Live-imaging of endogenous neurofascins reveals glial adhesion shapes developing nodes of Ranvier

The organisation of myelinated axons into specialised domains is essential for saltatory conduction and depends on the polarised distribution of neuronal and glial cell-adhesion molecules, including neurofascins. However, how these domains assemble and refine in vivo remains unclear, as longitudinal imaging of endogenous proteins within intact nervous systems remains challenging. Here, we generated knock-in zebrafish in which endogenous neuronal and glial neurofascins are fused to fluorescent proteins, which enabled live-imaging without perturbing node assembly or introducing overexpression artefacts. Using these reporters, we visualised neuronal and glial neurofascin dynamics, and the formation of nodes of Ranvier and paranodes in vivo. We find that nascent nodes progressively compact in developing peripheral nerves, and that this process depends on glial neurofascin. Our novel toolkit for imaging endogenous neurofascins reveals a glia-dependent mechanism that shapes the developmental refinement of nodal architecture after their initial assembly. Our findings imply that glia-driven modulation of paranodes can be a mechanism for nervous systems to regulate circuit function.