(A) Phylogenetically conserved serotonin biosynthetic pathway. C. elegans protein names appear in black case, mammalian in grey. AADC: aromatic L-amino acid decarboxylase; GCH: GTP cyclohydrolase; TPH: tryptophan hydroxylase; Trp: tryptophan; VMAT: vesicular monoamine transporter; 5HTP: 5-hydroxytryptophan; 5HT: serotonin. (B) C. elegans hermaphrodite serotonergic system is composed of three subclasses of bilateral neurons (NSM, ADF and HSN, L: left, R: right). See Figure 1—figure supplement 1 for expression profiles of serotonergic subclasses. (C) Micrographs showing HSN 5HT staining and tph-1::gfp reporter expression defects of ast-1(ot417), unc-86(n846), sem-4(n1971), hlh-3(tm1688), egl-46(sy628) and egl-18(ok290) mutant animals (quantified in E). Scale bar: 5 μm. (D) Micrographs showing expression defects in the K+/Cl- cotransporter kcc-2::gfp reporter, a terminal feature of HSN not related to 5HT signaling, and normal expression of the extracellular matrix gene kal-1, indicating HSN is still present. (E) Heatmap summary of single TF mutant characterization. Statistically significant expression defects compared to wild type are indicated with a black frame. flp-19: FMRF-like peptide; ida-1: Tyr phosphatase-like receptor; lgc-55: amine-gated Cl- channel; nlg-1: neuroligin; rab-3: ras GTPase; unc-17: vesicular acetylcholine transporter; unc-40: netrin receptor. n.a: not analyzed. See Source data 1 for primary data and Fisher's exact test p-values and Figure 1—figure supplement 2 and Supplementary file 1 for analysis of additional alleles. n > 100 cells per condition.