Development and application of optical microscopy tools for the study of axon guidance
During their development, neurons extend axons towards their target cells, where they branch to establish connections. The navigation of axons relies on the presence of chemical cues sensed through guidance cue receptors. Upon receptor activation, intracellular signalling pathways are initiated, one of which induces local protein synthesis (LPS), a key process to enable rapid navigational responses to guidance cues. Impairments of axon guidance and LPS are associated with several neurological disorders. In this thesis, state-of-the-art optical microscopy-based tools were developed to improve the efficiency and versatility of commonly used methods for studying axon guidance both *in vitro* and *in vivo*. Furthermore, imaging-based studies were performed on selected guidance cue receptors to investigate their regulation of cue-induced LPS. For the experimental investigations, *Xenopus laevis* retinal ganglion cells (RGCs) were utilised as a model system, enabling comprehensive studies on isolated outgrowing axons both *in vitro* and *in vivo*. Initially, an imaging method was established to examine intricate axonal structures within the highly complex physiological environment. To achieve this, expansion microscopy (ExM) was combined with light sheet fluorescence microscopy (LSFM) to visualise RGC branching *in vivo*. By tracing individual axons, this technique offers a valuable tool for studying cue-dependent arborisation within the brain. Then, ExM and structured illumination microscopy (SIM) were applied in a study aimed at investigating how guidance cue receptors facilitate cue-dependent responses through changes in specific mRNA translation. A mechanism was explored that involves the direct interaction of the guidance cue receptors deleted in colorectal cancer (DCC) and neuropilin-1 (Nrp1) with the translation machinery. This interaction was found to be mediated through RNA binding proteins (RBPs), enabling a receptor-specific mRNA subset to be rapidly and locally translated in response to cue stimulation. Further investigations focused on the cue-induced intracellular transport dynamics of DCC within the endosomal system. During these studies, DCC was observed to colocalise with ribonucleoprotein (RNP) granules on endosomes, suggesting a model in which DCC facilitates the association of RNP granules with endosomes through its affinity to RBPs. Finally, an optical system was developed to enhance the throughput of commonly employed assays for stimulating axons and enabling directed axonal outgrowth *in vitro*. This system employs surface-immobilisation of guidance cues and adhesion proteins. Protocols based on the principle of light-induced molecular adsorption of proteins (LIMAP) were established to guide outgrowing RGC axons towards their physiological target tissue. In summary, this work describes the development of highly sophisticated tools designed to facilitate the study of axon guidance both *in vitro* and *in vivo*. Additionally, valuable insights were gained into the cue-induced mechanisms that initiate LPS through guidance cue receptors. These advancements hold great potential for enhancing our comprehension of axon guidance and its implications for neurological disorders.
axon guidance, fluorescent microscopy, local translation, protein patterning, super-resolution microscopy, Xenopus laevis
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