Structure and expression of three Emx genes in the dogfish Scyliorhinus canicula: functional and evolutionary implications

We report the characterization of three Emx genes in a chondrichthyan, the dogfish Scyliorhinus canicula. Comparisons of these genes with their osteichthyan counterparts indicate that the gnathostome Emx genes belong to three distinct orthology classes, each containing one of the dogfish genes and either the tetrapod Emx1 genes (Emx1 class), the osteichthyan Emx2 genes (Emx2 class) or the zebrafish Emx1 gene (Emx3 class). While the three classes could be retrieved from the pufferfish genome data, no indication of an Emx3-related gene in tetrapods could be found in the databases, suggesting that this class may have been lost in this taxon. Expression pattern comparisons of the three dogfish Emx genes and their osteichthyan counterparts indicate that not only telencephalic, but also diencephalic Emx expression territories are highly conserved among gnathostomes. In particular, all gnathostomes share an early, dynamic phase of Emx expression, spanning presumptive dorsal diencephalic territories, which involves Emx3 in the dogfish, but another orthology class, Emx2, in tetrapods. In addition, the dogfish Emx2 gene shows a highly specific expression domain in the cephalic paraxial mesoderm from the end of gastrulation and throughout neurulation, which suggests a role in the segmentation of the cephalic mesoderm.     

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.     

Changes in heart rate during progressive hyperoxia in the dogfish Scyliorhinus canicula L.: evidence for a venous oxygen receptor

Progressive hyperoxia caused a gradual increase in arterial blood oxygen tension (PaO2). Initially there was no change in venous O2 tension (PvO2) but in extreme hyperoxia (PO2 650 mmHg) it increased to 2.5 times the normoxic (PO2 150 mmHg) level (Table 1). Ventilation frequency gradually decreased down to 73% of the normoxic value as PO2 rose towards a maximum at 700 mmHg (Fig. 1). In moderately hyperoxic water (mean PO2 233 mmHg) heart rate (fH) increased significantly above the normoxic level. Further increases in ambient PO2 caused a progressive reduction in fH to a level significantly below the normoxic rate in extreme hyperoxia (Fig. 2). Injection of atropine abolished these changes, and the atropinized fH was similar to that measured during moderate hyperoxia. The initial increase in fH during progressive hyperoxia is attributed to release of vagal tone, due to removal of normoxic stimulation of peripheral oxygen receptors; whereas, the secondary bradycardia is attributed to the stimulation of oxygen receptors located in the venous system. Injection of 5 ml of hyperoxaemic blood into the venous system of normoxic fish caused a transient bradycardia (Fig. 3), lasting a mean of 73 sec, which is the approximate time for passage of the blood volume of the venous system through the heart. This bradycardia was neither pH dependent nor a pressor response and provides supporting evidence for the existence of a venous oxygen receptor.     

Cell types and interactions in the spleen of the dogfish Scyliorhinus canicula L.: an electron microscopic study

The cell composition of the spleen of the dogfish, Scyliorhinus canicula L. was investigated by electron microscopy. It comprised areas of red and white blood cells. The red cells were observed in various stages of development and the spleen is probably the main erythropoietic organ, it is likely that thrombocytes are also produced in the spleen. The presence of plasma cells, and the appearance of rosette-like contacts between lymphocytes and phagocytic cells, probably macrophages, indicated that immunological processes were taking place. Destruction of effete blood cells, primarily erythrocytes, was indicated by the presence of macrophages containing residues of ingested blood cells.     

Binding sites for tachykinin peptides in the brain and stomach of the dogfish, Scyliorhinus canicula.

In membranes of dogfish brain and stomach, two binding sites for tachykinins were identified. One site specifically bound [125I]-Bolton-Hunter substance P (BH-SP) and the rank potency of tachykinins to compete for BH-SP binding revealed similarities with the rank potency of an NK1 receptor. The pharmacology of the other site, which specifically bound [125I]-Bolton-Hunter scyliorhinin II (BH-Scy II), did not resemble any of the mammalian tachykinin receptors. The rank potency to inhibit BH-Scy II binding to this second site was: scyliorhinin II approximately scyliorhinin I greater than eledoisin approximately substance P approximately neurokinin A greater than phyllomedusin approximately physalaemin greater than [Sar9Met(O2)11]substance P. Neurokinin B and senktide did not displace BH-Scy II binding. In addition, nucleotide analogues inhibited BH-SP binding but not BH-Scy II binding. Our binding data suggest the existence of a mammalian-like NK1 receptor and of a nonmammalian tachykinin receptor in the dogfish.     

Secretory glycoproteins of the subcommissural organ of the dogfish (Scyliorhinus canicula): evidence for the existence of precursor and processed forms

The probable presence of oxytocin in the hypothalamo-hypophysial system of two reptilian species, the snake Natrix maura and the turtle Mauremys caspica, was re-investigated. A high-pressure liquid chromatographic analysis of the turtle neural lobe revealed the existence of vasotocin, mesotocin, and a third compound co-eluting with oxytocin. Brains from both species were fixed by vascular perfusion with Bouin's fluid. Adjacent paraffin sections were immunostained using antisera against the following substances: (1) bovine oxytocin-neurophysin; (2) a mixture of bovine oxytocin-neurophysin and vasopressin-neurophysin; (3) dogfish neurophysins; (4) oxytocin; (5) arginine-vasotocin; (6) mesotocin; (7) somatostatin. Immunoreactivity against oxytocin was found in parvocellular neurons of the snake suprachiasmatic nucleus and cerebrospinal-fluid contacting neurons of the medial nucleus of the infundibular recess of both species, the latter immunoreactivity being much more conspicuous in the turtle. Numerous fibers containing immunoreactive oxytocin extended between the medial nucleus of the infundibular recess, and the internal region of the medium eminence and the neural lobe. The oxytocin-immunoreactivity in all locations was completely abolished by preabsorption of the anti-oxytocin serum with three different oxytocin preparations. None of the neurons of the suprachiasmatic and medial nucleus of the infundibular recess, including the oxytocin-immunoreactive elements, reacted with either the antineurophysin sera used, or the anti-vasotocin or anti-mesotocin antibodies. The possible existence of a reptilian oxytocin-neurophysin is discussed. The alternative that, in the reptilian hypothalamus, neurons synthesize a compound closely related to, but different from oxytocin is also considered.     

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.     

The mammalian Crx genes are highly divergent representatives of the Otx5 gene family,a gnathostome orthology class of orthodenticle-related homeogenes involved in the differentiation of retinal photoreceptors and circadian entrainment

The mammalian Crx genes are highly divergent orthodenticle (otd)-related homeogenes that play important roles in the differentiation of retinal photoreceptors and the circadian entrainment. However, their evolutionary origin and orthological relationships with other otd-related genes remain unclear. An orthology relationship of these genes with the highly conserved Otx5 genes identified in fish and amphibians, and also expressed in the eye and epiphysis, has been proposed previously but remains controversial. To test this hypothesis, we have identified Crx genes in a wide range of mammals, including three marsupials, and Otx5-related genes in a lizard, a turtle, and two archosaurs (crocodile and chick), as well as in the pufferfish. Phylogenetic analyses of the coding sequences show that the mammalian Crx genes are orthologous to the Otx5-related genes isolated in other gnathostomes. They also indicate that a duplication event has taken place in actinopterygians, after the splitting of the Cladistia, and that a relaxation of the structural constraints acting on the gene coding region has occurred early in the mammalian lineage. This process may be linked not only to the loss of ancestral Otx5/Crx functions during gastrulation or in the retinal pigmented epithelium, but also to the evolution of photic entrainment mechanisms in mammals.     

On the diel rhythms in metabolism and activity of post-hatching lesser spotted dogfish, Scyliorhinus canicula

The diel rhythms in metabolic rate ( M_R ) and activity level ( A_L ) were measured for single post-hatching dogfish (weight range, 2.76–10.61 g) at 15° C by the indirect calorimetric method of rate of oxygen consumption ( V O₂) and by video-observation respectively, over a period of 72 b. The mean VO ₂ increased from 62.0 (s.e. 2.9) mg O₂ kg⁻¹ h⁻¹ in the daylight hours to 85.5 (s.e. 3.1) mg O₂ kg⁻¹ h⁻¹ during the dark (light regíme, 12 h L: 12 h D). The simultaneous measurement of A _L also showed mean night elevation from 0.6 (s.e. 0.2) min h⁻¹ in the light phase to 14.5 (s.e. 1.6) min h⁻¹ during the darkness. Bimodal nocturnal activity (BNA) was exhibited by the post-hatching dogfish within the 12 h dark period, with V O₂ increasing from 71.4 (s.e. 2.8) mg O₂ kg⁻¹ h⁻¹ before 01.00 hours to 99.5 (s.e. 4.2) mg O₂ kg⁻¹ h⁻¹ after 01.00 hours. Similarly, A _L also increased from 8.9 (s.e. I.7)min h⁻¹ before 01.00 hours to 21.1 (s.e. 2.8) min h⁻¹ after 01.00 hours. The importance of the results presented to the natural behavioural ecology of the hatching dogfish are discussed.     

Effects of confinement stress and additional zinc treatment on some blood parameters in the dogfish Scyliorhinus canicula

Several blood parameters were studied in the dogfish Scyliorhinus canicula subjected to different confinement conditions (transport and maintenance) and zinc contamination. Confinement stress significantly decreased erythrocyte counts (RBCC), haematocrit (Ht), haemoglobin (Hb), leucocrit (Lt), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). Mean corpuscular volume (MCV) did not change with this stress and plasma glucose concentration significantly increased. In fish subjected to confinement stress conditions, Zn treatment significantly decreased Hb, MCH and MCHC and plasma glucose concentration. Ht and MCV did not change and Lt and RBCC significantly increased. Results are discussed related to other stress effects and effects of zinc treatment on fish confined in non-stressful conditions.     

Fatty acid composition and lipoxygenase metabolism in blood cells of the lesser spotted dogfish, Scyliorhinus canicula.

1. The fatty acid composition of erythrocytes and leucocytes of the elasmobranch, Scyliorhinus canicula, was determined so as to indicate substrate availability for eicosanoid formation. 2. Leucocytes showed a greater degree of fatty acid unsaturation than the erythrocytes, with particularly high levels of docosahexaenoic acid (22:6,n-3). 3. The major eicosanoid precursors, arachidonic acid (20:4,n-6) and eicosapentaenoic acid (20:5,n-3), represented 13.9% and 5.2% of the total fatty acid, respectively, in erythrocytes compared with 10.7% and 6% in leucocytes. 4. Whole blood and isolated leucocytes were stimulated with calcium ionophore, A23187 and the resulting lipoxygenase products separated by reverse phase high performance liquid chromatography. 5. The main lipoxygenase products formed were 6-trans-leukotriene B4, 6-trans-12-epi-leukotriene B4, 5(S),6(R) dihydroxyeicosatetraenoic acid and 5- and 15-hydroxyeicosatetraenoic acid. 6. No leukotriene B4, leukotriene B5, or lipoxins were detected.     

Ultrastructural study of the myocardium of the sinus venosus and sinoatrial valve in the dogfish (Scyliorhinus canicula)

The myocardium of the sinus venosus of the dogfish ( Scyliorhinus canicula ) is located between a thick subepicardial collagen-rich layer and a subendocardial network of nerve fibres and ganglion cells. The sinoatrial valve consists of two transversal folds of the cardiac wall which are separated by connective tissue, except in their free margins.
The myocardium of the sinus venosus and the sinusal face of the sinoatrial valve is arranged in bundles which are surrounded by a 40 nm-thick basal lamina. The myocardial cells measure about 7-9 μm in diameter at the nuclear level. Nerve terminals are frequent in the centre of the bundles. Most of the sinusal myocardiocytes have a scarce amount of myofibrils which are randomly orientated. The sarcoplasmic reticulum is relatively well developed and consists of peripheral couplings, subsarcolemmal vesicles, circular and longitudinal tubules. The scarce intercalated discs show only fasciae adhaerentes . Gap junctions, desmosomes or specific granules are not observed in the sinusal myocardiocytes of the dogfish. In contrast, the atrial myocardiocytes are smaller, about 5-6 μm in diameter at the nuclear level. The cytoplasm is denser and the myofibrils are abundant and orientated in parallel directions. Specific granules are present. although scarce. Subsarcolemmal vesicles are less frequent, while the atrial intercalated discs are larger and more abundant than those of the sinus venosus. Neural elements are scarce in the atrium.
The differences observed between sinus venosus and atrium might be related to the morphological criteria to distinguish between the nodal tissue and the working myocardiocytes of higher vertebrates. On the other hand, we think that the connective tissue placed between sinus venom and atrium means that the contraction impulse generated in the sinus venosus must reach the atrium through the free margin of the valve. It might play a role in the sinoatrial valve function.     

An ultrastructural and immunocytochemical study on the lymphomyeloid tissue in the embryonic kidney of the dogfish, Scyliorhinus canicula

The central role of the kidney during early development in the dogfish, Scyliorhinus canicula , is haemopoietic. The kidney is the first tissue to become lymphomyeloid and lymphocytes and developing granulocytes are found in the interstitial tissue surrounding the renal tubules. The lymphomyeloid role of the kidney is transient and does not persist after hatching. The kidney is a major immunoglobulin (Ig) containing tissue during early development as evidenced by increasing numbers of Ig–positive cells.     

Immunohistochemical localization of calbindin-D28K in the brain of a cartilaginous fish, the dogfish (Scyliorhinus canicula L.)

The occurrence and distribution of the vitamin-D-induced calcium-binding protein, calbindin-D28K, has been studied in the brain of a cartilaginous fish using immunohistochemical techniques. A strong immunoreactivity was found in the perikarya, dendrites and axons of neurons located in the nucleus interstitialis commissurae anterioris, the nucleus medialis of the left habenula, the thalamus dorsalis, the thalamus ventralis, the nucleus lobi lateralis, the nucleus interpeduncularis, the lobus vagi and the medial reticular zone. Fibre tracts associated with some of these neuronal groups, such as the fasciculus retroflexus, the stria medullaris and the commissura habenulae, also contained immunopositive fibres. Only a minor immunoreactivity could be detected in other brain areas such as the tectum mesencephali and some telencephalic zones. Interestingly, the cerebellum did not show any immunoreactivity in Purkinje cells nor in other neurons. The distribution of calbindin-D28K in the dogfish brain appears to be mainly related to the viscerosensory centres.     

Inositol lipid turnover and adenosine 3,5 cyclic monophosphate in the salt-secreting rectal gland of the dogfish (Scyliorhinus canicula)

The rectal gland of the dogfish is rich in inositol lipids. Total phospholipids from the gland contained 9.1 mol% of phosphatidylinositol (PtdIns), 1.0 mol% of phosphatidylinositol 4-phosphate (PtdIns4P) and 0.9 mol% of phosphatidylinositol 4,5-biphosphate (PtdIns4,5P2). [32P]Orthophosphate was readily incorporated into PtdIns, phosphatidic acid (PtdA) and especially into PtdIns4P and PtdIns4,5P2 in salt gland slices incubated in elasmobranch Ringer with glucose and no other additions over a 2 hr period. The calcium ionophore A23187 stimulated incorporation into PtdIns and PtdA, but not into PtdIns4P or PtdIns4,5P2. Oxygen uptake by rectal gland slices was maximally stimulated by 0.08mM forskolin, 2.5mM 8-chlorophenylthio cyclic AMP, 2.0mM dibutyryl cyclic AMP and 0.25mM theophylline. Stimulated oxygen uptake was inhibited by 0.1mM ouabain in all cases. Incorporation of [32P]orthophosphate into PtdIns, PtdA, PtdIns4P and PtdIns4,5P2 was inhibited by 0.08mM forskolin and 2.0mM dibutyryl cyclic AMP over a 2 hr period. The results are discussed in relation to the control of salt secretion by the rectal gland.     

Egg-case diversity among dogfish, Scyliorhinus canicula (L.): a study of egg laying rate and nidamental gland secretory activity

Egg-case size, shape and pigmentation are distinctive features of each female lesser-spotted dogfish, Scyliorhinus canicula. During 9 months captivity under constant conditions (water, temperature, photoperiod, food), a laying sequence reminiscent of an annual cycle was observed in three females. While oocyte size and (with exceptions) egg-case weight remained constant, there was a marked decrease of the'albumen'jelly secreted by the anterior halves of nidamental glands.     

Food and feeding habits of the lesser spotted dogfish, Scyliorhinus canicula (L.), in Isle of Man waters

Stomach contents of the lesser spotted dogfish, Scyliorhinus canicula , from Isle of Man waters were investigated. Dogfish fed opportunistically on a wide range of macrobenthic fauna with hermit crabs, cockles and whelks the dominant prey. Items of secondary importance included various crabs, callianassid shrimps, bivalve molluscs, holothurians, polychaetes and, when locally abundant, herring, Clupea harengus . Dogfish predated more heavily on molluscs than reported in previous studies, reflecting the abundance of molluscs in the associated benthic community.
Composition of the diet altered gradually with dogfish size, reliance on small crustaceans declined and consumption of hermit crabs and molluscs increased with growth. Seasonal changes in the composition of the diet were observed. Feeding intensity was greatest during summer, related in part to increased prey availability, and was least during autumn.     

The structure and ultrastructure of the sinus venosus in the mature dogfish (Scyliorhinus canicula): the endocardium, the epicardium and the subepicardial space

The sinus venosus of fish is the most caudal chamber of the heart. It is often reduced in teleosts but well developed in elasmobranchs. The sinus venosus of the dogfish (Scyliorhinus canicula) is vital, since it harbours key elements such as a little known neuroendocrine system and the nodal tissue. However, the study of its structure is still incomplete. We examined the endocardium, epicardium and subepicardium of the sinus venosus in mature dogfishes. The wall is 100-250 microm thick and comprises three main layers. Large bundles of myocardial cells occupy the middle layer. The endothelial ensheathing is composed of thin endocardial cells with prominent nuclei towards the lumen, whose cytoplasm contains numerous dense bodies and moderately dense bodies, 150-800 nm in diameter and large vacuoles. The possible functions of these organelles are discussed. The outermost layer is made of a robust sheet of cuboidal epicardial cells separated from the subepicardium by a conspicuous basal lamina. Numerous microvilli towards the pericardial cavity and elliptical vesicles are located in the apex of epicardial cells. A thick layer richly endowed with dense bundles of collagen fibres forms the subepicardial space. This structure should be contrasted with the venous return mechanism of elasmobranchs.