ORGANOID ASSEMBLOIDS REVEAL SEROTONERGIC REGULATION OF HUMAN CORTICAL DEVELOPMENT

Serotonin (5-HT) influences brain development long before it functions as a neurotransmitter, yet how early serotonergic signaling regulates human cortical progenitor populations remains poorly understood. It acts initially via maternal and peripheral sources on neuroepithelial (NE) cells and later through endogenous release from the raphe nuclei, with disruption implicated in neurodevelopmental disorders (NDDs).
Here, we combine controlled pharmacological approaches with organoid-based systems to dissect serotonergic regulation of human corticogenesis across developmental stages. Using human NE cells and forebrain organoids, we first defined receptor- and cell type–specific responses under controlled conditions. 5-HT selectively promoted proliferation of early NE cells as well as apical and basal radial glia (aRG and bRG) via distinct receptor subtypes: NE cells and aRG responded through 5-HTR2C, whereas bRG were regulated by 5-HTR2A. Intermediate progenitors were unaffected.
To model endogenous serotonergic input, we developed for the first time raphe organoids, enriched in serotonergic progenitors and neurons. Single-nucleus RNA-sequencing (snRNA-seq) validated serotonergic lineages, and functional imaging using the sDarken sensor demonstrated activity-dependent 5-HT release. Fusion of raphe and forebrain organoids produced raphe–forebrain assembloids that recapitulated physiological serotonergic innervation of the developing cortex, with increased progenitor proliferation in innervated regions. snRNA-seq profiling of raphe–forebrain assembloids revealed coordinated molecular programs across cortical progenitors and neurons, consistent with inter-regional signaling and emerging circuit integration.
Together, this work establishes human raphe organoids as a novel experimental system and reveals stage- and receptor-specific serotonergic control of human cortical progenitor expansion, providing insight into early developmental mechanisms relevant to NDDs. ​