Project Summary During development, the vast majority of the peripheral nervous system (PNS) is generated from neural crest cells that migrate extensively into the embryo and give rise to the sympathetic, parasympathetic, sensory and enteric nervous systems. Sympathetic nervous system (SNS) function is tightly linked to heart rate, blood pressure and temperature control. During development the sympathetic ganglia (SG) must move and interact with axons that project from preganglionic neurons (PGNs) in the ventral spinal cord to wire the circuit between the central nervous system (CNS) and PNS. Tissue repair or regeneration therapies for birth defective or damaged neural connections between the CNS and SNS offer a potential therapeutic strategy, however, will require the molecular signature of PGN and SG cell populations prior to clinical translation. Furthermore, one clinically significant example of mistakes in SNS development is neuroblastoma cancer in infants. Here, we explore an innovative strategy of novel spatial transcriptomics approaches, integrated multiplexed RNA/protein detection and visualization, and computational algorithms to uniquely identify and map molecular markers of the PGN and SG progenitor cell populations to the tissue architecture of the chick trunk anatomy. If successful, this novel platform will provide a transformative foundation for basic research of peripheral nervous system birth defects and repair using stem cell-based therapies, and future studies of neuroblastoma initiation.