Multicolour labelling of individual neural cells

The cerebral cortex, or the outer layer of neural tissue of the mammalian brain, has numerous functions, including memory, attention and language. Cortical mini-columns are the basic functional units of the cortex, each comprising about a hundred neurons. Mini-columns develop from the progenitor cells within the embryo. Until now, it was difficult to simultaneously mark multiple neural progenitors with distinct labels and track their descendants over long periods of time.

An ongoing EU-funded project, BRAINBOWAKT (Novel genetic engineering approaches for lineage analysis and exploration of Akt function in cortical development), uses a revolutionary method to track individual neurons. In the Brainbow process, the individual neurons in the brain can be distinguished from neighbouring neurons using fluorescent proteins.

Scientists developed novel genetic engineering techniques to mark multiple neighbouring progenitors and their descendants in vivo with unambiguous labels. Brainbow constructs expressing an expanded palette of trichromatic markers (red, yellow and cyan fluorescent proteins) were addressed to specific subcellular compartments. These transgenes were introduced into the embryonic mouse forebrain by electroporation. It was possible to label progenitor cells over several rounds of cell division, and to track their descendants to adult stages. Generating transgenic mice with the new Brainbow constructs permitted labelling of neural progenitors in stages and at locations where electroporation cannot be performed.

Brainbow was further developed to modulate the function of candidate proteins in vivo. The end result was a genetic mosaic in which the status of gene expression in the cells was colour coded. This approach allows for tracking neighbouring cells within the same sample with different gene expression levels. The method will help understand the influence of the key molecular regulators on cortical development.

The established multicolour multi-clonal strategy is currently being applied to the investigation of cortical mini-column formation and function of serine/threonine-specific protein kinase B (Akt), a key player in multiple cellular processes, including metabolism, survival and apoptosis.