Graeden Ellie, Sive Hazel
Department of Biology, Massachusetts Institute of Technology, USA.
J Vis Exp. 2009 Apr 1(26):1217. doi: 10.3791/1217.
In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 80-100 microm.
在本视频中，我们展示了我们实验室开发的一种方法，用于分析发育中的斑马鱼大脑弯曲和折叠所需的细胞形状变化和重排（古茨曼等人，2008年）。这种分析为脊椎动物大脑三维结构发育所需的潜在细胞生物学提供了新的理解，并显著提高了我们研究神经管形态发生的能力。受精后18小时（hpf）开始，随着神经上皮内的脑室膨胀，胚胎斑马鱼大脑开始成形。到24 hpf时，神经管形态发生的初始步骤完成。使用此处描述的方法，在单细胞阶段的胚胎中注射编码膜靶向绿色荧光蛋白（memGFP）的mRNA。注射和孵育后，将此时处于18至24 hpf之间的胚胎倒置固定在琼脂糖中，并用共聚焦显微镜成像。值得注意的是，斑马鱼胚胎是透明的，这使其成为荧光成像的理想系统。虽然我们的分析集中在中脑-后脑边界和后脑，但这种方法可以扩展用于分析斑马鱼中任何深度达80-100微米的区域。
