Selective muscle imaging reveals myopodia clustering in multiple ventral muscles during Drosophila neuromuscular development.
Precise neuromuscular connectivity depends on coordinated interactions between motor axons and their muscle targets during development. In the Drosophila embryo, dynamic muscle protrusions termed myopodia arise during early phases of muscle innervation and have been proposed to contribute to synaptic partner matching. However, whether similar structures occur across different muscle fibers and how they are developmentally regulated remain unclear. Here, we developed a genetic imaging toolkit based on GAL4 drivers that label defined subsets of embryonic muscle fibers or enable stochastic single-muscle visualization across the musculature. These reagents allow minimally invasive, high-resolution imaging of muscle membrane dynamics in both live and fixed embryos. Using this approach, we found that myopodial clustering is not restricted to a single muscle but is observed across multiple muscle fibers at presumptive synaptic contact sites. Importantly, clustered myopodia emerged during a defined developmental window coincident with motor axon arrival at target muscles, indicating that clustering is temporally associated with neuron-muscle interactions. While clustering was observed in multiple muscles, its frequency and morphology exhibited quantitative variation among muscle fibers. Together, these findings establish myopodial clustering as a recurrent and temporally regulated cellular behavior in the presumptive postsynaptic sites of ventral muscles during early neuromuscular contact development. This study provides a versatile toolkit and a framework for systematically analyzing muscle membrane dynamics during synaptic target recognition.