SummaryApical sensory neurones mediate developmental retardation induced by conspecific environmental stimuli in freshwater pulmonate snails

Many organisms survive in constantly changing environments, including cycling seasons. Developing embryos show remarkable instant adaptations to the variable environmental challenges they encounter during their adult life, despite having no direct contact with the changing environment until after birth or hatching. The mechanisms by which such non-genetic information is transferred to the developing embryos are largely unknown. Here, we address this question by using a freshwater pond snail (Lymnaea stagnalis) as a model system. This snail normally lives in a seasonal climate, and the seasons define its locomotion, feeding, and reproductive behavior. We discovered that the serotonergic system plays a crucial role in transmitting a non-genetic instructive signal from mother to progeny. This maternal serotonin-based signal functions in embryos during a short time window at exclusively early pre-neural developmental stages and modulates the dynamics of embryonic and juvenile growth, feeding behavior, and locomotion.

BackgroundSerotonin (5-HT) is well known as widely distributed modulator of developmental processes in both vertebrates and invertebrates. It is also the earliest neurotransmitter to appear during neuronal development. In aquatic invertebrates, which have larvae in their life cycle, 5-HT is involved in regulation of stages transition including larval metamorphosis and settlement. However, molecular and cellular mechanisms underlying developmental transition in aquatic invertebrate species are yet poorly understood. Earlier we demonstrated that in larvae of freshwater molluscs and marine polychaetes, endogenous 5-HT released from the neurons of the apical sensory organ (ASO) in response to external stimuli retarded larval development at premetamorphic stages, and accelerated it at metamorphic stages. Here we used a freshwater snail Helisoma trivolvis to study molecular mechanisms underlying these dual developmental effects of 5-HT.ResultsLarval development of H. trivolvis includes transition from premetamorphic to metamorphic stages and shares the main features of metamorphosis with free-swimming aquatic larvae. Three types of 5-HT receptors (5-HT1-, 5-HT4- and 5-HT7-like) are functionally active at premetamorphic (trochophore, veliger) and metamorphic (veliconcha) stages, and expression patterns of these receptors and respective G proteins undergo coordinated changes during development. Stimulation of these receptors modulated cAMP-dependent regulation of cell divisions. Expression of 5-HT4- and 5-HT7-like receptors and their downstream Gs protein was down-regulated during the transition of pre- to metamorphic stage, while expression of 5-HT1 -like receptor and its downstream Gi protein was upregulated. In accordance with relative amount of these receptors, stimulation of 5-HTRs at premetamorphic stages induces developmental retardation, while their stimulation at metamorphic stages induces developmental acceleration.ConclusionsWe present a novel molecular mechanism that underlies stage-specific changes in developmental tempo of H. trivolvis larvae in response to endogenous 5-HT produced by the neurons of the ASO. We suggest that consecutive changes in expression patterns of different receptors and their downstream partners in the course of larval development represent the molecular base of larval transition from premetamorphic (non-competent) to metamorphic (competent) state.

Serotonin (5-HT) is a key player in many physiological processes in both the adult organism and developing embryo. One of the mechanisms for 5-HT-mediated effects is covalent binding of 5-HT to the target proteins catalyzed by transglutaminases (serotonylation). Despite the implication in a variety of physiological processes, the involvement of serotonylation in embryonic development remains unclear. Here we tested the hypothesis that 5-HT serves as a substrate for transglutaminase-mediated transamidation of the nuclear proteins in the early embryos of both vertebrates and invertebrates. For this, we demonstrated that the level of serotonin immunoreactivity (5-HT-ir) in cell nuclei increases upon the elevation of 5-HT concentration in embryos of sea urchins, mollusks, and teleost fish. Consistently, pharmacological inhibition of transglutaminase activity resulted in the reduction of both brightness and nuclear localization of anti-5-HT staining. We identified specific and bright 5-HT-ir within nuclei attributed to a subset of different cell types: ectodermal and endodermal, macro-and micromeres, and blastoderm. Western blot and dot blot confirmed the presence of 5-HT-ir epitopes in the normal embryos of all the species examined. The experimental elevation of 5-HT level led to the enhancement of 5-HT-ir-related signal on blots in a species-specific manner. The obtained results demonstrate that 5-HT is involved in transglutaminase-dependent monoaminylation of nuclear proteins and suggest nuclear serotonylation as a possible regulatory mechanism during early embryonic development. The results reveal that this pathway is conserved in the development of both vertebrates and invertebrates.

The regulation of larval development by starved adults occurs in both freshwater snails, Helisoma trivolvis and marine polychaetes, Platynereis dumerilii. Serotonin (5-HT) links this environmental signal which is detected by larval apical sensory neurons to changes in larval development. A profile of the stage-dependent expression of 5-HT receptors and coupled G proteins is essential in this regulatory mechanism. The final effect on development depends on the modulation of the activity of the larval digestive system.