In Vivo Monitoring of Fabp7 Expression in Transgenic Zebrafish.

In zebrafish, like in mammals, radial glial cells (RGCs) can act as neural progenitors during development and regeneration in adults. However, the heterogeneity of glia subpopulations entails the need for different specific markers of zebrafish glia. Currently, fluorescent protein expression mediated by a regulatory element from the glial fibrillary acidic protein (gfap) gene is used as a prominent glia reporter. We now expand this tool by demonstrating that a regulatory element from the mouse Fatty acid binding protein 7 (Fabp7) gene drives reliable expression in fabp7-expressing zebrafish glial cells. By using three different Fabp7 regulatory element-mediated fluorescent protein reporter strains, we reveal in double transgenic zebrafish that progenitor cells expressing fluorescent proteins driven by the Fabp7 regulatory element give rise to radial glia, oligodendrocyte progenitors, and some neuronal precursors. Furthermore, Bergmann glia represent the almost only glial population of the zebrafish cerebellum (besides a few oligodendrocytes), and the radial glia also remain in the mature cerebellum. Fabp7 regulatory element-mediated reporter protein expression in Bergmann glia progenitors suggests their origin from the ventral cerebellar proliferation zone, the ventricular zone, but not from the dorsally positioned upper rhombic lip. These new Fabp7 reporters will be valuable for functional studies during development and regeneration.

Development, circuitry, and function of the zebrafish cerebellum.

The cerebellum represents a brain compartment that first appeared in gnathostomes (jawed vertebrates). Besides the addition of cell numbers, its development, cytoarchitecture, circuitry, physiology, and function have been highly conserved throughout avian and mammalian species. While cerebellar research in avian and mammals is extensive, systematic investigations on this brain compartment in zebrafish as a teleostian model organism started only about two decades ago, but has provided considerable insight into cerebellar development, physiology, and function since then. Zebrafish are genetically tractable with nearly transparent small-sized embryos, in which cerebellar development occurs within a few days. Therefore, genetic investigations accompanied with non-invasive high-resolution in vivo time-lapse imaging represents a powerful combination for interrogating the behavior and function of cerebellar cells in their complex native environment.

Lifelong regeneration of cerebellar Purkinje cells after induced cell ablation in zebrafish.

Zebrafish have an impressive capacity to regenerate neurons in the central nervous system. However, regeneration of the principal neuron of the evolutionary conserved cerebellum, the Purkinje cell (PC), is believed to be limited to developmental stages based on invasive lesions. In contrast, non-invasive cell type-specific ablation by induced apoptosis closely represents a process of neurodegeneration. We demonstrate that the ablated larval PC population entirely recovers in number, quickly reestablishes electrophysiological properties, and properly integrates into circuits to regulate cerebellum-controlled behavior. PC progenitors are present in larvae and adults, and PC ablation in adult cerebelli results in an impressive PC regeneration of different PC subtypes able to restore behavioral impairments. Interestingly, caudal PCs are more resistant to ablation and regenerate more efficiently, suggesting a rostro-caudal pattern of de- and regeneration properties. These findings demonstrate that the zebrafish cerebellum is able to regenerate functional PCs during all stages of the animal's life.

Culture and Transfection of Zebrafish Primary Cells.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue. In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

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.

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.

Development of the cerebellar afferent system in the shark Scyliorhinus canicula: insights into the basal organization of precerebellar nuclei in gnathostomes.

The cerebellum is recognized as an evolutionary innovation of jawed vertebrates, whose most primitive group is represented by the chondrichthyans, or cartilaginous fishes. A comprehensive knowledge of cerebellar connections in these fishes might shed light on the basal organization of the cerebellar system. Although the organization of the precerebellar system is known in adults, developmental studies are essential for understanding the origin and evolution of precerebellar nuclei. In the present work we performed a developmental study of cerebellar connections in embryos and juveniles of an advanced shark species, Scyliorhinus canicula, by application of tract tracing in combination with immunohistochemical techniques. Main precerebellar cell populations were located in the diencephalon (pretectum and thalamus), mesencephalon (reticular formation and nucleus ruber), rhombencephalon (cerebellar nucleus, reticular formation, and inferior olive), and spinal cord (ventral horn). The order of arrival of cerebellar afferent projections throughout development revealed a common pattern with other jawed vertebrates, which was helpful for comparison of stages of cerebellar development. The neurochemical study of the inferior olive and other precerebellar nuclei revealed many shared features with other gnathostomes. Furthermore, because many precerebellar nuclei originate from rhombic lips, the first analysis of neuronal migrations from these lips was performed with markers of neuroblasts. The shared features of development and organization of precerebellar connections observed between sharks and amniotes suggest that their basic pattern was established early in gnathostome evolution.

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.