F. De Bernardi

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.

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.

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.

Induction of Somites by Myosin mRNA

The effects of the messenger for myosin heavy chain (26S mRNA) on postnodal explants of chick embryo blastoderm were studied. Somites do not differentiate in the postnodal explants of chick embryo cultivated by News method. They are induced when postnodal pieces are cultivated in the presence of a 26S mRNA extracted from chick leg muscles or in the presence of myosin. 26S mRNA plus actinomycin D induces small somites. 26S mRNA of duck, rabbit, or trout induce somite structures often built up of cells separated by large spaces and joined around a large myocele. Crayfish 26S mRNA or chick myosin light chain induce columnar cells connected around a cavity. Liver or kidney mRNA do not induce. The induction process can be summarized as follows: 26S mRNA codes for myosin (heavy chain) and the myosin (heavy chain) induces the somites. Induction of somites by mRNA can occur in the presence of actinomycin D but not when there is mRNA plus puromycin. It does occur when myosin acts in the presence of puromycin. Myosin and its heavy chain are present in chick blastoderm before the appearance of somites. Induced somites are able to induce neural plates. We conclude that in normal development somites are induced by myosin.

The postnodal piece (PNP) of the chick embryo as a model system for studying organ differentiation

The postnodal piece of the chick embryo is prepared by cutting the blastoderm 0.6 mm behind Hensens node at the primitive streak stage. The expiants with their area opaca can be maintained in flat culture (News method) for 24-30 hr. With this method the polarity and the connections between various sheets are maintained, but the expiants are stretched and difficult to handle for histology and immunostaining. Several PNPs from which the area opaca had been trimmed were cultured on one vitelline membrane (Niu and Deshpandes method) for up to 4 days without stretching effects. The polarity and connections between the embryonic sheets are hard to recognize, but expiants can be easily processed for histology and immunofluorescence. In both culture types the expiants can be easily treated, even with high molecular weight substances. Although the flat culture was useful for the induction of somites and of neural plates, we describe the advantages of culture without the area opaca of neural plates induced by tubulin mRNA or by TPA, which can differentiate into neural tubes. We also demonstrated that TPA is a powerful neural inducer in the chick embryo and stimulates cell proliferation in ectoderm and endoderm.

Transition from Non-Muscle to Muscle Myosin Light Chains in Chick Embryo Development

In chick embryo blastoderm the electrophoretic pattern of myosin light chains changes between the 4-somite and the 19-somite stages (stages 8-13 of Hamburger and Hamilton) from that of non-muscle to muscle myosin. This transition seems to follow the differentiation of the myotomes and to be developmentally regulated.