We have been interested in uncovering the molecular events underlying the neurite "sprouting" defects observed in the Lim homeodomain mutant
lim-6 (Hobert et al. Development 126 : 1547-1562). We have focused our attention on identifying mutants that lead to similar sprouting defects in DVB, a GABAergic motorneuron whose development and function is crucial for execution of the defecation motor program in C. elegans. We found that mutations that affect communication of DVB with its target muscle cause DVB motor axon sprouting (see abstract by Mehta, Loria and Hobert). Moreover, we have discovered that two previously identified but mainly uncharacterized genes,
unc-122 and
unc-75 , cause low penetrant DVB sprouting defects. Both mutants are especially interesting in that they enhance the DVB sprouting defect seen in
unc-25 null animals and thus appear to act in a pathway parallel to GABAergic neurotransmission in DVB. Similar enhancement of
unc-25 sprouting is observed in
unc-31 mutants where peptidergic neurotransmission is presumably disabled. Based on this analysis, we hypothesize that DVB interacts with its target through GABA-mediated fast neurotransmission and a parallel aminergic/peptidergic pathway and that
unc-122 and
unc-75 may act in the latter. To test this hypothesis, we set out to clone
unc-122 and
unc-75. Since
unc-122 and
unc-75 mutants are also coilers, for cloning purposes we relied predominantly on their more obvious unc/coiling phenotypes. 3-factor and deficiency mapping, transformation rescue, and sequencing revealed that F11C3.2 corresponds to
unc-122 . UNC-122 is predicted to be a type II transmembrane protein, containing a 19 amino acid N-terminal intracellular domain. The extracellular portion of the protein contains a collagen-like repeat followed by a highly conserved olfactomedin (OLF) domain. Two independently isolated alleles of
unc-122 show premature stops within the OLF domain suggesting a crucial role for this region of the protein. The
unc-75 locus maps very close to
spe-9 on LGI and we have obtained transgenic rescue of
unc-75 using a YAC within this region (gift of Andrew Singson). SNP mapping has narrowed our focus and we expect cloning of
unc-75 in the very near future. The coiling phenotypes of
unc-122 and
unc-75 suggest defects in motorneurons or the muscles they innervate. Our mosaic analyses have clearly established that
unc-122 function is not required neuronally, but is in fact crucial within muscle lineages. In an effort to pinpoint the intracellular localization of UNC-122, antibody staining is currently underway using transgenic lines containing a rescuing epitope-tagged version of the protein. To delineate the pathway in which
unc-122 and
unc-75 act in regard to their coiling phenotype, we tested interaction with GABA- and cholinergic-neurotransmission. Double mutants of
unc-122 or
unc-75 with
unc-25 both coil and shrink. Thus, GABAergic neurotransmission is not involved in the coiling observed. We next investigated whether defects in cholinergic neurotransmission could account for the coiling phenotypes of each mutant. Drug studies show that
unc-122 is hypersensitive to both aldicarb and levamisole, indeed suggesting a defect in cholinergic synaptic transmission. But,
unc-29unc-122 double mutants still coil strongly indicating that levamisole-sensitive acetylcholine receptors are not involved. Similar drug studies are underway for
unc-75 and preliminary results suggest that
unc-75 is also slightly hypersensitive to acute doses of aldicarb and is thus likely involved in neurotransmission. Our current hypothesis is that as in DVB,
unc-122 and
unc-75 may also act in an aminergic/peptidergic pathway that interacts with cholinergic neurotransmission, an interaction that has been observed in other systems as well (Weiss et al. J. Physiology 87: 141-151). We anticipate that further analysis of
unc-122 and determination of the molecular identity of
unc-75 will soon provide us a much clearer understanding of both coiling and DVB neurite sprouting defects.