Worm Breeder's Gazette 14(2): 35 (February 1, 1996)
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.
Dept. of Genetics, Univ. of Washington, Seattle, WA 98195 USA
We are studying genes that function in the nematode nervous system using a dauer formation phenotype. The nematode senses environmental cues, such as dauer pheromone, the presence/absence of food, and temperature, and makes a decision to form a dauer. Dauer formation phenotype reflects, at least in part, neuronal activities and can be used as an indicator of a nervous system function. We are particularly focusing on the unc-31;aex-3 dauer constitutive (Daf-c) phenotype (1). Single mutants of either unc-31 or aex-3 are not Daf-c. However, unc-31 and aex-3 double mutants are strongly Daf-c at 25C. We made various combinations of unc-31 (e169, n928, and u280) and aex-3 (ad418, sa5, and ad696) alleles. The weakest is unc-31(e169);aex-3(ad418), which is 65% Daf-c at 25C and 30% at 15C. The strongest is unc-31(u280);aex-3(ad696), which is 100% Daf-c at 25C and 95% at 15C. Daf-c genes identified previously are classified into three groups based on genetic interactions among Daf-c and Daf-d genes. Group I includes daf-11 and daf-21, and Group II includes daf-1, daf-4, daf-7, daf-8, and daf-14. Group I and II genes are proposed to function in parallel to each other. Group III includes daf-2 and daf-23. We tested which Daf-c group unc-31 and aex-3 fall into, based on suppression of the Daf-c phenotype by 1) laser-ablation of ASJ, ASK, ADL (J/K/L) sensory neurons, 2) 8-bromo-cGMP, 3) a daf-5 mutation, and 4) a daf-12 mutation. Ablation of J/K/L cells suppresses the phenotype of Group I Daf-c mutations [90% to 5% in daf-11(sa195) worms], but not that of Group II and III (W. Schackwitz, pers. comm.). We ablated J/K/L cells in unc-31(u280);aex-3(ad696) animals and found that this operation only weakly suppressed this Daf-c phenotype (100% to 65% at 25C). daf-11 , a Group I gene, encodes a putative membrane-type guanylyl cyclase (GC) [D. Birnby, pers. comm.]. When daf-11 mutants are grown with 2.5 mM 8-Br-cGMP, a non-hydrolizable cGMP analog, their Daf-c phenotype is completely suppressed. This finding is consistent with daf-11 mutation lowering a GC activity. However, the phenotype of Group II and III Daf-c mutations is not suppressed at all by 8-Br-cGMP. We fed unc-31;aex-3 mutants this drug and found that this Daf-c phenotype was not suppressed at all. daf-5 is a dauer formation defective gene (Daf-d). daf-5 completely suppresses the phenotype of Group II Daf-c mutations and partially suppresses that of Group I, but does not suppress that of Group III at all. We made a daf-5;unc-31;aex-3 triple mutant strain and found that this Daf-c phenotype was epistatic to daf-5. daf-12 is another Daf-d gene. daf-12(m20) completely suppresses Group I and II mutations. Unlike daf-5, double mutants with daf-12 and Group III Daf-c mutations are lethal. We found that unc-31;aex-3;daf-12 worms were viable and daf-12 was epistatic to unc-31;aex-3. Based on these four criteria, we classify unc-31 and aex-3 in a new group, the Group IV genes. We are also interested in what molecules are encoded by Group IV genes. unc-31 encodes a homolog of p145 (D. Livingston, pers. comm.), which is a regulatory component of Ca++ -dependent synaptic vesicle (SV) release. We cloned the aex-3 gene by transformation rescue and are finishing the sequence of the gene. So far, we found no significant homology between AEX-3 and other known proteins. However, some evidence suggests that AEX-3 is also involved in SV release regulation. 1) The aex-3 gene is genetically redundant with unc-31 in the dauer formation pathway. 2) aex-3 mutants are resistant to Aldicarb, an acetylcholine esterase inhibitor (E. Jorgensen, M. Nonet, pers. comm., and our unpublished data). Mutants of synaptic vesicle component genes have been isolated using this phenotype. 3) RAB-3 protein, which is a component of SV, is mislocalized in aex-3 mutants (M. Nonet, pers. comm.). It is suggested that Rab3A has a role to recruit SV onto a presynaptic terminal membrane in a vertebrate system (2). 1. J. H. Thomas (1990) Genetics 124:855. L. Avery (1993) Genetics 133: 897 2. M. Geppert et al. (1994) Nature 369:493