Final Report Summary - BRAINBOWAKT (Novel genetic engineering approaches for lineage analysis and exploration of Akt function in cortical development) The mammalian brain develops through an elaborated molecular and cellular choreography. Imbalance in this process can lead to neurodevelopmental disorders. Key questions in neuroscience remain poorly explored regarding the contribution of multiple cortical progenitors to the formation of cerebral cortex basic functional units (minicolumns). To fully understand the development of minicolumns, it is essential to characterize the behavior of several neighboring family of cells simultaneously. However, due to technical limitations, it is difficult to mark multiple nearby neural progenitors with distinct labels and track their descendants over long periods of time. In departure from traditional single cell or clone labeling methods, new approaches using color markers allow one to distinguish multiple cells within a tissue and to compare their potentialities. In particular, a series of recent lineage studies performed in non-neural tissues have demonstrated the considerable potential of multicolor lineage tracing with brainbow-type transgenes. Yet the restricted palette of markers or the non-ubiquitous expression of current brainbow/confetti mouse lines prevents one from efficiently applying this strategy in the complex environment of the developing brain. To achieve this, one needs the largest possible number of heritable markers and a way to target them to cortical progenitors. The method that we propose tackles these issues, opening the way to simultaneous tracking of multiple neural stem cells and providing a methodological framework for these multiclonal analyses. During the four-year period of the funded project, the fellow has successfully established a multiclonal strategy critical to her investigation of cortical minicolumn formation by developing novel genetic engineering techniques to mark multiple neighboring progenitors and their descendants in vivo with unambiguous labels (Loulier et al, Neuron, 2014). New Brainbow constructs expressing an expanded palette of trichromatic markers (red, yellow and cyan fluorescent proteins) addressed to specific subcellular compartments have been generated and can be introduced by electroporation in the embryonic mouse forebrain. Coupling this approach with genome-integrative transposon vectors, color contrast and marker combinations allows one to label progenitors over several rounds of cell division, and to track neural stem cells and their descendants from embryonic to adult stages, achieving therefore the labeling of entire families of cortical cells. The need to implement novel strategies to overpass the limitations of multicolor tools available at the time the funded project was launched has led to deviations, and subsequent delays, that have forced us to reorient the objectives for the second period. In order to resolve and track clones originating from multiple neighboring progenitors, the fellow has generated transgenic mouse lines expressing cytoplasmic or nuclear versions of the Magic Markers constructs, or a combination of both, under the broadly active CAG promoter. Magic Markers mice display widespread expression of the blue fluorescent protein expressed by default in various embryonic and adult tissues and multiple distinct cell types. Intercrossing with tamoxifen-inducible Cre mice or in utero electroporation with a Cre expressing plasmid yields robust multicolor labeling of both early- (progenitors) and late-born neural cells (neurons, astrocytes, oligodendrocytes) from embryonic to adult stages. Following early and sparse recombination in the dorsal telencephalon, heterogeneous columns of clonally related cells displaying distinct combinations of color labels are obtained. Thus, these mice provide new insights on the heterogeneous behavior of neural progenitors and their progeny by shedding light on how distinct nearby cortical progenitors cooperate to build an operative brain. Besides its complex cellular events, the molecular actors involved in cortical cytoarchitecture formation remain also to be characterized. The fellow has successfully established the proof of principle of color-coded molecular mosaic by generating a Magic Markers construct whose genomic expression creates a genetic mosaic in which the status of cells regarding the molecular perturbation is color-coded. Before focusing on Akt signaling, she has undertaken further validation of the color-coded molecular mosaic by generating several Magic Markers constructs whose genomic expression creates a genetic mosaic inducing either loss or gain of function of two distinct signaling pathways involved in either the proliferation (dnLGN) or the tangential migration (caEphrinB1) of cortical progenitors and descendants. In conclusion, during the course of the funded project, the fellow has published a part of her work in a leading journal of her field, and has provided the scientific community with useful multicolor mice and strategies critical to investigate the clonal architecture of intact tissues in numerous biological contexts. Finally, the recipient of the present IRG, has obtained a permanent research associate position at INSERM in 2012 that offers her the opportunity to complete successfully in the upcoming years her studies aiming at deciphering the cellular and molecular events involved in physiological and pathological cortical development using multicolor lineage tracking strategies. This work meets strong expectations in the neurobiology field, first by transposing the brainbow approach to neurodevelopment, and second by providing a practical and broadly applicable clonal tracking methodology. This scheme is easy to implement and can have versatile applications including clonal tracking, cytoarchitecture studies, or genetic mosaic analysis and should therefore rapidly diffuse into the developmental and cancer biology communities.