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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.

Background The most abundant polychlorinated biphenyl (PCB) congeners found in the environment and in humans are the ortho-substituted, non-dioxin-like (NDL) congeners, especially PCB153. While evidence indicates that exposure to PCB153 and other NDL-PCBs in early vertebrate development can contribute to neurobehavioral disorders, the basis of neurobiological effects of NDL-PCBs is poorly understood. Methods We assessed a range of effects of several NDL-PCB congeners (PCB153, PCB52, PCB118, and PCB138) on cellular morphologies and synaptic transmission linked to the proper execution of a sensorimotor response in zebrafish, taking advantage of the well-established neural circuit in this model. Specifically, we imaged hair cells, neurons, and muscle tissue to evaluate their presence and morphology. We measured neurotransmitter levels in larvae and assessed the mechanosensory startle response in co-exposures to PCB153 and neurotransmitter modulators to evaluate the involvement of neurotransmitters in the behavioral response. The startle response was also assessed in dioxin-like (DL)-PCB126 exposed larvae. Results Startle responses were dramatically delayed in 6-day old larvae exposed to NDL-PCB. In contrast, exposure to the DL PCB126 did not delay startle response times. Morphological and biochemical data showed that exposure to NDL-PCBs during development induces swelling of afferent sensory neurons and disrupts dopaminergic and GABAergic signaling associated with motor movement. Discussion This study demonstrates that connections between neurotoxic mechanisms and processing of sensory stimuli occur at low concentrations of NDL-PCBs, similar to those present in human and animal samples. The effects on important and broadly conserved signaling mechanisms in vertebrates suggest that NDL-PCBs can contribute to neurodevelopmental abnormalities, relevant for both exposed human populations and wildlife.

Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification1. However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth1,2, but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period. Clonal genetic tracing is used to demonstrate that, in mice, longitudinal bone growth during fetal and neonatal periods relies on the gradual consumption of chondroprogenitors, whereas in adults, a stem cell niche is formed allowing renewing of chondroprogenitors and leading to formation of large, stable monoclonal columns of chondrocytes.

// Petr Dmitriev 1, 2, * , Ekaterina Kiseleva 2, 3, * , Olga Kharchenko 2, 3 , Evgeny Ivashkin 2, 3 , Andrei Pichugin 2, 3, 7 , Philippe Dessen 4 , Thomas Robert 4 , Frederique Coppee 5 , Alexandra Belayew 5 , Gilles Carnac 6 , Dalila Laoudj-Chenivesse 6 , Marc Lipinski 1, 2 , Andrei Vasiliev 3 , Yegor S. Vassetzky 1, 2, 3 1 UMR 8126, Univ. Paris-Sud, CNRS, Institut de Cancerologie Gustave-Roussy, Villejuif, France 2 LIA1066 Laboratoire Franco-Russe de Recherches en Oncologie, Villejuif, France 3 N.K. Koltzov Institute of Developmental Biology, RAS, Moscow, Russia 4 Functional Genomics Unit, Institut de Cancerologie Gustave-Roussy, Villejuif, France 5 Laboratory of Molecular Biology, Research Institute for Health Sciences and Technology, University of Mons, Mons, Belgium 6 PhyMedExp, University of Montpellier, INSERM U1046, CNRS UMR 9214, Montpellier, France 7 Peter the Great St. Petersburg Polytechnic University, St. Petersburg, Russia * These authors contributed equally to this work Correspondence to: Yegor S. Vassetzky, email: vassetzky@igr.fr Keywords: DUX4, CXCR4, SDF1, signalling, migration Received: April 01, 2016 Accepted: August 10, 2016 Published: August 18, 2016 ABSTRACT We performed transcriptome profiling of human immortalized myoblasts (MB) transiently expressing double homeobox transcription factor 4 (DUX4) and double homeobox transcription factor 4 centromeric (DUX4c) and identified 114 and 70 genes differentially expressed in DUX4- and DUX4c- transfected myoblasts, respectively. A significant number of differentially expressed genes were involved in inflammation, cellular migration and chemotaxis suggesting a role for DUX4 and DUX4c in these processes. DUX4 but not DUX4c overexpression resulted in upregulation of the CXCR4 (C-X-C motif Receptor 4) and CXCL12 (C-X-C motif ligand 12 also known as SDF1) expression in human immortalized myoblasts. In a Transwell cell migration assay, human bone marrow-derived mesenchymal stem cells (BMSCs) were migrating more efficiently towards human immortalized myoblasts overexpressing DUX4 as compared to controls; the migration efficiency of DUX4-transfected BMSCs was also increased. DUX4c overexpression in myoblasts or in BMSCs had no impact on the rate of BMSC migration. Antibodies against SDF1 and CXCR4 blocked the positive effect of DUX4 overexpression on BMSC migration. We propose that DUX4 controls the cellular migration of mesenchymal stem cells through the CXCR4 receptor.

Cultured cerebellar granule neurons (CGNs) are resistant to the toxic effect of ZnCl2 (0.005 mM, 3 h) and slightly sensitive to the effect of kainate (0.1 mM, 3 h). Simultaneous treatment of CGNs with kainate and ZnCl2 caused intensive neuronal death, which was attenuated by external acidosis (pH 6.5) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA, Na+/H+ exchange blocker, 0.03 mM). Intracellular zinc and calcium ion concentrations ([Zn2+]i and [Ca2+]i) were increased under the toxic action of kainate + ZnCl2, this effect being significantly decreased on external acidosis and increased in case of EIPA addition. Neuronal Zn2+ imaging demonstrated that EIPA increases the cytosolic concentration of free Zn2+ on incubation in Zn2+-containing solution. These data imply that acidosis reduces ZnCl2/kainate toxic effects by decreasing Zn2+ entry into neurons, and EIPA prevents zinc stores from being overloaded with zinc.

Cranial neural crest cells populate the future facial region and produce ectomesenchyme-derived tissues, such as cartilage, bone, dermis, smooth muscle, adipocytes, and many others. However, the contribution of individual neural crest cells to certain facial locations and the general spatial clonal organization of the ectomesenchyme have not been determined. We investigated how neural crest cells give rise to clonally organized ectomesenchyme and how this early ectomesenchyme behaves during the developmental processes that shape the face. Using a combination of mouse and zebrafish models, we analyzed individual migration, cell crowd movement, oriented cell division, clonal spatial overlapping, and multilineage differentiation. The early face appears to be built from multiple spatially defined overlapping ectomesenchymal clones. During early face development, these clones remain oligopotent and generate various tissues in a given location. By combining clonal analysis, computer simulations, mouse mutants, and live imaging, we show that facial shaping results from an array of local cellular activities in the ectomesenchyme. These activities mostly involve oriented divisions and crowd movements of cells during morphogenetic events. Cellular behavior that can be recognized as individual cell migration is very limited and short-ranged and likely results from cellular mixing due to the proliferation activity of the tissue. These cellular mechanisms resemble the strategy behind limb bud morphogenesis, suggesting the possibility of common principles and deep homology between facial and limb outgrowth., Science Advances, 2 (8), ISSN:2375-2548

Serotonin (5.HT) is known to be functionally active during early development in both vertebrates and invertebrates. However, the presence of 5-HT and its synthesis and transport system has not yet been demonstrated in bivalve early development. The presence of 5-HT was immunochemically demonstrated at the cleavage stage of bivalve Mytilus trossulus. 5-HT level dramatically increased within all embryonic cells after incubation with 5-HTP but not after incubation with tryptophan and 5-HT. The first 5-HT uptake by specific transporter was detected at 13 hpf blastula stage only and it was restricted to one distinct cell.

Serotonin (5-HT) is known to induce a wide range of short-term and long-term (or delayed) effects. In the present paper we demonstrated that short time-window application of the 5-HT precursor 5-hydroxytryptophan during early cleavage stages results in both irreversible morphological malformation (exogastrulation) and distinct changes in behavior of young animals of the freshwater snail, Lymnaea stagnalis (Mollusca: Gastropoda). Pharmacological and immunocytochemical analysis confirmed that both the increase of intracellular 5-HT level within the cleaved blastomers and activation of membrane 5-HT2-like type receptors are required for the appearence of these phenomena.

Commercial importance and ability to live in a wide range of salinities have made the common mussel, Mytilus trossulus, a relevant model to study modulation of larval growth and development. We investigated the effects of various salinities combined with neomycin and ampicillin application on Mytilus larvae survival and growth. Both neomycin and ampicillin enhanced trochophore and veliger survival under condition of low salinity. The average veliger size was increasing in accordance with the increase of salinity. In case of neomycin treatment 3.6% of the larvae reached the pediveliger stage. No abnormalities of larval morphology of the FMRFamide and 5-HT systems occurred after 7 days of culturing with both antibiotics.

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8–16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast), which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm-yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.

The state of gonads, age, structure of scales, and size of specimens of the resident lacustrine form of sock-eyed salmon—kokanee Onchorhynchus nerka—are analyzed. In stocked, previously fishless, lakes, there are specimens that have survived spawning and have remained active for a year or several years. No evidence was found of the possibility of repeated spawning. Thus, such fish do not belong to the spawning stock of the population, and their ecological function is not clear.

Chlorpromazine causes distinct defects in normal development of early cleaving embryos of gastropod pond snail (Lymnaea stagnalis L.) and teleost Eurasian weather loach (Misgurnus fossilis L.): a decrease in blastomere contact surfaces following the rounding in gastropod embryo and a reduction of cleaving blastodisk surface in teleost. Right after the application of chlorpromazine fluorescence is visible as bright spots on the surface of blastomeres. Then spherical vesicles appear within the whole volume of the cells. In loach, association of chlorpromazine with the plasma membrane was observed in blastodisk but not in the yolk cell membrane. Electron microscopy has shown that chlorpromazine induces folding of the cell membrane. Experiments with fluorescent dextran probe demonstrate that chlorpromazine modifies the rate and character of the dextran uptake. Our results indicate that chlorpromazine binds to specific sites of plasmalemma and stimulates endocytosis. Patterns of chlorpromazine binding to the membrane and its effects on endocytosis are similar in teleost and gastropod.

It has been demonstrated by us and other authors that first nervous cells in developing larvae from various trochozoan groups differentiate at the periphery. These pioneer neurons are distinguished by the set of characters. They are located outside the forming central ganglia; outgrowing fibers of central neurons use their processes as a “scaffolding” transmitter expression in these neurons is transient. On the one hand, pioneer neurons mark the “frame” of the adult nervous system and thus play a limiting role. On the other hand, pioneering navigation provides possible mechanisms for evolutional plasticity of the nervous system in adults. In addition, pioneer neurons can underlie functional adaptation of trochophore animals, which minimizes fitness decrease during the transition from the larval to the adult form during metamorphosis.

SummaryMany 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.

BACKGROUND: Serotonin (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. RESULTS: Larval 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. CONCLUSIONS: We 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. peerReviewed

The development of loach embryos is successfully regulated (normalized) after partial removal of the cytoplasm from one blastomere at the two- or four-cell stage or complete removal of one or two blastomeres at the stage of 8-16 cells. Using time-lapse video imaging and morphometric analysis, it has been shown that this regulation is a two-stage process. At the first stage, the ratio between the volumes of the blastodisk and yolk sac is rapidly (within one or two cell cycles) restored almost to the initial level; at the second stage, morphogenesis of the embryo is modified according to its new structural features acquired after the operation. After several rounds of cytokinesis, the cytoplasm remaining in the operated blastomere fuses with the marginal yolk syncytium (periblast),which at the blastula stage forms a distinct extension at the operation site. This extension marks the site of embryonic shield formation. The results of morphometric analysis show that restoration of the initial blastoderm volume in operated embryos leads to a reduction of active tension at the blastoderm--yolk boundary and an increase in the ratio of blastoderm surface to its volume at the moment of epiboly initiation. As a result, the convergence of blastoderm cells to the operation site and the embryonic shield formation begin at a lesser degree of epiboly, compared to the control.

Commercial importance and ability to live in a wide range of salinities have made the common mussel, Mytilus trossulus, a relevant model to study modulation of larval growth and development. We investigated the effects of various salinities combined with neomycin and ampicillin application on Mytilus larvae survival and growth. Both neomycin and ampicillin enhanced trochophore and veliger survival under condition of low salinity. The average veliger size was increasing in accordance with the increase of salinity. In case of neomycin treatment 3.6% of the larvae reached the pediveliger stage. No abnormalities of larval morphology of the FMRFamide and 5-HT systems occurred after 7 days of culturing with both antibiotics.

Serotonin (5.HT) is known to be functionally active during early development in both vertebrates and invertebrates. However, the presence of 5-HT and its synthesis and transport system has not yet been demonstrated in bivalve early development. The presence of 5-HT was immunochemically demonstrated at the cleavage stage of bivalve Mytilus trossulus. 5-HT level dramatically increased within all embryonic cells after incubation with 5-HTP but not after incubation with tryptophan and 5-HT. The first 5-HT uptake by specific transporter was detected at 13 hpf blastula stage only and it was restricted to one distinct cell.

In a case study on development of larvae of Trochozoa species of different systematic positions, it was shown that peripheral neurons differentiated firstly. According to the characters of early peripheral neurons, in particular their localization in parts that differed from known zones of appearance of central ganglia, the difficult periphery of processes used as a "frame" by differentiated neurons of definitive nervous system, and transient expression of specific markers, it is reputed that these cells are pioneer. On the one hand, pioneer neurons are the bottleneck of morphogenesis diversity in late stages of development which prepare, in early larvae, the framework of the further central nervous system. On the other hand, navigation and marking using pioneer neurons can be a mechanism of evolutionary lability of definitive neural structures. Functional adaptive significance of pioneer neurons of larvae of Trochozoa animals, probably, is in the maintenance of a fast change from larvae life-form to adult life-form in metamorphosis that decreases the time of animals at intermediate stages of morphogenesis, which are associated with a dramatic fall in adaptation.

Living beings are extremely complex. Multiple structures, especially evolutionarily young ones, develop or take their final shape during late stages of embryonic development, when the body of an embryo is large and comprised of a huge number of cells. Yet, these late structures frequently need cellular sources from other locations and, sometimes, developmental stages. During recent years it became obvious that nerves provide a perfect solution for transporting and hosting multipotent cells that are later recruited to become new cellular sources in the innervated organs. Moreover, the role of nerves and nerve-dwelling cells in morphogenesis and regeneration seems to be much broader than was previously appreciated in invertebrate and vertebrate animals. In a broader view, nerves can provide material for morphological plasticity and evolutional diversity.