Our lab has been investigating the function of VAB-1, an Eph receptor tyrosine kinase (RTK), in neuronal development.
vab-1 mutations result in abnormalities in epidermal morphogenesis, neuroblast movement, and also axon guidance [1-3]. We use the mechanosensory neurons with particular focus on the PLM to study the axon guidance roles of
vab-1 and related genes. Work on axon guidance has mainly utilized the L4 or young adult stage of worms to characterize defects in axon development. Recent work in our lab has shown that the L1 stage provides a greater degree of sensitivity. At the L1 stage
vab-1 null mutants display a 22% versus a 15% overgrowth defect, and for the hyperactive
vab-1 tyrosine kinase strain L1 worms achieved 97% versus a reported 85% axonal undergrowth defect [3]. With this improved PLM scoring phenotype we aim to focus on 1) studying the role of lethal genes in axon guidance , and 2) studying genes known to be synthetic lethal with
vab-1 null mutations. To overcome the issue of lethality we have used a tissue specific RNAi technique that is resistant to the spreading effects of RNAi [4]. The knockdown assay utilizes the
rde-1 mutant background to inhibit RNAi activity in the worm. These worms are then injected with a touch neuron
rde-1 specific rescuing construct via the
mec-4 promoter along with an RNAi construct consisting of a dual
mec-4 promoter flanking our gene of interest. We have created a number of constructs for lethal genes to study their role in axon guidance and will test whether they function in the
vab-1 signalling pathway. Our preliminary study focused on
arx-1 and
arx-2 which code for the Arp 2/3 complex for actin nucleation. Both
arx-1 and
arx-2 display penetrant embryonic lethality using standard RNAi feeding or injection methods. In contrast, our neuronal specific RNAi approach bypassed the embryonic lethality and
arx-1 or
arx-2 RNAi displayed ~80% PLM undergrowth, suggesting that
arx-1 and
arx-2 are required in PLM for axon outgrowth. Thus this technique allows us to study the function of essential genes or
vab-1 synthetic lethal genes in axon guidance. 1. Boulin, T., Pocock, R., and Hobert, O. (2006). Current Biology 16, 1871-1883. 2. George, S.E et al., (1998). Cell 92, 633-644. 3. Mohamed, A.M., and Chin-Sang, I.D. (2006). Dev. Biol. 290, 164-176. 4. Qadota, H et al., (2007). Gene 400, 166-173.