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Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos
Francisca Alcaraz-Pérez, Victoriano Mulero, María L Cayuela
BMC Biotechnology , 2008, DOI: 10.1186/1472-6750-8-81
Abstract: Here, we present a rapid and sensitive assay based on the classical dual-luciferase reporter technique which can be used as a new tool to characterize the minimum promoter region of a gene as well as the in vivo response of inducible promoters to different stimuli. We illustrate the usefulness of this system for studying both constitutive (telomerase) and inducible (NF-κB-dependent) promoters. The flexibility of this assay is demonstrated by induction of the NF-κB-dependent promoters using simultaneous microinjection of different pathogen-associated molecular patterns as well as with the use of morpholino-gene mediated knockdown.This assay has several advantages compared with the classical in vitro (cell lines) and in vivo (transgenic mice) approaches. Among others, the assay allows a rapid and quantitative measurement of the effects of particular genes or drugs in a given promoter in the context of a whole organism and it can also be used in high throughput screening experiments.The zebrafish has been established as an excellent model for studying any biological process. This organism possesses many advantages including ease of experimentation, optical clarity, drug administration, amenability to in vivo manipulation and feasibility of reverse and forward genetic approaches. The fish reach sexual maturity in only 3 to 4 months, and adult females are capable of producing 100 to 200 eggs weekly. Many thousands of animals can be kept in a fish facility requiring much less space than mice or other mammals, and hence the zebrafish is regarded as a cost-effective experimental vertebrate model for large-scale genetic screening [1]. Furthermore, the high degree of homology between the zebrafish genome and that of humans makes such discoveries especially pertinent to human disease and development [2,3].Morpholino antisense oligonucleotides (MO) have been widely used to inhibit gene function in zebrafish embryos [4-7] and are usually used as sequence-specific translation-blo
Proteomics of early zebrafish embryos
Vinzenz Link, Andrej Shevchenko, Carl-Philipp Heisenberg
BMC Developmental Biology , 2006, DOI: 10.1186/1471-213x-6-1
Abstract: As a prerequisite to carry out proteomic experiments with early zebrafish embryos, we developed a method to efficiently remove the yolk from large batches of embryos. This method enabled high resolution 2D gel electrophoresis and improved Western blotting considerably. Here, we provide detailed protocols for proteomics in zebrafish from sample preparation to mass spectrometry (MS), including a comparison of databases for MS identification of zebrafish proteins.The provided protocols for proteomic analysis of early embryos enable research to be taken in novel directions in embryogenesis.The zebrafish has become a widely used vertebrate model system for which a large tool-box of genetic and cell biological methods has been established [1,2]. Research using zebrafish is further supported by the zebrafish sequencing project, which has facilitated the generation of microarrays for large scale expression profiling. It has been proposed that proteomics should complement the genome-wide expression profiling [3]. However, a major obstacle in the application of proteomics has been the high proportion of yolk proteins in early embryos. Proteomic studies in zebrafish have therefore been limited to adult tissues [4]. One study targeted larval stages 48 or 72 hpf (hours post fertilization), when the yolk to cell mass ratio is already decreased [5], however, without identifying the proteins. Therefore, it remains unclear whether at this stage analysis without deyolking provides satisfactory information about cellular proteins. Thus, the development of a reliable method to remove the interfering yolk from cells on a large scale is required to apply proteomics to early embryos.Here, we provide detailed protocols for all zebrafish-specific steps of a proteomic experiment from dechorionation to mass spectrometry-based protein identification. As a key step, we present and validate a method for batch removal of the yolk from early embryos.In the early embryo, the cells forming the embry
A Comparative Analysis of Glomerulus Development in the Pronephros of Medaka and Zebrafish  [PDF]
Koichiro Ichimura, Ekaterina Bubenshchikova, Rebecca Powell, Yayoi Fukuyo, Tomomi Nakamura, Uyen Tran, Shoji Oda, Minoru Tanaka, Oliver Wessely, Hidetake Kurihara, Tatsuo Sakai, Tomoko Obara
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0045286
Abstract: The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects.
Medaka Cleavage Embryos Are Capable of Generating ES-Like Cell Cultures
Zhendong Li, Narayani Bhat, Dwarakanath Manali, Danke Wang, Ni Hong, Meisheng Yi, Ruowen Ge, Yunhan Hong
International Journal of Biological Sciences , 2011,
Abstract: Mammalian embryos at the blastocyst stage have three major lineages, which in culture can give rise to embryonic stem (ES) cells from the inner cell mass or epiblast, trophoblast stem cells from the trophectoderm, and primitive endoderm stem cells. None of these stem cells is totipotent, because they show gene expression profiles characteristic of their sources and usually contribute only to the lineages of their origins in chimeric embryos. It is unknown whether embryos prior to the blastocyst stage can be cultivated towards totipotent stem cell cultures. Medaka is an excellent model for stem cell research. This laboratory fish has generated diploid and even haploid ES cells from the midblastula embryo with ~2000 cells. Here we report in medaka that dispersed cells from earlier embryos can survive, proliferate and attach in culture. We show that even 32-cells embryos can be dissociated into individual cells capable of producing continuously growing ES-like cultures. Our data point to the possibility to derive stable cell culture from cleavage embryos in this organism.
Specification of primordial germ cells in medaka (Oryzias latipes)
Amaury Herpin, Stefan Rohr, Dietmar Riedel, Nils Kluever, Erez Raz, Manfred Schartl
BMC Developmental Biology , 2007, DOI: 10.1186/1471-213x-7-3
Abstract: PGCs specification in zebrafish appears to depend on inheritance of germ plasm in which several RNA molecules such as vasa and nanos reside. Whether the specification mode of PGCs found in zebrafish is general for other fish species was brought into question upon analysis of olvas expression – the vasa homologue in another teleost, medaka (Oryzias latipes). Here, in contrast to the findings in zebrafish, the PGCs are found in a predictable position relative to a somatic structure, the embryonic shield. This finding, coupled with the fact that vasa mRNA, which is localized to the germ plasm of zebrafish but does not label a similar structure in medaka opened the possibility of fundamentally different mechanisms governing PGC specification in these two fish species.In this study we addressed the question concerning the mode of PGC specification in medaka using embryological experiments, analysis of RNA stability in the PGCs and electron microscopy observations. Dramatic alterations in the somatic environment, i.e. induction of a secondary axis or mesoderm formation alteration, did not affect the PGC number. Furthermore, the PGCs of medaka are capable of protecting specific RNA molecules from degradation and could therefore exhibit a specific mRNA expression pattern controlled by posttrancriptional mechanisms. Subsequent analysis of 4-cell stage medaka embryos using electron microscopy revealed germ plasm-like structures located at a region corresponding to that of zebrafish germ plasm.Taken together, these results are consistent with the idea that in medaka the inheritance of maternally provided asymmetrically-localized cytoplasmic determinants directs cells to assume the germ line fate similar to zebrafish PGCs.Two basic strategies for the specification of primordial germ cells (PGCs) have been described. In Drosophila, C. elegans and Xenopus the inheritance of germ plasm with associated germ cell determinants appears to be required for PGC formation [1-3]. In these
Influence of carbon nanotube length on toxicity to zebrafish embryos
Cheng J, Cheng SH
International Journal of Nanomedicine , 2012, DOI: http://dx.doi.org/10.2147/IJN.S30459
Abstract: fluence of carbon nanotube length on toxicity to zebrafish embryos Original Research (2384) Total Article Views Authors: Cheng J, Cheng SH Published Date July 2012 Volume 2012:7 Pages 3731 - 3739 DOI: http://dx.doi.org/10.2147/IJN.S30459 Received: 01 February 2012 Accepted: 27 March 2012 Published: 20 July 2012 Jinping Cheng,1,2 Shuk Han Cheng1 1Department of Biology and Chemistry, City University of Hong Kong, Hong Kong; 2State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, China Abstract: There is currently a large difference of opinion in nanotoxicology studies of nanomaterials. There is concern about why some studies have indicated that there is strong toxicity, while others have not. In this study, the length of carbon nanotubes greatly affected their toxicity in zebrafish embryos. Multiwalled carbon nanotubes (MWCNTs) were sonicated in a nitric acid solution for 24 hours and 48 hours. The modified MWCNTs were tested in early developing zebrafish embryo. MWCNTs prepared with the longer sonication time resulted in severe developmental toxicity; however, the shorter sonication time did not induce any obvious toxicity in the tested developing zebrafish embryos. The cellular and molecular changes of the affected zebrafish embryos were studied and the observed phenotypes scored. This study suggests that length plays an important role in the in vivo toxicity of functionalized CNTs. This study will help in furthering the understanding on current differences in toxicity studies of nanomaterials.
Highly Asynchronous and Asymmetric Cleavage Divisions Accompany Early Transcriptional Activity in Pre-Blastula Medaka Embryos  [PDF]
Michael Kraeussling,Toni Ulrich Wagner,Manfred Schartl
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0021741
Abstract: In the initial phase of development of fish embryos, a prominent and critical event is the midblastula transition (MBT). Before MBT cell cycle is rapid, highly synchronous and zygotic gene transcription is turned off. Only during MBT the cell cycle desynchronizes and transcription is activated. Multiple mechanisms, primarily the nucleocytoplasmic ratio, are supposed to control MBT activation. Unexpectedly, we find in the small teleost fish medaka (Oryzias latipes) that at very early stages, well before midblastula, cell division becomes asynchronous and cell volumes diverge. Furthermore, zygotic transcription is extensively activated already after the 64-cell stage. Thus, at least in medaka, the transition from maternal to zygotic transcription is uncoupled from the midblastula stage and not solely controlled by the nucleocytoplasmic ratio.
Low Temperature Mitigates Cardia Bifida in Zebrafish Embryos  [PDF]
Che-Yi Lin, Cheng-Chen Huang, Wen-Der Wang, Chung-Der Hsiao, Ching-Feng Cheng, Yi-Ting Wu, Yu-Fen Lu, Sheng-Ping L. Hwang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0069788
Abstract: The coordinated migration of bilateral cardiomyocytes and the formation of the cardiac cone are essential for heart tube formation. We investigated gene regulatory mechanisms involved in myocardial migration, and regulation of the timing of cardiac cone formation in zebrafish embryos. Through screening of zebrafish treated with ethylnitrosourea, we isolated a mutant with a hypomorphic allele of mil (s1pr2)/edg5, called s1pr2as10 (as10). Mutant embryos with this allele expressed less mil/edg5 mRNA and exhibited cardia bifida prior to 28 hours post-fertilization. Although the bilateral hearts of the mutants gradually fused together, the resulting formation of two atria and one tightly-packed ventricle failed to support normal blood circulation. Interestingly, cardia bifida of s1pr2as10 embryos could be rescued and normal circulation could be restored by incubating the embryos at low temperature (22.5°C). Rescue was also observed in gata5 and bon cardia bifida morphants raised at 22.5°C. The use of DNA microarrays, digital gene expression analyses, loss-of-function, as well as mRNA and protein rescue experiments, revealed that low temperature mitigates cardia bifida by regulating the expression of genes encoding components of the extracellular matrix (fibronectin 1, tenascin-c, tenascin-w). Furthermore, the addition of N-acetyl cysteine (NAC), a reactive oxygen species (ROS) scavenger, significantly decreased the effect of low temperature on mitigating cardia bifida in s1pr2as10 embryos. Our study reveals that temperature coordinates the development of the heart tube and somitogenesis, and that extracellular matrix genes (fibronectin 1, tenascin-c and tenascin-w) are involved.
Toxic Effects of Silica Nanoparticles on Zebrafish Embryos and Larvae  [PDF]
Junchao Duan, Yongbo Yu, Huiqin Shi, Linwei Tian, Caixia Guo, Peili Huang, Xianqing Zhou, Shuangqing Peng, Zhiwei Sun
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0074606
Abstract: Silica nanoparticles (SiNPs) have been widely used in biomedical and biotechnological applications. Environmental exposure to nanomaterials is inevitable as they become part of our daily life. Therefore, it is necessary to investigate the possible toxic effects of SiNPs exposure. In this study, zebrafish embryos were treated with SiNPs (25, 50, 100, 200 μg/mL) during 4–96 hours post fertilization (hpf). Mortality, hatching rate, malformation and whole-embryo cellular death were detected. We also measured the larval behavior to analyze whether SiNPs had adverse effects on larvae locomotor activity. The results showed that as the exposure dosages increasing, the hatching rate of zebrafish embryos was decreased while the mortality and cell death were increased. Exposure to SiNPs caused embryonic malformations, including pericardial edema, yolk sac edema, tail and head malformation. The larval behavior testing showed that the total swimming distance was decreased in a dose-dependent manner. The lower dose (25 and 50 μg/mL SiNPs) produced substantial hyperactivity while the higher doses (100 and 200 μg/mL SiNPs) elicited remarkably hypoactivity in dark periods. In summary, our data indicated that SiNPs caused embryonic developmental toxicity, resulted in persistent effects on larval behavior.
Side chain modified peptide nucleic acids (PNA) for knock-down of six3 in medaka embryos  [cached]
Dorn Sebastian,Aghaallaei Narges,Jung Gerlinde,Bajoghli Baubak
BMC Biotechnology , 2012, DOI: 10.1186/1472-6750-12-50
Abstract: Background Synthetic antisense molecules have an enormous potential for therapeutic applications in humans. The major aim of such strategies is to specifically interfere with gene function, thus modulating cellular pathways according to the therapeutic demands. Among the molecules which can block mRNA function in a sequence specific manner are peptide nucleic acids (PNA). They are highly stable and efficiently and selectively interact with RNA. However, some properties of non-modified aminoethyl glycine PNAs (aegPNA) hamper their in vivo applications. Results We generated new backbone modifications of PNAs, which exhibit more hydrophilic properties. When we examined the activity and specificity of these novel phosphonic ester PNAs (pePNA) molecules in medaka (Oryzias latipes) embryos, high solubility and selective binding to mRNA was observed. In particular, mixing of the novel components with aegPNA components resulted in mixed PNAs with superior properties. Injection of mixed PNAs directed against the medaka six3 gene, which is important for eye and brain development, resulted in specific six3 phenotypes. Conclusions PNAs are well established as powerful antisense molecules. Modification of the backbone with phosphonic ester side chains further improves their properties and allows the efficient knock down of a single gene in fish embryos.
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