For the majority of developing neurons an essential step in proper guidance involves crossing the midline, and failure to do so often results in an inability to coordinate movement. This is further evidenced by the association between mirror movement disorders and mutations in the deleted in colorectal cancer (DCC) gene, which is a highly conserved guidance receptor that mediates midline crossing. However, DCC mutants (or frazzled in Drosophila) still exhibit significant midline crossing, implicating additional pro-crossing mechanisms. To identify these alternative pathways and their potential contribution to motor coordination, a genetic screen was performed and the transmembrane Sema, sema-1a, was identified as a novel pro-crossing gene. This proposal aims to determine the mechanism of sema-1a in midline crossing and the potential contribution of its canonical receptor, plexin A. Semas traditionally signal repulsion as ligands for Plexin receptors, however, transmembrane Semas can also act as receptors themselves and signal in reverse. Preliminary data indicates that Sema-1a requires its cytoplasmic domain to promote midline crossing, suggesting it may function through reverse signaling.
Aim 1 will determine if Sema-1a promotes midline crossing through forward or reverse signaling. Sema-1a reverse signaling would imply a cell autonomous requirement in commissural neurons, which can be tested in defined subsets through in vivo rescue assays. Additionally, the elements within the cytoplasmic region that confer midline crossing activity will be identified. Downstream effector molecules, which bind Sema-1a's cytoplasmic region, will also be examined for defects in midline crossing. Plexin A (PlexA) is the canonical binding partner of Sema-1a repulsive signaling, however, Sema- 1a can also function in a Plexin-independent manner. Mutations in plexA significantly enhance the crossing defects in the sensitized screening background suggesting that PlexA also promotes midline crossing. It is unclear whether PlexA functions as the binding partner for Sema-1a in this process.
Aim 2 will determine if PlexA acts as the binding partner for Sema-1a mediated midline crossing. It will be determined if Sema-1a mediated midline crossing is PlexA dependent. Furthermore, the signaling mechanism can be determined by defining which neurons require PlexA for midline crossing. As both Sema-1a and PlexA traditionally function through repulsive signaling, this mechanism will be tested with in vivo rescue assays. Together with the structural requirements identified in Aim1, these experiments will establish a mechanism for Sema-1a mediated midline crossing.
In order to establish a functioning nervous system, developing neurons must correctly navigate to their targets. For a majority of neurons this process is initiated by first crossing the midline and failure to do so results in an inability to coordinate movement. This proposal aims to understand the mechanisms that govern midline crossing during development and may provide insights for neural regeneration after injury.
| Hernandez-Fleming, Melissa; Rohrbach, Ethan W; Bashaw, Greg J (2017) Sema-1a Reverse Signaling Promotes Midline Crossing in Response to Secreted Semaphorins. Cell Rep 18:174-184 |
