Vasopressin-oxytocin-type signaling is ancient and has a conserved water homeostasis role in euryhaline marine planarians

Abstract

Vasopressin/oxytocin (VP/OT)-related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the "platytocin" system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an "antidiuretic hormone" and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses.

Fig. 1.. Platytocin (PT), a VP/OT-related neuropeptide, signals through a platytocin receptor (ptr).

(A) Domain structure/amino acid (aa) sequence of VP, OT, and PT precursors. (B) Amino acid sequence of PT and other VT/OT-type peptides. (C) and (D) Dose-response curves of COS-7 cells transfected with ptr when exposed to synthetic PT peptide (C) and OVTA (D), demonstrated by measuring the intracellular IP₁ responses. Error bars in all figures indicate mean ± SEM (n = 4). AN, annetocin; CP, conopressin; IT, inotocin.

Fig. 2.. Platytocin and a platytocin receptor (ptr) expression patterns.

(A) In situ hybridization reveals a pair of platytocin neurons on either side of the cranial ganglion (white arrows). (B) Immunohistochemistry shows the two neuronal cell bodies (white arrows) and some of their processes. Scale bars, 100 μm in the lower-magnification images and 10 μm in the higher magnification images in (A) and (B). (C) Electron microscopy shows a neuronal process containing a number of platytocin-immunoreactive dense-cored vesicles. The box shows at higher power the immunogold reactivity of a cluster of five such vesicles (arrows). Scale bars, 200 nm in the lower-magnification image and 50 nm in the higher-magnification image. (D) The whole worm picture for ptr expression by in situ hybridization. Scale bar, 1 mm. (E) The ptr gene is expressed in a number of neurons in the cranial ganglion and in the putative mushroom bodies, cells in the muscular layer of the pharynx, and interstitial cells scattered in the peripheral tissues including protonephridial and gonadal regions (arrowheads). Scale bars, 100 μm in the lower-magnification images and 10 μm in the higher-magnification images in (E).

Fig. 3.. Platytocin induces hyperosmolality resistance and egg-laying.

(A to C) Survival curves for control [gray dashed line, n = 20 (A), 45 (B), and 12 (C) planarians], 10⁻⁹ M platytocin-treated [light green, n = 20 (A) and 12 (C) planarians], 10⁻⁷ M platytocin-treated [green, n = 30 (A) and 12 (C) planarians], 10⁻⁵ M platytocin-treated [dark green, n = 40 (A) and 12 (C) planarians], and the antagonist of the platytocin pathway OVTA-treated (magenta, n = 45 planarians) S. pusilla in 70-ppt salinity (A and B) or 14-ppt salinity (C). (A) Hyperosmolality (70 ppt) resistance is induced by 10⁻⁷ M platytocin. (B) Hyperosmolality (70 ppt) resistance is blocked by 10⁻⁶ M OVTA. (C) There was no significant effect of platytocin on hypoosmolality resistance. Probably because of the effect of high pharmacological dose of platytocin on the endogenous platytocin system, 10⁻⁵ M platytocin has no significant effect on survival. n.s., not significant. (D) Relative platytocin expressions in animals exposed to 14-ppt salinity (white), 34-ppt salinity (blue), and 50-ppt salinity (dark blue) for 4, 8, and 24 hours. *P < 0.05; error bars indicate SEM (n ≥ 9 planarians; n ≥ 3 independent assays). (E) Acetylated α-tubulin immunohistochemistry shows the protonephridia (white arrowheads). In situ hybridization of serial sections shows the platytocin receptor (ptr) expression (magenta arrowheads). The merged image (right) shows that ptr is expressed in the protonephridia. Scale bar, 50 μm. (F) A classical AQP gene (spAQP) was also expressed in interstitial cells scattered in the peripheral tissues (magenta arrowheads) including protonephridial (marginal) region. Scale bars, 100 μm, and 10 μm in the enlarged image. (G) Cartoon shows previous segmentation model of the protonephridia (20).

Fig. 4.. Platytocin changes responses to attractive chemosensory cues and chemotaxis behavior.

(A) Diagram of the experimental protocol. (B) Behavioral track-tracing illustrated the effects of platytocin on chemotaxis behavior. (C) Chemotaxis behaviors of N. humilis. Planarians were deprived of food for a week before the start of the experiment. Planarians treated with platytocin approached the chemosensory attractant stimulus (diluted rat blood) more slowly than those that were untreated. (D) Chemosensory plasticity of platytocin-treated animals. Changes in relative chemotaxis index of animals after pre-exposure to blood (conditioned) or not (naive) are plotted. Statistical significance scores refer to the relative change in behavior of platytocin-treated animals compared with controls and were based on coefficients of a fitted linear model. All statistical analyses were based on these trajectory data presented in (B). *P < 0.05 and **P < 0.01; error bars indicate SEM (n ≥ 9 planarians; n ≥ 3 independent assays).