RESUMEN
The development and characterization of two novel humpback grouper (Cromileptes altivelis) fin cell lines are described in this study. The CA1F3Ex and CA1F4Tr cell lines were developed by explant and trypsinization methods, respectively, in Leibovitz's L15 (L-15) medium supplemented with 20% FBS (fetal bovine serum) and subcultured over 150 times. Cell lines exhibited high stability, as evidenced by the high revival rate (85-95%) and good attachment while seeding after one year of cryostorage. They displayed good seeding (91%) and plating efficiencies (15-25%). The optimum temperature for growth was recorded at 28ËC. Serum requirement decreased with increased passage and lowered to 2% FBS beyond 30-35 passages. However, higher serum concentration (2-20%) caused a concurrent increase in cell growth. Both the cell lines were fibroblast-type, and immunotyping results showed strong reactivity towards the fibroblast marker. Chromosome analysis of these cell lines revealed aneuploidy, and the authenticity was confirmed by mitochondrial Cytochrome C Oxidase Subunit I (COI) genotyping analysis. Cell cycle studies were performed utilizing the flow cytometric technique. CA1F3Ex and CA1F4Tr cell lines showed high transfection efficiency with pEGFP-N1 plasmid using Lipofectamine and cytotoxicity towards heavy metals (Hg and Cd) was also studied. Hence, these continuous cell lines could be employed as in vitro models for aquatic toxicological and genetic manipulation studies.
Asunto(s)
Aletas de Animales , Ciclo Celular , Transfección , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular , Aletas de Animales/citología , Proliferación Celular/efectos de los fármacos , Lubina/genética , Supervivencia Celular/efectos de los fármacosRESUMEN
Intending to compare in vitro cell growth in different conditions, we established cell cultures using fin biopsies of two freshwater fishes, Astyanax bimaculatus and Geophagus proximus. Three different culture media (Leibovitz-L-15, Dulbecco's Modified Eagle Medium [DMEM], and 199) were employed, with or without the addition of AmnioMax, maintaining a standard temperature of 29°C. Based on the results obtained, we standardized a cell growth protocol in which medium 199 was less efficient for both species. Notably, G. proximus cells exhibited superior proliferation in DMEM and L-15 media, whereas A. bimaculatus cells demonstrated better parameters exclusively in the DMEM medium. Successful subculturing of cells with good proliferation index was observed, accompanied by preserved morphological characteristics. Therefore, the methodology outlined in this study represents an advancement in establishing fish cell cultures.
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Técnicas de Cultivo de Célula , Characidae , Medios de Cultivo , Animales , Characidae/crecimiento & desarrollo , Línea Celular , Proliferación Celular , Aletas de Animales/citologíaRESUMEN
The lineage and developmental trajectory of a cell are key determinants of cellular identity. In the vascular system, endothelial cells (ECs) of blood and lymphatic vessels differentiate and specialize to cater to the unique physiological demands of each organ1,2. Although lymphatic vessels were shown to derive from multiple cellular origins, lymphatic ECs (LECs) are not known to generate other cell types3,4. Here we use recurrent imaging and lineage-tracing of ECs in zebrafish anal fins, from early development to adulthood, to uncover a mechanism of specialized blood vessel formation through the transdifferentiation of LECs. Moreover, we demonstrate that deriving anal-fin vessels from lymphatic versus blood ECs results in functional differences in the adult organism, uncovering a link between cell ontogeny and functionality. We further use single-cell RNA-sequencing analysis to characterize the different cellular populations and transition states involved in the transdifferentiation process. Finally, we show that, similar to normal development, the vasculature is rederived from lymphatics during anal-fin regeneration, demonstrating that LECs in adult fish retain both potency and plasticity for generating blood ECs. Overall, our research highlights an innate mechanism of blood vessel formation through LEC transdifferentiation, and provides in vivo evidence for a link between cell ontogeny and functionality in ECs.
Asunto(s)
Vasos Sanguíneos , Transdiferenciación Celular , Vasos Linfáticos , Aletas de Animales/citología , Animales , Vasos Sanguíneos/citología , Linaje de la Célula , Células Endoteliales/citología , Vasos Linfáticos/citología , Pez CebraRESUMEN
Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.
Asunto(s)
Aletas de Animales/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Esbozos de los Miembros/metabolismo , Aletas de Animales/citología , Aletas de Animales/crecimiento & desarrollo , Animales , Secuencia de Bases , Evolución Biológica , Boidae , Bovinos , Pollos , Embrión de Mamíferos , Embrión no Mamífero , Iguanas , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Esbozos de los Miembros/citología , Esbozos de los Miembros/crecimiento & desarrollo , Ratones , Ratones Transgénicos , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Conejos , Genética Inversa/métodos , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Tiburones , Transducción de Señal , PorcinosRESUMEN
NK-lysin, an effector of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), not only exhibits cytotoxic effect in fish cells, but also participates in the immune defense against pathogenic infection. In this study, ORF sequences of RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin were 369 bp. Tissue-specific analysis revealed that the highest expressions of RCC-NK-lysin and WCC-NK-lysin were observed in gill, while the peaked level of WR-NK-lysin mRNA was observed in spleen. A. hydrophila infection sharply increased RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin mRNA expression in liver, trunk kidney and spleen. In addition, elevated levels of NK-lysin mRNA were observed in cultured fin cell lines of red crucian carp (RCC), white crucian carp (WCC) and their hybrid offspring (WR) after Lipopolysaccharide (LPS) challenge. RCC-NK-lysin, WCC-NK-lysin and WR-NK-lysin exerted regulatory roles in inducing ROS generation, modulating mitochondrial membrane potential, decreasing fish cell viability and antagonizing survival signalings, respectively. RCC/WCC/WR-NK-lysin-overexpressing fish could up-regulate expressions of inflammatory cytokines and decrease bacterial loads in spleen. These results indicated that NK-lysin in hybrid fish contained close sequence similarity to those of its parents, possessing the capacities of cytotoxicity and immune defense against bacterial infection.
Asunto(s)
Aeromonas hydrophila , Carpas/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Proteolípidos/inmunología , Aletas de Animales/citología , Animales , Carpas/genética , Supervivencia Celular , Células Cultivadas , Quimera , Enfermedades de los Peces/genética , Enfermedades de los Peces/microbiología , Proteínas de Peces/genética , Expresión Génica , Infecciones por Bacterias Gramnegativas/genética , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/veterinaria , Riñón/metabolismo , Lipopolisacáridos/farmacología , Hígado/metabolismo , Potencial de la Membrana Mitocondrial , Proteolípidos/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Bazo/metabolismo , Bazo/microbiologíaRESUMEN
Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent 'tethering' to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis.
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Aletas de Animales/embriología , Células Epidérmicas/fisiología , Epidermis/embriología , Proteínas Hedgehog/fisiología , Morfogénesis , Proteínas de Pez Cebra/fisiología , Aletas de Animales/citología , Aletas de Animales/metabolismo , Animales , Benzamidas/farmacología , Movimiento Celular , Epidermis/metabolismo , Receptor Patched-2/metabolismo , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/fisiología , Pez CebraRESUMEN
Bacterial LPS is a heat-stable endotoxin and wall components of gram negative bacteria, which can exhibit a toxicological effect on physiology and biochemical activities of fish. In this study, we investigated the effect of LPS exposure on cell viability, oxidative stress, caspase activity and immune-related gene expressions in cultured fin cell lines of red crucian carp, white crucian carp and their hybrid offspring. LPS stimulation could reduce fish cell viability, whereas gene expression levels and promoter activities in inflammatory signals increased dramatically. Moreover, enhanced levels of intracellular oxidative stress and decreased levels of mitochondrial membrane potential (MMP) were observed in LPS-induced fish cells. N-Acetyl-L-cysteine (NAC) could alleviate LPS-stimulated reactive oxygen species (ROS) generation and caspase-3 activity in fish cells. These results suggested that ROS-mediated cytotoxic stress was involved in LPS-induced inflammation and mitochondrial damage in cultured fish cells.
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Apoptosis/efectos de los fármacos , Carpas/fisiología , Fibroblastos/efectos de los fármacos , Lipopolisacáridos/toxicidad , Estrés Oxidativo/efectos de los fármacos , Aletas de Animales/citología , Animales , Carpas/genética , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Hibridación Genética , Inflamación/inducido químicamente , Inflamación/metabolismo , Lipopolisacáridos/administración & dosificación , Mitocondrias/efectos de los fármacos , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de OxígenoRESUMEN
Faithful establishment and maintenance of proportion is seen across biological systems and provides a glimpse at fundamental rules of scaling that underlie development and evolution. Dysregulation of proportion is observed in a range of human diseases and growth disorders, indicating that proper scaling is an essential component of normal anatomy and physiology. However, when viewed through an evolutionary lens, shifts in the regulation of relative proportion are one of the most striking sources of morphological diversity among organisms. To date, the mechanisms via which relative proportion is specified and maintained remain unclear. Through the application of powerful experimental, genetic and molecular approaches, the teleost fin has provided an effective model to investigate the regulation of scaling, size, and relative growth in vertebrate organisms. This article is categorized under: Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration Comparative Development and Evolution > Regulation of Organ Diversity.
Asunto(s)
Aletas de Animales/fisiología , Tamaño Corporal , Aletas de Animales/citología , Animales , PecesRESUMEN
Zebrafish have high regenerative ability in several organs including the fin. Although various mechanisms underlying fin regeneration have been revealed, some mechanisms remain to be elucidated. Recently, extracellular vesicles (EVs) have been the focus of research with regard to their role in cell-to-cell communication. It has been suggested that cells in regenerating tissues communicate using EVs. In this study, we examined the involvement of EVs in the caudal fin regeneration of zebrafish using an in vivo electroporation method. The process of regeneration appeared normal after in vivo electroporation, and the transferred plasmid showed mosaic expression in the blastema. We took advantage of this mosaic expression to observe the distribution of exosomal markers in the blastema. We transferred exosomal markers by in vivo electroporation and identified EVs in the regenerating caudal fin. The results suggest that blastemal cells communicate with other cells via EVs during caudal fin regeneration.
Asunto(s)
Aletas de Animales/fisiología , Electroporación/métodos , Vesículas Extracelulares , Regeneración/fisiología , Pez Cebra/fisiología , Aletas de Animales/citología , Animales , Animales Modificados Genéticamente , Vesículas Extracelulares/metabolismo , Técnicas de Transferencia de Gen , Microscopía Fluorescente/instrumentación , Biología Molecular/instrumentación , Biología Molecular/métodos , Plásmidos/administración & dosificación , Plásmidos/genética , Tetraspanina 30/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Yangtze sturgeon (Acipenser dabryanus) is an endangered endemic freshwater fish of China. Cell-line is a potential means used for long-term preservation of germplasm resources and an ideal in vitro model in place of living organisms for biological studies. Here, culture condition and characterization of fin-derived cell in Yangtze sturgeon were carried out. Tissue explant techniques have been efficiently used in the Yangtze sturgeon caudal fin (YSCF) culture. The YSCF cell line showed a fibroblast-like morphology and stable growth in minimum essential medium eagle's (MEME) supplemented with 10-20% fetal bovine serum at 25°C. Cells were cryopreserved with preservative DMSO in liquid nitrogen and grew normally after recovery. No bacterial, fungal, or mycoplasma contamination was detected in the YSCF cells. Karyotype analysis of the YSCF cells showed that the chromosome numbers of the YSCF ranged from 242 to 273, and the modal chromosome number was identified as 264 at passage 9. The YSCF cells were confirmed from A. dabryanus by assay of 16S rRNA and COI. Furthermore, GFP reporter gene was successfully transferred into YSCF cells and expressed. The established YSCF cell lines will contribute to the preservation of germplasm resources and provide a useful vitro tool for further biological studies in sturgeon species.
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Aletas de Animales/citología , Técnicas de Cultivo de Célula/métodos , Línea Celular/citología , Especies en Peligro de Extinción , Peces/metabolismo , Animales , Secuencia de Bases , Proliferación Celular , Forma de la Célula , Cromosomas , Criopreservación , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Cariotipo , Metafase , ARN Ribosómico 16S/genéticaRESUMEN
As zebrafish develop, black and gold stripes form across their skin due to the interactions of brightly colored pigment cells. These characteristic patterns emerge on the growing fish body, as well as on the anal and caudal fins. While wild-type stripes form parallel to a horizontal marker on the body, patterns on the tailfin gradually extend distally outward. Interestingly, several mutations lead to altered body patterns without affecting fin stripes. Through an exploratory modeling approach, our goal is to help better understand these differences between body and fin patterns. By adapting a prior agent-based model of cell interactions on the fish body, we present an in silico study of stripe development on tailfins. Our main result is a demonstration that two cell types can produce stripes on the caudal fin. We highlight several ways that bone rays, growth, and the body-fin interface may be involved in patterning, and we raise questions for future work related to pattern robustness.
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Modelos Biológicos , Pez Cebra/crecimiento & desarrollo , Aletas de Animales/anatomía & histología , Aletas de Animales/citología , Aletas de Animales/crecimiento & desarrollo , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Simulación por Computador , Epitelio/crecimiento & desarrollo , Conceptos Matemáticos , Mutación , Pigmentación de la Piel/genética , Pigmentación de la Piel/fisiología , Análisis de Sistemas , Pez Cebra/genética , Pez Cebra/fisiologíaRESUMEN
Danio rerio, zebrafish, has been widely used as a non-mammalian vertebrate model organism in various studies. The present research describes to develop and characterize a new cell line from a wild strain Indian zebrafish native to Brahmaputra River, Assam, India. The new cell line designated as DRCF was developed from the caudal fin of D. rerio. The cell line was successfully subcultured up to 31 passages. Growth studies revealed that cell growth of DRCF was optimal at 28 °C in L-15 medium supplemented with 20% FBS. Molecular characterization of the DRCF cell line using mitochondrial genes namely cytochrome oxidase subunit I gene (COI) and 16S rRNA authenticated the true origin of the cell line. The chromosome analysis of the DRCF cell line expressed its 50 diploid chromosome number of D. rerio. The immunocytochemical characterization of the cell line exhibited its fibroblastic morphology. The expression of the green fluorescent protein (GFP) following transfection revealed the suitability of the cell line for transfection studies.
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Aletas de Animales/citología , Pez Cebra/anatomía & histología , Animales , Línea Celular , Proliferación Celular , Cromosomas , Complejo IV de Transporte de Electrones/genética , Genes Mitocondriales , Inmunohistoquímica , India , Microscopía de Contraste de Fase , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Ríos , Estaciones del Año , Células Madre/citología , Transfección , Pez Cebra/genéticaRESUMEN
BACKGROUND: Zebrafish can faithfully regenerate injured fins through the formation of a blastema, a mass of proliferative cells that can grow and develop into the lost body part. After amputation, various cell types contribute to blastema formation, where each cell type retains fate restriction and exclusively contributes to regeneration of its own lineage. Epigenetic changes that are associated with lineage restriction during regeneration remain underexplored. RESULTS: We produce epigenome maps, including DNA methylation and chromatin accessibility, as well as transcriptomes, of osteoblasts and other cells in uninjured and regenerating fins. This effort reveals regeneration as a process of highly dynamic and orchestrated transcriptomic and chromatin accessibility changes, coupled with stably maintained lineage-specific DNA methylation. The epigenetic signatures also reveal many novel regeneration-specific enhancers, which are experimentally validated. Regulatory networks important for regeneration are constructed through integrative analysis of the epigenome map, and a knockout of a predicted upstream regulator disrupts normal regeneration, validating our prediction. CONCLUSION: Our study shows that lineage-specific DNA methylation signatures are stably maintained during regeneration, and regeneration enhancers are preset as hypomethylated before injury. In contrast, chromatin accessibility is dynamically changed during regeneration. Many enhancers driving regeneration gene expression as well as upstream regulators of regeneration are identified and validated through integrative epigenome analysis.
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Aletas de Animales/metabolismo , Linaje de la Célula , Metilación de ADN , Epigenoma , Regeneración , Aletas de Animales/citología , Aletas de Animales/fisiología , Animales , Ensamble y Desensamble de Cromatina , Redes Reguladoras de Genes , Osteoblastos/citología , Osteoblastos/metabolismo , Pez CebraRESUMEN
Teleosts have the ability to regenerate their caudal fin upon amputation. A highly proliferative mass of undifferentiated cells called blastema forms beneath wound epidermis and differentiates to regenerate all missing parts of the fin. To date, the origin and fate of the blastema is not completely understood. However, current hypotheses suggest that the blastema is comprised of lineage-restricted dedifferentiated cells. To investigate the differentiation capacity of regenerating fin-derived cells, primary cultures were initiated from the explants of 2-days post-amputation (dpa) regenerates of juvenile gilthead seabream (Sparus aurata). These cells were subcultured for over 30 passages and were named as BSa2. After 10 passages they were characterized for their ability to differentiate towards different bone cell lineages and mineralize their extracellular matrix, through immunocytochemistry, histology, and RT-PCR. Exogenous DNA was efficiently delivered into these cells by nucleofection. Assessment of lineage-specific markers revealed that BSa2 cells were capable of osteo/chondroblastic differentiation. BSa2 cells were also found to be capable of osteoclastic differentiation, as demonstrated through TRAP-specific staining and pit resorption assay. Here, we describe the development of the first successful cell line viz., BSa2, from S. aurata 2-dpa regenerating caudal fins, which has the ability of multilineage differentiation and is capable of in vitro mineralization. The availability of such in vitro cell systems has the potential to stimulate research on the mechanisms of cell differentiation during fin regeneration and provide new insights into the mechanisms of bone formation.
Asunto(s)
Aletas de Animales/fisiopatología , Diferenciación Celular , Regeneración/fisiología , Dorada , Aletas de Animales/citología , Aletas de Animales/cirugía , Animales , Calcificación Fisiológica/fisiología , Línea Celular , OsteoblastosRESUMEN
Fish have a high ability to regenerate fins, including the caudal fin. After caudal fin amputation, original bi-lobed morphology is reconstructed during its rapid regrowth. It is still controversial whether positional memory in the blastema cells regulates reconstruction of fin morphology as in amphibian limb regeneration, in which limb blastema cells located at the same proximal-distal level have the same positional identity. We investigated growth period and growth rate in zebrafish caudal fin regeneration. We found that both the growth period and growth rate differed for fin rays that were amputated at the same proximal-distal level, indicating that it takes different periods of time for fin rays to restore their original lengths after straight amputation. We also show that more proximal amputation takes longer period to reconstruct the original morphology/size than more distal amputation. Statistical analysis suggested that both the growth period/rate are determined by amputated length (depth) regardless of the fin ray identity along dorsal-ventral axis. In addition, we suggest the possibility that the structural/physical condition such as width of the fin ray at the amputation site (niche at the stump) may determine the growth period/rate.
Asunto(s)
Muñones de Amputación/patología , Muñones de Amputación/fisiopatología , Aletas de Animales/citología , Aletas de Animales/fisiología , Proliferación Celular , Regeneración , Pez Cebra/fisiología , AnimalesRESUMEN
We describe a new cell line, Clarias dussumieri fin (ClDuF), from the caudal fin of C. dussumieri using the explant technique followed by cryopreservation. The cryopreserved CiDuF cells were validated for quality and other characteristics. They showed typical epithelial morphology in vitro and epithelial cells outgrew their fibroblast cells after the fifth passage. ClDuF cells had a characteristic sigmoid curve with population doubling in 24 h. Immunotyping of the ClDuF cells against cytokeratin suggested the epithelial lineage. Chromosome analysis showed normal diploid (2n = 50) numbers and the cells did not contain any contamination, including Mycoplasma and other microbes. Partial sequencing of fragments of mitochondrial 16s rRNA and COI genes of ClDuF confirmed that the cell line was initiated from C. dussumieri. Cells at the 10th and 25th passages had more than 80% and 70% viability in the culture, respectively, after 6 months of storage at LN2 . These ClDuF cells were morphologically identical to the cells before freezing and the genetic resource of C. dussumieri was preserved. The species-specific cells can serve as a valuable source for virus isolation, conservation and cloning of somatic cells.
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Aletas de Animales/citología , Línea Celular , Criopreservación/métodos , Células Epiteliales/citología , Animales , Bagres/genética , Complejo IV de Transporte de Electrones/genética , Congelación , ARN Ribosómico 16S/genéticaRESUMEN
A continuous cell line MPF derived from the fin of black carp Mylopharyngodon piceus was established and characterised in this study. Mylopharyngodon piceus fin (MPF) cells were subcultured for more than 80 passages with high viability recovery after long-term storage. The karyotyping analysis revealed that MPF had a modal diploid chromosome number (2n = 48) and identical ribosomal RNA sequence with black carp. In addition, the expression of pluripotency-associated markers including nanog, oct4 and vasa, were detected in MPF. The transient transfection efficiency of MPF reached 23% with a fluorescent reporter by modified electroporation and stable expression of red fluorescent MPF was established by the baculovirus system, indicating that MPF is an ideal platform for studying gene functions in vitro. Lastly, cytopathic effects were also observed and RNA transcripts of a viral gene increased after infection by spring viremia of carp virus (SVCV), suggesting that MPF could be an alternative tool for investigating pathogen-host interactions in black carp. In conclusion, a fin cell line that is susceptible to SVCV was established as a potential adult stem-cell line, providing a suitable tool for future genetic analyses and pathogen-host studies in black carp.
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Aletas de Animales/citología , Cyprinidae , Cultivo Primario de Células/métodos , Rhabdoviridae/crecimiento & desarrollo , Aletas de Animales/metabolismo , Aletas de Animales/virología , Animales , Línea Celular/metabolismo , Línea Celular/virología , Cyprinidae/metabolismo , Cyprinidae/virología , Enfermedades de los Peces/virología , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Peces , Expresión Génica , Marcadores Genéticos/genética , Marcadores Genéticos/fisiología , Predisposición Genética a la Enfermedad , Interacciones Microbiota-Huesped , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/virología , Infecciones por Rhabdoviridae/virología , Transfección/métodosRESUMEN
BACKGROUND: Matrix metalloproteinases 13 (MMP13) is a potent endopeptidase that regulate cell growth, migration, and extracellular matrix remodeling. However, its role in fin regeneration remains unclear. RESULTS: mmp13a expression is strongly upregulated during blastema formation and persists in the distal blastema. mmp13a knockdown via morpholino electroporation impairs regenerative outgrowth by decreasing cell proliferation, which correlates with a downregulation of fgf10a and sall4 expression in the blastema. Laminin distribution in the basement membrane is also affected in mmp13a MO-injected rays. Another impact of mmp13a knockdown is observed in the skeletal elements of the fin rays. Expression of two main components of actinotrichia, Collagen II and Actinodin 1 is highly reduced in mmp13a MO-injected rays leading to highly disorganized actinotrichia pattern. Inhibition of mmp13a strongly affects bone formation as shown by a reduction of Zns5 and sp7 expression and of bone matrix mineralization in rays. These defects are accompanied by a significant increase in apoptosis in mmp13a MO-injected fin regenerates. CONCLUSION: Defects of expression of this multifunctional proteinase drastically affects osteoblast differentiation, bone and actinotrichia formation as well as Laminin distribution in the basement membrane of the fin regenerate, suggesting the important role of Mmp13 during the regenerative process.
Asunto(s)
Osteoblastos/citología , Osteoblastos/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Aletas de Animales/citología , Aletas de Animales/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Laminina/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Retinoic acid (RA) signaling is an important regulator of chordate development. RA binds to nuclear RA receptors that control the transcriptional activity of target genes. Controlled local degradation of RA by enzymes of the Cyp26a gene family contributes to the establishment of transient RA signaling gradients that control patterning, cell fate decisions and differentiation. Several steps in the lineage leading to the induction and differentiation of neuromesodermal progenitors and bone-producing osteogenic cells are controlled by RA. Changes to RA signaling activity have effects on the formation of the bones of the skull, the vertebrae and the development of teeth and regeneration of fin rays in fish. This review focuses on recent advances in these areas, with predominant emphasis on zebrafish, and highlights previously unknown roles for RA signaling in developmental processes.
Asunto(s)
Aletas de Animales/metabolismo , Huesos/metabolismo , Diferenciación Celular , Diente/metabolismo , Tretinoina/metabolismo , Aletas de Animales/citología , Animales , Huesos/citología , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Transducción de Señal/genética , Diente/citología , Tretinoina/química , Pez CebraRESUMEN
The goal of this study was to develop and characterize a cell line from the caudal fin tissue of zebrafish and also its application as an in vitro model to study the effect of H2 O2 in wound healing. Fibroblastic cell line was developed using explant culture method from caudal fin tissue of zebrafish and characterized. This cell line was named as DrF cell line. The DrF cells treated with 0-10 µM/ml H2 O2 were tested for viability, proliferation and motility by MTT assay, trypan blue assay and chemotaxis assay, respectively. Among the different concentrations of H2 O2 , 4 µM was found to be nontoxic to study cell migration in in vitro scratch wound assay. Furthermore, the expression of proliferating cell nuclear antigen (PCNA) and chemokine receptor (CXCR4) genes was carried by qPCR. The cell survival, proliferation and migration were extremely enriched at 4 µM level of H2 O2 . We observed accelerated wound closure in DrF cells treated with H2 O2. The qPCR results indicated that H2 O2 markedly up-regulated mRNA expression of PCNA and CXCR4. The findings from our study suggest that H2 O2 at low levels promotes cell survival, proliferation, migration and wound healing in DrF cells.