PROJECT SUMMARY/ABSTRACT The majority of congenital hearing loss cases are sensorineural (SNHL), and at least half are associated with genetic defects. Unfortunately, regenerative medicine and gene therapy for inner ear diseases have generally not been realized in human patients due to the inability to ethically experiment on large numbers of human embryos in a scientifically rigorous manner. This also means that key knowledge gaps exist in the overall understanding of the early development of human sensory organs. In the case of the inner ear, it is known that proper cell fate commitment to the otic placode is essential for inner ear formation. Both sensory epithelia, where hair cells are located, and the vestibulocochlear nerve are derived from the otic vesicle, which is invaginated from the otic placode; defects in hair cells and/or the vestibulocochlear nerve result in SNHL. It is difficult to study early otic development, specifically during the induction and patterning of the pre-placodal ectodermal (PPE), where otic placodes are derived from, in mammalian embryos (not to mention human embryos). This study aims to 1) examine the role of WNT signaling pathways in early otic development, with a focus on the PPE, and 2) determine how cell fate decisions in the PPE culminate in the origin of the otic lineage in human inner ear organoids. In Aim 1, the effects of WNT signaling on otic development will be examined, with the goal of identifying the optimal level of WNT that maximizes inner ear organoid induction. Single-cell RNA sequencing (scRNA-seq) analyses in the human inner ear organoid system will reveal the early otic lineage progression and mechanisms underlying the processes mediated by WNT. In Aim 2, spatial gene expression profiles of organoids treated with the optimal level of WNT modulation will be created. Upon integrating these spatial data with the temporal scRNA-seq data from Aim 1, a cell atlas of the early otic lineage in human inner ear organoids will be established. These spatio-temporal transcriptomic data will then be used to create a genetic blueprint for early human otic development, with a focus on characterizing the gene regulatory networks that are critical to cell fate commitments underlying proper cranial sensory organ development. Such data will 1) advance human developmental biology, 2) generate a molecular database for understanding congenital disorders associated with ectodermal derivatives (including the inner ear), and 3) progress regenerative medicine and gene therapy for inner ear diseases.