Figure 2. The retromer complex is required for EGL-20 gradient formation. Whole-mount larvae carrying the integrated Pegl-20::
egl-20::proteinA transgene were fixed 1 to 5 hours after hatching and stained with a fluorescein isothiocyanate-coupled antibody. (A and B) In wild-type animals, EGL-20 forms a punctate anteroposterior gradient. (C) This gradient is not affected in
bar-1(
ga80) mutants. (D) In
vps-35(
hu68) mutants, the EGL-20 gradient is absent. Arrowheads indicate P11/12 (23). White bars delimit the group of
egl-20-expressing cells. Dashed lines represent the range of the gradient. A similar gradient was observed in live animals using an EGL-20::Venus fusion (fig. S4). (E) Schematic representation of the EGL-20 gradient. The punctate EGL-20 staining is indicated by green dots;
egl-20-expressing cells are in blue. (F) Loss of
vps-35 does not affect global levels of EGL-20. Western blots of total first and second larval stage lysates were stained for EGL-20::proteinA. Neg, nontransgenic animals. Equal loading was confirmed using an antibody to alpha-tubulin.