Project Summary The mammalian central nervous system contains unique projections from the cerebral cortex thought to underpin complex motor and cognitive skills. These projections include the corticospinal tract. involved in motor function, and the corpus callosum, involved in executive function. The cerebral cortical neurons that give rise to the corticospinal tract and corpus callosum are, respectively, corticospinal motor neurons and callosal projection neurons. These neurons develop from the same progenitor pool at the same time in development, but they acquire strikingly different projections to serve strikingly different behaviors. The mechanisms whereby corticospinal and callosal projection neurons develop represents a fundamental open question in basic neuroscience: how do the unique projections underlying complex mammalian behaviors arise? While it is known that transcription factors play a critical role in the fate of corticospinal, callosal, and other cortical projection neurons, the contribution of other gene regulatory mechanisms is poorly understood. We posit that a genomic cluster of selectively expressed microRNAs are epigenetically co-regulated in corticospinal motor neurons (Aim 1 ), where they in turn co-regulate axon guidance pathways (Aim 2) to favor corticospinal over callosal projection neuron fate (Aim 3). We will employ deep sequencing and in vivo functional perturbation studies to test our hypothesis. This microRNA-mediated specification of fate represents a novel mechanism of cortical projection neuron development, with implications not only for fundamental mechanisms of cortical development, but also for understanding the evolution of the complex connectivity of the mammalian central nervous system.