A niche-derived non-ribosomal peptide triggers planarian sexual development

Germ cells are regulated by local microenvironments (niches), which secrete instructive cues. Conserved developmental signaling molecules act as niche-derived regulatory factors, yet other types of niche signals remain to be identified. Single-cell RNA-sequencing of sexual planarians revealed niche cells expressing a non-ribosomal peptide synthetase (nrps). Inhibiting nrps led to loss of female reproductive organs and testis hyperplasia. Mass spectrometry detected the dipeptide β-alanyl-tryptamine (BATT), which is associated with reproductive system development and requires nrps and a monoamine-transmitter-synthetic enzyme (AADC) for its production. Exogenous BATT rescued the reproductive defects after nrps or aadc inhibition, restoring fertility. Thus, a non-ribosomal, monoamine-derived peptide provided by niche cells acts as a critical signal to trigger planarian reproductive development. These findings reveal an unexpected function for monoamines in niche-germ cell signaling. Furthermore, given the recently reported role for BATT as a male-derived factor required for reproductive maturation of female schistosomes, these results have important implications for the evolution of parasitic flatworms and suggest a potential role for non-ribosomal peptides as signaling molecules in other organisms.

Fig. S1.Single-cell RNA sequencing of cells from sexual S. mediterranea.(A) tSNE plot of 39 clusters.(B) t-SNE plots of representative genes for nine major planarian tissue classes previously characterized in asexual S. mediterranea (14).All somatic tissue classes are present except for pharyngeal cells, which are not detected in this sexual sc-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. 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. 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. S3 .
Fig. S3.nrps is expressed in somatic gonadal niche cells.(A) Projection of ventral head region (top) and confocal section of ovary (bottom) showing dFISH of nrps (magenta) and aadc (green).(B) Confocal section of testes with nrps (magenta; cytoplasmic localization) and ophis (green; nuclear) co-expressing cells.ophis RNA localizes mainly to the nucleus of somatic gonadal cells, which extend long nrps + cytoplasmic projections that encyst developing germ cells.(C) Projections of confocal sections showing dFISH of nrps (magenta) with aadc (green; top), or yolk cell marker surfactant b (green; bottom) in the ventral posterior region of sexually mature planarians.Nuclei are counterstained with DAPI (gray; A-C).Scale bars, 100 µm (A, top; C), 50 µm (A, bottom; B).

Fig. S5 .
Fig. S5.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.