Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.     

Clonal analysis by distinct viral vectors identifies bona fide neural stem cells in the adult zebrafish telencephalon and characterizes their division properties and fate

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.     

Adult neurogenesis and brain regeneration in zebrafish

Adult neurogenesis is a widespread trait of vertebrates; however, the degree of this ability and the underlying activity of the adult neural stem cells differ vastly among species. In contrast to mammals that have limited neurogenesis in their adult brains,zebrafish can constitutively produce new neurons along the whole rostrocaudal brain axis throughout its life.This feature of adult zebrafish brain relies on the presence of stem/progenitor cells that continuously proliferate,and the permissive environment of zebrafish brain for neurogenesis. Zebrafish has also an extensive regenerative capacity, which manifests itself in responding to central nervous system injuries by producing new neurons to replenish the lost ones. This ability makes zebrafish a useful model organism for understanding the stem cell activity in the brain, and the molecular programs required for central nervous system regeneration.In this review, we will discuss the current knowledge on the stem cell niches, the characteristics of the stem/progenitor cells, how they are regulated and their involvement in the regeneration response of the adult zebrafish brain. We will also emphasize the open questions that may help guide the future research.     

Extracellular Signal-regulated Kinase 5 (ERK5) Mediates Prolactin-stimulated Adult Neurogenesis in the Subventricular Zone and Olfactory Bulb

Prolactin-stimulated adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) mediates several reproductive behaviors including mating/pregnancy, dominant male pheromone preference in females, and paternal recognition of offspring. However, downstream signaling mechanisms underlying prolactin-induced adult neurogenesis are completely unknown. We report here for the first time that prolactin activates extracellular signal-regulated kinase 5 (ERK5), a MAP kinase that is specifically expressed in the neurogenic regions of the adult mouse brain. Knockdown of ERK5 by retroviral infection of shRNA attenuates prolactin-stimulated neurogenesis in SVZ-derived adult neural stem/progenitor cells (aNPCs). Inducible erk5 deletion in adult neural stem cells of transgenic mice inhibits neurogenesis in the SVZ and OB following prolactin infusion or mating/pregnancy. These results identify ERK5 as a novel and critical signaling mechanism underlying prolactin-induced adult neurogenesis.     

Neural stem cells and neurogenesis in the adult zebrafish brain: origin, proliferation dynamics, migration and cell fate

Lifelong neurogenesis in vertebrates relies on stem cells producing proliferation zones that contain neuronal precursors with distinct fates. Proliferation zones in the adult zebrafish brain are located in distinct regions along its entire anterior-posterior axis. We show a previously unappreciated degree of conservation of brain proliferation patterns among teleosts, suggestive of a teleost ground plan. Pulse chase labeling of proliferating populations reveals a centrifugal movement of cells away from their places of birth into the surrounding mantle zone. We observe tangential migration of cells born in the ventral telencephalon, but only a minor rostral migratory stream to the olfactory bulb. In contrast, the lateral telencephalic area, a domain considered homologous to the mammalian dentate gyrus, shows production of interneurons and migration as in mammals. After a 46-day chase, newborn highly mobile cells have moved into nuclear areas surrounding the proliferation zones. They often show HuC/D immunoreactivity but importantly also more specific neuronal identities as indicated by immunoreactivity for tyrosine hydroxylase, serotonin and parvalbumin. Application of a second proliferation marker allows us to recognize label-retaining, actively cycling cells that remain in the proliferation zones. The latter population meets two key criteria of neural stem cells: label retention and self renewal.     

Musashi1‐CreERT2: A new cre line for conditional mutagenesis in neural stem cells

The RNA‐binding protein Musashi1 (Msi1) is one of two mammalian homologues of DrosophilaMusashi, which is required for the asymmetric cell division of sensory organ precursor cells. In the mouse central nervous system (CNS), Msi1 is preferentially expressed in mitotically active progenitor cells in the ventricular zone (VZ) of the neural tube during embryonic development and in the subventricular zone (SVZ) of the postnatal brain. Previous studies showed that cells in the SVZ can contribute to long‐term neurogenesis in the olfactory bulb (OB), but it remains unclear whether Msi1‐expressing cells have self‐renewing potential and can contribute to neurogenesis in the adult. Here, we describe the generation of Msi1‐CreER^T2 knock‐in mice and show by cell lineage tracing that Msi1‐CreER^T2‐expressing cells mark neural stem cells (NSCs) in both the embryonic and adult brain. Msi1‐CreER^T2 mice thus represent a new tool in our arsenal for genetically manipulating NSCs, which will be essential for understanding the molecular mechanisms underlying neural development. genesis, 51:128–134, 2013. © 2012 Wiley Periodicals, Inc.     

Neurogenesis in the adult hippocampus

New neurons continue to be generated in two privileged areas of the adult brain: the dentate gyrus of the hippocampal formation and the olfactory bulb. Adult neurogenesis has been found in all mammals studied to date, including humans. The process of adult neurogenesis encompasses the proliferation of resident neural stem and progenitor cells and their subsequent differentiation, migration, and functional integration into the pre-existing circuitry. This article summarizes recent findings regarding the developmental steps involved in adult hippocampal neurogenesis and the possible functional roles that new hippocampal neurons might play.     

Cells in the astroglial lineage are neural stem cells

A common assumption of classical neuroscience was that neurons and glial cells were derived from separate pools of progenitor cells and that, once development was completed, no new neurons were produced. The subsequent disproving of the “no new neuron” dogma suggested that ongoing adult neurogenesis was supported by a population of multipotent neural stem cells. Two germinal regions within the adult mammalian brain were shown to contain neural progenitor cells: the subventricular zone (SVZ) along the walls of the lateral ventricles, and the subgranular zone (SGZ) within the dentate gyrus of the hippocampus. Surprisingly, when the primary progenitors (stem cells) of the new neurons in these regions were identified, they exhibited structural and biological markers of astrocytes. The architecture of these germinal regions and the pattern of division of neural stem cells have raised fundamental questions about the mechanism of adult neurogenesis. This review describes studies on the origin of adult neural stem cells, the features distinguishing them from astrocytes in non-germinal regions, and the control mechanisms of the proliferation and differentiation of these cells. Astrocytic adult neural stem cells are part of a developmental lineage extending from the neuroepithelium to radial glia to germinal astrocytes. Adult neural stem cells appear to be strongly influenced by their local microenvironment, while also contributing significantly to the architecture of these germinal zones. However, environment alone does not seem to be sufficient to induce non-germinal astrocytes to behave as neural stem cells. Although emerging evidence suggests that significant heterogeneity exists within populations of germinal zone astrocytes, the way that these differences are encoded remains unclear. The further characterization of these cells should eventually provide a body of knowledge central to the understanding of brain development and disease. Work in the Alvarez-Buylla laboratory is supported by grants from the NIH and the Goldhirsh Foundation and by a gift from John and Frances Bowes. Rebecca Ihrie is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation. Arturo Alvarez-Buylla holds the Heather and Melanie Muss Endowed Chair in Neurosurgery.     

Expression of the murine transcription factor SOX3 during embryonic and adult neurogenesis

Previous studies have shown that Sox3 is expressed in nascent neuroprogenitor cells and is functionally required in mammals for development of the dorsal telencephalon and hypothalamus. However, Sox3 expression during embryonic and adult neurogenesis has not been examined in detail. Using a SOX3-specific antibody, we show that murine SOX3 expression is maintained throughout telencephalic neurogenesis and is restricted to progenitor cells with neuroepithelial and radial glial morphologies. We also demonstrate that SOX3 is expressed within the adult neurogenic regions and is coexpressed extensively with the neural stem cell marker SOX2 indicating that it is a lifelong marker of neuroprogenitor cells. In contrast to the telencephalon, Sox3 expression within the developing hypothalamus is upregulated in developing neurons and is maintained in a subset of differentiated hypothalamic cells through to adulthood. Together, these data show that Sox3 regulation is region-specific, consistent with it playing distinct biological roles in the dorsal telencephalon and hypothalamus.     

Radial glial cell‐specific ablation in the adult Zebrafish brain

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. genesis 53:431–439, 2015. © 2015 Wiley Periodicals, Inc.     

Locating and labeling neural stem cells in the brain

The phenomenon of adult neurogenesis has been demonstrated in most mammals including humans. At least two regions of the adult brain maintain stem cells throughout life; the subgranular zone (SGZ) of the hippocampal dentate gyrus, and the subventricular zone (SVZ) of the lateral ventricle wall. Both regions continuously produce neurons that mature and become integrated into functional networks that are involved in learning and memory and odor discrimination, respectively. Apart from these well‐studied regions neurogenesis has been reported in a number of other brain regions, such as amygdala and cortex. However, these studies have been contested and there is currently no well‐postulated function for non‐SVZ/SGZ neurogenesis. The studies of the regional localization of neurogenesis in the brain have been made possible due to several methods for detecting adult neurogenesis including; bromodeoxyuridine labeling (BrdU) together with markers of mature neurons, genetic labeling, by mouse transgenesis, or with the use of viral vectors. These techniques are already put to creative use and will be essential for the discovery of the nature of the adult neural stem cells. In this mini‐review, we will discuss the localization of neural stem/progenitor cells in the brain and their implications as well as discussing the pro's and con's of stem cell labeling techniques. J. Cell. Physiol. 226: 1–7, 2010. © 2010 Wiley‐Liss, Inc.     

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish

Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2⁺ neural progenitor, A2B5⁺ astrocyte/ glial progenitor, NG2⁺ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.     

Adult stem cells in the knifefish cerebellum

Adult neurogenesis has been described in dozens of brain regions in teleost fish, with the largest number of new neurons being generated in the cerebellum. Here, we characterized the cerebellar neural stem/progenitor cells (NSPCs) in the brown ghost knifefish (Apteronotus leptorhynchus), an established model system of adult neurogenesis. The majority of the new cerebellar cells arise from neurogenic niches located medially, at the interface of the dorsal/ventral molecular layers and the granular layer. NSPCs within these niches give rise to transit‐amplifying progenitors which populate the molecular layer, where they continue to proliferate during their migration toward target areas in the granular layer. At any given time, the majority of proliferating cells are located in the molecular layer. Immunohistochemical staining revealed that the stem cell markers Sox2, Meis1/2/3, Islet1, and, to a lesser extent, Pax6, are widely expressed in all regions of the adult cerebellum. A large subpopulation of these NSPCs coexpress S100, GFAP, and/or vimentin, indicating astrocytic identity. This is further supported by the specific effect of the gliotoxin l ‐methionine sulfoximine, which leads to a targeted decrease in the number of GFAP+ cells that coexpress Sox2 or the proliferation marker PCNA. Pulse‐chase analysis of the label size associated with new cells after administration of 5‐bromo‐2′‐deoxyuridine demonstrated that, on average, two additional cell divisions occur after completion of the initial mitotic cycle. Overall numbers of NSPCs in the cerebellum niches increase consistently over time, presumably in parallel with the continuous growth of the brain. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 39–65, 2015     

Cellular and molecular attributes of neural stem cell niches in adult zebrafish brain

Adult neurogenesis is a complex, presumably conserved phenomenon in vertebrates with a broad range of variations regarding neural progenitor/stem cell niches, cellular composition of these niches, migratory patterns of progenitors and so forth among different species. Current understanding of the reasons underlying the inter‐species differences in adult neurogenic potential, the identification and characterization of various neural progenitors, characterization of the permissive environment of neural stem cell niches and other important aspects of adult neurogenesis is insufficient. In the last decade, zebrafish has emerged as a very useful model for addressing these questions. In this review, we have discussed the present knowledge regarding the neural stem cell niches in adult zebrafish brain as well as their cellular and molecular attributes. We have also highlighted their similarities and differences with other vertebrate species. In the end, we shed light on some of the known intrinsic and extrinsic factors that are assumed to regulate the neurogenic process in adult zebrafish brain. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1188–1205, 2017     

Human primordial germ cell-derived progenitors give rise to neurons and glia in vivo

We derived a cell population from cultured human primordial germ cells from early human embryos. The derivates, termed embryoid body-derived (EBD) cells, displayed an extensive capacity for proliferation and expressed a panel of markers in all three germ layers. Interestingly, EBD cells were also positive for markers of neural stem/progenitor cells, such as nestin and glial fibrillary acidic protein. When these cells were transplanted into the brain cavities of fetal sheep and postnatal NOD-SCID mice or nerve-degenerated tibialis anterior muscles, they readily gave rise to neurons or glial cells. To our knowledge, our data are the first to demonstrate that EBD cells can undergo further neurogenesis under suitable environments in vivo. Hence, with the abilities of extensive expansion, self-renewal, and differentiation, EBD cells may provide a useful donor source for neural stem/progenitor cells to be used in cell-replacement therapies for diseases of the nervous system.