A niche-derived nonribosomal peptide triggers planarian sexual development

Significance Throughout animals, germ cell development is regulated by extrinsic signals from somatic gonadal niche cells. Studies on traditional model organisms have shown that widely conserved signaling molecules, which activate developmental programs, also control germ cell development. Here, we show that a nonribosomal peptide can also act as a critical niche signal. The neurotransmitter-biosynthetic enzyme aadc plays a nonneuronal function in reproductive niche cells in conjunction with a nonribosomal peptide synthetase (nrps), to synthesize a tryptophan-derived metabolite: β-alanyl-tryptamine (BATT). Synthetic BATT rescues reproductive defects in nrps knockdown animals and triggers precocious sexualization in immature animals. This work provides an unanticipated niche-to-germline signaling paradigm and highlights the potentially broad involvement of nonribosomal peptides as signaling molecules across the animal kingdom.

Fig. S1.Single-cell RNA sequencing of cells from sexual S. mediterranea.(A) t-SNE plot of 39 clusters.(B) t-SNE plots of representative genes for nine major planarian tissue classes previously characterized in asexual S. mediterranea (1).All somatic tissue classes are present except for pharyngeal cells, which are not detected in this sexual scRNA-seq dataset since we enriched for reproductive tissues lacking this organ.(C) t-SNE plots showing expression of somatic gonadal gene markers dmd1, ophis, LamA, aadc, and nrps.

Fig. S2 .
Fig. S2.Smed-nrps encodes a non-ribosomal peptide synthetase.(A) Schematic showing adenylation (A), thiolation (T), and amine-selecting (AS) domains in D. melanogaster Ebony and the Schmidtea mediterranea homolog NRPS.Drosophila and S. mediterranea share a conserved serine residue in their thiolation domains.NRPS proteins like Ebony can conjugate β-alanine to various biogenic amines (e.g., dopamine, histamine, etc.).This enzymatic process involves three steps: adenylation of β-alanine catalyzed by the adenylation (A) domain; covalent attachment of β-alanine to a phosphopantetheinyl group on a conserved serine within the thiolation (T) domain; and binding of an amine in the amine-selecting (AS) domain, which facilitates nucleophilic attack of the NRPS-bound β-alanine resulting in a β-alanyl-amine dipeptide product.(B) Protein alignment of Drosophila Ebony, S. mediterranea NRPS, and S. mansoni NRPS.The serine thiolation site (marked by an asterisk) in the T domain (delineated by red lines) is conserved.

Fig. S4 .
Fig. S4.Testing nrps RNAi specificity and quantifying nrps expression in knockdown animals.(A) To test for nrps RNAi specificity and exclude the possibility of off-target effects, RNAi was performed with dsRNA targeting three ~1 kb regions of nrps: the original region used throughout this study, and 2 non-overlapping regions (N-terminus vs C-terminus).nrps gene (gray bar) is shown with positions for start (green) and stop (red) codons, exon-exon boundaries (dark gray), cloned regions (bottom), and qPCR amplicons (top: A-E).(B) qPCR analysis of nrps mRNA expression normalized to β-tubulin in control and nrps RNAi animals depicting efficient knockdown of nrps after RNAi.Top: dsRNA targeting the N-terminus of nrps was used for RNAimediated knockdown of nrps, and qPCR primers targeting regions C, D, and E were used to quantify nrps expression levels.Middle: dsRNA targeting the C-terminus of nrps was used for RNAi and qPCR primers targeting regions A, B, and E were used to quantify nrps expression levels.Bottom: dsRNA targeting the original cloned amplicon of nrps and qPCR primers targeting region E were used to quantify nrps expression levels.N = 4 biological replicates (3 technical replicates each).Bar graphs depict relative quantification (2 −ΔΔCt ) values normalized to control RNAi with 95% confidence intervals.

Fig. S6 .
Fig. S6.Colorimetric in situ hybridization of nrps paralogs.nrps transcript is expressed in the reproductive system and a paralogous nrps is expressed in the gut in adult sexual S. mediterranea.Scale bars, 1 mm.

Fig. S7 .
Fig. S7.BATT triggers sexual maturation.Quantification of sexual planarian length (mm; left Y axis; horizontal line represents median) and gonopore presence (right Y axis) during development.One-week old hatchlings were fed liver +/-BATT for 6 weeks.Supplementation with BATT did not affect growth but triggered precocious sexual maturation (evidenced by the presence of a gonopore) in +BATT individuals.n=10-18 planarians per time point.