Cristina Sotgia

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.

Adhesive Papillae of Phallusia mamillata Larvae: Morphology and Innervation

Abstract The swimming larvae of most solitary ascidians belonging to the Ascidiidae family bear three anterior, simple conic adhesive papillae. They secrete adhesive substances that are used to effect transitory settlement at the beginning of the metamorphosis. The adhesive papillae of newly hatched Phallusia mamillata larvae examined by the SEM are covered by the tunic. When the larvae are about to settle, the tunic becomes fenestrated over the central part of the papilla and bulb-ended microvilli protrude through the holes. These papillae have two types of elongated cells: many peripheral cells and few larger central cells with microvilli and bundles of microtubules oriented along the major axis of the cells. We have done immunofluorescence experiments with an anti-β-tubulin monoclonal antibody (clone 2-28-33) reacting with axonal microtubules. Only the central cells of the papillae were stained and the axons appeared to arise from the proximal ends of these cells. These axons form a long nerve that reaches the brain vesicle. Branches of the same nerve appear to connect to the basal ends of the peripheral cells. By confocal laser microscopy we were able to follow the course of the papillary nerve. The two nerves connecting the dorsal papillae fuse together into a single nerve that runs posteriorly. The nerve connecting the ventral papilla runs posteriorly for a long tract before fusing with the nerve of the dorsal papillae just near the brain. The reported observations raise the hypothesis that the central cells of the adhesive papillae might be primary sensory neurons and that they may have chemosensory function.

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.

The development of a bdelloid egg: a contribution after 100 years

Rotifer development has received very little attention: studies date back to the 19th century and to the first half of 20th century, and very limited contributions have been added in recent times. All information we have on rotifer embryology is mostly based on in vivo observation of developing embryos by light microscopy, and only in a minor way by classical histology. The study of rotifer embryogenesis is approached here using in vivo observation and laser confocal microscopy. We revealed cytoskeletal components (filamentous actin and tubulin) and nuclear DNA of the embryos to draw the pattern of the early development of Macrotrachela quadricornifera. Our results were then compared to the literature data, to determine a development pattern that can be generalized to the whole rotifer group. On the whole, our results agree with the general description provided by previous authors, i.e. the holoblastic unequal segmentation, the transverse furrow of the first division, the typical 16-cell stage, and the early gastrulation by epiboly. A peculiar pattern could also be seen that was interpreted as the formation of the mastax; it seemed to start from a mould of actin, visible by confocal only. The present study provides a preliminary contribution to a too-long-neglected aspect of rotifer biology.

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.

Settlement of the barnacle Balanus improvisus: The roles of dopamine and serotonin

The regulation of the settlement process in barnacles has attracted research interest due to their role as fouling organisms. The involvement of neurotransmitters in the regulation of settlement of marine invertebrate larvae has been described in several species. In this work, we reported the effects of the neurotransmitters dopamine and serotonin on the settlement of cyprids of the barnacle Balanus improvisus and described differences among cyprids of different ages. Also, we tested the effects of dopaminergic and serotonergic agonists and antagonists on settlement of cyprid larvae. We found that dopamine significantly stimulated settlement of 2‐ and 4‐day‐old cyprids, while serotonin exerted an inhibitory effect, regardless of cyprid age. The agonists and antagonists to the two neurotransmitters were not able to stimulate settlement but in general had either an inhibitory or no effect. We compared our results to those previously reported of the roles of dopamine and serotonin in the settlement of Balanus amphitrite. There appeared to be striking differences in the effects of these neurotransmitters between the two species because it has been reported that serotonin induces settlement in B. amphitrite and that dopamine inhibits it. This suggested that dopamine and serotonin play pivotal roles in settlement of barnacle but may act in different ways in the two species.

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.