Institut für Biologie

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Analyse des Flow-abhängigen Symmetriebruchs im Frosch Xenopus : die Funktion des Nodal-Inhibitors Coco
(2015) Getwan, Maike; Blum, Martin
The bilaterally symmetrical vertebrate body plan is characterized by the three body axes, anterior-posterior (AP), dorsal-ventral (DV) and the left-right (LR). The LR-axis is the last one to be specified during embryonic development. Its impact on the morphology of the developing organism is visible after a few days in Xenopus laevis, because of the orientation of the visceral organs, such as the heart, gut and the gall bladder. The first molecular differences between the left and right side can already be detected after one day during early neurulation. It is found at the gastrocoel-roof-plate (GRP), a ciliated epithelium which is essential for symmetry breakage. Cilia rotate to produce a leftward fluid movement, which represses the Cerberus/DAN gene Coco in the lateral cells of the epithelium. As Coco acts as an inhibitor of the coexpressed TGFß-type growth factor Nodal (Xnr1), Xnr1 is flow-dependently released from repression on the left side. Xnr1 is capable to induce a unilateral gene-cascade in the left lateral plate mesoderm (LPM) consisting of Nodal itself, its antagonist Lefty/ antivin and the homeobox gene Pitx2c. A central question in this setting concerns the mechanism by which flow results in the repression of Coco. The analysis of Coco transcription gave a first hint, indicating that Coco mRNA is post-transcriptionally degraded and/ or that its translation is blocked. Gene regulation at the level of mRNA usually occurs through the untranslated regions (UTR), in most cases via the 3UTR. To examine the role of the Coco 3UTR for its regulation, protector-RNAs were used which should protect endogenous Coco mRNA from potential inhibitors. Injections led to the interruption of the flow-dependent Coco repression, verifying regulation of Coco via the 3UTR. As 3UTRs are target sites for microRNAs, loss of function experiments of the processing enzyme Dicer were performed. These experiments verified the involvement of miRNAs in the regulation of Coco. Further analyses identified miR-15a as a central player. The interruption of its synthesis or the specific protection of its binding site within the Coco 3UTR prevented flow-dependent down-regulation of Coco. Epistatic experiments demonstrated that the LR-axis of embryos with inhibited flow could be rescued by addition of the miR-15a precursor on the left side. In summary this thesis work revealed miRNAs as a primary target of leftward flow, upstream of the Nodal inhibitor Coco.
Analyse relevanter Signalwege der strahleninduzierten COX-2 Expression in Tumorzellen
(2007) Krebiehl, Guido Klaus; Rodemann, H.-Peter
Summary: Cancer is a health problem worldwide and the number of new cases is rising. Surgery, radiotherapy and chemotherapy are the major treatment modalities. New developments in radiotherapy make radiation alone and in combination with chemotherapy to an important therapy becoming more and more mattering. The success of a therapy often depends on the genetic profile of a tumor. This makes analysis of molecular processes in cells after radiation an important aspect in radiotherapy developing an effective strategy for tumor treatment. COX-2 is overexpressed in a lot of tumors and correlates with a poor prognosis. Moreover COX-2 can be induced by ionizing radiation. This makes COX-2 an interesting molecular target in radiation therapy and in cancer therapy in general. Studies with specific COX-2 inhibitors came to different results in different cell lines. The aim of the presented study was to investigate the survival and the proliferation of prostate cancer cells after treatment with ionizing radiation alone and in combination with specific COX-2 inhibitor Celecoxib and the analysis of signaling pathways leading to radiation induced COX-2 expression. The following major results were obtained: 1. Treatment with Celecoxib had no influence on the radiosensitivity of the prostate cancer cell lines investigated. 2. The proliferation of different cell lines was inhibited by the treatment with Celecoxib. 3. The inhibition of the proliferation seems to be independent of the level of COX-2 of the cell lines. 4. Apoptosis can not be induced by Celecoxib in clinical relevant doses in the cell lines investigated. 5. Induction of COX-2 expression by ionizing radiation depends on the cell line investigated. 6. The MAPK-signaling pathways play a major role at COX-2 expression. In conclusion the results of the presented study indicate that COX-2 can be an important molecular target in radiation therapy. Although this depends on the cell line investigated. As well, the signaling pathways leading to a radiation induced expression of COX-2 are individual for each cell line. Thus the application of Celecoxib during radiation therapy can be positive on the treatment of different tumors.
Axes determination in the frog Xenopus laevis : the function of the goosecoid, myo1d and dmrt2
(2020) Tingler, Melanie Bianca; Schweickert, Axel
During early embryogenesis, pattern formation processes along the head-trunk (anteroposterior, AP), belly-back (dorsoventral, DV) and left-right (LR) body axis generate the fundamental body plan of the bilateria. The formation of the LR axis is exceptional because externally our body is bilateral symmetric whereas most inner organs are shaped and positioned asymmetrically. The three body axes are basically specified during gastrulation and neurulation by a set of developmental control genes. The aim of this work was to analyze the function of the highly conserved genes, goosecoid (gsc), myosin1d (myo1d) und dmrt2 during body axis determination in Xenopus. The first chapter of this work describes the activity of the homeobox transcription factor Goosecoid during AP- and DV-axis formation. Gsc acts as an autoregulatory transcriptional repressor and importantly is expressed in the Spemann Organizer (SO) of all vertebrate embryos. The SO represents the main dorsal signaling center for primary axis induction, regulates embryonic patterning and cell movements. It is further required for AP i.e. head and trunk development. Transferring of SO or gsc misexpression to ventral half of embryos resultes in secondary axis formation i.e. siamnese twins. However, SO function of Gsc was enigmatic, as gsc mutants showed no defects on early developmental processes what challenged Gsc function in the SO. In this chapter, gsc was characterized by conducting gain of function experiments in the embryonic midline of Xenopus embryos. Gsc was able to repress planar cell polarity (PCP) in a cell- and non-cell autonomous fashion leading to neural tube closure defects. In the early gastrulae, Gsc separates the head from the trunk mesoderm by repressing the mesodermal t-box gene transcription factor T (Tbxt). This inhibition allows the migration of the head mesodermal cells whereas the trunk notochord elongates by mediolateral intercalation. Gsc activity on PCP signaling seems to be specific for vertebrates only and correlates with the presence of two novel domains. The determination of the LR body axis is discussed in the second chapter of this work. At the so called left-right organizer (LRO) a cilia-mediated leftward-fluid flow initiates the symmetry breaking event in neurulae embryos. Lateral sensory cells (sLRO) of the LRO perceive flow on the left side and translate it into the left asymmetric induction of the highly conserved Nodal cascade. If and how the unconventional, actin-associated motor protein Myosin1d (Myo1d) as well as the transcription factor Doublesex and mab-3 related 2 (Dmrt2) intervene in LR specification was analyzed in this chapter. In evolutionary terms the study of myo1d was of high interest because in Drospohila, which lacks a ciliary flow mechanism, the homologous gene, myo31df, controls LR axis determination. Manipulations of myo1d in Xenopus demonstrated that in vertebrates Myo1d is involved in the cilia-based symmetry breakage event. By interacting with the PCP signaling pathway, Myo1d ensures leftward-fluid flow by regulating ciliary outgrowth and polarization. In Drosophila and Xenopus Myo1d interacts with PCP signaling and seems to link an ancestral symmetry breaking mechanism of the fly to the newly evolved leftward-fluid flow in vertebrates. Based on studies in zebrafish, which identified Dmrt2 as another factor involved in LR development and somitogenesis, we started the analysis of dmrt2 in Xenopus. Somitogenesis and laterality determination which on first sight are functionally distinct processes were analyzed in the context of dmrt2 function. In Xenopus, flow-sensing cells are affiliated to the somitic cell lineage and therefor paraxial mesoderm specification is crucial for setting up a functional LRO. Dmrt2 specifies the paraxial mesoderm and especially the sLRO by inducing the myogenic transcription factor myf5 in early gastrulae. This demonstrated for the first time experimentally how somitogenesis and laterality determination are intertwined and describes the genesis of the Xenopus sLRO cells in more detail.
Blood parasites of vangas and other corvoidea on Madagascar
(2022) Magaña Vázquez, Regina; Woog, Friederike; Dinkel, Anke; Mackenstedt, Ute; Musa, Sandrine
Madagascar hosts a great diversity of bird species. This study focuses on the description of the diversity and prevalence of blood parasites (Haemosporida, trypanosomes and filarioid nematodes) in 131 blood samples of 14 species of Corvoidea, namely vangas (Vangidae), Coracina cinerea (Campephagidae), Dicrurus forficatus (Dicruridae) and Terpsiphone mutata (Monarchidae) found in primary rainforests on Madagascar. Blood parasites were detected using both molecular and microscopic methods. Multiplex PCR was used to detect mixed haemosporidian infections and nested PCR was used to describe a 479 bp fragment of the haemosporidian cytochrome b (cytb) gene. Furthermore, a 770 bp SSU rRNA fragment of trypanosomes, and, for microfilariae, a 690 bp fragment of 28S rRNA, as well as a 770 bp fragment of 28S rRNA, were amplified for identification using nested PCRs. Phylogenetic analyses were carried out for all sequences obtained from all blood parasite taxa. Over half of the samples (54.2%; n = 71) were infected with Haemosporida, whereas only 21.4% (n = 28) were infected with Trypanosoma and 5.3% (n = 7) contained filarioid nematode DNA. Fourteen of 56 blood smears contained some of the above-mentioned parasite taxa. The results corroborate the great diversity of blood parasites in the different bird species studied, especially in vangas. Vangas had the greatest diversity of parasites found, as well as the highest number of multiple infections, which may be due to their morphological diversity and resulting habitat use. Fifteen haemosporidian lineages, seven Trypanosoma and five filarioid nematode isolates were newly discovered in the avian species studied, particularly in the vangas. Members of the other Corvoidea families on Madagascar showed a lower susceptibility for avian haemosporidian parasites than vangas, which could be attributed to possible resistance against those parasites. The study confirmed the host specificity of some Haemosporida and microfilariae; however, it demonstrated that this was not the case for Trypanosoma.
Causes and consequences of alterations in stress physiology, immunity and oxygen delivery in a small mammalian hibernator
(2017) Havenstein, Nadine; Fietz, Joanna
The different functions and activities of an organism require substantial amounts of energy and thus compete for the limited available resources. During demanding situations, allocation decisions potentially result in trade-offs between physiological processes that can have consequences on the performance and fitness of an individual. Endocrine mediators, especially glucocorticoid hormones (GCs) build the bridge between environmental stimuli and the responses of an individual via regulating energy allocation and numerous other physiological processes as well as behavior and are therefore of special interest in ecophysiological studies. The immune and the oxygen delivery system represent two vital body functions that are essential for survival and respond sensitively to altered environmental conditions, nutrient deficiencies and stress hormone levels, representing therefore further suitable targets of investigation in ecophysiological studies. Our study species, the edible dormouse (Glis glis) is a small arboreal rodent characterized by an extraordinarily long hibernation period. The aim of this study was to elucidate physiological mechanisms underlying the evolution of life history strategies and variations in fitness parameters associated with stressful and demanding situations like hibernation, reproduction, limited food availability and high population density. To achieve these goals urinary GC levels as well as white and red blood cell (WBC and RBC) parameters were investigated and urine samples were examined for haemoglobinuria of free ranging edible dormice in South Western Germany. Results of this study reveal that the post-hibernation period represents an extremely challenging period for edible dormice as their phagocytic cells, the immunological first line of defense, obviously become depleted during the extended hibernation period and recover only slowly at the beginning of the active season. The need to invest into the restoration of regressed organs and body functions directly after hibernation when high quality food is still limited, may explain the delayed recovery of these innate immune cells. Slightly elevated cortisol levels presumably reflect the mobilization of energy from body stores for these restoration processes. As the phenomenon of low phagocyte counts was even more pronounced at the beginning of a low food year and the few neutrophils present in the blood of dormice were primarily immature, preparatory mechanisms occurring during late arousals of the hibernation period seem to determine the regeneration of phagocytes before hibernation is terminated. This, in turn, indicates that dormice are able to predict upcoming food availability and, consequently, future reproductive effort and accordingly invest into physical-physiological recovery. Apparently edible dormice trade off restoration of regressed organs for immunity. Survival probabilities of edible dormice are lowest at that time of the annual cycle, suggesting that this post-hibernation impairment of the innate immune system may entail detrimental effects for their fitness. Elevated cortisol levels during mating and gestation-lactation, respectively, show that reproduction represents a stressful life history event in both sexes. This event furthermore coincides with drastic increases in the ratio of phagocyte to lymphocyte counts (P/L ratio), a stress response of the immune system, as well as distinct impairments in the oxygen delivery system. The latter seems to be in a large part due to energetic and nutrient deficits and large amounts of senescent RBCs. High frequency of haemoglobinuria in females support the notion of a nutrient-deficient anaemia. When all reproductive effort has ended, cortisol levels decrease whereas the strong increases in the P/L ratio persist until the end of the active season which gives notice of the prolonged immunological effects of chronic stress. As mortality is increased during reproductive years, the high cortisol levels measured during reproduction suggest an allostatic overload that has exceeded adaptive levels and the high P/L ratios might represent a pivotal indicator for the beginning of a stress-induced deprivation of immunity that contribute to reduced survival. Prolonged food limitation does not elevate cortisol levels and P/L ratios, supporting the hypothesis that food availability is predictable for edible dormice and that a restriction therefore does not cause considerable stress. During late summer of a low food year the oxygen delivery capacity is decreased. However, erythrocyte parameters furthermore indicate a senescent RBC pool, suggesting that a reduced erythrocyte production is part of the energy saving strategy. As survival is increased during years of low food availability, these findings indicate that dormice are able to perfectly adapt to prolonged periods of limited food availability.
Deskriptive und funktionelle Analyse der Mitglieder der Calponin-Genfamilie Xclp1, Xclp2 und Xclp3 während der Embryonalentwicklung von Xenopus laevis
(2008) Schmalholz, Silke; Blum, Martin
The embryonic development of vertebrates is characterized by controlled cell movements. During gastrulation and neurulation cells of the presumptive heart tissue and the neural crest after neural tube closure migrate towards their final position in the embryo. Cell intercalations, which drive the convergent extension (CE) movements to elongate the embryo also depend on active cell migration. The inhibition of CE leads to shortened body axis and neural tube closure defects (NTD). The motility of eukaryotic cells is finally based on the dynamic interaction of cytoskeletal components, which act on the actin filament. Secreted growthfactors of the Wnt family can regulate embryonic cell movement via the non canonical Wnt signaling pathways. The planar cell polarity (PCP) and the Wnt/Ca2+ pathway are thought to be crucial for the process of CE. Up to now there is a lack of knowledge about the cytoskeletal effectors of these signaling cascades. In the presented work, members of the calponin gene family (clp1 to 3) were analysed in this context. Calponins are actin binding proteins, which have been shown to inhibit actin-myosin-interactions and/or to stabilize the actin filament. Expression patterns provided first insights in the transcriptional activity of Xclp1, Xclp2 and Xclp3 during embryonic development. Two Xclp genes (Xclp2 and Xclp3) were already expressed broadly at the onset of gastrulation. Transcription, however, was not detected in the involuted cells, which form the mesodermal germlayer. At neurula stages Xclp2 mRNA was specifically found in the notochord, whereas Xclp3 was expressed in the neuroectoderm. Additionally the migrating cells of the embryonic heart and neural crest were positive for calponin expression. In summary the embryonic calponin pattern correlated with tissues in which cell movements occur. Over- or misexpression experiments were performed to manipulate embryonic calponin function in Xenopus laevis. Gain of function experiments however did not interfere with embryonic development. Probably calponin function was posttranslational negatively regulated in these experiments.The overexpression of calponin proteins, in which specific phosphorylation sites were mutated or known regulatory calponin domains deleted, again didn´t result in altered phenotypes. However, the misexpression of calponin actin binding domaine 2 (ABD2) inhibited the migration of Krox 20 positive neural crest cells, suggesting that in this tissue the Xclp ABD2 acts dominant negative. The presented data are not able to proof or disproof the hypothesis, that calponin proteins are effectors of the non canonical Wnt pathways.
DNA barcoding resolves quantitative multi‐trophic interaction networks and reveals pest species in trap nests
(2023) Fornoff, Felix; Halla, Wenzel; Geiger, Sarah; Klein, Alexandra‐Maria; Sann, Manuela
Insects, as one of the most species‐rich taxa with enormous taxonomic, behavioural and functional diversity, are in decline. Bees and wasps are especially crucial for ecosystems as pollinators or to control populations of other insects. To understand population drivers, comprehensive knowledge about top‐down and bottom‐up interactions, including all interaction partners, is needed. Nests of trap‐nesting bees and wasps include multi‐trophic interactions between bees, wasps, their food resources and natural enemies, simultaneously, however, up to today, all trophic interactions are not yet included in trap nest research because of challenges to identify the food used by nesting bees and wasps. Here, we reconstructed quantitative three‐ and four‐trophic interaction networks of species in three apoid wasp families using DNA barcoding. The obtained tripartite and quadripartite networks encompassed natural enemy‐wasp‐spider and natural enemy‐wasp‐herbivore‐plant interactions. Moreover, we identified so far undescribed Hymenoptera‐prey interactions, including prey species known as agricultural and forest pests. More extensive research on bee and wasp multitrophic interaction networks will provide valuable insights to better understand responses to environmental and biodiversity change, to investigate ecological theory and to reveal so far unknown feeding links.
Elucidating the megadiversity of Chalcidoidea (Hymenoptera) with a multi-taxonomic approach
(2022) Haas, Michael; Krogmann, Lars
With over 22,500 described and up to 500,000 estimated species, the jewel wasps (Chalcidoidea: Hymenoptera) are among the most species-rich insect lineages. Their evolutionary success is tightly linked to their parasitoid biology, having evolved to utilize a wide array of different arthropod hosts. Additionally, secondary phytophagy evolved several times within this superfamily. Although new approaches are employed in integrative taxonomic research, progress to decipher the megadiversity of this taxon, including their evolution, is still limited. With this work, the diversity of the superfamily is studied at two evolutionary key points in time. The evolutionary origin of Chalcidoidea is investigated in the Cretaceous and the resulting diversity since then is examined in the present. Different systematic levels will be elucidated with the help of integrative taxonomic methods. In the first chapter, the fossil origins of jewel wasps are addressed, around the middle of the Cretaceous period 110 million years ago. The morphology of a putative early chalcidoid specimen is studied, as it is highly informative for chalcidoid evolution due to its age. Based on those results, its phylogenetic placement is critically examined. The specimen is assumed to be one of the oldest described chalcidoid fossils, Parviformosus wohlrabeae Barling et al., 2013. It is a key fossil because of its age and putative assignment to the polyphyletic family Pteromalidae and could therefore be a valuable voucher for dating modern phylogenies. A precise redescription of the fossil was conducted and its morphology and phylogenetic position was discussed. No synapomorphic characters could be identified, warranting an inclusion in an already established chalcidoid family. In fact, none of the autapomorphies for Chalcidoidea could be recognized, necessitating a revised systematic placement in the Proctotrupomorpha. In the second chapter, several fossils in amber are described that grant insights in the early evolution of Chalcidoidea and the morphological diversity of Cretaceous lineages. Morphological characters are studied to answer the question of plesiomorphic character states in Chalcidoidea, aiding to understand their early evolution. The phylogenetic placement of these fossils is discussed, to provide hypotheses on the diversification of the superfamily, which so far has only few fossil representatives described from this time. Four fossils are made scientifically available that were found in 99 million year old Burmese amber. Those specimens are described in a new, extinct family, the Diversinitidae. This family exhibits a unique combination of plesiomorphic characters, not present in any other chalcidoid taxon, but lacks apomorphic characters. In total, three new genera and three new species are delimited and described. Phylogenetically relevant characters like the fully developed funicular segments, possessing multiporous plate sensilla, or the peg like cerci that improve our understanding of the early evolution of Chalcidoidea, are discussed based on the newly established family. A phylogenetic analysis based on morphological characters was performed. This analysis supported the monophyly of Diversinitidae, but left its exact systematic position within Chalcidoidea open. In the third chapter the focus shifts from the early evolution of Chalcidoidea towards the extant fauna, representing the diversity evolved since the Cretaceous. Exemplary, in the speciose family Pteromalidae the unknown diversity is examined to better understand the undiscovered species richness of parasitoid wasps. DNA barcoding is used to record and help identify previously unknown genera and species in Germany. Compared to the already known pteromalid fauna, 17 genera and 41 species are added as new records for Germany and the males of two species are described anew. The identified DNA barcodes were made available to enable the genetic identification of those species that have a high potential as indicators for nature conservation efforts due to their high host specificity. In the fourth chapter, the pertinent problem of cryptic diversity in Chalcidoidea is investigated. Via an extensive integrative taxonomic approach, the morphological species hypothesis is tested for one of the most abundant pteromalid species in Europe, Spintherus dubius. In this example, the benefit of combining different methods for species discovery and delimitation is highlighted. Genetic analyses of S. dubius reveal discrepancies between the morphological species concept and molecular data, indicating two potential species instead of one. The usage of an advanced morphological method, the multivariate ratio analysis, results in a confirmation of the molecular results, also exposing distinctive morphological characters per taxonomic unit. The examination of the host spectrum through rearing experiments further substantiates these findings, by revealing different host parasitoid affiliations. Altogether, this thesis showed that it is necessary to combine methods and examine different evolutionary points in time, to better understand the diversity of parasitoid lineages. Fossil taxa are important study subjects to examine the character evolution of any taxon, laying the base for phylogenetic research. The study of Diversinitidae highlights the plasticity of character states in Chalcidoidea, also providing evidence for plesiomorphic states. Their encompassing description and the redescription of P. wohlrabeae allow their incorporation into phylogenetic studies, to serve as solid anchor points in dating lineages and morphological evolution on the way towards extant diversity. Examining the extant fauna of Pteromalidae revealed the amount of diversity of species, for which the biology is often unknown. It is shown that molecular methods aid in the discovery of this diversity, opening possibilities for further research. It is affirmed that hidden diversity is even pertinent in abundant, well known species, with S. dubius being an example of cryptic diversity unveiled by integrative taxonomy.
Epidemiologie von Coxiella burnetii, Rickettsia spp., FSME- und Hantaviren in Süddeutschland unter Berücksichtigung klimatischer Veränderungen
(2011) Pluta, Silvia; Mackenstedt, Ute
Zoonoses, which are defined as human infections caused by animal pathogens, are of considerable medical importance. Therefore, it is important to evaluate the distribution of tick- and rodent borne pathogens and the risk of infections for humans. Hence, the epidemiology of specific human pathogens (Coxiella burnetii, Rickettsia spp., TBE-virus and Hantavirus) was studied. As climatic conditions are known to have a significant influence on ticks and rodents, the role of climate change in the epidemiology of the examined pathogens was considered. The role of natural foci in the epidemiology of Coxiella burnetii, the causative agent of Q fever, is widely unknown in Germany. Therefore, ticks of the genus Dermacentor and rodents from Q fever endemic areas in Southern Germany were examined for infections with C. burnetii. Altogether, 1120 ticks and 119 rodents were tested by PCR. However, no infections were detected. Rodent blood samples were additionally tested for antibodies against coxiellae, but likewise, all samples were found to be negative. Hence, no natural foci of Q fever were identified in the examined areas. A role of natural foci in the epidemiology of Q fever in Southern Germany is therefore unlikely. Both Dermacentor marginatus and D. reticulatus act as vectors for Rickettsia spp. To elucidate if human pathogenic rickettsiae are distributed in Southern Germany, Dermacentor ticks were investigated for infections by PCR. Moreover, rodents were screened by molecular and serological methods to identify potential reservoir hosts. The overall prevalence of Rickettsia spp. in 1120 ticks was 33.6%. By sequencing of the rOmpA-gene, R. raoultii was identified in 32.8% and R. slovaca in 0.8% of all examined ticks. Both species are pathogenic for humans; thus, there might be a considerable risk of infection. This was confirmed by the detection of an autochthonous case of R. slovaca-infection in Rhineland-Palatinate. Rickettsia spp. was not detected in any rodents by PCR. However, 48 animals (20.2%), muridae as well as arvicolidae, showed antibodies against rickettsiae. It was, therefore, shown that rodents can actually act as reservoir hosts for Rickettsia spp. To determine the current prevalence of TBE virus in ticks, 7842 Ixodes ricinus from Baden-Württemberg were examined by molecular methods. TBE virus was found in four out of 11 examined areas. Prevalences ranged from 0.13% to 0.66%. No infected ticks were found in other areas. The determined prevalences were lower compared to studies from the 1990ies in the same areas. However, reliable comparisons are difficult due to the low numbers of infected ticks. The viruses of ticks from Gengenbach, Hagnau am Bodensee and Hödinger Tobel were cultured and subsequently characterized by sequencing of the E and NS2a genes. Phylogenetic studies were conducted to identify the TBE virus strains present in Baden-Württemberg. The isolate from Hagnau am Bodensee was identified as strain Salem, whereas the isolates of the other two areas differed substantially from all strains found in the database. Therefore, no identification was possible for these isolates. In 2007, the number of notified hantavirus infections exceeded by far the average case numbers of the preceding years. To determine if the steep rise in human infections is associated with an unusual high virus prevalence in reservoir hosts, red bank voles from the endemic area of the Swabian Albs were examined for infections by molecular and serological methods. The overall prevalence of Puumala hantavirus was 57.9%. In a study in 2001 in the same area, the prevalence was only 10%. Therefore, there was a sixfold rise in virus prevalence which reflects the high number of human cases in the study year. Sequence comparisons showed a close relationship of the hantaviruses from the Swabian Alb to hantaviruses detected in other areas of Baden-Württemberg. The results presented in this study provide the basis for further continuous studies. These are necessary to estimate the actual influence of changes in climate conditions on the examined zoonoses and, thereby, the prospective infection risk for humans.
Establishment of a new in vitro culture system and functional analysis of sonic hedgehog and FGF8 in the determination of laterality in the rabbit embryo
(2008) Bitzer, Eva; Blum, Martin
Cilia-driven leftward flow plays a pivotal role in the determination of left-right (LR) asymmetry. In mammals, this extracellular fluid flow is produced by motile monocilia situated on the posterior notochordal plate (PNC). The PNC is homologous to superficial mesoderm derived structures of other species like the gastrocoel roof plate (GRP) in frog and Kupffer's vesicle (KV) in fish. Directional fluid flow created at these structures subsequently leads to the initiation of the left-specifying Nodal signalling cascade in the left lateral plate (LPM). The rabbit develops via a flat blastodisc phase representing the archetypical mode of mammalian embryogenesis. These specific advantages of the rabbit were employed in this study to further examine the role of two central determinants of laterality, namely Sonic hedgehog (Shh) and FGF8, and also extended by the design of a new in vitro culture technique. In this new method, the so-called ring culture, a medium-filled plastic ring was placed upon the extraembryonic tissue of the explanted embryo. In contrast to the semi-dry standard culture method, this setting corresponded more to the in vivo conditions of gastrulating/neurulating rabbit embryos in the uterus. It therefore facilitated stable development of laterality in most cases also when presomite stages were taken into culture. Subsequent analysis showed that this was due to improved development of the PNC. Conversely, in standard-cultured embryos showing altered LR marker gene expression, maturation of the PNC was impaired leading to a disturbance of leftward flow. This study also provided evidence that cilia-driven leftward flow is indispensable for the determination of laterality in rabbit embryos. When the flow was blocked during culture by methylcellulose-containing medium embryos displayed altered LR marker gene expression in a very high proportion. The unilateral gain-of-function of Shh revealed important differences between rabbit and chick embryos. In rabbit, Shh induced right-sided marker gene expression only in the 2 somite stage, whereas in chick this inductive effect lasted from stage 4 until up to the 1-2 somite stage. This indicated that in rabbit Shh works in conjunction with the flow, which has not been described up to now in chick. The systemic inhibition of Shh signalling by cyclopamine led to bilateral expression of LR marker genes in rabbit. This was due to disruption of the floor plate and therefore the loss of the restrictive midline barrier function of Lefty expression as described in Shh mutant mice. FGF8 has a right-sided repressive function in the rabbit implicated in the transfer of laterality cues. In the present study it could be shown that this repressive effect is epistatic to cilia-driven leftward flow, because it also functioned when the flow was blocked. The systemic inhibition of FGF8 signalling with SU5402 caused loss of LR marker gene expression prior to the 2 somite stage but did not influence ciliogenesis or the setup of cilia-driven leftward flow. Taken together, this suggested a dual function for FGF8 signalling: First, it is needed to confer competence to the lateral plate and second, during the 2 somite stage, it is needed for the transfer of LR cues.
Establishment of the body axes in Xenopus laevis through goosecoid, myosin 1d and bicaudal c
(2021) Maerker, Markus Ferdinand; Schweickert, Axel
The bilaterian body plan consists of three body axes: the anteroposterior (AP; head-trunk/tail), the dorsoventral (DV; back-belly) and the left-right (LR; placement of inner organs) axis. Axis formation occurs during early embryogenesis and is critical for further development and viability of the embryo. In this comprehensive study three highly conserved determinants were functionally analyzed in the context of axis development. The first chapter of this work covers the autoregulatory, homeodomain containing, repressor gene goosecoid (gsc), whose most prominent expression marks the Spemann-(Mangold) organizer (SO). The SO is the primary dorsal signaling center and is instructive for tissue patterning along the DV and AP axes. Transplanting the SO or misexpressing gsc on the opposite ventral side of an embryo is sufficient to establish a new/secondary AP axis. However, its function during normal development in the SO remained enigmatic as the gsc loss of function (LOF) lead to no severe early developmental defects. To elucidate the function of gsc, timed gain of function (GOF) experiments were performed. Gsc efficiently repressed the planar cell polarity (PCP)/Wnt signaling pathway leading to severe gastrulation and neurulation defects. This novel Gsc function was correlated with two vertebrate specific domains, suggesting an evolutionary new function of Gsc with the emergence of jaws/neural crests in vertebrates. The second chapter of this study addresses the functions of Myosin1d (Myo1d) and Bicaudal c1 (Bicc1) during the LR axis determination in vertebrates. In this group LR symmetry breakage takes place at a ciliated epithelium called LR organizer (LRO). The initial cue for the asymmetric LR axis development is a cilia-driven leftward fluid flow. These cilia have to be correctly polarized through PCP/Wnt signaling. Interestingly, the invertebrate Drosophila melanogaster also displays a distinct LR axis but uses a cilia independent, yet not fully understood, mechanism. It depends on a myo1d homologous gene, myo31DF, and PCP. To unravel a potential common evolutionary origin of the bilaterian LR axis myo1d was analyzed during Xenopus laevis lateralization. Myo1d LOF experiments disturbed LR axis formation by compromising PCP dependent outgrowth and polarization of LRO cilia. These experiments link the PCP/Myosin based mechanism of flies to the newly evolved cilia/flow dependent mode of vertebrate LR axis determination suggesting actomyosin as common ancestral LR determinant. Contrary to Myo1d, Bicc1 was already described for its function during polarization of flow producing LRO cilia. However bicc1s expression is most prominent in the sensory LRO cells (sLRO). These cells detect the fluid flow and translate it into left-sided signaling of the morphogen Nodal1 and consequently asymmetric LR axis formation. These cells downregulate the expression of the secreted Nodal1 antagonist DAN domain family member 5 (dand5) in response to flow. Bicc1s function was re-evaluated with respect to its function in sLRO cells. Ex vivo and in vivo experiments involving GOF as well as LOF experiments showed that Bicc1 regulates both dand5 and nodal1 via a direct and indirect post-transcriptional mechanism, respectively. In the process of dand5 regulation several other LR determinants and regulatory events were linked with the Bicc1 dependent mechanism: Dicer1 dependent microRNA repression of dand5 and a proposed cation channel Polycystin 2 mediated Bicc1 modification. These results highlight the importance of a tightly controlled Dand5 protein level as decisive for the overall outcome of the LR symmetry breakage in vertebrates.
Funktionelle Analyse der Gene Brachyury, Goosecoid und Myosin1d für die frühe Musterbildung und Etablierung der Körperachsen während der Embryogenese des Krallenfroschs Xenopus laevis
(2019) Kurz, Sabrina; Blum, Martin
During a fundamental phase of vertebrate embryonic development, gastrulation, the reorganization of the future body plan, is determined. This process controls the embryonic patterning of the antero-posterior (AP) and dorso-ventral (DV) axes, as well as the internal left-right (LR) axis. The establishment of the body axes is initially controlled by the dorsally localized Spemann organizer (SO), whereby the determination of the LR axis takes place the latest. The formation of laterality during subsequent neurulation is executed by a highly conserved mechanism of symmetry breakage within fishes, amphibians and mammals. An extracellular, monocilia-driven fluid-flow („Flow") eventually defines the laterality of the embryo, by left-asymmetric gene expression of the morphogen Nodal. Responsible for the Flow is a ciliated epithelium in the postero-dorsal region of the embryo, which undergoes morphological development and correct positioning during gastrulation and is termed GRP („gastrocoel roof plate") in the frog. Ultimately, this conserved tissue describes the left-right organizer (LRO) and guarantees proper organ situs. The origin and function of the LRO can be traced back to an epithelial organized cell structure on the dorsal surface of the gastrula, characterized by the expression of the canonical Wnt target gene and cilia marker foxj1. Following the specification of this Superficial Mesoderm (SM), the future cells of the LRO invaginate in a directed fashion during gastrulation. The patterns of cellular movement require a restructuring of the actin-cytoskeleton. These migrations, executing the future body plan, are ensured by the PCP- („planar cell polarity”) signaling pathway. This non-canonical Wnt pathway guarantees polarized alignment and migration of cells along the body axes, namely convergent extension (CE). As an organizer gene, the expression of Goosecoid (Gsc) characterizes SO and its’ properties. Contrary to the assumption that a Gsc loss of function thus inhibits gastrulation, no visible impairment was detected in the Knock-Out mouse and Knock-Down in Xenopus. Gain-of-function in the frog, which analyzed the role of Gsc during gastrulation finally demonstrated the homeobox-gene exerting a function in regulating cell movements. Overexpression resulted in impaired CE of dorsal tissue due to defective localization of nuclear proteins of the PCP signaling pathway. Gsc-induced malformations could be compensated by co-injections of associated components. In conclusion, a new function as an inhibitor of PCP-dependent CE during gastrulation was suggested. The morphogenetic movements of CE are responsible for the AP-elongation and LRO-positioning. Both, mechanical forces and cilia-based Flow functionally interact for that matter. In the invertebrate Drosophila, where neither Nodal nor cilia are expressed, organ asymmetry is ensured by internal chirality conducted via motor proteins, such as myosin1d, in a PCP-dependent manner. Similarly, in vertebrates such as Xenopus, myo1d mediates interactions of the actin cytoskeleton asymmetrically, that guarantee laterality of the organ system. The functional preservation of the non-canonical Wnt pathway could be demonstrated by co-injections of PCP core proteins, being able to restore disturbed LRO morphology. The obtained data clearly demonstrated the evolutionary, interspecific, regulation of axis asymmetry by myo1d. Since the strict spatio-temporal regulation of cell movements is fundamental for patterning, CE is propagated by another transcription factor: Brachyury (Tbxt in Xenopus). Its expression induces and controls differentiation of mesodermal cell populations, such as the notochordal cells of the dorsal midline, hence LRO. The necessity specifying progenitor cells in the SM could also be highlighted, showing to be mediated non-cell autonomously to guarantee the induction of foxj1. Additionally, the determination of both tissues is exerted by functional interactions of Tbxt with either PCP-, as well as ß-catenin-dependent Wnt signaling pathway. Loss of Brachyury affects laterality in mutants and morphants. The species-spanning Brachyury-FGF „feedback-loop" for the induction of Nodal and Foxj1 could already act in the SM and showed that function as well as the process of LR development, as suggested between Fgf8 and Brachyury, to be functionally conserved. In Xenopus, signal transduction for SM and foxj1 induction was extended by the function of the Wnt receptor frizzled-8, the ligand and Tbxt target gene wnt11b, and the ventrally acting wnt8a in Whole-Mount embryos and explant co-cultures.
Genetische Diversität von Echinococcus granulosus sensu stricto
(2017) Ebi, Dennis; Mackenstedt, Ute
The dog tapeworm Echinococcus granulosus sensu stricto, distributed worldwide, is the most important agent of human Cystic Echinococcosis, a zoonotic disease which is considered as one of the priority ‘neglected zoonotic diseases’ by the World Health Organisation. The parasite is known from a large number of host species and is conventionally subdivided into several genotypes. The epidemiological consequences of the variation, however, are still unknown. In the last years several studies on the intraspecific variation of this parasite were published. Most of these studies were based on small numbers of isolates from geographically restricted regions, so that the relevance of that diversity concerning pathogenicity, host specificity, transmission cycles and biogeography is still little understood. To achieve a comprehensive estimate of its genetic diversity, 1085 isolates of E. granulosus s.s. from humans and animal hosts originating from widely different regions of the world were analyzed for the sequence polymorphism of a 1609 bp fragment of the mitochondrial cox1 gene. For analysis haplotype networks, diversity and neutrality indices of subpopulations of different geographic origin were compared. To improve the analytical efficiency of the sequence-data, a LabVIEW application was written. It could be shown that most diverse sub-population is found in the Middle Eastern region and that the genetic diversity is lower in the other analyzed regions, which confirms a previous tentative hypothesis on the origin of the domestic cycle of E. granulosus s.s. in that region. Records of identical haplotypes in different regions, inclusion of published data, calculation of parameters of population genetics and haplotype network analysis led to hypotheses explaining the distribution history of E. granulosus s.s.. This in line with fossil and molecular data on domestic sheep as the principal final host. A set of new and easily calculable genetic diversity indices were developed and their added value was explained. Additionally, a statistical resampling experiment was programmed which showed that at least 40 well distributed samples are necessary to provide a reliable estimate of the genetic diversity of the parasite in a region. Using an extension for that software, it was shown that the newly developed diversity indices do not strongly depend on the number of analyzed samples, and provide appropriate tools to assess the genetic diversity of parasite populations.
Habitat use and morphological adaptations of endemic rodents (Muroidea: Nesomyinae) of East Madagascar
(2014) Marquart, Kathrin; Steidle, Johannes
In an adaptive radiation, Madagascar’s endemic rodents colonised a variety of different habitats. In this thesis, ecological and morphological aspects of the frequently sympatric nesomyines of East Madagascar were analysed. In the first part of the study, vegetation analyses were conducted in a model rainforest in East Madagascar and compared with species distribution to describe the species ecology. The results represent a preliminary ecological classification of the habitat use of nesomyines. In the second part of the study, the morphology of the hands and feet (chiridia) was correlated with ecological parameters and habitat use for the first time. Adaptive differentiations of the chiridia give evidence of a species’ ecological niche. Certain patterns of the volar integument, epidermal structures as well as length and area ratios are discussed in respect of their functional and ecological adaptive value. Seven endemic rodent species (Eliurus grandidieri, E. minor, E. tanala, E. webbi, Nesomys rufus, Gymnuromys roberti, Brachytarsomys albicauda) were trapped with live traps in different microhabitats. Ecological data and information on habitat structures were collected along five 1 km long trails. The specialised rodents were found in different ecological niches: on trees, lianas, underneath wood and thick roots, in dead wood or rotten logs. Some were found close to small rivers, some in humid valleys, whereas others seemed to prefer the higher mountain slopes or misty ridges, up to 1200 m a.s.l. Microhabitat analyses were conducted and trails where a species was significantly more abundant were compared with those where it was rare. The presence of an endemic rodent species was strongly connected with forest type, forest structure and vegetation density. In contrast, the deforested and cleared areas were species-poor. For the morphological studies, detailed microscopic drawings of the hands and feet were prepared of voucher specimens to be compared between the species. The chiridian structures differed clearly between the seven species. Slender hind feet with small thenar pads were significantly correlated with a terrestrial locomotion and a ground-dwelling ecology. Species with a predominantly arboreal locomotion exhibit large and prominent pads, covering a great area of the chiridian surface. Large-tree-climbers have larger pads in relation to foot size than bamboo-grass and small-branch-climbers which possess relatively longer feet with longer toes. Climbing on large trees needs good adhesive properties and climbing on grass or thin branches needs good grasping abilities. In conclusion, certain morphological structures of the rodents chiridia are evidence of a special way of life and foot-pad morphology in particular mirrors special adaptations to a species habitat. These adaptations are regarded as important to understand niche occupation, certain ecological aspects and locomotion forms of sympatric species. Thus, habitat preferences and special habitat requirements of the endemic nesomyines of East Madagascar could be analysed in details. In this thesis, it could be documented for the first time by the combination of these ecological and morphological results that the chiridian morphology allows to differentiate nesomyine taxa on ecological level.
Identifizierung und Charakterisierung der Succinsemialdehyd-Dehydrogenase aus parasitischen und nichtparasitischen Arthropoden
(2008) Rothacker, Boris; Ilg, Thomas
The objective of the PhD project was the molecular and enzymological characterization of SSADH in parasitic and nonparasitic insects and acarids, organism groups where this enzyme was virtually unexplored. In this project, the list of investigated non-arthropod organisms included the bacterium Escherichia coli (Ec) and the mammal Mus musculus (mouse). Their SSADH served as reference enzymes or were used for biophysical experiments. Amongst the arthropods, Drosophila melanogaster (Dm) (fruit fly) was chosen as nonparasitic model insect. Lucilia cuprina (Lc) (sheep blowfly) and Ctenocephalides felis (Cf) (cat flea) were included as important parasitic insects, as was the acarid Rhipicephalus microplus (Rm) as the economically most important tick species. At the start of this thesis, expressed sequence tag studies and whole genome sequencing on Dm suggested the existence of a single copy gene candidate that, based on amino acid sequence homology, was considered to be a candidate for a SSADH ortholog in this species (DmSSADH). The Dm SSADH gene candidate was cloned and expressed in Ec as a soluble protein. To compare the enzymological properties of DmSSADH, another so far uncharacterized Dm gene candidate for an acetaldehyde dehydrogenase (DmALDH) was cloned and expressed in Ec as a soluble protein. Both expression products showed the expected enzymological properties: a NAD+-dependent ssa-oxidizing activity for DmSSADH and a NAD+-dependent acetaldehyde-oxidizing activity for Dm-ALDH. Site-directed mutageneses on DmSSADH performed in this study demonstrated that two residues essential for catalysis are glutamate 277 and cysteine 311. The second part of the thesis encompassed the gene identification, full length gene cloning of SSADH in Lc (LcSSADH) and Cf (CfSSADH), as well as functional expression of one gene version, respectively. Substrate/cosubstrate specificity determinations combined with enzyme kinetics studies showed that both enzymes are predominantly NAD+-dependent SSADHs. Bioinformatics analyses detected N-terminal mitochondrial import sequences in both Lc and Cf SSADH suggesting that these enzymes are localized in vivo in the mitochondrial matrix. The investigation of the genomic structure of the LcSSADH and CfSSADH genes revealed significant differences to the previously known gene organizations: firstly, different to the single copy SSADH gene situation in Ec, Dm, Mus musculus and Homo sapiens, Lc appears to possess 2-3 SSADH genes, while in Cf up to 8 gene copies may be present. Secondly, compared to Dm (2 exons, 1 intron), the exon/intron structure of the SSADH genes in Lc and Cf is not conserved: the one SSADH genomic gene version investigated in detail in Lc contained 3 exons and 2 introns, while the genomic gene version of Cf analysed in this study was devoid of any introns. The central topic of the third part of this thesis was the gene identification and biochemical characterization of SSADH from the tick Rm. By a combination of different PCR methods a tick gene orthologous to insect and mammalian SSADH (RmSSADH) could be identified and isolated. The results of Southern blot analyses of Rm DNA are incompatible with a single copy situation and suggest the presence of 2-3 RmSSADH genes. To compare RmSSADH with a mammalian SSADH, the respective gene was isolated from mouse. Both the tick and the mouse SSADH genes were then expressed as soluble functional proteins in Ec. The initial comparison showed that both proteins are potent NAD+-dependent ssa-oxidizing enzymes with very similar enzyme kinetics. A more detailed comparison of both enzymes suggests that in general, the mouse enzyme appears to be more specific for succinic semialdehyde than the tick enzyme. In the fourth section of the experimental part of this thesis, saturation transfer difference nuclear magnetic resonance spectroscopy (STD-NMR) experiments were performed on three of the above SSADH preparations, to answer questions on the aldehyde substrate and cosubstrate binding to these enzymes. Importantly, the long-standing question whether the free aldehyde of succinic semialdehyde or its hydrated gem-diol form (present in aqueous solution in equimolar amounts) is the binding substrates was answered conclusively in favour of the aldehyde form for both the Ec and the Dm enzyme. Most remarkably, STD-NMR experimental investigation of the ssa interaction with the Dm SSADH cysteine311alanine mutant enzyme demonstrated binding of both the aldehyde and the gem-diol form. This experiment strongly suggests that cysteine311 is acting as an aldehyde versus gem-diol selectivity filter in the active site of the enzyme. Furthermore, STD-NMR epitope mapping of the NAD+/NADP+ binding to Ec SSADH and Dm SSADH was performed. In both cases, the data suggested that the dominant protein-ligand interactions are via the adenine and the nicotinamide ring systems, while the ribose moieties interact much less intensely with the enzymes.
Influence of ambient temperature on the phenology of the greater mouse‐eared bat (Myotis myotis)
(2023) Matthäus, Laura; Kugelschafter, Karl; Fietz, Joanna
In order to assess the consequences of climate change and evaluate its impacts on wildlife, it is essential to do so on a species-specific level. It is assumed that changes in the ambient temperature influence energy consumption as well as food availability and thus foraging behavior, reproduction, survival, and therefore population dynamics in bats. Based on this assumption, the present study aims to gain insights into the roosting and breeding behavior of the greater mouse-eared bat (Myotis myotis) in relation to changes of the ambient temperature. For this purpose, we investigated the effect of ambient temperature on the phenology of the greater mouse-eared bat by using activity data of the bats collected using light barriers at the maternity roosts. The light barrier used in this study is a system that detects the interruption of two light beams, for example, by a flying bat, and displays it as an electrical signal. The investigations have shown that 1. the higher the winter temperatures, the earlier the greater mouse-eared bats returned to the roosts to form the maternity colony; however, this was only true for ambient temperatures below 0.5°C, 2. birth season started earlier at higher spring temperatures, 3. the dissolution of maternity roosts occurred earlier with earlier birth season and at higher ambient temperatures during lactation. The results revealed that ambient temperature has an influence on the phenology of the greater mouse-eared bat. Depending on the respective life history stage, an increase in ambient temperature can have a positive or negative effect on the fitness of the animals. In recent years, mild winters have been recorded more frequently, which can have an influence on the behavior of bats. Warm winters within certain limits seem to lead to an earlier formation of the maternity colony, which can be positive or negative for the bats depending on persistent weather conditions and thus insect availability. In the course of climate change, we can also expect earlier spring events and an increase in spring temperature, as well as hot spells in summer. These warm springs and summers seem to lead to an earlier beginning of births, a faster development of the juveniles and an earlier dissolution of the maternity roost. An advance of reproductive activities can be assumed to increase the chance to survive the following winter in both mothers and their young, as they have more time to build up sufficient energy reserves for hibernation before winter starts. Due to the climatic changes, phenological changes of the bats be expected. This study highlights that in order to understand the impact of climate change on biodiversity, it is necessary to investigate in detail effects on a species-specific level and also to consider direct and indirect effects of ambient temperature on different life history stages.
Left-right asymmetry in Xenopus laevis : functional dissection of leftward flow
(2009) Vick, Philipp; Blum, Martin
Despite their external bilateral symmetry, vertebrates have a conserved left right (LR) asymmetry of their inner organs. For all vertebrates, it is well-known that the asymmetric organogenesis is preceded by the left-sided nodal signaling cascade during embryonic development. A question which has not been settled in detail is how the first asymmetrically directed signal arises, which activates nodal only on the left side. In mice and fish embryos an extracellular leftward fluid flow ? generated by rotating cilia ? was shown to be functionally necessary for gene activation. Recently, this process has also been demonstrated in frog embryos and its mechanic inhibition caused laterality defects. This raised the question if this process is also conserved among vertebrates. The aim of this study was to analyze the mechanism of flow in the frog in the context of the known models. Thereby, its prerequisites and the exact mode of activation of the left-sided genes should be assessed. Finally, general conclusions on the symmetry breakage of vertebrates were to be drawn. Loss of function of axonemal dynein heavy chains inhibited ciliary movement, fluid flow and laterality development of the embryos. By showing that flow was only necessary on the left half of the ciliated epithelium (GRP), definite statements could be made concerning origin, identity and possibility of a transported substance. Moreover, a function for GRP morphogenesis and thus for the generation of flow were proven for the serotonin receptor 3 and the calcium channel Pkd2. These results did not confirm the hypothesis that Pkd2 causes a flow-dependent left-sided calcium signal. Consequently, this contradicted the so-called "2-cilia model" in favor of an early morphogenetic function in frog. In the course of a collaboration it could be shown, that the RNA-binding protein xBic-C has a conserved function for cilia polarization and thus for the flow in both Xenopus and mice. Additionally, up to now, a right-sided nodal inhibitory function has been assigned to the protein coco. However, the exact mechanism was unknown. By specific, combined left- and right-sided loss of function experiments with coco, nodal and the above mentioned components, it could be demonstrated that coco but not nodal is directly dependent on leftward flow. With the flow, coco was downregulated on the left side only and could thus no longer inhibit nodal there. Loss of flow or xBic-C function ? but not that of Pkd2 ? could be rescued by coco inhibition; this revealed a clear hierarchy. Taken together a sequence of conditions could be formulated: Pkd2 and the serotonin receptor 3 are obligatory for the formation of the GRP and correct flow before neurulation. xBic-C also precedes the flow and is required for cilia polarization but seemed also to have a further function. coco is downstream of the fluid flow and is downregulated as its direct consequence on the left side. nodal, in turn, is downstream of this order and is only released on the left side where it can thus act as a putative mediator to transfer the generated signal into the lateral plate mesoderm. These results are discussed in terms of evolutionary origin and conservation.
Multiple Funktionen des FGF-Signalwegs regulieren die Lateralitätsentwicklung im Krallenfrosch Xenopus
(2013) Schneider, Isabelle; Blum, Martin
Early embryogenesis governs the formation of the three body axis. Like in a cartesian coordinate system, the LR-axis is defined by the generation of the anterior-posterior and the dorso-ventral axis. In the course of laterality specification, the original LR-symmetry has to be broken to enable the asymmetric arrangement of inner organs in a specific manner. This is mediated by the expression of conserved gene cascade, namely the Nodal gene cascade, which is expressed in the left but not in the right lateral plate mesoderm of the neurula stage embryo. Symmetry breakage, which leads up to this asymmetric Nodal gene cascade, is manifested by a cilia-based leftward fluid flow. The flow generating epithelium is located at the posterior end of the notochord and expresses Nodal in a bilateral symmetrical mode. This early Nodal domain is a prerequisite of the later asymmetric Nodal gene cascade. Despite the conserved nature of Nodal expression and of leftward flow, no conservation of the role of the FGF signaling has been described for mouse, chick, rabbit and zebrafish. In this work the role of FGF signaling in Xenopus laevis LR-development was investigated. Using of a receptor antagonist to inhibit FGF signaling revealed two temporally distinguishable functions. Firstly, FGF signaling in early gastrula stages is required for the proper expression of FoxJ1, the master control gene of motile cilia. Here, FGF signaling acts in the process of ciliogenesis of the symmetry-breaking epithelium, which is represented by the GRP (“gastrocoel roof plate”) in Xenopus. Secondly, FGF acts in a cilia-independent manner on the bilateral Nodal expression. A series of descriptive and functional studies revealed that these cells constitute the somitic part of the GRP and that inhibition of FGF signaling leads to the loss of these cells. Interestingly, the effect on ciliogenesis is consistent with the role of FGF signaling in zebrafish, whereas the loss of bilateral Nodal expression is in line with the hypomorpic Fgf8 mutant mouse. The description of these two successive functions in Xenopus indicates a higher degree of conservation of the role of FGF signaling than suggested so far. The FGF signaling pathway splits into several branches, two of which play important roles in the early development of Xenopus embryos. Activation of MAPK signaling is implicated in the induction of mesoderm, whereas the PLC/PKC/Calcium signaling branch impacts on morphogenetic movements. FGF-mediated control of Foxj1 expression was temporally correlated with FGF signaling that acts on mesoderm specification. As a consequence, mesodermal gene expression and blastopore closure was seriously affected by loss of FGF signaling at early gastrula stages. By starting inhibition experiments during gastrula stages, when mesoderm induction is almost finished, general mesoderm specification defects were avoided but the effect on the somitic GRP cells persisted. To unravel which FGF-induced signaling branch acted on the two different functions of FGF described here, the PLC/PKC/Calcium signaling branch was inhibited using the antagonist Sprouty1. Sprouty1 gain of function experiments had no effect on ciliogenesis, but caused loss of somitic GRP cells comparable to loss of function experiments using the FGF receptor antagonist. This suggests that the FGF-dependent formation of these cells is regulated by the PLC/PKC/Calcium pathway. A specific role of Calcium was supported by experiments using a calcium-permeable channel. Despite this, ciliogenesis was not affected by inhibition of PLC/PKC/Calcium, suggesting a role of MAPK for the early function of FGF. In conclusion, this work demonstrates two functions of FGF signaling in Xenopus LR-development, which furthermore are consistent with a conserved function of FGF signaling in vertebrate LR-axis determination. Novel insights into the role of FGF signalling in the very cells which sense leftward flow at the lateral margin of the GRP will open new approaches to analyse laterality specification in more detail.
New species based on the biological species concept within the complex of Lariophagus distinguendus (Hymenoptera, Chalcidoidea, Pteromalidae), a parasitoid of household pests
(2023) Pollmann, Marie; Kuhn, Denise; König, Christian; Homolka, Irmela; Paschke, Sina; Reinisch, Ronja; Schmidt, Anna; Schwabe, Noa; Weber, Justus; Gottlieb, Yuval; Steidle, Johannes Luitpold Maria
The pteromalid parasitoid Lariophagus distinguendus (Foerster) belongs to the Hymenoptera, a megadiverse insect order with high cryptic diversity. It attacks stored product pest beetles in human storage facilities. Recently, it has been shown to consist of two separate species. To further study its cryptic diversity, strains were collected to compare their relatedness using barcoding and nuclear genes. Nuclear genes identified two clusters which agree with the known two species, whereas the barcode fragment determined an additional third Clade. Total reproductive isolation (RI) according to the biological species concept (BSC) was investigated in crossing experiments within and between clusters using representative strains. Sexual isolation exists between all studied pairs, increasing from slight to strong with genetic distance. Postzygotic barriers mostly affected hybrid males, pointing to Haldane's rule. Hybrid females were only affected by unidirectional Spiroplasma‐induced cytoplasmic incompatibility and behavioural sterility, each in one specific strain combination. RI was virtually absent between strains separated by up to 2.8% COI difference, but strong or complete in three pairs from one Clade each, separated by at least 7.2%. Apparently, each of these clusters represents one separate species according to the BSC, highlighting cryptic diversity in direct vicinity to humans. In addition, these results challenge the recent ‘turbo‐taxonomy’ practice of using 2% COI differences to delimitate species, especially within parasitic Hymenoptera. The gradual increase in number and strength of reproductive barriers between strains with increasing genetic distance also sheds light on the emergence of barriers during the speciation process in L. distinguendus.
Reproductive success and escape behaviour in urban greylag geese (Anser anser)
(2023) Mai, Sabrina; Steidle, Johannes
Urbanisation and its effect on animals and plants is an important factor to analyse in behavioural studies. Warmer temperatures and an increased availability of food provide benefits to animals occurring in urban habitats. In contrast, these animals also have to tolerate the disturbance caused by artificial lights or noise. This work studies an urban local population of greylag geese Anser anser. Greylag geese live in wetland landscapes, but also in urban parks with wide lawns and water bodies. Since the nineties, the city of Stuttgart in southwest Germany is home to a breeding population of greylag geese. As a result of a long-term ringing project, a large percentage of the population is ringed. This data shows that the population is non-migratory, as the geese generally remain in the area year-round. The individual identification offers the opportunity of tracing data, such as reproduction or behaviour, for the same animal over a longer period. This data can then be individually connected to genetic information, which is a large benefit in behavioural studies. The first chapter of this work focuses on reproductive success as one of the two key parameters which influence population change. As reproductive success itself is influenced by a variety of factors, this study analysed the effect of factors such as population size, brood size or dispersal by using two different measures of reproductive success: fledging success (the relation between hatched and fledged young of a brood ) and hatchling survival (the likelihood of a hatchling to survive to fledging). Fledging success of pairs initially increased with the number of times pairs bred together but decreased again in later broods. While the experience of a pair is therefore beneficial for their reproductive success, the subsequent decrease may be caused by the increasing age of the parents. The brood size also influenced reproductive success, as fledging success was higher in larger broods and hatchling survival was also positively influenced by brood size. In some years, several goose families dispersed from the breeding ground to a different brood rearing area. Hatchling survival was higher in these families, though this effect decreased again when the broods were large. The total population size had a negative effect on hatchling survival, indicating density dependence. To determine if there is a genetic basis for escape behaviour in greylag geese, the second chapter of this work compares Stuttgart’s urban local population of greylag geese with a rural local population in the countryside near Ludwigsburg. Animals occurring in urban habitats need a higher tolerance towards disturbance. This tolerance may be due to the animal’s personality. Animal personality is defined as consistent behaviour across time or context and is expressed for example through risk-prone behaviour. Urban animals are likely more risk-prone than their rural counterparts and thus have a bolder personality. There is increasing evidence that personality and thus behaviour have a genetic basis. This study analyses the dopamine D4 receptor (DRD4) as a candidate gene associated with boldness / shyness and fear. The gene showed a high variation with eleven alleles and 35 genotypes being detected in the analysed greylag geese. Allele and genotype frequencies were skewed, with one allele and two genotypes occurring more often than the others. Based on the detected frequencies, these two genotypes were defined as common and the remaining 33 as rare genotypes. Common genotypes were found more frequently in geese from urban areas, while rare genotypes were found more frequently in geese from rural areas. To determine if the detected genotypes could be associated with behaviour, the flight initiation distance (FID) of individual geese was measured. As the geese are ringed, FID measurements could be directly assigned to a specific goose and thus to its genotype. A high FID indicates geese which are more cautious, while a low FID indicates boldness. Indeed, greylag geese of the rural location show increased FID, while those in urban areas in Stuttgart have lower FIDs and thus a bolder personality. Despite these results, there was no significant correlation between DRD4 genotype frequency and FID. This may be explained by methodological effects. Alternatively, DRD4 has also been associated with other behaviours such as novelty seeking and exploratory behaviour. These behaviours may also differ between urban and rural animals and may thus be the reason for the detected frequencies. It is therefore likely that local habitat selection may be influenced by the DRD4 genotype. Overall, this work demonstrates that different factors, including genetics, should be taken into account when managing urban populations of wild animals, as their behaviour can differ significantly from their rural counterparts, even within a single species.

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