Two pairs of mechanosensory neurons, ALM and PLM, mediate escape from mechanical stimuli. ALM responds to anterior touch, PLM to posterior touch. To respond appropriately, each anteriorly-directed sensory process must terminate precisely along the antero-posterior axis. Previously, we found that SAX-2 and the SAX-1 kinase, members of a conserved signaling pathway, play an important role in neurite termination. Specifically, the PLM neurite fails to terminate properly in
sax-1 and
sax-2 mutants, leading to overlap of anterior and posterior mechanosensory neurons. Furthermore, over-expression of
sax-2 in the mechanosensory neurons causes premature termination of the PLM neurite, suggesting that
sax-2 delivers an instructive, cell autonomous signal to inhibit neurite growth. To better understand how
sax-2 functions, we are currently using GFP fusions to characterize its sub cellular localization and expression pattern in whole animals and primary neuron culture. Initially, we co-injected
sax-2 genomic DNA with a C-terminal
sax-2 fragment fused to GFP. These two DNA pieces recombined in vivo to rescue the
sax-2 mutant. This reporter fusion shows that
sax-2 is expressed in a variety of cells and its sub cellular localization is punctate. Unfortunately, the punctate nature of SAX-2::GFP precludes identification of specific
sax-2 expressing cells. Our current approach involves the co-injection of
sax-2 genomic DNA with a C-terminal
sax-2 fragment fused to a dual reporter vector. In vivo recombination is expected to express both full-length
sax-2 fused to GFP and soluble mCherry. In theory, each reporter will be co-transcribed using
sax-2 regulatory sequences, while soluble mCherry will be translated independently due to the presence of a 5 SL2 recognition sequence (a technique pioneered by Coates and de Bono, 2002). We are also in the process of making this dual reporter vector Gateway compatible, TA clonable etc. in order to quickly build additional fusion genes of interest. Finally, we are taking a reverse genetic approach to identify additional candidate members of the SAX-1/SAX-2 molecular pathway. Specifically, we have built RNAi-sensitive strains expressing
mec-4::gfp and have begun to test worm orthologs of the S. cerevisiae RAM signaling pathway, a pathway that includes the orthologs of
sax-1 and
sax-2.