3D reconstruction of the mouse cochlea from scRNA-seq data suggests morphogen-based principles in apex-to-base specification

In the mammalian auditory system, frequency discrimination depends on numerous morphological and physiological properties of the organ of Corti that gradually change along the longitudinal (tonotopic) axis of the organ. For example, the basilar membrane stiffness changes tonotopically, thus affecting the tuning properties of individual hair cells. At the molecular level, those frequency-specific characteristics are mirrored by gene expression gradients; however, the molecular mechanisms controlling tonotopic gene expression in the mouse cochlea remain elusive. Through analyzing scRNA-seq data from two developmental time points, we predicted that morphogens, rather than a timing-associated mechanism, confer spatial identity in the cochlea. Subsequently, we reconstructed the developing cochlea in 3D space from scRNA-seq data to investigate the molecular pathways mediating tonotopic information. The retinoic acid and sonic hedgehog pathways were found to form opposing tonotopic gradients, and functional interrogation using mouse cochlear explants suggested that both pathways jointly specify the tonotopic axis during development. We investigated the transcriptional heterogeneity from the developing cochlear duct at embryonic day (E)12.5 and E14.5 via single-cell RNA sequencing (scRNA-seq). Inner ears samples were dissected from the Sox2-GFP mouse line and cochlea was separated from vestibular compartments. Cochlea samples were digested to single-cell level and subjected to fluorescence activated cell sorting of Sox2-GFP+ cells. For each time point, two independent libraries were generated using the 10x Genomics platform.