Summary

鸡小脑切片的体外培养和空间上有针对性的颗粒细胞前体电穿孔

Published: December 14, 2015
doi:

Summary

小脑外部颗粒层是最大的过境扩增在显影脑的部位。在这里,我们提出了一个协议,以目标基因改造这个层扩散采用体外电击和小脑切片文化从胚胎第14天鸡胚的高峰。

Abstract

小脑外部颗粒层(EGL)是最大的中转放大在发育中的大脑的网站,并为研究神经元的增殖和分化一个很好的典范。另外,其增殖能力的进化改进一直负责小脑大小在羊膜急剧扩张,使得小脑为脊椎动物脑的EVO发育生物学研究的优良模型。东瀛的组成细胞,小脑颗粒祖细胞,也代表来源的显著细胞的髓母细胞瘤,最常见的小儿神经肿瘤。以下中转扩增,颗粒的前体的径向迁移进入其中它们代表最大的神经元群中的成熟哺乳动物脑小脑的内部颗粒层。在小鸡,东瀛增殖高峰出现对妊娠的第二周结束。为了目标遗传修饰该层在泛滥的高峰期,我们已经制定了通过小脑切片的离体电穿孔的基因操纵从胚胎第14天鸡胚的方法。这种方法概括体内颗粒神经元发展的几个重要方面,并会生成一个透彻的了解小脑颗粒细胞的增殖和分化是有用的,因而小脑发育,进化和疾病。

Introduction

小脑坐在后脑的前端,并负责感觉和运动处理中的成熟大脑的整合以及调节更高的认知过程1。在哺乳动物和鸟类,它具有一个精心形态并被大量面理化,的祖细胞的广泛过境扩增产物产生超过一半在成人脑的神经元的发育过程中。小脑一直研究的课题的神经生物学家了几个世纪,在分子的时代也同样收到显著的关注。这不仅涉及其固有的吸引生物学,而且这样的事实,它在很大程度上与人类疾病包括发育的遗传性疾病如孤独症谱系障碍2,最突出的是小脑癌,髓母细胞瘤3,这是最普遍的儿童脑瘤。重要的是,它是内的wh的优良模型系统非物质文化遗产研究的命运分配和神经大脑发育过程中4。近年来,它也被确立为大脑发育的比较研究模型系统,由于对整个可见的脊椎动物系统发育5-10小脑形态的巨大差异。

小脑从菱1在后脑11背侧半开发和发育是由两个主要祖种群,菱形唇缘和脑室区的。周围的后脑的神经上皮的背部区域的菱形唇延伸与屋顶板的边界。它是小脑12-14的谷氨酸能神经元的兴奋性的发源地。脑室区产生了抑制GABA能神经元的小脑,最突出的是大浦肯野神经元14,15。后来在发展(约13.5小鼠胚胎天; E6的小鸡16),谷氨酸培罗成itors从菱形唇沿切线方向迁移,形成祖细胞的软脑膜层:所谓的外部颗粒层(EGL)二级祖区。正是这层经过广泛的过境放大,导致颗粒神经元的数量巨大,在成熟大脑中发现。

增殖在EGL早已被链接到子软脑膜位置而导致的切向迁移从菱形唇17,与交换机细胞周期出口和祖细胞的神经元分化正在与他们的出口从外EGL层到中间关联EGL 18。有丝分裂后的颗粒细胞的中间-侧向轴线广泛切线迁移发生在中间和内部的EGL 19,最终径向迁移进入成熟小脑皮层的内颗粒层之前。从菱形唇在小脑表面细胞的迁移是依赖于CXCL12信号从软20-22 </sup>和颗粒细胞表达CXCL12受体CXCR4。他们的切向迁移是这样回忆那新皮质切向迁移抑制中间神经元群体23-25。有趣的是,电子显微镜研究17表明,EGL细胞增殖形态保持软脑膜接触,连接细胞的行为具有增殖能力的让人联想到哺乳动物的大脑皮层26的基底祖细胞的方式。这反映在EGL的前述分层成三个子层是由不同的胞外环境,并在那里颗粒前体具有不同的基因表达签名18限定

在oEGL祖细胞增殖发生克隆大小正态分布使得当祖单独遗传标记在小鼠胚胎发育的末端,它们产生的有丝分裂后250-500克的中值平均ranule神经27,28。扩散是依赖于有丝分裂SHH信号来自底层的浦肯野神经元29-32。向SHH作出反应的能力已被证明是完全依赖于转录因子ATOH1的细胞中自主表达, 体外33 体内34,35。同样地,细胞周期出口和分化已被证明是取决于下游转录因子NEUROD1 36,这很可能ATOH1 37的直接阻遏的表达。

尽管这些进展,并且在解密的细胞周期出口38-42的细胞生物学的基础相当大的进步,根本的分子机制(S)所依据决定退出细胞周期和从祖过渡到微分神经元,并且在内侧的EGL关联的有丝分裂后的切向迁移以及后来的开关以放射状迁移,仍然不完全理解。这是为了由于EGL的实验棘手的很大程度:它是迟显影,而且很难基因靶向,因为许多相同的神经性分子,可以在颗粒的前体的寿命的关键早些时候在菱形唇。为了解决这个问题,许多作者已经开发出在体内和体外电作为一种方法,目标在啮齿类动物中43-48产后小脑。在这里,我们率先使用体外电穿孔在小鸡研究EGL,它代表在成本和便利方面相当大的优势。我们的电穿孔体外切片鸡小脑组织培养的方法使用组织解剖从胚胎第14天的雏鸡在EGL扩散的高峰期。这种方法允许菱形唇的EGL的遗传靶向独立地,将设置了阶段为从颗粒的过渡的遗传解剖祖在有丝分裂后小脑颗粒神经元。

Protocol

注:所有实验均用按照国王学院,英国伦敦和英国内政部的动物护理准则进行的。 1.解剖E14小脑在38℃孵育棕色受精母鸡的蛋胚胎第14天。 使用蛋剪刀斩首鸡胚卵内并取出头到含有冰冷的PBS(图1A)培养皿。 使用标准镊子,使切口每只眼睛后面所有的方式,通过组织,消除眼睛和上颚。让所有的方式,通过咽去除下颌( 图1B),<…

Representative Results

本节说明可使用切片电和小脑的文化胚胎第14天的小鸡得到的结果的例子。小脑的解剖示于图1和电穿孔室设置示于图2,我们表明,这是可能的电穿孔,并成功地培养小脑切片,仍保持其结构和细胞形态在体外 (图3A)。目标电穿孔到个别的Folia容易实现(图3B)。我们成功地电穿孔的一些?…

Discussion

这里所报告的协议描述了解剖,电穿孔和从小鸡培养的胚胎第14天小脑切片的方法。该协议使电的靶向东瀛小灶地区,包括个别的小脑叶片孤立的目标。它使遗传分析和成像以高分辨率和便利性,并以较低的成本相比,在啮齿类动物中43-47建立的技术。这样的分析是目前不可能在体内 ,由于延长的发育时间段,在小鼠EGL特异性遗传靶向的可能性很少,和的菱形唇与EGL之间的分子机制…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

在这篇文章中介绍的方法源于由BBSRC BB /资助的工作I021507 / 1(TB,RJTW)和MRC博士助学金(MH)。

Materials

McIlwain tissue chopper Mickle Laboratory Engineering Ltd Cut at 300μm for best results.
Basal Medium Eagle (Gibco) Life Technologies 41010-026
L-glutamine Sigma G7513
penicillin/streptomycin Sigma P4333
0.4μm culture insert Millipore PICM0RG50
TSS20 Ovodyne electroporator  Intracel 01-916-02 Use 3x10v, 10ms pulses for electroporation.

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