show Abstracthide AbstractThe brain executes control of nearly all bodily functions via spinal projecting neurons (SPNs) that carry command signals from numerous supraspinal regions to the spinal cord. Despite their physiological and clinical relevance, a comprehensive molecular characterization of SPNs is still lacking. Here, we use retrograde labeling, whole-brain imaging, and high-throughput transcriptional profiling to generate a unified brain-wide anatomic and transcriptomic atlas of adult mouse SPNs at single-cell resolution. We transcriptionally profiled a total of 65,002 SPNs, identified 76 region-specific SPN types, and mapped these types into a companion atlas of the whole mouse brain generated by the Allen Institute for Brain Science within the BRAIN Initiative Cell Census Network (BICCN). This SPN taxonomy reveals a three-component organization of SPNs: (1) molecularly homogeneous excitatory SPNs from the cortex, red nucleus, and cerebellum with somatotopic spinal terminations suitable for point-to-point communication; (2) highly heterogeneous excitatory and inhibitory populations in the reticular formation with broad spinal termination patterns, thus suitable for relaying commands related to the activities of the entire spinal cord; and (3) modulatory neurons expressing slow-acting neurotransmitters and/or neuropeptides in the hypothalamus, midbrain, and reticular formation for gain control of brain-spinal signals. Within each of these components, this atlas revealed additional insights. From components (2) and (3), we discovered a LIM homeobox transcription factor code that parcellates the most transcriptionally complex population, reticulospinal neurons, into five molecularly distinct and spatially segregated populations. For neurons in component (1), we found transcriptional signatures of a subset of SPNs with large soma size and correlated these with fast-firing electrophysiological properties; thus, at least two different cable lines (namely, Pvalb/Kcng4/Spp1 positive and negative) with different electrophysiological properties might underlie the transmission of brain signals to the spinal cord. Together, by integrating the anatomy, molecular identity, and physiological properties, this study establishes a comprehensive taxonomy of brain-wide SPNs and provides insight into the functional organization of SPNs in mediating brain control of bodily functions. Overall design: Spinal projecting neuron (SPN) nuclei of C57BL6/J mice were retrogradely labeled via injection of recombinant retrograde adeno associated virus (rAAV2/retro-Syn-H2B-fluorescent protein) into multiple segments centering the cervical (GFP) and lumbar (mScarlet) spinal cord of postnatal day 42 C57BL/6J mice. At postnatal day 56, the mice were sacrificed and nuclei were isolated via tissue dissection and dissociation of brain regions containing SPNs (i.e., rostral forelimb area cortex [RFA], primary and secondary motor cortex / primary somatosensory cortex [M1M2S1], secondary somatosensory cortex [S2], hypothalamus [HY], midbrain [MB], cerebellum [CB], pons [PONS], and medulla [MED]). Subsequently, SPNs were enriched via fluorescence activated nucleus sorting. Nuclei were then analyzed with single nucleus RNA sequencing using the 10x genomics and SmartSeq platforms.