In vivo imaging and quantitative analysis of changes in axon length using transgenic zebrafish embryos

We describe an imaging procedure to measure axon length in zebrafish embryos in vivo. Automated fluorescent image acquisition was performed with the ImageXpress Micro high content screening reader and further analysis of axon lengths was performed on archived images using AcuityXpress software. We utilized the Neurite Outgrowth Application module with a customized protocol (journal) to measure the axons. Since higher doses of ethanol (2–2.5%, v/v) have been shown to deform motor neurons and axons during development, here we used ethanol to treat transgenic [hb9:GFP (green fluorescent protein)] zebrafish embryos at 28 hpf (hours post-fertilization). These embryos express GFP in the motor neurons and their axons. Embryos after ethanol treatment were arrayed in 384-well plates for automated fluorescent image acquisition in vivo. Average axon lengths of high dose ethanol-treated embryos were significantly lower than the control. Another experiment showed that there was no significant difference in the axon lengths between the embryos grown for 24 h at 22° C and 28.5° C. These test experiments demonstrate that using axon development as an end-point, compound screening can be performed in a time-efficient manner.

► We used high throughput imaging to measure axon length in transgenic zebrafish.
► We quantitatively examined axon length in vivo using high content data analysis.
► This method will help assess drug toxicity rapidly and efficiently in zebrafish.