Deschene Elizabeth R, Barresi Michael J
Department of Biological Sciences, Smith College.
J Vis Exp. 2009 Sep 11(31):1422. doi: 10.3791/1422.
Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered. Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions. Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes. Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras. We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another. We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures. In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo. These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.
发育生物学领域的某些基本问题，只有当细胞被置于新环境中，或者当更大环境中的小细胞群被改变时才能得到解答。观察单个细胞在独特环境中的相互作用和行为，对于表征细胞功能至关重要。确定特定蛋白质的局部错误表达如何影响周围细胞，能为该蛋白质在各种发育过程中所起的作用提供有见地的信息。我们实验室利用斑马鱼模型系统，将遗传方法与经典移植技术独特地结合起来，以产生基因型或表型嵌合体。我们研究斑马鱼前脑连合形成过程中的神经元 - 胶质细胞相互作用。本视频描述了一种方法，使我们实验室能够研究间脑中星形胶质细胞群体的作用，以及影响投射轴突穿过中线的特定引导线索的作用。由于斑马鱼胚胎具有透明性，它们是进行此类异位细胞植入或局部基因错误表达的理想模型。可以使用活性染料或表达荧光蛋白的转基因鱼系来追踪移植细胞。我们在此展示如何制备带有活性染料示踪剂的供体胚胎用于移植，以及如何从一个原肠胚期胚胎中提取细胞并将其移植到另一个胚胎中。我们展示了斑马鱼胚胎前脑内异位 GFP + 转基因细胞的数据，并描述了这些细胞相对于前脑连合的位置。此外，我们展示了对移植到 GFP + 转基因宿主胚胎中的 Alexa 594 标记细胞进行激光扫描共聚焦延时显微镜观察的结果。这些数据提供了证据，表明原肠胚期移植能够实现异位细胞的靶向定位，以解决发育生物学中的各种问题。
