Generation of enhanced definitive endoderm from human embryonic stem cells under an albumin/insulin-free and chemically defined condition.

AIM: To enhance survival and generation of definitive endoderm cells from human embryonic stem cells in a simple and reproducible system. MAIN METHODS: Definitive endoderm (DE) differentiation from human embryonic stem cells (hESCs) was induced under a chemical-defined condition withdrawn insulin supplement and serum albumin. We dissected influence of "alternative growth factors", WNT3A, BMP4 and bFGF in activin A-driven differentiation by detection of DE-associated genes expression and cell viability. Expression of DE-associated SOX17 and FOXA2 genes was analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) and Western blot assays. Quantitative evaluation of DE efficiency was performed by flow cytometry analysis of CXCR4-expressed cell population. Cell viability during DE differentiation was analyzed by an Annexin V/PI double staining test. KEY FINDINGS: Supplementation with WNT3A, BMP4 or bFGF promoted DE generation in a dose- and time-dependent manner. Cell apoptosis elicited by activin A was significantly ameliorated by a cocktail with WNT3A, BMP4 and bFGF. This allowed for sustained cell viability without insulin-containing supplements, thereby indirectly improving the efficiency of DE generation. Therefore, the cocktail containing is optimal for efficient DE generation in the presence of activin A and an insulin/albumin-free condition. SIGNIFICANCE: This optimal condition facilitates the balance between the productivity and the viability maintenance, and could be valuable for mass production of DE with minimal variation.

A novel hirudin derivative inhibiting thrombin without bleeding for subcutaneous injection.

Currently, anticoagulants would be used to prevent thrombosis. Thrombin is an effector enzyme for haemostasis and thrombosis. We designed a direct thrombin inhibitor peptide (DTIP) using molecular simulation and homology modelling and demonstrated that the C-terminus of DTIP interacts with exosite I, and N-terminus with the activity site of thrombin, respectively. DTIP interfered with thrombin-mediated coagulation in human, rat and mouse plasma (n=10 per group) and blocked clotting in human whole blood in vitro. When administered subcutaneously, DTIP showed potent and dose-dependent extension of aPTT, PT, TT and CT in rats (n=10 per group). The antithrombotic dose of DTIP induced significantly less bleeding than bivalirudin determined by transecting distal tail assay in rats. Furthermore, DTIP reached peak blood concentration in 0.5-1 hour and did not cause increased bleeding after five days of dosing compared to dabigatran etexilate. The antithrombotic effect of DTIP was evaluated in mice using lethal pulmonary thromboembolism model and FeCl3-induced mesenteric arteriole thrombus model. DTIP (1.0 mg/kg, sc) prevented deep venous thrombosis and increased the survival rate associated with pulmonary thromboembolism from 30 % to 80 %. Intravital microscopy showed that DTIP (1.0 mg/kg, sc) decelerated mesenteric arteriole thrombosis caused by FeCl3 injury. These data establish that DTIP is a novel antithrombotic agent that could be used to prevent thrombosis without conferring an increased bleeding risk.

High-level expression, purification, and enzymatic characterization of truncated human plasminogen (Lys531-Asn791) in the methylotrophic yeast Pichia pastoris.

BACKGROUND: Plasmin is a serine protease that plays a critical role in fibrinolysis, which is a process that prevents blood clots from growing and becoming problematic. Recombinant human microplasminogen (rhµPlg) is a derivative of plasmin that solely consists of the catalytic domain of human plasmin and lacks the five kringle domains found in the native protein. Developing an industrial production method that provides high yields of this protein with high purity, quality, and potency is critical for preclinical research. RESULTS: The human microplasminogen gene was cloned into the pPIC9K vector, and the recombinant plasmid was transformed into Pichia pastoris strain GS115. The concentration of plasmin reached 510.1 mg/L of culture medium. Under fermentation conditions, the yield of rhµPlg was 1.0 g/L. We purified rhµPlg to 96% purity by gel-filtration and cation-exchange chromatography. The specific activity of rhµPlg reached 23.6 U/mg. The K m of substrate hydrolysis by recombinant human microplasmin was comparable to that of human plasmin, while rhµPlm had higher k cat /Km values than plasmin. The high purity and activity of the rhµPlg obtained here will likely prove to be a valuable tool for studies of its application in thrombotic diseases and vitreoretinopathies. CONCLUSIONS: Reliable rhµPlg production (for use in therapeutic applications) is feasible using genetically modified P. pastoris as a host strain. The successful expression of rhµPlg in P. pastoris lays a solid foundation for its downstream application.

Targeted Knockdown of RNA-Binding Protein TIAR for Promoting Self-Renewal and Attenuating Differentiation of Mouse Embryonic Stem Cells.

RNA-binding protein TIAR has been suggested to mediate the translational silencing of ARE-containing mRNAs. To analyze the functions of TIAR, we established RNAi and genetic rescue assays. We evaluated the expression of neuroectoderm markers Pax6 and nestin, mesoderm markers brachyury and Flk1, and hypoblast and definitive endoderm markers Sox17 and Gata6 during EB differentiation and found that knockdown TIAR expression restrained the differentiation of E14 cells. We assessed gene expression levels of Flk-1 and VE-cadherin and observed attenuated differentiation of E14 cells into endothelial cells upon downregulation of TIAR gene expression. As such, we hypothesized an essential role of TIAR related to EB differentiation. As TIAR inhibits the translation of c-myc, we proposed that downregulation of TIAR results in restrained differentiation of E14 cells, due in part to the function of c-myc. We found that TIAR inhibited c-myc expression at the translational level in E14 cells; accordingly, a reduction of TIAR expression promoted self-renewal of pluripotent cells and attenuated differentiation. Additionally, we established that TIAR inhibited TIA-1 expression at the translational level in E14 cells. Taken together, we have contributed to the understanding of the regulatory relationships between TIAR and both c-myc and TIA-1.

Structural basis of RGD-hirudin binding to thrombin: Tyr3 and five C-terminal residues are crucial for inhibiting thrombin activity.

BACKGROUND: Hirudin is an anti-coagulation protein produced by the salivary glands of the medicinal leech Hirudomedicinalis. It is a powerful and specific thrombin inhibitor. The novel recombinant hirudin, RGD-hirudin, which contains an RGD motif, competitively inhibits the binding of fibrinogen to GPIIb/IIIa on platelets, thus inhibiting platelet aggregation while maintaining its anticoagulant activity. RESULTS: Recombinant RGD-hirudin and six mutant variants (Y3A, S50A, Q53A, D55A, E57A and I59A), designed based on molecular simulations, were expressed in Pichia pastoris. The proteins were refolded and purified to homogeneity as monomers by gel filtration and anion exchange chromatography. The anti-thrombin activity of the six mutants and RGD-hirudin was tested. Further, we evaluated the binding of the mutant variants and RGD-hirudin to thrombin using BIAcore surface plasmon resonance analysis (SPR). Kinetics and affinity constants showed that the KD values of all six mutant proteins were higher than that of RGD-hirudin. CONCLUSIONS: These findings contribute to a novel understanding of the interaction between RGD-hirudin and thrombin.

Use of antagonists and morpholinos in loss-of-function analyses: estrogen receptor ESR2a mediates the effects of 17alpha-ethinylestradiol on primordial germ cell distribution in zebrafish.

BACKGROUND: Various chemicals released into the aquatic environment adversely affect the reproductive system of fish, particularly by changing gonad structure and function. 17alpha-ethinylestradiol (EE2) is a potent environmental estrogen that disrupts sexual differentiation and normal reproduction in fish. Previous studies have shown that exposure to endocrine-disrupting chemicals (EDCs) disrupts the migration of primordial germ cells (PGCs) in zebrafish. METHODS: To investigate the effects of EE2 exposure on PGC migration, zebrafish embryos were injected with gfp-nanos mRNA to label PGCs and subsequently exposed to different concentrations of EE2. Typical estrogen receptor antagonist treatment and morpholino knockdown experiments were used to identify functional estrogen receptors that mediate the effects of EE2. RESULTS: The migration of PGCs was disrupted after exposure to high concentrations of EE2 (1 mirog/L). Loss-of-function analyses were performed for estrogen receptor ESR1, ESR2a, and ESR2b, and only loss of ESR2a resulted in a decreased number of ectopic PGCs following exposure to 1 mirog/L EE2. CONCLUSIONS: EE2 exposure disrupts PGC migration and distribution, and this effect is mediated through the estrogen receptor ESR2a.

Parkin overexpression ameliorates hippocampal long-term potentiation and ß-amyloid load in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by a severe decline of memory performance. A widely studied AD mouse model is the APPswe/PSEN1ΔE9 (APP/PS1) strain, as mice exhibit amyloid plaques as well as impaired memory capacities. To test whether restoring synaptic plasticity and decreasing ß-amyloid load by Parkin could represent a potential therapeutic target for AD, we crossed APP/PS1 transgenic mice with transgenic mice overexpressing the ubiquitin ligase Parkin and analyzed offspring properties. Overexpression of Parkin in APP/PS1 transgenic mice restored activity-dependent synaptic plasticity and rescued behavioral abnormalities. Moreover, overexpression of Parkin was associated with down-regulation of APP protein expression, decreased ß-amyloid load and reduced inflammation. Our data suggest that Parkin could be a promising target for AD therapy.

Yes-associated protein (yap) is required for early embryonic development in zebrafish (danio rerio).

The hippo (Hpo) signaling pathway plays a critical role in regulation of organ size. The kinase cascade ultimately antagonizes the transcriptional co-activator Yki/YAP, which is a key regulator of cell proliferation and apoptosis. In this study, we performed a knocking down study using antisense morpholino (MO) reagents and found that zebrafish YAP, a key transcriptional co-activator of Hpo pathway, plays a critical role in early embryonic development. At the cellular level, yap inhibition increases apoptosis and decreases cell proliferation. Reduction of yap function severely delays several developmental events, including gastrulation, cardiogenesis and hematopoiesis. Knockdown of yap showed some evidence of ventralization, including reduction of dorsally expressed marker goosecoid (gsc), expansion of ventral marker gata2, disruption of the somites, and reduction in head size. Finally, we performed a preliminary analysis with real-time polymerase chain reaction (qPCR) for the candidate targets of zebrafish Hpo pathway. In conclusion, our results revealed that zebrafish yap coordinately regulates cell proliferation and apoptosis and is required for dorsoventral axis formation, gastrulation, cardiogenesis, hematopoiesis, and somitogenesis.

Retinol dehydrogenase, RDH1l, is essential for the heart development and cardiac performance in zebrafish.

BACKGROUND: Retinoic acid (RA) is a potent signaling molecule that plays pleiotropic roles in patterning, morphogenesis, and organogenesis during embryonic development. The synthesis from retinol (vitamin A) to retinoic acid requires two sequential oxidative steps. The first step involves the oxidation of retinol to retinal through the action of retinol dehydrogenases. Retinol dehydrogenases1l (RDH1l) is a novel zebrafish retinol dehydrogenase. Herein we investigated the role of zebrafish RDH1l in heart development and cardiac performance in detail. METHODS: RDH1l specific morpholino was used to reduce the function of RDH1l in zebrafish. The gene expressions were observed by using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction (VSF). RESULTS: The knock-down of RDH1l led to abnormal neural crest cells migration and reduced numbers of neural crest cells in RDH1l morphant embryos. The reduced numbers of cardiac neural crest cells also can be seen in RDH1l morphant embryos. Furthermore, the morpholino-mediated knock-down of RDH1l resulted in the abnormal heart loop. The left-right determining genes expression pattern was altered in RDH1l morphant embryos. The impaired cardiac performance was observed in RDH1l morphant embryos. Taken together, these data demonstrate that RDH1l is essential for the heart development and cardiac performance in zebrafish. CONCLUSIONS: RDH1l plays a important role in the neural crest cells development, and then ultimately affects the heart loop and cardiac performance. These results show for the first time that an enzyme involved in the retinol to retinaldehyde conversion participate in the heart development and cardiac performance in zebrafish.

MicroRNA-200a promotes anoikis resistance and metastasis by targeting YAP1 in human breast cancer.

PURPOSE: The process of metastases involves the dissociation of cells from the primary tumor, penetration into the basement membrane, invasion, and exiting from the vasculature to seed and colonize distant tissues. miR-200a is involved in this multistep metastatic cascade. This study aimed to test the hypothesis that miR-200a promotes metastasis through increased anoikis resistance in breast cancer. EXPERIMENTAL DESIGN: Breast cancer cells transfected with mimic or inhibitor for miR-200a were assayed for anoikis in vitro. miR-200a expression was assessed by quantitative real-time PCR (qRT-PCR). Luciferase assays, colony formation assays, and animal studies were conducted to identify the targets of miR-200a and the mechanism by which it promotes anoikis resistance. RESULTS: We found that overexpression of miR-200a promotes whereas inhibition of miR-200a suppresses anoikis resistance in breast cancer cells. We identified Yes-associated protein 1 (YAP1) as a novel target of miR-200a. Our data showed that targeting of YAP1 by miR-200a resulted in decreased expression of proapoptotic proteins, which leads to anoikis resistance. Overexpression of miR-200a protected tumor cells from anoikis and promoted metastases in vivo. Furthermore, knockdown of YAP1 phenocopied the effects of miR-200a overexpression, whereas restoration of YAP1 in miR-200a overexpressed breast cancer cells reversed the effects of miR-200a on anoikis and metastasis. Remarkably, we found that YAP1 expression was inversely correlated with miR-200a expression in breast cancer clinical specimens, and miR-200a expression was associated with distant metastasis in patients with breast cancer. CONCLUSIONS: Our data suggest that miR-200a functions as anoikis suppressor and contributes to metastasis in breast cancer.

The methylation of C/EBP ß gene promoter and regulated by GATA-2 protein.

Mammalian genomes are punctuated by DNA sequences containing an atypically high frequency of CpG sites (CpG islands; CGIs) that are associated with the majority of annotated gene promoters. Methylated C bases of CpG sites inhibit the expression of downstream genes. During the differentiation of 3T3-L1 preadipocytes, the CCAAT/enhancer-binding protein (C/EBP) ß gene plays an important role. We studied the CpG island methylation status of the C/EBP ß promoter and its relationship with the GATA-2 protein. We used computer analysis to determine that the C/EBP ß promoter sequence is rich in CGIs, and observed that two of seven methylated C bases were demethylated during the preadipocyte differentiation using bisulfite sequencing PCR (BSP). This corresponded with the onset of notable C/EBP ß gene expression. Immunofluorescence and molecular docking showed that the GATA-2 protein binds the C/EBP ß promoter in front of the first demethylated CpG site. We also found that expression of GATA-2 and C/EBP ß proteins is negatively correlated. These results indicate that the methylated C bases in the C/EBP ß promoter relate to expression of the C/EBP ß gene, and that its demethylation is linked with GATA-2 protein association.

Pleiotropy of glycogen synthase kinase-3 inhibition by CHIR99021 promotes self-renewal of embryonic stem cells from refractory mouse strains.

BACKGROUND: Inhibition of glycogen synthase kinase-3 (GSK-3) improves the efficiency of embryonic stem (ES) cell derivation from various strains of mice and rats, as well as dramatically promotes ES cell self-renewal potential. ß-catenin has been reported to be involved in the maintenance of self-renewal of ES cells through TCF dependent and independent pathway. But the intrinsic difference between ES cell lines from different species and strains has not been characterized. Here, we dissect the mechanism of GSK-3 inhibition by CHIR99021 in mouse ES cells from refractory mouse strains. METHODOLOGY/PRINCIPAL FINDINGS: We found that CHIR99021, a GSK-3 specific inhibitor, promotes self-renewal of ES cells from recalcitrant C57BL/6 (B6) and BALB/c mouse strains through stabilization of ß-catenin and c-Myc protein levels. Stabilized ß-catenin promoted ES self-renewal through two mechanisms. First, ß-catenin translocated into the nucleus to maintain stem cell pluripotency in a lymphoid-enhancing factor/T-cell factor-independent manner. Second, ß-catenin binds plasma membrane-localized E-cadherin, which ensures a compact, spherical morphology, a hallmark of ES cells. Further, elevated c-Myc protein levels did not contribute significantly to CH-mediated ES cell self-renewal. Instead, the role of c-Myc is dependent on its transformation activity and can be replaced by N-Myc but not L-Myc. ß-catenin and c-Myc have similar effects on ES cells derived from both B6 and BALB/c mice. CONCLUSIONS/SIGNIFICANCE: Our data demonstrated that GSK-3 inhibition by CH promotes self-renewal of mouse ES cells with non-permissive genetic backgrounds by regulation of multiple signaling pathways. These findings would be useful to improve the availability of normally non-permissive mouse strains as research tools.

Dihydrofolate reductase is required for the development of heart and outflow tract in zebrafish.

Folic acid is very important for embryonic development and folic acid inhibition can cause congenital heart defects in vertebrates. Dihydrofolate reductase (DHFR) is a key enzyme in folate-mediated metabolism. The dysfunction of DHFR disrupts the key biological processes which folic acid participates in. DHFR gene is conserved during vertebrate evolution. It is important to investigate the roles of DHFR in cardiac developments. In this study, we showed that DHFR knockdown resulted in the abnormal developments of zebrafish embryos in the early stages. Obvious malformations in heart and outflow tract (OFT) were also observed in DHFR knockdown embryos. DHFR overexpression rescued the abnormal phenotypes in the DHFR knockdown group. DHFR knockdown had negative impacts on the expressions of NKX2.5 (NK2 transcription factor-related 5), MEF2C (myocyte-specific enhancer factor 2C), TBX20 (T-box 20), and TBX1 (T-box 1) which are important transcriptional factors during cardiac development process, while DHFR overexpression had positive effects. DHFR was required for Hedgehog pathway. DHFR knockdown caused reduced cell proliferation and increased apoptosis, while its overexpression promoted cell proliferation and inhibited apoptosis. Taken together, our study suggested that DHFR plays crucial roles in the development of heart and OFT in zebrafish by regulating gene transcriptions and affecting cell proliferation and apoptosis.

Disruption of plasminogen activator inhibitor-1 gene enhances spontaneous enlargement of mouse airspace with increasing age.

Plasminogen activator inhibitor-1 (PAI-1) is the most effective protease inhibitor in the fibrinolysis system, and plays an important role in the remodeling of the extracellular matrix. We therefore explored whether PAI-1 is involved in the change of lung structure with increasing age. PAI-1 gene knockout mice and wild-type mice were sacrificed at age 3 weeks, 3 months, 6 months and 15 months for histopathology analysis, and assessed the relationship between PAI-1 and the change in lung structure with age. Six-month-old mice were chosen for further studies. Elastin in the lung was detected using Weigert staining. We measured the expression of matrix metalloproteinase-12 (MMP-12) that is a major protease in elastin degradation by real time PCR and immunostaining. Transforming growth factor-ß1 (TGF-ß1) expression was measured by western blot analysis. PAI-1 gene knockout mice showed significant increases in alveolar size with increasing age and damaged alveolar structure at the age of 15 months, compared with wild-type mice. At the age of 6 months, elastin protein was decreased in the lungs of PAI-1 gene knockout mice. PAI-1 null mice had higher MMP-12 mRNA expression, and lower expression level of active TGF-ß1 in the lung. Taken together, these results indicate that the emphysema-like change attributed to PAI-1 deficiency might be facilitated with increased MMP-12 expression that accelerates elastin degradation in mice lungs, and TGF-ß1 might be involved in the modulation of this process.

Effect of Tbx1 knock-down on cardiac performance in zebrafish.

BACKGROUND: Tbx1 is the major candidate gene for DiGeorge syndrome (DGS). Similar to defects observed in DGS patients, the structures disrupted in Tbx1(-/-) animal models are derived from the neural crest cells during development. Although the morphological phenotypes of some Tbx1 knock-down animal models have been well described, analysis of the cardiac performance is limited. Therefore, myocardial performance was explored in Tbx1 morpholino injected zebrafish embryos. METHODS: To elucidate these issues, Tbx1 specific morpholino was used to reduce the function of Tbx1 in zebrafish. The differentiation of the myocardial cells was observed using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction and atrial shortening fraction. RESULTS: Tbx1 morpholino injected embryos were characterized by defects in the pharyngeal arches, otic vesicle, aortic arches and thymus. In addition, Tbx1 knock down reduced the amount of pharyngeal neural crest cells in zebrafish. Abnormal cardiac morphology was visible in nearly 20% of the Tbx1 morpholino injected embryos. The hearts in these embryos did not loop or loop incompletely. Importantly, cardiac performance and heart rate were reduced in Tbx1 morpholino injected embryos. CONCLUSIONS: Tbx1 might play an essential role in the development of pharyngeal neural crest cells in zebrafish. Cardiac performance is impaired by Tbx1 knock down in zebrafish.

Overproduction of 15N-labeled r-RGD-hirudin in pichia pastoris for NMR studies.

The novel recombinant hirudin, r-RGD-hirudin, inhibits thrombin and platelet aggregation. Here, we reported over-expression of (15)N-labeled r-RGD-hirudin by Pichia pastoris in minimal medium. After extensive optimization, the yield of active r-RGD-hirudin reached ≈600 mg/L when the yeast cells were culture in a fermenter. The purified (15)N-labeled r-RGD-hirudin retained full biological activity and was uniformly labeled. Heteronuclear NMR of the (15)N-labeled r-RGD-hirudin was performed for the first time, and all signals in the heteronuclear single quantum coherence (HSQC) spectrum were successfully assigned.

Elevated glucose induces congenital heart defects by altering the expression of tbx5, tbx20, and has2 in developing zebrafish embryos.

BACKGROUND: Maternal diabetes increases the risk of congenital heart defects in infants, and hyperglycemia acts as a major teratogen. Multiple steps of cardiac development, including endocardial cushion morphogenesis and development of neural crest cells, are challenged under elevated glucose conditions. However, the direct effect of hyperglycemia on embryo heart organogenesis remains to be investigated. METHODS: Zebrafish embryos in different stages were exposed to D-glucose for 12 or 24 hr to determine the sensitive window during early heart development. In the subsequent study, 6 hr post-fertilization embryos were treated with either 25 mmol/liter D-glucose or L-glucose for 24 hr. The expression of genes was analyzed by whole-mount in situ hybridization. RESULTS: The highest incidence of cardiac malformations was found during 6-30 hpf exposure periods. After 24 hr exposure, D-glucose-treated embryos exhibited significant developmental delay and diverse cardiac malformations, but embryos exposed to L-glucose showed no apparent phenotype. Further investigation of the origin of heart defects showed that cardiac looping was affected earliest, while the specification of cardiac progenitors and heart tube assembly were complete. Moreover, the expression patterns of tbx5, tbx20, and has2 were altered in the defective hearts. CONCLUSIONS: Our data demonstrate that elevated glucose alone induces cardiac defects in zebrafish embryos by altering the expression pattern of tbx5, tbx20, and has2 in the heart. We also show the first evidence that cardiac looping is affected earliest during heart organogenesis. These research results are important for devising preventive and therapeutic strategies aimed at reducing the occurrence of congenital heart defects in diabetic pregnancy.

[Effects of folic acid on the development of heart of zebrafish].

OBJECTIVE: To construct the folic acid deficient model in zebrafish and observe the abnormal cardiac phenotypes, to find the optimal period for supplementing folic acid that can most effectively prevent the heart malformation induced by folic acid deficiency, and to investigate the possible mechanisms by which folic acid deficiency induces malformations of heart. METHOD: The folic acid deficient zebrafish model was constructed by using both the folic acid antagonist methotrexate (MTX) and knocking-down dhfr (dihydrofolate reductase gene). Exogenous tetrahydrofolic acid rescue experiment was performed. Folic acid was given to folic acid deficient groups in different periods. The percent of cardiac malformation, the cardiac phenotypes, the heart rate and the ventricular shortening fraction (VSF) were recorded. The out flow tract (OFT) was observed by using fluorescein micro-angiography. Whole-mount in situ hybridization and real-time PCR were performed to detect vmhc, amhc, tbx5 and nppa expressions. RESULT: About (78.00 ± 3.74)% embryos in MTX treated group and (68.00 ± 6.32)% embryos in dhfr knocking-down group had heart malformations, including the abnormal cardiac shapes, the hypogenesis of OFT and the reduced heart rate and VSF. Giving exogenous tetrahydrofolic acid rescued the above abnormalities. Given the folic acid on 8 - 12 hours post-fertilization (hpf), both the MTX treated group (20.20% ± 3.77%) and dhfr knocking-down group (43.40% ± 4.51%) showed the most significantly reduced percent of cardiac malformation and the most obviously improved cardiac development. In folic acid deficient group, the expressions of tbx5 and nppa were reduced while the expressions of vmhc and amhc appeared normal. After being given folic acid to MTX treated group and dhfr knocking-down group, the expressions of tbx5 and nppa were increased. CONCLUSIONS: The synthesis of tetrahydrofolic acid was decreased in our folic acid deficient model. Giving folic acid in the middle period, which is the early developmental stage, can best prevent the abnormal developments of hearts induced by folic acid deficiency. Folic acid deficiency did not disrupt the differentiations of myosins in ventricle and atrium. The cardiac malformations caused by folic acid deficiency were related with the reduced expressions of tbx5 and nppa.

Generation of a fluorescent transgenic zebrafish for detection of environmental estrogens.

To establish a novel in vivo test system for rapid detection of environmental estrogens, an ere-zvtg1: gfp transgenic zebrafish line has been generated. In this transgenic line, under control conditions, GFP was exclusively expressed in the liver of mature adult female fish. Male and larval transgenic fish did not express GFP but could be induced to express GFP in the liver after exposure to 17-alpha-ethynylestradiol (EE(2)). Concurrent accumulation of zvtg1 and gfp mRNAs in embryos and larvae after EE(2) exposure was observed, which indicated that the expression of gfp transgene was driven by the zvtg1 promoter. Green fluorescence was first observed in the liver at 53, 74, 100 or 131h post-fertilization (hpf) after exposure to 100, 10, 1 or 0.1ng/L EE(2) from 1 to 2 cell stage, respectively. As for mature male transgenic zebrafish, green fluorescence was observed after exposure to 100, 10, 1 or 0.1ng/L EE(2) for 2, 3, 4 or 7 days, respectively; as for mature female, fluorescence was increased after exposure to relatively high concentrations of EE(2) (10 and 100ng/L). Green fluorescence in the liver was increased with prolonging of exposure time and was repeatedly induced after removal and re-addition of EE(2). We also demonstrated that GFP expression could be induced by other estrogenic compounds, including beta-estradiol (E(2), 0.1microg/L), cadmium chloride (CdCl(2), 10microg/L), zearalenone (50microg/L), estriol (E(3), 1microg/L), diethylstilbestrol (DES, 50ng/L) bisphenol A (BPA, 1mg/L) but not by weakly estrogenic compounds such as nonylphenol (NP, up to 10mg/L), or non-estrogenic steroid hormones such as progesterone (up to 100mg/L) and 17-hydroxysteroid (up to 50mg/L). These data suggest the transgenic zebrafish is sensitive and specific for detection of estrogenic compounds. Because the observed-effect concentrations are as low as those of environment and the observed-effect exposure times are very short, this transgenic fish is a promising candidate system for monitoring environmental estrogens directly, rapidly and easily.