Cellular and molecular factors influencing mechanosensory circuit development in C. elegans

In C. elegans, response to mechanical stimuli is mediated by two mechanosensory circuits. One circuit functions in the anterior to promote backward movement and another circuit functions in the posterior to promote forward movement. The posterior neural circuit includes the mechanosensory neuron, PLM, which is required for detecting gentle touch within the posterior half of the animal. As expected for a spatially restricted function, the PLM dendrite extends from the tail and terminates just posterior to the anterior mechanosensory neuron, ALM. How is this termination point established? The cellular and molecular mechanisms that control this process are not well understood. I have used molecular genetics and microscopy to help identify these mechanisms. Interestingly, the dendrite tip of PLM makes contact with the neurite tip of BDU, an interneuron. Moreover, genetic ablation of BDU using a mosaic analysis strategy lead to significant PLM overextension in comparison to controls (Gallegos lab unpublished). In these initial experiments BDU was not visible. To determine if BDU makes and maintains contact with PLM in control animals, we re-made the mosaic analysis strain to allow visualization of both BDU and PLM. I found that BDU makes contact with PLM only in 70% of the control animals. Moreover, overexpression of unc-86 transcription factor in the extrachromosomal array likely leads to BDU guidance defects. Finally, my characterization of a new experimental system involving an unc-86 free duplication instead of an extrachromosomal array does not exhibit these defects and is better suited to confirm the role of BDU in PLM termination. I am also interested in molecular mechanisms that regulate PLM development. Importantly, sax-1 and sax-2 function cell autonomously to regulate PLM termination. cbk1, the sax-1 homolog in S. cerevisiae regulates cell growth and protein secretion via a rab GTPase, sec4. Interestingly, all three known mutant alleles of rab-10 (dx2, ok1494 and q373), a worm homolog of sec-4, are defective in endocytic recycling in C. elegans intestine. However, in PLM, we find that while the null alleles (dx2, ok1494) result in PLM overextension, PLM neurite length in the missense allele, q373, is not significantly different from wild type. We hypothesize that the missense mutation in allele q373 does not affect the pathways involved in PLM termination. To test this hypothesis, I developed a genetic strategy to cross a randomly integrated transgene carrying rab-10 (q373) allele into homozygous rab-10 (ok1494) animals. Interestingly, I found that the missense allele, q373, of rab-10, fails to rescue the mutant phenotype (PLM overextension) of the deletion allele, ok1494. This result could likely be due to misexpression of the randomly integrated rab-10 (q373) transgene and the future characterization of the localization of transgene expression could help solve this question.