to circumvent this problem.
Methods: We present a new semi-quantitative mathematical model of the metabolism of Arabidopsis thaliana. We used the Petri net formalism to express the complex reaction system in a mathematically unique manner. To verify the model for correctness and consistency we applied concepts of network decomposition and network reduction such as transition invariants, common transition pairs, and invariant transition pairs.
Results: We formulated the core metabolism of Arabidopsis thaliana based on recent knowledge from literature, including the Calvin cycle, glycolysis and citric acid cycle, glyoxylate cycle, urea cycle, sucrose synthesis, and the starch metabolism. By applying network decomposition and reduction techniques at steady-state conditions, we suggest a straightforward mathematical modeling process. We demonstrate that potential steady-state pathways exist, which provide the fixed carbon to nearly all parts of the network, especially to the citric acid cycle. There is a close cooperation of important metabolic pathways, e.g., the de novo synthesis of uridine-5-monophosphate, the γ-aminobutyric acid shunt, and the urea cycle. The presented approach extends the established methods for a feasible interpretation of biological network models, in particular of large and complex models.
The continuous conversion of natural wildlife habitats into agricultural areas, as well as the fragmentation of the last wildlife refuges, is increasing the interface between people and wildlife. When wildlife negatively impacts on people and vice versa, we speak about human-wildlife conflicts (HWCs). This definition includes losses on both sides and takes into consideration the rooting of most of these conflicts between different groups of interest, such as advocates for nature conservation and economic groups. The centres of highest biodiversity are located in developing countries, which are also characterized by poverty. In African and Asian countries, people living in the vicinity of national parks and other conservation areas mostly receive only little support through the government or conservation organisations. Especially for those people who are dependent on agriculture, damage to fields and harvests can have catastrophic consequences. If the species causing damage is protected by national or even international law, the farmer is not allowed to use lethal methods, but has to approach the authority in charge. If this agency, however, cannot offer appropriate support, resentment, anger or even hate develops, and the support for wildlife conservation activities declines. For this reason, HWCs were declared as one of the most important conservation topics today, being particularly relevant for large and threatened species such as the African and Asian elephant, hippopotamus and the greater one-horned rhino, as well as for large predators. Up to today, no general assessment scheme has been recommended for damage caused by protected wildlife species.
In my study, HWCs in Asia and Africa are compared, focussing on all herbivorous species identified which damaged crops. For the French NGO Awely, des animaux et des hommes, I developed a detailed assessment scheme suitable for all terrestrial ecosystems, and any type of HWCs and any species (Chapter 2). This HWC assessment scheme was used in four different study areas located in two African countries (South Luangwa/Zambia (SL), Tarangire/Tanzania (TA)) and two Asian countries (Bardia/Nepal (BA) and Manas/India (MA)). This scheme ran for six consecutive years (2009 to 2014) for Zambia, Nepal and India and two years (2010 to 2011) for Tanzania. To carry out the assessments, I trained local HWC officers (Awely Red Caps) to assess HWCs by field observations (measurement of damage, identification of species through signs of presence, landscape attributes etc.) and interviews with aggrieved parties (socio economic data). Results of this assessment are presented in Chapters 2-4.
To determine whether elephants prefer or avoid specific crop species, two field experiments were carried out, one in SL and one in BA (Chapter 5 and 6). For this, two test plots were set up and damage by elephants (and other herbivores) were quantified.
Within this doctoral thesis, 3306 damage events of 7408 aggrieved parties were analysed. In three out of the four study areas (SL, BA, MA), elephants caused the highest number of damage events compared to all other wildlife species, however, in TA, most fields were damaged by zebra. Furthermore, the greater one-horned rhino, hippopotamus, wild boar, bushpig, deer and antelope, as well as primates, caused damage to fields and harvests. Damage to houses and other property were nearly exclusively caused by elephants.
With this doctoral thesis I was able to show that season, crop availability, type and the phenological stage of the crop played an important role for crop damaging behavior of herbivores (Chapter 2). Elephants especially damaged rice, maize and wheat and preferred all crop types in a mature stage of growth. In contrast, rhinos preferred wheat to rice and similar to antelope and deer, they preferred crops at earlier stages of growth, before ripening. Crop damage by wildlife species varied strongly in size; most damages fell below 40% of the total harvest per farmer, but in several cases (3 to 8% depending on the study area), harvests were completely destroyed. Interestingly, during times of low nutritional availability in the natural habitat (dry season), crop damages in all four study areas were significantly less than during other seasons.
In all four study areas, crop protection strategies, such as active guarding in the fields, chasing wildlife with noise or fire torches or erecting barriers, were used. In some cases protection strategies were combined. Analysis of data revealed that traditional protection strategies did not reduce the costs of damage (Chapter 3). In some cases, costs of damage, on protected fields were even higher than for unprotected fields. Only in MA did strategic and cohesive guarding significantly reduce crop damage by wildlife species.
Besides damage in the fields, elephants also caused damage to properties in the villages. In search for stored staple crops, they damaged houses, grain stores and kitchens. Such damage was analysed in three study areas (SL, BA, MA) (Chapter 4). Although property damage occurred less frequently compared to crop damage in the fields, the mean cost of this damage was found to be double in BA/MA and four times higher in SL, compared to the costs of crop damage in the fields. It is further remarkable that property damage significantly increased towards the dry season, when the harvest was brought into the villages.
The findings of this study underpin the assumption that wildlife herbivores, especially elephants, are lured to fields and crops because the highly nutritional food (crop) being readily available. Traditional crop protection is cost and labour intensive and does not reduce the costs of damage. For this reason, crop types, which are thought to be not consumed by elephants were systematically tested on their attractiveness in field experiments in SL and BA (Chapter 5 and 6). In SL, lemon grass, ginger and garlic were proven to be less attractive to African elephants than maize and in BA, basil, turmeric, chamomile, coriander, mint, citronella and lemon grass were found to be less attractive to Asian elephants than rice.
The results of this doctoral thesis are relevant for the management of wildlife conservation as they can lead to new approaches to the mitigation of HWCs in African and Asian countries. Finally, specific needs for more scientific research in this field have been identified.
Endogenous AJAP1 associates with the cytoskeleton and attenuates angiogenesis in endothelial cells
(2017)
The adherens junction associated protein 1 (AJAP1, aka shrew-1) is presumably a type-I transmembrane protein localizing and interacting with the E-cadherin-catenin complex. In various tumors, AJAP1 expression is reduced or lost, including hepatocellular and esophageal squamous cell carcinoma, and glial-derived tumors. The aberrant expression of AJAP1 is associated with alterations in cell migration, invasion, increased tumor growth, and tumor vascularization, suggesting AJAP1 as a putative tumor suppressor. We show that AJAP1 attenuates sprouting angiogenesis by reducing endothelial migration and invasion capacities. Further, we show for the first time that endogenous AJAP1 is associated with the microtubule cytoskeleton. This linkage is independent from cell confluency and stable during angiogenic sprouting in vitro. Our work suggests that AJAP1 is a putative negative regulator of angiogenesis, reducing cell migration and invasion by interfering with the microtubule network. Based on our results and those of other authors, we suggest AJAP1 as a novel tumor suppressor and diagnostic marker.
The structural diversity of terpenoids is limited by the isoprene rule which states that all primary terpene synthase products derive from methyl-branched building blocks with five carbon atoms. With this study we discover a broad spectrum of novel terpenoids with eleven carbon atoms as byproducts of bacterial 2-methylisoborneol or 2-methylenebornane synthases. Both enzymes use 2-methyl-GPP as substrate, which is synthesized from GPP by the action of a methyltransferase. We used E. coli strains that heterologously produce different C11-terpene synthases together with the GPP methyltransferase and the mevalonate pathway enzymes. With this de novo approach, 35 different C11-terpenes could be produced. In addition to eleven known compounds, it was possible to detect 24 novel C11-terpenes which have not yet been described as terpene synthase products. Four of them, 3,4-dimethylcumene, 2-methylborneol and the two diastereomers of 2-methylcitronellol could be identified. Furthermore, we showed that an E. coli strain expressing the GPP-methyltransferase can produce the C16-terpene 6-methylfarnesol which indicates the condensation of 2-methyl-GPP and IPP to 6-methyl-FPP by the E. coli FPP-synthase. Our study demonstrates the broad range of unusual terpenes accessible by expression of GPP-methyltransferases and C11-terpene synthases in E. coli and provides an extended mechanism for C11-terpene synthases.
Rho GTPases control fundamental cellular processes and Cdc42 is a well-studied member of the family that controls filopodia formation and cell migration. Although the regulation of Cdc42 activity by nucleotide binding is well documented, the mechanisms driving its proteostasis are not clear. Here, we demonstrate that the highly conserved, RING domain containing E3 ubiquitin ligase XIAP controls the protein stability of Cdc42. XIAP binds to Cdc42 and directly conjugates poly ubiquitin chains to the Lysine 166 of Cdc42 targeting it for proteasomal degradation. Depletion of XIAP led to an increased protein stability and activity of Cdc42 in normal and tumor cells. Consistently, loss of XIAP enhances filopodia formation in a Cdc42-dependent manner and this phenomenon phenocopies EGF stimulation. Further, XIAP depletion promotes lung colonization of tumor cells in mice in a Cdc42-dependent manner. These observations shed molecular insights into ubiquitin-dependent regulation of Cdc42 and that of actin cytoskeleton.
Cells within a tissue form highly complex, cellular interactions. This architecture is lost in two-dimensional (2D) cell cultures. To close the gap between 2D cell cultures and in vivo tissues, three-dimensional (3D) cell cultures such as spheroids or embryoid bodies were developed. To fully take advantage of the third dimension, imaging techniques are essential. The emerging field of "image-based systems biology" exploits the information in images and builds a connection between experimental and theoretical investigation of biological processes. Such interdisciplinary approaches strongly depend on the development of protocols to establish 3D cell cultures, innovations in sample preparation, well-suited imaging techniques and quantitative segmentation methods.
Although 3D cell cultures and image-based systems biology provide a great potential, 2D methods are still not completely replaced by 3D methods. This is mainly due to methodical and technical hurdles. Therefore, this thesis provides a significant contribution to overcome these hurdles and to further develop 3D cell cultures. I established computational and experimental methods related to 3D aggregates and investigated fundamental, cellular processes such as adhesion, growth and differentiation.
The automatic segmentation method "PAS" and "LoS" were developed in the context of this thesis. They extract essential biological properties such as the projected area or features of cell nuclei from 2D or 3D images of 3D aggregates. Both algorithms show their accuracy robustly over image data from different samples and different microscopes. In addition, the superior performance of PAS and LoS was proven in a comparison with state-of-the-art methods.
The PAS approach served as an essential basis for investigating cellular processes such as adhesion and growth which are tightly regulated to contribute to tissue integrity. These processes are involved in the formation of spheroids. The temporally resolved data of spheroid formation of three mammary epithelial cell lines revealed differences in their formation dynamics as well as in the onset of spheroid formation phases (aggregation, compaction and growth). Despite these differences, adhesion- and growth-associated proteins such as E-cadherin, actin, microtubules, and the focal adhesion kinase show similar importance in a particular phase. Notably, certain proteins (e.g. E-Cadherin) contribute differently to spheroid formation of cells from different cell types in terms of cell adhesion and growth. Overall, analyses of the individual phases of spheroid formation revealed the temporal coordination of fundamental tissue-specific processes. The results contribute to a better understanding of the maintenance and disruption of tissue integrity.
An important but yet unknown process is how cells accomplish to arrange themselves against the gravitational force to form a spheroid. Live imaging with light sheet-based microscopy provides the best solution for a temporally and in particular spatially resolved investigation of spheroid formation. Although the imaging possibilities increase with this particular microscopy technique, available sample preparation methods are rare. Therefore, I have significantly optimized "agarose beaker" as preparation method for 3D long-term imaging of spheroid formation. The data show that upward movement of the cells takes place early. This movement is initiated in the centre of the initially flat cell layer. Subsequently, the cells move from the periphery of the cell layer toward the centre. Cells rearrange within the spheroid which is followed by growth. It is very likely that 3D aggregates form by adopting an energetically favoured, spherical shape by increasing cell-cell or cell-matrix contacts.
Besides the knowledge gained from the examination of the self-assembly process in different contexts, fully formed cellular aggregates can serve as basis to investigate differentiation processes. Differentiation guide cell fate specification during early embryonic development (i.e. preimplantation) and is not fully understood yet. Due to the lack of an in vitro system for preimplantation, I have developed "blastoids". These are 3D multicellular aggregates of mouse embryonic stem cells which represent important phases of preimplantation and beyond. In qualitative and quantitative analyses, a strong similarity was proven between blastoids and the inner cell mass of in vivo mouse embryos. Further results strongly suggest that both, the cell number and the trophectoderm play a subordinate role for cell fate decision during preimplantation. Furthermore, 3D neighbourhood analyses have shown that both, blastoids and mouse embryos, do not show a random "salt-and-pepper" pattern during differentiation. Instead, they show a yet unknown local clustering of cells with identical fates, suggesting local cell interactions that influence cell fate decision. Furthermore, the data indicate that the maturation of the epiblast in the later stages of preimplantation is initiated by an interaction between cells of the epiblast and the primitive endoderm.
Using image-based systems biology, I have investigated fundamental cellular processes such as adhesion, growth and differentiation in the context of tissue integrity and early embryonic development using 3D cellular aggregates. This highly interdisciplinary work is a major contribution to 3D cell biology and demonstrates how cells bind and interact within a complex system. The main methods developed in this thesis as well as the biological findings can be used not only in further biological but also in medical and pharmacological studies. They have the potential to advance our understanding of complex biological systems and to provide new opportunities for practical applications.
The pyrrolobenzodiazepine tilivalline (1) was originally identified in the human gut pathobiont Klebsiella oxytoca, the causative agent of antibiotic-associated hemorrhagic colitis. Here we show the identification of tilivalline and analogs thereof in the entomopathogenic bacterium Xenorhabdus eapokensis as well as the identification of its biosynthesis gene cluster encoding a bimodular non-ribosomal peptide synthetase. Heterologous expression of both genes in E. coli resulted in the production of 1 and from mutasynthesis and precursor directed biosynthesis 11 new tilivalline analogs were identified in X. eapokensis. These results allowed the prediction of the tilivalline biosynthesis being similar to that in K. oxytoca.
Im Kindes- und Jugendalter gehoert das Rhabdomyosarkom zu den haeufigsten Weichteilsarkomen. Bisher belaeuft sich das Therapieverfahren auf chirurgische Entfernung, gefolgt von Chemotherapie, bzw. bei nicht-operablen Faellen auf Radiotherapie und Chemotherapie, jedoch haben sich die Ueberlebenschancen fuer Patienten mit einer Erkrankung in metastasiertem oder rezidiviertem Stadium trotz intensiver Forschung ueber mehrere Jahrzehnte hinweg kaum gebessert und bleiben bei unter 30%. Neue therapeutische Strategien versuchen das Immunsystem des Patienten zu modulieren und dieses gezielter oder aggressiver gegen Tumorzellen zu machen. Nebst direkter Injektion von Zytokinen oder Antikoerpern bietet die adoptive Immunzelltherapie einen vielversprechenden Ansatz. In der vorliegenden Arbeit lag der Fokus auf Natuerlichen Killer- (NK) Zellen, da diese ein hohes zytotoxisches Potential gegenueber Tumorzellen aufweisen. Eine der groessten Herausforderungen der NK-Zellforschung ist die Breitstellung ausreichender Mengen an NK-Zellen mit optimaler antitumoraler Funktion fuer den klinischen Einsatz. Viele aktuell erprobte NK-Zellexpansionsstrategien basieren auf der Verwendung von Hilfs- oder Feeder-Zellen (Versorgerzellen), die jedoch vor der Applikation in Patienten aus dem finalen Produkt entfernt werden muessen. In der vorliegenden Arbeit sollten Feeder-zellfreie NK-Zellexpansionsprotokolle unter Verwendung von Gammakettenzytokinen getestet werden.
Interleukin (IL-) 15 erwies sich dabei vor allem fuer die Vermehrung der NK-Zellen als besonders foerderlich. Im Vergleich dazu fielen die Expansionsraten mit IL-2 oder IL-21 geringer aus. Interessanterweise wurde der expansionsfoerdernde Effekt von IL-15 durch dauerhafte Anwesenheit von IL-21 im Kulturmedium gehemmt. Ein kurzer, dreitaegiger IL-21-Boost am Ende der Expansionsphase wirkte sich wiederum positiv auf die NK-Zellexpansionsraten aus. Zudem zeigte sich durch IL-21 ein vermehrtes Auftreten von NK-Zellen des reiferen CD16posCD56dim Phaenotyps, der die zytotoxische Funktion vermittelt. Bei Degranulationsuntersuchungen wurden eine IL-21-induzierte Exozytoseaktivitaet und die vermehrte Ausschuettung von Perforin und Granzym B, welche Apoptose in den Zielzellen ausloesen, beobachtet. Vor allem der dreitaegige Boost mit IL-21 bewirkte eine gesteigerte Zytotoxizitaet gegenueber Tumorzellen, insbesondere gegenueber Rhabdomyosarkomzellen.
Auf dieser Grundlage bot es sich an fuer die NK-Zellexpansion ein Zwei-Phasen-Protokoll anzuwenden, bestehend aus einer initialen Proliferationsphase mit IL-15 und einem anschliessendem IL-21-Boost, durch den die antitumorale Funktionalitaet der NK-Zellen gesteigert wurde. Dieses IL-15+21boost-Protokoll wurde mit anderen Kombinationen aus den Gammakettenzytokinen IL-2, IL-15 und IL-21 verglichen und stellte sich hinsichtlich der NK-Zellexpansionsraten, der Degranulationskapazitaet und der damit verbundenen Zytotoxizitaet als den anderen Protokollen ueberlegen heraus.
Zytokinexpandierte NK-Zellen zeigten eine hoehere Rezeptorexpression an ihren Oberflaechen als unstimulierte Zellen. Die Expansion mit dem IL-15+21boost-Protokoll bewirkte die hoechste Dichte des Todesrezeptors TRAIL, jedoch auch der inhibitorischen KIR2D-Rezeptorfamilie. Fuer andere Oberflaechenmarker ergab sich jeweils eine mittlere Expressionsdichte verglichen mit dem IL-15- bzw. dem IL-15+21-Expansionsprotokoll. Die Sekretion von proinflammatorischen Zytokinen wie Interferon-gamma (IFN-g) und Tumor-Nekrose-Faktor-alpha (TNF-a) wurde zudem verstaerkt durch IL-21 angeregt, aber ebenso die Sekretion des immunsupprimierenden IL-10.
Weiter wurden die zytoinexpandierten NK-Zellen zur UEberpruefung ihrer in vivo Funktionalitaet anhand eines praeklinischen Xenograftmodells unter Verwendung von NOD SCID IL-2-Rgamma-/- (NSG) Maeusen und der Technologie der in-vivo-Biolumineszenzbildgebung getestet. Dabei konnte beobachtet werden, dass die NK-Zellen das Wachstum luciferaseexprimierender humaner Rhabdomyosarkome verlangsamten. Die Wirksamkeit der IL-15+21boost-expandierten NK-Zellen zeigte sich vor allem in einem kombinierten Ansatz, bei dem die Tumore zunaechst mit ionisierender Strahlung behandelt wurden und residuale Rhabdomyosarkomzellen anschliessend durch den adoptiven Transfer von humanen NK-Zellen in ihrem Wachstum gehemmt waren, solange die NK-Zelltherapie andauerte. Somit stellte sich die Kombination aus Bestrahlung und NK-Zelltransfer als wirksamer im Einsatz gegen Rhabdomyosarkome heraus als die alleinige Behandlung der Tumore durch Radiotherapie.
Zusammengefasst konnte in dieser Arbeit ein NK-Zellexpansionsprotokoll entwickelt werden, dass durch den ausschliesslichen Einsatz von Gammakettenzytokinen zu einem funktionalen NK-Zellprodukt fuehrte, welches auch in vivo lytische Aktivitaet gegenueber Rhabdomyosarkomzellen aufwies.
The major vault protein (MVP) is the predominant constituent of ubiquitous, evolutionarily conserved large cytoplasmic ribonucleoprotein particles of unknown function. Vaults are multimeric protein complexes with several copies of an untranslated RNA. Double labeling employing laser-assisted confocal microscopy and indirect immunofluorescence demonstrates partial colocalization of vaults with cytoskeletal elements in Chinese hamster ovary (CHO) and nerve growth factor (NGF)-treated neuronlike PC12 cells. Transfection of CHO and PC12 cells with a cDNA encoding the rat major vault protein containing a vesicular stomatitis virus glycoprotein epitope tag demonstrates that the recombinant protein is sorted into vault particles and targeted like endogenous MVPs. In neuritic extensions of differentiated PC12 cells, there is an almost complete overlap of the distribution of microtubules and vaults. A pronounced colocalization of vaults with filamentous actin can be seen in the tips of neurites. Moreover, in NGF-treated PC12 cells the location of vaults partially coincides with vesicular markers. Within the terminal tips of neurites vaults are located near secretory organelles. Our observations suggest that the vault particles are transported along cytoskeletal-based cellular tracks.
In der vorliegenden dreiteiligen Studie werden Mongolische Wüstenrennmäuse untersucht, deren Hörspektren im tieffrequenten Bereich und deren Unterscheidungsfähigkeiten von Kommunikationsrufen denen des Menschen ähneln. Die extrazelluläre Aktivität im primären auditorischen Kortex (AI) der narkotisierten Versuchstiere, evoziert durch Reintöne und arteigene Kommunikationsrufe, wird in der linken (LH) und rechten Gehirnhemisphäre (RH) aufgenommen. Es werden Multikanalelektroden (16 Eingangskanäle) verwendet, welche eine simultane Aufnahme der neuronalen Aktivitäten aller kortikalen Schichten ermöglichen. Zur Analyse der neuronalen Mechanismen werden Wellenformen einzelner Elektrodenkanäle und Aktivitätsprofile, bestehend aus den Wellenformen aller Elektrodenkanäle in einem Zeitfenster von 600 ms, auf Ebene von Aktionspotentialen (MUA), lokalen Feldpotentialen (LFP) und Current-source-density (CSD) Analysen, untersucht. Während MUAs die neuronalen Aktionspotentiale im Nahfeld der Elektrode reflektieren, umfassen die LFPs die summierten Potentiale (inhibitorisch und exzitatorisch) von Neuronen eines größeren Areals. Die CSDs hingegen werden durch die Integration von LFP-Wellenformen benachbarter, linear angeordneter Elektrodenkanäle berechnet und ermöglichen so eine Lokalisation der Ursprünge geräuschspezifischer Aktivitätsflüsse.
Im ersten Teilprojekt werden CSD-Profile in Antwort auf unterschiedliche Reintöne untersucht, um die Aktivitätskomponenten, die so genannten Sinks, für weiterführende Analysen zu quantifizieren. Es können zwei primäre (s1 und s2), drei mittlere (s3-s5) und vier späte (s6-s9) Sinks in einem Zeitfenster von 600 ms definiert werden. Eine Veränderung der Stimulusfrequenz eine Oktave über und unter der charakteristischen Frequenz (CF), beziehungsweise des Lautstärkepegels = 24 dB über der minimalen Schwelle, führt zu qualitativen Veränderungen in der CSD-Profilstruktur. Die Sink s7 wird durch Stimuli mit niedrigem Lautstärkepegel weniger verlässlich evoziert, wohingegen die Sink s9 bei Stimuli eine Oktave über der CF verlässlicher evoziert wird. Die Ergebnisse weisen darauf hin, dass im AI die spektralen Informationen eine Oktave über und unter der CF asymmetrisch integriert werden.
Auf Einzelschichtebene konnte bereits gezeigt werden, dass spektrotemporale Eigenschaften von Stimuli durch MUAs schlechter reflektiert wurden als durch LFPs, was vermutlich eine direkte Konsequenz der unterschiedlichen Ursprünge der Signaltypen ist. Daher werden im zweiten Teilprojekt die spezifischen Unterschiede der MUA-, LFP- und CSD-Antworten auf Ebene kortikaler Schichten und kompletter laminarer Profile untersucht, um die Unterschiede und den Informationsgehalt der drei Signaltypen zu charakterisieren. Signifikante Unterschiede, welche durch zwei Reintöne und sieben Kommunikationssignale evoziert werden, können verstärkt im mittleren und späten Latenzbereich und in granulären und infragranulären Schichten vorgefunden werden. Der Grad der Rufspezifizität ist in LFP und CSD-Antworten im Vergleich zu demjenigen in MUA-Antworten größer. Die Segregationsleistung ist im Vergleich zu einzelnen kortikalen Schichten in den von kortikalen Kolumnen abgeleiteten laminaren Profilen um den Faktor 1,8-2,6 erhöht. Die Neuronenpopulationen einzelner kortikaler Kolumnen sind vermutlich wichtig für die Kodierung von Geräuschen, welche sich in ihren spektrotemporalen Eigenschaften unterscheiden.
Viele vorangegangene Studien konnten zeigen, dass die Gehirnhemisphären akustische Signale asymmetrisch verarbeiten. Daher werden im dritten Hauptteil die laminaren Profile der LH und RH quantitativ und statistisch verglichen. Die MUA-, CSD-Profile und im geringeren Maße auch die LFP-Profile zeigen systematische Unterschiede auf signifikantem Niveau in der Dauer, Onset Latenz und vertikalen Ausdehnung bestimmter Aktivitäten. Kommunikationsrufe evozieren in der LH, welche beim Menschen auf Sprachstimuli spezialisiert ist, im Vergleich zur RH komplexere CSD-Profile. Die neuronale MUA-, LFP- und CSD-Aktivitätsstärke ist in der RH für weniger komplexe Stimuli teilweise signifikant erhöht. Die Asymmetrie in der Auftrittsverlässlichkeit der Sink s6 lässt vermuten, dass sich die intrakolumnäre Vernetzung in Schicht VIa zwischen der LH und RH unterscheidet. Die wenigen, signifikanten und nicht systematischen Unterschiede zwischen den Sink-Parametern der LH und RH nach kortikaler Ausschaltung mit dem GABAA-Rezeptor Agonist Muscimol weisen darauf hin, dass die Hemisphärenasymmetrie durch Prozesse des ipsilateralen Kortex maßgeblich beeinflusst wird.
The adult mammalian heart is unable to regenerate lost myocardial tissue after injury. In contrast, some lower vertebrates including zebrafish are able to undergo complete epimorphic regeneration following multiple types of cardiac injury. During the process of regeneration, spared zebrafish cardiomyocytes in the vicinity of the injured area undergo dedifferentiation and proliferation, thereby giving rise to new cardiomyocytes which replace the injured muscle. Insights into the molecular networks controlling these regenerative processes might help to develop novel therapeutic strategies to restore cardiac performance in humans.
While TGF-β signaling has been implicated in zebrafish cardiac regeneration, the role of individual TGF-β ligands remains to be determined. Here, I report the opposing expression response of two TGF-β ligand genes, mstnb and inhbaa, during zebrafish heart regeneration. Using gain- and loss-of-function approaches, I show that these ligands exert opposite effects on cardiac regeneration and specifically on cardiomyocyte proliferation. Notably, I show that overexpression of mstnb and loss of inhbaa negatively regulate cardiomyocyte proliferation and therefore disturb cardiac regeneration. In contrast, loss of mstnb and activation of inhbaa not only promote physiological cardiomyocyte proliferation but also enhance cardiac regeneration. I also identify Inhbaa as a mitogen which promotes cardiomyocyte proliferation independent of the well-established Nrg-ErbB signaling. Mechanistically, I unraveled that Mstnb and Inhbaa function through alternate Activin type 2 receptor complexes to control the activities of the signal transducers, Smad2 and Smad3, thereby regulating cardiomyocyte proliferation.
Altogether, I reveal novel and unidentified opposite functions of two TGF-β ligands during cardiac development and regeneration, resulting in a pro-mitogenic as well as an anti-mitogenic effect on cardiomyocytes. This study should therefore stimulate further research on targeting specific TGF-β family members to generate novel regenerative therapeutic strategies.
Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM‐CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM‐CF operations elaborated by the workgroups of the German network of ALM‐CFs, German Bio‐Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM‐CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences.
Establishing a yeast-based screening system for discovery of human GLUT5 inhibitors and activators
(2017)
Human GLUT5 is a fructose-specific transporter in the glucose transporter family (GLUT, SLC2 gene family). Its substrate-specificity and tissue-specific expression make it a promising target for treatment of diabetes, metabolic syndrome and cancer, but few GLUT5 inhibitors are known. To identify and characterize potential GLUT5 ligands, we developed a whole-cell system based on a yeast strain deficient in fructose uptake, in which GLUT5 transport activity is associated with cell growth in fructose-based media or assayed by fructose uptake in whole cells. The former method is convenient for high-throughput screening of potential GLUT5 inhibitors and activators, while the latter enables detailed kinetic characterization of identified GLUT5 ligands. We show that functional expression of GLUT5 in yeast requires mutations at specific positions of the transporter sequence. The mutated proteins exhibit kinetic properties similar to the wild-type transporter and are inhibited by established GLUT5 inhibitors N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA) and (−)-epicatechin-gallate (ECG). Thus, this system has the potential to greatly accelerate the discovery of compounds that modulate the fructose transport activity of GLUT5.
A full understanding of the origin, formation and degradation of volatile compounds that contribute to wine aroma is required before wine style can be effectively managed. Fractionation of grapes represents a convenient and robust method to simplify the grape matrix to enhance our understanding of the grape contribution to volatile compound production during yeast fermentation. In this study, acetone extracts of both Riesling and Cabernet Sauvignon grape berries were fractionated and model wines produced by spiking aliquots of these grape fractions into model grape juice must and fermented. Non-targeted SPME-GCMS analyses of the wines showed that several medium chain fatty acid ethyl esters were more abundant in wines made by fermenting model musts spiked with certain fractions. Further fractionation of the non-polar fractions and fermentation of model must after addition of these fractions led to the identification of a mixture of polyunsaturated triacylglycerides that, when added to fermenting model must, increase the concentration of medium chain fatty acid ethyl esters in wines. Dosage-response fermentation studies with commercially-available trilinolein revealed that the concentration of medium chain fatty acid ethyl esters can be increased by the addition of this triacylglyceride to model musts. This work suggests that grape triacylglycerides can enhance the production of fermentation-derived ethyl esters and show that this fractionation method is effective in segregating precursors or factors involved in altering the concentration of fermentation volatiles.
Characterizing the hologenome of Lasallia pustulata and tracing genomic footprints of lichenization
(2017)
The lichen symbiosis – consisting of fungal mycobionts and photoautotroph photobionts (green algae or cyanobacteria) – is globally successful. It covers an estimated 6% of the global surface with habitats ranging from deserts to the arctic. This success is reflected in the diversity of the mycobionts, with around 21% of all fungal species participating in lichen symbioses that can be facultative or obligate. Lichenization is furthermore evolutionary old, with fossil evidence for lichens reaching back 415 million years. For an individual fungal lineage, the Lecanoromycetes, the lichenization happened around 300 million years ago. This longstanding symbiotic relationship and the diversity of observed symbiotic dependency make them promising models to study the genomic consequences that follow the establishment of symbioses. Despite this, only little is known about the genomic effects of lichenization and extreme symbiotic dependency. To fill this gap we sequenced the hologenome of the lichen Lasallia pustulata, where the mycobiont could so far not been cultivated, suggesting that it might be more dependent on its symbionts.
As the poor culturability of lichen symbionts renders their genomes inaccessible to standard sequencing practices, we evaluated the extent to which different metagenome sequencing- and de novo assembly-strategies can be used to sequence and reconstruct the genomes of the individual symbionts. We find that the abundances of individual genomes present in the L. pustulata hologenome vary substantially, with the mycobiont being most abundant. Using in silico generated data sets and real Illumina sequencing data for L. pustulata we observe that the skewed abundances prevent a contiguous assembly of the underrepresented genomes when using only short-read sequencing. We conclude that short-read sequencing can offer first insights into lichen hologenomes. The fragmentation of the reconstructions hinders downstream analyses into the genomic consequences of lichenization though, as these are focused on identifying the gain and loss of genes.
We thus demonstrate a hybrid genome assembly strategy that is based on both short- and long-read sequencing. We show that this strategy is capable of creating highly contiguous genome reconstructions, not only for the L. pustulata mycobiont but also its photobiont Trebouxia sp., along with substantial amounts of the bacterial microbiome. A subsequent analysis of the microbiome of L. pustulata – performed over nine different samples collected in Germany and Italy – showed a stable taxonomic composition across the geographic range. We find that Acidobacteriaceae, which are known to thrive in nutrient poor habitats, are the dominant taxa. These would make them well adapted for the co-habitation with L. pustulata, which largely grows on rocks. Whether the Acidobacteriaceae are functionally involved in the lichen symbiosis is unclear so far.
As further comparative genomic studies rely on comprehensive genome annotations, we evaluate the completeness and fidelity of the gene annotations for the mycobiont L. pustulata as well as four further Lecanoromycetes. This reveals that un- and mis-annotated genes impact all evaluated genomes, with artificially joined genes and unannotated genes having the largest impact. In addition to these factors we find that the sequence composition – especially G/C-rich inverted repeats – lead to sequencing errors that interfere with the gene prediction. We minimize the effects of these artifacts through a rigorous curation.
Given the extremely sparse taxon sampling of available green alga genomes, we focus our search for the genomic footprints of lichenization on the mycobionts. We compare the genomes of the Lecanoromycetes to their closest relatives, the Eurotiomycetes and Dothideomycetes. This reveals that the last common ancestor of the Lecanoromycetes has lost around 10% of its genes after they split from the non-lichenized ancestor they share with the Eurotiomycetes. These losses are furthermore enriched, showing an excessive loss of genes involved with the degradation of polysaccharides. The loss of these genes fits a change from an ancestral saprotrophic lifestyle that depends on degrading complex plant matter, to the symbiotic lifestyle that relies on simpler nutrients provided by the photobionts. While the last common ancestor of the Lecanoromycetes additionally gained around 400 genes these could so far not be further characterized due to a lack of functionally annotated reference data.
As the mycobiont L. pustulata could so far not been grown in axenic culture, we initially expected to find an extensive genomic remodeling compared to the other mycobionts that easily grow in culture. We do not find evidence for this. Analyzing both the contraction of gene families and the loss of genes, we observe that L. pustulata and Umbilicaria muehlenbergii – its close relative that is easily grown in culture – share most of these. Furthermore, L. pustulata does not show an excessive loss of evolutionary old and well-conserved genes. These effects are mirrored on the functional level, as neither gene family contractions nor gene losses show a functional enrichment. This is partially due to the lack of functional reference data, analogous to the genes gained in the Lecanoromycetes, rendering their characterization hard. Thus, further studies on the genomic consequences of lichenization and differences in symbiotic dependence will have to be conducted, including larger taxon sets. This will be even more important for the photobionts, as the Chlorophyta are even more sparsely sampled today, hindering an effective functional and evolutionary study.
Tissue size regulation is critical for the normal functioning of the organ as well as to prevent unwanted pathogenesis such as cancer. The Hippo signaling pathway is well known for its robust regulation of tissue growth by the negative regulation of its nuclear effectors YAP1 and WWTR1. In this study, I have described the role of Yap1/Wwtr1 in zebrafish development, with a primary emphasis on the cardiovascular system.
I have generated zebrafish yap1 and wwtr1 mutants by CRISPR/CAS9. The mutant alleles are likely to be nonfunctional due to a premature stop codon and they show evidence of nonsense-mediated decay. Given that Yap1 and Wwtr1 are closely related proteins and have overlapping functions, I am given the opportunity to perform combinatorial analysis of the mutations on zebrafish development. Together with molecular probing tools, high-throughput sequencing and high-resolution imaging, I showed that
1. Double yap1;wwtr1 mutants exhibit severe posterior elongation phenotype, but somitogenesis appears to proceed as usual.
2. Yap1 and Wwtr1 may play an important role in PCV development and secondary angiogenic sprouting. However, key experiments will be needed to elucidate the direct role of Yap1 and Wwtr1 on these processes.
3. wwtr1-/- larvae hearts have a reduction in trabeculation, but in mosaic WT hearts, mutant cardiomyocytes prefer to populate the trabecular layer. My studies revealed that the mutant compact wall could not support trabeculation, which explains the hypotrabeculation phenotype of wwtr1-/- hearts. Additionally, Wwtr1 is required for myocardial Notch activity and can inhibit compact wall cardiomyocytes from entering the trabecular layer.
In summary, the Hippo signaling pathway, through Yap1/Wwtr1 has important regulatory functions in growth control. My work has revealed a surprising role for Yap1/Wwtr1 in tissue morphogenesis such as posterior tail morphogenesis and specific developmental processes of the cardiovascular system. It will be of interest to elucidate the regulation of Yap1/Wwtr1 in individual cells that translates into the complex cellular behaviors that drives morphogenesis.
NMR spectroscopy is a powerful technique to study ribonucleic acids (RNAs) which are key players in a plethora of cellular processes. Although the NMR toolbox for structural studies of RNAs expanded during the last decades, they often remain challenging. Here, we show that solvent paramagnetic relaxation enhancements (sPRE) induced by the soluble, paramagnetic compound Gd(DTPA-BMA) provide a quantitative measure for RNA solvent accessibility and encode distance-to-surface information that correlates well with RNA structure and improves accuracy and convergence of RNA structure determination. Moreover, we show that sPRE data can be easily obtained for RNAs with any isotope labeling scheme and is advantageous regarding sample preparation, stability and recovery. sPRE data show a large dynamic range and reflect the global fold of the RNA suggesting that they are well suited to identify interaction surfaces, to score structural models and as restraints in RNA structure determination.
Cardiovascular disease is the leading cause of death worldwide. Aging is among the greatest risk factors for cardiovascular disease. Cardiovascular disease comprises several diseases, for example myocardial infarction, elevated blood pressure and stroke. Many processes are known to promote or worsen cardiovascular disease and in the present study, cellular senescence and inflammatory activation were of special interest, as they have a strong association to aging and can be seen as hallmarks of cellular aging.
Long noncoding RNAs (lncRNAs) are noncoding RNAs with a length of more than 200 nucleotides. In recent years, numerous regulatory functions were shown for these transcripts and lncRNAs were shown to directly interact with DNA, RNA and proteins. The long noncoding RNA H19 was among the first described noncoding RNAs and was initially shown to act as a tumor suppressor. More recently, several studies showed oncogenic roles for H19. In regards to the cardiovascular system, H19 was not analyzed before.
We show that H19 is the most profoundly downregulated lncRNA in endothelial cells of aged mice compared to young littermates. Microarray analysis of human primary endothelial cells upon pharmacological H19 depletion revealed an involvement of H19 in cell cycle regulation. Loss of H19 in human endothelial cells in vitro led to reduced proliferation and to increased senescence. H19 depletion was shown to counteract proliferation before, but none of the described mechanisms applied to endothelial cells. We show that the reduction in proliferative capacity and the pro-senescent function of H19 is most probably mediated by an upregulation of p16ink4A and p21 upon H19 depletion.
When we compared the angiogenic capacity of aortic endothelial cells from young and aged mice in an aortic ring assay, rings from aged mice showed a reduced cumulative sprout length. Interestingly, pharmacological inhibition of H19 in aortic rings of young animals, where H19 is highly expressed, was sufficient to reduce the cumulative sprout length to levels we observed from aged animals. Furthermore, overexpression of human H19 in aortic rings of aged mice, where H19 is poorly expressed, rescued the impaired angiogenic capacity of aged endothelial cells.
We generated inducible endothelial-specific H19 knockout mice (H19iEC-KO) and subjected these animals to hind limb ischemia surgery followed by perfusion analysis in the hind limbs by laser-doppler velocimetry and histological analysis. Perfusion in the operated hind limb was increased in H19iEC-KO compared to Ctrl littermates, which was in contrast to a reduction in capillary density in the operated hind limbs of H19iEC-KO animals compared to Ctrl littermates and to our previous results. Analysis of arteriogenesis revealed an increase in collateral growth upon EC-specific H19 depletion in the ischemic hind limbs, which explains the increase in perfusion despite the reduction in capillary density. Further characterization of the animals revealed an increase in leukocyte infiltration into the tissue in the ischemic hind limbs upon endothelial-specific H19 depletion, indicating a potential role of H19 in inflammatory tissue activation.
Reanalysis of the microarray data from human primary endothelial cells upon H19 depletion revealed an association of H19 with inflammatory signaling and more specifically with IL-6/JAK2/STAT3 signaling. Analysis of cell surface adhesion molecule expression revealed an upregulation of ICAM-1 and VCAM-1 on mRNA level and an increase of the abundance of the two proteins on the cell surface of human primary endothelial cells. Consequently, adhesion of isolated human monocytes to human primary endothelial cells was increased upon H19 depletion in vitro. Interestingly, TNF-α mediated inflammatory activation of primary human endothelial cells repressed H19 expression. H19 did not function via previously described mechanisms. We excluded a competitive endogenous RNA (ceRNA) function for H19 in endothelial cells and showed that miR-675, which is processed from H19, does not play a role in the endothelium. Furthermore, H19 did not regulate previously described genes or pathways.
Analysis of transcription factor activity upon H19 depletion and overexpression revealed a differential activity of STAT3. STAT3 phosphorylation at TYR705 and thus activation was increased upon H19 depletion. Inhibition of STAT3 activation using a small compound inhibitor abolished the effects of H19 depletion on mRNA expression of p21, ICAM-1 and VCAM-1 and on proliferation, indicating that the effects of H19 are at least partially mediated via STAT3. STAT3 was shown to have positive effects on the cardiovascular system before, most likely due to upregulation of VEGF in a STAT3-dependent manner. We were not able to confirm previously described mechanisms for STAT3 in the present study and propose a new mechanism of action for the H19-dependent regulation of STAT3. Taken together, these results identify the long noncoding RNA H19 as a pivotal regulator of endothelial cell function. Figure 38 summarizes the described functions of H19 in endothelial cells.
We generated transgenic tomato plants with altered expression of heat stress transcription factor HsfA1. Plants with 10-fold overexpression of HsfA1 (OE plants) were characterized by a singleHsfA1 transgene cassette, whereas plants harboring a tandem inverted repeat of the cassette showed cosuppression (CS plants) by posttranscriptional silencing of the HsfA1 gene connected with formation of small interfering RNAs. Under normal growth conditions, major developmental parameters were similar for wild-type (WT), OE, and CS plants. However, CS plants and fruits were extremely sensitive to elevated temperatures, because heat stress-induced synthesis of chaperones and Hsfs was strongly reduced or lacking. Despite the complexity of the plant Hsf family with at least 17 members in tomato, HsfA1 has a unique function as master regulator for induced thermotolerance. Using transient reporter assays with mesophyll protoplasts from WT tomato, we demonstrated that plasmid-encoded HsfA1 and HsfA2 were well expressed. However, in CS protoplasts the cosuppression phenomenon was faithfully reproduced. Only transformation with HsfA2 expression plasmid led to normal expression of the transcription factor and reporter gene activation, whereas even high amounts of HsfA1 expression plasmids were silenced. Thermotolerance in CS protoplasts was restored by plasmid-borne HsfA2, resulting in expression of chaperones, thermoprotection of firefly luciferase, and assembly of heat stress granules.
We have recently shown that caspase-8 is a new substrate of Polo-like kinase 3 (Plk3) that phosphorylates the protein on residue T273 thereby promoting its pro-apoptotic function. In the present study we aimed to investigate the clinical relevance of Plk3 expression and phosphorylation of caspase-8 at T273 in patients with anal squamous cell carcinoma (SSC) treated with 5-fluorouracil and mitomycin C-based chemoradiotherapy (CRT). Immunohistochemical detection of the markers was performed in pretreatment biopsy specimens of 95 patients and was correlated with clinical/histopathologic characteristics including HPV-16 virus load/p16INK4a expression and cumulative incidence of local and distant failure, cancer specific survival (CSS), and overall survival (OS). We observed significant positive correlations between Plk3 expression, pT273 caspase-8 signal, and levels of HPV-16 virus DNA load/p16INK4a detection. Patients with high scores of Plk3 and pT273 caspase-8 showed increased local control (p = 0.011; p = 0.001), increased CSS (p = 0.011; p = 0.013) and OS (p = 0.024; p = 0.001), while the levels of pT273 caspase-8 were significantly associated (p = 0.033) with distant metastases. In multivariate analyses Plk3 expression remained significant for local failure (p = 0.018), CSS (p = 0.016) and OS (p = 0.023). Moreover, a combined HPV16 DNA load and Plk3 or pT273 caspase-8 variable revealed a significant correlation to decreased local failure (p = 0.001; p = 0.009), increased CSS (p = 0.016; p = 0.023) and OS (p = 0.003; p = 0.003). In conclusion these data indicate that elevated levels of Plk3 and pT273 caspase-8 are correlated with favorable clinical outcome in patients with anal SCC treated with concomitant CRT.
Regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in response to neuronal activity is critical for synaptic function and plasticity. Here, we show that neuronal activity induces the binding of ephrinB2 and ApoER2 receptors at the postsynapse to regulate de novo insertion of AMPA receptors. Mechanistically, the multi-PDZ adaptor glutamate-receptor-interacting protein 1 (GRIP1) binds ApoER2 and bridges a complex including ApoER2, ephrinB2, and AMPA receptors. Phosphorylation of ephrinB2 in a serine residue (Ser-9) is essential for the stability of such a complex. In vivo, a mutation on ephrinB2 Ser-9 in mice results in a complete disruption of the complex, absence of ApoER2 downstream signaling, and impaired activity-induced and ApoER2-mediated AMPA receptor insertion. Using compound genetics, we show the requirement of this complex for long-term potentiation (LTP). Together, our findings uncover a cooperative ephrinB2 and ApoER2 signaling at the synapse, which serves to modulate activity-dependent AMPA receptor dynamic changes during synaptic plasticity.
The cardiovascular system (CVS) consists of heart and blood vessels, forming a close circulatory loop. All tissues depend on the nutrients and molecular oxygen (O2) delivered by the blood. Therefore, it is not surprising that the CVS is one of the first working systems and the heart is the first functional organ in the forming embryo (Baldwin 1996). The building blocks of blood vessels are endothelial cells (ECs), which form the endothelium, a specialized epithelium that defines the luminal surface of the vessels (Pugsley and Tabrizchi 2000). The process of blood vessel development comprises several steps. The first events occurring are the formation of new vessels de novo to constitute the primary vascular loop known as vasculogenesis. During vasculogenesis the vascular precursors, known as angioblasts, migrate and coalesce to form the axial vessels. Subsequently, the main vessels undergo a specification step where they acquire either arterial or venous identity. As the embryo increases in size, the main vascular loop needs to increase in complexity. In order to reach all the different parts of the developing organs, new blood vessels are formed from pre-existing ones, a phenomenon known as angiogenesis (Gore et al. 2012).
Mature blood cells have a short lifespan. Therefore, hematopoietic stem cells (HSCs) are required throughout lifetime to constantly form new blood cells in a process called hematopoiesis. Interestingly, endothelial and immune cells development have been shown to converge at different points during their development, one of which is developmental hematopoiesis. During embryogenesis, definitive hematopoiesis occurs in a tissue called hemogenic endothelium (HE), a specialized subset of ECs at the ventral wall of the dorsal aorta (DA). HE acquires hematopoietic potentials and gives rise to HSCs, through a process known as endothelial-to-hematopoietic transition (EHT). During EHT, these specialized ECs extrude from DA and colonize the so-called aorta-gonadmesonephros (AGM) region, forming the native HSCs (Paik and Zon 2010).
As vascular development requires different steps, the molecular pathways involved are many. The Notch signaling pathway has been demonstrated to be one of the main players in vascular development. Among other functions, Notch signaling has been shown to be important during EHT. In the murine model, Runx1, a master regulator of HSC formation, has been shown to be transcriptionally regulated by NOTCH1 through GATA2 activation. This observation was later corroborated by knockdown studies for notch1a and notch1b in zebrafish (Butko, Pouget, and Traver 2016). Another essential pathway for vascular development is the HIF pathway. Hif-1α, Hif-1β and Hif-2α mouse mutants show severe vascular defects that result in early embryonic lethality (Simon and Keith 2008), which hinders a deep analysis of the phenotypes incurring in the mutant embryos. In addition, deletion of Hif-1α specifically in myeloid cells showed abnormalities in the motility, invasiveness, and adhesion of macrophages (Cramer et al. 2003). Intriguingly, Hif-1α deletion in vascular endothelial cadherin-expressing cells led to a significant but partial reduction of HSC number, suggesting that other players may be involved in this pathway (Imanirad et al. 2014).
Zebrafish embryos have been shown to be tolerant to hypoxia at very early stages of development (Padilla and Roth 2001). Also, zebrafish embryos develop externally and this allows to finely manipulate the environment where they grow (Lieschke and Currie 2007). These features make zebrafish an ideal model to investigate how hypoxia and Hif transcription factors affect vertebrate vascular development. In this study, I will examine the impact of hypoxia on zebrafish vascular development. Specifically, I will dissect the role of hif-1α in macrophage-EC interactions during vascular development and repair. Moreover, I show redundant functions for hif-1α and hif-2α in HSC development upstream of Notch signaling.