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Axon guidance, behavior and learning: The quest from Netrin to the brain
Biochemistry and Molecular Biology
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Axon guidance is fundamental to the initial organization of the brain, and as a result, the organization of behavior and learning in the mature animal. The impressive plasticity inherent in complex nervous systems allows developmental defects to frequently be overcome. Nevertheless, some faults can persist to adulthood and affect behavior. Analysis of these changes presents an opportunity to understand how developmental errors can lead to differences in the function of the adult. It is becoming increasingly important to conduct research that addresses both the development and function of the nervous system to understand how the distribution of cell populations progresses into a fully functional system. Here we report the generation of a viable Drosophila Netrin mutant line. Like previously characterized Netrin mutants, this line displays a number of axon guidance defects, including reduced or absent commissural and longitudinal axons in the embryonic central nervous system (CNS). As an adult, this line also displays several behavioral defects including general uncoordination, inability to fly, reduced negative geotaxis, reduced locomotion, reduced viability and fertility defects in both sexes. Female Netrin mutants lay fewer eggs than wild type. Male Netrin mutants fertilize fewer eggs and display defects in courtship including mirror-movement-like wing vibrations during the courtship song, although not all aspects of courtship are altered. The embryonic CNS disruptions are thought to occur because Netrins normally guide axons to the source of Netrin expression at the midline. The behavioral defects in the adult are also consistent with the idea that fewer connections between the two sides of the body lead to uncoordination. We were able to rescue axon guidance phenotypes in the embryo with directed expression of NetB in the midline and also by removal of one copy of enabled, as previously reported. Unexpectedly, we also observed rescue by expressing NetB with elav-gal4, which expresses in all mitotic neurons, but not midline glia. Pan-neuronal expression of NetB would be expected to enhance guidance defects if positional information at the midline were the only function of Netrin in the developing CNS. In the adult, only negative geotaxis and viability were rescued by midline expression of NetB. Disrupting one copy of enabled failed to rescue any behavioral measure. These results suggest that apparent rescue of the embryonic CNS does not necessarily lead to rescued behavior of the adult, and establishes adult behavior as a functional readout for manipulations of embryonic, larval, and pupal axon guidance. We hypothesized that expression of neuronal NetB rescued the embryonic CNS by increasing cell survival because Netrin has been found to be involved in neuronal survival in mammals. Therefore, we tested whether limiting cell death could rescue the embryonic CNS and adult behavior in Netrin mutants and found that limiting cell death strongly rescued CNS phenotypes. Locomotion and negative geotaxis were also rescued in the adult, through independent genetic manipulations of the cell death pathway. These data suggest that Netrin can promote cell survival in Drosophila in addition to canonical midline guidance functions. With the adult Netrin mutant, we attempted a directed screen based upon behavioral phenotypes to identify novel modifiers of the Netrin pathway. We were particularly interested in mutations that could suppress the behavioral defects as the underlying genes could harbor therapeutic potential for nerve injury and degeneration conditions, but this approach has thus far been unsuccessful. In addition, we characterized a small gene within the Netrin deletion, which we called hog. This gene has subtle phenotypes, unrelated to the Netrins, but hog function needs to be addressed when analyzing Netrin mutant phenotypes. In addition, I have applied the fundamentals of axon guidance and communication among specific neural groups to the problem of learning. First, I explored the literature on olfactory conditioning in Drosophila. Then, I applied these concepts to learning in mammals. In both cases, I arrived at different conclusions from those previously postulated in the literature.