RESUMEN
Adult zebrafish are able to heal large-sized cutaneous wounds in hours with little to no scarring. This rapid re-epithelialization is crucial for preventing infection and jumpstarting the subsequent regeneration of damaged tissues. Despite significant progress in understanding this process, it remains unclear how vast numbers of epithelial cells are orchestrated on an organismic scale to ensure the timely closure of millimeter-sized wounds. Here, we report an unexpected role of adult zebrafish appendages (fins) in accelerating the re-epithelialization process. Through whole-body monitoring of single-cell dynamics in live animals, we found that fin-resident epithelial cells (FECs) are highly mobile and migrate to cover wounds in nearby body regions. Upon injury, FECs readily undergo organ-level mobilization, allowing for coverage of body surfaces of up to 4.78 mm2 in less than 8 h. Intriguingly, long-term fate-tracking experiments revealed that the migratory FECs are not short-lived at the wound site; instead, the cells can persist on the body surface for more than a year. Our experiments on "fin-less" and "fin-gaining" individuals demonstrated that the fin structures are not only capable of promoting rapid re-epithelialization but are also necessary for the process. We further found that fin-enriched extracellular matrix laminins promote the active migration of FECs by facilitating lamellipodia formation. These findings lead us to conclude that appendage structures in regenerative vertebrates, such as fins, may possess a previously unrecognized function beyond serving as locomotor organs. The appendages may also act as a massive reservoir of healing cells, which speed up wound closure and tissue repair.
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BACKGROUND: Metabolic associated fatty liver disease (MAFLD), a prevalent liver disorder affecting one-third of the global population, encompasses a spectrum ranging from fatty liver to severe hepatic steatosis. Both genetic and lifestyle factors, particularly diet and nutrition, contribute to its etiology. Folate deficiency, a frequently encountered type of malnutrition, has been associated with the pathogenesis of MAFLD and shown to impact lipid deposition. However, the underlying mechanisms of this relationship remain incompletely understood. We investigated the impact of disturbed folate-mediated one-carbon metabolism (OCM) on hepatic lipid metabolism both in vitro using human hepatoma cells and in vivo using transgenic fluorescent zebrafish displaying extent-, stage-, and duration-controllable folate deficiency upon induction. RESULTS: Disturbed folate-mediated one-carbon metabolism, either by inducing folate deficiency or adding anti-folate drug, compromises autophagy and causes lipid accumulation in liver cells. Disturbed folate status down-regulates cathepsin L, a key enzyme involved in autophagy, through inhibiting mTOR signaling. Interfered mitochondrial biology, including mitochondria relocation and increased fusion-fission dynamics, also occurs in folate-deficient hepatocytes. Folate supplementation effectively mitigated the impaired autophagy and lipid accumulation caused by the inhibition of cathepsin L activity, even when the inhibition was not directly related to folate deficiency. CONCLUSIONS: Disruption of folate-mediated OCM diminishes cathepsin L expression and impedes autophagy via mTOR signaling, leading to lipid accumulation within hepatocytes. These findings underscore the crucial role of folate in modulating autophagic processes and regulating lipid metabolism in the liver.
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Autofagia , Ácido Fólico , Hepatocitos , Homeostasis , Metabolismo de los Lípidos , Pez Cebra , Autofagia/fisiología , Ácido Fólico/metabolismo , Humanos , Hepatocitos/metabolismo , Animales , Deficiencia de Ácido Fólico/metabolismoRESUMEN
The intricate interplay between biomechanical and biochemical pathways in modulating morphogenesis is an interesting research topic. How biomechanical force regulates epithelial cell tubulogenesis remains poorly understood. Here, we established a model of tubulogenesis by culturing renal proximal tubular epithelial cells on a collagen gel while manipulating contractile force. Epithelial cells were dynamically self-organized into tubule-like structures by augmentation of cell protrusions and cell-cell association. Reduction and asymmetric distribution of phosphorylated myosin light chain 2, the actomyosin contractility, in cells grown on soft matrix preceded tube connection. Notably, reducing matrix stiffness via sonication of collagen fibrils and inhibiting actomyosin contractility with blebbistatin promoted tubulogenesis, whereas inhibition of cytoskeleton polymerization suppressed it. CXC chemokine ligand 1 (CXCL1) expression was transcriptionally upregulated in cells undergoing tubulogenesis. Additionally, inhibiting actomyosin contractility facilitated CXCL1 polarization and cell protrusions preceding tube formation. Conversely, inhibiting the CXCL1-CXC receptor 1 pathway hindered cell protrusions and tubulogenesis. Mechanical property asymmetry with cell-collagen fibril interaction patterns at cell protrusions and along the tube structure supported the association of anisotropic contraction with tube formation. Furthermore, suppressing the mechanosensing machinery of integrin subunit beta 1 reduced CXCL1 expression, collagen remodeling, and impaired tubulogenesis. In summary, symmetry breaking of cell contractility on a soft collagen gel promotes CXCL1 polarization at cell protrusions which in turn facilitates cell-cell association and thus tubule connection.
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Actomiosina , Colágeno , Actomiosina/metabolismo , Matriz Extracelular/metabolismo , Morfogénesis , Células Epiteliales/metabolismoRESUMEN
Change in cell size may bring in profound impact to cell function and survival, hence the integrity of the organs consisting of those cells. Nevertheless, how cell size is regulated remains incompletely understood. We used the fluorescent zebrafish transgenic line Tg-GGH/LR that displays inducible folate deficiency (FD) and hepatomegaly upon FD induction as in vivo model. We found that FD caused hepatocytes enlargement and increased liver stiffness, which could not be prevented by nucleotides supplementations. Both in vitro and in vivo studies indicated that RIPK3/MLKL-dependent necroptotic pathway and Hippo signaling interactively participated in this FD-induced hepatocytic enlargement in a dual chronological and cooperative manner. FD also induced hepatic inflammation, which convenes a dialog of positive feedback loop between necroptotic and Hippo pathways. The increased MMP13 expression in response to FD elevated TNFα level and further aggravated the hepatocyte enlargement. Meanwhile, F-actin was circumferentially re-allocated at the edge under cell membrane in response to FD. Our results substantiate the interplay among intracellular folate status, pathways regulation, inflammatory responses, actin cytoskeleton and cell volume control, which can be best observed with in vivo platform. Our data also support the use of this Tg-GGH/LR transgenic line for the mechanistical and therapeutic research for the pathologic conditions related to cell size alteration.
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Necroptosis , Pez Cebra , Animales , Animales Modificados Genéticamente , Ácido Fólico/metabolismo , Hepatocitos/metabolismo , Hepatomegalia/metabolismo , Hipertrofia/metabolismo , Inflamación/patología , Pez Cebra/genéticaRESUMEN
Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule-cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions.
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Microtúbulos , Huso Acromático , Interfase , Microtúbulos/metabolismo , Huso Acromático/metabolismoAsunto(s)
Tejido Adiposo , Proteínas de la Matriz Extracelular/metabolismo , Inflamación , Macrófagos , Obesidad , Proteína-Lisina 6-Oxidasa/metabolismo , Tejido Adiposo/enzimología , Tejido Adiposo/metabolismo , Tejido Adiposo/fisiopatología , Animales , Femenino , Humanos , Inflamación/enzimología , Inflamación/metabolismo , Inflamación/fisiopatología , Macrófagos/citología , Macrófagos/enzimología , Macrófagos/fisiología , Masculino , Ratones , Ratones Obesos , Obesidad/enzimología , Obesidad/metabolismo , Obesidad/fisiopatologíaRESUMEN
OBJECTIVE: Congenital eye diseases are multi-factorial and usually cannot be cured. Therefore, proper preventive strategy and understanding the pathomechanism underlying these diseases become important. Deficiency in folate, a water-soluble vitamin B, has been associated with microphthalmia, a congenital eye disease characterized by abnormally small and malformed eyes. However, the causal-link and the underlying mechanism between folate and microphthalmia remain incompletely understood. METHODS: We examined the eye size, optomotor response, intracellular folate distribution, and the expression of folate-requiring enzymes in zebrafish larvae displaying folate deficiency (FD) and ocular defects. RESULTS: FD caused microphthalmia and impeded visual ability in zebrafish larvae, which were rescued by folate and dNTP supplementation. Cell cycle analysis revealed cell accumulation at S-phase and sub-G1 phase. Decreased cell proliferation and increased apoptosis were found in FD larvae during embryogenesis in a developmental timing-specific manner. Lowered methylenetetrahydrofolate reductase (mthfr) expression and up-regulated methylenetetrahydrofolate dehydrogenase (NADP+-dependent)-1-like (mthfd1L) expression were found in FD larvae. Knocking-down mthfd1L expression worsened FD-induced ocular anomalies; whereas increasing mthfd1L expression provided a protective effect. 5-CH3-THF is the most sensitive folate pool, whose levels were the most significantly reduced in response to FD; whereas 10-CHO-THF levels were less affected. 5-CHO-THF is the most effective folate adduct for rescuing FD-induced microphthalmia and defective visual ability. CONCLUSION: FD impeded nucleotides formation, impaired cell proliferation and differentiation, caused apoptosis and interfered active vitamin A production, contributing to ocular defects. The developmental timing-specific and incoherent fluctuation among folate adducts and increased expression of mthfd1L in response to FD reflect the context-dependent regulation of folate-mediated one-carbon metabolism, endowing the larvae to prioritize the essential biochemical pathways for supporting the continuous growth in response to folate depletion.
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[Figure: see text].
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Inductores de la Angiogénesis/farmacología , Células Endoteliales/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Podosomas/efectos de los fármacos , Trombomodulina/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Quinasas Asociadas a rho/metabolismo , Actinas/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/enzimología , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Hiperoxia/complicaciones , Masculino , Microdominios de Membrana/enzimología , Ratones Endogámicos C57BL , Ratones Noqueados , Podosomas/enzimología , Neovascularización Retiniana/enzimología , Neovascularización Retiniana/etiología , Neovascularización Retiniana/genética , Transducción de Señal , Trombomodulina/genética , Cicatrización de Heridas , Quinasas Asociadas a rho/genéticaRESUMEN
Lung injury is one of the pathological hallmarks of most respiratory tract diseases including asthma, acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). It involves progressive pulmonary tissue damages which are usually irreversible and incurable. Therefore, strategies to facilitate drug development against lung injury are needed. Here, we characterized the zebrafish folate-deficiency (FD) transgenic line that lacks a fully-developed swim bladder. Whole-mount in-situ hybridization revealed comparable distribution patterns of swim bladder tissue markers between wild-type and FD larvae, suggesting a proper development of swim bladder in early embryonic stages. Unexpectedly, neutrophils infiltration was not observed in the defective swim bladder. Microarray analysis revealed a significant increase and decrease of the transcripts for cathepsin L and a cystatin B (CSTB)-like (zCSTB-like) proteins, respectively, in FD larvae. The distribution of cathepsin L and the zCSTB-like transcripts was spatio-temporally specific in developing wild-type embryos and, in appropriate measure, correlated with their potential roles in maintaining swim bladder integrity. Supplementing with 5-formyltetrahydrofolate successfully prevented the swim bladder anomaly and the imbalanced expression of cathepsin L and the zCSTB-like protein induced by folate deficiency. Injecting the purified recombinant zebrafish zCSTB-like protein alleviated FD-induced swim bladder anomaly. We concluded that the imbalanced expression of cathepsin L and the zCSTB-like protein contributed to the swim bladder malformation induced by FD and suggested the potential application of this transgenic line to model the lung injury and ECM remodeling associated with protease/protease inhibitor imbalance.
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Sacos Aéreos/patología , Catepsina L/metabolismo , Cistatina B/metabolismo , Endopeptidasas/metabolismo , Deficiencia de Ácido Fólico/complicaciones , Lesión Pulmonar/etiología , Inhibidores de Proteasas/metabolismo , Pez Cebra/fisiología , Sacos Aéreos/metabolismo , Secuencia de Aminoácidos , Animales , Biomarcadores/metabolismo , Catepsina L/genética , Cistatina B/química , Cistatina B/genética , Modelos Animales de Enfermedad , Embrión no Mamífero/patología , Desarrollo Embrionario , Larva/metabolismo , Lesión Pulmonar/metabolismo , Lesión Pulmonar/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Pez Cebra/embriología , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: Thrombomodulin (TM), an integral membrane protein, has long been known for its anticoagulant activity. Recent studies showed that TM displays multifaceted activities, including the involvement in cell adhesion and collective cell migration in vitro. However, whether TM contributes similarly to these biological processes in vivo remains elusive. METHODS: We adapted zebrafish, a prominent animal model for studying molecular/cellular activity, embryonic development, diseases mechanism and drug discovery, to examine how TM functions in modulating cell migration during germ layer formation, a normal and crucial physiological process involving massive cell movement in the very early stages of life. In addition, an in vivo assay was developed to examine the anti-hemostatic activity of TM in zebrafish larva. RESULTS: We found that zebrafish TM-b, a zebrafish TM-like protein, was expressed mainly in vasculatures and displayed anti-hemostatic activity. Knocking-down TM-b led to malformation of multiple organs, including vessels, heart, blood cells and neural tissues. Delayed epiboly and incoherent movement of yolk syncytial layer were also observed in early TM-b morphants. Whole mount immunostaining revealed the co-localization of TM-b with both actin and microtubules in epibolic blastomeres. Single-cell tracking revealed impeded migration of blastomeres during epiboly in TM-b-deficient embryos. CONCLUSION: Our results showed that TM-b is crucial to the collective migration of blastomeres during germ layer formation. The structural and functional compatibility and conservation between zebrafish TM-b and mammalian TM support the properness of using zebrafish as an in vivo platform for studying the biological significance and medical use of TM.
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Estratos Germinativos/embriología , Morfogénesis , Organogénesis , Trombomodulina/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Blastómeros/metabolismo , Embrión no Mamífero/embriología , Trombomodulina/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Skeletal muscle development requires the cell-cell fusion of differentiated myoblasts to form muscle fibers. The actin cytoskeleton is known to be the main driving force for myoblast fusion; however, how actin is organized to direct intercellular fusion remains unclear. Here we show that an actin- and dynamin-2-enriched protrusive structure, the invadosome, is required for the fusion process of myogenesis. Upon differentiation, myoblasts acquire the ability to form invadosomes through isoform switching of a critical invadosome scaffold protein, Tks5. Tks5 directly interacts with and recruits dynamin-2 to the invadosome and regulates its assembly around actin filaments to strengthen the stiffness of dynamin-actin bundles and invadosomes. These findings provide a mechanistic framework for the acquisition of myogenic fusion machinery during myogenesis and reveal a novel structural function for Tks5 and dynamin-2 in organizing actin filaments in the invadosome to drive membrane fusion.
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Citoesqueleto de Actina/fisiología , Fusión Celular , Dinamina II/metabolismo , Fusión de Membrana , Mioblastos/fisiología , Proteínas de Unión a Fosfato/metabolismo , Podosomas/metabolismo , Animales , Comunicación Celular , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Ratones , Mioblastos/citologíaRESUMEN
Folate (vitamin B9) is an essential nutrient required for cell survival, proliferation, differentiation and therefore embryogenesis. Folate deficiency has been associated with many diseases, including congenital heart diseases and megaloblastic anemia, yet the mechanisms underlying these remains elusive. Here, we examine the impact of folate deficiency on the development of the circulation system using a zebrafish transgenic line which displays inducible folate deficiency. Impaired hematopoiesis includes decreased hemoglobin levels, decreased erythrocyte number, increased erythrocyte size and aberrant c-myb expression pattern were observed in folate deficient embryos. Cardiac defects, including smaller chamber size, aberrant cardiac function and cmlc2 expression pattern, were also apparent in folate deficient embryos. Characterization of intracellular folate content in folate deficiency revealed a differential fluctuation among the different folate derivatives that carry a single carbon group at different oxidation levels. Rescue attempts by folic acid and nucleotides resulted in differential responses among affected tissues, suggesting that different pathomechanisms are involved in folate deficiency-induced anomalies in a tissue-specific manner. The results of the current study provide an explanation for the inconsistent outcome observed clinically in patients suffering from folate deficiency and/or receiving folate supplementation. This study also supports the use of this model for further research on the defective cardiogenesis and hematopoiesis caused by folate deficiency.
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Circulación Sanguínea , Deficiencia de Ácido Fólico/fisiopatología , Larva/metabolismo , Pez Cebra/crecimiento & desarrollo , Animales , Animales Modificados Genéticamente , Movimiento Celular , Proliferación Celular , Desarrollo Embrionario , Corazón/embriología , Hematopoyesis , Pez Cebra/embriologíaRESUMEN
Folate is a nutrient essential for the development, function and regeneration of nervous systems. Folate deficiency has been linked to many neurological disorders including neural tube defects in fetus and Alzheimer's diseases in the elderly. However, the etiology underlying these folate deficiency-associated diseases is not completely understood. In this study, zebrafish transgenic lines with timing and duration-controllable folate deficiency were developed by ectopically overexpressing a recombinant EGFP-γ-glutamyl hydrolase (γGH). Impeded neural crest cell migration was observed in the transgenic embryos when folate deficiency was induced in early stages, leading to defective neural tube closure and hematopoiesis. Adding reduced folate or N-acetylcysteine reversed the phenotypic anomalies, supporting the causal link between the increased oxidative stress and the folate deficiency-induced abnormalities. When folate deficiency was induced in aged fish accumulation of beta-amyloid and phosphorylated Tau protein were found in the fish brain cryo-sections. Increased autophagy and accumulation of acidic autolysosome were apparent in folate deficient neuroblastoma cells, which were reversed by reduced folate or N-acetylcysteine supplementation. Decreased expression of cathepsin B, a lysosomal protease, was also observed in cells and tissue with folate deficiency. We concluded that folate deficiency-induced oxidative stress contributed to the folate deficiency-associated neuropathogenesis in both early and late stages of life.
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Envejecimiento/genética , Enfermedad de Alzheimer/etiología , Deficiencia de Ácido Fólico , Defectos del Tubo Neural/etiología , Estrés Oxidativo/genética , Acetilcisteína/metabolismo , Acetilcisteína/farmacología , Enfermedad de Alzheimer/genética , Animales , Animales Modificados Genéticamente , Catepsina B/genética , Catepsina B/metabolismo , Movimiento Celular/genética , Embrión no Mamífero , Ácido Fólico/metabolismo , Deficiencia de Ácido Fólico/complicaciones , Deficiencia de Ácido Fólico/genética , Deficiencia de Ácido Fólico/patología , Proteínas Fluorescentes Verdes/genética , Calor/efectos adversos , Proteínas Asociadas a Microtúbulos/metabolismo , Cresta Neural/fisiología , Defectos del Tubo Neural/genética , Estrés Oxidativo/efectos de los fármacos , Factores de Tiempo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , gamma-Glutamil Hidrolasa/metabolismoRESUMEN
Nanoparticles (NP) that target bone tissue were developed using PLGA-PEG (poly(lactic-co-glycolic acid)-polyethylene glycol) diblock copolymers and bone-targeting moieties based on aspartic acid, (Asp)(n(1,3)). These NP are expected to enable the transport of hydrophobic drugs. The molecular structures were examined by (1)H NMR or identified using mass spectrometry and Fourier transform infrared (FT-IR) spectra. The NP were prepared using the water miscible solvent displacement method, and their size characteristics were evaluated using transmission electron microscopy (TEM) and dynamic light scattering. The bone targeting potential of the NP was evaluated in vitro using hydroxyapatite affinity assays and in vivo using fluorescent imaging in zebrafish and rats. It was confirmed that the average particle size of the NP was <200 nm and that the dendritic Asp3 moiety of the PLGA-PEG-Asp3 NP exhibited the best apatite mineral binding ability. Preliminary findings in vivo bone affinity assays in zebrafish and rats indicated that the PLGA-PEG-ASP3 NP may display increased bone-targeting efficiency compared with other PLGA-PEG-based NP that lack a dendritic Asp3 moiety. These NP may act as a delivery system for hydrophobic drugs, warranting further evaluation of the treatment of bone disease.
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Ácido Aspártico/química , Huesos/metabolismo , Sistemas de Liberación de Medicamentos , Nanopartículas/química , Polietilenglicoles/química , Poliglactina 910/química , Adsorción , Animales , Ácido Aspártico/síntesis química , Ácido Aspártico/farmacología , Huesos/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Durapatita/química , Endocitosis/efectos de los fármacos , Larva/efectos de los fármacos , Ratones Endogámicos BALB C , Minerales/metabolismo , Nanopartículas/ultraestructura , Tamaño de la Partícula , Polietilenglicoles/síntesis química , Polietilenglicoles/farmacología , Poliglactina 910/síntesis química , Poliglactina 910/farmacología , Espectroscopía de Protones por Resonancia Magnética , Ratas , Espectrometría de Masa por Ionización de Electrospray , Electricidad Estática , Factores de Tiempo , Distribución Tisular/efectos de los fármacos , Pez CebraRESUMEN
BACKGROUND: Folate is an essential nutrient for cell survival and embryogenesis. 10-Formyltetrahydrofolate dehydrogenase (FDH) is the most abundant folate enzyme in folate-mediated one-carbon metabolism. 10-Formyltetrahydrofolate dehydrogenase converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2, the only pathway responsible for formate oxidation in methanol intoxication. 10-Formyltetrahydrofolate dehydrogenase has been considered a potential chemotherapeutic target because it was down-regulated in cancer cells. However, the normal physiological significance of 10-Formyltetrahydrofolate dehydrogenase is not completely understood, hampering the development of therapeutic drug/regimen targeting 10-Formyltetrahydrofolate dehydrogenase. METHODS: 10-Formyltetrahydrofolate dehydrogenase expression in zebrafish embryos was knocked-down using morpholino oligonucleotides. The morphological and biochemical characteristics of fdh morphants were examined using specific dye staining and whole-mount in-situ hybridization. Embryonic folate contents were determined by HPLC. RESULTS: The expression of 10-formyltetrahydrofolate dehydrogenase was consistent in whole embryos during early embryogenesis and became tissue-specific in later stages. Knocking-down fdh impeded morphogenetic movement and caused incorrect cardiac positioning, defective hematopoiesis, notochordmalformation and ultimate death of morphants. Obstructed F-actin polymerization and delayed epiboly were observed in fdh morphants. These abnormalities were reversed either by adding tetrahydrofolate or antioxidant or by co-injecting the mRNA encoding 10-formyltetrahydrofolate dehydrogenase N-terminal domain, supporting the anti-oxidative activity of 10-formyltetrahydrofolate dehydrogenase and the in vivo function of tetrahydrofolate conservation for 10-formyltetrahydrofolate dehydrogenase N-terminal domain. CONCLUSIONS: 10-Formyltetrahydrofolate dehydrogenase functioned in conserving the unstable tetrahydrofolate and contributing to the intracellular anti-oxidative capacity of embryos, which was crucial in promoting proper cell migration during embryogenesis. GENERAL SIGNIFICANCE: These newly reported tetrahydrofolate conserving and anti-oxidative activities of 10-formyltetrahydrofolate dehydrogenase shall be important for unraveling 10-formyltetrahydrofolate dehydrogenase biological significance and the drug development targeting 10-formyltetrahydrofolate dehydrogenase.
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Desarrollo Embrionario/genética , Ácido Fólico/metabolismo , Morfogénesis/genética , Estrés Oxidativo/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Secuencia de Aminoácidos , Animales , Ácido Fólico/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Morfolinos , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
Alcoholism induces folate deficiency and increases the risk for embryonic anomalies. However, the interplay between ethanol exposure and embryonic folate status remains unclear. To investigate how ethanol exposure affects embryonic folate status and one-carbon homeostasis, we incubated zebrafish embryos in ethanol and analyzed embryonic folate content and folate enzyme expression. Exposure to 2% ethanol did not change embryonic total folate content but increased the tetrahydrofolate level approximately 1.5-fold. The expression of 10-formyltetrahydrofolate dehydrogenase (FDH), a potential intracellular tetrahydrofolate reservoir, was increased in both mRNA and protein levels. Overexpressing recombinant FDH in embryos alleviated the ethanol-induced oxidative stress in ethanol-exposed embryos. Further characterization of the zebrafish fdh promoter revealed that the -124/+40 promoter fragment was the minimal region required for transactivational activity. The results of site-directed mutagenesis and binding analysis revealed that Sp1 is involved in the basal level of expression of fdh but not in ethanol-induced upregulation of fdh. On the other hand, CEBPα was the protein that mediated the ethanol-induced upregulation of fdh, with an approximately 40-fold increase of fdh promoter activity when overexpressed in vitro. We concluded that upregulation of fdh involving CEBPα helps relieve embryonic oxidative stress induced by ethanol exposure.
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Etanol/farmacología , Estrés Oxidativo/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteínas de Pez Cebra/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Depresores del Sistema Nervioso Central/farmacología , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Ácido Fólico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Regiones Promotoras Genéticas/genética , Tetrahidrofolatos/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Folate is a nutrient crucial for rapidly growing tissues, including developing embryos and cancer cells. Folate participates in the biosynthesis of nucleic acids, proteins, amino acids, S-adenosylmethionine, many neurotransmitters, and some vitamins. The intracellular folate pool consists of different folate adducts, which carry one-carbon units at three different oxidative states and participate in distinct biochemical reactions. Therefore, the content and dynamics of folate adducts will affect the homeostasis of the metabolites generated in these folate-mediated reactions. Currently, the knowledge on the level of each individual folate adduct in developing embryos is limited. With an improved high-performance liquid chromatography protocol, we found that tetrahydrofolate (THF), the backbone of one-carbon carrier, gradually increased and became dominant in developing zebrafish embryos. 5-methyl-tetrahydrofolate (5-CH3-THF) was abundant in unfertilized eggs but decreased rapidly when embryos started to proliferate and differentiate. 10-formyltetrahydrofolate at first increased after fertilization, and then dropped dramatically before reaching a sustained level at later stages. Dihydrofolate (DHF) slightly decreased initially and remained low throughout embryogenesis. Exposure to methotrexate significantly decreased 5-CH3-THF levels and increased DHF pools, besides causing brain ventricle anomaly. Rescuing with leucovorin partly reversed the abnormal phenotype. Unexpectedly, the level of 5-CH3-THF remained low even when leucovorin was added for rescue. Our results show that different folate adducts fluctuated significantly and differentially in concert with the physiological requirement specific for the corresponding developmental stages. Furthermore, methotrexate lowered the level of 5-CH3-THF in developing embryos, which could not be reversed with folate supplementation and might be more substantial to cellular methylation potential and epigenetic control than to nucleotide synthesis.
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Embrión no Mamífero/metabolismo , Tetrahidrofolatos/metabolismo , Animales , Desarrollo Embrionario , Leucovorina , Metotrexato , Tetrahidrofolatos/análisis , Pez CebraRESUMEN
The etiology of epilepsy is a very complicated, multifactorial process that is not completely understood. Therefore, the availability of epilepsy animal models induced by different mechanisms is crucial in advancing our knowledge and developing new therapeutic regimens for this disorder. Considering the advantages of zebrafish, we have developed a seizure model in zebrafish larvae using ginkgotoxin, a neurotoxin naturally occurring in Ginkgo biloba and hypothesized to inhibit the formation of the neurotransmitter γ-aminobutyric acid (GABA). We found that a 2-hour exposure to ginkgotoxin induced a seizure-like behavior in zebrafish larvae. This seizure-like swimming pattern was alleviated by the addition of either pyridoxal-5'-phosphate (PLP) or GABA and responded quickly to the anti-convulsing activity of gabapentin and phenytoin, two commonly prescribed anti-epileptic drugs (AEDs). Unexpectedly, the ginkgotoxin-induced PLP depletion in our experimental setting did not affect the homeostasis of folate-mediated one-carbon metabolism, another metabolic pathway playing a crucial role in neural function that also relies on the availability of PLP. This ginkgotoxin-induced seizure behavior was also relieved by primidone, which had been tested on a pentylenetetrazole-induced zebrafish seizure model but failed to rescue the seizure phenotype, highlighting the potential use and complementarity of this ginkgotoxin-induced seizure model for AED development. Structural and morphological characterization showed that a 2-hour ginkgotoxin exposure did not cause appreciable changes in larval morphology and tissues development. In conclusion, our data suggests that this ginkgotoxin-induced seizure in zebrafish larvae could serve as an in vivo model for epileptic seizure research and potential AED screening.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Conducta Animal , Neurotoxinas/toxicidad , Fosfato de Piridoxal/uso terapéutico , Piridoxina/análogos & derivados , Convulsiones/tratamiento farmacológico , Pez Cebra/fisiología , Ácido gamma-Aminobutírico/uso terapéutico , Animales , Anticonvulsivantes/farmacología , Conducta Animal/efectos de los fármacos , Carbono/metabolismo , Ácido Fólico/metabolismo , Larva/anatomía & histología , Larva/efectos de los fármacos , Modelos Neurológicos , Neuronas/efectos de los fármacos , Neuronas/patología , Pentilenotetrazol , Primidona/farmacología , Primidona/uso terapéutico , Fosfato de Piridoxal/farmacología , Piridoxina/toxicidad , Convulsiones/inducido químicamente , Convulsiones/patología , Natación , Pez Cebra/crecimiento & desarrollo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Capsulin and Musculin are basic helix-loop-helix transcription factors, but their biophysiological roles in zebrafish cranial myogenesis are unclear. Expressions of endogenous capsulin transcripts are detected at the central- (~24-hpf) and at dorsal- and ventral-mesoderm cores (~30-72 hpf) of branchial arches. In contrast, musculin transcripts are expressed as a two-phase manner: early phase (20-22 hpf) expressions of musculin are detected at the head mesoderm, whereas late-phase (36-72 hpf) are detected at all presumptive head-muscle precursors. Knockdown of either capsulin or musculin leads to loss of all cranial muscles without affecting trunk muscle development. The defective phenotypes of Capsulin- and Musculin-morphant can be rescued by co-injection of mRNA of each other. Both myf5 and myod transcripts are down-regulated in the Capsulin-morphant while myod transcripts are up-regulated in the Musculin-morphant. Therefore, we propose a putative regulatory network to understand how capsulin/musculin regulate distinctly either myf5 or myod during zebrafish craniofacial myogenesis.