P53 and p73 in neurogenesis of the adult zebrafish Danio rerio

The zebrafish brain can continue to produce new neurons in widespread neurogenic brain regions throughout life. In contrast, neurogenesis in the adult mammalian brain is restricted to the subventricular zone (SVZ) and dentate gyrus (DG). In neurogenic regions in the adult brain, radial glial cells (RGCs) are considered to function as neural stem cells (NSCs). We generated a Tg(gfap:Gal4FF) transgenic zebrafish line, which enabled us to express specific genes in RGCs. To study the function of RGCs in neurogenesis in the adult zebrafish brain, we also generated a Tg(gfap: Gal4FF; UAS:nfsB-mcherry) transgenic zebrafish line, which allowed us to induce cell death exclusively within RGCs upon addition of metronidazole (Mtz) to the media. RGCs expressing nitroreductase were specifically ablated by the Mtz treatment, decreasing the number of proliferative RGCs. Using the Tg(gfap:Gal4FF; UAS:nfsB-mcherry) transgenic zebrafish line, we found that RGCs were specifically ablated in the adult zebrafish telencephalon. The Tg(gfap:Gal4FF) line could be useful to study the function of RGCs.

Adult mammalian brain, including humans, has rather limited addition of new neurons and poor regenerative capacity. In contrast, neural stem cells (NSC) with glial identity and neurogenesis are highly abundant throughout the adult zebrafish brain. Importantly, the activation of NSC and production of new neurons in response to injuries lead to the brain regeneration in zebrafish brain. Therefore, understanding of the molecular pathways regulating NSC behavior in response to injury is crucial in order to set the basis for experimental modification of these pathways in glial cells after injury in the mammalian brain and to elicit neuronal regeneration. Here, we describe the procedure that we successfully used to prospectively isolate NSCs from adult zebrafish telencephalon, extract RNA, and prepare cDNA libraries for next generation sequencing (NGS) and full transcriptome analysis as the first step toward understanding regulatory mechanisms leading to restorative neurogenesis in zebrafish. Moreover, we describe an alternative approach to analyze antigenic properties of NSC in the adult zebrafish brain using intracellular fluorescence activated cell sorting (FACS). We employ this method to analyze the number of proliferating NSCs positive for proliferating cell nuclear antigen (PCNA) in the prospectively isolated population of stem cells.

Neurogenesis is widespread in the zebrafish adult brain through the maintenance of active germinal niches. To characterize which progenitor properties correlate with this extensive neurogenic potential, we set up a method that allows progenitor cell transduction and tracing in the adult zebrafish brain using GFP-encoding retro- and lentiviruses. The telencephalic germinal zone of the zebrafish comprises quiescent radial glial progenitors and actively dividing neuroblasts. Making use of the power of clonal viral vector-based analysis, we demonstrate that these progenitors follow different division modes and fates: neuroblasts primarily undergo a limited amplification phase followed by symmetric neurogenic divisions; by contrast, radial glia are capable at the single cell level of both self-renewing and generating different cell types, and hence exhibit bona fide neural stem cell (NSC) properties in vivo. We also show that radial glial cells predominantly undergo symmetric gliogenic divisions, which amplify this NSC pool and may account for its long-lasting maintenance. We further demonstrate that blocking Notch signaling results in a significant increase in proliferating cells and in the numbers of clones, but does not affect clone composition, demonstrating that Notch primarily controls proliferation rather than cell fate. Finally, through long-term tracing, we illustrate the functional integration of newborn neurons in forebrain adult circuitries. These results characterize fundamental aspects of adult progenitor cells and neurogenesis, and open the way to using virus-based technologies for stable genetic manipulations and clonal analyses in the zebrafish adult brain.

TrkB receptor antagonism inhibits stab injury induced proliferative response in adult zebrafish (Danio rerio) brain

Adult Neural Stem Cells Bridge Their Niche

ABSTRACTZebrafish display widespread and pronounced adult neurogenesis, which is fundamental for their regeneration capability after central nervous system injury. However, the cellular identity and the biological properties of adult newborn neurons are elusive for most brain areas. Here, we have used short-term lineage tracing of radial glia progeny to prospectively isolate newborn neurons from the her4.1+ radial glia lineage in the homeostatic adult forebrain. Transcriptome analysis of radial glia, newborn neurons and mature neurons using single cell sequencing identified distinct transcriptional profiles, including novel markers for each population. Specifically, we detected two separate newborn neuron types, which showed diversity of cell fate commitment and location. Further analyses showed that these cell types are homologous to neurogenic cells in the mammalian brain, identified neurogenic commitment in proliferating radial glia and indicated that glutamatergic projection neurons are generated in the adult zebrafish telencephalon. Thus, we prospectively isolated adult newborn neurons from the adult zebrafish forebrain, identified markers for newborn and mature neurons in the adult brain, and revealed intrinsic heterogeneity among adult newborn neurons and their homology with mammalian adult neurogenic cell types.

Insulin is a peptide hormone that plays a central role in the regulation of circulating blood glucose in vertebrates, including zebrafish. Increasing evidence has demonstrated the important role of insulin in many brain functions. In zebrafish, two insulin receptor genes (insra and insrb) have been identified. However, their biodistribution in the adult brain as well as their cell-specific expression pattern has not been well described. Using gene expression analysis, in situ hybridization and transgenic fish, we confirmed the expression of insra, insrb, and irs1 (insulin receptor substrate 1, the downstream effector of insulin receptor) in the brain of adult zebrafish and characterized their specific expression in neurons and neural stem cells (radial glia). After demonstrating that intracerebroventricular (ICV) injection resulted in the diffusion of the injected solution within the ventricular system, we analyzed the effect of insulin ICV injection on neurogenesis. We showed that insulin promotes ventricular cell proliferation 24h postinjection. This neurogenic effect appeared to be independent of neuroinflammatory processes. Also, after a mechanical telencephalic stab-wound injury, we highlighted the overexpression of irs1 gene 5 days postlesion notably in the ventricular zone where radial glial cells (RGCs) are localized, suggesting key roles of insulin signaling in regenerative processes. Finally, our results reinforced the expression of insulin-related proteins in the brain of adult zebrafish, highlighting the potential role of insulin signaling on neurogenesis.

The beta(2)-adrenergic receptors (ARs) are G-protein-coupled receptors that mediate the physiological responses to adrenaline and noradrenaline. The present study aimed to determine the regional distribution of beta(2)-ARs in the adult zebrafish (Danio rerio) brain by means of in vitro autoradiographic and immunohistochemical methods. The immunohistochemical localization of beta(2)-ARs, in agreement with the quantitative beta-adrenoceptor autoradiography, showed a wide distribution of beta(2)-ARs in the adult zebrafish brain. The cerebellum and the dorsal zone of periventricular hypothalamus exhibited the highest density of [(3)H]CGP-12177 binding sites and beta(2)-AR immunoreactivity. Neuronal cells strongly stained for beta(2)-ARs were found in the periventricular ventral telencephalic area, magnocellular and parvocellular superficial pretectal nuclei (PSm, PSp), occulomotor nucleus (NIII), locus coeruleus (LC), medial octavolateral nucleus (MON), magnocellular octaval nucleus (MaON) reticular formation (SRF, IMRF, IRF), and ganglionic cell layer of cerebellum. Interestingly, in most cases (NIII, LC, MON, MaON, SRF, IMRF, ganglionic cerebellar layer) beta(2)-ARs were colocalized with alpha(2A)-ARs in the same neuron, suggesting their interaction for mediating the physiological functions of nor/adrenaline. Moderate to low labeling of beta(2)-ARs was found in neurons in dorsal telencephalic area, optic tectum (TeO), torus semicircularis (TS), and periventricular gray zone of optic tectum (PGZ). In addition to neuronal, glial expression of beta(2)-ARs was found in astrocytic fibers located in the central gray and dorsal rhombencephalic midline, in close relation to the ventricle. The autoradiographic and immunohistochemical distribution pattern of beta(2)-ARs in the adult zebrafish brain further support the conserved profile of adrenergic/noradrenergic system through vertebrate brain evolution.

fezf2 expression delineates cells with proliferative potential and expressing markers of neural stem cells in the adult zebrafish brain

BackgroundUnlike mammals, zebrafish exhibits extensive neural regeneration after injury in adult stages of its lifetime due to the neurogenic activity of the radial glial cells. However, the genes involved in the regenerative neurogenesis response of the zebrafish brain are largely unknown. Thus, understanding the underlying principles of this regeneration capacity of the zebrafish brain is an interesting research realm that may offer vast clinical ramifications.ResultsIn this paper, we characterized the expression pattern of cxcr5 and analyzed the function of this gene during adult neurogenesis and regeneration of the zebrafish telencephalon. We found that cxcr5 was upregulated transiently in the RGCs and neurons, and the expression in the immune cells such as leukocytes was negligible during both adult neurogenesis and regeneration. We observed that the transgenic misexpression of cxcr5 in the ventricular cells using dominant negative and full-length variants of the gene resulted in altered proliferation and neurogenesis response of the RGCs. When we knocked down cxcr5 using antisense morpholinos and cerebroventricular microinjection, we observed outcomes similar to the overexpression of the dominant negative cxcr5 variant.ConclusionsThus, based on our results, we propose that cxcr5 imposes a proliferative permissiveness to the radial glial cells and is required for differentiation of the RGCs to neurons, highlighting novel roles of cxcr5 in the nervous system of vertebrates. We therefore suggest that cxcr5 is an important cue for ventricular cell proliferation and regenerative neurogenesis in the adult zebrafish telencephalon. Further studies on the role of cxcr5 in mediating neuronal replenishment have the potential to produce clinical ramifications in efforts for regenerative therapeutic applications for human neurological disorders or acute injuries.

Mapping of brain lipid binding protein (Blbp) in the brain of adult zebrafish, co-expression with aromatase B and links with proliferation

In mammals, glial cell line-derived neurotrophic factor (GDNF) is a growth factor of many neuronal populations in the central, peripheral and autonomous nervous system. GDNF may also function as a morphogen during kidney development and may regulate spermatogonial differentiation. GDNF has been characterised in zebrafish embryos and was demonstrated experimentally to be critical for the development of the enteric nervous system. However, in adult zebrafish, no data exist regarding GDNF expression and localisation in the brain and in different organs. Thus, the aim of the present study was to investigate the expression of GDNF in the brain of adult zebrafish (Danio rerio). Transcripts of GDNF mRNA were observed in brain extracts by a standard RT-PCR. The presence of the protein in the brain homogenates was confirmed by SDS-PAGE electrophoresis and Western blotting analysis. Immunohistochemistry and in situ hybridization experiments showed that GDNF protein and mRNA were localised in various nuclei of the telencephalon, diencephalon, mesencephalon, cerebellum and medulla oblongata of the zebrafish brain. In conclusion, this study showed that the expression of GDNF was not restricted to developmental periods but it seems that this factor might be involved in adult zebrafish brain physiology, as observed in mammals.

Nerve growth factor (NGF), a member of the neurotrophin family, was initially described as neuronal survival and growth factor, but successively has emerged as an active mediator in many essential functions in the central nervous system of mammals. NGF is synthesized as a precursor pro-NGF and is cleaved intracellularly into mature NGF. However, recent evidence demonstrates that pro-NGF is not a simple inactive precursor, but is also secreted outside the cells and can exert multiple roles. Despite the vast literature present in mammals, studies devoted to NGF in the brain of other vertebrate models are scarce. Zebrafish is a teleost fish widely known for developmental genetic studies and is well established as model for translational neuroscience research. Genomic organization of zebrafish and mouse NGF is highly similar, and zebrafish NGF protein has been reported in mature and two-precursors forms. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the NGF mRNA and protein distribution in the adult zebrafish brain and to characterize the phenotype of NGF-positive cells. NGF mRNA was visualized by insitu hybridization on whole-mount brains. NGF protein distribution was assessed on microtomic sections by using an antiserum against NGF, able to recognize pro-NGF in adult zebrafish brain as demonstrated also in previous studies. To characterize NGF-positive cells, anti-NGF was employed on microtomic slides of aromatase B transgenic zebrafish (where radial glial cells appeared fluorescent) and by means of double-immunolabeling against NGF/proliferative cell nuclear antigen (PCNA; proliferation marker) and NGF/microtube-associated protein2 (MAP2; neuronal marker). NGF mRNA and protein were widely distributed in the brain of adult zebrafish, and their pattern of distribution of positive perikaryal was overlapping, both in males and females, with few slight differences. Specifically, the immunoreactivity to the protein was observed in fibers over the entire encephalon. MAP2 immunoreactivity was present in the majority of NGF-positive cells, throughout the zebrafish brain. PCNA and aromatase B cells were not positive to NGF, but they were closely intermingled with NGF cells. In conclusion, our study demonstrated that mature neurons in the zebrafish brain express NGF mRNA and store pro-NGF.

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish. Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.

Multiphoton microscopy techniques, such as two-photon microscopy (2PM) and three-photon microscopy (3PM), are powerful tools for deep-tissue in vivo imaging with subcellular resolution. 3PM has two major advantages for deep-tissue imaging over 2PM that has been widely used in biology laboratories: (i) longer attenuation length in scattering tissues by employing ~1,300 nm or ~1,700 nm excitation laser; (ii) less background fluorescence generation due to higher-order nonlinear excitation. As a result, 3PM allows high-contrast structural and functional imaging deep within scattering tissues such as intact mouse brain from the cortical layers to the hippocampus and the entire forebrain of adult zebrafish. Today, laser sources suitable for 3PM are commercially available, enabling the conversion of an existing two-photon (2P) imaging system to a three-photon (3P) system. Additionally, multiple commercial 3P microscopes are available, which makes this technique readily available to biology research laboratories. This paper shows the optimization of a typical 3PM setup, particularly targeting biology groups that already have a 2P setup, and demonstrates intravital 3D imaging in intact mouse and adult zebrafish brains. This protocol covers the full experimental procedure of 3P imaging, including microscope alignment, prechirping of ~1,300 and ~1,700 nm laser pulses, animal preparation, and intravital 3P fluorescence imaging deep in adult zebrafish and mouse brains.