Imaging axon pathfinding in Xenopus in vivo.
Imaging axon pathfinding in Xenopus in vivo.
复制标题
非洲爪蟾体内轴突寻路成像。
DOI：
10.1101/pdb.prot070003
复制
发表时间：
2012
影响因子：
--
通讯作者：
C. Holt
中科院分区：
文献类型：
--
作者：
Louis C. Leung;C. Holt
在发育中的动物中，轴突寻路涉及一个高度动态的过程，其中轴突生长锥在朝向其目标导航时做出连续的决定。在生长锥中发生的关于从复杂的新区域缩回或延伸到复杂的新区域的变化可以在几分钟内发生。因此，在活的完整动物体内可视化轴突寻路策略的出现对于更好地理解生长锥如何响应多种线索做出如此快速的决定至关重要。将这些策略与功能丧失和/或功能获得技术相结合，可以让人们了解哪些分子对导航过程中特定选择点的特定生长锥行为至关重要。使用非洲爪蟾的主要优势在于主要轴突束的活体显微镜检查，因为它们的胚胎发育发生在子宫外。此外，健壮的胚胎在固定期间保持健康，并允许长期良好的成像。该方案描述了稳定和制备活非洲爪蟾胚胎的方法，用于在高空间和时间分辨率下对标记的轴突束进行长达72小时的成像。这种方法可以用来研究如何敲低某些基因的功能可以影响导航的速度，通过研究爪蟾retinotectal途径。它可以适用于可视化其他感兴趣的轴突束。
Axon pathfinding in the developing animal involves a highly dynamic process in which the axonal growth cone makes continuous decisions as it navigates toward its target. Changes occurring in the growth cone with respect to retracting from or extending into complex new territories can occur in minutes. Thus, the advent of strategies to visualize axon path-finding in vivo in a live intact animal is crucial for a better understanding of how the growth cone makes such rapid decisions in response to multiple cues. Combining these strategies with loss-of-function and/or gain-of-function techniques allows one to gain some insight as to which molecules are crucial to particular growth cone behaviors at specific choice points during navigation. The main advantage of using Xenopus lies in the accessibility of major axon tracts for live microscopy, as their embryonic development occurs ex utero. Furthermore, the robust embryos remain healthy during immobilization and allow for good imaging for long periods. This protocol describes the methods for stabilizing and preparing live Xenopus embryos for imaging labeled axonal tracts at high spatial and temporal resolution for up to 72 h. This approach can been used to investigate how the knockdown of certain gene functions can affect the speed of navigation through the well-studied Xenopus retinotectal pathway. It can be adapted to visualize other axon tracts of interest.