A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System
A Simple Method for 3D Analysis of Immunolabeled Axonal Tracts in a Transparent Nervous System
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DOI：
10.1016/j.celrep.2014.10.037
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发表时间：
2014-11-20
期刊：
影响因子：
8.8
通讯作者：
Chedotal, Alain
中科院分区：
文献类型：
--
作者：
Belle, Morgane;Godefroy, David;Chedotal, Alain
已经开发了清除技术来使小鼠大脑再生，从而保留3D结构，但其复杂性限制了其使用。在这里，我们表明，轴突束的免疫标记，然后用溶剂（3DISCO）和光片显微镜进行光学透明，揭示了小鼠胚胎和出生后大脑的大脑连接。我们表明Robo 3受体由形成反曲束（FR）的内侧缰核轴突选择性表达，并分析了Slit/Robo和DCC/Netrin途径突变体中该连合束的发育。Netrin-1和DCC需要吸引FR轴突到中线，但这两个突变体表现出特定的和异质性的轴突导向缺陷。此外，具有条件性Slit 2等位基因的Slit配体的底板特异性缺失不仅扰乱FR轴突的中线交叉，而且扰乱它们的前后分布。总之，这种方法是一种独特的和强大的成像工具，研究突变小鼠的轴突连接。
Clearing techniques have been developed to transparentize mouse brains, thereby preserving 3D structure, but their complexity has limited their use. Here, we show that immunolabeling of axonal tracts followed by optical clearing with solvents (3DISCO) and light-sheet microscopy reveals brain connectivity in mouse embryos and postnatal brains. We show that the Robo3 receptor is selectively expressed by medial habenula axons forming the fasciculus retroflexus (FR) and analyzed the development of this commissural tract in mutants of the Slit/Robo and DCC/Netrin pathways. Netrin-1 and DCC are required to attract FR axons to the midline, but the two mutants exhibit specific and heterogeneous axon guidance defects. Moreover, floor-plate-specific deletion of Slit ligands with a conditional Slit2 allele perturbs not only midline crossing by FR axons but also their anteroposterior distribution. In conclusion, this method represents a unique and powerful imaging tool to study axonal connectivity in mutant mice.