The nitric oxide (NO) -- cGMP signaling pathway is involved in many important physiological processes including neuronal signaling, axon guidance and maintenance of vascular tone. Nitric oxide synthase (NOS) synthesizes NO which diffuses to target cells, activating soluble guanylyl cyclase (sGC), generating cGMP. Soluble GC is classically a heterodimeric protein and requires an a and b subunit for catalytic activity. However, the discovery of a novel homodimeric, NO-insensitive sGC (MsGC b 3 ) from the tobacco hawk moth, Manduca sexta , implies that other control mechanisms may exist for these proteins. C. elegans appears to have a modified NO-cGMP pathway because no NOS gene is evident in its genome. Also, worm sGCs contain structural features likely to render them insensitive to NO. Of the seven sGC genes in C. elegans , we are primarily interested in
gcy-31 because it shows the highest homology to MsGC b 3 . We are using genetic and biochemical approaches to establish roles for
gcy-31 in C. elegans development and physiology. We previously localized
gcy-31 expression to a pair of bilaterally symmetric neurons in the worm head using extrachromosomal array line qaEx2201[
gcy-31(3' deletion) ::GFP
rol-6(
su1006) ]. Detailed analysis of this line revealed low level embryonic lethality, with some survivors showing deformations in the head similar to v ariable ab normal (vab) mutants. 4D-microscopy of developing qaEx2201 embryos identified defects in hypodermal enclosure, similar to those seen in
vab-1 , 2 and 3 mutants. Control experiments revealed these defects were caused by over expressing just the first 4 exons of
gcy-31 , as extrachromosomal array lines expressing full length
gcy-31 had almost no phenotype. These defects may be caused by titering out a transcription factor such as the Pax-6 homologue
vab-3 . However, array lines containing just the
gcy-31 promotor region were wild type. Furthermore, when qaEx2201 was crossed into
vab-3(
e643) worms their phenotypes were additive, implying that
vab-3 is not directly involved in
gcy-31 transcription. We hypothesize that the N-terminal fragment of
gcy-31 in qaEx2201 is dimerizing with wildtype
gcy-31 to generate a catalytically inactive species which is somehow responsible for the phenotype observed. We are currently screening for
gcy-31 deletion alleles to further characterize its role during C. elegans embryogenesis.