Identification of Neuronal Lineages in the Drosophila Peripheral Nervous System with a “Digital” Multi-spectral Lineage Tracing System

Elucidating cell lineages provides crucial understanding of development. Recently developed sequencing-based techniques enhance the scale of lineage tracing but eliminate the spatial information offered by conventional approaches. Multi-spectral labeling techniques, such as Brainbow, have the potential to identify lineage-related cells in situ. Here, we report nuclear Bitbow (nBitbow), a “digital” version of Brainbow that greatly expands the color diversity for scoring cells, and a suite of statistical methods for quantifying the lineage relationship of any two cells. Applying these tools to the Drosophila peripheral nervous system (PNS), we determined lineage relationship between all neuronal pairs. This study demonstrates nBitbow as an efficient tool for in situ lineage mapping, and the complete lineage relationship among larval PNS neurons opens new possibilities for studying how neurons gain specific features and circuit connectivity. [Display omitted] •A single nBitbow cassette generates up to 31 color variants•nBitbow enhances the efficiency and improves the accuracy of cell lineage tracing•nBitbow is suitable for lineage tracing in both the PNS and the CNS of Drosophila•The lineage relationship of all PNS neurons in Drosophila larvae is determined Veling et al. report a multi-color labeling system and statistical methods for mapping cell lineages. They identify the lineage relationship of all neurons in the peripheral nervous system of Drosophila larvae and show the utility of this technique in mapping neurons in the CNS.