Molecular analysis of UNC-4 pathway genes that regulate synaptic choice

Abstract

Neural function depends on the creation of synapses between specific neurons. Across species, transcription factor codes regulate the precise connectivity between neurons. In the C. elegans motor circuit, these critical features are controlled by the UNC-4 homeodomain protein. unc-4 mutants are unable to execute backward locomotion due to the miswiring of VA motor neurons with inputs normally reserved for VB sisters. Thus, we have proposed that UNC-4 preserves VA inputs and backward movement by repressing genes that promote VB-type wiring. Here we show that UNC-4 opposes the function of multiple downstream components, discovered in a genetic screen for UNC-4 pathway interactors. Specifically, we show that UNC-4 disables a signaling cascade involving the Frizzled proteins MOM-5 and MIG-1 to prevent VAs from responding to an EGL-20/Wnt cue that drives the creation of VB-type inputs. EGL-20/Wnt acts through a canonical Wnt signaling pathway to promote expression of the VB protein and transcription factor CEH-12/HB9 which in turn leads to the miswiring of unc-4 mutant VAs. This effect is regionally limited to VA motor neurons in the posterior nerve cord nearest the source of EGL-20/Wnt. The work also revealed unc-4 interactions with G-protein signaling pathways that regulate VA input specificity. UNC-4 antagonizes the Gαo homolog, GOA-1/Gαo. GOA-1 has been previously shown to inhibit acetylcholine (Ach) release in opposition to the Gαq homolog, EGL-30 and the Gαs homolog, GSA-1. Intriguingly, egl-30 and gsa-1 also antagonize goa-1 in the unc-4 pathway. However, the roles for these G-proteins in synaptic choice are independent of their function in regulating Ach secretion and thus are likely to involve other downstream components of the Gαo, Gαq and Gαs signaling pathways. Our results show that GOA-1 signaling functions in parallel to the egl-20/Wnt pathway to regulate VA inputs. Together, these findings demonstrate the critical role of precise transcriptional control in the regulation of responses to signaling pathways that specify connectivity in the nervous system.