Development of the Epicardium in the Dogfish (Scyliorhinus canicula)

The development of the epicardium has been described in mammals, including man, birds and amphibians. However, there is no information concerning this morphogenetic process in fishes. A study carried out in embryos of the dogfish ( Scyliorhinus canicula ) showed that, in this elasmobranch species, the precursors of the epicardium originate from two mesothelial anlagen, the right and left, that initially lie at the ventrolateral parts of the liver. These two anlagen, which will be referred to as the proepicardium, later shift to the right and left parts of the pericardial aspect of the transverse septum. The proepicardium comprises numerous spheric, smooth-contoured cells and a relatively small amount of extracellular matrix. The proepicardium is not covered by an epithelial layer.
Cells detaching from the proepicardium adhere to the surface of the heart and develop into epicardial cells. They firstly ensheathe the atrioventricular groove as well as the dorsal and lateral aspects of the ventricle, and the ventral and lateral aspects of the atrium. Both the sinus venosus and conus arteriosus become lined later.
In spite of the phylogenetic distance between elasmobranchs and mammals, the mechanism by which the epicardium develops is similar in both groups. This similarity relies principally on the arrangement and location of the proepicardium and the way in which the epicardial precursors reach and invest the heart.     

Phagocytic Cells in the Peripheral Blood of the Dogfish Scyliorhinus canicula L. I. In Vitro Studies

In vitro phagocytosis by peripheral blood leucocytes of the dogfish Scyliorhinus canicula L. was examined by exposing adherent cells to a variety of particulate and soluble antigens and inert material. Their subsequent uptake was monitored by light, scanning and transmission electron microscopy. The monocyte and the neutrophil-like granulocyte were found to be the major phagocytic cells. Larger particles like yeasts and erythrocytes were the most avidly phagocytosed. From studies on the effects of pH, temperature and the presence of plasma, metabolic inhibitors and divalent ions, it appeared that optimum phagocytosis occurred at pH 7.0 and between 10 and 20°C. Serum factors did not enhance the process in this species. Finally, the in vitro clearance of 5 bacterial species indicated that the presence of blood phagocytes had little or no effect on bacterial numbers.     

Phagocytic Cells in the Peripheral Blood of the Dogfish Scyliorhinus canicula L. II. In Vivo Studies

In vivo phagocytosis by peripheral blood leucocytes of the dogfish Scyliorhinus canicula L. was examined by monitoring the fate of a variety of injected materials, both particulate and soluble, in normal and immunised fish. Carbon, yeasts and bacteria were phagocytosed by monocytes, thrombocytes and type 1 granulocytes (neutrophils). Quantitative in vivo antigen clearance studies employed five species of bacteria, yeast and KLH. After an initial significant decrease of these antigens in the circulation, low numbers of viable bacteria and yeasts and low concentrations of KLH persisted for long periods after injection. Previous exposure to several of these antigens had little or no effect.     

Immunological Responses of the Dogfish (Scyliorhinus canicula L.) to Cellular Antigens

Humoral and cellular aspects of the immune response of the common dogfish (Scyliorhinus canicula L.) to a variety of cellular antigens have been examined. The fish made a relatively slow but positive antibody response to injected Salmonella typhi. Using the haemolytic plaque technique, antibody synthesis was shown to occur in the spleen after fish were challenged with sheep red blood cells, suggesting that this organ is a major site of antibody synthesis. Intraperitoneal and intravenous injections of allogeneic leucocytes stimulated marked histological changes in the spleens of recipients, indicative of a host-vs-graft reaction and suggesting that the fish are capable of alloimmune reactions.     

Immunohistochemical Study of the Origin of the Subepicardial Mesenchyme in the Dogfish (Scyliorhinus canicula)

A mesenchyme appears at the subepicardium of the vertebrate embryo closely following the epicardial ensheathing of the heart. We have suggested earlier that a localized epithelial-mesenchymal transition (= transdifferentialion) of the epicardial mesothelium might explain the origin of the subepicardial mesenchymal cells (SEMC) in a primitive vertebrate, the dogfish Scyliorhinus canicula. We have tested our hypothesis through the immunolocalization, in the embryonic dogfish heart, of three proteins involved in the epithelial—mesenchymal transitions. Fibronectin immunoreactivity (FN-IR) was present between epicardium and myocardium from the earliest stages of the epicardial development. However, in embryos ranging between 20 and 35 mm TL, FN-IR disappeared from the basal surface of the epicardial cells at the atrioventricular and conoventricular grooves and increased in association with the SEMC. Proliferating-cell nuclear antigen immunoreactivity was intense in most epicardial cells and SEMC in these areas and developmental stages, but it declined in later embryos. The JB3 antigen, a fibrillin-related protein, was delected in the cytoplasm and basal surface of the epicardial cells, as well as on the SEMC surface. These immunohistochemical patterns were remarkably similar at the subepicardium and at the endocardial cushions, which are populated by mesenchymal cells derived from the transdifferentiation of the endocardium. © 1998 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd. All rights reserved     

Cd-, Zn-, Cu-binding protein in the elasmobranch Scyliorhinus canicula

A Cd-, Zn-, Cu-binding protein was isolated from the liver of dogfish subjected to environmental experimental contamination (50 ppm Cd). It was also found in liver from untreated fish. This protein has a high absorption at 250 nm and a low absorption at 280 nm, suggesting a mercaptide bond and a lack of aromatic amino acids. The SDS-PAGE pattern and changes in absorption spectrum on adding Cd or lowering pH are found as for mouse MT. The protein contains Cu and Zn in control and Cu, Zn and Cd in treated fish. Cd levels in the protein are significantly higher in females and significantly increase with treatment duration. Copper levels decrease after 96 hr in males and 6 days in females.     

Functional Morphology of the Olfactory Organs in the Spiny Dogfish (Squalus acanthias L.) and the Small-spotted Catshark (Scyliorhinus canicula (L.))

The morphology of the olfactory organs in two sharks, the spiny dogfish and the small-spotted catshark, was studied by light microscopy and electron microscopy (TEM and SEM). The olfactory epithelium is arranged on olfactory lamellae which are provided with secondary folds. The epithelium mainly consists of microvillous receptor cells, multiciliated supporting cells and basal cells. The find of only one type of receptor cells, the microvillous type, is discussed and the condition considered a derived (apomorphic) character. The route of the water current through the olfactory organ and the different driving forces of the ventilation process are subject to discussion. In both the pelagic dogfish and the bottom-dwelling catshark the pressure difference between the incurrent and excurrent nostrils achieved by active swimming appears to be the driving force, whereas the role of the beating of the non-sensory cilia is not evident. In the bottom-dwelling catshark the ventilation of the olfactory organ is also supported by the respiratory activity.     

Studies on the reticulo-endothelial system of the dogfish,Scyliorhinus canicula

Summary Clearance and subsequent localisation of a range of materials, including colloidal carbon, latex beads, sheep erythrocytes, bacteria and dextran were followed in the lesser spotted dogfish, Scyliorhinus canicula. It was found that two populations of peripheral blood leucocytes — monocytes and thrombocytes, but not granulocytes — were involved in clearance of the circulation. In the case of carbon, this material was cleared from the plasma after 12 h, and both the colloid-containing thrombocytes and monocytes disappeared from circulation by 8 weeks post injection. Upon injection of some of the materials, and particularly bacteria, a settling out of monocytes containing phagocytosed material was seen in the secondary lamellae and cavernous bodies of the gills. Large clumps of monocytes were found in the gills as early as 30 min post injection and these increased in size for up to one week, after which they gradually dispersed. The lining cells of the cavernous body, known as CB cells, were also responsible for the sequestration of carbon, latex beads and probably erythrocytes, but dextran and bacteria were not internalised. The origin, functions and phylogenetic significance of the CB cells are discussed.     

Development of dentition and dermal skeleton in embryonic Scyliorhinus canicula

Serial sections ranging from very young embryos to hatched juveniles and whole embryos of Scyliorhinus show that dentition and dermal skeleton belong to two independent secondary developmental fields that differ both developmentally and structurally. The development of the dentition starts very early, with a thickening of the ectoderm in the region of the mouth (stage 04), the invagination of the dental lamina (stage 18), and the formation of the germs of the first generation (stage 20). Tooth replacement movements start only near the end of embryogenesis (stage 35). Scale germs, on the other hand, first begin to form at stage 24. Scales erupt shortly before the animal hatches (stage 43). Only one scale generation is formed during embryogenesis. The forces which erupt the scales may come from fluid pressures in vacuoles of the fibrous layer of the dermis. Those which erupt the teeth probably also result from similar fluid pressures. The crown and upper part of the base of scales and teeth are formed by cells of the inner dental epithelium which are differentiated from the ectoderm. They are also formed by odontoblasts which are derived from the vascular layer of the dermis. However, the basal plates of scales and teeth containing the anchoring fibers are formed by osteoblasts, which are derived from the fibrous layer of the dermis.     

Occurrence of bombesin-like immunoreactivity in the brain of the cartilaginous fish,Scyliorhinus canicula

Summary The presence and distribution of bombesin-like material were investigated in the brain of the cartilaginous fishScyliorhinus canicula using conventional immunocytochemical techniques. Perikarya containing bombesin-like immunoreactivity were identified in the hypothalamus, within the magnocellular component of the preoptic nucleus. Some immunopositive elements appeared to be of cerebrospinal fluid-contacting type. Beaded immunoreactive fibers were seen crossing the ventral telencephalon and the whole hypothalamus. An important tract of fibers was found in the infundibular floor and in the median eminence, in close contact with the vascular system of the pituitary portal plexus. A moderate number of positive fibers innervated the habenular complex and the dorsal wall of the posterior tuberculum. These findings indicate that a neuropeptide strictly related to amphibian bombesin is located in specific hypothalamic neurons ofS. canicula. The distribution of the immunoreactive fibers and terminals suggests that, in fish, this peptide, may be involved in neuroendocrine and neuromodulator functions.     

Structural studies on the thymus of the dogfish, Scyliorhinus canicula L.

Histological and ultrastructural studies of the thymus of the dogfish, Scyliorhinus canicula L., revealed paired lobular masses above the first two gill arches of embryonic and newly hatched dogfish, involuting at approximately 3 weeks. It did not have defined cortex and medullary regions and ultrastructural investigations showed the tissue to be composed mainly of a range of different sized lymphocytes within a connective tissue capsule and reticular epithelial cell framework. Lymphoblasts often in mitosis were also present being more electron lucent and larger than lymphocytes. Macrophages contained cellular debris and frequently whole small lymphocytes. A cell with cytoplasmic granules was occasionally observed.     

Ciliated cells on the surface of embryos of Scyliorhinus canicula

Monociliated surface cells were found for the first time in an elasmobranch and were common on embryos of Scyliorhinus canicula . They occurred mainly near the anterior end, until Ballard stage 30, but disappeared soon thereafter. The functions of surface ciliated cells suggested in other embryos ( e.g. surface currents in lungfishes and amphibians, and determination of left–right patterns in amniotes) seem unlikely in elasmobranchs.     

The developmental expression of foxl2 in the dogfish Scyliorhinus canicula

The FoxL2 genes are a subfamily of the Fox (forkhead box) gene family. FOXL2 is mutated in the disorder Blepharophimosis, Ptosis, and Epicanthus Inversus Syndrome (BPES), which is characterized by eyelid malformations, and Premature Ovarian Failure (POF). In the mouse expression is seen in the perioptic mesenchyme, developing eyelids, ovary and pituitary. We have isolated a foxl2 cDNA from the dogfish Scyliorhinus canicula (also known as the lesser spotted catshark), allowing the characterisation of this gene's sequence and expression from a lineage that diverged early in the evolution of gnathostomes. Molecular phylogenetic analysis strongly grouped this sequence with the gnathostomes within the FoxL2 subfamily. We demonstrate the early expression of Scyliorhinus canicula foxl2 in the mandibular head mesoderm and later in continuous populations of mandibular arch cells and mandibular head mesenchyme cells around the developing pituitary. As development proceeds expression decreases in the mesenchyme of the head but is seen in the mesenchyme around the eye and later in the developing eyelids. Additionally expression is seen in regions of pharyngeal arch mesoderm and in ectoderm from which gill buds will form. This expression is maintained in the developing and elongating gill buds. Thus, S. canicula foxl2 is a marker for the mandibular mesoderm and gill buds and its expression is conserved in the perioptic mesenchyme, developing eyelids and pituitary.     

Distribution of monoamines in the diencephalon and pituitary of the dogfish,Scyliorhinus canicula L.

Summary The distribution of monoamines in the diencephalon and pituitary of the dogfish, Scyliorhinus canicula, has been investigated using the histochemical fluorescence technique of Falck and Hillarp (Falck and Owman, 1965). Terminals of monoamine-containing axons were found in the neurointermediate lobe of the pituitary and the axons were traced, by means of nialamide and L-dopa treatment and lesions, to the nucleus medius hypothalamicus. A separate hypothalamic system converging on the anterior median eminence and the occurrence of aminergic cells in the nuclei lobi inferiores and nucleus medius hypothalamicus were similarly demonstrated. Normal fish show a bilateral uncrossed tegmental tract and two areas of catecholamine-containing neurones in modified ependymal organs. The organum vasculosum hypothalami includes both primary catecholamine and 5-hydroxytryptamine-containing cell types whilst the organum vasculosum praeopticum has only the former type. Both organs contain cells which send club-like processes into the third ventricle. The subcommissural organ does not contain monoamines.The role of hypothalamic catecholamine systems in the regulation of pituitary function is discussed.     

Metal accumulation and metallothionein induction in the spotted dogfish Scyliorhinus canicula

Recent studies indicate that elasmobranch fish respond differently to metal exposure than marine teleosts. Accumulation rates can be high, which despite the fact that normal background levels for metals in the marine environment are low, is worrying due to the long life span and late fecundity of most shark. The goals of the present study were to examine differences in accumulation rates and toxicity of a range of metals at equimolar concentrations (10 µM) in the Mediterranean or spotted dogfish, Scyliorhinus canicula. For this purpose, we exposed the dogfish to Ni (587 µg/L), Cd (1124 µg/L), Pb (2072 µg/L), Cu (635 µg/L), and Ag (1079 µg/L and two additional exposures at 10 µg/L and 1 µg/L) for one week and measured total metal accumulation, metallothionein induction, and parameters related to osmoregulation. Our study confirms the high toxicity and accumulation rates of Ag for elasmobranch fish, even at levels 100 to 1000 times lower than exposure levels of other metals. Also Pb accumulated readily in all organs, but did not cause any osmoregulatory disturbance at the exposure levels used. Ni and Cd seem to accumulate primarily in the kidney while Cu mainly accumulated in liver. In contrast to Ni and Cd, the three other metals Ag, Cu and Pb accumulated in the rectal gland, an important organ for osmoregulation and possible target organ for metal toxicity. Only Cu succeeded in initiating a protective response by inducing MT synthesis in liver and gills.