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Summary: We explore the changes in chromatin accessibility and transcriptional programs for cochlear hair cell differentiation from postmitotic supporting cells using organoids from postnatal cochlea. The organoids contain cells with transcriptional signatures of differentiating vestibular and cochlear hair cells. Construction of trajectories identifies Lgr5+ cells as progenitors for hair cells, and the genomic data reveal gene regulatory networks leading to hair cells. We validate these networks, demonstrating dynamic changes both in expression and predicted binding sites of transcription factors (TFs) during organoid differentiation. We identify known regulators of hair cell development, Atoh1, Pou4f3, and Gfi1, and the analysis predicts the regulatory factors Tcf4, an E-protein and heterodimerization partner of Atoh1, and Ddit3, a CCAAT/enhancer-binding protein (C/EBP) that represses Hes1 and activates transcription of Wnt-signaling-related genes. Deciphering the signals for hair cell regeneration from mammalian cochlear supporting cells reveals candidates for hair cell (HC) regeneration, which is limited in the adult.

Lgr5+ intestinal stem cells (ISCs) drive epithelial self-renewal, and their immediate progeny—intestinal bipotential progenitors—produce absorptive and secretory lineages via lateral inhibition. To define features of early transit from the ISC compartment, we used a microfluidics approach to measure selected stem- and lineage-specific transcripts in single Lgr5+ cells. We identified two distinct cell populations, one that expresses known ISC markers and a second, abundant population that simultaneously expresses markers of stem and mature absorptive and secretory cells. Single-molecule mRNA in situ hybridization and immunofluorescence verified expression of lineage-restricted genes in a subset of Lgr5+ cells in vivo. Transcriptional network analysis revealed that one group of Lgr5+ cells arises from the other and displays characteristics expected of bipotential progenitors, including activation of Notch ligand and cell-cycle-inhibitor genes. These findings define the earliest steps in ISC differentiation and reveal multilineage gene priming as a fundamental property of the process.