BHC80基因抑制对斑马鱼胚胎红细胞发育的影响

胚胎整体RNA原位杂交显示,BHC80基因表达主要集中在中枢神经系统部位． 应用吗啡啉修饰的反义寡核苷酸技术抑制BHC80基因表达,显示胚胎红细胞减少并堆积在PBI区,用胚胎期红细胞标志βe3 globin以及造血过程中的重要转录因子gata1、c-myb、lmo2的胚胎整体RNA原位杂交实验显示,BHC80基因表达下调使gata1标记胚胎红系前体细胞增殖增多并且分化延迟,导致红细胞减少和PBI区红细胞堆积． 血管内皮标志基因flk-1的RNA探针原位杂交和荧光显微造影显示,BHC80基因表达下调组血管与对照组相比清晰可见无明显差异．     

Connexin43基因抑制对斑马鱼心血管系统发育的影响

为了研究cx43基因抑制对斑马鱼胚胎心血管系统发育的影响,针对cx43的翻译起始位点设计两个吗啉修饰的反义寡核苷酸抑制其表达,在斑弓鱼受精卵一到两细胞期混合注射并且验证其有效性.注射后用原位杂交和原位免疫荧光检测心脏标志基因的表达以及心脏的表型,同时利用显微荧光造影和原位杂交检测血管的发育情况.用心室心房的标志基因vmhc和amhc反义RNA探针进行的原位杂交结果显示,vmhc表达抑制,而amhc表达上调.原位免疫荧光显示与原位杂交一致的结果表明:心房扩张心室缩小,并且心脏环化不全.用血管标志基凶flk-1的RNA探针原位杂交和显微荧光造影表明,cx43基因抑制的斑马鱼胚胎血管无明显缺陷.此外,cx43基因抑制的斑马鱼胚胎心脏功能也有明显改变,包括心脏搏动无力,有血液回流现象.抑制cx43的表达可能通过影响两个细胞群的迁移导致斑马鱼胚胎心脏的发育缺陷,从而影响了心脏的功能,但是未发现胚胎血管系统发育的明显缺陷.     

C型利钠肽基因调控斑马鱼胚胎血管发育

本研究旨在探索C型利钠肽前体蛋白基因(natriuretic peptide precursor C,nppc)在斑马鱼胚胎期的表达及其在血管发育过程中的功能。利用胚胎整体原位杂交技术检测nppc基因在斑马鱼胚胎期的表达。同时使用转基因斑马鱼系Tg(flk1:GFP)和Tg(fli1a:n GFP),显微注射nppc特异性吗啉基反义寡核苷酸(morpholino)和nppc m RNA调控nppc基因表达,利用激光共聚焦显微镜观察分析斑马鱼节间血管(intersegment vessel,ISV)表型,并统计内皮细胞数目。结果显示,在受精后24 h和48 h,nppc基因在斑马鱼脑、心脏、血管系统中都有表达。下调nppc基因表达导致ISV发育缺陷,ISV内皮细胞数目减少。以上结果表明下调nppc基因表达可能通过抑制内皮细胞增殖和内皮细胞迁移调控斑马鱼胚胎血管发育。     

金鱼雌核发育单倍体胚胎血液循环障碍产生机制

为研究鱼类单倍体血液循环障碍产生机制,人工诱导获得金鱼(Carassius auratus)雌核发育单倍体胚胎并进行活体观察及邻联茴香胺染色,结果显示金鱼雌核发育单倍体胚胎存在不同程度的血液循环不良和红细胞生成缺陷.为进一步探讨其发生的分子机制,利用反义RNA整胚原位杂交技术比较分析了原始造血和血管发生关键基因scl(...     

斑马鱼VEGF基因的siRNA表达载体构建、有效序列筛选及其对基因表达的干预性研究

为探索小干扰RNA（small interfering RNA,siRNA）表达质粒在研究斑马鱼血管内皮生长因子（vascular endothelial growth factor,VEGF）基因调控网络中的应用,构建了4个以斑马鱼VEGF基因为靶点的siRNA表达载体pSI—VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。通过显微注射的方法将载体导入1-2细胞期斑马鱼体内,于胚胎发育的48h采用RT-PCR的方法检测VEGF基因的表达量,研究不同干扰序列对VEGF基因表达的干涉作用。结果显示,成功地构建了siRNA表达载体。针对不同位点的寡核苷酸序列抑制VEGF基因表达的效率有显著差异,其中注射了ps1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状,同时肠下静脉、节间血管以及其它血管出现不同程度的发育缺陷。实验结果说明,pS1-VEGF可引起斑马鱼胚胎血管发育缺陷。     

地高辛标记Ucp2基因RNA探针的制备和应用

目的:制备用于检测小鼠胚胎早期Ucp2基因表达的地高辛标记的特异性RNA探针。方法:提取小鼠胚胎脑组织总RNA,设计引物,通过RT-PCR方法获取Ucp2基因片段,将其克隆到pGEM-T载体。分别利用Sp6、T7和Ucp2特异性引物,PCR扩增获得转录模板,通过Sp6及T7 RNA聚合酶,获得地高辛标记的正义、反义Ucp2 RNA原位杂交探针。检测标记探针的效价后,通过全胚胎原位杂交分析制备探针的特异性和杂交效果。结果:成功获得Ucp2基因正义、反义探针,反义探针能高效灵敏检测到Ucp2基因在小鼠胚胎Ed9.5、Ed10.5神经系统呈现高表达,而正义探针未能检测到表达信号。结论:成功制备了特异高效的地高辛标记Ucp2 RNA原位杂交探针,为进一步研究Ucp2基因在小鼠胚胎组织中的表达,尤其在神经组织的定位奠定基础。     

梅花鹿Fibulin 5 编码区cDNA的克隆及功能研究

为研究梅花鹿扣针蛋白5(Fibulin5, FBLN5)在鹿茸再生过程中的作用,本研究通过RT-PCR获得了梅花鹿FBLN5的cDNA编码区,并对其进行生物信息学分析。同时利用RNA干扰技术（RNA interference, RNAi）下调了梅花鹿角柄骨膜致敏区（Potentiated pedicle periosteum, PPP）细胞中FBLN5基因表达,并研究了基因表达下调后PPP细胞条件培养液对HUVEC细胞增殖的影响。结果显示：(1) 梅花鹿FBLN5 cDNA编码区长1347 bp,编码448个氨基酸;(2) 生物信息学软件分析显示,梅花鹿FBLN5蛋白含有一个跨膜区,一段23个氨基酸的信号肽序列,vWA_Matrilin、cEGF和EGF_CA等重要的功能结构域;(3) 构建了梅花鹿FBLN5基因的RNAi重组质粒并获得了一条能有效下调目的基因效率达88.72%的靶序列pLVTHM-FBLN2,MTT结果显示PPP细胞FBLN5基因表达量下调后,所获细胞条件培养液可显著促HUVEC细胞增殖,推测FBLN5可能参与了鹿茸再生过程中的血管生成及稳态维持等过程。     

Cdk6在维持ES细胞自我更新中的作用

细胞周期蛋白依赖性激酶6（cyclin dependent kinase 6,Cdk6）对胚胎早期发育有着重要的作用.然而,它在胚胎干（embryonic stem,ES）细胞中的生物学功能仍不清楚.在该研究中,我们运用RNA干扰技术和基因表达分析方法探索了Cdk6在小鼠胚胎干细胞中的功能及分子机制.结果表明：Cdk6的表达水平与小鼠ES细胞的自我更新密切相关.首先,维甲酸（RA）处理和白血病抑制因子（LIF）去除实验显示 ,随着ES细胞的分化,Cdk6的表达水平明显降低.更为重要的是,RNA干扰介导的Cdk6表达抑制导致ES细胞自我更新相关基因的显著下调,同时伴随细胞分化基因的表达激活,提示Cdk6对维持ES细胞自我更新至关重要.     

Tbx1基因功能下调影响斑马鱼Tbx20和Tbx2表达

目的采用吗啡啉修饰反义寡核苷酸显微注射方法下调斑马鱼Tbx1基因表达,研究斑马鱼Tbx1基因功能下调对其他两个T盒基因Tbx20和Tbx2表达的影响。方法采用吗啡啉修饰的反义寡核苷酸显微注射方法抑制斑马鱼Tbx1基因表达,分别将2.5、5、8、10 ng吗啡啉反义寡核苷酸在斑马鱼0-4细胞期注入胚胎,并构建Tbx20,骨形成蛋白2b（Bmp2b）和Tbx2反义RNA探针,进行整体原位杂交,观察Tbx1基因下调对Tbx20、Bmp2b及Tbx2表达的影响。结果Tbx1吗啡啉寡核苷酸显微注射组胚胎表现出鳃弓、耳囊、心血管系统和胸腺的发育异常。Tbx1基因下调导致Tbx20的表达出现改变,Tbx20在心脏的表达与对照组相比明显下调,神经元的表达范围明显缩小;Tbx1基因功能下调会导致Bmp2b在心脏和咽囊的表达减低,Bmp2b在后部咽囊的表达较前部咽囊减低得更为明显;Tbx1基因功能下调胚胎,Tbx2在鳃弓的表达模式发生改变,48 hpf,Tbx2在鳃弓的表达出现从后向前逐渐减低,鳃弓的表达范围较对照组明显缩小。结论Tbx1在发育过程中,会对其他T盒基因,如Tbx20和Tbx2具有激活或抑制的调控作用。Tbx1对Tbx20的作用可能是通过影响Bmp2b的途径,继发地影响Tbx20的表达。Tbx1基因功能下调,会改变Tbx2在鳃弓的表达模式。     

炎症免疫刺激对孕鼠胚胎基因表达谱影响的研究

目的:探讨妊娠期大鼠经炎症免疫刺激后对胚胎基因表达谱的影响.方法:选取6只孕鼠,分别腹腔注射LPS及Zymosan后,取胚胎抽提总RNA,经纯化反转录等过程合成生物素标记的cRNA,经片断化后与Rat Genome 230 2.0 Array芯片杂交,读取芯片结果并进行分析.结果:LPS组有183个基因上调、270个基因表达下调,Zymosan组有144个基因表达上调、417个基因表达下调.有50个基因在上述两组中均表达上调,其中已知功能的有9个;有173个基因在上述两组中表达均下调,已知功能的有85个.结论:应用全基因组芯片筛选了孕鼠经炎症刺激后胚胎差异表达的基因,为进一步研究孕鼠经炎症刺激后仔鼠血压升高的机制提供了新的思路.     

Definitive hematopoiesis initiates through a committed erythromyeloid progenitor in the zebrafish embryo

Shifting sites of blood cell production during development is common across widely divergent phyla. In zebrafish, like other vertebrates, hematopoietic development has been roughly divided into two waves, termed primitive and definitive. Primitive hematopoiesis is characterized by the generation of embryonic erythrocytes in the intermediate cell mass and a distinct population of macrophages that arises from cephalic mesoderm. Based on previous gene expression studies, definitive hematopoiesis has been suggested to begin with the generation of presumptive hematopoietic stem cells (HSCs) along the dorsal aorta that express c-myb and runx1. Here we show, using a combination of gene expression analyses, prospective isolation approaches, transplantation, and in vivo lineage-tracing experiments, that definitive hematopoiesis initiates through committed erythromyeloid progenitors (EMPs) in the posterior blood island (PBI) that arise independently of HSCs. EMPs isolated by coexpression of fluorescent transgenes driven by the lmo2 and gata1 promoters exhibit an immature, blastic morphology and express only erythroid and myeloid genes. Transplanted EMPs home to the PBI, show limited proliferative potential, and do not seed subsequent hematopoietic sites such as the thymus or pronephros. In vivo fate-mapping studies similarly demonstrate that EMPs possess only transient proliferative potential, with differentiated progeny remaining largely within caudal hematopoietic tissue. Additional fate mapping of mesodermal derivatives in mid-somitogenesis embryos suggests that EMPs are born directly in the PBI. These studies provide phenotypic and functional analyses of the first hematopoietic progenitors in the zebrafish embryo and demonstrate that definitive hematopoiesis proceeds through two distinct waves during embryonic development.     

Cloning, expression and functional study of translation elongation factor 2 (EF-2) in zebrafish

We have identified translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of the DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame between nucleotide 55 to 2631 and encodes a protein of 858 amino acids. Zebrafish EF-2 protein shares 92%, 93%, 93% and 92% identity with the corresponding amino acid sequence in human, mouse, Chinese hamster and Gallus EF-2, respectively. Whole-mount in situ hybridization showed that zebrafish EF-2 was a developmentally regulated gene and might play important roles during the early development of zebrafish embryos. Therefore, we further studied the function of EF-2 during early embryogenesis. Using morpholino antisense oligo knockdown assays, anti-MO injected embryos were found to display abnormal development. The yolk balls were larger than normal and the melanophores spreading on their bodies became fewer. Furthermore, their tails were incurvate and their lenses were much smaller than those of the normal embryos. However the EF-2 overexpression data showed that extra EF-2 protein had no obvious effect on zebrafish embryonic development.     

Hematopoietic regulatory domain of gata1 gene is positively regulated by GATA1 protein in zebrafish embryos

Expression of gata1 is regulated through multiple cis-acting GATA motifs. To elucidate regulatory mechanisms of the gata1 gene, we have used zebrafish. To this end, we isolated and analyzed zebrafish gata1 genomic DNA, which resulted in the discovery of a novel intron that was unknown in previous analyses. This intron corresponds to the first intron of other vertebrate Gata1 genes. GFP reporter analyses revealed that this intron and a distal double GATA motif in the regulatory region are important for the regulation of zebrafish gata1 gene expression. To examine whether GATA1 regulates its own gene expression, we microinjected into embryos a GFP reporter gene linked successively to the gata1 gene regulatory region and to GATA1 mRNA. Surprisingly, ectopic expression of the reporter gene was induced at the site of GATA1 overexpression and was dependent on the distal double GATA motif. Functional domain analyses using transgenic fish lines that harbor the gata1-GFP reporter construct revealed that both the N- and C-terminal zinc-finger domains of GATA1, hence intact GATA1 function, are required for the ectopic GFP expression. These results provide the first in vivo evidence that gata1 gene expression undergoes positive autoregulation.     

15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation

A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.     

Expression and characterization of a brain-specific protein kinase BSK146 from zebrafish

We have previously identified a novel protein kinase, pk146, in the brain of Tetraodon. In the present study, we cloned the homologous protein kinase gene encoding a protein of 385 amino acid residues from zebrafish. The overall amino acid sequence and the kinase domain of zebrafish BSK146 shows 48% and 69% identity to that of rat sbk, a SH3-containing serine/threonine protein kinase. By whole-mount in situ hybridization and RT-PCR, the expression of bsk146 mRNA was mainly in the brain. To explore the in vivo function of BSK146 during zebrafish development, we used morpholino knockdown approach and found that BSK146 morphants displayed enlarged hindbrain ventricle and smaller eyes. Whole-mount in situ hybridization was further performed to analyze the brain defects in BSK146-MO-injected embryos. The expression of brain-specific markers, such as otx2, pax2.1, and krox20, was found normal in morphant embryos at 24hpf, while expression of pax2.1 exerted changes in midbrain-hindbrain boundary and hindbrain in morphant embryos at 48hpf. These data suggest that BSK146 may play an important role in later ventricle expansion in zebrafish brain development. Although the recombinant BSK146 protein produced in insect cells was active and could phosphorylate both histone H1 and histone 2B, the endogenous substrate of BSK146 in the embryonic brain of zebrafish is not clear at the present time and needs further investigation.