'Paleontological Institute at The University of Kansas'
Abstract
Neuronal migration is essential to the formation of the central nervous system in vertebrates. In Caenorhabditis elegans, a screen was performed previously to identify mutations that affected the migration of the Q neuroblast descendants. One of the mutants isolated from this screen was mig-15. MIG-15, a Nck Interacting Kinase (NIK), is homologous to proteins found in a wide variety of organisms, including Drosophila, mice, and humans, in which NIK kinases have been implicated in cell migration. Interestingly, multiple components of the canonical Wnt signaling pathway had already been found to control the Q cell descendant migrations. Additionally, the MIG-15 homolog in Drosophila, Misshapen had also been found to work with Wnt signaling components in the non-canonical planar cell polarity pathway. To determine how MIG-15 was working to control the migrations of the Q cell descendants, a characterization of the Q neuroblast migration defects was performed. mig-15 mutants were found to affect the Q neuroblasts, along with their descendants as previously described. I carried this work further and found that MIG-15 is required for extension of lamellipodial protrusions, maintenance of the initial polarization directing these initial protrusions, and migration of the Q neuroblasts. Since the Wnt signaling pathway had been implicated in Q cell descendant migration as well, several Wnt signaling mutants were also examined in the Q neuroblasts. This analysis determined that for the Wnt signaling mutants that were observed, there was no effect on early Q neuroblast protrusion extension or migration. Therefore, MIG-15 does not appear to be acting with the Wnt signaling pathway to control Q neuroblast migration. Subsequently, the Q cell descendant migrations of the AQR and PQR neurons were also examined for both mig-15 and Wnt signaling mutants. Double mutants of mig-15 with Wnt signaling mutants resembled mig-15 mutants alone, further suggesting that MIG-15 is not working with the Wnt signaling pathway to control the Q neuroblast lineage migrations. In attempt to elucidate how MIG-15 is controlling the migrations of the Q neuroblasts and descendants, a candidate gene approach was taken to determine other possible proteins that are required for Q neuroblast migration. The C-terminal region of MIG-15 had previously been found to bind to PAT-3, the beta-integrin homolog in C. elegans. Since available mutants in pat-3 are not viable, INA-1/alpha-integrin was examined for defects in Q neuroblast migration. This analysis found that, like MIG-15, INA-1 is required for the extension of polarized protrusions and migration of the Q neuroblasts. Though, INA-1 was not involved in maintenance of polarization as was MIG-15. Another molecule that was examined was ERM-1, the C. elegans homolog of the ezrin, radixin, and moesin (ERM) family of proteins. Previous studies have found that ERM proteins bind to and are phosphorylated by Nck interacting kinases in cell culture. These studies found that removal of ERM-1 from mig-15 mutants suppressed the migration defects seen for the QL neuroblasts in the mig-15 mutants alone. Together, these results suggest that MIG-15 could be acting with the integrins for proper polarization and with both ERM-1 and the integrins to direct migration of the Q neuroblasts and descendants. Previous studies had suggested that MIG-15 works upstream of the Rac GTPases MIG-2 and CED-10 in axon pathfinding. Additionally, these molecules had been found to act in parallel to control axon pathfinding. In order to determine if these Rac GTPases are also required for migration of the Q neuroblasts, single and double mutants of the Rac GTPases and several other molecules that are known to work with these molecules in other systems, including CDC-42 and UNC-73, were examined for their effects on the Q neuroblasts and their migrations. Singly, mutants of mig-2, ced-10, and cdc-42 did not cause strong defects in the ability to extend protrusions or the migrations of the Q neuroblasts. When double mutants were analyzed, strong defects in the polarization and migration of the Q neuroblasts were observed for the mig-2;ced-10 double mutants. Previous data has suggested that UNC-73 acts as a guanine nucleotide exchange factor (GEF) for MIG-2 and CED-10. unc-73 mutants displayed polarization and migration defects like the mig-2; ced-10 double mutant, but the defects were less severe in the unc-73 mutants, suggesting that there is another GEF that facilitates GTP exchange for MIG-2 and CED-10. Observations of a second GEF, PIX-1, suggest that PIX-1 is involved in the protrusion extension and migration of the Q neuroblasts, suggesting that PIX-1 might act as the other GEF for MIG-2 and CED-10. Further studies found that PIX-1 might be functioning in a linear pathway with CED-10 and in parallel to the UNC-73/MIG-2 signaling pathway. In summary, my work reveals the roles of the Rac GTPases and MIG-15/NIK kinase in the migrations of the Q neuroblasts in Caenorhabditis elegans, which provides insight into the mechanisms that drive neuronal migration during nervous system development
