Genetically-defined lineage tracing of Nkx2.2-expressing cells in chick spinal cord

AbstractIn the spinal cord, generation of oligodendrocytes (OLs) is totally dependent on the presence of Olig2, a basic helix-loop-helix transcription factor. However, it also requires Nkx2.2 for its generation, whose expression follows the expression of Olig2. Although it is believed that oligodendrocytes originate from the pMN domain, Nkx2.2 is present in the p3 domain located ventral to the pMN domain. According to recent reports, it is possible that oligodendrocytes are directly derived from the p3 domain in addition to the pMN domain in the chick spinal cord. We examined this hypothesis in this paper.To analyze OL development in the spinal cord, chick embryos are widely used for genetic modification by electroporation or for transplantation experiments, because it is relatively easy to manipulate them compared with mouse embryos. However, genetic modification by electroporation is not appropriate for glial development analyses because glia proliferate vigorously before maturation. In order to overcome these problems, we established a novel method to permanently introduce exogenous gene into a specific cell type. We introduced the CAT1 gene, a murine retroviral receptor, by electroporation followed by injection of murine retrovirus. By using this method, we successfully transduced murine retrovirus into the chick neural tube. We analyzed cell lineage from the p3 domain by restricting CAT1 expression by Nkx2.2-enhancer and found that most of the labeled cells became OLs when the cells were labeled at cE4. Moreover, the labeled OLs were found throughout the white matter in the spinal cord including the most dorsal spinal cord. Thus p3 domain directly generates spinal cord OLs in the chick spinal cord