【作者】 张晖；

【导师】 陈秋生；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2009， 博士

【摘要】 采用体外转录的方法,分别在SP6和T7 RNA聚合酶的作用下合成地高辛(DIG)标记的Cdc25A, Cdc25B, Sox2和Mnb/Dyrk RNA探针。通过全胚胎原位杂交的方法检测早期鸡胚中上述四种基因mRNA的表达模式。结果表明,四种基因mRNA的表达模式是部分重叠或互补的,大部分表达于整个中枢神经系统(CNS),但也有表达于体节和肢芽。在特异的区域,四种基因的mRNA丰度是存在差异的。在鸡胚早期不同发育阶段,四种基因的表达模式支持了它们可能在早期的鸡胚发育中具有重要的调节功能,而且它们之间具有一定的相互调控作用。试验ⅠCdc25ARNA探针的制备本试验应用RT-PCR方法从鸡胚中扩增Cdc25A的基因片段,通过T-A克隆将其连接于pGM-T质粒载体中构成Cdc25A/pGM-T重组质粒。并将重组质粒转化入大肠杆菌DH5α株感受态细胞中扩增,经挑取阳性克隆进行扩增培养、小量提取质粒,再根据pGM-T的多克隆酶切位点,进行酶切鉴定,然后经测序验证。测序正确后,将阳性株扩增后提取的质粒分别用限制性内切酶NcoⅠ和SpeⅠ进行酶切,使其完全线性化。回收酶切片段,作为合成正、反义RNA探针的模板。分别利用pGM-T中T7和SP6的RNA聚合酶的启动子,在RNA聚合酶的作用下,以线性化的Cdc25A/pGM-T为模板,按Roche地高辛标记试剂盒说明书,利用试剂盒中含有的DIG-dUTP底物混合物,分别用Sp6和T7转录酶进行体外转录,成功转录得到长度为183nt的正、反义DIG标记的RNA探针,为后续的原位杂交试验做好了探针的准备。试验ⅡCdc25A mRNA在早期鸡胚中的分布本试验通过全胚胎原位杂交的方法,使用Cdc25A RNA探针检测其mRNA在早期鸡胚中的分布。结果表明,在9HH(Hamburger and Hamilton)阶段,脑部的杂交信号相对于后期阶段(10,11HH)不强,但中部神经管杂交信号相对于后期阶段较强。后部神经沟,尾部神经板杂交信号此时较强。体节部位信号最弱。在10HH阶段,脑部杂交信号此时很强,但中部神经管杂交信号很弱。体节处的杂交信号也较少。后部神经沟,尾部神经板杂交信号依然较强。在11HH阶段,头部区域的杂交信号仍然最强,中部神经管杂交信号有所加强,后部神经沟,尾部神经板杂交信号有所减弱。此时,在心原基中未见杂交信号。这些结果说明,Cdc25A mRNA的表达模式与增殖中的神经上皮分布区域基本一致。试验ⅢCdc25B RNA探针的制备及其mRNA在早期鸡胚神经管、体节和肢芽中的分布本试验使用分子克隆的方法获得特异性Cdc25B基因目的片段,以线性化的Cdc25B/pGM-T重组质粒为模板,应用体外转录的方法合成长度为575nt的正、反义地高辛标记的Cdc25B RNA探针。通过全胚胎原位杂交方法,用体外转录的Cdc25BRNA探针探查Cdc25B mRNA在早期鸡胚中的动态表达模式。结果表明,在5HH阶段,Cdc25B原位杂交信号在整个胚胎中分布是微弱的,特别是在头部区域,但在亨氏节(Hensen’s node)中杂交信号可见。此时,Cdc25B杂交信号不对称地分布在亨氏节的左侧。在6HH阶段,脑部杂交信号已经清晰可见。杂交信号在亨氏节和原条两侧是不对称的。到了8ˉHH期,脑部杂交信号很强,并且在神经褶中Cdc25B原位杂交信号也较强。此时,神经褶两侧杂交信号分布是对称的,但亨氏节两侧信号仍然是不对称的。在全胚胎原位杂交的横断切片中,Cdc25B原位杂交信号分布在底板。在即将封闭的神经管中,杂交信号呈现出腹背递减的浓度梯度,而且,在大部分尾部神经上皮中缺乏杂交信号。体节中的杂交信号很弱。直到8HH阶段,Cdc25B原位杂交信号在脑部很强。在体节中的杂交信号也较强。从Cdc25B原位杂交的横断切片来看,在8HH阶段,杂交信号在神经管和神经沟两侧是对称的,但在即将封闭的神经管和神经沟中,杂交信号仍然呈现出腹背递减的浓度梯度。从9ˉ到10HH阶段,杂交信号分布于CNS,包括脑部,神经管和神经沟。特别是,脑部的杂交信号很强,但是在10HH阶段,在尾部神经板中没有杂交信号。此外,体节中的信号较强。在12HH阶段,Cdc25B原位杂交信号分布于CNS,特别是脑部比先前发育阶段信号更强。此时,体节中的杂交信号较强。在三天的鸡胚(18HH阶段)切片中,Cdc25B原位杂交信号仍然分布在神经管的腹部区域。体节中信号很强。同时在肢芽的顶端,即AER区域,Cdc25B原位杂交信号很强。在后期的23HH阶段,杂交信号广泛分布于发育中的脊髓,主要分布在腹部区域,并且在腹部区域的外侧缘检测到强烈的信号。此时,体节中信号也较强。然而,自始至终,在早期鸡胚不同发育阶段,脊索都是阴性的。所有这些结果说明,在早期鸡胚不同发育阶段,Cdc25B mRNA主要分布于CNS,在体节和肢芽中也有不同程度地分布。但是,大部分尾部神经上皮中缺乏Cdc25B mRNA分布。试验ⅣSox2 RNA探针的制备及其mRNA在早期鸡胚中的原位杂交检测本试验应用体外转录的方法合成正、反义地高辛标记的Sox2 RNA探针。通过全胚胎原位杂交方法,使用体外转录的探针检测Sox2 mRNA在10HH阶段鸡胚中的表达情况。结果表明,Sox2原位杂交信号沿着CNS区域分布。杂交信号在脑部很强。中部神经管的杂交信号较弱。然而,在尾部神经板杂交信号较强。此外,在体节中并未见到杂交信号。这些结果说明,Sox2 mRNA主要分布在CNS未成熟的神经上皮中。Sox2 mRNA与Cdc25A mRNA在早期鸡胚中的分布模式具有相似性。试验ⅤMnb/Dyrk RNA探针的制备及其全鸡胚原位杂交反应本试验应用体外转录的方法合成正、反义地高辛标记Mnb/Dyrk的RNA探针。通过全胚胎原位杂交方法,用体外转录的RNA探针检测了10HH阶段的鸡胚Mnb/Dyrk mRNA的表达模式。结果表明,Mnb/Dyrk原位杂交信号在CNS中很强。在封闭的神经管中可检测到杂交信号,包括将来发育为前脑,中脑和菱脑的区域,以及脊髓。在头部有很强的杂交信号。尾部神经板区域杂交信号很弱。此外,在体节中杂交信号很弱。这些结果说明,Mnb/Dyrk mRNA主要分布于CNS中。Mnb/Dyrk mRNA在早期鸡胚中的分布与Cdc25B是相似的。更多还原

【Abstract】 The sense and antisense digoxigenin-labeled RNA probes of Cdc25A, Cdc25B, Sox2 and Mnb/Dyrk four genes were produced by using SP6 and T7 RNA polymerases respectively and in vitro transcription. Expression patterns of four genes were detected by in situ hybridization in various developmental HH(Hamburger and Hamilton) stages of the early chick embryo. The results indicate that expression patterns of four genes were similar or complementary. Four genes mRNA was mostly restricted to the entire CNS (central nervous system). All of them have become confined to an identical region, neural tube, neural groove or caudal neural plate, corresponding to spinal cord. But there was some distinction in specific region or in concentration, for example in somites. Four genes mRNA was overlapping in the head region. Cdc25A and Cdc25B mRNA was complementary in CNS except head. The overlap in expression in the same developmental stage in CNS suggests that four genes may be functional similar or relation in CNS development. Expression patterns of four genes support specific roles of these regulators in the developing CNS.ExperimentⅠPreparation of chicken Cdc25A RNA probe To study the distribution of Cdc25A mRNA in early chicken embryo, the sense and anti-sense digoxigenin (DIG) labeled RNA probe were prepared. The fragment of Cdc25A gene was obtained by RT-PCR through total RNA of chicken embryos. Amplified cDNA fragment was subcloned into pGM-T vector, and the plasmid was transformed into E. coli DH5a and chosen by "white-blue plaque selection". The recombinant plasmid was identified by EcoR I restriction enzyme digestion and sequencing, then Cdc25A/pGM-T vector was linearized with the restriction enzyme of Nco I and Spe I respectively. The sense and anti-sense DIG labeled RNA probe were producted by SP6 and T7 RNA polymerase respectively and transcription in vitro according to the protocol of "DIG RNA Labeling Kit (SP6/T7)".ExperimentⅡCdc25A mRNA was detected in chicken embryos by whole mount in situ hybridization histochemistry The distribution of Cdc25A mRNA in chicken embryo was examined by whole mount in situ hybridization histochemistry (ISHH). In stage 9HH, Cdc25A mRNA was weak in rostral head and little in the somites. But there was fruitful in the neural tube, neural groove and neural plate. In stage 10HH, Cdc25A mRNA expression was most redundant in rostral head fold. The caudal neural plate was fairly plentiful, but the middle neural tube was little. In addition, the mRNA was weak in somites. In stage 11HH, Cdc25A mRNA expression was most redundant in rostral head fold. The middle neural tube had more mRNA than prior stages. The mRNA was weak in neural groove and caudal neural plate. In addition, no Cdc25A mRNA was detected in heat primordium. The distribution of Cdc25A mRNA was in neuroepithelium.ExperimentⅢPreparation of chicken Cdc25B RNA probe and its detection in neural tube、somites and limb bud in chicken embryos by whole mount in situ hybridization histochemistry In order to study the distributions of Cdc25B mRNA in chicken embryos, the sense and anti-sense digoxigenin (DIG) labeled RNA probes were prepared. The distributions of Cdc25B mRNA in the embryos were examined by means of whole mount in situ hybridization histochemistry (ISHH). In stage 5HH, Cdc25B mRNA was only emerged in the left of Hensen’s node. In stage 6HH, Cdc25B mRNA was visible in head fold; distribution of Cdc25B mRNA was asymmetrical in Hensen’s node. In stage 8-HH embryos, Cdc25B mRNA was conspicuous in the rostral head fold, and the mRNA of Cdc25B was very abundant in the neural fold; the staining of the neural fold was symmetrical, but that of Hensen’s node was still asymmetrical. Cdc25B transcripts were present on both sides of the floor plate, displayed a ventro-dorsally decreasing gradient in the closing neural tube. The somites were faintly visible. In stage 8HH, the transcripts of Cdc25B were plentiful in the head fold. The somites were made clearly visible by staining. From stage 9- to 10HH, mRNA of Cdc25B was confined to the CNS, including the brain, neural tube, and neural groove. But in stage 10HH, the staining was scarce in the caudal neural plate. Cdc25B mRNA was conspicuous in the somites. In stage 12HH, the staining was stronger than in the previous stages in the CNS, especially in the head fold. Cdc25B mRNA was obvious in the somites. In stage 18HH embryos, Cdc25B was found to be still expressed in a discrete ventral domain in the neural tube. Cdc25B mRNA was detected in the tip of the limb bud. In stage 23HH embryos, Cdc25B was broadly expressed in the developing spinal cord. In general, expression patterns of Cdc25B were restricted in the CNS, somites and the tip of the limb bud in different HH stages during embryo development.ExperimentⅣDistribution of Sox2 in CNS of chicken embryo by whole mount in situ hybridization In order to study the distributions of Sox2 mRNA in chicken embryos, the sense and anti-sense digoxigenin (DIG) labeled RNA probes were prepared. The distributions of Sox2 mRNA in the stage 10HH in chicken embryos were examined by means of whole mount in situ hybridization histochemistry (ISHH). Sox2 was predominantly expressed in the immature neural epithelium of the entire CNS. Sox2 express in a band along the length of most of the CNS and in all compartments of the brain. Sox2 mRNA expression was redundant in rostral head fold according to the staining. The middle neural tube was weak of expression. The caudal neural plate was fairly plentiful. In addition, Sox2 expression was not visible in somites.In general, Sox2 mRNA was detected in the proliferative neural epithelium. The distribution of Sox2 and Cdc25A mRNA was similar in stage 10HH in chicken embryos.Experiment V Distribution of Mnb/Dyrk in CNS of chicken embryo by whole mount in situ hybridization In order to study the distributions of Mnb/Dyrk mRNA in chicken embryos, the sense and anti-sense digoxigenin (DIG) labeled RNA probes were prepared. The distributions of Mnb/Dyrk mRNA in the stage 10HH in chicken embryos were examined by means of whole mount in situ hybridization histochemistry (ISHH). Mnb/Dyrk mRNA was considerable in CNS. In HH stage 10, Mnb/Dyrk mRNA can be detected at different levels of the neural tube:the prospective prosencephalon, mesencephalon, rhombencephalon, and spinal cord. The mRNA was redundant in rostral head fold. But it was weak in caudal neural plate. Moreover, Mnb/Dyrk mRNA was very poor in somites. The distribution of Mnb/Dyrk and Cdc25B mRNA was similar in stage 10HH in chicken embryos.更多还原