Project summary Understanding brain function critically depends on knowing the identities of neural cell types. The olfactory (piriform, PCx) cortex is the main recipient of afferent inputs from the olfactory bulb and plays key roles in odor perception and memory. However, we lack a detailed molecular description of piriform cell types and the molecular machinery that defines their functional properties. Furthermore, and in contrast to sensory and motor areas in the neocortex, the genetic programs underlying PCx cell lineage specification during development remain largely unknown. A comparative analysis of cell types in the PCx and neocortex will reveal piriform- specific cell types and molecular features. Here, we propose to determine the gene expression and chromatin accessibility states of PCx neurons in the adult mouse, and to characterize the dynamic interaction of domains of regulatory chromatin and transcription factor activity that drive cell lineage specification during development. Our central hypothesis is that cell type identity in PCx is specified during development by the activity of piriform-specific gene regulatory network (GRN) activity. We further posit that GRN activity in piriform is distinct from neocortex due to the scarcity of epigenetic mechanisms for transcriptional repression. Aim 1: to identify molecularly distinct cell types in the adult olfactory cortex of mice. We will simultaneously measure gene expression and chromatin accessibility states of single cells in the mouse PCx. We predict that the molecular diversity of piriform neurons matches the diversity of their functional properties. Aim 2: to determine epigenetic mechanisms of piriform cell lineage specification during development. We will measure changes in gene expression and chromatin accessibility in developing piriform neurons and computationally reconstruct their differentiation trajectories. We predict that cell lineage specification in PCx is driven by piriform-specific dynamic interactions between domains of regulatory chromatin and associated transcription factor activity. Aim 3: to reverse engineer a gene regulatory network model for piriform cell type specification. We will infer data-driven, mechanistic GRN models for piriform and neocortex (somatosensory cortex S1) development. We will compare GRNs for PCx and neocortex using dynamical systems theory to test our model that the scarcity of repressive interactions between transcription factors drives the specification of piriform cell types. Our work will provide mechanistic insight into the epigenetic control of TF activity during neuronal differentiation and test the role of TF cross-repression in cell type specification. By integrating experimental data across PCx and neocortex, we will reveal piriform-specific cell types and the molecular features that specify the functional properties of the olfactory cortex.