Browsing by Subject "Musterbildung"

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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.
Oszillation und Musterbildung in reinen und Polypeptid-dotierten Bilayern
(2017) Gaugele, Sebastian; Hanke, Wolfgang
The question of how lipids and proteins interact in bilayers and membranes and why cells and organelles have different lipid compositions in their membranes has been in focus of research for decades. Which role these different lipid compositions play for interaction between integrated and associated proteins has not yet been clarified until today. This thesis with the title "Oszillation und Musterbildung in reinen und Polypeptid-dotierten Bilayern" focuses on forces that influence the interaction of lipids and proteins. In both, living cells as well as in bilayer experiments, the protein-lipid interaction is a complex process involving a huge number of molecules. Different test series with temperature and amperage have been carried out using electrophysiological experiments with bilayers and fluorescence in vesicles. The results were used to determine similarities and differences that indicate certain characteristics of the setup and the interaction among the molecules. Since the question of the interaction between proteins and lipids is a very complex process, the already well-known pore former alamethicin has been used for this work. Alamethicin was used in the both types of experiments, bilayer and vesicles, with different lipid compositions consisting of DOPC, DOPE, DOPS and DMPC. The lipids were solved in ethanol first and then added with certain concentrations of alamethicin in two test chambers which were connected to each other by a small hole. In this hole the bilayer was formed by the addition of aqueous KCl solution. After that the current-clamp-method with different currents was used and subjected to the bilayer. The resulting voltage profiles were observed for oscillation patterns. Subsequently, the obtained data were subjected to the method of Fourier-Transformation to separate the results from noise and interference as well as overlays which resulted from the multi-channel system of alamethicin. The Fourier-Transformation has shown a large number of heterogeneous oscillation patterns. Due to the great heterogeneity of the results that did not allow direct comparisons of individual data with each other the average time-period of oscillation was calculated. This data were used to form data clouds where regression lines were generated. In addition the slopes of the lines were calculated for comparison. The results show that the orientation and slope angle of the slopes directly dependent on the concentrations of alamethicin and KCl. It was found that the addition of a small amount of negatively charged lipid like DOPS to a mixture of DOPC and DOPE has a big impact on the results and can change the orientation of the slopes of the average time-period of oscillation. Similar results were found while using asolectin bilayer containing inositol that is negatively charged, too. As a result, it was concluded that contrary to previous assumptions of some earlier work groups, bilayers are not pure hydrophobic barrier layers between two aqueous phases but highly complex structures that affect the gating of its integrated pores in different ways. The insert and operation of alamethicin strongly depends on the used lipids. Moreover a major dependency from the used salt concentration of the alamethicin pores and bilayer stability was found. The experiments with DMPC and temperature showed that the chain length of the lipids play an important role for integrated proteins on the one hand. On the other hand it could be confirmed that the phase transition of lipids can comprise a wide range of temperature of several degree Celsius depending on the experimental conditions. It could also be confirmed that alamethicin remains active in the liquid-crystal domains during phase transition of a bilayer. In addition it was found that temperature dependent changes appear in voltage oscillation patterns that affect both the average time-period and its resulting slopes in angle and orientation.
Vergleichende Untersuchungen zur Musterbildung in erregbaren Medien mit Vermerken zum Einfluss schwacher magnetischer Felder - Schwerpunkt: Belousov-Zhabotinsky-Reaktion
(2007) Dolzmann, Kerstin; Hanke, Wolfgang
In this work we did some research on the influence of a weak external magnetic field (MF) on the creation of patterns in excitable media (duration field (DC) and alternating field (AC)). As examples we chose the well known Belousov-Zhabotinsky reaction (BZR) and ferrofluids. If ferrofluids are stimulated mechanically by vertical vibration they show changes of phases in the building of patterns while raising the induced energy (here by different hights of amplitudes). The viscosity of the magnetic fluid is increasing in a DC-field. Because of this the changing of the phase is different from the ones without an external force. A non-stationary stirred BZR shows a periodic change of colour between yellow and colourless ? or red and blue, if ferroin is added as a catalyst. This oscillation is described as a simple curve in literature. We, however, found a much more complex behaviour in the experiment. The intrinsic optical signals (IOS) of a ferroin-catalysed, stirred BZR show a double-peak at the beginning of the reaction, which is fading after a few further oscillations. This behaviour depends on the concentration of ferroin and resembles very much electrical and optical signals known from neuronal processes (e.g. retinal spreading depression). This basic similarity makes the BZR an ideal model for a variety of neurophysiological signals, even if the underlying mechanisms are completely different. If this system is put to the influence of a weak external magnetic DC-field a further inner oscillation is added to the double-peak behaviour of the IOS. Also the double-peak itself looks different from the one without external field. If further components are added to the system it gradually changes to chaotic behaviour. This could be the induced little currents in an AC-field. Each expansion of the system is followed by further bifurcation culminating in a transition from pattern to chaos. BRZ gels show bright propagating concentric rings or spirals as a pattern. With the used materials and methods of measurement we were not able to record changes in the formation of patterns if the system was expanded. But one can assume that the behaviour of the gel is changing in the DC-field: all in all it seems to get faster.