The neurogenic fate of the hindbrain boundaries relies on Notch3-dependent asymmetric cell divisions.

Elucidating the cellular and molecular mechanisms that regulate the balance between progenitor cell proliferation and neuronal differentiation in the construction of the embryonic brain demands the combination of cell lineage and functional approaches. Here, we generate the comprehensive lineage of hindbrain boundary cells by using a CRISPR-based knockin zebrafish transgenic line that specifically labels the boundaries. We unveil that boundary cells asynchronously engage in neurogenesis undergoing a functional transition from neuroepithelial progenitors to radial glia cells, coinciding with the onset of Notch3 signaling that triggers their asymmetrical cell division. Upon notch3 loss of function, boundary cells lose radial glia properties and symmetrically divide undergoing neuronal differentiation. Finally, we show that the fate of boundary cells is to become neurons, the subtype of which relies on their axial position, suggesting that boundary cells contribute to refine the number and proportion of the distinct neuronal populations.