Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.
Development. 2012 May;139(9):1691-9. doi: 10.1242/dev.072702.
To understand the molecular mechanisms of development it is essential to be able to turn genes on and off at will and in a spatially restricted fashion. Morpholino oligonucleotides (MOs) are very common tools used in several model organisms with which it is possible to block gene expression. Recently developed photo-activated MOs allow control over the onset of MO activity. However, deactivation of photo-cleavable MO activity has remained elusive. Here, we describe photo-cleavable MOs with which it is possible to activate or de-activate MO function by UV exposure in a temporal and spatial manner. We show, using several different genes as examples, that it is possible to turn gene expression on or off both in the entire zebrafish embryo and in single cells. We use these tools to demonstrate the sufficiency of no tail expression as late as tailbud stage to drive medial precursor cells towards the notochord cell fate. As a broader approach for the use of photo-cleavable MOs, we show temporal control over gal4 function, which has many potential applications in multiple transgenic lines. We demonstrate temporal manipulation of Gal4 transgene expression in only primary motoneurons and not secondary motoneurons, heretofore impossible with conventional transgenic approaches. In another example, we follow and analyze neural crest cells that regained sox10 function after deactivation of a photo-cleavable sox10-MO at different time points. Our results suggest that sox10 function might not be critical during neural crest formation.
为了理解发育的分子机制，能够随心所欲且在空间上受到限制地开启和关闭基因至关重要。 形态发生素寡核苷酸（MOs）是几种模式生物中非常常用的工具，可用于阻断基因表达。 最近开发的光激活 MO 允许控制 MO 活性的开始。 然而，光可裂解 MO 活性的失活仍然难以捉摸。 在这里，我们描述了光可裂解 MO，通过紫外线照射可以以时间和空间方式激活或去激活 MO 功能。 我们以几个不同的基因为例表明，有可能在整个斑马鱼胚胎和单个细胞中开启或关闭基因表达。 我们使用这些工具来证明尾端表达的充分性，直到尾芽阶段都足以驱动中胚层前体细胞向脊索细胞命运发展。 作为光可裂解 MO 的更广泛应用方法，我们展示了对 gal4 功能的时间控制，这在多个转基因系中有许多潜在的应用。 我们证明了 Gal4 转基因表达在初级运动神经元中的时间操纵，而不是在次级运动神经元中，这是传统的转基因方法以前不可能实现的。 在另一个例子中，我们在不同时间点失活光可裂解 sox10-MO 后，追踪并分析了恢复 sox10 功能的神经嵴细胞。 我们的结果表明，sox10 功能在神经嵴形成过程中可能不是关键的。
