Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium

Abstract

Endocytosis is a vital process, required during development and for maintenance of tissue homeostasis, that allows cells to internalize a wide range of molecules from their environment as well maintain their plasma membrane composition. The ability to visualise endocytosis in vivo requires suitable assays to monitor the process. Here, we describe imaging-based assays to visualize endocytosis in the neuroepithelium of living zebrafish embryos. These assays rely on injection of fluorescent tracers into the brain ventricles followed by live imaging and can be used to study fluid-phase or receptor-mediated endocytosis, for which we use receptor-associated protein (RAP) as a ligand for LDL receptor-related protein (LRP) receptors expressed at the neuroepithelium. Using dual colour imaging combined with transient or stable expression of endocytic markers, it is possible to track the progression of endocytosed tracers and to monitor trafficking dynamics. Using these assays, we reveal a role for the Lowe syndrome protein Ocrl in endocytic trafficking within the neuroepithelium. We also find that the RAP binding receptor Lrp2 appears to only partially contribute to neuroepithelial RAP endocytosis. Altogether, our results provide a basis to track endocytosis within the neuroepithelium in vivo, and support a role for Ocrl in this process. Summary statementWe describe live imaging assays to analyse endocytosis in the zebrafish neuroepithelium and show involvement of the inositol phosphatase OCRL in this process