The AGEF-1/ARF-1 GTPase/AP-1 trafficking pathway differentially regulates LIN-12/Notch signaling in a tissue specific manner in C. elegans

LIN-12/Notch signaling regulates C. elegans vulval development via cell fate specifications in the gonad and epidermis. In the somatic gonad LIN-12/Notch activity specifies the anchor cell (AC) versus ventral uterine cell (VU) fates, with VU receiving more signal. The AC secretes epidermal growth factor (EGF) which induces the underlying vulval precursor cells (VPCs) to adopt vulval fates. In the VPCs the secondary vulval fates are specified by LIN-12/Notch activity. We previously reported that the AGEF-1, an Arf GEF homologous to ArfGEF1 and ArfGEF2, the ARF-1 GTPase, and the adaptor protein complex 1 (AP-1) inhibit LET-23/EGF receptor (EGFR) signaling in the VPCs by antagonizing LET-23/EGFR basolateral localization. Here we report that AGEF-1, ARF-1 and AP-1 regulate LIN-12/Notch signaling during somatic gonad and vulval development. The lin-12(n302) partial gain-of-function causes a potent Vulvaless phenotype due to a lack of AC specification. We demonstrate that loss of AGEF-1, ARF-1 or AP-1 restored the AC fate in lin-12(n302) animals, indicating that AGEF-1/ARF-1/AP-1 promotes LIN-12/Notch signaling in the somatic gonad. Interestingly, loss of AGEF-1, ARF-1 or AP-1 also induced ectopic vulval secondary fates in lin-12(n302) animals, indicating that AGEF-1/ARF-1/AP-1 inhibits LIN-12/Notch in the VPCs. Using a LIN-12/Notch biosensor we demonstrate that loss of UNC-101/AP-1 results in decreased signaling in the VU cell and increased signaling in the VPCs that correspond with decreased expression levels of LIN-12/Notch and LAG-1/DSL ligand in the presumptive AC and VU while also causing increased apical localization of LIN-12/Notch in the VPCs. We hypothesize that the differential regulation of LIN-12/Notch signaling could reflect different trafficking pathways in epithelial cells (VPCs) versus non-epithelial cells (AC and VU). Our results indicate that the AGEF-1/ARF-1/AP-1 trafficking pathway maintains the VPC cell fate patterning by limiting both LET-23/EGFR and LIN-12/Notch signaling. Author summaryCell signaling and membrane trafficking are highly interconnected processes whereby membrane trafficking can regulate signal transduction pathways and vice versa. We previously demonstrated that the ARF-1 GTPase, the downstream AP-1 clathrin adaptor and upstream activator AGEF-1 antagonize the membrane trafficking of the Epidermal Growth Factor Receptor (EGFR) and hence signaling during C. elegans vulva induction. Strong loss of the ARF-1 GTPase pathway resulted in ectopic vulval induction. Here we demonstrate that the ARF-1 GTPase pathway differentially regulates Notch signaling to regulate vulva induction. In the somatic gonad it promotes Notch signaling to regulate the specification of the anchor cell which secretes the inductive signal. In the vulva precursor cells, the ARF-1 GTPase pathway antagonizes Notch signaling which cooperates with EGFR signaling to induce the vulval cell fates. We hypothesize that the differential regulation of Notch signaling by the ARF-1 GTPase pathway could be a result of more complex membrane trafficking pathways in polarized epithelial cells (vulva precursors) versus non-epithelial cells in the developing somatic gonad. Thus, the AGEF-1/ARF-1/AP-1 antagonizes both EGFR and Notch signaling in ensuring that only three of the six vulval precursor cells adopt are induced.