Efficient extraction, antioxidant activities and anti-inflammation of polysaccharides from Notopterygium franchetii Boiss.

To optimize the extraction of polysaccharides from Notopterygium franchetii Boiss (NFP), microwave-assisted aqueous two phase system (MAATPS) and response surface methodology (RSM) were employed. Results showed that the optimum conditions of MAATPS were as follows: ethanol concentration of 32 % (w/w), (NH4)2SO4 concentration of 24 % (w/w) and extraction time of 15 min. Under such condition, YS (the recovery of polysaccharides) was 80.57 % with 90.40 % of YP (the recovery of proteins). In addition, analysis of the physicochemical properties of extracted polysaccharides (NFP-MAATPS) under the optimal conditions indicated that compared to polysaccharides extracted by traditional extraction method (NFP-HWE), NFP-MAATPS exhibited higher extraction efficiency, decreased average molecular weight, different monosaccharide compositions and mole ratios. Moreover, NFP-MAATPS exhibited stronger antioxidant activity in vitro and more potent anti-inflammatory activity in zebrafish embryos than NFP-HWE. Our results suggest that purified NFP-MAATPS could be potentially used in complementary medicines or functional foods.

The role of PI3K/Akt/mTOR signaling in dose-dependent biphasic effects of glycine on vascular development.

Glycine, a non-essential amino acid, exerts concentration-dependent biphasic effects on angiogenesis. Low-doses of glycine promote angiogenesis, whereas high-doses cause anti-angiogenesis. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling participates in angiogenesis of both physiological development, and pathological events including tumor and inflammation. We assessed the role of PI3K/Akt/mTOR signaling in vascular development, and the interaction with glycine, using transgenic zebrafish Tg(fli1a:Myr-mCherry)ncv1 embryos expressing fluorescent proteins in vascular endothelial cells. Treatment with inhibitors of mTORC1 (rapamycin and everolimus), mTORC1/mTORC2 (KU0063794), PI3K (LY29400), and Akt (Akt inhibitor) decreased the development of intersegmental vessels (ISVs). These inhibitors cancelled the angiogenic effects of a low-dose of glycine, while acted synergistically with a high-dose of glycine in anti-angiogenesis. mTOR signaling regulates the gene expression of vascular endothelial growth factor (VEGF), a major angiogenic factor, and nitric oxide (NO) synthase (NOS), an enzyme for the synthesis of an angiogenic mediator NO. Expressions of VEGF and NOS were consistent with the vascular features induced by glycine and an mTOR inhibitor. Our results suggest that PI3K/Akt/mTOR signaling may interact with dose-dependent biphasic effects of exogenous glycine on in vivo angiogenesis. mTOR signaling is a key target for cancer therapy, thus, the combining mTOR inhibitors with glycine may be a potential approach for controlling angiogenesis.

Folic acid supplement rescues ethanol-induced developmental defects in the zebrafish embryos.

Fetal alcohol syndrome (FASD) describes a range of birth defects. Mechanisms of FASD-associated defects are not well understood. It has great significance to investigate whether nutrient supplements like folic acid (FA) can effectively rescue ethanol-induced defects. Moreover, it is very important to determine the optimal time for FA supplementation when it can most effectively antagonize the teratogenic effects of ethanol during embryonic development. Our results indicated that ethanol exposure interrupted the development of zebrafish embryos and induced multiple defects in cardiac function, pharyngeal arch arteries, vessel, craniofacial cartilage, pharyngeal arches, brain, somite and hemoglobin formation. The expressions of critical genes that play important roles in above organs such as tbx1, flk-1, hand2, ngn1, huc, titin, gata-1 and c-myb were reduced, and the apoptosis was increased in ethanol-treated group. FA supplementation could reverse ethanol-induced defects, improve the decreased expressions of above genes and reduce the apoptosis. We also found that giving FA at 6-12 h post-fertilization (hpf), which is at the gastrula period (5.25-10 hpf), can obviously prevent the teratogenicity of ethanol. This research provides clues for elucidating the mechanism of fetal abnormalities caused by alcohol intake and for preventing FASD.

The Early Ediacaran Caveasphaera Foreshadows the Evolutionary Origin of Animal-like Embryology.

The Ediacaran Weng'an Biota (Doushantuo Formation, 609 Ma old) is a rich microfossil assemblage that preserves biological structure to a subcellular level of fidelity and encompasses a range of developmental stages [1]. However, the animal embryo interpretation of the main components of the biota has been the subject of controversy [2, 3]. Here, we describe the development of Caveasphaera, which varies in morphology from lensoid to a hollow spheroidal cage [4] to a solid spheroid [5] but has largely evaded description and interpretation. Caveasphaera is demonstrably cellular and develops within an envelope by cell division and migration, first defining the spheroidal perimeter via anastomosing cell masses that thicken and ingress as strands of cells that detach and subsequently aggregate in a polar region. Concomitantly, the overall diameter increases as does the volume of the cell mass, but after an initial phase of reductive palinotomy, the volume of individual cells remains the same through development. The process of cell ingression, detachment, and polar aggregation is analogous to gastrulation; together with evidence of functional cell adhesion and development within an envelope, this is suggestive of a holozoan affinity. Parental investment in the embryonic development of Caveasphaera and co-occurring Tianzhushania and Spiralicellula, as well as delayed onset of later development, may reflect an adaptation to the heterogeneous nature of the early Ediacaran nearshore marine environments in which early animals evolved.

Cardiac remodeling in response to embryonic crude oil exposure involves unconventional NKX family members and innate immunity genes.

Cardiac remodeling results from both physiological and pathological stimuli. Compared with mammalian hearts, fish hearts show a broader array of remodeling changes in response to environmental influences, providing exceptional models for dissecting the molecular and cellular bases of cardiac remodeling. We recently characterized a form of pathological remodeling in juvenile pink salmon (Oncorhynchus gorbuscha) in response to crude oil exposure during embryonic cardiogenesis. In the absence of overt pathology (cardiomyocyte death or inflammatory infiltrate), cardiac ventricles in exposed fish showed altered shape, reduced thickness of compact myocardium and hypertrophic changes in spongy, trabeculated myocardium. Here, we used RNA sequencing to characterize molecular pathways underlying these defects. In juvenile ventricular cardiomyocytes, antecedent embryonic oil exposure led to dose-dependent upregulation of genes involved in innate immunity and two NKX homeobox transcription factors not previously associated with cardiomyocytes, nkx2.3 and nkx3.3 Absent from mammalian genomes, the latter is largely uncharacterized. In zebrafish embryos, nkx3.3 demonstrated a potent effect on cardiac morphogenesis, equivalent to that of nkx2.5, the primary transcription factor associated with ventricular cardiomyocyte identity. The role of nkx3.3 in heart growth is potentially linked to the unique regenerative capacity of fish and amphibians. Moreover, these findings support a cardiomyocyte-intrinsic role for innate immune response genes in pathological hypertrophy. This study demonstrates how an expanding mechanistic understanding of environmental pollution impacts - i.e. the chemical perturbation of biological systems - can ultimately yield new insights into fundamental biological processes.

Effects of hyperthyroidism in the development of the appendicular skeleton and muscles of zebrafish, with notes on evolutionary developmental pathology (Evo-Devo-Path).

The hypothalamus-pituitary-thyroid (HPT) axis plays a crucial role in the metabolism, homeostasis, somatic growth and development of teleostean fishes. Thyroid hormones regulate essential biological functions such as growth and development, regulation of stress, energy expenditure, tissue compound, and psychological processes. Teleost thyroid follicles produce the same thyroid hormones as in other vertebrates: thyroxin (T4) and triiodothyronine (T3), making the zebrafish a very useful model to study hypo- and hyperthyroidism in other vertebrate taxa, including humans. Here we investigate morphological changes in T3 hyperthyroid cases in the zebrafish to better understand malformations provoked by alterations of T3 levels. In particular, we describe musculoskeletal abnormalities during the development of the zebrafish appendicular skeleton and muscles, compare our observations with those recently done by us on the normal developmental of the zebrafish, and discuss these comparisons within the context of evolutionary developmental pathology (Evo-Devo-Path), including human pathologies.

In vivo assay of the ethanol-induced embryonic hair cell loss and the protective role of the retinoic and folic acid in zebrafish larvae (Danio rerio).

In reference to the auditory manifestation of fetal alcohol syndrome, previous work has preferentially focused on the deviant neural development of the auditory system. Changes in the sensory hair cell, the ultimate sensory organ, were not well understood. In this study, we carried out an in vivo assessment of the embryonic hair cell changes on the lateral line of zebrafish upon exposure to various ethanol concentrations (0.25%, 0.5%, 0.75%, and 1.0%). A significant decrease in the hair cell count was confirmed as the ethanol concentration increased. Long-term observation (up to 240 hours post-fertilization [hpf]) suggested an irreversible hair cell loss with little chance of a simple delayed development. For an underlying biological process, a significant increase of hair cell apoptosis and a significant decrease of cytoplasmic mitochondria were confirmed as the ethanol concentration increased. Co-treatment with retinoic (0.1 nM) or folic (0.1 mM) acid with the same concentrations of ethanol resulted in significant increases in the remaining hair cells, compared to the ethanol-only treatment group, for every ethanol concentration. The retinoic acid provided more effective protection over folic acid, resulting in no significant changes in hair cell counts for every ethanol concentration (except 1.0%), compared with that of the negative control (without chemical treatment). Hair cell counts in every ethanol concentration were significantly lower than those in negative controls without chemical treatment after folic acid co-treatment. In conclusion, gestational ethanol exposure causes developmental sensory hair cell loss. Potential underlying mechanisms include retinoic or folic acid deficiency, and mitochondrial damage with subsequent hair cell apoptosis. Hair cell loss could possibly be prevented by administering either retinoic or folic acid, with retinoic acid supplementation as the preferred treatment.

Identification of Novel Ezrin Inhibitors Targeting Metastatic Osteosarcoma by Screening Open Access Malaria Box.

Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins and functions as a linker between the plasma membrane and the actin cytoskeleton. Ezrin is a key driver of tumor progression and metastatic spread of osteosarcoma. We discovered a quinoline-based small molecule, NSC305787, that directly binds to ezrin and inhibits its functions in promoting invasive phenotype. NSC305787 possesses a very close structural similarity to commonly used quinoline-containing antimalarial drugs. On the basis of this similarity and of recent findings that ezrin has a likely role in the pathogenesis of malaria infection, we screened antimalarial compounds in an attempt to identify novel ezrin inhibitors with better efficacy and drug properties. Screening of Medicines for Malaria Venture (MMV) Malaria Box compounds for their ability to bind to recombinant ezrin protein yielded 12 primary hits with high selective binding activity. The specificity of the hits on ezrin function was confirmed by inhibition of the ezrin-mediated cell motility of osteosarcoma cells. Compounds were further tested for phenocopying the morphologic defects associated with ezrin suppression in zebrafish embryos as well as for inhibiting the lung metastasis of high ezrin-expressing osteosarcoma cells. The compound MMV667492 exhibited potent anti-ezrin activity in all biologic assays and had better physicochemical properties for drug-likeness than NSC305787. The drug-like compounds MMV020549 and MMV666069 also showed promising activities in functional assays. Thus, our study suggests further evaluation of antimalarial compounds as a novel class of antimetastatic agents for the treatment of metastatic osteosarcoma.

Lipid Abundance in Zebrafish Embryos Is Regulated by Complementary Actions of the Endocannabinoid System and Retinoic Acid Pathway.

The endocannabinoid system (ECS) and retinoic acid (RA) signaling have been associated with influencing lipid metabolism. We hypothesized that modulation of these pathways could modify lipid abundance in developing vertebrates and that these pathways could have a combinatorial effect on lipid levels. Zebrafish embryos were exposed to chemical treatments altering the activity of the ECS and RA pathway. Embryos were stained with the neutral lipid dye Oil-Red-O (ORO) and underwent whole-mount in situ hybridization (WISH). Mouse 3T3-L1 fibroblasts were differentiated under exposure to RA-modulating chemicals and subsequently stained with ORO and analyzed for gene expression by qRT-PCR. ECS activation and RA exposure increased lipid abundance and the expression of lipoprotein lipase. In addition, RA treatment increased expression of CCAAT/enhancer-binding protein alpha. Both ECS receptors and RA receptor subtypes were separately involved in modulating lipid abundance. Finally, increased ECS or RA activity ameliorated the reduced lipid abundance caused by peroxisome proliferator-activated receptor gamma (PPARγ) inhibition. Therefore, the ECS and RA pathway influence lipid abundance in zebrafish embryos and have an additive effect when treated simultaneously. Furthermore, we demonstrated that these pathways act downstream or independently of PPARγ to influence lipid levels. Our study shows for the first time that the RA and ECS pathways have additive function in lipid abundance during vertebrate development.

A novel whole-embryo culture model for pharmaceutical and developmental studies.

INTRODUCTION: This report introduces a new vertebrate whole-embryo culture model for the direct application of pharmaceuticals and/or toxins into developing embryos. This method uses a terrestrial amphibian system and therefore has eliminated the problem of mammalian placental and uterine concerns. To test the utility and effectiveness of this method, we investigated the effect of fluoxetine on craniofacial development. Fluoxetine is one of the most commonly prescribed selective serotonin reuptake inhibitor (SSRI) on the market and treatment of depression during pregnancy is commonly deemed necessary. Previous studies have shown that SSRIs may promote developmental defects and congenital malformations of the heart. METHODS: This model utilized the egg/embryos of the directly developing Puerto Rican coquí frog, Eleutherodactylus coqui. The E. coqui embryo clutches were placed on filter paper in a Petri dish and were directly exposed (chronically) to fluoxetine concentrations ranging from 0.10mM to 1.0mM. Traditional whole-mount bone (Alizarin red) and cartilage (Alcian blue) staining was utilized to show the effect of fluoxetine on craniofacial development. RESULTS: Whole-mount staining revealed profound defects in cartilage development, particularly in the nasal capsule, mandible, and the brain case. Further, fluoxetine-treated embryos developed significantly slower compared to control animals. DISCUSSION: We found that the E. coqui culture model was an effective and sensitive technique for pharmaceutical studies, particularly since it allows the direct application of drugs and toxins into the developing embryo without the hindrance of the uterus and placenta. Chromatographic analysis revealed that fluoxetine infiltrated and penetrated embryonic tissue. It was found that altering serotonergic activity during development, via fluoxetine, stunted craniofacial development and organization.

Pharmacologic rescue of an enzyme-trafficking defect in primary hyperoxaluria 1.

Primary hyperoxaluria 1 (PH1; Online Mendelian Inheritance in Man no. 259900), a typically lethal biochemical disorder, may be caused by the AGT(P11LG170R) allele in which the alanine:glyoxylate aminotransferase (AGT) enzyme is mistargeted from peroxisomes to mitochondria. AGT contains a C-terminal peroxisomal targeting sequence, but mutations generate an N-terminal mitochondrial targeting sequence that directs AGT from peroxisomes to mitochondria. Although AGT(P11LG170R) is functional, the enzyme must be in the peroxisome to detoxify glyoxylate by conversion to alanine; in disease, amassed glyoxylate in the peroxisome is transported to the cytosol and converted to oxalate by lactate dehydrogenase, leading to kidney failure. From a chemical genetic screen, we have identified small molecules that inhibit mitochondrial protein import. We tested whether one promising candidate, Food and Drug Administration (FDA)-approved dequalinium chloride (DECA), could restore proper peroxisomal trafficking of AGT(P11LG170R). Indeed, treatment with DECA inhibited AGT(P11LG170R) translocation into mitochondria and subsequently restored trafficking to peroxisomes. Previous studies have suggested that a mitochondrial uncoupler might work in a similar manner. Although the uncoupler carbonyl cyanide m-chlorophenyl hydrazone inhibited AGT(P11LG170R) import into mitochondria, AGT(P11LG170R) aggregated in the cytosol, and cells subsequently died. In a cellular model system that recapitulated oxalate accumulation, exposure to DECA reduced oxalate accumulation, similar to pyridoxine treatment that works in a small subset of PH1 patients. Moreover, treatment with both DECA and pyridoxine was additive in reducing oxalate levels. Thus, repurposing the FDA-approved DECA may be a pharmacologic strategy to treat PH1 patients with mutations in AGT because an additional 75 missense mutations in AGT may also result in mistrafficking.

Generalized estimating equation approach for analyzing the effects of metal-derived products on survival and hatching of zebrafish embryos.

Zebrafish embryos are widely used as a model to monitor the effect of chemicals on their survival and hatching at different time epochs. This experimental design generates longitudinal data in which the observations for a given subject are correlated and they are statistically independent across the subjects. This particular nature of the observations suggests the use of generalized estimating equation (GEE) methodology for performing meaningful statistical analysis. However, it has been observed that the researchers working in this area have been routinely employing statistical methodologies such as analysis of variance (ANOVA) if the data are continuous and logit or probit models if the data are discrete. In our opinion, it is grossly incorrect to use these methods as they do not take into account the correlation structure mentioned above. The sole purpose of this article is to bring out this serious flaw clearly to the attention of the researchers. For illustration, we have studied the effects of two Ayurvedic bhasmas-Tamra bhasma and Suwarnamakshik bhasma-on survival and hatching of zebrafish embryos over certain time duration. The statistical analysis using GEE reveals a weak promotional effect of Suwarnamakshik bhasma and an inhibitory effect of Tamra bhasma on hatching.

Toxicity of pesticides associated with potato production, including soil fumigants, to snapping turtle eggs (Chelydra serpentina).

Turtles frequently oviposit in soils associated with agriculture and, thus, may be exposed to pesticides or fertilizers. The toxicity of a pesticide regime that is used for potato production in Ontario on the survivorship of snapping turtle (Chelydra serpentina) eggs was evaluated. The following treatments were applied to clean soil: 1) a mixture of the pesticides chlorothalonil, S-metolachlor, metribuzin, and chlorpyrifos, and 2) the soil fumigant metam sodium. Turtle eggs were incubated in soil in outdoor plots in which these mixtures were applied at typical and higher field application rates, where the eggs were subject to ambient temperature and weather conditions. The pesticide mixture consisting of chlorothalonil, S-metolachlor, metribuzin, and chlorpyrifos did not affect survivorship, deformities, or body size at applications up to 10 times the typical field application rates. Hatching success ranged between 87% and 100% for these treatments. Metam sodium was applied at 0.1¯ times, 0.3¯ times, 1 times, and 3 times field application rates. Eggs exposed to any application of metam sodium had 100% mortality. At typical field application rates, the chemical regime associated with potato production does not appear to have any detrimental impacts on turtle egg development, except for the use of the soil fumigant metam sodium, which is highly toxic to turtle eggs at the lowest recommended application rate.

A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling.

Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen - Jasminum gilgianum, an Oleaceae species native to Papua New Guinea - induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME) as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125) phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME's mechanism of action will help determine this compound's pharmacological utility.

Effects of prestorage application of propolis and storage time on eggshell microbial activity, hatchability, and chick performance in Japanese quail (Coturnix coturnix japonica) eggs.

Propolis, a resinous mixture produced by honeybees from substances collected from plants, has strong antibacterial and antifungal properties. The purpose of the current study was to establish the effects of prestorage application of propolis and storage time on eggshell microbial activity, egg weight loss, hatchability, and chick performance in quail hatching eggs. Treatments were compared in a 2 × 5 factorial design with 2 different storage times (7 and 14 d) and 5 prestorage applications (control, ethyl alcohol 70%, 5% propolis, 10% propolis, and 15% propolis solution). After application, the eggs were stored for 7 or 14 d at 13°C and 75 to 80% RH before incubation. Eggs sprayed with propolis had lower levels of total aerobic mesophilic bacteria, coliform, Salmonella spp., Staphylococcus spp., and mold yeasts than control eggs over the storage period and incubation period. Microbial activity in eggs stored for 7 d was significantly higher than in eggs stored for 14 d at the end of the storage. The lowest egg weight loss during storage was obtained in P15 treatment eggs stored for 7 d, whereas the highest egg weight loss was found in the treatment A after storage for 14 d. Although propolis treatment at 3 different doses was not effective on relative growth, only P15 decreased the BW at d 10, compared with the control. Hatchability in eggs stored for 14 d was significantly lower than in eggs stored for 7 d. No significant differences were observed for hatchability and embryonic mortality among propolis treatment groups. Propolis did not have a detrimental effect on hatchability, embryonic mortality, or BW gain. Our results indicate that propolis may be used to effectively reduce microbial activity on the surface of quail hatching eggs during storage and incubation without any detrimental effects on hatchability.

Dihydroartemisinin promotes angiogenesis during the early embryonic development of zebrafish.

AIM: To investigate the embryotoxicity of dihydroartemisinin (DHA), the main active metabolite of artemisinin, in zebrafish, and explore the corresponding mechanisms. METHODS: The embryos of wild type and TG (flk1:GFP) transgenic zebrafish were exposed to DHA. Developmental phenotypes of the embryos were observed. Development of blood vessels was directly observed in living embryos of TG (flk1:GFP) transgenic zebrafish under fluorescence microscope. The expression of angiogenesis marker genes vegfa, flk1, and flt1 in the embryos was detected using real-time PCR and RNA in situ hybridization assays. RESULTS: Exposure to DHA (1-10 mg/L) dose-dependently caused abnormal zebrafish embryonic phenotypes in the early developmental stage. Furthermore, exposure to DHA (10 mg/L) resulted in more pronounced embryonic angiogenesis in TG (flk1:GFP) zebrafish line. Exposure to DHA (10 mg/L) significantly increased the mRNA expression of vegfa, flk1, and flt1 in the embryos. Knockdown of the flk1 protein partially blocked the effects of DHA on embryogenesis. CONCLUSION: DHA causes abnormal embryonic phenotypes and promotes angiogenesis in zebrafish early embryonic development, demonstrating the potential embryotoxicity of DHA.

Marked toxicity of Albizia bernieri extracts on embryo-larval development in the medaka fish (Oryzias latipes).

Previous phytochemical studies have shown that the plants of the Albizia genus (Fabaceae) contain bioactive saponins, lignans, spermine alkaloids, flavonoids, glycosides phenols and pyridoxine derivatives. Their extracts sometimes display medical properties, but can have also toxic effects. The purpose of our study was to determine the in vivo toxicity of Albizia bernieri seeds in the experimental model of the medaka fish embryo, which is recommended for use in toxicity studies. Our results show clearly that incubating the embryos or larvae of the medaka fish in a medium containing A. bernieri extracts caused a dose-dependent reduction in embryo or larvae survival. Embryos exposed to an extract of A. bernieri displayed cerebral lesions, such as cell lysis and the emergence of lysosomes in the glial tissue. We conclude that when comparing with data obtained with different plant extracts tested on medaka development in our laboratory, A. bernieri displays an unusually high toxicity. Focussing on the cerebral target as well as the fish behaviour could bring more specific informations.

Studying the effects of genistein on gene expression of fish embryos as an alternative testing approach for endocrine disruption.

Assessment of endocrine disruption currently relies on testing strategies involving adult vertebrates. In order to minimize the use of animal tests according to the 3Rs principle of replacement, reduction and refinement, we propose a transcriptomics and fish embryo based approach as an alternative to identify and analyze an estrogenic activity of environmental chemicals. For this purpose, the suitability of 48 h and 7 days post-fertilization zebrafish and medaka embryos to test for estrogenic disruption was evaluated. The embryos were exposed to the phytoestrogen genistein and subsequently analyzed by microarrays and quantitative real-time PCR. The functional analysis showed that the genes affected related to multiple metabolic and signaling pathways in the early fish embryo, which reflect the known components of genistein's mode of actions, like apoptosis, estrogenic response, hox gene expression and steroid hormone synthesis. Moreover, the transcriptomic data also suggested a thyroidal mode of action and disruption of the nervous system development. The parallel testing of two fish species provided complementary data on the effects of genistein at gene expression level and facilitated the separation of common from species-dependent effects. Overall, the study demonstrated that combining fish embryo testing with transcriptomics can deliver abundant information about the mechanistic effects of endocrine disrupting chemicals, rendering this strategy a promising alternative approach to test for endocrine disruption in a whole organism in-vitro scale system.

Visualization of retinoic acid signaling in transgenic axolotls during limb development and regeneration.

Retinoic acid (RA) plays a necessary role in limb development and regeneration, but the precise mechanism by which it acts during these processes is unclear. The role of RA in limb regeneration was first highlighted by the remarkable effect that it has on respecifying the proximodistal axis of the regenerating limb so that serially repeated limbs are produced. To facilitate the study of RA signaling during development and then during regeneration of the same structure we have turned to the axolotl, the master of vertebrate regeneration, and generated transgenic animals that fluorescently report RA signaling in vivo. Characterization of these animals identified an anterior segment of the developing embryo where RA signaling occurs revealing conserved features of the early vertebrate embryo. During limb development RA signaling was present in the developing forelimb bud mesenchyme, but was not detected during hindlimb development. During limb regeneration, RA signaling was surprisingly almost exclusively observed in the apical epithelium suggesting a different role of RA during limb regeneration. After the addition of supplemental RA to regenerating limbs that leads to pattern duplications, the fibroblast stem cells of the blastema responded showing that they are capable of transcriptionally responding to RA. These findings are significant because it means that RA signaling may play a multifunctional role during forelimb development and regeneration and that the fibroblast stem cells that regulate proximodistal limb patterning during regeneration are targets of RA signaling.

Scube activity is necessary for Hedgehog signal transduction in vivo.

The Hedgehog (HH) signaling pathway is a central regulator of embryonic development, controlling the pattern and proliferation of a wide variety of organs. Previous studies have implicated the secreted protein, Scube2, in HH signal transduction in the zebrafish embryo (Hollway et al., 2006; Kawakami et al., 2005; Woods and Talbot, 2005) although the nature of the molecular function of Scube2 in this process has remained undefined. This analysis has been compounded by the fact that removal of Scube2 activity in the zebrafish embryo leads to only subtle defects in HH signal transduction in vivo (Barresi et al., 2000; Hollway et al., 2006; Ochi and Westerfield, 2007; van Eeden et al., 1996; Wolff et al., 2003). Here we present the discovery of two additional scube genes in zebrafish, scube1 and scube3, and demonstrate their roles in facilitating HH signal transduction. Knocking down the function of all three scube genes simultaneously phenocopies a complete loss of HH signal transduction in the embryo, revealing that Scube signaling is essential for HH signal transduction in vivo. We further define the molecular role of scube2 in HH signaling.