Minimal essential requirements for neural tube self-organisation

The reliable generation of diverse cell types in precise proportions is essential for the formation of functional tissues during embryonic development. Three-dimensional organoid models derived from pluripotent stem cells (PSCs) provide powerful systems for identifying principles governing tissue self-organisation. Neural tube organoids (NTOs), initiated from single PSCs with a pulse of retinoic acid (RA), self-organise into structures containing floorplate cells that secrete SHH morphogen to pattern adjacent neural tissue. Yet, how the initial cellular diversity arises and how appropriate cell-type proportions are allocated has remained unclear. Here, using time-resolved single-cell transcriptomics, quantitative immunofluorescence, and dynamical systems modelling we show that RA triggers a transient co-expression state of the transcription factors PAX6 and FOXA2 from which cells asynchronously resolve into two opposing fates: PAX6 neural precursors and FOXA2 floorplate precursors. PAX6 and FOXA2 are both necessary and sufficient to reconstitute self-organisation, establishing these transcription factors as key determinants of emergent tissue pattern. Rather than operating as a simple feed-forward system in which cells are guided solely by RA, feedback between the alternative cell fates, mediated by BMP signalling from floorplate precursors, determines cell type proportions and ensures reproducible cell-type diversity in each NTO. This dual expression state was also identified in mouse embryos, demonstrating how in vitro models inform in vivo biology. These findings establish a general design strategy - symmetry breaking through opposing fate determinants coupled to proportioning via signal feedback control - that may operate broadly across developmental contexts to generate tissues with predictable cellular compositions.