Temporal patterning is an evolutionarily conserved means to generate the proper complement of neurons during brain development. Our long-term goals are to resolve temporal fate specification in the Drosophila brain and discern which mechanisms are evolutionarily conserved in mammals. Temporal patterning has been best studied in Drosophila, where temporal features of the cycling neural stem cells (NSC) are passed to the progeny. Despite decades of study on temporal pattering of the mouse neocortex, the mechanisms that drive temporal progression of cortical fates are not well understood. The Drosophila model system is a proven source for fundamental gene and mechanistic discovery and many aspects of brain development and NSC behavior are conserved between flies and mammals. Thus, investigating Drosophila temporal factors is a fitting strategy to discover candidates for mammalian study. Insulin-like growth factor 2 mRNA-binding proteins (IMP) are expressed in fly and mouse NSCs in a descending temporal gradient. By binding mRNA targets and regulating their protein levels, Drosophila Imp (together with another RNA binding protein, Syp) controls both neuronal temporal fate and the cycling and termination of NSCs. Mouse IMP1 and IMP2 similarly regulate temporal changes in NSC behavior. Nonetheless, a role for IMPs in neuronal temporal fating has not been explored in the mouse. The objectives of this proposal are to 1) utilize the Drosophila model system to gain deeper insight into temporal fating and 2) explore roles for mouse IMPs in neocortical neuronal temporal fating. This proposal combines discovery science and targeted experiments to achieve an in-depth understanding of fating mechanisms at the single-cell level. Specific Aim 1 will uncover the detailed molecular, genetic signatures of a diverse Drosophila neuronal lineage. The developmental code of neurons as they undergo temporal-fate specification will be matched with the resulting terminal code. Specific Aim 2 will explore how Imp/Syp temporal gradients and periodic Notch signaling coordinate to produce terminal neuron fates. Aims 1 & 2 exploit innovative, sophisticated genetic tools to target a specific neuronal lineage for single-cell RNAseq, reporter-based birth-order analysis, spatial transcriptomics, and targeted genetic perturbations. Specific Aim 3 will explore mouse IMPs in neocortical neuronal temporal fating and test the hypothesis that, like Drosophila Imp, mouse IMPs control temporal fate. Aim 3 exploits spatial transcriptomics to elucidate temporal changes occurring in neocortical progenitors. To test the functions of IMPs, in-utero electroporation will allow TEMPO (a tool that tracks lineage and birth order) and IMP overexpression constructs to be expressed in the neocortical progenitors. Our results will help lay the foundation to tailor neurogenesis, critical for cell-replacement therapies to treat neurological disorders. Moreover, as IMPs are implicated in other stem cells an...