Stowers Institute for Medical Research; Kansas City, MO USA.
Organogenesis. 2009 Oct;5(4):238-47. doi: 10.4161/org.5.4.10482.
Tracing cell movements in vivo yields important clues to organogenesis, yet it has been challenging to accurately and reproducibly fluorescently mark single and small groups of cells to build a picture of tissue assembly. In the early embryo, the small size (hundreds of cells) of progenitor cell regions has made it easier to identify and selectively mark superficially located cells by glass needle injection. However,during early organogenesis,subregions of interest may be several millions of cells in volume located deeper within the embryo requiring an alternative approach. Here, we combined (confocal and 2-photon) photoactivation cell labeling and multi-position, multi-time imaging to trace single cell and small subgroups of cells in the developing brain and spinal cord. We compared the photostability and photoefficiency of a photoswitchable fluorescent protein, PSCFP2, with a novel nuclear localized H2B-PSCFP2 protein. We showed that both fluorescent proteins have similar photophysical properties and H2B-PSCFP2 is more effective in single cell identification in dense tissue. To accurately and reproducibly fluorescently trace subregions of cells in a 3D tissue volume, we developed a protocol for multi-position photoactivation and multi-time acquisition in the chick spinal cord in up to eight tissue sections. We applied our techniques to address the formation of the sympathetic ganglia,a major component of the autonomic nervous system,and showed there are phenotypic differences between early and later emerging neural crest cells and their positions in the developing ganglia. Thus, targeted fluorescent cell marking by confocal or 2-photon multi-position photoactivation and multi-time acquisition offer a more efficient, less invasive technique to trace cell movements in large regions of interest and move us closer towards mapping the cellular events of organogenesis.
在体内追踪细胞运动为器官发生提供了重要线索，但准确和可重复地荧光标记单个和小群细胞以构建组织组装的图像一直具有挑战性。在早期胚胎中，祖细胞区域的小尺寸（数百个细胞）使得更容易通过玻璃针注射来识别和选择性标记位于表面的细胞。然而，在早期器官发生过程中，感兴趣的亚区可能是体积为数百万个细胞，位于胚胎内部更深的位置，需要采用替代方法。在这里，我们结合（共聚焦和双光子）光激活细胞标记和多位置、多次成像来追踪发育中的大脑和脊髓中的单个细胞和小亚群细胞。我们比较了光可切换荧光蛋白 PSCFP2 和新型核定位 H2B-PSCFP2 蛋白的光稳定性和光效率。我们表明，这两种荧光蛋白具有相似的光物理性质，并且 H2B-PSCFP2 在密集组织中单细胞识别中更有效。为了在 3D 组织体积中准确和可重复地荧光追踪细胞亚区，我们开发了在鸡脊髓中进行多位置光激活和多次获取的方案，最多可在八个组织切片中进行。我们应用我们的技术来解决交感神经节的形成问题，这是自主神经系统的主要组成部分，并表明在发育中的神经节中，早期和晚期出现的神经嵴细胞及其位置之间存在表型差异。因此，通过共聚焦或双光子多位置光激活和多次采集进行靶向荧光细胞标记提供了一种更有效、侵入性更小的技术，可用于追踪大感兴趣区域中的细胞运动，并使我们更接近于绘制器官发生的细胞事件图谱。
