Dev-ResNet: automated developmental event detection using deep learning.

Delineating developmental events is central to experimental research using early life stages, permitting widespread identification of changes in event timing between species and environments. Yet, identifying developmental events is incredibly challenging, limiting the scale, reproducibility and throughput of using early life stages in experimental biology. We introduce Dev-ResNet, a small and efficient 3D convolutional neural network capable of detecting developmental events characterised by both spatial and temporal features, such as the onset of cardiac function and radula activity. We demonstrate the efficacy of Dev-ResNet using 10 diverse functional events throughout the embryonic development of the great pond snail, Lymnaea stagnalis. Dev-ResNet was highly effective in detecting the onset of all events, including the identification of thermally induced decoupling of event timings. Dev-ResNet has broad applicability given the ubiquity of bioimaging in developmental biology, and the transferability of deep learning, and so we provide comprehensive scripts and documentation for applying Dev-ResNet to different biological systems.

GSK3ß controls the timing and pattern of the fifth spiral cleavage at the 2-4 cell stage in Lymnaea stagnalis.

Establishment of the body plan of multicellular organisms by the primary body axis determination and cell-fate specification is a key issue in biology. We have examined the mRNA localization of three Wnt pathway components gsk3ß, ß-catenin, and disheveled and investigated the effects of four selective inhibitors of these proteins on the early developmental stages of the spiral cleavage embryo of the fresh water snail Lymnaea (L.) stagnalis. mRNAs for gsk3ß and ß-catenin were distributed uniformly throughout the embryo during development whereas disheveled mRNA showed specific localization with intra- and inter-blastomere differences in concentration along the A-V axis during spiral cleavages. Remarkably, through inhibitor studies, we identified a short sensitive period from the 2- to 4-cell stage in which GSK3ß inhibition by the highly specific 1-azakenpaullone (AZ) and by LiCl induced a subsequent dramatic developmental delay and alteration of the cleavage patterns of blastomeres at the fifth cleavage (16- to 24-cell stage) resulting in exogastrulation and other abnormalities in later stages. Inhibition of ß-Catenin or Disheveled had no effect. Our inhibitor experiments establish a novel role for GSK3ß in the developmental timing and orientated cell division of the snail embryo. Further work will be needed to identify the downstream targets of the kinase.

Diaphanous gene mutation affects spiral cleavage and chirality in snails.

L-R (left and right) symmetry breaking during embryogenesis and the establishment of asymmetric body plan are key issues in developmental biology, but the onset including the handedness-determining gene locus still remains unknown. Using pure dextral (DD) and sinistral (dd) strains of the pond snail Lymnaea stagnalis as well as its F2 through to F10 backcrossed lines, the single handedness-determining-gene locus was mapped by genetic linkage analysis, BAC cloning and chromosome walking. We have identified the actin-related diaphanous gene Lsdia1 as the strongest candidate. Although the cDNA and derived amino acid sequences of the tandemly duplicated Lsdia1 and Lsdia2 genes are very similar, we could discriminate the two genes/proteins in our molecular biology experiments. The Lsdia1 gene of the sinistral strain carries a frameshift mutation that abrogates full-length LsDia1 protein expression. In the dextral strain, it is already translated prior to oviposition. Expression of Lsdia1 (only in the dextral strain) and Lsdia2 (in both chirality) decreases after the 1-cell stage, with no asymmetric localization throughout. The evolutionary relationships among body handedness, SD/SI (spiral deformation/spindle inclination) at the third cleavage, and expression of diaphanous proteins are discussed in comparison with three other pond snails (L. peregra, Physa acuta and Indoplanorbis exustus).

Formin Is Associated with Left-Right Asymmetry in the Pond Snail and the Frog.

While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signaling of Nodal, downstream of symmetry breaking, may be an ancestral feature of the Bilateria [1 and 2]. Here, we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or overexpression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models [3, 4 and 5], we discovered asymmetric gene expression in 2- and 4-cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro [6 and 7], together these results are consistent with the view that animals with diverse body plans may derive their asymmetries from the same intracellular chiral elements [8].

Multiple roles for protein kinase C in gastropod embryogenesis.

Protein kinase C (PKC) contributes to the correct development of organisms, but its importance to the embryogenesis of molluscs is not yet known. We report here that PKC activation is cyclic within early developing embryos of the gastropod snail Lymnaea stagnalis, and that activation with phorbol myristate acetate (PMA) results in disorganised and developmentally arrested embryos within 24 h. Moreover, chronic modulation of PKC activation by PMA or by the PKC inhibitor GF109203X in early embryos results in altered rotation and gliding behaviours and heartbeat during development. Finally, dis-regulation of PKC activity during early development significantly increased the duration to hatching. Our findings thus support novel roles for PKC in L. stagnalis embryos, in several physiological contexts, providing further insights into the importance of protein kinases for gastropod development in general.

Developmental toxicity of metaldehyde in the embryos of Lymnaea stagnalis (Gastropoda: Pulmonata) co-exposed to the synergist piperonyl butoxide.

Metaldehyde is a widely used molluscicide in countries where damage to crops from slugs and snails is a major problem associated with warm and wet winters. In the UK it is estimated that over 8% of the area covered by arable crops is treated with formulated granular bait pellets containing metaldehyde as the main active ingredient. Metaldehyde is hydrophilic (log Kow=0.12), water soluble (200 mg·L(-1) at 17 °C) and has been detected in UK surface waters in the concentration range of typically 0.2-0.6 µg·L(-1) (maximum 2.7 µg·L(-1)) during 2008-2011. In the absence of chronic data on potential hazards to non-target freshwater molluscs, a laboratory study was conducted to investigate the impact of metaldehyde on embryo development in the gastropod Lymnaea stagnalis (RENILYS strain) and using zinc as a positive control. L. stagnalis embryos were exposed to metaldehyde under semi-static conditions at 20±1 °C and hatching success and growth (measured as shell height and intraocular distance) examined after 21 d. Exposure concentrations were verified using HPLC and gave 21 d (hatching)NOEC and (hatching)LOEC mean measured values of 36 and 116 mg MET·L(-1), respectively (equal to the 21 d (shell height)NOEC and (shell height)LOEC values). For basic research purposes, a second group of L. stagnalis embryos was co-exposed to metaldehyde and the pesticide synergist piperonyl butoxide (PBO). Co-exposure to the PBO (measured concentrations between 0.47-0.56 mg·L(-1)) reduced hatching success from 100% to 47% and resulted in a 30% reduction in embryo growth (shell height) in snail embryos co-exposed to metaldehyde at 34-36 mg·L(-1) over 21 d. In conclusion, these data suggest mollusc embryos may have some metabolic detoxication capacity for metaldehyde and further work is warranted to explore this aspect in order to support the recent initiative to include molluscs in the OECD test guideline programme.

[ENGAGEMENT OF SEROTONIN-MODULATING ANTICONSOLIDATION PROTEIN IN REGULATION OF EMBRYOGENESIS OF LYMNEAE STAGNALIS AND LEWIS SARCOMA IN HYBRID MICE Fl C57B2/6 X DBA].

The article concerns study of the effects of a novel serotonin-modulating anticonsolidation protein (SMAP) being in a linear relationship with serotonin level, on embryogenesis of Lymneae stagnalis and Lewis sarcoma in hybrid mice Fl C57B2/6 X DBA. Inhibition of embryogenesis of Lymneae stagnalis on the stage of four blastomers and late blastula, lack of changes on the stage of trochofora and acceleration of metamorphosis under the effects of SMAP in a dose-dependent manner was observed. Short-term retardation (during the first 10 days) of development of Lewis sarcoma in mice and survival of 25% of transferring animals under high doses of SMAP was revealed. Cytostatic activity for high doses of SMAP and their effects on the duration of single phases of the cell cycle is proposed.

An optimised whole mount in situ hybridisation protocol for the mollusc Lymnaea stagnalis.

BACKGROUND: The ability to visualise the expression of individual genes in situ is an invaluable tool for developmental and evolutionary biologists; it allows for the characterisation of gene function, gene regulation and through inter-specific comparisons, the evolutionary history of unique morphological features. For well-established model organisms (e.g., flies, worms, sea urchins) this technique has been optimised to an extent where it can be automated for high-throughput analyses. While the overall concept of in situ hybridisation is simple (hybridise a single-stranded, labelled nucleic acid probe complementary to a target of interest, and then detect the label immunologically using colorimetric or fluorescent methods), there are many parameters in the technique that can significantly affect the final result. Furthermore, due to variation in the biochemical and biophysical properties of different cells and tissues, an in situ technique optimised for one species is often not suitable for another, and often varies depending on the ontogenetic stage within a species. RESULTS: Using a variety of pre-hybridisation treatments we have identified a set of treatments that greatly increases both whole mount in situ hybridisation (WMISH) signal intensity and consistency while maintaining morphological integrity for early larval stages of Lymnaea stagnalis. These treatments function well for a set of genes with presumably significantly different levels of expression (beta tubulin, engrailed and COE) and for colorimetric as well as fluorescent WMISH. We also identify a tissue-specific background stain in the larval shell field of L. stagnalis and a treatment, which eliminates this signal. CONCLUSIONS: This method that we present here will be of value to investigators employing L. stagnalis as a model for a variety of research themes (e.g. evolutionary biology, developmental biology, neurobiology, ecotoxicology), and brings a valuable tool to a species in a much understudied clade of animals collectively known as the Spiralia.

A conserved set of maternal genes? Insights from a molluscan transcriptome.

The early animal embryo is entirely reliant on maternal gene products for a 'jump-start' that transforms a transcriptionally inactive embryo into a fully functioning zygote. Despite extensive work on model species, it has not been possible to perform a comprehensive comparison of maternally-provisioned transcripts across the Bilateria because of the absence of a suitable dataset from the Lophotrochozoa. As part of an ongoing effort to identify the maternal gene that determines left-right asymmetry in snails, we have generated transcriptome data from 1 to 2-cell and ~32-cell pond snail (Lymnaea stagnalis) embryos. Here, we compare these data to maternal transcript datasets from other bilaterian metazoan groups, including representatives of the Ecydysozoa and Deuterostomia. We found that between 5 and 10% of all L. stagnalis maternal transcripts (~300-400 genes) are also present in the equivalent arthropod (Drosophila melanogaster), nematode (Caenorhabditis elegans), urochordate (Ciona intestinalis) and chordate (Homo sapiens, Mus musculus, Danio rerio) datasets. While the majority of these conserved maternal transcripts ("COMATs") have housekeeping gene functions, they are a non-random subset of all housekeeping genes, with an overrepresentation of functions associated with nucleotide binding, protein degradation and activities associated with the cell cycle. We conclude that a conserved set of maternal transcripts and their associated functions may be a necessary starting point of early development in the Bilateria. For the wider community interested in discovering conservation of gene expression in early bilaterian development, the list of putative COMATs may be useful resource.

Growth inhibition in early life-stage tests predicts full life-cycle toxicity effects of lead in the freshwater pulmonate snail, Lymnaea stagnalis.

The freshwater pulmonate snail, Lymnaea stagnalis, is the most sensitive freshwater organism tested to date for several metals (Co, Cu, Pb, Ni) based on 28 d early life-stage (ELS) tests in which growth was the most sensitive endpoint. The United States Environmental Protection Agency (USEPA) has expressed concern that growth in 28 d ELS tests with mollusks may overpredict toxicity because of the potential for recovery in a full life-cycle (LC) test. Consequently, the USEPA only accepts the survival endpoint for these tests in establishing water quality criteria (WQC). To address this concern, the current study aimed to test the sensitivity of L. stagnalis to Pb in a 56 d full LC test evaluating survival, growth, reproductive and embryonic growth endpoints and compare the estimated effect levels to those established using the 28 d ELS test design. The most sensitive endpoints in this study were 28 d growth and 56 d egg mass production, both with a NOEC of <1.0 µg L(-1) and a LOEC of 1.0 µg L(-1), showing that the ELS growth endpoint is predictive of the 56 d reproduction endpoint. Snails exposed to 1.0 and 2.7 µg L(-1) Pb showed full and partial recovery from growth inhibition between 28 and 56 d. While this recovery supports the USEPA's concern about the 28 d growth endpoint; considering the reproductive lifespan of L. stagnalis and the recovery dose-response, we conclude that the 28 d growth endpoint will be within a factor of 3 of full LC endpoints. This is consistent with the level of precision previously determined for fish ELS tests, which the USEPA accepts for WQC derivation, and suggests that tests using 28 d ELS growth endpoint for L. stagnalis may be acceptable for inclusion in WQC derivation.

Neuronal control of pedal sole cilia in the pond snail Lymnaea stagnalis appressa.

5-HT (serotonin) is a ubiquitous neurotransmitter that produces ciliary beating in gastropods when applied topically, but ciliary beating caused by gastropod serotonergic neurons has been described in only three neuron pairs. We extend these results to the North American Lymnaea stagnalis appressa, which is a different species from the European Lymnaea stagnalis. We describe a non-serotonergic neuron pair, PeV1, which accelerates pedal sole mucociliary transport and a serotonergic neuron pair, PeD7, which slows mucociliary transport. We compare and discuss development and identified neurons in L. s. appressa and in L. stagnalis, which have homologs to L. s. appressa PeD7 and PeV1 neurons. In addition to PeD7 and PeV1 neurons, we test neurons immunoreactive to Tritonia pedal peptide antibodies with negative results for mucociliary transport. In characterizing PeD7 and PeV1 neurons, we find that PeV1 does not excite PeD7. In semi-intact preparations, a strong increase in PeD7 neuron activity occurs during tactile stimulation, but V1 neurons are inhibited during tactile stimulation. Following tactile stimulation, PeV1 neurons show strong activity. This suggests a distinct difference in function of the two neuron pairs, which both have their axons overlying pedal sole ciliary cells. Application of 5-HT to the pedal sole initiates mucociliary transport in 1.4-1.9 s with a time course similar to that seen when stimulating a PeV1 neuron. This result appears to be through a 5-HT(1A)-like receptor on the pedal sole. We describe a possible external source of 5-HT on the pedal sole from 5-HT immunoreactive granules that are released with mucus.

[Endogenous biorhythms of the oxygen consumption rate in individual development of Lymnaea stagnalis (Lymnaeidae, Gastropoda)].

Two endogenous biorhythms of the oxygen consumption rate with periods of 10.3 and 7.2 weeks have been revealed in the late postlarval ontogenesis of freshwater gastropods Lymnaea stagnalis by singular spectrum analysis. It has been determined that local maxima of both biorhythms in different individuals occur at the same age; in addition to that, the periods of biorhythms are approximately the same in all studied animals and remain unchanged during the whole individual development. It has been noted that the biorhythm with a period of 10.3 weeks is damped (its amplitude decreases from 8 µL O2/(h x g) at the age of 20 weeks to 3 µL O2/(h x g) at the time of death), and the biorhythm with the period of 7.2 weeks is sustained (its average amplitude is 2.4 ± 0.4 µL O2(h x g).

Development of an embryo toxicity test with the pond snail Lymnaea stagnalis using the model substance tributyltin and common solvents.

The development of a chronic mollusc toxicity test is a current work item on the agenda of the OECD. The freshwater pond snail Lymnaea stagnalis is one of the candidate snail species for such a test. This paper presents a 21-day chronic toxicity test with L. stagnalis, focussing on embryonic development. Eggs were collected from freshly laid egg masses and exposed individually until hatching. The endpoints were hatching success and mean hatching time. Tributyltin (TBT), added as TBT-chloride, was chosen as model substance. The selected exposure concentrations ranged from 0.03 to 10 µg TBT/L (all as nominal values) and induced the full range of responses. The embryos were sensitive to TBT (the NOEC for mean hatching time was 0.03 µg TBT/L and the NOEC for hatching success was 0.1 µg TBT/L). In addition, data on maximum limit concentrations of seven common solvents, recommended in OECD aquatic toxicity testing guidelines, are presented. Among the results, further findings as average embryonic growth and mean hatching time of control groups are provided. In conclusion, the test presented here could easily be standardised and is considered useful as a potential trigger to judge if further studies, e.g. a (partial) life-cycle study with molluscs, should be conducted.

Delayed action of serotonin in molluscan development.

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.

Organization of the serotonergic innervation of the feeding (buccal) musculature during the maturation of the pond snail Lymnaea stagnalis: a morphological and biochemical study.

The serotonergic innervation of the buccal musculature responsible for feeding (radula protraction) was investigated during the maturation of the pond snail, Lymnaea stagnalis L., applying light and electron microscopic immunohistochemistry and biochemical approaches. According to epifluorescence and laser confocal microscopy, the first 5-HT-like-immunoreactive (5-HTLIR) processes appeared on the surface of the musculature at the postmetamorphic E80% embryonic stage. Until hatching, the innervation continued to increase in density, showing axon arborizations with projections into the deeper muscle levels. An adult-like pattern of 5-HTLIR innervation appeared at P2-P3 juvenile stages. At the ultrastructural level, close (16-20 nm) but mostly unspecialized neuromuscular contacts were formed by both unlabeled and 5-HTLIR axon profiles from the E80% embryonic stage. Labeled processes were also found located relatively far from the muscle cells. An HPLC assay showed a gradual increase of the 5-HT level in the buccal mass during development. The buccal mass was characterized by a single-component high-affinity 5-HT uptake system, and 5-HT release could be evoked by 100 mM K(+) and blocked in Ca(2+) -free medium. It is suggested that 5-HT plays a wide modulatory role in the peripheral feeding system and is also involved in the functional maturation of the muscle system.

[Effect of hormonal compounds on embryogenesis of the pond snail Lymnaea stagnalis (L., 1758)].

Effect of preparations ofa peptide nature (pituitrin and oxytocin) and of a steroid nature (progesterone and hydrocortisone) on embryonic development of freshwater gastropod Lymnaea stagnalis (Mollusca, Gastropoda, Pulmonata) is described. The hormonal preparations used, which differed in chemical nature and physiological activity, may render diverse effects on embryogenesis of the studied mollusk. Of neurohormones, pituitrin rendered the most noticeable and principally stimulating effect. Oxytocin was incorporated in regulatory processes much later and its effect on the rate of realization of particular stages depended more on the quality of occurring changes. In final stages of development, this hormone principally inhibited growth and development of embryos. The female sex hormone progesterone rendered an expressed stimulatory effect, especially notable in later developmental stages of embryos. The hormone hydrocortisone stimulated initial stages of embryogenesis. Its effect was almost not expressed in the final stages. The discovered differences seem to be related both to the functional specificity of the investigated compounds and to specific traits of mechanisms of realization of their effects. A hypothesis is formulated: in gastropods, similarly to vertebrates, the hormones are systemic embryonic and postnatal inducers of differentiation processes.

Identification and evolutionary implications of neurotransmitter-ciliary interactions underlying the behavioral response to hypoxia in Lymnaea stagnalis embryos.

Acceleration of embryonic rotation is a common response to hypoxia among pond snails. It was first characterized in Helisoma trivolvis embryos, which have a pair of sensorimotor neurons that detect hypoxia and release serotonin onto postsynaptic ciliary cells. The objective of the present study was to determine how the hypoxia response is mediated in Lymnaea stagnalis, which differ from H. trivolvis by having both serotonergic and dopaminergic neurons, and morphologically distinct ciliated structures at comparative stages of embryonic development. Time-lapse video recordings of the rotational behavior in L. stagnalis revealed similar rotational features to those previously observed in H. trivolvis, including rotational surges and rotational responses to hypoxia. Serotonin and dopamine increased the rate of rotation with similar potency. In contrast, serotonin was more potent than dopamine in stimulating the ciliary beat frequency of isolated pedal cilia. Isolated apical plate cilia displayed an irregular pattern of ciliary beating that precluded the measurement of ciliary beat frequency. A qualitative assessment of ciliary beating revealed that both serotonin and dopamine were able to stimulate apical plate cilia. The ciliary responses to dopamine were reversible in both pedal and apical plate cilia, whereas the responses to serotonin were only reversible at concentrations below 100 µmol l(-1). Mianserin, a serotonin receptor antagonist, and SKF83566, a dopamine receptor antagonist, effectively blocked the rotational responses to serotonin and dopamine, respectively. The rotational response to hypoxia was only partially blocked by mianserin, but was fully blocked by SKF83566. These data suggest that, despite the ability of serotonin to stimulate ciliary beating in L. stagnalis embryos, the rotational response to hypoxia is primarily mediated by the transient apical catecholaminergic neurons that innervate the ciliated apical plate.

Possible functions of Dpp in gastropod shell formation and shell coiling.

We examined dpp expression patterns in the pulmonate snail Lymnaea stagnalis and analyzed the functions of dpp using the Dpp signal inhibitor dorsomorphin in order to understand developmental mechanisms and evolution of shell formation in gastropods. The dpp gene is expressed in the right half of the circular area around the shell gland at the trochophore stage and at the right-hand side of the mantle at the veliger stage in the dextral snails. Two types of shell malformations were observed when the Dpp signals were inhibited by dorsomorphin. When the embryos were treated with dorsomorphin at the 2-cell and blastula stages before the shell gland is formed, the juvenile shells grew imperfectly and were not mineralized. On the other hand, when treated at the trochophore and veliger stage after the shell gland formation, juvenile shells grew to show a cone-like form rather than a normal coiled form. These results indicated that dpp plays important roles in the formation and coiling of the shell in this gastropod species.

Effects of CdO nanoparticles on the development and hatching of a freshwater pulmonate snail Lymnaea luteola L.

The existing information and data on ecotoxicological impact of nanomaterials is limited. Therefore the present study was undertaken to study the effects of CdO nanoparticles on an Indian freshwater pulmonate snail Lymnaea luteola L. The present study demonstrated that even at 1.0 ppm CdO nanoparticles blocked the development of pond snail.

Predator cues alter the timing of developmental events in gastropod embryos.

Heterochrony, differences in the timing of developmental events between descendent species and their ancestors, is a pervasive evolutionary pattern. However, the origins of such timing changes are still not resolved. Here we show, using sequence analysis, that exposure to predator cues altered the timing of onset of several developmental events in embryos of two closely related gastropod species: Radix balthica and Radix auricularia. These timing alterations were limited to certain events and were species-specific. Compared with controls, over half (62%) of exposed R. auricularia embryos had a later onset of body flexing and an earlier occurrence of the eyes and the heart; in R. balthica, 67 per cent of exposed embryos showed a later occurrence of mantle muscle flexing and an earlier attachment to, and crawling on, the egg capsule wall. The resultant developmental sequences in treated embryos converged, and were more similar to one another than were the sequences of the controls for both species. We conclude that biotic agents can elicit altered event timing in developing gastropod embryos. These changes were species-specific, but did not occur in all individuals. Such developmental plasticity in the timing of developmental events could be an important step in generating interspecific heterochrony.