Morphogenesis of the saccus vasculosus of turbot Scophthalmus maximus: assessment of cell proliferation and distribution of parvalbumin and calretinin during ontogeny.

The ontogenesis of the saccus vasculosus (SV) of turbot Scophthalmus maximus is described using histological and immunohistochemical methods to assess the general morphology, as well as the distribution of proliferative cells and several calcium-binding proteins (CaBP). The results reveal that the SV begins to differentiate on hatching, when immature coronet cells are morphologically distinguishable. Further morphogenesis involves the formation of a tubular avascular SV, which remains until premetamorphic larval stages. Folding and vascularization of the SV occurs mostly during metamorphosis, when S. maximus settle down on the bottom. Proliferative cells were placed within the SV itself and in the neighbouring infundibular hypothalamus. Their putative relationship with the growth of the SV is discussed. The CaBPs analysed are expressed in coronet cells. Parvalbumin is expressed in these cells from the beginning of their differentiation, while calretinin expression arises in the tubular SV and becomes more widespread over time. These data emphasize the importance of calcium buffering in the function of coronet cells.

Development of the olfactory system in turbot (Psetta maxima L.).

We have examined the histogenesis of the olfactory system during turbot development using histological and immunohistochemical methods. Proliferating cell nuclear antigen (PCNA) immunohistochemistry was used to detect dividing cells, whereas calretinin (CR) immunohistochemistry was used to distinguish some neuronal components of the olfactory system. Around hatching, the olfactory placode of embryos transforms into an olfactory pit, which enlarges progressively during development. In metamorphic turbots, the right olfactory organ moves to the tip of the head. Each olfactory chamber opens to the external medium by two nostrils and accessory nasal sacs develop during metamorphosis. The order of birth of olfactory receptor cells in the sensory epithelium follows the pattern of most teleosts: ciliated cells differentiate prior to microvillous cells in turbot larvae, and crypt cells are generated during metamorphosis. Axons of olfactory sensory neurons reach the rostral forebrain by hatching, and calretinin-immunoreactive (CR-ir) glomerular fields were apparent during the subsequent larval development. During metamorphosis olfactory bulbs become strongly distorted by head torsion and glomeruli acquire asymmetric organization. The spatio-temporal course of proliferation in the olfactory system reveals changes in the distribution of dividing cells in the sensory epithelium throughout the developmental period investigated. In the olfactory bulb, proliferative activity becomes restricted to the ventral periventricular zone in turbot larvae, as well as in metamorphic specimens.

The terminal nerve in turbot, Psetta maxima: a developmental immunocytochemical study.

The ontogeny and organization of the terminal nerve (TN) during turbot development was studied using an antiserum to neuropeptide Y. First immunoreactive cells were detected in the olfactory placode at hatching time. At 1 day after hatching, a loose group of labeled neurons form an extracranial primordial ganglion of the TN. During the subsequent larval development, more perikarya displaying increased immunoreactivity were found along the course of the olfactory nerve. Moreover, labeled cells cross the meninx of the forebrain gathering in the olfactory bulb of larval turbot. Projections from these cells, directed both to the caudal brain and to the retina, develop when the cells become established in the olfactory bulb. The generation of immunoreactive cells in the olfactory organ extends into the metamorphic period, when a pronounced asymmetry affects the turbot morphology. At this time, the topological location of the immunoreactive cells in the TN becomes distorted. This developmental pattern was compared with those found in other teleosts and in other vertebrates. Preabsorption experiments of anti-neuropeptide Y serum with neuropeptide Y and FMRF-amide suggests that immunoreactive material observed in TN cells was not neuropeptide Y, and raises the possibility that other peptides, e.g. FMRF-amide-like peptides, exist in this neural system.

Immunochemical localization of calretinin in the retina of the turbot (Psetta maxima) during development.

The expression of the calcium-binding protein calretinin was analysed by immunohistochemistry techniques in the retina of turbot (Psetta maxima) from embryonic to juvenile stages. Calretinin immunoreactivity was first detected in retinae from newly hatched larvae, in which the anlage of the inner plexiform layer and a subset of amacrine and ganglion cells displayed a faint immunolabelling. First appearance of photoreceptors during larval life coincided with an increase in the intensity of the labelling. During subsequent larval development, the expression of calretinin affected distinctive retinal components. The inner plexiform layer, optic fiber layer, and a population of amacrine and ganglion cells were invariably labelled. Occasional bipolar cells were labelled at the end of the larval period. By metamorphosis, calretinin is sequentially expressed in horizontal cells, and bipolar immunoreactive cells become numerous. The pattern of calretinin immunoreactivity of the inner plexiform layer changes from the larval to juvenile period. In all cases, calretinin immunoreactivity exhibited variations between the peripheral retina, which contains the most recently differentiated retinal components, and the remainder of the differentiated retina. Our results suggest that the progressive expression of calretinin in the turbot retina appears associated with some degree of neuronal differentiation. Once the definitive pattern of calretinin immunoreactivity is established in the turbot retina, both similarities and differences with the calretinin location in the retina of other vertebrates can be demonstrated.