Abstract:
Development and growth of many tissues and organs takes place in parallel during the embryonal development. Therefore a very high number of processes in the body have to be organized and coordinated. Besides numerous other systems, which are indispensable for the body, the nervous system and its proper connection is essential for proper functioning of an organism. The connections from origin and target region via the axons of nerve cells form so-called topographic maps. The implementation of these maps is due to guidance molecules, which insure, that axons find their appropriate way and are guided to the matching target cell. The retinotectal projection of the chick embryo serves as one model system to investigate the underlying molecular mechanisms of map formation and axon guidance. In recent years numerous molecules and mechanisms, which were found to be important for the development of the retinotectal projection, have also been found in other systems. Nonetheless, many questions remain to be answered. The results obtained in the experimental part of this thesis, give answers to open questions and help to better understand the molecular mechanisms of axon guidance. Thereby the focus of this work was the identification and characterization of a novel ligand receptor system, that is involved in the formation of the retinotectal projection.
First a of all, essential discoveries were provided for the characterization of RGM (the repulsive guidance molecule, which was isolated biochemically 1990 (Stahl et al.) and cloned by PD Dr. B. K. Mueller an colleagues 1999). Not only that examination of the temporal expression pattern in the retinotectal system as well as the enthorinal projection confirmed a potential participation of RGM during the development of both topographic maps but in addition, also the expression patterns, on RNA- and protein level, in the chick embryo tissue suggested, together with data from in vitro experiments, that RGM is a major player in the retinotectal projection development. The results of the first part of this thesis were integrated into the publication Monnier et al. 2002.
Furthermore, tools suitable to isolate RGM binding molecules, in the form of RGM-AP fusion proteins, were developed, which were then produced, characterized and, successfully applied in experiments. Additionally, the characterization of the RGM-AP fusion protein proofed to be an important step in the finer molecular analysis of RGM. Due to this construct, that shares identical properties with native RGM, the posttranslational processing by proteolytic cleavage could be shown. Farther using the fusion protein, not only a binding partner for RGM was found, but also further characterization of the binding specificity was determined.
Finally the isolation of a RGM-binding molecule was successfully carried out in cooperation with Prof. Dr. S. M. Strittmatter from Yale University. Concerning the scientific discoveries, which are described in this thesis and the resulting specificity, activity and its characteristics is most likely that this isolated molecule functions as RGM receptor.
Summarizing the results from this thesis, a novel ligand receptor system, besides the well known Ephrin Eph-receptor family, could be characterized on the molecular level, and seems to be a major component during the development of the retinotectal system in the chick embryo. Therefore the basis for further investigations was set, which goes even beyond developmental biology. Recent results show, that RGM is active in the formation of other topographic maps in other species and it was also shown that in adult animals after lesioning the CNS, RGM is up regulated. Therefore it is very likely that RGM functions as well as a regeneration inhibitor.