Acoustic signals are relayed from the ear to the brain via spiral ganglion neurons (SGNs) that receive auditory information from the cochlear inner hair cells (IHCs) and transmit that information to the cochlear nucleus of the brainstem. Physiologically distinct classes of SGNs have been characterized by their spontaneous firing rate and responses to sound and those physiological distinctions are thought to correspond to stereotyped synaptic positions on the IHC. More recently, single-cell profiling has identified multiple groups of SGNs based on transcriptional profiling; however, correlations between any of these groups and distinct neuronal physiology have not been determined. In this study, we show that expression of the POU (Pit-Oct-Unc) transcription factor Pou4f1 in type I SGNs in mice of both sexes correlates with a synaptic location on the modiolar side of IHCs. Conditional deletion of Pou4f1 in SGNs beginning in mice at embryonic day 13 rescues the early path-finding and apoptotic phenotypes reported for germline deletion of Pou4f1, resulting in a phenotypically normal development of SGN patterning. However, conditional deletion of Pou4f1 in SGNs alters the activation of Ca 2ϩ channels in IHCs primarily by increasing their voltage sensitivity. Moreover, the modiolar to pillar gradient of active zone Ca 2ϩ influx strength is eliminated. These results demonstrate that a subset of modiolar-targeted SGNs retain expression of Pou4f1 beyond the onset of hearing and suggest that this transcription factor plays an instructive role in presynaptic Ca 2ϩ signaling in IHCs.

Single cell RNA sequencing (scRNA-seq) has been recently developed to study the gene expression profile of individual cells. This high-resolution transcriptomic analysis is enabling deep insights into biological systems and disease mechanisms. Multiple techniques have been used to capture single cells, including microfluidics platforms, microtiter plates, and droplet-based technologies. These are followed by cell lysis and reverse-transcription (RT) of RNA from the individual cells. Barcoded and multiplexed cDNA are then subjected to high throughput sequencing. To apply this powerful approach for the study of skin biology, we have developed a protocol for the isolation of labeled single cells from neonatal mouse skin. Dispase is used to separate the epidermis from the dermis, with hair follicles remaining in the dermis. Enzymatic digestion then generates single cell suspension, and fluorescently-labeled single cells are sorted into microtiter plates using fluorescence-activated cell sorting (FACS). We have optimized this approach to maximize post-sort cell viability and cDNA purification for scRNA-seq library preparation. Taken together, this approach allows for the collection of high quality scRNA-seq data to analyze the biology of lineage specific cells from neonatal mouse skin.