A spatially-resolved transcriptional atlas of the murine dorsal pons at single-cell resolution

The “dorsal pons”, or “dorsal pontine tegmentum” (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard (http://harvard.heavy.ai:6273/) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data. Overall design: Single nuclei were isolated from 1mm thick bilateral dissections of the dorsal pontine tegmentum (PnTg). To guide the dissection of the dorsal PnTg, we selectively labeled two different brain regions, the Parabrachial nucleus (PB) and the Barrington nucleus (Bar) (also known as PMC), that define its extension. A total of 9 and 8 batches (3-5 mice each) of male and female mice, respectively, of age between 8 and 10 weeks old, were used for the DroNc-seq assay. In total, 447,833 nuclei were recovered.