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Abstract Since its debut in the biomedical research fields in 1981, zebrafish have been used as a vertebrate model organism in more than 40,000 biomedical research studies. Especially useful are zebrafish lines expressing fluorescent proteins in a molecule, intracellular organelle, cell or tissue specific manner because they allow the visualization and tracking of molecules, intracellular organelles, cells or tissues of interest in real time and in vivo. In this review, we summarize representative transgenic fluorescent zebrafish lines that have revolutionized biomedical research on signal transduction, the craniofacial skeletal system, the hematopoietic system, the nervous system, the urogenital system, the digestive system and intracellular organelles.

Abstract Background IK is a splicing factor that promotes spliceosome activation and contributes to pre-mRNA splicing. Although the molecular mechanism of IK has been previously reported in vitro, the physiological role of IK has not been fully understood in any animal model. Here, we generate an ik knock-out (KO) zebrafish using the CRISPR/Cas9 system to investigate the physiological roles of IK in vivo. Results The ik KO embryos display severe pleiotropic phenotypes, implying an essential role of IK in embryonic development in vertebrates. RNA-seq analysis reveals downregulation of genes involved in skeletal muscle differentiation in ik KO embryos, and there exist genes having improper pre-mRNA splicing among downregulated genes. The ik KO embryos display impaired neuromuscular junction (NMJ) and fast-twitch muscle development. Depletion of ik reduces myod1 expression and upregulates pax7a, preventing normal fast muscle development in a non-cell-autonomous manner. Moreover, when differentiation is induced in IK-depleted C2C12 myoblasts, myoblasts show a reduced ability to form myotubes. However, inhibition of IK does not influence either muscle cell proliferation or apoptosis in zebrafish and C2C12 cells. Conclusion This study provides that the splicing factor IK contributes to normal skeletal muscle development in vivo and myogenic differentiation in vitro.

Ependymal cells are supporting cells in the central nervous system. Here the authors elucidate a signalling axis in zebrafish spinal cord ependymal cells that is important for motile cilia assembly and maintenance, demonstrating that it depends on intercellular propagation of calcium ions via connexin 43.

Anti-melanoma differentiation-associated gene 5 (anti-MDA5) antibody is a myositis-specific marker detected in clinically amyopathic dermatomyositis (DM). DM with anti-MDA5 antibody can be accompanied by rapidly progressive interstitial lung disease (RP-ILD) and other autoimmune disorders. Until now, only one case of neuromyelitis optica (NMO) with anti-MDA5-positive DM has been reported worldwide, in which the patient achieved a favorable outcome with intensive immunotherapy. We report a case of NMO in a patient with anti-MDA5-positive DM complicated by ILD and rheumatoid arthritis. Our patient experienced a fulminant course of NMO, rather than RP-ILD, in the presence of hyperferritinemia, which resulted in profound neurological sequelae despite immunotherapy including rituximab.

Background Knowledge of the genetic etiology of epilepsy can provide essential prognostic information and influence decisions regarding treatment and management, leading us into the era of precision medicine. However, the genetic basis underlying epileptogenesis or epilepsy pharmacoresistance is not well-understood, particularly in non-familial epilepsies with heterogeneous phenotypes that last until or start in adulthood. Methods We sought to determine the contribution of known epilepsy-associated genes (EAGs) to the causation of non-familial epilepsies with heterogeneous phenotypes and to the genetic basis underlying epilepsy pharmacoresistance. We performed a multi-center study for whole exome sequencing-based screening of 178 selected EAGs in 243 non-familial adult patients with primarily focal epilepsy (122 drug-resistant and 121 drug-responsive epilepsies). The pathogenicity of each variant was assessed through a customized stringent filtering process and classified according to the American College of Medical Genetics and Genomics guidelines. Results Possible causal genetic variants of epilepsy were uncovered in 13.2% of non-familial patients with primarily focal epilepsy. The diagnostic yield according to the seizure onset age was 25% (2/8) in the neonatal and infantile period, 11.1% (14/126) in childhood and 14.7% (16/109) in adulthood. The higher diagnostic yields were from ion channel-related genes and mTOR pathway-related genes, which does not significantly differ from the results of previous studies on familial or early-onset epilepsies. These potentially pathogenic variants, which were identified in genes that have been mainly associated with early-onset epilepsies with severe phenotypes, were also linked to epilepsies that start in or last until adulthood in this study. This finding suggested the presence of one or more disease-modifying factors that regulate the onset time or severity of epileptogenesis. The target hypothesis of epilepsy pharmacoresistance was not verified in our study. Instead, neurodevelopment-associated epilepsy genes, such as TSC2 or RELN, or structural brain lesions were more strongly associated with epilepsy pharmacoresistance. Conclusions We revealed a fraction of possible causal genetic variants of non-familial epilepsies in which genetic testing is usually overlooked. In this study, we highlight the importance of earlier identification of the genetic etiology of non-familial epilepsies, which leads us to the best treatment options in terms of precision medicine and to future neurobiological research for novel drug development. This should be considered a justification for physicians determining the hidden genetics of non-familial epilepsies that last until or start in adulthood.

Abstract Background Alexander disease (AxD) is an astrogliopathy that predominantly affects the white matter of the central nervous system (CNS), and is caused by a mutation in the gene encoding the glial fibrillary acidic protein (GFAP), an intermediate filament primarily expressed in astrocytes and ependymal cells. The main pathologic feature of AxD is the presence of Rosenthal fibers (RFs), homogeneous eosinophilic inclusions found in astrocytes. Because of difficulties in procuring patient’ CNS tissues and the presence of RFs in other pathologic conditions, there is a need to develop an in vivo assay that can determine whether a mutation in the GFAP results in aggregation and is thus disease-causing. Methods We found a GFAP mutation (c.382G > A, p.Asp128Asn) in a 68-year-old man with slowly progressive gait disturbance with tendency to fall. The patient was tentatively diagnosed with AxD based on clinical and radiological findings. To develop a vertebrate model to assess the aggregation tendency of GFAP, we expressed several previously reported mutant GFAPs and p.Asp128Asn GFAP in zebrafish embryos. Results The most common GFAP mutations in AxD, p.Arg79Cys, p.Arg79His, p.Arg239Cys and p.Arg239His, and p.Asp128Asn induced a significantly higher number of GFAP aggregates in zebrafish embryos than wild-type GFAP. Conclusions The p.Asp128Asn GFAP mutation is likely to be a disease-causing mutation. Although it needs to be tested more extensively in larger case series, the zebrafish assay system presented here would help clinicians determine whether GFAP mutations identified in putative AxD patients are disease-causing.

Abstract Despite recent interest in using zebrafish in human disease studies, sparked by their economics, fecundity, easy handling, and homologies to humans, the electrophysiological tools or methods for zebrafish are still inaccessible. Although zebrafish exhibit more significant larval–adult duality than any other animal, most electrophysiological studies using zebrafish are biased by using larvae these days. The results of larval studies not only differ from those conducted with adults but also are unable to delicately manage electroencephalographic montages due to their small size. Hence, we enabled non-invasive long-term multichannel electroencephalographic recording on adult zebrafish using custom-designed electrodes and perfusion system. First, we exploited demonstration of long-term recording on pentylenetetrazole-induced seizure models, and the results were quantified. Second, we studied skin–electrode impedance, which is crucial to the quality of signals. Then, seizure propagations and gender differences in adult zebrafish were exhibited for the first time. Our results provide a new pathway for future neuroscience research using zebrafish by overcoming the challenges for aquatic organisms such as precision, serviceability, and continuous water seepage.

Mitochondria maintain a web-shaped network in cells through a balance between fusion and fission. Under certain physiological and pathological conditions, this balance is breached, and as a result, change in mitochondrial morphology ensues. Real-time monitoring of such change is of significant importance for studying mitochondrial physiology and pathology, such as apoptosis, aging, and neurodegeneration. Numerous studies have been conducted in animal cell culture systems concerning mitochondrial morphology change. However, very little is known to date about the real-time changes in mitochondrial morphology at the organism level due to difficulties in observation and administration of mitochondria-disrupting drugs. Here we report the generation of transgenic zebrafish (Danio rerio) expressing mitochondrially targeted green fluorescent protein (GFP). The transparency of transgenic zebrafish embryos make it possible to monitor mitochondrial morphology in real time and in vivo. Since zebrafish inhabit fresh water, incubating zebrafish in drug-dissolved water sufficed to administer drugs to the zebrafish. We observed real-time and in vivo fragmentation of mitochondria in the transgenic embryos upon incubation in water with the following apoptosis-inducing drugs: valinomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and staurosporine. Thus, the transgenic zebrafish we generated could provide a platform for research on apoptosis and mitochondrial physiology and a screen for apoptosis-modulating drugs. It could also facilitate study of the pathogenesis of apoptosis-related diseases.

When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.

Zebrafish models have recently been highlighted as a valuable tool in studying the molecular basis of neuromuscular diseases and developing new pharmacological treatments. Needle electromyography (EMG) is needed not only for validating transgenic zebrafish models with muscular dystrophies (MD), but also for assessing the efficacy of therapeutics. However, performing needle EMG on larval zebrafish has not been feasible due to the lack of proper EMG sensors and systems for such small animals. We introduce a new type of EMG needle electrode to measure intramuscular activities of larval zebrafish, together with a method to hold the animal in position during EMG, without anesthetization. The silicon-based needle electrode was found to be sufficiently strong and sharp to penetrate the skin and muscles of zebrafish larvae, and its shape and performance did not change after multiple insertions. With the use of the proposed needle electrode and measurement system, EMG was successfully performed on zebrafish at 30 days postfertilization (dpf) and at 5 dpf. Burst patterns and spike morphology of the recorded EMG signals were analyzed. The measured single spikes were triphasic with an initial positive deflection, which is typical for motor unit action potentials, with durations of ∼10 ms, whereas the muscle activity was silent during the anesthetized condition. These findings confirmed the capability of this system of detecting EMG signals from very small animals such as 5 dpf zebrafish. The developed EMG sensor and system are expected to become a helpful tool in validating zebrafish MD models and further developing therapeutics. [ABSTRACT FROM AUTHOR]

Background: Mutations in genes that are part of the splicing machinery for myelodysplastic syndromes (MDS), including MDS without ring sideroblasts (RS), have been widely investigated. The effects of these mutations on clinical outcomes have been diverse and contrasting. Methods: We examined a cohort of 129 de novo MDS patients, who did not harbor RS, for mutations affecting three spliceosomal genes (SF3B1, U2AF1, and SRSF2). Results: The mutation rates of SF3B1, U2AF1, and SRSF2 were 7.0 %, 7.8 %, and 10.1 %, respectively. Compared with previously reported results, these rates were relatively infrequent. The SRSF2 mutation strongly correlated with old age (P < 0.001), while the mutation status of SF3B1 did not affect overall survival (OS), progression-free survival (PFS), or acute myeloid leukemia (AML) transformation. In contrast, MDS patients with mutations in U2AF1 or SRSF2 exhibited inferior PFS. The U2AF1 mutation was associated with inferior OS in low-risk MDS patients (P = 0.035). The SRSF2 mutation was somewhat associated with AML transformation (P = 0.083). Conclusion: Our findings suggest that the frequencies of the SF3B1, U2AF1, and SRSF2 splicing gene mutations in MDS without RS were relatively low. We also demonstrated that the U2AF1 and SRSF2 mutations were associated with an unfavorable prognostic impact in MDS patients without RS. [ABSTRACT FROM AUTHOR]

Background: The proliferating activity of a single leukemia stem cell and the molecular mechanisms for their quiescent property remain unknown, and also their prognostic value remains a matter of debate. Therefore, this study aimed to demonstrate the quiescence property and molecular signature of leukemia stem cell and their clinicopathological implications. Methods: Single cell sorting and culture were performed in the various sets of hematopoietic stem cells including CD34+CD38- acute myeloid leukemia (AML) cell population (ASCs) from a total of 60 patients with AML, and 11 healthy controls. Their quiescence related-molecular signatures and clinicopathological parameters were evaluated in AML patients. Results: Single cell plating efficiency of ASCs was significantly lower (8.6%) than those of normal hematopoietic stem cells i.e.: cord blood, 79.0%; peripheral blood, 45.3%; and bone marrow stem cell, 31.1%. Members of the TGFβ super-family signaling pathway were most significantly decreased; as well as members of the Wnt, Notch, pluripotency maintenance and hedgehog pathways, compared with non ASC populations. mtDNA copy number of ASCs was significantly lower than that of corresponding other cell populations. However, our data couldn't support the prognostic value of the ASCs in AML. Conclusions: ASCs showed remarkable lower plating efficiency and slower dividing properties at the single cell level. This quiescence is represented as a marked decrease in the mtDNA copy number and also linked with down-regulation of genes in various molecular pathways. [ABSTRACT FROM AUTHOR]

Background: Mitochondria are highly dynamic organelles whose morphology and position within the cell is tightly coupled to metabolic function. There is a limited list of essential proteins that regulate mitochondrial morphology and the mechanisms that govern mitochondrial dynamics are poorly understood. However, recent evidence indicates that the core machinery that governs mitochondrial dynamics is linked within complex intracellular signalling cascades, including apoptotic pathways, cell cycle transitions and nuclear factor kappa B activation. Given the emerging importance of mitochondrial plasticity in cell signalling pathways and metabolism, it is essential that we develop tools to quantitatively analyse the processes of fission and fusion. In terms of mitochondrial fusion, the field currently relies upon on semi-quantitative assays which, even under optimal conditions, are labour-intensive, low-throughput and require complex imaging techniques. Results: In order to overcome these technical limitations, we have developed a new, highly quantitative cell-free assay for mitochondrial fusion in mammalian cells. This assay system has allowed us to establish the energetic requirements for mitochondrial fusion. In addition, our data reveal a dependence on active protein phosphorylation for mitochondrial fusion, confirming emerging evidence that mitochondrial fusion is tightly integrated within the global cellular response to signaling events. Indeed, we have shown that cytosol derived from cells stimulated with different triggers either enhance or inhibit the cell-free fusion reaction. Conclusions: The adaptation of this system to high-throughput analysis will provide an unprecedented opportunity to identify and characterize novel regulatory factors. In addition, it provides a framework for a detailed mechanistic analysis of the process of mitochondrial fusion and the various axis of regulation that impinge upon this process in a wide range of cellular conditions. See Commentary: http://www.biomedcentral.com/1741-7007/8/99 [ABSTRACT FROM AUTHOR]

Fusion of vesicles into target membranes during many types of regulated exocytosis requires both SNARE-complex proteins and fusogenic lipids, such as phosphatidic acid. Mitochondrial fusion is less well understood but distinct, as it is mediated instead by the protein Mitofusin (Mfn). Here, we identify an ancestral member of the phospholipase D (PLD) superfamily of lipid-modifying enzymes that is required for mitochondrial fusion. Mitochondrial PLD (MitoPLD) targets to the external face of mitochondria and promotes trans-mitochondrial membrane adherence in a Mfn-dependent manner by hydrolysing cardiolipin to generate phosphatidic acid. These findings reveal that although mitochondrial fusion and regulated exocytic fusion are mediated by distinct sets of protein machinery, the underlying processes are unexpectedly linked by the generation of a common fusogenic lipid. Moreover, our findings suggest a novel basis for the mitochondrial fragmentation observed during apoptosis. [ABSTRACT FROM AUTHOR]

Histone lysine methylation, as one of the most important factors in transcriptional regulation, is associated with a various physiological conditions. Using a bioinformatics search, we identified and subsequently cloned mouse SET domain containing 3 (SETD3) with SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) and Rubis-subs-bind domains. SETD3 is a novel histone H3K4 and H3K36 methyltransferase with transcriptional activation activity. SETD3 is expressed abundantly in muscular tissues and, when overexpressed, activates transcription of muscle-related genes, myogenin, muscle creatine kinase (MCK), and myogenic factor 6 (Myf6), thereby inducing muscle cell differentiation. Conversely, knockdown of SETD3 by shRNA significantly retards muscle cell differentiation. In this study, SETD3 was recruited to the myogenin gene promoter along with MyoD where it activated transcription. Together, these data indicate that SETD3 is a H3K4/K36 methyltransferase and plays an important role in the transcriptional regulation of muscle cell differentiation. [ABSTRACT FROM AUTHOR]