Live Imaging of Neuroblast Lineages within Intact Larval Brains in Drosophila

Neuroblasts are the precursors of the Drosophila central nervous system and undergo repeated physical and molecular asymmetric cell divisions. Live imaging of neuroblast lineages within intact Drosophila larval brains has dramatically improved our current understanding of basic cellular processes such as the establishment of cell polarity, spindle orientation, and cytokinesis. The analysis of mutant phenotypes using live imaging can enlarge our understanding of asymmetric neuroblast division and self-renewal. Although much live neuroblast imaging is performed using green fluorescent protein only, the generation of improved fluorescent proteins has led to an increase in the use of two-color imaging. Here we present a simple protocol for isolating and imaging larval brain neuroblasts. We describe procedures for the dissection and mounting of brains from third-instar Drosophila larvae in explant solution and their subsequent live imaging. The method provides a close approximation to the in vivo environment and produces data with high temporal and spatial resolutions. We also discuss potential problems and pitfalls and provide examples of how this technique is used.