Neurogenesis In Zebrafish Research Articles

Oscillatory DeltaC Expression in Neural Progenitors Primes the Prototype of Forebrain Development.

Notch signaling plays a pivotal role in regulating various developmental processes, particularly in controlling the timing of neuronal production within the developing neocortex. Central to this regulatory mechanism is the oscillatory pattern of Delta, which functions as a developmental clock modulator. Its deficiency profoundly impairs mammalian brain formation, highlighting its fundamental role in brain development. However, zebrafish carrying a mutation in the functional ortholog DeltaC (dlc) within their functional ortholog exhibit an intact forebrain structure, implying evolutionary variations in Notch signaling within the forebrain. In this study, we unveil the distinct yet analogous expression profiles of Delta and Her genes in the developing vertebrate forebrain. Specifically, for the first time, we detected the oscillatory expression of the Delta gene dlc in the developing zebrafish forebrain. Although this oscillatory pattern appeared irregular and was not pervasive among the progenitor population, attenuation of the dlc-involved Notch pathway using a γ-secretase inhibitor impaired neuronal differentiation in the developing zebrafish forebrain, revealing the indispensable role of the dlc-involved Notch pathway in regulating early zebrafish neurogenesis. Taken together, our results demonstrate the foundational prototype of dlc-involved Notch signaling in the developing zebrafish forebrains, upon which the intricate patterns of the mammalian neocortex may have been sculpted.

Telencephalic stab wound injury induces regenerative angiogenesis and neurogenesis in zebrafish: unveiling the role of vascular endothelial growth factor signaling and microglia.

After brain damage, regenerative angiogenesis and neurogenesis have been shown to occur simultaneously in mammals, suggesting a close link between these processes. However, the mechanisms by which these processes interact are not well understood. In this work, we aimed to study the correlation between angiogenesis and neurogenesis after a telencephalic stab wound injury. To this end, we used zebrafish as a relevant model of neuroplasticity and brain repair mechanisms. First, using the Tg(fli1:EGFP × mpeg1.1:mCherry) zebrafish line, which enables visualization of blood vessels and microglia respectively, we analyzed regenerative angiogenesis from 1 to 21 days post-lesion. In parallel, we monitored brain cell proliferation in neurogenic niches localized in the ventricular zone by using immunohistochemistry. We found that after brain damage, the blood vessel area and width as well as expression of the fli1 transgene and vascular endothelial growth factor (vegfaa and vegfbb) were increased. At the same time, neural stem cell proliferation was also increased, peaking between 3 and 5 days post-lesion in a manner similar to angiogenesis, along with the recruitment of microglia. Then, through pharmacological manipulation by injecting an anti-angiogenic drug (Tivozanib) or Vegf at the lesion site, we demonstrated that blocking or activating Vegf signaling modulated both angiogenic and neurogenic processes, as well as microglial recruitment. Finally, we showed that inhibition of microglia by clodronate-containing liposome injection or dexamethasone treatment impairs regenerative neurogenesis, as previously described, as well as injury-induced angiogenesis. In conclusion, we have described regenerative angiogenesis in zebrafish for the first time and have highlighted the role of inflammation in this process. In addition, we have shown that both angiogenesis and neurogenesis are involved in brain repair and that microglia and inflammation-dependent mechanisms activated by Vegf signaling are important contributors to these processes. This study paves the way for a better understanding of the effect of Vegf on microglia and for studies aimed at promoting angiogenesis to improve brain plasticity after brain injury.

Elevated glucocorticoid alters the developmental dynamics of hypothalamic neurogenesis in zebrafish

Exposure to excess glucocorticoid (GC) during early development is implicated in adult dysfunctions. Reduced adult hippocampal neurogenesis is a well-known consequence of exposure to early life stress or elevated GC, however the effects on neurogenesis during development and effects on other brain regions are not well understood. Using an optogenetic zebrafish model, here we analyse the effects of GC exposure on neurogenesis during development in the whole brain. We identify that the hypothalamus is a highly GC-sensitive region where elevated GC causes precocious development. This is followed by failed maturation and early decline accompanied by impaired feeding, growth, and survival. In GC-exposed animals, the developmental trajectory of hypothalamic progenitor cells is strikingly altered, potentially mediated by direct regulation of transcription factors such as rx3 by GC. Our data provide cellular and molecular level insight into GC-induced alteration of the hypothalamic developmental trajectory, a process crucial for health across the life-course.

The Roles of Histamine Receptor 1 (hrh1) in Neurotransmitter System Regulation, Behavior, and Neurogenesis in Zebrafish

Histamine receptors mediate important physiological processes and take part in the pathophysiology of different brain disorders. Histamine receptor 1 (HRH1) is involved in the development of neurotransmitter systems, and its role in neurogenesis has been proposed. Altered HRH1 binding and expression have been detected in the brains of patients with schizophrenia, depression, and autism. Our goal was to assess the role of hrh1 in zebrafish development and neurotransmitter system regulation through the characterization of hrh1−/− fish generated by the CRISPR/Cas9 system. Quantitative PCR, in situ hybridization, and immunocytochemistry were used to study neurotransmitter systems and genes essential for brain development. Additionally, we wanted to reveal the role of this histamine receptor in larval and adult fish behavior using several quantitative behavioral methods including locomotion, thigmotaxis, dark flash and startle response, novel tank diving, and shoaling behavior. Hrh1−/− larvae displayed normal behavior in comparison with hrh1+/+ siblings. Interestingly, a transient abnormal expression of important neurodevelopmental markers was evident in these larvae, as well as a reduction in the number of tyrosine hydroxylase 1 (Th1)–positive cells, th1 mRNA, and hypocretin (hcrt)-positive cells. These abnormalities were not detected in adulthood. In summary, we verified that zebrafish lacking hrh1 present deficits in the dopaminergic and hypocretin systems during early development, but those are compensated by the time fish reach adulthood. However, impaired sociability and anxious-like behavior, along with downregulation of choline O-acetyltransferase a and LIM homeodomain transcription factor Islet1, were displayed by adult fish.

Hydrogen peroxide signaling modulates neuronal differentiation via microglial polarization and Wnt/β-catenin pathway.

Reactive oxygen species (ROS) are generated within the cell and serve as second messengers in fundamental cellular processes under physiologic conditions. Although the deleterious effects of high-level ROS associated with oxidative stress are well established, it is unclear how the developing brain reacts to redox changes. Our aim is to investigate how redox alteration affects neurogenesis and the mechanism that underlies it.We investigated in vivo microglial polarization and neurogenesis in zebrafish after hydrogen peroxide (H2O2) incubation. To quantify intracellular H2O2 levels in vivo, a transgenic zebrafish line that expresses Hyper and termed Tg(actb2:hyper3)ka8 was used. Then, in vitro studies with N9 microglial cells, 3-dimensional neural stem cell (NSC)-microglia coculture, and conditioned medium experiments are carried out to comprehend the mechanism underlying the changes in neurogenesis upon redox modulation.In zebrafish, exposure to H2O2 altered embryonic neurogenesis, induced M1 polarization in microglia, and triggered the Wnt/β-catenin pathway. N9 microglial cell culture experiments revealed that exposure to H2O2 resulted in M1 polarization in microglial cells, and this polarization was mediated by the Wnt/β-catenin pathway. Redox modulation of microglia interfered with NSC differentiation in coculture experiments. Neuronal differentiation was significantly higher in NSCs cocultured with H2O2-treated microglia when compared to control microglia. Wnt inhibition prevented the effects of H2O2-treated microglia on NSCs. No significant alterations were observed in conditioned medium experiments.Our findings point to a robust interplay between microglia and neural progenitors influenced by the redox state. Intracellular H2O2 levels can interfere with neurogenesis by altering the phenotypic state of the microglia via the Wnt/β-catenin system.

Acrolein-inducing ferroptosis contributes to impaired peripheral neurogenesis in zebrafish.

Diabetes mellitus (DM) is associated with physiological disorders such as delayed wound healing, diabetic retinopathy, diabetic nephropathy, and diabetic peripheral neuropathy (DPN). Over 50% of diabetic patients will develop DPN, characterized by motor dysfunction and impaired sensory nerve function. In a previous study, we have uncovered acrolein (ACR) as an upstream initiator which induced impaired glucose homeostasis and microvascular alterations in zebrafish. Whether ACR has specific effects on peripheral neurogenesis and mediates DPN, is still waiting for clarification. To evaluate the function of ACR in peripheral nerve development, in vivo experiments were performed in Tg(hb9:GFP) zebrafish. In addition, a series of rescue experiments, metabolomics assessment, and bioinformatics analysis was performed aimed at identifying the molecular mechanisms behind ACR's function and impaired neurogenesis. Impaired motor neuron development was confirmed in wild-type embryos treated with external ACR. ACR treated embryos displayed ferroptosis and reduction of several amino acids and increased glutathione (GSH). Furthermore, ferroptosis inducer caused similarly suppressed neurogenesis in zebrafish embryos, while anti-ACR treatment or ferroptosis inhibitor could successfully reverse the detrimental phenotypes of ACR on neurogenesis in zebrafish. Our data indicate that ACR could directly activate ferroptosis and impairs peripheral neurogenesis. The data strongly suggest ACR and activated ferroptosis as inducers and promising therapeutic targets for future DPN studies.

Social Enhancement of Adult Neurogenesis in Zebrafish is Not Regulated by Cortisol

In Mammals adult neurogenesis is influenced by environmental conditions, and the glucocorticoid hormones (GC) play a major role in this regulation. In contrast in fish, the study of the effects of cortisol on the regulation of environmental driven adult neurogenesis has produced conflicting results. While in some species elevated cortisol levels impair cell proliferation, in others, it promotes cell proliferation and differentiation. This lack of consistency may be explained by methodological differences across studies, namely in the stimuli and/or cortisol treatments used. Here, we tested the effects of the social environment on adult neurogenesis, considering a positive and a negative social context, and different durations of cortisol exposure. We hypothesise that there is an interaction between the valence of the social environment and cortisol, such that elevated acute cortisol experienced during social interactions only have a detrimental effect on neurogenesis in negative social contexts. Therefore, fish were exposed to a positive (conspecific shoal) or negative (predator) social experience, and the interaction between the valence of the social context and cortisol exposure (acute and chronic) was tested. Our results indicate that adult neurogenesis is modulated by the social environment, with the number of newly generated cells being dependent on the valence of the social information (positive > negative). These effects were independent of cortisol, either for acute or chronic exposure, highlighting the social environment as a key factor in the modulation of cell proliferation in the adult zebrafish brain, and rejecting a role for cortisol in this modulation.

Distribution of microglia/immune cells in the brain of adult zebrafish in homeostatic and regenerative conditions: Focus on oxidative stress during brain repair.

Microglia are macrophage-like cells exerting determinant roles in neuroinflammatory and oxidative stress processes during brain regeneration. We used zebrafish as a model of brain plasticity and repair. First, by performing L-plastin (Lcp1) immunohistochemistry and using transgenic Tg(mpeg1.1:GFP) or Tg(mpeg1.1:mCherry) fish, we analyzed the distribution of microglia/immune cells in the whole brain. Specific regional differences were evidenced in terms of microglia/immune cell density and morphology (elongated, branched, highly branched, and amoeboid). Taking advantage of Tg(fli:GFP) and Tg(GFAP::GFP) enabling the detection of endothelial cells and neural stem cells (NSCs), we highlighted the association of elongated microglia/immune cells with blood vessels and rounded/amoeboid microglia with NSCs. Second, after telencephalic injury, we showed that L-plastin cells were still abundantly present at 5 days post-lesion (dpl) and were associated with regenerative neurogenesis. Finally, RNA-sequencing analysis from injured telencephalon (5 dpl) confirmed the upregulation of microglia/immune cell markers and highlighted a significant increase of genes involved in oxidative stress (nox2, nrf2a, and gsr). The analysis of antioxidant activities at 5 dpl also revealed an upregulation of superoxide dismutase and persistent H2 O2 generation in the injured telencephalon. Also, microglia/immune cells were shown to be a source of oxidative stress at 5 dpl. Overall, our data provide a better characterization of microglia/immune cell distribution in the healthy zebrafish brain, highlighting some evolutionarily conserved features with mammals. They also emphasize that 5 days after injury, microglia/immune cells are still activated and are associated to a persistent redox imbalance. Together, these data raise the question of the role of oxidative stress in regenerative neurogenesis in zebrafish.

Transcriptomic networks of gba3 governing specification of the dopaminergic neurons in zebrafish embryos.

Molecular networks associated with dopaminergic (DA) neurogenesis remain undefined within mammalian models. To address this issue, the transient zebrafish model lmx1al: EGFP was generated, which expresses GFP in the DA precursor cells as well as in the DA neurons of the ventral diencephalon (VD). We found that the novel pseudogene gba3 has not been well studied in zebrafish neurogenesis. Crucial networks associated with gba3 transcripts were investigated because the biological functions of these networks have not been reported in DA neurogenesis in zebrafish. RNA isolation and sequencing were employed with GFP-expressing cells from 20-, 22-, and 24h post-fertilization (hpf), while subsequent transcriptomic analysis generated differentially expressed genes with DA neurogenesis (DEG-DA) list. Hierarchical cluster analysis provided absolute guidance for the collection of gba3, an essential transcript that is strictly spatiotemporally expressed during DA neurogenesis, which was proven with whole-mount in situ hybridization (WISH) and knockdown and rescue of the gba3 transcripts in zebrafish embryos. The gba3 transcripts were restricted to the midbrain at 24 hpf and the midbrain and pectoral fins at 30 hpf in zebrafish embryos. Functional studies with knockdown of gba3 found a diminished state in the midbrain and midbrain-hindbrain boundary (MHB) and an underdeveloped condition in the anteroposterior and dorsolateral axis relative to the wild type (WT) at 24 hpf. However, it was recovered after forced expression of gba3 transcripts at 24 hpf. Molecular markers for the DA precursors and mature neurons lmx1al, nurr1,th, and pitx3 were analyzed in the gba3 MOs. The levels of transcripts lmx1al, nurr1, and th were significantly reduced in the midbrain ventral diencephalon (VD) and hindbrain of gba3 morphants compared to the WT at 24 hpf, while expression patterns of pitx3 transcripts showed no differences in the identical regions between gba3 MOs and the controls. We discuss transcriptional networks in which transcripts of gba3 plausibly govern the specification of dopaminergic neurogenesis in zebrafish embryos.

Sequential and additive expression of miR-9 precursors control timing of neurogenesis.

MicroRNAs (miRs) have an important role in tuning dynamic gene expression. However, the mechanism by which they are quantitatively controlled is unknown. We show that the amount of mature miR-9, a key regulator of neuronal development, increases during zebrafish neurogenesis in a sharp stepwise manner. We characterize the spatiotemporal profile of seven distinct microRNA primary transcripts (pri-mir)-9s that produce the same mature miR-9 and show that they are sequentially expressed during hindbrain neurogenesis. Expression of late-onset pri-mir-9-1 is added on to, rather than replacing, the expression of early onset pri-mir-9-4 and -9-5 in single cells. CRISPR/Cas9 mutation of the late-onset pri-mir-9-1 prevents the developmental increase of mature miR-9, reduces late neuronal differentiation and fails to downregulate Her6 at late stages. Mathematical modelling shows that an adaptive network containing Her6 is insensitive to linear increases in miR-9 but responds to stepwise increases of miR-9. We suggest that a sharp stepwise increase of mature miR-9 is created by sequential and additive temporal activation of distinct loci. This may be a strategy to overcome adaptation and facilitate a transition of Her6 to a new dynamic regime or steady state.

Rbbp4 loss disrupts neural progenitor cell cycle regulation independent of Rb and leads to Tp53 acetylation and apoptosis.

BackgroundRetinoblastoma binding protein 4 (Rbbp4) is a component of transcription regulatory complexes that control cell cycle gene expression. Previous work indicated that Rbbp4 cooperates with the Rb tumor suppressor to block cell cycle entry. Here, we use genetic analysis to examine the interactions of Rbbp4, Rb, and Tp53 in zebrafish neural progenitor cell cycle regulation and survival.ResultsRbbp4 is upregulated across the spectrum of human embryonal and glial brain cancers. Transgenic rescue of rbbp4 mutant embryos shows Rbbp4 is essential for zebrafish neurogenesis. Rbbp4 loss leads to apoptosis and γ‐H2AX in the developing brain that is suppressed by tp53 knockdown or maternal zygotic deletion. Mutant retinal neural precursors accumulate in M phase and fail to initiate G0 gene expression. rbbp4; rb1 mutants show an additive effect on the number of M phase cells. In rbbp4 mutants, Tp53 acetylation is detected; however, Rbbp4 overexpression did not rescue DNA damage‐induced apoptosis.ConclusionRbbp4 is necessary for neural progenitor cell cycle progression and initiation of G0 independent of Rb. Tp53‐dependent apoptosis in the absence of Rbpb4 correlates with Tp53 acetylation. Together these results suggest that Rbbp4 is required for cell cycle exit and contributes to neural progenitor survival through the regulation of Tp53 acetylation.

Zebrafish Embryo as an Emerging Model Organism in Neurodevelopmental Toxicity Research

Zebrafish is a model organism that has become increasingly popular in recent years due to some of the advantages it has when compared to traditional model organisms. Its genetic similarity with humans has contributed significantly to the elucidation of the molecular mechanisms underlying diseases. Moreover, external fertilization and rapid embryonic development of zebrafish embryos have made it attractive in many research areas. The genome of humans and zebrafish are found to be highly conserved having 76-82 % of the disease genes in humans that are also present in zebrafish. Zebrafish have been used in different studies in several concepts of neurogenesis. Unlike mammals, the external development of a zebrafish embryo makes it accessible for experimental manipulation in central nervous system research. It was observed that neurotoxic agents induced similar responses to other vertebral models in zebrafish embryos, whose brain development and blood-brain barrier were similar to those of other vertebrates. This review provides brief information about the availability of zebrafish embryos in neurodevelopmental toxicity research while giving brief information on embryogenesis and neurogenesis in zebrafish. Evaluation of neurotoxicity and the specific effects of various neurotoxins on motor and dopaminergic neurons, neuronal proliferation, mobility, and neurodevelopment are also explained.

The miR-26 family regulates neural differentiation-associated microRNAs and mRNAs by directly targeting REST.

Repressor element 1-silencing transcription factor (REST) plays a crucial role in the differentiation of neural progenitor cells (NPCs). C-terminal domain small phosphatases (CTDSPs) are REST effector proteins that reduce RNA polymerase II activity on genes required for neurogenesis. miR-26b regulates neurogenesis in zebrafish by targeting ctdsp2 mRNA, but the molecular events triggered by this microRNA (miR) remain unknown. Here, we show in a murine embryonic stem cell differentiation paradigm that inactivation of miR-26 family members disrupts the formation of neurons and astroglia and arrests neurogenesis at the neural progenitor level. Furthermore, we show that miR-26 directly targets Rest, thereby inducing the expression of a large set of REST complex-repressed neuronal genes, including miRs required for induction of the neuronal gene expression program. Our data identify the miR-26 family as the trigger of a self-amplifying system required for neural differentiation that acts upstream of REST-controlled miRs.

A unique macrophage subpopulation signals directly to progenitor cells to promote regenerative neurogenesis in the zebrafish spinal cord.

Central nervous system injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. Activation of the innate immune system promotes regenerative neurogenesis, but it is fundamentally unknown whether this is indirect through the activation of known developmental signaling pathways or whether immune cells directly signal to progenitor cells using mechanisms that are unique to regeneration. Using single-cell RNA-seq of progenitor cells and macrophages, as well as cell-type-specific manipulations, we provide evidence for a direct signaling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, TNFa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This establishes the principle that macrophages directly communicate to spinal progenitor cells via non-developmental signals after injury, providing potential targets for future interventions in the regeneration-deficient spinal cord of mammals.

Cfdp1 controls the cell cycle and neural differentiation in the zebrafish cerebellum and retina.

Although the cell cycle and cell differentiation should be coordinately regulated to generate a variety of neurons in the brain, the molecules that are involved in this coordination still remain largely unknown. In this study, we analyzed the roles of a nuclear protein Cfdp1, which is thought to be involved in chromatin remodeling, in zebrafish neurogenesis. Zebrafish cfdp1 mutants maintained the progenitors of granule cells (GCs) in the cerebellum, but showed defects in their differentiation to GCs. cfdp1 mutants showed an increase in phospho-histone 3 (pH3)-positive cells and apoptotic cells, as well as a delayed cell cycle transition from the G2 to the M phase in the cerebellum. The inhibition of tp53 prevented apoptosis but not GC differentiation in the cfdp1 mutant cerebellum. A similar increase in apoptotic cells and pH3-positive cells, and defective cell differentiation, were observed in the cfdp1 mutant retina. Although mitotic spindles formed, mitosis was blocked before anaphase in both the cerebellum and retina of cfdp1 mutant larvae. Furthermore, expression of the G2/mitotic-specific cyclin B1 gene increased in the cfdp1 mutant cerebellum. Our findings suggest that Cfdp1 regulates the cell cycle of neural progenitors, thereby promoting neural differentiation in the brain.

A Unique Macrophage Subpopulation Signals Directly to Progenitor Cells to Promote Regenerative Neurogenesis in the Zebrafish Spinal Cord

During ontogeny, neural stem cells in the spinal cord cease production of neurons. Spinal cord injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. It is unclear whether regenerative neurogenesis from spinal progenitor cells simply depends on recapitulation of developmental signals and intracellular signal transduction or is driven by signals and mechanisms that are unique to regeneration. Using single cell RNAseq of progenitor cells and macrophages, as well as cell type-specific manipulations, we provide evidence for a direct signalling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, Tnfa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This demonstrates regeneration-specific signalling mechanisms that provide targets for future interventions in the non-regenerating spinal cord of mammals.

Sensory Neuroblast Quiescence Depends on Vascular Cytoneme Contacts and Sensory Neuronal Differentiation Requires Initiation of Blood Flow

In many organs, stem cell function depends on communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels; however, whether vasculature is an integral component of their niches is yet unknown. Here, two separate roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves precise spatiotemporal endothelial-neuroblast cytoneme contacts and Dll4-Notch signaling to restrain neuroblast proliferation. The second, instead, requires blood flow to trigger a transcriptional response that modifies neuroblast metabolic status and induces sensory neuron differentiation. In contrast, no role of sensory neurogenesis in vascular development is found, suggesting unidirectional signaling from vasculature to sensory neuroblasts. Altogether, we demonstrate that the cranial vasculature constitutes a niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.

Conserved and Divergent Features of Adult Neurogenesis in Zebrafish.

Adult neurogenesis, i.e., the generation of neurons from neural stem cells (NSCs) in the adult brain, contributes to brain plasticity in all vertebrates. It varies, however, greatly in extent, location and physiological characteristics between species. During the last decade, the teleost zebrafish (D. rerio) was increasingly used to study the molecular and cellular properties of adult NSCs, in particular as a prominent NSC population was discovered at the ventricular surface of the dorsal telencephalon (pallium), in territories homologous to the adult neurogenic niches of rodents. This model, for its specific features (large NSC population, amenability to intravital imaging, high regenerative capacity) allowed rapid progress in the characterization of basic adult NSC features. We review here these findings, with specific comparisons with the situation in rodents. We specifically discuss the cellular nature of NSCs (astroglial or neuroepithelial cells), their heterogeneities and their neurogenic lineages, and the mechanisms controlling NSC quiescence and fate choices, which all impact the neurogenic output. We further discuss the regulation of NSC activity in response to physiological triggers and non-physiological conditions such as regenerative contexts.

Chemical Genetics Screen Identifies Epigenetic Mechanisms Involved in Dopaminergic and Noradrenergic Neurogenesis in Zebrafish.

The cell type diversity and complexity of the nervous system is generated by a network of signaling events, transcription factors, and epigenetic regulators. Signaling and transcriptional control have been easily amenable to forward genetic screens in model organisms like zebrafish. In contrast, epigenetic mechanisms have been somewhat elusive in genetic screens, likely caused by broad action in multiple developmental pathways that masks specific phenotypes, but also by genetic redundancies of epigenetic factors. Here, we performed a screen using small molecule inhibitors of epigenetic mechanisms to reveal contributions to specific aspects of neurogenesis in zebrafish. We chose development of dopaminergic and noradrenergic neurons from neural progenitors as target of epigenetic regulation. We performed the screen in two phases: First, we tested a small molecule inhibitor library that targets a broad range of epigenetic protein classes and mechanisms, using expression of the dopaminergic and noradrenergic marker tyrosine hydroxylase as readout. We identified 10 compounds, including HDAC, Bromodomain and HAT inhibitors, which interfered with dopaminergic and noradrenergic development in larval zebrafish. In the second screening phase, we aimed to identify neurogenesis stages affected by these 10 inhibitors. We analyzed treated embryos for effects on neural stem cells, growth progression of the retina, and apoptosis in neural tissues. In addition, we analyzed effects on islet1 expressing neuronal populations to determine potential selectivity of compounds for transmitter phenotypes. In summary, our targeted screen of epigenetic inhibitors identified specific compounds, which reveal chromatin regulator classes that contribute to dopaminergic and noradrenergic neurogenesis in vivo.

Neuro-protective effects of nano-formulated hesperetin in a traumatic brain injury model of Danio rerio

Understanding the mechanism behind neuronal regeneration is critical for treating ischemic stroke and traumatic brain injury. The presence of neural stem cells in and around the sub-ventricular zone of human and also in zebrafish is evidenced. In this current study, the neuro-protective potential of nano-formulated hesperetin on injury-induced neurogenesis in zebrafish was assessed. Nanoformulation of hesperetin was prepared by anti-solvent precipitation technique using sodium dodecyl sulfate (SDS) as the stabilizing agent. The synthesized particles were characterized using SEM, DLS, XRD and FT-IR. Anti-oxidant capacity of nano hesperetin (nHST) in in vitro followed by in vivo studies in a traumatic brain injury (TBI) model of adult zebrafish (Danio rerio), catalase activity, histological analysis and gene expression studies for the genes Sox2, Nestin, Fabp7a and HuC were carried out. The synthesized particles were found to be in nanoscale and SDS had successfully integrated with hesperetin. Moreover, nHST had a significantly higher anti-oxidant capacity in vitro. Catalase levels in nHST treated group were significantly restored compared to other groups. Histological studies supported reduced tissue damage on oral administration of nano-hesperetin as compared to other groups. Gene expression studies showed that nano-hesperetin at a concentration of 10 μM when administered orally induced proliferation of neural stem cells without inducing cell death.