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[Methods in Molecular Biology] Astrocytes Volume 1938 (Methods and Protocols) || Fluorescence-Activated Cell Sorting-Based Isolation and Characterization of Neural Stem Cells from the Adult Zebrafish Telencephalon
摘要： Adult mammalian brain, including humans, has rather limited addition of new neurons and poor regenerative capacity. In contrast, neural stem cells (NSC) with glial identity and neurogenesis are highly abundant throughout the adult zebrafish brain. Importantly, the activation of NSC and production of new neurons in response to injuries lead to the brain regeneration in zebrafish brain. Therefore, understanding of the molecular pathways regulating NSC behavior in response to injury is crucial in order to set the basis for experimental modification of these pathways in glial cells after injury in the mammalian brain and to elicit neuronal regeneration. Here, we describe the procedure that we successfully used to prospectively isolate NSCs from adult zebrafish telencephalon, extract RNA, and prepare cDNA libraries for next generation sequencing (NGS) and full transcriptome analysis as the first step toward understanding regulatory mechanisms leading to restorative neurogenesis in zebrafish. Moreover, we describe an alternative approach to analyze antigenic properties of NSC in the adult zebrafish brain using intracellular fluorescence activated cell sorting (FACS). We employ this method to analyze the number of proliferating NSCs positive for proliferating cell nuclear antigen (PCNA) in the prospectively isolated population of stem cells.
关键词： Neural stem cells,Neural stem cell purification,Intracellular FACS,Zebrafish
更新于2025-09-23 15:23:52
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Ultrasmall Au-Ag Alloy Nanoparticles: Protein-directed Synthesis, Biocompatibility and X-ray Computed Tomography Imaging ?
摘要： The ultrasmall sizes of nanoparticles have attracted significant attention for potential application in the fields of catalysis and nanomedicine. Herein, we reported on the green preparation and X-ray computed tomography (CT) imaging of ultrasmall bimetallic bovine serum albumin-directed gold-silver (Au-Ag@BSA) nanoparticles (2–4 nm) using BSA as a stabilizing and template-directed agent. Further, the effects of synthesis condition were systematically explored to prepare products by adjusting the different molar ratios of Au/Ag. The resulting Au-Ag@BSA nanoparticles exhibited the spherical shape, well-dispersed ability, as well as long-term room-temperature stability. The cytotoxicity effects of Au-Ag@BSA nanoparticles on A549 and MCF-7 cells were compared with those of individual Ag nanoparticles, and the results indicated lower cytotoxicity effect by Au-Ag@BSA nanoparticles. Furthermore, the in vivo toxicity of Au-Ag@BSA nanoparticles was investigated in the early-stage zebrafish embryos. The results indicate that there are not any obvious changes of survival and hatching percentages at multiple growth stages (4-120 hpf) even a high level of Au-Ag@BSA nanoparticles (up to 80 mM), revealing the good biocompatibility. Interestingly, a rational design of Au/Ag molar ratio (3:2) surprisingly possessed the enhanced CT performances compared to the Au nanoparticles and iohexol. Accordingly, this study highlights a new prospect in the green preparation of ultrasmall alloy nanomaterials with good biocompatibility and will be of great interest in developing CT contrast agent, catalyst as well as drug delivery carrier.
关键词： green synthesis,Au-Ag@BSA nanoparticles,biocompatibility,zebrafish embryos,enhanced CT imaging
更新于2025-09-23 15:23:52
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Visualize Embryogenesis and Cell Fate Using Fluorescent Probes with Aggregation-Induced Emission
摘要： Horseradish peroxidase (HRP) and fluorogen-dextran conjugate are tracers extensively used for injection-based lineage tracing. However, HRP is sensitive to proteolytic digestion while the high-molecular-weight dextran may have antigenicity. Small molecular tracers can overcome these problems, but they usually diffuse from labeled cells, causing inaccurate information. Herein, we developed a small-molecular-weight fluorogen with aggregation-induced emission (AIEgen) for embryonic cell tracing with strong signals against tracer dilution caused by cell division. Once injected into the ancestor cells, the AIEgen can be entrapped in the cells without leakage because of the two hydrophilic and neutral arms. Consequently, it can specifically trace the progenies of the treated ancestor cells. More importantly, the operating concentration of AIEgen can be much higher than those of fluorogens with aggregation-caused quenching (ACQ), which provides bright signals in daughter cells during embryonic cell tracing, thus overcoming the problem of fast signal degrading typically encountered with the use of traditional cell tracers.
关键词： bright signals,small molecule,aggregation-induced emission,cell entrapped,embryonic cell,zebrafish lineage tracing
更新于2025-09-23 15:23:52
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Live imaging of angiogenesis during cutaneous wound healing in adult zebrafish
摘要： Angiogenesis, the growth of new blood vessels from pre-existing vessels, is critical for cutaneous wound healing. However, it remains elusive how endothelial cells (ECs) and pericytes (PCs) establish new blood vessels during cutaneous angiogenesis. We set up a live-imaging system to analyze cutaneous angiogenesis in adult zebrafish. First, we characterized basic structures of cutaneous vasculature. In normal skin tissues, ECs and PCs remained dormant to maintain quiescent blood vessels, whereas cutaneous injury immediately induced angiogenesis through the vascular endothelial growth factor signaling pathway. Tortuous and disorganized vessel networks formed within a few weeks after the injury and subsequently normalized through vessel regression in a few months. Analyses of the repair process of injured single blood vessels revealed that severed vessels elongated upon injury and anastomosed with each other. Thereafter, repaired vessels and adjacent uninjured vessels became tortuous by increasing the number of ECs. In parallel, PCs divided and migrated to cover the tortuous blood vessels. ECs sprouted from the PC-covered tortuous vessels, suggesting that EC sprouting does not require PC detachment from the vessel wall. Thus, live imaging of cutaneous angiogenesis in adult zebrafish enables us to clarify how ECs and PCs develop new blood vessels during cutaneous angiogenesis.
关键词： Pericytes,Cutaneous wound healing,Angiogenesis,Endothelial cells,Zebrafish
更新于2025-09-23 15:23:52
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Cardiac Electrophysiological Effects of Light-Activated Chloride Channels
摘要： During the last decade, optogenetics has emerged as a paradigm-shifting technique to monitor and steer the behavior of specific cell types in excitable tissues, including the heart. Activation of cation-conducting channelrhodopsins (ChR) leads to membrane depolarization, allowing one to effectively trigger action potentials (AP) in cardiomyocytes. In contrast, the quest for optogenetic tools for hyperpolarization-induced inhibition of AP generation has remained challenging. The green-light activated ChR from Guillardia theta (GtACR1) mediates Cl?-driven photocurrents that have been shown to silence AP generation in different types of neurons. It has been suggested, therefore, to be a suitable tool for inhibition of cardiomyocyte activity. Using single-cell electrophysiological recordings and contraction tracking, as well as intracellular microelectrode recordings and in vivo optical recordings of whole hearts, we find that GtACR1 activation by prolonged illumination arrests cardiac cells in a depolarized state, thus inhibiting re-excitation. In line with this, GtACR1 activation by transient light pulses elicits AP in rabbit isolated cardiomyocytes and in spontaneously beating intact hearts of zebrafish. Our results show that GtACR1 inhibition of AP generation is caused by cell depolarization. While this does not address the need for optogenetic silencing through physiological means (i.e., hyperpolarization), GtACR1 is a potentially attractive tool for activating cardiomyocytes by transient light-induced depolarization.
关键词： heart,action potential,zebrafish,optogenetics,GtACR1,natural anion channelrhodopsin
更新于2025-09-23 15:22:29
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Arl13b Interacts With Vangl2 to Regulate Cilia and Photoreceptor Outer Segment Length in Zebrafish
摘要： Mutations in the gene ARL13B cause the classical form of Joubert syndrome, an autosomal recessive ciliopathy with variable degrees of retinal degeneration. As second-site modifier alleles can contribute to retinal pathology in ciliopathies, animal models provide a unique platform to test how genetic interactions modulate specific phenotypes. In this study, we analyzed the zebrafish arl13b mutant for retinal degeneration and for epistatic relationships with the planar cell polarity protein (PCP) component vangl2. Photoreceptor and cilia structure was examined by light and electron microscopy. Immunohistochemistry was performed to examine ciliary markers. Genetic interactions were tested by pairwise crosses of heterozygous animals. Genetic mosaic animals were generated by blastula transplantation and analyzed by fluorescence microscopy. At 5 days after fertilization, photoreceptor outer segments were shorter in zebrafish arl13b?/? mutants compared to wild-type larvae, no overt signs of retinal degeneration were observed by light or electron microscopy. Starting at 14 days after fertilization (dpf) and continuing through 30 dpf, cells lacking Arl13b died following transplantation into wild-type host animals. Photoreceptors of arl13b?/?;vangl2?/? mutants were more compromised than the photoreceptors of single mutants. Finally, when grown within a wild-type retina, the vangl2?/? mutant cone photoreceptors displayed normal basal body positioning. We show that arl13b?/? mutants have shortened cilia and photoreceptor outer segments and exhibit a slow, progressive photoreceptor degeneration that occurs over weeks. The data suggest that loss of Arl13b leads to slow photoreceptor degeneration, but can be exacerbated by the loss of vangl2. Importantly, the data show that Arl13b can genetically and physically interact with Vangl2 and this association is important for normal photoreceptor structure. The loss of vangl2, however, does not affect basal body positioning.
关键词： Arl13b,planar polarity,photoreceptor,cilia,zebrafish,retina,Vangl2
更新于2025-09-23 15:21:21
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Anionic Quantum Dots reveal actin-microridges in zebrafish epidermis
摘要： Enhancement of the aqueous solubility and functionalization of CdTe-QDs (Quantum Dots) via surface modifications have made them suitable to be used as specific probes for cell imaging. Applications for targeting cell surfaces have been widely demonstrated in vitro but their use in animal models is not trivial. Here, we reported the interaction of mercaptosuccinic-coated (MSA) CdTe-QDs with the epidermis of living and Carnoy-fixed zebrafish embryos, providing a faster approach compared with immunodetection or standard Phalloidin staining of actin for visualization by fluorescence microscopy. In our study, labeling with anionic QDs is attained within minutes at nanomolar concentrations in whole mounted Carnoy-fixed zebrafish embryos, providing a faster approach compared with immunodetection or standard Phalloidin staining of actin for visualization by fluorescence microscopy. QDs concentrate along adherent junctions and reveal the characteristic pattern of actin microridges at the apical surface of the enveloping layer.
关键词： actin-microridges,anionic quantum dots,zebrafish epidermis
更新于2025-09-23 15:21:01
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Frontline Science: Dynamic cellular and subcellular features of migrating leukocytes revealed by in vivo lattice lightsheet microscopy
摘要： Neutrophil and macrophage (M??) migration underpin the inflammatory response. However, the fast velocity, multidirectional instantaneous movement, and plastic, ever-changing shape of phagocytes confound high-resolution intravital imaging. Lattice lightsheet microscopy (LLSM) captures highly dynamic cell morphology at exceptional spatiotemporal resolution. We demonstrate the first extensive application of LLSM to leukocytes in vivo, utilizing optically transparent zebrafish, leukocyte-specific reporter lines that highlighted subcellular structure, and a wounding assay for leukocyte migration. LLSM revealed details of migrating leukocyte morphology, and permitted intricate, volumetric interrogation of highly dynamic activities within their native physiological setting. Very thin, recurrent uropod extensions must now be considered a characteristic feature of migrating neutrophils. LLSM resolved trailing uropod extensions, demonstrating their surprising length, and permitting quantitative assessment of cytoskeletal contributions to their evanescent form. Imaging leukocytes in blood vessel microenvironments at LLSM’s spatiotemporal resolution displayed blood-flow-induced neutrophil dynamics and demonstrated unexpected leukocyte-endothelial interactions such as leukocyte-induced endothelial deformation against the intravascular pressure. LLSM of phagocytosis and cell death provided subcellular insights and uncovered novel behaviors. Collectively, we provide high-resolution LLSM examples of leukocyte structures (filopodia lamellipodia, uropod extensions, vesicles), and activities (interstitial and intravascular migration, leukocyte rolling, phagocytosis, cell death, and cytoplasmic ballooning). Application of LLSM to intravital leukocyte imaging sets the stage for transformative studies into the cellular and subcellular complexities of phagocyte biology.
关键词： uropod,leukocytes,migration,zebrafish,lattice lightsheet microscopy,phagocytes
更新于2025-09-23 15:19:57
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The Effect of Fluence on Macrophage Kinetics, Oxidative Stress, and Wound Closure Using Real-Time <i>In Vivo</i> Imaging
摘要： Objective: The aim of our study was to quantify the effect of doses delivered by a He:Ne laser on individual macrophage kinetics, tissue oxidative stress, and wound closure using real-time in vivo imaging. Background: Photobiomodulation has been reported to reduce tissue inflammation and accelerate wound closure; however, precise parameters of laser settings to optimize macrophage behavior have not been established. We hypothesized that quantitative and real-time in vivo imaging could identify optimal fluence for macrophage migration, reduction of reactive oxygen species, and acceleration of wound closure. Methods: Larval zebrafish Tg(mpeg-dendra2) were loaded with dihydroethidium for oxidative stress detection. Fish were caudal fin injured, treated with 635 nm continuous 5 mW He:Ne laser irradiation at 3, 9, or 18 J/cm2 and time-lapsed imaged within the first 120 min postinjury. Images taken 1 and 24-h postinjury were compared for percentage wound closure. Results: A fluence of 3 J/cm2 demonstrated significant increases in macrophage migration speed, fewer stops along the way, and greatest directed migration toward the wound. These findings were associated with a significant reduction in wound content reactive oxygen species when compared with control wounded fins. Both 3 and 9 J/cm2 significantly accelerated wound closure when compared with nonirradiated control fish. Conclusions: Wound macrophage activity could be manipulated by applied fluence, leading to reduced levels of wound reactive oxygen species and accelerated wound closure. The zebrafish model provides a means to quantitatively compare wound macrophage behavior in response to a variety of laser treatment parameters in real time.
关键词： zebrafish,macrophage,He:Ne laser,oxidative stress,time-lapse imaging
更新于2025-09-19 17:15:36
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Photoreceptor Progenitors Depend Upon Coordination of <i>gdf6a</i> , <i>thrβ</i> , and <i>tbx2b</i> to Generate Precise Populations of Cone Photoreceptor Subtypes
摘要： PURPOSE. Replacing cone photoreceptors, the units of the retina necessary for daytime vision, depends upon the successful production of a full variety of new cones from, for example, stem cells. Using genetic experiments in a model organism with high cone diversity, zebrafish, we map the intersecting effects of cone development factors gdf6a, tbx2b, and thrb. METHODS. We investigated these genes of interest by using genetic combinations of mutants, gene knockdown, and dominant negative gene expression, and then quantified cone subtype outcomes (which normally develop in tightly regulated ratios). RESULTS. Gdf6a mutants have reduced blue cones and, discovered here, reduced red cones. In combined gdf6a/tbx2b disruption, the loss of gdf6a in heterozygous tbx2b mutants reduced UV cones. Intriguingly, when we disrupted thrb in gdf6a mutants by using a thrb morpholino, their combined early disruption revealed a lamination phenotype. Disrupting thrb activity via expression of a dominant negative thrb (dnthrb) at either early or late retinal development had differential outcomes on red cones (reduced abundance), versus UV and blue cones (increased abundance). By using dnthrb in gdf6a mutants, we revealed that disrupting thrb activity did not change gdf6a mutant cone phenotypes. CONCLUSIONS. Gdf6a loss directly affects blue and red cones and indirectly affects UV cones by increasing sensitivity to additional disruption, such as reduced tbx2b, resulting in fewer UV cones. The effects of thrb change through photoreceptor development, first promoting red cones and restricting UV cones, and later restricting UV and blue cones. The effects of gdf6a on UV, blue, and red cone development overlap with, but likely supersede, those of thrb.
关键词： BMP signaling,progenitor,color vision,retinal lamination,thyroid signaling,zebrafish,retinal development,determination,regeneration,cone photoreceptor
更新于2025-09-19 17:15:36