Piggybac Transposon-Based Lineage Tracing to Reveal Ependymogenesis

The lateral walls of the lateral ventricle of the brain are home to one of the two main neurogenic niches of the postnatal and adult mammalian brain, the ventricular-subventricular zone (V-SVZ). During late embryonic development (E14 to E16 in mouse brain), a subpopulation of radial glial cells from the neuroepithelial lining of lateral ventricle wall starts ependymogenesis to establish an ependymal cell lining of the ventricular system. Ependymal cell maturation and ciliogenesis continues after birth and the remaining radial glia, designated neural stem cells after birth, are established within the ependymal lining by retention of a thin apical process. The postmitotic multi-ciliated ependymal cells provide structural and trophic support to the V-SVZ stem cell niche. We sought to investigate changes to the V-SVZ stem cell niche over the course of mouse brain development and in particular to determine the association between ependymogenesis, stem cell number and stem cell niche organization at the ventricle surface. Our piggybac transposon-based lineage tracing method, through sparse labeling, shows neural stem cells and ependymal cells have a common origin and ependymal cells can arise from radial glial cells via both symmetric and asymmetric division. Using IUE in conjunction with thymidine analog double-labeling, we propose that from a single radial glial cell multiple rounds of division are involved in the process of ependymogenesis and terminal symmetric ependymal generation from neural stem cells reduces neural stem cell numbers. At the time of ependymal cell formation, the new ependymal cell lining displaces the remaining radial glia/stem cell somata to the subventricular zone (SVZ). These remaining ventricular subventricular zone (V-SVZ) stem cells, are arrayed in clusters and maintain only a thin apical process at the ventricle surface. In the en face wholemount view of the lateral wall of the lateral ventricle, the stem cell apical processes surrounded by ependymal cells are referred to as ‘pinwheels’ and represent regenerative units. To further understand the composition of the distinct stem cell retention pattern in the form of a pinwheel in the lateral wall of the lateral ventricle of the rodent brain, we have used the piggybac multicolor fluorophore together with in utero electroporation technique for lineage tracing analysis