Inhibition of Renal Stellate Cell Activation Reduces Renal Fibrosis.

Interstitial fibrosis is a common feature of chronic kidney disease, and platelet-derived growth factor receptor-ß (PDGFR-ß)-positive mesenchymal cells are reportedly the major source of scar-producing myofibroblasts. We had previously demonstrated that albumin and its derivative R-III (a retinol-binding protein-albumin domain III fusion protein) inhibited the transdifferentiation/activation of hepatic stellate cells (HSCs) to myofibroblasts and that R-III administration reduced liver fibrosis. In this study, we isolated cells (referred to as renal stellate cells, RSCs) from rat kidney tissues using the HSC isolation protocol and compared their morphological and biochemical characteristics with those of HSCs. RSCs shared many characteristics with HSCs, such as storage of vitamin A-containing lipid droplets and expression of HSC markers as well as pericyte markers. RSCs underwent spontaneous transdifferentiation into myofibroblasts in in vitro culture, which was inhibited by albumin expression or R-III treatment. We also evaluated the therapeutic effects of R-III in unilateral ureteral obstruction (UUO)-induced renal fibrosis in mice. Injected R-III localized predominantly in cytoglobin/stellate cell activation-associated protein (Cygb/STAP)-positive cells in the kidney and reduced renal fibrosis. These findings suggest that RSCs can be recognized as the renal counterparts of HSCs and that RSCs represent an attractive therapeutic target for anti-fibrotic therapy.

Prevention of mitochondrial impairment by inhibition of protein phosphatase 1 activity in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of motor neurons (MNs) and subsequent muscle weakness. These pathological features are associated with numerous cellular changes, including alteration in mitochondrial morphology and function. However, the molecular mechanisms associating mitochondrial structure with ALS pathology are poorly understood. In this study, we found that Dynamin-related protein 1 (Drp1) was dephosphorylated in several ALS models, including those with SOD1 and TDP-43 mutations, and the dephosphorylation was mediated by the pathological induction of protein phosphatase 1 (PP1) activity in these models. Suppression of the PP1-Drp1 cascade effectively prevented ALS-related symptoms, including mitochondrial fragmentation, mitochondrial complex I impairment, axonal degeneration, and cell death, in primary neuronal culture models, iPSC-derived human MNs, and zebrafish models in vivo. These results suggest that modulation of PP1-Drp1 activity may be a therapeutic target for multiple pathological features of ALS.

Myelin degeneration induced by mutant superoxide dismutase 1 accumulation promotes amyotrophic lateral sclerosis.

Myelin is a specialized membrane that wraps around nerve fibers and is essential for normal axonal conduction in neurons. In the central nervous system, oligodendrocytes are responsible for myelin formation. Recent studies have reported pathological abnormalities in oligodendrocytes in human patients with amyotrophic lateral sclerosis (ALS) and a mouse model of ALS expressing the G93A mutation of the human superoxide dismutase 1 (mtSOD1). However, it is unclear whether oligodendrocyte pathology in ALS represents the primary dysfunction induced by mtSOD1 and how mtSOD1 contributes to oligodendrocyte degeneration and ALS pathogenesis. We analyzed GAL4-VP16-UAS transgenic zebrafish selectively expressing mtSOD1 in mature oligodendrocytes. We observed that mtSOD1 directly induced oligodendrocyte degeneration by disrupting the myelin sheath and downregulating monocarboxylate transporter 1 (MCT1), thereby causing spinal motor neuron degeneration. Pathological changes observed in this transgenic zebrafish were similar to the pathology observed in the SOD1G93A mouse model of ALS, which is characterized by expression of mtSOD1 in all cells. In addition, oligodendrocyte dysfunction induced by mtSOD1 was associated with anxiety-related behavioral abnormalities, learning impairments, and motor defects in the early symptomatic stage. We also found that treatment with potassium channel inhibitors rescued behavioral abnormalities without rescuing MCT1 expression, suggesting that myelin disruption induces behavioral abnormalities independently of MCT1. These results indicate that mtSOD1-induced dysfunction of mature oligodendrocytes is sufficient to induce motor neuron degeneration, thus informing future therapeutic strategies targeted at oligodendrocytes in ALS.

Distribution and neuronal circuit of spexin 1/2 neurons in the zebrafish CNS.

Spexin (SPX) is a highly conserved neuropeptide that is widely expressed in mammalian brain and peripheral tissue. In teleost, SPX1 is mainly expressed in the brain and ovary, and is involved in reproduction and food intake. A second form of SPX, SPX2, was recently identified in chick, Xenopus, and zebrafish. The expression pattern and roles of SPX2 are unknown. SPX (spx1) is highly expressed in the vertebrate brain, but its distribution, circuits, and interactions with its putative receptor are unknown. Here, we observed expression of spx1 in the midbrain and hindbrain, and spx2 in the hypothalamic preoptic area using in situ RNA hybridization in zebrafish. Analysis of transgenic reporter zebrafish revealed that hindbrain SPX1 neurons are PAX2+ inhibitory interneurons and project to the spinal cord, where they interact with galanin receptor 2b (GALR2b) neurons, suggesting that hindbrain SPX1 neurons are reticulospinal neurons. spx1 mRNA and SPX1 reporter expression were observed in dorsal habenula (dHb). SPX1 neurons in the dHb project to the interpeduncular nucleus (IPN), where GALR2a and GALR2b expression was also observed, suggesting that habenula SPX1 neurons may interact with GALR2a/2b in the IPN.

Yeast genetic interaction screen of human genes associated with amyotrophic lateral sclerosis: identification of MAP2K5 kinase as a potential drug target.

To understand disease mechanisms, a large-scale analysis of human-yeast genetic interactions was performed. Of 1305 human disease genes assayed, 20 genes exhibited strong toxicity in yeast. Human-yeast genetic interactions were identified by en masse transformation of the human disease genes into a pool of 4653 homozygous diploid yeast deletion mutants with unique barcode sequences, followed by multiplexed barcode sequencing to identify yeast toxicity modifiers. Subsequent network analyses focusing on amyotrophic lateral sclerosis (ALS)-associated genes, such as optineurin (OPTN) and angiogenin (ANG), showed that the human orthologs of the yeast toxicity modifiers of these ALS genes are enriched for several biological processes, such as cell death, lipid metabolism, and molecular transport. When yeast genetic interaction partners held in common between human OPTN and ANG were validated in mammalian cells and zebrafish, MAP2K5 kinase emerged as a potential drug target for ALS therapy. The toxicity modifiers identified in this study may deepen our understanding of the pathogenic mechanisms of ALS and other devastating diseases.

Repurpose terbutaline sulfate for amyotrophic lateral sclerosis using electronic medical records.

Prediction of new disease indications for approved drugs by computational methods has been based largely on the genomics signatures of drugs and diseases. We propose a method for drug repositioning that uses the clinical signatures extracted from over 13 years of electronic medical records from a tertiary hospital, including >9.4 M laboratory tests from >530,000 patients, in addition to diverse genomics signatures. Cross-validation using over 17,000 known drug-disease associations shows this approach outperforms various predictive models based on genomics signatures and a well-known "guilt-by-association" method. Interestingly, the prediction suggests that terbutaline sulfate, which is widely used for asthma, is a promising candidate for amyotrophic lateral sclerosis for which there are few therapeutic options. In vivo tests using zebrafish models found that terbutaline sulfate prevents defects in axons and neuromuscular junction degeneration in a dose-dependent manner. A therapeutic potential of terbutaline sulfate was also observed when axonal and neuromuscular junction degeneration have already occurred in zebrafish model. Cotreatment with a ß2-adrenergic receptor antagonist, butoxamine, suggests that the effect of terbutaline is mediated by activation of ß2-adrenergic receptors.

Protective effects of caffeic acid phenethyl ester (CAPE) against neomycin-induced hair cell damage in zebrafish.

OBJECTIVE: Caffeic acid phenethyl ester (CAPE) is known to reduce the generation of oxygen-derived free radicals, which is a major mechanism of aminoglycoside-induced ototoxicity. The objective of the present study was to evaluate the effects of CAPE on neomycin-induced ototoxicity in zebrafish (Brn3c: EGFP). METHODS: Five-day post-fertilization zebrafish larvae (n=10) were exposed to 125 µM neomycin and one of the following CAPE concentrations for 1h: 50, 100, 250, 500, or 1000 µM. Ultrastructural changes were evaluated using scanning electron microscopy (SEM). The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) assay and 2-[4-(dimethylamino)styryl]-N-ethylpyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. RESULTS: CAPE decreased neomycin-induced hair cell loss in the neuromasts (500 µM CAPE: 12.7 ± 1.1 cells, 125 µM neomycin only: 6.3 ± 1.1 cells; n = 10, P < 0.05). In the ultrastructural analysis, structures of mitochondria and hair cells were preserved when exposed to 125 µM neomycin and 500 µM CAPE. CAPE decreased apoptosis and mitochondrial damage. CONCLUSION: In the present study, CAPE attenuated neomycin-induced hair cell damage in zebrafish. The results of the current study suggest that neomycin induces apoptosis, and the apoptotic cell death can be prevented by treatment with CAPE in zebrafish.

Protective role of edaravone against neomycin-induced ototoxicity in zebrafish.

Aminoglycosides such as neomycin are one of the most commonly prescribed types of antibiotics worldwide. However, these drugs appear to generate free radicals within the inner ear, which can result in permanent hearing loss. We evaluated the effects of edaravone, a neuroprotective agent, on neomycin-induced ototoxicity in transgenic zebrafish. The 5-day post fertilization (dpf) zebrafish larvae were exposed to 125 µM neomycin and various concentrations of edaravone for 1 h. Hair cell survival was calculated as average numbers of the hair cells in the control group, which was not exposed to neomycin. Ultrastructural changes were evaluated using a scanning electron microscope (SEM) and transmission electron microscope (TEM). Edaravone protected against neomycin-induced hair cell loss in the neuromasts (1000 µM: 11.6 ± 1.1 cells, neomycin only: 5.5 ± 0.5 cells; n = 10, P<0.05) and decreased the TUNEL reaction for detecting apoptosis. In ultrastructural analysis, structures of mitochondria and hair cells within neuromasts were preserved in zebrafish exposed to 125 µM neomycin and 1000 µM edaravone for 1 h. Edaravone protected against neomycin-induced hair cell loss by preventing apoptosis.

Protective role of NecroX-5 against neomycin-induced hair cell damage in zebrafish.

NecroX-5, one of the derivatives of NecroX series compounds, is a mitochondrial reactive oxygen species and reactive nitrogen species scavenger that inhibits cell death against various kinds of oxidative stresses. The objective of the present study was to evaluate the effects of NecroX-5 on neomycin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five days post-fertilization, zebrafish larvae were exposed to 125 µM neomycin and one of the following NecroX-5 concentrations for 1 h: 10, 25, 50, and 75 µM. Hair cells within the neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed using fluorescence microscopy (n = 10). The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and 2-[4-(dimethylamino) styryl]-N-ethylpyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. Ultrastructural changes were evaluated using scanning electron microscopy. NecroX-5 decreased neomycin-induced hair cell loss in the neuromasts (NecroX-5 50 µM: 13.4 ± 2.0 cells, 125 µM neomycin only: 8.1 ± 1.2 cells; n = 10, P < 0.05) and decreased the TUNEL reaction. The ultrastructural analysis showed that the structures of mitochondria and hair cells within the neuromasts were preserved in zebrafish exposed to 125 µM neomycin and 50 µM NecroX-5. NecroX-5 decreased apoptosis and mitochondrial damage. In conclusion, NecroX-5 attenuated neomycin-induced hair cell loss in zebrafish.

Protective Role of Trimetazidine Against Neomycin-induced Hair Cell Damage in Zebrafish.

OBJECTIVES: Trimetazidine (TMZ) is known to reduce the generation of oxygen-derived free radicals. The objective of the present study was to evaluate the effects of TMZ on neomycin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). METHODS: Five-day, postfertilization zebrafish larvae were exposed to 125 µM neomycin and one of the following TMZ concentrations for 1 hour: 10 µM, 100 µM, 500 µM, 1,000 µM, 1,500 µM, or 2,000 µM. Hair cells within the neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed using fluorescence microscopy and confocal microscopy (n=10). Hair cell survival was calculated as a percentage of hair cells in the control group that were not exposed to neomycin. Ultrastructural changes were evaluated using scanning electron microscopy. RESULTS: TMZ protected against neomycin-induced hair cell loss in the neuromasts (TMZ 1,000 µM, 11.2±0.4 cells; 125 µM neomycin only, 4.2±0.5 cells; n=10; P<0.05) and decreased the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction. In the ultrastructural analysis, structures of mitochondria and hair cells within the neuromasts were preserved in zebrafish exposed to 125 µM neomycin and 1,000 µM TMZ. CONCLUSION: TMZ attenuated neomycin-induced hair cell loss in zebrafish. The results of this study suggest that neomycin induces apoptosis, and that apoptotic cell death can be prevented by treatment with tremetazidine.

Generation of demyelination models by targeted ablation of oligodendrocytes in the zebrafish CNS.

Demyelination is the pathological process by which myelin sheaths are lost from around axons, and is usually caused by a direct insult targeted at the oligodendrocytes in the vertebrate central nervous system (CNS). A demyelinated CNS is usually remyelinated by a population of oligodendrocyte progenitor cells, which are widely distributed throughout the adult CNS. However, myelin disruption and remyelination failure affect the normal function of the nervous system, causing human diseases such as multiple sclerosis. In spite of numerous studies aimed at understanding the remyelination process, many questions still remain unanswered. Therefore, to study remyelination mechanisms in vivo, a demyelination animal model was generated using a transgenic zebrafish system in which oligodendrocytes are conditionally ablated in the larval and adult CNS. In this transgenic system, bacterial nitroreductase enzyme (NTR), which converts the prodrug metronidazole (Mtz) into a cytotoxic DNA cross-linking agent, is expressed in oligodendrocyte lineage cells under the control of the mbp and sox10 promoter. Exposure of transgenic zebrafish to Mtz-containing media resulted in rapid ablation of oligodendrocytes and CNS demyelination within 48 h, but removal of Mtz medium led to efficient remyelination of the demyelinated CNS within 7 days. In addition, the demyelination and remyelination processes could be easily observed in living transgenic zebrafish by detecting the fluorescent protein, mCherry, indicating that this transgenic system can be used as a valuable animal model to study the remyelination process in vivo, and to conduct high-throughput primary screens for new drugs that facilitate remyelination.

Protective effects of edaravone against cisplatin-induced hair cell damage in zebrafish.

OBJECTIVE: Edaravone is known to have a potent free radical scavenging effect. The objective of the present study was to evaluate the effects of edaravone on cisplatin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). METHODS: Five day post-fertilization zebrafish larvae were exposed to 1000 µM cisplatin and 50 µM, 100 µM, 250 µM, 500 µM, 750 µM, and 1000 µM concentrations of edaravone for 4h. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed by fluorescence microscopy and confocal microscopy (n=10). Hair cell survival was calculated as a percentage of the hair cells in the control group that were not exposed to cisplatin. Ultrastructural changes were evaluated using scanning electron microscopy and transmission electron microscopy. RESULTS: Edaravone protected cisplatin-induced hair cell loss of neuromasts (edaravone 750 µM: 8.7 ± 1.5 cells, cisplatin 1000 µM only: 3.7 ± 0.9 cells; n=10, p<0.0001) and decreased the Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction. Structures of mitochondria and hair cell within neuromasts in ultrastructural analysis were preserved in zebrafish exposed to 1000 µM cisplatin and 750 µM edaravone for 4h. CONCLUSIONS: Edaravone attenuated cisplatin-induced hair cell damage in zebrafish. The results of the current study suggest that cisplatin induces apoptosis, and the apoptotic cell death can be prevented by treatment with edaravone in zebrafish.

Indian hedgehog B function is required for the specification of oligodendrocyte progenitor cells in the zebrafish CNS.

A subset of ventral spinal cord precursors, known as pMN precursor cells, initially generate motor neurons and then oligodendrocyte progenitor cells (OPCs), which migrate and differentiate as myelinating oligodendrocytes in the developing neural tube. The switch between motor neuron and oligodendrocyte production by the pMN neural precursors is an important step in building a functional nervous system. However, the precise mechanism that orchestrates the sequential generation of motor neurons and oligodendrocytes within the common population of pMN precursors is still unclear. The current study demonstrates that Indian Hedgehog b (Ihhb), previously known as Echidna Hedgehog, begins to be expressed in the floor plate cells of the ventral spinal cord at the time of OPC specification in zebrafish embryos. Ihhb loss-of-function analysis revealed that Ihhb function is required for OPC specification from pMN precursors by negatively regulating the proliferation of neural precursors. Finally, results showed that Sonic Hedgehog (Shh) could not replace Ihhb function in OPC specification, suggesting that Ihhb and Shh play separate roles in OPC specification. Altogether, data from the present study suggested a novel mechanism, mediated by Ihhb, for the sequential generation of motor neurons and oligodendrocytes from pMN precursors in the ventral spinal cord of zebrafish embryos.

Protective effects of apocynin on cisplatin-induced ototoxicity in an auditory cell line and in zebrafish.

Cisplatin is a very effective anticancer drug and generates reactive oxygen species (ROS) such as superoxide anions that can deplete antioxidant protective molecules in the cochlea. These processes result in the death of cochlear hair cells by induction of apoptosis. Apocynin, which is used as a specific nicotinamide adenine dinucleotide phosphate oxidase inhibitor, has a preventive effect for intracellular ROS generation. In this study, the effect of apocynin was investigated in a cochlear organ of Corti-derived cell line, HEI-OC1 cells, and in transgenic zebrafish (Brn3C: EGFP). To investigate the protective effects of apocynin, HEI-OC1 cells were treated with various concentrations of apocynin and a 20 µm concentration of cisplatin, simultaneously. An in vivo study of transgenic zebrafish (Brn3C: EGFP) was used to investigate the protective effects of apocynin on cisplatin-induced hair cell death. In an in vitro study, apocynin appeared to protect against cisplatin-induced apoptotic features on Hoechst 33258 staining in the HEI-OC1 cells. Treatment of the HEI-OC1 cells with 100 µm of apocynin, significantly decreased caspase-3 activity. Treatment of the cells with a 100 µm concentration of apocynin and a 20 µm concentration of cisplatin significantly decreased the intracellular ROS production. In the in vivo study, apocynin significantly decreased the TUNEL reaction and prevented cisplatin-induced hair cell loss of the neuromasts in the transgenic zebrafish at low concentrations (125 and 250 µm). These findings suggest that apocynin has antioxidative effects and prevents cisplatin-induced apoptotic cell death in HEI-OC1 cells as well as in zebrafish.

Antagonistic regulation of PAF1C and p-TEFb is required for oligodendrocyte differentiation.

Oligodendrocytes are myelinating glial cells in the CNS and are essential for proper neuronal function. During development, oligodendrocyte progenitor cells (OPCs) are specified from the motor neuron precursor domain of the ventral spinal cord and differentiate into myelinating oligodendrocytes after migration to the white matter of the neural tube. Cell cycle control of OPCs influences the balance between immature OPCs and myelinating oligodendrocytes, but the precise mechanism regulating the differentiation of OPCs into myelinating oligodendrocytes is unclear. To understand the mechanisms underlying oligodendrocyte differentiation, an N-ethyl-N-nitrosourea-based mutagenesis screen was performed and a zebrafish leo1 mutant, dalmuri (dal(knu6)) was identified in the current study. Leo1 is a component of the evolutionarily conserved RNA polymerase II-associated factor 1 complex (PAF1C), which is a positive regulator of transcription elongation. The dal(knu6) mutant embryos specified motor neurons and OPCs normally, and at the appropriate time, but OPCs subsequently failed to differentiate into myelinating oligodendrocytes and were eliminated by apoptosis. A loss-of-function study of cdc73, another member of PAF1C, showed the same phenotype in the CNS, indicating that PAF1C function is required for oligodendrocyte differentiation. Interestingly, inhibition of positive transcription elongation factor b (p-TEFb), rescued downregulated gene expression and impaired oligodendrocyte differentiation in the dal(knu6) mutant and Cdc73-deficient embryos. Together, these results indicate that antagonistic regulation of gene expression by PAF1C and p-TEFb plays a crucial role in oligodendrocyte development in the CNS.

Microarray screening for genes involved in oligodendrocyte differentiation in the zebrafish CNS.

Within the vertebrate nervous system, myelination is required for the normal function of neurons by facilitating the rapid conduction of action potentials along axons. Oligodendrocytes are glial cells which myelinate axons in the central nervous system. Disruption of myelination and remyelination failure can cause human diseases such as multiple sclerosis. Despite the importance of myelination, the molecular basis of oligodendrocyte differentiation and myelination are still poorly understood. To understand the molecular mechanisms which regulate oligodendrocyte differentiation and myelination, novel genes were identified using a microarray analysis. The analysis used oligodendrocyte lineage cells isolated from transgenic zebrafish expressing fluorescent proteins in the oligodendrocyte lineage cells. Seven genes not previously known to be involved in oligodendrocyte differentiation were identified, and their expression during oligodendrocyte development was validated.

Tcf3 function is required for the inhibition of oligodendroglial fate specification in the spinal cord of zebrafish embryos.

The generation of various subtypes of neurons and glial cells at the right time and place is crucial for the proper development of the vertebrate CNS. Although the mechanisms and factors for the regulation of neuronal diversity in the CNS have been well studied, the mechanisms regulating the sequential production of neuronal and glial cells from neural precursors remain poorly understood. This study shows that Tcf3, a member of the Lef/Tcf family of proteins, is required to inhibit the premature oligodendroglial fate specification of spinal cord precursors using the transgenic zebrafish, which expresses a dominant repressor form of Tcf3 under the control of a heat-shock inducible promoter. In addition, the data revealed that Tcf3 function in oligodendroglial fate specification is mediated independently of canonical Wnt signaling. Altogether, these results show a novel function for Tcf3 in regulating the timing of oligodendroglial fate specification in the spinal cord.

Neuron-specific expression of atp6v0c2 in zebrafish CNS.

Vacuolar ATPase (V-ATPase) is a multi-subunit enzyme that plays an important role in the acidification of a variety of intracellular compartments. ATP6V0C is subunit c of the V(0) domain that forms the proteolipid pore of the enzyme. In the present study, we investigated the neuron-specific expression of atp6v0c2, a novel isoform of the V-ATPase c-subunit, during the development of the zebrafish CNS. Zebrafish atp6v0c2 was isolated from a genome-wide analysis of the zebrafish mib(ta52b) mutant designed to identify genes differentially regulated by Notch signaling. Whole-mount in situ hybridization revealed that atp6v0c2 is expressed in a subset of CNS neurons beginning several hours after the emergence of post-mitotic neurons. The ATP6V0C2 protein is co-localized with the presynaptic vesicle marker, SV2, suggesting that it is involved in neurotransmitter storage and/or secretion in neurons. In addition, the loss-of-function experiment suggests that ATP6V0C2 is involved in the control of neuronal excitability.

Visualization of myelination in GFP-transgenic zebrafish.

The insulation of axons in the vertebrate nervous system by myelin is essential for efficient axonal conduction. Myelination disruption and remyelination failure can cause human diseases, such as multiple sclerosis and hereditary myelin diseases. However, despite progress in understanding myelination regulation, many important questions remain unanswered. To investigate the mechanisms underlying myelination in vivo, we generated transgenic zebrafish expressing enhanced green fluorescent protein (EGFP) under the control of the mbp promoter. This transgenic fish displayed faithful EGFP expression in oligodendrocytes and Schwann cells in embryonic and adult zebrafish. Interestingly, although myelination progressed continuously in the postembryonic central nervous system, some of the spinal cord regions were filled with unmyelinated axons even in the adult spinal cord, suggesting functional differences between myelinated and unmyelinated axons. Our results suggest that this transgenic zebrafish could be a valuable animal model to study oligodendrocyte differentiation and myelination in vivo.

Intravital imaging in zebrafish using quantum dots.

BACKGROUND/AIMS: Fluorescent quantum dots (QDs) are powerful multipurpose interfaces of nanotechnology providing long-term and multicolor imaging of cellular and molecular interactions. The application of QDs in living organisms is just beginning to be explored, and zebrafish embryos may be suitable vertebrate model organisms for intravital imaging with QDs. To investigate their potential in skin research, we used QDs as microangiography contrast agents and attempted to visualize the cardiovascular system in zebrafish. We also attempted to find the pathway relationship between the cardiovascular system and the nerve network using QDs together with the transgenic zebrafish line. METHOD: Quantum Dot QD605, which reveals green color under the fluorescent microscope, was used as a microangiography contrast agent. The olig2-Dsred transgenic zebrafish line, which expresses motor neurons in red color, was used together with QDs. Images of QD605-injected embryos were recorded with a digital camera. RESULTS: Combining the green fluorescence of QD605 and the red fluorescence of olig2-Dsred transgenic zebrafish, we could obtain detailed images manifesting the spatial relationship between the vascular and the nervous system of zebrafish CONCLUSION: QDs could easily be used as a bright microangiography agent in living embryos. Our image of the vascular and motor nervous system in zebrafish showed a similar pattern of trajectory overall. However, their segmented repetitive networks along the dorsoventral axis were not completely overlapped.