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.

unc-31 and aex-3 are a new class of Daf-c genes.

Kouichi Iwasaki , James H. Thomas.

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:
2. M. Geppert et al. (1994) Nature 369:493