Molecular Characterization of Dendritic Regeneration in Gryllus bimaculatus
Prof. Hadley Horch
Dept. of Neurobiology
This study focuses on determining molecular cues responsible for the guidance of regenerating neurons in Gryllus bimaculatus (cricket). Observations of the adult cricket central nervous system (CNS) show that when the ear on one side is severed, the auditory dendrites will regenerate rather than retract or die (Schildberger et al., 1986). The regenerating dendrites then grow across the CNS midline and synapse with nerves from the contralateral ear of the same frequency. It is likely that molecular cues serve as guidance for these growing dendrites.
In Drosophila melanogaster the proteins Slit and Robo serve as neuronal guidance cues. Slit is an extracellular molecule secreted by cells at the CNS midline and serves as a repellant ligand for the Robo receptors found on axon growth cones (Bashaw et al., 2000). Axons appear to use the expression of Robo receptors to control midline crossing\downregulating Robo to cross and upregulating Robo to avoid crossing (Bashaw et al., 1999).
This midline guidance property of the Slit/Robo pair in D. melanogaster has led us to hypothesize that these proteins may be used in adult G. bimaculatus to allow the midline crossing phenomenon. The long-term goal of this study is to observe whether expression patterns of Robo and Slit genes correlate with dendrite regeneration. In the short-term I am working to identify the genetic sequence for Slit in G. bimaculatus.
The first step toward sequencing the gene was to determine regions of Slit that are most conserved between species (in this case two Drosophila paralogs for Slit and two homologs in Apis mellifera and Anopheles gambiae). Primers were then designed based on these regions and used to amplify and sequence pieces of DNA likely to be part of the Slit gene. At this point a small portion (200 base pairs) of the Slit gene has been sequenced in the cricket. Future work will include the complete sequencing of Slit and Robo, as well as using pieces of the sequences to visualize Slit/Robo expression patterns using in situ hybridization.