Midline assembloids reveal regulators of human axon guidance

Organizers orchestrate cell patterning and axon guidance in the developing nervous system. Although nonhuman models have led to fundamental discoveries about floor plate (FP)–mediated midline organization, an experimental model of the human FP would enable insights into human neurodevelopment and midline connectivity. Here, we developed organoids resembling human FP (hFpOs) and assembled them with human spinal cord organoids (hSpOs) to generate midline assembloids (hMAs). We demonstrate that hFpOs promote ventral patterning, commissural axon guidance, and bilateral connectivity. To investigate midline regulators, we profiled the hFpO secretome, identifying 27 human-enriched genes compared with mouse. In an arrayed CRISPR screen of hMAs, we discovered that loss of GALNT2 and PLD3 impaired FP–mediated guidance of axons. This platform holds promise for revealing aspects of human-specific neurobiology and disease.