Study of the glial cytoarchitecture of the developing olfactory bulb of a shark using immunochemical markers of radial glia.

During development of the olfactory bulb (OB), glial cells play key roles in axonal guiding/targeting, glomerular formation and synaptic plasticity. Studies in mammals have shown that radial glial cells and peripheral olfactory glia (olfactory ensheathing cells, OECs) are involved in the development of the OB. Most studies about the OB glia were carried out in mammals, but data are lacking in most non-mammalian vertebrates. In the present work, we studied the development of the OB glial system in the cartilaginous fish Scyliorhinus canicula (catshark) using antibodies against glial markers, such as glial fibrillary acidic protein (GFAP), brain lipid-binding protein (BLBP), and glutamine synthase (GS). These glial markers were expressed in cells with radial morphology lining the OB ventricle of embryos and this expression continues in ependymal cells (tanycytes) in early juveniles. Astrocyte-like cells were also observed in the granular layer and surrounding glomeruli. Numerous GS-positive cells were present in the primary olfactory pathway of embryos. In the developmental stages analysed, the olfactory nerve layer and the glomerular layer were the regions with higher GFAP, BLBP and GS immuno-reactivity. In addition, numerous BLBP-expressing cells (a marker of mammalian OECs) showing proliferative activity were present in the olfactory nerve layer. Our findings suggest that glial cells of peripheral and central origin coexist in the OB of catshark embryos and early juveniles. These results open the path for future studies about the differential roles of glial cells in the catshark OB during embryonic development and in adulthood.

Characterization of neurogenic niches in the telencephalon of juvenile and adult sharks.

Neurogenesis is a multistep process by which progenitor cells become terminally differentiated neurons. Adult neurogenesis has gathered increasing interest with the aim of developing new cell-based treatments for neurodegenerative diseases in humans. Active sites of adult neurogenesis exist from fish to mammals, although in the adult mammalian brain the number and extension of neurogenic areas is considerably reduced in comparison to non-mammalian vertebrates and they become mostly reduced to the telencephalon. Much of our understanding in this field is based in studies on mammals and zebrafish, a modern bony fish. The use of the cartilaginous fish Scyliorhinus canicula (representative of basal gnathostomes) as a model expands the comparative framework to a species that shows highly neurogenic activity in the adult brain. In this work, we studied the proliferation pattern in the telencephalon of juvenile and adult specimens of S. canicula using antibodies against the proliferation marker proliferating cell nuclear antigen (PCNA). We have characterized proliferating niches using stem cell markers (Sex determining region Y-box 2), glial markers (glial fibrillary acidic protein, brain lipid binding protein and glutamine synthase), intermediate progenitor cell markers (Dlx2 and Tbr2) and markers for migrating neuroblasts (Doublecortin). Based in the expression pattern of these markers, we demonstrate the existence of different cell subtypes within the PCNA immunoreactive zones including non-glial stem cells, glial progenitors, intermediate progenitor-like cells and migratory neuroblasts, which were widely distributed in the ventricular zone of the pallium, suggesting that the main progenitor types that constitute the neurogenic niche in mammals are already present in cartilaginous fishes.

Mitral cell development in the olfactory bulb of sharks: evidences of a conserved pattern of glutamatergic neurogenesis.

In mammals, the development of the olfactory bulb (OB) relies in part on the expression of transcription factors involved in the specifications/differentiation of glutamatergic cells. In a previous study from our group, a high molecular similarity was reported between mammals and cartilaginous fishes regarding the neurogenic mechanisms underlying the development of glutamatergic cells in the telencephalon. However, information about the transcriptional program operating in the development of the glutamatergic system (mainly represented by mitral cells) in the OB is lacking in the catshark Scyliorhinus canicula, a cartilaginous fish. Using immunohistochemistry and in situ hybridization techniques, we have found that, previously to the appearance of the olfactory primordium (OP), proliferating cells expressing Pax6 with molecular hallmarks of progenitor radial glia were located in the ventrolateral pallial ventricular zone. Later in development, when the OP is recognizable, a stream of Pax6-positive cells were observed between the ventricular zone and the OP, where transcription factors involved in mitral cell development in mammals (ScTbr2, ScNeuroD, Tbr1) are expressed. Later in development, these transcription factors became expressed in a layered-like structure where ScVglut1, a marker of mitral cells, is also present. Our data suggest that the transcriptional program related with the specification/differentiation of glutamatergic cells in the telencephalon has been conserved throughout the evolution of vertebrates. These results, in combination with previous studies concerning GABAergic neurogenesis in sharks, have evidenced that the OB of mammals and sharks shares similarities in the timing and molecular programs of development.

Expression of radial glial markers (GFAP, BLBP and GS) during telencephalic development in the catshark (Scyliorhinus canicula).

Radial glial cells (RGCs) are the first cell populations of glial nature to appear during brain ontogeny. They act as primary progenitor (stem) cells as well as a scaffold for neuronal migration. The proliferative capacity of these cells, both in development and in adulthood, has been subject of interest during past decades. In contrast with mammals where RGCs are restricted to specific ventricular areas in the adult brain, RGCs are the predominant glial element in fishes. However, developmental studies on the RGCs of cartilaginous fishes are scant. We have studied the expression patterns of RGCs markers including glial fibrillary acidic protein (GFAP), brain lipid binding protein (BLBP), and glutamine synthase (GS) in the telencephalic hemispheres of catshark (Scyliorhinus canicula) from early embryos to post-hatch juveniles. GFAP, BLBP and GS are first detected, respectively, in early, intermediate and late embryos. Expression of these glial markers was observed in cells with radial glia morphology lining the telencephalic ventricles, as well as in their radial processes and endfeet at the pial surface and their expression continue in ependymal cells (or tanycytes) in early juveniles. In addition, BLBP- and GS-immunoreactive cells morphologically resembling oligodendrocytes were observed. In late embryos, most of the GFAP- and BLBP-positive RGCs also coexpress GS and show proliferative activity. Our results indicate the existence of different proliferating subpopulations of RGCs in the embryonic ventricular zone of catshark. Further investigations are needed to determine whether these proliferative RGCs could act as neurogenic and/or gliogenic precursors.

Study of pallial neurogenesis in shark embryos and the evolutionary origin of the subventricular zone.

The dorsal part of the developing telencephalon is one of the brain areas that has suffered most drastic changes throughout vertebrate evolution. Its evolutionary increase in complexity was thought to be partly achieved by the appearance of a new neurogenic niche in the embryonic subventricular zone (SVZ). Here, a new kind of amplifying progenitors (basal progenitors) expressing Tbr2, undergo a second round of divisions, which is believed to have contributed to the expansion of the neocortex. Accordingly, the existence of a pallial SVZ has been classically considered exclusive of mammals. However, the lack of studies in ancient vertebrates precludes any clear conclusion about the evolutionary origin of the SVZ and the neurogenic mechanisms that rule pallial development. In this work, we explore pallial neurogenesis in a basal vertebrate, the shark Scyliorhinus canicula, through the study of the expression patterns of several neurogenic markers. We found that apical progenitors and radial migration are present in sharks, and therefore, their presence must be highly conserved throughout evolution. Surprisingly, we detected a subventricular band of ScTbr2-expressing cells, some of which also expressed mitotic markers, indicating that the existence of basal progenitors should be considered an ancestral condition rather than a novelty of mammals or amniotes. Finally, we report that the transcriptional program for the specification of glutamatergic pallial cells (Pax6, Tbr2, NeuroD, Tbr1) is also present in sharks. However, the segregation of these markers into different cell types is not clear yet, which may be linked to the lack of layering in anamniotes.