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Abstract

In order to establish a proper functioning nervous system, several guidance molecules provide signals to allow precise pathfinding and patterning of axons. Many proteins regulating axonal guidance have been identified and characterized successfully by the analysis of dissociated neurons in vitro. However, these cultures neither allowed to investigate adhesive interactions between the growth cones and the surrounding tissue nor to analyze the role of the membrane anchorage of the guidance molecules and the receptor complex compositions which can induce various cell responses. It is therefore important to manipulate nerve outgrowth in situ and to observe the effects in real time. The aim of my thesis was to establish a slice culture system that allows us to follow the outgrowth of peripheral nerves into the developing forelimb of the mouse, and to validate this ex situ model by the analysis of slices prepared from mice lacking the gene encoding the chemorepulsive guidance molecule semaphorin3A. The first experiments were performed using a mouse line expressing the enhanced green fluorescent protein (EGFP) from the gene locus Mapt, resulting in an ectopic EGFP expression in postmitotic neurons of the central and peripheral nervous system. The slice culture system proved to be suitable to reproduce endogenous forelimb innervation and to image spinal nerve outgrowth into the murine forelimb as it starts from embryonic day (E) 10.5. Analysing the specification of non-neural tissues (chondrogenesis and myogenesis) confirmed that they retained a developmentally normal morphology over the course of the culture, and employing assays for the quantification of cell death revealed a very low mortality rate of neural and non-neural cells. To solve the imaging restrictions resulting of the weak GFP intensity at the nerve ends, we planned to improve the system by employing a mouse line that was expected to have a stronger GFP signal intensity specifically in the growth cone. Furthermore, we wanted to analyse the limb innervation in a mouse line deficient for the guidance protein semaphorin3A. Applying our system, we could clearly confirm premature outgrowth and a strong defasciculation of spinal nerves in homozygous mutant embryos. Furthermore, with the aid of the EGFP expression we were able to detect cells that had not been previously reported for this mouse line. Characterising these cells by immunohistochemistry, two distinct populations, namely a sensory and a sympathetic lineage, were detected. These cells were shown to be located ectopically dur to the absence of semaphorin3A, revealing also an impact of this guidance molecule on neural crest migration. Our established slice culture system allows the observation and analysis of spinal nerve outgrowth as it occurs in situ and therefore provides a system to determine the effect of guidance molecules on axonal outgrowth ex vivo.