Project Summary/Abstract Learning how spontaneous neuronal activity shapes embryonic brain development is critical for understanding neurodevelopmental processes, with implications in neurological or neuropsychiatric disorders. Early sporadic activity is essential for the formation of mature correlated neuronal networks. To date, we have had success in identifying the motor circuit as the first circuit to function in a developing zebrafish embryo. In addition, a few neurons, the “leader cells”, are the first neurons to obtain spontaneous neuronal activity. This spontaneous activity is followed by the formation of small synchronized neuronal networks that elaborate into complex circuits as development progresses. However, due to their sparsity and transient nature, molecular characterization of leader cells has been a challenge. This proposal aims to combine zebrafish genetics with powerful new methods for single-cell transcriptomic profiling to deliver a high-resolution map of activity-dependent development and to use it to identify activity-dependent gene programs in a cell-type specific manner and profile leader cells. The proposal focuses on integrating new technologies into discovering how the embryonic vertebrate brain develops in an activity-dependent manner. The aims are to perform comprehensive molecular profiling of the leader cells as they acquire spontaneous activity in the developing vertebrate nervous system, identify potential genetic regulators (Aim 1), and assess how perturbations to leader cell activity shape circuit maturation and brain development (Aim 2). The proposed work leverages recent developments from several disciplines: single-cell RNA sequencing (scRNA-seq), zebrafish genetics, and optogenetics. Specifically assessing how the brain develops during health and when the motor circuit, the first circuit to function in the zebrafish nervous system, is disrupted. The ideal setting to carry out this proposal is with the co-mentoring of Dr. Wagner, who developed TRACERSEQ and STITCH to map the quantitative relationship between cell states and lineages in healthy and perturbed contexts using quantitative biology techniques and Dr. Kriegstein, an expert in development, genomics, and stem cell biology. Overall, these studies will lead to general insights into how transcriptional profiles of neuronal and non-neuronal cells coordinate to create a healthy brain with proper cell proliferation and differentiation in an activity-dependent manner.