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

Abstract

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 24h 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.

Fig. 1

A schematic diagram of the trunk segment of a 48 hpf embryo showing moto-neurons and axons. The spinal cord and notochord are indicated. The layer in between (not marked) on which motor neurons reside is the floor plate. Their axons descend from the spinal cord at the same ventral root, cross the horizontal myoseptum (dashed line) through the trajectory point, and then diverge along axon-specific pathways corresponding to each hemisegment. Three populations of secondary motoneurons are the CaP-like, the MiP-like and the RoP-like that reside in the spinal cord.

Fig. 2

A low resolution montage of a region of the 384-well plate arrayed with the zebrafish embryos in the wells is shown. Embryos are manually arrayed into the plate using a transfer pipet. Using a multi-channel pipet, excess water is aspirated from the wells and 25 μl fish water containing tricaine (0.016%) is added to each well.

Fig. 3

Representative images from a 384-well plate used for high content automatic image capture and analysis of hb9:GFP transgenic zebrafish embryos. A panel of control (untreated) and ethanol-treated embryos (48 hpf) are presented. Representative images of GFP expression captured by FITC filter (green), overaly (red) and overlay (red plus green) are shown, respectively for each group. Control (A, B, C); 2% ethanol (D, E, F); and 2.5% ethanol (G, H, I). EtOH=Ethanol.

Fig. 4

Average axon lengths in the control and ethanol-treated embryos derived from the acquired images. Using a custom journal, the Select Axon journal following the method detailed in the “Materials and methods” section, lengths of axons in the specific regions (marked in dotted red lines) were calculated from the archived images Representative images for control, 1%, 1.5%, 2% and 2.5% ETOH (ethanol) are shown (A). Values for average axon lengths are presented as mean +/− SD (B). Significance (*) level was set at p<0.05. NS=no significant difference.

Fig. 5

High content automatic image capture and analysis of axon lengths of hb9:GFP transgenic zebrafish embryos grown at two different temperatures. A panel of embryos grown for 24 h starting at 24 hpf at 22 °C and 28.5 °C were analyzed for axon growth. The analysis was at the actual age of 48 hpf. Representative images for 22 °C and 28.5 °C (A) and average axon lengths (B) are shown. Data are calculated from the archived images following the method detailed in the “Materials and methods” section. Values are presented as mean +/− SD. Significance (*) level was set at p<0.05.