Single-cell multi-omic atlas and morphogen screening informs midbrain and hindbrain organoid engineering.

Patterning of the neural tube establishes midbrain and hindbrain structures that coordinate motor movement, process sensory input and integrate cognitive functions. Cellular impairment within these structures underlies diverse neurological disorders, and in vitro organoid models promise inroads to understanding development and modeling disease. Here, we use paired single-cell transcriptome and accessible chromatin sequencing to map cell composition and regulatory mechanisms in organoid models of midbrain and hindbrain. We find that existing midbrain organoid protocols generate ventral and dorsal cell types, covering regions including floor plate, dorsal and ventral midbrain and adjacent hindbrain regions. Gene regulatory network inference and transcription factor perturbation resolve mechanisms underlying neuronal differentiation. A single-cell multiplexed patterning screen identifies morphogen concentrations that expand existing organoid models, including conditions generating medulla glycinergic neurons and cerebellum glutamatergic subtypes. Together, the multi-omic atlas and morphogen screen reveal morphogen-regulon relationships guiding region-specific progenitor differentiation towards diverse neuron types of the posterior brain.