In Situ Hybridization to Characterize Neurulation and Midbrain–Hindbrain Boundary Formation in Zebrafish

Whole-mount in situ hybridization is able to harness the inherent advantages of zebrafish as a model organism for developmental biology, particularly when visualizing the formation of the neural tube, specifically at the level of the midbrain–hindbrain boundary. The size and transparency of developing zebrafish embryos allow for the visualization of neural markers in vivo along the length of the developing zebrafish central nervous system. In practice, this technique is useful for examining defects in neurulation and midbrain–hindbrain boundary formation that may arise following gene manipulation, for example, CRISPR mutagenesis. This method describes the process of embryo collection and preparation, RNA probe transcription, probe hybridization in vivo, as well as the process of probe detection and visualization.