The prenatal period of cortical development is important for the establishment of neural circuitry and functional connectivity of the brain; however, the molecular mechanisms underlying this process remain unclear. Here we report that disruption of the actin-cytoskeletal network in the developing mouse prefrontal cortex alters dendritic morphogenesis and synapse formation, leading to enhanced formation of fear-related memory in adulthood. These effects are mediated by a brain-enriched microRNA, miR-9, through its negative regulation of diaphanous homologous protein 1 (Diap1), a key organizer of the actin cytoskeletal assembly. Our findings not only revealed important regulation of dendritogenesis and synaptogenesis during early brain development but also demonstrated a tight link between these early developmental events and cognitive functions later in life.miR-9 | learning | memory | Diap1 | dendritogenesis P erturbation during the critical period of perinatal cortical development influences the functional connectivity of the brain and can lead to an increased propensity toward neurological disorders such as anxiety, schizophrenia, and autism spectrum disorders (1-3). Neuronal maturation involves distinct, highly regulated events, including neuronal differentiation and migration, dendritogenesis, axon formation/guidance, and synaptogenesis, among others. However, precisely how these events are regulated and how they orchestrate brain development and cognitive function remain largely unknown.Fear-related learning and memory play a significant role in the development of anxiety disorders. Cortical dysfunction is associated with emotional disturbances, which are underpinned by impaired fear extinction, and an inefficient termination of physiological stress responses (4, 5). The medial prefrontal cortex (mPFC) is a primary mediator of fear-related learning and memory (6, 7). It is evident that the actin cytoskeleton is involved in synaptic plasticity and neuronal morphogenesis underlying the formation of fear memory. For example, a disruption in the actin cytoskeleton assembly in the adult brain impairs both cued and contextual fear conditioning (8-10), and several actin-regulatory proteins have been shown to be involved in synaptic plasticity and neuronal morphogenesis associated with memory formation (11-15).Small noncoding RNAs, and miRNAs in particular, have emerged as a major regulatory mechanism that precisely controls the level of gene expression. In invertebrates, miRNAs play essential roles in regulating developmental timing. For example, in Caenorhabditis elegans the succession of certain cell fates from first to second larval stage relies on the induction of miRNA lin-4 expression at the first larval stage and reduction of lin-14 activity via base-pairing interactions with its 3′ UTR (16,17). In mice, miRNAs have been shown to be involved in rapidly fine-tuning the expression of their target mRNAs and in regulating cognitive function (18,19). In our study, we found that the expression of the actin polyme...