Species-specific contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates.

Posterior body elongation is a widespread mechanism propelling the generation of the metazoan body plan. The posterior growth model predicts that a posterior growth zone generates sufficient tissue volume to elongate the posterior body. However, there are energy supply-related differences between vertebrates in the degree to which growth occurs concomitantly with embryogenesis. By applying a multi-scalar morphometric analysis in zebrafish embryos, we show that posterior body elongation is generated by an influx of cells from lateral regions, by convergence-extension of cells as they exit the tailbud, and finally by a late volumetric growth in the spinal cord and notochord. Importantly, the unsegmented region does not generate additional tissue volume. Fibroblast growth factor inhibition blocks tissue convergence rather than volumetric growth, showing that a conserved molecular mechanism can control convergent morphogenesis through different cell behaviours. Finally, via a comparative morphometric analysis in lamprey, dogfish, zebrafish and mouse, we propose that elongation via posterior volumetric growth is linked to increased energy supply and is associated with an overall increase in volumetric growth and elongation.

A Developmental Study of the Cerebellar Nucleus in the Catshark, a Basal Gnathostome.

The output of the cerebellar cortex is mainly released via cerebellar nuclei which vary in number and complexity among gnathostomes, extant vertebrates with a cerebellum. Cartilaginous fishes, a basal gnathostome lineage, show a conspicuous, well-organized cerebellar nucleus, unlike ray-finned fishes. To gain insight into the evolution and development of the cerebellar nucleus, we analyzed in the shark Scyliorhinus canicula (a chondrichthyan model species) the developmental expression of several genes coding for transcription factors (ScLhx5,ScLhx9,ScTbr1, and ScEn2) and the distribution of the protein calbindin, since all appear to be involved in cerebellar nuclei patterning in other gnathostomes. Three regions (subventricular, medial or central, and lateral or superficial) became recognizable in the cerebellar nucleus of this shark during development. Present genoarchitectonic and neurochemical data in embryos provide insight into the origin of the cerebellar nucleus in chondrichthyans and support a tripartite mediolateral organization of the cerebellar nucleus, as previously described in adult sharks. Furthermore, the expression pattern of ScLhx5,ScLhx9, and ScTbr1 in this shark, together with that of markers of proliferation, migration, and early differentiation of neurons, is compatible with the hypothesis that, as in mammals, different subsets of cerebellar nucleus neurons are originated from progenitors of 2 different sources: the ventricular zone of the cerebellar plate and the rhombic lip. We also present suggestive evidence that Lhx9 expression is involved in cerebellar nuclei patterning early on in gnathostome evolution, rather than representing an evolutionary innovation of the dentate nucleus in mammals, as previously hypothesized.

Early patterning in a chondrichthyan model, the small spotted dogfish: towards the gnathostome ancestral state.

In the past few years, the small spotted dogfish has become the primary model for analyses of early development in chondrichthyans. Its phylogenetic position makes it an ideal outgroup to reconstruct the ancestral gnathostome state by comparisons with established vertebrate model organisms. It is also a suitable model to address the molecular bases of lineage-specific diversifications such as the rise of extraembryonic tissues, as it is endowed with a distinct extraembryonic yolk sac and yolk duct ensuring exchanges between the embryo and a large undivided vitelline mass. Experimental or functional approaches such as cell marking or in ovo pharmacological treatments are emerging in this species, but recent analyses of early development in this species have primarily concentrated on molecular descriptions. These data show the dogfish embryo exhibits early polarities reflecting the dorso-ventral axis of amphibians and teleosts at early blastula stages and an atypical anamniote molecular pattern during gastrulation, independently of the presence of extraembryonic tissues. They also highlight unexpected relationships with amniotes, with a strikingly similar Nodal-dependent regional pattern in the extraembryonic endoderm. In this species, extraembryonic cell fates seem to be determined by differential cell behaviors, which lead to cell allocation in extraembryonic and embryonic tissues, rather than by cell regional identity. We suggest that this may exemplify an early evolutionary step in the rise of extraembryonic tissues, possibly related to quantitative differences in the signaling activities, which shape the early embryo. These results highlight the conservation across gnathostomes of a highly constrained core genetic program controlling early patterning. This conservation may be obscured in some lineages by taxa-specific diversifications such as specializations of extraembryonic nutritive tissues.

Development of head and trunk mesoderm in the dogfish, Scyliorhinus torazame: I. Embryology and morphology of the head cavities and related structures.

Vertebrate head segmentation has attracted the attention of comparative and evolutionary morphologists for centuries, given its importance for understanding the developmental body plan of vertebrates and its evolutionary origin. In particular, the segmentation of the mesoderm is central to the problem. The shark embryo has provided a canonical morphological scheme of the head, with its epithelialized coelomic cavities (head cavities), which have often been regarded as head somites. To understand the evolutionary significance of the head cavities, the embryonic development of the mesoderm was investigated at the morphological and histological levels in the shark, Scyliorhinus torazame. Unlike somites and some enterocoelic mesodermal components in other vertebrates, the head cavities in S. torazame appeared as irregular cyst(s) in the originally unsegmented mesenchymal head mesoderm, and not via segmentation of an undivided coelom. The mandibular cavity appeared first in the paraxial part of the mandibular mesoderm, followed by the hyoid cavity, and the premandibular cavity was the last to form. The prechordal plate was recognized as a rhomboid roof of the preoral gut, continuous with the rostral notochord, and was divided anteroposteriorly into two parts by the growth of the hypothalamic primordium. Of those, the posterior part was likely to differentiate into the premandibular cavity, and the anterior part disappeared later. The head cavities and somites in the trunk exhibited significant differences, in terms of histological appearance and timing of differentiation. The mandibular cavity developed a rostral process secondarily; its homology to the anterior cavity reported in some elasmobranch embryos is discussed.

Development of the head and trunk mesoderm in the dogfish, Scyliorhinus torazame: II. Comparison of gene expression between the head mesoderm and somites with reference to the origin of the vertebrate head.

The vertebrate mesoderm differs distinctly between the head and trunk, and the evolutionary origin of the head mesoderm remains enigmatic. Although the presence of somite-like segmentation in the head mesoderm of model animals is generally denied at molecular developmental levels, the appearance of head cavities in elasmobranch embryos has not been explained, and the possibility that they may represent vestigial head somites once present in an amphioxus-like ancestor has not been ruled out entirely. To examine whether the head cavities in the shark embryo exhibit any molecular signatures reminiscent of trunk somites, we isolated several developmentally key genes, including Pax1, Pax3, Pax7, Pax9, Myf5, Sonic hedgehog, and Patched2, which are involved in myogenic and chondrogenic differentiation in somites, and Pitx2, Tbx1, and Engrailed2, which are related to the patterning of the head mesoderm, from an elasmobranch species, Scyliorhinus torazame. Observation of the expression patterns of these genes revealed that most were expressed in patterns that resembled those found in amniote embryos. In addition, the head cavities did not exhibit an overt similarity to somites; that is, the similarity was no greater than that of the unsegmented head mesoderm in other vertebrates. Moreover, the shark head mesoderm showed an amniote-like somatic/visceral distinction according to the expression of Pitx2, Tbx1, and Engrailed2. We conclude that the head cavities do not represent a manifestation of ancestral head somites; rather, they are more likely to represent a derived trait obtained in the lineage of gnathostomes.

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.

Absence of an external germinal layer in zebrafish and shark reveals a distinct, anamniote ground plan of cerebellum development.

The granule cell layer of the cerebellum comprises the largest population of neurons in the vertebrate CNS. In amniotes, its precursors undergo a unique phase of transit amplification, regulated by Sonic hedgehog. They do so within a prominent but transient secondary proliferative epithelium, the external germinal layer, which is formed by tangential migration of precursor cells from the rhombic lip. This behavior is a hallmark of bird and mammal cerebellum development. Despite its significance for both development and disease, it is unclear whether an external germinal layer is a requirement for granule cell production or an expedient of transit amplification. Evidence for its existence in more basal vertebrates is contradictory. We therefore examined cerebellum development in the zebrafish, specifically in relation to the expression of the basic helix-loop-helix gene Atonal 1, which definitively characterizes granule cell precursors. The expression of Atoh1a-Atoh1c, in combination with patterns of proliferation and fate maps, define precursor pools at the rhombic lip and cerebellar midline but demonstrate that an external germinal layer is absent. Sonic hedgehog signaling is correspondingly absent in the zebrafish cerebellum. Sustained roof-plate-derived signals suggest that, in the absence of transit amplification, primary granule cell precursor pools are maintained throughout development. To determine whether this pattern is specific to zebrafish or reflects a more general anamniote organization, we examined the expression of similar genes in the dogfish, Scylliorhinus canicula. We show that these anamniotes share a common ground plan of granule cell production that does not include an external germinal layer.

The homology of odontodes in gnathostomes: insights from Dlx gene expression in the dogfish, Scyliorhinus canicula.

BACKGROUND: Teeth and tooth-like structures, together named odontodes, are repeated organs thought to share a common evolutionary origin. These structures can be found in gnathostomes at different locations along the body: oral teeth in the jaws, teeth and denticles in the oral-pharyngeal cavity, and dermal denticles on elasmobranch skin. We, and other colleagues, had previously shown that teeth in any location were serially homologous because: i) pharyngeal and oral teeth develop through a common developmental module; and ii) the expression patterns of the Dlx genes during odontogenesis were highly divergent between species but almost identical between oral and pharyngeal dentitions within the same species. Here we examine Dlx gene expression in oral teeth and dermal denticles in order to test the hypothesis of serial homology between these odontodes. RESULTS: We present a detailed comparison of the first developing teeth and dermal denticles (caudal primary scales) of the dogfish (Scyliorhinus canicula) and show that both odontodes develop through identical stages that correspond to the common stages of oral and pharyngeal odontogenesis. We identified six Dlx paralogs in the dogfish and found that three showed strong transcription in teeth and dermal denticles (Dlx3, Dlx4 and Dlx5) whereas a weak expression was detected for Dlx1 in dermal denticles and teeth, and for Dlx2 in dermal denticles. Very few differences in Dlx expression patterns could be detected between tooth and dermal denticle development, except for the absence of Dlx2 expression in teeth. CONCLUSIONS: Taken together, our histological and expression data strongly suggest that teeth and dermal denticles develop from the same developmental module and under the control of the same set of Dlx genes. Teeth and dermal denticles should therefore be considered as serial homologs developing through the initiation of a common gene regulatory network (GRN) at several body locations. This mechanism of heterotopy supports the 'inside and out' model that has been recently proposed for odontode evolution.

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.

A developmental approach to forebrain organization in elasmobranchs: new perspectives on the regionalization of the telencephalon.

It is essential to consider chondrichthyans (cartilaginous fishes) in analyzing ancestral brain organization because this radiation represents the out-group to all other living gnathostomes (jawed vertebrates). It is particularly crucial to understand the evolution of the telencephalon in chondrichthyans, as this structure develops by evagination (as in most other vertebrates), whereas in most osteichthyans (bony fishes), it develops by eversion, a markedly different process. Among chondrichthyans, the Lesser Spotted Dogfish Scyliorhinus canicula (Elasmobranchii) appears to offer the most potential as a model species for study. Developmental studies of Scyliorhinus have revealed a segmentary pattern in the developing forebrain, similar to that described in other vertebrates, as well as the occurrence of tangential cell migration within the telencephalon, especially in relation to the pallial-subpallial boundary. These observations indicate that major morphogenetic processes thought to be a hallmark of mammalian brains actually existed much earlier in vertebrate phylogeny. In addition, analysis of telencephalic development in Scyliorhinus indicates the existence of telencephalic structures that are probably related to the ganglionic eminences of mammals.

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.

Accumulation and transport behaviour of 241americium, 60cobalt and 134cesium by eggs of the spotted dogfish Scyliorhinus canicula.

An experimental study examined the 96-h net influx from seawater of the anthropogenic radionuclides (241)Am, (60)Co and (134)Cs through the egg-case of the spotted dogfish Scyliorhinus canicula. Net influx directly through the wall of the egg-case was greatest for (134)Cs, then (241)Am and lastly (60)Co. Within the egg-case wall itself the measured concentration factors (CFs) and their gradients in the external, median and internal layers showed that for both (241)Am and (60)Co they were >10(3) in the external layer and declined by an order of magnitude in the interior layer. In contrast (134)Cs had a CF of only about three in the external layer which declined by a factor of 2 towards the two more internal layers of the egg-case. The egg-case apertures, that open within the prehatching stage of embryological development, significantly (P<0.05) increased the net influx of (241)Am and (60)Co to the interior of the egg-case, although their water concentrations were still lower than those in the labelled seawater bath. In contrast, the aperture did not increase the net influx of (134)Cs whose water concentrations equilibrated with those in seawater. Together these results indicate that the egg-case wall is very permeable to (134)Cs, representing little barrier to its movement, and hence consistent with the lack of importance of the aperture in determining its internal water concentrations in the egg-case. In contrast, (241)Am and (60)Co show much higher rates of accumulation by the egg-case, consistent with the measured reduced permeability of its wall, and therefore giving greater prominence to its aperture in the net transfer of these two radionuclides to the egg case's interior. The presence of the embryo within its egg-case did not significantly (P>0.05) affect the rates of influx of radioisotopes, with the exception of an interactive effect for (60)Co with the egg-case aperture (P<0.05). The CF of only (241)Am in the embryo itself relative to the external seawater concentration was significantly (P<0.05) enhanced by the presence of the aperture.

Genoarchitecture of the rostral hindbrain of a shark: basis for understanding the emergence of the cerebellum at the agnathan-gnathostome transition.

The cerebellum is present in all extant gnathostomes or jawed vertebrates, of which cartilaginous fishes represent the most ancient radiation. Since the isthmic organizer induces the formation of the cerebellum, comparative genoarchitectonic analysis on the meso-isthmo-cerebellar region of cartilaginous fishes with respect to that of jawless vertebrates could reveal why the isthmic organizer acquires the ability to induce the formation of the cerebellum in gnathostomes. In the present work we analyzed the expression pattern of a variety of genes related to the cerebellar formation and patterning (ScOtx2, ScGbx2, ScFgf8, ScLmx1b, ScIrx1, ScIrx3, ScEn2, ScPax6 and ScLhx9) by in situ hybridization, and the distribution of Pax6 protein in the developing hindbrain of the shark Scyliorhinus canicula. The genoarchitectonic code in this species revealed high degree of conservation with respect to that of other gnathostomes. This resemblance may reveal the features of the ancestral condition of the gene network operating for specification of the rostral hindbrain patterning. Accordingly, the main subdivisions of the rostral hindbrain of S. canicula could be recognized. Our results support the existence of a rhombomere 0, identified as the ScFgf8/ScGbx2/ScEn2-positive and mainly negative ScIrx3 domain just caudal to the midbrain ScIrx1/ScOtx2/ScLmx1b-positive domain. The differential ScEn2 and Pax6 expression in the rhombomere 1 revealed anterior and posterior subdivisions. Interestingly, dissimilarities between S. canicula and lampreys (jawless vertebrates) were noted in the expression of Irx, Lhx and Pax genes, which could be part of significant gene network changes through evolution that caused the emergence of the cerebellum.

Morphogenesis of the cerebellum and cerebellum-related structures in the shark Scyliorhinus canicula: insights on the ground pattern of the cerebellar ontogeny.

Because the cerebellum emerged at the agnathan-gnathostome transition and cartilaginous fishes are at the base of the gnathostome lineage, this group is crucial to determine the basic developmental pattern of the cerebellum and to gain insights into its origin. We have systematically analyzed key events in the development of cerebellum and cerebellum-related structures of the shark Scyliorhinus canicula. Three developmental periods are distinguished based on anatomical observations combined with molecular analysis. We present neurochemical and genoarchitectonic evidence on the onset of cerebellar development, the rostral and caudal cerebellar boundaries, the compartmentalization of the cerebellum, and correspondence of cerebellar domains to rhombomeric segmentation of the rostral hindbrain. Our observations, mainly based on the expression pattern of ScHoxA2, support the origin of both the upper and lower auricular leaves from r1 and exclude any cerebellar origin from r2. Correlation between subrhombomeres r1a/r1b and cerebellar domains is proposed based on the ScEn2 expression. The ScEn2 and ScOtx2 expression patterns revealed an antero-posterior cerebellar compartmentalization similar to that of mammals, and supported certain fissures (commonly used to define cerebellar domains) as reliable anatomical landmarks. At difference from mammals, the expression of ScEn2 along the cerebellar median-lateral axis does not reveal a multiple-banded pattern. The present study provides an atlas of cerebellar development in one of the most basal extant gnathostome lineages and emphasizes the importance of combining classic descriptive with modern molecular studies to gain knowledge on the ancestral condition of cerebellar developmental processes and the origins and evolution of the cerebellum.

Ultrasound and physical models shed light on the respiratory system of embryonic dogfishes.

Embryos of live-bearing elasmobranchs (sharks, skates, and rays) must acquire oxygen in the uterus for several months to more than a year, but the mechanisms of delivery and uptake are still largely unknown. Diagnostic sonography performed on a captive Japanese dogfish (Squalus japonicus) showed that a late-stage embryo used buccal movement to pump uterine fluid, suggesting that the embryo acquires oxygen from uterine fluid via gill ventilation. It has been assumed that embryonic respiration in aplacental sharks depends on oxygen supplied by the uterine wall. To test this hypothesis, the rate of oxygen diffusion was estimated by applying a physical model to the uterine wall of two dogfish species (Squalus cf. mitsukurii and Squalus cubensis). The model calculations indicate that the supply of oxygen via diffusion through the uterine villi contributes less than 15-30% of the total oxygen demand of late-stage embryos. Some previous authors have suggested that pregnant dogfish intermittently exchange uterine fluid with external seawater during late gestation. Thus, late-stage embryos may acquire oxygen primarily from uterine seawater introduced from the external environment.

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.

Rostral floor plate (flexural organ) secretes glycoproteins immunologically similar to subcommissural organ glycoproteins in dogfish (Scyliorhinus canicula) embryos.

The subcommissural organ of vertebrates secretes glycoproteins into the cerebrospinal fluid of the third cerebral ventricle. This material polymerizes in Reissner's fiber. During ontogenetic development, besides the subcommissural organ, the ependyma lining the pontine flexure constitutes an additional Reissner's fiber-secreting gland named flexural organ. We have studied the secretion of the flexural organ and the subcommissural organ in dogfish (Scyliorhinus canicula) embryos using three different antisera and the lectins concanavalin A and wheat germ agglutinin. AFRU is an antiserum against the bovine Reissner's fiber, Ab-600 is an antiserum against 600 kDa dogfish subcommissural organ glycoproteins; and APSO is an antiserum against immunoaffinity purified bovine subcommissural organ secretory glycoproteins. These three antisera immunostained the flexural organ indicating that it contains epitopes similar to those present in bovine and dogfish subcommissural organ glycoproteins. It seems highly probable that the flexural organ and the subcommissural organ of dogfish embryos secrete similar compound(s). Other ependymal regions were also immunostained with Ab-600 and APSO antisera. Then, Reissner's fiber-like glycoproteins were transiently expressed by most embryonary ependymal cells. These glycoproteins might play a role in the development of the central nervous system of vertebrates.

Development of catecholaminergic systems in the spinal cord of the dogfish Scyliorhinus canicula (Elasmobranchs).

The development of catecholamine-synthesizing cells and fibers in the spinal cord of dogfish (Scyliorhinus canicula L.) was studied by means of immunohistochemistry using antibodies against tyrosine hydroxylase (TH). The only TH-immunoreactive (TH-ir) cells already present in the spinal cord of stage 26 embryos were of cerebrospinal fluid-contacting (CSF-c) type. These cells were the first catecholaminergic neurons of the dogfish CNS. The number of these TH-ir cells increased very considerably in later embryos and adult dogfish. In later embryos (stage 33; prehatching), faintly TH-ir non-CSF-contacting neurons were observed in the ventral horn throughout most of the spinal cord. In adult dogfish, some non-CSF-contacting TH-ir cells were observed ventral or lateral to the central canal. In the rostral spinal cord, the catecholaminergic neurons observed in dorsal regions were continuous with caudal rhombencephalic populations. Numerous TH-ir fibers were observed in the spinal cord of later embryos and in adults, both intrinsic and descending from the brain, innervating many regions of the cord including the dorsal and ventral horns. In addition, some TH-ir fibers innervated the marginal nucleus of the spinal cord. The early appearance of catecholaminergic cells and fibers in the embryonic spinal cord of the dogfish, and the large number of these elements observed in adults, suggests an important role for catecholamines through development and adulthood in sensory and motor functions.

Pax6 expression patterns in Lampetra fluviatilis and Scyliorhinus canicula embryos suggest highly conserved roles in the early regionalization of the vertebrate brain.

We report expression patterns of the Pax6 gene in the dogfish Scyliorhinus canicula and the lamprey Lampetra fluviatilis during neurulation and at the beginning of organogenesis. At the stages studied, both genes display very similar expression domains in the dorsal forebrain, with a sharp posterior boundary at the diencephalon-mesencephalon border, in the hindbrain, excluding the floor plate and the roof plate, and in the spinal cord. The comparison of these expression patterns with those reported in osteichthyans suggests that the roles played by Pax6 in early brain regionalization have been highly conserved during vertebrate evolution.