Susana Ferreiro-Galve

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.

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.

Patterns of cell proliferation and rod photoreceptor differentiation in shark retinas

We studied the pattern of cell proliferation and its relation with photoreceptor differentiation in the embryonic and postembryonic retina of two elasmobranchs, the lesser spotted dogfish (Scyliorhinus canicula) and the brown shyshark (Haploblepharus fuscus). Cell proliferation was studied with antibodies raised against proliferating cell nuclear antigen (PCNA) and phospho-histone-H3, and early photoreceptor differentiation with an antibody raised against rod opsin. As regards the spatiotemporal distribution of PCNA-immunoreactive cells, our results reveal a gradual loss of PCNA that coincides in a spatiotemporal sequence with the gradient of layer maturation. The presence of a peripheral growth zone containing pure-proliferating retinal progenitors (the ciliary marginal zone) in the adult retina matches with the general pattern observed in other groups of gnathostomous fishes. However, in the shark retina the generation of new cells is not restricted to the ciliary marginal zone but also occurs in retinal areas that contain differentiated cells: (1) in a transition zone that lies between the pure-proliferating ciliary marginal zone and the central (layered) retina; (2) in the differentiating central area up to prehatching embryos where large amounts of PCNA-positive cells were observed even in the inner and outer nuclear layers; (3) and in the retinal pigment epithelium of prehatching embryos. Rod opsin immunoreactivity was observed in both species when the outer plexiform layer begins to be recognized in the central retina and, as we previously observed in trout, coincided temporally with the weakening in PCNA labelling.

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.

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.

Pax6 expression during retinogenesis in sharks: comparison with markers of cell proliferation and neuronal differentiation.

Pax6 is a highly conserved transcription factor that appears involved in the entire process of retinogenesis, including maintenance of proliferation of retinal progenitors and differentiation of particular neuron fates. To gain insight into the retinogenesis in fish, we study the dynamics of Pax6 expression in the developing and mature retina of two sharks that inhabit in particular environments, and compare it with the dynamics of a marker of cell proliferation (proliferating cell nuclear antigen, PCNA) and markers of neuronal differentiation, such as glutamic acid decarboxylase (GAD), calretinin (CR), tyrosine-hydroxylase, and serotonin (5-HT). Our results reveal that Pax6 is expressed in PCNA-immunoreactive cells within the nonlayered retina, suggesting a role for Pax6 in proliferating progenitors. Pax6 expression decays as development proceeds and eventually remains in some postmitotic cells, which points to additional roles of Pax6 following neurogenesis. Double immunofluorescence reveals Pax6/CR colocalization in the ganglion cell layer, Pax6/5-HT in the inner part of the inner nuclear layer (INLi), and Pax6/GAD in the INLi and horizontal cell layer. Our results suggest that Pax6 may contribute to neuron diversification in the neural retina.

Morphogenesis in the retina of a slow-developing teleost: Emergence of the GABAergic system in relation to cell proliferation and differentiation

Gamma-aminobutyric acid (GABA) has been implicated in cell proliferation and differentiation during development. In the present study, immunohistochemical techniques were used to investigate the development of the GABAergic system in the retina of the trout and its relation to markers of differentiation [calretinin (CR), and tyrosine hydroxylase (TH)]. The expression of Pax6, an eye-patterning protein involved in the proliferation and emergence of specific retinal cell types, was also studied. Retinal layering was observed to begin centrally in prehatching embryos, as the first GABAergic cells appeared in the ganglion cell layer (GCL) and inner part of the inner nuclear layer (INL). At hatching, GABAergic cells were also observed in the horizontal cell layer (HCL). In alevins, GABAergic cells and processes spread laterally following retinal growth although they did not invade neuroblastic retinal regions. CR- and Pax6-immunoreactive (ir) cells were first seen in the GCL and the inner part of the INL, whereas sparse TH-ir cells appeared in the INL. In juveniles, GABAergic cells were observed in the GCL, inner part of the INL and HCL, whereas CR-ir cells spread to the outer part of the INL and HCL. A subset of CR-ir in the GCL and of Pax6-ir cells in the GCL and INL showed colocalization with GABAergic markers. This study provides further comparative knowledge about the development of GABAergic system of the retina in teleosts and shows differences and similarities with that reported in fast-developing species such as zebrafish, in which retinal expression of GABA was transient in some populations.

Calretinin-immunoreactive systems in the cerebellum and cerebellum-related lateral-line medullary nuclei of an elasmobranch, Scyliorhinus canicula

Calretinin immunohistochemistry was used to study the organization of some cerebellar structures and lateral line medullary nuclei of an elasmobranch, the lesser-spotted dogfish Scyliorhinus canicula. In the cerebellar molecular layer, stellate cells are strongly calretinin-immunoreactive (CR-ir). Perikarya and dendrites of Purkinje cells are contacted by numerous stellate cell small CR-ir boutons. Some Purkinje cell perikarya are contacted by CR-ir climbing fibers forming complex axo-somatic contacts. In the granular layer, numerous CR-ir mossy fibers exhibited large swellings. Notable differences in density and diameter of mossy fibers are observed between the auricles and cerebellar body. Thin beaded CR-ir fibers are also present in the granular layer of the body. The lateral line nuclei of the octavolateralis region are comprised of a molecular-like cerebellar crest that covers the dorsal (electroreceptive) and the medial octavolateralis nuclei (mechanoreceptive). The cerebellar crest exhibited numerous CR-ir stellate cells. In the dorsal octavolateralis nucleus, the presence of conspicuous CR-ir cells and neuropil closely associated to the region of primary fiber terminals distinguishes it clearly from the medial nucleus, revealing major differences between the electroreceptive and mechanoreceptive primary nuclei of elasmobranchs. Moreover, CR distribution in the dogfish cerebellum showed interesting differences with those reported in cerebella of other vertebrates, indicating a high variability of cerebellar CR expression in phylogeny.