micromanipulation-gene-expression-adult-zebrafish-brain-using

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

modeling-amyloid-42-toxicity-neurodegeneration-adult-zebrafish

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Two hallmarks of vertebrate epimorphic regeneration are a significant increase in the proliferation of normally quiescent cells and a re-activation of genes that are active during embryonic development. It is unclear what the molecular determinants are that regulate these events and how they are coordinated. Zebrafish have the ability to regenerate several compound structures by regulating cell proliferation and gene transcription. We report that fam53b/simplet (smp) regulates both cell proliferation and the transcription of specific genes. In situ hybridization and quantitative RT-PCR experiments showed that amputation of zebrafish hearts and fins resulted in strong up-regulation of the smp gene. In regenerating adult fin, smp expression remained strong in the distal mesenchyme which later expanded to the basal layers of the distal epidermis and distal tip epithelium. Morpholino knockdown of smp reduced regenerative outgrowth by decreasing cell proliferation as measured by BrdU incorporation and histone H3 phosphorylation. In addition, smp knockdown increased the expression of msxb, msxc, and shh, as well as the later formation of ectopic bone. Taken together, these data indicate a requirement for smp in fin regeneration through control of cell proliferation, the regulation of specific genes and proper bone patterning.

Simplet controls cell proliferation and gene transcription during zebrafish caudal fin regeneration.

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.

Adult neurogenesis and brain regeneration in zebrafish.

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The teleost fish Danio rerio (zebrafish) has a remarkable ability to generate newborn neurons in its brain at adult stages of its lifespan-a process called adult neurogenesis. This ability relies on proliferating ventricular progenitors and is in striking contrast to mammalian brains that have rather restricted capacity for adult neurogenesis. Therefore, investigating the zebrafish brain can help not only to elucidate the molecular mechanisms of widespread adult neurogenesis in a vertebrate species, but also to design therapies in humans with what we learn from this teleost. Yet, understanding the cellular behavior and molecular programs underlying different biological processes in the adult zebrafish brain requires techniques that allow manipulation of gene function. As a complementary method to the currently used misexpression techniques in zebrafish, such as transgenic approaches or electroporation-based delivery of DNA, we devised a cerebroventricular microinjection (CVMI)-assisted knockdown protocol that relies on vivo morpholino oligonucleotides, which do not require electroporation for cellular uptake. This rapid method allows uniform and efficient knockdown of genes in the ventricular cells of the zebrafish brain, which contain the neurogenic progenitors. We also provide data on the use of CVMI for growth factor administration to the brain--in our case FGF8, which modulates the proliferation rate of the ventricular cells. In this paper, we describe the CVMI method and discuss its potential uses in zebrafish.

Cerebroventricular microinjection (CVMI) into adult zebrafish brain is an efficient misexpression method for forebrain ventricular cells.

Unlike 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.

The chemokine receptor cxcr5 regulates the regenerative neurogenesis response in the adult zebrafish brain.

The zebrafish regenerates its brain after injury and hence is a useful model organism to study the mechanisms enabling regenerative neurogenesis, which is poorly manifested in mammals. Yet the signaling mechanisms initiating such a regenerative response in fish are unknown. Using cerebroventricular microinjection of immunogenic particles and immunosuppression assays, we showed that inflammation is required and sufficient for enhancing the proliferation of neural progenitors and subsequent neurogenesis by activating injury-induced molecular programs that can be observed after traumatic brain injury. We also identified cysteinyl leukotriene signaling as an essential component of inflammation in the regenerative process of the adult zebrafish brain. Thus, our results demonstrate that in zebrafish, in contrast to mammals, inflammation is a positive regulator of neuronal regeneration in the central nervous system.

Acute inflammation initiates the regenerative response in the adult zebrafish brain.

The adult zebrafish brain, unlike mammalian counterparts, can regenerate after injury owing to the neurogenic capacity of stem cells with radial glial character. We hypothesized that injury-induced regenerative programs might be turned on after injury in zebrafish brain and enable regenerative neurogenesis. Here we identify one such gene-the transcription factor gata3-which is expressed only after injury in different zebrafish organs. Gata3 is required for reactive proliferation of radial glia cells, subsequent regenerative neurogenesis, and migration of the newborn neurons. We found that these regeneration-specific roles of Gata3 are dependent on the injury because Gata3 overexpression in the unlesioned adult zebrafish brain is not sufficient to induce neurogenesis. Thus, gata3 acts as a specific injury-induced proregenerative factor that is essential for the regenerative capacity in vertebrates.

Regenerative neurogenesis from neural progenitor cells requires injury-induced expression of Gata3.

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Injuries in the central nervous system (CNS) are one of the leading causes of mortality or persistent disabilities in humans. One of the reasons why humans cannot recover from neuronal loss is the limited regenerative capacity of their CNS. By contrast, non-mammalian vertebrates exhibit widespread regeneration in diverse tissues including the CNS. Understanding those mechanisms activated during regeneration may improve the regenerative outcome in the severed mammalian CNS. Of those mechanisms, recent evidence suggests that inflammation may be important in regeneration. In this review we compare the different events following acute CNS injury in mammals and non-mammalian vertebrates. We also discuss the involvement of the immune response in initiating regenerative programs and how immune cells and neural stem/progenitor cells (NSPCs) communicate.

Neuroinflammation and central nervous system regeneration in vertebrates.

Canonical β-catenin-dependent Wnt signal transduction is important for several biological phenomena, such as cell fate determination, cell proliferation, stem cell maintenance and anterior-posterior axis formation. The hallmark of canonical Wnt signaling is the translocation of β-catenin into the nucleus where it activates gene transcription. However, the mechanisms regulating β-catenin nuclear localization are poorly understood. We show that Simplet/Fam53B (Smp) is required for Wnt signaling by positively regulating β-catenin nuclear localization. In the zebrafish embryo, the loss of smp blocks the activity of two β-catenin-dependent reporters and the expression of Wnt target genes, and prevents nuclear accumulation of β-catenin. Conversely, overexpression of smp increases β-catenin nuclear localization and transcriptional activity in vitro and in vivo. Expression of mutant Smp proteins lacking either the nuclear localization signal or the β-catenin interaction domain reveal that the translocation of Smp into the nucleus is essential for β-catenin nuclear localization and Wnt signaling in vivo. We also provide evidence that mammalian Smp is involved in regulating β-catenin nuclear localization: the protein colocalizes with β-catenin-dependent gene expression in mouse intestinal crypts; siRNA knockdown of Smp reduces β-catenin nuclear localization and transcriptional activity; human SMP mediates β-catenin transcriptional activity in a dose-dependent manner; and the human SMP protein interacts with human β-catenin primarily in the nucleus. Thus, our findings identify the evolutionary conserved SMP protein as a regulator of β-catenin-dependent Wnt signal transduction.

Simplet/Fam53b is required for Wnt signal transduction by regulating β-catenin nuclear localization.

Inflammation entails a complex set of defense mechanisms acting in concert to restore the homeostatic balance in organisms after damage or pathogen invasion. This immune response consists of the activity of various immune cells in a highly complex manner. Inflammation is a double-edged sword as it is reported to have both detrimental and beneficial consequences. In this review, we discuss the effects of inflammation on stem cell activity, focusing primarily on neural stem/progenitor cells in mammals and zebrafish. We also give a brief overview of the effects of inflammation on other stem cell compartments, exemplifying the positive and negative role of inflammation on stemness. The majority of the chronic diseases involve an unremitting phase of inflammation due to improper resolution of the initial pro-inflammatory response that impinges on the stem cell behavior. Thus, understanding the mechanisms of crosstalk between the inflammatory milieu and tissue-resident stem cells is an important basis for clinical efforts. Not only is it important to understand the effect of inflammation on stem cell activity for further defining the etiology of the diseases, but also better mechanistic understanding is essential to design regenerative therapies that aim at micromanipulating the inflammatory milieu to offset the negative effects and maximize the beneficial outcomes.

Effects of inflammation on stem cells: together they strive?

Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs) that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI), RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs) and identified two- polyR and Trans - that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael's addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.

Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides.

Regenerative capacity of the brain is a variable trait within animals. Aquatic vertebrates such as zebrafish have widespread ability to renew their brains upon damage, while mammals have--if not none--very limited overall regenerative competence. Underlying cause of such a disparity is not fully evident; however, one of the reasons could be activation of peculiar molecular programs, which might have specific roles after injury or damage, by the organisms that regenerate. If this hypothesis is correct, then there must be genes and pathways that (a) are expressed only after injury or damage in tissues, (b) are biologically and functionally relevant to restoration of neural tissue, and (c) are not detected in regenerating organisms. Presence of such programs might circumvent the initial detrimental effects of the damage and subsequently set up the stage for tissue redevelopment to take place by modulating the plasticity of the neural stem/progenitor cells. Additionally, if transferable, those "molecular mechanisms of regeneration" could open up new avenues for regenerative therapies of humans in clinical settings. This review focuses on the recent studies addressing injury/damage-induced molecular programs in zebrafish brain, underscoring the possibility of the presence of genes that could be used as biomarkers of neural plasticity and regeneration.

Regeneration, Plasticity, and Induced Molecular Programs in Adult Zebrafish Brain.

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Alzheimer's disease (AD) is the most prevalent neurodegenerative disease and a worldwide health challenge. Different therapeutic approaches are being developed to reverse or slow the loss of affected neurons. Another plausible therapeutic way that may complement the studies is to increase the survival of existing neurons by mobilizing the existing neural stem/progenitor cells (NSPCs) - i.e. "induce their plasticity" - to regenerate lost neurons despite the existing pathology and unfavorable environment. However, there is controversy about how NSPCs are affected by the unfavorable toxic environment during AD. In this review, we will discuss the use of stem cells in neurodegenerative diseases and in particular how NSPCs affect the AD pathology and how neurodegeneration affects NSPCs. In the end of this review, we will discuss how zebrafish as a useful model organism with extensive regenerative ability in the brain might help to address the molecular programs needed for NSPCs to respond to neurodegeneration by enhanced neurogenesis.

Neural stem/progenitor cells in Alzheimer's disease.

Human brains are prone to neurodegeneration, given that endogenous neural stem/progenitor cells (NSPCs) fail to support neurogenesis. To investigate the molecular programs potentially mediating neurodegeneration-induced NSPC plasticity in regenerating organisms, we generated an Amyloid-β42 (Aβ42)-dependent neurotoxic model in adult zebrafish brain through cerebroventricular microinjection of cell-penetrating Aβ42 derivatives. Aβ42 deposits in neurons and causes phenotypes reminiscent of amyloid pathophysiology: apoptosis, microglial activation, synaptic degeneration, and learning deficits. Aβ42 also induces NSPC proliferation and enhanced neurogenesis. Interleukin-4 (IL4) is activated primarily in neurons and microglia/macrophages in response to Aβ42 and is sufficient to increase NSPC proliferation and neurogenesis via STAT6 phosphorylation through the IL4 receptor in NSPCs. Our results reveal a crosstalk between neurons and immune cells mediated by IL4/STAT6 signaling, which induces NSPC plasticity in zebrafish brains.

IL4/STAT6 Signaling Activates Neural Stem Cell Proliferation and Neurogenesis upon Amyloid-β42 Aggregation in Adult Zebrafish Brain.

Alzheimer disease is the most prevalent neurodegenerative disease and is associated with aggregation of Amyloid-β42 peptides. In mammals, Amyloid-β42 causes impaired neural stem/progenitor cell (NSPC) proliferation and neurogenesis, which exacerbate with aging. The molecular programs necessary to enhance NSPC proliferation and neurogenesis in our brains to mount successful regeneration are largely unknown. Therefore, to identify the molecular basis of effective brain regeneration, we previously established an Amyloid-β42 model in adult zebrafish that displayed Alzheimer-like phenotypes reminiscent of humans. Interestingly, zebrafish exhibited enhanced NSPC proliferation and neurogenesis after microinjection of Amyloid-β42 peptide. Here, we compare old and young fish to address the effects of aging on regenerative ability after Amyloid-β42 deposition. We found that aging does not affect the rate of NSPC proliferation but reduces the neurogenic response and microglia/macrophage activation after microinjection of Amyloid-β42 in zebrafish, suggesting an important link between aging, neuroinflammation, regenerative neurogenesis and neural stem cell plasticity.

The effects of aging on Amyloid-β42-induced neurodegeneration and regeneration in adult zebrafish brain.

Microtubule-associated TAU protein is a pathological hallmark in Alzheimer's disease (AD), where hyperphosphorylation of TAU generates neurofibrillary tangles. To investigate the effects of TAU in a regenerative adult vertebrate brain system, we generated a cre/lox-based transgenic model of zebrafish that chronically expresses human TAU(P301L), which is a variant of human TAU protein that forms neurofibrillary tangles in mouse models and humans. Interestingly, we found that although chronic and abundant expression of TAU(P301L) starting from early embryonic development led to hyperphosphorylation, TAU(P301L) did not form oligomers and neurofibrillary tangles, and did not cause elevated apoptosis and microglial activation, which are classical symptoms of tauopathies in mammals. Additionally, TAU(P301L) neither increased neural stem cell proliferation nor activated the expression of regenerative factor Interleukin-4, indicating that TAU(P301L) toxicity is prevented in the adult zebrafish brain. By combining TAU(P301L) expression with our established Aβ42 toxicity model, we found that Aβ42 ceases to initiate neurofibrillary tangle formation by TAU(P301L), and TAU(P301L) does not exacerbate the toxicity of Aβ42. Therefore, our results propose a cellular mechanism that protects the adult zebrafish brain against tauopathies, and our model can be used to understand how TAU toxicity can be prevented in humans.

ArticleTotal : 2	Year
Publication title Cited by 44	2013
Publication title Cited by 50	2017

Article	Year
Simplet controls cell proliferation and gene transcription during zebrafish caudal fin regeneration. Developmental biology\| PubMed ID: 19014929	2009
Adult neurogenesis and brain regeneration in zebrafish. Developmental neurobiology\| PubMed ID: 21595047	2012
Academic autonomy: more freedom for Turkish science. Nature\| PubMed ID: 21956319	2011
Turkish science seeks freedom. Science (New York, N.Y.)\| PubMed ID: 22034413	2011
Cerebroventricular microinjection (CVMI) into adult zebrafish brain is an efficient misexpression method for forebrain ventricular cells. PloS one\| PubMed ID: 22076157	2011
The chemokine receptor cxcr5 regulates the regenerative neurogenesis response in the adult zebrafish brain. Neural development\| PubMed ID: 22824261	2012
Acute inflammation initiates the regenerative response in the adult zebrafish brain. Science (New York, N.Y.)\| PubMed ID: 23138980	2012
Regenerative neurogenesis from neural progenitor cells requires injury-induced expression of Gata3. Developmental cell\| PubMed ID: 23168169	2012
Turkey must end violent response to protests. Science (New York, N.Y.)\| PubMed ID: 23869001	2013
Neuroinflammation and central nervous system regeneration in vertebrates. Trends in cell biology\| PubMed ID: 24029244	2014
Simplet/Fam53b is required for Wnt signal transduction by regulating β-catenin nuclear localization. Development (Cambridge, England)\| PubMed ID: 25183871	2014
Effects of inflammation on stem cells: together they strive? EMBO reports\| PubMed ID: 25739812	2015
Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides. PloS one\| PubMed ID: 25894337	2015
Regeneration, Plasticity, and Induced Molecular Programs in Adult Zebrafish Brain. BioMed research international\| PubMed ID: 26417601	2015
Government: Concern grows for Turkey's academics. Nature\| PubMed ID: 26819036	2016
Neural stem/progenitor cells in Alzheimer's disease. The Yale journal of biology and medicine\| PubMed ID: 27505014	2016
IL4/STAT6 Signaling Activates Neural Stem Cell Proliferation and Neurogenesis upon Amyloid-β42 Aggregation in Adult Zebrafish Brain. Cell reports\| PubMed ID: 27760324	2016
The effects of aging on Amyloid-β42-induced neurodegeneration and regeneration in adult zebrafish brain. Neurogenesis (Austin, Tex.)\| PubMed ID: 28656156	2017
Human TAU(P301L) overexpression results in TAU hyperphosphorylation without neurofibrillary tangles in adult zebrafish brain. Scientific reports\| PubMed ID: 29021554	2017

Caghan Kizil

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