Browsing by Subject "In-vitro-Kultur"

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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.
Odorantrezeptoren in Axonen olfaktorischer Sinneszellen : in vitro Studien an Explantatkulturen
(2008) Luxenhofer, Georg; Breer, Heinz
Olfactory sensory neurons (OSN) expressing a particular odorant receptor (OR) are widely scattered throughout the olfactory epithelium (OE) of the nose and send their axon into a small number of common glomeruli in the olfactory bulb (OB). In a spatially well conserved pattern these axons establish synaptic contacts to second order neurons. The molecular mechanisms underlying the precise wiring are still not well understood. To generate a system which may facilitate the investigation of distinct aspects of this complex process, an in vitro culture with tissue explants from the olfactory system was established in the present work. The use of tissues from transgenic mice which enabled the visualisation of OSN and their processes by intrinsic fluorescence allowed a continuous observation of distinct axonal populations under defined and manipulable conditions. Cells within an explant from the OE harvested at the embryonic stage 14 (E14) extended numerous axonal processes within a few days which grew out radially and without fasciculation. During the initial culture period the explants contained mainly progenitor cells; after several days in culture cells differentiated to OMP-positive, thus mature OSN. Using receptor specific transgenic mouse lines the expression of distinct OR genes in a subpopulation of OSN could be detected. Altogether, the culture conditions thus allowed the differentiation of progenitor cells into OSN with characteristic gene expression. Concerning the key question of how axons of OSN interact with their target tissue, co-culture experiments with OB tissue were performed; they showed that axons were initially repelled by their target. A precultivation of OB tissue, however, resulted in an attraction of axons even from larger distances. Moreover, the bulb tissue exerted a positive effect on the growth rate of OSN axons. During their growth these axons formed bundles which defasciculated in the vicinity of the OB explants. These results showed that characteristic parameters in the generation of OSN, their axonal growth and interactions with the target tissue were recapitulated by the in vitro culture system, thus, providing optimal conditions for the examination of key questions regarding the molecular mechanisms involved in establishing the unique projection pattern. Subsequently, the explant culture system was used to investigate the role of the odorant receptor protein in the process of path finding. Expression of genetically modified receptor variants in the explants revealed novel insights into the subcellular localisation of the odorant receptor mOR256-17. An mOR256-17-EGFP fusion protein could be detected in vesicles transported into the dendrite of OSN, resulting in an accumulation of the OR in the cilia. Using this technique it was possible to observe for the first time OR proteins in vesicles which were transported anterogradely and retrogradely along the entire axon. The OR could be visualised within the growth cones and the attached filopodia. Taking advantage of a novel detection method in which proteins integrated into the plasma membrane were selectively marked, retrogadely transported vesicles containing internalised mOR256-17 protein could be observed. The generation of an OR variant, in which the G-protein binding domain was mutated resulted in a disturbed localisation of the OR protein within OSN. Hence, by developing an improved in vitro explant system, an important tool was generated that allowed novel insights into the function of distinct molecular components and should be valuable for future studies aimed at understanding the complex processes that lead to the precise connection of OSN with their target.