The kinesin-like protein ZEN-4/KIF23 and CYK-4/MgcRacGAP comprise the centralspindlin complex known to be required for cytokinesis. KIF23 is highly expressed in several human cancers [1-3], underscoring the importance of understanding its function. We have found a novel centralspindlin-independent role for C. elegans ZEN-4 in vulval development. During C. elegans larval development, the conserved EGFR/Ras/MAPK pathway relays an EGF signal from the gonadal anchor cell (AC) and induces vulval precursor cells (VPCs) to develop into the vulva. Loss of LET-23 EGFR signaling results in a Vulvaless (Vul) phenotype, while increased signaling causes a Multivulva (Muv) phenotype. Before VPC patterning, the AC is also specified through a LIN-12 Notch signaling cascade. Using temperature sensitive mutants and RNAi, I bypassed the embryonic lethality of
zen-4 and
cyk-4 loss of functions and found that loss of
zen-4 during larval development resulted in a strong (94%) Vul phenotype, while loss of
cyk-4 did not affect development. I hypothesized that ZEN-4 functions in either the VPCs or the AC to promote EGFR-mediated vulval development. To determine ZEN-4's position relative to the LET-23 EGFR pathway, I performed genetic epistasis of
zen-4(RNAi) with LET-23 EGFR negative regulators
lin-1 and
lin-15A/B. I found that
zen-4(RNAi) does not suppress the Muv phenotypes of either
lin-1(
n304) or
lin-15A/B
(n765). This suggests that ZEN-4 is not required for VPC division and that it functions upstream of LET-23 EGFR signaling. Therefore, I investigated ZEN-4's potential role in AC specification by analyzing expressions of the GFP::
hlh-2 AC reporter [4] before and after specification. Unlike the empty vector control which specified a single GFP::
hlh-2 positive cell from 4 progenitors,
zen-4(RNAi) and
cyk-4(RNAi) animals often had 0 or 2 positive cells with fewer progenitors before the specification event, suggesting a possible defect in progenitor cell division. Interestingly,
zen-4(RNAi) alone also affected the localization of GFP::
hlh-2 positive cells. I performed temperature upshifts of
zen-4(
or153) exclusively at the L2 stage and observed that it is sufficient to induce the Vul phenotype. Altogether, these results suggest that ZEN-4 promotes vulval development through its regulations of AC specification and localization. References: [1] Takahashi, S., et al., J Neurooncol, 2012. [2] Kato, T., et al., Lung Cancer, 2016. [3] Li, X., et al., Dis Markers, 2019. [4] Attner, M.A., et al., Curr Biol, 2019.