Figure 4. Anterior Lateral Microtubule cell (ALMR) exophergenesis levels correlate with proximity to the egg zone.

(A) ALMR is positioned close to the uterus and Anterior Lateral Microtubule cell (ALML) is situated on the opposite side, close to the intestine. A diagram of ALMR and ALML positions relative to major body organs in cross-section that is anterior to the ‘egg zone.’ Image drawing based on the EM pictures of adult hermaphrodite slice #273 of WormAtlas. DNC: dorsal nerve cord; VNC: ventral nerve cord; Hyp7: hypodermal cell 7; CAN(L/R): canal-associated neurons (left/right); neurons in red. (B) Electron microscopy cross-section image of the uterus region indicating ALMR soma and eggs within the adult uterus. Note ALMR is close to the egg-filled uterus, ALML is on the opposite side closer to the intestine. The ALML soma is not evident in this cross section. Scale bar = 10 μm. (C) Illustration of the egg zone definition, distance between outermost eggs. The measure of this distance corresponds to uterine length. (D) ALMRs positioned close to the filled gonad produce exophers more frequently than ALMRs that are positioned a bit more distally. We selected Ad2 mCherry animals at random, then identified whether or not ALMR had produced an exopher, and subsequently determined whether ALMR was positioned within the egg zone or outside the egg zone as indicated (neuronal soma positioning differences are a consequence of developmental variation). Neurons with somas positioned further from the egg area produced fewer exophers than neurons within the egg zone indicated; total of 91 worms for the ‘No Exopher’ group and 37 for the ‘Exopher +’ group; **p<0.01 in Chi-Square test. (E) The Aamodt group (Toms et al., 2001) previously reported that high copy numbers of plasmid pJC4 containing the mec-3 promoter region (−1 to –563, and –1898 to –2372 of the mec-3 translational start) exhibited increased abnormal positioning of ALM neurons anterior to AVM. We introduced plasmid pJC4 along with transformation reporter pRF4 rol-6(su1006) in the background of mCherryAg2 (note this revealed that rol-6(su1006) is a strong exopher enhancer) and identified neurons that were positioned posterior to AVM (normal, close to the uterus) and those that were positioned anterior to AVM (further away from the uterus). (F) ALMR neurons genetically induced to adopt positions further away from the uterus generate fewer exophers then those close to the uterus. We counted numbers of exophers produced in Ad2 Rol hermaphrodites for each position type. Strain ZB5046: Ex [(pJC4) P_mec-3::GFP +pRF4]; bzIs166[P_mec-4::mCherry] II. Total of three trials (61(34a + 27p); 39(19a + 20p); 51(20a + 31p) animals per trial); ****p<0.0001 in Cochran–Mantel–Haenszel test. (G) When eggs cannot be laid in the sem-2(rf) mutant, the eggs that accumulate in the body are brought in closer proximity to ALML, AVM, and PVM touch neurons with a resulting increase in their exopher production. Strain ZB4757: bzIs166[P_mec-4::mCherry] II vs. strain ZB4902: sem-2(n1343) I; bzIs166[P_mec-4::mCherry] II. Left, Exopher scoring (Mean ± SEM) of all six touch receptor neurons in Ad1 wild-type hermaphrodite; Right, Exopher scoring (Mean ± SEM) of all six touch receptor neurons in Ad1 sem-2(rf) hermaphrodite. Total of 4 trials (50 worms per trial) for each. Wild-type, egg laying proficieint animals on Ad1 exhibit low exopher levels, but when eggs accumulate early in the sem-2 mutant, exophers markedly increase in ALMR and other touch neurons that are in the vicinity of an expanded uterus. PLM neurons are situated posterior to the anus and are not subject to uterine squeezing effects.