The live imaging of motor neurons in sensor zebrafish.

(A) The structure of sensor C3 was predicted by using Rosetta software. (B) The principle of sensor C3 for apoptosis detection. (C and 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.

Visualizing caspase-3 activation and single motor neuron apoptosis during zebrafish early development.

(A−D) FRET images of four somites showing motor neurons underwent apoptosis during the development of Tg(mnx1:sensor C3) zebrafish. Two dead motor neurons in (C) are indicated with yellow and white arrowheads, respectively. The axon in (D) 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. (E) Z-stack imaging showed the apoptosis of the motor neuron in (D). 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 caspase-3 activation occurred quickly and almost at the same time between the cell body and axon in apoptotic motor neurons.

(A) Tracking 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) Tracking 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 a small percentage of motor neurons died during zebrafish early development.

(A) The tracking of motor neuron apoptosis by FRET imaging during zebrafish early development. White arrowheads indicate apoptotic motor neurons. (B) 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). (C) The percentages of apoptotic motor neurons among total motor neurons at 30 hpf and 36 hpf. (D) 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. (E) The percentages of apoptotic motor neurons detected by TUNEL assays.

Most dead motor neurons were not colocalized with macrophages.

(A) Live imaging showing the distribution of macrophages in zebrafish body at 36 hpf. (B) An example showing the colocalization between apoptotic bodies derived from the axon of a motor neuron and 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 colocalization is indicated with a yellow arrowhead in the right image. The anatomical structures are indicated. (C) The percentage of apoptotic motor neurons colocalized with macrophages. The total number of apoptotic motor neurons observed and the number of apoptotic motor neurons colocalized with macrophages are listed.