Live Cell Imaging to Monitor Axonal Pruning in Drosophila Motor Neurons

Abstract

Over the lifespan of an individual, brain function requires adjustments in response to environmental changes and learning experiences. During early development, neurons overproduce neurite branches, and neuronal pruning removes the unnecessary neurite branches to make a more accurate neural circuit. Drosophila motoneurons prune their intermediate axon bundles rather than the terminal neuromuscular junction (NMJ) by degeneration, which provides a unique advantage for studying axon pruning. The pruning process of motor axon bundles can be directly analyzed by real-time imaging, and this protocol provides a straightforward method for monitoring the developmental process of Drosophila motor neurons using live cell imaging. Key features • Long-range projecting axon bundles of Drosophila motor neurons extending from soma on the ventral nerve cord (VNC) undergo degeneration rather than retraction during metamorphosis. • The pruning process of motor axon bundles can be directly observed by real-time live-cell imaging. • The complete clearance of axon bundles occurs approximately 22 h after pupal formation (22 h APF). • Mushroom body (MB) γ neuron axon pruning regulatory genes are conserved for motor neurons.

Keywords: Axonal pruning; Drosophila melanogaster; Live cell imaging; Motor neurons; Neurodevelopment.

Figure 1.. Process of embryo collection

Figure 2.. Process of sample processing at the white pupa (WP) phase

Figure 3.. Process of sample processing at the after-pupal-formation (APF) phase

Figure 4.. Schematic diagram of axon bundle pruning in Drosophila motor neurons.

The upper part of this figure reveals the morphology and distribution of a Drosophila motoneuron, with the soma situated at the surface of the ventral nerve cord (VNC), extending long axon bundles, and terminating in contact with the muscle to form the neuromuscular junction (NMJ). The red dotted box reveals the region we examined. The bottom portion of this figure shows that the axon bundles of motor neurons undergo pruning at different stages of development in Drosophila.

Figure 5.. Drosophila motor neuron axon bundles undergo pruning during development.

(A–D) Representative confocal live images of the development of mCD8-GFP-labeled motor neurons at WP (A), 14 h APF (B), 18 h APF (C), and 22 h APF (D), showing that Drosophila motor neuron axon bundles experience pruning processes during development. Scale bar, 200 μm.

Figure 6.. Uba1 and EcR-B1 are involved in regulating axon bundle pruning in motor neurons.

(A–D) Representative confocal live images of the development of mCD8-GFP-labeled motor neurons of control RNAi (A), uba1 RNAi (B), EcR-B1 RNAi (C), and O/E EcR-B1 ^DN (D) at 22 h APF. (E) Quantification of total length of unpruned motor axon bundles at 22 h APF. O/E represent as overexpress. Data are presented as mean ± SEM. One-way ANOVA with Bonferroni’s test was applied to determine statistical significance. ****p < 0.0001. Scale bars, 200 μm.