Hadjieconomou, D; (2013) Development of a genetic multicolor cell labeling approach for neural circuit analysis in Drosophila. Doctoral thesis (PhD), UCL (University College London).

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Abstract

The assembly of functional neural circuits during development is pivotal for the ability of the brain to generate complex behaviors. To facilitate the analysis of the underlying molecular mechanisms in Drosophila, we have developed a genetic multicolor cell labeling approach called Flybow (FB), which is based on the vertebrate Brainbow-2 system. FB relies on the stochastic expression of membrane tethered fluorescent proteins (FPs). FP encoding sequences were arranged in pairs within one or two cassettes each flanked by recombination sites. Recombination mediated by an inducible modified Flp/FRT system results in both excisions and inversions of the flanked cassettes providing temporal control of FP expression. Moreover, FB employs the GAL4/UAS system and hence can be used to investigate distinct cell populations in the tissue of interest. We have generated three FB variants. FB 1.0 consists of one cassette driving expression of either mCherry or V5-tagged Cerulean. FB 1.1 contains a second cassette with opposing enhanced green fluorescent protein (EGFP) and mCitrine cDNAs leading to stochastic expression of four FPs. Finally, FB2.0 contains an additional excisable cassette flanked by classical FRT sites to refine transgene expression in specific cell types, in which Gal4 and Flp activities overlap. The FB approach was validated by investigating neural circuit assembly and connectivity in the visual system. FB makes it possible to visualize dendritic and axonal arborizations of different neuron subtypes and the morphology of glial cells with single cell resolution in one sample. Using live and fixed embryonic tissue, we could show that FB is suitable for studies of this early developmental stage. Additionally, we demonstrated that the approach can be used in non-neural tissues. Finally, combining the mosaic analysis with a repressible cell marker (MARCM) and FB approaches, we demonstrate that our technique is compatible with available Drosophila tools for genetic dissection of neural circuit formation.

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