A simple method for 3D analysis of immunolabeled axonal tracts in a transparent nervous system

Cell Rep. 2014 Nov 20;9(4):1191-201. doi: 10.1016/j.celrep.2014.10.037. Epub 2014 Nov 6.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Axons / metabolism*
  • Biomarkers / metabolism
  • Brain / metabolism*
  • DCC Receptor
  • Embryo, Mammalian / metabolism
  • Imaging, Three-Dimensional / methods*
  • Membrane Proteins / metabolism
  • Mice, Knockout
  • Mutation
  • Nerve Growth Factors / metabolism
  • Nerve Tissue Proteins / metabolism
  • Netrin-1
  • Receptors, Cell Surface / deficiency
  • Receptors, Cell Surface / metabolism
  • Staining and Labeling*
  • Tumor Suppressor Proteins / deficiency
  • Tumor Suppressor Proteins / metabolism


  • Biomarkers
  • DCC Receptor
  • Dcc protein, mouse
  • Membrane Proteins
  • Nerve Growth Factors
  • Nerve Tissue Proteins
  • Ntn1 protein, mouse
  • Receptors, Cell Surface
  • Robo3 protein, mouse
  • Tumor Suppressor Proteins
  • Netrin-1