Figure 4.

Wnt pathway asymmetry characterization in posterior seam cell lineages at the L4 stage. (A) Representative images of egl-18 smFISH (black dots correspond to mRNAs) in V6papp and V6pppp daughter cells at 20° and 25°. Seam cells are marked green due to expression of the scm::GFP marker. (B) Quantification of egl-18 mRNA levels in V5pppp, V6papp, and V6pppp daughters at 20° and 25°. Expression was significantly lower in posterior cells, i.e., V5ppppp, V6pappp, and V6ppppp at 25° (red circles) compared to the same seam cells grown at 20° (blue circles, **P < 0.01 and ***P < 0.001 with a two-sample Student’s t-test, n > 20 per cell). At low frequency, animals grown at 25° showed extreme expression values in V6pappa and V5ppppa cells, which were even higher than their posterior counterparts (cell pairs are indicated by black lines). (C) Seam cell duplications at anterior V6 lineage are suppressed in the egl-18(ga97) mutant at 25° (***P < 0.001 with a binomial test, n = 120). (D) Representative images of nuclear POP-1::GFP expression at 20° and 25° at the L4 stage. (E) The ratio of nuclear POP-1::GFP expression between V6papp and V6pppp daughter cells at 20° and 25°, n ≥ 22 (*P < 0.05 and ***P < 0.001, two-sample Student’s t-test, error bars indicate 95% C.I.s of the mean). In both cases, anterior/posterior cell fluorescence intensity ratio is shown (simplified as “aa”/“ap” for V6papp daughters and “pa”/“pp” for V6pppp). (F) Average POP-1::GFP intensity in V6papp and V6pppp daughter cells at 20° and 25° (error bars represent SEM, *P < 0.05 and **P < 0.001, with a two-sample Student’s t-test, n ≥ 22). L4, fourth larval; mRNA, messenger RNA; RNAi, RNA interference; smFISH, single-molecule fluorescence in situ hybridization; WT, wild-type.