Houyan Song

Exposure to 17α-ethynylestradiol impairs reproductive functions of both male and female zebrafish (Danio rerio)

In this study, the impact of 17alpha-ethynylestradiol (EE2) on reproduction in zebrafish (Danio rerio) was evaluated using vitellogenin (Vtg) induction, mortality rate, growth, sex ratio, gonad histology, fecundity, and sperm parameters as endpoints. Two days post-hatch (2dph) zebrafish were exposed to solvent control or EE2 at 0.4, 2, and 10ng/l for 3 months. At 21dph, Vtg mRNA expression was detected only in fish exposed to 10ng/l EE2. At 90dph, increased mortality rate and sex ratio (female:male) were observed in fish exposed to 2 and 10ng/l EE2. A dose-dependent increase in gonads with underdeveloped gametes was observed in fish exposed to EE2. At 180dph, malformation of the sperm duct and reduced number of spermatozoa were found in fish exposed to 2ng/l and 10ng/l EE2. Reduced fecundity and 12hpf egg viability were found in EE2-exposed males and females. The number of fish with no expressible milt was elevated dose dependently in EE2-exposed males, although no difference in sperm density was found. After a 3-month recovery period, growth and sex ratio were partially recovered. Our findings suggest that EE2 can adversely affect the fecundity, sex differentiation, gametes development, and other reproductive functions of both male and female zebrafish, and some of the toxic effects persist.

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.

Exogenous tissue plasminogen activator enhances peripheral nerve regeneration and functional recovery after injury in mice.

Tissue plasminogen activator (tPA) is an essential component of the proteolytic cascade that lyses blood clots. Various studies also suggest that tPA plays important roles in the nervous system. We show that exogenous tPA or tPA/plasminogen (plg) promotes axonal regeneration, remyelination, and functional recovery after sciatic nerve injury in the mouse. Local application of tPA or tPA/plg 7 days after sciatic nerve crush significantly increased the total number of axons and myelinated axons, which is accompanied by enhanced expression of neurofilament. Treatment with tPA or tPA/plg reduced the deposition of fibrin(ogen) after nerve injury. Moreover, tPA or tPA/plg increased the number of macrophages and induced MMP-9 expression at the injury site, coincident with reduced collagen scar formation and accelerated clearance of myelin and lipid debris after treatment. Consequently, tPA or tPA/plg treatment protected muscles from atrophy after nerve injury, indicating better functional recovery. These results suggest that administration of exogenous tPA or tPA/plg promotes axonal regeneration and remyelination through removal of fibrin deposition and activation of MMP-9-positive macrophages, which may be responsible for myelin debris clearance and preventing collagen scar formation. Therefore, tPA may be useful for treatment of peripheral nerve injury.

yap is required for the development of brain, eyes, and neural crest in zebrafish

The Yes-associated protein (YAP) is a small protein that binds to many transcription factors and modulates their activity. Bioinformatics analysis indicated that zebrafish Yap shares high identity with its orthologs in fruit fly, chicken, mouse, and human. Expression analysis revealed that maternal transcripts of yap are ubiquitous, and endogenous yap is chronologically expressed in the notochord, brain, eyes, branchial arches, and pectoral fins. Knockdown of yap causes distinct morphological defects in embryos, which display a small head with smaller eyes than normal and fewer cartilages in the branchial arches. Proneural and neuronal gene expression in yap morphant brain is significantly reduced. The expression of crestin is also markedly reduced in all recognizable arch-associated regions of yap morphants. Furthermore, TUNEL analysis revealed that there is a marked increase in cell death in yap morphant brain. In conclusion, zebrafish yap is required for the development of the brain, eyes, and neural crest during embryogenesis.

Requirements of myocyte-specific enhancer factor 2A in zebrafish cardiac contractility

Myocyte‐specific enhancer factor 2A (MEF2A) regulates a broad range of fundamental cellular processes including cell division, differentiation and death. Here, we tested the hypothesis that MEF2A is required in cardiac contractility employing zebrafish as a model organism. MEF2A is highly expressed in heart as well as somites during zebrafish embryogenesis. Knock‐down of MEF2A in zebrafish impaires the cardiac contractility and results in sarcomere assembly defects. Dysregulation of cardiac genes in MEF2A morphants suggests that sarcomere assembly disturbances account for the cardiac contractile deficiency. Our studies suggested that MEF2A is essential in cardiac contractility.

Disruption of tissue-type plasminogen activator gene in mice aggravated liver fibrosis

Background and Aim:  Tissue‐type plasminogen activator (tPA) is one of the major components in the matrix proteolytic network whose role in the pathogenesis of liver fibrosis remains unknown. The aim of this study is to investigate the role of tPA in carbon tetrachloride (CCl4)‐induced liver fibrosis.

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.

Myocyte-specific enhancer factor 2A is essential for zebrafish posterior somite development

Somite development is governed tightly by genetic factors. In the large-scale mutagenesis screens of zebrafish, no mutations were linked to myocyte enhancer factor 2A (MEF2A) locus. In this study, we find that MEF2A knock-down embryos display a downward tail curvature and have U-shaped posterior somites. Furthermore, we demonstrate that MEF2A is required for Hedgehog signaling. MEF2A inhibition results in induction of apoptosis in the posterior somites. We further find that Hedgehog signaling can negatively regulate MEF2A expression in the somites. Microarray studies reveal a number of genes that are differentially expressed in the MEF2A morphants. Our studies suggest that MEF2A is essential for zebrafish posterior somite development.

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.

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

BackgroundHirudin 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.ResultsRecombinant 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.ConclusionsThese findings contribute to a novel understanding of the interaction between RGD-hirudin and thrombin.