Garcia-Urbano, A.; Yang, J.; Pearton, D. J.; Ahmad, M.; Cocks, G. D.; Thiery, A. P.; Zheng, T.; Streit, A.

During development axial patterning of the otic vesicle sets up the blueprint for the adult inner ear. While human inner ear organoids recapitulate the initial steps of ear formation, whether and how vesicles are properly regionalised remains unknown. Here, we combine re analysis of published single cell transcriptomic datasets with 2D and 3D imaging to assess axial patterning in human stem cell derived otic vesicles using a new PAX2-reporter line. We find that the transcriptional signatures of individual cells do not conform to clearly defined regional characteristics, with some cells co-expressing conflicting regional markers, while markers that should be co-expressed are not. Some vesicles establish spatially segregated molecular domains. However, patterning is incomplete, stochastic, and restricted to a subset of vesicles. Within the same aggregates, patterned axes lack consistent orientation. Together, our findings point to incomplete segregation of transcriptional programs that are mutually exclusive in vivo and to the absence of morphogen gradients that are crucial for otic vesicle patterning.