Wilson Nicole H, Stoeckli Esther T
Institute of Molecular Life Sciences, University of Zurich, Switzerland.
J Vis Exp. 2012 Oct 16(68):4384. doi: 10.3791/4384.
Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development(1,2). These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development. Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors(3) harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript(4) (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown(3). Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations(5), this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development(6). This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.
连合dI1神经元已被广泛研究，以阐明发育过程中轴突导向的潜在机制(1,2)。这些神经元位于脊髓背侧，其轴突沿着固定的轨迹延伸。连合轴突最初向腹侧投射，然后穿过底板。在穿过中线后，这些轴突急剧转向头侧并纵向向脑投射。这些步骤中的每一步都受到吸引和排斥导向线索的协同活动的调节。对这些线索的正确解读对于轴突沿着其划定的路径导向至关重要。因此，理想情况下，应在活胚胎的背景下研究特定分子对连合轴突导向的生理贡献。因此，体内基因敲低必须精确控制，以便仔细区分在发育过程中可能发挥多种作用的基因的轴突导向活性。在这里，我们描述了一种以细胞类型特异性、可追踪的方式在鸡神经管中敲低基因表达的方法。我们使用新型质粒载体(3)，其含有驱动荧光蛋白标记物表达的细胞类型特异性启动子/增强子，紧接着是miR30-RNAi转录本(4)(位于编码荧光蛋白的cDNA的3'-UTR内)(图1)。当电穿孔导入发育中的神经管时，这些载体可有效下调基因表达，并表达明亮的荧光标记蛋白，以便直接追踪经历敲低的细胞(3)。在电穿孔之前混合不同的RNAi载体，可以同时在脊髓的独立区域敲低两个或更多基因。这使得在发育过程中能够以快速、简单、精确和廉价的方式研究复杂的细胞和分子相互作用。结合在开放书标本中对连合轴突轨迹的DiI追踪(5)，该方法是体内研究连合轴突生长和导向的细胞和分子机制的有用工具。原则上，可以使用任何启动子/增强子，这可能使该技术更广泛地应用于发育过程中基因功能的体内研究(6)。本视频首先演示如何处理鸡蛋并开窗、将DNA质粒注射到神经管中以及电穿孔过程。为了研究连合轴突导向，将脊髓从胚胎中取出制成开放书标本，固定后注射DiI以追踪轴突路径。将脊髓置于盖玻片之间，并用共聚焦显微镜观察。
