Contributions of developmental studies in the dogfish Scyliorhinus canicula to the brain anatomy of elasmobranchs: insights on the basal ganglia.

The basic anatomy of the elasmobranch brain has been previously established after studying the organization of the different subdivisions in the adult brain. However, despite the relatively abundant immunohistochemical and hodologic studies performed in different species of sharks and skates, the organization of some brain subdivisions remains unclear. The present study focuses on some brain regions in which subdivisions established on the basis of anatomical data in adults remain controversial, such as the subpallium, mainly the striatal subdivision. Taking advantage of the great potential of the lesser spotted dogfish, Scyliorhinus canicula, as a model for developmental studies, we have characterized the subpallium throughout development and postembryonic stages by analyzing the distribution of immunomarkers for GABA, catecholamines, and neuropeptides, such as substance P. Moreover, we have analyzed the expression pattern of regulatory genes involved in the regionalization of the telencephalon, such as Dlx2, Nkx2.1, and Shh, and followed their derivatives throughout development in relation to the distribution of such neurochemical markers. For further characterization, we have also analyzed the patterns of innervation of the subpallium after applying tract-tracing techniques. Our observations may shed light on postulate equivalences of regions and nuclei among elasmobranchs and support homologies with other vertebrates.

Calretinin immunoreactivity in the developing retina of sharks: comparison with cell proliferation and GABAergic system markers.

The calcium-binding protein calretinin (CR) has been widely used as a marker of neuronal differentiation. In the present study we analyzed the distribution of CR-immunoreactive (CR-ir) elements in the embryonic and postembryonic retina of two elasmobranchs, the lesser spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus). We compared the distribution of CR with that of a proliferation marker (the proliferating cell nuclear antigen, PCNA) in order to investigate the time course of CR expression during retinogenesis and explored the relationship between CR and glutamic acid decarboxylase (GAD), the synthesizing enzyme of the gamma-aminobutyric acid (GABA), which has been reported to play a role in shark retinogenesis. The earliest CR immunoreactivity was concurrently observed in subsets of: a) ganglion cells in the ganglion cell layer; b) displaced ganglion cells in the inner plexiform layer and inner part of the inner nuclear layer (INLi); c) amacrine cells in the INLi, and d) horizontal cells. This pattern of CR distribution is established in the developing retina from early stage 32, long after the appearance of a layered retinal organization in the inner retina, and coinciding with photoreceptor maturation in the outer retina. We also demonstrated that CR is expressed in postmitotic cells long after they have exited the cell cycle and in a subset of GABAergic horizontal cells. Overall our results provide insights into the differentiation patterns in the elasmobranch retina and supply further comparative data on the development of CR distribution in the retina of vertebrates. This study may help in understanding the possible involvement of CR in aspects of retinal morphogenesis.

Development of the serotoninergic system in the central nervous system of a shark, the lesser spotted dogfish Scyliorhinus canicula.

Chondrychthyans (cartilaginous fishes) are key to understanding the ancestral gnathostome condition since they provide an outgroup to sarcopterygians and actinopterygians. To gain comparative knowledge about the development of the vertebrate serotoninergic systems, we studied by immunohistochemistry the origin, spatiotemporal organization, and migration patterns of serotonin-containing neurons and the growth of axonal pathways in the central nervous system of a shark, the lesser spotted dogfish. Hindbrain serotonin-immunoreactive cells arose close to the floor plate and most populations migrated ventrally and mediolaterally to form the various raphe and reticular groups. The order of appearance of serotoninergic populations in the rhombencephalon and spinal cord (first the superior groups and then the inferior and spinal populations) roughly matched with that reported in other vertebrates but important differences were noted in the formation of prosencephalic groups in fishes. In addition to preoptic and hypothalamic areas, serotoninergic cerebrospinal fluid-contacting cells were observed in the isthmus (raphe dorsalis anterioris). Transient serotonin-immunoreactive cells were noted in the pineal organ, habenula, and pretectum. Further, we provide a revised anatomical framework for reticular and raphe serotoninergic populations considering their origin and segmental organization. Two distinct phases of development of the serotoninergic innervation were distinguished, that of the formation of the main axonal pathways and that of the branching of fibers. The development of main serotoninergic ascending pathways in dogfish was notably similar to that described in mammals. Our findings suggest the conservation of developmental patterns in serotoninergic systems and enhance the importance of elasmobranchs for understanding the early evolution of this system in vertebrates.

Evolution of a core gene network for skeletogenesis in chordates.

The skeleton is one of the most important features for the reconstruction of vertebrate phylogeny but few data are available to understand its molecular origin. In mammals the Runt genes are central regulators of skeletogenesis. Runx2 was shown to be essential for osteoblast differentiation, tooth development, and bone formation. Both Runx2 and Runx3 are essential for chondrocyte maturation. Furthermore, Runx2 directly regulates Indian hedgehog expression, a master coordinator of skeletal development. To clarify the correlation of Runt gene evolution and the emergence of cartilage and bone in vertebrates, we cloned the Runt genes from hagfish as representative of jawless fish (MgRunxA, MgRunxB) and from dogfish as representative of jawed cartilaginous fish (ScRunx1-3). According to our phylogenetic reconstruction the stem species of chordates harboured a single Runt gene and thereafter Runt locus duplications occurred during early vertebrate evolution. All newly isolated Runt genes were expressed in cartilage according to quantitative PCR. In situ hybridisation confirmed high MgRunxA expression in hard cartilage of hagfish. In dogfish ScRunx2 and ScRunx3 were expressed in embryonal cartilage whereas all three Runt genes were detected in teeth and placoid scales. In cephalochordates (lancelets) Runt, Hedgehog and SoxE were strongly expressed in the gill bars and expression of Runt and Hedgehog was found in endo- as well as ectodermal cells. Furthermore we demonstrate that the lancelet Runt protein binds to Runt binding sites in the lancelet Hedgehog promoter and regulates its activity. Together, these results suggest that Runt and Hedgehog were part of a core gene network for cartilage formation, which was already active in the gill bars of the common ancestor of cephalochordates and vertebrates and diversified after Runt duplications had occurred during vertebrate evolution. The similarities in expression patterns of Runt genes support the view that teeth and placoid scales evolved from a homologous developmental module.

Epicardial development in lamprey supports an evolutionary origin of the vertebrate epicardium from an ancestral pronephric external glomerulus.

The epicardium is the outer layer of the vertebrate heart. Both the embryonic epicardium and its derived mesenchyme are critical to heart development, contributing to the coronary vasculature and modulating the proliferation of the ventricular myocardium. The embryonic epicardium arises from an extracardiac, originally paired progenitor tissue called the proepicardium, a proliferation of coelomic cells found at the limit between the liver and the sinus venosus. Proepicardial cells attach to and spread over the cardiac surface giving rise to the epicardium. Invertebrate hearts always lack of epicardium, and no hypothesis has been proposed about the origin of this tissue and its proepicardial progenitor in vertebrates. We herein describe the epicardial development in a representative of the most basal living lineage of vertebrates, the agnathan Petromyzon marinus (lamprey). The epicardium in lampreys develops by migration of coelomic cells clustered in a paired structure at the roof of the coelomic cavity, between the pronephros and the gut. Later on, these outgrowths differentiate into the pronephric external glomerulus (PEG), a structure composed of capillary networks, mesangial cells, and podocytes. This observation is consistent with the conclusion that the primordia of the most anterior pair of PEG in agnathans have been retained and transformed into the proepicardium in gnathostomes. Glomerular progenitor cells are highly vasculogenic and probably allowed for the vascularization of a cardiac tube primarily devoid of coronary vessels. This new hypothesis accounts for the striking epicardial expression of Wt1 and Pod1, two transcription factors essential for development of the excretory system.

The accumulation of lead and mercury from seawater and their depuration by eggs of the spotted dogfish Scyliorhinus canicula (Chondrichthys).

Radiotracer experiments using (210)Pb and (203)Hg demonstrated that eggs of the spotted dogfish Scyliorhinus canicula absorbed lead and inorganic mercury directly from seawater over 21 days of experimental exposure, attaining total egg concentration factors (CFs) relative to water of approximately 400 for Pb and 180 for Hg, predominantly (> or =98%) due to their accumulation by the collagenous egg case. The rates of accumulation of both Pb and Hg by the total egg were significantly (P < 0.0001) reduced by its increasing age since parturition, whereas only the rate of depuration of Pb was reduced (P < 0.0001) with increasing age; these effects indicate a declining chemical reactivity of the egg case that may be due to the continued tanning of the case following parturition. The egg case per se, attained average CFs of about 1,500 and 850 for Pb and Hg, respectively. Both Pb and Hg showed declining concentration gradients from the exterior to the interior membranes of the wall of the egg case; CFs for Pb declined from 3,500 to 2,000 and for Hg from 5,000 to 500. Comparison of concentrations in separate membranes also demonstrated significant (P < or = 0.01) depurations of Hg from the external and internal membranes during the loss experiments. The presence of radiotracers of Pb and Hg in the internal components of the egg at the end of uptake phase, and prior to the opening of the apertures, confirmed the permeability of the egg case wall to them, consistent with their observed gradients in it. The average CFs for all embryos at the end of the uptake experiment were 34 and 44 for Pb and Hg, respectively, but were significantly (P < 0.001) enhanced for Hg by a factor of 6 in the older eggs. The accumulatory and kinetic characteristics of the egg-case may operate to optimize the exposure of embryos to Pb and Hg following episodic contaminant events in coastal habitats.

Antioxidant system of Black Sea animals in early development.

Activities of lipoxygenase, catalase, superoxide dismutase, peroxidase, glutathione reductase and content of low molecular weight antioxidants were determined in eggs and larvae of some molluscs, crustaceans, elasmobranchs and teleost fish of the Black Sea. The enzyme activities and concentrations of low molecular weight antioxidants showed marked interspecies differences, depending on specific developmental peculiarities. During marine animal embryogenesis the activities of lipoxygenase and most of the examined antioxidant enzymes tended to increase in eggs and especially in hatching larvae, while the contents of low molecular weight antioxidants were decreased. High correlations between antioxidant enzyme activities (0.52 < r < 0.96), content of low molecular weight antioxidants (0.58 < r < 0.99) and developmental stages of examined marine animals were established.

Ontogeny of serotonin-immunoreactive cells in the gut epithelium of the cloudy dogfish, Scyliorhinus torazame, with reference to coexistence of serotonin and neuropeptide Y.

The ontogeny of serotonin (5-HT)-immunoreactive (IR) cells in the gut epithelium of an oviparous elasmobranch, Scyliorhinus torazame, was examined immunohistochemically. 5-HT-IR cells first appeared in the proximal part of the vitellointestinal duct (VID) and in the anterior part of the midgut of the embryo (30 mm in total length). At the 40-mm stage, the cells slightly increased in number and spread to the posterior part of the midgut, but no labeled cells were found in the foregut or hindgut. By the late embryonic (74- and 80-mm) stages, 5-HT-IR cells were markedly increased in number in the spiral intestine and stomach, whereas they were few in the VID and rectum. During these stages, the density of the cells in the inner yolk sac, the derivative of the VID, tended to be increased. This tendency seemed to be consistent in the posthatching juveniles at the 95-mm stage. In juveniles, 125 mm in length and 1.7 months after hatching, the cells further increased in number in the spiral intestine, reaching their adult value. Double immunostaining by the use of anti-5-HT and -neuropeptide Y (NPY) antisera demonstrated that some of the 5-HT-IR cells were also positive for NPY.

Ontogenetic development of neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system of the dogfish.

This immunocytochemical study was carried out to elucidate the ontogenetic development of neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system of the cloudy dogfish, Scyliorhinus torazame. Immunostained cells first appeared in the pancreas of the embryo at the 15-mm stage, and were also detected in the vitello-intestinal duct of the yolk stalk at the 20-mm stage. These cells were polymorphic, with occasional processes that were sometimes directed toward the vascular wall or into the cavity of the vitello-intestinal duct. At the 34-mm stage, immunostained cells could also be found in the proximal part of the spiral intestine and, by the 74-mm stage, immunopositive cells were present in the gastric mucosa. In the gut and pancreas, the cells gradually increased in number with development, whereas in the vitello-intestinal duct and internal yolk sac, they decreased and seemed to disappear following hatching. Thus, in juveniles, the distribution of the neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system had attained that of adults. Electron-microscopic immunocytochemistry demonstrated that, in the labeled cells of the vitellointestinal duct, the neuropeptide Y-like antigen was located in cytoplasmic granules, as in the cells of the gut and pancreas.

Development of the lymphomyeloid system in the dogfish, Scyliorhinus canicula.

Previous studies on the morphology of the lymphomyeloid tissues in the dogfish, Scyliorhinus canicula, have been confined to adults. This study was restricted to the structure and functioning of the developing immune system in embryonic and post-hatch dogfish. A major feature of the developing immune system in S. canicula, is the succession of haemopoietic/lymphoid tissues. The liver is the first tissue to contain immunoglobulin positive cells at 2 months, followed by the interstitial kidney at 3 months. The thymus, spleen, and Leydig organ appears at 4 months while the epigonal and gut-associated lymphomyeloid tissues are the last tissues to differentiate. The haemopoietic/lymphoid nature of the kidney and thymus disappear at post-hatch and the other lymphomyeloid tissues persist through adult life. By the time of egg case splitting (ca. 6 months), when embryos receive massive exposure to water-borne antigens, the structural development of most of the lymphomyeloid tissues is well advanced.

Development of the coronary arteries and cardiac veins in the dogfish (Scyliorhinus canicula).

The development of the coronary system in the Dogfish was studied using light microscopy. The sample examined consisted of 44 embryos and four newborns. The chronology of events occurring during the process was referred to the total length (TL) of the specimens. The nourishment of the developing myocardium first takes place by means of intertrabecular sinusoids. This system is later switched to a circulation through coronary vessels. The cardiac veins develop earlier than the coronary arteries. The earliest evidence of development of heart vessels in the present sample was the appearance of a diverticulum from the sinus venosus in three embryos of 31 mm TL. This diverticulum outlined the future coronary sinus. Both the atrioventricular and conoventricular venous rings were completely developed in an embryo of 36 mm TL. Coronary artery vessels appeared for the first time in embryos of 40 mm TL. In these specimens, two arteries arose from the midventral hypobranchial artery and divide to give rise to the four coronary artery conal trunks. In a 51-mm TL embryo it was already possible to follow the course of the coronary arteries, from the hypobranchial artery to the conoventricular groove. All main coronary vessels were fully developed in embryos of more than 58 mm TL. However, the arteries supplying the atrium were firstly recorded in a newborn of 77 mm TL. Birth usually occurs when the shark reaches about 72-76 mm TL.

Developing nephrons in adolescent dogfish, Scyliorhinus caniculus (L.), with reference to ultrastructure of early stages, histogenesis of the renal countercurrent system, and nephron segmentation in marine elasmobranchs.

Light and electron microscopy of the excretory kidney of adolescent dogfish, Scyliorhinus caniculus (L.), revealed immature and mature nephrons as well as four developmental stages of nephrons. At stage I the nephron was characterized by a condensed mass of mesenchymal cells in the center of several concentric layers of connective tissue. At stage II of the nephron, the S-shaped body was an elongate cyst with a high prismatic epithelium that was connected by a developing collecting tubule with the collecting duct system. At stage III, the developing nephrons already possess the essential features of the mature nephron but lack complete differentiation. Developing renal corpuscles had one afferent arteriole and two efferent vessels. Developing tubules ran four times between the lateral bundle zone and the mesial tissue zone before they joined the collecting duct system. A continuous sheath of flat cells, encompassing the collecting duct system, extended around the developing lateral bundle. A rudimentary central vessel ran from the developing lateral bundle to the venous sinusoid capillaries between the mesial convolutions. Developmental stage IV was similar to the mature nephron, however, renal corpuscles and tubular segments were smaller than those of mature nephrons. Conclusive evidence for morphological homology of elasmobranch nephron segments and collecting tubule-collecting duct system with those of other vertebrates is provided. The origin and nature of the central vessel and the bundle sheath is clarified. These specific structures of marine elasmobranch kidney supposedly are of great functional relevance for the renal countercurrent system that in turn is essential for ion- and osmo-regulation.

Distribution and ontogeny of glucagon-like cells in the gastrointestinal tract of the cartilaginous fish Scyliorhinus stellaris (L.).

The ontogeny and distribution of glucagon-like cells were studied in the gastrointestinal tract of embryos, neonates, and adults of the cartilaginous fish Scyliorhinus stellaris (L.) by immunocytochemistry. The results indicate that they appear early during embryonic development, and, in some portion of the gastrointestinal tract, even before the mucosa morphological differentiation. Immunoreactive glucagon-like cells were observed both in gastric and intestinal epithelium, being present in the pyloric portion only at a particular period of its differentiation. Some differences were observed between the embryonic and adult distributive pattern. They were more numerous in proliferative zone and sometimes were situated near other endocrine epithelial cells. These findings together with available information on trophic effects of some gastrointestinal hormonal peptides suggest a possible regulatory role of this peptide on the growth and differentiation of the gastrointestinal tract.

A quantitative analysis of the spinal motor pool and its target muscle during growth in the dogfish, Scyliorhinus canicula.

In order to relate the phenomenon of continuous growth in fishes to the development of the neuromuscular system, we established the numbers and sizes of spinal motoneurons and red and white axial muscle fibers in late- and post-embryonic dogfish within the size range 6-71 cm. We found that motoneuron somata, ventral root axons, and red and white muscle fibers increase their size throughout the life of the animal; there is an initial increase in the number of motoneurons that ceases as the fish reaches a length of about 40 cm; white muscle fibers initially decrease in number during post-embryonic life; and red muscle fibers increase in number, but this increase levels off at a fish length of about 40 cm. Spinal motoneurons innervating red myotomal muscle fibers or centrally located white muscle fibers were identified after retrograde labeling with horseradish peroxidase, which was injected in small amounts into the respective muscle areas. The motoneurons supplying the red muscle are smaller and occupy a more lateral position in the ventral horn than the white muscle motoneurons. The number of motoneurons in the ventral horn per unit area increases from medial to lateral and this is associated with a reduction in the sizes of the somata. Values for the ratio of number of muscle fibers to number of supplying motoneurons in the various muscle areas were based on axon counts within the peripheral nerves. This ratio was higher for the centrally located white muscle fibers than for the ventral or dorsal white fibers, but was highest for the red fibers.

Selachian tooth development: II. Immunolocalization of amelogenin polypeptides in epithelium during secretory amelogenesis in Squalus acanthias.

We have determined the distribution of amelogenin polypeptides in an order of elasmobranchs using indirect immunofluorescence with rabbit polyclonal antibodies prepared to purified murine amelogenins. We find that amelogenins are definitely present within the inner enamel epithelium prior to the production of the extracellular matrix component termed "enameloid" (row II developing tooth organs). During subsequent stages of selachian tooth development (row III tooth organs), immunofluorescence staining data indicated localization of amelogenin antigens within epithelium as well as the enameloid extracellular matrix. The results from these immunohistochemical studies suggest that the 16-20 kdalton amelogenins, which are characteristic of murine inner enamel epithelial cells undergoing terminal biochemical differentiation into secretory ameloblasts, may also be regarded as molecular markers for amelogenesis in developing teeth in the spiny dogfish, Squalus acanthias.

Selachian tooth development: I. Histogenesis, morphogenesis, and anatomical features in Squalus acanthias.

We have determined the general features of epithelial-mesenchymal interactions during fetal and mature adult stages of odontogenesis in the selachian spiny dogfish, Squalus acanthias. The general features of odontogenesis included the formation of an extended dental lamina, bud, cap, bell, and crown stages as identified and described using serial sections from fetal as well as adult specimens and light microscopy. Fetal and adult lengths, the rostrum-caudal fin distance, were correlated with the number of tooth rows and columns present in each specimen. This positional information and terminology was extremely valuable in attempting to acquire reproducible data for analyses. Whereas a number of histologic features of fetal and adult odontogenesis were comparable to those described in Mammalia, we found a number of epithelial-mesenchymal interaction characteristics unique to elasmobranch tooth development, including the persistence of the dental lamina in the adult stages, the coupling of cervical inner enamel epithelia between sequential stages of tooth formation, and the patterns of odontogenesis in the adult dentition.

Selachian tooth development: III. Ultrastructural features of secretory amelogenesis in Squalus acanthias.

Ultrastructural features of secretory amelogenesis during selachian tooth development show several similarities to mammalian amelogenesis. However, the following critical differences were noticed: 1) subcellular organelles associated with merocrine-type protein synthesis and secretion were located in both the infranuclear as well as supranuclear regions of the selachian ameloblasts; 2) no evidence for Tomes process formation was found; 3) the basal lamina was not removed during epithelial differentiation into ameloblasts in the selachian model, and the structural features of the basal lamina were significantly altered during amelogenesis in rows III, IV, and VI; and 4) no dentine-enameloid junction was detected. It is suggested that enameloid is an extracellular matrix which is derived from the selachian inner enamel epithelium and appears to be secreted from both the lateral and apical surfaces of ameloblasts.

[Influence of thyroidectomy and PTU treatment on cartilage ultrastructure in the embryo and very young dogfish (scyllium canicula, chondrichthyes) (author's transl)]. / Influence de la thyroïdectomie et du traitement par le propylthiouracile sur l'ultrastructure du cartilage de l'embryon et de la très jeune roussette (Scyllium canicula, chondrichthyens).

In embryos and very young dogfish made hypothyroidian by various treatments (radiothyroidectomy, surgical thyroidectomy and PTU treatment), ultrastructural abnormalities have been observed in all tested cartilages : Meckel's, branchial and pelvic cartilages. Many chondrocytes show a dark nucleus, enlarged REG vesicles and very electron dense hyaloplasm giving the cell a very peculiar reticulated aspect. Other chondrocytes appear unaffected. Cartilage ultrastructure is almost fully restored after a 21 days' recovery time. Around abnormal chondrocytes, extracellular matrix components are scarce. Moreover, matrix mineralization occurs around some abnormal chondrocytes by hydroxyapatite crystalization, never seen in normal embryos. During recovery, these crystals are preserved. When the antithyroid effects of PTU are compensated for by a simultaneous thyroxine treatment, cartilage abnormalities are minimized. If radiothyroidectomy is performed on young embryos, chondrocyte differentiation is delayed. It is concluded from these various experiments that thyroid hormones are required for the normal differentiation of cartilage and for the maintenance of its integrity in this fish.

Postembryonic production and aging in inner ear hair cells in sharks.

In many animals the sensory hair cells of the inner ear are ultrastructurally variable within individual epithelia. This variation has been hypothetically related to both the function and the age of the individual cells. In this study, growth-related changes in hair cell populations were examined in the macula neglecta sensory epithelia of juvenile and adult sharks. Scanning electron microscopy demonstrated that more than 80% of the 200,000 hair cells in the adult's macula negecta are produced postembryonically. Tritiated thymidine autoradiography and histological descriptions of the hair cells in this sound detector indicate that new sensory cells are produced in growth zones at the edges of the epithelia. The hair cells in those zones have small cell bodies, small and heterogeneous cilia complexes, and associations with small numbers of particularly thin nerve terminals. Their cytological features and their sparse innervation contrast with the features of the more numerous central cells in each epithelium, but appear to resemble the published descriptions of embryonically developing hair cells. Thus, a germinal zone at the leading edge of sensory epithelium growth appears to persist into adult life in sharks. Published reports reinterpreted in light of this evidence suggest that such hair cell population growth may be expected in other anamniotes and that latent growth zones might persist in the ears of amniotes.

[Biology of Squalus blainvillei (Risso, 1826) (Pisces, Squalidae) from the Tunisian coast: relation of body, liver and gonadal weight. Relative coefficients of condition and of hepato and gonosomatic processes. Embryonic development]. / Recherches sur la biologie de Squalus blainvillei (Risso, 1826) (Pisces, Squalidae) des côtes tunisiennes: relations taille-poids du corps, du foie et des gonades. Coefficients de condition rapports hépato et gonosomatique. Croissance embryonnaire.

The relative increase of body-weight size, liver-weight size and gonad weight size relationships is pointed out in all individuals and chiefly in the young ones of Squalus blainvillei (Risso, 1826) of Tunisian coasts. The evolution of the cubic and relative coefficients of condition, and of hepato and gonosomatic relationships are depending of reproductive processes. During their growth, the embryos eat only vitellin reserves, the female uterin secretions interfere only on the ending gestation.