Alcohol Affects The Neural Development Of Zebrafish Embryos By Notch Signaling Pathways

BackgroundFASD is one of the most severe conditions caused by early ethanol exposure,which has facial abnormalities,cardiac enlargement,severe cognitive and behavioral deficits.Based on alcohol's effects on the fetus is so serious,we want to explore the effects of alcohol on the fetus.Up to l in 100 children born in the Canada and US are diagnosed with FASD.In embryonic period,if pregnant women were exposed to alcohol,it will affect the neural development of the babys,especially the influence of neurons and glial cells development.Despite the research had reported,but the specific mechanism is unclear.The choice of animal model is very important to the study of FASD.Zebrafish embryos offer certain advantages over other mammalian models.Zebrafish embryos develop outside the mother,thereby allowing us to accurately control the concentration and time of adding ethanol.In contrast with mammals,the development of fish larvae occurs externally,making the zebrafish CNS development accessible for observation and recording.Another advantage of zebrafish as a laboratory animal is the high spawning quantity.A pair of females and males can produce 100-200 at one time and can mate once a week.Compared with other animals,the zebrafish study period is shorter.It is important to note that zebrafish embryos exposed to ethanol exhibit similar phenotypes to children with FASD.Many of the advantages of zebrafish cannot be replaced by other animals.Zebrafish is an experimental animal that has been gradually recognized.In view of the above advantages,we chose the zebrafish as an experimental animal.The Notch signaling pathways are very important to neural developmental processes during the embryonic stages.These signaling pathways were highly conservative in the chain of the evolution from zebrafish to people.In embryonic brain development,it controls maintenance and cell differentiation.It not only controled the cell proliferation and cell differentiation,and was also closely related to the growth of neuron development.The Notch signaling pathway is activated by an interaction between the Notch receptor and its canonical ligands Delta or Serrate(JAGGED in mammals),causing gamma-secretase-mediated release of the Notch Intracellular Domain(NICD).NICD translocates to the nucleus where it interacts with the DNA binding protein CSL(CBF/RBP-J,Su(H),LAG-1/CSL),leading to the transcription of Target genes,Enhancer-of-split[Hairy/E(spl)]and Hairy to produce the corresponding biological effect..For example,it inhibits the expression of neuronal genes,neurogenesis and neural development in zebrafish.N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester(DAPT)efficiently blocks the presenilin-?-secretase complex and,as a consequence,efficiently prevents activation of the Notch response.So this experiment adopts the DAPT to study the role of Notch signaling in zebrafish neural development process.We first observed the effects of alcohol on the neural development in zebrafish.then we observed the effects of alcohol on the Notch signaling pathways.To further clarify the role of Notch signaling pathways,we observe the effects of alcohol on the nervous system development after Notch signal blocking,We compared the effects of the combination of signal blocking and alcohol treated and the separate effects of alcohol aim to preliminarily discuss the event that alcohol affects the neural development of zebrafish embryos by Notch signaling pathways.For further study the specific mechanism of the influence of alcohol,we chose the mature glial factor as the target.Glia maturation factor,a 17-kDa protein which was composed of 142 amino acid residues,mainly includes glia maturation factor-beta(GMFB)and glia maturation factor-gamma(GMFG).GMFB mainly express in vertebrate brain,thymus and small intestine.GMFB play an important role in regulating the growth and differentiation of neurons and glial cells,But the specific mechanism,especially the function of the gene is not clear.Therefore,welooked at effects of alcohol on GMFB gene expression after alcohol treatment,studied the GMFB expressed in zebrafish development and built GMFB knockout models.we again observed the changes of zebrafish phenotypic,nervous system and the blood system after GMFB knockout.At the same time,We analyze the gene expression patterns of zebrafish embryos using RNA-seq technical.Part oneEthanol reduces the neural precursor cells and inhibits neuronal and glial differentiation in zebrafish embryoOBJECTIVEThis paper focuses on the influence of different concentrations of ethanol(1%,2%,and 2.5%)on neural progenitor cells,neuronal and glial cells in vivo.Through exploring its possible molecular mechanism,we can more clearly understand the effects of alcohol on zebrafish neural development.METHODS1.Zebrafish breeding under standard conditions The embryos were staged according to the number of somites,hours post fertilization(hpf),and days post fertilization(dpf).For the embryos of a pair of male and female fish spawning.We were randomly divided into four groups:control,1%,2%,and 2.5%alcohol group.Embryos were exposed to various ethanol concentrations(1%,2%,and 2.5%[v/v])at 5hpf by adding ethanol to the egg water.The exposure medium was then replaced with fresh egg water without ethanol at 24hpf.we chose 5-24hpf as the exposure time.2.Probe preparation We designd probe primer and established the probe of different markers.the probe was confirmed by whole-moun in situ hybridization.3.In situ hybridization The neural progenitors markers is sox2.The proneural marker is neurogeninl.The postmitotic neuronal marker elavl3(encoding HuC).The early glial marker is slcla3a.The mature glial cell marker is myelin-associated glycoprotein(mag)markers.We established the probe of different markers to detect the corresponding cell change.4.Real-time PCR Primers were designed for different markers of neural development to detect the change of corresponding cells.RESULTS1.Ethanol exposure caused embryonic death and morphologic anomalies With an increase in ethanol concentration,the rate of mortality increased significantly at 4dpf(Fig.1A),and there were significant upregulated in the mortality rate when the ethanol concentration exceeded 2%.The hatching rate decreased significantly at 4dpf(Fig.1B),the heart rate decreased gradually at 4 dpf(Fig.1C),and the malformation rate was significantly higher with an increase in ethanol concentration(Fig.1D).In addition,some deformities appeared,such as cardiac enlargement,slow blood flow,or even blood stasis,when the ethanol concentration was more than 1.5%(data unshown)2.Ethanol reduce the neural precursor cells The result of whole mount in situ hybridization at the 75%epiboly stage showed that sox2 expression decreased slightly after ethanol exposure for 3.5h.The higher the concentration of ethanol,the stronger the inhibitory effect of ethanol on neural development.Quantitative real-time PCR(qPCR)analysis confirmed a reduction in sox2 expression.The expression of sox2 increased following ethanol exposure compared with control at 24hpf(Fig.2B).Quantitative real-time PCR(qPCR)analysis confirmed the increased sox2 expression.3.Ethanol exposure inhibits neuronal differentiation The result showed that embryos exhibited significantly down-regulated HuC expression following ethanol exposure4.Ethanol exposure inhibits glial differentiation Whole-mount in situ hybridization showed that the expression of mag was significantly decreased following ethanol exposure.However,the expression of the early glial marker slcla3a was downregulated in the spinal cord,while upregulated in the brain with an increasing ethanol concentrationCONCLUSIONSOverall,we inferred that ethanol can reduce the neural precursor cells and inhibits neuronal and glial differentiation.Part twoAlcohol affects the neural development of zebrafish embryos by Notch signaling pathwaysOBJECTIVEAlcohol inhibits the neural development in zebrafish.Notch signals also play an important role in zebrafish neural development.We want to know whether alcohol could influential to Notch signals.In addition,we want to know that alcohol could affect neural development via regulation of the Notch signaling pathway in zebrafish embryoMETHODS1.zebrafish breeding Breeding zebrafish were maintained at 28.5?on a 14 h light/10h dark cycle.Zebrafish spawning according to the standard operating methods.2.Ethanol and drug treatment Embryos at 5hpf were treated with r DAPT(N?[N?(3,5_difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester)at a final concentration of 100 ?M in eggwater.We chose 5-24hpf as the exposure time.Control embryos were treated with an equal amount of DMSO in eggwater.Pigment formation was blocked by adding 0.003%phenylthiourea(PTU)to the egg water at 24hpf.3.In situ hybridization Probe According to cell markers of neural development in different stages,we do the corresponding probe to detect the neural development changes.4.Real-time PCR Primers were designed for different markers of neural development to detect the change of corresponding cells.RESULTS 1.1.5%alcohol could upregulated Notch signaling pathway in zebrafishembryos zebrafish embryo were dealed with 1.5%alcohol,we found that the expression of the related genes of Notch signaling pathway were increased.2.Inhibition of Notch signaling is to promote the neural development of zebrafish,respectivelyAfter treated with Notch signal inhibitors DAPT,we found that Cells at different stages of neural development is up-regulated.After treated with Notch signal inhibitors DAPT could induce premature neuronal differentiation At different time points,we detect the expression of neural precursor cells,proneural genes and neurons with in situ hybridization technique in zebrafish.we found that DAPT could induce premature neuronal differentiation and promote the differentiation of neural progenitor cells.3.The effects of 1.5%alcohol with different concentration of DAPT on zebrafish neurons(HUC)expression The number of neurons was increasing with the increased concentration of the notch signal inhibitors.CONCLUSIONSAlcohol could upregulated Notch signaling pathway in zebrafish.Alcohol affects zebrafish neural development via regulation of the Notch and Hedgehog signaling pathway.Part threeThe effect of ethanol on GMFB and the effect of GMFB knockout on zebrafish developmentOBJECTIVEThe first one is the effect of ethanol on GMFB.The second one is the establishment and validation of the knockout GMFB zebrafish model.The next one is that we want to observe the change of small neutrophils and macrophages in zebrafish blood system after knockout GMFB gene.The last one is to reveal the gene expression profile in GMFB mutant.METHODS1.Whole-mount in situ hybridization In order to confirm the effect of GMFB knockout,we prepared the GMFB probe.2.sudan black B stain In order to observe the effect of GMFB knockout on neutrophils in zebrafish3.Neutral red staining In order to observe the effect of GMFB knockout on small macrophages in zebrafish4.Western We want to confirm whether GMFB knockout was success from protein levels5.Real-time PCR We want to observe the interested gene expression from mRNA levelRESULTS1.The effect of ethanol on GMFB There is no signicant change after ethanol treatment.2.Spatial and Temporal expression of GMFB.In situ hybridization results showed that GMFB expression began in 3 h and has expressed in the body,but it only expressed in head after 3 days.The Western results showed that GMFB protein reached the highest in 2 to 3 days.The GMFB expression gradually reduced after 3 days.3.GMFB gene knockout is successful Confirmed by western and in situ hybridization In situ hybridization,western and rt-pcr results showed that GMFB gene is knock out successfully.4.The effect of GMFB gene knockout on zebrafish neurons and glial cells Results show that there is no statistical difference.5.The Sudan black B staining of GMFB Homozygous,GMFB heterozygote and AB zebrafish The change of Sudan black B stain mainly represents the change of neutrophils,We make two heterozygote mating and get eggs.A quarter is homozygous eggs,a quarter is AB eggs,half is heterozygous fish eggs.Because of their male and female are the same,so the results are more credible.Each group has thirty eggs.The result in three groups was not statistically difference(the results repeated three times).6.The Neutral red staining of GMFB Homozygous,GMFB heterozygote and AB zebrafish The neutral red staining an represent small macrophages in zebrafish,we did neutral red staining to detect the change of GMFB gene knockout on small macrophages,The result is no difference through statistical analysis.The GMFB gene knockout has no effects on small macrophages in zebrafish embryos.7.The gene expression profile in GMFB mutant.RNA-seq revealed that the expression of 9 genes were upregulated and 24 genes downregulated.CONCLUSIONSGMFB gene knockout has been successful in protein level and mRNA level tests.The knockout of GMFB gene has no effects on the number of zebrafish neutrophils and Small macrophages.