A screen for axon guidance genes in parallel to heparan sulfate proteoglycans in C. elegans

During animal development the establishment of a functional nervous system is an absolutely crucial step. Neurons send axons, sometimes over long distances to reach a certain target with very high accuracy. If errors occur during this process a wide range of neuronal defects can appear ranging from simple motor dysfunction to severe psychological diseases in humans. While increasing anatomical complexity is usually accompanied by increasing complexity in the nervous system the developmental cues that orchestrate the establishment of nervous systems are amazingly conserved from animals as simple as Caenorhabditis elegans all the way to humans. Therefore studies in model organisms have revealed a fair number of conserved major axon guidance cues. However it seems unlikely that these major cues are the only ones responsible for building the nervous system. In order to achieve the complexity of an adult nervous system there must be other mechanisms that fine-tune the major cues. We have identified Syndecan and Glypican, two Heparan Sulfate Proteoglycan (HSPG) core proteins, plus a number of Heparan Sulfate (HS) modifying enzymes as minor modulators of axon guidance. Typically mutations in genes coding for these proteins do not lead to severe defects in axon guidance. However, since there are multiple HS related pathways acting in parallel mutations in more than one branch of these pathways lead to clear defects. Since little is known about downstream factor of HS signalling we designed screens to find such components, e.g. axon guidance cues that could interact with HS or other components of established axon guidance pathways. We were able to find a large number of candidates with a possible link to axon guidance (Chapter III) and still did not saturate the genome with the mutagenesis. We also found a mutation in the gene called zfp-1 for which there has not been any indication so far that it would play a role in axon guidance. Furthermore a mutation in the C. elegans homologue of the Retinoblastoma protein lin-35 results in comparable defects. Both genes play roles in vulval development and the RNAi pathway. We decided to focus on these two genes (Chapter IV) because they were the most unexpected ones and a possible involvement of the RNAi pathway in nervous system development certainly seemed to be an interesting topic to explore. In order to visualize the neurons that we were focusing our study on (D-type motor neurons) we used the transgene oxIs12 labelling these neurons with green fluorescent protein (gfp). Initially we assumed that this transgene is not interfering with the phenotype we screened for and for the average candidate this also seems to be true. However for mutations in zfp-1 and lin35 we found that they only exhibited axon guidance defects if combined with oxIs12 and not with other transgenes labelling the same neurons. Our efforts to shed light onto this surprising effect led to the conclusion that the integration site of oxIs12 is playing a role. We also gained considerable insight to the nature of oxIs12 being a large multi copy transgene spanning roughly 3.6Mb enlarging the X chromosome by about 20%. Nevertheless, mechanistically the transgene dependent effect of zfp-1 and lin-35 mutations remains a mystery. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-59152