Extended Data Figure 1: Trans-synaptic labeling by GRASP.

(a) The normally hermaphrodite-specific PHB>AVA and PHA>AVG synaptic connections fail to prune in post-dauer adult males, while the normally male-specific PHB>AVG and AVG>DA9 connections are pruned in post-dauer adult hermaphrodites. Top panel shows red cytoplasmic axon label, middle panel shows GRASP (PHB>AVA, PHB>AVG, AVG>DA9) or iBLINC signal (PHA>AVG), bottom panel shows magnified inset of color-inverted synaptic puncta with arrowheads to indicate puncta. Intestinal auto-fluorescence is labeled “gut.” Representative images shown, for quantification and replication see Fig. 1b,c and Methods.

(b) Starvation does not affect expression of the cell-specific promoters used for GRASP, and thus the effects on synaptic pruning are not an artifact of changes in promoter expression. Representative maximum intensity projection images of control animals and animals recovered from 24 hours of L1 starvation are shown. mu=muscle (srg-13p is variably expressed in some muscle cells in addition to PHA).

(c) Neither L1 heat shock (30min at 35C) nor L1 osmotic stress (24 hours on plates with 200mM NaCl) affect the male-specific pruning of the PHB>AVA connection. Each dot represents one animal (red=hermaphrodite, cyan=male in all figures, n=number of animals, shown in each column), blue bars show median, black boxes represent quartiles, vertical black lines show range (c, d). p-values shown by two-sided Wilcoxon rank-sum test with Bonferroni corrections for multiple testing (where applicable; see Methods).

(d) L1 starvation does not affect the normally male-specific PHB>AVG and AVG>DA9 synapses. Control animals are the progeny of starved animals.