Giuliana Zega

Complex neural architecture in the diploblastic larva of Clava multicornis (Hydrozoa, Cnidaria).

The organization of the cnidarian nervous system has been widely documented in polyps and medusae, but little is known about the nervous system of planula larvae, which give rise to adult forms after settling and metamorphosis. We describe histological and cytological features of the nervous system in planulae of the hydrozoan Clava multicornis. These planulae do not swim freely in the water column but rather crawl on the substrate by means of directional, coordinated ciliary movement coupled to lateral muscular bending movements associated with positive phototaxis. Histological analysis shows pronounced anteroposterior regionalization of the planulas nervous system, with different neural cell types highly concentrated at the anterior pole. Transmission electron microscopy of planulae shows the nervous system to be unusually complex, with a large, orderly array of sensory cells at the anterior pole. In the anterior half of the planula, the basiectodermal plexus of neurites forms an extensive orthogonal network, whereas more posteriorly neurites extend longitudinally along the body axis. Additional levels of nervous system complexity are uncovered by neuropeptide‐specific immunocytochemistry, which reveals distinct neural subsets having specific molecular phenotypes. Together these observations imply that the nervous system of the planula of Clava multicornis manifests a remarkable level of histological, cytological, and functional organization, the features of which may be reminiscent of those present in early bilaterian animals. J. Comp. Neurol. 519:1931–1951, 2011.

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.

Immunohistochemical study of the nervous system of the tunicate Thalia democratica (Forsskal, 1775)

Thalia democratica is a cosmopolitan tunicate belonging to the Thaliacea class. To further investigate the anatomy of this species, immunohistochemical labelling was performed using anti-tubulin and anti-serotonin antibodies on specimens collected in the Mediterranean Sea. The anti-tubulin antibody stained the cilia of the endostyle, the pericoronal bands and of the gill bar, enabling a detailed description of these structures. Moreover, immunolabelling of the nervous system showed the presence of eight pairs of nerve fibres emerging from the neural ganglion. Serotonergic cells were observed in the distal tract of the intestine, along the pericoronal bands, and in the placenta of gravid blastozooids, as well as in the neural ganglion. The presence of serotonin in the central nervous system has also been reported in the larvae of ascidians and may be linked to the planktonic life of these animals, a condition shared by adult thaliaceans and ascidian larvae. This work improves our knowledge of the anatomy of T. democratica and demonstrates the presence of a complex serotonergic system.

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.

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.

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.

Immunohistochemical analysis of the adhesive papillae of Botrylloides leachi (Chordata, Tunicata, Ascidiacea): Implications for their sensory function

Most ascidian larvae settle and begin adhesion by means of three mucus secreting and sensory organs, the adhesive papillae or palps. However, the adhesive papillae of Botrylloides genus larvae, despite their name, have only a sensory function. By immunohistochemical localization of serotonin and β‐tubulin, we demonstrated that the adhesive papillae of Botrylloides leachi contain two distinct types of neurons with different localization and possibly a different function. The central neurons emerge from the tunic at the apex of papillae and probably have a mechanosensory function. The lateral neurons contain serotonin and may play a role in the mechanism of metamorphosis triggering. Moreover, by histological analysis we found numerous secreting cells, clustered in the centre of the area among the three papillae, forming a glandular organ. This organ could perform the attachment of the larva to the substrate, which is traditionally considered to be operated by the anterior epidermis of the larva, with a sucker‐like mechanism.

Immunohistochemical analysis of adhesive papillae of Clavelina lepadiformis (Müller, 1776) and Clavelina phlegraea (Salfi, 1929) (Tunicata, Ascidiacea)

Almost all ascidian larvae bear three mucus secreting and sensory organs, the adhesive papillae, at the anterior end of the trunk, which play an important role during the settlement phase. The morphology and the cellular composition of these organs varies greatly in the different species. The larvae of the Clavelina genus bear simple bulbous papillae, which are considered to have only a secretory function. We analysed the adhesive papillae of two species belonging to this genus, C. lepadiformis and C. phlegraea, by histological sections and by immunolocalisation of β-tubulin and serotonin, in order to better clarify the cellular composition of these organs. We demonstrated that they contain at least two types of neurons: central neurons, bearing microvilli, and peripheral ciliated neurons. Peripheral neurons of C. lepadiformis contain serotonin. We suggest that these two neurons play different roles during settlement: the central ones may be chemo- or mechanoreceptors that sense the substratum, and the peripheral ones may be involved in the mechanism that triggers metamorphosis.