Wnt signaling regulates proliferation and differentiation of radial glia in regenerative processes after stab injury in the optic tectum of adult zebrafish

Zebrafish have superior abilities to generate new neurons in the adult brain and to regenerate brain tissue after brain injury compared with mammals. There exist two types of neural stem cells (NSCs): neuroepithelial‐like stem cells (NE) and radial glia (RG) in the optic tectum. We established an optic tectum stab injury model to analyze the function of NSCs in the regenerative condition and confirmed that the injury induced the proliferation of RG, but not NE and that the proliferated RG differentiated into new neurons after the injury. We then analyzed the involvement of Wnt signaling after the injury, using a Wnt reporter line in which canonical Wnt signaling activation induced GFP expression and confirmed that GFP expression was induced specifically in RG after the injury. We also analyzed the expression level of genes related to Wnt signaling, and confirmed that endogenous Wnt antagonist dkk1b expression was significantly decreased after the injury. We observed that Wnt signal inhibitor IWR1 treatment suppressed the proliferation and differentiation of RG after the injury, suggesting that up‐regulation of Wnt signaling in RG after the stab injury was required for optic tectum regeneration. We also confirmed that Wnt activation by treatment with GSK3β inhibitor BIO in uninjured zebrafish induced proliferation of RG in the optic tectum. This optic tectum stab injury model is useful for the study of the molecular mechanisms of brain regeneration and analysis of the RG functions in physiological and regenerative conditions.     

Heterogeneity in progenitor cell subtypes in the ventricular zone of the zebrafish adult telencephalon

The zebrafish has become a new model for adult neurogenesis, owing to its abundant neurogenic areas in most brain subdivisions. Radial glia‐like cells, actively proliferating cells, and label‐retaining progenitors have been described in these areas. In the telencephalon, this complexity is enhanced by an organization of the ventricular zone (VZ) in fast and slow‐dividing domains, suggesting the existence of heterogeneous progenitor types. In this work, we studied the expression of various transgenic or immunocytochemical markers for glial cells (gfap:gfp, cyp19a1b:gfp, BLBP, and S100β), progenitors (nestin:gfp and Sox2), and neuroblasts (PSA‐NCAM) in cycling progenitors of the adult zebrafish telencephalon (identified by expression of proliferating cell nuclear antigen (PCNA), MCM5, or bromodeoxyuridine incorporation). We demonstrate the existence of distinct populations of dividing cells at the adult telencephalic VZ. Progenitors of the overall slow‐cycling domains express high levels of Sox2 and nestin:gfp as well as all glial markers tested. In contrast, domains with an overall fast division rate are characterized by low or missing expression of glial markers. PCNA‐positive cells in fast domains further display a morphology distinct from radial glia and co‐express PSA‐NCAM, suggesting that they are early neuronal precursors. In addition, the VZ contains cycling progenitors that express neither glial markers nor nestin:gfp, but are positive for Sox2 and PSA‐NCAM, identifying them as committed neuroblasts. On the basis of the marker gene expression and distinct cell morphologies, we propose a classification for the dividing cell states at the zebrafish adult telencephalic VZ. © 2010 Wiley‐Liss, Inc.     

Excitotoxic brain injury in adult zebrafish stimulates neurogenesis and long‐distance neuronal integration

Zebrafish maintain a greater capacity than mammals for central nervous system repair after injury. Understanding differences in regenerative responses between different vertebrate species may shed light on mechanisms to improve repair in humans. Quinolinic acid is an excitotoxin that has been used to induce brain injury in rodents for modeling Huntington's disease and stroke. When injected into the adult rodent striatum, this toxin stimulates subventricular zone neurogenesis and neuroblast migration to injury. However, most new neurons fail to survive and lesion repair is minimal. We used quinolinic acid to lesion the adult zebrafish telencephalon to study reparative processes. We also used conditional transgenic lineage mapping of adult radial glial stem cells to explore survival and integration of neurons generated after injury. Telencephalic lesioning with quinolinic acid, and to a lesser extent vehicle injection, produced cell death, microglial infiltration, increased cell proliferation, and enhanced neurogenesis in the injured hemisphere. Lesion repair was more complete with quinolinic acid injection than after vehicle injection. Fate mapping of her4‐expressing radial glia showed injury‐induced expansion of radial glial stem cells that gave rise to neurons which migrated to injury, survived at least 8 weeks and formed long‐distance projections that crossed the anterior commissure and synapsed in the contralateral hemisphere. These findings suggest that quinolinic acid lesioning of the zebrafish brain stimulates adult neural stem cells to produce robust regeneration with long‐distance integration of new neurons. This model should prove useful for elucidating reparative mechanisms that can be applied to restorative therapies for mammalian brain injury. GLIA 2014;62:2061–2079     

Gfap‐positive radial glial cells are an essential progenitor population for later‐born neurons and glia in the zebrafish spinal cord

Radial glial cells are presumptive neural stem cells (NSCs) in the developing nervous system. The direct requirement of radial glia for the generation of a diverse array of neuronal and glial subtypes, however, has not been tested. We employed two novel transgenic zebrafish lines and endogenous markers of NSCs and radial glia to show for the first time that radial glia are essential for neurogenesis during development. By using the gfap promoter to drive expression of nuclear localized mCherry we discerned two distinct radial glial‐derived cell types: a major nestin+/Sox2+ subtype with strong gfap promoter activity and a minor Sox2+ subtype lacking this activity. Fate mapping studies in this line indicate that gfap+ radial glia generate later‐born CoSA interneurons, secondary motorneurons, and oligodendroglia. In another transgenic line using the gfap promoter‐driven expression of the nitroreductase enzyme, we induced cell autonomous ablation of gfap+ radial glia and observed a reduction in their specific derived lineages, but not Blbp+ and Sox2+/gfap‐negative NSCs, which were retained and expanded at later larval stages. Moreover, we provide evidence supporting classical roles of radial glial in axon patterning, blood–brain barrier formation, and locomotion. Our results suggest that gfap+ radial glia represent the major NSC during late neurogenesis for specific lineages, and possess diverse roles to sustain the structure and function of the spinal cord. These new tools will both corroborate the predicted roles of astroglia and reveal novel roles related to development, physiology, and regeneration in the vertebrate nervous system. GLIA 2016;64:1170–1189     

Expression of hairy/enhancer of split genes in neural progenitors and neurogenesis domains of the adult zebrafish brain

All subdivisions of the adult zebrafish brain maintain niches of constitutive neurogenesis, sustained by quiescent and multipotent progenitor populations. In the telencephalon, the latter potential neural stem cells take the shape of radial glia aligned along the ventricle and are controlled by Notch signalling. With the aim of identifying new markers of this cell type and of comparing the effectors of embryonic and adult neurogenesis, we focused on the family of hairy/enhancer of split [E(spl)] genes. We report the expression of seven hairy/E(spl) (her) genes and the new helt gene in three neurogenic areas of the adult zebrafish brain (telencephalon, hypothalamus, and midbrain) in relation to radial glia, proliferation, and neurogenesis. We show that the expression of most her genes in the adult brain characterizes quiescent radial glia, whereas only few are expressed in progenitor domains engaged in active proliferation or neurogenesis. The low proliferation status of most her-positive progenitors contrasts with the embryonic nervous system, in which her genes are expressed in actively dividing progenitors. Likewise, we demonstrate largely overlapping expression domains of a set of her genes in the adult brain, which is in striking contrast to their distinct embryonic expression profiles. Overall, our data provide a consolidated map of her expression, quiescent glia, proliferation, and neurogenesis in these various subdivisions of the adult brain and suggest distinct regulation and function of Her factors in the embryonic and adult contexts.     

Deafferentation enhances neurogenesis in the young and middle aged hippocampus but not in the aged hippocampus

Increased neurogenesis in the dentate gyrus (DG) after brain insults such as excitotoxic lesions, seizures, or stroke is a well known phenomenon in the young hippocampus. This plasticity reflects an innate compensatory response of neural stem cells (NSCs) in the young hippocampus to preserve function or minimize damage after injury. However, injuries to the middle‐aged and aged hippocampi elicit either no or dampened neurogenesis response, which could be due to an altered plasticity of NSCs and/or the hippocampus with age. We examined whether the plasticity of NSCs to increase neurogenesis in response to a milder injury such as partial deafferentation is preserved during aging. We quantified DG neurogenesis in the hippocampus of young, middle‐aged, and aged F344 rats after partial deafferentation. A partial deafferentation of the left hippocampus without any apparent cell loss was induced via administration of Kainic acid (0.5 μg in 1.0 μl) into the right lateral ventricle of the brain. In this model, degeneration of CA3 pyramidal neurons and dentate hilar neurons in the right hippocampus results in loss of commissural axons which leads to partial deafferentation of the dendrites of dentate granule cells and CA1‐CA3 pyramidal neurons in the left hippocampus. Quantification of newly born cells that are added to the dentate granule cell layer at postdeafferentation days 4–15 using 5′‐bromodeoxyuridine (BrdU) labeling revealed greatly increased addition of newly born cells (～three fold increase) in the deafferented young and middle‐aged hippocampi but not in the deafferented aged hippocampus. Measurement of newly born neurons using doublecortin (DCX) immunostaining also revealed similar findings. Analyses using BrdU‐DCX dual immunofluorescence demonstrated no changes in neuronal fate‐choice decision of newly born cells after deafferentation, in comparison to the age‐matched naive hippocampus in all age groups. Thus, the plasticity of hippocampal NSCs to increase DG neurogenesis in response to a milder injury such as partial hippocampal deafferentation is preserved until middle age but lost at old age. © 2010 Wiley‐Liss, Inc.     

Impaired constitutive and regenerative neurogenesis in adult hyperglycemic zebrafish

A growing body of evidence supports hyperglycemia as a putative contributor to several brain dysfunctions observed in diabetes patients, such as impaired memory capacity, neural plasticity, and neurogenic processes. Thanks to the persistence of radial glial cells acting as neural stem cells, the brain of the adult zebrafish constitutes a relevant model to investigate constitutive and injury‐induced neurogenesis in adult vertebrates. However, there is limited understanding of the impact of hyperglycemia on brain dysfunction in the zebrafish model. This work aimed at exploring the impact of acute and chronic hyperglycemia on brain homeostasis and neurogenesis. Acute hyperglycemia was shown to promote gene expression of proinflammatory cytokines (il1β, il6, il8, and tnfα) in the brain and chronic hyperglycemia to impair expression of genes involved in the establishment of the blood–brain barrier (claudin 5a, zona occludens 1a and b). Chronic hyperglycemia also decreased brain cell proliferation in most neurogenic niches throughout the forebrain and the midbrain. By using a stab wound telencephalic injury model, the impact of hyperglycemia on brain repair mechanisms was investigated. Whereas the initial step of parenchymal cell proliferation was not affected by acute hyperglycemia, later proliferation of neural progenitors was significantly decreased by chronic hyperglycemia in the injured brain of fish. Taken together, these data offer new evidence highlighting the evolutionary conserved adverse effects of hyperglycemia on neurogenesis and brain healing in zebrafish. In addition, our study reinforces the utility of zebrafish as a robust model for studying the effects of metabolic disorders on the central nervous system. J. Comp. Neurol. 525:442–458, 2017. © 2016 Wiley Periodicals, Inc.     

Natural and lesion‐induced decrease in cell proliferation in the medial nucleus of the trapezoid body during hearing development

The functional interactions between neurons and glial cells that are important for nervous system function are presumably established during development from the activity of progenitor cells. In this study we examined proliferation of progenitor cells in the medial nucleus of the trapezoid body (MNTB) located in the rat auditory brainstem. We performed DNA synthesis labeling experiments to demonstrate changes in cell proliferation activity during postnatal stages of development. An increase in cell proliferation correlated with MNTB growth and the presence of S100β‐positive astrocytes among MNTB neurons. In additional experiments we analyzed the fate of newly born cells. At perinatal ages, newly born cells colabeled with the astrocyte marker S100β in higher numbers than when cells were generated at postnatal day 6. Furthermore, we identified newly born cells that were colabeled with caspase‐3 immunohistochemistry and performed comparative experiments to demonstrate that there is a natural decrease in cell proliferation activity during postnatal development in rats, mice, gerbils, and ferrets. Lastly, we found that there is a stronger decrease in MNTB cell proliferation after performing bilateral lesions of the auditory periphery in rats. Altogether, these results identify important stages in the development of astrocytes in the MNTB and provide evidence that the proliferative activity of the progenitor cells is developmentally regulated. We propose that the developmental reduction in cell proliferation may reflect coordinated signaling between the auditory brainstem and the auditory periphery. J. Comp. Neurol. 522:971–985, 2014. © 2013 Wiley Periodicals, Inc.     

Increased expression of specific recognition molecules by retinal ganglion cells and by optic pathway glia accompanies the successful regeneration of retinal axons in adult zebrafish

Retinal ganglion cells (RGCs) in adult zebrafish can regenerate their axons. We show that successful axonal regeneration is accompanied by the re-expression by RGCs of mRNAs encoding specific recognition molecules that are expressed at high levels in the larval retina but are down-regulated in the adult. Message levels for l1.1 and l1.2 (two homologs of mammalian L1), n-cam (homologous to mammalian N-CAM), beta3 (related to the beta3 and beta2 subunits of mammalian Na, K-ATPase), and tn-c (homologous to mammalian tenascin-C) were high in larval RGCs undergoing axonogenesis and low in adult RGCs. After an optic nerve crush, axotomized adult RGCs showed increased levels of l1.1, l1.2, and n-cam mRNA expression, whereas the levels of beta3 and tn-cmRNA remained unchanged. The optic nerve crush also induced the expression of some of these mRNAs in the optic nerve and tract where they are not normally detectable. This lesion induced up-regulation by presumptive glia was observed for l1.1, l1.2, n-cam and beta3 but not for tn-c. The combination of a neuronal (intrinsic) response to axotomy with an environmental (extrinsic) response may be an important determinant allowing for the successful axonal regeneration. © 1996 Wiley-Liss, Inc.     

Proliferation, migration, neuronal differentiation, and long-term survival of new cells in the adult zebrafish brain

In contrast to mammals, fish exhibit an enormous potential to produce new cells in the adult brain. By labeling mitotically dividing cells with 5-bromo-2'-deoxyuridine (BrdU), we have characterized the development of these cells in the zebrafish (Danio rerio). Proliferation zones were located in specific regions of the olfactory bulb, dorsal telencephalon (including a region presumably homologous to the mammalian hippocampus), preoptic area, dorsal zone of the periventricular hypothalamus, optic tectum, torus longitudinalis, vagal lobe, parenchyma near the rhombencephalic ventricle, and in a region of the medulla oblongata lateral to the vagal motor nucleus, as well as in all three subdivisions of the cerebellum, the valvula cerebelli, the corpus cerebelli, and the lobus caudalis cerebelli. In the valvula cerebelli and the corpus cerebelli, the young cells migrated from their site of origin in the molecular layers to the corresponding granule cell layers. By contrast, in the lobus caudalis cerebelli and optic tectum, no indication of a migration of the newly generated cells over wider distances could be obtained. BrdU-labeled cells remained present in the brain over at least 292 days post-BrdU administration, indicating a long-term survival of a significant portion of the newly generated cells. The combination of BrdU immunohistochemistry with immunolabeling against the neural marker protein Hu, or with retrograde tracing, suggested a neuronal differentiation in a large portion of the young cells.     

Brain‐derived neurotrophic factor stimulates proliferation and differentiation of neural stem cells,possibly by triggering the Wnt/β‐catenin signaling pathway

Brain‐derived neurotrophic factor (BDNF) has critical functions in promoting survival, expansion, and differentiation of neural stem cells (NSCs), but its downstream regulation mechanism is still not fully understood. The role of BDNF in proliferation and differentiation of NSCs through Wnt/β‐catenin signaling was studied via cell culture of cortical NSCs, Western blotting, immunocytochemistry, and TOPgal (Wnt reporter) analysis in mice. First, BDNF stimulated NSC proliferation dose dependently in cultured neurospheres that exhibited BrdU incorporation and neuronal and glial differentiation abilities. Second, BDNF effectively enhanced cell commitment to neuronal and oligodendrocytic fates, as indicated by increased differentiation marker Tuj‐1 (neuronal marker), CNPase (oligodendrocyte marker), and neuronal process extension. Third, BDNF upregulated expression of Wnt/β‐catenin signaling (Wnt1 and free β‐catenin) molecules. Moreover, these promoting effects were significantly inhibited by application of IWR1, a Wnt signaling‐specific blocker in culture. The TOPgal mouse experiment further confirmed BDNF‐triggered Wnt signaling activation by β‐gal labeling. Finally, an MEK inhibition experiment showed a mediating role of the microtubule‐associated protein kinase pathway in BDNF‐triggered Wnt/β‐catenin signaling cascades. This study overall has revealed that BDNF might contribute to proliferation and neuronal and oligodendrocytic differentiation of NSCs in vitro, most possibly by triggering the Wnt/β‐catenin signaling pathway. Nevertheless, determining the exact cross‐talk points at which BDNF might stimulate Wnt/β‐catenin signaling pathway in NSC activity requires further investigation. © 2012 Wiley Periodicals, Inc.     

Neurogenesis is required for behavioral recovery after injury in the visual system of Xenopus laevis

Nonmammalian vertebrates have a remarkable capacity to regenerate brain tissue in response to central nervous system (CNS) injury. Nevertheless, it is not clear whether animals recover lost function after injury or whether injury‐induced cell proliferation mediates recovery. We address these questions using the visual system and visually‐guided behavior in Xenopus laevis tadpoles. We established a reproducible means to produce a unilateral focal injury to optic tectal neurons without damaging retinotectal axons. We then assayed a tectally‐mediated visual avoidance behavior to evaluate behavioral impairment and recovery. Focal ablation of part of the optic tectum prevents the visual avoidance response to moving stimuli. Animals recover the behavior over the week following injury. Injury induces a burst of proliferation of tectal progenitor cells based on phospho‐histone H3 immunolabeling and experiments showing that Musashi‐immunoreactive tectal progenitors incorporate the thymidine analog chlorodeoxyuridine after injury. Pulse chase experiments indicate that the newly‐generated cells differentiate into N‐β‐tubulin‐immunoreactive neurons. Furthermore, in vivo time‐lapse imaging shows that Sox2‐expressing neural progenitors divide in response to injury and generate neurons with elaborate dendritic arbors. These experiments indicate that new neurons are generated in response to injury. To test if neurogenesis is necessary for recovery from injury, we blocked cell proliferation in vivo and found that recovery of the visual avoidance behavior is inhibited by drugs that block cell proliferation. Moreover, behavioral recovery is facilitated by changes in visual experience that increase tectal progenitor cell proliferation. Our data indicate that neurogenesis in the optic tectum is critical for recovery of visually‐guided behavior after injury. J. Comp. Neurol. 521:2262–2278, 2013. © 2012 Wiley Periodicals, Inc.