Development of an Embryonic Zebrafish Oligodendrocyte-Neuron Mixed Coculture System

During vertebrate neural development, oligodendrocytes insulate nerve axons with myelin sheaths. Zebrafish (Danio rerio) has emerged as a useful model organism for studying oligodendrocyte development. However, the absence of an in vitro culture system necessitates in vivo manipulations and analyses, which, in some instances, limits the questions that can be addressed. To fill this gap we developed a mixed coculture system for embryonic zebrafish neurons and oligodendrocyte-lineage cells. Cultures harvested from embryos ≥30 hours postfertilization (hpf) yielded oligodendrocyte progenitor cells (OPCs) positive for olig2 and sox10 transgenic reporters. Cultured OPCs exhibited dynamic, exploratory membrane processes, and cell morphologies resembled those established in vivo. Cells harvested from advanced stage embryos possessed more arborized processes than those from early stage embryos. Advanced stage (>60 hpf) embryo culture produced differentiated, mbp⁺ oligodendrocytes. Genetically tractable neuron subtypes extended neurites when harvested from embryos ≥19 hpf. Coculture produced juxtaposed oligodendrocytes and neurons, demonstrating the practical usefulness of this technique for future studies examining axon-oligodendrocyte interactions under defined conditions. We expect that zebrafish oligodendrocyte culture will complement existing in vivo strengths and may facilitate future studies elucidating the mechanisms of oligodendrocyte specification, proliferation, differentiation, motility, and axon-oligodendrocyte interactions that shape adult myelination patterns.