PROJECT SUMMARY During inner ear development, multipotent otic progenitors give rise to mechanosensitive hair cells through a series of cell-fate transition points. The transcriptional cascades dictating hair cell specification have not been defined although a number of transcription factors essential for inner ear development have been identified. Using a human pluripotent stem cell-derived vestibular organoid system that allows mechanistic investigation due to its scalability, we performed a trajectory analysis of otic lineage cells using scRNA-seq. Our results suggest that supporting cells are precursors of hair cells in human vestibular organoids. Additionally, we identified LHX3 as a candidate hair cell driver gene, whose expression precedes ATOH1, a previously perceived hair cell driver. Moreover, our transcriptomic analysis between human vestibular and cochlear organoids revealed that NR2F1 is among the most differentially expressed genes in both otic progenitors and hair cells in cochlear organoids than in vestibular organoids. Based on these findings, we propose a series of experiments to define the role for LHX3 and NR2F1 in hair cell and cochlear specification, respectively. In Aim 1, using a longitudinal single-cell multi-omics approach, we will test if supporting cells are primary hair cell precursors in human cochlear organoids and if LHX3 is a pioneer transcription factor that promotes hair cell differentiation from supporting cells. To test the pioneer activity of LHX3, we will identify its target genes with CUT&RUN. We will also test if inducible expression of LHX3 alone or with another transcription factor can promote hair cell differentiation from supporting cells or prosensory cells. In Aim 2, we will define the mode of action of NR2F1 during cochlear specification. We will test if NR2F1 occupies overlapping cis-regulatory regions of target genes with GATA3. Additionally, we will test if the DNA-binding domain of NR2F1 is essential for cochlear specification and if NR2F1 regulates the timing of cochlear hair cell differentiation. Moreover, we will test if NR2F1 regulates thyroid hormone signaling. By accomplishing these aims, we will not only advance our understanding of the biology of human inner ear development, but also develop a strategy to realize hair cell regeneration in the human inner ear. Additionally, information gained from this study will be integrated into a synthetic biology and bottom-up engineering approach to recapitulate developmental trajectories and achieve better engineering control with human inner ear organoids.