Nitric oxide acts as a positive regulator to induce metamorphosis of the ascidian Herdmania momus.
ABSTRACT: Marine invertebrates commonly have a biphasic life cycle in which the metamorphic transition from a pelagic larva to a benthic post-larva is mediated by the nitric oxide signalling pathway. Nitric oxide (NO) is synthesised by nitric oxide synthase (NOS), which is a client protein of the molecular chaperon heat shock protein 90 (HSP90). It is notable, then, that both NO and HSP90 have been implicated in regulating metamorphosis in marine invertebrates as diverse as urochordates, echinoderms, molluscs, annelids, and crustaceans. Specifically, the suppression of NOS activity by the application of either NOS- or HSP90-inhibiting pharmacological agents has been shown consistently to induce the initiation of metamorphosis, leading to the hypothesis that a negative regulatory role of NO is widely conserved in biphasic life cycles. Further, the induction of metamorphosis by heat-shock has been demonstrated for multiple species. Here, we investigate the regulatory role of NO in induction of metamorphosis of the solitary tropical ascidian, Herdmania momus. By coupling pharmacological treatments with analysis of HmNOS and HmHSP90 gene expression, we present compelling evidence of a positive regulatory role for NO in metamorphosis of this species, in contrast to all existing ascidian data that supports the hypothesis of NO as a conserved negative regulator of metamorphosis. The exposure of competent H. momus larvae to a NOS inhibitor or an NO donor results in an up-regulation of NOS and HSP90 genes. Heat shock of competent larvae induces metamorphosis in a temperature dependent manner, up to a thermal tolerance that approaches 35°C. Both larval/post-larval survival and the appearance of abnormal morphologies in H. momus post-larvae reflect the magnitude of up-regulation of the HSP90 gene in response to heat-shock. The demonstrated role of NO as a positive metamorphic regulator in H. momus suggests the existence of inter-specific adaptations of NO regulation in ascidian metamorphosis.
Project description:Tunicates are the only animals that perform cellulose biosynthesis. The tunicate gene for cellulose synthase, Ci-CesA, was likely acquired by horizontal transfer from bacteria and was a key innovation in the evolution of tunicates. Transposon-based mutagenesis in an ascidian, Ciona intestinalis, has generated a mutant, swimming juvenile (sj). Ci-CesA is the gene responsible for the sj mutant, in which a drastic reduction in cellulose was observed in the tunic. Furthermore, during metamorphosis, which in ascidians convert the vertebrate-like larva into a sessile filter feeder, sj showed abnormalities in the order of metamorphic events. In normal larvae, the metamorphic events in the trunk region are initiated after tail resorption. In contrast, sj mutant larvae initiated the metamorphic events in the trunk without tail resorption. Thus, sj larvae show a "swimming juvenile" phenotype, the juvenile-like trunk structure with a complete tail and the ability to swim. It is likely that ascidian cellulose synthase is required for the coordination of the metamorphic events in the trunk and tail in addition to cellulose biosynthesis.
Project description:Metamorphosis takes place within the life cycle of most marine invertebrates. The marine ascidian is a classical model to study complex cellular processes and underlying molecular mechanisms involved in its larval metamorphosis. The detailed molecular signaling pathways remain elusive, though extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinase (JNK) have been revealed to regulate cell migration, differentiation, and apoptosis in ascidian larval organ regression and juvenile organ development. MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression at the post-transcriptional level. Large numbers of miRNAs have been demonstrated to be involved in many developmental and metamorphic processes. However, the identification of miRNAs in ascidian larval metamorphosis has not yet been investigated.Totally, 106 known and 59 novel miRNAs were screened out through RNA-sequencing of three small RNA libraries from 18 to 21-h post-fertilization (hpf) tailbud embryos as well as from 42 hpf larvae (after tail regression) in Ciona savignyi. Expression profiling of miRNAs was confirmed by quantitative real-time PCR, showing that the expression levels of csa-miR-4040, csa-miR-4086, csa-miR-4055, csa-miR-4060, csa-miR-216a, csa-miR-216b, csa-miR-217, csa-miR-183, and csa-miR-92c were significantly higher in 42 hpf larvae, whereas those of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were higher in 18 and 21 hpf embryos; then, their expression in 42 hpf larvae became significantly low. For these 12 miRNAs, whose expression levels significantly changed, we predicted their target genes through the combination of miRanda and TargetScan. This prediction analysis revealed 332 miRNA-target gene pairs that were associated with the ERK, JNK, and transforming growth factor beta signaling pathways, suggesting that the identified miRNAs are involved in the regulation of C. savignyi larval metamorphosis via controlling the expression of their target genes. Furthermore, we validated the expression of five selected miRNAs by northern blotting. Among the selected miRNAs, the expression patterns of csa-miR-4018a, csa-miR-4018b, and csa-miR-4000f were further examined by whole-mount in situ hybridization. The results showed that all three miRNAs were specifically expressed in a cell population resembling mesenchymal cells at the head and trunk part in swimming larvae but not in metamorphic larvae. Utilizing the luciferase assay, we also confirmed that miR-4000f targeted Mapk1, suggesting that the csa-miR-4018a/csa-miR-4018b/csa-miR-4000f cluster regulates larval metamorphosis through the Mapk1-mediated signaling pathway.Totally, 165 miRNAs, including 59 novel ones, were identified from the embryos and larvae of C. savignyi. Twelve of them showed significant changes in expression before and during metamorphosis. In situ hybridization and northern blotting results revealed that three miRNAs are potentially involved in the signaling regulatory network for the migration and differentiation of mesenchymal cells in larval metamorphosis. Furthermore, the luciferase reporter assay revealed that Mapk1 is a target of csa-miR-4000f. Our results not only present a list and profile of miRNAs involved in Ciona metamorphosis but also provide informative cues to further understand their function in ascidian larval metamorphosis.
Project description:Nitric oxide (NO) is known to regulate mitochondrial respiration, especially during metabolic stress and disease, by nitrosation of the mitochondrial electron transport chain (ETC) complexes (irreversible) and by a competitive binding at O2 binding site of cytochrome c oxidase (CcO) in complex IV (reversible). In this study, by using bovine aortic endothelial cells, we demonstrate that the inhibitory effect of endogenously generated NO by nitric oxide synthase (NOS) activation, by either NOS stimulators or association with heat shock protein 90 (Hsp90), is significant only at high prevailing pO2 through nitrosation of mitochondrial ETC complexes, but it does not inhibit the respiration by competitive binding at CcO at very low pO2. ETC complexes activity measurements confirmed that significant reduction in complex IV activity was noticed at higher pO2, but it was unaffected at low pO2 in these cells. This was further extended to heat-shocked cells, where NOS was activated by the induction/activation of (Hsp90) through heat shock at an elevated temperature of 42 degrees C. From these results, we conclude that the entire attenuation of respiration by endogenous NO is due to irreversible inhibition by nitrosation of ETC complexes but not through reversible inhibition by competing with O2 binding at CcO at complex IV.
Project description:Cytoskeletal proteins are crucial in maintaining cellular structure and, in certain cell types, also play an essential role in motility and shape change. Nitric oxide (NO) is an important paracrine mediator of vascular and platelet function and is produced in the vasculature by the enzyme NO synthase type 3 (NOS-3). Here, we demonstrate in human platelets that the polymerization state of beta-actin crucially regulates the activation state of NOS-3, and hence NO formation, through altering its binding of heat shock protein 90 (Hsp90). We found that NOS-3 binds to the globular, but not the filamentous, form of beta-actin, and the affinity of NOS-3 for globular beta-actin is, in turn, increased by Hsp90. Formation of this ternary complex among NOS-3, globular beta-actin, and Hsp90, in turn, results in an increase in both NOS activity and cyclic guanosine-3',5'-monophosphate, an index of bioactive NO, as well as an increased rate of Hsp90 degradation, thus limiting the duration for which NOS-3 remains activated. These observations suggest that beta-actin plays a critical role in regulating NO formation and signaling in platelets.
Project description:In this study, the effects of environmental variables on larval metamorphosis of the solitary ascidian Ciona savignyi were investigated in a laboratory setting. The progression of metamorphic changes were tracked under various temperature, photoperiod, substrate, larval density, and vessel size regimes. Metamorphosis was maximised at 18 °C, 12:12 h subdued light:dark, smooth polystyrene substrate, and 10 larvae mL(-1) in a twelve-well tissue culture plate. Eliminating the air-water interface by filling culture vessels to capacity further increased the proportion of metamorphosed larvae; 87 ± 5% of larvae completed metamorphosis within 5 days compared to 45 ± 5% in control wells. The effects of the reference antifouling compounds polygodial, portimine, oroidin, chlorothalonil, and tolylfluanid on C. savignyi were subsequently determined, highlighting (1) the sensitivity of C. savignyi metamorphosis to chemical exposure and (2) the potential to use C. savignyi larvae to screen for bioactivity in an optimised laboratory setting. The compounds were bioactive in the low ng mL(-1) to high µg mL(-1) range. Polygodial was chosen for additional investigations, where it was shown that mean reductions in the proportions of larvae reaching stage E were highly repeatable both within (repeatability = 14 ± 9%) and between (intermediate precision = 17 ± 3%) independent experiments. An environmental extract had no effect on the larvae but exposing larvae to both the extract and polygodial reduced potency relative to polygodial alone. This change in potency stresses the need for caution when working with complex samples, as is routinely implemented when isolating natural compounds from their biological source. Overall, the outcomes of this study highlight the sensitivity of C. savignyi metamorphosis to environmental variations and chemical exposure.
Project description:Settlement is a rapid process in many marine invertebrate species, transitioning a planktonic larva into a benthic juvenile. In indirectly developing sea urchins, this ecological transition correlates with a morphological, developmental and physiological transition (metamorphosis) during which apoptosis is essential for the resorption and remodelling of larval and juvenile structures. While settlement is initiated by environmental cues (i.e. habitat-specific or benthic substrate cues), metamorphosis is regulated by developmental endocrine signals, such as histamine (HA), thyroid hormones (THs) and nitric oxide (NO). In the purple sea urchin, Strongylocentrotus purpuratus, we found that suH1R mRNA levels increase during larval development and peak during metamorphic competence. SuH1R positive cell clusters are prominently visible in the mouth region of sea urchin larvae, but the protein appears to be expressed at low levels throughout the larval arms and epidermis. SuH1R knock-down experiments in larval stages show that the function of suH1R is in inhibiting apoptosis. Our results therefore suggest that suH1R is regulating the metamorphic transition by inhibiting apoptosis. These results provide new insights into metamorphic mechanisms and have implications for our understanding of settlement and metamorphosis in the marine environment.
Project description:In many marine invertebrates, larval metamorphosis is induced by environmental cues that activate sensory receptors and signalling pathways. Nitric oxide (NO) is a gaseous signalling molecule that regulates metamorphosis in diverse bilaterians. In most cases NO inhibits or represses this process, although it functions as an activator in some species. Here we demonstrate that NO positively regulates metamorphosis in the poriferan Amphimedon queenslandica. High rates of A. queenslandica metamorphosis normally induced by a coralline alga are inhibited by an inhibitor of nitric oxide synthase (NOS) and by a NO scavenger. Consistent with this, an artificial donor of NO induces metamorphosis even in the absence of the alga. Inhibition of the ERK signalling pathway prevents metamorphosis in concert with, or downstream of, NO signalling; a NO donor cannot override the ERK inhibitor. NOS gene expression is activated late in embryogenesis and in larvae, and is enriched in specific epithelial and subepithelial cell types, including a putative sensory cell, the globular cell; DAF-FM staining supports these cells being primary sources of NO. Together, these results are consistent with NO playing an activating role in induction of A. queenslandica metamorphosis, evidence of its highly conserved regulatory role in metamorphosis throughout the Metazoa.
Project description:<h4>Background</h4>A metamorphic life-history is present in the majority of animal phyla. This developmental mode is particularly prominent among marine invertebrates with a bentho-planktonic life cycle, where a pelagic larval form transforms into a benthic adult. Metamorphic competence (the stage at which a larva is capable to undergo the metamorphic transformation and settlement) is an important adaptation both ecologically and physiologically. The competence period maintains the larval state until suitable settlement sites are encountered, at which point the larvae settle in response to settlement cues. The mechanistic basis for metamorphosis (the morphogenetic transition from a larva to a juvenile including settlement), i.e. the molecular and cellular processes underlying metamorphosis in marine invertebrate species, is poorly understood. Histamine (HA), a neurotransmitter used for various physiological and developmental functions among animals, has a critical role in sea urchin fertilization and in the induction of metamorphosis. Here we test the premise that HA functions as a developmental modulator of metamorphic competence in the sea urchin Strongylocentrotus purpuratus.<h4>Results</h4>Our results provide strong evidence that HA leads to the acquisition of metamorphic competence in S. purpuratus larvae. Pharmacological analysis of several HA receptor antagonists and an inhibitor of HA synthesis indicates a function of HA in metamorphic competence as well as programmed cell death (PCD) during arm retraction. Furthermore we identified an extensive network of histaminergic neurons in pre-metamorphic and metamorphically competent larvae. Analysis of this network throughout larval development indicates that the maturation of specific neuronal clusters correlates with the acquisition of metamorphic competence. Moreover, histamine receptor antagonist treatment leads to the induction of caspase mediated apoptosis in competent larvae.<h4>Conclusions</h4>We conclude that HA is a modulator of metamorphic competence in S. purpuratus development and hypothesize that HA may have played an important role in the evolution of settlement strategies in echinoids. Our findings provide novel insights into the evolution of HA signalling and its function in one of the most important and widespread life history transitions in the animal kingdom--metamorphosis.
Project description:In the ascidian Ciona intestinalis larval development and metamorphosis require a complex interplay of events, including nitric oxide (NO) production, MAP kinases (ERK, JNK) and caspase-3 activation. We have previously shown that NO levels affect the rate of metamorphosis, regulate caspase activity and promote an oxidative stress pathway, resulting in protein nitration. Here, we report that NO down-regulates MAP kinase phosphatases (mkps) expression affecting positively ERK signaling. By pharmacological approach, we observed that the reduction of endogenous NO levels caused a decrease of ERK phosphorylation, whereas increasing levels of NO induced ERK activation. We have also identified the ERK gene network affected by NO, including mpk1, mpk3 and some key developmental genes by quantitative gene expression analysis. We demonstrate that NO induces an ERK-independent down-regulation of mkp1 and mkp3, responsible for maintaining the ERK phosphorylation levels necessary for transcription of key metamorphic genes, such as the hormone receptor rev-erb and the van willebrand protein vwa1c. These results add new insights into the role played by NO during larval development and metamorphosis in Ciona, highlighting the cross-talk between different signaling pathways.
Project description:Uncoupling of NO production from NADPH oxidation by endothelial nitric-oxide synthase (eNOS) is enhanced in hyperglycemic endothelium, potentially due to dissociation of heat shock proteins 90 (Hsp90), and cellular glucose homeostasis is enhanced by a ROS-induced positive feed back mechanism. In this study we investigated how such an uncoupling impacts oxygen metabolism and how the oxidative phosphorylation can be preserved by heat shock (42 °C for 2 h, hyperthermia) in bovine aortic endothelial cells. Normal and heat-shocked bovine aortic endothelial cells were exposed to normoglycemia (NG, 5.0 mM) or hyperglycemia (30 mM). With hyperglycemia treatment, O(2) consumption rate was reduced (from V(O(2)max) = 7.51 ± 0.54 to 2.35 ± 0.27 mm Hg/min/10(6) cells), whereas in heat-shocked cells, O(2) consumption rate remained unaltered (8.19 ± 1.01 mm Hg/min/10 × 10(6) cells). Heat shock was found to enhance Hsp90/endothelial NOS interactions and produce higher NO. Moreover, ROS generation in the hyperglycemic condition was also reduced in heat-shocked cells. Interestingly, glucose uptake was reduced in heat-shocked cells as a result of decrease in Glut-1 protein level. Glucose phosphate dehydrogenase activity that gives rise to NADPH generation was increased by hyperthermia, and mitochondrial oxidative metabolism was preserved. In conclusion, the present study provides a novel mechanism wherein the reduced oxidative stress in heat-shocked hyperglycemic cells down-regulates Glut-1 and glucose uptake, and fine-tuning of this pathway may be a potential approach to use for therapeutic benefit of diabetes mellitus.