Silvia Groppelli

Serotonin localization in Phallusia mammillata larvae and effects of 5‐HT antagonists during larval development

The neurotransmitter 5‐hydroxytryptamine (5‐HT, serotonin) plays an important role in a wide range of non‐neural processes. Using immunofluorescence with an antiserotonin antibody, 5‐HT was localized in the brain and in some neurons of the larval tail of Phallusia mammillata. To test the effect of 5‐HT on development, we treated embryos with two different 5‐HT receptor subtype antagonists. Treatment at the gastrula stage with 10 μM ondansetron, an antagonist of the 5‐HT3 receptor, induced anterior truncation and a short tail. At 10 μM, ritanserin, a 5‐HT2B receptor antagonist, induced larval phenotypes characterized by a roundish trunk region with flat papillae. The juveniles developed from these larvae had an abnormal cardiocirculatory system: their heart contractions were ineffective and their blood cells accumulated in the heart cavity. We conclude that an appropriate level of 5‐HT is necessary for correct development and morphogenesis. Moreover, a different key role for multiple receptors in modulating the morphogenetic effects of 5‐HT is suggested.

Developmental expression of glutamic acid decarboxylase and of γ‐aminobutyric acid type B receptors in the ascidian Ciona intestinalis

We describe Ciona intestinalis γ‐aminobutyric acid (GABA)‐ergic neurons during development, studying the expression pattern of Ci‐GAD (glutamic acid decarboxylase: GABA synthesizing enzyme) by in situ hybridization. Moreover, we cloned two GABAB receptor subunits (Ci‐GABABRs), and a phylogenetic analysis (neighbor‐joining method) suggested that they clustered with their vertebrate counterparts. We compared Ci‐GAD and Ci‐GABABRs expression patterns in C. intestinalis embryos and larvae. At the tailbud stage, Ci‐GAD expression was widely detected in central and peripheral nervous system (CNS/PNS) precursors, whereas Ci‐GABABRs expression was evident at the level of the precursors of the visceral ganglion. GABA was localized by immunohistochemistry at the same developmental stage. In the larva, Ci‐GAD transcripts and GABA immunofluorescence were also detected throughout the CNS and in some neurons of the PNS, whereas transcripts of both GABAB receptor subunits were found mainly in the CNS. The expression pattern of Ci‐GABABRs appeared restricted to Ci‐GAD‐positive territories in the sensory vesicle, whereas, in the visceral ganglion, Ci‐GABABRs transcripts were found in ventral motoneurons that did not express Ci‐GAD. Insofar as GABAergic neurons are widely distributed also in the CNS and PNS of vertebrates and other invertebrate chordates, it seems likely that GABA signaling was extensively present in the protochordate nervous system. Results from this work show that GABA is the most widespread inhibitory neurotransmitter in C. intestinalis nervous system and that it can signal through GABAB receptors both pre‐ and postsynaptically to modulate different sensory inputs and subsequent swimming activity. J. Comp. Neurol. 506:489–505, 2008.

Effects of the azole fungicide Imazalil on the development of the ascidian Ciona intestinalis (Chordata, Tunicata): morphological and molecular characterization of the induced phenotype.

Imazalil (IMA) is a fungicide that is used extensively in fruit plantations and post-harvest treatments, but has teratogenic effects on vertebrate development, possibly due to the perturbation of retinoic acid (RA) levels in the embryo. Ascidians are sessile marine invertebrate chordates that develop through a tadpole larva, with a body plan that shares basic homologies with vertebrates. In this work, we tested the effects of IMA on the development of the solitary ascidian Ciona intestinalis by treating two-cell stage embryos with a range of concentrations (0.1, 0.5, 1, 2.5, 5, 10, 20 and 50microThe fungicide significantly altered ascidian development even at low concentrations and its effects were dose-dependent. Probit analysis revealed that the median lethal concentration, LC(50), was 4.87microM and the median teratogenic concentration, TC(50), was 0.73microM. Larvae developing from embryos exposed to IMA showed malformations of the anterior structures, which became more severe as IMA concentration increased. In particular, the anterior nervous system and the sensory vesicle were reduced, and the pigmented organs (the ocellus and the otolith) progressively lost their pigmentation. The larval phenotype induced by 5microM IMA exposure was further characterized by means of molecular analysis, through whole mount in situ hybridization with probes for genes related to the nervous system: Ci-Otp, Ci-GAD, Ci-POU IV, which are markers of the anterior neuro-ectoderm, the central nervous system and the peripheral nervous system respectively, and Ci-Hox-1, a gene specifically activated by RA, and Ci-Aldh2, a gene for aldehyde dehydrogenase, which is involved in RA synthesis. The altered expression of Ci-Otp, Ci-GAD, Ci-POU IV in 5microM IMA-exposed larvae compared to control larvae showed that this fungicide could affect the differentiation of the anterior nervous system, particularly of the sensory vesicle neurons. Recent studies suggest a similarity between IMA- and RA-induced phenotypes in tunicates, indicating that triazoles may also alter RA metabolism in ascidians. The observed Ci-Hox-1 and Ci-Aldh2 expression in control and treated larvae did not allow a direct link between IMA teratogenic potential and RA-dependent morphogenesis to be identified. It is likely that the fungicidal teratogenic mechanism involved RA signalling but that its effects on ascidian development depend on a more complex mechanism.

Observations on the settlement of phallusia mammillata larvae: Effects of different lithological substrata

Abstract In the sessile marine tunicates, the selection of a suitable substratum by the larvae is an important and critical factor determining the distribution of species. The present paper investigated, under laboratory conditions, the role played by mineral content of the substratum in settlement of the ascidian Phallusia mammillata, using siliceous and carbonaceous stones. Individuals that attached to the different substrata and metamorphosed were scored. The data indicate that larvae could discriminate between the substrata on the basis of their silica content. Under the same laboratory conditions, the larvae that attached to siliceous stones grew faster and had a wider area of contact with the substratum than those that grew on carbonaceous stones. The present study concludes that silica is a mineral factor that can be discriminated by chemosensory palps of ascidian larvae during the choice of substratum. It is suggested that the mineral composition of the habitat can contribute, with other environmental factors, to regulate the spatial distribution of tunicate communities.

Action of valproic acid on Xenopus laevis development: teratogenic effects on eyes.

Valproic acid (VPA) is an anticonvulsive drug used in the treatment of epilepsy. Teratogenic effects of VPA have been described in different animal species. In this study, we investigate the effects of VPA on the development of Xenopus laevis embryos, by short pulse treatments (4 h) with relation to the dose and the stage of exposure to the drug. We exposed Xenopus embryos from blastula to stage 32 to three different doses of VPA (0.25, 5, and 10 mM) and we allowed these to develop until the controls reached stage 47. The embryos became more sensitive during the stages of neurulation, as observed in mouse and differently from Amblystoma, in which the more severe effects were produced by treatments at blastula stage. The malformations observed were similar to those described in mammals and other amphibians and consisted in developmental delay, perturbation of neural crest migration, and somite segmentation. We also observed abnormal development of the retina, which had never been described for VPA treatments. Therefore we analyzed the relation between VPA-induced eye malformations and the expression of Pax-6. We examined VPA-treated Xenopus embryos by whole mount in situ hybridization for mis-expression of Pax-6 in correlation with eye anomalies. Our results are consistent with the hypothesis that different members of Pax gene family are candidate target of VPA teratogenic action and in particular the decreased level of Pax-6 expression, shown by Northern blot analysis, is responsible for the retinal malformations we observed in VPA-treated Xenopus embryos. Teratogenesis Carcinog. Mutagen. 21:121-133, 2001.

WAY-100635, an antagonist of 5-HT1A receptor, causes malformations of the CNS in ascidian embryos

Serotonin (5-HT) is a neurotransmitter which is supposed to play a key role during development. In the last few years 5-HT receptors have been cloned in many animal species, and there is evidence that different 5-HT receptors are also present in ascidians. Ascidians and vertebrates are both members of the phylum Chordata and both have a dorsal tubular central nervous system. Embryos of the ascidian Phallusia mammillata have been treated with WAY-100635, a potent and selective 5-HT1A receptor antagonist. The larvae developed from treated embryos showed a dramatic reduction of their anterior sensory vesicles and the pigment of two sensory organs, the ocellus and the otolith. Immunofluorescence experiments with an anti β-tubulin monoclonal antibody specific for the neural system showed that the anterior neural system of treated animals was radically altered by the action of the drug in a dose-dependent way. These results suggest that 5-HT plays a role in the development of the neural system in ascidians and its action is mediated by receptors similar to the members of the 5-HT1A receptor subtype of mammals.

Developmental expression of tryptophan hydroxylase gene in Ciona intestinalis

To describe the serotonergic system in a tunicate larva, we cloned a gene encoding for tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin synthesis, in the ascidian Ciona intestinalis and studied its expression pattern during development. Ci-TPH expression was found from tailbud stage in the precursor cells of the visceral ganglion and in the tail. In the larva, TPH-expressing neurons formed two clusters in the anterior central nervous system at the level of the visceral ganglion. Moreover, we found Ci-TPH expression at the level of the muscle cells of the tail and suggested that this localisation might be at the level of neuro-muscolar junctions. Moreover, we discussed the involvement of serotonin in the control of larval locomotory activity.

AChE localization in adhesive papillae of ascidian larva: effects of citral, a retinoic acid synthesis inhibitor

Summary Acetylcholinesterase (AChE) activity has been localized in the primary neurons of the adhesive papillae of Ciona intestinalis and Phallusia mammillata larvae using the histochemical staining method described by Karnovsky and Roots (1964) after short fixation. The shape and cell structure of the papillae and the arrangement of primary neurons are different in the two species. Anterior truncation caused by RA or retinol suppresses the differentiation of the papillae and of papillary primary neurons, but combined treatment with retinol and citral can rescue the formation of rather normal papillae with AChE positive primary neurons. The AChE staining method has proved to be a useful tool in revealing differences in the cellular structure of adhesive papillae of the two species and to investigate the differentiation of anterior ectodermal structures.

The adhesive organs of Anura: A histological and molecular study

Abstract The cement gland is a secreting organ located at the anterior end of the larva of anuran Amphibia. It is a highly conserved structure even if it has different shapes in the different species. The morphology and the histology of the cement glands of seven species are analysed: Xenopus laevis, Discoglossus sardus, Bufo bufo, Bufo viridis, Rana esculenta, Rana latastei and Hyla intermedia. XCG and XAG are genes specifically expressed in the cement gland of Xenopus laevis and can be used as markers to identify cement gland territory. By Northern analysis and whole mount ’in situ’ hybridization, the expressions of these genes are tested in the studied species, the presence of homolog transcripts is detected in all the species, except Discoglossus sardus. Histological and molecular analysis show a substantial uniformity among the different types of adhesive organs. On the basis of the preliminary results obtained, some phylogenetic relations are inferred according to a recent phylogenetic tree based on molecular characters. The results seem to agree with the hypothesis that Pipi‐dae, to which Xenopus laevis belongs, are nearer to Neobatrachia than to Archeobatrachia.

Fluconazole induces teratogenic effects in the tunicate Phallusia mammillata

Fluconazole (FLUCO) is an azole derivative used to treat fungal and yeast infections. Embryotoxicity tests on the ascidian Phallusia mammillata were performed to evaluate the effects of this drug. FLUCO proved to have strong consequences on P. mammillata development. Incidence of malformations and of lethality increased in a dose dependent way. Probit analysis showed that FLUCO had a high TI value (Teratogenic Index, LC(50)/TC(50)), thus this substance could be classified as a teratogenic compound for ascidians. Larvae exposed to FLUCO showed a typical phenotype characterized by malformations restricted to the trunk region: the trunk appeared round in shape with flat palps, the sensory vesicle cavity was absent or reduced and the anterior central nervous system failed to correctly differentiate. These anomalies resulted similar to those induced by retinoic acid (RA) treatment. Thus, it could be hypothesized that FLUCO and RA may act with a similar pathogenic mechanism in ascidian larvae, as it has been proposed for mammals.