The Drosophila Brainbow technique enables observation of the projections of many different neurons individually within their native context and allows interactions between lineages to be visualized in the same brain rather than relying on computational alignments of neurons or lineages from different preparations. The combination of neuronal targeting with sparse GAL4 lines and faithful color expression throughout neurites allows us to follow processes over long distances and even through discontinuous samples such as the brain and peripheral NMJ in the proboscis.
The dBrainbow construct can be used in two modes: for live imaging with two useable colors from endogenous fluorescence and for imaging fixed tissue with six colors derived from antibody-epitope combinations. To accomplish this, we expanded the repertoire of fluorescent proteins and epitope-antibody options usable in flies. Live imaging using endogenous fluorescence may be best suited to the more transparent embryonic or larval stages. New developments in bright, photostable blue and far-red fluorescent proteins should be incorporated.
The vertebrate Brainbow system was a huge leap forward in visualizing many individual neurons simultaneously; it allowed direct comparisons between similar neurons in their native context. dBrainbow adds two new innovations. Because the dBrainbow
construct is under the control of a binary expression system, it can be targeted to neurons of interest using the extensive collections of available GAL4 lines. (The vertebrate Brainbow is currently fused to a single Thy1
promoter and so can only visualize neurons within the Thy1
.) Secondly, the use of epitopes and antibodies permits signal amplification and spectrally discrete, photostable fluorescent dyes. This makes color assignment and fine process tracing easier. Future versions may use membrane targeted proteins to improve neurite tracing. A synaptically localized version of dBrainbow
would also have applications for circuit mapping. The UAS-dBrainbow
construct can be used to anatomically subdivide complex GAL4 patterns and study individual neurons and lineages in context. We have focused entirely on the nervous system, but the construct could be used in other tissues.
The current version of dBrainbow works well for labeling neurons from the same lineage in a common color because of the early expression of the Cre recombinase in the neuroblasts. Biological questions remain about how lineages develop in relation to one another. The lineages may act as functional units within neural circuits; understanding how they project and interact is key for testing this hypothesis. Some GAL4 lines express in multiple lineages and dBrainbow represents an efficient way to image individual lineages within them. For labeling individual neurons, a GAL4 line that does not express throughout whole lineages can be chosen. Although the recombination to select a specific fluorescent protein occurs in the neuroblast, only the cells expressing GAL4 produce this color. To subdivide the neurons within a lineage into different colors would require a truly inducible source of Cre recombinase. All of the existing Drosophila melanogaster Cre reagents suffer from drawbacks – leaky expression, poor inducibility, and/or toxicity. The development of better Cre reagents would be a great contribution to the field. We routinely compare the total expression pattern of a GAL4 labeled with UAS-dBrainbow to the expression pattern revealed by cytoplasmic UAS-EGFP (Bloomington #1521; B. J. Dickson 1996 unpublished) or UAS-mCD8-GFP to determine what fraction of the neurons we are detecting and whether their projections appear normal. With appropriate controls, we have been able to obtain biologically interesting results with the existing hs-Cre lines.
One of the next major goals in Drosophilamelanogaster neuroanatomy is to map each individual neuron in the fly brain. Techniques such as dBrainbow facilitate this by speeding up imaging (multiple individual neurons can be seen in each brain), but also because labeling one neuron in the context of others makes assigning it to a class or type less ambiguous.