Project Summary The principal goal of this new grant cycle is to uncover the origin and nature of uniqueness of the cerebral cortex of the human, and non-human primates (NHP), particularly the association areas such as the prefrontal cortex, that underlie the highest level of our cognitive capacities and are thought to be undermined in neuropsychiatric disorders including drug abuse. Although basic principles of cortical development in all mammals are similar, there are important quantitative and qualitative evolutionary innovations that were introduced in primates since their split from the rodent lineage about 60 million years ago. Thus, our strategy will be to study in parallel the developmental events in the NHP (macaque) and human skin-cell-derived iPSC cortex-mimicking cultures by using the most advanced molecular and cell biological methods available, including comparative high resolution single-cell mRNA sequencing, advanced confocal and light-sheet imaging, and in utero and ex utero functional manipulation of neural stem cells. We will complete and further augment our ongoing high-resolution single-cell mRNA sequencing and gain insight through bioinformatic gene network analysis in three species: macaque, human, and mouse. We will first investigate the genetic determinants of primate- specific stem cell subtypes, using gene clustering analysis at the transcriptome level, and determine how this impacts the cortical expansion process through functional studies (Aim #1). As a next step, we will examine genes and regulatory elements involved in emergence of areal and laminar specific distinction in monkey embryonic cortex and human iPSC 3D cortex mimicking cultures, generated from skin fibroblasts, to identify unique signatures of human and nonhuman primate-specific gene expression in evolutionarily new cortical areas such as prefrontal cortex (Aim #2). Finally, we will examine how cortical neuropil and the growth and patterning of white matter contribute to cortical area differences and drive primate-specific cortical gyrification (Aim #3). Although the proposed research is time-consuming, logistically difficult and costly, it is essential if we are to understand the biological basis of our humanity and the pathogenesis of some high order cognitive disorders, including drug abuse. We have established primate precisely timed breeding facilities at Yale, as well as mastered and modified essential methodology and already have obtained a substantial amount of high impact information which alone, or in combination, can give insight into elusive neuropsychiatric disorders and neuronal susceptibility to prenatal exposure to drugs of therapy and abuse.