Cerebellar folding is initiated by mechanical constraints on a fluid-like layer without a cellular pre-pattern

Models based in differential expansion of elastic material, axonal constraints, directed growth, or multi-phasic combinations have all been proposed to explain brain folding. However, the cellular and physical processes at the time of folding have not been defined. We used the murine cerebellum to challenge the standard folding models with in vivo data from the time of folding initiation. We show that at folding initiation differential expansion is created by the outer layer of proliferating progenitors expanding faster than the core. However, the stiffness differential, compressive forces, and emergent thickness variations required by elastic material models are not present. We find that folding occurs without an obvious cellular pre-pattern, that the outer layer expansion is uniform and fluid-like, and that the cerebellum is under radial and circumferential constraints. Lastly, we find that a multi-phase model incorporating differential expansion of a fluid outer layer and radial and circumferential constraints approximates the in vivo shape evolution observed during initiation of cerebellar folding. We discuss how our findings provide a new mechanistic framework to understand brain folding.