Worm Breeder's Gazette 16(5): 36 (February 1, 2001)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Reverse genetic approach to identify genes required for thermotaxis in C. elegans

Hitoshi Inada, Hidetoshi Komatsu, Mikihiro Kosaki, Ikue Mori

Laboratory of Molecular Neurobiology, Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan, and PRESTO, JST

C. elegans can sense and memorize temperature, and change its temperature response, depending on the past experience. For example, after cultivation with sufficient food at 20 degree, C. elegans migrates to its cultivation temperature (i.e. 20 degree C) and then moves isothermally on a thermal gradient without food. In contrast, after cultivation without food for a few hours, C. elegans becomes to avoid its cultivation temperature. This behavior is called thermotaxis. It has been previously shown by laser ablation experiments that AFD thermosensory neurons, and their downstream neurons, AIY, AIZ and RIA interneurons, are required for execution of normal thermotaxis. For example, killing AFD thermosensory neurons results in athermotactic or cryophilic phenotype, and killing AIY or AIZ interneurons results in cryophylic or thermophilic phenotype, respectively. Several mutants defective in thermotaxis have been isolated. Of these, tax-4 and tax-2 show athermotactic phenotype, and both tax-4 and tax-2 gene were found to encode two different subunits of cyclic nucleotide-gated cation channel (CNG channel), which functions in several sensory neurons including AFD thermosensory neurons. The TAX-4/TAX-2 CNG channel shows higher affinity to cGMP than cAMP, suggesting that cGMP acts as a second messenger in thermosensory signal transduction. Thermotaxis must also require execution of some form of associative learning between temperature and food. Recently, our laboratory has been isolating mutants designated aho(abnormal hunger orientation), which are likely to be defective in mechanism of associative learning between temperature and food.

The completion of C. elegans genome project makes us possible to systematically analyze the function and development of the nervous system by reverse genetic approach. Among 19,000 genes that exist in the C. elegans genome, about 1,700 genes are thought to function in the nervous system. In order to dissect the molecular mechanism of thermotaxis, we have started to construct TMP/UV-induced deletion mutants for the genes that likely function in neurons required for thermotaxis. Since (1) cGMP could be a second messenger in thermosensation and (2) associative learning in thermotaxis can be established only in a few hours (0.5 ~ 4 hr), implicating the importance of change in neuronal activity, we focus on genes for (1) molecules related to cGMP signal transduction and (2) ion channels required for modulation of neuronal activity. For the first aim, we plan to knockout genes for guanylyl cyclase (GC), cyclic nucleotide phosphodiesterase (PDE), and cGMP dependent protein kinase (PKG). In C. elegans, there are at least 33 genes for GC, and at least two of these genes, gcy-8 and gcy-12, are expressed in AFD thermosensory neurons. Also, there are several genes for PDE and a single gene for PKG. For the second aim, we are paying attention to the following genes; 6 genes for voltage-gated chloride channel, and 11 genes for transient receptor potential (TRP) ion channel. In any case, we first express GFP-tagged proteins encoded by these genes in wild type animals and look for genes that are expressed in neurons involved in thermotaxis circuit, before undertaking gene-knockout experiment. To date, we screened about 1,200,000 genomes and obtained deletion mutants for GCY-12 and TRP ion channel. These mutants showed nearly normal thermotaxis, but the gcy-12 mutant showed patial defect in chemotaxis to AWC-sensed isoamyl alcohol. Normal thermotaxis in the gcy-12 mutant would probably result from redundancy of gcy genes, which are expressed in AFD thermosensory neurons.