Vincent Tropepe

Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism.

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.

Separate Proliferation Kinetics of Fibroblast Growth Factor-Responsive and Epidermal Growth Factor-Responsive Neural Stem Cells within the Embryonic Forebrain Germinal Zone

The embryonic forebrain germinal zone contains two separate and additive populations of epidermal growth factor (EGF)- and fibroblast growth factor (FGF)-responsive stem cells that both exhibit self-renewal and multipotentiality. Although cumulative S phase labeling studies have investigated the proliferation kinetics of the overall population of precursor cells within the forebrain germinal zone through brain development, little is known about when and how (symmetrically or asymmetrically) the small subpopulations of stem cells are proliferating in vivo. This has been determined by injecting timed-pregnant mice with high doses of tritiated thymidine (3H-thy) to kill any stem cells proliferating within the striatal germinal zone in vivoand then by assaying for neurosphere formation in vitro. Injections of 0.8 mCi of 3H-thy given every 2 hr for 12 hr to timed-pregnant mice at E11, E14, and E17 resulted in significant depletions in the number of neurospheres generated by FGF-responsive stem cells at E11 and by EGF-responsive and FGF-responsive stem cells at E14 and E17. With increasing embryonic age, the depletions observed in the number of neurospheres generated in vitro in response to FGF2 after exposure to 3H-thy in vivo decreased, suggesting there is an increase in the length of the cell cycle of FGF-responsive neural stem cells through embryonic development. The results suggest that the FGF-responsive stem cell population expands between E11 and E14 by dividing symmetrically, but switches to primarily asymmetric division between E14 and E17. The EGF-responsive stem cells arise after E11, and their population expands through symmetric divisions and through asymmetric divisions of FGF-responsive stem cells.

Dopamine D2 Receptor Activity Modulates Akt Signaling and Alters GABAergic Neuron Development and Motor Behavior in Zebrafish Larvae

An imbalance in dopamine-mediated neurotransmission is a hallmark physiological feature of neuropsychiatric disorders, such as schizophrenia. Recent evidence demonstrates that dopamine D2 receptors, which are the main target of antipsychotics, modulate the activity of the protein kinase Akt, which is known to be downregulated in the brain of patients with schizophrenia. Akt has an important role in the regulation of cellular processes that are critical for neurodevelopment, including gene transcription, cell proliferation, and neuronal migration. Thus, it is possible that during brain development, altered Akt-dependent dopamine signaling itself may lead to defects in neural circuit formation. Here, we used a zebrafish model to assess the direct impact of altered dopamine signaling on brain development and larval motor behavior. We demonstrate that D2 receptor activation acutely suppresses Akt activity by decreasing the level of pAkt(Thr308) in the larval zebrafish brain. This D2-dependent reduction in Akt activity negatively regulates larval movement and is distinct from a D1-dependent pathway with opposing affects on motor behavior. In addition, we show that D2-dependent suppression of Akt activity causes a late onset change in GSK3b activity, a known downstream target of Akt signaling. Finally, altered D2 receptor signaling, or direct inhibition of Akt activity, causes a significant decrease in the size of the GABAergic neuron population throughout most of the brain. Our observations suggest that D2 receptor signaling suppresses Akt-GSK3b activity, which regulates GABAergic neuron development and motor behavior.

The cellular composition of neurogenic periventricular zones in the adult zebrafish forebrain

A central goal of adult neurogenesis research is to characterize the cellular constituents of a neurogenic niche and to understand how these cells regulate the production of new neurons. Because the generation of adult‐born neurons may be tightly coupled to their functional requirement, the organization and output of neurogenic niches may vary across different regions of the brain or between species. We have undertaken a comparative study of six (D, Vd, Vv, Dm, Dl, Ppa) periventricular zones (PVZs) harboring proliferative cells present in the adult forebrain of the zebrafish (Danio rerio), a species known to possess widespread neurogenesis throughout life. Using electron microscopy, we have documented for the first time the detailed cytoarchitecture of these zones, and propose a model of the cellular composition of pallial and subpallial PVZs, as well as a classification scheme for identifying morphologically distinct cell types. Immunolabeling of resin‐embedded tissue confirmed the phenotype of three constitutively proliferating (bromodeoxyuridine [BrdU]+) cell populations, including a radial glial‐like (type IIa) cell immunopositive for both S100β and glutamine synthetase (GS). Our data revealed rostrocaudal differences in the density of distinct proliferative populations, and cumulative labeling studies suggested that the cell cycle kinetics of these populations are not uniform between PVZs. Although the peak numbers of differentiated neurons were generated after ∼2 weeks among most PVZs, niche‐specific decline in the number of newborn neurons in some regions occurred after 4 weeks. Our data suggest that the cytoarchitecture of neurogenic niches and the tempo of neuronal production are regionally distinct in the adult zebrafish forebrain. J. Comp. Neurol. 520:2275–2316, 2012.

The role of dopaminergic signalling during larval zebrafish brain development: a tool for investigating the developmental basis of neuropsychiatric disorders.

Abstract Neurodevelopment depends on intrinsic and extrinsic factors that influence the overall pattern of neurogenesis and neural circuit formation, which has a direct impact on behaviour. Defects in dopamine signalling and brain morphology at a relatively early age, and mutations in neurodevelopmental genes are strongly correlated with several neuropsychiatric disorders. This evidence supports the hypothesis of a neurodevelopmental origin of at least some forms of mental illness. Zebrafish (Danio rerio) has emerged as an important vertebrate model system in biomedical research. The ease with which intrinsic and extrinsic factors can be altered during early development, the relatively conserved dopaminergic circuit organisation in the larval brain, and the emergence of simple sensorimotor behaviours very early in development are some of the appealing features that make this organism advantageous for developmental brain and behaviour research. Thus, examining the impact of altered dopamine signalling and disease related genetic aberrations during zebrafish development presents a unique opportunity to holistically analyse the in vivo biochemical, morphological and behavioural significance of altered dopamine signalling during a crucial period of development using a highly tractable vertebrate model organism. Ultimately, this information will shed new light on potential therapeutic targets for the treatment of schizophrenia and perhaps serve as a paradigm for investigating the neurodevelopmental origin of other psychiatric disorders.

The PI3K/Akt/mTOR pathway mediates retinal progenitor cell survival under hypoxic and superoxide stress

Oxygen (O₂) tension has emerged as a major regulator of stem cell (SC) biology. Low O₂ concentrations that are toxic to mature cells can confer advantage to stem and early progenitors, while superoxide stress remains a constant threat in aerobic biology and may be partially avoided through sequestration of SCs in the relatively hypoxic stem or regenerative niche. Using primary retina-derived retinal progenitor cells (RPCs) and the R28 progenitor cell line in vitro, we show that RPCs are sensitive to hydrogen peroxide (H₂O₂) induced damage and resistant to moderate levels of low oxygen stress (1% O₂). Under hypoxic conditions, multipotent RPCs upregulate Epo receptors, and Epo, along with insulin, protects against both superoxide- and severe hypoxia- (0.25% O₂) induced apoptosis through activation of the canonical PI3K/Akt/mTOR pathway. This survival advantage is sensitive to inhibitors of PI3K and mTOR. We further demonstrate phosphorylation of the p70S6 ribosomal kinase, a downstream mediator of PI3K/Akt/mTOR and translational activator. Overall, these data confirm that RPCs are sensitive to superoxide stress and resistant to hypoxia and that this resistance is mediated in part by Epo. They further suggest that manipulation of RPCs ex vivo prior to ocular delivery, or the in vivo delivery of exogenous survival factors at the time of cell implantation, could enhance the success of regenerative therapies aimed to restore sight.

Evolutionary transformation of rod photoreceptors in the all-cone retina of a diurnal garter snake

Significance This study provides compelling evidence that the previously reported all-cone retina of a diurnal garter snake in fact contains a population of rod photoreceptors with the appearance, and presumably function, of cones. Our results suggest that the evolution of all-cone retinas occurred not through loss of rods but rather via the evolutionary transmutation of ancestral rods into more “cone-like” photoreceptors, to regain functionality that was lost during the early, possibly fossorial, origin of snakes. This study provides a better understanding of the process by which complex molecular/cellular structures and tissue types can evolve, and how, particularly for sensory systems, physiological constraints can be shaped by selective forces to produce evolutionary novelty. Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the “transmutation” theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single “cones.” Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality.

Changes in the social environment induce neurogenic plasticity predominantly in niches residing in sensory structures of the zebrafish brain independently of cortisol levels

The social environment is known to modulate adult neurogenesis. Studies in mammals and birds have shown a strong correlation between social isolation and decreases in neurogenesis, whereas time spent in an enriched environment has been shown to restore these deficits and enhance neurogenesis. These data suggest that there exists a common adaptive response among neurogenic niches to each extreme of the social environment. We sought to further test this hypothesis in zebrafish, a social species with distinct neurogenic niches within primary sensory structures and telencephalic nuclei of the brain. By examining stages of adult neurogenesis, including the proliferating stem/progenitor population, their surviving cohort, and the resulting newly differentiated neuronal population, we show that niches residing in sensory structures are most sensitive to changes in the social context, and that social isolation or novelty are both capable of decreasing the number of proliferating cells while increasing the number of newborn neurons within a single niche. Contrary to observations in rodents, we demonstrate that social novelty, a form of enrichment, does not consistently rescue deficits in cell proliferation following social isolation, and that cortisol levels do not negatively regulate changes in adult neurogenesis, but are correlated with the social context. We propose that enhancement or suppression of adult neurogenesis in the zebrafish brain under different social contexts depends largely on the type of niche (sensory or telencephalic), experience from the preceding social environment, and occurs independently of changes in cortisol levels.

Sensory‐specific modulation of adult neurogenesis in sensory structures is associated with the type of stem cell present in the neurogenic niche of the zebrafish brain

Teleost fishes retain populations of adult stem/progenitor cells within multiple primary sensory processing structures of the mature brain. Though it has commonly been thought that their ability to give rise to adult‐born neurons is mainly associated with continuous growth throughout life, whether a relationship exists between the processing function of these structures and the addition of new neurons remains unexplored. We investigated the ultrastructural organisation and modality‐specific neurogenic plasticity of niches located in chemosensory (olfactory bulb, vagal lobe) and visual processing (periventricular grey zone, torus longitudinalis) structures of the adult zebrafish (Danio rerio) brain. Transmission electron microscopy showed that the cytoarchitecture of sensory niches includes many of the same cellular morphologies described in forebrain niches. We demonstrate that cells with a radial‐glial phenotype are present in chemosensory niches, while the niche of the caudal tectum contains putative neuroepithelial‐like cells instead. This was supported by immunohistochemical evidence showing an absence of glial markers, including glial fibrillary acidic protein, glutamine synthetase, and S100β in the tectum. By exposing animals to sensory assays we further illustrate that stem/progenitor cells and their neuronal progeny within sensory structures respond to modality‐specific stimulation at distinct stages in the process of adult neurogenesis – chemosensory niches at the level of neuronal survival and visual niches in the size of the stem/progenitor population. Our data suggest that the adult brain has the capacity for sensory‐specific modulation of adult neurogenesis and that this property may be associated with the type of stem cell present in the niche.

FGF dependent regulation of Zfhx1b gene expression promotes the formation of definitive neural stem cells in the mouse anterior neurectoderm

BackgroundMouse definitive neural stem cells (NSCs) are derived from a population of LIF-responsive primitive neural stem cells (pNSCs) within the neurectoderm, yet details on the early signaling and transcriptional mechanisms that control this lineage transition are lacking. Here we tested whether FGF and Wnt signaling pathways can regulate Zfhx1b expression to control early neural stem cell development.ResultsBy microinjecting FGF8b into the pro-amniotic cavity ex vivo at 7.0 days post-coitum (dpc) and culturing whole embryos, we demonstrate that neurectoderm-specific gene expression (for example, Sox2, Nestin, Zfhx1b) is increased, whereas Wnt3a represses neurectoderm gene expression. To determine whether FGF signaling also mediates the lineage transition from a pNSC to a NSC, 7.0-dpc embryos were microinjected with either FGF8b or inhibitors of the FGF receptor-MAP kinase signaling pathway ex vivo, cultured as whole embryos to approximately 8.5 dpc and assayed for clonal NSC colony formation. We show that pre-activation of FGF signaling in the anterior neurectoderm causes an increase in the number of colony forming NSCs derived later from the anterior neural plate, whereas inhibition of FGF signaling significantly reduces the number of NSC colonies. Interestingly, inhibition of FGF signaling causes the persistence of LIF-responsive pNSCs within the anterior neural plate and over-expression of Zfhx1b in these cells is sufficient to rescue the transition from a LIF-responsive pNSC to an FGF-responsive NSC.ConclusionOur data suggest that definitive NSC fate specification in the mouse neurectoderm is facilitated by FGF activation of Zfhx1b.