Iván Carrera

New insights on Saccus vasculosus evolution: a developmental and immunohistochemical study in elasmobranchs.

The saccus vasculosus (SV) is a circumventricular organ of the hypothalamus of many jawed fishes whose functions have not yet been clarified. It is a vascularized neuroepithelium that consists of coronet cells, cerebrospinal fluid-contacting (CSF-c) neurons and supporting cells. To assess the organization, development and evolution of the SV, the expression of glial fibrillary acidic protein (GFAP) and the neuronal markers γ-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD; the GABA synthesizing enzyme), neuropeptide Y (NPY), neurophysin II (NPH), tyrosine hydroxylase (TH; the rate-limiting catecholamine-synthesizing enzyme) and serotonin (5-HT), were investigated by immunohistochemistry in developing and adult sharks. Coronet cells showed GFAP immunoreactivity from embryos at stage 31 to adults, indicating a glial nature. GABAergic CSF-c neurons were evidenced just when the primordium of the SV becomes detectable (at stage 29). Double immunolabeling revealed colocalization of NPY and GAD in these cells. Some CSF-c cells showed TH immunoreactivity in postembryonic stages. Saccofugal GABAergic fibers formed a defined SV tract from the stage 30 and scattered neurosecretory (NPH-immunoreactive) and monoaminergic (5-HT- and TH-immunoreactive) saccopetal fibers were first detected at stages 31 and 32, respectively. The early differentiation of GABAergic neurons and the presence of a conspicuous GABAergic saccofugal system are shared by elasmobranch and teleosts (trout), suggesting that GABA plays a key function in the SV circuitry. Monoaminergic structures have not been reported in the SV of bony fishes, and were probably acquired secondarily in sharks. The existence of saccopetal monoaminergic and neurosecretory fibers reveals reciprocal connections between the SV and hypothalamic structures which have not been previously detected in teleosts.

Development of the cerebellar body in sharks: spatiotemporal relations of Pax6 expression, cell proliferation and differentiation.

We have studied the patterns of cell proliferation, regional organization and differentiation in the cerebellar body of embryos and juveniles of two shark species by immunohistochemistry with antibodies against proliferating cell nuclear antigen (PCNA), Pax6, reelin (RELN), GABA, glutamic acid decarboxylase (GAD) and calretinin (CR). The organization of Pax6-expressing cells was also studied by in situ hybridization. Our results reveal that a transient secondary matrix zone, the external germinal layer, is formed in sharks at early stages of cerebellar development and is the source of the earliest Pax6-expressing (granule) cells. Later in development, new granule Pax6-expressing cells arise from medial proliferation zones and accumulate medially in the granular eminences. The GABAergic components appear very early, and show clear regional differences. The medial proliferation zones remain active even in adults. Taken together, the proliferation and differentiation markers used in the present study highlight striking similarities during development between the cerebellar body of elasmobranchs and the cerebella of tetrapods. These results show the importance of elasmobranch models to reconstruct the evolutionary developmental history of the vertebrate cerebellum.

Tangentially migrating GABAergic cells of subpallial origin invade massively the pallium in developing sharks

We studied the development of the GABAergic system in the telencephalon of the dogfish Scyliorhinus canicula using GABA and glutamate decarboxylase (GAD) immunocytochemistry. The earliest GABA-expressing cells appeared in the basal telencephalon (subpallium) of stage 24 embryos. Shortly after, the subpallium showed abundant GABA-expressing neuroblasts near the meningeal surface or migrating radially in the neuroepithelium. The limit between the GABA-expressing region and the remainder of the telencephalon (pallium) was sharp and coincides with the pallial/subpallial boundary. At stage 28, GABA-expressing cells with the morphology of tangentially migrating cells (showing a thick growth cone-like leading process) migrate from a dome-shaped protrusion of the lateral subpallium and extended laterally and rostrodorsally into the pallium following either a superficial route or coursing periventricularly. At later stages, abundant GABA-expressing cells were seen in various pallial regions and strings of GABA-expressing cells, possibly migrating, were also noted. The colonization of the dogfish pallium by GABA-expressing cells, originating from the subpallium, is strongly reminiscent of the palliopetal tangential migrations of GABA-expressing cells demonstrated in the telencephalon of mammals and follows similar routes. These results strongly suggest that tangential migrations of GABA-expressing cells appeared very early in vertebrate forebrain evolution.

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. J. Comp. Neurol. 511:804–831, 2008.

Distribution and development of glutamic acid decarboxylase immunoreactivity in the spinal cord of the dogfish Scyliorhinus canicula (elasmobranchs)

The adult distribution and development of γ‐aminobutyric acid (GABA)‐synthesizing cells and fibers in the spinal cord of the lesser spotted dogfish (Scyliorhinus canicula L.) was studied by means of immunohistochemistry using antibodies against glutamic acid decarboxylase (GAD). Complementary immunostaining with antibodies against GABA, tyrosine hydroxylase (TH), and HuC/HuD (members of the Hu/Elav family of RNA‐associated proteins) and staining with a reduced silver procedure (“en bloc” Bielschowski method), Nissl, and hematoxylin were also used. In adults, GAD‐immunoreactive (GAD‐ir) cells were observed in the ventral horns, in the spinal nucleus of the dorsal horn, at the base of the dorsal horns, and around the central canal, where some GAD‐ir cells were cerebrospinal fluid‐contacting (CSF‐c). In addition, a few GAD‐ir cells were observed in the lateral funiculus between the ventral horn and the marginal nucleus. The adult spinal cord was richly innervated by GAD‐ir fibers. Large numbers of GAD‐ir fibers and boutons were observed in the dorsal and ventral horns and also interstitially in the dorsal, lateral, and ventral funiculi. In addition, a rich GAD‐ir innervation was observed in the marginal nucleus of the spinal cord. In the embryonic spinal cord, GAD‐ir cells develop very early: The earliest cells were observed in the very thin mantle/marginal layer of stage 22 embryos in a short length of the spinal cord. At stages 25 and 26, several types of GAD‐ir cells (commissural and noncommissural) were distinguished, and two of these cells were of CSF‐c type. At stages 28, 30, and 31, the GAD‐ir populations exhibited a marked longitudinal columnar organization. Double‐immunolabeling experiments in embryos showed the presence of two different GAD‐ir CSF‐c cell populations, one ventral that is simultaneously TH‐ir and other more dorsal that is TH‐negative. By stage 33 (prehatching), GAD‐expressing cells are present in virtually all loci, as in adults, especially in the ventral horn and base of the dorsal horn. The present results for the lesser spotted dogfish suggest an important role for γ‐aminobutyric acid in sensory and motor circuits of the spinal cord. J. Comp. Neurol. 478:189–206, 2004.

The segmental organization of the developing shark brain based on neurochemical markers, with special attention to the prosencephalon.

Brain regionalization has been extensively studied in tetrapods, teleosts and cyclostomes. In contrast, it has not been investigated in elasmobranchs, despite their key phylogenetic position to understand brain evolution in jawed vertebrates. In this study we provide a schematic view of the segmental pattern of the developing shark brain based on mapping of the expression of Pax6 and neurochemical markers such as calretinin, tyrosine hydroxylase, serotonin, and glutamic acid decarboxylase. By correlating the cytoarchitectonic limits with the specific location of these markers, we identify transverse and longitudinal boundaries and domains, which suggest a segmental pattern, reminiscent of the one described in other vertebrates. Taken together, these data provide an initial scheme, which will be further tested and refined using a broader range of genetic markers involved in patterning and differentiation.

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.

Regionalization of the Shark Hindbrain: A Survey of an Ancestral Organization

Cartilaginous fishes (chondrichthyans) represent an ancient radiation of vertebrates currently considered the sister group of the group of gnathostomes with a bony skeleton that gave rise to land vertebrates. This out-group position makes chondrichthyans essential in assessing the ancestral organization of the brain of jawed vertebrates. To gain knowledge about hindbrain evolution we have studied its development in a shark, the lesser spotted dogfish Scyliorhinus canicula by analyzing the expression of some developmental genes and the origin and distribution of specific neuronal populations, which may help to identify hindbrain subdivisions and boundaries and the topology of specific cell groups. We have characterized three developmental periods that will serve as a framework to compare the development of different neuronal systems and may represent a suitable tool for comparing the absolute chronology of development among vertebrates. The expression patterns of Pax6, Wnt8, and HoxA2 genes in early embryos of S. canicula showed close correspondence to what has been described in other vertebrates and helped to identify the anterior rhombomeres. Also in these early embryos, the combination of Pax6 with protein markers of migrating neuroblasts (DCX) and early differentiating neurons (general: HuC/D; neuron type specific: GAD, the GABA synthesizing enzyme) revealed the organization of S. canicula hindbrain in both transverse segmental units corresponding to visible rhombomeres and longitudinal columns. Later in development, when the interrhombomeric boundaries fade away, accurate information about S. canicula hindbrain subdivisions was achieved by comparing the expression patterns of Pax6 and GAD, serotonin (serotoninergic neurons), tyrosine hydroxylase (catecholaminergic neurons), choline acetyltransferase (cholinergic neurons), and calretinin (a calcium-binding protein). The patterns observed revealed many topological correspondences with other vertebrates and led to reconsideration of the current view of the elasmobranch hindbrain segmentation as peculiar among vertebrates.

Development of tyrosine hydroxylase-immunoreactive cell populations and fiber pathways in the brain of the dogfish Scyliorhinus canicula: new perspectives on the evolution of the vertebrate catecholaminergic system.

Developmental studies of the central catecholaminergic (CA) system are essential for understanding its evolution. To obtain knowledge about the CA system in chondrichthyans, an ancient gnathostome group, we used immunohistochemical techniques for detecting tyrosine hydroxylase (TH), the initial rate‐limiting enzyme of the CA synthesis, to study: 1) the neuromery of developing TH‐immunoreactive (ir) neuronal populations, 2) the development of TH‐ir innervation, and 3) the organization of TH‐ir cells and fibers in the brain of postembryonic stages of the shark Scyliorhinus canicula. The first TH‐ir cells appeared in the hypothalamus and rostral diencephalon (suprachiasmatic, posterior recess and posterior tubercle nuclei at embryonic stage 26, and dorsomedial hypothalamus at stage 28); then in more caudal basal regions of the diencephalon and rostral mesencephalon (substantia nigra/ventral tegmental area); and later on in the anterior (locus coeruleus/nucleus subcoeruleus) and posterior (vagal lobe and reticular formation) rhombencephalon. The appearance of TH‐ir cells in the telencephalon (pallium) was rather late (stage [S]31) with respect to the other TH‐ir prosencephalic populations. The first TH‐ir fibers arose from cells of the posterior tubercle (S30) and formed recognizable ascending (toward dorsal and rostral territories) and descending pathways at S31. When the second half of embryonic development started (S32), TH‐ir fibers innervated most brain areas, and nearly all TH‐ir cell groups of the postembryonic brain were already established. This study provides key information about the evolution of the developmental patterns of central CA systems in fishes and thus may help in understanding how the vertebrate CA systems have evolved. J. Comp. Neurol. 520:3574–3603, 2012.

Early development of GABAergic cells of the retina in sharks: An immunohistochemical study with GABA and GAD antibodies

We studied the ontogeny and organization of GABAergic cells in the retina of two elasmobranches, the lesser-spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus) by using immunohistochemistry for gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD). Both antibodies revealed the same pattern of immunoreactivity and both species showed similar organization of GABAergic cells. GABAergic cells were first detected in neural retina of embryos at stage 26, which showed a neuroepithelial appearance without any layering. In stages 27-29 the retina showed similar organization but the number of neuroblastic GABAergic cells increased. When layering became apparent in the central retina (stage-30 embryos), GABAergic cells mainly appeared organized in the outer and inner retina, and GABAergic processes and fibres were seen in the primordial inner plexiform layer (IPL), optic fibre layer and optic nerve stalk. In stage-32 embryos, layering was completed in the central retina, where immunoreactivity appeared in perikarya of the horizontal cell layer, inner nuclear layer and ganglion cell layer, and in numerous processes coursing in the IPL, optic fibre layer and optic nerve. From stage 32 to hatching (stage 34), the layered retina extends from centre-to-periphery, recapitulating that observed in the central retina at earlier stages. In adults, GABA/GAD immunoreactivity disappears from the horizontal cell layer except in the marginal retina. Our results indicate that the source of GABA in the shark retina can be explained by its synthesis by GAD. Such synthesis precedes layering and synaptogenesis, thus supporting a developmental role for GABA in addition to act as neurotransmitter and neuromodulator.