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Culture of cells from zebrafish (Brachydanio rerio) blastula-stage embryos
Abstract
The zebrafish has become a popular model for studies of vertebrate development and toxicology. However, in vitro approaches utilizing this organism have not been fully exploited due to the absence of suitable cell culture systems. Previously, we developed methods for the culture of cells derived from zebrafish blastula-stage embryos. One of these cultures, ZEM-2, was derived in a complex medium containing trout embryo extract, trout serum and medium conditioned by buffalo rat liver cells. In this study we describe a zebrafish embryo cell line, ZEM-2A, derived from ZEM-2 following selection for growth in a simplified medium. Optimal growth of ZEM-2A cells is attained in nutrient medium supplemented with 5% fetal bovine serum.
... Similar approaches have not been employed with the zebrafish due to the absence of appropriate cell culture systems. Previously, we established methods for the culture of cells derived from blastula stage zebrafish embryos (Collodi et al., 1992a; Ghosh and Collodi, 1994). The zebrafish blastula is comprised of pluripotent cells (Ho and Kimmel, 1993; Lin et al., 1992). ...
... cyto555.tex; 3/03/1997; 13:13; v.7; p.3 Figure 2A. Figure 2B. embryos (ZEM-2) (Ghosh and Collodi, 1994 ...
... ) and liver (ZF-L) (Ghosh et al., 1994) were used as controls. ...
The zebrafish is a polular nonmammalian model for studies of neural development. We have derived cell cultures, initiated from blastula-stage zebrafish embryos, that differentiate in vitro into neurons and astrocytes. Cultures were initiated in basal nutrient medium supplemented with bovine insulin, trout serum, trout embryo extract and fetal bovine serum. After two weeks in culture the cells exhibited extensive neurite outgrowth and possessed elevated levels of acetylcholinesterase enzyme activity. Ultrastructural analysis revealed that the neurites possessed microtubules, synaptic vessicles and areas exhibiting growth cone morphology. The cultures expressed proteins recognized by antibodies to the neuronal and astrocyte-specific markers, neurofilament and glial fibrillary acidic protein (GFAP). Poly-D-lysine substrate stimulated neurite outgrowth in the cultures and inhibited the growth of nonneuronal cells. Medium conditioned by the buffalo rat liver line, BRL, promoted the growth and survival of the cells in culture. Mitotically active cells were identified in cultures that had undergone extensive differentiation. The embryo cell cultures provide an in vitro system for investigations of biochemical parameters influencing zebrafish neuronal cell growth and differentiation.
... Primary cell cultures from embryonic blastomeres have been used to examine mechanisms of differentiation in species as diverse as sea urchins 9 and humans. 10 In the zebrafish, primary cell culture methods using blastula-stage embryos have been previously described, [11][12][13][14][15] but are not often used in developmental research, despite the usefulness of examining differentiating cells in cultured monolayers. This is largely because zebrafish embryos are both small and transparent enough for the resolution of individual cells, [16][17][18] and so the differential benefits of cell culture systems are reduced when compared to other organisms. ...
... However, existing methods for primary embryonic cell culture in zebrafish require the dissociation of large numbers of early blastula-stage embryos, well before phenotypic identification of mutants or transgene carriers would be possible. 11,14,15 This effectively prohibits the use of heterozygous carrier strains for embryonic lethal mutations, which comprise the majority of useful mutants, since genetically uniform sets of embryos cannot be obtained without phenotypic screening. We have therefore explored the feasibility of generating differentiated cell cultures from single zebrafish embryos, which provide genetically identical populations of cells that can be individually genotyped from unattached cells in the culture medium. ...
... As proof of principle, we have examined spontaneous myogenic differentiation of our single-embryo cultures by both phenotypic and genotypic analysis. Spontaneous myogenesis of dissociated blastula cultures has been previously reported by expression of muscle-specific markers in culture, 14,22 though not at the subcellular level. We examined single-embryo cultures for similar marker expression, particularly the subcellular patterns of actin and myosin expression characteristic of differentiated myocytes. ...
Culturing cells in vitro can produce a uniform population for the study of cellular differentiation, which is especially useful for the quantification of gene expression or the observation of subcellular structures. In zebrafish, a handful of immortalized cell lines have been used for these purposes, despite being heavily selected by passaging. Methods for primary cell culture of zebrafish embryonic blastomeres have been previously reported, but require combining a large number of genetically heterogeneous embryos, meaning that subsequent cell cultures are not clonal. Without genetically uniform cultures, this model system cannot exploit the wealth of available embryonic lethal mutants in zebrafish. We therefore describe methods for the generation of zebrafish embryonic blastomere cell cultures from single genetically characterized embryos. We examined myogenic differentiation and gene expression in single-embryo cultures from early wild-type embryos, as well as embryos containing an embryonic lethal mutation of unc45b, a myosin chaperone known to be required for sarcomere organization during myogenesis. We also demonstrated the practical usefulness of this technique by experimentally manipulating expression of specific genes in individual embryos before cell culture using standard tools of zebrafish biology such as morpholino-oligonucleotide gene knockdown and transgene-mediated gene expression.
... Cell cultures of marine fish and invertebrate origin (Tables 3 and 4) are also provided. Table 2: List of cell lines developed from warmwater and cold-water fish species and currently available from American Type Culture Collection (ATCC) and the European Collection of Authenticated Cell Cultures (ECACC) [157][158][159]. This is not an exhaustive list. ...
... In research involving fish cell cultures (in virology and toxicology), a common practice observed is to use non-specific cells [29,30,47] unlike in mammalian biology studies. Utilizing fish cell lines with specific functions (originated from specific tissue type) will greatly advance fundamental knowledge in the respective fields [151][152][153][154][155][156][157][158][159]. ...
Fish cell cultures are employed in diverse research fields such as virology, physiology, toxicology, immunology, genetics, and pharmacology. These systems can be utilized for pathogen detection, confirmation, propagation, and characterization, especially of viruses. Cell cultures are also utilized in the case of intracellular bacteria, Myxosporean, or Microsporidian parasites. Fish cell cultures have gained more popularity in recent years and have prominent roles as model systems and in the large-scale production of biologicals. The recent swift growth observed in research employing cell cultures is definitely an outcome of the progress in this sector and also due to increasing ethical demands for reduction and replacement of animals used in research. In vitro fish cell cultures are excellent research models in simulating host animal in vivo. The diverse applications of fish cell cultures in various research fields are attributed to their versatility, cost-effectiveness, convenience in handling, and ease in genetic manipulation. For several infectious viral diseases, as therapeutic options are limited, early disease diagnosis and prophylactic measures are crucial for efficient fish health management. In this scenario, a better understanding of the viral pathogenesis and mechanisms utilizing in vitro cell lines are essential to facilitate disease management strategies such as vaccines and antiviral agents. Moreover, host preferences of pathogens, virus-host cell interactions, and virus localization can also be studied using cell cultures. Availability of host-specific or host-susceptible fish cell cultures is very limited, which is a major concern in this area. In near future, innovations in 3D cell culture, stem cells, and genome editing will further enhance the research prospects of fish cell cultures.
... SAEC-A3 and SAEC-H7 cells were maintained in conventional Leibovitz's 15 medium supplemented with 10% FBS at 22 • C in constant darkness, similar to other marine fish cells reported to date (Béjar et al., 2002;Buonocore et al., 2006;Gignac et al., 2014;Molino et al., 2019;Olivares-Ferretti et al., 2019;Parameswaran et al., 2012;Parameswaran et al., 2007;Servili et al., 2009). Altogether, these facts indicate the establishment of two viable cell populations, each derived from one cell that can be considered immortalised, permanent ES cell lines with selfrenewal and differentiation capabilities, and suitable for early vertebrate development assays (Collodi et al., 1992;Ghosh and Collodi, 1994). ...
... Therefore, EBs formation from Sparus aurata ES cell lines A3 and H7 confirmed the pluripotency of both embryonic cell lines. This has also been demonstrated in mammalian ES cells (Robertson, 1987) and other ES cell lines developed from fish (Fan and Collodi, 2006;Ghosh and Collodi, 1994;Hong et al., 1996;Li et al., 2019). Another important criteria for evaluation of pluripotency is the potential to differentiate into distinct cell lineages (Brickman and Serup, 2017;Evans and Kaufman, 1981;Martin, 1981). ...
An important bottleneck in fish aquaculture research is the supply and maintenance of embryos, larvae, juvenile and adult specimens. In this context, cell lines represent alternative experimental models for in vitro studies that complement in vivo assays. This allows us to perform easier experimental design and sampling and avoid the sacrifice of animals. Embryonic stem (ES) cell lines have attracted increasing attention because they have the capability to proliferate indefinitely and could be differentiated into any cell type of the organism. To minimise cell heterogeneity and increase uniformity of in vitro studies results, in this manuscript we report the development and characterisation of two single cell-derived ES cell lines (monoclonal) from the morula stage embryos of the gilthead seabream, Sparus aurata, named as SAEC-A3 and SAEC-H7. Both cell lines have been passaged for over 100 times, indicating the establishment of long-term, immortalised ES cell cultures. Sequence analyses confirmed the seabream origin of the cell lines, and growth analyses evidenced their high viability and proliferating activity, particularly in culture medium supplemented with 10–15% fetal bovine serum and 22 °C. Both cell lines showed the ability to generate embryoid bodies and show different sensitivity and response to all-trans retinoic acid. The analysis of epithelial (col1α1) and neuronal (sox3) markers in differentiated cultures revealed that SAEC-A3 tended to differentiate towards epithelial-like cells whereas SAEC-H7 tended to differentiate towards neuronal-like cells. Both cell lines were efficiently transfected with pDsRed2-ER and/or pEGFP-N1 plasmids, indicating that they could represent useful biotechnological tools. Daily expression of pcna showed significant expression rhythms, with maximum levels of cell proliferation during the day-night transition. Currently, these cell lines are being successfully used as experimental models for the study of cellular metabolism, physiology and rhythms as well as for toxicological, pharmacological and gene expression analyses.
... To determine whether Pcdh19 interacts with classical cadherins in vivo, we used antibodies against Pcdh19 to perform immunoprecipitations from extracts prepared from five-somite stage embryos and Zem2S cells (Fig. 4 H). The Zem2S cell line was derived from blastula-stage embryos (Collodi et al., 1992; Ghosh and Collodi, 1994) and expresses both Ncad and Pcdh19, as determined by RT-PCR (Fig. S1). Immunoprecipitation of endogenous Pcdh19 was able to coimmunoprecipitate classical cadherins from both embryo and Zem2S cell extracts. ...
... possibility, we performed cell aggregation assays using Zem2S cells (Collodi et al., 1992; Ghosh and Collodi, 1994). Zem2S cells were scrape loaded with morpholinos against either ncad or pcdh19 and used in a standard cell aggregation assay (Takeichi, 1977). ...
The protocadherins comprise the largest subgroup within the cadherin superfamily, yet their cellular and developmental functions are not well understood. In this study, we demonstrate that pcdh 19 (protocadherin 19) acts synergistically with n-cadherin (ncad) during anterior neurulation in zebrafish. In addition, Pcdh 19 and Ncad interact directly, forming a protein-protein complex both in vitro and in vivo. Although both molecules are required for calcium-dependent adhesion in a zebrafish cell line, the extracellular domain of Pcdh 19 does not exhibit adhesive activity, suggesting that the involvement of Pcdh 19 in cell adhesion is indirect. Quantitative analysis of in vivo two-photon time-lapse image sequences reveals that loss of either pcdh 19 or ncad impairs cell movements during neurulation, disrupting both the directedness of cell movements and the coherence of movements among neighboring cells. Our results suggest that Pcdh 19 and Ncad function together to regulate cell adhesion and to mediate morphogenetic movements during brain development.
... Previously, a zebrafish embryonic cell line was established in a growth medium with complex additions, such as trout embryo extract, trout serum, and bovine insulin. 24,47 When grown in a simpler medium, the resulting cell line, ZEM-2A, attained a wide chromosome range like ZEB2J and a modal number near triploidy. 47 These cells had limited pluripotency when transplanted into embryos. ...
... 24,47 When grown in a simpler medium, the resulting cell line, ZEM-2A, attained a wide chromosome range like ZEB2J and a modal number near triploidy. 47 These cells had limited pluripotency when transplanted into embryos. 48 Many human and murine EC cell lines are aneuploid but retain the ability to differentiate in vitro. ...
A continuous cell line, ZEB2, was developed from zebrafish blastula-stage embryos expressing enhanced green fluorescent protein (GFP). Originally the rainbow trout spleen cell line, RTS34st, was used as feeders to initiate and maintain the cells through several passages. ZEB2 was then grown for 2 years without feeders in L-15 with 15% fetal bovine serum (FBS) for 120 population doublings. This new cell line, ZEB2J, was heteroploid, had detectable telomerase activity, and was adherent. After growing into monolayers, some cells continued to grow into mounds. Cultures expressed Pou-2 mRNA and contained many alkaline phosphatase and a few stage-specific embryonic antigen-1-positive cells. In dishes coated with a phospholipid polymer (2-methacryloxyloxyethyl phosphorylcholine, MPC), ZEB2J formed spherical aggregates. Aggregates attached to conventional culture plastic, and most cells that emerged from aggregates had typical epithelial-like shapes of ZEB2J, which suggests that ZEB2J had limited differentiation potential, despite expressing some stem cell properties. The fluorescence of ZEB2J allowed relationships with feeder cells to be studied. In MPC dishes, ZEB2J formed mixed spheroids with RTS34st. In adherent cocultures, RTS34st and other fish cell lines strongly stimulated the ZEB2J growth, which could be quantified specifically because ZEB2J expressed GFP. ZEB2J should be useful for optimizing culture conditions for zebrafish embryonic stem cells.
... One criterion used in the classification of media is the level of supplemented serum. FBS is the most common serum added to the basal medium, which is richer in growth factors, contains a low level of γ-globulin (a cell growth inhibitor), acts as a carrier for lipids and enzymes, and is involved in the transportation of micronutrients and trace elements (Ghosh & Collodi, 1994;Subbiahanadar Chelladurai et al., 2021;Yao & Asayama, 2017). FBS contains a standard formulation based on amino acids, vitamins, glucose, and inorganic salts. ...
Investigations were conducted to develop a general protocol for the isolation and in vitro culture of ovary-derived cells from black crappie, Pomoxis nigromaculatus, and white crappie, P. annularis. Five digestive enzymes: 500 U/mL collagenase type I, 500 U/mL collagenase type IV, 0.05% trypsin-EDTA, 0.25% trypsin-EDTA, and 1X TrypLE™ Express were evaluated for live cell isolation. The isolated cells were cultured in 10 or 20% fetal bovine serum (FBS) in L-15 growth media. In addition , four incubation temperatures (15 C, 20 C, 25 C, and 30 C) were also evaluated. The number of live cells obtained from the 0.25% trypsin and TrypLE™ Express treatments was significantly higher than other treatments. No difference in cell growth was observed between the two FBS treatments. Cells isolated using TrypLE™ Express and 0.25% trypsin reached 80% to 90% confluency in 12.5 cm 2 cell culture flasks within 5 days of inoculation at 20 and 25 C. At 15 C, 10 days were required to reach 80%-90% confluency. Morphologically, cells incubated at 15 C, 20 C, and 25 C appeared healthier than cells incu-bated at 30 C, where irregular cell shape and substrate detachment were observed. We concluded TrypLE™ Sujan Bhattarai and Dayan A. Perera are the primary contributors and Co-First Authors of this Manuscript.
... Quick embryonic development in 96 hours (Ghosh and Collodi 1994) helps to reduce time period of toxicity studies when compared to rodents or other mammals. ...
The aim of this PhD thesis was to develop and assess the potential of novel zebrafish (zf)- based in vitro and in vivo reporter gene assays as bio-analytical tools to monitor estrogenic activity and their implementation in effect-directed analysis (EDA) approach to identify fishspecific
estrogenic compounds in complex mixtures. For this purpose, we first characterized the response of the assays towards a panel of (xeno)estrogens, revealing differences in the affinity of zebrafish estrogen receptor (zfER) subtypes to (xeno)-estrogens. Comparison with human cell-based (MELN-hERα) assay further highlighted inter-species differences showing different chemical ranking towards different classes of known ER ligands. Then, application of these tools to different environmental matrices demonstrated for the first time their functionality to detect and quantify estrogenic activity in complex mixtures, highlighting the zfERβ2 assay as the most sensitive among the different in vitro zfER assays. The above in vitro estrogenic activity was also confirmed in vivo at the most contaminated sites by using the EASZY assay, further adding eco-toxicological relevance. Interestingly, we also reported zebrafish-specific activities at several sites that were not active by the human MELN assay, suggesting the occurrence of fish-specific ER ligands. To address this hypothesis, we applied our zebrafish tools in specific higher tier-EDA studies that allowed isolating zebrafish-specific active fractions by multi-step sample fractionation procedures. Chemical analyses of these specific fractions so far identified several candidate compounds, of which few showed higher selectivity towards zfERβ2 than hERα, hence confirming inter-species differences. This work supports recommendations for the integration of these effect-based tools in future water monitoring strategies.
... The Table 7.1 List of commercially available established fish cell lines from the cell repositories, American Type Culture Collection (ATCC), and European Collection of Authenticated Cell Cultures (ECACC). Species of origin, family, tissue of origin, and morphology of commercially available cell lines are provided (Driever and Rangini 1993;Ghosh and Collodi 1994;Paw and Zon 1999 ...
Cell culture serves as a reliable and proficient tool in diverse research fields such as virology, physiology, toxicology, immunology, oncology, genetics, and pharmacology. These systems can be employed for pathogen detection, confirmation, and characterization especially of viruses. It is also applicable in the case of intracellular bacteria, myxosporean or microsporean parasites as well. Fish cell cultures have gained more popularity in recent years and have prominent roles in viral disease diagnosis. Since treatment options are limited for many viral diseases, early disease diagnosis and proactive management measures are key for successful fish health management. The ability to propagate fish viruses in vitro using cell cultures is imperative in advancing research on viruses and to facilitate disease management strategies such as vaccines and antiviral agents. Moreover, potential host range of pathogens via susceptibility to cell cultures, virus-host cell interactions, and virus localization studies using cell cultures provide a better understanding of the viral pathogenesis. Availability of suitable fish cell cultures for propagation of viruses and disease diagnosis is very limited, which is a major concern in this area. The wide array of applications exemplifies the versatility, cost-effectiveness, and high potential of fish cell cultures in various research fields. The recent swift growth observed in research employing cell cultures is definitely an outcome of the progress in this sector and also due to increasing ethical demands for reduction and replacement of animal use in research. In the near future, innovations in 3D cell culture and CRISPER-Cas9 genome editing will further enhance the research prospects of fish cell culture systems.
... Cells were permitted to attach for 2 h at 28 1C and then fed with 200 ml of culture medium (25/25/ 50% DMEM/L-15/Hank's saline) containing 10% FBS, 5% fish serum (GenWay), 2 mM L-glutamine, 0.8 mM CaCl 2 , plus HEPES/saline buffer (final medium pH of 7.4), 5 mg/ml bovine insulin, and 10% embryo extract. Embryo extract was made in culture medium as previously described and filter-sterilized before use (Ghosh and Collodi, 1994;Myhre and Pilgrim, 2010). Media were also supplemented with antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, 100 mg/ml kanamycin, 50 mg/ml gentamycin and 25 mg/ml amphotericin B). ...
The sarcomeres of skeletal and cardiac muscle are highly structured protein arrays, consisting of thick and thin filaments aligned precisely to one another and to their surrounding matrix. The contractile mechanisms of sarcomeres are generally well understood, but how the patterning of sarcomeres is initiated during early skeletal muscle and cardiac development remains uncertain. Two of the most widely-accepted hypotheses for this process include the "molecular ruler" model, in which the massive protein titin defines the length of the sarcomere and provides a scaffold along which the myosin thick filament is assembled, and the "premyofibril" model, which proposes that thick filament formation does not require titin, but that a "premyofibril" consisting of non-muscle myosin, α-actinin and cytoskeletal actin is used as a template. Each model posits a different order of necessity of the various components, but these have been difficult to test in vivo. Zebrafish motility mutants with developmental defects in sarcomere patterning are useful for the elucidation of such mechanisms, and here we report the analysis of the herzschlag mutant, which shows deficits in both cardiac and skeletal muscle. The herzschlag mutant produces a truncated titin protein, lacking the C-terminal rod domain that is proposed to act as a thick filament scaffold, yet muscle patterning is still initiated, with grossly normal thick and thin filament assembly. Only after embryonic muscle contraction begins is breakdown of sarcomeric myosin patterning observed, consistent with the previously noted role of titin in maintaining the contractile integrity of mature sarcomeres. This conflicts with the "molecular ruler" model of early sarcomere patterning and supports a titin-independent model of thick filament organization during sarcomerogenesis. These findings are also consistent with the symptoms of human titin myopathies that exhibit a late onset, such as tibial muscular dystrophy.
... 13,[25][26][27][28][29] However, studies with zebrafish in the field of bone biology have been hampered by the absence of in vitro cell systems suitable to study the cellular mechanisms associated with bone cell (e.g., osteoblasts and chondrocytes) differentiation and extracellular matrix (ECM) mineralization. During the last decades, few zebrafish cell lines have been generated, [30][31][32][33][34][35] mostly derived from embryonic tissues and none of them were reported to be capable of in vitro mineralization. We describe here the establishment, for the first time, of a stable cell culture derived from a pool of zebrafish calcified tissues and the characterization of its mineralogenic capacity by histological staining, immunocytochemistry, and gene expression analysis. ...
Abstract Mechanisms of bone formation and skeletal development have been successfully investigated in zebrafish using a variety of in vivo approaches, but in vitro studies have been hindered due to a lack of homologous cell lines capable of producing an extracellular matrix (ECM) suitable for mineral deposition. Here we describe the development and characterization of a new cell line termed ZFB1, derived from zebrafish calcified tissues. ZFB1 cells have an epithelium-like phenotype, grow at 28°C in a regular L-15 medium supplemented with 15% of fetal bovine serum, and are maintained and manipulated using standard methods (e.g., trypsinization, cryopreservation, and transfection). They can therefore be propagated and maintained easily in most cell culture facilities. ZFB1 cells show aneuploidy with 2n=78 chromosomes, indicative of cell transformation. Furthermore, because DNA can be efficiently delivered into their intracellular space by nucleofection, ZFB1 cells are suitable for gene targeting approaches and for assessing gene promoter activity. ZFB1 cells can also differentiate toward osteoblast or chondroblast lineages, as demonstrated by expression of osteoblast- and chondrocyte-specific markers, they exhibit an alkaline phosphatase activity, a marker of bone formation in vivo, and they can mineralize their ECM. Therefore, they represent a valuable zebrafish-derived in vitro system for investigating bone cell differentiation and extracellular matrix mineralization.
... Apart from that, fish cell lines are grown similarly to mammalian cell lines using the same growth media, i.e. basal culture media supplemented with mammalian sera, such as fetal calf serum (FCS). Several researchers have worked on cell and tissue cultures derived from fish to estimate the optimal growth conditions (Bradford et al. 1994, Ghosh & Collodi 1994, Ciba et al. 2008. ...
We describe a method for fast and easy isolation of cells via trypsin digestion from larvae of Atlantic sturgeon Acipenser oxyrinchus oxyrinchus resulting in a stable, well-proliferating cell culture. The culture conditions for these cells were optimized with the aim of supporting the production of high amounts of biomass. To enhance cell growth and cell density, 4 different cultivation temperatures as well as commercially available carp serum (CS) and fetal calf serum (FCS) at different concentrations were tested and evaluated. Cell growth was measured via an impedance-based online cell-monitoring system (xCELLigence). These results showed the best cultivation temperature to be at 25 C and a media composition of Dulbecco's modified Eagle's medium (DMEM) supplemented with either 10 or 20% FCS or 5% CS. The cells were stable in the process of long-term cultivation over 33 passages and could be cryo-preserved. Immunocytochemical analysis revealed that the cells expressed proteins of different blastodermic layers. Ectodermic glia fibrilliary acid protein, vigilin (mRNA transport protein), and pan cytokeratin were abundant. This fast-growing cell culture provides an important tool for research on Atlantic sturgeon populations.
... Recently, techniques similar to those used for producing knockout mice, such as chimera production ( Lin et al., 1992; Wakamatsu et al., 1993) and establishment of embryonic stem (ES) cells (Ghosh and Collodi, 1994;Wakamatsu et al., 1994;Sun et al., 1995;Hong et al., 1996), have been applied to small experimental fish (e.g., medaka and zebra fish) for the purpose of knockout fish production. However, the ES-like cells transplanted into early fish embryos have never contributed to the germline, despite their resemblance to mouse ES cells ( Sun et al., 1995;Hong et al., 1998;Hyodo et al., 1998). ...
Green fluorescent protein (GFP) has been used as an indicator of transgene expression in living cells and organisms. For
testing the utility of GFP in rainbow trout, we microinjected fertilized eggs with four types of supercoiled constructs containing
two variants of GFP complementary DNA (S65T and EGFP), driven by two ubiquitous regulatory elements, human cytomegalovirus
immediate early enhancer-promoter (CMV) and Xenopus laevis elongation factor 1α enhancer-promoter (EF1).
Green fluorescence was first observed at 3 days postfertilization, when the embryo was in the mid-blastula stage. Fluorescence
could be detected mosaically in various types of embryonic cells and tissues of swim-up fry. Both the percentage of fluorescent
cells and the fluorescence intensity of GFP-expressing cells on blastoderms, measured with a microscopic photometry system,
were highest in CMV-EGFP-microinjected embryos. We conclude that GFP is capable of producing detectable fluorescence in rainbow
trout, and can be a powerful tool as a cell marker and reporter gene for cold-water fish, and that analysis of GFP expression
in living cells is useful for characterizing the activity of cis-elements in vivo.
... ES cells might provide a common assay for answering these questions. There is evidence for ES-like cells derived from horse (Saito et al. 2002), chick (Pain et al. 1996, but see also Soodeen-Karamath and Gibbins, 2001), mink (Sukoyan et al., 1993), pig (Chen et al. 1999), zebrafish (Ghosh and Collodi, 1994, Sun et al. 1995), and a relative of zebrafish called medakafish (Hong et al. 1996; Hong et al. 1998). Despite intensive efforts, ES cells have not yet been obtained from rats (Buehr et al., 2003) as well as many other species. ...
... Growth was best at 26 to 28 °C and required at least some kind of sera. The only other cell lines from an internal organ are from the liver, and the most studied of these is ZFL (Ghosh et al., 1994). Like ZFL, ZSSJ had an epithelial-like morphology. ...
A zebrafish spleen cell line, ZSSJ, was developed and its growth arrest by gamma radiation determined and its capacity to stimulate the proliferation of the zebrafish blastula cell line, ZEB2J, measured. ZSSJ was initiated by explant outgrowth, grew adherent with mainly an epithelial-like morphology, and stained strongly for alkaline phosphatase. ZSSJ was not only grown in L-15 with 15% fetal bovine serum at 26 degrees C to 28 degrees degrees C but also grew at room temperature. Cultures of ZSSJ have undergone approximately 40 population doublings, had few cells staining for b-galactosidase activity, which is commonly present in senescent cultures, and many cells with an aneuploid karyotype, which is frequently associated with immortalization. ZSSJ growth was arrested by 30 to 50 Gy of g-irradiation, whereas after 20 Gy, some slight growth was observed. By contrast, growth of the rainbow trout spleen stromal cell line, RTS34st, which has been used as a feeder for zebrafish ES cell cultures, was arrested completely by 20 Gy. In cocultures, nongrowth-arrested ZSSJ stimulated ZEB2J proliferation better than growth-arrested ZSSJ and better than RTS34st. ZSSJ should be useful as a feeder cell line for zebrafish ES cell cultures.
... These constitute useful tools for studying the expression of gene of interest and protein export at a cellular level. The cell lines ZEM2S [101] and ZF4 [102] were established from zebrafish embryos (blastula and 1-day old, respectively). The ZF4 cell line was derived from adult liver [103]. ...
Cell surface glycans, such as glycocoproteins and glycolipids, encode information that modulates interactions between cells, or between cells and the extracellular matrix, by specifically regulating the binding to cell surface-associated or soluble carbohydrate-binding receptors, such as lectins. Rapid modifications of exposed carbohydrate moieties by glycosidases and glycosyltransferases, and the equally dynamic patterns of expression of their receptors during early development, suggest that both play important roles during embryogenesis. Among a variety of biological roles, galectins have been proposed to mediate developmental processes, such as embryo implantation and myogenesis. However, the high functional "redundancy" of the galectin repertoire in mammals has hindered the rigorous characterization of their specific roles by gene knockout approaches in murine models. In recent years, the use of teleost fish as alternative models for addressing developmental questions in mammals has expanded dramatically, and we propose their use for the elucidation of biological roles of galectins in embryogenesis and innate immunity. All three major galectin types, proto, chimera, and tandem-repeat, are present in teleost fish, and phylogenetic topologies confirm the expected clustering with their mammalian orthologues. As a model organism, the zebrafish (Danio rerio) may help to overcome limitations imposed by the murine models because it offers substantial advantages: external fertilization, transparent embryos that develop rapidly in vitro, a diverse toolbox of established methods to manipulate early gene expression, a growing collection of mutations that affect early embryonic development, availability of cell lines, and most importantly, an apparently less diversified galectin repertoire.
... 8 They derived the ZFL cell line from a pool of ϳ10 normal adult zebrafish livers 9,10 and the ZEM2 cell line from blastula-stage embryos. 11 ZEM2S cells were derived from ZEM2 by selection for growth in a basal nutrient medium. It was reported that zebrafish embryonic cell lines derived from blastula and gastrula stages remained pluripotent and germ-line competent for multiple passages in culture. ...
Compared with the increasing use of zebrafish as a model organism in many laboratories, zebrafish cell lines are still unexploited and limited in application, partly due to their unknown genetic and physiological properties. We characterize two zebrafish embryonic fibroblast cell lines, ZF4 and PAC2. We demonstrate the genetic stability of these two zebrafish cell lines and achieved genetic manipulation by either lipid-mediated transfection or an electroporation- based nucleofection method. Data from zebrafish chip analysis (Affymetrix) demonstrate unique characteristics of these two cell lines in gene expression levels, showing that different zebrafish cell lines can be classified by their transcriptome profile. Their transcriptional responses to serum growth factor exposure suggest that zebrafish fibroblast cell lines may be used to study processes related to wound-healing or cancer.
The “toxicology in the twenty-first century” paradigm shift demands the development of alternative in vitro test systems. Especially in the field of ecotoxicology, coverage of aquatic species-specific assays is relatively scarce. Transient reporter gene assays could be a quick, economical, and reliable bridging technology. However, the user should be aware of potential pitfalls that are influenced by reporter vector geometry. Here, we report the development of an AhR-responsive transient reporter-gene assay in the permanent zebrafish hepatocytes cell line (ZFL). Additionally, we disclose how viral, constitutive promoters within reporter-gene assay cassettes induce squelching of the primary signal. To counter this, we designed a novel normalization vector, bearing an endogenous zebrafish-derived genomic promoter (zfEF1aPro), which rescues the squelching-delimited system, thus, giving new insights into the modulation of transient reporter systems under xenobiotic stress. Finally, we uncovered how the ubiquitously used ligand BNF promiscuously activates multiple toxicity pathways of the xenobiotic metabolism and cellular stress response in an orchestral manner, presumably leading to a concentration-related inhibition of the AhR/ARNT/XRE-toxicity pathway and non-monotonous concentration–response curves. We named such a multi-level inhibitory mechanism that might mask effects as “maisonette squelching . ”
Graphical abstract
A transient reporter gene assay in zebrafish cell lines utilizing endogenous regulatory gene elements shows increased in vitro toxicity testing performance.
Synthetic and constitutive promotors interfere with signal transduction (“squelching”) and might increase cellular stress (cytotoxicity).
The squelching phenomenon might occur on multiple levels (toxicity pathway crosstalk and normalization vector), leading to a complete silencing of the reporter signal.
The field of neurotoxicology is confronted with two significant demands: the testing of an ever increasing list of chemicals, and resource limitations/ethical concerns associated with testing using traditional mammalian species. National and international government agencies have well-defined a need to reduce, refine or replace mammalian species in toxicological testing with alternative testing methods and non-mammalian models. Toxicological assays using alternative animal models may relieve some of this pressure by allowing testing of more compounds while reducing expense and using fewer mammals. Recent advances in genetic technologies and the strong conservation between human and non-mammalian genomes allow for the dissection of the molecular pathways involved in neurotoxicological responses using genetically tractable organisms such as zebrafish (Danio rerio). A constantly increasing database on basic developmental biology, gene transfers, and the rich foundation of molecular genetic and genomic data make zebrafish a powerful modeling system for revealing mechanisms in neurotoxicology. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, the databases regarding pathologic lesions in zebrafish lag far behind the information available on most other domestic mammalian and avian species, particularly rodents. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding neurotoxicological influences on human development and disease we must greatly advance our knowledge on zebrafish diseases and pathology.
This chapter describes methods for derivation, maintenance, and genetic manipulation of zebra fish embryonic stem (ES) cell cultures. A key component of the cell culture system is the use of feeder layers, derived from rainbow trout spleen cell lines. Methods are described for the introduction of a targeting vector by electro oration with procedures for the efficient selection of homologous recombinants using a visual marker screen. Also, a protocol is presented for the introduction of the ES cells into host embryos by microinjection to generate zebra fish germ-line chimeras. Reverse genetic methods have also been used to study zebra fish gene function. Colonies of ES cells that harbor targeted mutation are selected and expanded in culture and the cells are introduced into a host embryo, where they participate in development and contribute to the germ cell lineage. Development of an ES cell-based gene targeting system in zebra fish requires the cells to be able to incorporate vector DNA in a targeted fashion by homologous recombination and those methods are available to identify and select the colonies of homologous recombinants.
This is the sixth edition of the leading text in the basic methodology of cell culture, worldwide. Rigorously revised, it features updates on specialized techniques in stem cell research and tissue engineering; updates on molecular hybridization, somatic cell fusion, hybridomas, and DNA transfer; new sections on vitrification and Organotypic Culture, and new chapters on epithelial, mesenchymal, neurectodermal, and hematopoietic cells; germs cells/stemcells/amniocytes; and non-mammalian/avian cells. It is written for graduate students, research and clinical scientists, and technicians and laboratory managers in cell and molecular biology labs and genetics labs. PowerPoint slides of the figures as well as other supplementary materials are available at a companion website: www.wiley.com/go/freshney/cellculture.
This chapter presents a description of the methodology applied to zebrafish. With the general approaches of animal cell culture, the chapter provides an overview of the basic equipment, techniques, and concepts. Zebrafish embryo cell cultures can be initiated using techniques adapted from mammalian cell culture techniques, although several methodological aspects require approaches somewhat unique to fish species. The culture techniques that have benefitted other model systems can thus be applied to zebrafish developmental biology including genomic manipulation and cell selection in vitro. The chapter also discusses the application of the techniques to other interesting models, such as the Japanese pufferfish, Fugu. In principle, the approaches for zebrafish can be applied to any fish species. The Japanese pufferfish possesses a highly compact genome, and has gained interest among those involved in the sequencing and mapping of vertebrate genomes. Cell cultures from these fish provide a biological complement to the genomic libraries derived to study the molecular biology of Fugu.
Zebrafish have been used predominantly in developmental biology and molecular genetics, but their value in toxicology as well as drug discovery has been recognized. To evaluate the toxicity of a chemical, it is essential to identify the endpoints of toxicity and their dose-response relationships, elucidate the mechanisms of toxicity, and determine the toxicodynamics of the chemical. In addition to detailed toxicological investigations of a single chemical, there also is a need for high-throughput large- scale screening for toxicity of several hundreds of chemicals at a time. In both cases, the zebrafish has numerous attributes. More is probably known about ''what is normal'' in the zebrafish than any other fish species. This includes morpho- logical, biochemical, and physiological information at all stages of early development and in juveniles and adults of both sexes. This makes using the zebrafish ideal for toxicology research where the objective is to identify adverse effects of chemical exposure.
This chapter presents a number of techniques that are associated with cell culture though not necessarily culture techniques per se such as the protocols provided for autoradiography, in situ hybridization, FISH, cell hybridization, production of monoclonal antibodies, DNA transfer by lipofection and electroporation, and time-lapse video recording. Specialized culture techniques include culture of amniocytes for chromosome analysis, lymphocyte preparation and PHA stimulation, cultures from zebra fish embryos, and propagation of insect cell lines.
Keywords: autoradiography; in situ hybridization; FISH; DNA transfer; electroporation; time lapse video; amniocytes; chromosome analysis; lymphocytes; zebra fish; feeder layers; insect cell lines
Dechorionation or partial digestion of the egg chorion is necessary for introducing embryonic stem cells into blastulas for chimera production and for harvesting blastulas. Several methods to digest the Nile tilapia Oreochromis niloticus chorion were tried and it was found that the chorions of most clutches of eggs were digested in <3 h using hatching medium [produced by allowing embryos to hatch in Hanks balanced salt solution (HBSS) in an incubator], or 2 mg ml−1 pronase P6911 in 10% Ca/Mg free HBSS. The chorion of Nile tilapia possesses multiple lamellae as found in most teleost species that have been studied. It was found to be thinner than that of medaka Oryzias latipes and thicker than that of zebrafish Danio rerio. During natural hatching the chorion was digested from the inner surface, and tail movements helped to break the remaining chorion; however, chorion digestion has to be complete for experimental dechorionation, because digestion starts at the external surface. The zona radiata externa remained intact after experimental digestion with hatching media but was disrupted by pronase. Embryos dechorionated at the cleavage or blastula stage only survived for 2 or 3 days, but some dechorionated at the gastrula stage or early segmentation stage developed until the natural hatching time. If the chorion was partially digested at the cleavage or blastula stage, some embryos survived to hatch.
The zebrafish is increasingly popular as a nonmammalian model for studies of vertebrate developmental biology, genetics, and toxicology. The availability of cell culture systems makes it possible to address many basic questions using in vitro approaches. Here we describe materials and procedures for initiating cell cultures from zebrafish early (blastula- and gastrula-stage) diploid and haploid embryos and adult tissues (gills, fins, liver, viscera). Zebrafish cells are grown in a complex basal nutrient medium supplemented with insulin, selenite, fetal bovine serum, trout serum, and an extract prepared from rainbow trout embryos. The procedure for preparing trout embryo extract (demonstrated to be mitogenic for a variety of piscine cell lines), is also described.
Methods were developed for the culture of cells derived from tissues of the sea lamprey (Petromyzon marinus). Cultures were initiated from gill, liver, muscle and gut from larvae and newly transformed individuals and brain, heart, kidney and ovary from sexually mature adults. The lamprey cells were viable for up to six months in culture and cells from ovary, muscle, gut, gill and liver were propagated for multiple passages. For all cultures except liver, optimal cell attachment and spreading was obtained on surfaces coated with fibronectin and collagen. Optimal liver cell attachment was achieved on basement membrane. Cells synthesizing DNA were detected by precursor incorporation in five week-old cultures derived from adult and larval tissues. Metabolic labeling experiments with [(35)S]-methionine demonstrated that cultures initiated from liver and ovary continued to synthesize and release proteins into the medium for several weeks. Ultrastructural examination revealed the presence of ciliated cells in cultures from brain and the accumulation of lipid in epithelial cells derived from liver and gill.
As the zebrafish, Danio rerio, has been increasingly used as an animal model for biomedical research, we aimed to establish zebrafish cell line models for inflammation and cancer studies in this thesis. Several zebrafish cell lines were characterized and their genetic and physiological properties were compared. We also developed a set of tool methods to investigate cellular signaling events in zebrafish cell lines. Our case studies illustrated that zebrafish cell lines are as reliable models as the widely used mammalian cell cultures. Taking advantage of the transparency of zebrafish embryos and cell implantation protocols, zebrafish cell lines can serve as a bridge platform between in vitro, in silico, ex vivo and in vivo studies in order to enhance our understanding of molecular mechanisms underlying disease progression.
The use of Zebrafish as a model-organism is growing exponentially. Somatic Cell Nuclear Transfer could potentially enhance the way gene modifications are done and facilitate the selection and preservation of specific strains and mutant lines. However, since first reported in 2002, SCNT failed to be implemented widely. The reason for the lack of widespread use is likely the difficulty to perform the protocol as was described. This chapter addresses all the critical parameters in the procedure including handling of the recipient eggs, donor cells, and care of the reconstructed embryos. We describe a novel protocol that overcomes common hindrances and incorporates a new approach for enucleation and cell transfer, parameters that can greatly influence SCNT success. We predict that the simplicity of this protocol will entice other laboratories to use it.
Transgenic fish, owing to a number of advantages which they offer over other species, are proving to be valuable model systems for the study of gene regulation and development genetics in addition to being useful targets for the genetic manipulation of commercially important traits. Despite having begun only a decade ago, the production of transgenic fish has become commonplace in a number of laboratories world-wide and considerable progress has been made. In this review, we initially consider the various regulatory elements and coding genes which have been used in fish, and subsequently discuss and compare both the transient and long-term fate and expression patterns of injected DNA sequences in the context of the different factors which are likely to have an effect on the expression of transgenes.
Using the golden mutant zebrafish having a decrease in interfering pigmentation, we are developing transgenic lines in which DNA motifs that respond to selected environmental pollutants are capable of activating a reporter gene that can be easily assayed. We have begun with three response elements that recognize three important classes of foreign chemicals. Aromatic hydrocarbon response elements (AHREs) respond to numerous polycyclic hydrocarbons and halogenated coplanar molecules such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and polychlorinated biphenyls. Electrophile response elements (EPREs) respond to quinones and numerous other potent electrophilic oxidants. Metal response elements (MREs) respond to heavy metal cations such as mercury, copper, nickel, cadmium, and zinc. Soon, we will include estrogen response elements (EREs) to detect the effects of environmental endocrine disruptors, and retinoic acid response elements (RARE, RXRE) to detect the effects of retinoids in the environment. Each of these substances is known to be bioconcentrated in fish to varying degrees; for example, 10(-17) M TCDD in a body of water becomes concentrated to approximately 10(-12) M TCDD in a fish, where it would act upon the AHRE motif and turn on the luciferase (LUC) reporter gene. The living fish as a sentinel will not only be assayed intact in the luminometer, but--upon several days or weeks of depuration--would be usable again. To date, we have established that zebrafish transcription factors are able to recognize both mammalian and trout AHRE, EPRE, and MRE sequences in a dose-dependent and chemical-class-specific manner, and that expression of both the LUC and jellyfish green fluorescent protein (GFP) reporter genes is easily detected in zebrafish cell cultures and in the intact live zebrafish. Variations in sensitivity of this model system can be achieved by increasing the copy number of response elements and perhaps by altering the sequence of each core consensus response element and flanking regions. This transgenic technology should allow for a simple, exquisitely sensitive, and inexpensive assay for monitoring aquatic pollution. We have already initiated studies using sentinel zebrafish to monitor a public drinking water source.
The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1-2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.
The zebrafish is an established model for the genetic analysis of vertebrate development. Forward-genetic screens have generated thousands of mutations, and antisense-based methods have been used to transiently knockdown gene expression during embryogenesis. Although these methods have made the zebrafish a valuable system for the identification and functional characterization of developmentally important genes, one deficiency of the zebrafish model is the absence of methods to introduce targeted mutations to generate knockout lines of fish. Application of gene-targeting methods has been limited in nonmurine species due to the absence of germ-line competent embryonic stem (ES) cell lines. Recently, progress was made in addressing this problem by the derivation of zebrafish embryo cell lines that remain pluripotent and germ-line competent for multiple passages in culture. Zebrafish germ-line chimeras were generated using cultures derived from embryos at two different developmental stages, and targeted insertion of vector DNA by homologous recombination was demonstrated in both cultures. Several strategies are being used to optimize the production and identification of germ-line chimeras. The zebrafish embryo cell culture system should provide the basis of a gene-targeting approach that will complement other genetic strategies and improve the utility of the zebrafish model for studies of development and disease.
This chapter describes the culture of Embryoniv stem cell lines from Zebrafish. Despite its many advantages for studies of embryo development and human disease, one deficiency of the zebrafish model has been the lack of methods for targeted mutagenesis using embryonic stem (ES) cells. A similar strategy applied to zebrafish would complement other genetic methods currently available, such as large-scale random mutagenesis antisensebased gene knockdown and target selected mutagenesis approaches to increase the utility of this model system. To address this problem, our laboratory has been working to establish zebrafish ES cell lines that are suitable for use in a gene targeting approach. This chapter illustrates that the germline competent ES cells are genetically altered in culture by targeted incorporation of foreign DNA by homologous recombination followed by in vitro selection of cell colonies that have undergone the targeting event. This chapter describes the methods for the derivation of germline competent zebrafish ES cell cultures along with a protocol for the efficient introduction of plasmid DNA into the cells by electroporation and in vitro selection of homologous recombinants.
Zebrafish (Danio rerio) has been a prominent model vertebrate in a variety of biological disciplines. Substantial information gathered from developmental and genetic research, together with near-completion of the zebrafish genome project, has placed zebrafish in an attractive position for use as a toxicological model. Although still in its infancy, there is a clear potential for zebrafish to provide valuable new insights into chemical toxicity, drug discovery, and human disease using recent advances in forward and reverse genetic techniques coupled with large-scale, high-throughput screening. Here we present an overview of the rapidly increasing use of zebrafish in toxicology. Advantages of the zebrafish both in identifying endpoints of toxicity and in elucidating mechanisms of toxicity are highlighted.
Murine embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo which can contribute differentiated progeny to all adult tissues, including the germ-cell lineage, after re-incorporation into the normal embryo. They provide both a cellular vector for the generation of transgenic animals and a useful system for the identification of polypeptide factors controlling differentiation processes in early development. In particular, medium conditioned by Buffalo rat liver cells contains a polypeptide factor, ES cell differentiation inhibitory activity (DIA), which specifically suppresses the spontaneous differentiation of ES cells in vitro, thereby permitting their growth as homogeneous stem cell populations in the absence of heterologous feeder cells. ES cell pluripotentiality, including the ability to give rise to functional gametes, is preserved after prolonged culture in Buffalo rat liver media as a source of DIA. Here, we report that purified DIA is related in structure and function to the recently identified hematopoietic regulatory factors human interleukin for DA cells and leukaemia inhibitory factor. DIA and human interleukin DA/leukaemia inhibitory factor have thus been identified as related multifunctional regulatory factors with distinct biological activities in both early embryonic and hematopoietic stem cell systems.
Recent advances in tissue culture and endocrinology have made possible the growth of established cell lines in hormone-supplemented serum-free media. The hormone requirements differ for different cell types but are similar or identical for the same cell types. The hormone supplements derived for four different cell types, a melanoma, GH3 pituitary tumor, and testicular cell lines TM3 and TM4 are used in preparing primary cultures for organs to detect melanoma metastasis, and grow normal pituitary and normal Leydig and Sertoli cells, respectively. This hormone supplementation and the concomitant elimination or reduction of the serum requirement is shown to have several advantages in the preparation of primary cultures including prolonged viability and function, partial or total selection of the desired cell type and inhibition of fibroblast overgrowth.It is felt that such culture systems will significantly expand the range of problems which can be approached using primary culture systems.
It has been demonstrated in mammalian systems that techniques using embryonal stem cells provide advantages over conventional injection of DNA into embryos for generation of transgenic animals. We employed cell culture approaches in an attempt to develop this technology for fish transgenesis. Using a trout embryo-derived mitogenic preparation in a specialized culture medium, we initiated replication of zebrafish blastula-derived cell cultures and expressed marker genes introduced into the cells by plasmid transfection. Reintroduction of cells from the cultures into blastula-stage embryos indicated that the cultured cells survived and may contribute to the developing organism.
To determine whether embryonic cells transplanted from one zebrafish embryo to another can contribute to the germ line of the recipient, and to determine whether pigmentation can be used as a dominant visible marker to monitor cell transplants, we introduced cells from genetically pigmented (donor) embryos to albino recipients at midblastula stage. By 48 hr many of the resulting chimeras expressed dark pigment in their eyes and bodies, characteristics of donor but not albino embryos. By 4-6 weeks of age pigmentation was observed on the body of 23 of 70 chimeras. In contrast to fully pigmented wild-type fish, pigmentation in chimeras appeared within transverse bands running from dorsal to ventral. Pigmentation patterns differed from one fish to another and in almost every case were different on each side of a single fish. At 2-3 months of age chimeras were mated to albino fish to determine whether pigmented donor cells had contributed to the germ line. Of 28 chimeric fish that have yielded at least 50 offspring each, 5 have given rise to pigmented progeny at frequencies of 1-40%. The donor cells for some chimeras were derived from embryos that, in addition to being pigmented, were transgenic for a lacZ plasmid. Pigmented offspring of some germ-line chimeras inherited the transgene, confirming that they descended from transplanted donor cells. Our ability to make germ-line chimeras suggests that it is possible to introduce genetically engineered cells into zebrafish embryos and to identify the offspring of these cells by pigmentation at 2 days of age.
The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1. Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.
Recessive lethal mutations and mutations at the gol-1 locus were induced in the zebrafish by exposure of mature sperm to the alkylating agent ethyl nitrosourea (ENU). Embryonic lethal phenotypes were recognized among the parthenogenetic progeny of mutagenized animals or among the progeny of daughters of mutagenized animals. Novel specific locus mutations were identified by the failure of mutagenized chromosomes to complement pre-existing mutant alleles at the gol-1 locus. Each mutagenized individual harboured approximately 10 embryonic lethal mutations in its germ line and about 1 in 500 mutagenized animals harboured a new mutation at the gol-1 locus. Three lines of evidence indicate that the majority of mutations that were recovered following treatment of mature sperm with ENU were probably point mutations. First, the soma and germ lines of mutagenized animals were mosaic, as expected following simple alkylation of sperm DNA. Second, mutations induced by ENU at the gol-1 locus affected pigmentation but not viability, unlike the majority of mutations induced at this locus with gamma-irradiation. Third, the ratio of specific locus:recessive lethal mutations induced by ENU was approximately 50-fold lower than the ratio observed following mutagenesis with gamma-rays. Comparison of the incidence with which embryonic recessive lethal mutations were induced with the incidence with which specific locus mutations arose indicates that there are greater than 5000 genes essential to the development and viability of the zebrafish embryo.
Serum-free mouse embryo (SFME) cells, derived in medium in which serum is replaced with growth factors and other supplements, display distinctive properties: (i) SFME cells do not lose proliferative potential or show gross chromosomal aberration upon extended culture, (ii) these cells depend on epidermal growth factor for survival; and (iii) SFME cell proliferation is reversibly inhibited by serum. Treatment of SFME cells with serum or transforming growth factor beta led to the appearance of glial fibrillary acidic protein, a specific marker for astrocytes. The appearance of glial fibrillary acidic protein in cultures was reversed upon removal of transforming growth factor beta or serum. Cells with properties similar to SFME cells were also isolated from adult mouse brain. These results suggest a role for transforming growth factor beta in astrocyte differentiation in developing organisms and in response to injury and identify the cell type that has the unusual properties of SFME cells.
Vertebrate embryonic development has traditionally been studied in the amphibian and the chick. In these organisms extensive experimental manipulations, and, more recently, molecular characterization have helped to illuminate mechanisms of cell-cell interactions that lead to lineage development and pattern formation. But neither Xenopus nor chick is readily amenable to genetic studies. The power of mutational analysis in understand ing developmental events in invertebrates has led re searchers to consider other vertebrate species in which embryology and genetics might be combined to provide insights into the genetic control of early development. An obvious candidate is the mouse. The early embryol ogy of the mouse is fairly well understood (Rossant and Pedersen 1986), and there is already a storehouse of spon taneous and radiation-induced mutations affecting de velopmental processes (Green 1989). A number of these
An extract of 21-day rainbow trout embryos stimulated growth of several piscine cell lines in the absence of added serum. Established lines from trout (RTG-2 and STE-137), salmon (CHSE-214), carp (EPC), and goldfish (CAR) and early-passage cells initiated from trout embryos grew in serum-free medium containing the embryo extract. In addition the extract was sufficient for maintaining long-term cultures of CHSE-214 cells for several months through a minimum of 20 passages (approximately 50 population doublings) in the absence of serum. Optimal response was achieved with 100 micrograms of extract protein per ml, but a significant growth-promoting effect was observed with as little as 2.5 micrograms/ml. The activity was nondialyzable, protease-sensitive, and stable in 200 mM acetic acid. The level of mitogenic response induced by the extract could not be duplicated with purified mammalian growth factors added individually or in combination, and the extract did not stimulate DNA synthesis in quiescent mouse fibroblasts. These results suggest that trout embryo extract may contain a novel growth-promoting activity for fish cells.
We have analyzed lineages of cells labeled by intracellular injection of tracer dye during early zebrafish development to learn when cells become allocated to particular fates during development, and how the fate map is organized. The earliest lineage restriction was described previously, and segregates the yolk cell from the blastoderm in the midblastula. After one or two more cell divisions, the lineages of epithelial enveloping layer (EVL) cells become restricted to generate exclusively periderm. Following an additional division in the late blastula, deep layer (DEL) cells generate clones that are restricted to single deep embryonic tissues. The appearance of both the EVL and DEL restrictions could be causally linked to blastoderm morphogenesis during epiboly. A fate map emerges as the DEL cell lineages become restricted in the late blastula. It is similar in organization to that of an amphibian embryo. DEL cells located near the animal pole of the early gastrula give rise to ectodermal fates (including the definitive epidermis). Cells located near the blastoderm margin give rise to mesodermal and endodermal fates. Dorsal cells in the gastrula form dorsal and anterior structures in the embryo, and ventral cells in the gastrula form dorsal, ventral and posterior structures. The exact locations of progenitors of single cell types and of local regions of the embryo cannot be mapped at the stages we examined, because of variable cell rearrangements during gastrulation.
Fish represent the largest and most diverse group of vertebrates. Their evolutionary position relative to other vertebrates
and their ability to adapt to a wide variety of environments make them ideal for studying both organismic and molecular evolution.
A number of other characteristics make them excellent experimental models for studies in embryology, neurobiology, endocrinology,
environmental biology, and other areas. In fact, they have played a critical role in the development of several of these disciplines.
Research techniques that enable scientists to make isogenic lines in a single generation, create and maintain mutants, culture
cells, and transfer cloned genes into embryos signal an increasing role for fish as experimental models.
Zebrafish genes and development are being studied in a growing number of laboratories. Given that many other organisms are already being exploited by large numbers of investigators, and that our general knowledge about the zebrafish embryo and genome is at present rather sketchy, why should we now concern ourselves with how this tropical fish develops? Whereas the zebrafish embryo is similar in important ways to other vertebrate embryos, it is relatively simple and unusually accessible for both cellular and genetic analyses.
Leukaemia inhibitory factor (LIF) is a cytokine that induces macrophage differentiation of the murine M1 myeloid leukaemia cell line. We have isolated a cDNA clone encoding a novel human haemopoietic growth factor, human interleukin for DA cells (HILDA) that supports the proliferation of the murine interleukin-3-dependent leukaemic cell line, DA-la (refs 3-5). HILDA proved to be identical to LIF. The demonstration that the differentiation factor LIF will also serve as a growth factor for at least one myeloid leukaemic cell line provides further evidence that the distinction between growth-promoting and differentiation-inducing activities are largely determined by the target cell type.
Homozygous diploid zebra fish have been produced on a large scale by the application of simple physical treatments. Clones of homozygous fish have been produced from individual homozygotes. These clones and associated genetic methods will facilitate genetic analyses of this vertebrate.
During the last decade, zebrafish (Brachydanio rerio) have emerged as a novel and attractive system to study embryogenesis and organogenesis in vertebrates. The main reason is that both extensive genetic studies and detailed embryologic analysis are possible using this small tropical fresh water teleost. However, in vitro analysis using cell culture or molecular genetics are still far less advanced than in other vertebrate systems. Here we report the generation and characterization of a fibroblast like cell line, ZF4, derived from 1-day-old zebrafish embryos. The hyperploid cell line has been stable in multiple passages for more than 2 yr now and is the first zebrafish cell line that can be maintained in conventional medium containing mammalian serum. Using a series of plasmids for expression of a marker gene, we evaluate in ZF4 cells the relative strength of expression from several different viral, fish, and mammalian promoters. Stable integration can be obtained by using G418 selection. We hope that our cell line will be a useful tool for the analysis of gene regulation in zebrafish.
In the zebrafish, cells of a clone derived from a single blastomere migrate away from one another during gastrulation. Later in development their descendants are usually found scattered within several different types of tissues of embryo. The divisions and migrations of individual cells were monitored during early development, revealing that in most cases the lineal descendants of single cells present at gastrula stage exclusively populate only single tissues, and may have stereotyped positional relationships within these tissues. Thus the gastrula stage is the first stage when heritable restrictions in cell type might arise in the zebrafish.
Chromosomes of the zebrafish
- A Endo
- T Ingalls