Acute toxic effects of 8‐epidiosbulbin E,a 19‐norclerodane diterpene from yam Dioscorea antaly,on medaka Oryzias latipes embryos

The fractionation of an aqueous extract of yam Dioscorea antaly from Madagascar led to the isolation of terpenoids and flavonoids. Compounds were identified on the basis of modern mass spectrometry and two‐dimensional nuclear magnetic resonance (2D‐NMR). Toxicological effects of the most abundant isolated compound, 8‐epidiosbulbin E were studied on medaka Oryzias latipes embryo‐larval development. The lethal concentration (killing 50%; LC₅₀) to embryos treated 24 h before hatching and for 3 days after hatching was estimated to be 0·56 mg ml^?1 (P< 0·05). No mortality was observed with O. latipes larvae exposed after hatching until day 4. Anatomo‐pathological studies of embryos exposed to 0·56 mg ml^?1 showed development anomalies of the central nervous system, liver, muscle and intestine. The present data thus extend the model of O. latipes embryos as a useful animal model to analyse the effects of food toxins.     

Gonadal sex differentiation and expression of Sox9a2, Dmrt1, and Foxl2 in Oryzias luzonensis

Oryzias luzonensis is closely related to the medaka, O. latipes. The sex of both species is determined by an XX‐XY system. However, the testis determining gene (DMY/Dmrt1bY) found in O. latipes does not exist in O. luzonensis. Instead, a different gene is thought to act as a testis determining gene. In this study, we focused the gonadal sex differentiation process in O. luzonensis under different testis determining gene. First, we observed the gonadal development of O. luzonensis histologically. We then analyzed the expression of Sox9a2/Sox9b, Dmrt1, and Foxl2 during early development. Our results suggest that the sexual differentiation of germ cells in O. luzonensis is initiated later than in O. latipes. However, the timing of the sexual differentiation of the supporting cell linage is similar between the species. genesis 47:289–299, 2009. © 2009 Wiley‐Liss, Inc.     

Establishment and growth responses of Nile tilapia embryonic stem‐like cell lines under feeder‐free condition

Embryonic stem (ES) cells provide an invaluable tool for molecular analysis of vertebrate development and a bridge linking genomic manipulations in vitro and functional analysis of target genes in vivo. Work towards fish ES cells so far has focused on zebrafish (Danio renio) and medaka (Oryzias latipes). Here we describe the derivation, pluripotency, differentiation and growth responses of ES cell lines from Nile tilapia (Oreochromis niloticus), a world‐wide commercial farmed fish. These cell lines, designated as TES1‐3, were initiated from blastomeres of Nile tilapia middle blastula embryos (MBE). One representative line, TES1, showed stable growth and phenotypic characteristics of ES cells over 200 days of culture with more than 59 passages under feeder‐free conditions. They exhibited high alkaline phosphatase activity and expression of pluripotency genes including pou5f3 (the pou5f1/oct4 homologue), sox2, myc and klf4. In suspension culture together with retinoic acid treatment, TES1 cells formed embryoid bodies, which exhibited expression profile of differentiation genes characteristics of all three germ cell layers. Notably, PKH26‐labeled TES1 cells introduced into Nile tilapia MBE could contribute to body compartment development and led to hatched chimera formation with an efficacy of 13%. These results suggest that TES1 cells have pluripotency and differentiation potential in vitro and in vivo. In the conditioned DMEM, all of the supplements including the fetal bovine serum, fish embryonic extract, fish serum, basic fibroblast growth factor and non‐protein supplement combination 5N were mitogenic for TES1 cell growth. This study will promote ES‐based biotechnology in commercial fish.     

Expression profile of oestrogen receptors and oestrogen‐related receptors is organ specific and sex dependent: the Japanese medaka Oryzias latipes model

Gene expression of all known subtypes of oestrogen receptor (ER) and oestrogen‐related receptor (ERR) in multiple organs and both sexes of the Japanese medaka Oryzias latipes was profiled and systematically analysed. As revealed by statistical analyses and low‐dimensional projections, the expressions of ERRs proved to be organ and sex dependent, which is in contrast with the ubiquitous nature of ERs. Moreover, expressions of specific ERR isoforms (ERRγ1, ERRγ2) were strongly correlated with that of all ERs (ERα, ERβ1 and ERβ2), suggesting the existence of potential interactions. Findings of this study shed light on the co‐regulatory role of particular ERRs in oestrogen‐ERs signalling and highlight the potential importance of ERRs in determining organ and sex‐specific oestrogen responses. Using O. latipes as an alternative vertebrate model, this study provides new directions that call for collective efforts from the scientific community to unravel the mechanistic action of ER‐ERR cross‐talks, and their intertwining functions, in a cell and sex‐specific manner in vivo.     

Infrared laser‐mediated local gene induction in medaka,zebrafish and Arabidopsis thaliana

Heat shock promoters are powerful tools for the precise control of exogenous gene induction in living organisms. In addition to the temporal control of gene expression, the analysis of gene function can also require spatial restriction. Recently, we reported a new method for in vivo, single‐cell gene induction using an infrared laser‐evoked gene operator (IR‐LEGO) system in living nematodes (Caenorhabditis elegans). It was demonstrated that infrared (IR) irradiation could induce gene expression in single cells without incurring cellular damage. Here, we report the application of IR‐LEGO to the small fish, medaka (Japanese killifish; Oryzias latipes) and zebrafish (Danio rerio), and a higher plant (Arabidopsis thaliana). Using easily observable reporter genes, we successfully induced gene expression in various tissues in these living organisms. IR‐LEGO has the potential to be a useful tool in extensive research fields for cell/tissue marking or targeted gene expression in local tissues of small fish and plants.     

Evolution of growth rates under the constraint of growth-development trade-off in a fish

Intrinsic growth rates often vary greatly among populations within a single species, implying that trade-offs with fast growth are present. It has been hypothesized that such a trade-off exists between growth rate and development rate. Growth-development trade-offs have been considered from observations of a negative correlation between growth and development rates among populations. In this study, we examined not only interpopulation but also intrapopulation correlations in a fish Oryzias latipes. Rearing experiments revealed that larvae from a high-latitude population grew faster but metamorphosed at larger sizes than larvae from a low-latitude population. Moreover, within each population, individuals that grew faster tended to delay metamorphosis. The parallelism of the negative interpopulation and intrapopulation correlations between growth and development rates strongly support a growth-development trade-off. Observations of swimming behaviors revealed that high-latitude, fast-growing juveniles showed lower steady-swimming and burst-swimming speeds, probably reflecting that their underdeveloped skeletal and muscular structures translated into the poorer swimming performances. These results suggest that the higher growth capacity of high-latitude O. latipes has evolved at the expense of fast development.     

Length–weight relationships for four small fish species caught in wetlands of central Yangtze River,China

Length–weight relationships for four native species (Oryzias latipes Temminck & Schlegel, 1846; Rhodeus ocellatus Kner, 1866; Rhinogobius giurinus Rutter, 1897; and Micropercops swinhonis Günther, 1873) were determined. The fish were collected from the wetlands of the central Yangtze River, in Wuhan, Hubei Province, China. Samples were obtained from May 2012 to January 2013. Length–weight relationships for O. latipes were determined for the first time.     

Electroshock‐induced mortality in freshwater fish embryos increases with embryo diameter: a model based on results from 10 species

The purpose of this study was to develop a model to predict electroshock‐induced mortality in embryos of freshwater fishes. Herein, electroshock‐induced mortality was evaluated for sauger Sander canadensis and rainbow trout Oncorhynchus mykiss, and results were combined with published results obtained for eight other species in studies that used consistent methodology. The model was based on embryo diameter (1·1–4·1 mm among species), the most sensitive stage of embryonic development (near completion of epiboly for all species), and mortality (to hatching or eyed stage) after exposure to a homogeneous electric field [direct current (DC) or 60 Hz pulsed DC (PDC)]. Embryo diameter was positively related to electroshock‐induced mortality for DC (P < 0·001) and 60 Hz PDC (P < 0·05). Results suggested that vulnerability to electroshock‐induced mortality was related to physical characteristics (i.e. size) of embryos rather than species differences, and indicated that prediction of vulnerability in untested species is possible. This is particularly important for protection of endangered species, and those species with larger embryo sizes may be at most risk from electroshock. Conversely, results also suggested that it may be possible to assist in removal of invasive non‐native fish species with large embryo diameters (e.g. O. mykiss) by application of electroshock to target and kill embryos of these species in some freshwater habitats.     

Design of novel artemisinin‐like derivatives with cytotoxic and anti‐angiogenic properties

Artemisinins are plant products with a wide range of medicinal applications. Most prominently, artesunate is a well tolerated and effective drug for treating malaria, but is also active against several protozoal and schistosomal infections, and additionally exhibits anti‐angiogenic, anti‐tumorigenic and anti‐viral properties. The array of activities of the artemisinins, and the recent emergence of malaria resistance to artesunate, prompted us to synthesize and evaluate several novel artemisinin‐like derivatives. Sixteen distinct derivatives were therefore synthesized and the in vitro cytotoxic effects of each were tested with different cell lines. The in vivo anti‐angiogenic properties were evaluated using a zebrafish embryo model. We herein report the identification of several novel artemisinin‐like compounds that are easily synthesized, stable at room temperature, may overcome drug‐resistance pathways and are more active in vitro and in vivo than the commonly used artesunate. These promising findings raise the hopes of identifying safer and more effective strategies to treat a range of infections and cancer.     

Normal development of the tomato clownfish Amphiprion frenatus: live imaging and in situ hybridization analyses of mesodermal and neurectodermal development

The normal embryonic development of the tomato clownfish Amphiprion frenatus was analysed using live imaging and by in situ hybridization for detection of mesodermal and neurectodermal development. Both morphology of live embryos and tissue‐specific staining revealed significant differences in the gross developmental programme of A. frenatus compared with better‐known teleost fish models, in particular, initiation of somitogenesis before complete epiboly, initiation of narrowing of the neurectoderm (neurulation) before somitogenesis, relatively early pigmentation of melanophores at the 10–15 somite stage and a distinctive pattern of melanophore distribution. These results suggest evolutionary adaptability of the teleost developmental programme. The ease of obtaining eggs, in vitro culture of the embryo, in situ staining analyses and these reported characteristics make A. frenatus a potentially important model marine fish species for studying embryonic development, physiology, ecology and evolution.     

The toxicity of endod extract to the early life stages of Dreissena bugensis

Toxicity bioassays were conducted on embryos and early larvae of quagga mussels, Dreissena bugensis, using a filtered aqueous extract and a lyophilized butanol extract of the soap berry plant Phytolacca dodecandra. Developmental stages exposed to each extract were embryos to trochophores (ca 3h‐17h), trochophores to D‐hinge larvae (ca 17h‐40h) and embryos to D‐hinge (3h‐ca 40). Over the whole embryo to D‐hinge exposure period, the aqueous extract resulted in a lowest observed effective concentration (LOEC) of 5mgl^‐1 although mortality did not exceed 50%. For the butanol extract, the LOEC was 2mgl^‐1 and the LC₅₀ was 2.1 mgl^‐1. For the aqueous extract, most of the endod toxicity was seen at the embryo stage, whereas for the butanol extract the toxicity was associated with the trochophore stage. Compared with other non‐oxidizing commercial molluscicides, endod has only moderate toxicity to early dreissenid life stages.     

Influence of cortisol along the pituitary‐ovary axis in the cichlid fish Oreochromis mossambicus

Cortisol is the principal glucocorticoid released due to various forms of environmental as well as aquacultural stressors in fish. The aim of the present investigation was to determine cortisol‐induced alterations along the luteinizing hormone (LH)‐secreting cells–ovary axis in the tilapia Oreochromis mossambicus. Administration of cortisol to stripped O. mossambicus for a period of 22 days during the ovarian cycle caused significantly higher number of follicles with chromatin nucleoli (stage I) compared to those of initial controls and controls. Whereas the number of follicles at perinucleolar (stage II) and vitellogenic (stage IV) stages did not differ significantly between controls and cortisol‐treated fish, the number of follicles at cortical alveolar stage (stage III) was significantly lower in cortisol‐treated fish than in controls. While the stage V follicles (maturation stage) were absent in initial controls, their presence in controls was concomitant with intensely labelled LH‐secreting cells in the proximal pars distalis (PPD) region of the pituitary gland during prespawning phase. However, cortisol‐treatment resulted in complete absence of stage V follicles associated with weakly immunoreactive LH‐content in the PPD region of the pituitary gland during prespawning phase. These results suggest that chronic cortisol‐ treatment causes suppression of LH‐secreting cells activity and blocks progression of vitellogenic follicular development process in O. mossambicus.     

Colored medaka and zebrafish: Transgenics with ubiquitous and strong transgene expression driven by the medaka β‐actin promoter

Conditional cell labeling, cell tracing, and genetic manipulation approaches are becoming increasingly important in developmental and regenerative biology. Such approaches in zebrafish research are hampered by the lack of an ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the medaka fish (Oryzias latipes) β‐actin (Olactb) promoter, which drives constitutive transgene expression during all developmental stages, and the analysis of adult organs except blood cell types. Taking advantage of the compact medaka promoter, we succeeded in generating a zebrafish transgenic (Tg) line with unprecedentedly strong and widespread transgene expression from embryonic to adult stages. Moreover, the Tg carries a pair of loxP sites, which enables the reporter fluorophore to switch from DsRed2 to enhanced green fluorescent protein (EGFP). We induced Cre/loxP recombination with Tg(hsp70l: mCherry‐t2a‐Cre^ERt2) in the double Tg embryo and generated a Tg line that constitutively expresses EGFP. We further demonstrate the powerful application of Olactb‐driven Tgs for cell lineage tracing using transplantation experiments with embryonic cells at the shield stage and adult cells of regenerating fin. Thus, the use of promoter elements from medaka is an alternative approach to generate Tgs with stronger and even novel expression patterns in zebrafish. The Olactb promoter and the Tg lines presented here represent an important advancement for the broader use of Cre/loxP‐based Tg applications in zebrafish.     

The accumulation of manganese from the environmental medium by the egg of Oryzias latipes

The manganese content of the egg and embryo of the Medaka, Oryzias latipes was determined by activation analysis. A remarkable increase in the amount of manganese in the egg was observed within one hour after fertilization. The rate of increase was reduced by the gastrula stage and the concentration of manganese remained unchanged at a later stage. The accumulation of manganese by the Oryzias egg was discussed in relation to the effect of manganese on respiratory enzyme systems.     

Improved in vivo gene transfer into tumor tissue by stabilization of pseudodendritic oligoethylenimine‐based polyplexes

Background

HD O is a low molecular weight pseudodendrimer containing oligoethylenimine and degradable hexanediol diacrylate diesters. DNA polyplexes display encouraging gene transfer efficiency in vitro and in vivo but also a limited stability under physiological conditions. This limitation must be overcome for further development into more sophisticated formulations.

Methods

HD O polyplexes were laterally stabilized by crosslinking surface amines via bifunctional crosslinkers, bioreducible dithiobis(succimidyl propionate) (DSP) or the nonreducible analog disuccinimidyl suberate (DSS). Optionally, in a subsequent step, the targeting ligand transferrin (Tf) was attached to DSP‐linked HD O polyplexes via Schiff base formation between HD O amino groups and Tf aldehyde groups, which were introduced into Tf by periodate oxidation of the glycosylation sites.

Results

Crosslinked DNA polyplexes showed an increased stability against exchange reaction by salt or heparin. Disulfide bond containing DSP‐linked polyplexes were susceptible to reducing conditions. These polyplexes displayed the highest gene expression levels in vitro and in vivo (upon intratumoral application in mice), and these were significantly elevated and prolonged over standard or DSS‐stabilized HD O formulations. DSP‐stabilized HD O polyplexes with or without Tf coating were well‐tolerated after intravenous application. High gene expression levels were found in tumor tissue, with negligible gene expression in any other organ.

Conclusions

Lateral stabilization of HD O polyplexes with DSP crosslinker enhanced gene transfer efficacy and was essential for the incorporation of a ligand (Tf) into a stable particle formulation. Copyright © 2010 John Wiley & Sons, Ltd.