Identifying Amygdala-Like Territories in Scyliorhinus canicula (Chondrichthyan): Evidence for a Pallial Amygdala.

To identify the putative amygdalar complex in cartilaginous fishes, our first step was to obtain evidence that supports the existence of a pallial amygdala in the catshark Scyliorhinus canicula, at present the prevailing chondrichthyan model in comparative neurobiology and developmental biology. To this end, we analyzed the organization of the lateral walls of the telencephalic hemispheres of adults, juveniles, and early prehatching embryos by immunohistochemistry against tyrosine hydroxylase (TH), somatostatin (SOM), Pax6, serotonin (5HT), substance P (SP), and Met-enkephalin (MetEnk), calbindin-28k (CB), and calretinin (CR), and by in situ hybridization against regulatory genes such as Tbr1, Lhx9, Emx1, and Dlx2. Our data were integrated with those available from the literature related to the secondary olfactory projections in this shark species. We have characterized two possible amygdalar territories. One, which may represent a ventropallial component, was identified by its chemical signature (moderate density of Pax6-ir cells, scarce TH-ir and SOM-ir cells, and absence of CR-ir and CB-ir cells) and gene expressions (Tbr1 and Lhx9 expressions in an Emx1 negative domain, as the ventral pallium of amniotes). It is perhaps comparable to the lateral amygdala of amphibians and the pallial amygdala of teleosts. The second was a territory related to the pallial-subpallial boundary with abundant Pax6-ir and CR-ir cells, and 5HT-ir, SP-ir, and MetEnk-ir fibers capping dorsally the area superficialis basalis. This olfactory-related region at the neighborhood of the pallial-subpallial boundary may represent a subpallial amygdala subdivision that possibly contains migrated cells of ventropallial origin.

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.

Development of the terminal nerve system in the shark Scyliorhinus canicula.

The nervus terminalis (or terminal nerve) system was discovered in an elasmobranch species more than a century ago. Over the past century, it has also been recognized in other vertebrate groups, from agnathans to mammals. However, its origin, functions or relationship with the olfactory system are still under debate. Despite the abundant literature about the nervus terminalis system in adult elasmobranchs, its development has been overlooked. Studies in other vertebrates have reported newly differentiated neurons of the terminal nerve system migrating from the olfactory epithelium to the telencephalon as part of a 'migratory mass' of cells associated with the olfactory nerve. Whether the same occurs in developing elasmobranchs (adults showing anatomically separated nervus terminalis and olfactory systems) has not yet been determined. In this work we characterized for the first time the development of the terminal nerve and ganglia in an elasmobranch, the lesser spotted dogfish (Scyliorhinus canicula), by means of tract-tracing techniques combined with immunohistochemical markers for the terminal nerve (such as FMRF-amide peptide), for the developing components of the olfactory system (Gα0 protein, GFAP, Pax6), and markers for early postmitotic neurons (HuC/D) and migrating immature neurons (DCX). We discriminated between embryonic olfactory and terminal nerve systems and determined that both components may share a common origin in the migratory mass. We also localized the exact point where they split off near the olfactory nerve-olfactory bulb junction. The study of the development of the terminal nerve system in a basal gnathostome contributes to the knowledge of the ancestral features of this system in vertebrates, shedding light on its evolution and highlighting the importance of elasmobranchs for developmental and evolutionary studies.

Distribution of gamma-aminobutyric acid immunoreactivity in the brain of the Siberian sturgeon (Acipenser baeri): Comparison with other fishes.

Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates. Immunohistochemical techniques with specific antibodies against GABA or against its synthesizing enzyme, glutamic acid decarboxylase (GAD) allowed characterizing GABAergic neurons and fibers in the CNS. However, studies on the CNS distribution of GABAergic neurons and fibers of bony fishes are scant and were done in teleost species. With the aim of understanding the early evolution of this system in bony vertebrates, we analyzed the distribution of GABA-immunoreactive (-ir) and GAD-ir neurons and fibers in the CNS of a basal ray-finned fish, the Siberian sturgeon (Chondrostei, Acipenseriformes), using immunohistochemical techniques. Our results revealed the presence and distribution of GABA/GAD-ir cells in different regions of the CNS such as olfactory bulbs, pallium and subpallium, hypothalamus, thalamus, pretectum, optic tectum, tegmentum, cerebellum, central grey, octavolateralis area, vagal lobe, rhombencephalic reticular areas, and the spinal cord. Abundant GABAergic innervation was observed in most brain regions, and GABAergic fibers were very abundant in the hypothalamic floor along the hypothalamo-hypophyseal tract and neurohypophysis. In addition, GABA-ir cerebrospinal fluid-contacting cells were observed in the alar and basal hypothalamus, saccus vasculosus, and spinal cord central canal. The distribution of GABAergic systems in the sturgeon brain shows numerous similarities to that observed in lampreys, but also to those of teleosts and tetrapods.

Physical stimuli-emitting scaffolds: The role of piezoelectricity in tissue regeneration.

The imbalance between life expectancy and quality of life is increasing due to the raising prevalence of chronic diseases. Musculoskeletal disorders and chronic wounds affect a growing percentage of people and demand more efficient tools for regenerative medicine. Scaffolds that can better mimic the natural physical stimuli that tissues receive under healthy conditions and during healing may significantly aid the regeneration process. Shape, mechanical properties, pore size and interconnectivity have already been demonstrated to be relevant scaffold features that can determine cell adhesion and differentiation. Much less attention has been paid to scaffolds that can deliver more dynamic physical stimuli, such as electrical signals. Recent developments in the precise measurement of electrical fields in vivo have revealed their key role in cell movement (galvanotaxis), growth, activation of secondary cascades, and differentiation to different lineages in a variety of tissues, not just neural. Piezoelectric scaffolds can mimic the natural bioelectric potentials and gradients in an autonomous way by generating the electric stimuli themselves when subjected to mechanical loads or, if the patient or the tissue lacks mobility, ultrasound irradiation. This review provides an analysis on endogenous bioelectrical signals, recent developments on piezoelectric scaffolds for bone, cartilage, tendon and nerve regeneration, and their main outcomes in vivo. Wound healing with piezoelectric dressings is addressed in the last section with relevant examples of performance in animal models. Results evidence that a fine adjustment of material composition and processing (electrospinning, corona poling, 3D printing, annealing) provides scaffolds that act as true emitters of electrical stimuli that activate endogenous signaling pathways for more efficient and long-term tissue repair.

Dynamic expression of Pax6 in the shark olfactory system: evidence for the presence of Pax6 cells along the olfactory nerve pathway.

Pax6 is involved in the control of neuronal specification, migration, and differentiation in the olfactory epithelium and in the generation of different interneuron subtypes in the olfactory bulb. Whether these roles are conserved during evolution is not known. Cartilaginous fish are extremely useful models for assessing the ancestral condition of brain organization because of their phylogenetic position. To shed light on the evolution of development of the olfactory system in vertebrates and on the involvement of Pax6 in this process, we analyzed by in situ hybridization and immunohistochemistry the expression pattern of Pax6 in the developing olfactory system in a basal vertebrate, the lesser spotted dogfish Scyliorhinus canicula. This small shark is becoming an important fish model in studies of vertebrate development. We report Pax6 expression in cells of the olfactory epithelium and olfactory bulb, and present the first evidence in vertebrates of strings of Pax6-expressing cells extending along the developing olfactory nerve. The results indicate the olfactory epithelium as the origin of these cells. These data are compatible with a role for Pax6 in the development of the olfactory epithelium and fibers, and provide a basis for future investigations into the mechanisms that regulate development of the olfactory system throughout evolution.

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.

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.

Developmental genoarchitectonics as a key tool to interpret the mature anatomy of the chondrichthyan hypothalamus according to the prosomeric model.

The hypothalamus is a key vertebrate brain region involved in survival and physiological functions. Understanding hypothalamic organization and evolution is important to deciphering many aspects of vertebrate biology. Recent comparative studies based on gene expression patterns have proposed the existence of hypothalamic histogenetic domains (paraventricular, TPa/PPa; subparaventricular, TSPa/PSPa; tuberal, Tu/RTu; perimamillary, PM/PRM; and mamillary, MM/RM), revealing conserved evolutionary trends. To shed light on the functional relevance of these histogenetic domains, this work aims to interpret the location of developed cell groups according to the prosomeric model in the hypothalamus of the catshark Scyliorhinus canicula, a representative of Chondrichthyans (the sister group of Osteichthyes, at the base of the gnathostome lineage). To this end, we review in detail the expression patterns of ScOtp, ScDlx2, and ScPitx2, as well as Pax6-immunoreactivity in embryos at stage 32, when the morphology of the adult catshark hypothalamus is already organized. We also propose homologies with mammals when possible. This study provides a comprehensive tool to better understand previous and novel data on hypothalamic development and 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.

Differential expression of five prosomatostatin genes in the central nervous system of the catshark Scyliorhinus canicula.

Five prosomatostatin genes (PSST1, PSST2, PSST3, PSST5, and PSST6) have been recently identified in elasmobranchs (Tostivint et al., General and Comparative Endocrinology, 2019, 279, 139-147). In order to gain insight into the contribution of each somatostatin to specific nervous systems circuits and behaviors in this important jawed vertebrate group, we studied the distribution of neurons expressing PSST mRNAs in the central nervous system (CNS) of Scyliorhinus canicula using in situ hybridization. Additionally, we combined in situ hybridization with tyrosine hydroxylase (TH) immunochemistry for better characterization of PSST1 and PSST6 expressing populations. We observed differential expression of PSST1 and PSST6, which are the most widely expressed PSST transcripts, in cell populations of many CNS regions, including the pallium, subpallium, hypothalamus, diencephalon, optic tectum, midbrain tegmentum, and rhombencephalon. Interestingly, numerous small pallial neurons express PSST1 and PSST6, although in different populations judging from the colocalization of TH immunoreactivity and PSST6 expression but not with PSST1. We observed expression of PSST1 in cerebrospinal fluid-contacting (CSF-c) neurons of the hypothalamic paraventricular organ and the central canal of the spinal cord. Unlike PSST1 and PSST6, PSST2, and PSST3 are only expressed in cells of the hypothalamus and in some hindbrain lateral reticular neurons, and PSST5 in cells of the region of the entopeduncular nucleus. Comparative data of brain expression of PSST genes indicate that the somatostatinergic system of sharks is the most complex reported in any fish.

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.

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.

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.

Distribution of somatostatin immunoreactive neurons and fibres in the central nervous system of a chondrostean, the Siberian sturgeon (Acipenser baeri).

Somatostatin (SOM) is a neuropeptide that is widely distributed in the central nervous system of vertebrates. Two isoforms of somatostatin (SS1 and SS2) have been characterized in sturgeon and in situ hybridisation studies in the sturgeon brain have demonstrated that mRNAs of the two somatostatin precursors (PSS1 and PSS2) are differentially expressed in neurons [Trabucchi, M., Tostivint, H., Lihrmann, I., Sollars, C., Vallarino, M., Dores, R.M., Vaudry, H., 2002. Polygenic expression of somatostatin in the sturgeon Acipenser transmontanus: molecular cloning and distribution of the mRNAs encoding two somatostatin precursors. J. Comp. Neurol. 443, 332-345.]. However, neither the morphology of somatostatinergic neurons nor the patterns of innervation have yet been characterized. To gain further insight into the evolution of this system in primitive bony fishes, we studied the distribution of somatostatin-immunoreactive (SOM-ir) cells and fibres in the brain of the Siberian sturgeon (Acipenser baeri). Most SOM-ir cells were found in the preoptic area and hypothalamus and abundant SOM-ir fibres coursed along the hypothalamic floor towards the median eminence, suggesting a hypophysiotrophic role for SOM in sturgeon. In addition, SOM-ir cells and fibres were observed in extrahypothalamic regions such as the telencephalon thalamus, rhombencephalon and spinal cord, which also suggests neuromodulatory and/or neurotransmitter functions for this peptide. Overall there was a good correlation between the distribution of SOM-ir neurons throughout the brain of A. baeri and that of PSS1 mRNA in Acipenser transmontanus. Comparative analysis of the results with those obtained in other groups of fishes and tetrapods indicates that widespread distribution of this peptide in the brain is shared by early vertebrate lines and that the general organization of the somatostatinergic systems has been well-conserved during evolution.

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.

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.

The Shark Basal Hypothalamus: Molecular Prosomeric Subdivisions and Evolutionary Trends.

The hypothalamus is a key integrative center of the vertebrate brain. To better understand its ancestral morphological organization and evolution, we previously analyzed the segmental organization of alar subdivisions in the catshark Scyliorhinus canicula, a cartilaginous fish and thus a basal representative of gnathostomes (jawed vertebrates). With the same aim, we deepen here in the segmental organization of the catshark basal hypothalamus by revisiting previous data on ScOtp, ScDlx2/5, ScNkx2.1, ScShh expression and Shh immunoreactivity jointly with new data on ScLhx5, ScEmx2, ScLmx1b, ScPitx2, ScPitx3a, ScFoxa1, ScFoxa2 and ScNeurog2 expression and proliferating cell nuclear antigen (PCNA) immunoreactivity. Our study reveals a complex genoarchitecture for chondrichthyan basal hypothalamus on which a total of 21 microdomains were identified. Six belong to the basal acroterminal region, the rostral-most point of the basal neural tube; seven are described in the tuberal region (Tu/RTu); four in the perimamillar region (PM/PRM) and four in the mamillar one (MM/RM). Interestingly, the same set of genes does not necessarily describe the same microdomains in mice, which in part contributes to explain how forebrain diversity is achieved. This study stresses the importance of analyzing data from basal vertebrates to better understand forebrain diversity and hypothalamic evolution.