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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.
Taxonomy, phylogeny and zoogeography of the hexaploid Torini of the Middle East and North Africa
(2017)
Fishes of the tribe Torini Karaman, 1971 (Teleostei: Cyprinidae) are a diverse group of primary freshwater fishes, distributed in Africa, the Middle East, and Indomalaya. They are an important component of the native freshwater-fish fauna of the Middle East and North Africa, and occur in most large river systems of the Levant, Arabia, Mesopotamia, southern Iran, and Morocco. They belong to the subfamily Cyprininae, are characterised by being tetraploid or hexaploid, having large scales, and a smooth and ossified last unbranched ray in the dorsal fin. As primary freshwater fishes they are not able to tolerate marine conditions and depend on direct freshwater connections for their dispersal. This makes them an ideal model for zoogeographic studies.
Prior to this study, the diversity of the Torini species in the Middle East and North Africa was not well understood. The validity of several genera and species was unclear, and the generic assignment of several species changed frequently.
In this PhD project the taxonomy, phylogeny, and zoogeography of the Torini of the Middle East and North Africa were investigated with morphological, as well as molecular methods. More than 1550 fish specimens were examined morphologically. Some of the specimens, including the types of most nominal species, were already available from museum collections. The remaining specimens were collected during expeditions to Ethiopia, Iran, Jordan, Morocco and Syria. Tissue samples were collected for molecular genetic analyses. The mitochondrial genes for cytochrome b, NADH dehydrogenase subunit 4 and the tRNAs for serine and histidine were sequenced from more than 120 specimens, representing 20 species of Torini and two small, diploid African barbs (Cyprinidae, tribe Smiliogastrini). Molecular data were analysed with Bayesian inference and other methods.
The analyses confirmed that the hexaploid Torini of Africa and the Middle East form a monophyletic group. In the Middle East and North Africa the Torini are represented by the genera Arabibarbus, Carasobarbus, Mesopotamichthys, and Pterocapoeta. These genera are each morphologically diagnosable, monophyletic, and genetically distinct. The species 'Labeobarbus' reinii cannot be assigned to any of these genera, because it is morphologically dissimilar and genetically clearly separated from each of them. A generic name for this species is presently not available and until the description of a new genus it is preliminarily assigned to the genus 'Labeobarbus'.
Out of the 28 species-group taxa described from the Middle East and North Africa until now, 15 are valid: Arabibarbus arabicus, A. grypus, A. hadhrami, Carasobarbus apoensis, C. canis, C. chantrei, C. exulatus, C. fritschii, C. harterti, C. kosswigi, C. luteus, C. sublimus, Mesopotamichthys sharpeyi, Pterocapoeta maroccana, and 'Labeobarbus' reinii.
The phylogenetic relationships between the Middle Eastern and North African Torini are well resolved, based on the analysis of mitochondrial DNA sequences from nearly all relevant species.
The interspecific and intraspecific morphological and genetic diversity is shaped by the zoogeographic history. Conclusions can be drawn about the events that shaped the evolution of this group. The Torini originated in the Indomalayan biogeographical realm and colonised the Middle East and Africa during the Miocene via the Gomphotherium landbridge. The Indomalayan Torini are tetraploid, whereas those of the Middle East and Africa are hexaploid. Molecular phylogenetic analyses showed that the hexaploid Torini cluster within the tetraploid Torini. This makes the tetraploid Torini a paraphyletic group with respect to the hexaploid Torini. Morocco was colonised in two independent waves. The first came from sub-Saharan Africa and is represented by Pterocapoeta maroccana. The second originated in the Middle East and gave rise to C. fritschii, C. harterti, and probably 'L.' reinii. The Tigris-Euphrates system is the largest freshwater system in the Middle East. Its central position between the Orontes River and Jordan River in the West, the Iranian tributaries to the Persian Gulf in the East, and the Arabian Peninsula in the South made it an important crossroad for the colonisation of the Middle East by Torini and other freshwater biota. During the Miocene the predecessors of the Jordan and Orontes rivers were connected to the Tigris-Euphrates system. The Jordan River was separated from the Euphrates before the Orontes. Arabia was colonised in two waves. The first (A. arabicus, A. hadhrami, C. exulatus) dates to the Pliocene, whereas the second (C. apoensis) ended as recently as the late Pleistocene or early Holocene.
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.
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.
Deciphering the ecological functions of fungal root endophytes based on their natural occurrence
(2017)
Plants are colonized by a large diversity of fungi, some residing on the surface and others penetrating the plant tissues, the latter referred to as fungal endophytes (endon Gr., within; phyton, plant; de Bary 1879). Despite the saprotrophic potential of fungal endophytes, they are not found to cause visible disease symptoms to the host. Plants are colonized simultaneously by various fungal species, which form rich and diverse endophytic assemblages. Although it is hypothesized that fungal endophytes contribute to the fitness of their hosts and to the functioning of ecosystems, the ecological function of fungal endophytic assemblages remains cryptic. The aims of this doctoral thesis are to gain insight to the ecological functions of root fungal endophytes, by deciphering their roles in ecosystems based on their natural occurrence and the structure of their assemblages. The thesis focuses on studying the diversity and structure of the endophytic mycobiome within roots of two annual and widespread plant hosts Microthlapsi perfoliatum and M. erraticum (Brassicaceae) in several locations across northern Mediterranean and central Europe. The thesis is composed by six Chapters, with a primary focus on Chapter 1, 2 and 3.
Chapter 1 (Glynou et al., 2016) aimed at characterizing the diversity of fungal endophytes in roots at a continental scale and at assessing the factors affecting the structure of endophytic assemblages with the use of cultivation-based methods. For that, root samples were collected from 52 plant populations, along with a collection of soil, bioclimatic, geographic and host data. Cultivation of surface-sterilized root samples on culture media and isolation of fungal colonies in pure culture generated 1,998 fungal colonies. Grouping of sequences into Operational Taxonomic Units (OTUs), based on the 97% similarity of the isolates’ rDNA Internal Transcribed Spacer (ITS) sequence, generated in total 296 OTUs, representing taxa mostly within the phylum Ascomycota with a minor representation of Basidiomycota. Endophytic assemblages were mostly correlated with variation in bioclimatic conditions. Interestingly, despite the large diversity revealed, the assemblages were dominated by only six OTUs related to the orders Hypocreales, Pleosporales and Helotiales, which had a widespread distribution across populations but with some following patterns of ecological preferences.
Chapter 2 aimed at characterizing the uncultivable fraction of the root fungal endophytic diversity, which was not possible to capture in Chapter 1. High-throughput sequencing via the
Illumina Miseq platform was implemented in 43 of the 52 original populations and mostly in the same root samples. In comparison with the cultivation-based approach, the HTS managed to cover the overall diversity within samples. It revealed a large non-cultivated endophytic diversity but the same cultivable fungi dominated assemblages. Moreover, the endophytic diversity was grouped mostly within fungal orders with demonstrated ability to grow in culture and taxonomically related groups were found to have divergent ecological preferences.
The genetic identity of the most abundant OTUs was further investigated in Chapter 3 (Glynou et al., 2017), aiming to unravel genotypic variability, which was possibly overlooked due to the use of lTS, as a universal genetic marker, and could explain their high abundance and widespread distribution. Multi-locus gene sequencing and AFLP profiling for the five most abundant OTUs suggested a low within-OTU genetic variability and show that these fungi have ubiquitous distribution and are not limited by environmental conditions within the ecological ranges of the study. A selection of endophytes frequently isolated in Chapter 1 was functionally characterized in Chapter 4 (Kia et al., 2017) based on the isolates’ traits and interactions with plants. In Chapter 5 (Cheikh-Ali et al., 2015) fungal cultures of Exophiala sp. with differential colony structure where investigated for their production of secondary metabolites. Moreover, Chapter 6 (Maciá-Vicente et al., 2016) comprises the description of the new species Exophiala radicis based on morphological and molecular characteristics.
Compilation of all results shows that the fungal endophytic diversity in roots of Microthlaspi spp. is high but few widespread OTUs dominate the assemblages, and have unlimited dispersal ability. These fungi seem also to have a wide niche breadth and are not affected by environmental filtering. The findings indicate that the local environment but also processes of competitive exclusion determine the structure of endophytic assemblages. In addition, the fungal endophytes associated with Microthlapsi spp. likely have saprotrophic activity however the interactions with plants are likely context-dependent. Further research is needed to assess the biotic interactions among endophytes and their effect on the structure of fungal endophytic assemblages. Ultimately, the findings of this thesis are useful to shed light on the processes underlying the structure of endophytic assemblages. They also upraise the need to describe diversity by combining genetic, metabolic and physiological data, in order to disentangle the elusive ecological roles of the endophytic mycobiome.
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.
Heat stress transcription factors (Hsfs) play essential role in heat stress response and thermotolerance by controlling the transcriptional activation of heat stress response (HSR) genes including molecular chaperones. Plant Hsf families show a striking multiplicity, with more than 20 members in the many plant species. Among Hsfs, HsfA1s act as the master regulators of heat stress (HS) response and HsfA2 becomes one of the most abundant Hsfs during HS. Using transgenic plans with suppressed expression of HsfA2 we have shown that this Hsf is involved in acquired thermotolerance of S. lycopersicum cv Moneymaker as HsfA2 is required for high expression and maintenance of increased levels of Hsps during repeated cycles of HS treatment.
Interestingly, HsfA2 undergoes temperature-dependent alternative splicing (AS) which results in the generation of seven transcript variants. Three of these transcripts (HsfA2-Iα-γ), generated due to alternative splicing of a second, newly identified intron encode for the full length protein involved in acquired thermotolerance. Another 3 transcripts (HsfA2-IIIα-γ) are generated due to alternative splicing in intron 1, leading in all cases to a premature termination codon and targeting of these transcripts for degradation via the non-sense mRNA decay mechanism (NMD).
Interestingly, excision of intron 2, results into the generation of a second previously unreported protein isoform, annotated as HsfA2-II. HsfA2-II shows similar transcriptional activity to the full-length protein HsfA2-I in the presence of HsfA1a but lacks the nuclear export signal (NES) required for nucleocytoplasmic shuttling which allows efficient nuclear retention and stimulation of transcription of HS-induced genes. Furthermore, stability assays showed that HsfA2-II exhibits lower protein stability compared to HsfA2-I.
The presence of a second intron and the generation of a second protein isoform we identified in other Solanaceae species as well. Remarkably, we observed major differences in the splicing efficiency of HsfA2 intron 2 among different tomato species. Several wild tomato accessions exhibit higher splicing efficiency that favors the generation of HsfA2-II, while in these species the splice variant HsfA2-Iγ is absent. This natural variation in splicing efficiency specifically occurring at temperatures around 37.5oC is associated with the presence of 3 intronic polymorphisms. In the case of wild species these polymorphisms seemingly restrict the binding of RS2Z36, identified as a putative splicing silencer for HsfA2 intron 2.
Tomato accessions with the polymorphic “wild” HsfA2 show enhanced thermotolerance against a direct severe heat stress incident due to the stronger increase of Hsps and other stress induced genes. Introgression of the “wild” S. pennellii HsfA2 locus into the cultivar M82, resulted in enhanced seedling thermotolerance highlighting the potential use of the polymorphic HsfA2 for breeding.
We conclude that alterations in the splicing efficiency of HsfA2 have contributed to the adaption of tomato species to different environments and these differences might be directly related to natural variation in their thermotolerance.
Der Gyrus dentatus ist eine anatomische Region im Hippocampus und besitzt die einzigartige Fähigkeit auch im adulten Gehirn lebenslang neue Nervenzellen zu generieren. Dieser Prozess wird als adulte Neurogenese bezeichnet, stellt eine besondere Form struktureller Plastizität dar und es wurde gezeigt, dass adult neugebildete Körnerzellen im Gyrus dentatus essentiell am Prozess des hippocampalen Lernens und der Gedächtnisausbildung beteiligt sind. Es wird vermutet, dass neue Körnerzellen aufgrund ihrer charakteristischen Eigenschaften verstärkt auf neue Informationsmuster reagieren können und darauf spezialisiert sind Muster, die eine hohe Ähnlichkeit zueinander haben zu separieren und diese Unterschiede zu kodieren. Obwohl bereits eine Vielzahl von wissenschaftlichen Studien zum Verständnis der Entwicklung und Funktion adult neugebildeter Körnerzellen beitragen konnte, bestehen immer noch Unklarheiten darin, wie sich diese neuen Nervenzellen strukturell entwickeln, wann es zu einer funktionellen Integration kommt und wie diese beiden Prozesse miteinander zusammenhängen. In den vorliegenden Arbeiten wurde die strukturelle Entwicklung und synaptische Integration adult neugebildeter Körnerzellen in das bestehende hippocampale Netzwerk der Ratte und Maus unter in vivo Bedingungen untersucht. Zur Beantwortung dieser Fragen wurden Methoden aus der Anatomie, Histologie und in vivo Elektrophysiologie kombiniert. Der Nachweis neuer Körnerzellen erfolgte entweder durch immunhistologische Färbungen gegen spezifische Marker für unreife und reife Körnerzellen, Markierungen mit Bromdesoxyuridin oder retro- bzw. adenovirale intrazerebrale Injektionen und Expression von GFP. Es wurde eine in vivo Stimulation des Tractus perforans in der anästhesierten Ratte zur Langzeitpotenzierung der Körnerzellsynapsen und anschließend eine immunhistologische Analyse der Expression von synaptischen Aktivitäts- und Plastizitätsmarkern in neugebildeten und reifen Körnerzellen nach der Stimulation durchgeführt. Zusätzlich wurden detaillierte drei-dimensionale Rekonstruktion dendritischer Bäume erstellt und dendritische Dornenfortsätze an retroviral markierten Zellen analysiert.
Die vorliegenden Daten belegen den generellen Verlauf der Entwicklung neugeborener Körnerzellen in zwei unterschiedliche Phasen: eine frühe dendritische Reifung und eine späte funktionelle und synaptische Integration. Neugeborene Körnerzellen zeigten ein rasches dendritisches Auswachsen, dass innerhalb der ersten drei bis vier Wochen abgeschlossen war. Während dieses Wachstumsprozesses passieren Dendriten nacheinander die Körnerzellschicht und anschließend die innere, mittlere und äußere Molekularschicht. Dadurch sind sie innerhalb ihrer morphologischen Entwicklungsphasen anatomisch auf spezifische präsynaptische Partner limitiert. In der wissenschaftlichen Literatur wird eine transiente kritische Phase beschrieben, in der neugeborene Körnerzellen eine starke Plastizität und sensitivere synaptische Erregbarkeit aufweisen. Obwohl die vorliegenden Resultate keine direkten Hinweise auf eine stärkere bzw. sensitivere Plastizität neugeborener Körnerzellen liefern, konnte eine Phase zwischen vier und fünf Wochen identifiziert werden, in der neue Körnerzellen einen sprunghaften Anstieg in ihrer Fähigkeit zur Expression synaptischer Aktivitätsmarker (z.B. Arc und c-fos) und Ausbildung struktureller Plastizität (Dendriten und Dornenfortsätze) zeigten. Die präsentierten Resultate machen deutlich, dass Dornenfortsätze neuer Körnerzellen nach elf Wochen eine vergleichbare Dichte, Größenverteilung und Plastizität aufzeigen, die vergleichbar mit denen vorhandener Körnerzellen sind. Die Fähigkeit zur dendritischen Plastizität nach synaptischer Aktivierung zeigten jedoch nur neugeborene Körnerzellen zwischen der vierten und fünften Woche. Diese Ergebnisse implizieren, dass die Integration neugebildeter Körnerzellen kontinuierlich verläuft und obwohl die vorliegenden Daten die Existenz einer dendritischen Plastizität und einen sprunghaften Anstieg synaptischer Plastizität in der vierten und fünften Woche belegen, wurden keine weiteren Hinweise auf eine transiente kritische Phase gefunden. Des Weiteren zeigten dendritische Bäume von gereiften adult neugeborenen und reifen Körnerzellen Unterschiede, die daraufhin deuten, dass neue Körnerzellen eine eigene Subpopulation darstellen.
Glucose homeostasis is tightly regulated by insulin production from ß-cells and glucagon production from α-cells. Changes in the balance of these hormones lead to Diabetes Mellitus (DM), which is foreseen to be the 7th leading cause of death by 2030, warranting a high demand to identify new therapeutics. DM is characterized by a reduction in ß-cell mass and reduced insulin production from ß-cells. α-cell development and fate mainly depend on the activity of the homeodomain-containing transcription factor Aristaless related homeobox (Arx). Conditional loss- of- function of Arx in α-cells leads to their conversion into functional insulin-producing ß-cells and thus an expansion of ß-cell mass. Therefore, inhibition of Arx is an interesting target for the expansion of ß-cells. The zebrafish model provides a fast, cost-effective and reliable translational platform for drug discovery in an in vivo setting. Here, we screened ~6217 small molecules on a transgenic zebrafish line (TgBAC(arxa:Luc2)) in which the arx promoter drives the expression of the luciferase gene which allows a sensitive and quantitative readout of promoter activity. Small molecule screening allowed us to identify 36 candidate repressors of arxa promoter activity. Furthermore, we started to validate these candidates in other assays. Preliminary results showed that DMAT (a potent CK2 inhibitor) and CNS-1102 (NMDA receptor inhibitor) increase functional ß-cell regeneration. By lineage tracing α-cells during ß-cell regeneration, we could show that both DMAT and CNS-1102 promote α- to ß-cell transdifferentiation. Here, we propose that Casein kinase II and NMDA receptor as potential molecular targets that could be exploited for the treatment of diabetes by generating functional beta-cells from the non-beta-cell progenitor, particularly alpha-cells in situ.