Figure 7. Anterior lateral microtubule cell (ALMR) exophergenesis can be induced by uterine compartment distortion that accompanies fluid injection.
(A) Summary of timing of ALMR exophergenesis from age synchronized spermless + vulvaless hermaphrodite. Total of three trials and 50 hermaphrodites in each trial. Strain ZB4749: fxIs1[Ppie-1::TIR1::mRuby] zdIs5[Pmec-4::GFP] I; bzIs166[Pmec-4::mCherry] II; spe-44(fx110[spe-44::degron]) IV, cultured on the 1 mM auxin treated nematode growth media (NGM) agar plates seeded with HT115 E. coli expressing dsRNA against the C. elegans lin-39 gene. Eventually, oocytes accumulate in this strain, but at worst very few are evident in the timeframe in which we performed injections. Data shown are mean ± SEM at each time point. The data demonstrate that there is no ALMR exophergenesis in this background before the 18th hr after L4 sync. Testing the impact of injection on ALMR exophergenesis before the 17th hr after L4 thus monitors injection consequences during a timeframe in which no exophers are normally produced. (B) Illustrated experimental design for testing the ALMR exophergenesis response to physically expanding the gonad via 2 min continuous fluid injection. We performed 2+ min duration injections of M9 buffer mixed with food color dye 1:10 or 1.5:10 dye/M9 ratio (to verify successful injection; dye contains water, propylene glycol, FD&C reds 40 and 3, propylparaben) into the uteri of sperm-less only (EV control) or sperm-less + vulvaless (treated with lin-39 RNAi) animals. Strain: ZB4749 as in panel A, treated with either empty vector (EV) or RNAi against lin-39 in the presence of 1 mM auxin. lin-39 RNAi disrupts vulval development such that injected fluids are retained to expand the uterus. In injections with animals that have functioning egg-laying apparatus, fluids can exit the animal and do not expand the uterus. (C) 2 min sustained injection into egg-laying blocked, reproduction blocked animals can induce rapid exophergenesis: representative picture of an ALMR exophergenesis event consequent to injection. (D) 2 min sustained injection into egg-laying blocked, reproduction blocked animals can induce rapid exophergenesis: exopher scoring of the control mock injected and 2 min injected animals. Strain ZB4749: fxIs1[Ppie-1::TIR1::mRuby] zdIs5[Pmec-4::GFP] I; bzIs166[Pmec-4::mCherry] II; spe-44(fx110[spe-44::degron]) IV treated with auxin and lin-39 RNAi to induce the sperm-less and vulva-less status, respectively. Data represent a total of three trials (6–10 worms in each trial). p<0.05 in Cochran–Mantel–Haenszel test, as compared to the no-injection fluid control. (E) 2 min sustained injection induces rapid ALMR exophergenesis only from vulvaless animals. Data showing the exopher scoring of the sperm-less EV control (with functional vulva) or sperm-less +vulvaless (treated with lin-39 RNAi) worms. Strain: ZB4749 (genotype in panel A legend) treated with either EV or RNAi against lin-39 in the presence of 1 mM auxin to disrupt sperm maturation. Data represent a total of 6 trials (6–10 worms in each trial). 3 out of 6 trials showed ALMR exopher induction by M9 injection to the vulvaless worms; while not a single trial produced ALMR exopher induction by M9 injection to animals with WT vulvae. If the vulva is intact, injected fluids are observed to leak out, consistent with the assumption that the gonads of egg-laying proficient animals will not sustain required expansion. (F) Summary. Eggs, dead egg accumulation, oocyte accumulation, or injection pressure all lead to ALMR exophergenesis. These varied interventions have a similar impact on the uterus, which is the uterine distortion by mechanical forces. We propose that early adult ALMR exophergenesis requires mechanical or stretch-associated force generated by the uterine cargos.