(A–C) Whole-mount in situ hybridization to detect pc2 mRNA in sexual animals. (A) Ventral view, expression in CNS, pharynx, and copulatory apparatus. (B and C) Dorsal view, expression in testes. (D, E) DAPI staining showing the distribution of testes in (D) control and (E) pc2(RNAi) animals fixed 17 d after the initiation of RNAi treatment. (F and G) Single confocal sections showing expression of nanos (magenta) and gH4 (green) in testes of (F) control and (G) pc2(RNAi) animals. DAPI staining is shown in grey. Orange and yellow arrows indicate spermatids and mature sperm, respectively. White arrows indicate germ line stem cells expressing both gH4 and nanos. Scale bars: (A–C) 300 µm; (D and E) 500 µm; (F and G) 50 µm. Abbreviations: CG, Cephalic Ganglia; VNC, ventral nerve cord; PH, pharynx; CA, copulatory apparatus.

(A) Schematic representation of the methodology used for the identification and confirmation of planarian prohormones and their respective peptides. We performed homology and pattern searches for preliminary annotation of peptide prohormone genes and subsequently verified these predictions with molecular techniques. The post-translational processing of verified prohormones to bioactive peptides was then predicted in silico using Neuropred and the sequences of mature peptides were then confirmed in whole animal tissue extracts from sexual and asexual planarians by LC-MS/MS and/or MALDI-TOF MS. This approach is depicted in blue ovals. To complement the bioinformatics-driven discovery, de novo sequencing of unassigned MS peaks was used to characterize novel neuropeptides (red ovals). The sequences of such peptides were then mapped to the S. mediterranea genome and new prohormone genes were annotated. These prohormone genes were then analyzed further, leading to the characterization of additional peptides. (B) Full sequence coverage of prohormones SPP-1B, SPP-3, SPP-4, NPP-18, and PPP-1 by mass spectrometry. Underlined sequences indicate peptides identified by MS/MS sequencing and the shaded sequence indicates a peptide detected by MS mass match. Signal peptides for each prohormone are italicized. (C and D) S. mediterranea possesses an expanded NPY family. (C) ClustalW alignment of two vertebrate NPY-family peptides, Pancreatic Polypeptide (PP), with a variety of invertebrate NPY family members. Matching residues are shown in yellow and a conserved α-amidation site is shown in green. C-terminal tyrosine and phenylalanine are highlighted in magenta and blue, respectively. (D) Gene structure of vertebrate and S. mediterranea npy genes. These prohormone genes have an intron within the arginine codon preceding the aromatic amino acid residue (blue), the α-amidation site (green), and the dibasic cleavage site (magenta). npy-11 lacks a C-terminal aromatic residue but also shares this gene organization. (E and F) MALDI-MS analysis of pc2 RNAi in sexual animals. (E) Comparison of peptide profiles for control and pc2(RNAi)-treated sexual animals 16 d after the initiation of RNAi treatment. MALDI-TOF MS spectra (limited to m/z 1150–1450) comparing control and pc2(RNAi) groups (n = 7 for each group); stars indicate peaks that were significantly different (p<0.05). (F) Characterized peptides and their respective prohormones that were detected at significantly different levels (p<0.05) following pc2 RNAi. The pc2 RNAi/control column reports the ratio of peak intensities of pc2 RNAi relative to control.

Prohormone genes are displayed alphabetically. Full gene names are provided in . No expression was detected for npy-8 in asexual animals. Arrow for npy-11 indicates expression at the distal region of the pharynx. Gene names in bold indicate prohormones with at least one peptide confirmed by MS analysis. Ventral views, anterior towards top. Scale bars (to right of images), 300 µm.

(A) Three-color FISH for ppp-1, npp-2, and spp-1. (B and C) Merged images, colors indicated in panel A. Prohormone genes ppp-1, npp-2, and spp-1 are not predicted to encode any related peptides and do not appear to have overlapping distributions within the CNS. (D) Three-color FISH for prohormone genes spp-6, spp-7, and spp-9. (E and F) Merged images, colors indicated in panel D. Prohormones genes spp-6, spp-7, and spp-9 encode related prohormones that are co-expressed in cells between the VNCs; the expression of these genes is not co-localized in the CNS. Images from (A–F) are confocal projections; whole animal views in (A) and (D) are derived from tiled stacks. Ventral views, anterior towards left. Scale bars, 100 µm.

(A) Double FISH showing expression of prohormone genes in anterior and posterior photoreceptor neurons. Top left, DAPI staining (magenta) and immunofluorescence with VC-1 antibody that recognizes arrestin (green) to show the photoreceptor cell bodies (magenta surrounded by green) and their projections (green); image also indicates orientation for the other images in the panel. Remaining images are a matrix showing FISH for each prohormone gene expressed in the photoreceptors in comparison to the other three genes. All panels are shown overlaid with differential interference contrast optics. Dorsal view, anterior towards top. (B) Prohormones mpl-2 and eye53-2 are expressed differentially along the dorsal-ventral (D-V) axis of the photoreceptors. Shown is a maximum projection of a confocal XZ-series through the photoreceptors. Left, staining with the VC-1 antibody showing the photoreceptor cell bodies (pseudocolored red) and their rhabdomeric projections (pseudocolored yellow). Lateral (L) and medial (M) domains are indicated. Middle three panels, FISH with mpl-2 and eye53-2; colors are indicated at bottom. Right, FISH and immunofluorescence with the VC-1 antibody (grey). Posterior view, dorsal towards top, medial towards right. Scale bars, 25 µm.

(A) Diagram depicting the location of various organs in sexual S. mediterranea. Right, enlarged view of the copulatory apparatus. Abbreviations: SV, Seminal vesicles; CB, copulatory bursa; BC, bursa canal; PP, penis papilla; GP, gonopore; G, cement glands. (B–H) Genes are listed with their sexual-specific expression pattern; (B–F) expression in asexual (As) and sexual (S) animals is shown. (B) cpp-1; oviducts and penis papilla. (C) npp-22; oviducts and penis papilla. (D) npp-2; penis papilla. (E) npy-9; penis papilla and cement glands. (F) npp-18; gland cells surrounding copulatory apparatus. (G) spp-10; testes, (H) ilp-1; testes. Scale bars, 300 µm.

Comparison of the ventral expression of (A) ppl-1 or (B) npy-8 between asexual, immature sexual hatchlings, and mature sexual animals. (C) Dorsal expression of npy-8 in immature sexual hatchlings (left) and mature sexual animals (right). (D) Transparency rendering showing expression of npy-8 in a cell in close proximity to testes lobes. Inset shows higher magnification of npy-8-expressing cell. (E) Northern blot comparing expression of npy-8 in asexual “As,” immature sexual hatchlings “H,” juvenile sexual animals “J,” and mature sexual animals “M.” grb-2 (GB: DN305385) is expressed at similar levels in asexual and sexual animals (J. Stary and P. Newmark, unpublished observations) and is shown as a loading control. Scale bars: (A–C) 300 µm; (D) 10 µm.

(A) Sequence of the prohormone and predicted peptides encoded by npy-8. Following removal of the signal peptide (italics) the NPY-8 prohormone is predicted to be processed at two consensus prohormone convertase cleavage sites (red). This cleavage would result in two peptides: the C-terminally amidated peptide NPY-8A (potential amidation site is shown in purple) and the 15–16 AA peptide NPY-8B. (B) DAPI staining showing distribution of testes in control and npy-8(RNAi) animals at 4 wk after the first RNAi treatment. Arrows show region of the copulatory organs. (C) Penis papilla of control and npy-8(RNAi) animals visualized by DAPI staining (red) and differential interference contrast microscopy. Anterior towards top. (D) Ventral view of live control and npy-8(RNAi) animals showing the pharyngeal opening (PH) and the gonopore (GP). Anterior towards left. (E) Single confocal sections showing expression of nanos (magenta) and gH4 (green) RNAs in testes of control (top) and npy-8(RNAi) animals that display either an intermediate or severe level of testes regression. DAPI staining is shown in gray. Animals were fixed ∼7 wk after the initiation of RNAi treatment. (F) Maximum confocal projection showing the localization of the npy-8 and pc2 transcripts surrounding the nucleus (gray) of a neuron at the level of the ventral sub-muscular neural plexus in a mature sexual animal. Similar co-localization was seen in other parts of the central and peripheral nervous systems (unpublished data). Scale bars: (B) 500 µm; (C–D) 300 µm; (E) 20 µm; (F) 10 µm.