To enable powerful genetic mosaic investigation of gene function in complex systems? without current limitations of gene location or toxicity? in particular for development and diversity of neuronal subtypes and their complex circuitry in cerebral cortex, we propose to develop and implement two entirely novel, innovative, inter-related systems for genetic mosaic functional analysis. These systems enable binary? all-or-none, neuron-by-neuron ?aleatory?? random, mosaic analysis with control over ratios of wt and genetically manipulated cells. The proposed work will substantially extend the range of tools available for mosaic analysis. We first propose to further develop and adapt for AAV viral use a new plasmid-based transfection system, BEAM (for binary expression aleatory mosaic), which relies on sparse recombinase activation to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Since all requisite plasmids can be delivered by electroporation or viral transduction, BEAM can be used directly on wild-type or floxed mice, without the need for complex breeding schemes. We also propose to engineer a Rosa26 reporter allele in mice, R26BEACON (for binary expression aleatory cre-operated nested mosaic), which will use Flp to stochastically recombine incompatible frt-site variants, thereby generating green cells that express Cre (EGFP-positive;Cre-positive) and red cells that do not express Cre (tdTomato-positive;Cre-negative). Because green cells express Cre, R26BEACON can be used to delete genes of interest using existing floxed alleles, and to activate expression of effector molecules using existing Rosa26 alleles for cell ablation, modulation of membrane potential, and transcellular labeling. In addition, unlike existing systems of MADM and MASTR, which can be activated only sparsely, R26BEACON will be able to be activated efficiently throughout the entire organism, or in a specific organ or cell type of interest. The motivating biological goals of the proposed work are both to elucidate central molecular controls and regulatory mechanisms over development, subtype diversity, circuit formation, and potential regeneration of cortical projection neurons (PN), and to identify potential causes and therapeutic approaches to dysgenesis and disease involving PN. These innovative BEAM and R26BEACON mosaic systems will uniquely enable new discovery in multiple fields. Toward Aim 1, we have completed highly motivating studies developing BEAM as an already successful approach for electroporation. We now propose to further develop and validate the BEAM plasmid system, and to generate an AAV virus-based BEAM system, 1) testing fidelity of reporters and recombination status, 2) analyzing cell autonomy, and 3) generating an AAV-based BEAM. Toward Aim 2, we have already generated a R26BEACON targeting construct by placing an operator module in tandem with a reporter module, which labels cells red in the presence of Flp, and green in either the presence of Cre alone or Cre and Flp in combination. We propose to 1) generate R26BEACON mice, and 2) validate with floxed alleles. BEAM and R26BEACON are novel and innovative. This work advances the tradition of cutting edge recombinase-based genetic approaches such as MADM, Brainbow, and other genetic analysis tools generated by the neuroscience community, enabling investigation of CNS complexity and diversity. Our deep investigation of PN development, diversity, and connectivity provide the foundation to innovatively and rigorously develop and implement BEAM and R26BEACON.