The live imaging of motor neurons in sensor zebrafish. (A) The structure of sensor C3 predicted by using Rosetta software. (B) The principle of sensor C3 for apoptosis detection. (C-D) The expression of sensor C3 in the motor neurons of Tg(mnx1:sensor C3) zebrafish. The bright field image and fluorescent image were merged in (C) to show the location of motor neurons and their axons. The anatomical structures and developmental time are indicated.

Both the cell body and axon of motor neurons can undergo apoptosis during zebrafish development. (A-C) FRET images of three somites showing motor neurons underwent apoptosis during the development of Tg(mnx1:sensor C3) zebrafish. The axon in (C) is enlarged for a better illustration. Blue apoptotic signals from cell bodies and axons are indicated with arrowheads. The developmental time is indicated in each image. (D) Z-stack imaging showed the apoptosis of the motor neuron in (C). The axon region in the stack of 13.8 µm is enlarged for a better illustration. The depth of each stack is indicated in each image.

The apoptosis of motor neurons occurred quickly and almost at the same time between the cell body and axon. (A) Real-time imaging of motor neuron apoptosis during zebrafish development. During tracking, one motor neuron changed from green to blue. The cell body is indicated with white arrowheads. The axon is enlarged for a better illustration and indicated with yellow arrowheads. The developmental time is indicated in each image. (B) Real-time imaging of the apoptotic process in another motor neuron during zebrafish development. The cell body is indicated with white arrowheads, and the axon is indicated with yellow arrowheads. The developmental time is indicated in each image.

Only around 2% of motor neurons died during zebrafish early development. (A) The quantified results show the number of apoptotic motor neurons at each time point during zebrafish early development (n = 30 zebrafish for each time point). (B) The percentages of apoptotic motor neurons among total motor neurons at 30 hpf and 36 hpf. (C) TUNEL assays showing apoptotic motor neurons at 30 hpf and 36 hpf. Immunofluorescence staining with an anti-GFP antibody was used to indicate motor neurons expressing sensor C3 proteins. White arrowheads indicate TUNEL-positive motor neurons. (D) The percentages of apoptotic motor neurons detected by TUNEL assays. (E) The quantified results showing the number of apoptotic motor neurons at 30 hpf after the knockdown of GDNF or BDNF gene using morpholinos (n = 20 zebrafish for each time point). ns, no significant difference.

Most dead motor neurons could not be engulfed by macrophages. (A) Live imaging showing the distribution of macrophages in zebrafish body at 36 hpf. (B) An example showing the engulfment of apoptotic bodies derived from the axon of a motor neuron by a macrophage in a somite at 36 hpf. The apoptotic bodies are indicated with a white arrowhead and enlarged in the box in the left image. The engulfment by the macrophage is indicated with a yellow arrowhead in the right image. The anatomical structures are indicated. (C) The percentage of apoptotic motor neurons engulfed by macrophages. The total number of apoptotic motor neurons observed, and the number of engulfed apoptotic motor neurons are listed.