Extended Data Figure 6: Regulation and neuronal migration effects of serotonin signaling.

(a) Effects of L1 starvation on male-specific synaptic pruning can also be rescued by exogenous serotonin during L3 (while animals are feeding). Each dot represents one adult male animal (n= number of animals, shown in each column), blue bar represents median, black box represents quartiles, vertical black bars represent range (a, b, e). p-values shown by two-sided Wilcoxon rank-sum test (a, b, e).

(b) tph-1 overexpression in NSM does not rescue starvation effects on pruning. Quantification of PHB>AVA and PHA>AVG synaptic connectivity in L1 starved adult males overexpressing tph-1 in NSM. Two independent transgenic lines were tested for each experiment, L1 starved animals without transgenic lines are siblings of transgenic animals, controls are non-starved adult males with transgenic arrays. None of the transgenic lines resulted in partial or complete rescue.

(c) Overexpression of tph-1 under an ADF-specific promoter during L1 starvation rescues the male-specific pruning of the PHB>AVA and PHA>AVG synaptic connections. Representative images shown, for quantification and replication see Fig. 3e and Methods.

(d) tph-1 (n4622) and ttx-3 (ot22); unc-86 (n846) mutants (in which the NSM neuron does not express tph-1 or produce serotonin²⁹) have cell body displacement, dendrite, and axon fasciculation defects in the phasmids. Overexpression of tph-1 under an NSM-specific promoter in the tph-1 (n4622) mutant background rescues the severity and penetrance of these defects. Representative images of defects in the PHB neuron are shown here as inverted black and white fluorescence images. * indicates dendrite defect, arrow shows anteriorly shifted cell body, arrowhead shows fasciculation defect. Scale bars 10μm. Percent animals with visible defects categorized and quantified to the right, n= number of animals, shown in each column. p-values shown by Freeman-Halton extension of one-sided Fisher exact test.

(e) Overexpressing tph-1 under an NSM-specific promoter in the tph-1 (n4622) mutant background (essentially, an ADF-specific tph-1 null) results in male-specific PHB>AVA pruning defects. Of two independent NSM::tph-1 transgenic lines, one resulted in a slight but insignificant defect in pruning, and one resulted in a significant defect in pruning.